responses of winged bean (psophocarpus tetragonolobus ... papers/jtas vol. 19 (1) apr... ·...

ISSN: 0126-6128Pertanika J. Trop. Agric. Sci. 19(1): 17-31 (1996) © Penerbit Universiti Pertanian Malaysia

Responses of Winged Bean (Psophocarpus tetragonolobus) toMycorrhiza Inoculation in Pot and Field Trials

AZIZAH HASHIM1, M. OMAR2 and I.R. HALL3

department of Soil ScienceFaculty of Agriculture

Universiti Pertanian Malaysia,43400 UPM Serdang, Selangor Darul Ehsan Malaysia

2Department of MicrobiologyUniversiti Kebangsaan Malaysia

43600 Bangi, Selangor Darul Ehsan Malaysia

3Invermay Agricultural Research Institute,Mosgiel New Zealand

Keywords: vesicular-arbuscular mycorrhiza, winged bean, Munchong, Serdang, sandy soil

ABSTRAK

Dua kajian di rumahijau dan satu kajian di ladang telah dijalankan untuk mendapat maklumat kemungkinanmengeksploit faedah kehadiran kulat mikoriza vesikul arbuskul dalam pertanian di Malaysia. Dalam kajianpertama, kacang kelisa (Psophocarpus tetragonolobus) telah ditanam dalam tanah Serdang disucihama.Perkembangan endofit ini dinilai setiap dua minggu. Kdjian-kedua menggunakan tanah Serdang dan Munchongyang tidak disucihama, dengan 3 paras baja fosforus (P) dan diben ifWkulasi dengan Acaulospora laevis,Glomus macrocarpum, Glomus mosseae, campuran spesies Glomus dan Scutellospora calospora.Kesemua inokula kecuali S. calospora meningkatkan pertumbuhan kacang kelisa dengan bererti, disampingmeningkatkan kepekatan tisu JV, P dan K. Glomus mosseae merupakan spesies yang superior. Dalam kajian diladang, Acaulospora laevis, Scutellospora calospora dan Glomus mosseae telah diinokulatkan kepadakacang kelisa ditanam pada tanah yang tidak disucihama, dengan atau tanpa baja fosforus. Kehadiran Glomusmosseae mampu meningkatkan pembentukan bunga secara bererti (P < 0.05) (4.5 bungajpokok), khususnyapada kadar baja Pyang sederhana (60 kg P ha'1).

ABSTRACT

Two greenhouse experiments and one field trial were conducted to provide information on the possibility of exploitingthe beneficial effects of vesicular-arbuscular mycorrhizal fungi in Malaysian agriculture. In the first study, wingedbean (Psophocarpus tetragonolobus) was grown in steam-sterilized Serdang soils and the development of theendophytes evaluated fortnightly. The second experiment was conducted in unsterilized Serdang and Munchong soilsrespectively with three levels of P and/or inoculated with Acaulospora laevis, Glomus macrocarpum, Glomusmosseae, a mixture of Glomus species and Scutellospora calospora. All inocula, except for S. calospora,significantly enhanced growth throughout the course of the experiments and increased N, P and K concentrations inthe plant tissues. G. mosseae was superior to the rest. In the field trial, Acaulospora laevis, Scutellosporacalospora and Glomus mosseae were inoculated into winged bean grown in unsterilized field soil, with orwithout phosphate fertilizer. Inoculation of winged bean with G. mosseae significantly (P < 0.05) increasedinflorescence formation (4.5/plant), particularly at an intermediate level (60 kg ha'1) of P fertilizer.

INTRODUCTION lar mycorrhizal (VAM) fungi on plant

The beneficial effects of vesicular-arbuscu- growth are well documented (Abbott and

AZIZAH HASHIM, M. OMAR AND I.R. HALL

Robson 1982; Harley and Smith 1983;Mosse 1986; Hall 1988). There is also agrowing body of evidence that shows it maybe possible to exploit the beneficial effects ofVAM in agriculture (Daniels et al. 1981;Ganry et al. 1982; Abbott and Robson1984). In developing countries, the relativecost of chemical fertilizer is high andconsequently restricts their use. Therefore,there is considerable interest in the use ofalternative fertilizers and the exploitation ofsymbionts such as rhizobia and VAM(Bagyaraj et al 1979; Harris et al. 1985;Hall 1988; Azizah 1991; Kumaran andAzizah 1995). Reproducibility of resultsobtained from pot studies under controlledconditions is of prime importance if mycor-rhiza inoculation is to be successfullyintroduced under uncontrolled conditions.The experiments described here weredesigned to screen a number of VAMfungi for effectiveness in enhancing growthof winged bean (Psophocarpus tetragonolobus)in Malaysian soils.

