ORIGINAL PAPER

Screening and selecting arbuscular mycorrhizal fungi forinoculating micropropagated apple rootstocks in acid soils

Jose Renato Pereira Cavallazzi ÆOsmar Klauberg Filho Æ Sidney Luiz Sturmer ÆPaul T. Rygiewicz Æ Margarida Matos de Mendonca

Received: 8 March 2006 / Accepted: 5 September 2006 / Published online: 17 July 2007

� Springer Science+Business Media B.V. 2007

Abstract Santa Catarina state is the largest pro-

ducer of apples in Brazil. Soils in this region have

low pH and high levels of aluminum and manga-

nese, requiring high inputs of fertilizers and

amendments increasing costs of apple production.

Inoculation of arbuscular mycorrhizal fungi can

improve the establishment of micropropagated

apple plants in such adverse soil conditions. Soil

samples were collected from apple orchards in the

Cacador, Fraiburgo and Sao Joaquim regions to

develop a corn bioassay to identify mycorrhizal

communities with high infectivity. Eleven fungal

species were identified from one Cacador soil with

the highest infectivity. Glomus etunicatum SCT110,

Scutellospora pellucida SCT111, Acaulospora scro-

biculata SCT112 and Scutellospora heterogama

SCT113 were brought into single-species culture

and used in a plant growth and nutrient uptake

experiment using micropropagated apple (Malus

prunifolia), cultivated at three soil pH. Colonization

by fungal isolates significantly affected plant

height, shoot and root dry weights, and root:shoot

ratio. Soil pH also significantly affected all growth

parameters except shoot dry weight. Mycorrhizal

inoculation also significantly altered tissue concen-

trations of P, Zn, Cu, Ca, S, Na, N, K, Fe and Al.

Association with mycorrhizal fungi increased P

concentration and also decreased Al concentrations

in the shoots. Overall, G. etunicatum and S.

pellucida were the most effective isolates to

promote plant growth and nutrient uptake.

Inoculation of apple rootstock with selected fun-

gal isolates during the acclimatization stage

represents a useful strategy for producing micro-

propagated apples that can withstand acidic soil

conditions.

Keywords Acid soils � Aluminum � Brazil �Micropropagated apple � Mycorrhizae �Plant–microorganisms interaction �Nutrient content � Plant growth

J. R. P. Cavallazzi � M. M. de Mendonca (&)

Departamento de Microbiologia e Parasitologia,

Universidade Federal de Santa Catarina, Cx. P. 476,

Florianopolis, SC, 88040-900, Brazil

e-mail: margarid1@terra.com.br

O. K. Filho

Departamento de Solos, Universidade do Estado de Santa

Catarina, Cx. P. 281, Lages, SC, 88520-000, Brazil

S. L. Sturmer

Departamento de Ciencias Naturais, Universidade

Regional de Blumenau, Cx. P. 1507,

Blumenau, SC, 89010-971, Brazil

P. T. Rygiewicz

Western Ecology Division, National Health and

Environmental Effects Research Laboratory, U.S.

Environmental Protection Agency, Corvallis,

OR, 97333, USA

123

Plant Cell Tiss Organ Cult (2007) 90:117–129

DOI 10.1007/s11240-006-9163-6

Introduction

The State of Santa Catarina is the largest producer of

apples in Brazil, with an annual production of

750,000 tons, representing ca 53% of the country’s

production (ICEPA/SC 1999). In the 1970s, apples

ranked as the third most important agricultural

product in the country. Consequently, areas support-

ing apple production were greatly increased due to

governmental subsidies. This was especially the case

in Santa Catarina. Brazil reached self-sufficiency in

apple production in 1990, and today produces an

excess of apples, which are exported to Europe and

North America. During the period of rapid expansion

of apple orchards, demand for rootstock was higher

than production rates. As a result, reproductive

material of inferior health was used which contrib-

uted to high rates of viral diseases that caused

reduced orchard productivity leading to extensive

replanting of orchards. In Santa Catarina, most

orchards and apple nurseries are located on soils

with low pH and high aluminum levels (Basso and

Suzuki 1992). Plant growth in these soils is limited by

several factors including high concentrations of Mn

and H and low solubilities of P, Mg and Ca

(Marschner 1991). As a consequence, large inputs

of fertilizers and lime are required, making apples an

agricultural product in the state having among the

highest costs of production.

It is well known that the endomycorrhizal associ-

ation, established by arbuscular mycorrhizal fungi

(AMF) colonizing roots, promotes growth and nutri-

tion of plants by increasing uptake of minerals from

soil and protects plants against potentially phytotoxic

elements, e.g., Cd and Pb (Weissenhorn and Leyal

1995; Weissenhorn et al. 1995). This type of

mycorrhizal fungi occurs naturally in soils and

colonizes the roots of the vast majority of plant

species. Colonization by AMF in acidic soils

increases plant biomass production (O’Donnell

et al. 1992; Soedarjo and Habte 1993) and uptake

of P (Habte and Soedarjo 1996), K and Mg (Borie

and Rubio 1999). The endomycorrhizal association

also decreases uptake of toxic elements such as Cd

(El-Kherbawy et al. 1989), Mn (Arines et al. 1989)

and Al (Borie and Rubio 1999), reducing plant tissue

concentrations of these elements.

Several reports have shown that the arbuscular

endomycorrhizal association is more efficient in

promoting growth of apple plants than is applying

fertilizers only. Inoculation with AMF isolates

resulted in mean increases in plant height and

biomass of 735 and 1,161%, respectively, compared

with non-inoculated plants (Geddeda et al. 1984).

The authors also observed a negative correlation

between soil P levels and root colonization rates,

reflecting the consequences of adding fertilizers to

the soil. In a P fertilizer experiment, Miller et al.

(1985) inoculated apple plants with one of six

isolates of Glomus or an isolate of Gigaspora, and

found that only the plants growing in soils not

receiving the P fertilizer treatment were responsive

to the mycorrhizal association. Plenchette et al.

