cowpea rhizobia producing dark nodules: use in competition … · cultivar(cv.) vita3 wasroutinely...

Vol. 44, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1982, p. 611-6180099-2240/82/090611-08$02.00/0Copyright C 1982, American Society for Microbiology

Cowpea Rhizobia Producing Dark Nodules: Use inCompetition Studies

ALLAN R. J. EAGLESHAM,* MOHAMED H. AHMAD,t SALAH HASSOUNA, AND BARBARA J.GOLDMAN

Boyce Thompson Institute for Plant Research at Cornell, Ithaca, New York 14853

Received 30 November 1981/Accepted 12 May 1982

During a program of screening rhizobia from West Africa, it was found thatsome strains produced nodules of unusually dark appearance on cowpeas, but noton peanuts, soybeans, pigeon peas, or mung beans. The dark pigmentation was inthe bacteroid zone, was not correlated with nodule effectiveness, and was

additional to the leghemoglobin pigment. Only rhizobial strains with a nongummy

("dry") colony morphology produced dark nodules. Visually distinguishable pinkand dark nodules formed on the same root when a mixture of pink and dark strainswas applied as inoculum. The dark-nodule phenotype was therefore appraised as a

marker and found to be useful for studying nodulation competition with strains ofthe orthodox pink-nodule type. The competitiveness of 10 pink-nodule strains wasexamined relative to a black-nodule strain, IRc 256; a range of competitivenesswas obtained of less competitive than, equally competitive to, or more competi-tive than IRc 256. Patterns of primary (early) nodulation were generally the same

as patterns of secondary (later) nodulation. Mixed infections by dark and pinkstrains produced piebald nodules, the frequency of occurrence of which was muchgreater among primary than among secondary nodules.

The internal pigmentation in the root nodulesof legumes is indicative of their N2-fixing effec-tiveness. Actively fixing nodules are pink to red-brown due to the presence of leghemoglobin,and nonfixing nodules are normally white orgreen. When the nodule cortex of a species, e.g.,cowpea, is translucent, the external nodule coloris pale pink, white, or pale green.While screening rhizobial isolates from three

West African locations for effectiveness withcowpea, we found that a significant proportionproduced dark-colored nodules. Instances ofunusually dark-colored nodules have been re-ported for several tropical legumes: Vigna mari-na (2), Dolichos lablab (7), Centrosema pubes-cens and Phaseolus atropurpureus (17), Mimosaspp. and Leucaena glauca (6), and Vigna sinen-sis (16). Although it has been suggested that thedark-nodule phenotype could be a very conve-nient marker for ecological studies (17), thephenomenon has been regarded merely as anoddity, and its utility has received little atten-tion.Here we report some of the characteristics of

these rhizobia and examine their use for thestudy of competition between strains for nodula-tion.

t Present address: Department of Biochemistry, Universityof the West Indies, Kingston, Jamaica.

MATERIALS AND METHODSRhizobia. The rhizobia were isolated as described

before (la) from a range of cowpea (Vigna unguicu-lata) cultivars growing at Maradi (Niger), Ibadan (Ni-geria), or Onne (Nigeria) by A. Ayanaba and K.Mulongoy, both of the International Institute of Tropi-cal Agriculture (IITA), Ibadan, Nigeria (Table 1).Cultures were maintained on yeast extract-mannitolagar (YEMA) slopes (17). For rhizobia-plant interac-tion studies, rhizobia were cultured in yeast extract-mannitol (YEM) broths (17) at 30°C for 12 days whencell numbers were in excess of 109 per ml. To examinefor the ability of rhizobia to produce a melanin-likepigment in culture, as do some strains of Rhizobiumphaseoli (5), 14 strains (Table 1) were grown for 21days at 30°C on tryptone-yeast agar (TYA) (4) or on adefined medium (la) supplemented with tyrosine at afinal concentration of 30 ,ug/ml. To check for mixedinfections in the first competition experiment, rhizobiawere isolated from nodules (17) and cultured onYEMA supplemented with gentamicin at a final con-centration of 50 jLg/ml (YEMA + Gm5O).

Plants. Cowpeas were grown in pots in the green-house, using sandculture techniques as described be-fore (la) and were harvested at 35 days after planting(DAP). Acetylene reduction assays were done onexcised roots (13). Cultivar (cv.) VITA 3 was routinelyused to score nodule color and also to determineRhizobium strain effectiveness.To examine the stability of the dark-nodule pheno-

type within the cowpea species, 10 cultivars (VITA 3,VITA 5, TVu 382, TVu 3945, TVu 5132, KnucklePurple-Hull, Brown Crowder, Bush Purple-Hull, Chi-nese Red, and Calico Crowder) were inoculated with

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612 EAGLESHAM ET AL.

