J, Agronomy & Crop Science 180, 249—258 (1998)© 1998 Blackwell Wissenschafts-Verlag, BerlinISSN 0931-2250

Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut, Lebanon, and Inlernational Centerfor Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria

Interactive Effects of Salinity and Biological Nitrogen Fixation on Chickpea(Cicer arietinum L.) Growth

R, Zurayk, M, Adlan, R, Baalbaki and M, C, Saxena

Authors' addresses: Dr R, A, Zurayk (corresponding author) and Dr R, Baalbaki, Faculty of Agriculturai and Food Sciences,American University of Beirut, Beirut, Lebanon; M, Adlan, Soil Science and Biochemistry Department, Agricultural ResearchCorporation, Gezira Research Station, Wad Medani, Sudan; Dr M, C, Saxena, International Center for Agricultural Researchin the Dry Areas (ICARDA), PO Box 5466, Aleppo, Syria

With 7 tahlE.s

Received April 16, 1997; accepted July 8, 1997

AbstractThe effect of salinity on the noduiation, N-fixation andplant growth of selected chickpea-,R/i/zo6/wm symbiontswas studied. Eighteen chickpea rhizobial strains wereevaluated for their growth in a broth culture at salinitylevels of 0 to 20 dS m^' of NaCl -!-Na2SO4, Variability inresponse was higb. Salinity generally reduced the lagphase and/or slowed the log phase of multiplication ofRhizobium. Nine chickpea genotypes were also evaluatedfor salt tolerance during germination and early seedlinggrowth in Petri dishes at five salinity levels (0-32 dS m~').Chickpea genotypes ILC-205 and ILC-1919 were themost salt-tolerant genotypes. The selected rhizobialstrains and chickpea cultivars were combined in a potejtperiment aimed at investigating the interactive effect ofsalinity (3, 6 and 9 dS m ') and N source (symbiosis vs,inorganic N) on plant growth. Symbiotic plants weremore sensitive to salinity than plants fed mineral N, Sig-nificant reductions in nodule dry weight (59,8 %) and N-fixation (63,5 %) were evident even at the lowest salinitylevel of 3 dS m ' ' . Although nodules were observed ininoculated plants grown at 6 dS m~', N-fixation wascompletely inhibited. The findings indicate that symbiosisis more salt-sensitive than both Rhizobium and the hostplant, probably due to a breakdown in one of the pro-cesses involved in symbiotic-N fixation. Improvement ofsalinity tolerance in field grown chickpea may be achievedby application of sufficient amounts of mineral nitrogen.

Key words: Cicer arietinum L, — germination —Rhizobium — salinity — N fixation — noduiation

IntroductionChickpea (Cicer arietinum L,) is a major source ofprotein in the arid and semi-arid world where soiland water salinity can severely limit cropproduction. In Sudan, for instance, chickpea is

mainly grown in the northern region, where land islimited. Any expansion in cropped area will have totake place on the marginal soils, commonly affectedby NaCl and Na,S04 salinity (Yousif 1978), Chick-pea is highly sensitive to salinity (Saxena et al,1980), Identification of salt-tolerant lines is there-fore a prerequisite for the improvement of foodproduction.

Because the productivity of chickpea depends onthe successful establishment of symbiosis with N-fixing bacteria, making productivity gains in salineenvironments would require a different agronomicmanagement approach from that commonly usedfor non-nodulating crops. Indeed, the salt toleranceof the plant-bacteria system is detennined by theability of (1) the rhizobia to survive, multiply andeventually infect plant root hairs in saline soils; (2)chickpea seed to germinate and grow sufficiently toenable infection, (3) the establishment of a suc-cessful symbiosis, and (4) the ability of the symbiontto endure the deleterious effects of salinity. Thus,salinity tolerance in chickpea has to be identified inplant, Rhizobium and in their association. Althoughthere have been major efforts in addressing the issueof salt tolerance in chickpea (Balasubramanian andSinha 1976, Elsheikh and Wood 1990a,b, 1989, Dua1992, Ashraf and Waheed 1993) few studies haveaimed at the integration of rhizobia-plant symbiosisin a single endeavour.

