Arbuscular mycorrhizal status and root phosphatase activities in vegetative Carica papaya L....

ORIGINAL PAPER

Arbuscular mycorrhizal status and root phosphatase activitiesin vegetative Carica papaya L. varieties

Sharda W. Khade • Bernard F. Rodrigues •

Prabhat K. Sharma

Received: 27 July 2009 / Revised: 10 October 2009 / Accepted: 26 November 2009 / Published online: 23 December 2009

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2009

Abstract The arbuscular mycorrhizal (AM) status and

root phosphatase activities were studied in four vegetative

Carica papaya L. varieties viz., CO-1, CO-2, Honey Dew

and Washington. Standard techniques were used to ascertain

information on spore density and species diversity of AM

fungi. Although in case of estimation of root colonization

and root phosphatase activities, the existing methods were

slightly modified. Root colonization and spore density of

AM fungi along with root phosphatase (acid and alkaline)

activities varied significantly in four papaya varieties. The

present study recorded higher acid root phosphatase activity

when compared with alkaline root phosphatase activity

under P-deficient, acidic soil conditions. The present study

revealed that the root colonization of AM fungi influenced

acid root phosphatase activity positively and significantly

under P-deficient, acidic soil conditions. A total of 11 spe-

cies of AM fungi belonging to five genera viz., Acaulospora,

Dentiscutata, Gigaspora, Glomus and Racocetra were

recovered from the rhizosphere of four papaya varieties.

Keywords Arbuscular mycorrhizal fungi �Carica papaya L. � Root colonization �Root phosphatase activity � Spore density

Introduction

Phosphatases represent a broad range of intracellular as well

as soil accumulated activities that catalyze the hydrolysis of

both esters and anhydrides of phosphoric acid (Speir and

Ross 1978). Phosphatase enzymes are also directly involved

in the acquisition of phosphorus by plants. However, their

importance is not always obvious. The proposition that

plants with lower activities of root phosphatases may gain

and use phosphorus more readily than plants with higher

ones has been put forward by McLachlan (1980) who found

that acid phosphatase activity was lower in plants with more

efficient in P-uptake than grown under P-deficient condi-

tions. During the last few years, evidence has accumulated,

which suggests that microbial activity plays a significant

role in soil phosphorus transformations (Chauhan et al.

1981). Similarly, Helal and Sauerbeck (1984, 1987) have

demonstrated that soil organic phosphorus (Po), which is a

product of microbial activity, is transformed rather rapidly

in the plant rhizosphere. In agreement with this, the plant

availability of phosphorus from organic phosphates added

to soil has been frequently demonstrated. However, the

factors controlling the plant availability of soil Po are not

yet clear. Of special interest is the role of root phosphatases

and their genetic dependence (Helal 1990) in this context.

Because action of phosphatases, release inorganic phos-

phorus (Pi) from Po (Joner et al. 2000). Colonization by soil

arbuscular mycorrhizal (AM) fungi have shown to influence

phosphatase activity (Krishna et al. 1983). These AM fungi

are known to be a major microbial component of plant and

Communicated by W. Filek.

S. W. Khade � B. F. Rodrigues � P. K. Sharma

Department of Botany, Goa University,

Taleigao Plateau, Panaji 403206, Goa, India

B. F. Rodrigues

e-mail: [email protected]

P. K. Sharma


Present Address:S. W. Khade (&)

IInd Floor, Darshan Apts, Vidhyanagar Colony,

Carenzalem Post, Miramar, Panaji 403002, Goa, India


123

Acta Physiol Plant (2010) 32:565–574

DOI 10.1007/s11738-009-0433-x

soil ecology. They form highly evolved, mutualistic asso-

ciations with plant roots. The AM symbiosis, influences

several aspects of plant physiology such as mineral nutri-

tion, plant development and plant protection (Gianinazzi

et al. 1990). The AM fungi have direct influence on plant

growth because of their effect on soil structure stabilization,

i.e. soil aggregate formation and accumulation of humic

substances (Bethlenfalvay and Linderman 1992; Bethlen-

falvay and Schuepp 1994). The primary effect of AM

symbiosis is to increase the supply of mineral nutrients

particularly those whose ionic forms have a poor mobility

rate or those which are present in low concentration in the

soil solution. This mainly concerns phosphate, ammonium,

zinc and copper (Barea 1991). The interconnected network

of external hyphae acts as additional catchments and

absorbing surface in the soil beyond the depletion zone

that would otherwise remain inaccessible (Rhodes and

Gerdemann 1975).

The association between AM fungi and papaya has been

reported (Khade et al. 2002; Khade and Rodrigues 2008a,

b, 2009). Papaya belongs to family Caricaceae. The genus

Carica has about 48 species of which only Carica papaya

L. is grown for its edible fruits. This paper presents results

of preliminary work, where commercially available varie-

ties of papaya were screened for their root phosphatase

activities in relation to their AM status. The activity of the

enzyme phosphatase is a physiological characteristic rela-

ted to plant efficiency in relation to P acquisition and uti-

lization, and is genetically variable (Machado and Furlani

2004). Therefore, as part of a study on papaya genotype

characterization, experiments were set-up to measure

phosphatase activity in roots of four dioecious papaya

varieties and to investigate its AM status.

