pertanikaj. trop. agric. sci. 20(2/3): issn: 0126-6128 papers/jtas vol... · ujian terhadap kultur...

PertanikaJ. Trop. Agric. Sci. 20(2/3): 135-146(1997) ISSN: 0126-6128© Universiti Putra Malaysia Press

Possible Role of Arbuscular Mycorrhizal (AM) Fungi on Drought Tolerancein Vigna unguiculata subsp. unguiculata (L.) Walp and

Leucaena latisiliqua L.

K. UDAIYAN, A.P. GOWSALYA DEVI, A. CHITRA and S. GREEPMicrobiology Division

Department of Botany, Bharathiar University,Coimbatore-641 046, Tamil Nadu, India

Keywords: AMF, Rhizobium sp., drought tolerance, proline, Vigna unguiculata, Leucaena latisiliqua

ABSTRAK

Ujian terhadap kultur pot telah dibuat dalam tanah yang disteril bagi menentukan kesan tiga kulat AM,Acaulospora scrobiculata, Glomas aggregatum dan G. etunicatum, kedua-duanya secara bersendiri sertadicantumkan, pada pertumbuhan tanaman serta rintangan kemarau Vigna unguiculata dan Leucaenalatisiliqua. Semua tumbuhan diinokulat bersama Rhizobium sp. Semasa 45 hari pertama, semua pot diairkankepada kapasiti lapangan dan tanaman kemudiannya bergantung kepada pusingan kemarau melalui airtertahan selama 7 hari. Tanaman tersebut seterusnya bergantung kepada 7 pusingan kemarau. Penginokulatanendofit tunggalG. etunicatum dalamV. unguiculata dan A. scrobiculata dalamL. latisliqua menghasilkanpertumbuhan, biojisim, bilangan nodula, dan peratus pengkolonian akar yang lebih baik. Kandungan prolinalebih tinggi dalam semua tanaman yang diinokulat bersama G.etunicatum. Sebaliknya penginokulatan G.aggregatum dalam Vigna unguiculata dan G. etunicatum dalam L. latisliqua terhasil dalam kandungantisu N, P dan K yang lebih tinggi. Dalam penginokulatan endofit berganda, A. scrobiculata dan G.aggregatum dalam V. unguiculata; A. latisilaqua dan G. etunicatum dalam cantuman L. latisiliquadidapati menjadi sangat berkesan.

ABSTRACT

Pot culture experiments were carried out in sterilized soil to determine the effects of three AM fungi, Acaulosporascrobiculata, Glomus aggregatum and G. etunicatum, both individually as well as in combinations, on theplant growth and drought resistance of Vigna unguiculata and Leucaena latisiliqua. All plants wereinoculated with Rhizobium sp. During the first 45 days all pots were watered to field capacity and the plantswere then subjected to drought cycles by withholding water for 7 days. The plants were further subjected to 7drought cycles. Inoculation of single endophyte G. etunicatum in V. unguiculata and A. scrobiculata in L.latisiliqua produced higher growth, biomass, nodule number, and percentage of root colonization. Proline contentwas higher in all the plants inoculated with G. etunicatum, whereas inoculation ofG. aggregatum in Vignaunguiculata and G. etunicatum in L. latisiliqua resulted in higher tissue N, P and K contents. In doubleendophyte inoculations, A. scrobiculata and G. aggregatum in V. unguiculata; A. scrobiculata and (i.etunicatum in L. latisiliqua combinations were found to be highly effective.

