Litter quality inuences on decomposition ectomycorrhizal

Soil Biology & Biochemistry 32 (2000) 489±496

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Litter quality in¯uences on decomposition, ectomycorrhizal community structure and mycorrhizal root surface acid phosphatase activity Christine Conn, John Dighton* Rutgers University Pinelands Field Station, P.O. Box 206, New Lisbon, NJ, 08064, USA Accepted 24 September 1999

Abstract The in¯uence of litter quality on root growth, ectomycorrhizal communities and decay processes was investigated through a litter bag experiment. Litter bags containing either pine needles, oak leaves or oak+pine mix were placed within the O horizon of a lowland pitch pine (Pinus rigida ) forest in the New Jersey Pinelands. Upon retrieval, ingrown pine roots were removed and quanti®ed for total length and percent ectomycorrhizal colonization by morphotype. Phosphatase activity was determined for dominant morphotypes. In addition, litter decay rates and N and P litter content were measured. Mixed litter (oak+pine) had highest total pine root ingrowth. Dominant ectomycorrhizal morphotypes di€ered in response to litter type. A tuberculate form dominated (35%) in pine litters while distinctly di€erent nontuberculate morphotypes dominated in oak and mixed litters. High phosphatase activity of morphotypes was correlated with high phosphorus immobilization during oak leaf decay. Results indicate that a mix of forest litters (oak and pine) optimizes retention of scarce nutrients such as nitrogen and phosphorus. The diverse chemical environment of these di€erent litter types induces di€erent ectomycorrhizal community development which show functional di€erences in the way phosphorus is likely to be cycled. The in¯uence of litter type on diversity and function of ectomycorhizae is an important step in identifying linkages between biodiversity of this group and ecosystem functions. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Litter decay; Oak+pine forest; Phosphorus cycling; Mycorrhiza

1. Introduction The development of mycorrhizae increases the ability of roots to absorb nutrients and water from the soil environment. The degree to which this ability is enhanced depends both on the extent of mycorrhizal colonization and the unique functional attributes of the mycorrhizal fungal species involved. Community structure of ectomycorrhizas on a root system may be related to nutrient uptake eciency if di€erent mycorrhizal species have di€ering abilities to sequester nutri* Corresponding author. Tel.: +1-609-894-8849; fax: +1-609-8940472. E-mail address: [email protected] (J. Dighton).

ents from the soil solution (Dighton, 1995). The structure of the ectomycorrhizal community is in¯uenced by competitive interactions between the colonizing mycorrhizal fungi and, possibly, between mycorrhizal and saprotrophic fungal species (Shaw et al., 1995). Edaphic factors, such as anthropogenic pollution, also have been shown to in¯uence mycorrhizal abundance and community structure. Dighton and Skengton (1985) showed that acidifying pollutants altered ectomycorrhizal community structure by reducing abundance of species producing large amounts of extramatrical mycelium. Other e€ects of acidifying pollutants on mycorrhizal communities are reported in Jansen et al. (1988) and are modeled in Dighton and Jansen (1991). Nitrogen amendment in a spruce forest produced di€erent ectomycorrhizal species composition

