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Phosphate solubilization potential and stress toleranceof Eupenicillium parvum from tea soil

Pratibha VYAS, Praveen RAHI, Anjali CHAUHAN, Arvind GULATI*

Plant Pathology and Microbiology, Hill Area Tea Sciences, Institute of Himalayan Bioresource Technology,

Post Box No. 06, Palampur, Himachal Pradesh 176 061, India

a r t i c l e i n f o

Article history:

Received 6 November 2006

Received in revised form

1 May 2007

Accepted 10 June 2007

Published online 29 June 2007

Corresponding Editor:

Stephen W. Peterson

Keywords:

Aluminium stress

Desiccation tolerance

Eupenicillium parvum

Inorganic phosphate solubilization

Iron stress

Tea

a b s t r a c t

Eupenicillium parvum was recorded for first time during isolation of phosphate-solubilizing

microorganisms from the tea rhizosphere. The fungus developed a phosphate solubiliza-

tion zone on modified Pikovskaya agar, supplemented with tricalcium phosphate. Quanti-

tative estimation of phosphate solubilization in Pikovskaya broth showed high

solubilization of tricalcium phosphate and aluminium phosphate. The fungus also solubi-

lized North Carolina rock phosphate and Mussoorie rock phosphate, and exhibited high

levels of tolerance against desiccation, acidity, salinity, aluminium, and iron. Solubilization

of inorganic phosphates by the fungus was also observed under high stress levels of alu-

minium, iron, and desiccation, though the significant decline in phosphate solubilization

was marked in the presence of aluminium than iron. The fungal isolate showed 100 % iden-

tity with E. parvum strain NRRL 2095 ITS 1, 5.8S rRNA gene and ITS 2, complete sequence;

and 28S rRNA gene, partial sequence.

ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction

Tea is an economically important plantation crop. With little

scope to increase the area under cultivation, improving tea

productivity is imperative to meet the increasing consumer

demand. The availability of phosphorus is a limiting factor in

the growth and production of tea (Verma & Palani 1997). The

acidic conditions of the soil required for tea growth favour

the formation of insoluble phosphate complexes through

binding of phosphate anions with metal cations. Conse-

quently, a large portion of the inorganic phosphates applied

as fertilizers is rendered inaccessible to the plant due to rapid

binding into complex forms (Venktesan & Murugesan 2004).

The complex dynamic equilibrium of solubilization and im-

mobilization of both macro- and micronutrients and their

availability to the plant are greatly influenced by the activity

of microorganisms (van Elsas et al. 1997). The ability of several

microorganisms, including actinomycetes, other bacteria, and

fungi, to dissolve relatively insoluble phosphate compounds

has introduced the possibility that microbial inoculants could

be used to induce solubilization of phosphates in the soil

(Whitelaw 2000). Although exotic strains of the microbial

agents can increase growth and yield of plants, evidence sug-

gests that genotypes of beneficial microbes may be endemic to

a biogeographical region (Cho & Tiedje 2000). The endemic mi-

crobial pool of a region may contain highly efficient genotypes

* Corresponding author.E-mail address: gal_arvind@yahoo.co.in

0953-7562/$ – see front matter ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.mycres.2007.06.003

932 P. Vyas et al.

and the indigenous strains are also likely to perform better

than the exotic strains. However, little work has been done

on the aspects of phosphate solubilization by microorganisms

from the tea soil. The microorganisms growing in tea soil are

subject to acidic conditions, high concentrations of aluminium

and iron, and desiccation through periodic spells of drought.

The present paper reports in vitro solubilization of inorganic

mineral phosphates and rock phosphates and tolerance to

high levels of desiccation and salt concentration of Eupenicil-

lium parvum, which has been isolated from tea rhizosphere.

The results have introduced the feasibility of assessing plant

growth-promoting activity through inoculation of the tea soil

with phosphate-solubilizing fungus.

Methods and materials

Isolation and identification of the fungus

The fungus was isolated from soil samples collected at the Tea

Experimental Farm Banuri, Palampur in Himachal Pradesh

(India) from 15–25 cm depth from the rhizosphere of feeder

roots of tea (Camellia sinensis). The serial soil dilutions were

spread plated on modified Pikovskaya (PVK) agar containing

0.5 % tricalcium phosphate (TCP) as the source of insoluble

phosphate (Gupta et al. 1994). The fungal colonies producing

distinct zones of TCP solubilization were raised into pure

cultures, maintained on potato dextrose agar slants at 4 �C,

and identified on the basis of cultural and microscopic features

(Subramanian 1971; Barnett & Hunter 1972). The phosphate-

solubilizing fungus identified as Eupenicillium parvum at the

Microbial Type Culture Collection and Gene Bank, Institute of

Microbial Technology, Chandigarh (MTCC accession no.