Materials and methods

Study site and management regimes

Papaya plantations in agricultural farm located at Old Goa

(North Goa) was selected for the study. Four dioecious

varieties viz., CO-1, CO-2, Honey Dew and Washington

were planted in monoculture. Papaya plantations were

managed conventionally. Generally, papaya seedlings are

raised in the nursery for 2 months and then they are

transplanted in the field. The early vegetative stage mainly

comprises of tap root system with feeble primary branches

making it difficult to collect root (feeder roots) samples for

mycorrhizal studies. Hence, the study was initiated during

late vegetative stage (here after referred to as ‘vegetative

stage’) when the plants were 6-month old and exhibited

extensive root system with ample of feeder roots during the

monsoons (Fig. 1a).

Collection of samples

During vegetative stage, two healthy plants per variety were

randomly selected for the collection of rhizosphere soil and

root samples. For each plant, three random soil cores were

collected from within 60 cm of each plant, at the depth of

0–25 cm. Sub-samples collected from both plants were then

combined to make composite sample after thorough mixing.

From this composite sample, five sub-samples were made

for quantification of spore density, two sub-samples for

nutrient analysis and two sub-samples for establishment of

pot cultures. Roots were packed separately for assay of

phosphatases and for estimation of root colonization per

variety in the field. Root samples collected for assay of

phosphatases were transported in ice to the laboratory.

These root samples (feeder roots) were washed in ice-cold

water and rinsed with double distilled water, packed in

polyethylene bags, labeled and stored at -80�C. For esti-

mation of root colonization, the root samples of both the

plants were combined. From this composite sample, five

sub-samples were made and they were utilized for estima-

tion of degree of root colonization and these root samples

were processed freshly. Sampling procedures were carried

out according to Tews and Koske (1986) except that the

core size was bigger (15 cm in diameter). This was carried

out to avoid non-normal distribution of spores recorded in

counts from small core samples (St. John and Koske 1988).

Soil sample analysis

Two rhizosphere soil samples per variety were employed

for analysis of nine edaphic factors during the vegetative

stage. Soil pH was measured in 1:2 soil water suspension

using pH meter (LI-120 Elico, India). Electrical conduc-

tivity was measured at room temperature in 1:5 soil sus-

pension using conductivity meter (CM-180 Elico, India).

Standard soil analysis techniques viz., micro-Kjeldahl

method (Jackson 1971) and Bray and Kurtz (1945) method

were employed for the determination of total nitrogen and

available phosphorus, respectively. Available potassium

was estimated by ammonium acetate method (Hanway and

Heidel 1952) using Flame Photometer (CL-361, Elico,

India). Available zinc, copper, manganese and iron were

quantified by DTPA–CaCl2–TEA method (Lindsay and

Norvell 1978) using Atomic Absorption Spectrophotometer

(AAS 4139, Electronic Corporation of India Ltd, India).

Establishment of pot cultures

Baiting of native AM fungi were carried out using open pot

cultures (Gilmore 1968). Two pot cultures were maintained

per variety. The roots of host species were checked for AM

colonization after 45 days. Pots showing successful

566 Acta Physiol Plant (2010) 32:565–574

123

mycorrhization were maintained for period of 6 months

and application of water was reduced at final 3 weeks to

maximize spore production (Menge 1982). At the end of

6 months, the plants were cut near the base and the cultures

were air-dried and checked for the presence of spores.

Spores isolated from pot cultures were used for verifying

the identification of AM fungi recovered from original field

samples.

Estimation of root colonization by AM fungi

Roots were cleared in 10% KOH, acidified in 1N HCl and

stained in 0.05% trypan blue in lactoglycerol (Phillips and

Hayman 1970). Total root colonization and length of the

root colonized by hyphae, arbuscules and vesicles by AM

fungi were estimated by modification of grid intersection

method (Mc Gonigle et al. 1990) and slide method

(Giovannetti and Mosse 1980). Stained root bits were

placed on the microscopic slides and the field of the view

was moved across the slide. The hairline graticule inserted

in the eyepiece acted as a line of intersection. According to

grid intersection method, at each intersection, presence of

hyphae, arbuscule and/or vesicle was noted. Hundred and

fifty intersections were examined per sample under com-

pound microscope at 2009 magnification and degree of

root colonization of AM fungi was expressed in percentage

Fig. 1 Scale bar denotes

50 lm. a Habitat of Caricapapaya L., b extraradial hyphae

(arrow) of AM fungi (9100),

c hyphal colonization of AM

fungi (9100), d hyphal coils of

AM fungi (9400), e a single

matured, arbuscule (arrow)

inside the host cell (91,000),

f vesicular colonization of AM

fungi (9100) [note the arrowpointing single vesicle]

Acta Physiol Plant (2010) 32:565–574 567

123

according to slide method. Five replicates were considered

per plant per variety

Assay of phosphatase enzyme activities

This experiment comprises two sets of observations with

five replications each per type of assay per variety.

Activities were assayed separately for acid phosphatase

and alkaline phosphatase. In all 40 readings, each was

recorded for acid and alkaline phosphatase, respectively.

One gram of root tissue was weighed per set. Methods for

quantification of activity of root phosphatases were stan-

dardized for papaya based on the procedures provided by

Kapoor et al. (1989) and Sukhada (1992).

Extraction of enzyme

Enzyme was extracted by macerating 1 g of detached root

tissue at 4�C using 20 ml of phosphate (0.1 M KH2PO4, pH

6.6) buffer. The homogenate was filtered through muslin

cloth and the filtrate was centrifuged at 5,000 rpm for 15 min

using a cooling centrifuge (Remy C-24, India). The super-

natant was stored at 4�C and further employed for protein

estimation of enzyme and assay of root phosphatases.