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 7 8 - 9

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and decreased root tip number in relation to an unamended community as observed by mycorrhizal morphology (Alexander and Fairley, 1983) and molecular taxonomy (KaÊreÂn and Nylund, 1997). Increased soil nitrogen and phosphorus can also reduce ectomycorrhizal development (Brun et al., 1995) and increase root growth (Pregitzer et al., 1993; Weber and Day, 1996). Heterogeneity of resource units in soil as an in¯uencing factor has been less well documented. Pregitzer et al. (1993) have shown that patchiness of nutrient addition to soil altered root growth patterns and Baar et al. (1994) showed that removal of litter and humus from the forest ¯oor altered the pattern of distribution of Laccaria bicolor sporophore production. This e€ect could be manifest through altered nutrient availability or as a result of the in¯uence of leaf litter extracts on fungal growth. Soluble litter extracts have been shown to stimulate or inhibit mycorrhizal fungal activity (Baar et al., 1994; Michelsen et al., 1995; Koide et al., 1998). Di€erences in ectomycorrhizal eciency have been suspected, but not clearly demonstrated in the ®eld. Dighton et al. (1990) found di€erent phosphorus uptake rates from soil pools to the tree canopy among various ectomycorrhizal communities. In addition, mycorrhizal enzymes may be able to directly in¯uence nutrient cycling by acquiring both nitrogen and phosphorus from complex organic forms (Dighton, 1991; Read, 1991). Plant communities of the New Jersey pine barrens have a nearly total reliance on mycorrhizal associations to obtain and conserve limiting nutrients and water. We have speci®cally addressed the role litter quality has on root growth and ectomycorrhizal development and how these factors integrate to a€ect ecosystem nutrient cycling. Speci®c null hypotheses were (i) leaf litter decomposition rate is the same between leaf litter species, (ii) leaf litter type does not a€ect root growth into that litter and (iii) leaf litter type does not in¯uence ectomycorrhizal species composition and their enzyme expression. 2. Material and methods 2.1. Site description The New Jersey Pinelands are located on the southeastern coastal plain of New Jersey, USA. Soils are sandy and oligotrophic (total N: 0.5±3 g kgÿ1, Olson extractable P: 70±140 mg kgÿ1); see Tedrow (1979) and Markley (1979). Pinelands communities consist of upland forests dominated by pitch pine (Pinus rigida ) and various oak species (Quercus alba, Q. velutina, Q. ilicifolia, Q. marilandica, etc.) with lowland stands

dominated by pitch pine forests, red maple swamps (Acer rubrum ) or Atlantic white cedar swamps (Chamaecyparis thyoides ). Understory ¯ora is primarily ericaceous, represented by various huckleberry (Gaylussacia ) and blueberry (Vaccinium ) species (McCormick, 1979). Mycorrhizal regulation of nutrient cycles is considered very important in this nutrient poor ecosystem, where interception of mineralized nutrients in the organic horizons is of great importance. The study site was located in a lowland pitch pine forest. Soil pH ranged from 3.4 to 3.8 with a 5± 10 cm deep organic horizon over sand. 2.2. Litter decomposition experiments and root ingrowth Oak and pine litter was gathered from the forest ¯oor in January 1995, dried at 708C for 48 h and placed in 1 mm mesh nylon litter bags. Bags contained a total of 5 g of leaf material comprised of either pine needles (pure pine), oak leaves (pure oak) or a mix of oak and pine (2.5 g oak, 2.5 g pine). The mixed species bags were included because these two litters do cooccur extensively and it was hypothesized that important interactions may be present that could not be quanti®ed by the single species approach. Seven 2.25 m2 plots were located in close proximity to mature pine trees in order to maximize encounters between pine roots and litter bags. Equal numbers of bags for each litter treatment were randomly located in each plot. Half of the bags were placed on the forest ¯oor and the remaining half were buried at the interface between the organic and mineral horizon on 23 February 1995. Subsets of bags were recovered on 25 May, 17 August and 9 November (13, 27 and 41 weeks, respectively). A total of seven replicate bags were harvested on each sampling occasion. For each sampling event, we only retrieved the number of bags on a daily basis that could be processed that day in order to reduce any experimental error due to root aging in the phosphatase enzyme assay. The entire sampling e€ort lasted for less than 1 week. Extracted litter bags were brie¯y rinsed with tap water to remove any adhering sand. Roots that had grown into bags were removed by careful dissection of the litter after opening the bags. Litter was dried at 708C for 48 h and weighed to determine percent mass loss. Phosphorus and nitrogen content of initial litter and decayed litter were determined by the Kjeldahl (N) and molybdate blue (P) methods (Allen, 1989). Oak and pine fractions from mixed species bags were analyzed separately in order to determine changes in chemistry (nutrient loss or gain) in each leaf species within the mixed litter bags. Total length of ingrown pine roots from the litter bags was measured using the line intersect method of Tennant (1975). Ectomycorrhizal community structure