6487), on the basis of phenotypic characters, was selected for

further studies on inorganic phosphate solubilization and

stress tolerance parameters typical of tea soil.

Five-day-old mycelium was scraped from the Petri dishes,

frozen in liquid nitrogen and ground to a fine powder. DNA

was extracted using Qiagen Plant DNeasy Kit (Qiagen Gmb H,

Hiden). The amplification of ITS 1, the 5.8 rRNA gene and ITS

2 was achieved using primers ITS1: 50 TCC GTA GGT GAA

CCT GCG G and ITS4: GCT GCG TTC ATC GAT GC (White et al.

1990). The PCR reaction was performed in 50 ml total volume in-

cluding 50 ng genomic DNA, 10 pmol each primer, 0.5 mM

dNTPs, 1� PCR buffer with 1.5 mM MgCl2, and 3 U taq polymer-

ase. The thermocycling conditions consisted of an initial dena-

turation at 94 �C for 5 min, followed by 35 amplification cycles

at 94 �C for 1 min, 54 �C for 1 min and 72 �C for 2 min, and a final

extension at 72 �C for 8 min. The sequence of the PCR-product

was determined by employing the ABI Prism Big Dye Termina-

tor v. 3.1 Cycle Sequencing Kit. The sequence was analysed us-

ing the gapped BLASTn (http://www.ncbi.nlm.nih.gov) search

algorithm and aligned to the nearest neighbours. The evolu-

tionary distances among E. parvum and related taxa were

calculated with TREECON software package version 1.3b

(Copyright Yves van de Peer, University of Antwerp, 1994,

1998) using Kimura’s two-parameter model, after aligning

the sequences with ClustalW. The ITS region sequence of

Aspergillus flavipes strain ATCC 1030 was used as an outgroup.

Solubilization of phosphate sources by the fungus

The solubilization of different inorganic phosphate sources

was studied by replacing TCP from modified PVK agar and un-

modified PVK agar with 0.5 % aluminium phosphate (AP), iron

phosphate (FP), Mussoorie rock phosphate (MRP), North Caro-

lina phosphate (NCRP) or Udaipur rock phosphate (URP). The

Petri dishes were incubated for 12 d at 28 �C after point inocu-

lations with the fungus and observed every third day for the

presence of a clear zone around the colony indicating phos-

phate solubilization.

Quantitative estimation of TCP solubilization was under-

taken in PVK broth. The solubilization of AP, FP, MRP, NCRP

or URP was studied by replacing TCP (0.5 % w/v) from PVK

broth. The rock phosphates were washed to remove the solu-

ble phosphate and dried at 40 �C for 24 h before use as the

phosphate source. Five-day-old fungal colonies were homoge-

nized and suspended in normal saline (0.85 % NaCl) for prep-

aration of the fungal suspension. The flasks containing

broth were inoculated by 1 ml fungal suspension (105 CFU

ml�1) and incubated at 28 �C in a Innova Model 4230 refriger-

ated incubator shaker (New Brunswick Scientific, Edison, NJ)

at 180 rev min�1. The phosphorus in the culture filtrate was

estimated on days 3, 6, 9 and 12 of incubation by the vanado-

molybdophosphoric yellow colour method (Jackson 1973). The

uninoculated autoclaved medium with different phosphate

substrates was incubated under similar conditions as those

employed for incubation of the inoculated medium to serve

as the controls for solubilization of various phosphate sub-

strates by the fungus. The pH of the liquid medium was mea-

sured using CyberScan 510 pH meter (Merck and Cp., Inc.,

Whitehouse Station, NJ).

Screening the fungus for stress tolerance

The tolerance of the fungus toward different abiotic factors

was studied by growing the fungus for 9 d in potato dextrose

broth modified to produce stressful conditions. The prelimi-

nary experiments on discerning the optimum temperature

and pH for fungal growth were performed by measuring the

radial growth on solid medium (data not shown). The

optimum temperature for the fungal growth was 36 �C and

optimum pH was 4.5. The effect of temperature was studied

by incubating the cultures over a range of 16-40 �C at pH

4.5. The influence of pH on fungal growth was studied by

growing the fungus at 36 �C in the medium made in citrate–

phosphate buffer, Tris-HCl, and glycine-NaOH buffer for

maintaining pHs of 3-7, 7.5 and 8, and 8.5-11, respectively.

