AUTHORS

M. J. Haddad � Department of Earth andEnvironmental Science, The University of Texasat San Antonio, 6900 N Loop 1604 W, SanAntonio, Texas 78249-0663

Melissa J. Haddad received her M.S. degreein environmental science from the Universityof Texas at San Antonio. Her thesis researchfocused on glomalin and its relationship withsoil properties. Prior to this, Haddad served inthe U.S. Army as a Medical Service Corps officer,completing assignments in Germany, Bosnia–Herzegovina, and Fort Bragg, North Carolina.She completed her undergraduate studies atMercer University, where she received a B.S.E.degree in biomedical engineering with minorsin environmental engineering and mathematics.

D. Sarkar � Earth and Environmental ScienceDepartment, The University of Texas at SanAntonio, 6900 N Loop 1604 W, San Antonio,Texas 78249-0663; dsarkar@utsa.edu

Dibyendu Sarkar is an assistant professor anddirector of the Environmental GeochemistryLaboratory at the University of Texas at SanAntonio. Sarkar received his Ph.D. from theUniversity of Tennessee and did his postdoc-toral training at the University of Florida. Hisareas of expertise include soil chemistry,environmental quality and remediation, andrisk assessment. Sarkar is also an associateeditor of Environmental Geosciences.

ACKNOWLEDGEMENTS

This research was made possible by a graduatestudent research grant from the GeologicalSociety of America and laboratory support fromthe Environmental Geochemistry Laboratory atthe University of Texas at San Antonio (UTSA).We would like to acknowledge the Center forWater Research, UTSA for providing the seniorauthor with a Graduate Research Assistantship.Additionally, we would like to thank the UnitedStates Department of Agriculture and the NationalResources Conservation Service laboratory inLincoln, Nebraska, for their assistance in thecollection of soil samples. In addition, our thanksgo to S. F. Wright and Kris Nichols for providingthe primary monoclonal antibody.

Glomalin, a newly discoveredcomponent of soil organicmatter: Part II—Relationshipwith soil propertiesM. J. Haddad and D. Sarkar

ABSTRACT

Soil organic matter (SOM) is one of the most important com-

ponents of soil when evaluating soil structure and its impact upon

the environment. The discovery of a unique glycoprotein, glomalin,

in 1996 challenged what was known about the composition of

SOM. In an effort to better understand the presence of glomalin in

SOM, the current research focused on glomalin concentrations in

soils as it relates to the key soil properties, such as pH, clay (%),

total C (%), C/N ratio, organic C/N ratio, inorganic C (%), and soil

organic matter (%). Twenty soils collected from undisturbed range-

lands throughout the United States, representing a wide range of the

aforementioned soil properties, were analyzed for easily extractable

glomalin (G1) and for total glomalin (G2). The G1 and G2 extracts

were analyzed for protein content using the Bradford assay, and their

respective immunoreactive fractions (IR-G1 and IR-G2) were deter-

mined using the enzyme-linked immunosorbent assay (ELISA). Sta-

tistical correlations indicated that pH, total C, and SOM were the

best predictors of glomalin in soils (R2 values of 0.43, 0.42, and

0.46, respectively). Inorganic carbon and C/N ratio were deter-

mined to be the best predictors of the immunoreactive fractions of

soil glomalin (R2 values of 0.63 and 0.55, respectively). Multiple

regression analysis revealed that 75% of variation in the G2 fraction

and 73% of variation in the IR-G2 fraction could be explained by

the collective soil properties. Overall, the results indicate that G1

and IR-G1 measurements may be ambiguous and that G2 and IR-

G2 are the most accurate representation of total glomalin and im-

munoreactive glomalin in soils.

INTRODUCTION

Soil organic matter (SOM) is a key component of soil that greatly

influences its structure. In 1996, the somewhat accidental discov-

ery of glomalin by Sara Wright of the United States Department of

Environmental Geosciences, v. 10, no. 3, pp. 99–106 99

Copyright #2003. The American Association of Petroleum Geologists/Division of EnvironmentalGeosciences. All rights reserved.

Agriculture (USDA) challenged what was then known

about the general composition of soil organic matter.