INTRODUCTION

Arbuscular mycorrhizas (AM) confer severalbenefits on host plants. The AM fungi helpplants not only in the better utilization of soilphosphorus (Hayman 1982; Koide 1991) throughincreased uptake but also of other elementssuch as N, K, Zn, Mg, Cu and S (Lambert et al1979; Abbott and Robson 1984; Stribley 1987;Barea 1991). Arbuscular mycorrhizas are also

known to increase resistance of plants to condi-tions such as drought (Safir et al. 1972) andextreme soil acidity (Mosse 1973). The improve-ment in the water relations of plants as a resultof mycorrhizal infection has been reviewed byCooper (1983) and Harley and Smith (1983).'But it is difficult to distinguish direct mycorrhizaleffects from those that could be mediated viaimproved mineral nutrition (Nye and Tinker

K. UDAIYAN, A.P. GOWSALYA DEVI, A. CHITRA AND S. GREEP

1977; Nelson and Safir 1982). In addition, otherfactors associated with AM colonization includechanges in leaf elasticity, improved leaf waterand turgor potential and maintenance ofstomatal opening and transpiration (Auge et al1987). Increased root length and developmentof external hyphae may influence water rela-tions of mycorrhizal plants (Kothari et al 1990).

Although most studies on AM fungi haveconcentrated on their effect on plant nutrition,there is substantial evidence that the mycorrhizalassociations may alter plant-water relations.Higher transpiration rates have been found inarbuscular mycorrhizal red clover, rangelandgrass (Bouteloua gratilis), rose and apple, cowpea and cassava plants (Hardie and Leyton 1981;Allen 1982; Pai et al 1994; Sundaresan andSudhakaran 1996). In addition, more rapid re-covery from water stress and higher soil mois-ture extraction at low soil water potential havealso been observed in mycorrhizal plants (Safiret al 1971; Hardie and Leyton 1981). Thesechanges were broadly attributed to higher roothydraulic conductivity because of improved Pnutrition of mycorrhizal plants, although thesizes of mycorrhizal and non-mycorrhizal plantswere altogether different (Hardie and Leyton1981). However, only a few studies have com-pared the role of different AM fungal species/isolates on imparting drought tolerance to hostplants (Simpson and Daft 1990). The purpose ofthe present study was to compare the effects ofAM fungal species, both individually as well as incombinations, in imparting drought resistanceand also growth enhancement in Vigna unguiculatasubsp. unguiculata and leucaena latisiliqua.

MATERIALS AND METHODS

Plant Sources

Seeds of Vigna unguiculata subsp. unguiculata(L.) Walp and Leucaena latisiliqua (L.) Gills wereprocured from Tamil Nadu Agricultural Univer-sity and Institute of Forest Genetics and TreeBreeding, Coimbatore, Tamil Nadu, India, re-spectively. Seeds were weighed and selected foruniformity, surface sterilized in 5% H2O2 for 5min, treated with boiling water for 30 sec (V.unguiculata) or scarified in concentrated H2O2

for 30 min (L. latisiliqua) and then soaked inwater for 24 h.

Substrate

Seeds were directly sown into 10 x 15 cm poly-thene bags each filled with ca 1.5 kg sterilized

soil:sand mixture (3:1). The soil had pH 8.1 andelectric conductivity of 0.2 mScnr1. The contentof nitrogen, phosphorus and potassium was 104,4 and 380 kg ha"1 respectively.

Inoculum

Soil with infected root bits, hyphae and sporeswas collected from the pot cultures of AMfungi, Acaulospora scrobiculata Trappe, Glomusaggregatum Schenck and Smith Emend. Koskeand Glomus etunicatum Becker and Gerde., main-tained in sterilized sand:soil (1:1 by volume)mixture, with cowpea as the host plant in thegreenhouse of the Department of Botany,Bharathiar University, Coimbatore - 46, TamilNadu, India. Inocula of single and double andtriple combinations were added respectively atthe rate of 20, 10 + 10 and 6.7 + 6.7 + 6.7 gmsoil per pot 5 cm below the soil surface. All theplants were additionally inoculated with a 20-ml suspension of nodulating bacteria whichwere grown in a yeast extract mannitol broth(Subbarao 1986). The bacteria were obtainedfrom nodules of Vigna unguiculata and Leucaenalatisiliqua growing at Bharathiar University cam-pus, Coimbatore.

