Identification of nodule-dominant Rhizobium meliloti strains carrying pRmeGR4b-type plasmid within indigenous soil populations by PCR using primers derived from specific DNA sequences

Abstract: Rhizobium meliloti strain GR4 is a highly infective and competitive bacteria which was isolated in 1975 from a field site in Granada (Spain) and which has a high potential as an inoculant. R. meliloti isolates from alfalfa plants grown in this field site were characterized using polymerase chain reaction. Characterization was based on primers derived from insertion sequence elements (IS Rm3 and IS Rm4 ), plasmid origin of replication (pRmeGR4a repC locus) and plasmid pRmeGR4b specific DNA sequences. Soil isolates harbouring plasmid type pRmeGR4b represented the major infective population in this field site. A direct correlation between the presence of pRmeGR4b-like plasmid and the competitiveness of the strains was found. In addition, four different R. meliloti field populations isolated from Spanish soils were analyzed for the presence of pRmeGR4b related plasmids. Our results indicate that this plasmid type is widespread among R. meliloti field populations and that its frequency within the infective isolates depends on the host plant.     

Genetic basis of enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprint pattern in Sinorhizobium meliloti and identification of S. meliloti employing PCR primers derived from an ERIC-PCR fragment

The enterobacterial repetitive intergenic consensus (ERIC)-PCR method was employed to generate genomic amplification products of Sinorhizobium meliloti strain 2011. Eleven distinctive PCR fragments obtained in PCR reactions by using the ERIC2 primer were cloned and their partial or complete nucleotide sequences established. DNA sequences that extended past the ERIC2 primer region were not conserved among the 11 PCR fragments and showed no sequence similarity to the enterobacterial ERIC consensus sequence. Thus, repetitive ERIC or ERIC-like sequences seem not to be an integral part of the S. meliloti genome. An amplification product of S. meliloti 2011 was identified which was present in S. meliloti strains but absent in other rhizobial species. Based on the nucleotide sequence information, a pair of PCR primers was designed and used for PCR amplification of sequences of S. meliloti laboratory strains 2011, L5–30, AK631 and 102F34. Nucleotide sequence analysis of the amplification products revealed a 100% DNA sequence conservation. Database searches showed that the DNA fragment putatively encodes the C-terminal part of a protein displaying similarity to 2-hydroxyacid dehydrogenases of various organisms. The newly designed PCR primers should be useful for the rapid identification of S. meliloti isolates. Received: 17 February 1999 / Accepted: 9 April 1999     

Monitoring the diversity of Rhizobium meliloti field and microcosm isolates with a novel rapid genotyping method using insertion elements

Rhizobium meliloti strains isolated from alfalfa plants grown in a mining recultivation field, in a model ecosystem (microcosm) and in soil core containers were characterized by two new taxonomic methods, fingerprinting and handprinting, using insertion sequence elements (IS) as hybridization probes. The diversity of strains within the field population could first be detected with IS-fingerprinting, whereby nearly three times more groups of Rhizobium meliloti strains could be identified in comparison to the groups according to plasmid profiles. This complexity and diversity of the rhizobial population was also detected in microcosm studies. Strains identified among the field population were also detected in the microcosm studies. The persistence of rhizobia in soil was demonstrated in soil core samples held in a cold room for 2 years. A decrease in the genomic diversity of the R. meliloti population upon soil storage was observed. A novel monitoring method, IS-handprinting, in which the presence of certain endogenous insertion elements within a strain is registered, was successfully employed to characterize genetically the field R. meliloti strains with simplicity and speed. In contrast to IS-fingerprinting, IS-handprinting is based on a simple plus-or-minus detection, which is sufficient for a taxonomic characterization. Both methods, using a non-radioactive detection system, are sensitive enough to detect one copy of an insertion element in a strain's genome. IS-fingerprinting, with its fine resolution, would be suitable for ecological studies of individual strains in any complex ecosystem, whereas IS-handprinting would be suitable for monitoring strains and characterizing large numbers of strains.     

