Ecology and genetics of tropical rhizobia species

Ecology and genetics of tropical rhizobia species

Biotech Advs Vol. 3, pp 155-170, 1985 Printed in Great Britain. All Rights Reserved. 0734-9750/85 $0.00 + .50 Copyright © Pergamon Press Ltd ECOLOGY...

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Biotech Advs Vol. 3, pp 155-170, 1985 Printed in Great Britain. All Rights Reserved.

0734-9750/85 $0.00 + .50 Copyright © Pergamon Press Ltd

ECOLOGY AND GENETICS OF T R O P I C A L R H I Z O B I A SPECIES M. H. AHMAD* and WAYNE MCLAUGHLIN** Departmetl! of Biochemislry, University of the West Indies, Mona, Kingston 7, Jamaica

ABSTRACT Biological nitrogen fixation (BNF) technology with special reference to Rhizobium -legume symbiosis is growing very rapidly with the hope of combatting world hunger by producing cheaper protein for animal and human consumption in the Third World. One can see rapid progress made in the biochemistry and molecular biology of symbiotic nitrogen fixation in general; however, less progress has been made on the ecological aspects despite the fact that an enormous amount of l i t e r a t u r e is available on inoculation problems and on agronomic aspects of symbiotic nitrogen fixation.

So far most information on Rhizobium concerns fast-growing

rhizobia and their host legume. Although i t is essential that food production using BNF technology should be maximized in the Third World, the least work has been done on slow-growing rhizobia, which are generally found in tropical and sub-tropical soils.

The majority of the developing countriesare in tropical

and sub-tropical regions.

Except for R. japonicum, a microsymbiont partner of

soybean (Glycine max), the majority of the slow-growing rhizobia belong to the cowpea group, and we refer to cowpea rhizobia as tropical rhizobia species.

In

this review we have tried to consolidate the recent progress madeon ecology and genetics of tropical rhizobia.

By using recombinant DNA technology techniques

i t is expected that super strains of rhizobia with desirable characteristics can be produced. One must evaluate the efficiency and effectiveness of these

*For reprint request **Permanent address:

Scientific Research Council, Hope, Kingston 6

Jamaica.

155

156

~.H. AIIqAD AND W. M C L A U G H L I N

g e n e t i c a l l y manipulated laboratory strains under f i e l d conditions.

In conclu-

sion, i f one aims at combatting hunger in the Third World using BNF technology, an intensive research programme on fundamental and applied aspects of t r o p i c a l rhizobia species is suggested.

This involves close cooperation between molecular

b i o l o g i s t s and microbial e c o l o g i s t s .

KEY WORDS Biological nitrogen f i x a t i o n (BNF), cowpea r h i z o b i a , d i v e r s i t y , fast-growing r h i z o b i a , hydrogenase uptake system (Hup), inoculant, n i f and nod genes, Rplasmid, slow-growing r h i z o b i a , Sym plasmid, transposon Tn5, t r o p i c a l r h i z o b i a , recombinant DNA technology.

INTRODUCTION Leguminous plants are d i s t r i b u t e d throughout the world but the m a j o r i t y of them grow in the tropics and sub-tropics.

So f a r , thousands of legume species are

known but less than twenty are used extensively. legumes are peanuts, soybean, peas, l e n t i l s , cowpeas, a l f a l f a , clover and vetches.

Some of the common grain

pigeon peas, mungbeans, kidney beans

Many legumes are able to f i x nitrogen as

a r e s u l t of symbiosis between Rhizobium (a microsymbiont) and the legume host (a macrosymbiont).

A leguminous crop, i f inoculated with e f f e c t i v e s t r a i n ( s ) of

r h i z o b i a , can add up to 200 kg. of nitrogen to the s o i l per hectare per year (32,39).

In the United States alone legumes l i k e a l f a l f a ,

contribute about 2.4 m i l l i o n tons of nitrogen a year.

soybean and peanuts

This is one of the

reasons why annual worldwide support of b i o l o g i c a l nitrogen f i x a t i o n (BNF) research exceeds 25 m i l l i o n d o l l a r s , which is small compared to 20 b i l l i o n d o l l a r s spent annually on the cost of N - f e r t i l i z e r .

I t is expected that the

results from BNF research may stimulate and expand the market f o r the Rhizobium inoculant industry, which at present values 15 m i l l i o n d o l l a r s per year in the USA; however, there is a market p o t e n t i a l of 350 m i l l i o n d o l l a r s per year (67). To improve the y i e l d of legumes by b i o l o g i c a l nitrogen f i x a t i o n , e f f e c t i v e , e f f i c i e n t and competitive strains of rhizobia need to be i d e n t i f i e d and the legume seeds should be inoculated by such r h i z o b i a l s t r a i n s (21,78).

While

genetic studies are important to improve the strains of rhizobia in respect of desired characters, the ecological aspects cannot be overlooked because event u a l l y the g e n e t i c a l l y manipulated strains need to be f i e l d tested f o r t h e i r a b i l i t y to (a) nodulate and f i x nitrogen e f f e c t i v e l y on the recommended legume host, (b) compete, survive and p e r s i s t in the presence of indigenous r h i z o b i a , and (c) t o l e r a t e a wide range of soil temperatures and stresses l i k e a c i d i t y ,

ECOLOGY AND GENETICS OF TROPICAL RHIZOBIA SPECIES

157

a l k a l i n i t y , s a l i n i t y and high levels of aluminium and manganese. The rhizobia are divided into fast and slow-growing on the basis of their generation time (46) and acid/alkali production in the culture

medium(58).

Fast-

growing rhizobia nodulate a limited host species whereas slow-growing rhizobia generally nodu|ate a broad host range. While much progress has been made on genetics and ecology of fast-growing rhizobia, the published work on slow-growing rhizobia is limited.

Amongslow-growers R. japonicum (a microsymbiont partner

of soybean) is relatively better studied than another class of slow-grower, so called cowpea rhizobia.

Cowpearhizobia are promiscuous in their host range for

nodulation and are widely distributed in tropical and sub-tropical soils.

They

can nodulate more than 50 tropical legumes (6,34) and also one non-legume Parasponia (72).

Someof the grain legumes nodulated by cowpea rhizobia include

Vigna spp (cowpeas, mungbeans), Arachis spp (peanuts), Phaseolus spp (limabean), Cajanus cajan (pigeon peas) and sometimes Glycin spp (soybean).