Water Regime

During the first 45 days of the experiment, allplants were watered at field capacity. The plantswere subjected to drought cycle by withholdingwater for 7 days. Similarly, plants were subjectedto seven drought cycles.

Treatments

The pot culture experiments comprised a2 x 8 x 5 factorial of the following treatments: 2sources of plant materials, 8 combinations ofendophytes and 5 replicates. The treatmentswere arranged in randomized block design.

Plants were harvested along with their entireroot system 101 days after sowing. Growth param-eters such as root length, shoot length, leaf area,nodule and plant dry weights were measured.

Known weight of fresh soil sample (50 g)was dried in an oven at 105 °C until a constantweight was attained. It was cooled in a desiccatorand weighed. The loss in weight denotes themoisture content

Percentagemoisturecontent

Wt. of fresh soil - Wt.of dry soil

Turgid wt. - Dry wt.100

136 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 2/3, 1997

POSSIBLE ROLE OF ARBUSCULAR MYCORRHIZAL (AM) FUNGI ON DROUGHT TOLERANCE

Relative Water Content (RWC)

Relativewatercontent(RWC)

Fresh wt. - Dry wt.

Turgid wt. - Dry wt.x 100

Proline Estimation (Chinard 1952)Fifty mg. of plant materials were homogenizedin 10 ml of 3% aqueous sulphosalicylic acid, andfiltered through Whatman No. 1 filter paper. To2 ml of the filtrate, 2 ml of acid-ninhydrin and2 ml of glacial acetic acid were added. Themixture was heated in a water bath at 100°C for1 h and cooled in running water. Then 4 ml oftoluene was added and mixed vigorously with atest tube stirrer for 15 to 20 seconds and readwith a spectrophotometer at 520 nm.

Plant Tissue N, P and K

Dry matter of 101-day-old plants was ground anddigested in a triple acid mixture, and tissue Pwas determined by the molybdenum blue methodas described by Jackson (1973). N was estimatedfollowing micro-kjeldahl digestion of the sam-ples (Humphries 1956) and K was estimated bya flame photometric method (David 1962).

lactophenol). The percentage of root lengthinfected was evaluated using the magnified in-tersection method as described by McGonigle etal (1990).

RESULTSGrowth and Biomass

Growth, as measured by root and shoot lengthsand dry weights, was invariably enhanced byendophyte inoculations, although the increasewas not always statistically significant. Infectionwith either Glomus etunicatum or Acaulosporascrobiculata significantly increased the growth ofVigna unguiculata and I^ucaena latisiliqua, re-spectively (Table 1, 2) while in double inocula-tions, A. scrobiculata and G. etunicatum on V.unguiculata; G. aggregatum and G. etunicatum onL. latisiliqua combinations gave maximum plantgrowth and biomass. The double and triple com-binations of endophytes tend to eliminate thepoor performance by a single species althoughsome single species produced the largest plants.In general, the more consistent results wereobtained from plants with mixed inoculations.

Root: Shoot Ratio

Though the double and triple endophyte inocu-lation for V. unguiculata (Table 1) and singleendophyte G. etunicatum for L. latisiliqua (Table2) increased root to shoot ratio, it generallydecreased with endophyte inoculations but were

TABLE 1Growth and biomass of Vigna unguiculata subsp. unguiculata treated with various combinations of AMF and

Rhizobium sp. under drought-stressed conditions

Root Colonization

The roots were cleared in 2.5% KOH at 90°Cand stained with trypan blue (0.05% in

Treatment

ControlVIV2V3VI + V2VI + V3V2 + V3VI + V2 + V3

ShootLength

(cm)(plant"1)

21.90d52.30 a31.40 c52.80 a30.40 c52.66 a22.54 d37.60 b

RootLength

(cm)(planr1)

24.00 d30.20 abed36.60 a34.80 ab28.00 cd33.20 abc35.30 ab29.60 cd

Leaf area(cm*)

(plant"1)