TABLE 1. Rhizobial strains, their origin, nodulephenotype on cowpea (V. unguiculata) cy. VITA 3,and ability to synthesize a brown pigment on TYAStrain Ca Nodule Pigmentorigin IRc no. phenotype on TYAb

Onne 252 Pink ND253 Dark256 Dark ND261 Dark282 Dark291 Pink ND299 Pink ND

Maradi 334A Pink ND338B Dark ND345A Dark ND348C Dark ND369D Dark +370A Dark +383C Pink ND389B Pink +390A Dark +393A Dark +400A Pink ND401A Pink411B Dark +428B Pink430A Pink ND432B Dark

Ibadan 464B Pink484B Pink489B Pink ND500A Pink ND506C Pink ND

a IRc is the prefix denoting the IITA Rhizobiumculture collection.

b ND, Not determined; +, brown pigment pro-duced; -, no pigment produced.

each of four dark-nodule strains (IRc 256, IRc 345A,IRc 338B, and IRc 348C), with each treatment beingreplicated three times.The competition experiments were done with cow-

pea cvs. VITA 3 and TVu 3945.Strain promiscuity was examined with four diverse

hosts, peanut (Arachis hypogaea cv. NC 7), soybean(Glycine max cv. Wilkin), pigeon pea (Cajanus cajancv. CITA 1), and mung bean (Vigna radiata cv. MB33) as described before (la).Column chromatography. Crude extracts of black

and pink nodules were prepared separately by homog-enizing (in a mortar and pestle) 1.0 g (fresh weight) in2.5 ml of 10 mM phosphate buffer with 5 mM dithio-threitol, pH 6.8. After being centrifuged at 3,000 x g,each supernatant was added directly to an 85-mlcolumn of Sephadex G-50 equilibrated in 10 mMphosphate buffer, pH 6.8. Samples of 2 ml werecollected, and the 300- to 700-nm spectra were exam-ined with a Gilford-250 spectrophotometer.

Competition experiments. The enumeration of rhizo-bia was done with a Petroff-Hausser chamber. Asnecessary, bacterial cultures were diluted with steril-ized YEM broth to prepare inocula of desired celldensity and stored at 4°C until use.

In the first competition experiment, two effective

strains from Onne were used: IRc 256, a gentamicin-sensitive strain, and IRc 291, a strain intrinsicallyresistant to 50 ,ug of gentamicin per ml. All inoculawere prepared at final cell densities of 106 cells per mland 103 cells per ml. Six treatments were prepared asfollows: IRc 256 alone, IRc 291 alone, IRc 256:IRc 291at 9:1, IRc 256:IRc 291 at 1:1, IRc 256:IRc 291 at 1:9,and uninoculated. The experiment had a randomizedblock design with four replicates. Cowpea (cv. VITA3) seeds were surface sterilized (la), three were plant-ed per pot, and each was inoculated with 1 ml of broth.At 7 DAP, plants were thinned to two per pot, and at35 DAP, the experiment was harvested. All of thenodules were picked off, separated into black and pinktypes, and counted.

In the second experiment, 11 effective strains wereused. IRc 256 as the standard and 10 pink-nodule teststrains. All YEM cultures were enumerated and dilut-ed as appropriate to 3 x 108 cells per ml. Mixtures ofeach of the 10 pink-nodule strains with IRc 256 wereprepared at a 1:1 ratio. Twenty-two treatments wereused: inocula of each strain alone, inocula of each ofthe 10 mixtures of standard plus the test strain, and anuninoculated control. The experiment had a random-ized block design of five replicates. Two cowpea cvs.(VITA 3 and TVu 3945) were used in this experimentand were grown as described above. At 35 DAP, theplants were harvested, and shoot dry weights (80°C)were taken. Nodules were picked off, separated aspink or black types, and counted.Analyses of variance were performed on all data to

detect significant differences between treatmentmeans.

RESULTSOccurrence of dark nodules on cowpea. Unusu-

ally dark colors-brown, dark brown, dark pur-ple, and black-were noted in the external ap-

FIG. 1. Primary nodulation on the roots of cow-peas (V. unguiculata cv. VITA 3). (A) Inoculated witha pink-nodule strain (left) and a black-nodule strain(right); (B) thin sections of a pink nodule (left) and ablack nodule (right). Scale bars = 1 cm.