I t is estimated

that 86% of the world's cowpea production is concentrated in the savannah region of West Africa between I0 ° and 20°N (7), where temperatures are high; because of this, we refer to cowpea rhizobia as tropical rhizobia.

In this review we con-

centrate on the recent ecological and genetical studies done on cowpea rhizobia, which is one of the largest groups of tropical rhizobia and has great potential in tropical agriculture.

BIOCHEMICAL & ECOLOGICAL DIVERSITY On the basis of nodulation a b i l i t i e s , tropical rhizobia are promiscuous. Because of this particular character, they offer an excellent tool for geneticists and biochemists to understand the mechanismof their a b i l i t y to nodulate a wide range of legume hosts.

While host promiscuity suggests genetic heterogeneity, studies

on the diversity of cowpea rhizobia are limited (2).

Recently, an attempt was

made to examine the diversity within and between populations of indigenous tropical rhizobia isolated from cowpeas grown in three different locations in West Africa: (a) Onne, Southeastern Nigeria (tropical rainforest), (b) Ibadan, Western Nigeria (rainforest/savannah/transition zone), and (c) Maradi, in Niger Republic (savannah/sahel zone), and in two locations in Jamaica. Serological profiles of tropical rhizobia showed a high degree of antigenic heterogeneity, each population had its own general characteristics (3,5). Serological relatedness of tropical rhizobia was correlated with colony morphologies but not with their nodulating a b i l i t i e s in different hosts.

Most of the strains isolated from

Maradi nodulated peanuts (Arachis hypogaea) while few from Ibadan and Onne nodulated peanuts. On the contrary, most of the strains from Ibadan and Onne nodulated pigeon peas (Cajanus cajan) and mungbeans (Vigna radiata) than the

158

M.H. AH}IAD AND W. MCLAUGHLIN

s t r a i n s i s o l a t e d from Maradi ( 2 , 3 ) . D i v e r s i t y among t r o p i c a l rhizobia strains in terms of i n t r i n s i c tance (IAR) patterns has been examined.

antibiotic

resis-

The populations as a whole were r e s i s -

t a n t to gentamycin but varied in t h e i r resistance to streptomycin, r i f a m p i c i n , kanamycin, p e n i c i l l i n , between a n t i b i o t i c

a m p i c i l l i n and t e t r a c y c l i n e (54,63).

The c o r r e l a t i o n s

resistance pattern and colony morphology were examined.

Most

of the r e s i s t a n t s t r a i n s showed wet colony morphologies whereas s t r a i n s showing dry colony morphologies were generally s e n s i t i v e to a n t i b i o t i c s . between high i n t r i n s i c

antibiotic

A correlation

resistance and reduced rate of drug uptake by

cowpea r h i z o b i a was also shown (54).

In terms of IAR p a t t e r n , each population

was d i v e r s e ; however, less d i v e r s i t y was found w i t h i n the population than between the populations i s o l a t e d from d i f f e r e n t l o c a t i o n s (63). DNA:DNA

h y b r i d i z a t i o n studies of cowpea r h i z o b i a s t r a i n s have also shown a

significant diversity.

Some of the cowpea r h i z o b i a s t r a i n s examined were not

c l o s e l y r e l a t e d to the cowpea s t r a i n 32-HI with respect to t h e i r DNA homology (43).

Recently, Chakarbarti et al (15) showed a wide n u t r i t i o n a l

diversity

among cowpea r h i z o b i a in t h e i r requirement f o r v i t a m i n s , carbon and nitrogen sources.

This was in contrast to e a r l i e r findings of West and Wilson (79,80)

and Graham (37) t h a t the growth of cowpea r h i z o b i a did not require vitamin supplements in t h e i r media.

Recently, Stowers and Elkan (69) examined 25 s t r a i n s

of cowpea r h i z o b i a and showed t h a t these s t r a i n s did not a b s o l u t e l y require v i t a m i n s , although b e t t e r growth was observed when a f u l l added.

Likewise, a heterogenous pattern of u t i l i z a t i o n

cowpea r h i z o b i a was found (15). determined a c i d / a l k a l i

vitamin supplement was of carbon sources by

However, carbon as well as nitrogen sources

production in c u l t u r e medium by cowpea r h i z o b i a (4,22,69).

This r e p o r t was again in contrast to e a r l i e r findings t h a t cowpea r h i z o b i a always produced a l k a l i (34,36,45).

in c u l t u r e media regardless of the media composition

I t was independently shown in d i f f e r e n t l a b o r a t o r i e s t h a t nitrogen

sources l i k e ammonium c h l o r i d e or ammonium sulphate s t r o n g l y i n f l u e n c e a c i d / a l k a l i production by cowpea rhizobia (4,69).

Tan and Broughton (71) suggested

t h a t pH change o f the media by cowpea r h i z o b i a was due to s e l e c t i v e u t i l i z a t i o n of nitrogen sources, t h i s was l a t e r confirmed in two independent studies (4,69). The old b e l i e f ~hat a l k a l i production in c u l t u r e media by t r o p i c a l r h i z o b i a was thought to make d i f f i c u l t acid and i n f e r t i l e

the s e l e c t i o n of acid t o l e r a n t s t r a i n s s u i t a b l e f o r

s o i l s of the t r o p i c s (22).

Recent findings are t h a t a c i d /

a l k a l i production by t r o p i c a l rhizobia can be affected by carbon or nitrogen substrates in the c u l t u r e view t h a t a l k a l i

media (4,22,69).

While these findings challenge the

production by t r o p i c a l r h i z o b i a is a d i s t i n g u i s h i n g c h a r a c t e r s -

t i c with ecological s i g n i f i c a n c e (58), microbial p h y s i o l o g i s t s should undertake

ECOLOGY AND GENETICS OF TROPICAL RHIZOBIA SPECIES

159

a detailed study in examining the regulation of a c i d / a l k a l i production and t h e i r effects on the survival and growth of Rhizobium. The physiological d i v e r s i t y in respect of minimal vitamin requirement, a b i l i t y to u t i l i z e a wide range of carbon and nitrogen sources and heterogenous pattern for a c i d / a l k a l i production may give an ecological advantage to select rhizobia by ensuring better survival and persistence in s o i l .

