Inhibitory effects of Bromelia pinguin (Bromeliaceae) on soil ecosystems in primary forests
of Costa Rica
Looby, C.1 and Eaton, W.D.2a
1Kean
University, NJ Center for STEM, 1000 Morris Ave, Union, New Jersey, 07083
2 Kean University, School of Environmental and Life Science, 1000 Morris Ave, Union, New Jersey, 07083
aCorresponding author, [email protected], 908-737-3611
This project was supported by NSF grant DBI 1034896, and the Kean University Presidential Scholars Challenge Grant Program
Abstract: Few studies have been performed on the ecology of Bromelia pinguin (Bromeliaceae), a bromeliad commonly found in large patches within primary forests of Central America and the Caribbean. Previously, Camacho-Hernández et al.
(2001) found that the fruit pulp of this bromeliad possessed antifungal activity, and Looby et al. (2011) found decreased fungal DNA and differences in C and N nutrient levels in bromeliad soils in comparison to adjacent primary forest soils. The
current study showed that, although the standing pools of Total N and Dissolved Organic C did not differ much between the bromeliad and primary forest soils, the bromeliad soils had lower rates of N and C biomass development, less laccase
activity (suggesting less lignin degradation activity), and much less fungal 18s rRNA and ITS region DNA than found in the adjacent primary forest soils. The results of T-RFLP analyses showed that the community diversity and abundance of fungal
DNA was also less in the bromeliad forest. Metagenomic studies of the soil community DNA showed clear differences in the populations of the Basidiomycota (Tremellales, Agricales, Thelephorales, Boletales representatives), the Ascomycota
(Helotiales representatives), and Zygomycota between the bromeliad and primary forest soils. Data suggest that the presence of the bromeliad is altering the fungal community structure and therefore C and N nutrient dynamics within these
soils.
INTRODUCTION: Bromelia pinguin (Bromeliaceae) is a bromeliad with unique characteristics found throughout Central America and the Caribbean. Two studies were performed on this bromeliad in the Maquenque Reserve located in the
lowland forest of Northern Zone of Costa Rica present in the San Juan-La Selva Biological Corridor. Camacho-Hernández et al. (2001) found that methanol extracts from the fruit pulp had antifungal activity. Looby et al. (2011) suggested
that fungal inhibition is occurring within the soil when compared to adjacent primary forest soil in that the bromeliad soil showed: (1) decreased fungal target DNA (universal fungal 18s rRNA and fungal ITS); (2) increased soil dissolved
organic C; (3) decreased efficiency of C use; (4) decreased amount of standing C biomass. However, it is unclear how the reduced fungal DNA mass, observed by Looby et al (2011) and likely due to the anti-fungal effects of this
bromeliad, impact the rate of C and N biomass development and decomposition activity, and the fungal community diversity within these soils. This current study began to address these issues by comparing the amounts of soil
dissolved organic C (DOC), total N, C and N biomass, and the rates of production of these, and the amount of laccase activity as an indicator of fungal decomposition. T-RFLP and fungal ITS sequence analysis were performed to assess
diversity and groups of fungi present in both habitats.
METHODS:
Soil was collected aseptically from twenty 15 m x 20 m along two 20 m transects each in bromeliad patches and adjacent primary forests and composited by habitat.
The primary forest was used as a control and was defined by the lack of B. pinguin.
Bromeliad habitats consisted of 32 or more plants were subplot.
The soil dissolved organic C (DOC; modified Walkly-Black methods) and total N (potassium thiosulfate oxidation methods) were determined, and from this, the biomass C and N and rates of development of these were determined as
the difference in these values from chloroform-fumigated and unfumigated samples. These were measured spectrophotometrically.
Laccase activity, as an indicator of basidiomycete fungi, was performed via standard NaC2H3O2 extractions and spectrophotometric methods.
Fungal ITS target DNA was amplified using ITS 1F and ITS 4 primer pairs
T-RFLP and sequencing was performed to determine fungal ITS diversity
T-RFLP was done using FAM labeled primers on a 3130 Genetic Analyzer. Analysis was done using T-REX software (citation).
Cloning was performed using the pGEM T-Easy system. Sequencing of 35-40 clones was done on a 3130 Genetic Analyzer using ABI Big Dye methods.
Sequence taxonomic identity was determined by comparing our sequences to those known sequences in GenBank.
Diversity analysis of bands and sequences was done using EstimateS Version 8.2
Figure 1. Rates of (a) N biomass development, (b) C biomass
development, and (c) laccase activity in bromeliad and adjacent
primary forest soils.