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TABLE 2. Effectiveness, in terms of acetylenereduction activity of six strains of rhizobiaa

Strain Acetylene reduction activityb(,umol/plant per h)

Dark-noduleIRc 298 93.6 ± 8.48IRc 261 38.2 ± 9.53IRc 253 9.57 ± 7.38

Pink-noduleIRc 254 92.9 ± 26.4IRc 274 58.3 ±11.8IRc 257 26.5 ± 21.5

a Three dark-nodule and three pink-nodule strainsof rhizobia from Onne were grown on cowpeas (V.unguiculata cv. VITA 3) in the greenhouse at 35 DAP.

b Means of three replicates ± standard deviation.

pearance of nodules on cowpea cv. VITA 3when inoculated with particular strains (Fig.1A). The unusual colors resulted from darkpigmentation in the nodule bacteroid zone (Fig.1B).A total of 260 strains were screened for nodule

color. Of 85 from Maradi, 29% formed darknodules; of 102 from Onne, 24% formed darknodules; and of 73 from Ibadan, none wasscored as dark. On 35-day-old cowpeas, nodulesboth at the crown and on secondary roots weredark; however, sometimes a few small (<2 mm),young, secondary nodules with a pale-pink ap-pearance were seen. Nodules of intermediateage and size were intermediate in color. Occa-sionally, a strain produced brown nodules onone or two of the three replicate plants and pinknodules on the other replicate(s), but these werenot scored as dark types.When 10 cowpea cultivars were inoculated

with each of four dark-nodule strains, all threereplicate plants of each of the 40 combinationshad dark nodules; nodule color with any onestrain did not differ significantly between culti-vars.Some of the dark-nodule strains which effec-

tively nodulated peanut, pigeon pea, mung bean,and soybean produced a darker internal nodulepigmentation of brown in pigeon pea and mung

DARK-NODULE RHIZOBIA 613

bean and dark pink in soybean and peanut.Black nodules were not found, and externalnodule color was not affected in these particularhosts.

Effectiveness and host-specificity of dark-nod-ule strains. The dark-nodule strains did not havepatterns of effectiveness with cowpea whichwere significantly different from pink-nodulestrains from the same location. Ranges of effec-tiveness were found with both nodule types(Table 2).

Strains were screened for the ability to nodu-late peanut, soybean, pigeon pea, and mung

bean plants. Marked differences were foundbetween dark- and pink-nodule strains fromMaradi in their ability to nodulate mung beanplants and between those from Onne in theirability to nodulate peanut and soybean plants(Table 3). On the other hand, it is interesting tonote that marked differences were not seen inthe potential of dark- and pink-nodule strainsfrom Maradi to nodulate peanut, soybean, andpigeon pea plants nor of those from Onne tonodulate pigeon pea and mung bean plants.

Correlation of nodule phenotype with colonymorphology. The West African rhizobia fell intotwo groupings according to colony morphologyafter 12 days of growth on YEMA: (i) a dry typewith colonies <2 mm, circular and convex withentire margin and (ii) a wet type with flattercolonies usually >2 mm, irregular and entirewith gum production, causing confluence in ex-treme cases. Maradi strains were 98% of the drytype, Ibadan strains were 96% wet, whereasOnne strains were 60:40 of wet/dry. All of thedark-nodule strains had a dry colony type,which was particularly surprising in the case ofthe Onne strains, of which it would be expectedthat approximately 60% of the dark-nodulestrains would be of the wet colony type.

Preliminary characterization of the dark pig-ment. The crude extract of pink nodules gave a

single visible band on Sephadex G-50, whichwhen eluted absorbed with twin peaks in the550-nm range of the spectrum, characteristic ofleghemoglobin (3). The dark-nodule extract gavetwo visible bands. The first band to elute ab-sorbed with twin peaks in the 550-nm range,

TABLE 3. Fractions of dark- and pink-nodule strains able to nodulate various legumes'Fraction (%) nodulating on

Strain No. of strainsStriin No. of strains Peanut Soybean Pigeon pea Mung beanorigin

Dark Pink Dark Pink Dark Pink Dark Pink Dark Pink

Maradi 24 61 96 90 33 47 63 58 4 18Ibadan 0 73 0 16 0 33 0 99 0 99Onne 14 46 43 20 86 48 93 98 86 93

a See the text.