Strains with these characters can easily adapt to a new en-

vironment and readily colonise the soil or the rhizosphere. The removal of fixed nitrogen from the soil by rhizobial d e n i t r i f i c a t i o n has been demonstrated (19) and cowpea rhizobia show considerable variation in d e n i t r i f i c a tion a b i l i t y and for the anaerobic growth in the presence of n i t r a t e (20,83). Likewise, exopolysaccharide production and composition in cowpea rhizobia strains is variable and does not correlate with nodulating a b i l i t i e s (41).

During

screening of cowpea rhizobia for t h e i r a b i l i t y to nodulate various host species, some of the hosts show symptoms of f o l i a r chlorisis in mungbean (Phaseolus radiata) and soybean ( G l _ ~ m a x ) plants inoculated with certain strains of cowpea rhizobia (29).

However,there was no correlation between chlorisis

symptoms and the presence of nodules.

In another report two rhizobia strains,

IHP324 and IHP342, belonging to the cowpea group were reported to induce leaf r o l l symptoms in pigeon pea (Cajanus cajan). induced disease in pigeon pea.

Thesesymptoms resembled a virus-

These strains formed normal nodules on pigeon

pea and were moderately effective in nitrogen f i x a t i o n (49). The presence or absence of hydrogenase uptake system (HUP) in rhizobia has significance in agriculture in terms of economic benefits (67,84).

During the pro-

cess of nitrogen f i x a t i o n , 25 - 30% of the reducing power is used for the production of hydrogen gas which is supposed to be a wasteful product.

The strains

that possess an uptake hydrogenase system (HUP+) can recycle this hydrogen for the synthesis of ATP. Thus, rhizobia strains having HUP+ are more e f f i c i e n t in t h e i r energy metabolism; they are known to produce 50% more plant dry matter than HUP- strains.

Significant increase in y i e l d could be obtained by inoculating

legumes with HUP+ strains.

Someof the inoculant industries are supporting the

idea that HUP- rhizobia should be genetically constructed to HUP+, which can eventually be used for inoculum production.

The HUP+ strains can also oxidize

hydrogen to reduce the i n t r a - c e l l u l a r 02 concentrations, thus protecting the nitrogenase from oxygen.

So far only few rhizobium strains are known to have

the hydrogenase uptake system (HUP+). Of the hundreds of R. japonicum strains examined, 25% were HUP+ whereas a l l of the R. m e l i l o t i or R. t r i f o l i i examined were HUP- (30,75).

strains

Recent studies with cowpea rhizobia have shown that

the majority of them are HUP+ (51).

However,more data on hydrogenase a c t i v i t y

of tropical rhizobia are needed to confirm this statement.

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M.H. AF~LA.DAND W. MCLAUGHLIN

Recently, a few cowpea r h i z o b i a s t r a i n s were found to be exceptional in respect of

t h e i r growth r a t e and nodulating a b i l i t y ,

such as (a) s t r a i n NGR234 i s o l a t e d

from Lablab purpureus nodulates cowpea but is a f a s t grower (73),

(b) stem

n o d u l a t i n g s t r a i n s i s o l a t e d from a t r o p i c a l legume Aeschynomene is also a f a s t grower, and (c) s t r a i n 0RS57 i s o l a t e d from stem and r o o t nodules of t r o p i c a l legumes Sesbania r o s t r a t a (25) grows f a s t and cannot u t i l i z e

disaccharides (23).

These recent findings have a t t r a c t e d r h i z o b i o l o g i s t s to undertake d e t a i l e d genet i c and biochemical i n v e s t i g a t i o n s to understand the basic mechanism of nodulat i o n and n i t r o g e n f i x a t i o n in cowpea r h i z o b i a and t h e i r e v o l u t i o n a r y p a t t e r n s . Generally speaking, biochemical c h a r a c t e r i z a t i o n of cowpea r h i z o b i a has been done as a part of work along with other r h i z o b i a species.

We have noted t h a t too many

g e n e r a l i z a t i o n s about cowpea r h i z o b i a have been made based on f i n d i n g s f o r an individual strain. SURVIVAL STUDIES In t r o p i c a l s o i l s the indigenous populations of r h i z o b i a in general are not large enough; the number of indigenous cowpea r h i z o b i a examined in Maradi s o i l s of West A f r i c a were low (4.9 x i02/g s o i l )

(2); the highest number of indigenous cowpea

r h i z o b i a was only 2.8 x 102/g s o i l

in one of the many s o i l s examined in Jamaica

using the method of Vincent (78) (McLaughlin and Ahmad, unpublished).

Table

I

shows the number of nodules on kidney beans (Phaseolus v u l g a r i s ) produced by indigenous r h i z o b i a in Jamaican s o i l s , which i n d i c a t e s an extremely low number of i n f e c t i v e indigenous r h i z o b i a l populations. t r o p i c a l s o i l s encourage f i e l d

Such low r h i z o b i a l population in

inoculation trials

to examine the e f f e c t s of

i n o c u l a t i o n on y i e l d s of host legumes. Table I

Nodulation of kidney beans (Phaseolus v u l g a r i s ) by indigenous Rhizobia in Jamaican s o i l s (Thetford Farm).

Cultivars

Age of the Plant (DAP)

No. of p l a n t s sampled

Round Red

30

30

0.43

50

30

0.0

65

30

0.04

30

30

0.76

50

30

0.05

65

30

0.0

Miss K e l l y

Source:

McLaughlin and Ahmad, unpublished

Average No. o f nodules

ECOLOGY AND GENETICS OF TROP1CAL RHIZOB[A SPECIES

161

Ahmad et al (2) have shown the potential for the use of legume inoculants in West African soils.

However,benefits from legume inoculation depend on the survival

and persistence of introduced strains of rhizobia in the soil (21).

The rhizobia

strains should be evaluated for t h e i r survival before an inoculation programme is launched.

So far, work on the survival of rhizobia is mainly limited to fast-

growing rhizobia and very l i t t l e is available.

information on the survival of tropical rhizobia

Tropical soils fluctuate widely in temperature and moisture which

greatly influence the survival of rhizobia in the soil (4,59).

In the arid

tropics, rhizobia need to survive at a high soil temperature while seeds are geminating.

Persistence in the tropical soil from crop to crop means that the

strains need to be temperature tolerant.

Recently, temperature tolerant

strains of cowpea rhizobia on YEM (yeast extract mannitoI) were identified (2, 28).