4.65 c6.83 cd6,14 de9.26 ab8.39 abc7.57 bed8.05 abc9.58 a

Dry Weight (g plant"1)

Leaf

0.024 d0.040 c0.430 be0.470 ab0.048 ab0.044 be0.045 abc0.052 a

Shoot

0.298 c0.638 b0.588 b0.767 a0.526 b0.867 a0.542 b0.615 b

Root

0.049 b0.088 b0.092 b0.109 b0.198 a0.198 a0.109 b0.094 b

R/S ratio

0.178 a0.142 a0.157 a0.141 a0.200 a0.200 a0.170 a0.200 a

(VI: Acaulospora scrobiculata; V2: Glomus aggcegatum; V3: G. etunicatum)

Mean values followed by the same letter are not significant according to Duncan's new multiple range test atP< 0.05.

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 2/3, 1997 137


TABLE 2Growth and biomass of I^ucaena latisiliqua treated with various combinations of AMF and Rhizobium sp.

under drought-stressed conditions

Treatment


ShootLength

(cm)(planr')

12.58 d16.30 a13.90 bed14.80 abc13.20 cd13,80 bed15.30 ab13.40 cd

RootLength

(cm)(plant"1)

32.80 c40,86 ab37,80 b42,80 a41.60 ab42.60 a39.50 ab37.60 cd

Leaf area(cm2)

(planr1)

9.00 ab9.54 a5.82 e6.22 de5.83 de8.30 abc8.00 be7.50 cd

Dry

Leaf

0.044 a0.052 a0.042 a0.048 a0.049 a0.057 a0.058 a0.056 a

Weight (g planr1)

Shoot

0.176 c0.297 ab0.265 ab0.285 ab0.222 be0.324 a0.319 a0.310 a

Root

0.160 c0.264 b0.236 c0.268 b0.213 d0.238 c0.296 a0.266 b

R/S ratio

0.950 a0.910 a0.940 a1.010 a0.960 a0.730 a0.940 a0.890 a

(VI: Acaulospora scrobiculata; V2: Glomus aggregatum; V3: G etunicatum)

Mean values followed by the same letter are not significant according to Duncans' new multiple range test atP< 0.05.

not statistically significant. Single inoculation ofG. etunicatum on V. unguiculata and double in-oculation of A. scrobiculata and G. etunicatumcombinations on L. latisiliqua resulted in thelowest root to shoot ratio.

Moisture Content (teaf, Shoot and Soil)Leaf moisture content was lower in endophyte-noculated V. unguiculata than the control plants(Fig. 1) whereas in L. latisiliqua (Fig. 2) doubleendophyte inoculations, with A. scrobiculata andG. aggregatum combinations, had higher leafmoisture content.

Single endophyte A. scrobiculata in V.unguiculata and double endophyte combinations,especially A. scrobiculata and G. etunicatum in Llatisiliqua, had higher shoot moisture contentthan other inoculations but was statistically sig-nificant {Fig. I 2).

Higher soil moisture content was observedin plants with single endophyte inoculation:A. scrobiculata on V. unguiculata and doubleendophytes, G. aggregatum and G. etunicatum, onL latisiliqua (Fig. 1, 2).

Proline Accumulation

Proline accumulation was significantly higher inG. etunicatum-\nocu\2ite& V. unguiculata andL. latisiliqua. While double endophytesG. aggregatum and G. etunicatum on V. unguiculata(Fig. 1) and triple endophytes A. scrobiculata, G.

aggregatum and G. etunicatum on L. latisiliqua (Fig.2) resulted in higher accumulation of proline.Nodulation

Nodule number was higher in G. etunicatum andA. scrobiculata-\nocu\2Lted V. unguiculata (Table3) and L. latisiliqua (Table 4) seedlings respec-tively. Double endophytes, G. aggregatum and G.etunicatum and triple endophytes A. scrobiculata,G. aggregatum and G. etunicatum, combinationsinoculated seedlings had significantly highernodule number in V. unguiculata, but not inL. latisiliqua.