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FIG. 2. A cowpea (V. unguiculata cv. VITA 3)root inoculated with a mixture of a pink-nodule strainand a black-nodule strain. (A) Pink and black (ar-rowed) nodules on the same root; (B) piebald nodules,intact (left) and dissected (right). Scale bars = 1 cm.

whereas the second band did not absorb at 550nm. The results showed that the dark pigmentwas additional to leghemoglobin and was signifi-

cantly different in molecular weight and, hence,unlikely to be a modified form of leghemoglobin.No dark pigmentation was observed during

the routine culturing of our rhizobia on YEMAor in YEM broths. When 14 strains were incu-bated on TYA for 21 days, 6 of the Maradistrains, 1 of which was a pink-nodule type,produced a dark-brown pigment (Table 1),which may be similar to the melanin-like pig-ment produced by strains of R. phaseoli on TYA(5). None of the dark-nodule strains from Onneproduced pigment. A result consistent withthese findings was obtained when nine dark-nodule strains (three from Maradi and six fromOnne) were cultured on a defined medium sup-plemented with tyrosine; only two (from Mar-adi) had brown pigmentation after 15 days (datanot shown). Clearly there was no consistentcorrelation between the production of the brownpigment in culture with the ability to producedark nodules. Moreover, melanin-producing R.phaseoli strains did not form dark nodules;hence, we concluded that the dark-nodule phe-notype was not associated with the productionof a melanin-like brown pigment.

Dark-nodule phenotype as a competitivenessmarker. When a mixture ofYEM broth culturesof dark- and pink-nodule strains were applied tocowpeas, nodules of both types formed on thesame root. The nodules were easily distin-guished visually (Fig. 2A), suggesting the possi-bility that the dark phenotype could be used toinvestigate competitive interactions betweenrhizobial strains. When cowpeas were inoculat-ed with mixtures of a black-nodule strain (IRc256) and a pink-nodule strain (IRc 291), therelative numbers of the two nodule phenotypescorrelated with the relative numbers of the twostrains in the inoculum at cell densities of 106and 103 cells per ml (Table 4). Uninoculatedplants remained nodule free. Nodulation pat-terns were essentially the same at both celldensities except that at 106 cells per ml, IRc 256

TABLE 4. Competitive interactions between black-nodule strain IRc 256 and pink-nodule strain IRc 291 attwo cell densities and three cell ratios on cowpea (V. unguiculata) cv. VITA 3

Nodule no. per plant at cell densities of:" Fraction (%) ofnodules of piebald

Inoculum 106/ml 103/ml phenotype at celldensities of:

Dark Pink Dark Pink 106/ml 103/mlnodules nodules nodules nodules

IRc 256 (alone) 37.3a 0 39.5a 0 bIRc 291 (alone) 0 28.9bc 0 34.1abIRc 256 + IRc 291 (9:1) 20.5de 4.12g 28.5bc 9.87fg 2.4 2.8IRc 256 + IRc 291 (1:1) 19.1de 12.8ef 23.9cd 19.9de 4.1 5.1IRc 256 + IRc 291 (1:9) 8.12fg 19.Ode 13.4ef 20.Ode 4.0 7.9

a Nodule numbers followed by the same letter are not significantly different at P = 0.05, by Duncan's multiple-range test.

b -, Not applicable.



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TABLE 5. Shoot dry weight accumulation and patterns of primary and secondary nodulation by 11 strains ofrhizobia from West Africa on cowpea (V. unguiculata) cvs. VITA 3 and TVu 3945 at 35 DAP

Nodule no. per plant on cultivar:Shoot dry weight" (g/plant)

Strain VITA 3 TVu 3945

VITA 3 TVu 3945 Primary Secondary Primary Secondary

IRc 252 5.97bcd 7.16ab 25.5bc 32.3bcd 31.3c 75.5aIRc 256 5.95bcd 6.51b 42.0a 37.5bc 58.5a 31.OcdIRc 291 5.35d 7.56ab 25.5bc 19.0d 34.0c 62.8abIRc 299 5.52cd 6.88ab 31.5abc 64.3a 54.3ab 50.5bcIRc 334A 6.30b 7.64ab 33.5abc 35.3bcd 47.Oabc 44.3bcIRc 383C 6.01bcd 7.50ab 28.3bc 43.3b 38.Obc 49.5bcIRc 400A 6.49b 7.96a 26.Obc 17.3d 42.8bc 19.5dIRc 430A 6.77ab 7.69ab 21.8c 21.5cd 39.Obc 42.ObcIRc 489B 6.92ab 7.75ab 43.Oa 43.Ob 44.Oabc 38.8cdIRc 500A 6.87ab 7.15ab 37.8ab 29.Obcd 37.Obc 62.8abIRc 506C 7.44a 8.13a 31.Oabc 28.3bcd 38.Obc 46.8bcUninoculated 0.74e 1.08c

I Statistical comparisons are within columns; numbers followed by the same letter are not significantlydifferent at P = 0.05 by Duncan's multiple-range test.

