Intrinsic multiple antibiotic resistance markers for competitive and effectiveness studies with various strains of mung bean rhizobia

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1 Biosci., Vol. 5, Number 3, September 1983, pp Printed in India. Intrinsic multiple antibiotic resistance markers for competitive and effectiveness studies with various strains of mung bean rhizobia Introduction R. P. GUPTA, M. S. KAIRA, S. C. BHANDARI and A. S. KHURANA Department of Microbiology, Punjab Agricultural University, Ludhiana MS received 28 January 1983; revised 1 July 1983 Abstract. Ten strains of Rhizobium sp. with multiple antibiotic resistance markers were used for competitive and efficiency studies with mung bean var. ML 5. All the strains showed significant increase in grain yield and so also for nitrogenase activity except MO 5. Nitrogenase activity correlated very well with grain yeild. The compatibility of strains varied from 17 to 50%. The intrinsic multiple antibiotic markers for strain identification were found to be stable after passing through soil and host conditions and could be used for ecological studies. It was further revealed that the overall efficiency of a strain is the combined effect of characters like compatability, competitiveness and inherent capacity to fix nitrogen. Keywords. Intrinsic multiple antibiotic markers; competitiveness; ecological studies; effectiveness. Of the various nitrogen fixing systems, the symbiosis of Rhizobium with leguminous plants is the most effective system contributing annually at the rate of tonnes to the nitrogen balance of the ecosystem (Burris, 1977). The major controlling factors contributing to the efficiency of inoculated strains are reported to be host genotype (Minchin et al., 1978; Mytton, 1978; Poi and Kabi, 1979) and ability of strain to dominate over the native rhizobia for nodulation (Herridge and Roughley, 1975; Gibson et al., 1976), in addition to its inherent capacity to fix nitrogen (Johnson and Beringer, 1975). Various valid methods such as serological technique (Dudmen and Brockwell, 1968), immunfluorescence microscopy (Bolhool and Schimdt, 1970), antibiotic resistance markers (Obaton, 1971) have been employed to understand the contribution of introduced rhizobia with respect to nodulation. Due to close correlation between results obtained using immunological and antibiotic resistance characters (Schwinghamer and Dudman, 1973) as well as the convenience of nodule sampling procedure, the intrinsic multiple antibiotic resistance markers were used to study the competitive and effectiveness of the introduced strains of Rhizobium. Abbreviations used: Amp, Ampicillin; Ery, erythromycin; Str, streptomycin; Gen, gentamycin; Nal, nalidixic acid; Tet, tetracycline; Can, chloramphenicol; kan, kanamycin. 253

2 254 Gupta et al. Materials and methods Screening Ten strains of Rhizobium sp. (cowpea group) used for competitive studies were screened for their intrinsic antibiotic resistance markers (table 1) using antibiotic disc assay method on Bergersen s medium (Bergersen, 1961). The discs (Span Diagnostics, Surat) used were streptomycin, 10; kanamycin, 30; nalidixic acid 30; chloramphenicol, 30; tetracycline, 30; gentamycin, 10; erythromycin, 15; and ampicillin, 10 µg/disc. Experimental layout Seeds of mung bean var. ML 5 were treated with charcoal based culture of 10-strains of Rhizobium sp., and sown in randomised block design (net plot area 5.4 sq.m.). Uninoculated seeds served as control. Phosphorus was applied at 40 kg P 2 O 5 /ha through single super phosphate as a basal dose. Observations At 45 days of growth: Five plants from each plot were uprooted and observations for the number of nodules, dry weight (wt) of nodules, plant height, dry wt of plants were recorded. Nitrogenase activity was measured by the acetylene reducing technique (Hardy et al., 1968) by gas liquid Chromatograph (Gas Chromatograph, Nucon Model 5560) using a porapeak R column. Competitiveness: The isolates of thirty nodules from five plants were screened for intrinsic antibiotic markers as explained already. The per cent competitiveness was calculated according to formula % Competitiveness= % marked nodules % marked nodules in experiment in control At harvest: Crop was harvested at maturity and observations for total dry matter and grain yield were recorded. Results Intrinsic antibiotic spectra of Rhizobium strains Out of ten strains used, strains Μ 10 and GMBS 1 were resistant to only one antibiotic; strains KM - 1, MO 5 5, PL 1, Μ 1 and Niftal (Tal 169) to two antibiotics; 32 HI to three antibiotics and strains Μ and Μ to four and five of the antibiotics tested (table 1). The frequency of resistance to different antibiotics was in the order of Amp>Ery = Str>Gen = Nal>Tet=Can>kan. Nitrogen fixation of efficiency in mung bean under field conditions The efficiency of ten inoculated strains in relation to number of nodules per plant, nodule dry wt per plant, plant height, dry wt per plant and nitrogenase activity was observed. The data are given in table 2. Out of these five parameters, nodule number, nodule weight and nitrogenase activity were found to be significantly higher with inoculation. The inoculation resulted in higher number of nodules with all the

3 Antibiotic markers for host-rhizobium interactions 255 Table 1. Antibiotic resistance spectra of different strains of Rhizobium sp. (cowpea group). s = Sensitive; r = resistant. Table 2. Effect of Rhizobium inoculation on ancillary characters of mung bean ML5.

