Fort Ellis inoculation day

Today was a big day out in the field at Fort Ellis: virus inoculation day for the project I’ve been part of, on how farming system can alter reactions to adverse growing conditions (like climate change, weed competition, and disease).  This is the second year of the project, and the fifth year of the larger crop rotation study, so a lot is riding on being able to keep to the schedule.

Spring has been cool and wet here in Montana, which has presented us from being able to do work in the muddy fields but hasn’t slowed down the wheat or the weeds.  If the wheat is too developed when the virus is sprayed, the infection won’t manifest well enough to measure.  Thanks to carefully prepared protocols, seasoned personnel, and a stretch of sunny, dry days, we treated our plots and went home early!

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Draft twice, submit once: the grant writing process

Today, the research team that I am a part of submitted a grant which I co-wrote with Dr. Tim Seipel, along with Dr. Fabian Menalled, Dr. Pat Carr, and Dr. Zach Miller. We submitted to the Organic Transitions Program (ORG) through the US Department of Agriculture’s (USDA) National Institute of Food and Agriculture (NIFA).  The culmination of months of work, and some 12+ hour days this past week to meet today’s deadline, this grant will hopefully fund some very exciting work in agriculture!

Research relies on grant money to fund projects, regardless of the type of institution performing the research, though commercial research centers may partially self-fund projects.  Most new research hires to universities will receive a “start-up package” which includes some funding for a few years to buy equipment, pay for a small, preliminary project, or temporarily hire a technician. Start-up funds are designed to hold a researcher over for a year or two until they may apply for and receive grant funding of their own.  Sooner or later, everyone in academia writes a grant.

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Grants may be available for application on a regular basis throughout the year, but some grant calls are specific to a topic and are made annually.  These have one submission date during the year, and a large number of federal grants are due during in the first quarter of the year, a.k.a. Grant Season.  University researchers find themselves incredibly pressed for time from January to March and will hole up in their office for days at a time to write complex grants.  Despite the intention of starting your writing early, and taking the time to thoroughly discuss your project design with all your co-PDs well before you start writing to avoid having to rewrite it all again, most researchers can attest that these 20-30 pages grants can get written over from scratch 2 or 3 times, even before going through a dozen rounds of group editing.

The Bright Idea

Most large grants, providing several hundred thousand to over a million in funding over several years, require project teams with multiple primary researchers (called Principal Investigators or Project Directors) to oversee various aspects of research, in addition to other personnel (students, technicians, subcontractors).  One researcher may conceptualize the project and approach other researchers (usually people they have worked with in the past, or new hires) to join the project.  Project ideas may get mulled over for several years before they mature into full grant submissions, or go through multiple versions and submissions before they are perfected.

The grant I just co-wrote investigates the use of cover crops in Montana grain production.  Briefly, cover crops are plant species which improve the soil quality but which you aren’t necessarily intending to eat or sell.  They are grown in fields before or after the cash crop (ex. wheat) has been grown and harvested.  Legumes like peas, beans, or alfalfa, are a popular choice because they fix nitrogen from its gaseous form in the atmosphere into a solid form in soil which other plants (like wheat) can use.  Other popular cover crop plants are great at bio-remediation of contaminated soils, like those in the mustard family (1, 2, 3). Planting cover crops in an otherwise empty (fallow) field can out-compete weeds that may grow up later in the year, and they can prevent soil erosion from being blown or washed away (taking the nutrients with it).  For our project, we wanted to know how different cover crop species affect the soil microbial diversity, reduce weeds, put nutrients back into soil, and improve the production of our crop.

We designed this project in conjunction with the Montana Organic Association, the Organic Advisory and Research Council, and Montana organic wheat farmers who wanted research done on specific cover crops that they might use, in order to create a portfolio of cover crops that each farmer could use in specific situations.  As these organizations comprise producers from across the state, our research team was able to get perspective on which cover crops are being used already, what growing conditions they will and won’t work in (as much of Montana is extremely dry), and what production challenges growers face inherent to planting, managing, and harvesting different plant types.