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was determined by morphotype identi®cation of all root tips and expressed as a percentage of the total number of root tips. Classi®cation was based on color and gross sheath morphology, following the methods of Agerer (1987) and Ingleby et al. (1990). 2.3. Acid phosphatase activity of ectomycorrhizas Immediately following ectomycorrhizal classi®cation, acid phosphatase activity of root tips of each of the dominant ectomycorrhizal morphotypes were assayed following the methods used by Dighton and Coleman (1992). The phosphatase assay was used as an indicator for linkages between functional attributes of mycorrhizae and their chemical environment. The phosphatase assay measures the amount of phosphate cleaved from an organophosphate ( p-nitrophenol phosphate). Root tip volume (diameter and mycorrhizal unit length measured using an eyepiece graticule in a steromicroscope) was measured in order to express enzyme activity as mg nitrophenol per mm2 root tip produced. 2.4. Litter decay measurements and fungal development The decay dynamics of litter in bags placed aboveground were compared against bags of litter placed below-ground under the assumption that fungal in¯uences on decay and nutrient ¯ux from litter would be greater below-ground, especially within the mycorrhizal group. Subsamples of the decomposed litter were analyzed for hyphal length. Fungal hyphal lengths were measured by homogenizing a 1-g sample of leaf material in a blender in 1 l of sterile water. Five 1-ml aliquots were ®ltered through membrane ®lters (0.25 mm pore size), stained with Loe¯er's methylene blue, mounted in immersion oil. Fungal hyphal length was measured using a grid-line intersect procedure (Frankland et al., 1990). Litter hyphal length may suggest how desirable these substrates were for fungal utilization.

491

3. Results Analysis of variance (three-way ANOVA) showed that leaf litter decomposition was signi®cantly greater in leaf litter placed below-ground (P < 0.001; F ˆ 71:09† (Fig. 1). Overall, there was a signi®cant loss of mass over time (P < 0.01; F ˆ 62:2). Although there were no signi®cant di€erences between decomposition rates of litter types overall (P > 0.05; F ˆ 1:52). The presence of pine needles aboveground increased the rate of oak leaf decomposition (mixed oak). The presence of oak leaves had no e€ect on the decay of pine needles. However, the opposite pattern occurred below-ground. Oak leaves increased pine needle decay, while the presence of pine needles had no e€ect on the decay of oak leaves. These di€erences in response account for the signi®cant (P < 0.05; F ˆ 4:5† depth  time interaction of the ANOVA. Initial nitrogen content was higher (P < 0.01 by ttest) in oak leaves (1.61 2 0.22 mg gÿ1) than pine needles (0.2920.03 mg gÿ1). When changing nutrient content is coupled with decay rate, patterns of nutrient immobilization or mineralization can be detected. Nitrogen immobilization refers to a net increase in the absolute amounts of nitrogen (>100% initial nitrogen remaining). This generally occurs in litters that are

2.5. Statistics All data were analyzed using three-way ANOVA or GLM sequences of SAS (SAS, 1990). Pair-wise mean separation was determined using Tukey's Honestly Signi®cant Di€erence Test. Data pertaining to leaf litter mass loss and litter chemistry were separated into component litter species (i.e. oak, pine, oak in oak/pine mixture and pine in oak/pine mixture) yielding a total of 168 samples. Data regarding root length were analyzed on a per litter bag basis, yielding 126 samples and phosphatase activity based on the number (variable) of root tips of each morphotype available within any one litter bag.

Fig. 1. Leaf litter decomposition (expressed as percent mass remaining) of single and mixed pine and oak litter in litter bags placed on the soil surface or buried in the organic horizon of New Jersey pine barrens soil. Data points are means of seven replicates 2 standard error.