MATERIAL AND METHODS

Soils

Two greenhouse trials were established inunsterilized Serdang and Munchong soils

TABLE 1Physicochemical properties of Serdang and

Munchong soils

Physicochemicalproperty

Serdang Munchong

Horizon+Depth (cm)+Coarse sand (%)Tine sand (%)+ Silt (%)+Clay (%)Organic C (%)Total N (%)Extractable P (fig/g)pH (H2O)

Ap0-1514.059.0

3.025.6

1.080.116.315.10

Ap0 - 15

7.727.813.351.2

1.680.132.974.78

+ Paramananthan (Pers. comm.)

(Table 1) and in Serdang soil, which hadbeen steam-sterilized for 1 h at 100°C. ThepH of the Serdang and Munchong soils wasraised from the respective values of 4.6 and4.4 to pH 6.0 by incorporating groundmagnesium limestone (GML) at the rates of2.3 and 2.7 g kg"1 soil respectively.

In Experiment 1, 1 kg sterilizedSerdang soil was used to fill each 12-cmdiameter pot and the following basalfertilizers were added: urea, at the equiva-lent rate of 14 kg N ha"1, triplesuperphos-phate (TSP), at 12 kg P ha"1 and muriate ofpotash (MOP), at 30 kg K ha"1.

In Experiment 2, unsterilized Serdangand Munchpng soils were used; 5 kg of eachsoil type in each 25-cm diameter pot linedwith plastic. The following basal fertilizerswere added: urea, at the equivalent rate of14 kg N ha"1, MOP at 60 kg K ha"1, andthree levels of TSP at 0, 30 and 60 kg P ha"1

respectively.In Experiment 3, the field trial was

conducted 4 months later in ExperimentalPlot No. 2 of Universiti Pertanian Malay-sia, Serdang where the mean annualrainfall is 2000 mm and the mean annualair temperature is 24°C. The soil is analluvium (old mining land) with pH(H2O)4.8 and 20ug bicarbonate extractable P.The site selected was overgrown with mixedweed species and had not received any formof fertilizer for the previous two years. Afterit had been ploughed three times in order toreduce soil heterogeneity, 2.3 t/ha GMLwas added to raise the pH to 6.0.

Endophytes

Five endophyte treatments were used inExperiment 1. They were: Acaulospora laevi$>Glomus macrocarpum, Glomus mosseae, a mix-ture of Glomus species, and Scutellosporacalospora. All species except Glomusmacrocarpum were obtained from Dr. I.R.Hall of New Zealand. G. macrocarpum wassupplied by Dr. K.R. Krishna of ICRI-

18 PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 1, 1996

RESPONSES OF WINGED BEAN TO MYCORRHIZA INOCULATION

SAT, India. These species were propagatedin the greenhouse of Universiti PertanianMalaysia for a period of 9 months usingSetana anceps var. splendida as the host plant(Azizah and Omar 1987). All the VAMspecies above except G. macrocarpum wereused in Experiment II, which was set up 1month later.

Experiment 1: 50 g soil plus spores,mycelium and colonized root segments ofSetana were added to each inoculated pot.The inoculum was spread in a thin layer 5cm below the level where the seeds were tobe planted. Control plants were similarlytreated using soil inocula grown withuninoculated Setana. The plants werewatered with distilled water twice dailyfor the first 14 days of growth and later asneeded for the duration of the experiment.

Experiment 2: 100 g soil inoculum wasused per pot; otherwise the procedure wasidentical to Experiment 1.

Experiment 3: 50 g soil inoculumcontaining either A. laevis, G. mosseae, S.calospora or sterilized soil was thinly spread 8cm below each planting hole. In theRhizobium-inoculated plots, a suspension ofa Rhizobium culture was applied with awatering-can after the seeds were sown.Two pre-soaked seeds were sown perplanting hole with 40 cm between plantsand 45 cm between rows. Thinning wascarried out one week after sowing. As theseedlings grew they were supported onstring hung from a network of 1.5-m highwire trellises supported with woodenbeams.

Experimental Design

The pots in each experiment were placedon wooden benches and randomized inblocks in the greenhouse.