Subsequently, the fungus was grown over different concen-

trations of NaCl (2.5, 5, 7.5, and 10 %), AlCl3 (2.5, 5, 7.5, 10,

25, 50, and 100 mM), FeCl3 (2.5, 5, 7.5, 10, 25, 50, and

100 mM), and polyethylene glycol (PEG) 6000 (20, 30, 40, and

50 %) at pH 4.5 and 36 �C.

Phosphate solubilization under stress

The solubilization of TCP, AP, and NCRP by the fungus was

studied under different temperatures (15, 20, 25, 30, and

36 �C) at pH 4.5 and pH (4.5, 5.5, and 7) at 36 �C. The acidic

pH of medium was adjusted with 1 N HCl. Solubilization of

Phosphate solubilization potential and stress tolerance 933

inorganic phosphates was also quantified under desiccation

of 30 % PEG, and aluminium and iron concentrations of

2.5-5 mM at pH 4.5 and 36 �C.

Experimental design and data analysis

Randomized block design with two factor factorial arrange-

ment was adopted for conducting the experiments. The data

were checked for normality and subjected to two-way analy-

sis of variance (ANOVA) using the STATISTICA data analysis

software system version 7 (StatSoft� Inc., Tulsa, USA 2004).

All values are means of three replicates. The mean of the

treatments were compared by CD value at P¼ 0.01.

Results

Identification of the fungus

The fungal colonies were radially sulcate, surface texture

dense, with deep floccose overlay; margins low, somewhat

irregular; mycelium white at the margins, centrally maize

yellow, deep red-brown exudates abundant; cleistothecia

abundant and submerged at 37 �C, not observed below 24 �C;

reddish brown soluble pigment produced; reverse deep red-

dish brown. Conidia sparse, more on PVK and modified PVK

than potato dextrose agar, and monoverticillate; stipes non-

vesiculate; conidia and ascospores small. A single band of ca

600 bp was obtained on amplification of the ITS region of the

fungus. The sequence of 439 bp ITS region of the fungus

showed 100 % identity with the Eupenicillium parvum strain

NRRL 2095 ITS 1, 5.8S rRNA gene and ITS 2 complete sequence,

and 28S rRNA gene partial sequence. The phylogenetic tree

constructed with the TREECON software is shown in Fig 1. In

the tree, sequences of reference strains were obtained from

the NCBI GenBank. The newly generated sequence was depos-

ited at GenBank (accession no. DQ536524).

Solubilization of phosphate sources by the fungus

The zone of TCP solubilization by Eupenicillium parvum

appeared on third day, which became prominent and sharp

after 7 d of incubation both on PVK agar and modified PVK

agar. However, no phosphate solubilization zone was ob-

served in the medium supplemented with AP, FP, MRP, URP,

and NCRP. The quantification of the phosphate liberated

showed that TCP solubilization was significantly higher, fol-

lowed by the solubilization of NCRP, AP, and MRP in the de-

creasing order, as compared with FP and URP (Table 1).

However, no significant difference was recorded in the solu-

bilization of FP and URP. The incubation period also exhibited

significant influence on the quantities of phosphate solubili-

zation. The solubilization increased significantly with in-

creases in the incubation period from 3-9 d, followed by

a significant decline at 12 d of incubation (Table 1). Likewise,

a significant increase in the reduction of the pH of the me-

dium was also registered with advancement in the incuba-

tion up to 9 d, followed by a significant decline at 12 d of

incubation. The decline in the pH of the culture medium

was highest in the solubilization of AP, followed by the solu-

bilization of FP. Solubilization of URP showed the lowest

reduction in the pH of the medium. The reduction in the

pH of the medium was comparable during the solubilization

of TCP and NCRP. The data were found to be normally

distributed.

Fungus growth under stress

The results of the effects of temperature, pH, and salt concen-

trations on fungal growth, as well as ability to tolerate desicca-

tion, aluminium, and iron are given in Fig 2. The maximum

fungal biomass was obtained at 36 �C and pH 4.5. The fungus

was able to grow in up to 40 % PEG 6000, 10 % NaCl, 100 mM al-

uminium, and 100 mM iron for up to 9 d of incubation in potato

dextrose broth.

Distance 0.02

Aspergillus flavipes strain ATCC 1030

Penicillium indicum strain NRRL 3387

Eupenicillium hirayamae strain NRRL 143

Eupenicillium erubescens strain NRRL 6223

Eupenicillium rubidurum strain NRRL 6033

Penicillium vinaceum strain NRRL 739

Eupenicillium parvum strain NRRL 2095

Eupenicillium parvum strain FIHB 539

Fig 1 – Phylogenetic tree showing the relationships among Eupenicillium parvum (FIHB 539) and representatives of related

taxa, based on the ITS region sequences. Bar [ 0.02 substitutions per site.