Typically, soil organic matter is considered to be pre-

dominantly composed of particulate organic matter,

humic acid, fulvic acid, and insoluble humin (Weber

and Michalczyk, 1997). However, since the discovery

of the soil glycoprotein, glomalin, numerous tests have

suggested that it is, in fact, distinctly different from

other components in soil organic matter (Nichols et al.,

2000) and has tremendous environmental significance.

The most distinguishable characteristic of glomalin

is its unusual toughness. Solubilization of glomalin is

quite difficult and requires soil extractions at tempera-

tures of 121jC for a minimum period of 1 hr. These

are extremely unusual conditions for studying soil pro-

teins (Wright and Upadhyaya, 1996). The stringent

conditions associated with the extraction of glomalin

are possibly what prevented the discovery of this soil

protein for so many years because soil extractions are

uncommonly conducted at temperatures as high as those

required to extract glomalin. However, solubilizing glo-

malin is necessary to study the chemistry of the protein

after it is sloughed from the arbuscular mycorrhizal

(AM) fungi to the soil. To date, the chemical structure

of the glomalin protein remains unknown. However,

research efforts have identified glomalin to be a com-

plex of amino acids, carbohydrates, iron, and other

ions (Comis, 2002). The recalcitrant behavior and ap-

parent hydrophobic characteristics of glomalin also

suggest that this molecule is very stable (Wright and

Upadhyaya, 1998).

Glomalin has shown evidence of being an important

part in many aspects that benefit soil science (Comis,

2002; Wright, 2000), both from agronomic and en-

vironmental points of view, particularly from the angle

of decreased soil erosion. The physical and chemical

properties of glomalin and its environmental signifi-

cance have been reviewed elsewhere (Haddad and

Sarkar, 2003). As the authors point out, despite the

significant amount of research performed since its rel-

atively recent discovery, there is a world of unknowns

about this unique, uncommonly tough soil protein,

which has now been unequivocally recognized as a key

player in maintaining soil aggregate stability. One of

the more pertinent issues that still need to be addressed

is the influence that soil properties have on glomalin

concentrations (Haddad and Sarkar, 2003). The objec-

tive of the present research was to investigate glomalin

concentrations in soil as a function of the following soil

properties: pH, clay (%), total C (%), C/N ratio, organic

C/N ratio, inorganic C (%), and organic matter (%).

Determining the relationships between glomalin and

the key physicochemical properties of soils has the

potential of predicting the soil types that have the

greatest glomalin retention capabilities. Hence, the re-

sults from this study will potentially enable soil sci-

entists to better evaluate what direction to take in the

study of glomalin and its importance to agronomic and

environmental soil chemistry.

MATERIALS AND METHODS

Soil Properties

Twenty surface soil samples were obtained from un-

disturbed rangelands located throughout the United

States. The soils were selected based on the associated

USDA characterization data to cover a wide range of

physicochemical properties. All samples were of ap-

proximately the same age and were dried and sieved

upon receipt. Soil pH data were experimentally ver-

ified using a soil/water ratio of 1:2. Total carbon and

total nitrogen were measured using a CHN analyzer

and then compared to the values obtained from the

USDA characterization data; the values were compa-

rable. Organic carbon and nitrogen values were mea-

sured after pretreating the samples with cold 10% HCl,

followed by analysis using the CHN analyzer (Loeppert

and Suarez, 1996). The organic C and N values were

subtracted from the total concentrations, resulting in

the inorganic C and N content of each soil sample. The

organic C values were normalized to the organic nitro-

gen values to yield the organic C/N ratio. Organic mat-

ter content was calculated from the organic C values

following the empirical relations stated in Nelson and

Sommers, 1996. Clay contents were taken from the

USDA sample characterization data. Values of soil

properties of the samples evaluated are given in Table 1.

Glomalin Extraction

Both total (G2) and easily extractable fractions (G1)

of glomalin were extracted from the soils following

the procedures outlined in Wright et al. (1996). The

easily extractable glomalin fraction was extracted using

20 mM sodium citrate at a pH of 7.0 (Wright et al.,

1996). A 1.0-g sample of soil was mixed with 8 mL of

20 mM citrate solution and then autoclaved at 121jC

for 30 min. The samples were then centrifuged at

2500 rpm for 15 min at room temperature. The super-

natant was saved as the easily extractable fraction of

100 Glomalin in Soil Organic Matter as a Function of Soil Properties

glomalin (G1) and refrigerated until further use. The

residual pellet was used in the process of obtaining the

total glomalin protein (G2) fraction. For this fraction,

8 mL of a 50 mM sodium citrate solution at pH 8.0

was mixed with the pellet and autoclaved at 121jC

for 60 min, then centrifuged at 2500 rpm for 15 min at

room temperature. The supernatant was saved in a sep-

arate vial as a portion of the total glomalin fraction.

