Bacterial Diversity in Tree Canopies of the Atlantic Forest
KeywordsMicrobial Community Bacterial Community Leaf Surface Microbial Diversity Bacterial Community Structure
16S rRNA gene profiling is one of the main approaches used for the study of microbial communities that are associated with plants and animals, which are mostly comprised of species unable to grow under laboratory conditions. Even though plants harbor an enormous microbial diversity on their various surfaces, the functions of these microorganisms, except for a few that are pathogens or symbionts, are largely unknown, but are speculated to modify plant chemical signals, alter root exudation patterns, and provide protection against pathogens. Understanding of the factors that shape the structure of microbial communities, and the functions of microorganisms that are associated with plants, will likely be essential for establishing conservation strategies for protecting endangered plant species. The large reservoir of microbial diversity on plant surfaces also represents a largely untapped bank of microbial products that may be of interest for pharmaceutical, agricultural, and environmental applications.
Plant surfaces in natural and agricultural ecosystems are colonized by a variety of epiphytic microorganisms that have been examined in relation to their diversity, ecology, and genetics using culture-dependent and culture-independent approaches. Among the various surfaces that are presented by plants, the leaf surface, also known as the phyllosphere (Ruinen 1956), is one of the most common habitats for terrestrial microorganisms. The phyllosphere may be colonized by bacterial cells at an average density of 106–107 cells cm−2 on plants from temperate regions (Lindow and Brandl 2003) and may be even higher on tropical plants where dense canopies and a moist shaded environment are conducive for bacterial growth. Considering that the estimated total leaf area of terrestrial plants is approximately 6.4 × 108 km2 (Morris and Kinkel 2002), the number of bacterial cells on leaf surfaces globally has been estimated to be as high as 1026 cells. Despite the importance of plant-microbe interactions in plant disease, almost nothing is known about the indigenous, nonpathogenic bacteria that colonize plant leaf surfaces and their functions in terrestrial ecosystems.
The Phyllosphere Habitat
One of the major selection factors for microbial colonization of leaf surfaces is the ability to tolerate or grow on the myriad chemical substances that are released from plant leaf tissues and/or produced by other microorganisms. This includes many thousands of secondary metabolites, such as monoterpenes that serve as signal factors and defense compounds, as well as chemical attractants and deterrents for insects, herbivores, and pathogens. However, the specific secondary metabolites driving the structure of bacterial communities in the phyllosphere are unknown.
Bacterial Communities in the Phyllosphere
Many early surveys of phyllosphere communities have relied on descriptions of bacteria that can be cultivated on agar media and isolated as individual colonies. Using various types of growth media, 85 species of culturable microorganisms from 37 genera have been reported in the phyllospheres of rye, olive, sugar beet, and wheat (Ercolani 1991; Legard et al. 1994; Thompson et al. 1993). While this is an impressive number of species, studies using molecular methods have revealed that the actual microbial species richness in the phyllosphere of agricultural plants is much greater than this and suggest that different plant species harbor unique communities that are similar for individuals of the same plant species (Yang et al. 2001). The discovery of high levels of bacterial species richness associated with different agronomic plants has prompted many questions about the true extent of microbial diversity that may be associated with the phyllosphere of different plants in natural ecosystems around the world. It has been speculated that since bacteria can be transported across the globe in dust (Griffin et al. 2002), only a small number of bacterial species may be adapted to grow on leaf surfaces. On the other hand, if each plant species selects for its own microbial community, the microbial species diversity that is associated with all of the different plant species on earth could be enormous. This question can only be answered by systematic surveys of phyllosphere microbial diversity in different ecosystems. Considering the current rate of extinction of plant species, it is especially urgent to begin surveys of phyllosphere microorganisms that are associated with endangered biomes.
Bacterial Community in the Phyllosphere of the Atlantic Forest
Many tropical forests and biodiversity hotspots contain endemic plant species that are preserved only in a few remnant areas. The Atlantic forest of Brazil is an example of a forest with high levels of biodiversity that is struggling to survive. The Atlantic forest used to be the second largest tropical forest in South America and represented 1.3 million km2 in the 1500s, when the Portuguese first arrived in Brazil. Today, approximately 7 % of the original Atlantic forest remains, since most of it has been converted to agricultural or urban areas, leaving a patchwork of fragmented remnants. The remnants of the Atlantic forest are considered to be some of the oldest undisturbed forests on the planet, containing approximately 20,000 plant species, of which nearly half are endemic (Tabarelli et al. 2003). Several research projects have been developed in the Atlantic forest as part of the ongoing BIOTA-FAPESP (São Paulo Research Foundation) program, which has been successfully established to examine the biodiversity of the São Paulo State (Brazil).