Experiment 1: A 6 x 6 factorial com-bination consisting of the following treat-ments: five VAM fungal species plus onecontrol, and six harvests. Only one plant

was planted per pot, with each treatmentreplicated three times. Three plants fromeach treatment were harvested at fort-nightly intervals.

Experiment 2: A randomized completeblock design comprising the followingtreatments: four VAM fungal species(Acaulospora laevis, Scutellospora calospora,Glomus mosseae and mixed Glomus species,designated as Alae, Seal, Gmos and Gmixrespectively in the text); two soil types(Serdang and Munchong), and three levelsof P fertilizer - 0, 30 and 60 kg TSP ha"1

(designated as Po, Pi and P2 respectively).There was also one plant per pot. withthree replications per treatment. Themycorrhizal treatment (+ R) was treatedwith a Rhizobium strain, RRIM 56; thecontrol (-R) was not.

Experiment 3 (conducted four monthslater): The experimental design was a 3 x 5factorial laid out in 4 randomized completeblocks giving a total of 60 plots. Each plotmeasured 2.6 x 2.7 m. The treatmentsincluded inoculation with: A. laevis + Rhi-zobium, G. mosseae + Rhizobium, S. calospora +Rhizobium, no inoculation with either VAMor Rhizobium, and inoculation withRhizobium alone.

Harvests and Plant AnalysisExperiment 1: Successive harvests of threerandomly selected replicates from eachtreatment were made 14 days after sow-ing, and continued thereafter at fortnightlyintervals until 12 weeks after inoculation.At each harvest, fresh weights of root andshoots and also shoot dry weights wererecorded. Randomly selected root sampleswere cleared of adhering debris, rinsedthoroughly and then stored in 10%formalin acetic acid (FAA). These rootsegments were then cleared in 10% KOHand stained with trypan blue (Phillips andHayman 1970). A total of 90% lmm-rootsections per treatment was assessed. The

PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. 1, 1996 19


percentage of root length in the stainedsample colonized by mycorrhizal fungi wasrecorded as positive and calculated usingthe following formula:

% VAM colonization =s

No. of VAM positive segmentsTotal no. of segments scored

x 100

Experiment 2: The plants were harvested16 weeks after sowing. Plant parts wereweighed immediately after harvest, andagain after drying to constant weights at72°C (approx. 2 days). The dried shootswere then ground. Twenty-five mg of theground material was then wet ashed bydigesting with 5 ml concentrated H2SO4

and oxidised with H2O2 (Thomas et al.1967). The digest was subsequently madeup to 250 ml, and N and P levelsdetermined with a TechniconR autoanaly-ser using the ascorbic acid method for Pand the alkaline salicylate method for N. Kconcentrations were measured with a flamephotometer.

Experiment 3: Fifty days after sowing,leaf numbers from 12 plants in each plotwere recorded and after a further 20 days,numbers of fully opened flowers in each plotwere counted. Untimely heavy rainfallproduced over-luxuriant plant growthwhich broke the main trellis support and

the experiment had to be terminated threeweeks after the flower count was completed.

All the data obtained except percentageroot colonization were subjected to analysisof variance (ANOVA) using GENSTAT.Percentage of root colonization was subjec-ted to a chi-square test for test of significance.

RESULTS

Formation of MycorrhizaeGood infection of winged bean roots as aresult of inoculation with the five mycor-rhizal species was observed (Experiment 1).However, positive signs of infection by theseendophytes only became obvious 4 weeksafter inoculation, after which infection ofthe roots increased with increase in sam-pling time (Table 2).

There was heavy infection (63%) of theroots inoculated with A. laevis from week 8onwards. At age 4 weeks, development ofmycorrhiza was greater in plants inoculatedwith G. macrocarpum, with 58% of the rootscolonized. Plants inoculated with G. mosseaehad 49% and 70% (with internal hyphaeand arbuscules) mycorrhizal infection at 4and 6 weeks after inoculation, respectively.

