Final Report - Center for Invasive Plant Management

Number of text pages: 22Number of tables: 3Number of figures: 65 Shorlrunnins title: Seasonality of AM hyphae and glomalinAuthor of correspondence: Dr. Matthias C. Rillig, The University of Montana, Division ofBiological Sciences, HS 104, Missoula, Montana 5981?, U.S.A.; +l (406) 243'2389. FAX +1(406) 243-4184, email matthias@mso umt.edui0t520

Seasonality of arbuscular mycorrhizal hyphae and glomalin in awestern Montana grasslandEmily R. Lutgenr, Deborah Muir-Clairmontz, Jon Graham3, Matthias C. RilligttMicrobial Ecolog; Program, Dilr.rlon of Biological S'ilelces., 7-he {hiversit-v- of A,{ontana,Missoula, MT 59812, {/SA:tSalish Kootenai College, I).O. Rox 117, Pablo,lvtT' 59555, USA:sDepartment af lu{athematicctl Sciences, 7-he (hir'{rsity af Montana, Missoula, MT 598}2, {iS.,ll0Kelnvords', root solonization, soil moisture, AMF hyphal products, root lengthAbstractt5In order to more fully understand the basic biology of arbuscular mycorrhizal fungi (AMF), andtheir role in natural ecosystems, it is necessary to document seasonal changes of various aspectsof the life history of these fungi. Due to their unique position at the root-soil interface, AMFhave been described as "keystone mutualists" in ecosystems. Despite the importance of AMF inecosystems, few studies exist that examine the seasonality of external hyphae and their exudedz0products (e.g. glomalin), the AMF parameters directly related to ecosystem function throughtheir contributions to soil aggregation. This study examined seasonal dynamics of several soilparameters, with a specific interest in the seasonality of external hyphae and glomalin" aglycoprotein produced by AMF fungi (which is correlated with soil aggregate stability). Here

we measured glomalin concentrations and external AMF and non-AMF hyphal length. as well assoil moisture, percent fungal colonization (AMF and non-AMF), and root length in soil in anintermountain grassland in western Montana over one growing season (13 time points). Of theglomalin pools and hyphal lenghs measured, significant seasonal changes occurred for totalglomalin (TG;2a.5% change), immunoreactive easily extractable glomalin (IREEG; 53.8%change), and AM hyphal length (10790 change). Prior studies on glomalin in natural systemshave not considered seasonal effects on the measured glomalin. The seasonality of glomalinvalues observed in this study highlights the importance of implementing a sampling regime thatwill capture this seasonal variation. These results also provide valuable information for thel0development of future studies in this type of natural ecosystem.IntroductionAMF are obligately biotrophic fungi that are closely associated with both host plants and the soil15environment, functioning as a quasi-extension of the root system, and having numerous effectson plant physiology and plant communities (e.9. Allen 1991, Smith and Read 1997, van derHeijden et al 1998). AMF play an integral role in the translocation of carbon to soil, havingdirect access to root carbon (Smith and Read 1997). due to their unique position at the root-soilinterface, AMF have been described as "keystone mutualists" in ecosystems (O'Neill et al.2A1991).In order to more fully comprehend the role of AMF in natural ecosystems, as well as theirbasic biology, it is important to document seasonal changes of various aspects of the life historyof these fungi. While numerous studies have examined seasonality of AMF spore production