(1981) inoculated apple (Malus pumila) in sterile

soils (pH 4.7) and demonstrated that mycorrhizal

plants were significantly taller and more uniform in

height compared with non-inoculated plants. Dry

mass of the stem and leaves, and the leaf surface

area per plant of the mycorrhizal plants increased

335, 204 and 153%, respectively, compared with

non-inoculated plants.

In vitro micropropagation can be used to

produce pathogen-free and, especially, virus-free

apple plantlets while also achieving increases in

scale and speed of production (Grattapaglia and

Machado 1991). However, several problems limit

the widespread use of micropropagated plants.

Upon transfer of plantlets from in vitro to post

vitro conditions, high mortality frequently occurs

since the photosynthetic capability needs to im-

prove and root systems need to develop further

(George 1996). This conditioning of the plants to

new growth conditions leads to an extended

weaning stage that is often accompanied by high

losses of plantlets, and large inputs of fertilizers,

pesticides and other chemicals. Additionally, woody

plants can pose a particular problem in that the root

systems produced in vitro may not develop further

leading to plantlet death or severe reductions in

growth (George 1996). This characteristic might

limit commercial production of some genotypes of

apples. Sterilized substrates used during plantlet

acclimation impair establishment of the symbiotic

association, causing problems for survival and

118 Plant Cell Tiss Organ Cult (2007) 90:117–129

123

growth of seedlings after transplant (Ravolanirina

et al. 1989). Branzanti et al. (1992) observed that

AMF colonization yielded micropropagated apple

plants significantly more uniform and taller than

those plants growing in fertilized substrates.

Granger et al. (1983) observed that clones of apple

Malling 7 micropropagated and inoculated with

Glomus epigaeum produced higher biomass and had

higher P and Cu concentrations when compared

with non-mycorrhizal plants. The endomycorrhizal

association also increased tolerance of microprop-

agated apples to several stresses during rooting and

acclimation stages, reducing the time necessary to

produce seedlings (Uosukainen and Vestberg 1994).

However, the majority of studies involving micro-

propagated apples and AMF used fungal isolates

originating from areas with varying soil conditions

supporting different plant communities, paying little

attention to controlling or assessing soil conditions

such as pH.

Several factors influence the efficiency of the

endomycorrhizal association, e.g., genotype of the

fungal isolate (Uosukainen and Vestberg 1994),

plant host species (Branzanti et al. 1992; Guillemin

et al. 1992) and soil physical, chemical and biotic

properties (Habte and Soedarjo 1996). Therefore,

targeted and specific screening and selecting of

AMF isolates should precede large-scale inoculation

programs and need to consider the host plant

species and the specific environmental conditions

under which the inoculated plants will be grown.

The work we report here is part of a larger project

which had the objective of selecting appropriate

technologies to produce healthy propagated apple

rootstock that would be adapted to the acidic soil

conditions found in Santa Catarina. A multidisci-

plinary approach was undertaken, encompassing

microbiological- and plant-based strategies. The

specific aim of the research was to integrate the

use of AMF isolates and the techniques of micro-

propagation to produce apple rootstock that would

tolerate transplanting into acidic soil conditions. If

successful, the integrated approach could lead to

producing large amounts of plant material that

would be readily available to nurseries. Specifi-

cally, we strove to define a strategy to recover

native AMF isolates from orchard soils and assess

the inoculation of the most competitive species/

isolates on the growth of micropropagated apple

rootstocks, using sterilized substrates adjusted to

pH values likely to be found in large-scale

production operations.

Methods and materials

Sampling sites

The study was conducted in three areas producing

apple in the plateau region (900–1,500 m) in the

central part of the state of Santa Catarina, South

Brazil (Table 1). The climate is Koeppen type Cfb

(i.e., mesothermic humid with cool summers and

harsh winters), with precipitation evenly distributed

throughout the year in the form of rain (annual total:

1,400–1,800 mm). Mean annual temperature ranges

from 14 to 18�C, and relative humidity ranges from

75 to 85%. Soils are often acidic and have high

aluminum content.

In each of the two orchards in each of the three

apple producing areas, ten trees were selected from

under which four soil samples (500 g each) were

taken and combined, resulting in ten samples per

orchard. The four sub-samples were collected from

the soil surface to 20 cm depth, at 20–40 cm from the

stem with one sub-sample coming from each of the

four cardinal directions. Orchards were maintained

free of weeds and, therefore, no other plants were

found growing near each sampled apple tree. Soil was

collected exclusively from around apple feeder roots

that were detected by following a main root. Each

pooled sample was placed in a plastic bag and then

stored at 4�C before processing.

Mycorrhizal infectivity

A corn bioassay was used to estimate mycorrhizal

infectivity of the soil from each orchard (Moorman

and Reeves 1979). Soil samples from each orchard

were homogenized, mixed (1:1, vol:vol) with

washed, sterilized sand and then the mixture placed

in 0.5 l pots. One 6-day-old seedling of hybrid corn

(Agroeste—AS 532), pre-germinated in sterilized

sand was transplanted to each pot. Six pots were set

up for each orchard and sampled 40 days after

transplanting. Corn roots were gently separated from

the soil, washed and stained according to Koske and

Plant Cell Tiss Organ Cult (2007) 90:117–129 119

123

Gemma (1989). Roots were scored for percent

mycorrhizal colonization following Giovannetti and

Mosse (1980) and the value used as an estimate of

mycorrhizal infectivity in each orchard.

AMF species diversity and establishing fungal

isolates

Only soil samples from the orchard having the

greatest mycorrhizal infectivity were used to assess

species diversity associated with apples and to

establish fungal isolates in pure culture. Spores were

recovered from 50 g of soil from each of the ten

pooled samples by wet sieving (Gerdemann and

Nicolson 1963) followed by centrifugation in a 50%

sucrose solution. Spores were grouped into morpho-

types, mounted in PVLG (polyvinyl-lacto-glycerol)

and Melzer’s reagent and then identified either

according to original species description protocols

or reference cultures described on the International

Culture Collection of Arbuscular Mycorrhizal Fungi

web site (http://invam.caf.wvu.edu).