934 P. Vyas et al.

Table 1 – Effect of incubation on solubilization of phosphate substrates by Eupenicillium parvum at 28 �C in Pikovskaya brothwith initial pH 7

P source Phosphate solubilization over control (mg ml�1) Reduction in pH of medium (%)

Days TCP AP FP MRP URP NCRP Mean TCP AP FP MRP URP NCRP Mean

3 120.8 27.4 1.3 5.7 2 6.9 27.4 17.6 35.3 25 8.8 5.9 17.6 18.4

6 150.5 58.4 2.9 20.5 3.2 65.3 50.1 19.1 50 35.3 13.2 7.4 22.1 24.5

9 213.7 89.6 7.7 51.3 6.3 110.4 79.8 27.9 52.9 55.9 16.2 11.8 26.5 31.9

12 197.3 45.7 5.7 24.9 3 72.5 58.2 20.6 48.5 51.5 13.2 7.4 23.5 27.5

Mean 170.6 55.3 4.4 25.6 3.6 63.8 21.3 46.7 41.9 12.9 8.1 22.4

Variant S.E.M. � CD (P¼ 0.01) S.E.M. � CD (P¼ 0.01)

P source 0.8 3.1 0.8 3

Days 0.7 2.6 0.6 2.4

Interactions 1.6 6.3 1.6 6

TCP, tricalcium phosphate; AP, aluminium phosphate; FP, ferrous phosphate; MRP, Mussoorie rock phosphate; URP, Udaipur rock phosphate;

NCRP, North Carolina rock phosphate.

Phosphate solubilization by the fungus underdifferent stress conditions

The fungus was tested for the ability to solubilize inorganic

phosphates at different pHs and temperatures. The pH of

the culture medium influenced phosphate solubilization by

the fungus. The solubilization of various substrates was

significantly higher in the medium with a neutral pH than

those with acidic pHs (Table 2). The pH of the medium also de-

creased significantly on solubilization of phosphate substrates

(Table 2). A significant increase in the solubilization of phos-

phate substrates was recorded in the cultures incubated at

various temperature intervals through 15-30 �C. Maximum

phosphate solubilization occurred at 36 �C, which is at par

with 30 �C (Table 3). The increase in incubation temperature

also influenced the reduction in the pH of the culture medium,

with the minimum reduction at 15 �C and the maximum re-

duction at 36 �C. The percent reduction at 15 �C was statisti-

cally comparable with the reduction at 20, 25, and 30 �C.

However, there was no significant difference in the pH reduc-

tion at 25, 30, and 36 �C. The presence of aluminium and iron

at stressful concentrations caused a reduction in phosphate

solubilization, with a significantly higher reduction at higher

concentrations of these metals in the medium (Table 4). The

reduction in phosphate solubilization was significantly higher

in the presence of aluminum as compared with the presence

of iron in the growth medium. The fungus was also able to sol-

ubilize inorganic phosphates in the presence of 30 % PEG 6000.

The lowered phosphate solubilization in the presence of PEG

30 %, aluminium, and iron was accompanied by a smaller de-

crease in the pH as compared with the decrease in pH during

phosphate solubilization in the absence of stress conditions

(Table 4).

Discussion

The previously unreported fungus from tea soil was identified

as Eupenicillium parvum on the basis of morphological features

and ITS region sequencing. The fungus was isolated from the

soil around tea rhizospheres, which were acidic and had high

aluminium levels. The tea soil is also subject to desiccation

under periodic spells of drought. The phosphate-solubilizing

microbial isolates from tea soils are likely to be more success-

ful as microbial inoculants than the microorganisms isolated

from other soils because of their ability to survive the stress

factors that occur in tea culture. Fungi are generally more tol-

erant to acidity than bacteria and account for most of the

highly aluminium-resistant microorganisms (Myrold & Nason

1992; Kanazawa & Kunito 1996).

The appearance of a clear halo around the colony of E. par-

vum indicated phosphate solubilization by the fungus (Gupta

et al. 1994; Kang et al. 2002). Quantitative estimation of phos-

phate solubilization by the fungus in PVK broth containing

TCP showed high phosphate solubilization (Table 1). This is

the first report on the solubilization of a phosphate source

by E. parvum isolated from tea soil. Phosphate solubilization

by isolates of E. parvum from cultivated and forest soils has

not been previously recorded, although E. shearli from the soils

of Brazil has been reported to solubilize phosphate (Nahas

1996).