The chickpea rhizobial strains show a wide vari-ation in growth in saline environments. Both thelag and log phases of growth are affected, causingdelays, or the growth is totally halted. The delayingeffect can be observed at moderate levels of salinitythat would, otherwise, cause no effect on total

U,S Copyrighl Clearance &n,er Code statement: 0931-2250/98/8004-0249 $14,00/0

250 Zurayk et al.

growth (Elsheikh and Wood 1990a), For instance,the onset of turbidity in yeast-mannitol broth offour chickpea Rhizobium strains was delayed by 1day at 176 mM NaCl (st 17,6 dS m"'), while slowgrowth was noted at 352 mM NaCl (%35,2 dS m^')(Lauter et al, 1981), Saxena and Rewari (1992)screened 10 strains of chickpea Rhizobium at sal-inities ranging from 0,1 % to 2 % (=K 1,7-34,2 dSm"'), Only two strains, F-75 and KG 31, were tol-erant to 1,5 % NaCI (*25,6 dS m~')- Elsheikh andWood (1990a) showed tolerance to 32,5 dS m" ' in15 out of 30 chickpea and soybean rhizobia strains,but all strains were sensitive to 39,2 dS m ', Thus,genetic variability in salt tolerance exists within therhizobia, and may significantly affect crop per-formance. For instance, the use of rhizobia isolatedfrom saline environments improved chickpea nodu-iation in saline soils (Ibrahim and Salih 1980); sen-sitive Rhizobium strains produced poor noduiationunder salinity (Saxena and Rewari 1992),

Chickpea germination under salinity appears to bevery variable. Germination in vitro was reportedlyunaffected by a salinity level of 8-10 dS m ' andwas reduced by over 50 Vo at 15 dS m "' (Goal andVarshney 1987, Murumkar and Chavan 1987,Kheradnam and Ghorashy 1973), Saxena et al,(1980) reported that a salinity level as low as 2dS m ' couid inhibit germination. This level wasdesignated as the response threshold. Salinity wasalso reported to affect the speed of germination.Salinity levels of 8,5 dS m"' delayed germinationfor 10 days and resulted in 65% inhibition of ger-mination (Yadav et al, 1989), Salinity also causedconsiderable reductions in both shoot and rootgrowth of chickpea during the early seedling stage.Considerable adverse effects were noted at 8 dS m"'and higher levels of salinity (Goal and Varshney1987. Murumkar and Chavan 1987),

The salt-sensitivity of chickpea decreases withontogeny. Reports indicate that vegetative growth,reproduction and yield might be severely curtailedat levels ranging from 2,5 dS m~' to 8 dS m~'depending on genotype, salt species and culturalpractices (Singh and Singh 1980, Sekhan and Singh1983, Icrisat 1986, Johansen etal, 1988, Khandewalet al, 1990, Elsheikh and Wood 1990b, Dau 1992,Ashraf and Waheed 1993), Lauter and Munns(1986) found that among 160 genotypes only onecultivar, L 550, survived at 50 mM NaCl ( * 5 dSm '), The tolerance of L 550 was also reported byChandra (1980),

The interactive effect of salinity and biologicalnitrogen fixation on chickpea growth is further con-

firmed by the differential response of plants receiv-ing their N from inorganic sources or symbiosis.Chickpea plants dependent on symbiosis showedpoorer salt tolerance than N supplied plants, indi-cating that poor salt tolerance might be due to theimpairment of the N nutrition (Lauter et al. 1981),This response is not common to all legumes, since asimilar response was documented for soybean butnot for alfalfa (Bernstein and Ogata 1996, Singletonand Bohlool 1983), This is supported by severalreports of reduced N fixation attributed to poornoduiation, and nodule function under salinity(Balasubramanian and Sinha 1976, Elsheikh andWood 1990b, Saxena and Rewari 1992, Ram et al,1989),

It follows from the above that improvement inchickpea production in sait-affected soils wouldrequire the optimization of symbiosis throughimproved tolerance of both symbionts and theirsymbiotic association to salinity, A series of experi-ments was conducted to study the impact of salinityon the chickpea-rhizobia association. The specificobjectives were to:

1 evaluate the growth response of a large numberof strains of chickpea Rhizobium to salinity levelin the medium,

2 examine the effect of salinity on the germinationand early seedling growth of chickpea and

3 investigate the impact of salinity on the symbiontby assessing infection, nodule function and plantgrowth.

