Lair of the Round Worm

For one of America’s costliest pests, a self-made sanctum is also its chief vulnerability. By Melody Kroll

The path of Heterodera glycines, a pallid roundworm known to farmers everywhere as the soybean cyst nematode, leaves a trail of financial hardship way out of proportion to its ultra-diminutive size. How much?

Try $1.286 billion in crop losses each year, estimates nematode expert J. Allen Wrather.

Wrather is a professor and plant pathologist at MU’s Delta Research Center. He recently conducted a study of diseases affecting soybean production in the United States from 2006 to 2009. When it comes to soybean loss, he found, the cyst nematode is truly in a league of its own.

“It causes more yield loss than any other pest of soybean in the U.S. It far surpasses the damage caused by the second most damaging pest, Phytophthora root rot,” says Wrather.

Still, if it weren’t so costly—and violently destructive—one could almost admire the worm’s parasitic modus operandi. Each microscopic creature hatches from a soil-embedded egg, and then worms its way toward a vulnerable soybean root. On arrival, it extends a hollow, spear-like “stylet” from its mouth, piercing through cell walls as it stabs its way into the root’s core. Once inside, the worm selects a single cell and spits into it. The spit alters the chemistry of the cell’s cytoplasm, causing it to draw nourishment away from the rest of the plant.

Microscopic view of the soybean cyst nematode.

Like the chewing-gum-addicted Violet Beauregarde in Charlie and the Chocolate Factory, the female nematode sucks up the plant’s vital nutrients, growing bigger and bigger until she eventually bursts through the outer surface of the root. At this point she mates, fills with eggs, and dies, her egg-filled corpse hardening into a cyst. These cysts are visible to the human eye, resembling a pinhead-sized lemon on the root. The above-ground portion of the plant, now starved for nutrition, becomes stunted and yellow as the nematodes divert the plant’s resources for their own benefit.

For some growers, stunting and discoloration might suggest any number of soybean problems. But not to a fifth-generation farmer and commodities trader like Jason Bean. “You just know because you’ve had that legume crop on [the field] forever and ever, and you can just tell when the nematode population is getting high,” Bean says.

Bean farms 4,000 acres in rice, soybeans, corn, and cotton near Gideon, a small town near the top of Missouri’s Bootheel. “We’ve had the cyst nematode down here for a long time,” he says.

He’s not alone. The first confirmed case of the soybean cyst nematode in Missouri occurred in the Bootheel in 1956, just two years after the first U.S. infestation was identified in North Carolina. The pest likely arrived from Japan, where scientists say it was identified more than 75 years ago. It is now confirmed in more than 25 states.

Bean says nematodes are just one factor he takes into account when deciding how many acres to plant in soybeans each year. But it’s an important one. When it comes to their appetite for destruction, he says, nematodes never sleep. That is why as vice chair of the production committee of the United Soybean Board, a farmer-funded organization focused on soybean research and marketing issues, Bean thinks it is important to look further ahead than the next growing season.

“You don’t drive looking at the hood, but 100 miles down the road. You’ve got to be on top of things,” says Bean.

For Bean and other producers, the fight against cyst nematodes boils down to something of a “survival of the fittest” contest between the soybean and pest, the outcome of which will be decided by whether the plant’s genetic variability can triumph over the pathogen’s amazing adaptability. Until now, the soybean has had the upper hand. But that is changing.

Adaptability, says Wrather, makes the cyst nematode special: “It’s one of the very few pests that is adapted to the entire soybean production area in the United States.”

Most pathogens are restricted to one agricultural region or another. The cyst nematode is less particular. In climates wet and dry, temperatures cold and hot, soils sand and clay, the worm thrives. This extreme adaptability fueled the cyst nematode’s quick spread across the United States and Canada.

Melissa Mitchum explains the soybean cyst nematode’s destructive life-cycle.

Conqueror Worm The USDA estimates soybean cyst nematodes, illustrated on this page from nematode images created for Illumination by Melissa Mitchum and MU’s Electron Microscopy Core Facility, are responsible for more than 31,539,690 tons of soybean loss in North America each year.