Drafting Your Team

When you assemble a research team, you want to choose Project Directors who have different experiences and focuses and who will oversee different parts of the project.  A well-crafted research team can bring their respective expertise to bear in designing a large and multi-faceted project.  For our grant, I am the co-PD representing the microbial ecology and plant-microbe interaction facet, about a third of the scope of the grant.  We will also be investigating these interactions under field settings, which requires a crop production and agroecology background, as well as expanding the MSU field days to include organic-specific workshops and webinars, which requires an extension specialty.

Because grant project teams are made up of researchers with their own projects and goals, in addition to providing valuable perspective they may also change the scope or design of your project.  This can be extremely beneficial early on in the grant-writing phase, especially as you may not have considered the limitations of your study, or your goals are too unambitious or too lofty.  For example, the cover crop species you want to test may not grow well under dry Montana conditions, do you have a back-up plan?  However, as the submission deadline looms larger, changing the focus of your study can cost you precious writing time.  Working in a research team requires a high degree of organization, a flair for communication, and an ability to work flexibly with others.

Identifying the research question

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All grants center around a Project Narrative, and funding agencies will provide detailed instructions on how to format your project grant.  Pay strict attention- in very competitive pools your grant can be flagged or rejected for not having the appropriate file names or section headers.  The Narrative gives introductory background on your topic that details the research that has previously been published.  Ideally, it also includes related studies that you and your team have published, and/or preliminary data from projects you are still working on.  The aim is to provide a reasoned argument that you have correctly identified a problem, and that your project will fill in the knowledge gaps to work towards a solution.  Grant panels are made up of researchers in a related field, but they may not be intimately aware of your type of research.  So, you need to be very specific in explaining  your reasoning for doing this study.  If your justification seems weak, your project may be designated as “low priority” work and won’t get funded.

In our case, cover crops have been used by farmers already, but not much basic research has been done on the impacts of picking one species over another to plant.  Thus, when cover crops fail, it may be unclear if it was because of unfavorable weather, because the previous crop influenced the soil in ways which were detrimental to your new crop, because you seeded your crop too sparsely and weeds were able to sneak in and out-compete, because you seeded too densely and your crop was competing with itself, or something else entirely.

You also need to identify the specific benefits of your project.  Will you answer questions? Will you create a new product for research or commercial use?  Will organic producers be able to use what you have learned to improve their farm production?  Will you teach students?  When you are identifying a need for knowledge and describing who or what will benefit from this study, you need to identify “stakeholders”.  These are people who are interested in your work, not people who are directly financially invested.  For us, our stakeholders are organic wheat farmers in Montana and the Northern Great Plains who want to integrate cover crops into their farming as an organic and sustainable way to improve crops and reduce environmental impact.  Not only did our stakeholders directly inform our project design, but we will be working closely with them to host Field Day workshops, film informative webinars, and disseminate our results and recommendations to producers.

Crafting Your Experimental Plan

Once you have identified a problem or research question, you need to explain exactly how you will answer it.  For experiments in the laboratory or field, you need to be incredibly specific about your design.  How many samples will you take and when?  Will you have biological replicates?  Biological replicates are identical treatments on multiple individual organisms (like growing a single cover crop species in four different pots) to help you differentiate if the results you see are because of variation in how the individual grows or because of the treatment you used.  Do you have technical replicates?  Technical replication is when you analyze the same sample multiple times, like sequencing it twice to make sure that your technology creates reproducible results.  Will you collect samples which will provide the right type of information to answer your question?  Do your collection methods prevent sample deterioration, and how long will you keep your samples in case you need to repeat a test?