4 256 Gupta et al. strains except KM 1 which was, however, at par with control and Μ 1. Seven strains viz. M 1, M 73001, MO 5, Μ 73002, PL 1, Niftal (Tal 169) and M 10 were at par with control whereas strains GMBS 1 and 32 HI were significantly higher than control but at par between themselves. The per cent increase in nodule number in strains GMBS 1 and 32 HI was 48.3 and 62.9 respectively. In terms of nodule dry weight, Μ 10, Niftal (Tal 169) and 32 HI gave significantly higher nodule dry weight over KM 1, Μ and PL 1. Other strains, however, were at par amongst themselves. A significant increase in nitrogenase activity with all the strains over uninoculated control except strain MO 5 which showed non-significant increase of 27.6% was observed. Strains PL 1 and M 10 were superior over MO 5 whereas strain GMBS 1, KM 1, Μ and Μ were at par among themselves but significantly higher than Μ 10. Strain Μ 1 showed highest activity followed by Niftal (Tal 169) and 32 HI. Strain Μ 1 and Niftal (Tal 169) showed significantly higher activities over all other treatments. The effect of inoculation on final dry matter and grain yield is given in table 3. The final dry matter was significantly higher with four strains viz. GMBS 1, PL 1, KM 1 and Μ 1 with a percentage increase of 28.4, 28.9, 36.4 and 43.8 respectively, whereas strains Niftal (Tal 169), Μ 73001, and 32 HI with per cent increase of 7.5, 7.5 and 9.5 were at par with control. However, strains Μ 73002, MO 5, Μ 10 showed a decrease in final dry matter. All the test strains resulted in significantly higher grain yield with percentage increase ranging from 15 60%. Strain Μ 1 gave highest grain yield (875 kg/ha) followed by Niftal (Tal 169) (823 kg/ha). Table 3. Performance of various strains of Rhizobium sp. on yield of mung bean ML 5.

5 Antibiotic markers for host-rhizobium interactions 257 Correlation coefficient and regression between yield and yield attributing characters The correlation coefficient values and regression equations between yield and yield attributing characters are given in table 4. The grain yield was found to be the best correlated with dry matter having r value of 0.58 with a regression of Y = X followed by nitrogenase activity and the nodule weight with r value of and respectively, whereas, the nodule number was found to be poorly correlated (r=0.059). Similarly, a poor correlation of was observed in case of nodule number vs nitrogenase activity, but on the other hand nodule weight vs nitrogenase activity showed a correlation of A good correlation (r=0.4) was observed between nitrogenase activity and dry matter with a regression of Y = X. Table 4. Correlation coefficients and regression equations for yield and yield attributing characters in mung bean. Table 5. Competitive ability of introduced strains of Rhizobium sp. with native rhizobia (mung bean ML 5).

6 258 Gupta et al. Competitive ability The competitive ability of introduced strains over native population in forming nodules is given in table 5 which ranged from 17 to 50.0%. The highest competitiveness was found with GMBS 1 and Niftal (Tal 169) (50.0%) followed by Μ and Μ 1 (40% each), whereas strains MO 5 and PL 1 could compete only to the extent of 17%. The antibiotic spectra of isolates obtained during competition studies showed that a large number of native rhizobia were sensitive to all the test antibiotics (53%). It was further observed that frequency of resistance to single and double antibiotics was higher than multiple resistance (figure 1). Figure 1. Frequency of multiple antibiotic resistance in cultures of Rhizobium sp. isolated from root nodules of mung bean. Discussion Screening for multiple drug resistance and also for antibiotic resistance revealed, that most of the strains were resistant to ampicillin and streptomycin, and least to chloramphenicol, tetracycline and kanamycin. Similarly reports on occurrence of multiple drug resistance and higher resistance to antibiotics like penicillin, streptomycin, erythromycin and low frequency to tetracycline and chloroamphenicol have been reported by Cole and Elkan (1979) and Kahlon (1980). Frequency distribution of isolates made from root nodules of mung bean (figure 1) during competitive studies revealed that with the increase in resistance to number of antibiotic markers their frequency decreased. In an extensive screening of soil bacterial isolates, Partiskaya and Corelova (1976) and Cole and Elkan (1979) also demonstrated low frequency of multiple drug resistance. This is in confirmation with the theory of probability, according to which chances of an incidence decreases with the increase in limitations to which it is subjected. Similarly, the susceptibility of majority of isolates to antibiotics may be due to the fact that these bacteria have not been exposed to antibiotics in the natural habitat. The low degree of increase in dry matter production in some of the inoculated strains could possibly be on account of comparatively low efficiency of these