I t was also observed that cowpea rhizobia isolated from hot dry areas in

West Africa were more temperature and dessication tolerant than those isolated from cooler regions (2,38). rhizobia

Boonkerdand Weaver ( l l ) ,

proposed that cowpea

strains have different rate of survival in soil when exposed to the

different moisture and temperature conditions.

Theyalso showed that survival

of cowpea rhizobia in dry soil at 35°C was poorer than in moist s o i l .

Hartel

and Alexander (38) also found that cowpea rhizobia isolated in West Africa grew well in moist soils at 30°C.

We have examined the survival of cowpea rhizobia

in soils incubated at 30°C and 37°C and found no significant difference in t h e i r survival (1).

Also, a significant difference in survival of cowpea rhizobia in

s t e r i l e and non-sterile soils was found (1,11).

Survival of tropical rhizobia

in soils undergoing drying (a characteristic of tropical soils)

tends to be

biphasic; the f i r s t phase corresponds to the greatest loss of moisture whereas the second phase begins when the soil has dried (60).

While Pena-Cabriales and

Alexander (60) found that the rate of drying had no effect on survival,

Hartel

and Alexander (38) showed that slow-drying increased the survival of cowpea rhizobia.

However,i t has been suggested that the rhizobia able to survive at

high temperatures under laboratory conditions may not tolerate high temperatures in the s o i l .

Choice of c a r r i e r plays a v i t a l role in the production of legume inoculants. Before selection of c a r r i e r , survival of the rhizobia strains in the c a r r i e r must be examined. Various workers have studied the survival of rhizobia in different carriers (1,33,50,6~and found that peat is s t i l l

the best carrier (1,64).Recent-

l y effects of temperature on the survival of tropical rhizobia in peat inoculants .were examined (65,82).

Cowpearhizobia survived very well over the 21 weeks

period at 25°C and 35°C. There was a slight decline in rhizobial number (2 log)

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M.H. A ~ D

AND W. MCLAUGHLIN

at 4b°C whereas the death rate was much greater (4 Log) at 55°C during 15 weeks. In other studies, exposure of the peat inoculant to high temperature revealed that except for two strains a l l rhizobia strains survived at 37°C (82).

The

rhizobia strains multiplied and survived for a longer period at 28°C than at 37°C. All rhizobia strains examined died in one week at 46°C (64,65).

Somase-

garan et al (65) examined the effect of temperature to which the inoculants were exposed during shipment on the survival of rhizobia.

Temperatureduring trans-

portation was varied from 26°C to 45°C and the duration of transportation varied from 6 to 44 days.

The number of rhizobia which survived in the inoculants a~er

going through the postal t r a n s i t was in excess of 108 cells/g of peat in more than 89% of the cases (65). There are d i f f e r e n t views regarding the mechanism of survival of rhizobia in soils undergoing dessication.

Tropical rhizobia species were able to survive

better in soil undergoing drying than in moist s o i l , maybe because they possess low internal water content (76).

Chao and Alexander (16) found a relationship

between exopolysaccharides (EPS) production by cowpea rhizobia and s e n s i t i v i t y to drying.

We also found that EPS production by cowpea rhizobia was higher at

40°C than 30°C (41).

I t was also postulated that survival a b i l i t y of rhizobia

in soils undergoing drying may depend on the clay content of the soils (70). However, so f a r , no d e f i n i t e mechanism of survival of rhizobia in soil has been postulated.

This may be because of a lack of s u f f i c i e n t information on the bio-

chemistry and genetics of tropical rhizobia species.

I d e n t i f i c a t i o n , expression

and regulation of genes responsible for survival under d i f f e r e n t ecological conditions w i l l be one of the challenging tasks for molecular biologists to explain the mechanism of survival.

As stated before, understanding of rhizobial ecology

is necessary to achieve successful application of recombinant DNA technology to produce super strains of rhizobia.

While sophisticated technology to improve

rhizobia strains by genetic engineering is available in the laboratory, there is a lack of suitable techniques available for identifying specific strains d i r e c t l y recovered from soils.

Presently, immunofluorescence antibody technique (IF)(9),

of enzyme-linked immunosorbent

assay (ELISA) (3) or i n t r i n s i c a n t i b i o t i c resis-

tance pattern (63) are commonly used for detection and i d e n t i f i c a t i o n of p a r t i cular strains of rhizobia in s o i l , rhizosphere and nodules. We w i l l not review the survival of tropical rhizobia under d i f f e r e n t stress conditions l i k e soil a c i d i t y , aluminium tolerance, s a l i n i t y , a l k a l i n i t y and waterlogged conditions, etc. because there is already an excellent review readily available on this topic (27).

ECOLOGY AND CENETICS OF TROPICAL RHIZOBIA SPECIES

163

GENETIC STUDIES Symbiotic biological nitrogen fixation is a complex interaction between rhizobia and the legume host plant.

Recently, some of the bacterial and the host plant

genes involved in symbiosis have been identified and characterized (8,17,18,24, 35,40,53,62,77).

However, such studies were mainly of fast-growing rhizobia with

a few studies on slow-growing R. japonicum and their host soybean. More recent work on genetic analysis of tropical rhizobia species has been i n i t i a t e d in different ]aboratories including

that of the authors.

During the course of

these studies some unusual features were noted, such as the case of Rhizobium strains isolated from Sesbania rostrata, which forms both stem and root nodules on the host plant (25) and are able to grow rapidly using N2 as the sole source of nitrogen.

Elmerich et al (31) have done genetic analysis of Sesbania rhizobia

and showed the p o s s i b i l i t y of studying n i f genes explanta using the methodology previously used for K. pneumoniae. Recently, fast-growing rhizobia strains from different tropical legumes were isolated.

These strains were grouped as inter-

mediates between classical fast- and slow-growing rhizobia (12).

Someof these

strains nodulate wing bean (Psophocarpus tetragenolubus), cowpeas (Vigna unguiculata),and soybean (Glycin max).

Hybridization studies with n i f a n d nod probes

showed that nif-nod genes of these fast-growing tropical rhizobia are linked on a single Sym plasmid (12,13).

One of such unusual strains is NGR234which

nodulates a broad range of tropical legumes and also a non-legume Parasponia (13, 72).

Genetic studies showed that strain NGR234 has a large Sym plasmid which

carries genes for nodulation and nitrogen fixation.

Such Sym plasmids have not

so far been reported in slow-growing tropical rhizobia.

Sym plasmid from NGR234

was mobilized by cointegration with plasmid pSUP 1011 (57).