These extraction steps for total glomalin were repeated

until the supernatant became transparent. All the

aliquots specific to each sample were pooled to obtain

the total volume of G2 fraction.

Protein Assays

Protein concentrations for the G1 and G2 fractions

were determined using the Bradford assay. This meth-

od measures total protein and is not specific to glo-

malin; however, it does provide a total measurement

of all soil proteins in the samples (Wright et al., 1996).

In this case, the concentrations determined for each

sample were completely attributed to glomalin because

other proteins are likely to be eliminated during the

harsh conditions of the extraction procedure (Wright

and Upadhyaya, 1996). The volume of extractant used

for each sample was determined based on the intensity

of the color of the extracts, with the darker, almost

black extracts requiring much less volume. Phosphate-

buffered saline (PBS) was used to dilute the plate wells

to the final volume of 250 mL. All samples were com-

pared to a known curve using bovine serum albumin as

the standard (Wright et al., 1996). Concentrations of

glomalin (A590) were extrapolated to milligrams per

gram of soil using the volume of extract and the initial

weight of soil.

Enzyme-linked immunosorbent assay (ELISA) was

used to determine the amount of immunoreactive glo-

malin in the G1 and G2 fractions. The easily extractable

fraction of soil from the Mattapex series was used for

the standard curve in the ELISA analysis of all samples

because of its highly immunoreactive glomalin fraction

(Table 2). This series was identified in previous re-

search as one containing glomalin that is nearly 100%

immunoreactive (Wright and Upadhyaya, 1998). In

setting up the plates, a 0.4-mg concentration of protein,

determined from the Bradford assay, was added to a

series of plate wells in triplicate for each soil sample.

The ELISA procedure involved using the MAb32B11

antibody as the primary antibody and biotinylated

anti-mouse IgM antibody as the secondary antibody.

MAb32B11 was developed from Glomus intraradicesFL208 spores that were used to immunize a BALB/c

Table 1. Average Values of Soil Properties for each of the 20 Soil Samples

Sample pH Total C Clay (%) C/N Ratio SOM (%) Organic C/N Inorganic C (%)

A 6.4 2.93 12.8 10.07 7.93 28.00 0.97

B 6.9 0.91 21.0 11.52 5.01 100.00 0.10

C 5.3 1.82 19.6 9.38 6.08 139.00 0.43

D 7.3 1.55 39.9 8.52 11.72 45.67 0.18

E 6.1 2.60 44.2 9.70 9.81 34.40 0.88

F 4.8 2.21 12.0 10.28 5.61 143.00 0.78

G 7.1 1.03 14.1 9.20 4.23 16.40 0.21

H 5.0 1.81 37.0 10.11 11.56 55.33 0.15

I 4.5 6.03 32.6 10.47 17.61 14.05 0.41

J 7.2 1.35 18.8 9.38 6.02 99.00 0.36

K 7.3 2.40 32.2 10.86 9.72 44.75 0.61

L 7.9 1.34 39.0 8.70 11.34 24.00 0.38

M 5.8 1.56 12.7 9.45 5.07 33.00 0.57

N 5.8 1.97 10.5 11.13 4.93 35.67 0.90

O 5.5 2.53 11.0 11.67 5.77 13.00 0.06

P 4.9 10.9 0.0 17.25 26.90 17.44 0.96

Q 5.1 26.3 0.0 1.65 50.02 15.13 3.00

R 7.9 4.87 7.9 27.99 4.96 16.09 3.10

S 5.0 6.19 26.9 10.51 13.02 15.16 1.34

T 7.4 9.28 18.6 29.94 11.07 15.69 5.20

Haddad and Sarkar 101

mouse (Wright et al., 1996) and is specific to glomalin.

The determined concentrations (A405) were again ex-

trapolated to milligrams per gram of soil using the vol-

ume of extract used to obtain a protein concentration

of 0.4 mg per plate well and the initial weight of the soil

used in the extraction. The percentage of this value to

the value of total soil protein obtained from the Brad-

ford assay was recorded as the percent immunoreac-

tive fraction of glomalin for G1 and G2 (IR-G1 and IR-

G2), specific to each particular sample. These values,

along with the total protein values for both glomalin

fractions, are shown in Table 3.