Trap cultures were set up according to Stutz and

Morton (1996), by mixing field soil (1:1, vol:vol)

with sterilized sand. The mixture was placed in 1.5 kg

pots; seeds (50–60) of Sorghum bicolor L. were sown

in each pot and then covered with 1 cm sterilized

vermiculite. After 4 months in the greenhouse, three

100 g sub-samples of each pot were collected to

isolate and identify spores as described above.

Pure cultures of AMF were obtained following

procedures of Morton et al. (1993). Fresh spores

obtained from trap cultures were separated into

morphotypes using a dissecting microscope. Spores

of each morphotype were inoculated onto the root

systems of germinated Sorghum bicolor and Pasp-

alum notatum. Host plants were grown in a sterilized

mixture of sand and a red–yellow spodosol (2:1,

vol:vol), maintained in the greenhouse and watered

with distilled water. After 4 months, watering was

stopped, and plants and the planting mix were

allowed to dry in situ for 4–10 days. Spores were

extracted from the planting mix as described above

and fungal cultures were checked for purity. Suffi-

cient inocula, derived from the single-species cul-

tures, were produced to use in the plant growth

experiment. The volumes of inocula were increased

by mixing the soil inoculum from each single culture

with sterilized substrate (sand: red–yellow spodosol,

1:1, vol:vol). Each inoculum mixture was placed in

1.5 kg pots, seeded with Sorghum and Paspalum, and

maintained in the greenhouse. Pots filled only with

sterilized substrate and seeded with both hosts as

described above served as the non-inoculated control

treatment. After 4 months, plant tops were discarded

and the soil associated with the root balls was

chopped, homogenized and stored at 4�C.

Efficiency of AMF isolates in acid soils

Efficiency of AMF isolates was studied using the

Marubakaido rootstock of apple (Malus prunifolia)

growing in acidic soil conditions. Micropropagated

plantlets were produced in the Plant Morphogenesis

Laboratory at the Universidade Federal de Santa

Catarina from apical meristems grown in modified

Murashige–Skoog medium, following the methodol-

ogy of Nunes et al. (1991).

Plants were transferred from in vitro conditions to

Styrofoam trays containing 60 ml cells filled with a

mixture of sterilized carbonized rice shells and

vermiculite (1:1, vol:vol). Styrofoam trays containing

the micropropagated plants were placed inside a

plastic tray holding ca 1 cm of water. A glass cover

with a 2-cm opening on one side was placed over

both the Styrofoam and plastic trays. Plants were

Table 1. Location and

characteristics of apple

orchards sampled

Region Location Age (year) Apple cultivar Code

Cacador EPAGRI Experimental

Station

3 Fuji and Gala CA1

18 Fuji CA2

Sao Joaquim EPAGRI Experimental

Station

17 Rome Beauty

and Fuji

SJ1

4 Fuji SJ2

Fraiburgo Fischer Company 12 Belgolden FR1

7 Fuji FR2

120 Plant Cell Tiss Organ Cult (2007) 90:117–129

123

maintained in a growth chamber (27�C) until roots

developed.

After 20 days, plants with roots were individually

transplanted into the 120 ml cells of another Styro-

foam tray that contained a sterilized mixture of

soil:sand:feedstock (1:1:1, vol:vol:vol). This mixture

had the following characteristics: pH 6.0,

P > 50 mg kg�1, 9.8% organic matter, and 7.5 and

2.8 cmolc kg�1 of Ca and Mg, respectively. During

the transplanting procedure, soil inoculum (spores,

hyphae, pieces of colonized roots) derived from one

single-species isolate of AMF was added to each cell

as a 30-cm layer between two layers of the sterilized

planting mixture. Control plants received the same

amount of substrate without AMF. Plants were grown

in greenhouse conditions for 30 days and were

irrigated only with distilled water. When plants

reached 6–10 cm height, the mycorrhizal efficiency

experiment was initiated and the plants and planting

substrate were transferred to 1.5 kg pots containing

soil with different pH.

The planting substrate was prepared by mixing a

latossolo bruno alico (EMBRAPA 1999) from the

Fraiburgo region, with a native pH of 4.0. This soil

was mixed (1:1, vol:vol) with washed, sterilized sand.

The mixture was divided into three portions: one was

kept at its native pH and the other two portions were

adjusted to either pH 5.0 or 6.0 by adding CaCO3

using incubation with neutralizing curves as de-

scribed by Melo (1985). Substrate was sterilized

(121�C, 1 h) twice with a 24-h interval between

autoclaving sessions. No fertilizer was added during

the experiment and soil chemical characteristics of

the three substrates are presented in Table 2. After

transplanting, a layer of sterilized vermiculite was

added to the substrate surface.

A completely randomized design was used con-

sisting of three pH levels (4.0, 5.0 and 6.0) and five

mycorrhizal inoculation treatments (four single-spe-

cies AMF isolates and the non-inoculated control)

with eight replicates per treatment combination.

Plants were maintained under greenhouse conditions

and watered only with distilled water to field

capacity. After 70 days, plant height was measured

and shoots were separated from roots. Mycorrhizal

colonization was assessed using 0.3–0.5 g roots that

were randomly sampled, stained according to Koske

and Gemma (1989) and then scored as percent root

colonization following Giovanetti and Mosse (1980).

Shoots and the remaining root fractions were oven

dried (60�C, 3 days) and weighed to determine dry

mass. The equivalent amounts of root dry mass

removed to determine mycorrhizal colonization were

added to the mass measured for the remaining root

fractions to calculate total root weights. Total dry

mass of mycorrhizal and non-mycorrhizal plants was

used to calculate relative mycorrhizal dependency

(RMD) according to Plenchette et al. (1983):

RMD = [(dry mass myc plants – dry mass non-myc

plants)/dry mass myc plants] · 100. Mineral concen-

trations of N, P, K, Ca, Mg, S, Cu, Zn, Fe, Mn, Na, B

and Al were measured using the shoots according to

Tedesco et al. (1995).