Protein estimation

Protein content of the enzyme extract was estimated by

Lowry’s method (Lowry et al. 1951) using bovine serum

albumin as standard.

Assay of root phosphatases

For alkaline root phosphatase activity, 5 lg protein equiv-

alent enzyme extract was incubated with 2 ml of 15 mM

p-nitrophenyl phosphate (pNPP) and 0.8 ml 0.25 M

Tris–HCl (buffer pH 9.8). For acid root phosphatase activity,

5 lg protein equivalent enzyme extract was incubated with

2 ml of 15 mM p-nitrophenyl phosphate (pNPP) and 0.8 ml

0.25 M sodium acetate (buffer pH 6.0). The reactions in both

the above-mentioned cases were terminated by adding 2 ml

of 1 N NaOH. For each set, T00 (zero time of incubation) and

T030 (30 min after incubation) were taken separately for

alkaline and acid root phosphatase activity. Optical density

was read at 410 nm using uv-1201 Shimadzu spectropho-

tometer (Japan) against a blank solvent (distilled water).

Root phosphatase activity was expressed in terms of n moles

of p-nitrophenol released per min per lg protein.

Quantification of spore density of AM fungi

Spores and sporocarps of AM fungi were isolated by wet

sieving and decanting method (Gerdemann and Nicolson

1963) and quantification of spore density of AM fungi was

carried out by method described by Gaur and Adholeya

(1994). Five replicates were considered per plant per

variety.

Identification of AM fungi

Diagnostic slides containing intact and crushed spores and

sporocarps of AM fungi were prepared in polyvinyl alcohol

lactoglycerol (Koske and Tessier1983). Spore morphology

and wall characteristics were considered for the identifi-

cation of AM fungi and these characteristics were ascer-

tained using compound microscope. The AM fungi were

identified to species level using manual identification of

AM fungi by Schenck and Perez (1990). Taxonomic

identification of spores was also carried out by matching

the descriptions provided by International Collection of

Vesicular-Arbuscular Mycorrhizal fungi (http://invam.

caf.wvu.edu) and Oehl et al. (2008).

Frequency of occurrence

Frequency of occurrence of AM fungi was calculated using

the following formula (Beena et al. 2000).

Relative abundance

Relative abundance of AM fungi was calculated using the

following formula (Beena et al. 2000).

Frequency ð%Þ ¼ Number of soil samples that possess spores of particular species

Total number of soil samples analyzed� 100:


123

http://invam.caf.wvu.edu

http://invam.caf.wvu.edu

Statistical analysis

Data on root colonization, spore density and root phos-

phatase activity was subjected to one-way analysis of

variance to investigate the variations in these parameters

with respect to four varieties of papaya. Before analysis of

variance, root colonization values were subjected to arcsine

transformations. In addition, multiple linear regression

analysis was carried out to determine the relationship

between root colonization and root phosphatase activity.

Data were statistically analyzed using Web Agri Stat

Package (WASP) 1.0.

Results

Edaphic factors

Data on edaphic factors are recorded in Table 1. The soil

pH was acidic, while electrical conductivity was ranging

from low to optimum. Available phosphorus recorded low

levels, whereas total nitrogen and potassium levels were

ranging from low to optimum. Micronutrients in general

were present in high concentrations.

Root colonization and spore density of AM fungi

All the root samples collected during the study period

exhibited the presence of AM colonization that was char-

acterized by the presence of extramatrical hyphae

(Fig. 1b), hyphae (Fig. 1c), hyphal coils (Fig. 1d), arbus-

cules (Fig. 1c, e) and vesicles (Fig. 1f). Data on root col-

onization of AM fungi in four papaya varieties are

presented in Fig. 2a. In the present study, CO-2 variety

recorded the lowest hyphal (13%), vesicular (2%) and total

(14.2%) root colonization of AM fungi. Although the

highest hyphal (22%) and vesicular (6%) colonization was

recorded in Honey Dew variety. The highest total root

colonization (30.6%) was recorded in Washington variety.

In the study, arbuscular colonization was recorded only in

Washington variety (6%) (Fig. 2a). The average hyphal,

arbuscular, vesicular and total root colonization recorded

during the study was 17.5, 2.45, 4.3 and 24.5%, respec-

tively. Further, the hyphal (CD = 3.488; P = 0.05),

arbuscular (CD = 1.202; P = 0.05), vesicular (CD =

2.599; P = 0.05) and total root colonization (CD = 4.067;

P = 0.05) of AM fungi varied significantly in four varieties

of papaya during vegetative stage.

Data on spore density of AM fungi in four papaya

varieties are presented in Fig. 2b. The lowest spore density

was recorded in Washington (8 spores 50 g-1 soil) and the

highest was recorded in CO-1 and Honey Dew (20 spores

50 g-1 soil) variety, respectively. The average spore den-

sity recorded during the study was 15.5 spores 50 g-1 soil.

Spore density of AM fungi also varied significantly within

the four varieties of papaya during vegetative stage

(CD = 25.961; P = 0.05).