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nitrogen poor and can re¯ect nitrogen limited decay. Mineralization refers to the net release of a nutrient (<100% initial nitrogen remaining) and generally occurs once that nutrient is available in amounts over that needed by decomposers. Pine needles initially immobilized signi®cantly more nitrogen (Fig. 2) than oak leaves in both aboveground and below-ground locations (P < 0.01 by three-way ANOVA; F ˆ 37:33). Pair-wise comparison of means show that pine alone or pine in the oak/pine mix immobilize more N than oak alone or oak in the oak/pine mix. There is a signi®cant reduction in immobilized N over time (P < 0.001; F ˆ 16:92† but all other factors and interactions are not signi®cant. By November, the pine needles still exhibit net N immobilization, while oak leaves demonstrate nitrogen mineralization. Placement e€ects are also signi®cant in pine needle N immobilization. Aboveground, mixed pine needles show less nitrogen mineralization than pure pine needles while belowground, the presence of oak leaves has no e€ect on percent nitrogen remaining in pine needles. Initial phosphorus content in pine needles (71 2 11 mg gÿ1) was greater (P < 0.05 by t-test) than in oak leaves (36 2 5 mg gÿ1). Oak leaves show a pattern of net phosphorus immobilization throughout the ®rst two sampling periods and appear to enter a phase of phosphorus mineralization at the ®nal sampling inter-

val. The pattern of percent P remaining is signi®cantly di€erent between pine and oak as revealed by ANOVA (P < 0.01; F ˆ 20:45). Pair-wise comparison of means show that oak alone or in the oak/pine mix immobilize more P than pine alone or pine in the oak/ pine mix. Percent phosphorus remaining showed a signi®cant decrease over time for all litter types (Fig. 3; P < 0.001; F ˆ 63:85 by three-way ANOVA). Signi®cantly greater hyphal colonization was observed on litter decaying below-ground (P < 0.01; F ˆ 6:8 by three-way ANOVA) suggesting greater fungal utilization of leaf substrate resources below-ground than aboveground (Fig. 4). This trend continued over time among oak leaves decaying below-ground vs. aboveground. There were no signi®cant di€erences between litter types. Three-way ANOVA showed no overall di€erences in fungal colonization between leaf litters (P > 0.05; F ˆ 2:18), but two-way analysis within the belowground litter bags showed that fungal hyphal colonization of pine litter alone was signi®cantly less than oak or oak in oak/pine mix (P < 0.01; F ˆ 4:34). There was signi®cantly greater fungal colonization of litter decaying below-ground (P < 0.01; F ˆ 6:6), suggesting greater fungal utilization of leaf substrate resources below-ground than aboveground (Fig. 4). There were

Fig. 2. Nitrogen content of single and mixed pine and oak litter in litter bags placed on the soil surface or buried in the organic horizon of New Jersey pine barrens soil. Data points are means of seven replicates2standard error.

Fig. 3. Phosphorus content of single and mixed pine and oak litter in litter bags placed on the soil surface or buried in the organic horizon of New Jersey pine barrens soil. Data points are means of seven replicates2standard error.

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Fig. 5. Ectomycorrhizal morphotype categories associated with roots colonizing single and mixed pine and oak litter in litter bags buried in the organic horizon of New Jersey pine barrens soil after 41 weeks.

Fig. 4. Fungal hyphal length associated with single and mixed pine and oak litter in litter bags placed on the soil surface or buried in the organic horizon of New Jersey pine barrens soil. Data points are means of seven replicates2standard error.

signi®cantly more hyphae in the ®nal harvest than either of the ®rst or second (P < 0.001; F ˆ 83:98). Discussion of pine root colonization and mycorrhizal classi®cation is necessarily con®ned to litter bags placed below-ground since root ingrowth did not occur aboveground. The root ingrowth data presented is limited to the third sampling interval since root ingrowth was universally absent during the ®rst sampling date and absent from many litter bags for the second sampling date. More root ingrowth occurred in mixed species litter bags (425.02155.5 mm per bag) than in single species litter bags (oak 218.6 2