Different approaches can be used to survey the microbial diversity in the phyllosphere. The first approach is using DNA fingerprinting methods. A low-resolution DNA fingerprinting method referred to as PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis), through which amplified fragments of highly variable regions of the bacterial 16S rRNA gene are separated by electrophoresis in a denaturing gradient polyacrylamide gel, has been used for studying the bacterial community structures in the phyllosphere of tree species of the Atlantic forest. This methodology generates a distinctive fingerprint that can be used to compare the relative similarities of communities, but does not provide information on the identities of the bacterial species within the communities. To compare the phylogenetic diversity in the phyllosphere and generate diversity indices for different phyllosphere communities, sequencing of specific regions of the bacterial 16S rRNA gene is normally used.
With these combined approaches, it has been shown that the 16S rRNA gene band patterns for the bacterial communities from different tree species of the Atlantic forest are distinct from each other (Lambais et al. 2006). Communities from replicates for different individuals of the same tree species showed some expected variation, but overall are highly similar to each other. The similarities between the leaf bacterial communities within and between species were further measured statistically and showed that the trees could be segregated into groups according to tree species, family, and order, suggesting a coevolution between trees and microbial populations associated with the phyllosphere (Lambais et al. data not published). Evidence of coevolution of microbial populations associated with the bark (dermosphere) and rhizosphere of trees of the Atlantic forest also has been observed, suggesting that plants coevolved with specific microbiomes (Lambais et al. data not published). An estimate of the bacterial species richness associated with the phyllosphere of trees in the Atlantic forest suggests the existence of 2–13 million undescribed bacterial species that colonize the collective phyllosphere of the Atlantic forest (Lambais et al. 2006). Interestingly, studies of the phyllosphere of different individuals of the same tree species in the Atlantic forest over a range of distances and at different times show that the similarities between bacterial community structures in the phyllosphere of the same plant species decrease with the increasing distance between individual trees, even though they still share high levels of similarity (Lambais et al. data not published). Over larger scales, such as when the bacterial communities of the individuals of the same plant species are separated by hundreds of kilometers, significant differences in community structure are observed. These data suggest that the bacterial diversity in the phyllosphere of plants of the Atlantic forest may be even higher than the predicted 2–13 million species estimate that does not take into account beta diversity.
While still in an early phase, research aimed at measurements of beta diversity includes a survey of Tamarix trees in Mediterranean and Dead Sea regions in Israel and two locations in the USA (Finkel et al. 2011). These studies suggest that besides the plant genetic component driving the bacterial community structure in the phyllosphere, environmental conditions associated with particular geographical locations are also important. On the other hand, the high levels of similarity of the bacterial communities in the phyllosphere of Pinus ponderosa over transcontinental distances (Redford et al. 2010) suggest a strong genetic component in the regulation of the phyllosphere associated microbiome.
The majority of bacterial OTUs in the phyllosphere of the trees of the Atlantic forest have been assigned to the phylum Proteobacteria. Based on a survey of several tree species in the Atlantic forest, including Ocotea dispersa, Ocotea teleiandra, Mollinedia schottiana, Mollinedia uleana, Eugenia cuprea, Eugenia melanogyna, and Tabebuia serratifolia, it has been shown that, in general, approximately half of the bacteria in the phyllosphere are phylogenetically related to Gammaproteobacteria, whereas 20 % are related to Alphaproteobacteria and 5 % to Flavobacteria, even though interspecific variation may occur (Lambais et al. data not published). For instance, in the phyllosphere of Ocotea teleiandra, a high frequency of Alphaproteobacteria and a low frequency of Gammaproteobacteria have been detected, in contrast to other tree species.
Altogether, these results show that every tree species that has been examined in the Atlantic forest contains its own unique bacterial community and that spatially separated individuals of the same tree species have similar bacterial communities, within the same environment (forest physiognomy). The variations in bacterial community structures in the phyllosphere that were observed using the PCR-DGGE and sequencing approaches to compare similarities among individuals indicate that the community compositions may vary on different leaves. This may correspond with different leaf ages, location in the canopy, light incidence, and microclimate conditions that influence the leaf environment and types of chemical substances that are secreted by the plant leaves. The bacteria may also interact with various fungi and algae that colonize the leaf surfaces and change the chemical and physical environment of the leaf habitat. In future studies, it will be necessary to examine the microbial communities on leaf surfaces at the microsite scale to determine changes in species composition and the ecology of different habitats on the leaf surface, for example, on the adaxial and abaxial leaf surfaces or within biofilms and microcolonies at distinct physical locations on the leaf surface.