Aluminium, iron, and clay, which are abundant in tea soils,

render the applied phosphorus unavailable to the plants by

forming insoluble complexes (Ranganathan 1976). The micro-

bial solubilization of phosphates is not restricted to calcium

salts as microorganisms also act upon iron, aluminium, and

other phosphates (Gaur 1990; Kang et al. 2002). In our study,

the development of clear halos was not observed around the

colonies of E. parvum, grown on PVK agar and modified PVK

agar, with aluminium phosphate or iron phosphate as the

sole phosphate source. However, the isolate exhibited solubi-

lization of both aluminium phosphate and iron phosphate in

PVK broth (Table 1). Thus solubilization of aluminium phos-

phate and iron phosphate in the broth was independent of

the appearance of a phosphate solubilization zone on the solid

medium, as also reported previously for many microorgan-

isms (Ahmad & Jha, 1968; Gupta et al. 1994). The majority of

phosphate-solubilizing microorganisms are able to solubilize

calcium-phosphorus complexes but only a few can solubilize

iron–phosphorus and aluminium-phosphorus complexes

(Banik & Dey 1983; Gaur & Gaind 1983; Kucey et al. 1989).

Phosphate solubilization potential and stress tolerance 935

0

0.2

0.4

0.6

0.8

1

1.2

0 2.5 5 7.5 10Fu

ngal

Dry

Wt.

(g/5

0ml b

roth

)

0

0.2

0.4

0.6

0.8

1

1.2

0 20 30 40 50

Fung

al D

ry W

t. (g

/50m

l bro

th)

A B

DC

E

0

0.2

0.4

0.6

0.8

1

1.2

16 20 24 28 32 36 40

Temperature (ºC)

Fung

al D

ry W

t. (g

/50m

l bro

th)

0

0.2

0.4

0.6

0.8

1

1.2

0 2.5 5 7.5 10 25 50 100

Al3+/Fe3+ Concentration (mM)

Fung

al D

ry W

t. (g

/50m

l bro

th)

Al Fe

0

0.2

0.4

0.6

0.8

1

1.2

3 4 5 6 7 8 9 10 11pH

Fung

al D

ry W

t. (g

/50m

l bro

th)

Fig 2 – Effect of temperature (A), PEG 6000 (desiccant) (B), metal concentration (C), salt concentration (D), and pH

(E) on growth of Eupenicillium parvum in potato-dextrose broth after 9 d of incubation. The results are mean of three

replicates, error bars indicate standard deviation.

Table 2 – Effect of pH of the medium on the solubilization of phosphate substrates by Eupenicillium parvum at 36 �C inPikovskaya broth after 9 d of incubation

P source Phosphate solubilizationover control (mg ml�1)

Reduction in pH of medium (%)

pH TCP AP NCRP Mean TCP AP NCRP Mean

4.5 190.4 52.8 64.4 102.5 13.3 24.4 4.4 14.1

5.5 207 55.9 71.7 111.5 5.5 36.4 3 14.9

7 231.4 92.3 116.4 146.7 15.7 50 10 25.2

Mean 209.6 67 84.2 11.5 36.9 5.8

Variant S.E.M. � CD (P¼ 0.01) S.E.M. � CD (P¼ 0.01)

P source 1.4 5.7 1.5 6.2

pH 1.4 5.7 1.5 6.2

Interactions 2.4 9.9 2.6 10.7

TCP, tricalcium phosphate; AP, aluminium phosphate; NCRP, North Carolina rock phosphate.

936 P. Vyas et al.

Table 3 – Effect of temperature on the solubilization of phosphate substrates by Eupenicillium parvum in Pikovskaya brothwith initial pH 4.5 after 9 d of incubation

P source Phosphate solubilizationover control (mg ml�1)

Reduction in pH of medium (%)

Temperature TCP AP NCRP Mean TCP AP NCRP Mean

15 �C 139.4 22.5 29.8 63.9 2.2 22.2 2.2 8.9

20 �C 143.7 26.1 51.8 73.9 4.4 24.4 2.2 10.4

25 �C 166.2 51.2 57.8 91.7 8.9 24.4 7.4 13.6

30 �C 186.6 52.3 62.1 100.3 11.1 22.2 9.6 14.3

36 �C 190.4 52.8 64.7 102.6 14.1 22.2 12.6 16.3

Mean 165.3 41 53.2 8.1 23.1 6.8

Variant S.E.M. � CD (P¼ 0.01) S.E.M. � CD (P¼ 0.01)

P source 0.9 3.5 1.1 4.2

Temperature 1.2 4.5 1.4 5.4

Interactions 2 7.9 2.1 9.4

TCP, tricalcium phosphate; AP, aluminium phosphate; NCRP, North Carolina rock phosphate.

