Indian Journal of BiotechnologyVol 2, October 2003, pp 577-582

Use of Heavy Metals for Quantification of Rhizobia and Suppressionof Bacterial Contaminants in Carrier Based Inoculants

R P Jeevan Kumar and K S Jauhri*Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110 012, India

Received 17 October 2001; revised received 31 January 2003; accepted 18 March 2003

Quality analysis of commercial legume inoculants involves determination of number of live rhizobia ofappropriate species per unit weight of carrier. Conventional analysis is performed by means of plate counts ofrhizobia on CRYEMA, but contaminants sometimes suppress/mask the growth of rhizobial colonies and make theircounts unrealistic. Plant infection-most probable number (MPN) assay is considered to be more reliable, but isrelatively expensive and time consuming. In the present study, a selective medium is devised, which permits growthof selected strains of rhizobia and at the same time refrains the growth of contaminants on plates. To inhibit non-rhizobial contaminants in carrier based inoculants, heavy metals were added in different combinations. Acombination of Zn and Co at a concentration of SOppm each was found to inhibit bacterial contaminants withoutimpeding the growth of rhizobial colonies on CRYEMA. However, these additions were not effective in refraining theproliferation of contaminants in charcoal-soil based rhizobial inoculants at this concentration. These metals whenused in higher concentrations in charcoal-soil based inoculants, adversely affected nodulation and the dry matteryield of soybean.

Keywords: heavy-metal, bacterial contaminant, inhibition, Rhizobium-inoculant

IntroductionBio-inoculants are used to provide leguminous

crops with rhizobia for increasing their yields. A goodquality inoculant must maintain and deliver a highnumber of effective rhizobia in rhizosphere of theinoculated crop (Smith, 1992). However, severalcommercial inoculants of dubious quality have beenmarketed especially in tropical countries where highquality peat is not available to enhance biologicalnitrogen fixation (Brockwell & Bottomley, 1995).These inoculants apart from having high levels ofcontaminants, which adversely affect their quality andin turn effectiveness in field, also contaminate soilwith unknown microorganisms (Olsen et al, 1994).Many countries have overcome the potential risk byplacing legislation that prohibits the use of non-sterilecarriers in the manufacture of legume inoculants.Indian Standards Institution (1986) also prescribedthat there should be no contaminant at 10-6 dilution ofRhizobium inoculant when enumerated by dilutionplate method. However, carriers in India are generallysterilized once in an autoclave and then impregnatedwith rhizobial broth culture in open trays. This

*Author for correspondence:Tel:91-l1-25842112, 25847649; Fax: 91-11-25846420E-mail:ksjauhri@yahoo.co.in

procedure permits the proliferation of a large numberof contaminants in carrier based inoculants(Thompson, 1984).

Attempts have been made to reduce the level ofcontamination and to enumerate rhizobia in theirpresence In carrier based inoculants. Certainfungicides and antibiotics when added Incombination, suppressed fungal and actinomycetecontaminants in charcoal-soil based inoculants toacceptable limits. However, bacterial contaminantssurvived well till 16,000 ppm of each antibiotic usedin combination in the carrier (Jauhri & Gupta, 1989).

Metal tolerance among soil microorganisms hasbeen extensively studied and marked differences inthe levels of resistance have been reported even fordifferent rhizobial strains within a single species andother organisms (Giller et al; 1984; Silver & Mishra,1988; Dean-Ross & Mills, 1989; El-Aziz et al, 1991).If metal resistant strains of rhizobia are used in carrierbased inoculants, the problem of suppression ofrhizobial colonies by contaminants may be overcomeby incorporation of heavy metals in the CRYEMA.This will make rhizobial counts more realistic.

In the present investigation, the utility of heavymetals was explored for direct quantification ofrhizobia from commercial inoculants. The feasibilityof adding heavy metals in carrier was also examined

578 INDIAN J BIOTECHNOL, OCTOBER 2003

for reducing contaminants and improving symbioticperformance of inoculants.

Materials and Methods

Organisms and Cultural ConditionsThe strains of Bradyrhizobium and Rhizobium

species listed in Table 1 were obtained from theCulture Collection of the Division of Microbiology,Indian Agricultural Research Institute, New Delhi 110012, India. Yeast-ex tract-mannitol (YEM) medium(Vincent, 1970) was used for the cultivation ofrhizobial strains. Wherever maintenance of thesestrains was required 15 g of agar was added into themedium to solidify it and it was referred to as YEMA.For enumeration purpose, sterilized solution of congored (0.25%) was added at 10 mlrl of medium justbefore pouring into the plates and the medium wasreferred to as CR YEMA.