Statistical analyses

All dependent variable data were checked for homo-

geneity of variance according to the test of Burtlett.

Mineral concentrations of N, P, K, Ca, Mg and S

were square root transformed, and percent root

colonization for the corn bioassay and apple plants

were square root + 1 transformed. Root colonization

for the corn bioassay was analyzed by one-way

ANOVA, while colonization of apple plants in the

efficiency experiment was analyzed by two-way

ANOVA. Means were separated by the least signif-

icance difference test (P = 0.05). Statistical analyses

were performed using STATGRAPHICS1 Plus,

version 2.1.

Results

Mycorrhizal inoculum potential of apple orchards

After 40 days, mycorrhizal colonization of corn

growing in soil from Cacador (CA1) was significantly

Table 2. Soil chemical properties of the native soil (pH 4.0)

and limed soil (pH 5.0 and 6.0) used in the AMF efficiency

experiment

Soil pH P K OM Al Ca Mg

mg kg�1 (%) cmolc kg�1

4.0 5.2 99 6.4 2.7 2.0 1.0

5.0 2.7 78 6.0 0.3 2.7 1.6

6.0 4.2 72 5.6 – 4.7 2.0

Plant Cell Tiss Organ Cult (2007) 90:117–129 121

123

higher (35%) then levels found in the other soils,

followed by orchard SJ1. Therefore, the soil from

CA1, corresponding to the mycorrhizal community

with the highest inoculum potential, was selected to

set up trap cultures and obtain single-isolate fungal

cultures (Fig. 1).

Taxonomic characterization of AMF populations

Spores of four AMF species were recovered from the

soil at CA1. The highest concentration of spores

(number 100 g soil�1) was found for Acaulospora

mellea Spain and Schenck (8), followed by Scutel-

lospora heterogama (Nicol. and Gerd.) Koske and

Walker (6), Gigaspora decipiens Hall and Abbott (4)

and Acaulospora spinosa Walker and Trappe (2).

Both Acaulospora species were recovered only from

field samples, while S. heterogama and Gigaspora

decipiens spores were recovered from field samples

and trap cultures. Spores were found only after one

growth cycle of the trap cultures for Scutellospora

pellucida Nicol. and Schenck, Scutellospora persica

Koske and Walker, Acaulospora morrowiae Spain

and Schenck, Acaulospora scrobiculata Trappe,

Glomus etunicatum Becker and Gerdemann, Glomus

clarum Nicol. and Schenck, and Paraglomus occul-

tum Walker (Morton and Redecker).

Trap cultures yielded abundant spores (number

100 g soil�1) of S. heterogama (4,524), S. pellucida

(311), A. scrobiculata (54) and G. etunicatum (37).

These species were successfully established as single-

isolate cultures and used in the efficiency experiment.

Efficiency of AMF isolates to promote apple

growth in acidic soils

Mycorrhizal colonization

Mycorrhizal colonization on apple plants was signif-

icantly influenced by fungal isolates and by the

fungus · soil pH interaction. After 70 days, all plants

were mycorrhizal ranging from 24 to 68% (Table 3).

The higher levels of colonization were in plants

inoculated with S. pellucida SCT111, which had 68,

66 and 65% colonization at pH 4.0, 5.0 and 6.0,

respectively. Soil pH affected colonization only for

S. heterogama SCT113: colonization was signifi-

cantly higher (62%) at pH 4.0 then at pH 5.0 and

6.0 (44 and 46%, respectively). Non-inoculated

control plants showed no evidence of mycorrhizal

colonization.

Plant growth parameters

Soil pH significantly influenced plant height, total dry

weight (TDW), root dry weight (RDW) but it did not

influence shoot dry weight (SDW) and root:shoot

ratio (RSR) (Table 4). Fungal isolates had a signif-

icant effect on all growth parameters measured, while

the fungus · soil pH interaction significantly affected

only RSR.

Glomus etunicatum SCT110 and S. pellucida

SCT111 increased plant height 132 and 146%,

respectively, compared with non-inoculated plants

0CA1 CA2 FR1 FR2 SJ1 SJ2

10

20

30

40

Roo

t Col

oniz

atio

n (%

)

Orchards

a

c

d d

b

d

Fig. 1 Root colonization (%) of corn plants in the infectivity

assay for all six orchards 40 days after planting. Means

followed by the same letter are not statistically significant

(P > 0.05)

Table 3. Percent mycorrhizal colonization on roots of mi-

cropropagated apple plants inoculated with arbuscular mycor-

rhizal fungi (AMF) isolates at three soil pH levels

AMF isolates Soil pH

4.0 5.0 6.0

Glomus etunicatumSCT110

27 b Aa 36 b A 28 c A

Scutellospora pellucidaSCT111

65 a A 66 a A 68 a A

Acaulospora scrobiculataSCT112

24 b A 26 c A 32 c A

Scutellospora heterogamaSCT113

62 a A 44 b B 47 b B

Control 0 c A 0 d A 0 d A

a Values are mean of eight replicates. Means followed by the

same lower case letters within columns and by capital letters

within rows are not significantly different (P > 0.05)

122 Plant Cell Tiss Organ Cult (2007) 90:117–129

123

(Table 5). Plants inoculated with these isolates also

had greater TDW and SDW (128 and 134%),

respectively, compared with non-inoculated plants.

Respective increases in TDW and SDW by

G. etunicatum SCT110 were 128 and 134% while

for S. pellucida SCT111 increases were 187 and

219%. The smallest SDW (1.46 g), found for the non-

inoculated plants, was not significantly different from

the values for plants inoculated with A. scrobiculata

SCT112 (Table 5). G. etunicatum and S. pellucida

produced the greatest RDW, while RDW did not

differ between non-inoculated plants and those col-

onized by S. heterogama and A. scrobiculata. Soil pH

did not affect SDW and RSR. However, plant height,

TDW and RDW were significantly greater at pH 4.0

(Table 5). Non-inoculated plants had significantly

greater RSR compared with mycorrhizal treatments.