There was a significant variation in the quantities of phos-

phorus liberated by E. parvum from the different inorganic

phosphates tested (Table 1). The quantity of phosphate solubi-

lized was by far the highest for TCP among the inorganic phos-

phates. The solubilization of aluminium phosphate was also

high, whereas the solubilization of iron phosphate was low.

The results on solubilization of inorganic phosphates by the

test fungus agreed with reports that rock phosphates, FP and

AP are less amenable to microbial solubilization than TCP

(Gaur 1990; Shin et al. 2006).

In acidic soils with a low availability of phosphorus, rock

phosphates could be used through the action of microbial sol-

ubilization. The ability of E. parvum to solubilize various rock

phosphates from PVK broth, which contains rock phosphates

as the sole phosphate source, was evident from the results

(Table 1). The highest solubilization was obtained for NCRP

among the three rock phosphates tested. Although solubiliza-

tion of NCRP and MRP was appreciable, solubilization of URP

was very low. The solubilization of rock phosphates have

been reported to depend on their structural complexity and

particle size as well as the nature and quantity of organic acids

secreted by the microorganisms (Gaur 1990; Narsian & Patel

2000; Pradhan & Sukla 2005). MRP was found to be more vul-

nerable to solubilization as compared with URP (Illmer &

Schinner 1992). The maximum solubilization of different

phosphate sources was obtained at day 9 of incubation, which

decreased with further incubation (Table 1). A similar trend of

decreasing phosphate solubilization with advancement in the

incubation period has been reported for some microorgan-

isms, which could be attributed to the depletion of nutrients

in the culture medium (Ortuno et al. 1978; Goenadi et al.

2000; Kang et al. 2002).

Phosphate solubilization was accompanied by the reduc-

tion in the pH of the medium, with the highest reduction co-

inciding with the day of highest solubilization of phosphate

source (Table 1). A significant reduction in the pH of the

Table 4 – Effect of different stress levels on the solubilization of phosphate substrates by Eupenicillium parvum inPikovskaya broth with initial pH 4.5 after 9 d of incubation at 36 �C

P source Phosphate solubilizationover control (mg ml�1)

Reduction in pH of medium (%)

Stress level TCP AP NCRP Mean TCP AP NCRP Mean

Controla 190 46.4 64.4 100.3 14.1 22.2 11.1 15.8

2.5 mM aluminium 125.6 16.2 9 50.3 9.6 17.4 11.1 12.7

5 mM aluminium 88.8 11.2 4.6 34.9 7.4 22.2 15.5 15

2.5 mM iron 165.9 37.26 35.4 79.5 10.4 17.9 8.9 12.4

5 mM iron 117.6 20.58 32 56.7 8.1 24.4 4.4 12.3

30 % polyethylene

glycol 6000

125.5 32.6 4.7 54.3 11.2 20.7 9.6 13.8

Mean 135.6 27.4 25 10.1 20.8 10.1

Variant S.E.M. � CD (P¼ 0.01) S.E.M. � CD (P¼ 0.01)

P source 0.7 2.7 0.5 2.2

Stress level 1 3.8 0.8 3.1

Interactions 2 6.6 1.3 5.4

TCP, tricalcium phosphate; AP, aluminium phosphate; NCRP, North Carolina rock phosphate.a Medium without stress.

Phosphate solubilization potential and stress tolerance 937

culture filtrates containing various inorganic phosphates sug-

gested secretion of organic acids by the fungal strain (Nahas

1996; Pradhan & Sukla 2005). However, in the present study,

the phosphate substrates appear to influence the solubiliza-

tion irrespective of the corresponding reduction in the pH

of the medium, as a significantly higher reduction in the pH

of the medium was observed in AP and FP solubilization,

though the phosphate solubilization was significantly low, in

comparison with the other substrates (Table 1). Earlier find-

ings on the solubilization of rock phosphates by some effi-

cient strains of Aspergillus and Penicillium also indicated that

phosphate solubilization varies greatly with the nature of

phosphate substrates (Bardiya & Gaur 1974).

