Introduction
We have initiated an undergraduate research program that includes a two-week expedition to the Amazonian rainforest and a ten-week summer research experience. Our objective is the discovery of (i) novel microbes that are endophytically associated with plants and (ii) the potentially biologically active molecules that these microbes produce. Although the foreign travel is a strong enticement for student participation, we have found that the opportunity to own and independently direct their research program is key to program success.
Intensive research is often used as a capstone experience to successfully enrich an undergraduate student's science education1. Typically, a student joins a research group and participates in an ongoing project under the supervision of a professor, or a senior member of the laboratory. This experience provides personalized training that can be inspirational and highly motivating. Undergraduate students become exposed to a group of people engaged in inquiry-based research who communicate in the 'foreign language' of that scientific discipline. Some students realize that science is not beyond their abilities and that it is much more exciting than the vocabulary-focused courses they have taken, particularly at the introductory levels. Several studies have demonstrated that an independent research experience is the best way to get undergraduates excited about a career in science. In fact, the vast majority of science faculty in US universities had an undergraduate research experience during their training.
Despite the critical role hands-on research has in scientific career development, some very good students still come away from the experience frustrated and discouraged. In our experience working with undergraduate students in a variety of different laboratories, their frustration often resulted from the nature of the project to which they had been assigned. Undergraduates were assigned work that was primarily technical support and were provided insufficient opportunity to formulate hypotheses and design experiments to test their ideas. This might reflect the need to provide projects designed to fit within the short timescale available to students, whose time is often interrupted by classes, vacations and other activities.
Given that project design is critical to a successful undergraduate research experience, what are the elements of a research project that make it well suited to the abilities and time constraints of a motivated undergraduate student? Graham Hatfull, a Howard Hughes Medical Institute professor at the University of Pittsburgh, has proposed a series of criteria based on the successful implementation of his phage hunters program2. He argues that undergraduate research projects should be technically feasible and should not require a large body of prior knowledge. The projects should include multiple success points, allowing confidence to be gained as the project grows to greater levels of complexity. For projects involving large numbers of students, the projects should be parallel in nature, thereby simplifying mentoring and maximizing the number of students that can be involved. This also provides an opportunity to publish the collective observations made by individual students. The projects must be scientifically 'real'—that is, capable of producing scientific results and providing motivation through scientific discovery. Most importantly, the project must provide a sense of intellectual ownership, with sufficient freedom for the student to pursue his or her own experimental questions and observations. This feeling of empowerment to control the scientific direction of an inquiry is critical to the success of any research experience, regardless of the student's academic level.
Hatfull's program, which has involved both undergraduates and high school students, has focused on the isolation, sequencing and annotation of mycobacteriophage from soil samples. The initial stages of the work are technically simple, but invoke the students' imaginations as they consider where to collect their soil samples. The abundance and diversity of phage provide a high likelihood that each student can isolate, name and characterize a new virus, thus creating a strong sense of intellectual ownership and project control. Though not all high school or college students who participated in the phage hunt concluded that a scientific career is in their future, every student achieved at least the first success point and several went on to scientific publication3, 4.
We have set out to incorporate the conceptual elements of Hatfull's phage hunters program in an intensive undergraduate research experience of our own. Our focus is the discovery of biologically active natural products produced by endophytic microorganisms associated with plants. Endophytes represent a diverse potential source of new products for use in medicine, agriculture and industry5, 6. They colonize living tissues of plants without inflicting negative effects and can be either fungal or bacterial organisms. Of the nearly 300,000 plant species on earth, each is likely to be host to at least one endophyte7; but relatively few of these organisms have been characterized. Many endophytes make bioactive natural products to inhibit the growth of other organisms or provide a selective advantage to their plant host5, 6, 7, 8. In some cases they can even acquire the ability to synthesize the same defensive natural products produced by the plant9. Our focus is on endophytes associated with plants of the neotropics, an ecosystem in which the hypervariability of the plants is expected to be matched by the hypervariability of their associated microbes10. Many plants have an extensive ethnobotanical history of use by indigenous people that provides an intriguing starting point for each student's project design. For example, students may wish to explore microbes associated with the cinchona (the "Fever tree" or Jesuits' Bark), which is responsible for quinine production. Quinine was used worldwide for the treatment malaria, based on original observations of its utility by the Malacatos Indians of present-day Ecuador in the early 1600s11. Alternatively, they may wish to explore plants that were indigenous to ancient Gondwanaland, given the potential for long-term and potentially beneficial association between plant and microbes. No matter how the students design their projects, bioprospecting for endophytic natural products in the rainforest presents an open-ended, inquiry-based opportunity for students to make original discoveries about the natural world.
We are halfway through the first year of a four-year bioprospecting program funded by the Howard Hughes Medical Institute and the US National Science Foundation (NSF). The program includes three major components: a semester-long seminar course designed to prepare the students for the expedition and laboratory work, a two-week expedition to one of the world's rainforests during spring break, and an intensive summer laboratory experience isolating and characterizing endophytic microbes associated with the collected plant samples (Fig. 1).
The course is designed to cover a breadth of topics relevant to both the field and the laboratory experience, including conservation, ethnobotany, plant phylogenetics, mycology, pharmacology, patenting and intellectual property, and even a lecture on ants by Mark Moffett, a National Geographic photographer. The intention is to prepare students to see more than just plants when they visit the jungle. Many of these subjects are whole courses unto themselves, so the goal is not for them to master each discipline, but instead to expose the students to key ideas that drive these fields. Students were selected for participation in the course based primarily on two questions: what can you contribute to the course, and what do you wish to learn from this experience? Some answers to the first question were particularly helpful, making it possible to include two Spanish translators and an EMT among the students on the expedition.