Results obtained from the chi-squaretest showed that differences in root coloni-zation pwrcentage by the different VAMspecies were only significant at the firstsampling period, i.e. 4 weeks after inocula-

TABLE 2Percentage colonization of winged bean roots by five mycorrhizal fungi with time of harvest

Sampling weeks

+A. laevis+G mosseae

G macrocarphum

"'"Mixed Glomus spp.+S. calospora

W4*

38.049.058.034.032.3

W6

53.570.063.152.255.0

W8

63.073.364.326757.8

W10

66.374.568.769.462.2

W12

70.278.874.475.164.4

*For sampling week, Significant at P < 0.05+ For VAM species, Significant at P < 0.05



tion. Differences in root colonization per-centage were not significant at 6, 8, 10 and12 weeks of sampling due to the gradualincrease in roots being colonized by all theVAM fungi.

Plant Growth

In Experiment 1, mycorrhiza, time ofharvest (week) and interaction of these twofactors significantly (P < 0.05) influencedgrowth and dry matter production of wingedbean. Owing to heterogeneity in the sam-pling time variance data, each sampling timehad to be analysed separately. However,applying a loge transformation allowed allthe data to be analysed together.

All five VA mycorrhizal fungal speciessignificantly (P < 0.001) stimulated growthof winged bean compared to control plants.Shoot dry weights of mycorrhizal plantsincreased curvilinearly at all harvestsexcept for plants treated with S. calospora.

In these plants, shoot dry weights reached amaximum value of 2.7 g at week 10 andthen declined to 2.4 g at the final harvest atweek 12 {Fig. 1).

Fungal effectiveness was in the order: A.laevis = G. mosseae — mixed Glomus = G.macro-carpum » S. calospora » Control.

The root/shoot ratio values of mycor-rhizal plants seemed to fluctuate with eachharvest (Fig. 2). However, at the finalharvest, all the mycorrhizal plants exceptfor those treated with S. calospora had root/shoot ratios lower than the control plants.Both the S. calospora-trtated and controlplants showed a sharp increase in the root/shoot ratios from week 8 onwards.

In Experiment 2, soil, mycorrhiza andrate of phosphorus fertilization and inter-actions between these variables had sig-nificant (P < 0.05) effects on shoot (Fig. 3)and root dry weights (Fig. 4) in bothSerdang and Munchong soils.

6 8TIME (week)

12

O A. laevis • Mixed Glomus speciesA G. macrocarpum % G. calospora• G. mosseae A Control

Fig L Shoot dry weight of Psophocarpus tetragonolobus as affected by VAM inoculum and time of harvest

PERTANIKA J. TROP. AGRIC. SCI. VOL. 19 NO. I, 1996 21


(25.08)

4 6 8

WEEKS AFTER PLANTING

10

O A. laevisA G. macrocarpumQ G. mosseae

• Mixed Glomus species• G. calospora

A Control

Fig 2. Root/shoot ratio of Psophocarpus tetragonolobus as affected by VAM inoculum and time of havest

33

30

-. 2 7

fc 24I

- 21HI ^ '

c 18

Q

I- 15

I 12.

LSD at 5% - 3.03

30

P LEVELS60

O A. laevis• G. mosseae• Mixed Glomus species

• G. calospora

A ControlSerdang series

Munchong series

Fig 3. Shoot dry weight (g) of Psophocarpus tetragonolobus as affected by VAM inoculum and P levels in Serdangand Munchong soils



12

60

O A. laevis

Q G. mosseae

• Mixed Glomus species

# G. calospora

A Control— Serdang series

Munchong series

Fig 4. Root dry weights (g) o/Psophocarpus tetragonolobus as affected by VAM inoculum and P levels in Serdangand Munchong soils

All the mycorrhizal species testedstimulated plant growth significantly (P< 0.001) compared to non-inoculated con-trols. However, differences in growth incre-ments between the four species studied werenot significant. Effectiveness of the fungalspecies in enhancing plant growth was inthe order: G. mosseae > A. laevis = S. calos-pora > mixed Glomus. Addition ofRhizobium RRIM 56 had little effect onplant growth at Po and P2. However, thesynergistic effects between RRIM 56 andthe four VAM species studied becameevident with the significant (P < 0.05)increase in plant growth at the intermediateP level of 30 kg Pha"1 (Table 3).

The best overall plant growth wasrecorded in Serdang soil; shoot dry weightsincreased with increasing P level except forplants inoculated with G. mosseae and mixedGlomus species. In G. mosseae-tve&ted plants,shoot dry weight decreased at Pj while in

mixed G/omza-inoculated plants, shoot dryweight was highest at this level of Pfertilization. Overall, the highest shoot dryweight (29.4 g) was obtained from G.mosseae-treated plants at P2, followed by27.6 g in A. laevis-treated plants also at thesame P level. Mixed Glomus also appearedto work as well as the other species, with27.1 g shoot dry weight.