Materials and MethodsRhizobium growth

Eighteen chickpea rhizobial strains of different origins(Table I), obtained from the collection of the Inter-national Center for Agricultural Research in the DryAreas (ICARDA), were used. Yeast extract-mannitolbroth (Vincent 1970) was salinized with a mixture ofNaCI and Na3SO4 at a 1 :1 ratio on equivalent basis togive a salinity level of 20 dS m~', This level was selectedbased on both the literature and our preliminary testswhich showed that at this level a clear eifect on growthcould be readily observed, without complete inhibition.The pH was adjusted to 6,8 with 1 N HCl (Mohammedet al, 1991) and 70 ml aliquots were then dispensed into125 volumetric flasks, autoclaved for 20 min at 120 Cand left to cool. Afterwards, 0,1 ml from each of the 18chickpea Rkizobium strain cultures, previously grown onyeast extract-mannitol for five days, were used to inocu-late the flasks. The flasks were then arranged in a com-pletely randomized design (CRD) with two replicates andplaced on a rotatory shaker at 120 rpm. The ambienttemperature was kept at 27 + 2''C, The growth of the

Interactive Effects of Salinity and Biological Nitrogen Fixation on Chickpea Growth

Table 1: Response of 18 chickpea Rhizobium strains to salinity

251

Straiti

CP-31CP-36CP-39CP-51CP-54CP-92CP-10CP-I7CP-29CP-32CP-85CP-93CP-5CP-15CP-24CP-28CP-90CP-109

Origin

UtiknownSpainUSACyprusMoroccoSyriaJordanLebanonIndiaMoroccoFranceTurkeyBangladeshIndiaSyriaTunisiaNepalArgentina

LengthOdSm

27d30cd36b30cd80a30cd32c32c2gcd30cd32c30cd28cd38b32c32c26d32c

of lag phase (h)- ' 2 0 d S m - '

45g52e-g48g48g

•• 1 5 0 a

44g120c94d34h50fg56ef46g585852efg

142b]48ab154a

SlopeOdSm^'

0.0256ab0.0245ab0.0143d-g0.0178b-eO.O155c-f0.0162C'f0.0176b^0.0158c-f0.0208a-<l0.0159c f0.0195b^i00140d-g0.0t79bH-e0.0095e-j0.0133d-i0.0177b-e0.0285a0.0096e-j

of log phase2 0 d S m - '

0.0190b-d0.0137d-hO.OO58g-k0,0I83b^0.0000k0.0048i-kO.OO52h-k0.001 IjkO.OI62c-fO.O153d-f0.0165c--f0.0082f-kO.OO38jk0.0045i-k0.0013jk0.0003k0.0008jk0.00000k

Final growthO d S m - '

0.95cdl.lOb0.7If^0.8e-g0.75e-i0.94cd0.66hi0.65hi0.79e-hl.Olbc0.78e-h0.95cd1.24a0.71f-i0.63i0.66g-i0.88de0.76e-h

(absorbance)2 0 d S n i - '

0.86deO.SOef0.49J0.82d f0.0010.40jk0.0710.0710,8 lef0.94cd0.80efg0.80J0.28k0.091O.iOI0.0410.0410.001

Means followed by the same letter are not statistically diiferent according to Duncan's Multiple Range Test.

strains was assessed by measuring the optical density(absorbance) twice a day for 7 days at 540 nm (Somase-garan and Hoben 1985). A sterilized series of saline andnonsaline media served as blanks. Slopes at the log phaseof rhizobial growth were estimated following logisticcurves to quantify the exponential increase in growth.

Chickpea seed germination and early seedling growth

Nine chickpea genotypes, six of which were of Sudaneseorigin (Table 2), were subjected to five salinity levels (0,8, 16, 24 and 36 dS m" ' of NaCl + Na^SOi mixture at1 : 1 ratio on equivalent basis). Twenty-four seeds pertreatment were grown in Petri dishes lined with filterpaper and incubated at 20 C for 8 days. The treatmentswere arranged in a randomized complete biock design(RCBD) replicated thrice over time. Water loss was com-pensated by the addition of distilled water (EC = 0.003dS m" ' ) . Germination counts, based on radicle emerg-ence, were taken daily. Seedling fresh weight was re-corded 8 days from sowing. The speed of germinationindex (SGI) was calculated for the entire period of growthusing the formula developed by Maguire (1962):

where i is the germination count day, Af, is the number ofseeds germinating on day ; and c is the total number ofdays. Arc sine transformation of the germination datawas performed to attain homogeneity in variance (Gomezand Gomez 1984).

Chickpea plant growth and symbiosis

This experiment studied the eflect of salinity, strains ofRhizohium and nitrogen source on the vegetative growthand noduiation of two chickpea cultivars. The chickpeagenotypes ILC-205 and ILC-1919, and the rhizobialstrains CP-29 and CP-32 were selected for use in thisexperiment, based on the results of the germination andrhizobial growth experiments described earlier in thispaper. The genotype ILC-1919 is the same as L-550,which has been noted for its sah tolerance by Chandra(1980) and Lauter and Munns (J986).