Root ColonizationMycorrhizal root colonization varied amongstdifferent endophyte inoculations and host plants.Infection levels in V. unguiculata (Table 3)ranged from 20.0-97.7% of total root length.Higher root colonization was observed in singleendophyte, G. etunicatum inoculation, whereasL latisiliqua (Table 4) infection levels rangedfrom 10.4-48.7%. Combination of A. scrobiculata,G aggregatum and G. etunicatum gave higherroot colonization. No AM fungal contaminationwas evident in the control plants.

Mycorrhizal Dependency (MD)The mycorrhizal dependency of the host spe-cies varied depending upon the endophytesand their combinations (Table 3 and 4). In asingle endophyte, inoculation with G. etunicatum



40

30

5 20

H

j .

70

6 0 -

_ := 30

10

oL

Fig. 1

CD Leaf moisture

H Shoot moisture

S3 Soil moisture

O Proline

60

40 i

I30 I

20

Effect of VAM fungi (VI, Acaulospora scrobiculata; V2 Glomus aggregatum; V3 G.etunicatumj with Rhizobium sp. in different combinations on plant / soil moisture contentand proline accumulation in Vigna unguiculata. subsp. unguiculata. Bars with the sameletter(s) are not statistically significant according to DMRT at P ^ 0.05

I - 1 -

- ^ 30

L o

ab

HIUtfIIIIIIifIIIItlitliI!II

0 1 !TlMTi11UII

D Leaf moistureH Shoot moistureH Soil moistureS3 Proline

gb

iff iif

V 1 . V 2

320

260

240

I|o

£

80

Fig. 2 Effect of VAM fungi (V,, Acaulospora scrobiculata; V2 Glomus aggregatum; V, G.etunicatumj zvith Rhizobium sp. in different combinations on plant / soil moisture contentand proline accumulation in Leucaena latisiliqua. Bars with the same letter(s) are notstatistically significant according to DMRT at P ^ 0.05

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 2 /3 , 1997 139

K. UDAIYAN, A.P. GOWSALYA DEVI, A. CHITRA AND S. CREEP

and A. scrobiculata, there was higher mycorrhizaldependency on V. unguiculata and L latisiliqua,respectively. Co-inoculation with two or threeendophytes, A. scrobiculata and G. etunicatumonto V. unguiculata; G. aggregatum and G.etunicatum on L. latisiliqua combinations resultedin highest mycorrhizal dependency.

Relative Water Content (RWC)Single endophyte inoculation of G, aggregatumon V. unguiculata (Table 3) and G. etunicatumon L latisiliqua (Table 4) had higher relativewater content than other inoculation treatments.This increase was significant only on L, latisiliqua,but not on V. unguiculata.

Tissue N, P and K ContentNitrogen content (shoot and root) was highestin A. scrobiculata-mocuteted V. unguiculata (Fig.

3) plants. On L latisiliqua, G. etunicatum hadhighest tissue N content, while in double andtriple endophyte inoculations A. scrobiculata andG. aggregatum combinations had higher tissueN content than single inoculation (Fig. 4).

On V. unguiculata single endophyte G.aggregatum inoculation had higher tissue K con-tent than double or triple endophyte inocula-tions (Fig. 3). Whereas on L latisiliqua all singleendophytes and their combinations had a slightlyhigher tissue K content compare to the control(Fig. 4).