The mobile Sym

plasmid was transferred to fast-growing strains (R. leguminosarumand R. t r i f o l i i ) in which nodulation and nitrogen fixation functions of the host species belon~ng to cowpea group were expressed. The transconjugants of R. leguminosarumand R. trifolii

were able to nodulate siratro, a commonlegumehost nodulated by cowpea

rhizobia (57).

This was the f i r s t report that genes for symbiosis with the host

species nodulated by cowpea rhizobia could be introduced into fast-growing rhizobia. There are two recent reports of biochemical and genetic studies of a stem rhizobium strain BTAiL which forms nodules on the stem of Aeschynomene, a tropical legume (52,68).

Isolation and cloning of n i f genes from stem nodulating

rhizobia and hybridization of their DNA with n i f KDH probe of Klebsiella pneumoniaeindicated by the presence of n i f genes on two DNA fragments about 28 kb and 6 kb in size (52).

Two leghaemoglobin species, Lb~ and LbB were reported in

this strain whose expressions were different in stem and root nodules.

These

164

M.H. A ~ D

AND W. MCLAUGHLIN

unusual features of t r o p i c a l rhizobia were regarded as an important discovery in plant biotechnology. Very l i t t l e

genetic studies on slow-growing t r o p i c a l rhizobia species (referred

to as cowpea r h i z o b i a ) have been done.

Information on genetic t r a n s f e r systems

in cowpea r h i z o b i a , which is an important step to characterize any system genetically,

is l i m i t e d .

Recently, plasmids R68.45 and RP4 were transferred from

E. c o l i to cowpea rhizobia and t h e i r maintenance and expression were examined (47,55).

We have shown that the frequency of plasmid t r a n s f e r from E. c o l i to

cowpea rhizobia was in the range of 4 x 10-2 to 4 x 10-6 per r e c i p i e n t c e l l . Plasmid RP4 was transferred to cowpea rhizobia at a higher frequency than plasmid R68.45.

The plasmids expressed t h e i r a n t i b i o t i c resistance genes in cowpea

rhizobia f o r kanamycin and t e t r a c y c l i n e but not for a m p i c i l l i n .

Transfer of R-

plasmids within and between strains of cowpea rhizobia was also examined.

The

frequency of R-plasmid t r a n s f e r was higher in isogenic than non-isogenic strains of cowpea rhizobia (55). The genetic analysis of the molecular cloning of b a c t e r i a l genes have been f a c i l i t a t e d by the use of transposons (48).

Transposon directed mutagenesis was used

to i d e n t i f y the symbiotic genes in fast-growing rhizobia (10,24) and recently the transposon Tn5 was used to induce a v a r i e t y of mutants of R. japonicum (42, 61,65).

However, only one report of transposon Tn5 induced mutagenesis of cowpea

rhizobia is a v a i l a b l e (44).

We have i s o l a t e d a number of transposon Tn5 induced

mutants of cowpea rhizobia with d i f f e r e n t phenotypes.

Our aim was to i s o l a t e a

large number of mutants derived from two wild type cowpea rhizobia s t r a i n s , IRC256 and JRC23. One of the wild type s t r a i n s , IRC256, produced dark nodules on cowpeas (26,74).

We have isolated Tn5 induced mutants of t h i s s t r a i n which do

not form dark nodules on cowpeas (56).

The Tn5 induced mutants of cowpea rhizo~a

were also screened f o r auxotrophy and symbiotic d e f e c t i v e phenotypes.

Of I000

mutants (derived from s t r a i n IRC256) tested for auxotrophy, only three auxotrophs

were i d e n t i f i e d .

On the other hand, none of the I000 Tn5 mutants (derived

from s t r a i n JRC23) examined yielded any single auxotroph. auxotrophy in cowpea rhizobia is d i f f i c u l t

to e x p l a i n .

The low frequency of

I t may be possible that

more than one gene is involved in maintaining prototrophy.

We have also examined

about 400 Tn5 induced mutants of cowpea rhizobia f o r d e f e c t i v e symbiosis phenotypes.

Various mutants with Nod+ Fix- and Nod- phenotypes were obtained, though

the frequency was very low (56).

Tn5 i n s e r t i o n in DNA of cowpea rhizobia was

stable even a f t e r plant passage (44,56). cowpea rhizobia mutagenesis (81).

E f f e c t i v e and i n e f f e c t i v e mutants of

were also i s o l a t e d by NTG ( N - m e t h y l - N - n i t r o - n i t r o s o guanidine) I t is hoped that these mutants w i l l

serve as useful tools for

genetic analysis of cowpea rhizobia which is presently l i m i t e d due to the e x t r e -

ECOLOGY AND GENETICS OF TROPICAL RIIIZOBIA SPECIES

mely low frequency rate at which these mutants are obtained.

165

Someof the recent

results obtained on genetics of tropical rhizobia can be summarised: (a) plasmids are present in some of the fast-growing tropical rhizobia but generally absent in slow-growing tropical rhizobia.

There is no evidence that these

plasmids contain n i f gene, (b) i t has been shown that symbiotic functions ( n i f and nod) are located on the chromosome in slow-growing tropical rhizobia, (c) that a group of tropical rhizobia intermediate between classical fast- and slowgrowing types exists.

ACKNOWLEDGEMENTS

We thank Ms. Sharon Aarons and Mr. Eustace Smith for their help in l i t e r a t u r e search. The financial support from the Postgraduate Board, Research and Publication Fund of the University of the West Indies; Melcoe Group of Companies, Jamaica; and the Scientific Research Council is acknowledged. We are obliged to Prof. H.G. Coore, Head of the Biochemistry Department, for reading the manuscript and for his constructive c r i t i c i s m and valuable comments. We also thank Ms. Carol Thompson for typing the manuscript.

REFERENCES I.

S. Aarons and M.H. Ahmad, Survival of cowpea rhizobia in Jamaican Peat and Jamaican soils. 6th Intr. Cong. Nitrogen Fixation (Abstr.) (1985).

2.

M.H. Ahmad, A.R.J. Eaglesham, S. Hassouna, B. Seaman, A. Ayanaba, K. Mulongoy and E.L. Pulver, Examining the potential for inoculant use of cowpeas in West African soils.

3.

Trop. Agric. (Trin.) 5_88,325-335 (1981).

M.H. Ahmad, A.R.J. Eagleshamand S. Hassouna, Examining serological diversity of cowpea rhizobia by the ELISA technique.