was more infective than IRc 291, and at the 1: 9ratio, the numbers of black and pink noduleswere not significantly different than at 103 cellsper ml.Most of the plants inoculated with a mixture

of IRc 256 and IRc 291 had piebald nodules (Fig.2B), which were partly black and partly pink andoccurred at frequencies of 2.4 to 7.9% of thetotal nodule number in the different treatments(Table 4). Piebald nodules were not obtainedwith the single-strain inocula. Four piebald nod-ules were selected, surface sterilized, andcrushed, and isolates were made separately fromthe dark side and the pink side of each noduleand plated on YEMA and YEMA + Gm5O. Thedark isolates all had a dry colony morphology onYEMA and did not grow on YEMA + Gm5O,characteristic of IRc 256, whereas the pink iso-lates all had a wet colony morphology on YEMAand grew on YEMA + GmSO, characteristic ofIRc 291. We concluded that the piebald nodulephenotype resulted from simultaneous infectionby IRc 256 and IRc 291.To examine for mixed infections in nonpiebald

nodules and for whether the dark phenotype wasdominant or recessive where IRc 256 and IRc291 shared the same bacteroid zone, 50 nodules(20 black, 30 pink) were selected from two plantsof the 1:1 mixture at 106 cells per ml treatment.Isolates made from these nodules were culturedindividually on YEMA and YEMA + Gm5O. Onthe basis of colony morphology and resistanceor susceptibility to gentamicin, we concludedthat the black nodules resulted from infection byIRc 256 exclusively and the pink nodules result-ed from infection by IRc 291 exclusively. Theseresults indicated that double infections producedpiebald nodules and that mixed infection result-

ing in purely dark or pink nodules was, if itoccurred at all, insignificant in frequency.The relative competitiveness of 10 effective

pink-nodule strains was examined, using IRc 256as the standard. Although there were differencesin effectiveness among the strains with bothhosts, VITA 3 and TVu 3945, with the minimumdry weight accumulation being 5.35 g/plant at 35DAP, none was poorly effective (Table 5). Inthis experiment, we made a distinction betweenprimary and secondary nodules. The primarynodules were around the root crown on theupper part of the tap root and on the oldest partsof the first-formed lateral roots and were in theform of a contiguous mass. The secondary nod-ules were smaller, on the younger lateral roots,and infrequently, low on the tap root. Uninocu-lated plants remained nodule free. Strain IRc 256was, perhaps, a fortunate choice as the standardsince as a single-strain inoculum it was highlyinfective for primary nodulation on both hosts(Table 5). The patterns of relative competitive-ness for primary nodulation were the same onboth hosts with the exception of IRc 252, whichwas equally competitive with IRc 256 on VITA 3but less competitive on TVu 3945 (Table 6). Thethree strains from Maradi, IRc 383C, IRc 400A,and IRc 430A, were poorly competitive with IRc256; strains IRc 291, IRc 299, IRc 334A, IRc489B, and IRc 506C were equally competitive,whereas IRc 500A was more competitive. It isinteresting to note that as single-strain inocula,the Maradi strains were relatively poorly infec-tive for primary nodulation (Table 5). However,there was no correlation of poor infectiveness orpoor competitiveness with poor effectiveness,since the Maradi strains were effective as single-strain inocula (Table 5). Secondary nodulation

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TABLE 6. Patterns of primary nodulation, in termsof numbers of dark and pink nodules produced by

combinations of the black-nodule strain IRc 256 with10 pink-nodule strains on cowpea (V. unguiculata)

cvs. VITA 3 and TVu 3945 at 35 DAPPrimary nodule no. per plant on cultivar:

Straincombined with VITA 3a TVu 3945

IRc 256 Dark Pink Dark Pink

IRc 252 14.7 120NS 30.7 12.0*IRc 291 25.2 14.8NS 16.4 18.6NSIRc 299 22.4 18.4NS 16.6 22.8NIRc 334A 11.4 21.8NS 18.0 26.0NSIRc 383C 26.3 5.25* 40.5 10.8**IRc 400A 45.5 1.50** 46.8 6.75**IRc 430A 41.8 1.00** 34.0 16.6*IRc 489B 19.0 23.4NS 17.6 25.6NSIRc 500A 7.00 16.8** 13.3 28.0*IRc 506C 14.8 23.8NS 13.8 18.8NSa Statistical comparisons are within strain combina-

tion and within cultivar. *, Numbers are significantlydifferent at P = 0.05; ** numbers are significantlydifferent at P = 0.01; NS, numbers are not significantlydifferent by student's t test.

