Novel technique reveals insights into soil microbe alarm clock

UNIVERSITY PARK, Pa. — Soil microbes benefit plants by helping with nutrient uptake and disease resistance. Modulating these communities of bacteria and fungi could potentially sustainably improve agriculture, according to the Food and Agriculture Organization of the United Nations. But a major obstacle to this approach has been that many of these microbes are dormant, or inactive, in the soil — and microbes must become active to colonize plant roots, moving into and living inside plant tissues. Until now, it hasn’t been clear how dormancy affects which microbes make it into a plant, but in a new study using a novel technique, a team of researchers at Penn State found that a microbe’s activity appears to matter more than abundancy.

They published their results in mSystems, a journal published by the American Society for Microbiology. The researchers found that microbial activity was 10 times higher inside the plant — the endosphere — compared to nearby soil or even soil right around the root — the rhizosphere. They hypothesized that the disparity likely resulted because plants provide more nutrients inside their tissues. Active microbes in the rhizosphere — soil right around the root — were more likely to colonize the plant than microbes that were abundant but more dormant.

There is an enormous diversity of microbes in soils but only a small minority seem to be able to make it into plants, explained study first author Jennifer Harris, a doctoral degree candidate in Penn State’s Intercollege Graduate Degree Program in Ecology.

“We saw that there seems to be a couple of families and groups that more commonly make it into the plant, but most soil microbes are dormant, so they must ‘wake up’ and exit dormancy to have the potential to carry out plant-beneficial functions,” she said. “It is unclear at this point if dormant microbes revive in proximity to plant-produced resources and if overcoming dormancy in the soil is important for successful plant colonization. Finding the answer to that question is the next phase of this line of research.”

To determine which microbes were active, the researchers used a technique called BONCAT — bioorthogonal non-canonical amino acid tagging — to mark only active microbes. BONCAT is a chemical-labeling technique used to probe proteins that have been newly synthesized during a specific, defined window of time.

By coupling BONCAT to flow cytometry — a laboratory technique used to analyze and characterize individual cells or particles in a fluid sample to sort active cells — and the sequencing of a marker gene to identify microbes, researchers were able to get a snapshot of the complete make-up of the active sub-fraction of a microbial community rather than the whole community where most organisms are dormant. The team also confirmed the BONCAT approach worked with microscopy to visualize the tagged microbes inside plant tissues.

“This is the first time BONCAT has been used to study microbes along the gradient from nearby soil to root surface to inside the root,” said team leader Estelle Couradeau, assistant professor of soils and environmental microbiology, and senior author on the study. “Perhaps the biggest innovation here is that we used this method to actually look inside the plant and see microbes and then identify which ones are active.”

To see how microbial activity changes near and inside roots, the researchers chose crimson clover, or Trifolium incarnatum, as a test plant. It’s a legume commonly grown as a cover crop in the U.S. Northeast that forms root nodules with bacteria.

This soil microbes research indicates that proximity to plant roots may help microbes become active, and that microbial activity is a key predictor of whether a microbe will successfully enter and live in the plant, Couradeau pointed out.

“This knowledge helps in designing better microbial inoculants — microbes added to soil to help crops, often applied as crop seed coatings,” she said. “Instead of just choosing microbes that grow well in the lab, we might choose those that become active near roots in real soils. This research offers a new way to identify which microbes actually ‘work’ in the soil-plant system by looking at their activity, not just their presence. This could make microbial products more effective and agriculture more sustainable.”

This research was enabled by use of the Flow Cytometry Core Facility and the Genomics Core Facility at Penn State’s Huck Institutes of the Life Sciences. Contributing to the study at Penn State were: Sharifa Crandall, assistant professor of soilborne disease dynamics and management; Regina Bledsoe, former lab technician in the Burghardt lab; Sohini Guha, postdoctoral scholar in the Department of Plant Science; and Haneen Omari, former lab technician in the Couradeau lab.

This work was supported by the U.S. Department of Agriculture’s National Institute of Food and Agriculture (USDA-NIFA). Funding was provided by USDA-NIFA through an Agriculture and Food Research Initiative Predoctoral Fellowship to Harris and an Agricultural Microbiomes in Plant Systems and Natural Resources grant to Burghardt. Seed grant funding was provided by the Penn State Ecology Institute.