Effects of inoculation of phosphate-solubilizing microorganisms and an arbuscular mycorrhizal fungus on mungbean grown under natural soil conditions

Mycorrhiza (1998) 7 :249–253 Q Springer-Verlag 1998

ORIGINAL PAPER

Satpal Singh 7 K. K. Kapoor

Effects of inoculation of phosphate-solubilizing microorganisms

and an arbuscular mycorrhizal fungus on mungbean grown

under natural soil conditions

Accepted: 13 October 1997

S. Singh 7 K. K. Kapoor (Y)Department of Microbiology,CCS Haryana Agricultural University,Hisar, 125004, IndiaFax: c91-1662-33257

Abstract The effect of inoculation of the phosphate-solubilizing microorganisms (PSM) Bacillus circulansand Cladosporium herbarum and the arbuscular my-corrhizal (AM) fungus Glomus fasciculatum with orwithout Mussoorie rockphosphate (MRP) was studiedin a P-deficient natural non-disinfected sandy soil onmungbean (Vigna radiata). The AM levels increasedfollowing the addition of MRP or inoculation with PSMor G. fasciculatum. Both grain and straw yield of mung-bean increased following inoculation with PSM or theAM fungus. In general, the increase in yield was higherin the presence of MRP and inoculation with a combi-nation of PSM and AM fungus. Highest N and P up-take by mungbean was recorded after treatment with acombination of B. circulans, C. herbarum and G. fasci-culatum in the presence of MRP. Generally the PSMpopulation increased after AM fungus inoculation.

Key words Rockphosphate 7 Phosphate-solubilizingmicroorganisms 7 Arbuscular mycorrhizae 7 Mungbean

Introduction

Much of the inorganic P applied to soil as fertilizer israpidly converted to unavailable forms with low solu-bility (Sanyal and De Datta 1991). Soluble P is releasedfrom insoluble phosphates by a variety of solubilizationreactions involving rhizosphere microorganisms (Ka-poor et al. 1989; Kucey et al. 1989). Mycorrhizal plantscan take up more P than non-mycorrhizal plants, main-ly from the same soluble P pool (Barea 1991). Inocula-tion with phosphate-solubilizing microorganisms(PSM) may help to solubilize native soil P as well as Pfrom rockphosphates. The soluble P released by the ac-

tivity of PSM is actively taken up by mycorrhizal roots(Barea et al. 1983; Kucey et al. 1989). Coinoculation ofPSM and arbuscular mycorrhizas (AM) may enhanceplant acquisition of P from insoluble P sources (Azconet al. 1976; Azcon-Aguilar et al. 1986; Piccini and Az-con 1987). Most studies involving inoculation of PSMand AM fungi have been conducted in sterile soilswhere competition from native microflora is eliminated(Kucey 1987; Piccini and Azcon 1987; Toro et al. 1996).However, a few studies carried out with non-disinfectedsoils under pot conditions and involving coinoculationof PSM and AM fungi demonstrated an increase in drymatter production and P uptake (Singh and Singh 1993;Tilak et al. 1995). During the present investigation, weevaluated the effect of PSM and AM with and withoutMussoorie rockphosphate (MRP) on the mycorrhiza-tion, dry matter production, and N and P uptake ofmungbean under pot conditions in a natural unsterilesandy P-deficient soil. This was done to assess the po-tential of PSM and AM fungi for increasing availabilityof P from rockphosphate in alkaline soils.

Material and methods

To study the interaction of P-solubilizing microorganisms Bacilluscirculans and Cladosporium herbarum, and the AM fungus Glo-mus fasciculatum (Thaxter sensu Gerd) (Gerd & Trappe) agreenhouse experiment was carried out in a randomized block de-sign. There were eight treatments with five replications each.

Soil

The soil used in the study was taken from Haryana AgriculturalUniversity dryland farm, Hisar and had clay 5.6%, silt 4.0%, sand90.4%, 0.10% organic C, 2.5 ppm NaHCO3-extractable P, 0.001%total P, 4.9 c mole (pc) kgP1 cation exchange capacity and pH7.6.

The AM inoculum

The AM endophyte G. fasciculatum (Thaxter sensu Gerd) (Gerd& Trappe) was obtained from Dr. D. J. Bagyaraj, University of

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Agricultural Sciences, Bangalore, India was found to performwell with mungbean (Vigna radiata L. Wilczek cv. Asha). Theinoculum consisted of chopped root segments and soil from a 10-week-old pot culture of G. fasciculatum grown on pearl millet(Pennisetum typhoides) in a sterile sandy soil.