To counter this adaptive advantage plant scientists such as MU professors David Sleper, Grover Shannon, and Henry Nguyen, all of whom specialize in soybean breeding and genetics, are sifting through soybean germplasm—collections reflecting the bean’s natural genetic diversity—to identify cultivars with cyst nematode resistance. The goal is to pinpoint specific genes that confer this resistance, and then breed these genes into high-yielding lines.

“A lot of varieties that have some resistance have been made available to farmers, and farmers have been planting those in fields that are infested,” says Wrather. The availability and use of resistant soybean cultivars, he says, has made a huge difference in reducing soybean cyst nematode yield loss.

Unfortunately, the genetic diversity of resistant soybean cultivars is limited. About 90 percent of soybeans available to farmers are derived from one plant introduction, called 88788. Since growers always plant something with soybean cyst resistance, they are essentially creating a monoculture of 88788.

Cyst nematodes, being a varied bunch, love this arrangement. A single field of soybeans is typically infested with a genetically diverse population of cyst nematodes. Some infect resistant soybean plants; others can’t. Over time, the successful nematodes grow more populous, making resistant soybean cultivars less effective. Recent surveys show that nearly 80 percent of nematode populations in Missouri, irrespective of region, were able to attack 88788. Clearly, says Wrather, “what we need are soybean varieties with broad, durable resistance to soybean cyst nematode.”

Melissa Mitchum, a molecular plant nematologist and associate professor in MU’s Division of Plant Sciences, couldn’t agree more.

“The problem with having a single source of resistance is that we end up selecting for the individuals that can grow on that type of resistance,” Mitchum says. “We can deploy new resistant cultivars, but the same thing is going to happen: the nematode will continue to adapt.”

To move beyond the cycle, Mitchum says, involves asking a different set of questions: What molecular weaponry makes the cyst nematode so effective at attacking soybeans in the first place? And how might we neutralize it?

The answer to both queries, Mitchum believes, lies hidden in the nematode’s spit. When a cyst nematode selects a soybean root from which to feed, proteins in its spit induce developmental and metabolic changes. These include replacement of the root cell’s central vacuole—a sac that provides structural support and storage for the plant—with a nutritious gel meant to perpetuate the nematode’s own growth.

Gradually, the walls of the cell under nematode attack begin to fuse with neighboring cells. The result is the formation of one huge feeding cell, called a syncytium, that provides the cyst nematode with a ready source of nourishment.

Once the nematode has committed to feeding, it remains sedentary in the root. “It’s 100 percent dependent upon the successful establishment of the syncytium,” says Mitchum.

In resistant soybean cultivars, she explains, nematodes ably penetrate and migrate through the roots, but they cannot set up the feeding cell. The feeding cell dies and the nematode, now no longer able to move or feed, dies as well.

This is not news. Scientists have long known that the syncytium is necessary to the nematode’s survival. More novel is the focus on the proteins in nematode spit that enable the worm to set up the feeding site. “We think these are the proteins that are really the major players in signal exchange between the plant and the nematode to establish successful infection,” Mitchum says.

A breakthrough occurred about ten years ago after scientists were able to extract contents of the gland cells responsible for spit production directly from the nematode.

Microscopic view of the soybean cyst nematode.

“This began back when I was a Ph.D. student in Eric Davis’s lab at North Carolina State University,” says Mitchum. “Working in collaboration with Richard Hussey’s group at the University of Georgia, we started making gland-enriched gene libraries that we subsequently mined for secreted proteins that could end up in the worm’s spit. Those became our candidates for parasitism proteins.” These proteins are now called “effector proteins” because they exert some effect on soybean plants.

Over the past decade, Mitchum has been working to collect more information about the role these nematode effector proteins play both in the nematode’s feeding habits and, more specifically, the development of its syncytium.

Since arriving at MU in 2003, Mitchum and her lab team, in collaboration with Xiaohong Wang’s group at Cornell University, have been examining a specific family of effector proteins termed CLAVATA3/ESR-like peptides, or CLEs. Her lab confirmed the presence of multiple forms of soybean cyst nematode CLEs, and also isolated the CLE genes from the beet cyst nematode. The beet cyst nematode is a species closely related to the soybean cyst nematode, but one that infects Arabidopsis, the quick-to-reproduce plant that scientists use as a research model.