In addition to describing exactly what you will do, you need to explain what might go wrong and how you will deal with that.  This is called the Pitfalls and Limitations section.  Because basic research needs to be done in controlled environments, your study may be limited by a “laboratory effect”: plants grown in a greenhouse will develop differently than they will in a field.  Or, you might not be able to afford the gold-standard of data analysis (RNA sequencing of the transcriptome still costs hundreds of dollars per sample and we anticipate over 1,200 samples from this project) so you need to justify how other methods will still answer the question.

Supporting Documents

Even after explaining your research question in the Narrative and your design in the Methods sections, your grant-writing work is still far from complete.  You will need to list all of the Equipment and research Facilities currently available to you to prove that your team can physically perform the experiment.  If you will have graduate students, you need a Mentoring Plan to describe how the research team will train and develop the career of said student.  If you will be working with people outside of the research team, you will need Letters of Support to show that your collaborators are aware of the project and have agreed to work with you, or that you have involved your stakeholders and they support your work.  I was delighted by the enthusiasm shown towards this project by Montana organic producers and their willingness to write us letters of support with only a few days’ notice!  You’ll also need a detailed timeline and plan for disseminating your results to make sure that you can meet project goals and inform your stakeholders.

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Poster presentation at ASM 2016.

Perhaps the most difficult accessory document is the Budget, for which you must price out almost all the items you will be spending money on.  Salary, benefits (ex. health insurance), tuition assistance, travel to scientific conferences, journal publication costs, travel to your research locations, research materials (ex. seeds, collection tubes, gloves, etc.), cost to analyze samples (ex. cost of sequencing or soil nutrient chemical analysis) cost to produce webinars, and every other large item must be priced out for each year of the grant.  The Budget Narrative goes along with that, where you explain why you are requesting the dollar amount for each category and show that you have priced them out properly.  For large pieces of equipment, you may need to include quotes from companies, or for travel to scientific conferences you may need airline and hotel prices to justify the costs.

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On top of what you need to complete the study, called Direct Costs, you also need to request money for Indirect Costs.  This is overhead that is paid to the institution that you will be working at to pay for the electricity, water, heating, building space, building security, or other utilities that you will use, as well as for the administrative support staff at the institution. Since nearly all grants are submitted through an organization (like universities), instead of as an individual, the university will handle the money and do all the accounting for you. Indirect costs pay for vital research support, but they run between 10-44% of the dollar amount that you ask for depending on the type of grant and institution, potentially creating a hefty financial burden that dramatically reduces the available funding for the project.  On a $100,000 grant, you may find yourself paying $44,000 of that directly to the university.

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Draft Twice, Submit Once

The Budget is by far the most difficult piece to put together, because the amount of money you have available for different experiments will determine how many, how large, and how intensive they are.  Often, specific methods or whole experiments are redesigned multiple times to fit within the financial constraints you have.  If you factor in the experimental design changes that all your co-PDs are making on the fly, having to balance the budget and reconstruct your narrative on an hourly basis to reflect these changes, and the knowledge that some grants only fund 6-8 projects a year and if you miss this opportunity you may not have future salary to continue working at your job, it’s easy to see why so many researchers find Grant Season to be extremely stressful.

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2016 Year In Review

Looking back

2016 started with a bang when I launched this site and joined Twitter for the first time!  For the first quarter of the year, I was a post-doctoral researcher in the Yeoman Lab in the Department of Animal and Range Sciences at Montana State University.  I was working on a total of eight grants, ranging from small fellowships to million dollar projects, both as a principal investigator and as a co-PI.  I was also doing the bioinformatic analysis for multiple projects, totaling nearly 1,000 samples, as well as consulting with several graduate students about their own bioinformatic analyses.

In late spring, my position in the Yeoman lab concluded, and I began a post-doctoral position in the Menalled Lab in the Department of Land Resources and Environmental Sciences at MSU.  This position gave me the opportunity to dramatically increase my skill-set and learn about plant-microbe interactions in agricultural fields.  My main project over the summer was studying the effect of climate and other stresses on wheat production and soil microbial diversity, and this fall I have been investigating the legacy effects of these stressors on new plant growth and microbial communities.  I have extracted the DNA from all of my Fort Ellis summer trial soil samples, and look forward to having new microbial data to work with in the new year.  Based on the preliminary data, we are going to see some cool treatment effects!