Root phosphatase activity

Comparative account of alkaline and acid root phosphatase

activities in four papaya varieties is recorded in Table 2. In

the present study, Washington variety recorded the highest

root phosphatase (alkaline and acid) activity followed by

Honey Dew, CO-2 and CO-1 variety (Table 2). In addition,

in the present study, acid root phosphatase activity was

consistently higher as compared to alkaline root phospha-

tase activity in four papaya varieties (Table 2). The

Table 1 Comparative account of the edaphic factors in papaya varieties

Varieties aEdaphic factors

pH EC

(m mos cm-1)

Total N (%) Available P

(kg Ha-1)

Available K

(kg Ha-1)

Zn

(ppm)

Cu

(ppm)

Fe

(ppm)

Mn

(ppm)

CO-1 4.80 (0.218) 0.08 (0.001) 0.51 (0.002) 6.00 (0.861) 110.00 (5.486) 2.29 (0.000) 5.31 (0.088) 64.28 (0.451) 67.64 (1.337)

CO-2 4.90 (0.165) 0.40 (0.032) 0.35 (0.001) 4.00 (0.014) 129.00 (4.448) 1.27 (0.001) 4.31 (0.841) 22.94 (0.469) 97.41 (1.580)

Honey Dew 5.00 (0.222) 0.29 (0.035) 0.51 (0.000) 4.00 (0.028) 200.00 (3.231) 1.48 (0.002) 5.69 (0.457) 16.88 (0.587) 10.70 (1.452)

Washington 4.40 (0.234) 0.42 (0.033) 0.51 (0.008) 6.00 (0.027) 200.00 (4.520) 2.03 (0.003) 5.11 (0.358) 34.64 (0.521) 78.49 (1.741)

a Values presented are mean of two readings and values in parenthesis indicate ±SE

Relative abundance (% ) ¼ Number of AM fungal spores of particular species

Total number of AM fungal spores of all species� 100:


123

average alkaline and acid root phosphates activity recorded

was 5.92 (n moles of p-NPP released per min per lg pro-

tein) and 33.30 (n moles of p-NPP released per min per lg

protein), respectively. Both alkaline and acid root phos-

phatase activity varied significantly among four papaya

varieties (Table 2).

Relationship between stages of root colonization

and root phosphatase activities

Multiple regression analysis was carried out to study the

relationship between root colonization and root phospha-

tase activities. This analysis revealed that all the four root

colonization parameters viz., hyphal, arbuscular, vesicular

and total root colonization contributed to both alkaline and

acid root phosphatase activities. However, coefficient of

regression was significant only for acid root phosphatase

activity (Table 3).

Distribution of AM fungi

A total of 11 species of AM fungi belonging to five genera

viz., Acaulospora, Dentiscutata, Gigaspora, Glomus and

Racocetra were recovered from the rhizosphere of four

papaya varieties. They were Acaulospora mellea Spain &

Schenck, Glomus fasciculatum (Thaxter) Gerd. & Trappe

emend. Walker & Koske, Glomus sinuosum (Gerd. &

Bakshi) Almeida, Racocetra gregaria (Schenck & Nicol.)

Oehl, De Souza & Sieverding and Racocetra verrucosa

(Koske & C. Walker) Oehl, De Souza & Sieverding. The

remaining six species viz., Acaulospora myriocarpa Spain,

Sieverding & Schenck, Acaulospora spinosa, Walker &

Trappe (Fig. 3a, b), Gigaspora margarita Becker & Hall,

Glomus claroideum (Smith & Schenck) Vestberg &

Walker, Glomus coremioides (Berk. & Broome) Redecker

& Morton and Dentiscutata reticulata (Koske & Walker)

Sieverd., Souza & Oehl, were commonly occurring in the

four varieties of papaya. The frequency of occurrence and

relative abundance of commonly occurring species of AM

fungi of represented in Table 4. It was observed that Glo-

mus coremioides (Berk. & Broome) Redecker & Morton,

recorded the lowest frequency of occurrence (23%) and

minimum relative abundance (1.48), whereas the highest

frequency of occurrence (100%) and maximum relative

abundance (38.96) was reported in Acaulospora myrio-

carpa Spain. Dentiscutata reticulata (Koske & Walker)

Sieverd., Souza & Oehl also recorded the highest fre-

quency of occurrence (100%) with relative abundance of

17.26% (Table 4).

Discussion

The study recorded significant variations in degree of root

colonization by native AM fungi, in four varieties of

papaya. These results exhibited the differential preference

of AM fungi towards the papaya varieties. Similar results

were reported by Karangiannidis et al. (1997) in four grape

vine root stocks grown in an 8-year-old experimental

vineyard near Messimbria in Greece. In the present study,

out of the different stages of root colonization, total colo-

nization recorded the highest levels, followed by hyphal,

arbuscular and vesicular colonization, respectively. Thus,

the proportion of AM fungal structures inside the host

plant reflected to certain extent, the fungal growth stage

(Bonfante-Fasalo 1984). The present study also supports

Table 2 Comparative account of root phosphatase activities in

different papaya varieties

Variety Mean alkaline root

phosphatase activity

(n moles of p-NPP

released per min

per lg protein)a

Mean acid root

phosphatase activity

(n moles of p-NPP

released per min

per lg protein)a

CO-1 1.46 ± 0.53 9.62 ± 2.50

CO-2 1.88 ± 0.63 9.62 ± 2.50

Honey Dew 7.86 ± 2.69 24.71 ± 3.21

Washington 8.35 ± 0.72 49.19 ± 2.61

Average 5.92 ± 1.81 33.30 ± 6.26bCDP = 0.05 2.53 6.15

a Values presented are mean of ten readings and values indicate ±SEb F test significant at 0.05 level of probability

(a)

0

5

10

15

20

25

30

35

CO-1 CO-2 Honey dew Washington

Varieties

Ro

ot

colo

niz

atio

n (

%)

Hyphal Arbuscular Vesicular Total

(b)

0

5

10

15

20

25

30

CO-1 CO-2 Honey Dew WashigtonVarieties

Sp

ore

den

sity

/ 50

g s

oil

Fig. 2 Root colonization (a) and spore density (b) of AM fungi

associated with papaya varieties. Error bar indicates ±SE


123

the contention that root colonization is genetically con-

trolled (Kesava Rao et al. 1990; Mercy et al. 1990; Raju

et al. 1990). Throughout the study, feeder roots were

sampled because they generally reported greater incidence

and intensity of colonization than the main root axis

(Mosse 1975; Smith and Walker 1981). In present study,

although the spore density of AM fungi exhibited narrow

fluctuation, it varied significantly in four papaya varieties.

