Summer 2019


Tomatoes grow in the King Hall greenhouse as part of a plant pathology study. They will be used to harvest seeds for additional experiments. Photo by Michael P. King

At some point in your life, you’ve probably gone to class knowing you left an assignment unfinished. The knot in your stomach grew as homework was collected. And, as you practically crawled under your desk to avoid the teacher’s gaze, a ridiculous, clichéd excuse may have flitted through your mind: “The dog ate my homework.”

For a team of researchers in the Department of Plant Pathology, the destroyed assignment was a plot of test tomatoes in northern Florida. But they didn’t need to devise a hungry canine. For them, the villain was a swarm of voracious whiteflies. And then came not one, but two howling hurricanes.

These scientists aren’t making any excuses, though. Instead, they’re treating the unfortunate events as opportunities to better understand how, in real-world settings, tomato genetics influence the microbes that live on them, even under the pressure of ravenous insects and 80-mile-per-hour winds.

“If we’ve learned anything in the project yet, it’s that fieldwork is very challenging,” says professor of plant pathology Jeri Barak with a smile. “It’s important to us that we are doing the work in the real environment in the field.”

In this real environment, Barak is working with plant pathology colleagues Caitilyn Allen and Rick Lankau to study the microbiomes of tomato plants. They’re exploring how a plant’s genetics influence its microbiome and, in turn, how microbiomes may alter the behavior of pathogens. And they’re finding that the ability of plants to ward off diseases may also be a key component in protecting human health.

Where Outbreaks Start
Raw eggs or undercooked chicken typically get the bad rap as food poisoning sources, but over the last 10 years, fresh produce has been the more likely culprit in cases of salmonellosis. And the romaine lettuce episode of Thanksgiving 2018 showed that fresh produce can also harbor a toxic form of E. coli (as opposed to the mostly harmless strains of the bacteria that humans need to live). It’s a risky pathogen. Of those who get infected with the toxic strain of E. coli, 1% will die and another 3%–6% will develop hemolytic uremic syndrome, an illness that shuts down kidneys and requires dialysis for the life of the patient. Those percentages increase in children and immunocompromised people.

Although these pathogens prove dangerous for humans on the consumer side of the food system, epidemiology studies show that contamination of fresh produce happens in the fields, not during processing or transportation. Animals and water both bring pathogens to areas where crops grow. So Barak and her colleagues went to the field to study the microbes and the circumstances around them.

“We’re having outbreaks annually with these organisms now,” says Barak. “So we’re looking at the agricultural system, trying to figure out the factors that come together to create a perfect storm that results in outbreaks in humans.”

Inside Microbial Communities
Although their investigation is system-wide, much of the researchers’ focus is on the many microbiomes found in plants. Microbiomes, or communities of microbes that live in a particular environment, are often thought of in humans as part of a healthy gut. We take probiotics to build up healthy microbes and read about how our diets can affect our microbiomes. Plants, too, have microbiomes, where human pathogens, such as Salmonella and the toxic strain of E. coli, although rare, are sometimes present.

From left, members of the plant pathology department — assistant professor Rick Lankau, professor Caitilyn Allen, and associate professor Jeri Barak — gather in the King Hall greenhouse where tomato plants grow as part of their study. Photo by Michael P. King

“There are different groups of microbes in plants, though there’s not rigid, distinct separations,” explains Lankau, an assistant professor of plant pathology. “We talk about endophytic microbes, those that live inside the plant — for instance, in the vascular system — and epipthytic ones, those that live on the surface, such as on the leaves. There are also microbes associated with the rhizosphere, the zone of soil around a root that’s under the influence of that root.”

The different environments where microbes live on a plant vary greatly, and they each pose their own challenges. The roots are leaky and buried under soil, and microbes that live on or near them have to contend with antimicrobials that the roots exude. These microbes also may have to partake in intermicrobial warfare to defend their place in a crowded community.

The leaves, on the other hand, are bombarded by sunlight for much of the day and act as tiny fortresses set up to defend the plant against invaders. There are some cracks in the walls, though, in the form of small holes called stomata, which open and close to allow the plant to “breathe” or exchange gases. Plant pathogens sneak through these stomata to infect the leaf and live more comfortably in small chambers, where they can avoid drying out in harsh UV rays.

With so many niches for microbiomes in the plant, researchers want to better understand which microbes live where and how they might affect the presence of human pathogens in these environments. In 2017, with funding from the Wisconsin Alumni Research Foundation, UW–Madison launched the Microbiome Initiative, a strategic effort to fund research that galvanizes the research community and allows UW faculty to be more competitive when applying for federal grants. Barak, Lankau, and Allen saw the initiative as an ideal opportunity and came together with Barak’s colleagues in the Sunshine State, including Gary Vallad PhD’03, associate professor of plant pathology at the University of Florida, to propose a winning project using tomatoes as their research model.

