Dart, P J

Josey, D P

Mittal, S

Rupela, O P

Thompsan, J A

Toomsan, B

ICRISAT Open Access Repository

SNF Technology for Tropical Agriculture APPLICATION OF IN H E R E N T  ANTIBIOTIC RESISTANCETO ECOLOGICAL S T U D I E S  O F  K H I Z O B I A  O.P. Rupe1i.l D.P. Josey,z B. Toomsm,' S. Mittal,] P.J. Dart,' and J.A. Thompson] Summary The  inherent antibiotic t-csisti~i~cr: technique shows promise for use in ecological studies. 1 h c  t cc l~nique  involves the use of readily available chemicals I'or grnwiny r h i ~ o b i a ,  antibiotics and a simple, easily manufactured multi-inoculator. I-he main requirement of the method i s  precise rnaintcniincc of conditions th roughout  the  experiment for  all va r l ; ~  hics a n d  laboratory processes. -1'hesc precautions a re  discusscd. In a study of 473 l'icld ihirlitt~s l ' r on~  a trial inuculated with a streptomycin (s t r  200) rnarkcd nlututll. the  use of 30charactcrist ics (10 antibiotics x 2 4  conccntri~tiorls)  classit'ied the strains into 203 groups  when all charartrrihlics hi~d  to match perfectly, and into I19 groups  when o n e  mismi~tch ing  characteristic was allowed. The 22 isolates having s t r  200 rcsiat;~ncc wcrc placed in three groups with n o  mismatches. and  inti ,  two groups  with one mismatching character allowed. INTRODUCTION The success of introduced Rftirohium strains in field trials has bccn monitored using both serology and strains marked with antibiotic resistance (Read, 1953; Dudman & Brockwell, 1968; Schwinghamcr & Dudman, 1973). Neither of these techniques, however. gives much information concerning the composition of the indigenous population of Rhizobiurn. Using the inherent antibiotic resistance technique described by Josey er a/. ( 1979). the indigenous soil population of Rhizohiunt has been shown to be hcterageneous(Beynon & Joscy, 1980). This technique could be used in examining some current I CP No. 43, ICRISAT. Patancheru P.O., Andra Frrdesh M2-324. India, 2 John Innet Institute, Colney Lone. Nomich NR4 74H, Englnnd. probkms in field inoculation trials: the heterogeneity of indigenous populations, changes in the population over cropping season and with particular sgricultur~l practices. and,in some cases, the reason for the success of particular inoculant* st rains. This paper discusses precautions to be observed in adapting the technique to local conditions, and reports on application of the technique to study rhizobia nodulating chickpea (Cicer arierinum t.) at ICRISAT. " SOURCES OF ERROR IN THE INHERENT ANTIBIOTIC RESISTANCE METHOD Every effort must be made to maintain rigid control of the experimental conditions. If the test conditions are not completely standardized, it will be very difficult to draw any conclusions about the relationship between strains examined in different tc5t ieries. Potential soums of error using this technique are: Medium campostfion: The concentration of all constituents of the growth medium must be conrtant, and media should always be prepared using the same grade and brand of reagents. The number of ions available in one make of yeast extract may be widely different from those in another brand, and this can affect strain growth and antibiotic resistance differentially, Medium s/~rifiza/ion and melting: Growth medium should always be sterilized and melted in the same way. If  the medium is heated for different periods of time or at different temperatures, its composition may also vary, and hence af'f'cct growth. Antibiorirs: The same supplier shbuld be used, as the strength and formulation of' antibiotics can vary between manufacturers. The potency of nearly all antibiotics will decrease with age, particularly when made up into stock salution, so large amounts of stock solution should not be prepared. Repeated freezing and thawing of antibiotics should also be avoided as much as possible. Mixing oj' an/ihiolit:t it1 media: When mixing the antibiotic with agar medium the temperature should be kept at 60° in a water bath. Once the plates arc pouted oome antibiotics also will start to lose activity; thus, plates should be inoculated as soon as the whole set of plates is ready. If the agar is cool during mixing (c. 4S°C), an even distribution of antibiotic throughout the medium may not be achieved, and strict comparisons between plates will not be powible, I t  is very important that the antibiotic concentrations in the medium m accurately reproduced on each occasion. InocslI~t  condiriun: Scneitivity to some antibiotics (such as penicillin) may depend on cell growth phaoc, and it is very desirable to ust inocula of a rearonrb~'uniform 11ta8e ~ I ~ r o w t h .  