Phosphate-solubilizing microorganisms

The P-solubilizing bacterium B. circulans and fungus C. herbarumwere earlier isolated from soil and tested for phosphate solubili-zation under in vitro conditions using tricalcium phosphate andMRP as P sources. Inoculum was prepared in Pikovskaya me-dium (Pikovskaya 1948) at 30 7C for 5 days using tricalcium phos-phate as a P source. Inoculation was carried out by dipping themungbean seeds in cells/spore suspension for 10 min containingabout 108 colony-forming units per ml.

Rockphosphate

Mussoorie rockphosphate was obtained from M/S Pyrites, Phos-phates and Chemicals Ltd., Noida, India as a 100-mesh-size pow-der. It contained total P 8.1%, water-soluble P 0.056 mg gP1,0.5 M NaHCO3-soluble P 0.18 mg gP1, 2% citric acid-soluble P2.3 mg gP1, and pH 8.7. Phosphorus in MRP is present as carbon-ate fluorapatite, which consists of about 67% total rock phos-phate and is of sedimentary origin.

Plant growth conditions

Four kg sieved (2 mm) soil was placed into earthenware pots.MRP was added to each pot in a uniform layer at a depth of 5 cmso as to supply 40 kg P2O5 haP1. A basal dose of nitrogen wasapplied as ammonium sulphate at 20 kg N haP1 in the upper 5 cmprior to sowing. The mycorrhizal inoculum (5 g potP1) was addedat a depth of 5 cm. Uniform inoculation with Rhizobium sp. Vig-na S32 strain was obtained by putting 1 ml of culture (about 108

cells) over the seed hole. Ten seeds were sown at a depth of2.5 cm and after germination thinned to 5 plants per pot. The potswere distributed at random in a greenhouse under natural condi-tions and the crop grown during June to August 1995. The meanambient temperature during this period was 31.8 7C with a meanminimum of 26.3 and mean maximum of 37.3 7C. The mean brightsunshine was 6.2 hours and relative humidity 72%. Plants werewatered daily to maintain moisture at field capacity and harvestedat maturity after a growth period of 11 weeks for grain and strawyield.

Measurements

At harvest, the dry matter yield was determined after drying thegrain and straw for 20 h at 70 7C. The N and P uptake were re-corded. The microkjeldahl method was used for N determination(Bremner 1960) and total P was determined by the method ofJohn (1970). The root clearing and staining method of Phillipsand Hayman (1970) was used for studying root colonization byAM. Mycorrhizal colonization in cortical root tissue was esti-mated by examining microscopically stained root segments (1 cmlong). Fifty root segments per pot were examined and the per-centage of root segments colonized scored.

The pour-plate technique was applied for estimation of PSMpopulations in the rhizosphere soil (Pikovskaya 1948). The rhi-zosphere soil was collected by uprooting the plants and soil ad-hering to roots was serially diluted; 0.1 ml aliquots of appropriatedilution were spread on Pikovskaya medium plates. The plateswere incubated at 30 7C for 1 week and colonies showing a clearzone of tricalcium phosphate dissolution were counted.

Table 1 Effects of the P-solubilizing microorganisms Bacillus cir-culans and Cladosporium herbarum alone or in combination withGlomus fasciculatum on root colonization of mungbean with andwithout Mussoorie rockphosphate (MRP)

Microbialtreatment

Root colonization (%)

WithoutMRP

WithMRP

ControlBacillus circulansCladosporium herbarumBacillus circulans cCladosporium herbarumGlomus fasciculatumBacillus circulans cGlomus fasciculatumCladosporium herbarum cGlomus fasciculatumBacillus circulans cCladosporium herbarum cGlomus fasciculatum

6.017.022.027.0

66.673.3

72.3

73.2

21.629.327.534.7

68.279.8

82.0

88.2

LSD (5%) 4.2 4.0

Results and discussion

AM root colonization

The AM root colonization at harvest ranged from 6.0 to88.2% (Table 1) and it was poor without AM inocula-tion. Improvement of AM colonization was observedwith B. circulans or C. herbarum either singly or in com-bination. Inoculation with G. fasciculatum alone gave66.6% root colonization and this was further improvedin the presence of PSM. In general, root colonizationby the AM fungus in the presence of MRP was higher.The significant increase in the degree of mycorrhizalcolonization by PSM inoculation alone may be due tophytohormone production by these microorganisms,which in turn may stimulate mycorrhizal colonization.These results confirm the findings of Azcon et al.(1978), who reported enhanced mycorrhizal develop-ment with Lavandula, Lycopersicum and Medicagoplants due to phytohormones in the culture filtrate ofP-solubilizing bacterium Pseudomonas. The enhance-ment of mycorrhizal root colonization by addition ofMRP and inoculation with PSM may be attributed toeffects on root morphology or on the physiology of thefungal symbionts, or could be due to the creation ofmore acidic conditions by an increasing population ofnative as well as the introduced PSM. Similar increasesin root colonization by AM fungi in presence of rock-phosphate have been observed previously (Barea andAzcon-Aguilar 1982; Piccini and Azcon 1987; Singh andSingh 1993).