Four salinity treatments were used: 0.5 dS m" ' (tap-water control), 3 dS m' ', 6 dS n i" ' and 9 dS m"' ofNaCI-l-Na2SO4 mixture at 1 :1 ratio on equivalent basis.The two Rhizobium strains and a mineral N treatmentconstituted the inoculum and added nitrogen ( + N) treat-ments. An N and rhizobial-free control ( —N) was alsoincluded. These factors were combined in a full factorialexperiment with 32 treatments arranged in an RCBDwith four replicates.

Chickpea seeds were surface-sterilized with 0.2 %HgCl2 (Vincent 1970) and germinated for 3 days in Petridishes between filter papers moistened with distilledwater. Concurrently, Rhizohium strains CP-32 and CP-29 were cultured on yeast extract-mannitol for 3 days(Vincent 1970). Three-day-old seedlings from each treat-ment were then transplanted into 20 litre perlite-fiUedplastic pots on wire mesh tables to prevent cross-con-tamination by the leachate. Inoculated seedlings received1 ml of inoculum culture at transplanting. Nitrogen in theform of NH4NOJ was supplied to the treatment receiving

252

Table 2:m-')

Cultivar

Germination

Origin

percentage of nine

0.5

chickpea cultivars

8

at four sahnity

Salinity level16

levels and

(dSm ')24

Zurayk

one freshwater control {0

32

et

.5

aL

dS

ILC-205ILC-2609ILC-206ILC-203ILC-204ILC-1919ILC-3872ILC-482ILC-3279

SudanSudanSudanSudanTurkeyIndiaSudanUSSRSudan

lOOa,,)100a(a,98.667,b(,

100,,a,93.333,,,,

96|x(a)100,,,,lOO,,,,

98.667,,,IOO,,,,IOO,,,,

100,,,,

100,,,,97.333,,,,

94.333,,,,94.333,,,,

100,,,,80.333,,,,

9L667,,,,,80.333,,,,

30.333,79.33

93a'b(b)

,a)

5.333^,,,58.333,,,,27.667^,,,

1.333 ,,,,

* Means followed by the same letter in each column are not significantly different (P < 5 %) according to Duncan'sMultiple Range Tests.** Means followed by the same letter in each row are not significantly different (P < 5 %) according to Duncan'sMultiple Range Tests.

mineral N in increasing quantities to meet the plantrequirements (Beck et al. 1993, Lauter et al. 1981). Thefirst dose was applied at week three at a rate of 14 mgN/pot spht into two equal quantities supplied at the firstand fourth day of the week. The second was added atweek four at a rate of 28 mg N/pot, and thereafter N wasadded weekly at a rate of 56 mg N/pot. Salinized, N-free modified Broughton and Dillworth fourth strengthnutrient solution (Beck et al. 1993) was fed daily to theplants through a drip system in amounts sufficient tocause free drainage. After 26 days from sowing, thestrength of the nutrient solution was doubled.

The experiment was terminated at flowering tocoincide with the peak N fixation (Hooda et al. 1986).The plants were harvested, roots were cleaned and thenodules were picked and counted. The dry weights ofthe above-ground biomass and of the nodules were mea-sured. Shoot N content was determined by theKjeldahl method (Bremner 1965).

ResultsRhizobium growth

Salinity affected the lag and log phases, often result-ing in a marked efFect on the overall rhizobialgrovv'th, and the variability in response was clearamong the 18 rhizobial strains (Table 1). The slopesof the log phase of eight of the strains (CP-31, 39,51, 29, 32, 85, 93 and 15) were not significantlyaffected by salinity (P = 0.05). Salinity significantlyextended the lag phase of all strains. The leastaffected was CP-29, while the lag phases of strainsCP-28, 90, 109, 10 and 17 were extended the most.

Five out of the 18 strains (CP-31, 51. 29, 32 and85) suffered no significant salinity effect on the

growth and log phases at 20 dS tn ' (Table 1). StrainCP-32 had the highest growth in the salinized mediawhile strains CP-51, 29 and 85 showed the highestrelative growth (102%). Strains CP-54, 10, 17, 15,24,28,90 and 109 were extremely sensitive to salinityand their growth was almost completely inhibited.

