Alagely et al 1
THE RELATIONSHIP OF MYCORRHIZAE TO ARSENIC UPTAKE IN THE
HYPERACCUMULATOR Pteris vittata L (BRAKE FERN)
Abid Alagely, David M Sylvia, Lena Q Ma, and Nandita Singh
Abid Alagely* and Lena Ma
Department of Soil and Water Science, P.O. Box 110290
University of Florida, Gainesville, FL 32611
David Sylvia
Department of Crop and Soil Sciences
116 ASI, The Pennsylvania State Univ., University Park, PA 16801
Nandita Singh
National Botanical Research Institute,
Rana Pratap Marg, Lucknow 226001, India
Abid Alagely (correspondence)*
Department of Soil and Water Science, P.O. Box 110290
University of Florida, Gainesville, FL 32611
Phone: 352 392 1951 EXT 220, FAX: 352 392 3902
E-mail:

ABSTRACT
Pteris vittata L (Brake fern) is a hyperaccumulator of arsenic (As) that grows voluntarily
on arsenic-contaminated soil in the Southern United States (Florida). It is the only fern
species being found as an active hyperaccumulator. Directly related to plant nutritional
needs, mycorrhizae are important for arsenic hyperaccumulators. Brake ferns are known
to be colonized by arbuscular mycorrhizal fungi. Mycorrhizae have a well-documented
role in increasing plant uptake of phosphorus (P) and other poorly mobile elements and
are recognized as important components of bioremediation strategies for heavy metals.
Mycorrhizal symbioses are the best examples of compatibility between plants and
microorganisms; however, we still have a poor understanding of the interactions of plant
and fungal factors that contributes to these associations. As and P share the same
chemical properties, thus this study assumed that mycorrhizae may be important for As

uptake. Objectives of this research were to determine if brake fern growth or arsenic
Alagely et al 2
uptake is enhanced by mycorrhizal colonization and if natural As contaminated soil is
important for mycorrhizal establishment. Four mycorrhizal treatments were used to test
these objectives. In each assessment, brake ferns associated with mycorrhizal fungi
produced higher responses when compared to the brake ferns of no mycorrhizal
association. Statistical analyses of the results proved that mycorrhizal fungi are essential
for As hyperaccumulator growth and development, but no significant differences were
found in As uptake. Further studies are underway to evaluate if different compensation
of As and P concentrations in soil, or AM fungal isolate adaptation, are important for
growth and As uptake in brake ferns.

Key words: Indigenous AM mycorrhizal fungi, phosphorus, arsenic, inoculum, fungi,
brake fern, hyperaccumulator
INTRODUCTION
Arsenic (As) is one of the most toxic substances for living organisms. Nonetheless,
Pteris vittata L (brake fern) was found an active As hyperaccumulator (Ma et al., 2001).
Sources of As in the environment are both natural and anthropogenic.
Toxicological and ecological studies on As were accelerated in the recent years due to
health and environmental concerns (Smith and others 1992; Chappell 2001; Chappell and
others 1997); (Chiou and others 1995); ([Anon] 1999); and (Oremland and Stolz 2003).
In an attempt to protect public health, the United States Environmental Protection
Agency (USEPA) is in the process of lowering the drinking water standard of arsenic
from 50 to 10 ppm (USEPA, 2001), making the issue of cleaning up arsenic-
contaminated soils one a high priority.
Mycorrhizae are important for As hyperaccumulator growth and development.
Arbuscular mycorrhizae (AM) ability to acquire phosphate (P) and other poorly mobile
elements has been well-documented (Smith and Read 1997), and are recognized as
important components of bioremediation strategies for heavy metals (Khan and others
2000). Chemically, As and P share the same pathway during microbial mobilization