Dry matter production

Both grain and straw yield of mungbean increased sig-nificantly after inoculation with PSM, particularly after

251

Table 2 Effects of the P-solubilizing microorganisms Bacillus circulans and Cladosporium herbarum alone or in combination withGlomus fasciculatum on dry matter yield (g potP1) in mungbean with and without Mussoorie rockphosphate (MRP)

Microbial treatment Without MRP With MRP

Grain Straw Total Grain Straw Total

ControlBacillus circulansCladosporium herbarumBacillus circulans c Cladosporium herbarumGlomus fasciculatumBacillus circulans c Glomus fasciculatumCladosporium herbarum c Glomus fasciculatumBacillus circulans c Cladosporium herbarum cGlomus fasciculatum

0.6300.7420.7650.8090.6480.7920.8090.868

1.7022.4462.9043.5202.1612.6072.9503.800

2.3323.1883.6944.3292.8093.3933.7394.668

0.6420.7860.7950.8240.7800.9370.9550.966

1.7682.7033.2183.4052.6053.5073.6314.282

2.4103.4894.0134.2293.3854.4344.5866.664

LSD (5%) 0.048 0.260 0.291 0.122 0.028 0.029

Table 3 Effects of the P-solubilizing microorganisms Bacillus cir-culans and Cladosporium herbarum alone or in combination withGlomus fasciculatum on P uptake (mg potP1) and P content (%

dry wt.) of mungbean with and without Mussoorie rockphosphate(MRP)

Microbialtreatment

Without MRPP uptake P content

With MRPP uptake P content



0.0510.0660.0740.1000.0590.0690.0790.106

0.0840.1470.1620.2180.1860.2760.2860.375

0.1350.2130.2360.3780.2450.3450.3650.491

0.0060.0070.0090.0090.0090.0100.0100.010

0.0660.0740.0900.1120.1100.1360.1330.195

0.2100.3020.3100.4120.3500.3820.4620.518

0.2760.3750.4000.5220.4600.5170.5950.713

0.0110.0110.0100.0120.0140.0120.0130.011

LSD (5%) 0.048 0.089 0.078 0.001 0.044 0.068 0.072 0.001

combined inoculation (Table 2). Total plant biomassincreased in the presence of MRP except with the com-bined inoculation. The effectivity of PSM on grain pro-duction was similar both in the presence and absence ofMRP but straw production was higher (207%) withoutMRP than with MRP (192%). Thus addition of MRPand microbial inoculation affected partioning of bio-mass between grain and straw (Azcon-Aguilar et al.1986). There was a synergistic effect of triple inocula-tion with B. circulans, C. herbarum and G. fasciculatum.Both grain and straw yield were higher when G. fasci-culatum and PSM were inoculated in combination thanin treatments with only PSM or G. fasciculatum. Theincrease in dry matter may be attributed to better P uti-lization from soil as well as from rockphosphate in thepresence of these organisms (Raj et al. 1981; Picciniand Azcon 1987).

Nutrient uptake and the population of PSM in the soil

The concentration of P in plant biomass was higher inthe presence of MRP (Table 3). Inoculation with PSMor the AM fungus significantly improved the concentra-tion of P in the plant biomass in absence of rockphos-

phate. However, in the presence of rockphosphate, Pconcentration increased only in treatments receivingthe AM fungus or the AM fungus in combination withC. herbarum. This may be due to better utilization of Pfrom the pool of native soil P as well as from the MRPby the action of PSM and G. fasciculatum.