Glomus etunicatum SCT110 and S. pellucida

SCT111 produced the highest values of RMD (ranging

from approximately 52–58%, and 50–64%, respec-

tively, Fig. 2). Conversely, RMD values did not exceed

38% when apples were inoculated with S. heterogama

SCT113 and A. scrobiculata SCT112. Higher RMD

values were found for plants colonized by isolates

SCT110 and SCT111 at pH 5.0, whereas RMD values

were higher for SCT112 and SCT113 at pH 4.0 (Fig. 2).

Mineral nutrition

Only N, Mg and Fe contents in tissues were not

significantly influenced by soil pH. AMF isolates

significantly altered concentrations of P, Zn, Cu, Ca,

S, Na, N, K, Fe and Al. The soil pH · fungal

Table 4. Results of two-way ANOVA on height, total dry

weight (TDW), shoot dry weight (SDW), root dry weight

(RDW) and root:shoot ratio (RSR) of micropropagated apple

plants inoculated with arbuscular mycorrhizal fungi (AMF)

isolates at three soil pH levels

Factors

Soil pH (A) AMF (B) A · B Residual

Height ** ** ns 38.77

TDW * ** ns 1.87

SDW ns ** ns 1.22

RDW * ** ns 0.14

RSR ns ** * 0.028

Degrees of freedom: soil pH = 2; fungus = 4; interaction = 8;

residual = 105

ns not significant

* P = 0.05

** P = 0.01

Table 5. Height, total dry weight (TDW), shoot dry weight

(SDW), root dry weight (RDW) and root:shoot ratio (RSR) of

micropropagated apple plants inoculated with arbuscular

mycorrhizal fungi (AMF) isolates at three soil pH levels

Treatments Height

(cm)

TDW

(g)

SDW

(g)

RDW

(g)

RSR

AMF

GlomusetunicatumSCT110

31.74a ab 6.19 a 4.20 a 1.99 a 0.47 a

ScutellosporapellucidaSCT111

33.63 a 6.35 a 4.65 a 1.70 b 0.42 a

AcaulosporascrobiculataSCT112

18.58 c 3.05 bc 1.85 c 1.19 c 0.72 b

ScutellosporaheterogamaSCT113

23.89 b 3.72 b 2.53 b 1.19 c 0.52 a

Control 13.65 d 2.71 c 1.46 c 1.25 c 0.89 c

Soil pH

4.0 27.47 a 4.87 a 3.28 a 1.59 a 0.54 a

5.0 23.25 b 4.18 b 2.79 a 1.40 b 0.64 a

6.0 22.18 b 4.16 b 2.75 a 1.41 b 0.63 a

a Values represent means of 24 replicates of mycorrhizal

inoculation treatment and 40 replicates of soil pH treatmentb Within columns, values followed by the same letters do not

differ significantly according to the LSD test (P > 0.05)

G. etunicatumSCT110

S. pellucidaSCT111

Fungal isolates

0

10

20

30

40

50

60

70pH 4pH 5pH 6

Rel

ativ

e M

ycor

rhiz

al D

epen

denc

y (%

)

A. scrobiculataSCT112

S. heterogama SCT113

Fig. 2 Relative mycorrhizal dependency of micropropagated

apple plants inoculated with AMF isolates at three soil pH

levels

Plant Cell Tiss Organ Cult (2007) 90:117–129 123

123

inoculation interaction was significant for P, Zn, Cu,

Ca, S, Na, Mn and Al (Table 6).

Mycorrhizal apple plants had higher concentra-

tions of P at all soil pH values compared with non-

inoculated plants. At pH 5.0, P concentrations were

not significantly different among isolates (Table 7).

The largest difference in P concentrations occurred

between plants inoculated with S. pellucida at pH 6.0

and the non-inoculated plants (146% difference,

P = 1,600 and 650 mg kg�1, respectively). Phospho-

rus concentrations of mycorrhizal plants were not

influenced by pH, except at pH 6.0 where S.

heterogama plants had the lowest P concentration

compared with the other fungal treatments. Zinc

concentrations at pH 6.0 for G. etunicatum plants

were significantly greater than values found in the

other treatments (Table 7). Zinc concentrations at pH

6.0 were similar between non-inoculated plants and

plants inoculated with S. heterogama; likewise,

similar respective values were found at pH 5.0 for

plants inoculated with A. scrobiculata. Inoculation

with S. heterogama at pH 5.0 increased Zn concen-

trations 83% relative to control plants. Inoculation

with S. heterogama and S. pellucida resulted in a

twofold increase in Cu concentrations at all soil pH

levels. Copper concentrations were significantly

higher at pH 4.0 than at pH 5.0 and 6.0 in all

mycorrhizal treatments, except for G. etunicatum

(Table 7).

Calcium concentrations were significantly higher

in mycorrhizal plants than non-inoculated plants at

pH 5.0 (Table 7). At pH 6.0, plants inoculated with G.

etunicatum and S. pellucida had Ca levels 23 and

36% higher than in control plants, while similar

values were found between plants inoculated with S.

heterogama and A. scrobiculata and non-inoculated

plants. Soil pH increased Ca concentrations in shoots,

except for non-inoculated plants. Plants inoculated

with G. etunicatum and S. heterogama and cultivated

at pH 6.0 had S concentrations significantly higher

than plants of other treatments, increasing 42 and

67% relative to control plants, respectively (Table 7).