Over the summer, I attended the American Society for Microbiology in Boston, MA in June, where I presented a poster on the microbial diversity in organic and conventional farm soil, and the Joint Annual Meeting for three different animal science professional societies in Salt Lake City, UT in July, where I gave my first two oral conference presentations. One was on the effect of a juniper-based diet on rumen bacteria in lambs, and the other was on the biogeography of the calf digestive system and how location-specific bacteria correlate to immune-factor expression.

Thanks to a lot of hard work from myself and many collaborators, a number of research projects were accepted for publication in scientific journals, including the microbial diversity of agricultural soils, in reindeer on a lichen diet, and in relation to high-fat diets in mice, it also included work on virulent strains of Streptococcus pyogenes, and a review chapter on the role of methanogens in human gastrointestinal disease.

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Looking forward

A whopping thirteen manuscripts are still in review at scientific journals or are in preparation waiting to be submitted! Some of those are primarily my projects, and for others I added my skills to the work of other researchers.  Editing all those is going to keep me plenty busy for the next few months. I’ll also be writing several more grants in early 2017, and writing a blog post about the Herculean task that can be.

I’ll be concluding my greenhouse study by March of 2017, just in time to prepare for another field season at Fort Ellis, on the aforementioned climate change study that is my main focus. In January, I’ll be spending time in the lab helping to process and sequence DNA from my 270 soil samples, and begin the long task of data quality assurance, processing, and analysis.  I’m not worried, though, 270 samples isn’t the most I’ve worked with and bioinformatic analysis is my favorite part of the project!

This year, I am hoping to attend two conferences that I have never previously attended, and present data at both of them.  The first will be the 2017 Congress on Gut Function in Chicago, IL in April, and the second will be the Ecological Society of America’s Annual Meeting in Portland, OR in August.  Both conferences will give me the opportunity to showcase my work, network with researchers, and catch up with old friends.

If 2017 is anything like the past few years, it’s going to be full of new projects, new collaborators, new skills, and new opportunities for me, and I can’t wait!  So much of what I’ve accomplished over the last year has been possible because of the hard work, enthusiasm, and creativity of my colleagues, students, friends, and family, and I continue to be grateful for their support.  I’d also like to thank anyone who has been kind enough to read my posts throughout the last year; it’s been a pleasure putting my experiences into words for you and I appreciate the time and interest you put in.  I look forward to sharing more science with you next year!

Preparing for my first greenhouse trial

As the 2016 growing season comes to a close in Montana, here in the lab we aren’t preparing to overwinter just yet.  In the last few weeks, I have been setting up my first greenhouse trial to expand upon the work we were doing in the field.  My ongoing project is to look at changes in microbial diversity in response to climate change.  The greenhouse trial will expand on that by looking at the potential legacy effects of soil diversity following climate change, as well as other agricultural factors.

First, though, we had to prep all of our materials, and since we are looking at microbial diversity, we wanted to minimize the potential for microbial influences.  This meant that the entire greenhouse bay needed to be cleaned and decontaminated.  To mitigate the environmental impact of our research, we washed and reused nearly 700 plant pots and tags in order to reduce the amount of plastic that will end up in the Bozeman landfill.

We also needed to autoclave all our soil before we could use it, to make sure we are starting with only the microorganisms we are intentionally putting in.  These came directly from my plots in the field study, and are being used as an inoculum, or probiotic, into soil as we grow a new crop of wheat.

This is trial one of three, each of which has three phases, so by the end of 2016 I’ll have cleaned and put soil into 648 pots with 648 tags; planted, harvested, dried and weighed 11,664 plants; and sampled, extracted DNA from, sequenced, and analyzed 330 soil and environmental samples!

After only a few days, seedlings are beginning to emerge.