patterns of competitiveness, in general, correlat-ed with primary nodulation patterns (Table 7).On VITA 3, the exceptions were IRc 299, IRc383C, and IRc 430A, whereas on TVu 3945, theexceptions were IRc 252, IRc 299, IRc 383C,IRc 400A, and IRc 430A. In no case did a poorlycompetitive rating for primary nodulation be-come a highly competitive rating for secondarynodulation or vice-versa.Up to 28% of the primary nodules were pie-

bald (Table 8), with an overall mean of 16%. Thelowest frequencies of piebald nodulation oc-curred with the Maradi strains, IRc 383C, IRc400A, and IRc 430A. Among the secondarynodules, the mean occurrence of piebald nod-ules was only 0.7%.

DISCUSSIONAlthough the predominant color among the

dark nodules was black, others of brown, darkbrown, and dark purple were produced. There-fore, different pigments may be produced bydifferent strains. Thin sections of black nodules(Fig. 1B) revealed the pigment to be purple-redin color, perhaps the same pigment which wasdescribed in cowpea nodules infected by strainCB756 (14). This latter pigment was resolvedinto two colored components, neither of whichwas a heme-containing compound or an antho-cyanin (14), but possibly an anthro- or naphtho-quinone (8). Our column chromatography workconfirmed that the dark pigment is probably nota modified leghemoglobin, although previousattempts to separate a black-nodule pigmentfrom leghemoglobin were unsuccessful (7).

Whether the pigment was synthesized by themicro- or macrosymbiont is an interesting ques-tion. Although the pigment was strain specific,we have as yet no evidence that it can be formedoutside the nodule. Whether pigment synthesisresults from a reading of the plant or microbialgenome or both remains to be determined. Inany case, study of the process involved may leadto a better understanding of rhizobia-host inter-actions within the nodule. It is intriguing that aparticular strain produced black nodules withgenetically diverse cultivars of cowpea, but al-tered the pigmentation in nodules of other spe-cies rblatively little.The strict correlation between the dark geno-

type and the dry colony morphology was unex-pected. Although not all dry types formed darknodules, so far we have not found a single darkstrain of the wet type. A strain from Ibadanwhich produced brown nodules on two of threereplicate cowpeas belonged to the 4% having adry colony morphology. We have identified oth-er characteristics which correlated with colonymorphology. Toxin-producing strains whichcaused chlorosis in legume hosts were all of thedry type (10; unpublished data); strains of thedry type were intrinsically sensitive to antibiot-ics (la) and highly reactive serologically (1). Theinterrelation of these features is the subject ofcurrent study.The identification of strains of rhizobia in the

nodules of legumes in pot and field experimentsis a task often involving the typing of hundredsof nodules requiring surface sterilization, extrac-

TABLE 7. Patterns of secondary nodulation, interms of dark and pink nodules produced by

combinations of the black-nodule strain IRc 256 with10 pink-nodule strains on cowpea (V. unguiculata)

cvs. VITA 3 and TVu 3945 at 35 DAP

Secondary nodule no. per plant oncultivar:

Strain combinedwith IRc 256 VITA 3a TVu 3945

Dark Pink Dark Pink

IRc 252 31.3 15.8NS 21.3 32.3NSIRc 291 30.2 15.2NS 37.0 22.6NSIRc 299 14.2 59.6** 14.4 58.2**IRc 334A 17.4 21.6NS 38.2 23.2NSIRc 383C 25.8 26.5N 21.5 27.0NIRc 400A 51.8 14.3** 12.8 27.5NSIRc 430A 27.6 21.2NS 20.8 41 ONSIRc 489B 31.4 32.4NS 20.0 31 ONSIRc 500A 14.6 28.4* 14.4 34.4**IRc 506C 20.3 14.8N 26.8 36.3NSa Statistical comparisons are within strain combina-

tion and within cultivar. *, Numbers are significantlydifferent at P = 0.05; **, numbers are significantlydifferent at P = 0.01; NS, numbers are not significantlydifferent by Student's t test.



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TABLE 8. Occurrence of piebald nodules as afraction of primary and secondary nodules on

cowpea (V. unguiculata) cvs. VITA 3 and TVu 3945when inoculated with a combination of the black-nodule strain IRc 256 and one of 10 pink-nodule

strainsFraction (%) of nodules of piebald

Strain combined phenotype on cultivar:

with IRc 256 VITA 3 TVu 3945

Primary Secondary Primary Secondary

IRc 252 17 0.0 14 0.0IRc 291 14 0.5 13 1.0IRc 299 17 0.3 22 0.5IRc 334A 28 2.0 17 0.0IRc 383C 10 2.0 12 2.0IRc 400A 0.0 0.0 10 0.0IRc 430A 3.0 0.0 16 0.0IRc 489B 21 2.0 21 0.0IRc 500A 17 1.0 19 1.0IRc 506C 25 2.0 21 0.0

tion of rhizobia from the bacteroid tissue, andassaying for antibiotic resistance or serologicalaffinity. At least with cowpea and some othertropical legume hosts, the dark-nodule pheno-type offers a very simple alternative for studiesinvolving nodule typing. In the two experimentsdescribed here, we have demonstrated the utilityof the method for nodulation-competition work.