Chickpea seed germination and early seedling growth

Sahnity caused a significant reduction in the ger-mination and speed of germination index (SGI) ofall cultivars. The onset of this reduction varied withcultivars and salinity levels (Tables 2 and 3). Theinteraction between cultivars and salinity was sig-nificant. The differential response in the germinationpercentage among the nine cultivars became sig-nificant at 24 dS m ', at which level ILC-3872showed the poorest performance (30.33 %) whileILC 205 maintained a 1 0 0 % germination. Differ-ential response in the speed of germination, whichwas apparent even in the absence of salinity, becamemore conspicuous as sahnity was increased. Slow-germinating cultivars in the absence of salt generallyperformed poorly under salinity. ILC-205 retainedthe highest SGI at all salinity levels, with no sig-nificant effect until 24 dS m " ' , while ILC-3872 wasamong the poorest perfotmers and showed a sig-nificant reduction even at the lowest salinity level.

Genotypic differences of the early seedling-growth in response to salt were also clear. Thegrowth was completely inhibited at 32 dS m ~ '(Table 4). Significant reduction in fresh weight wasobserved at the lowest salinity level (8 dS m~ ' ) for

Interactive Eifects of Salinity and Biological Nitrogen Fixation on Chickpea Growth 253

Table 3: Speed(0.5 d S m ' )

Cultivar

ILC-205ILC-2609ILC-206ILC-203ILC-204ILC-1919ILC-3872ILC-482ILC-3279

of germination

0.5

24,*, , , '*22.667,,̂ ,̂ )23.6673,,,,23.667ab(a,22.1,aa)22.167|K(a,,18. Id,,,24a(a)21.167,,,,

index of nine chickpea cultivars at four

8

23.833,,,,19.167,,,,,22.667,,(^,23.167;-,,,21.433,,,,,)23.167,,,,13.767,,,,18.6,,b,10.767,,,,

Salinity levels (dS16

23.5.,.)15.733,,,,21,h,a,15.367,,,,17.933,,,,18.5,^,,,10.333,,,,] lj,,)9.73,,,,

salinity levels and

24

15.6,,,,8-2b,d)8.433,.,,,8.2b,c)7.833h,,)7.333,,,,2.167,,,,4.633,,,,,2.667,,,,

one freshwater control

32

5.933.,c)l . l c d ( t )4.633,,«,,

2.3bod(d)

0.4d(d>

3-lhcidl

0.033,,,,0*c,

* Means followed by the same letter in each column are not significantly different (P < 5 %) according to Duncan'sMultiple Range Tests.** Means followed by the same letter in each row are not significantly different (P < 5 %) according to Duncan'sMultiple Range Tests.

Table 4: Seedling fresh weight (mg per plant) of nine chickpea cuitivars grown for eight days at four salinity levelsand one freshwater control (0.5 dS m ')

Cultivar

ILC-205ILC-2609ILC-206ILC-203ILC-204ILC-1919ILC-3872ILC-482ILC-3279

0.5

217,*,,,**184.3,,,,,,209.6,b,,,160b<;{,l

179,,,,,155.3bc(ii)162.3|x;(a)181.3,,,,)118.,^,

8

196.6,,,,173.3,,,,,238.3,,,,162.6,,d,^,1 78bctai

143.6cd(,,119137,dft)137,,(,,

Salinity levels (df16

118,,,,95.,iKih,

117.6,,,,)02.3,b(i,i90.6,,,,b,

116a(,,68,ic,90,,,,,)78.3,,,,,

lm-')24

45.6,,,,28.3ah,j|c,42.6,h,b,,34.3^1^1,,15.6,j,,,

12.3,,,,17.6,d,d,

10.6,,,,

32

5.667,,,,Oiifcl

5.667,,,,5.333,,,Oa(r»

Oa(b)

0,(d)0,(d)

0,,,,

* Means followed by the same letter in each column are not significantly different (P < 5 %) according to Duncan'sMultiple Range Tests.** Means followed by the same letter in each row are not significantly different (P < 5 %) according to Duncan'sMultiple Range Tests.