Statistical Analysis

Pearson correlation coefficients were derived for all pos-

sible pairs of variables. Linear regression models were

employed using StatPlus (Berk and Carey, 2000) in Excel

for each of the soil properties and the different glomalin

fractions. Multiple regression models enabled the eval-

uation of all measured soil properties collectively with

each glomalin fraction. A 0.05 level of significance was

used for all statistical analyses. Total C (%) was not

examined in combination with SOM (%) to avoid prob-

lems with collinearity.

RESULTS AND DISCUSSION

Careful consideration was taken in the selection of 20

geographically different, chemically variant, undisturbed

rangeland soil samples. Soil samples were identified

based on their pH, clay content, and total C (USDA soil

characterization database), making sure the final sample

group precluded the excessive influence of one or a few

observations on the overall conclusions. Soil pH ranged

from 4.8 to 7.9; clay content varied between 0 and

44.2%, and total C values ranged from 0.9 to 26.3%

(Table 1). In addition to these precharacterized soil

properties, C/N ratio, organic C/N ratio, inorganic C

contents, and organic matter contents were analyzed in

the laboratory upon receipt of the soils and were in-

cluded in the evaluation of the relationship between

soil properties with glomalin concentrations. The Pear-

son correlation coefficients are shown in Table 4.

Soil pH, Total C Content, Organic Matter Contentand Glomalin

There was a significant negative correlation between

pH and both easily extractable (G1) and total glomalin

protein (G2), shown in Table 4 and also in Figure 1.

These results indicate that as pH increases, there is a

decrease in the amount of glomalin present in the soils.

This observation is not consistent with data for the

immunoreactive fractions of glomalin (IR-G1 and IR-

G2) that appear to be unaffected by soil pH (Table 4).

Soils low in pH may have an increase in total glomalin

protein as a result of increased organic activity (be-

cause of increased concentrations of organic matter) at

lower pH values (Table 4). Previous studies have also

linked an increase in total glomalin to an increase in

soil organic matter content (Bird et al., 2002). Fungi

tend to predominate in more acidic soils. In higher pH

soil environments, fungi still grow, but they meet

Table 2. Measurement of Total Glomalin Protein and Their

Corresponding Immunoreactive Fractions for the Mattapex Soil

Used in Generating the Standard Curve for ELISA

Mattapex soil G1 (mg/g) IR-G1 (%) G2 (mg/g) IR-G2 (%)

Replicate 1 0.94 96.7 0.53 84.8

Replicate 2 1.01 100.0 0.55 80.9

Replicate 3 1.01 95.9 0.58 70.6

Table 3. Mean Values for G1, IR-G1, G2, and IR-G2 with

Standard Deviation in Parenthesis for All Samples

Sample G1 (mg/g) IR-G1 (%) G2 (mg/g) IR-G2 (%)

A 3.477 (0.032) 20.0 (0.7) 3.719 (0.116) 35.3 (0.4)

B 1.018 (0.011) 12.9 (0.4) 2.821 (0.072) 15.7 (0.2)

C 2.906 (0.122) 24.9 (0.4) 7.078 (0.130) 10.7 (1.3)

D 0.340 (0.004) 41.3 (4.9) 4.574 (0.321) 7.0 (0.5)

E 4.352 (0.314) 7.9 (0.7) 7.838 (0.391) 2.0 (0.1)

F 3.945 (0.170) 71.5 (2.8) 11.657 (0.088) 57.3 (2.5)

G 0.949 (0.026) 33.9 (0.5) 0.734 (0.065) 29.1 (0.8)

H 0.722 (0.018) 91.4 (7.6) 4.024 (0.231) 9.4 (0.5)

I 4.516 (0.146) 59.3 (1.2) 18.488 (0.188) 23.1 (0.1)

J 0.437 (0.014) 39.4 (1.3) 1.810 (0.314) 34.7 (4.5)

K 1.006 (0.024) 85.4 (1.6) 6.471 (0.272) 18.7 (0.6)

L 0.199 (0.016) 62.8 (4.2) 2.132 (0.111) 12.7 (1.9)