In the first experiment, the patterns of nodula-tion were essentially the same at 103 cells per mlas at 106 cells per ml, showing that there was no

preferential rhizosphere effect on either strain,which was particularly likely at the lower celldensity (Table 4). However, the fact that theratios of dark nodules to pink nodules werealways narrower than the 1: 9 ratio of cell num-bers indicated the favoring of whichever strainwas in the minority, a phenomenon which hasbeen observed before (11). This supports theconcept that, when assessing strain competitive-ness for nodulation, the relative numbers ofrhizobia at the root surface are more importantthan the relative numbers in the inoculum (11).Nevertheless, this first experiment clearly illus-trated that the dark-nodule phenotype is indeeda useful marker, e.g., the dark strains mighthave been extremely poorly competitive but, infact, were comparable to the pink strains.The second experiment demonstrated that

pink-nodule-producing strains could be rated interms of competitiveness relative to a singledark-nodule strain. High-density inocula of 3 x108 cells per ml were used to minimize differen-tial rhizosphere effects on the strains in themixtures, and the fact that the same competi-tiveness ratings were obtained for 9 of the 10strains with both hosts indicated that we were


successful. The competitiveness ratings for sec-ondary nodulation were not necessarily thesame as for primary nodulation. It has beenshown with other species in the cowpea cross-inoculation group that rhizobial numbers onlateral roots were much lower than on the taproot after both were inoculated (11). In ourwork, the inocula were added directly to theseed in the pot before germination. It is likely,therefore, that rhizobial numbers on the lateralroots were significantly lower than at the rootcrown and dependent on how mobile strainswere in traveling with the growing roots. A highfrequency of mixed-strain infections on soy-beans has been correlated with high cell densityin the inoculum (9), therefore, the greatly differ-ent frequencies of piebald nodulation among theprimary and secondary nodules (16 versus 0.7%,respectively) are evidence of relatively noncom-petitive conditions on the lateral roots. We be-lieve, therefore, that the primary nodulationdata reflect the relative competitiveness of thestrains more accurately than do the secondarynodulation data, thus emphasizing the need toconsider the method of inoculum application inconjunction with nodule age and position on theroot when interpreting data from rhizobia com-petition experiments.The poorly competitive nature of all three

Maradi strains merits investigation. Althoughthree strains do not constitute a representativesample for the Maradi rhizobial population as awhole, these strains were otherwise of diversecharacteristics (unpublished data). The poorcompetitiveness could not be directly linked totheir dry colony morphology per se, since IRc299 was also a dry type (the other six strainswere wet). It has been suggested that there is alink between infectiveness and competitivenessof rhizobia (12), and such a correlation existedwith these Maradi strains (Table 5) and with IRc252 on TVu 3945 (Tables 5 and 6); on the otherhand, IRc 252 was poorly infective with VITA 3but equally competitive with IRc 256 on thishost, and IRc 291 was poorly infective with bothhosts but competed well on both with IRc 256(Tables 5 and 6). In the same vein, whether thereis a link between poor competitive ability and apaucity of piebald nodules awaits further workfor elucidation.The discovery of piebald nodules is evidence

that mixed infections of cowpea plants resultedfrom two infection threads, in either the same ordifferent root hairs, each carrying a differentstrain of Rhizobium sp., rather than a singlethread carrying two strains; we have observedroot hairs carrying two infection threads in cow-peas (unpublished data). There also occurred afew "harlequin" nodules, in which the bacteroidzone was in quadrants, diagonally opposite ar-


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eas being of the same pigmentation, either blackor pink. This phenomenon may be allied to themultiple infections reported in soybeans (15).The question of whether these ratings of com-

petitiveness for primary nodulation have anyrelevance in the soil environment is presentlybeing addressed by our colleagues at IITA withcowpea inoculant trials, using the same 10strains, in Nigeria, Upper Volta, and Niger.Whatever are the results of these field trials,however, the dark-nodule phenotype clearly of-fers a useful tool for fundamental studies of theinteractions between rhizobia leading to nodula-tion and for ecological studies in the field, insoils where dark-nodule rhizobia are not indige-nous.