Alagely et al 3
(Meharg and others 1994), for that reason mycorrhizal fungi may have an important role
in As uptake from soil.
Among hyperaccumulators, mycorrhizal plants have some advantages in mobilizing
heavy metals in soil because they have larger symbiotic interface areas than the non-
mycorrhizal plants (Sanders and Tinker 1971). Also, mycorrhizal plant roots have
additional mechanisms to improve uptake more than the non-mycorrhizal plant roots as
described in (O'Keefe and Sylvia 1991).
Although mycorrhizal association is one of the prevalent symbiotic associations in fern
plants (Berch and Kendrick 1982; Iqbal and others 1981; LaFerriere and Koske 1981;
Ponton and others 1990; Schmid and Oberwinkler 1995; Sharma 1998), brake ferns
required more investigation because it is the only fern being found as an active
hyperaccumulator. Brake ferns grow rapidly and distribute widely in low fertility soil
(Chen and others 2002), thus we hypothesized that AM fungi have a critical role in their
productivity and contribute significantly to arsenic uptake.
Removing As from soil by biological means will ultimately require large-scale
production of well-adapted mycorrhizal brake ferns. Here we studied the (I) mycorrhizal
status of brake fern in an As-contaminated site in northern Florida, (II) influence of
mycorrhizal fungi on growth and As uptake of brake fern, and (III) differences among
adapted and nonadapted AM fungi for remediation potential. This study is part of larger
study to understand and to enhance arsenic hyperaccumulation by a fern Plant.
MATERIALS AND METHODS
The experiment was complete randomization of four (inoculations) and ten replications
per treatment. Experiment was terminated at May 2003 after 20 weeks of growing period
in greenhouse at maximum and minimum temperatures were 32 and 18
o
C, respectively,
and average maximum photosynthetic photon flux density (PPFD) was 1235 µmol/m
2
/s.

The sampling was conducted on an abandoned copper-chromium-arsenic (CCA) site in
central Florida on SR 24, west of US 27 (Fig 1). This site was the location of pressure
wood treatments that used an aqueous solution of arsenic pentoxide, copper sulfate, and
Alagely et al 4
sodium or potassium chromate as a pesticide from 1951 to 1962. Because of this activity,
the site has become heavily contaminated with As and other heavy metals (Woodward-
Clyde, 1992). Physical and chemical analyses of soil in this site were completed prior to
the onset of this study and are described in a previous study (Ma et al., 2001).
Soil samples were collected randomly from the rhizosphere of the top 20-cm depth of 30
locations. One-liter soil samples from each location were sealed in plastic bags, placed in
cooling box, and transferred to the research laboratory. Upon arrival at the laboratory,
soil samples were mixed and subsampled for pH, water-extractable P, and mycorrhizal
inoculum potential (MIP) bioassay tests. The extra soil samples were used as “soil
inoculum” for the respective mycorrhizal treatments.
Culture media was 1:1:1 CCA site soil, acid-washed sand, and coarse vermiculite.
Culture media was pasteurized twice, 48 hours a part, at 85 °C for 8 hr with dry heat.
Each replicate according to their respective treatment received either 100-g (dry mass
basis) “soil inoculum” of Glomus etunicatum (Nicol & Gerd.) Gerdemman & Trappe
(Isolate S3029) of an agricultural field in north Florida, Glomus sp. (Isolate S3065) of
CCA site, nonpasteurized soil from CCA site (AM fungal community treatments), or
pasteurized soil from CCA site (control treatment). “Soil inoculum” of each replicate of
the respective treatment was mixed with culture media and placed into 1.5-liter pot.
The host plant was Pteris vittata L (brake fern). Laboratory seedlings were initiated from
spores on sandy soil to insure nonmycorrhizal plant production for the control treatment.
Each replicate received one healthy brake fern of 5-6 fronds.
Roots were removed from the culture media by wet sieving, and their fresh weights were
determined. Subsample of 0.5 g from each root system was cleared with 10% (w/v)
KOH and stained with 0.05% (v/v) trypan blue in lactophenol as described by Phillips
and Hayman (1970). Percent colonization of root length was determined by the gridline-
intersect technique (Giovannetti and Mosse, 1980). Math calculation based on root fresh

and dry weight was used to estimate the dry weight of the subsamples used for AM
colonization tests and added to the total root dry weight.
Alagely et al 5
Ground fronds or roots were digested with concentrated HNO
3
and Distilled water (1:1,
v: v), followed by 30% H
2
O
2
for As determination or with HNO
3
, H
2
SO
4
, and 30% H
2
O
2
for P determination. As concentration was determined using Perkin Elmer SIMMA 6000,
while P concentration was determined using a colorimetric assay by BIO-RAD
Microplate Reader model 550. In either procedure, blanks and internal standards were
included for quality assurance.
The experiment was randomized in complete blocks of four treatments of mycorrhizal
inoculation (CCA community AM fungi, S3029, S3065, and Control). Each treatment
has 10 replications. Results were statistically analyzed and plotted by PC SAS (SAS
Inst., 2003). Significant differences were achieved at the 0.05 level. Population
normality was tested for each variable before using parametric statistics for comparisons
and testing.