and root colonization (e.g., Gay et al. 1982, Anderson et al. 1984, Johnson et al. 1991, Sandersand Fitter T992, and Mullen and Schmidt 1993), studies examining the seasonality of AMFextraradical hyphae and their exuded products are sparse. Although fungal spore production androot colonization are important for elucidating fungal life histories (Hart and Reader 20QZ),neither of these characteristics directly relate to ecosystem function. Conversely, extraradicalhyphae and their products, such as glomalin. can be directly related to ecosystem processes, e.g.by virtue of their contributions to soil aggregate stability (Jastrow and Miller 1997, Wright andUpadhvahya 1998). More specifically, AMF contribute to soil aggregation through the hyphalentanglement process, assisting in soil aggregate formation (Jastrow and Miller 1997), and10through the production of extracellular polyrneric compounds on hyphal surfaces, which can sorbto inorganic materials, helping to stabilize soil aggregates (Jastrow and Miller 1997). As anexample, extraradical hyphae of AMF produce glomalin" a glycoprotein that is highly correlatedw-ith the percentage of water-stable aggregates (WSA) in soil (Wright and Upadhyahya 1998,Rillig et al. 2001)l5To our knowledge, only two studies have examined the seasonality of extraradicalhyphae in the field, and only one of these studies examined hyphae from a natural system. Kabiret al. (1997) examined the seasonal changes ofextraradical and intraradical arbuscularmycorrhizal hyphae affected by tillage and fertilization in an agricultural soil over a growing20 season (n=4). Abundance of AN{ hyphae fluctuated significantly within a growing season, withlowest hlphal densities found in the spring. Seasonal variation in mycorrhizal root colonizationfollowed corn plant development. increasing up to silking and decreasing thereafter. Miller et al(1995) examined external hyphal production and its relation to gross root morphology (specific

oot length: SRL) over a season in two temperate grassland communities (n:4). SRL wasstrongly associated with external hyphal lengths, where root systems with low SRL had greaterlengths of external hyphae. From these studies it is clear that important seasonal patterns inextraradical hyphal length may exist, but the database is far too small to draw firm conclusions.Seasonal dynamics of AMF hyphal products, such as glomalin, in the soil are unknown.The field study described herein examines the seasonality of AMF extraradical hyphaeand glomalin in a spotted knapweed invaded grassland. To relate these parameters to intraradicalcolonization, we also measured AMF root colonization. Total root length and the root length ofl0 two root diameter size classes were also measured to examine the relationship of hyphae andtheir products to plant root morphology.Materials and Methods15Site descriptionThis research site was located in a grassland with an initial invasion of spotted knapweedapproximately I km north of Missoula, Montana, in the North Hills area" The plant communityof this area is an Idaho fescue/bluebunch wheatgrass community type (b'estucaidahoenisislAgropyroft spicatam) (Mueggler and Stuart 1980). The soil at this site is a sandy20loam with pH 6.6 (Table l).Field experiment and samplingSoil cores (2 cm diameter) were extracted systematically along a 5-meter transect, with 3-4 soilsamples taken within a l5 x l5 cm area and pooled every meter (n=5) Samples were taken

eginning on May 13, 2001, and then approximately bi-weekly until the last sampling date,November 30, 2001, lor a total of l3 time points. Samples were repeatedly taken within thesame i 5 x 15 cm area and 5-m transect through time. Gravimetric soil moisture was determinedon a subsample of soil (5 g) from each sample at each time point. All soil samples were thendried overnight at 70 'C. Soil samples were sealed in polyethylene bags and stored at *20 oCuntil analysis.lt{onthly precipitation and temperature data were obtained from the MissoulaInternational Airport rveather station, approximately I I km southwest of the study site (WesternRegional Climate Center and National Climatic Data Center).l0Extraradi cal hyphal and glomali n nreasurementsExtraradical hyphae were extracted from soil samples (4 g) using an aqueous extraction andfiltration method (Rillig et al. 1999). Arbuscular mycorrhizal (AM) hyphae were distinguishedfrom non-mycorrhizal hyphae at 200x magnification using similar criteria to Miller et al. (1995).l5Hyphal length was determined using the line intersect method as described in Jakobsen et al.(1992) and Tennant (1975).Two detection methods are used to quantifu glomaiin: the Bradlord protein assay,yielding the easily extractable -qlomalin(EEG) and the total glomalin (TG) liactions, and anELISA assay (employing the monoclonal antibody developed against crushed spores of Glnmus2Aintrsradices; Wright and Upadhyahya 1998), yielding the irnrnunoreactive easily extractableglomalin (IREEG) and immunoreactive total glomalin (IRTC) fractions. These glomalinfractions are operationally defined based on their extractabilitylsolubility and detection methods(much like other soil fractions" such as humic acids). While the ELISA assay is a very specific