59.0; pine 230.6 2 68.6 mm per bag), but this was not statistically signi®cant …P ˆ 0:31; F ˆ 1:24). Of the ectomycorrhizal morphotypes identi®ed, the ®ve most abundant (90% of all ectomycorrhizae) are described in Table 1. The other mycorrhizae were lumped together in a ``miscellaneous'' category. Each litter type was dominated by a di€erent mycorrhizal morphotype (Fig. 5). Types 2 and 3 dominated root tips invading pure oak leaves while Type 10 dominated root tips invading pure pine needles. Roots in mixed oak and pine litter supported a mycorrhizal ¯ora dominated by type 4. Phosphatase activity measures the potential to cleave inorganic phosphate from an organic molecule. Table 2 shows the phosphatase activity of the main mycorrhizal morphotypes. The highest rates of phosphatase activity were associated with the morphotypes (types 2, 3 and 4) most common on root tips in¯uenced by oak leaves and, consequently, to litter that immobilized phosphate in organic forms. Root tips associated with pure pine needles, which show rapid losses of phosphorus, were dominated by type 10 mycorrhizae which had signi®cantly lower phosphatase activity than type 4 and slightly less phosphatase activity than types 2 and 3. Type 1 was non-mycorrhizal and had the lowest phosphatase activity. The in¯uence of rapid P mineralization from pine leaves on the phosphatase enzyme production by roots is suggested by the signi®cant reduction in phosphatase production in oak/pine mixed

Table 1 Mycorrhizal morphotype classi®cation Morphotype

Description

Type Type Type Type Type Type

non-mycorrhizal cream mantle, white granular hyphae with clamp connections, outer sheath loosely woven cream mantle, cream rhizomorphs, slightly granular hyphae with clamp connections, hyphal dia > type 2 Black mantle, black wirey hyphae, club shaped tip rusty brown mantle, no hyphae, sheath loose prosenchyma tuberculate, frosty white mantle, black non-granular hyphae

1 2 3 6 4 10

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litter compared to oak alone (226 2 125, 301 2 165 ng p-nitrophenol mmÿ2 root surface, respectively, P < 0.05) and a lower, though not signi®cant, activity in pine alone (2712194 ng p-nitrophenol mmÿ2 root surface). From the analysis of variance, there was a signi®cant litter  mycorrhizal interaction for phosphatase activity. This could be explained by the di€ering response of the same mycorrhizal type to di€erent leaf litters. For example, type 1 showed similar enzyme activity between leaf litter types (125 2 52, 121256, 121277 ng p-nitrophenol mmÿ2 root surface in oak, oak±pine and pine, respectively). Type 6 is a mycorrhizal morphotype that occurred in low abundance in all leaf litter types. Type 6 showed highest levels of activity when in the presence of oak leaves alone (300 2 160, 135 2 49, 153 2 40 ng p-nitrophenol mmÿ2 root surface in oak, oak±pine and pine, respectively). Type 4 exhibited lowest activity on oak±pine mixed litter (4712115, 196236, 5642296 ng p-nitrophenol mmÿ2 root surface in oak, oak±pine and pine, respectively). 4. Discussion Our study has shown that, aboveground, decomposition of oak leaves is slower than that of pine needles or of oak in combination with pine needles. However, there is no di€erence below-ground in the decomposition rates. Pine needles represent a source of easily mobilized phosphorus while oak leaves serve as a phosphorus sink. Both litter types immobilize nitrogen, but this immobilization is signi®cantly greater in pine than oak. Litter quality di€erences in¯uence the turnover of organic matter in the Pinelands ecosystem. Pure and mixed litters decompose at di€erent rates and these patterns di€er in response to placement above or below ground. It was hypothesized that faster decay would be related to greater hyphal length. Indeed there was a signi®cantly higher fungal biomass on litter placed below ground, where decomposition was accelerated, but there were no di€erences between leaf litter types. Resources of di€ering nutrient content