ILC-3872 and ILC-482, and at 16 dS m'-' for ILC- inputs. In the absence of salinity, the combination205 and ILC-206, both of which produced the high- ILC-205-CP-29 resulted in best growth. The lowestest fresh weight, ILC-1919 was the only cultivar to salinity reduced the above-ground dry biomass yieldshow no fresh weight reduction at 16dSm '. of the inoculated plants to the leve! of the - N

treatment, while the -I-N plants remained unaffected.Chickpea plant growth and symbiosis The better performance of the + N treatmentSalinity caused a significant reduction in the dry was also apparent at 6 dS m^'. The differentialweight of the above-ground biomass, shoot N response to salinity because ofN source disappearedcontent, and nodule number and dry weight (Tables at the highest salinity level (9 dS m" ')•4-6). Plants in the - N treatment grew very poorly Total N content showed the same trend as the dryeven in the absence of salt, and there was no nodu- weight of the above-ground biomass in response tolation, indicating that cross-contamination was sue- treatments. The N content of the inoculated plantscessfuUy avoided, and that there were no external N was appreciably decreased even at the lowest salinity

254

Table 5: Shoot dry weight (mg persalinity and one freshwater control

CultivarSource ofnitrogen

plant)(0.5 d

0.5

of two chickpea

3

cultivars grown with different N

Salinity levels (dSm"')6

sources at

9

Zurayk et al

three levels of

iLC-205

ILC-1919

+ NCP-32CP-29

- N-fN

CP-32CP-29

298.75e-i637.50bc693.00b836.75a283.00f-j549.00c645.25bc547.50c

282.50f^609.00bc373.00def379.25def360.50d-g599.75bc345.75d-h406.75deLSD (0.05)

222.00i-l368.25d-fI90.50i 1200.00i-l273.50f-j433.25d250.00g-k235.75h-l

= 98.97

BS.OOkl144.00kl125.001127.501122.251128.751180.75J-I121.001

Means followed by the same letter are not significantly different (P < 5 %) according to Duncan's Multiple RangeTest.

Table 6: Shoot N content (mg per plant) of two chickpea cultivars grown with different N sources at three levels ofsalinity and one freshwater control (0.5 dS m ')

Cultivar

ILC-205

ILC-1919

Source ofnitrogen

- N+ N

CP-32CP-29

- N+ N

CP-32CP-29

0.5

3.65i14.25cde18.20b23.02a

3.78i15.35bcd17.15bc16.05bcd

Salinity levels (d:3

3.72]14.25cde6.70ghi9.48fg4.15i

15.45bcd6.25hi9.18fgh

LSD (0.05) = 2.92

5m-')6

3.971!1.23ef3.5Oi3.95i4.85i

12.77de3.90i3.80i

9

4.03i5.50i3.65i3.9Oi4.07i5.88i4.80i3.28i

Means followed by the same letter are not significantly different (P < 5 %) according to Duncan's Multiple RangeTest.

level, but was higher than that of the - N treatment,indicating that some N fixation must be taking placeat 3 dS m"'. Combinations including CP-29 hadhigher N in the above-ground biotnass at the lowestsahnity. The N content of the N-fed plants was onlysignificantly reduced at 9 dS m '.

The Rhizobium strain effect on nodule numberper plant was not significant, hence the results arepresented pooled over both strains (Table 7). ILC-1919 showed better noduiation in the absence ofsalinity. Nodule number of both genotypesdecreased with increasing salinity, and noduiationwas totally inhibited at 9 dS m"'. Nodule dry weightwas unaffected by either rhizobial strain or hostcultivar, but was significantly reduced by each suc-cessive increase in the level of salinity, falhng from

106.3 mg plant"' at 0.5 dS m~' to 55.6 mg plant"'a t 3 d S m " ' and 7.8 mg plant"' a t 6 d S m '.

DiscussionRhizobium growth

Overall growth reduction resulted from a com-bination of extended lag phase and reduced growthrate because of sahnity. A longer lag period and areduced growth rate of chickpea rhizobia as a resultof increased osmotic pressure were also reported byElsheikh and Wood (1989a, 1990). The lag time is afunction of the time needed for the culture to adaptto a new environment, and may last for as long as 26days at high sahnity levels (Steinborn and Roughly1975). A long lag time is undesirable, as it can delay

Interax:tive Effects of Salinity and Biological Nitrogen Fixation on Chickpea Growth 255

Table 7: Nodule number per plant of two chickpea cul-tivars grown at three levels of salinity and one freshwatercontrol (0.5 dSm"' )

Type 0.5Salinity levels

3

ILC-205 95.5a 55.6b 18.2c O.OdILC-1919 58.2b 52.0b 18.2c O.Od

LSD (0.05) = 6.79

Means followed by the same letter are not significantlydifferent (P < 5 %) according to Duncan's MultipleRange Test.

noduiation, which can result in N deficiency in theplant. Indirect effects on noduiation may occur, asroot hair damage caused by prolonged exposure tosalinity will reduce the chances of effective infection.