Drivers of Community Structure in the Phyllosphere
The development of different bacterial communities in the phyllosphere of different tree species demonstrates the strong effect of leaf surface environment as a selection factor. The initial inoculation of leaves of different trees very likely begins with the growth of opportunistic microorganisms that are transported in dust, by insects, or that are splashed from adjacent trees by rain. Inheriting a minimal microbiome through the seeds may also be a possibility. Further selection then occurs depending on differences in the types of carbon substrates that are available for growth, as well as various physical and environmental factors and interactions within the microbial community. The primary carbon substrates that are used for microbial growth include carbohydrates, amino acids, and organic acids. The composition and amounts of these substances may vary for different plant species, but may also vary over time depending on leaf age, insect damage, and rainfall, for instance. Another potentially important selective factor is the production of different types and quantities of monoterpenes and other volatile substances that are released from the leaf surfaces. These substances may be both toxic to some microorganisms and used as growth substrates by others. Phytochemistry research has shown that tree species have species-specific differences in their biochemical signatures for volatile molecules (Arey et al. 1995). If terpenes act as selective substances, certain types of bacteria may be predicted to occur in relation to the biochemical signatures of volatile organic compounds released by the leaves. Very little work has been conducted on this research topic, but bacteria are known to contain enzymes that convert terpenes to derivative substances. In this manner, the phyllosphere bacteria may influence chemical signaling to insects and other microorganisms or between plants. Terpenes and other plant secondary metabolites produced in plant leaves are also important feedstocks for various biochemicals that are used in the industry and for pharmacology. Future studies should investigate the genomes and genes encoding enzymes in the phyllosphere that may have broad application for industrial biotechnology, as in the work described by Delmotte et al. (2009), which used proteogenomics to study the microbial community associated with the phyllosphere of soybean, clover, and Arabidopsis.
Recent studies have provided only a glimpse into the microbial diversity that is associated with the tree canopies in the Atlantic forest, and there are many new questions that arise from this research. For example, to what degree do soil, nutritional, and other environmental factors affect the composition and structure of microbial communities in the phyllosphere? What is the diversity of fungi and Archaea on the plant leaf surfaces, and how do these microorganisms interact? Future research should also examine the functional aspects of phyllosphere microbial communities and the interactions that occur between phyllosphere bacteria and their host plants using metagenomics, metaproteomics, and metabolomics. As we begin to survey these bacterial communities through systematic study of different plant species, there will be exciting opportunities for studies of the metabolic capabilities and ecological functions of phyllosphere microorganisms in terrestrial ecosystems.
Each plant species is able to select its own bacterial community, and probably its own microbiome, which may be affected by plant genomic components and the environment. Altogether, the phyllosphere of plant species of the Atlantic forest may harbor several million species of bacteria that remain to be described. The roles of the microbial communities of the phyllosphere in forest ecology are not yet known, but are likely to include chemical signaling, nitrogen fixation, and plant protection, among other functions. This immense microbial diversity may also provide new biomolecules of interest for pharmaceutical, agricultural, and environmental applications.
- Legard DE, McQuilken MP, Whipps JM, Fenlon JS, Fermor TR, Thompson IP, Bailey MJ, Lynch JM. Studies of seasonal changes in the microbial populations on the phyllosphere of spring wheat as a prelude to the release of a genetically modified microorganism. Agric Ecosyst Environ. 1994;50:87–101.CrossRefGoogle Scholar
- Morris CE, Kinkel LL. Fifty years of phylosphere microbiology: significant contributions to research in related fields. In: Lindow SE, Hecht-Poinar EI, Elliott V, editors. Phylosphere microbiology. St Paul: APS Press; 2002. p. 365–75.Google Scholar
- Tabarelli M, Pinto LP, Silva JMC, Costa CMR. Endangered species and conservation planning. In: Galindo-Leal C, Câmara IG, editors. The Atlantic forest of South America: biodiversity, status, threats and outlooks. Washington, DC: Island Press; 2003. p. 86–94.Google Scholar