What Tomatoes Teach
Why tomatoes? For the past 15 years or so, this widely cultivated fruit, which is often enjoyed raw, has been a common source of salmonellosis. Each year, tomatoes, usually in the form of salsa, take the blame for foodborne illnesses. They are a cash crop in many developing countries, and they grow almost everywhere in the tropics, clear signs of their worldwide importance. All these factors, combined with Barak’s standing collaboration with researchers in Florida who study tomato diseases, made the plant an ideal fit for the project.

To address the question of how microbiomes and human pathogens interact, Barak and her colleagues are starting with plant diseases. Specifically, they are using a variety of tomatoes that has been engineered to resist certain types of plant pathogens. One plant cultivar, Bs2, is named after a resistance gene borrowed from peppers. Bs2 plants have resistance specifically to Xanthomonas, a microbe that causes bacterial spot. The other cultivar, EFR, is named after the elongation factor receptor gene that was cloned and put into the tomato. EFR plants have resistance to a number of pathogens, including Xanthomonas and another bacterium called Ralstonia solanacearum, which causes bacterial wilt.

“We have plants with two different types of defenses,” says Lankau. “We have plants that basically always have their defenses on, their shield up all the time. And then we’ve got another version that has this narrow defense against just the Xanthomonas pathogen.”

With these two models of tomato plants growing in the sun-kissed Florida fields alongside “normal,” unmodified tomatoes, the researchers challenged each with either Ralstonia or Xanthomonas to put them on alert and kick-start their defense mechanisms. How would the microbiomes of the unmodified tomatoes versus those with either the broad or narrow resistance change in response to a plant pathogen? And what would those changes mean for human pathogens?

Barak already had some clues from previous work. “What we’ve found, starting in about 2014 in greenhouse experiments, is that when tomato plants are infected with Xanthomonas, Salmonella does really well,” she says. “And if it’s doing well in the plant, that means the fruit gets contaminated.”

The researchers started growing both tomato models in fields in the same area of Florida. But an invasion of whiteflies, which carry a devastating pathogenic plant virus, decimated their plots. Luckily, their collaborators were growing the same plants in a field unaffected by whiteflies and were able to supply leaf samples that season. To avoid losing all their research in the event of another catastrophe, the UW scientists decided to split the research into two places. The group studying the EFR tomatoes stayed in the north, where Ralstonia is a common problem, and the group studying Bs2 tomatoes and Xanthomonas moved south.

Tomato plants in a field plot in Florida display signs of bacterial spot, which is caused by the pathogen Xanthomonas euvesicatoria. Photo courtesy of Adam Bigott

Then, more proverbial homework-eating dogs showed up. In 2017, Hurricane Maria hit. The researchers were astonished to find that some of the plants survived the natural disaster, but they couldn’t sort out which were inoculated and which should have been disease-free. And in 2018, the northern group suffered a direct hit from Hurricane Michael. Luckily, amid the upheaval, some samples and data were salvageable.

Plant Disease and Food Safety
Adam Bigott, a plant pathology graduate student in Barak’s lab, works on the ground gathering plants and data for the Bs2 model system. “I travel to Florida twice a year to collect leaf samples,” he says. “It’s imperative to process the samples and extract DNA quickly so it doesn’t degrade. Then I prepare the samples for DNA sequencing so we can find out which microbes are in the sample. My end product is a very large spreadsheet that I spend a lot of my time processing.”

Even with all that data to decipher and hurricanes and insects to face, researchers are starting to uncover answers to some of their burning questions. Bigott and Lankau, for example, have discovered fascinating distinctions between how plants with the Bs2 narrow resistance and those without react to Xanthomonas.

“On the leaf surface, the different plants weren’t that different in terms of their microbe communities,” says Lankau. “But when the pathogen came in, the whole microbial community shifted and became less diverse. We saw that the two most common members of a healthy community decreased. The Bs2 gene did a very good job of preventing the infection, and the microbial communities on those leaves did not shift like those on the plants vulnerable to the pathogen.”

The number of changes in the microbial communities that seem to occur when a plant gets infected suggests that the entire microbial state could become more vulnerable to harmful microbes and, possibly, human pathogens.

“Again, our greenhouse data has shown over and over again that with plant diseases like bacterial spot, Salmonella does better,” Barak says. “But we could show in the field that, with resistance like the Bs2 model, you can decrease the human pathogens. If that’s true, then we need to increase our investment in reducing plant disease to increase food safety.”