detection method for glomalin, the rnore general Bradford protein assay is also utilized. Thisprotein assay may capture glomalin protein that has undergone small (perhaps microbiallymediated)changes" possibly resulting in the destruction or concealment of the epitope for themonoclonal antibody. Because of well-documented and strong correlations with soil aggregatestability (e.g., Wright and Upadhyahya 1998). these glomalin fractions continue to be quantified.Glomalin extractions from soil (1 gram) were carried out as described by Wright andUpadhyahya (1998). The EEG fraction was extracted with 20 mM sodium citrate, pH 7.0 at121o C for 30 minutes. Fotlowing the EEG extraction, the TG tiaction was extracted with 50mM sodium citrate, pH 8.0 at l2l nC for 60 minute cycles until the supernatant showed none of10the red-brown color typical of glomalin. Both fractions of glomalin were analyzed using theBradford Protein Assay (Bio-Rad, Melville, NY). The glomalin ttactions were further analyzedusing an enzyme-linked immunosorbent assay (ELISA) using the monoclonal antibodyMAb32Bl1. After all glomalin analyses were completed, four fractions of glomalin values wereobtained: EEG. TG. IREEG. and IRTG.l5Root extracti on and rpt anti fi cal ionRoots were removed from the soil samples by a hand flotation and sieving method modifiedfrom Cook et al. (1988) and Miller et al. (1995). A l0 g subsample fron'r each soil sample wassoaked in 100 ml of tap water and20 ml sodium hexametaphosphate (35 g L-t) for 30 minutes.?nThe soil suspension was then added to 880 n{ tap water (total volume: 1000 ml), manuallyagitated to suspend roots, and poured through 212 pm and 0.5 mm sieves to retain roots. Thisprocess was repeated five times with all soil samples to maximize retrieval of roots. Separatedroots were washed with tap water several times to remove any attached soil. Obvious organic

material and other debris were removed. Extracted roots were dried in a drying oven at 70 oCovernight and stored at room temperature until analysis. Total root length and root lengths oftwo root diameter size classes, fine roots (>0.35 mm diameter) and very fine roots (

Pearson product-moment correlations (r) on the means (n=13) were determined in the procedureof JMP (version 3.1.6.2,1996). The coefficient of variation (cov) was calculated by dividing thestandard deviation of means by the grand mean for each response variable. The percent changefor response variables was calculated as ((mean X*o, - mean X'*) / mean X*6)*100.ResultsRepeated measures multivariate analysis of the response rtariables measured revealed asignificant difference (RM-MANOVA: Frso.rrr: 2.413, P

Variation in the IREEG fiaction, but not the IRTG fraction, was significant through time(Table 3). and also had a low coefficient of variation (Figure 2b)" There was a 53.8% changebetween the lowest and highest average concentrations of [REEG. An initial decrease in theconcentration of IREEG occurred from May to June (Julian day 133 to 162), with IREEGconcentrations generally rising thereafter until October (Julian day 286). After October. theconcentration of IREEG again decreased (Figure 2b). The IRTG pattern through time wassimilar to that of the IREEG fraction (Figure 2b). However" due to large error bars of someIRTG data points, there was not enough power to observe a significant variance of the IRTGl0concentrations throu gh time.External arbuscular mycorrhizal (AM) hyphal length varied significantly through time(Table 3), with a large increase from late September to early October (Julian day 265 to 286),followed by a decrease in hyphal length through November (Julian day 286 to 334, Figure 3).15 The decrease in AM hyphal length at the end of the sampling period was similar to the demeaseobserved in the IREEG fraction. The percent change between the highest and lowest AM hyphallengths measured was 1079/o. Non-AM hyphal length did not vary significantly through time(Table 3).20Percent AM hyphal colonization decreased in November (Julian days 307 to 334), similarto the decrease in external AM hyphal length (Figures 3 and 4; Table 3). Percent vesiclecolonization did not vary significantly, while percent arbuscule colonization varied significantlythrough time (Table 3). with great fluctuation from May to the beginning of August (Julian dayst0

133 to 215), followed by a considerable decrease (Julian day 230;Figure 4) until the end of thesampling period. with colonization leveling out at 2% (Figure 4).Fine root length (>0.25 mrn diameter) changed significantly through time, while very fineroot length (