Sulfur concentrations were comparatively higher at

pH 6.0 but only for plants inoculated with G.

etunicatum and S. pellucida. Sodium concentrations

of plants inoculated with A. scrobiculata were

significantly higher relative to the other treatments

at pH 4.0; increasing 560% relative to control plants

(Table 7). Conversely, at pH 5.0 control plants had

Na concentrations 270% higher than plants inocu-

lated with A. scrobiculata. Soil pH did not affect Na

shoot concentrations of plants colonized by S.

heterogama and S. pellucida.

Concentrations of Mn were not significantly

different between mycorrhizal and non-inoculated

plants at soil pH 4.0 and 6.0 (Table 7). At pH 5.0,

plants inoculated with S. heterogama had signifi-

cantly greater Mn concentrations compared with the

other fungal treatments. At pH 4.0, Al concentrations

of control plants were similar to levels in plants

inoculated with G. etunicatum, S. pellucida and S.

heterogama, but values were significantly lower than

for plants inoculated with A. scrobiculata (Table 7).

At pH 5.0 and 6.0, mycorrhizal inoculation signifi-

cantly decreased Al concentrations in apple shoots;

non-inoculated plant Al concentrations were 75–

200% larger than the amounts found in mycorrhizal

plants.

The soil pH · mycorrhizal inoculation interaction

was not significant for tissue concentrations of N,

Table 6 Results of two-way ANOVA on 13 mineral elements

in shoots of micropropagated apple inoculated with arbuscular

mycorrhizal fungi (AMF) isolates at three soil pH levels

Factors

Soil pH

(A)

AMF

(B)

A · B Residual

Phosphorus (P) * ** * 0.0018

Zinc (Zn) ** ** ** 41.03

Copper (Cu) ** ** ** 2.15

Calcium (Ca) ** ** ** 0.001

Sulfur (S) ** ** ** 0.0013

Sodium (Na) ** ** ** 1,713.12

Manganese (Mn) ** ns ** 13,013

Aluminum (Al) ** ** ** 5.98

Nitrogen (N) ns * ns 0.011

Magnesium (Mg) ns ns ns 0.009

Potassium (K) ** ** ns 0.006

Iron (Fe) ns ** ns 2,885.7

Boron (B) ns ns ns 125.5

Degrees of freedom: soil pH = 2; fungus = 4; interaction = 8;

residual = 105. Degrees of freedom of the residue for

Mg = 97; K = 95; B = 87; N = 95 and Na = 104

ns not significant

* P = 0.05

** P = 0.01

124 Plant Cell Tiss Organ Cult (2007) 90:117–129

123

Mg, K, Fe and B (data not shown). Nevertheless,

plants inoculated with S. pellucida, S. heterogama

and A. scrobiculata had significantly greater N

concentrations compared with control plants. No

significant differences were observed in shoot N

contents between G. etunicatum and control plants.

Table 7. Concentrations of

eight elements in shoots of

micropropagated apple

inoculated with AMF

isolates at three soil pH

levels

a Values represent mean of

eight replicatesb Means followed by the

same lower case letter

within a column, and by the

same capital letter within a

row, are not significantly

different (P > 0.05)

mg kg�1 Mycorrhizal treatments pH

4.0 5.0 6.0

P Ge SCT110 1,211a bb A 1,330 a A 1,330 a A

Sp SCT111 1,600 a A 1,570 a A 1,600 a A

As SCT112 1,260 b A 1,300 a A 1,010 b A

Sh SCT113 1,660 a A 1,460 a A 1,230 b B

Control 850 c A 680 b B 650 c B

Zn Ge SCT110 37.13 bc B 35.88 b B 44.13 a A

Sp SCT111 40.38 ab A 30.63 bc B 31.00 b B

As SCT112 36.38 bc A 27.75 c B 33.50 b AB

Sh SCT113 44.25 a B 52.00 a A 24.00 c C

Control 33.00 c A 28.50 c AB 26.13 c B

Cu Ge SCT110 7.21 c B 9.05 bc A 8.13 b AB

Sp SCT111 15.14 a A 10.88 a B 9.88 a B

As SCT112 8.83 b A 7.20 c B 4.70 c C

Sh SCT113 16.18 a A 9.88 ab B 7.85 b C

Control 7.60 bc A 4.93 d B 3.64 c B

Ca Ge SCT110 3,730 b C 5,410 a B 5,740 b A

Sp SCT111 4,030 ab C 5,640 a B 6,380 a A

As SCT112 4,240 a B 5,260 a A 5,080 c A

Sh SCT113 4,040 ab B 5,460 a A 5,110 c A

Control 3,880 ab A 4,680 b A 4,680 c A

S Ge SCT110 700 b B 880 ab B 1,210 a A

Sp SCT111 830 ab B 700 b B 1,390 a A

As SCT112 790 b A 760 ab A 890 bc A

Sh SCT113 1,000 a A 900 a A 910 b A

Control 750b A 780 ab A 850 c A

Na Ge SCT110 49.00 bc B 56.88 a B 148.63 a A

Sp SCT111 67.50 b B 46.25 ab B 130.38 a A

As SCT112 163.75 a A 13.00 b B 143.14 a A

Sh SCT113 64.13 bc A 61.63 a A 54.63 b A

Control 24.75 bc B 48.13 ab AB 79.25 b A

Mn Ge SCT110 306.13 a A 210.13 b AB 127.88 a B

Sp SCT111 338.75 a A 147.88 b B 113.00 a B

As SCT112 262.50 a A 131.50 b B 119.71 a B

Sh SCT113 250.75 a A 359.13 a A 114.25 a B

Control 240.75 a A 191.50 b AB 124.00 a B

Al Ge SCT110 5.15 ab A 5.65 b A 1.62 b B

Sp SCT111 5.38 ab A 4.28 b A 3.05 b A

As SCT112 7.56 a A 4.78 b B 2.35 b B

Sh SCT113 3.17 b B 5.68 b A 3.31 b AB

Control 4.03 b B 8.80 a A 7.14 a A

Plant Cell Tiss Organ Cult (2007) 90:117–129 125

123

Concentrations of Mg and B were not affected by

mycorrhizal inoculation. Relative to non-inoculated

plants, inoculation with AMF isolates increased K

concentrations and decreased Fe concentrations in

shoots. Soil pH did not influence shoot concentrations

of N, Mg and Fe.