DISCUSSIONThe results of the study indicate that endophyteinoculations improve growth and biomass,though the performance varied with host plantsand AM fungal species. Inoculation of A.scrobiculata and G. etunicatum individually and in

SHOOT

ESNttrogm • Phosphofout

Fig. 3. Plant tissue nutrient contents of Vigna unguiculata subsp.unguiculata inoculated with (V}t Acaulospora scrobiculata; V2

Glomus aggregatum; V3 g. Etunicatum) with Rhizobium sp. indifferent combination on plant / soil moisture content and prolineaccumulation in Vigna unguiculata subsp. unguiculata. Barshatting the same letter(s) are not statistically significant according toDMRT at P ^ 0.05

140 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 2/3 , 1997


TABLE 3Estimate of total VAM root colonization and growth of nodules in Vigna unguiculata subsp. unguiculata

inoculated with various combinations of AMF and Rhizobium sp. under drought-stressed conditions

Treatment


No. ofnodules(plant"')

2.00 d5.80 c3.00 d

6,00 be2.20 d

7.00 ab7.80 a7.80 a

Total rootcolonization

(%)

00.00 e88,35 ab82.52 ab

97.70 a20.62 d78.72 b78.68 b48.62 c

Mycorrhizaldependency

(%)

00.00 e51.72 bed

47.26 cd59.75 ab42.91 cd

67.15 a41.26 d

52.90 be

Relativewater content

(%)

71,29 a83,97 a93.02 a88.23 a88.72 a80.55 a86.47 a86.47 a

(VI: Acaulospara scrobiculata; V2: Glomus aggregatum; V3: G. etunicatum)

Mean values followed by the same letter are not significant according to Duncan's new multiple range test atP< 0.05.

TABLE 4Estimate of total VAM root colonization and growth of nodules in I^eucaena latisiliqua inoculated with various

combinations of AMF and Rhizobium sp. under drought-stressed conditions

Treatment


No. ofnodules(plant"1)

7.40 ab8.00 a6.80 abc7.80 a5.40 de6.40 bed6.00 cd4.20 e

Total rootcolonization

(%)

00.00 e37.72 ab21.67 bed24.56 bed27.98 be10.49 de14.85 cde48.73 a

Mycorrhizaldependency

(%)

00.00 c39.39 a32.71 ab37.97 a22.72 b39.84 a45.89 a40.82 a

Relativewater content

(%)

38.93 b42.83 b56.17 b8974 a42.84 b44.87 b51.47 b61.16 b

(VI: Acaulospora scrobiculata; V2: Glomus aggregatum; V3: G. etunicatum)

Mean values followed by the same letter are not significant according to Duncan's new multiple range test atP< 0.05

combinations on Vigna unguiculata andAcaulospora scrobiculata, G. aggregatum and G.etunicatum on Leucaena latisiliqua was found tobe highly effective. Simpson and Daft (1990)also reported that AM fungi inoculation onmaize and sorghum increased plant dry weightunder water-stress conditions. It may in part bedue to improvements in mineral nutrition andpartly to the direct water-absorbing capacity ofthe mycelium, which acts to increase the con-ductivity of the plant root system and to main-tain water flow to the plant even under condi-tions of water stress. Some inocula reduced leaf

area and dry weight suggesting in these cases, aparasitic effect due to limited carbohydrate avail-ability in the plant (Bethlenfalvay et al 1982).These results emphasize the variation in theeffectiveness of different AM fungi (Abbott andRobson 1978; Carling and Brown 1980) andthat not all AM fungi may be equally beneficialto all host species.

The root to shoot ratio of V. unguiculatareflects the high resistance of this species todrought stress. V. unguiculata is able to signifi-cantly increase the root to shoot ratio underdrought stress, which is considered to be im-

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 2/3,1997 141


SHOOT

E3 Nitrogen 0 Phosphorus I B Potassium

Fig. 4. Plant tissue nutrient contents of Vigna unguiculata subsp.unguiculata inoculated with (V}, Acaulospora scrobiculata; V2

Glomus aggregatum; V3 G. etunicatumj with Rhizobium sp. indifferent combination on plant / soil moisture content and prolineaccumulation in Leucaena latisiliqua. Bars having the same letter(s) are not statistically significant according to DMRT at P ^ 0.05

portant for drought prone areas. The root toshoot ratio of L. latisiliqua was high, but thisspecies showed no capability to adapt to theenvironment. AM-inoculated seedlings of bothplant species had a lower root to shoot ratiothan uninfected plants, but the decreased rootmass of the inoculated plants was probablyfunctionally substituted by the externalmycelium of the AM fungi. The length andbiomass of the extraradical mycelium have beenshown to increase under drought stress(Bethlenfalvay et al 1988), and this could be akey factor in AM mediated drought stress.