 Stay tuned for more updates and results (eventually) from this and my field study!

Wrapping up summer projects

After a hot, dry summer growing season in Montana, the samples have all been collected and the crop harvested for my project investigating wheat production under farming system (organic vs. conventional), climate change (hot or hot and dry), disease (wheat streak virus), and weed competition (cheatgrass) conditions.  We collected wheat and weed biomass from every subplot, totaling 108 bags of wheat and an estimated 500 bags of weeds!  This will be weighed to determine production, and diversity (number of different weed species) will be assessed.

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The biomass bags were temporarily stored in my office until we could find enough shelf space in the lab.

At the end of July, we also collected our final soil samples, which required over 500 grams of aseptically collected of soil in each subplot.  With the extremely dry, clay-containing soil on the farm, this was no quick undertaking, and it took 6-7 lab members a total of 9 hours to collect all 108 samples!  Those soil samples will be used for DNA sequencing to determine what microorganisms are present, and compared to other time points to see how they changed over the summer in response to our treatment conditions.  The soil will also be measured for essential nutrients, such as nitrogen and carbon content, and saved to be used in a greenhouse experiment to look at the legacy effects of microbial change.

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The samples might be collected, but we aren’t done yet.  This was year one of a two-year project, and as winter wheat and cheatgrass need to be sown soon, before it gets cold, we have a lot of prep work to do.  This includes resetting our data collection tools, including gypsum blocks for soil moisture and ibuttons for soil temperature.  We will also need to set up our climate chamber equipment in all new subplots, since we are interested in third-year winter wheat that is part of a five-year crop rotation.  We also plan to start a greenhouse experiment looking at the legacy effects of our soil this fall.  Not to mention all of data to analyze over the winter months!

What I do for a living Part 3: Soil Ecology and Weed Management

In May, 2016, I started a post doctoral position in a laboratory that focuses on weed management in agricultural systems, especially organic farms which don’t use chemical fertilizer or herbicide. My role is to integrate microbial ecology. For example, is the soil microorganism diversity different in fields that compete better against weeds than in fields that can’t? Are there certain microorganisms that make it easier for weeds to grow, and how do they do that? Can we suppress weeds by manipulating bacteria or fungi in the soil?

So far, I’ve been doing field work for my project, as well as assisting other lab members in their own projects, as many large scale greenhouse or field experiments require large groups of helpers to accomplish certain tasks. I’m also new to weed ecology, and I wanted to learn as much as possible. Thus, I put on some sunscreen and one of those vendor t-shirts you get when you order a certain dollar amount, and got to work.

Some of our projects investigate the link between crop health and climate change.  To simulate climate change, we create rain-out shelters to mimic dry conditions, and plastic shielding to mimic hotter conditions.

One of the treatments is to infect crops with wheat streak mosaic virus, to determine whether climate change will affect the plant’s ability to fight infection, and whether it will change soil microbiota. To do this, we needed to infect our crops, which meant growing infected plants in the laboratory and selectively spreading them in the field as a slurry.

 


A similar project is using mites as a virus transmission vector, so we attached mite-infected wheat to healthy wheat.


Another project is collecting data about ground beetle diversity in organic versus conventionally farmed soil. For this, we planted pitfalls traps in fields to collect and identify beetles.


Throughout many of the ongoing lab projects, I’ll be investigating the effect of treatments on soil health and diversity.

My project is part of a larger experiment, which also involves assessing crop and weed communities.  For this, we need to randomly sample plants in the field and collect all above-ground plant material (to measure biomass as weight), as well as the biomass of each individual weed species to measure diversity (number of different weed species) and density (how large the plants are actually growing).

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And, of course, there is plenty of weed species identification!