(a)
(b)
FINDINGS:
NUTRIENTS COMPOSITION AND DYNAMICS
Bromeliad soil had:
30.50% lower rate of N biomass (Nmic) development (p=0.1; large Effect size d= 0.87)
21.20% lower rate of C biomass (Cmic) development (p= 0.15; moderate Effect size d= 0.75)
52.00 % less laccase activity (p=0.01; large Effect size d=1.54)
a similar standing pool of soil dissolved organic C as the primary forest (322.1 µg/g and 355 µg/g respectively; 9.30% difference, p= 0.52, small Effect size d=0.33)
a similar standing pool of total N as the primary forest (1.01 µg/g and 1.29 µg/g respectively; 21.7 % difference, p= 0.29, moderate Effect size d=0.549)
FUNGAL COMMUNITY STRUCTURE
Diversity, richness, and Bray-Curtis indices for T-RFLP and DNA sequences show that there are different decomposers present in the bromeliad and primary forest soils
(Figures/Tables???). There is increased diversity and richness in the primary forest, with a Bray-Curtis index showing only 5.1 % similarity between the two habitats. Relative
abundance of fungi showed that different phyla responsible for decomposition of organic matter within the bromeliad soils were dominated by Basidiomycetes and primary
forest soils were dominated by Ascomycetes. These data suggest that the bromeliads, whether due to anti-fungal materials produced or due to overtaking the ground cover, are
associated with changes in the structure of the decomposer community.
Different fungal orders are dominant in each habitat. Bromeliad forest soils were dominated by the Basidiomycete orders Tremellales (class Tremellomycetes) and Agricales
(class Agaricomycetes). Tremellales are common saprotrophic decomposers, and the Agricales in these soils are also likely functioning as decomposers. In the primary forest,
the dominant Order in Ascomycota Hypocreales (class Sordariomycetes), that is considered a common decomposers of plants and fungi in forest soils, was found in great
abundance in the primary forests, but not the bromeliad soils. Members of the Ascomycota Orders Sordariales and Xylariales, also common decomposers similar to
Hypocreales, were present in the bromeliad but not the primary forest soils. It is possible that Hypocreales is inhibited by the presence of the bromeliads, thus, allowing these
other groups of fungi to fill this niche.
16% of the fungi present in the primary forest were from groups containing significant plant pathogens including the order Pleosporales, the order Eurotiales, and the class
Exobasidiomycetes. These types of fungi were not found in bromeliad forest soil, possi bly due to the relative absence of understory plants, replaced by high densities of
bromeliads, and/or the fungal inhibition by the bromeliads.
A moderate amount of fungi in the phylum Zygomycota were only found in bromeliad soil. Some fungi in this phylum include arbuscular mycorrhizae and soil saprobes, such as
the Glomermycota and Chytridiomycota, which were present in the bromeliad soil and absent in the primary forest soil; again highlighting the differences in fungal community
structure in these two habitats.
Decrease in diversity in the bromeliad soils may be due to fungal inhibition. There are many fungal orders in both Basidiomycota and Ascomycota present in the primary forest
soils that are in low abundance in the bromeliad soils, which could be associated with the fungal inhibition activity of the bromeliads. It also appears that the decomposition
activity is spread out among a more diverse community in the primary forest.
Decrease in rates of C and N biomass development and laccase activity suggests that the decomposition activity due to the fungi in the bromeliad soils is slower than that in
the primary forest soils—possibly due to the shift in community structure that is likely due to the presence of the bromeliads.
CONCLUSIONS AND FUTURE AND CURRENT WORK:
(c)
REFERENCES:
CULMAN, S.W., BUKOWSKI, R., GAUCH, H.G., CADILLO-QUIROZ, H., BUCKLEY, D.H. 2009. T-REX: Software for the Processing and Analysis of T-RFLP data. BMC Bioinformatics 10:171
CAMACHO-HERNÁNDEZ, I. L., J. A. CHÁVEZ-VELÁQUEZ, M. J. URIBE-BELTRÁN, A. RÍOS-MORGAN, DELGADO-VARGAS, F. 2002. Antifungal activity of fruit pulp extract from Bromelia pinguin. Fitoterapia 73: 411-13.
COLWELL, R. K. 2009. EstimateS, Version 8.2: Statistical Estimation of Species Richness and Shared Species from Samples; http://viceroy.eeb.uconn.edu/EstimateS
LOOBY, C., HAUGE, J.B., BARRY, D., EATON, W.D. 2011. Fungal inhibition by Bromelia pinguin (Bromeliaceae) and its effect on nutrient cycle dynamics. Accepted for Tropical Ecology 53:3.