Results also indicated that differences existamong the Rhizobium strains in tolerating a salinitylevel of 20 dS m"'. These results do not agree withthe findings of Elsheikh and Wood (1990a) whoshowed variability at 32.5 dS m"' but not at 20.6dS m"'. The difference in the level of the salinityat which the variability was detected in the twoexperiments may be due to the difference in thetime and method of measurement. The results ofElsheikh and Wood (1990a) were based on visibleturbidity 14 days after salt exposure, while in ourexperiment spectrophotometric readings taken after7 days were used to evaluate total growth. Saxenaand Rewari (1992) found that 2 out of 10 chickpeaRhizobium strains evaluated were tolerant to a sal-inity level of 1.5 % NaCl (^25.6 dS m ') imposedfor 4 days. Thus, variability in salt tolerance ofchickpea rhizobia can be observed at lower salt con-centrations if sampling is performed earlier.

In the final analysis, five strains emerged clearlyas potentially successful candidates for furtherstudies on the estabhshment of symbiosis in salt-affected fields. These were CP-32, 85, 29, 31 and 51,which showed good growth in saline and non-salinemedia, and whose lag and log phases of growth wereleast affected by salinity. Strain CP-29 showed theshortest lag phase under salinity, and CP-32 showedthe highest total growth in both saline and non-saline media.

Chickpea seed gennination and early seedling growth

The wide range of response indicates the variabilityin chickpea genotypes for gennination and early

seedling growth under salinity. Our observationsconfirm the reports of Saxena and Rewari (1992)who found that the germination of 10 chickpea cul-tivars ranged between 0 and 82 % at 1 % NaCl (s= 17dS m~') with no gennination occuning in any ofthe genotypes at 2 % NaCl ( x 34.2 dS m"'). Similarresults were also reported by JCheradnam and Gho-rashy (1973) and Saxena et al. (1980). The existenceof high variabihty in chickpea genotypes for salttolerance at germination provides opportunities forselection for this trait.

The speed of gennination index was reduced insome genotypes at the lowest sahnity treatment,while the percentage germination remained un-affected. It was also noted that cultivars with lowSGI in the absence of salt retained this characteristicunder salinity. Rapid germination is an importantattribute in saline agriculture, as it will enhancethe plant's tolerance to salinity by avoiding stresscaused by zonal salinization due to the upwardmovement of salts. The speed of gennination index,which appears to be more sensitive to salinity thandoes percentage germination, may provide a betterselection tool to improve adaptation of chickpea tosaline conditions. The same applies for improvingthe adaptation of wheat for drought-prone environ-ments (Read and Beaton 1963, Ashraf and Abu-Shakra 1978, Bleik 1994).

Of all the tested cultivars, ILC-205, 206 and 1919performed best under salinity. These cultivarsappear, however, to have different strategies foroptimizing germination and early seedling devel-opment under salt stress. ILC-205 and ILC-206maintained higher germination percentage and SGIunder salinity, while ILC-1919 showed better tol-erance to salinity at the early seedling growth stage.Both the strategies eventually lead to the sameresult: a larger number of surviving seedlings (Chan-dra 1980, Dua 1992, Zurayk et al. 1993). That ILC-1919 (the same as L-550) has a better salt tolerancethan other chickpea cultivars has been reported earl-ier (Chandra 1980, Lauter and Munns 1986).

Chickpea plant growth and symbiosis

The results confirmed earlier reports that chickpeais highly sensitive to salinity, with symbiosis-depen-dent plants showing severe biomass yield reductionsat salinity levels as low as 3 dS m"'. The thresholdof tolerance in symbiotic chickjwas could not bedetermined in this study but may well be below 3 dSm"'. This threshold may be significantly increasedby the addition of mineral N. The issue of threshold

256 Zurayfc et al.

of salt tolerance in chickpea is controversial. Lauterand Munns (1987) rejected its existence, while Dua(1992) showed thresholds ranging between 1 and 4dSm- ' .

The decrease in the dry weight of the above-ground biomass paralleled the decline in N contentin the symbiotic plants, indicating that higher sen-sitivity to salinity was associated with poorsymbiosis. The establishment of efficient symbiosisinvolves a number of steps, beginning with rhizobia-host recognition and ending with biochemical N-fixation. The sharp decrease in both nodule numberand dry weight in this experiment indicate that sym-biosis breaks down at the early stages of symbiosisestablishment. There are several possible reasons forthis. Islam and Ghoulam (1981) showed that salinityaffected root exudates which altered chemotaxisratios of the rhizobia, resulting in poor noduiation.Failure of noduiation has also been attributed todamaged root hairs (Tu 1981, Elsheikh and Wood1990b).

