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.
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
~.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.
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).
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
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
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.
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
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.
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.
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
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
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
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
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
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).
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.
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).
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.
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),
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
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).
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
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).
Age of the Plant (DAP)
No. of p l a n t s sampled
Miss K e l l y
McLaughlin and Ahmad, unpublished
Average No. o f nodules
ECOLOGY AND GENETICS OF TROP1CAL RHIZOB[A SPECIES
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).
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 .
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,
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)
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).
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).
garan et al (65) examined the effect of temperature to which the inoculants were exposed during shipment on the survival of rhizobia.
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
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).
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
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.
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.
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.
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.
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.
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.
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