Arch. Microbiol.

130, 281-287 (1981). 4.

M.H. Ahmad and E. Smith, U t i l i z a t i o n of carbon and nitrogen sources and acid/alkali production by cowpea rhizobia.

5.

Plant & Soil (in press) (1985).

M.H. Ahmad, R. Uddin and W. McLaughlin, Characterization of indigenous rhizobia from wild legumes. FEMSMicrobiol. Letters, 24, 197-203 (1984).

6.

E.K. Allen and O. Allen,

The Leguminosae. University of Wisconsin Press.

Madison (1981). 7.

Anonymous. Production Yearbook 1974. Vol. 29. Food & Agricultural Organisation Rome, p. 79 (1975).

8.

Z. Banfalvi, V. Sakanyan, C. Konez, A. Kiss, I. Dusha, A. Kondorsi, Location of nodulation and nitrogen fixation genes on a high molecular weight plasmids

166

M.H.

of R. m e l i l o t i . 9.

A H M A D AND W. M C L A U G H L I N

Mol. Gen. Genet. 184, 318-325 (1981).

B. Ben Bohlool, R. Kosslak and R. Woolfenden,

The ecology of Rhizobium in

the rhizosphere: Survival, growth and competition.

In:

gen f i x a t i o n research eds. D. Veeger and W.E. Newton.

Advances in n i t r o Martinus N i j h o f f

Press (1984). I0.

J.E. Beringer, J.L. Beynon, A.V. Buchanan-Wollaston and F. Canon, Transfer of the d r u g - r e s i s t a n t Tn5 to Rhizobium. Nature 270, 633-634 (1978)

11.

N. Boonkerd and R.W. Weaver, Survival of cowpea rhizobia in s o i l s as affected by soil temperature and moisture.

Appl. Environ, M i c r o b i o l .

43, 585-589 (1982). 12.

W.J. Broughton, B.B. Bohlool, C.H. Shaw, H.J. Bohnert and C.E. Pankurst, Conserved plasmid chromosome sequences in f a s t - and slow-growing rhizobia that nodulate the same plant.

13.

Arch. M i c r o b i o l . 141, 14-21 (1985).

W.J. Broughton, N. Heycke, Z.A. Heinermeyer and C.E. Pankurst, Plasmidlinked n i f and nod genes in fast-growing rhizobia that nodulate Glycine max, Psophocarpus

tetragenolubus and Vigna unguiculata.

Proc. Natl. Acad. Sci.

USA, 81, 3093-3093 (1984). 14.

H.V.A. Bushby and K.C. Marshall, Some factors a f f e c t i n g the survival of root

15.

S. Chakarbarti, M.S. Lee and A.H. Gibson, D i v e r s i t y in the n u t r i t i o n a l

nodula bacteria on dessication.

Soil B i o l . Biochemistry 9, 143-147 (1977)

requirements of strains of various rhizobium s t r a i n s .

Soil B i o l . Biochem.

I__33, 349-354 (1981). 16.

W. Chao and M. Alexander, Influence of soil c h a r a c t e r i s t i c s on the survival of rhizobium in s o i l s undergoing drying.

Soil Sci. Soc. Am. J. 46, 949-952

(1982). 17.

D. Corbin, L. Barran and G. D i t t a , Organisation and expression of Rhizobium m e l i l o t i nitrogen f i x a t i o n genes.

Proc. Natl. Acad. Sci. U.S.A. 80, 3005-

3009 (1983). 18.

D. Corbin, G. D i t t a , D.R. H e l i n s k i , Clustering of nitrogen f i x a t i o n ( n i f ) genes in Rhizobium m e l i l o t i .

19.

J. B a c t e r i o l . 149, 221-228 (1982).

R.M. Daniel, K.W. Steele and A.W.Limmer, D e n i t r i f i c a t i o n by r h i z o b i a . possible f a c t o r c o n t r i b u t i n g to nitrogen losses from s o i l s .

A

New Zealand

A g r i c u l t u r a l Science. 14, 109-112 (1980). 20.

R.M. Daniel, A.W. Limmer, K.W. Steele and I.M. Smith,

21.

M i c r o b i o l . 128, 1811-1815 (1982). R.A.Date, Principles of Rhizobium s t r a i n selection In: symbiotic nitrogen

n i t r a t e reduction and) ~ e n i t r i f i c a t i o n

Anaerobic growth

in 46 Rhizobium s t r a i n s . J. Gen.

f i x a t i o n in plants ed. P.S. Nutman, Cambridge Univ. Press. PP (1976) 22.

R.A. Date and J. H a l l i d a y , Selection rhizobium f o r acid i n f e r t i l e

23.

the t r o p i c s . Nature 277, 62-64 (1979). R.G. Donald and R.A. Ludwig, Rhizobium sp. ORS 571 ammonium a s s i m i l a t i o n

s o i l s of

ECOLOGY AND GENETICS OF TROPICAL RHIZOBIA SPECIES

167

24.

and nitrogen fixation. J. Bact. 158, 1144-1151 (1984). J.A. Downie, G. Hombrecher, Q,S. Ma, C.D. Knight, B. Wells, W.B. Johnston,

25.

Cloned nodulation genes of Rhizobium leguminosarumdetermine host-range specificity. Mol. Gen. Genet. 190, 395-365 (1983). B.L. Dreyfuss and Y.R. Dommergues, Nitrogen fixing nodules induced by

26.

rhizobium on the stem of tropical legume Sesbania rostrata. FEMSMicrobiol. Lett. _5, 369-372 (1981). A.R.J. Eaglesham, M.H. Ahmad, S. Hassouna, B. Seaman,B. Goldman, Cowpea "

rhizobia producing dark nodules: Use in competition studies. Environ. Microbiol. 4_44,611-618 (1982). 27.

Appl.

A.R.J. Eagleshamand A. Ayanaba, Tropical stress ecology of rhizobia root nodulation and legume fixation. In: Current Developments in Biological Nitrogen Fixations. ed. N.S. Subba Rao, Oxford and IBH Publishing Co.(1984).

28.

A.R.J. Eaglesham, B. Seaman, M.H. Ahmad, S. Hassouna, A. Ayanabaand K. Mulongoy, High temperature tolerant "cowpea" rhizobia. In: Current Perspectives in Nitrogen Fixation. eds. A.H. Gibson and W.E. Newton, Australian Acad. Sci. Canberra, p. 436 (1982).