Cells in the stationary phase may survive cxparu~to an antibiotic to which they are normally considered sensitive and thm C O ~ ~ I I C M  to grow again after concentration of&tibiotic in the medium fi " 1  has decreased below the threshold value for activity a p h t  that particular strain. Thickness ofplar~:  If  the thickness of medium in the platea wr&, then the colony morphology will be influenced; e.g., slime may only be produced by large colonies making it difficult to assess the difference between control and antibiotic plates. if they are of different thickness. Plates with bubblesshould also be discarded because of lack of homogeneity, Drying ojpkates: As plates should be used immediately, drying may not be practical unless a laminar llow sterile hood is available. If plater arc dried the same procedure must be used on every occasion. Contamination: Contaminated plates should be discarded, at waste products from the contaminants may be synergistic with, or destroy, the antibiotics, and these effects may permeate the whole plate; notjust the region - where the contaminant is growing. For similar reasons, fast- and slow- growing strains of Rhizuhiirm should not be tested on the same phte. AN APPLICATION OF THE TECHNIQUE TO RHIZOBIA NODULATING CHICKPEA AT ICRISAT In an experiment with chickpea at ICRISAT, in which the mutant strain 9036, resistant to 200/ug/ ml streptomycin was used, 473 nodule isolates werc obtained that lacked this resistance, and 22 were found with resistance to streptomycin. In herent anti biotic resistance studies werc undertaken to characterize these strains. The technique used was as follows: Yeast extract mannitol agar (YMA), was made up accurately, sterilized, and kept at 60-6S°C ready for use. Just before pouring the plates, the required volume of antibiotic stock solution (see Table I )  was added to the known volume of medium in the flask and swirl- mixed to ensure its proper distribution in the medium. The same volume of medium was added to each petri dish, using either a sterile measuring cylinder or a sterile graduated beaker; for 9 cm diameter glass petri dishes. 30 mi was used. The plates were then poured on a flat surface to achieve uniform thickness. Rhizobium strains used for strain typing were grown on YMA slopes in McCartney bottles, and as soon as growth war seen (usually four to five dop with Cicer rhizobia) the cells were suspended in sterile, 2046 (vlv) gfyarol, dispensed in small quantities suitable for one day's testing, and stored in a deep freeze at - 10°C. The antibiotic test plates and controls were inoculated with a pronged, multiple inoculator (Josey err dl.. 1979). Glycerol-stored cultum were diluted with sterile distilled water to give approximately 10Jctlla/ mi and plaotd in the wells of the pin inoculator. At this concentration each prong of the inmalator transferred aproximately 10' rhizobia to the test plater. After incukaon at  about - 2B°C for 7 days the growth on antibiotic medium was c a m p e d  with TABLE 1 : Ut of antibiotics and their cancentradom. ' AntiWoti~s Cancen trationa (mg/ 1 ) 1 Cubeaidllin 1,0, 2.5, 5.0 kytbomyda 1'25, 2.5, 10 Krnmycin 2.5, 10, 20 NdidMc add 2.5, 10, 15 Nsomyein 2.5, 10, 15 Pol y myxh 5, 10, 20 Rifuapicin 0125, 0.5, 23 Stmp tomy& 2.5, 10, 20, 200 Tstncycljnn 0,1, 0 s  Vancamycln 1.25, 2.5, 10 'AU 8~tibbW arc frbm Sigma except ~rbenleillin which &I from 'Pyopcn', B8whun. I *Antibbtlc ldvtbn' wore made in it~rlla deionized water, except cnthmrn y - cin (in e tho l )  and nJldixic acid (in 1 M NaOH). that on control plates. Colony growth was scored numerically (1: no growth; 2: weak growth: 3: good growth) go that the results w e n  amenable to computer mrlysi~.  Wheo the 473 N t e r  wm lcod on the bani of rcrbtance to 10 antibiotics at 24 co~l~~atmtiou. $03 ditinct groups were found. When one mismatch in the amy of (;ltl rm pumittod, 1 19 goup8 were distinguihcd. Samples of ~ t n i a  PO36 (nfcten~ culture) could be separated into two Broups by inherent antibiotic mhtm#, while streptomycin-resistant field isolates fell into these two p u p , ,  plus onrother, Whcn one W t c h  w u  allowed, all nfcrence culturcr fell into one group u dM 21 of tbe 22 r$mptoetlyCirr nrirtant field icolatec. Josey, D. P., Beynon, J. L., Johnston, A. W. B., & Bcringa, J .E .  (1979) J,  Appl. Ba(-~eriol. 46. 34.1-.150. Read. M.P. (1953) 3. Gurr. Mic.rohirr1. 9, 1-3. Schwinghamer, E.A. & Dudman, W.F. (1973) J.  Appl, Bacraiol. 36,263-272. 6: ! , t  J q < , '  Tbc rmMf,iiA#norut@d; @I the heterogeneity of roil populations. 3ku ~ 1 n of the pmptrth of the different groups of bolates, and of *aIs wib w@d hip explain the buh for their competition with 

Application Of Inherent Antibiotic Resistanceto Ecological Studies Of Rhizobia

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