M 1.813 (0.124) 9.0 (1.7) 2.259 (0.258) 10.1 (0.3)

N 2.018 (0.087) 5.7 (1.1) 3.424 (0.120) 8.3 (0.8)

O 7.325 (0.370) 6.7 (0.3) 13.479 (0.372) 13.5 (1.1)

P 5.702 (0.191) 21.2 (1.0) 28.647 (5.187) 58.1 (9.5)

Q 3.370 (0.066) 28.0 (2.7) 20.378 (0.644) 59.5 (0.5)

R 1.041 (0.014) 96.4 (0.8) 4.279 (0.312) 49.1 (6.1)

S 6.133 (0.154) 44.7 (0.3) 20.352 (0.529) 39.0 (0.9)

T 1.355 (0.112) 99.3 (6.5) 8.079 (1.540) 96.7 (15.9)

102 Glomalin in Soil Organic Matter as a Function of Soil Properties

competition from bacteria and other organisms and

therefore may not be as active as they are in acid soils

(Brady, 1990). Because glomalin is produced by AM

fungi, more protein is expected in the more acidic soils

because of an increase in the activity of AM fungi and

lower competition. However, the immunoreactive frac-

tion of glomalin, which is considered to be responsible

for the gluelike properties of the protein, is not influ-

enced by soil pH.

Total C demonstrated a positive correlation with

all fractions of glomalin (Table 4). The correlations

were significant and more robust for the G2 and IR-G2

fractions than for the easily extractable fractions of glo-

malin. The linear regression of total glomalin with total

C is shown in Figure 2. The G2 fraction yielded much

higher regression coefficient and much larger slope

compared to the G1 fraction; a trend that appeared to

be quite common and reflected by other soil properties

under consideration in the study. This suggests that

perhaps the G2 fraction of glomalin, despite being more

tedious in its extraction, is a more thorough (and more

complete) means of accounting for and explaining the

relationship of glomalin to various soil properties. The

results obtained from this study describing the relation-

ship of G2 to total C in soils support previous research

that recognized glomalin as a distinct component of soil

organic matter that can account for as much as 30% of

soil carbon (Nichols et al., 2000). Results from this

study indicate that total C content in soils alone can

explain in excess of 40% variability in soil glomalin con-

centration. Apparently, total soil C is a robust indicator

of the presence of glomalin protein in soils.

Organic matter was positively correlated with the

protein fractions of glomalin (Table 4; Figure 3). This

relationship was similar to that seen with total soil C;

the correlation of the G2 fraction with SOM was much

greater than that of the G1 fraction. Again, this implies

that G2 may be a much better representation of glomalin

Table 4. Correlation Matrix Representing the Measured Soil Properties and G1, IR-G1, G2, and IR-G2y

pH Clay (%) Total C (%) SOM (%) C/N Ratio Organic C/N Inorganic C (%) G1 IR-G1 G2 IR-G2

pH 1.00 0.21 �0.30* �0.39* 0.27* �0.11 0.20 �0.71* 0.28* �0.66* 0.01

Clay (%) 1.00 �0.46* �0.43* �0.39* 0.04 �0.33* �0.27* 0.25 �0.30* �0.52*

Total C (%) 1.00 0.43* �0.35* 0.59* 0.28* 0.02 0.65* 0.58*

SOM (%) 1.00 0.26* �0.32* 0.42* 0.32* �0.08 0.68* 0.45*

C/N ratio 1.00 �0.32 0.89* �0.05 0.48* 0.18 0.74*

Organic C/N 1.00 �0.32* �0.20 �0.02 �0.26* �0.13

Inorganic C (%) 1.00 �0.04 0.43* 0.19 0.80*

G1 1.00 �0.39* 0.76* 0.12

IR-G1 1.00 �0.08 0.43*

G2 1.00 0.39*

IR-G2 1.00

yyValues marked with * are significant at p < 0.05. Total C (%) and SOM (%) were not evaluated simultaneously.

Figure 1. Regression analysis of total glomalin protein and pH(* denotes significance at p < 0.05).

Figure 2. Regression analysis of total glomalin protein andtotal C (ns and * denote nonsignificance and significance atp < 0.05, respectively).