Discussion

The corn bioassay effectively identified orchard

soils having the highest mycorrhizal inoculum

potential. After 40 days, colonization on corn was

37% in orchard CA1 (despite the low spore count,

20 spores 100 g soil�1) compared with 10–19% in

the other orchard soils. Considering that inoculum

potential depends on fungal infectivity and propa-

gule density, AMF in CA1 were highly infective

and quickly colonized roots, a desirable selection

characteristic in screening processes (Abbott and

Robson 1981). Due to low spore counts in this soil,

mycorrhizal infectivity was caused mainly by other

fungal propagules, e.g., pieces of colonized roots

and extraradical mycelia. The low spore densities

in apple plantations were not expected as Siqueira

et al. (1989) recovered twice the number of spores

in agrosystems compared with natural systems, and

Miller et al. (1985) found spore densities in

orchards in the USA ranging from 62 to 2,150

per 100 g soil�1.

Low spore counts partially compromised identify-

ing AMF species and hindered establishing single-

species cultures. Trapping yielded seven additional

species not found sporulating in the field, indicating

that absence of sporulation should not suggest

absence of fungus (Stutz and Morton 1996). Trapping

is efficient, especially in apple orchards: 17 of 41

AMF species detected by Miller et al. (1985) were

recovered only by trap culture. In this study, the most

efficient isolates, G. etunicatum and S. pellucida,

were not detected from spores in field samples, but

found by trapping. It is possible that in the field, both

fungi allocated resources to structures enhancing

fungal efficiency such as internal and external

mycelia and arbuscules (Graham et al. 1982; Smith

and Read 1997). Sporulation can depend on the plant

species used for trapping (Bever et al. 1996);

therefore, the corn we used may have differentially

stimulated AMF species.

Increasing soil pH from 4.0 to 5.0 and 6.0 did not

influence root colonization, except for S. heterogama

where colonization declined as pH increased sug-

gesting that the isolates were adapted to abrupt

changes in pH. This plasticity may be attributable to

the long-term management of soil in the apple

orchards. In Cacador, native soils are acidic and

were limed prior to establishing orchards perhaps

promoting increased resiliency of the mycorrhizal

community to changes in soil pH as found by Mamo

and Killham (1987). According to Sieverding (1991),

sensitivity of AMF isolates to liming might be a

characteristic of the fungal species. In our study, the

highest rates of colonization was found for S.

pellucida, supporting the possibility that this fungus

invests resources preferentially to mycelial produc-

tion rather than to sporulation.

Inoculation of micropropagated apple in the post

vitro stage stimulated shoot height. After 70 days,

plants inoculated with G. etunicatum and S. pellucida

were taller than those colonized by other isolates. The

mean daily growth increment was 0.045 cm for the

first 28 days, and 0.26 cm from 29 to 42 days (an

increase of 478%). The maximal mean daily growth

rate of 0.52 cm occurred after 70 days (data not

shown). Fortuna et al. (1992) suggest that initial slow

growth rates might be related to either the: (1) lag

phase of fungal growth, reducing root colonization, or

(2) cessation of apical growth of the micropropagated

plants caused by transplanting. In this study, inocu-

lum potential was not measured for all isolates, and

only spore numbers were recorded per 100 g soil�1

(37 for G. etunicatum, 311 for S. pellucida, 4,524 for

S. heterogama and 54 for A. scrobiculata). Appar-

ently, spore numbers did not limit initiation of root

colonization. Perhaps then, the slow growth rates may

be due to transplanting the seedlings from a fertile

substrate to a low nutrient, acidic substrate.

Despite detecting 11 fungal species in CA1, 4

single-isolate cultures were obtained, and only 3 of

them were found to promote plant growth. These

results support findings of Branzanti et al. (1992) that

inoculation with AMF ensures maximum growth of

apple seedlings under low fertility, and efficiency

varies according to the isolate. Guillemin et al. (1992)

inoculated micropropagated pineapple plants with

one isolate of S. pellucida (LPA20), three isolates of

Glomus sp. (LPA21, LPA22 and LPA25) and one of

G. clarum (LPA16) and found that S. pellucida

126 Plant Cell Tiss Organ Cult (2007) 90:117–129

123

LPA20 did not promote plant growth compared with

the other isolates. Conversely, our isolate of S.

pellucida was one of the more efficacious fungi,

promoting both vegetative growth and P uptake,

supporting the hypothesis that the AMF species is not

the most appropriate taxonomic descriptor to link

physiological processes of the mycorrhizal associa-

tion with increases in plant growth (Morton 1993).

Although apple orchards receive substantial annual

inputs of fertilizers, 75% of isolates tested still

increased plant biomass contradicting the hypothesis

that long-term fertilization selects for less efficient

AMF isolates (Johnson 1993). According to Johnson,

the fungus is adapted to a rhizosphere low in root

exudates, as occurs with fertilized plants, and which

results in lower allocations to external mycelia. Even

though we did not test all species that were detected

in CA1, our results do not support the hypothesis that

fertilization selects for less mutualistic fungi.

Plant concentrations of P, Zn and Cu generally

increase from AMF inoculation (Bolan 1991). Inoc-

ulation increased P concentrations at all pH levels,

contradicting results of Branzanti et al. (1992) where

inoculation did not alter P concentrations. Increased P

uptake can be explained by the overall low levels of P

in our soil (mg kg soil�1), 5.1 at pH 4, 2.7 at pH 5 and

4.1 at pH 6, which as suspected did not inhibit

colonization and mycorrhizal activity. Inhibition has

been observed for Acacia mangium (Habte and

Soedarjo 1996), manihot (Sieverding and Howeler

1985) and red clover (Sainz and Arines 1988); P

concentrations in mycorrhizal and non-inoculated

plants averaged 1,400 and 700 mg kg�1, respectively.