have been shown to in¯uence fungal colonization (Rayner, 1991; Ritz, 1995) by the in¯uence of di€erent hyphal growth rates and branching patterns. Although root growth into litter bags containing a mixture of oak and pine was greater than single litter species, the di€erence was not statistically signi®cant. This enhanced root growth suggests that patches of high resource diversity encourage root production. Di€erential rates of root growth into patches of soil with di€erent nutrient contents has been shown by Pregitzer et al. (1993), Eissenstat and Van Rees (1994) and Barber (1995). In the case of di€erences in root growth into litter bags, a number of interacting factors may be present, including availability of nutrients, water relations, physical constraints on root growth and allelopathic chemical leaching from the litter (Michelsen et al., 1995). We found that ectomycorrhizal community structure also responds to patches of resource diversity. Ectomycorrhizal species composition was di€erent between leaf litter types. J. Baar (unpublished Ph.D. thesis, University of Wageningen, 1995) and Baar et al. (1994) showed signi®cant e€ects of leaf litter and humus on the ectomycorrhizal community structure of Scots pine. Koide et al. (1998) has shown di€erential response of ectomycorrhizal fungal hyphal growth to leaf litter extracts, with growth of some species being suppressed whilst being promoted in other species. Michelsen et al. (1995) showed that leaf litter extracts a€ected nutrient acquisition by mycorrhizal plants. Ectomycorrhizal root surface acid phosphatase activity has been shown to di€er between fungal species. In this study, we have shown that our morphotype 4 produced signi®cantly more phosphatase than types 1, 6 and 10. When combined with the distribution of morphotypes within the di€erent litter types, a pattern emerges of the presence of greater abundance of phosphatase producing morphotypes (especially type 4 in oak litter and types 2 and 3 in the oak/pine litter mixture) where P immobilization is greatest. It is suggested, therefore, that the greater amounts of organically-bound phosphorus in decaying oak leaves may be a factor selecting for phosphatase producing ecto-

Table 2 Acid phosphatase activity (ng p-nitrophenol mmÿ2 root surface area) of the main ectomycorrhizal morphotypes from litter bags and from litter types. Means within columns sharing identical superscripted letters are not signi®cantly di€erent from each other p ˆ 0:05: (Tukey's Honestly Signi®cant Di€erences test) Mycorrhizal morphotype

Phosphatase mean2S.E.

Litter type

Phosphatase mean2S.E.

1 2 3 4 6 10

100211a 270215b 370238bc 420254c 204243ab 192228ab

oak oak/pine pine

301225a 226219bc 194230ac

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mycorrhizal species. The di€erent litters of this experiment demonstrated very di€erent nutrient dynamics. Pine needles are low in nitrogen and high in phosphorus. Phosphorus is rapidly lost, presumably as leachate, while nitrogen is conserved and accumulated via immobilization. Oak leaves, initially higher in nitrogen and lower in phosphorus, did not exhibit a net loss of nitrogen and accumulated phosphorus. It may be that decaying oak leaves represent a biological sink for phosphorus leached from pine leaves. These varying chemical environments are likely to select for di€erent ectomycorrhizal species. Mycorrhizae with high phosphatase activity are better suited to utilize immobilized phosphorus and were, therefore, associated more strongly with oak litters. In mixed species forest, the distribution of leaf litter on the forest ¯oor is heterogeneous. The nature of litter patches may have e€ects on the soil biota below it. In an unpublished study, we (Dighton et al.) have found that the distribution of litter patches in the New Jersey upland oak±pine habitats of the pine barrens is related to litter dams created by stems of the ericaceous understory community. Litter patch size (area) was positively correlated …r2 ˆ 0:908† with the density of ericaceous stems. Small litter patches contained a higher percentage contribution (by weight) of pine needles and large patches contained a higher percentage contribution of oak leaves. Large litter patches were also found to have a higher Simpson's diversity index of ectomycorrhizal species than small patches. Our results suggest one mechanism (selective chemical environment) by which litter patches of varying composition could in¯uence mycorrhizal diversity. Forming links between biodiversity and function was one of the main areas of perceived future research in soil microbiology (Coleman et al., 1994). In this paper we have shown that environmental factors can signi®cantly a€ect ectomycorrhizal community structure. These micro-spatial changes in communities are related to changes in physical and chemical properties of the environment. We have also provided preliminary evidence of di€ering functional attributes of the resultant ectomycorrhizal community developing within leaf litter patches of di€ering resource quality. This function has been expressed as root surface phosphatase enzyme production, but represents possible wider overall physiological competence of di€erent ectomycorrhizal communities (see also Dighton et al., 1990). Acknowledgements We would like to thank The Victoria Foundation for supporting Christine Conn's postdoctoral fellowship and Dennis Gray for his assistance with the

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chemical analyses. We would also like to thank two anonymous reviewers for their constructive comments on the original draft of this paper.

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