Similar results were reported by Karangiannidis et al.

(1997) in four grape vine root stocks grown in an 8-year-

old experimental vineyard near Messimbria in Greece.

The present study was carried out during the monsoons

that support the findings of Clarholm and Rosengren-

Brinck (1995) who reported that estimations of acid

phosphatase made during moist soil conditions were much

more informative than those made during dry conditions.

Further, in present the study, acid root phosphatase activity

and alkaline root phosphatase activity varied significantly

within different varieties of papaya under P-deficient,

acidic (pH 4.4–5.0) soil conditions. Similarly, Helal (1990)

reported that root phosphatase activity is pH-dependent and

varietal differences in root phosphatase activities are

especially evident in the more acid range (pH 4–5) and

diminish at higher pH (6). Furthermore, Helal (1990)

reported that the pronounced pH dependency of root

phosphatase activity is indicative of the significance of the

rhizosphere pH not only for the availability of inorganic

phosphorus, but also for the activity of root enzymes and

the related turnover of organically bound plant nutrients.

The practical significance of root phosphatase for the

nutritional efficiency of plants under field conditions is not

yet clear. However, the results are indicative that plant

roots with high phosphatase activity obviously have the

potential to utilize soil organic phosphorus. Contradictory

to the findings of present study, Rubio et al. (1990) in a

greenhouse pot experiments conducted at the Universidad

de la Frontera, Tamuco, Chile, reported that root surface

acid phosphatase activity did not vary significantly in

wheat cultivars grown in the typical low phosphorus vol-

canic soils.

The present study reported consistently higher acid root

phosphatase activity when compared with alkaline root

phosphatase activity in four papaya varieties under

P-deficient, acidic soil conditions. Therefore, our study

Fig. 3 Scale bar denotes 50 lm. a Spore of Acaulospora spinosaWalker & Trappe laterally attached to hyaline sporiferous saccule.

b A portion of spore wall of Acaulospora spinosa Walker & Trappe

with spines

Table 4 Frequency of occurrence and relative abundance of AM

fungi common to four papaya varieties

AM fungal species Frequency of

occurrence (%)

Relative

abundance (%)

Acaulospora spinosa 50 2.96

Acaulospora myriocarpa 100 38.96

Gigaspora margarita 50 11.85

Glomus claroideum 62.50 2.90

Glomus coremioides 25 1.48

Dentiscutata reticulata 100 17.26

Table 3 Relationship between root phosphatase activity (Y) and root colonization of AM fungi (X) in papaya varieties

Root phosphatase activity (n moles of p-NPP

released per min per lg protein)

Equations Regression

coefficient (R2)a

Alkaline Y = -5.12 - 0.33H - 0.53 A ? 0.47V ? 1.09T 0.373

Acid Y = 3.75 ? 0.10H ? 1.84A ? 0.83V - 1.73T 0.747a

T total root colonization and root length colonized by H hyphae, A arbuscules, and V vesiclesa Regression significant at 0.05 level of probability


123

upholds the view that one of the initial responses of plants

to P deficiency stress is an increase in root acid phospha-

tase activity (Goldstein et al. 1988, Duff et al. 1994)

because its function is the hydrolysis of inorganic phos-

phorous (Pi) from orthophosphate monoesters used for

plant nutrition in the soil (Huttova et al. 2002). Further, the

findings of the present study is in accordance with Sukhada

(1992) who reported higher acid phosphatase activity in

comparison to alkaline phosphatase activity in roots of

4-month-old papayas inoculated with Glomus mosseae

(Nicol. & Gerd.) Gerd. & Trappe and Glomus fasciculatum

(Thaxt.) Gerd. & Trappe in acidic soil conditions. Similarly

Krishna et al. (1983) reported higher acid root phosphatase

activity when compared with alkaline root phosphatase

activity in different growth stages of Arachis hypogea L.

inoculated with Glomus fasciculatum (Thaxt.) Gerd. &

Trappe under P-deficient, acidic soil conditions. Garcıa-

Gomez et al. (2002) also reported increased acid root

phosphatase activity in papaya plants inoculated with

Glomus claroideum as compared to non-mycorrhizal

plants. Contradictory to this, Allen et al. (2006) reported

that Bouteloua gracilis colonized with Glomus fascicula-

tum and grown in the phytate medium had substantially

higher alkaline phosphatase activity than non-mycorrhizal

plants and that acid phosphatase activity was not affected

by mycorrhizal condition.