ACKNOWLEDGMENTS

This work was supported by grant GLO/77/013 from theUnited Nations Development Programme and was done inconjunction with A. Ayanaba (IITA).We are grateful to Barbara Seaman, Martha Sinclair, Jenne

McGinnis, and Stanley Scharf for technical assistance, to JayPeterson for advice on column chromatography, to William G.Smith Jr. for photography, and to Suzette Payne for typing themanuscript.

LITERATURE CITED1. Ahmad, M. H., A. R. J. Eaglesham, and S. Hassouna.

1981. Examining serological diversity of "cowpea," rhi-zobia by the ELISA technique. Arch. Microbiol. 130:281-287.

la.Ahmad, M. H., A. R. J. Eaglesham, S. Hassouna, B. Sea-man, A. Ayanaba, K. Mulongoy, and E. L. Pulver. 1981.Examining the potential for inoculant use with cowpeas inWest African soils. Trop. Agric. (Trinidad) 58:325-335.

2. Allen, 0. N., and E. K. Allen. 1936. Root nodule bacteriaof some tropical leguminous plants. I. Cross-inoculationstudies with Vigna sinensis L. Soil Sci. 42:61-77.

3. Appleby, C. A. 1974. Leghemoglobin, p. 521-554. In A.Quispel (ed.), The biology of nitrogen fixation. Elsevier/North-Holland Publishing Co., Amsterdam.

4. Beringer, J. E. 1974. R factor transfer in Rhizobiumleguminosarum. J. Gen. Microbiol. 84:188-198.

5. Beynon, J. L., J. E. Beringer, and A. W. B. Johnston.1980. Plasmids and host-range in Rhizobium legumino-sarum. J. Gen. Microbiol. 120:421-429.

6. Campelo, A. B., and C. R. Campelo. 1970. Efficiency ofcross inoculation between species of the subfamily Mimo-soideae. Pesqui. Agropecu. Bras. 5:333-337.

7. Cloonan, M. J. 1963. Black nodules on Dolichos. Aust. J.Sci. 26:121.

8. Dart, P. J. 1977. Infection and development of legumi-nous nodules, p. 367-472. In R. W. F. Hardy and W. S.Silver (ed.), A treatise on dinitrogen fixation, section III:biology. John Wiley & Sons, Inc., New York.

9. Devine, T. E., L. D. Kuykendall, and B. H. Breithaupt.1980. Nodulation of soybeans carrying the nodulation-restrictive gene, ijI, by an incompatible Rhizobiumjapon-icum strain upon mixed inoculation with a compatiblestrain. Can. J. Microbiol. 26:179-182.

10. Eaglesham, A., B. Seaman, H. Ahmad, S. Hassouna, A.Ayanaba, and K. Mulongoy. 1981. High-temperature toler-ant "cowpea" rhizobia, p. 436. In A. H. Gibson andW. E. Newton (ed.), Current perspectives in nitrogenfixation. Australian Academy of Science, Canberra, Aus-tralia.

11. Franco, A. A., and J. M. Vincent. 1976. Competitionamongst rhizobial strains for the colonization and nodula-tion of two tropical legumes. Plant Soil 45:27-48.

12. Graham, R. A. 1981. Demonstration of a constant rankingfor Rhizobium pathogenicity and host susceptibility.Trop. Agric. (Trinidad) 58:319-323.

13. Hardy, R. W. F., R. D. Holsten, E. K. Jackson, and R. C.Burns. 1968. The acetylene-ethylene assay for N2-fixa-tion. Laboratory and field evaluation. Plant Physiol.43:1185-1207.

14. Maskali, S. M., P. J. Dart, and J. Carpenter. 1972. Blacknodule pigments. Rep. Rothamsted Exp. Stat. 1971 1:98-99.

15. Ranga Rao, V., and D. L. Keister. 1978. Infection threadsin the root hairs of soybean (Glycine max) inoculated withRhizobium japonicum. Protoplasma 97:311-316.

16. Stamford, N. P., and A. M. L. Neptune. 1978. Host speci-ficity and strain competition in the Rhizobium-Vignasinensis (L.) Endl. symbiosis, p. 337-338. In J. Doberei-ner, R. H. Burris, and A. Hollaender (ed.), Limitationsand potentials for biological nitrogen fixation in the trop-ics. Plenum Publishing Corp., New York.

17. Vincent, J. M. 1970. A manual for the practical study ofroot-nodule bacteria. Blackwell Scientific Publications,Oxford, England.



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