In Munchong soil, increase in shoot dryweight was also seen to parallel the increasein the level of P fertilizer added. This holdstrue for all treatments including thecontrol. Differences in shoot dry weightbetween inoculated and uninoculatedplants were also significant (P < 0.005).As in Serdang soil, G. mosseae and A. laevis-treated plants recorded the highest shootdry weights of 14.8 g and 13.6 g respec-tively. Plant growth was stimulated less inMunchong than in Serdang soil.

Results obtained from the two pot trials



TABLE 3Shoot dry weight (g) of Psophocarpus tetragonolobus as affected by VAM inoculum and

Rhizobium inoculation in Serdang and Munchong soils

Phosphorus

Rhizobium

Soil

Serdang

Munchong

pot)

?

1 up

tat

M

O A. laevisD G. mosseae• Mixed Glomus

Mycorrhiza

AlaeSealGmosGmixControlAlaeSealGmosGmixControl

DOU

600

550

500

450

400

350

300MM

250

200150

100

en

-_*—--—

-

-

-

\ * - '

t

0

Aspecies

0

+

24.0523.7730.3926.188.814.697.554.733.284.58

.——r

L S

_ — - — —

G. calospora

Control

-

25.5526.9028.9426.89

7.0814.8412.7615.4612.002.71

•

^ — — '

D at 5%

-—-—

+

30.5832.3330.9031.4610.1817.2121.3320.4017.834.78

-c—• "•o

• *

= 85.1

i a

30

P LEVELS

Serdang series

30

-

25.5222.8626.1425.239.774.647.086.072.933.61

_

Munchong

60

+

31.4726.3727.8524.3711.8716.9613.2119.9710.936.73

- —

—

series

-

27.6727.6732.33 SED28.33 2.13811.6522.3418.1222.23 LSD5%17.33 4.285.43

— O

—•

si

.a

—•»

•

60

Fig 5. Uptake of N (mgjplant) by Psophocarpus tetragonolobus as affected by VAM inoculum and P levels inSerdang and Munchong soils

showed the same trends as those for leafnumber and initiation of flowering inExperiment 3 (Table 4 and 5 respectively).

Overall, P had a significant (P < 0.05)

effect on leaf number (Experiment 3).Analysis of the data using linear comparisonof means showed that G. mosseae significantlystimulated growth, particularly at 60 kg P



45

35

25

15

5

L S D at 5% - 13.6

O A. laevis• G. mosseae• Mixed Glomus species

• G. calosporaA Control

Serdang series

Munchong series

Fig 6. Uptake of P (mgjplant) by Psophocarpus tetragonobus as affected by VAM inoculum and P levels inSerdang and Munchong soils

oCL

1

60

O A. laevisD G. mosseae• Mixed Glomus species

# G. calosporaA Control

— Serdang series

Munchong series

Fig 7. Uptake of K (mgjplant) by Psophocarpus tetragonolobus as affected by VAM inoculum and P levels inSerdang and Munchong soils



LJLJ

o

8

60

O A. laevisQ G. mosseae• Mixed Glomus species

• G. calospora

A Control— Serdang series

Munchong series

Fig 8. Percentage of N Concentrations in Psophocarpus tetragonolobus shoots as affected by VAM inoculum and Plevels in Serdang and Munchong soils

0.40

0.10

O A. laevisD G. mosseae• Mixed Glomus species

60

• G. calosporaA Control

Serdang series

Munchong series

Fig 9. Percentage of P concentrations in Psophocarpus tetragonolobus shoots as affected by VAM inoculum and Plevels in Serdang and Munchong soils



TABLE 4Leaf number per plant of Psophocarpus tetragonolobus

as affected by VAM inoculum and levels of Pfertilingers

Applied P (kg ha-1)Inoculum 0 60 150

TABLE 5Initiation of flowering in Psophocarpus tetragonolobus

as affected by VAM inoculum and levels of Pfertilizers (number of flowers per plant)

Applied P (kg ha'1)Inoculum 0 60 150

Acaulospora laevis

Glomus mosseae

Scutellospora calospora

ControlRhizobium

LSD (5%): 13.02

28.821.328.519.021.8

28.841.325.834.027.0

38.339.037.544.830.5

Acaulospora laevis

Glomus mosseae

Scutellospora calospora

ControlRhizobium

LSD (5%): 3.19

3.752.002.001.001.75

0.754.500.252.501.25

1.211.000.752.001.00

ha"1 whilst the effects of A. laevis and S.calospora approached significance withoutany P fertilizer (Table 4).