The shoot and soil moisture contents werehigher in mycorrhizal plants. Mycorrhizalplants, which frequently appear to be less proneto wilting and transplanting shock thanuninfected plants (Barrows and Roncadori1977; Levy and Krikun 1980; Janos 1980; Hardieand Leyton 1981; Cooper 1983). Safir et al(1972) reported that soybean root resistanceto water uptake was reduced by about 40%with mycorrhizal infection. This was made pos-

sible as a result of increase in surface area ofthe root system and absorption of thehygroscopic water by external hyphae. Nye andTinker (1977) also suggested that hyphae rami-fying into the soil are likely to increase theabsorbing area for water uptake even further,and may also be able to bypass the dry zonesthat often surround the slow-growing rootsduring periods of drought. Allen (1982) calcu-lated that the rate of fungus root water trans-port was 2.5 x 10~3 mg S"1 per hyphal entrypoint. Tisdall (1994) suggested that the hyphaepresent in the soil are produced by extracellularpolysaccharides, to which microaggregates areattached and bound into stablemacroaggregates, so that they do not collapsein water.

It is known that a number of physio-chemi-cal factors are associated with drought toler-ance in plants. Proline accumulation correlateswith resistance to water stress in various plantspecies (Thakur 1980; Singh and Rai 1981:Fukutoku and Yoshio 1981). In the present

142 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 2 /3 , 1997


study, proline accumulation was higher in G.etunicatum-inoculated V. unguiculata and L.latisiliqua plants. Ramakrishnan et al (1988)also reported that mycorrhizal maize plantsyielded considerably greater amounts of prolineaccumulation under water stress. Differentmechanisms have been suggested for prolineaccumulation, which occurred in desiccatedleaves due to protein degradation. However,Singh et al (1973) observed that the net pro-tein synthesis continued slowly even as prolinewas accumulating under osmotic stress, suggest*ing that pre-existing protein is not the majorsource of proline. Therefore, some de novosynthesis seems to take place in water stressedleaves through inter conversion from otheramino acids, especially glutamic acid (Srivastavaand Kooner 1974). Some inocula yielded lowerproline accumulation, suggesting that undermoisture stress conditions, abscisic acid accu-mulation increases and transpiration rate isreduced, thus increasing the level of water inplants. Indirectly, proline has been shown toincrease that water level by binding its activegroups with water (Palfi et al 1974).

Nodule water potential plays an importantrole in the nodular activity. Soil based stress,either directly affects the infection process and/or nodule functioning or indirectly plant growthand available photosynthates by acting upon thesymbiosis (Singleton 1983). Plants inoculatedwith G. etunicatum and in combination with otherendophytes on V. unguiculata and A. scrobiculataon L latisiliqua had higher nodule number.However, the higher number of nodules formedin control plants were smaller. Similar resultswere reported in Medicago saliva and Trifoliumalexandricum (Patterson et al 1990). It may bedue to the absorption of water by the externalhyphae beyond the water depletion zone aroundroots and root hairs.

Percentage of AMF root colonization var-ied with different endophyte inoculations. Ingeneral, mycorrhizal inoculation had a higherroot colonization than uninoculated control.Simpson and Daft (1990) reported similar in-creases in mycorrhizal root colonization in in-oculated maize and sorghum plants. Higherroot colonization makes more fungal-host con-tact and exchange of nutrients and water forbetter plant growth.