Relative genetic structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of alfalfa (Medicago sativa) and sweet clover (Melilotus alba)

Insertion sequence (IS) hybridization was used to define the structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown under controlled conditions and inoculated with a suspension of the same soil. The detection of R. meliloti isolated from soil on agar plates was facilitated by use of a highly species specific DNA probe derived from ISRm5. All R. meliloti obtained directly from soil proved to be symbiotic (i.e. nodulated and fixed nitrogen with alfalfa). Analysis of 293 R. meliloti isolates revealed a total of 17 distinct IS genotypes of which 9, 9 and 15 were from soil, M. alba and M. sativa, respectively; 8 genotypes were common to soil and both plant species. The frequency of R. meliloti genotypes from soil differed markedly from that sampled from nodules of both legume species: 5 genotypes represented about 90% of the isolates from soil whereas a single genotype predominated among isolates from nodules accounting for more than 55% of the total. The distribution of genotypes differed between M. sativa and M. alba indicating species variation in nodulation preferences for indigenous R. meliloti. The data are discussed in the context of competition for nodulation of the host plant and the selection of Rhizobium strains for use in legume inoculants. This study has ecological implications and suggests that the composition of R. meliloti populations sampled by the traditionally used host legume may not be representative of that actually present in soil.     

The persistence of bioluminescent Rhizobium meliloti strains L1 (RecA-) and L33 (RecA+) in non-sterile microcosms depends on the soil type,on the co-cultivation of the host legume alfalfa and on the presence of an indigenous R. meliloti population

In comparative analyses the influence of soil type, the rhizosphere of plants and the presence of an indigenous R. meliloti population on the population dynamics of bioluminescent R. meliloti strains L1 (RecA-) and L33 (RecA+) was assessed in microcosm studies. Both strains established better in a loamy and a clayey soil compared to a sandy soil. RecA- strain L1 showed a slightly but statistically significant reduced survival ability compared to RecA+ strain L33 (p 0.05). The presence of the host plant alfalfa stimulated the growth of both strains in non-sterile soil and no differences in the survival between both strains were observed. Co-cultivation of clover or wheat plants, respectively, neither positively nor negatively influenced the strains' survival. The most pronounced effect on the survival of both strains was exerted by the presence of an indigenous R. meliloti population. RecA- strain L1 showed a significantly impaired survival compared to RecA+ strain L33 (p 0.002). Moreover, no growth stimulation of strains L1 and L33 by the presence of the host plant alfalfa could be observed. These results indicate that the recA mutation affects the long-term rather than the short-term persistence of R. meliloti after environmental release.     

Genetic structure analysis of a highly inbred captive population of the African antelope Addax nasomaculatus. Conservation and management implications

The African antelope Addax nasomaculatus is a rare mammal at high risk of extinction, with no more than 300 individuals in the wild and 1,700 captive animals distributed in zoos around the world. In this work, we combine genetic data and genealogical information to assess the structure and genetic diversity of a captive population located at Parque Lecocq Zoo (N=27), originated from only two founders. We amplified 39 microsatellites previously described in other Artiodactyls but new to this species. Seventeen markers were polymorphic, with 2–4 alleles per locus (mean=2.71). Mean expected heterozygosity (He) per locus was between 0.050 (marker ETH3) and 0.650 (marker D5S2), with a global He of 0.43. The mean inbreeding coefficient of the population computed from pedigree records of all registered individuals (N=53) was 0.222. The mean coancestry of the population was 0.298 and F_IS index was ?0.108. These results reflect the importance of an adequate breeding management on a severely bottlenecked captive population, which would benefit by the incorporation of unrelated individuals. Thanks to the successful amplification of a large number of microsatellites commonly used in domestic bovids, this study will provide useful information for the management of this population and serve as future reference for similar studies in other captive populations of this species. Zoo Biol 30:399–411, 2011. © 2010 Wiley‐Liss, Inc.