CarrierLocally available materials, such as farm-yard

manure (FYM), wood charcoal, vermiculite, soil andpeat, which are commonly used as carriers forcommercial production of inoculant in India, were air-dried, powdered and passed through a 100-meshsieve. Charcoal and vermiculite were separately addedto soil in a 3:1 proportion. Soil when used as carrier,was amended with FYM in a 3: 1 proportion beforeuse.

Heavy MetalSeven heavy metals of analytical grade were added

as ZnS04.7H20, CuS04.5H20, CoS04.7H20, PbS04,CdS04, HgCh and NaMo04. A stock solution of eachmetal was prepared at 10 gl' in distilled water exceptfor PbS04 which was prepared at 5 gl'. All stocksolutions were filter-sterilized before addition tosterile molten media. Jensen's N-free solution(Vincent 1970) was used for irrigation of plantsgrown for enumeration of bacteria by the mostprobable numbers (MPN) method. Seeds of soybean(Glycine max) cultivar Pusa-22 obtained from PulseLaboratory, IARI, New Delhi, were used in this study.Strains of rhizobia and bradyrhizobia were tested forintrinsic resistance to heavy metals by spotting aloopful of cell suspension (max lx103 cells ml") onCRYEMA plates containing a range of heavy metalconcentrations, including control without heavy metaladdition. The level of resistance was assigned to thehighest concentration of heavy metal whereunimpeded growth of the test strains occurred.

Minimal Inhibitory Concentration (MIC) TestHeavy metals were tested singly and in

combination for their ability to inhibit the growth ofcommon bacterial contaminants of charcoal-soi Icarrier on CRYEMA. Dilutions of carrier basedinoculants above 10-6 were checked for inhibition ofcontaminants by heavy metals. The heavy metalconcentration at which no bacterial contaminantappeared in 10-6 dilution, was taken as its MIC.

Preparation of Carrier Based InoculantA total of 100 g each of non-sterile carrier VlZ.

charcoal-soil (3:1), soil-FYM (3:1), vermiculite-soil(3:1), peat and charcoal was weighed separately inpolythene bags. Wherever required, charcoal-soil(3:1) carrier was amended with zinc and cobalt incombination at different concentrations ranging 0-5000 ppm on a dry weight basis. An appropriate andequal quantity of fresh broth culture ofBradyrhizobium japonicum (SB-103) was addedseparately. For optimum aeration, final moisturecontent of each carrier was then adjusted to 35% oftheir water-holding capacity by mixing with sterilewater. The inoculant bags were then sealed. Thecontents of the bags were mixed thoroughly bymassaging the packets from outside. The inoculantpackets were stored at ambient temperature(30o±2°C).

Quantification of Rhizobia from InoculantThe rhizobial population was assessed in inoculants

30 days after inoculation by the three methods ofenumeration, dilution and plate count usingCRYEMA (Vincent, 1970), plating on selectivemedium (CRYEMA containing 50 ppm each of zincand cobalt) and plant infection most probable numberassay (Somasegaran & Hoben, 1985).

Symbiotic Performance of Bradyrhizobial InoculantContaining Heavy Metals

Symbiotic performance of heavy metal amendedinoculant of Bradyrhizobium japonicum (SB-l 03) wasexamined with soybean (cv Pusa-22) in a potexperiment. The soil was treated with a basal doseof finely ground single superphosphate at 60 kg P20Sha-l and well decomposed FYM at 10 tonnes ha'. Soilwas moistened to one third of its saturation capacityand earthen pots of appropriate size were filled inwith 12 kg of this soil. The pots were wrapped withpaper and steam sterilized for 2 hrs at 121°C.

Surface-sterilized seeds of soybean (cv Pusa-22)were inoculated with charcoal-soil (3: 1) based

JEEVAN KUMAR & JAUHRI: USE OF HEAVY METALS IN CARRIER BASED INOCULANTS

inoculants of Bradyrhizobium japonicum (SB-103)containing Zn and Co applied at differentconcentrations in combination using a sterile sucrosesolution (10%) as adhesive. Five seeds were sown ineach pot and each treatment was in triplicate. Afteremergence, the seedlings were thinned to three plantsin each pot. Plants were irrigated with sterile tapwater. The data on nodulation and dry matter yield ofsoybean were recorded after 45 days of plant growth.The data on counts of colony forming units ofrhizobia were transformed to 10glO. Standardstatistical procedures were used for analysis (Gomez& Gomez, 1984).