The mycorrhizal dependency varied amongendophyte inoculations. Similar differences in

mycorrhizal dependency of other plant speciessuch as citrus, wheat, forest plants, hardwoodtrees and crop plants have been reported byother workers (Menge et al 1978; Janos 1980;Azcon and Ocampo 1981; Plenchette et al 1983;Pope et al. 1983). It has been proposed that thelength of the root hairs is indicative of thedegree of mycorrhizal dependency (Baylis 1975).However, in the present study, it was observedthat although mycorrhizal plants had higherroot length, they were more dependent onmycorrhiza. This clearly indicates that root lengthalone can not be used as an index for assessingmycorrhizal dependency. Root production, rootfibrosity and root geometry may be even moreimportant factors in mycorrhizal dependency(Mosse et al 1973; Plenchette et al 1983).

Inoculation of A. scrobiculata in V. unguiculataand G, etunicatum in L. latisiliqua resulted inhigher tissue P content. Michelsen andRosendahl (1990) also reported similar resultsin Acacia nilotica and L. leucocephala underdrought stress conditions. This can be attributedto the increase in surface area for absorptiondue to the extensive extramatrical network ofmycelium produced by the mycorrhizal fungi inassociation with the host root system (Hayman1978; Howeler et al 1981).

Increased uptake of other nutrients, espe-cially N and K, by mycorrhizal plants has alsobeen reported by some workers. Dhillion andAmpornpan (1992) reported that inoculation ofthe AM fungi significantly increased the concen-trations of N in rice than the control. Thepresent study, in addition to confirming theabove findings, adds that uptake of N and K byplants is greatly influenced by AM fungal speciescolonizing the roots. Stribley (1987) reportedthat P seems to be the most important nutrientinvolved, Other nutrients, such as N, K, S, Zn,Cu and Mn, are translocated along AM hyphae.Besides this increasing content of tissue N bydirect absorption of extramatrical hyphae(Johanson et al 1992, 1993; Frey and Schuepp1993) which may also be increased indirectlythrough Rhizobium. The decrease in the nutrientcontent of V. unguiculata inoculated with doubleand triple endophytes could be due to the dilu-tion effect of growth because dry matter usuallyaccumulates faster than nutrient uptake (Jarreland Beverly 1981).

The plants inoculated with multiple AMfungi in general had higher growth, proline and

PERTANIKAJ. TROP. AGRIC. SCI. VOL, 20 NO. 2/3, 1997 143


nutrient content. Similar results were reportedby Daft and Hogarth (1983) in maize and on-ion. In these cases, two situations may occur; twoor more AM fungal species colonize the rootsand add their P uptake abilities to give the planta better phosphorus nutrition and water uptake.An alternative suggested by Daft and Hogarth(1983) claimed that each endophyte may beimportant at different times during the growingseason. The results of the present study suggestthat the inoculations of selected AM fungi offerresistance to drought . The in t roducedendophytes have to compete with the indig-enous flora. Using a mixed inoculum, contain-ing endophytes with differing strategies, mightreduce variation and give more consistent ben-efits to the host plants. An ideal endophytewould need to possess several properties. Forexample, the ability to infect plants early in theirgrowth period, efficiency in exploiting the soil,easy transfer of materials to the host quick,spread and multiplication, effective infection ona range of host plants under different environ-mental conditions. All these properties may notbe found in one single endophyte and so amultiple inoculum would perhaps give moreeffective compromise.

CONCLUSION

The present study clearly reveals that inoculationwith Glomus etunicatum in Vigna unguiculata andAcnulospora scrobiculata in I^eucaena latisiliqua wouldbe the best for producing better growth, nodulenumber and percentage of root colonization.Proline content was higher in both plants inocu-lated with Glomus etunicatum. Multiple AM fungiinoculation had a positive role on drought toler-ance in both plants in terms of growth, prolineand nutrient content. Work is in progress toselect the most effective AMF for drought toler-ance under field conditions.

ACKNOWLEDGEMENT

S. Greep thanks the University Grants Commis-sion (UGC), New Delhi, for providing financialassistance.

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(Received 26 June 1997)(Accepted 20 October 1997)


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