Field bindweed study sampling

Field bindweed (Convolvulus arvensis) is an invasive plant related to morning glories.  Their winding vines grow into a tangled mass which can strangle other plants, and a single plant can produce hundreds of seeds.  The plants can also store nutrients in the roots which allow them to regrow from fragments, thus it can be very difficult to get rid of field bindweed.  It will return even after chemical or physical control (tilling or livestock grazing), but it does not tolerate shade very well.  Thus, a more competitive crop, such as a taller wheat which will shade out nearby shorter plants, reduce the viability of bindweed.

First seen in the US in 1739, Field bindweed is native to the Mediterranean. By 1891, it had made its way west and was identified in Missoula, Montana.  As of 2016, it has been reported from all but two counties in Montana, where it has been deemed “noxious” by the state department (meaning that it has been designated as harmful to agriculture (or public health, wildlife, property).  In the field, this can be visually striking, as pictured below.  In the foreground, MSU graduate student Tessa Scott (lead researcher on this project) is standing in a patch of wheat infested with bindweed. Just seven feet away in the background, undergraduate Lazarro Vinola is standing in non-infested wheat, with soil core samplers used for height reference.
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In agricultural fields, bindweed infestations severely inhibit crop growth and health.

 

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Last week, Tessa, Lazarro, and I went to several farms in and around Big Sandy and Lewistown, Montana in order to sample fields battling field bindweed. To do so, we harvested wheat, field bindweed, and other weed biomass by cutting all above-ground plant material inside a harvesting frame.  These will be dried and weighed, to measure infestation load and the effect on wheat production.

The sampling locations are consistent with previous years to track how different farm management practices influence infestations.  This means using GPS coordinates to hike out to spots in the middle of large fields.

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It also means getting very dirty driving and walking through dusty fields!

Counting seedlings

Today I went to the MSU Post Farm, one of the several agricultural farms affiliated with MSU Bozeman, along with several other members of the Menalled lab. We were going to count seedlings of the agricultural crop winter wheat, and a competitive weed, cheat grass.

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The plots are left out in the field for ambient rain and temperature conditions, or put into one of two treatments, or both combined, to mimic climate change: increased temperatures and reduced rainfall. This is similar to the project I will be working on, so it’s good job training. And, those study cards that my mentee made me last week really did come in handy!

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I Accepted a New Position in Soil Microbiology and Agroeconomy!

As my current post-doctoral position winds down in the Yeoman Lab in the Department of Animal and Range Sciences, I am pleased to announce that I have accepted a post-doctoral position in the Menalled Lab in the Land Resources and Environmental Sciences Department! Dr. Menalled’s work focuses on agricultural weed ecology and management, particularly with respect to plant-plant interactions, changing climate (water and temperature changes), and now plant-microbe interactions!

I’ll primarily be working on a new two-year project that recently got funded through the USDA, entitled “Assessing the vulnerability and resiliency of integrated crop-livestock organic systems in water-limited environments under current and predicted climate scenarios”, but I’ll also be working collaboratively on several other similar projects in the lab.

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A little pre-job job training: I’m helping to make structures to keep rain out (rain-out shelters) of plots to simulate drier climate conditions.  Photo: Tim Seipel

My new responsibilities will include comparing agronomic performance and weed-crop-pathogen interactions between organic-tilled and organic-grazed systems, evaluating the impact of management and biophysical variables on soil microbial communities, and collaborating in modeling the long-term consequences of these interactions under current and predicted climate scenarios.  It’ll mean a lot more field work, and a lot of new skills to learn!  In fact, to help me study for my new job working with agricultural plants, my mentee and her friend made me flash cards:

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My mentee made my study cards so I could learn to identify common crop and weed species.

In addition to my new skills, I’ll be integrating my background in microbial ecology and bioinformatics, in order to study agricultural ecosystems more holistically and measure plant-microbe interactions.  In the same way that humans eat probiotics to promote a healthy gut microbiome, plants foster good relationships with specific soil microorganisms. The most exciting part is that I will act as an interdisciplinary bridge between the agroecology of the Menalled lab and the microbial ecology of the Yeoman lab, which will allow for more effective collaborations!