identification.
4. DISCUSSION
4 . 1 Isolation of Gram negative soi l bacteria
In this study a collection of soil bacteria were isolated and purified from four different soil types n amely Ramiha silt loam, Tokomaru silt loam, Kairanga silt loam (under w h i te clover-ryegrass pastures) a n d M a n aw atu sandy loam (under fallow l and with shrubs of Lupinus sp.). The soil bacteria w hich showed v arying colony morphology, n umbered about 200 were checked for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia. Only four strains nodul ated. The rem aining 1 96 strains did not nodulate. The medium used i n our study was not selectiv e for rhizobia. The experi mental design of the project w as i n tended to allow detection of any soil b ac teria ( n o t only rhizo b i a) w h i c h c o u l d accept a n d e x pres s p S y m fol low i n g c o nj u g a t i o n w i t h E s c h e r i c h i a c o li s t r a i n P N 2 0 0 c o n t a i n i n g p l a s m i d pPN l (pRtr5 1 4a: : R68.45).
The present work differed from earlier studies where the object was to i solate non symbiotic Rhizobium strain s from soil. I n these, Sober6n-Chavez and N ajera, ( 1 989) and Jarvis et ai . .. ( 1 989) i sol ated strains from soil that were chromosomally similar to R. leguminosarwn. Laguerre et al. , ( 1 993a) isolated from soil both symbiotic and non symbiotic Rhizobium Ieglilninosarum. I n an earlier study non -symbiotic strain s were also isolated from the rhizosphere of bean plants (Segovia et al., 1 99 1 ). These authors specifically wen t looking for non-nodulating rhizobia whereas in the present study we screened for any Gram negative soil bacteria unable to nodulate white clover.
The existence of self-transmissible plasmids coding for symbiotic functions in strains of R hizobium ieg Llminosarum is also well k nown and established (Djordjevic et al. , 1 9 8 3 ;
Hooykaas
et al. , 1 98 1 ; John ston e t a l . , 1978; Lamb et al., 198 2) and there is i ndirect evidence for plasmid transfer be tween s trains of R. Ieguminosarum in soil (Schofield et al. , 1 9 87). Conjugative plasmids like RP4 and its derivative R68.45 are readily transmitted between bacterial strains i n n atural environments such as soil and water (Sayre and Miller, 1 99 1 ) . Plasmids confe rring different plant specificities can occur in rhizobia of the same electrophoretic type (Young, 1 9 85 ) . When Rhizobium Ieguminosarum biovar trifoli i i solates from a field population were characterized with DNA probes, the same symbiotic plasmids were found i n related strains (Schofield et a I . , 1 9 8 7 ) . Y o u n g a n d W e x ler, ( 1 9 8 8 ) e x am i n ed t w o fie l d popul a t i o n s of R . leguminosarum biovar v iceae and found that although the distribution o f symbiotic1 1 0 p l a smids across chromosomal backgrounds was far from random, indistinguishable p l asmids c o u ld be found in geneti c a l l y u nrel ated bacteria . Each of these a uthors concluded that genetic exchange occurs between rhi zobia i n soil. Transfer of the symbiotic plasmid pJBSJI between strains of R hizobium at frequencies of upto 1 0-4 per rec ipient i n sterile and non- sterile soil has also been observed (Kinkle and S chmit,
1 99 1 ).
Interspecies transfer of symbiotic plasmids has been reponed from R . Zeguminosarum b i o v ar trifo l i i ( p R trSa) (Hoo y k a a s e t aZ. , 1 98 1 ) and fro m R hizo bium meliloti (pRMe4 1 6) (Kondorosi et al., 1 982) to Agrobacterium tumefa ciens. These species are as closely related as are some rhizobi a (Jarvis et ai. , 1 986), and there is evidence for a close ancestral relations hip between the plasmids of Rhizobium and Agrobacterium species based on plasmid incompati bility studies (O' Connell et al., 1 987) as well a s on phylogenetic analyses of the family Rhizobiaceae and related bacteria by sequencing of 1 6S r R N A genes ( W i l lems and Col l i n s, 1 99 3 ; Y anagi and Y amasato, 1 99 3 ) . N ev ertheless despi te t h i s c l o se rel ated n e s s , nod u l e s forme d b y A g robacte r ium transconjugants have been found to be ineffective (Kondorosi et al. , 1 982).
Conj ugative transfer of symbiotic plasmids to bacteria not normally associated with root nodules has been reported for A grobacteriwn tumefaciens (Hooykaas et al. , 1 98 1 ; Kondorosi et af., 1 9 8 2), Enterobac ter agg lomerans (Dohler and Kli ngmuller, 1988), Pseudomonas aeruginosa and a Lignobacter sp. (Plasin ski and Rolfe, 1 985) and soil bacteria (Jarvi s et al. , 1 9 89). These and similar observations have stimulated the pres e n t s tudy to a s se s s the d i versity of G ram negative soil bacteria cap a b l e of expressing a p S ym plasmid pRtrS 1 4a.
4. 2 T ransfer of pPN l
In this study, transfer of pPN 1 (pSym : : R68 .4S) to soil bacteria was demonstrated by (i) growth on an ti b iotic med i a , ( i i ) p l ant tes t ( V i ncent, 1 97 0 ) , ( i i i ) E c k h ardt g e l s (Eckhardt, 1 97 8 ) , (iv) the prese nce of bacteroids i n nodules (Pankhurst e t al. , 1 979) and ( v ) by probing transconj ugan t soi l bacteria i solated from the nodules with a radioactive nodA gene sequence.
4. 2. 1 Bacteria from several soi l types can express pRtrS 1 4a
In earlier work, Segovia et al. , ( 1 99 1 ) investi gated the soil population of symbiotic and non-symbiotic rhizobia isolated from the root region of six week old nodulated p lants h arves t e d from a field i n Tepoztl an , More lo s , M exic o. They reported th at the
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