Tolerance to acidity, aluminium, and iron is important in

the growth, establishment and survival of microorganisms in

tea soils. The ability to adapt to desiccation and temperature

stresses may also be important in the survival of the microor-

ganisms during droughts. An understanding of the physiology

of E. parvum under similar stress conditions is required for the

successful application as a bioinoculant in tea. The fungus

exhibited good growth over a temperature range of 20-36 �C

(Fig 2), which is ideal for tea growth (Eden 1976). The acidic cul-

ture conditions were found suitable for fungal growth, coincid-

ing with the pH of the soil required for good tea growth (Eden

1976). The fungus also showed high tolerance to drought as it

could grow in the presence of 20-40 % PEG 6000 in broth, which

generates -0.49 and -1.76 MPa of osmotic pressures, respec-

tively (Michel & Kaufmann 1973). Likewise, the fungus could

also tolerate high salt concentrations, though a decline was

observed in the growth with an increasing salt concentration.

The fungus also showed tolerance to aluminium and iron un-

der strongly acidic conditions of the culture, as it could grow at

pH 4.5 in the presence of 100 mM aluminium and 100 mM iron.

A fairly good growth of the fungus was recorded in aluminium-

supplemented medium at concentrations of exchangeable al-

uminium reported for the acidic soils of tea (Sharma & Tripathi

1989). Aluminium and iron added to the medium under

strongly acidic conditions are toxic to microorganisms (Haug

1984). Previously six strains identified as Aspergillus flavus,

Penicillium sp., Penicillium janthinellum, Trichoderma asperellum,

Cryptococcus humicola and Rhodotorula glutinis from tea fields

were reported to tolerate 100-200 mM aluminium under

strongly acidic conditions, whereas the growth of most micro-

organisms was almost completely inhibited by 1-2 mM inor-

ganic monomeric aluminium (Kawai et al. 2000).

The results revealed the ability of E. parvum to solubilize

phosphate substrates over a wide range of pHs, temperatures,

and stress levels,althoughadeclinewasregistered insolubiliza-

tion under stress conditions for the fungal growth (Tables 2–4).

The phosphate-solubilization by the fungus appears to be

affected by the availability of phosphorus at the time of inocu-

lations in the culture medium. This is evident from the results

obtained in the present study regarding the solubilization of

phosphate substrates at acidic pH. The low phosphate solubili-

zation by the fungus under acidic pH could be attributed to the

availability of higher initial content of soluble phosphate in the

medium, which was estimated at 153.3, 12.7 and 3.6 mg ml�1 for

TCP, AP and NCRP at pH 4.5 in comparison with 25, 2.7 and

0.8 mg ml�1 for TCP, AP and NCRP at pH 7, respectively. The

results are in agreement with earlier studies on the effect of

the concentration of soluble phosphate on the solubilization

of fluorapatite by Aspergillus niger (Nahas & Assis 1992).

Eupenicillium parvum appears to adapt well to the stress

conditions and has the ability to solubilize inorganic phos-

phates under high stress conditions. The results have shown

the potential for further testing the fungus as a phosphate-

solubilizing inoculant in soil where conditions are much

more complex than those prevailing in vitro.

Acknowledgements

We acknowledge Dharamrajan Ananthapadamanabhan, In-

stitute of Microbial Technology, for his help in identification

of the fungus, and Kamlesh Singh, Department of Statistics,

Mathematics and Physics, Himachal Pradesh Krishi Vishva-

vidyalaya and Rakesh Deosharan. Singh, Biodiversity Divi-

sion, Institute of Himalayan Bioresource Technology and

for their help in statistical analysis. Thanks are also due

to Paramvir Singh Ahuja, Institute of Himalayan Biore-

source Technology for providing necessary facilities.

r e f e r e n c e s

Ahmad N, Jha KK, 1968. Solubilization of rock phosphate bymicroorganisms isolated from Bihar soils. Journal of General andApplied Microbiology 14: 89–95.

Barnett HL, Hunter BB, 1972. Illustrated Genera of Imperfect Fungi.Burgess Publishing, Minneapolis.

Banik S, Dey BK, 1983. Phosphate solubilization potentiality of themicroorganisms capable of utilizing AlPO4 as sole phosphatesource. Zentraalblatt fur Bakteriologie 138: 17–23.

Bardiya S, Gaur AC, 1974. Isolation and screening of microorgan-isms dissolving low grade rock phosphate. Folia Microbiology 19:386–389.

Cho JC, Tiedje JM, 2000. Biogeography and degree of endemicity offluorescent pseudomonas strains in soil. Applied and Environ-mental Microbiology 66: 5446–5448.

Eden T, 1976. Tea, third edn. Longman Group, London, 236 pp.Gaur AC, Gaind S, 1983. Microbial solubilization of phosphates

with particular reference to ferrous phosphate and aluminumphosphate. Science and Culture 49: 110–112.

Gaur AC, 1990. Phosphate Solubilizing Microorganisms as Biofertil-izers. Omega Scientific, New Delhi.