Further, it is evident from multiple regression analysis

that the root colonization of AM fungi influenced acid and

alkaline root phosphatase activity positively. However,

acid root phosphatase activity was significantly influenced

by root colonization by AM fungi, while alkaline root

phosphatase activity was marginally influenced by root

colonization of AM fungi. Similarly, McArthur and

Knowles (1993) reported that the specific activities of root

acid phosphatases were enhanced in P-deficient potato

(Solanum tuberosum L.) plants inoculated by Glomus fas-

ciculatum [Thaxt. sensu Gerdemann] Gerdemann and

Trappe). Here, the establishment of AM symbiosis by low-

P plants was essential for efficient P acquisition and a

greater root colonization levels for P-stressed plants indi-

cated increased compatibility to the AM fungus. However,

the AM fungus only, partially alleviated P deficiency stress

and did not completely compensate for inadequate abiotic

P supply. In another studies conducted at the Department of

Botany and Plant Pathology, Michigan State University, E

Lansing, USA, maize (Zea mays cv. Great Lakes 586)

plants were grown under five different levels of soil

phosphorus, either in the presence or absence of formo-

nonetin or the VAM fungus, Glomus intraradices. Here,

the ACP (acid phosphatase) and ALP (alkaline phospha-

tase) activities were closely related to the level of fungal

colonization in maize roots. Acid phosphatase activity in

maize roots responded more to soil phosphorus availability

than ALP activity (38% more). These results suggest that

ACP was involved in the increased uptake of phosphorus

from the soil, while ALP may be linked to active phosphate

assimilation or transport in mycorrhizal roots. Thus, soil

phosphorus directly affected a number of enzymes essen-

tial in host–endophyte interplay (Fries et al. 1998). These

findings of Fries et al. (1998) explains the results of the

present study that recorded higher acid phosphatase activ-

ity in papaya roots when compared with alkaline phos-

phatase activity under P-deficient, acidic soil conditions.

The study recorded eleven species of AM fungi in

association with four papaya varieties. The AM fungal

species belonging to genus Glomus were the most repre-

sentative type. Similarly, maximum numbers of Glomus

species were found to be associated with papayas (Khade

and Rodrigues 2008a, b, 2009), medicinal plants (Khade

et al. 2002), pteridophytes (Khade and Rodrigues 2002) and

forest tree species (Khade and Rodrigues 2003) of Goa,

India. In the present study, few species were frequently

occurring and abundant when compared with other species

of AM fungi. Thus, the occurrence of one species sporu-

lating at the expense of others may be regulated by factors,

such as interspecific competition, spatial restriction and/or

edaphic factors (Gemma et al. 1989). Allen et al. (2003)

have pointed out that AM fungal root colonization is

mediated by interspecific fungal interactions, such as

competition, antagonism and dominance. The results from

other studies (Koske 1987; Sylvia 1986) suggest that indi-

vidual AM fungal species compete for resources through a

combination of strategies resulting in the maintenance of a

diverse AM fungal community. Therefore, a possible

explanation for the patterns found in our experiment is the

competitive interaction among AM fungal species (Schal-

amuk et al. 2006). Open pot culture technique in present

study was not successful for sporocarpic species of Glomus

that were placed earlier under genus Sclerocystis as they

failed to produce new sporocarps. This is accordance with

the findings of Muthukumar and Udaiyan (2002).

In conclusion, production of root phosphatase enzymes

specifically acid root phosphatase are inducible under

P-deficient, acidic soil conditions and this activity consid-

erably varies among four papaya varieties. The study

highlighted considerable varietal differences in root colo-

nization and spore density of AM fungi associated with

papayas. The higher acid and alkaline root phosphatase

activity along with higher total root colonization values in

Honey and Washington variety proves high P demand of

these varieties when compared to CO varieties. The present

study reported consistent higher acid root phosphatase

activity as compared to alkaline root phosphatase activity

in four papaya varieties under P-deficient, acidic soil

conditions. Therefore, the study emphasizes the fact that

tropical soils are acidic and, therefore, P-deficient that


123

induces the secretion of root phosphatase activity, espe-

cially high amount of acid phosphatase that results in the

availability of phosphorus and is, therefore, taken up by

extraradical fungal hyphae. This P is translocated passively

by intra-radical fungal hypha or actively transferred to the

plant through arbuscules; the key feature of AM symbiosis.

The study, therefore, shows the relationship between the

root length colonized by hyphae, arbuscules, vesicles and

total root colonization of symbiotic AM fungi with root

phosphatase activity and highlights the fact that under

acidic soil conditions, acidic root phosphatase activity

increases with the root colonization of AM fungi. The

present study has new approach since it was carried out

under field conditions and under the influence of all envi-

ronmental pressures that to in monsoons and still exhibited

a consistency in the findings of data pooled from four

varieties, which were generalized to logical conclusions.

Acknowledgments Shri Waman M. Khade Ex-director of Agri-

culture Department and Directorate of Agriculture, State Government

of Goa are thanked for their assistance to carry out research work.

References

Allen EB, Swenson W, Querejeta JI, Egerton-Waburton LM, Treseder

KK et al (2003) Ecology of mycorrhizae: a conceptual frame-

work for complex interactions among plants and fungi. Ann Rev

Phytopathol 41:271–303

Allen MF, Sexton JC, Moore TS Jr, Christensen M et al (2006)

Influence of phosphate source on vesicular-arbuscular mycor-

rhizae of Bouteloua gracilis. New Phytol 87(4):687–694

Barea JM (1991) Vesicular-arbuscular mycorrhizae as modifiers of

soil fertility. In: Stewart BA (ed) Advances in soil science.