7 Antibiotic markers for host-rhizobium interactions 259 strains in terms of nitrogen fixation, thereby overall less contribution to the pool of source but the extent of transfer towards the sink remaining the same. The low efficiency of these strains is evident from the low values of nitrogenase activities (2848 and 1672 units for Μ 10 and MO 5 respectively) as compared to other strains. In case of strains Μ 73002, although the activity was reasonably good but its nodule weight was found to be less as compared to uninoculated control. Similarly, low values were also observed in few other strains like KM 1, Μ and PL 1 which might be due to small size and fewer number of nodules. The distribution pattern of root nodule on root system might be governing the utilization of biologically fixed nitrogen by the host. The beneficial effects of Rhizobium inoculation towards increasing the nitrogenase activity and yield have also been observed by various workers (Subba Rao, 1976; Sinha, 1978; Minchin et al., 1978). The correlation coefficient between yield and yield attributing characters indicated that yield is correlated to dry matter (r=0.58) which in turn is dependent on nitrogenase activity (r=0.4) hence the nitrogenase activity would be most reliable parameter for estimating the yield. Moreover, nitrogenase activity can be assessed after a short period of crop growth and the possibility of handling large number of samples at a time makes it a more feasible parameter for assessing the efficiency of inoculation strain and in computing the yield. Results reveal that higher yield of the crop is dependent not only on the inherent nitrogen fixing capacity (nodulation on nitrogenase activity) on the strains but also on their competitive ability. For instance strains Μ 1 and Niftal (Tal 169) show high degree of nitrogenase activity as well as competitive ability (40 50%) and consequently highest grain yield were also obtained due to these strains: High yield obtained through strains GMBS 1 is also attributed to its υ. high competitive ability and moderate nitrogenase activity. On the other hand strain 32 HI showed moderate nitrogenase activity but extremely poor competitive ability and consequently resulted in poor grain yeild. As an extreme case, the two strains PL 1 and MO 5 showed very low nitrogenase activity as well as competitive ability and these two strains produced the lowest grain yield. Thus it is seen that presence of both the qualities, viz., high nitrogenase activity as well as high competitive ability is essential for obtaining optimum symbiotic association leading to high yield of the crop. Consideration of competitive ability (or dominance against native rhizobia) especially, in case of highly promiscuous legume such as groundnut (Arachis hypogea) and other cowpea group of legumes, while screening for the most efficient rhizobial strains was also advocated by Gaur et al (1974). The reisolation of multiple antibiotic resistance marked strains from the root of nodules of mung bean after passing through the soil environment and host indicate that these markers are stable and could be used successfully for such ecological studies. References Bergersen, F. J. (1961) Aust. Biol. Sci, 14, 349. Bolhool, Β. Β. and Schmidt, Ε. L. (1970) Soil Sci, 110, 229. Burris, R. Η. (1977) in Genetic Engineering for Nitrogen Fixation, ed. Α. Hollaender, (NewYork: Plenum Press), pp Cole, Μ. A. and Elkan, G. Η. (1979) Appl. Environ. Microbiol., 37, 867. Dudman, W. F. and Brockwell, J. (1968) Aust. Agric. Res., 19, 739. Gaur, Υ. D., Sen, A. N. and Subba Rao, N. S. (1974) Zbl. Bakt. II Abt, 129.

8 260 Gupta et al. Gibson, Α. H., Date, R. Α., Ireland, J. A. and Brockwell, J. (1976) Soil Biol. Biochem., 8, 395. Hardy, R. W. F., Holsten, R. D., Jackson, E. K. and Burns, R. C. (1968) Plant Physiol., 43,1185. Herridge, D. F. and Roughly, R. J. (1975) Appl. Bacteriol., 38, 19. Johnson, A. W. Β. and Beringer, J. Ε. (1975) Gen. Microbiol., 87, 343. Kahlon, G. Κ. (1980) Study of symbiotic nitrogen fixation with Rhizobia mutants, M. Sc., Thesis, Punjab Agricultural University, Ludhiana. Minchin, F. R., Summerfield, R. J. and Eaglesharn, A. R. J. (1978) Trop. Agric., 55, 107. Mytton, L. R. (1978) Ann. Appl. Biol., 88, 443. Obaton, M. (1971) C.R. Acad. Bulg. Sci., Ser. D, 272, Partiskaya, A. N. and Corelova, O. R. (1976) Mikrobiologiya, 45, 869. Poi, S. C. and Kabi, M. C. (1979) Indian Agric., 23, 49. Schwinghamer, E. A. and Dudman, W. F. (1973) Appl. Bacteriol., 36, 263. Sinha, S. K. (1978) in Nitrogen Assimulation and Crop Productivity, eds S. P. Sen, Y. P. Abrol and S.K. Sinha (New Delhi: Associated Pub. Co.), pp Subba Rao, N, S. (1976) in Symbiotic Nitrogen Fixation in Plant, ed. P. S. Nutman, (Cambridge: Cambridge University Press), pp