A key assignment in the class was for each student to define the theme for his or her plant collection based on the published ethnobotanical history of various plants and his or her own imagination and interests. This maximized each student's sense of project ownership. One student collected plants used by native people to treat tuberculosis infections; another selected plants likely to produce antioxidants. One targeted carnivorous plants, while another selected plants used in the treatment of wounds. One particularly creative student selected trees related to Tabernanthe iboga, a tree indigenous to Africa that produces ibogaine, an indole alkaloid that has been postulated as a treatment for drug addictions12. He hopes to identify a microbial source for this natural product. The extensive scientific possibilities and the intellectual license to design their own experimental plan has been highly motivating to the students.
In March, 15 students (most in their sophomore year) and one high school biology teacher traveled to the upper Amazonian basin of South America, where we collected plant samples (Fig. 2). With the help of Percy Nunez, a field biologist with an encyclopedic knowledge of neotropical botany, each student found most of the plants on his or her list (Fig. 2). "I found MY plant!" was a common cry heard along the jungle trail. Even more encouraging was the call "We found YOUR plant" over the group's walkie-talkie network. The group developed a strong sense of collegiality and cooperation during the expedition that was manifest in the willingness of students to collect samples from each other's lists. Since our return, the plant stems have been dissected and the segments placed in Petri dishes. Growth is evident on all of the plates, as almost every plant collected had several endophytic associations. We will spend the rest of the summer characterizing these microbes and screening for any bioactive compounds they may produce. The program is designed to provide students with multiple success points, beginning with plant identification and collection, and followed soon after with microbial isolation. The objectives then build in complexity as they phylogenetically classify the microbes by molecular analysis, screen them for bioactivity, and undertake chemical fractionation (Fig. 1).
This is the first large-scale attempt to use endophyte isolation and characterization as a tool for empowering undergraduate research. Though student enthusiasm is anticipatory of success, it is still too soon to judge the value of our experiment in science education. However, work done on a more limited scale has established that undergraduates can use this platform to successfully design and implement their own scientific plan. In the three cases listed below, the students embarked on the laboratory studies after the fieldwork was completed by others, but the examples demonstrate the feasibility of using natural product isolation as a format for inquiry-based undergraduate research. These three students were undergraduates at Montana State University working in the laboratory of G.A.S. during summer break and subsequent semesters.
A novel endophytic fungus (Muscodor albus) was isolated from a cinnamon tree growing along the Caribbean coast of Honduras (Fig. 3a). Emily Dirkse focused on this organism during an NSF-sponsored summer undergraduate program. M. albus produces volatile compounds that inhibit and/or kill a wide spectrum of fungi and bacteria. Emily identified 28 of these natural products by GC/MS and prepared artificial chemical mixtures that mimicked the antibiotic effects of the fungus. Individual fungal compounds had some inhibitory effect against test fungi and bacteria, but none was lethal. However, when added in combination they acted synergistically to cause death in a broad range of plant and human pathogenic fungi and bacteria, including Escherichia coli. A report describing the "mycofumigation" effects of M. albus was published in the journal Microbiology and highlighted in the editor's choice section of Science13, 14, and a patent was issued for its use in the treatment of human waste.
Figure 3: Endophytic microbes isolated from plants.
(a) M. albus from Honduras. (b) One of the streptomycetes isolated from the Australian snakevine.
Full size image (23 KB)Plant materials isolated from the jungles of the Peruvian Amazon were given to Bryn Daisy, another undergraduate working on endophytic microorganisms. Her goal was to identify relatives of M. albus by virtue of their resistance to its antimicrobial volatiles when grown on the same Petri plate. She isolated and characterized a novel fungus, Muscodor vitigenus, that produces naphthalene as its sole volatile compound, and she demonstrated that the fungus produces naphthalene at sufficient levels to repel insects15, 16. This provided a potential clue about the selective advantage provided to the plant by its microbial association.
Lindsey Browne became particularly adept at isolating endophytes from plants collected throughout the world. She recovered six novel bioactive streptomycetes from Notofagus spp. growing in Patagonian Chile. The isolates were characterized by molecular and morphological features and found to be active against such plant pathogens as Phytophthora cinnamomi and Pythium ultimum17. She isolated endophytes from plants collected on Socotra, an island off the coast of Yemen in the Arabic Sea18, and had a major role in the isolation of 139 endophytic streptomycetes from an Australian snakevine plant (Fig. 3b). Many of these organisms were found to be novel based on their recombinant DNA sequences, unusual morphologies and biological activities19.
Our hope is that a group of students involved in an institutionalized program will also feel intellectually empowered to become creatively engaged in science. Because the program includes both plant collection and microbial characterization, students will have the opportunity to take the project from the field to the laboratory. The program is a fishing expedition by its very design, so it is uncertain what will be found, but the diversity of microbial life in these environments is sufficiently rich that each student is all but assured to 'catch' something interesting. The program provides a holistic view of scientific training that incorporates experience in the subjects of ecology, microbiology, biochemistry, bioinformatics, pharmacology and chemistry.
Though funding for students to visit a rainforest is beyond the budget of most college courses, a less expensive variation on the program could still be implemented at other institutions. Plant materials can be collected from many locations that do not require extensive travel. There are many old-growth forests or other unique ecosystems within driving distance of most campuses that could provide the source of materials for endophyte-targeted programs. An enormous amount of biological and chemical diversity remains to be discovered20. We anticipate that the range of scientific possibilities will inspire the scientific imagination of most of the students who participate.