Significant increases in total N and P uptake due toinoculation of PSM and G. fasciculatum were observed(Tables 3, 4) in the crop at maturity. Inoculation withB. circulans or C. herbarum alone or with G. fascicula-tum enhanced P uptake into both grain and straw, ascompared with uninoculated controls. P uptake washigher when the soil was amended with MRP. Thehigher P uptake in plants inoculated with PSM and theAM fungus in presence of MRP may be attributed togreater absorption of P by AM (Barea 1991). Manystudies have shown the synergistic effect of coinocula-tion of PSM and AM fungi on growth and nutrition ofplants. Azcon et al. (1976) using lavender as test crop ina steam-sterilized soil observed higher P uptake withmycorrhiza plus P-solubilizing bacteria than plants witheither mycorrhiza or bacteria separately. In a green-house experiment on wheat and beans, better utiliza-tion of P from rockphosphate was observed in the pres-ence of an AM fungus and a P-solubilizing Penicillium

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Table 4 Effects of the P solubilizing microorganisms Bacillus circulans and Cladosporium herbarum alone or in combination withGlomus fasciculatum on N uptake (mg potP1) of mungbean with and without Mussoorie rockphosphate (MRP)

Microbialtreatment

Without MRP With MRP



0.8901.1421.2161.3401.1351.2931.3701.586

0.3360.8591.1391.5761.5251.9292.0262.416

1.2262.0012.3552.9162.6603.2223.3964.002

1.1681.3051.4151.6401.4841.5601.6952.220

1.6892.1022.1702.4791.9552.3452.4852.908

2.8573.3173.5864.1193.4393.9054.1805.128

LSD (5%) 0.108 0.168 0.208 0.210 0.215 0.672

Table 5 Effect of the P-solubilizing microorganisms Bacillus cir-culans and Cladosporium herbarum alone or in combination withGlomus fasciculatum on the population of P-solubilizing microor-

ganisms (gP1 soil) in the rhizosphere soil of mungbean with andwithout Mussoorie rocksphosphate (MRP)

Microbialtreatment

Without MRP With MRP

P-solubilizingbacteria

P-solubilizingfungi

P-solubilizingbacteria

P-solubilizingfungi


3.3!104

5.0!106

2.7!104

8.8!106

3.0!104

15.0!106

3.8!104

17.8!106

2.0!102

3.7!102

5.0!103

6.3!103

1.3!102

5.3!102

11.5!103

15.3!103

3.8!104

19.2!106

5.1!104

21.2!106

2.6!104

29.8!106

8.8!104

42.3!106

2.3!102

4.3!102

16.2!103

19.0!103

3.4!102

6.3!102

28.0!103

36.3!103

bilaji isolate (Kucey 1987). Similar positive interactionsof a calcium phosphate-solubilizing isolate of Bacillussp. and G. mossae on the growth of Pueraria phaseo-loides and nutrient uptake have also been observed(Toro et al. 1996). On the other hand, Azcon-Aguilar etal. (1986) reported no increase in the utilization of Pfertilizer by soybean after inoculation with AM, Rhizo-bium japonicum and P-solubilizing bacteria in a gam-ma-irradiated soil amended with tricalcium phosphate.However, there was an increase in shoot N concentra-tion.

There was no significant effect of PSM or AM aloneor in combination on nodulation of mungbean, eitherin the presence or absence of MRP (the number of no-dules varied from 21 to 30 per pot). This is in contrastto earlier studies where AM inoculation was shown toenhance nodulation and nitrogen fixation of legumesby rhizobia (Manjunath and Bagyaraj 1986; Barea et al.1988). The higher N uptake by mungbean due to inocu-lation with PSM and G. fasciculatum may be due to bet-ter P nutrition of mungbean, which subsequently im-proved utilization of soil N. The significant increase intotal N uptake by the crop due to MRP application andinoculation with PSM along with the AM fungus couldalso have been due to AM hyphae translocating soil N

to the plant. Johansen et al. (1994) observed increasedtranslocation of soil N to cucumber plants inoculatedwith G. intraradices when 15N-labelled NH4NO3 wasadded to soil, indicating hyphal transport of inorganicN.

There was no change in the population of P-solubil-izing bacteria in the rhizosphere of mungbean after ino-culation of the fungi (Table 5). Addition of MRP stim-ulated the population of both PSM in the rhizosphereof mungbean. A larger population of PSM was main-tained in the presence of G. fasciculatum. These effectsmay be attributed to higher metabolic activities of PSMfor longer periods in the rhizosphere after MRPamendment and AM inoculation (Singh and Singh1993).

The results of the present study show that yield ofmungbean can be improved by a combination of anAM fungal symbiont and saprophytic PSM in a non-disinfected soll. This double inoculation helps theplants to obtain P from insoluble P sources such asrockphosphate and also leads to better N nutrition ofthe legume. The potential of using this dual inoculationto enhance utilization of P from rockphosphate in aneutral soil is important, since P availability from rock-phosphate is low in such a soil.

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Acknowledgement The senior author gratefully acknowledgesfinancial assistance from the Council of Scientific and IndustrialResearch, New Delhi.

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Documents

Effects of inoculation of phosphate-solubilizing microorganisms and an arbuscular mycorrhizal fungus on mungbean grown under natural soil conditions