For apple plants, Plenchette et al. (1981), Geddeda

et al. (1984) and Branzanti et al. (1992) found

maximum P concentrations of 1,800, 1,900 and

1,800 mg kg�1, respectively. Our P values are below

ideal foliar concentrations (1,500–3,000 mg kg�1)

recommended by Basso et al. (1986). Only plants

inoculated with S. heterogama SCT113 attained these

recommended concentrations at all pH levels. We

analyzed leaves and stems while the recommended

concentrations are for leaves. Additionally, ideal

concentrations are expected in plants growing under

optimal relative levels of all nutrients; our substrates

were low in P and certain other nutrients.

Calcium is important for producing apples as

deficient plants reduce Ca allocation to fruits (Basso

et al. 1986). In this work, inoculation tended to

increase Ca concentrations at pH 5.0 and 6.0, but not

at pH 4.0 where inoculation did not alter amounts.

This pattern may be explained by K and Mg soil

levels, which are considered medium to high at pH

5.0 and 6.0, and sufficient to medium at pH 4.0. High

availability of K and Mg can induce Ca-deficiency in

apples (Basso et al. 1986), as what likely occurred at

pH 5.0 and 6.0 where the mycobiont promoted Ca

uptake. Despite the inoculation effect for Ca concen-

trations, we measured plant levels considered insuf-

ficient by Basso et al. (1986), i.e., below

8,000 mg kg�1.

The fungal isolates appeared to reduce Al uptake

at pH 5.0 and 6.0 where non-inoculated plants had

greater shoot Al concentrations than levels found for

plants inoculated with any of the isolates. Isolate S.

pellucida SCT111 was highly efficient at reducing Al

toxicity at all acidity levels. A protective effect

against Al was observed by Mendoza and Borie

(1998) for oat plants inoculated with an isolate of G.

etunicatum and grown in soils having high levels of

Al. Clark and Zeto (1996) found that inoculation with

Glomus isolates decreased Al concentrations and

contents of maize plants growing in two acid soils

from West Virginia. However, Borie and Rubio

(1999) studied oat varieties sensitive and tolerant to

Al and inoculated with G. etunicatum. Inoculation

increased Al concentrations in sensitive plants rela-

tive to non-inoculated plants, but not in tolerant

plants. After liming, Al concentrations in sensitive

mycorrhizal plants were half the levels measured in

non-inoculated plants. In general, these results sug-

gest that protection against high Al levels in soil

depends on the mycorrhizal community, the ability of

the host plant to tolerate this element, and on the pH

of the substrate.

Although the mycorrhizal association frequently is

considered a mutualistic symbiosis, there is a con-

tinuum of plant responses to mycorrhizal colonization

ranging from positive (mutualism) to negative (par-

asitism) (Johnson et al. 1997). We have evidence for

this continuum when apple biomass and nutrient

uptake are considered. A. scrobiculata plants exhib-

ited parasitic behavior; its effect on these processes

did not differ from responses measured for non-

inoculated plants. Alternatively, S. heterogama plants

attained greater relative shoot and total mass, but not

root mass, and Zn and Mn uptake were optimized at

pH 5.0. Finally, isolates G. etunicatum and S.

Plant Cell Tiss Organ Cult (2007) 90:117–129 127

123

pellucida behaved as mutualists. Plants inoculated by

them, at all pH levels, achieved the greatest heights,

produced greater mass than non-inoculated plants,

and attained normal shoot contents of P, Zn, Cu, Fe

and Mn. An overall analysis scheme of this sort is

paramount to identify relative abundances of efficient

and non-efficient fungi composing a mycorrhizal

community and allows for a greater understanding of

how the symbiosis works in natural systems, a

necessary requirement to utilize AMF in agriculture

(Johnson et al. 1997).

Event though the fungal isolates originated from

acidic soils, and they produced significant differ-

ences among them in attaining plant biomass and

nutrient uptake, they generally were well adapted to

the range in soil pH studied. Knowing these relative

responses is important when the goal is to produce

commercial fungal inocula for wide-scale use on

plants grown in the field, where conditions are highly

variable. The ideal inoculum should be efficient

under the diverse of conditions found among field

settings (Lovato et al. 1992). In the field, attributes of

the mycorrhizal inoculum other than colonization

efficiency are important such as reproductive capac-

ity and ability to compete with indigenous soil biota

(Lambert et al. 1980). We demonstrated that AMF

isolates stimulate plant biomass, and nutrient uptake

by micropropagated apple plants growing under a

range of acidity. Further work is needed to investi-

gate the capabilities of the same isolates directly

under field conditions. Combinations of isolates must

be evaluated striving to produce multi-isolate inoc-

ulants that are superior to single-isolate inoculants,

potentially optimizing plant performance in highly

variable field conditions.

Acknowledgments This work is part of the requirements of

the senior author for a M.S. degree in biotechnology. This

project was funded by FINEP (Financiadora de Estudos e

Projetos, Brazil). We thank CAPES (Coordenacao de

Aperfeicoamento de Pessoal de Nıvel Superior, Ministerio de

Ciencia e Tecnologia, Brazil) and CNPq (Conselho Nacional

de Desenvolvimento Cientifico e Tecnologico) for providing

scholarships to JRPC during his graduate studies and to SLS as

a Post-doctoral researcher. We are grateful to Dr Enio Pedrotii

for providing micropropagated apple seedlings, and to Dr Clori

Basso (EPAGRI, Cacador, SC) for technical support while the

soils were sampled. We thank Drs P.E. Lovato and R.G.

Linderman for valuable suggestions made to an earlier version

of the manuscript. The document was subjected to the peer and

administrative reviews of the U.S. EPA at the National Health

and Environmental Effects Research Laboratory, Western

Ecology Division, and was approved for publication.

Mention of trade names or commercial products in this paper

does not constitute endorsement or recommendation of use.

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