DiscussionThe main objective of this study rvas to test whether seasonal changes in glomalin ti'actionsoccur o\,'er a growing season. ln a previous studv. glomalin had a long tumover time (up to 40years), much longer than the turnover time expected for AM hyphae (Rillig et al. 2001, Frieseand Aiien i llq l). ln a lab incr-rbation comparing glomalin and AMF extraradical hypiraedecomposition, glomalin concentrations only decreased by 250",ii over 150 days, while AMFextraradical hyphal length declined 6096 (Steinberg and Rillig 2003). Currently there is nointbrmation on tire seasonalitv of slomalin.lnConcentrations of the TG and IREEG glomalin fractions changed significantly throughtime, while the EEG and IRTG fi actions did not. This is the first report of seasonal changes ofpools. At this time, it is unclear how these operationally-elomalindetined glornalin pools differfrom each other in terms of biochemistry, function in soil, and age. or how the production ofglomalin is controlled (Ri[ig et al. 2001). fhe patterns of change in the TG and IREEGl5concentrations through time are generally dissimilar.Several fungal parameters fluctuated significantlv through time in the soil studied.External AM hyphal length changed significantly over the growing season, with a decrease inNovember. This decrease is sirnilar to the decrease noted in the IREEG fraction. Hyphal length20values measured in this study were fairly high, w-ith the highest hvphal length measured to be 82m g-1 soil in October. and an average hyphal length throughout the season of 50 m g'r soil. Ourhyphal length values were higher than hyphal lengths from several studies reported in Smith andGaninazi-Pearson (1988). It is not uncommon for hyphal lengths to vary considerably from12

study to study, due to varying fungal species and different geographic areas (Smith andGianinazi-Pearson i988). Hor.vever, external hyphal iengths from this study are consistent withhyphal lengths measured in a diflbrent intermountain grassland in westem Montana (Lutgen andRillig submitted). where hyphal lengths reached 45 m -c-rsoil.Percent AM and non-AIVI hyphal colonization in roots decreased in November, similar tothe decrease in AM hyphal length. However, the decrease in fungal hyphal colonization beginsearlier in the sampling period. during August. A dramatic decline in percent AM arbusculecolonization also oceurs at this time. It is likely that plants were stres$ed from the drought10 conditions during the sampling period" and the carbon allocation from plant to fungus may havebeen diminished as a consequence. Because AM arbuscules are the structures involved innutrient exchange r.vith plants, the drought response in plants could be reflected quitedramatically in percent arbuscule colonization.15 Root length parameters measured did not exhibit much significant change through time,with only the fine root length (>0.25 mm diameter) parameter changing through time. Themajority of root iength measured was found in the very fine root length (

observed in the IREEG protein fraction. Interestingly, the trade-offexperienced bv plant rootsystems between the production of fine roots and external ltyphal associations (Miller et al.1995) was not observed, For example. an increase in external AM hyphal length did not occurwhen fine root length decreased. Instead" both parameters seemed to decline together. Althoughmycorrhizal infections are generally considered beneficial, mycorhizal fungi can represent acarbon drain on plants (Allen 1991). ln tbct. it has been fcrund that some species r"rf arbuscularmycorrhizao can astilally reduce plant tolerance to drought (Allen and Boosalis 1983). By latesummer during the sampling period, both plant and fungus could have suffered from a carbondeficit due to the severe drought conditions experienced during the sampling period.10Correlations between glomalin tiactions and root parameters were only obsen'ed betweentlre IREEG fraction and all root length paran"teters. As previously mentioned, it is unclear horvthese operationally defined glomalin pools differ lrom each other. It has been suggested that theEEG and IREEG pools are more readily available in the soils, either by being the most recentlyl5deposited fractions (Wright and Upadhyahya 1998) or by being the rnost recently deconipasedfractions (Steinberg and Rillig 2003). It has been suggested that degradation of glomalin in thesoil also decreases its adsorption to soil particles (Steinberg and Rillig ?003).Plants produce more roots during certain times in a growing season due to environmental20 factors. During this active root production, plant roots release exudates that are a labile source ofcarbon tbr the soil microbial community, enhancing the environment fbr decomposers (saprobesand bacteria). Priha et al. (1998/1999) lound that two species of trees had a stimulative effect onsoil microbes. possibly due to the tree species having more roots and releasing more root14