Results with the EFR tomato model are proving to be a bit more complex. The researchers did not see any effect of the EFR gene on microbes living on the leaves, but the microbe communities in the roots of EFR plants were different than those found on the normal tomato plants. Specifically, there were more Actinobacteria, a group of bacteria that play an important role in decomposing organic material and in producing antibiotics, and fewer Proteobacteria, which include a wide variety of pathogens, such as Salmonella, and many of the bacteria responsible for nitrogen fixation. And this was true even when the plants weren’t challenged by a pathogen.

The team is still teasing apart what these changes mean. Although it is by no means a conclusive result, a pattern in their data prompts speculation that an abundance of Proteobacteria is linked with greater yield. It seems that the EFR genetic resistance reduces the infection by pathogens when they’re present. But it may come at a cost: The resistance shifts the communities in the roots to a state that harbors fewer microbes that support or promote growth.

Plant pathology research assistant Adam Bigott poses with tomato plants in a grow room at Russell Labs. Photo by Michael P. King

“In the end, the effects sort of cancel out in the field,” explains Lankau. “This broad spectrum resistance is a useful disease control tool, and I think it is protecting plants from pathogens. But it also is potentially affecting the nonpathogenic, beneficial microbes in a way that’s dragging yield down.”

Lankau finds the results associated with the EFR broad resistance tomato plants especially intriguing. It may answer a long-standing question of his: If plant pathogens are such a nuisance and detriment to plants generally, why don’t we have plants that always guard against all diseases? It would make targeting defenses at specific diseases unnecessary.

“Yet in all of evolutionary history, we don’t have plants that are defended against every pathogen,” says Lankau. “So we’re interested in what might be the trade-off behind putting this broad spectrum resistance out there.”

Plant-Soil Feedback
One way in which those trade-offs may be felt is if farmers start to dial back on the water and nutrients, or inputs, they put on their fields. Over the years, breeders have selected for plants that give higher yields. But they’ve been selecting for those plants in well-irrigated and fertilized fields — comfy places for crops to grow. In other words, they may have been selecting for wimpy plants that no longer need the help of beneficial microbes. They’ve become poor hosts.

But if farmers want to save money and energy and decrease their environmental impact, they will have to move toward agricultural systems with fewer inputs and more variabilities. In response, breeders may need to make the plants resilient again by “teaching” them how to be proper hosts for the helpful microbes they’ve taken for granted.

“Relying on a microbe comes at a cost to a plant,” explains Lankau. “So if the plant can get that thing directly from the field, why pay a microbe? In fact, the plants that do pay microbes are probably smaller, since it’s a yield drag. We may have selected against plants that host beneficial microbes. We have to go backward now and figure out what we bred away. It’s possible we bred past a plant that could have been extremely useful when we were only worried about yield in irrigated, fertilized conditions.”

Researchers can get insights into useful plants that may have been overlooked in the breeding process in what Lankau calls plant-soil feedback experiments. Lankau and his team take soil from various fields, grow plants in the different soils, and expose the plants to a stressor, such as disease or drought.

Since researchers can sequence the genome of microbe communities found in the soil in each pot, they can figure out which microbes in particular are best for surviving drought or fighting off a disease. In this way, they could define the best “probiotics” for plants under different circumstances. Perhaps in the future, we can have the equivalent of a probiotic supplement for crops to keep them healthy.

“I don’t think of these plants as individual organisms anymore,” says Bigott. “I’ve come to see them as a much more complex assemblage. There is interplay between plants and the pathogens and microbes that associate with them.”

Understanding that interplay and the balance between inhibiting plant pathogens while supporting beneficial microbes and making plants as strong as possible will be extremely useful as foodborne illnesses continue to cause problems.

“On the human gut side, people are really interested in what a healthy microbiome is and what we can do to promote that,” says Barak. “I’d like to know that on the plant side too. When you see a decrease in human pathogens, do you see an increase in something else? Can we decrease the chance of foodborne illness by increasing the amount of other particular microbes [on plants]?”

Rows of tomato plants stand in a commercial grower’s field not far from the CALS–University of Florida team’s test site in northern Florida in 2017. Photo courtesy of Jeri Barak

All these experiments could lead to a range of healthier and more robust crops, not just tomatoes. Studies on the leaf microbiomes of soybeans, rice, and clover have found that all three crops have the same three most abundant microbes. It’s possible that a healthy prescription for one plant could help researchers write healthy prescriptions for others. They are, after all, faced with many of the same challenges in the field.

For Barak, this is the power of their tomato microbiome project — not just that they’re posing questions about healthy plant microbiomes but that they’re digging up answers in the field, where plants grow and where crops encounter environmental challenges, diseases, and pests.

“Our agriculture is outside, and so we have to go where our agriculture is to really understand it,” she explains. “That is where we will see how defense mechanisms and genetics can affect microbial communities and human health. We have to do it outside, insects or no insects, hurricanes or no hurricanes.”

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