Springer, New York, pp 1–40

Beena KR, Raviraja NS, Arun AD, Sridhar KR et al (2000) Diversity

of arbuscular mycorrhizal fungi on coastal sand dunes of the

West Coast of India. Curr Sci 79(10):1459–1465

Bethlenfalvay GJ, Linderman RG (1992) Mycorrhizae in sustainable

agriculture. ASA Special Publication, Madison, p 124

Bethlenfalvay GJ, Schuepp H (1994) Arbuscular mycorrhizas and

agrosystem stability. In: Gianinazzi S, Scheupp H (eds) Impact

of arbuscular mycorrhiza on sustainable agriculture and natural

ecosystems. ALS, Birkhauser, Basel, pp 117–131

Bonfante-Fasalo P (1984) Anatomy and morphology. In: Powell CL,

Bagyaraj DJ (eds) VA mycorrhiza. CRC Press Inc, Boca Raton,

pp 5–33

Bray RH, Kurtz LT (1945) Determination of total organic carbon and

available forms of phosphorus in soils. Soil Sci 59:39–45

Chauhan BS, Stewart JWB, Paul EA (1981) Effect of labile inorganic

phosphate status and organic carbon additions on the microbial

uptake of phosphorus in soils. Can J Soil Sci 61:373–385

Clarholm M, Rosengren-Brinck U (1995) Phosphorous and nitrogen

fertilization of a Norway spruce forest-effect on needle concen-

trations and acid phosphatase activity in the humus layer. Plant

Soil 175:239–249

Duff SMG, Sarath G, Plaxton WC (1994) The role of acid

phosphatase in plant phosphorus metabolism. Physiol Plant

90:791–800

Fries LLM, Pacovsky RS, Safir GR, Kaminski J et al (1998)

Phosphorus effect on phosphatase activity in endomycorrhizal

maize. Physiol Plant 103(2):162–171

Garcıa-Gomez R, Chavez-Espinosa J, Mejıa-Chavez A, Duran BC

et al (2002) Short term effects of Glomus claroideum and

Azospirillum brasilense on growth and root acid phosphatase

activity of Carica papaya L. under phosphorus stress. Rev

Latinoam Microbiol 44(1):31–37

Gaur A, Adholeya A (1994) Estimation of VAM spores in the soil—a

modified method. Mycorrhiza News 6(1):10–11

Gemma JN, Koske RE, Carreiro M et al (1989) Seasonal dynamics of

selected species of VA mycorrhizal fungi in a sand dune. Mycol

Res 92:317–321

Gerdemann JW, Nicolson TH (1963) Spore density of Endogonespecies extracted from soil wet sieving and decanting. Trans Bri

Mycol Soc 46:235–244

Gianinazzi S, Trouvelot A, Gianiazzi-Pearson V et al (1990) Role and

use of mycorrhizas in horticulture crop production. XXII.

International Horticulture Congress Florence, pp 25–30

Gilmore AE (1968) Phycomycetous mycorrhizal organisms collected

by open pot cultures. Hilgardia 39:87–105

Giovannetti M, Mosse B (1980) An evaluation of techniques for

measuring vesicular arbuscular mycorrhizal infection in roots.

New Phytol 84:489–500

Goldstein AH, Baertlein DA, Mcdaniel RG (1988) Phosphate

starvation inducible metabolism in Lycopersicon esculentum. I.

Excretion of acid phosphatase by tomato plants and suspension-

cultured cells. Plant Physiol 87:711–715

Hanway JJ, Heidel H (1952) Soil analysis method as used in

Iowa State College Soil Testing Laboratory. Iowa Agric

57:1–31

Helal HM (1990) Varietal differences in root phosphatase activity as

related to the utilization of organic phosphates. Plant Soil

123:161–163

Helal HM, Sauerbeck DR (1984) Influence of plant roots on carbon

and phosphorus metabolism in soil. Plant Soil 76:175–182

Helal HM, Sauerbeck DR (1987) Direct and indirect influences of

plant roots on organic matter and phosphorus turnover in soil.

INTECOL Bull 15:49–58

Huttova J, Tamas L, Mistrık I (2002) Aluminum induced acid

phosphatase activity in roots of Al-sensitive and Al-tolerant

barley varieties. Rostlinna Vyroba 48(12):556–559

Jackson ML (1971) Soil chemical analysis. Prentice Hall, New Delhi

Joner EJ, Van Aarle IM, Vosatka M et al (2000) Phosphatase activity

of extraradical arbuscular mycorrhizal hyphae: a review. Plant

Soil 226:199–210

Kapoor A, Singh VP, Mukerji KG (1989) Studies on the phosphatase

of mycorrhizal and non mycorrhizal Trigonella roots. In:

Mahadeva A, Raman A, Natarajan K et al (eds) Mycorrhizae

for Green Asia. CAS, Madras, pp 125–127

Karangiannidis N, Velmis D, Stravropoulos N et al (1997) Root

colonization and spore population by VA-mycorrhizal fungi in

four grapevine rootstock. Vitis 36(2):57–60

Kesava Rao PS, Tilak KVBR, Arunachalam V et al (1990) Genetic

variation of mycorrhiza-dependent phosphate mobilization in

ground nut (Arachis hypogea L.). Plant Soil 121:291–294

Khade SW, Rodrigues BF (2002) Arbuscular mycorrhizal fungi

associated with some pteridophytes from Western Ghat region of

Goa. Trop Eco 43(2):251–256

Khade SW, Rodrigues BF (2003) Occurrence of arbuscular mycor-

rhizal fungi in tree species from Western Ghats of Goa India.