Compared with the control + Rhizo-bium, treatment, the G. mosseae treatmentsignificantly (P < 0.05) initiated earlierflowering in winged bean plants receiving60 kg P ha"1 (Table 5). No other effectswere significant.

N, P, K Uptake and Concentrations in Shoots

N, P and K uptake by the non-inoculatedplants was significantly lower than theinoculated ones. Uptake of all threenutrients increased with increased P levels(Fig. 5, Fig, 6 and Fig, 7 respectively).

As with shoot and root dry weights, theN, P and K uptake by plant shoots wassignificantly (P < 0.05) influenced by soil,mycorrhiza and applied P. The uptake ofall three elements was higher from Serdangthan from Munchong soil.

Concentrations of N (Fig. 8 ), P (Fig. 9)and K (Table 6) in winged bean shootswere increased twofold through symbiosiswith VAM in both plants grown at Po fromSerdang and Munchong soils. In Serdangsoil, the highest shoot P and K concentrations occurred in the G. mosseae-inocula&edplants, while in the Munchong soils, plantsinoculated with mixed Glomus species hadthe highest concentrations of P and K.

TABLE 6K concentration (% dry weight) in winged bean as affected byVAM inoculum and P levels for Serdang and Munchong soils

Phosphorus

Soil

Serdang

Munchong

(kg TSP/ha)

Mycorrhiza

AlaeSealGmosGmixControl

AlaeSealGmosGmixControl

0

1.1121.0851.1621.1680.105

1.3151.3751.1071.6920.192

30

0.9731.0631.0231.0000.213

1.2631.3081.2321.3380.197

60

0.8600.9751.0230.9920.298

0.7501,0630.9231.1980.218

SED0.0855

LSD 5%0.17



Shoot N concentrations were highest inplants inoculated with mixed species forboth Serdang and Munchong soils.

DISCUSSION

The growth of winged bean was greatlyenhanced as a result of mycorrhizalassociations and addition of P fertilizers inP-deficient soils. As expected, there was,however, variability in effectivity of thedifferent VAM species tested. Abbott andRobson (1981) and Schubert and Hayman(1986) had earlier made similar observa-tions. Several factors have been attributedas being responsible for the differences inthe degree of infectivity between species.One factor affecting effectiveness of aparticular VAM species is its ability toinfect roots of the host plant at a time mostappropriate for increased uptake of adeficient nutrient (Abbott and Robson1984).

In Experiment 1, the decline in shootproduction after the fifth harvest in plantstreated with S. calospora was probably theresult of inter-root competition for phos-phate, which becomes more serious withtime as the roots become clumped at thesides and bottoms of the pot (Sander et al.1977). This effect was successfully overcomeby using a larger pot with 5 kg soil(Experiment 2). In this experiment, allfive VAM species tested significantly in-creased dry matter production, especially at30 kg P ha"1. Dry matter production ofwinged bean in Serdang soil was increasedby 169-197% following treatment with S.calospora and G. mosseae^ respectively. In thehigher P-fixing Munchong soil, increase indry matter was in the range of 131-220%following infection with the mixture ofGlomus species, and G. mosseae, respectively.This clearly shows that mycorrhizal plantsin the Serdang soil were larger, but theactual increase as a result of inoculationwith VAM was greater in Munchong soil.

The effect of mycorrhiza was less inSerdang Soil because, being a better soil,it allows better growth of the control plants,thus reducing comparatively the effects ofinoculation with the mycorrhiza.

The effect otRhizobium in both soils wasevident at an intermediate level (Pj) of Pfertilization. However, the effect of inocula-tion was less at Po and P2. This is probablydue to inefficient nitrogen fixation at Pobecause of insufficient P in the soil.However, the high rates of P applied (P2)had adverse effects on Rhizobium, resultingin the lower yield of these plants at this level(60 kh TSP ha_i) of P fertilization.