Goenadi DH, Siswanto S, Sugiarto Y, 2000. Bioactivation of poorlysoluble phosphate rocks with phosphorus solubilizing fungus.Soil Science Society of America Journal 64: 927–932.

Gupta R, Singal R, Shanker A, Kuhad RC, Saxena RK, 1994.A modified plate assay for screening phosphate-solubilisingmicroorganisms. Journal of General and Applied Microbiology 40:255–260.

Haug A, 1984. Molecular aspects of aluminium toxicity. CriticalReviews In Plant Sciences 1: 345–373.

Illmer P, Schinner F, 1992. Solubilization of inorganic phosphatesby microorganisms isolated from forest soils. Soil Biology andBiochemistry 24: 389–395.

Jackson ML, 1973. Soil Chemical Analysis. Prentice Hall, New Delhi.Kanazawa S, Kunito T, 1996. Preparation of pH 3 agar plate,

enumeration of acid-tolerant and Al-resistant microorgan-isms in acid soils. Soil Science and Plant Nutrition 42: 165–173.

Kawai F, Zhang D, Sugimoto M, 2000. Isolation and characteriza-tion of acid- and Al-tolerant microorganisms. FEMS Microbiol-ogy Letters 189: 143–147.

938 P. Vyas et al.

Kang SC, Ha CG, Lee TG, Maheshwari DK, 2002. Solubilization ofinsoluble inorganic phosphates by a soil-inhabiting fungusFomitopsis sp. PS 102. Current Science 82: 439–441.

Kucey RMN, Janzen HH, Legett ME, 1989. Microbially mediatedincreases in plant-available phosphorus. Advances In Agronomy42: 198–228.

Michel BE, Kaufmann MR, 1973. The osmotic potential of poly-ethylene glycol 6000. Plant Physiology 51: 914–916.

Myrold DD, Nason GE, 1992. Effect of acid rain on soil microbialprocesses. In: Mitchell R (ed.), Environmental Microbiology.Wiley-Liss, New York, pp. 59–81.

Nahas E, 1996. Factors determining rock phosphate solubilizationby microorganisms. World Journal of Microbiology & Biotechnology12: 567–572.

Nahas E, Assis LC, 1992. Phosphate rock solubilization by Asper-gillus niger in different type of vinasse. Pesquisa AgropecuariaBrasileira 27: 325–331.

Narsian V, Patel HH, 2000. Aspergillus aculeatus as a rock phosphatesolubilizer. Soil Biology and Biochemistry 32: 559–565.

Ortuno A, Hernansaez A, Noguera J, Morales V, Armero T, 1978.Phosphorus solubilizing effect of Aspergillus niger and Pseudo-monas fluorescens. Microbiologıa Espanola 31: 113–120.

Pradhan N, Sukla LB, 2005. Solubilization of inorganic phosphatesby fungi isolated from agriculture soil. African Journal ofBiotechnology 5: 850–854.

Ranganathan V, 1976. Tea soils of South India with referenceto nutritional and manuring problems. In: Handbook of TeaCulture. UPASI Tea Research Institute, Valparai.

Shin W, Ryu J, Kim Y, Yang J, Madhaiyan M, Sa T, 2006. Phosphatesolubilization and growth promotion of maize (Zea mays L.) bythe rhizosphere soil fungus Penicillium oxalicum. 18th WorldConference of Soil Science, 9–15 July, Philadelphia.

Sharma SP, Tripathi BR, 1989. Studies on lime requirement of acidsoils of India. Journal of the Indian Society of Soil Science 37: 363–370.

Subramanian CV, 1971. Hyphomycetes. Indian Council of Agricul-tural Research, New Delhi.

van Elsas JD, Trevors JT, Wellington EMH, 1997. Modern SoilMicrobiology. Marcel Dekker, New York.

Verma DP, Palani N, 1997. Manuring of tea in South India (revisedrecommendations). In: Handbook of Tea Culture. UPASI TeaResearch Institute, Valparai, p. 33.

Venktesan S, Murugesan S, 2004. Phosphorus fixing capacity of teasoilsofAnamallaisandNilgiris. Journal ofPlantationCrops32: 65–69.

White TJ, Bruns T, Lee S, Taylor J, 1990. Amplification and directsequencing of fungal ribosomal genes for phylogenies. In:Innis MA, Gelfand DH, Sninsky J, White TJ (eds), PCR Protocols:a guide to methods and applications. Academic Press, San Diego,pp. 315–322.

Whitelaw MA, 2000. Growth promotion of plants inoculated withphosphate solubilizing fungi. Advances In Agronomy 69: 99–151.