Figure 1: Soil Survey Map of CCA site, Sources: USDA Soil Survey of Alachua County
1985
Alagely et al 6
RESULTS AND DISCUSSION
Physical analyses showed that soil in the study site was an Arredondo-urban land
complex with taxonomic classification of loamy, siliceous, and hyperthermic Grossarenic
Paleudult. Soil particle distribution was determined to be 88% sand, 8% silt, and 4% clay
(Ma et al., 2001).
The pH and water-extractable P (µgml
-1
) concentration of the study site were 7.6 and
10.6, respectively. This soil pH was relatively high for Florida soils, thus it may play an
important role in As uptake by the hyperaccumulator brake fern. Other soil chemical
analyses indicated that the site was heavily contaminated with As, as well as moderately
contaminated with Cu and Cr (Table 1). These values are also well above the natural
background levels for Florida soils. Soil organic matter (OM) was found to be very low
(0.8%) in the study site soil. MIP bioassay resulted in a mean of 43% for the CCA
community AM fungi, 41% for the S3029 AM fungal isolate, and 37% for the S3065 AM
fungal isolate.

As Cu Cr Zn Fe Ca
SAMPLE
mgkg
-1

OM
20-cm Depth 184 252 84 58 1328 33,655 0.8%
Table 1: Soil characteristic of the study site, Sources: Ma et al., 2001
Analysis of variance revealed that percent colonized root length was significant (P =

0.001) variable between the experiment treatments. The AM fungal community
colonized brake ferns more aggressively than AM S3065 or S3029 isolates. S3029 AM
isolate established the least percent colonization of root length; maybe because it did not
adapt to As contamination or brake ferns as a host as well as the other AM fungal
isolates.
Alagely et al 7

Figure 1. Mean percent root colonization of brake fern cultured with arsenic
contaminated soil for 20 weeks.
The MA fungal community had significant (P = 0.04) impacts on frond biomass than
other mycorrhizal treatments did. It was significantly different from the control ferns but
it was not significantly different from ferns associated with S3029 or S3065 AM fungal
isolates (Figure 2). The high production of frond biomass did not reflect a significant
variation in frond-As content or the amount of As uptake per gram biomass (Figure 3 and
4). However, there was a significant (P = 0.001) variation in frond-P content and P
uptake per gram biomass between the different mycorrhizal treatments (Figure 3 and 4).
0
5
10
15
20
25
30
35
40
45
50
COMMUNITY S3029 S3065 CONTROL
COLONIZED ROOT LENGTH (Percentages)
A

C
B
Alagely et al 8

Figure 2: Total shoot biomass dry weight (g) of brake fern growing with arsenic
contaminated soil for 20 weeks.

Figure 3 Arsenic and phosphorus concentration: A, mg / plant and B, mg / gram in shoot
of brake ferns cultured for 20 weeks
The degree of adaptation of AM fungi to their native soil in general and specially to As
contamination is still an open question. The best performance of the indigenous AM
fungi in their native soil was the conclusion of many researchers (Sylvia and others 2002;
Porter and others 1987; Henkel and others 1989; Schultz and others 2001; Ronsheim and
0
2
4
6
8
10
12
14
16
18
COMMUNITY S3029 S3065 CONTROL
SHOOT BIOMASS Dry WEIGHT (g)
A
AB AB
B
ARSENIC
PHOSPHATE

0.0
0.5
1.0
1.5
2.0
2.5
3.0
COMMUNITY S3029 S3065 CONTROL
MYCORRHIZAL TREATMENTS
CONCENTRATION (mg/g)
A
A
A
A
A
B
B
BC
ARSENIC
PHOSPHATE
0
5
10
15
20
25
30
35
COMMUNITY S3029 S3065 CONTROL
MYCORRHIZAL TREATMENTS

CONCENTRATION (mg/Plant)
A
A
A
A
A
B
B
BC
A B
Alagely et al 9
Anderson 2001). The finding in this research that AM fungal communities extensively
colonized and increased brake fern frond biomass suggested a greater capability of
response, possibly because of less available P in the soil, or other factors related to
ecological adaptation with the native soil.
While the soil under investigation has low concentration of P and high concentration of
As, the tendency of brake fern to uptake these elements reflects their competition for
sorption sites. When brake ferns uptake more As, they uptake less P. This is true for all
treatments except the control (Figures 3). This finding is supported by early studies by
(Gao and Mucci 2001) and by (Smith and others 2002) when they found that As and P
interaction in soil increased their concentration in soil solution as a result of competition
for sorption sites. The AM fungal association may magnify these competition statues.
Further studies ar in progress to evaluate adaptation strategies of both AM fungal isolates
and brake ferns toward the combination of different concentrations of As and P in order
to improve our ability to predict growth response in the field. Further research is also in
progress to include evaluating the amount and speciation of As in mycorrhizal structures.
ACKNOWLEDGMENT
This research was partially funded by the National Science Foundation (Grant ).
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