J Trop For Sci 15(2):320–331

Khade SW, Rodrigues BF (2008a) Ecology of arbuscular mycorrhizal

fungi associated with Carica papaya L. in agro-based ecosystem

of Goa, India. Trop Subtrop Agroecosyst 8:265–278


123

Khade SW, Rodrigues BF (2008b) Spatial variations in arbuscular

mycorrhizal (AM) fungi associated with Carica papaya L. in a

tropical agro-based ecosystem. Bio Agric Hort 26:149–174

Khade SW, Rodrigues BF (2009) Arbuscular mycorrhizal fungi

associated with varieties of Carica papaya L. in tropical agro-

based ecosystem of Goa, India. Trop Subtrop Agroecosyst

10(3):369–381

Khade SW, Bukhari MJ, Jaiswal V, Gaonkar UC, Rodrigues BF et al

(2002) Arbuscular mycorrhizal status of medicinal plants: a field

survey of AM fungal association in shrubs and trees. J Eco Tax

Bot 26(3):571–578

Koske RE (1987) Distribution of VA mycorrhizal fungi along a

latitudinal temperature gradient. Mycologia 79:55–68

Koske RE, Tessier B (1983) A convenient permanent slide mounting

medium. Mycol Soc Am Newslett 34:59

Krishna KR, Bagyaraj DJ, Papavinashasundaram KG et al (1983)

Acid and alkaline phosphatase activities in mycorrhizal and

uninfected roots of Arachis hypogaea L. Ann Bot 51:551–553

Lindsay WL, Norvell WA (1978) Development of DPTA soil test for

zinc, iron, manganese and copper. Am Soil Sci Soc J 42:421–488

Lowry OH, Rosebrough NJ, Fan AL, Randall RJ et al (1951) Protein

measurement with the Folin-phenol reagent. J Biol Chem

193:265–275

Machado CTT, Furlani AMC (2004) Root phosphatase activity, plant

growth and phosphorus accumulation of maize genotypes. Sci

Agric 61(2):216–223

Mc Gonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA et al

(1990) A new method which gives an objective measure of

colonization of roots by vesicular-arbuscular mycorrhizal fungi.

New Phytol 115:495–501

McArthur DAJ, Knowles NR (1993) Influence of vesicular-arbuscular

mycorrhizal fungi on the response of potato to phosphorus

deficiency. Plant Physiol 101(1):147–160

McLachlan KE (1980) Acid phosphate activity of intact roots and

phosphorous nutrition in plants II: variation among wheat roots.

Aust J Agric Res 31:441–448

Menge JA (1982) Utilization of vesicular arbuscular mycorrhizal

fungi in agriculture. Can J Bot 61:1015–1024

Mercy MA, Shivashanker G, Bagyaraj DJ et al (1990) Mycorrhizal

colonization in cowpea is dependent and heritable. Plant Soil

121:291–294

Mosse B (1975) A microbiologist’s view of root anatomy. In: Walker

N (ed) Soil microbiology: a critical review. Butterworths,

London, pp 39–66

Muthukumar T, Udaiyan K (2002) Arbuscular mycorrhizal fungal

composition in semi-arid soils of Western Ghats, southern India.

Curr Sci 82(6):625–628

Oehl F, de Souza FA, Sieverding E (2008) Revision of Scutellosporaand description of five new genera and three new families in the

arbuscular mycorrhiza-forming Glomeromycetes. Mycotaxon

106:311–360

Phillips JM, Hayman DS (1970) Improved procedure for clearing

roots and staining of mycorrhizal fungi for rapid assessment of

infection. Trans Bri Mycol Soc 55:158–161

Raju PS, Clark RB, Duncan JR, Maranville JW et al (1990) Benefit

and cost analysis and phosphorus efficiency of VA-mycorrhizal

fungi colonization with Sorghum (Sorghum bicolor) genotypes

grown at varied phosphorus levels. Plant Soil 124:199–204

Rhodes LH, Gerdemann JW (1975) Phosphate uptake zones of

mycorrhizal and non-mycorrhizal onions. New Phytol 75:555–

561

Rubio R, Moraga E, Borie F et al (1990) Acid phosphatase activity

and vesicular-arbuscular mycorrhizal infection associated with

roots of 4 wheat cultivars. J Plant Nutr 13(5):585–598

Schalamuk S, Velazquez H, Cabello CM et al (2006) Fungal spore

diversity of arbuscular mycorrhizal fungi associated with spring

wheat: effects of tillage. Mycologia 98(1):16–22

Schenck NC, Perez Y (1990) Manual for identification of VA

mycorrhizal fungi. INVAM, University of Florida, Gainesville,

USA, pp 1–283

Smith SE, Walker NA (1981) A qualitative study of mycorrhizal

plants in Trifolium: separate determination of the rates of

infection and of mycelial growth. New Phytol 89:225–240

Speir TW, Ross DJ (1978) Soil phosphatase and sulphatase. In: Burns

RG (ed) Soil enzymes. Academic Press, New York, USA,

pp 197–250

St. John TV, Koske RE (1988) Statistical treatment of endogonaceous

spore counts. Trans Bri Mycol Soc 91:117–121

Sukhada M (1992) Effect of VAM inoculation on plant growth,

nutrient level and root phosphatase activity in papaya (Caricapapaya cv. Coorg Honey Dew). Fert Res 31:263–267

Sylvia DM (1986) Spatial and temporal distribution of vesicular-

arbuscular mycorrhizal fungi associated with Uniola paniculatain Florida foredunes. Mycologia 78:728–734

Tews LL, Koske RE (1986) Towards a sampling strategy for vesicular

arbuscular mycorrhizas. Trans Bri Mycol Soc 87(8):353–358


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