Haddad and Sarkar 103

in soils, as the G1 fraction does not appear to account

for glomalin in its entirety. Glomalin has already been

shown to be a substantial component of soil organic

matter (Nichols et al., 2000), and therefore, its con-

centration in soils was expected to be strongly corre-

lated with SOM. There was a small positive correlation

with IR-G2, which might be a result of AM fungi ac-

commodating to the environment by modifying the

surrounding soil to benefit the symbiotic relationship

between itself and the host plant (Rillig and Steinberg,

2002). Because it is possible that the immunoreactive

fraction of glomalin is a product of active hyphae, it is

conceivable that this production of fresh glomalin ac-

counts for its close association with SOM and soil car-

bon to create an environment favorable to nutrient

transport to the host plant.

C/N Ratio, Inorganic C Content andImmunoreactive Glomalin

The C/N ratio demonstrated a significant positive cor-

relation with the immunoreactive fractions of glomalin

(Table 4; Figure 4) and suggests that the C/N ratio can

explain in excess of 55% variability in immunoreactive

glomalin concentrations in soils. Bird et al. (2002) iden-

tified the C/N ratio as the best indicator of soil aggregate

stability and demonstrated a strong, positive correlation

between the C/N ratio and immunoreactive glomalin.

The results of the current study are in agreement with

those of Bird et al. (2002) and also support the findings

of earlier research (Wright and Upadhyaya, 1998) that

suggests a strong correlation between IR-TG and soil

aggregate stability.

Inorganic carbon demonstrated a significant posi-

tive correlation with the immunoreactive fractions of

glomalin (Table 4; Figure 5). The symbiotic association

of AM fungi with plant roots may explain this obser-

vation. Plants take in inorganic carbon in the form of

carbon dioxide from the atmosphere. Plants then al-

locate a portion of this fixed carbon to their roots and

the soil (Rillig et al., 1999). The fixed carbon is used

to develop further AM fungi that are attached to the

plant roots (Rouhier and Read, 1998). This allows for

increased nutrient uptake from the soil, causing the

hyphae to become more active. These active hyphae

produce fresh glomalin, which, in turn, helps to im-

prove soil structure, easing the passage of air and water

and increasing resistance to erosion. This also leads to

an increased ability for the soil to hold on to valuable

organic matter and soil carbon. This newly generated

glomalin from the fresh hyphae is believed to be in a

conformational state that results in glomalin’s gluelike

properties (Wright et al., 1999). This is associated with

the immunoreactive fraction of glomalin and therefore

offers a possible explanation to the significant influ-

ence of inorganic carbon on immunoreactive glomalin

concentrations in soil. In areas with soils containing

greater amounts of inorganic carbon, it is possible that

the relationship between the AM fungi and plant roots

is more active, resulting in glomalin that is highly

immunoreactive.

Clay Content, Organic C/N and Glomalin

The correlation of clay content with the glomalin pro-

tein fractions yielded small negative values (Table 4),

indicating that as clay content increases, glomalin con-

centration decreases in soils. A regression analysis

(data not shown) revealed an R2 value of 0.07 for the

G2 fraction, and that of 0.09 for G1 (0.05 level of

significance). These results are similar to those obtained

by Rillig and Steinberg (2002) from the angle of soil

Figure 3. Regression analysis of total glomalin protein andSOM (ns and * denote nonsignificance and significance atp < 0.05, respectively).

Figure 4. Regression analysis of immunoreactive fractions ofglomalin and C/N ratio (* denotes significance at p < 0.05).

104 Glomalin in Soil Organic Matter as a Function of Soil Properties

aggregate stability, because clays are considered to in-

crease soil aggregation. In their study, Rillig and Stein-

berg (2002) used different-sized glass beads to repre-

sent different levels of aggregate stability and demon-

strated that the representative nonaggregated soil had

the maximum increase in glomalin protein. They ex-

plained that in the nonoptimal conditions of a non-

aggregated environment, AM fungi circum to their

surroundings by becoming more efficient at producing

what they need. In their case, they viewed the increase

in glomalin necessary to potentially lead to an increase

in aggregate stability. In the current study, as clay con-

tent increased, there was a decrease in glomalin protein.

Applying the above explanation here implies that per-

haps the decrease in protein was caused by an asso-

ciated increase in optimal aggregate conditions with an

increase in clay content.