Greater effectiveness of G. mosseae and A.laevis over the other inocula in the Serdangsoil could be indicative of (the respective)soil-endophyte and host-endophyte specifi-city as suggested earlier by Hayman (1982).Superiority of G. mosseae over the otherinocula could also be attributed to theability of this fungus to maintain low sporeproduction for a long period of plant growth(Wilson 1984) as well as to its ability toproduce rapidly growing and extensiveexternal hyphae (Aidwell and Hall 1986).The less competitive A. laevis loses out to G.mosseae because of its slower production ofexternal hyphae. Similar observations forthese two species have been reported byAldwell and Hall (1986). A. laevis has alsobeen suspected to have a limited life cycle inthe host plant (L.K. Abbott and A.D.Robson, pers. comm. to I.R. Hall).

In Serdang soil, the beneficial effects ofinoculation with G. mosseae were furtherevidenced by the high shoot P and Kcontent of these plants. In Munchong soil,the mixture of Glomus species was the mostsuitable inoculum in stimulating N, P andK shoot concentrations, and hence thegrowth of winged bean. The higher shootK concentrations of plants in Munchongthan in Serdang soil gave a clear indicationof the "dilution effect" (as defined by



Jarrell and Beverly 1981) of plants in thelatter soil. Positive growth responses as aresult of inoculation with mixed Glomusspecies in Munchong soil indicates probablesynergistic effects between these species.The use of mixed inocula (containing morethan one endophyte species) has earlierbeen shown to give more consistent resultsthan those containing a single species (Daftand Hogarth 1983).

VAM and P Recovery

Successful inoculation with these VAMfungi was also shown by the higherrecovery of phosphate from highly phos-phate-fixing Serdang and Munchong soils,with 437 and 256% P recovery respectively.The growth of winged bean was enhancedby a factor of four as a result of inoculation(Experiment 2). Greater phosphate absorp-tion of VAM arises because of a superiorefficiency of P uptake by these fungi fromthe labile forms of soil phosphate (Mosse etal 1973; Azizah 1991).

Pot Experiments Versus the Field TrialAlthough the field trial could only beregarded as a partial success, the resultsobtained were consistent with resultsobtained from the pot experiments. This issignificant as conditions in the field are verydifferent to those in pots. The volume ofavailable soil is different, and there may beother growth-limiting factors besides insuf-ficient P (Schubert and Hayman 1986).Consistency of results obtained from potand field trials could also indicate thatfactors affecting the growth parameters inboth these experiments are similar.

Results obtained from the chi-square testindicate two important points: First, therewere significant differences in colonization ofroots by the four VAM species four weeksafter inoculation, indicating variability be-tween VAM species in the rate of rootcolonization. Second, from six weeks on-

wards, there was no significant difference inroot colonization between these four species,showing the ability of the slow colonizers tocompete with the fast colonizers.

The ability to colonize roots rapidly wasrepeatedly exhibited by G. mosseae underboth pot and field conditions. In the lattertrial, significant growth stimulation ofwinged bean as a result of association withthe mycorrhizal fungi was recorded at 60 kgP ha"1. Significant growth responses fromintroduced VAM species strongly indicatedthe ineffectiveness and slow growth of theindigenous VAM fungal species, since theefficiency of these species is a majordeterminant governing growth responses ofplants to VAM treatment in non-sterile soils.

In view of the successful field response tomycorrhizal inoculations it appears feasiblein future trials to inoculate legumes inunsterilized soils. The use of mycorrhizaalso seems warranted in future trialsbecause: 1. Most Malaysian soils have lowinoculum levels of the indigenous VAMspecies, which cannot compete with super-ior, introduced mycorrhiza species (Azizah1986, 1991), 2. In winged bean, optimumperformance was demonstrated at the inter-mediate level of P fertilization, indicating thepotential of these mycorrhizal fungi inlowering mineral fertilizer input and hencea form of saving for the farmers. However,more evidence is required of successful fieldtrials in different soil types before thebiotechnology of mycorrhizal inoculationcan be applied by farmers. Work is now inprogress to achieve this goal.

CONCLUSION

These experiments clearly show the impor-tance of time-course studies in elucidatingthe interactions between mycorrhizal fungiand the host plant. The significant andappreciable growth increases over theentire experiment obtained by inoculatingwith mycorrhizal fungi, especially G. mosseae



and a mixture of Glomus species, aresufficiently encouraging to warrant furtherutilization of these mycorrhizal fungi inother field studies in the humid tropics.

ACKNOWLEDGEMENTS

The senior author thanks the InternationalFoundation for Science (IFS, Grant NO.D684/2) for financial support and SurayahAdam for typing the manuscript.

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