Results and DiscussionTo be an effective agent for enumeration of

rhizobial and bradyrhizobial strains, heavy metalsmust inhibit the growth of contaminants on CRYEMAwithout impeding the growth of rhizobial strains.

579

Therefore, an effort was made to determine theminimal inhibitory concentrations of heavy metals toinhibit common bacterial contaminants of charcoal-soil based inoculants of rhizobia.

Since no fungal or actinomycete contaminants wereobserved in charcoal-soil based inoculants at 10-6

dilution, MIC of each metal was determined only forbacterial contaminants. In comparision to rhizobia,the bacterial contaminants of the carrier were found tobe more tolerant to high concentrations of Mo (>7,000ppm), Pb (>2,000 ppm) and Hg (5 ppm). Thus, thesemetals were found to be ineffective for inhibitingbacterial contaminants tested on growth medium. TheMICs of the other metals tested were more or lesssimilar to those of the rhizobial and bradyrhizobiaistrains except Zn and Co. From these results theauthors concluded that it was not possible to inhibitthe growth of contaminants by the addition of heavymetals individually to CRYEMA during enumeration

Table I-Tolerance of rhizobial strains and charcoal-soil based bacterial contaminants to heavy metals in CRYEMA

MetalZnCoCuCdPbHgMo

MIC*(ppm)150751501002,0005>7,000

Zn+CuZn+CoZn+CdCo+Cd

100+10050+5075+4030+30

Heavy metal resistant strains of rhizobia**81,2,3,4,5,6,7,8,9NoneNoneNoneNoneNone

None1,2,3,4,5,6,7,8,9,10,11,12,13,14NoneNone

Zn+Cu+CoCu+Co+CdZn+Cd+CdZn+Co+Cd+Cu

*MIC of heavy metal for bacterial contaminants of charcoal-soil (3: 1) based inoculants of rhizobia**Rhizobial strains tested:

50+50+2525+25+2525+25+25<25+25+25+25

Group/speciesl. Bradyrhizobium sp. (Cajanus)2. Bradyrhizobium sp. (Vigna)3. Bradyrhizobium sp. (Vigna)4. Bradyrhizobium sp. (Arachis)5. Bradyrhizobium sp. (Crotolaria)6. Bradyrhizobium sp. (Cyamopsis)7. Bradyrhizobium japonicum8. Azorhizobium caulinodans9. Mesorhizobium ciceri10. Rhizobium leguminosarum bv viceae11. Rhizobium leguminosarum bv trifoli12. Rhizobium leguminosarum bv viceae13. Rhizobium phaseoli bv viceae14. Rhizobium meliloti

NoneNoneNoneNone

StrainCC-5lO-BM-4-93GN-lSH-3S-24SB-103DL-6SPA

'L-77RCL-4L-2-88FB-77LA-l

Host-legumeCajanus cajanVigna mungoVigna radiataArachis hypogaeaCrotolaria junceaCyamopsis tetragonalabaGlycine maxSesbania rostrataCice rarietinumPisum sativumTrifolium alexandrinumLens esculentaPhaseolus vulgarisMedicago sativa

580 INDIAN J BIOTECHNOL, OCTOBER 2003

of the rhizobia. Therefore, these metals were tried indifferent combinations to suppress growth of thecontaminants. The heavy metals, when added incombination were found to be more toxic to rhizobiaand contaminants than incorporated individually inthe medium (Table 1). This might be due to thesynergistic effect of these metals in combination.

Cd with Zn, Cu or Co were found to be highlydeleterious both to rhizobia and contaminants andhence their combinations were not suitable for thispurpose (Table 1). Of the other two metalcombinations, Zn and Co was the only one whichbesides allowing unimpeded growth of all the teststrains of rhizobia and bradyrhizobia, couldeffectively inhibit the growth of common bacterialcontaminants of charcoal-soil carrier at a lowerconcentration (50 ppm each) in CRYEMA. Theusefulness of Zn and Co with other selective agentsalso has been suggested for isolation of bradyrhizobiafrom soil and commercial inoculants (Tong &Sadowsky, 1994).

For reliability test, plate counts of bradyrhizobiataken on the devised semi-selective medium werecompared with those enumerated by plating onconventional CRYEMA medium and plant infection-MPN assay (Figs 1 & 2). The plate counts ofbradyrhizobia on conventional CRYEMA mediumwere higher than those enumerated by the other twomethods in the present study. This is in conformitywith the observations of earlier workers whoattributed such variation in counts to problemsassociated with plating deficiencies, countingcontaminant colonies looking like rhizobia orlimitation of plant infection assay which is based onthe hypothesis that a single bacterium is capable offorming a nodule (Jauhri & Iswaran, 1976; Kingsley& Bohlool, 1981; Somasegaran & Hoben, 1985;Woomer et al, 1988; Tong & Sadowsky, 1994).