Organic C/N ratio was also examined but was

deemed not significant in pairwise comparisons with

the different glomalin fractions. All correlations yielded

small negative values (Table 4). This observation, in

conjunction with the results for inorganic carbon, sug-

gests that perhaps other fractions of carbon aside from

organic carbon are influencing the presence and the

immunoreactivity of glomalin protein in soils.

Collective Evaluation of Properties

A multiple regression analysis of the soil properties

with G1 and G2, as well as the respective immuno-

reactive fractions (IR-G1 and IR-G2), indicated that

collectively, all the soil properties considered in the

study (pH, clay content, total C, total C/N, organic

C/N, inorganic C, and SOM) can account for a signif-

icant amount of the glomalin found in soils. The result-

ing equations explaining each of these fractions and

their respective R2 values are shown in Table 5. The R2

values for the easily extractable fractions (G1 and IR-

G1) were much lower than those corresponding to the

G2 and IR-G2 fractions. This analysis supported the

pairwise comparisons which found total C and inor-

ganic C to be the strongest predictors for immunore-

active glomalin in soils. In addition, total C, inorganic

C, and pH were all found to be significant in their

predicting capabilities for total glomalin protein in

soils. Whereas clay content and organic C/N were not

strongly correlated with glomalin in the individual

pairwise comparisons, the multiple regression analysis

demonstrated that when the properties are collectively

evaluated, both of these properties are important in

predicting the presence of glomalin.

SUMMARY AND CONCLUSIONS

Soil organic matter is the most important component

of soil when it comes to understanding soil structure

and its potential impact on the environment. Gloma-

lin is a critical portion of SOM that, despite its proven

abundance, continues to structurally and functionally

perplex those who realize its enormous agronomic and

environmental significance. The current study focused

on the basics of understanding glomalin as it relates

to various soil properties. The results indicated the

following:

Figure 5. Regression analysis of immunoreactive glomalin frac-tions and inorganic carbon (* denotes significance at p < 0.05).

Table 5. Predicting Equations Determined from Multiple Regression Analysis for G1, IR-G1, G2, and IR-G2 with Corresponding

Regression Coefficients*

Fraction Predicting equation R 2

G1 = 13.06 � 1.45a � 0.027b � 0.036d � 0.017f 0.60

IR-G1 = �49.76 + 1.24b � 376.2c + 219.1d + 5.31e + 0.131f + 369g 0.55

G2 = 18.39 � 3.50a + 0.107b + 60.4c � 34.6d + 0.712e � 62.5g 0.75

IR-G2 = 19.50 � 0.461b + 13.9c � 7.84d + 0.085f 0.73

*a = pH, b = clay (%), c = total C (%), d = SOM (%), e = C/N ratio, f = organic C/N, and g = inorganic C (%). R 2 values are significant at p < 0.05.

Haddad and Sarkar 105

1. Soil pH is negatively correlated with glomalin pro-

tein in soils. As soil becomes more acidic, glomalin

concentrations increase, and as soil pH increases,

glomalin concentrations decrease.

2. Total C and SOM are positively correlated with

glomalin protein in soils. As the values of total C and

SOM increased, the concentrations of glomalin pro-

tein increased as well. The relationship of both of

these properties is similar in that the easily extrac-

table (G1) fractions did not represent the complete

glomalin fraction that existed in the soil sample. The

G2 fractions yielded much stronger correlations.

3. C/N ratio and inorganic C are positively correlated

with the immunoreactive fractions of glomalin in

soils. Inorganic C serves as an energy source for AM

fungi to increase the production of glomalin; freshly

produced glomalin is more immunoreactive and

more characteristic of a soil-glue. In addition, if C/N

ratio is considered to represent aggregate stability,

the results from this study support earlier research

that found immunoreactive glomalin to be strongly

correlated with soil aggregate stability.

4. In individual pairwise comparisons, clay content and

organic C/N ratio were not as strongly correlated

with soil glomalin fractions as the other soil prop-

erties evaluated. However, When evaluating all soil

properties collectively, both clay content and or-

ganic C/N were deemed important factors in pre-

dicting the presence of glomalin in soils.

5. For evaluations of glomalin as it relates to other

variables in the soil environment, it is not necessary

to measure easily extractable glomalin as is the

current trend. These results strongly suggest that the

easily extractable fraction is not an accurate rep-

resentation of glomalin in soil.

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106 Glomalin in Soil Organic Matter as a Function of Soil Properties