The exact stage of symbiosis breakdown maydiffer depending on rhizobial strains and hosts. Thelower relative decrease in nodule number in ILC1919 than other genotypes may be due to differencesin root growth. It is clear that symbiosis broke downat a salinity of 3 dS m"' because at this salinity levelsymbiotic plants showed poorer growth than theplants supplied with inorganic nitrogen. Thus, thesymbiotic association appears to be more sensitiveto sahnity than either rhizobia or host taken indi-vidually. This is in agreement with the work ofElsheikh and Wood (1990b) and Yousef andSprent (1983), who reported that N-fixation is moresensitive to salinity than plant growth, rhizobia orNH4NO3 assimilation. The interacting phenomenaof symbiotic N fixation underly this complexity, andindicate that there must be, in the chain of reactionsleading to N transfer to plant, a link that is moresensitive to salinity than plant or bacteria. It is pos-sible that Rhizobium strain and host plant have anumber of genes that are expressed only in symbiosis(Rai and Prasad 1986).

The poor growth of the plants in ~ N treatmentunder salinity can be only partly attributed to poorN nutrition. This is clearly indicated by the decreasein dry weight of plants in the N-fed treatment at 6dS m~', while the N content remained stable. Theresults, however, underscore the importance of pro-viding adequate amounts of combined nitrogen tochickpea for salinity tolerance, and indicate that thedetrimental impact of moderate levels of salinity onthe growth of chickpea can be alleviated, and yields

improved by relying on mineral nitrogen fertil-ization rather than on symbiotic N fixation. Therelative economics will, however, dictate whetherthe practice would be acceptable to farmers.

ZusammenfassungWechselwirkungen von Versalzung und biologischerStickstoflixierung auf das Wachstum von Kichererbse(Cicer arietinum L.)

Der EinfluB der Versalzung auf die Knollchenbildung,N-Fixierung und das Pflanzenwachstum selektierterK.\ch.trerhsen-Rhizobium Symbionten wurde untersucht.18 K'lchsTerbsen-Rkizohium Linien wurden hinsichtlichihres Wachstums in Brutkultur bei einer Versalzung von0 und 20 dS m"' von NaCl-i-Na2SO4 ausgewertet. DieVariabilitat der Reaktion war hoch. Versalzung redu-zierte grundsatzlich die Verzogerungsphase und/oder ver-langsamte die Verzogerungsphase der Vermehrung vonRhizohium. Neun Kichererbsen-Genotypen wurden aus-gewertet hinsichtiich Salztoleranz wahrend der Keimungund des frUhen Samlingswachstums in Petrischalen bei 5Versalzungskonzentrationen (0-32 dS m"'). Die Kicher-erbsen-Genotypen ILC-205 und ILC-1919 erwiesen sichals die am deutlichsten salztoleranten Genotypen. Dieausgelesenen Rhizobium-Linien und Kichererbsen-Kul-tivare wurden in einem GefaBexperiment kombiniert, umdie interaktiven Wirkungen der Versalzung (3, 6 und 9dS m ') und der N-Quelle (Symbiose gegenuber anorga-nischen N) auf das Pflanzenwachstum zu untersuchen.Die symbiotisch infizierten Pflanzen waren sensitiver gegen-uber Versalzung als die tiber mineralisches N ernahrtenPflanzen. Signifikante Reduktionen im Knollchen-Trok-engewicht (59.8%) und N-Fixierung (63.5%) wurdenauch bei der geringsten Versalzungskonzentration von 3dS m"' deutlich. Obwohl Knoilchen bei den inokuliertenPflanzen, die bei 6 dS m"' beobachtet wurden, war dieN-Fixierung vollstandig inhibiert. Die Ergebnisse weisendarauf hin, daB die Symbiose starker salzempfindlich istals Rhizobium und die Wirtspflanze, was wahrscheinlichauf einen Zusammenbruch in einem Teil der Vorgange,die in der symbiotischen N-Fixierung vorliegen, zuruck-zufuhren ist. Eine Verbesserung der Salztoleranz imFeldanbau von Kichererbse konnte durch eine Anwen-dung ausreichender Mengen mineralischen Stickstoffserreicht werden.

Acknowledgement

This research is part of a project funded by a grant fromthe Royal Government of the Netherlands through theNile Regional Program/ICARDA.

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