exudates in soil. Perhaps root production in the current study (represented here by root length)seln*'ed as a priming etlect fbr the microbial community by producing abr-rndant exudates andenhancing the environrnent for decomposers. As a result, these abundant decomposers degradedthe more readily available EEG and IREEG fractions, decreasing their adsorption to soilparticles. Hence. an increase in root length may have resulted in an increase in either of thesefractions. as observed with the IRHEG l'i'action.The sampling schedule for this study was quite intense (soil cores extracted bi-weeklyfrom N{ay through November 2001 , I3 time points) compared to other iield studies on AM10hyphal dvnamics, partioularly those that measured external hyphal lengfh (four time points;Miller et al. 1995, Kabir et al. 1997). Our sampling frequency allowed for a more detailedresolution of the seasonal patterns of AM hyphal dvnamics and glornalin pools.Although glomalin has a slow turnover time in soil, it appears that some _elomalin15fractions can fluctuate through a growing season" Changes in AM fungal and root parametersdid not exactlv niatch changes observed in glomalin concentrations, However, the decline inIREEG concentration in November is comparable to the general decrease in AN{ fungal and rootparameters in late sumnler through the end of the sampling period. The overall lack ofcorrelation betrveen AM fungal parameters and glomalin is important. as it points out tlrat20glomalin fractions, as cuffently detined, may not be useful as indicators of AMF hyphal lengthand fungal activity in general.l5

Although this study was conduoted only over one growing season, it is the first study toexamine glomaiin concentrations (an AM hyphal product) over a grorving season in auinterrnountain grassland community. Every studv on glomalin in natural ecosvstellls thus thr hasnot considered the importance of seasonal fluctuations in glomalin (since only one time point5 was examined). Our study indicates that. i.vhile fluctr-rations in this seemingly stable soil pool arerelatively small, they were nevertheless significant itr our study. Hence a sampling regintecapturing seasonal variation may have to be employed in future studies measuring glomalin.Acknowledgmentsl0E.R.L. and N{.C.R. acknowledge funding fbr this project by the Center for lnvasive PlantManagement, located at Montana State University, the National Science Foundation, and thet-i.S. Department of Energy. D.M-C. acknowledges funding from Project TRAIN (TrainingAmerican Indians in Environmental Biology) at the University of Montana. We thank Dr S Fl5 Wrisht for NfAb32B11.l6

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Figure captionsFigure 1. Average percent soil moisture through time (Julian days). Standard errors of the mean(n:5) and coefficient of variation (cov) are shown. Univariate repeated measures ANOVA p-5 value is presented in Table 3. cov:variability of the means (n:13)Figure 2a. N{ean concentrations of glomalin fractions through time (Julian days) Note scale andaxis break Standard errors of the meal {n:5) and coefficient of variation (cov) are shown.Univariate repeated measures A].{OVA p-values are presented in Table 3. cov:variability of thei0 means (n:13)Figure 2b. Mean concentrations of immunoreactive glomalin fractions through time (Juliandays). Note scale and axis break. Standard errors of the mean (n-5) and coefficient of variation(cov) are shor,vn. Univariate repeated measures ANOVA p-values are presented in Table 3.l5 cov:variability of the means (n:13)Figure 3. Mean external hyphal length of arbuscular and non-arbuscular mycorrhizae throughtime (Julian days). Standard effors of the mean (n=5) and coetEcient of variation are shown.Univariate repeated measures ANOVA p-values are presented in Table 3. cov:variability of the20means {n:13)Figure 4. Average percent colonization of arbuscular mycorrhizal (AM) hyphae, vesicles, andnon-AM hyphae through tine (Julian davs). Standard erors of the mean (n:5) and coefiicient of

variation (cov) are shown. Univariate repeated measures ANOVA p-values are presented inTable 3. cov:variability of the means (n:13) Inset: Mean arbuscular colonization through time(Julian days). Standard errors of the ntean (n:5) and coetXcient of variation (cov) are shown.Univariate repeated measures AI'IOVA p-value is presented in Table 3. cov:variability of themeans (n:13)Figure 5. Mean total root length, fine root length (>0.25 mm dianieter), and very fine root length(