29.

A.R.J. Eagleshamand S. Hassouna, Foliar chlorosis in legume induced by cowpea rhizobia.

30.

Plant & Soil 65, 425-428 (1982).

G. Eisbienner and H.j. Evans, Aspects of hydrogen metabolism in nitrogen fixing legumes and other plant microbe association. Ann. Rev. Pl. Physiol. 3_44, 105-136 (1983).

31.

C.E. Elmerich, B.L. Dreyfuss, G. Reysett and J.P. Aubert, Genetic Analysis of nitrogen fixation in a tropical fast-growing Rhizobium, EMBOJ. _I, 499503 (1982).

32.

L.W. Erdman, Legume inoculation, US Dept. Agr. Farmers Bull. 2003 (1959),

33.

A.Wo Faizah, W.J. Broughtonand C.K. John. Rhizobia in tropical legumes X.

34.

Growth in coir-dust-soil. Soil Biol. Biochem. 12, 219-227 (1980). E.D. Fred, I.R. Baldwin and E. McCoy, Root nodule bacteria and leguminous plants.

35.

University of Wisconsin Press, Madison, W.I. (1932).

F. Fuller, P.W. Kunstner; T. Ngnyenand D.P.S .Verma, Soybeannodulin'genes: Analysis of DNA clones reveals several major tissue specific sequences in nitrogen fixing nodules. Proc. Natl. Acad. Sci. U.S.A. 80, 2594-2598 (1983)

36.

C.E. Georgi and J.M. Ettinger, Utilisation of carbohydrates and sugar acids

37.

by rhizobia. J. Bacteriol. 41, 323-340 (1941). P.H. Graham, Vitamin requirement of root nodule bacteria.

38.

30, 245-248 (1963). P.G. Hartel and M. Alexander, Temperatureand dessication tolerance of

39.

cowpea rhizobia. Can. J. Microbiol. 30, 820-823 (1984) E.F. Hanzell and D.O. Norris, Processes by which nitrogen is added to the

J. Gen. Microbiol

soil/plant system. CommonwealthAgr. Bur. Bull. 46, 1-18 (1962).

168 40.

M.H. ARMAD AND W. ~CLAUGHLI~ A.M. Hirsch, M. Bang, F.M. Ausubel, U l t r a - s t r u c t u r a l tive alfalfa Bacteriol.

41.

a n a l y s i s of i n e f f e c -

nodules formed by nif:Tn5 mutants of Rhizobium m e l i l o t i .

J.

155, 367-380 (1983).

R. H o l l i n g s w o r t h , E. Smith and M.H. Ahmad, Chemical composition of e x t r a c e l l u l a r polysaccharides of cowpea r h i z o b i a .

Arch. M i c r o b i o l .

(in press)

(1985). 42.

S.S. Hom, S.L. Uratsu and F. Hoang, Transposon Tn5-induced mutagenesis of R. japonicum yielding a wide variety of mutants. J. Bacteriol. 159, 335-340 (1984).

43.

A.B. H o l l i s , W.E. Kloos and G.H. Elkan, DNA: DNA h y b r i d i z a t i o n studies of Rhizobium japonicum and r e l a t e d Rhizobiaceae. J. Gen. M i c r o b i o l . 123, 215222 (1981).

44.

M.N. Jagadish and A.A. Szalay, Directed transposon Tn5 mutagenesis and complementation in slow-growing broad host range cowpea r h i z o b i a .

Mol. Gen.

Genet. 196, 290-300 (1984). 45.

M.D. Johnson and OoN. A l l e n , Cultural reactions of r h i z o b i a with special reference to s t r a i n s i s o l a t e d with Sesbania species.

Antoine Van Leeuwen-

hoeck. J. M i c r o b i o l . Serol. 18, 1-2 (1952). 46.

D.C. Jordan, Transfer of Rhizobium japonicum

Buchanan 1980 to Brady-

rhizobium gen. Nov. a genus of slow-growing root nodule b a c t e r i a from leguminous plants. I n t . J. Syst. B a c t e r i o l . 32, 136-139 (1982). 47.

C. Kennedy, B. Dreyfuss and J. Brockwell, Transfer, maintenance and expression of P-plasmids in s t r a i n s of cowpea r h i z o b i a .

J. Gen. M i c r o b i o l . 125,

233-240 48.

N. Kleckner, J. Roth and D. B o t s t e i n , Genetic engineering in v i v o using t r a n s l o c a t a b l e drug r e s i s t a n t elements: New methods in B a c t e r i a l Genetics. J. Mol. B i o l . 116, 125-159 (1977).

49.

J.D.V.K. Kumar Rao, P.J. Dart and M. Ushakiran, Rhizobia-induced l e a f r o l l

50.

R.J. Kremer and H.L. Peterson, Effect of i n o c u l a n t c a r r i e r on s u r v i v a l of

in pigeon pea (Cajanus c a j a n ) .

Soil B i o l . Biochem. 16, 88-91 (1984).

Rhizobium on inoculated seed. Soil Sci. 134, 117-125 (1982). 51.

J.S. LaFavre and D.D. Focht, Conservation in s o i l o f H2 l i b e r a t e d from nitrogen f i x a t i o n by Hup- nodules.

Appl. Environ. M i c r o b i o l . 46, 304-311

(1983). 52.

R.P. Legocki, A.R.J. Eaglesham and A.A. Szalay, Stem n o d u l a t i o n in Aeschynomene: A model system f o r bacterium-plant i n t e r a c t i o n s Genetics of the B a c t e r i a - P l a n t I n t e r a c t i o n

In: Molecular

ed. A. Puhler Springer-Verlag.

B e r l i n p. 210 (1983). 53.

S.R. Long, H.M. Heade, S.E. Brown and F.M. Ausubel, Transposon induced symbiotic mutants of Rhizobium m e l i l o t i .

In: Genetic Engineering in Plant

Sciences. ed. N.G. Panopoules, Prager P u b l i c a t i o n s U.S.A. 129-143 (1981)

ECOLOGY AND GENETICS OF TROPICAL RHIZOBIA SPECIES

54.

169

W. McLaughIin and M.H. Ahmad, Intrinsic antibiotic resistance and streptomycin uptake in cowpea rhizobia. FEMSMicrobiol. Lett. 21, 299-303 (1984).

55.