These results recommend the use of heavy metalsin quantification of rhizobia from commercialinoculants. Since plant infection (MPN) assay is veryexpensive and time consuming, and often delayacquisition of results when used in large-scale studiesand quality control programme (Tong & Sadowsky,1994), the newly devised semiselective medium mayprove to be the medium of choice for rapidenumeration of rhizobia from charcoal-soil basedcommercial inocu1ants.

The combination of Zn and Co was effective forsuppression of bacterial contaminants only when theywere used in higher concentration (2,500-5,000 ppm

12

m:lCRYEMA~Selective medium

DMPN assay

[3LSD (P=O.05)

..CI>"Ecou-o~:0o 6N

t"E.Q~01o...J

CharcQal Peat Soil Vermiculite Charcoal+ FYM (3:1) + soil (3:1) + soil (3:i)

Fig. l--Comparison of rhizobial counts assessed by threemethods of enumeration

1K1B.cowpea (10·B)

GilIB. japonicum (5B.l03)

E] LSD (P=0.05)

CRYEMA Selective medium MPN assay

Fig. 2---Comparison of number of viable bradyrhizobia incommercial inoculants of moongbean and soybean assessed bythree methods of enumeration

of each) in charcoal-soil carrier based inoculants(Figs 3&4). It is more cost effective than antibioticsused for this purpose (Jauhri & Gupta, 1989).However, sensitivity of rhizobial strains to high

JEEVAN KUMAR & JAUHRI: USE OF HEAVY METALS IN CARRIER BASED INOCULANTS

10 IlliJContamjnants~B. japonicum (SB-103)

rnLSD (P=0.05)

50+50 500+500 2500+2500 5000+5000

Zn+Co (ppm)

Fig. 3--Effect of Zn and Co concentrations on the survival ofbacterial contaminants and B. japonicum in charcoal-soil carrier

12YEM (Zn+Co)(ppm)+0+0*50+50-500+500*2500+2500+5000+5000IJ LSD (P-0.05)

•..••"E 10 -:J S

"0 Gi

i .;;'•..c !coc '"·e E.l!! e-c C0u

-58CI

0-I

6 1-------------------,-----------------~o 15

Days after Inoculation

Fig. 4---Effect of Zn and Co concentration on the survival ofbacterial contaminants in charcoal-soil carrier

581

15 0.4

IlliJNpmber of nodule~Nodule dry wt.GJLSD (P=0.05)

12

50+50 2500+ 2500 5000+5000500+500

Zn+Co (ppm)

Fig. ~Nodulation of soybean in response to differentconcentrations of Zn and Co used in charcoal-soil carrier basedinoculant for suppression of contaminants

25rs::JUninoculated,~InoculatedEZJLSD (P=0.05)

20

15

10

5

oo 50+50 500+500 2500+2500 5000+5000

30Zn+Co (ppm)

Fig 6--Dry matter yield of soybean in response to differentconcentrations of Zn and Co used in charcoal-soil carrier basedinoculants for suppression of contaminants

582 INDIAN J BIOTECHNOL, OCTOBER 2003

concentrations of Zn and Co suggests that rhizobialstrains of higher resistance should be used forinoculant preparation. Moreover, the possibility ofusing other naturally occurring elements which cansuppress bacterial contaminants more effectivelywithout impeding the growth of rhizobia as well asnodulation of legumes when used in carrier basedinoculants, cannot be ignored. Kinkle et al (1994)used tellurium and selenium resistance in Rhizobiumfor direct isolation of Rhizobium meliloti from soil.

Although higher concentrations of Zn and Co (500ppm and above) in charcoal-soil based inoculant werenot favourable for nodulation when used for seedinoculation, their effect was significant on the drymatter yield of soybean (Figs 5&6). Likewise,Skipper et al (1980) reported a significant decrease innodulation of soybean with peat-based inoculantscontaining fungicide or Mo. Rother et al (1983) alsoreported a reduction in nodule and plant size and innitrogenase activity of clovers at sites heavilycontaminated with Cd and Pb. The results of thisstudy suggest that selected metals may be used atconcentrations not inhibitory to nodulation andgrowth of legumes to restrain proliferation of bacterialcontaminants in charcoal-soil carrier.

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