W. McLaughlin and M.H. Ahmad, R-plasmids transfer and chromosomal mobilization in cowpea rhizobia. 6th Int. Symposiumon nitrogen fixation. (Abstr.) (1985).

56.

W. McLaughlin and M.H. Ahmad, Transposon Tn5 induced mutagenesis in cowpea

57.

N.A. Morrison, Y. Huacen, H. Cai Chen, J. Planzinski, R..Ridge and G.B.

rhizobia (unpublished) (1985). Rolfe, M o b i l i z a t i o n of Sym plasmid from a fast-growing cowpea Rhizobium s t r a i n . J. B a c t e r i o l . 16(], 483-487 (1984). 58.

D.O. N o r r i s , Acid production by Rhizobium. A unifying concept. Plant and

59.

L.O. Osa-Afiana and M. Alexander, Clays and the survival of Rhizobium in

Soil 22, 143-166 (1965). soil during dessication. 60.

Soil Sci. Soc. Am. J. 46, 285-288 (1983).

J.J. Pena-Cabriales and M. Alexander, Survival of rhizobium in s o i l s undergoing drying. Soil Sci. Soc. Am. J. 43, 962-922 (1979).

61.

K. Rostas, P.R. Sista, J. Stanley, D.P.S. Verma, Transposon mutagenesis of Rbizobium japonicum. Mol. Gen. Genet. 19__2_7,230-235 (1984).

62.

K.F. Scott, J.E. Hughes, P.M. Gresshoff, J.E. Beringer, B.G. Rolfe, J. Shine, Molecular cloning of Rhizobium t r i f o l i i

gene involved in symbiotic

nitrogen f i x a t i o n . J. MoI. Appl. Genet. ! , 315-326 (1982). 63.

M.J. S i n c l a i r and A.R.J. Eaglesham, I n t r i n s i c a n t i b i o t i c resistance in r e l a t i o n to colony morphology in three populations of West African cowpea rhizobia.

64.

Soil B i o l . Biochem. I___6,247-251 (1984).

S.D. Sparrow and G.E. Ham, Survival of Rhizobium phaseoli in six c a r r i e r m a t e r i a l . Agron. Jo 75, 181-184 (1983).

65.

P. Somasegaran, V.G. Reyes and H.J. Hoben, The influence of high temperature on the growth and survival of Rhizobium sp~ in peat inoculants during preparation, storage and d i s t r i b u t i o n . Can. J. Microbiol. 29, 23-30 (1984).

66.

G. Stacey, A.S. Paau, K.D. Noel, R.J. Maier, L.E. S i l v e r and W.G. B r i l l , Mutants of Rhizobium japonicum defective in nodulation.

Arch. Microbio1.

132, 219-224 (1982). 67.

G. Stacey and R.G. Upchurch, Rhizobium inoculation of legumes.

Trend in

8iotech. 2, 65-70 (1984). 68.

M.D. Stowers and A.R.J. Eaglesham, A stem-nodulating Rhizobium with physiol o g i c a l c h a r a c t e r i s t i c s of both f a s t - and slow growers. J. Gen. Microbiol. 129, 3651-3655 (1983)

69.

M.D. Stowers and G.H. Elkan, Growth and n u t r i t i o n a l c h a r a c t e r i s t i c s of cow-

70.

N.S. Subba Rao, Current developments in b i o l o g i c a l nitrogen f i x a t i o n s .

pea r h i z o b i a .

Plant and Soil 800, 191-200 (1984).

Oxford and IBH Publishing Co. (1984).

170

71.

M.H.

A H M A D AND W. M C L A U G H L I N

I.K.P. Tan and W.J. Broughton, Rhizobia in tropical legumes Xll Biochemical basis of acid and alkali reactions.

72.

Soil Biol. Biochem.13, 389-393 (1981).

M'.J. Trinick, Symbiosis between Rhizobium and the non-legume Trema aspera Nature 244, 459-460 (1973)

73.

M.J. Trinick, Relationships amongst the fast-growing rhizobia of Lablab purpureus, Leucaena ]eucocephala, Mimosa ~ . ,

Acacia farnesiana and Ses-

bania 9randiflora and their a f f i n i t i e s with other rhizobial groups. J. Appl. Bacteriol. 49, 39-53 (1980). 74.

R. Uddin, W. McLaughlin and M.H. Ahmad, Competition between inoculum and native rhizobia and nodulation of cowpea: Use of a dark nodule strain. Plant and Soil 81, 305-307 (1984).

75.

S.L. Uratsu, H.H. Keyser, D.F. Weber and S.T. Lim, Hydrooen uptake (HUP) a c t i v i t y of Rhizobium japonicum from major U.S. soybean production areas. Crop Sci. 22, 600-602 (1982)

76.

H.J. van Rensburg and B.W. Strijdom, Survival of fast- and slow-growing Rhizobium s_p_Eunder conditions of r e l a t i v e l y mild dessication. Soil.Biol. 12, 353-356 (1980).

77.

D.P.S. Verma and S. Long, The molecular biology of Rhizobium legume

78.

J.M. Vincent, A manual for the practical study of root-nodule bacteria.

symbiosis. Int. Rev. Cytol. Suppl. 14, 211-245 (1983). International Biological Programme,Blackwell Scientific Publication, Oxford (1970). 79.

P.M. West and P.W. Wilson, Growth factor requirements of the root nodule bacteria.

80.

J. Bacteriol. 3_7_7,161-185 (1939).

P.M. West and P.W. Wilson, Biotin as a growth stimulant for the root nodule bacteria.

Enzymologia8, 152-162 (1940).

81.

P.M. Williams, The isolation of effective and ineffective mutants of cowpea

g2.

D.O. Wilson and K.M. Trang, Effects of storage temperature and enumeration

rhizobium.

Plant & Soil 60, 349-356 (1981).

method on Rhizobium spp numbers in peat inoculants Trop. Agric.(Trin.) 57, 83.

233-238 (Ig80). R.M. Zablotowicz and D.D. Focht, Physiological characteristics of cowpea rhizobia evaluation of symbiotic efficiency on Vigna unguiculata.

Appl.

Environ. Microbiol. 41, 679-685 (1978). 84.

R.M. Zablotowicz, S.A. Russel and H.J. Evans, Effect of the hydrogenase system in Rhizobium japonicum on the nitrogen fixation and growth of soybeans at different stages of development. Agron. J. 72, 555-559 (1980).