Plants use CLEs to influence the development of adjacent cells by activating receptive proteins on the cell’s surface. This well-understood process is part of the molecular cross talk needed for normal growth. What is more interesting, says Mitchum, is that nematode CLEs are the first to be identified outside of plants.

She hypothesized that the CLE’s presence among effector proteins may mean the nematodes are attacking its host by mimicking plant proteins. Simply put, CLEs are, after a fashion, reprogramming the soybean root cells using the plant’s existing developmental pathways.

Recent results from Mitchum’s lab, published in the September 2010 edition of the journal New Phytologist, confirm this mimicry. One of the researchers working with Mitchum on the project, MU postdoctoral fellow Jianying Wang, discovered that after the nematode CLEs are delivered into a plant cell a specific sequence of the protein, called the “variable domain,” re-targets the protein back outside the cell. Once outside, the nematode CLE mimics the plant CLE and binds to a plant receptor protein on the cell’s surface. When activated in this way, it triggers reactions inside the cell leading to the formation of the syncytium.

“The next step is to identify exactly which receptors these nematode CLEs are actually binding to in the plant. Mitchum says that Amy Replogle, a graduate research assistant working on her lab team, is making progress toward this result.

“We’ve identified a number of candidates that show, if you knock out the receptor in the plant, it can’t perceive the nematode CLEs and the infection is blocked,” says Mitchum.

Identification of receptors for nematode CLE peptides is key to unveiling the role peptides play in the formation of feeding cells, a development that could turn the tide on the cycle of plant resistance and nematode adaptations.

“If we can block the nematode CLE from interacting with the soybean root cell receptor, that could be a potential mechanism for engineering durable resistance,” Mitchum says.

This is just one effector protein and one strategy. Mitchum and her colleagues are also confident that additional spit proteins are good targets for syncytium disruption. And not just CLEs. Mitchum is also involved in a collaborative project with Iowa State University, North Carolina State University and the University of Georgia to characterize the function of 30 additional effector proteins.

Unlike the CLEs, several of these other effector proteins are like nothing else anyone has seen: “They match nothing in the database. They don’t match to other pathogen effectors or nematode sequences. Nothing. They’re completely novel,” says Mitchum.

Such “pioneer” effector proteins are potential game changers, she adds. “The exciting thing about these effector proteins is that they’re very specific to soybean cyst nematodes. This means that transgenic strategies targeting these proteins for disruption will be less likely to harm beneficial organisms or other nematodes.”

Across the midwest, millions of acres are planted in neat green rows of soybeans. Plumes of water blast skyward from spray irrigation systems gleaming in the sun.

U.S. farmers harvested more acres of soybeans in 2010 than either corn or hay, according to the U.S. Department of Agriculture. In Missouri, revenue from soybean production will earn farmers some $2 billion.

Pull Quote: ‘We can deploy new types of resistance using naturally resistant cultivars, but the same thing is going to happen: the nematode is going to adapt.’

Much of this success can be attributed to pest management, say experts like Allen Wrather. And controlling the cyst nematode, largely through the use of resistant soybean varieties, has been a big part of the pest management strategy.

“Because of the efforts of soybean breeders, like Drs. Sleper and Shannon, and our extension efforts to inform soybean farmers about the problems in Missouri, cyst nematode control has improved, and soybean farmers in Missouri realized $15 million more income compared to the late 90s.”

Still, Wrather says, problems remain. Currently the most effective method for reducing the effects of the pest is through crop rotation with a non-host plant, such as corn, along with resistant cultivars.

Such rotations have worked well for farmers like Jason Bean, but he’s among those growers holding out for something better. “It is frustrating for me as a farmer that, while advances are being made, rotation is still the best strategy,” says Bean.

Change may be coming soon, Mitchum says, in both nematode reduction and soybean improvement.

“We’re to the point where this is going to speed up. In the last five years, the soybean community has generated a lot of new tools. We now have the entire soybean genome sequence, new reverse genetic tools, improved transgenic technologies, and more efficient phenotyping methods and gene mapping tools in place.”

Mitchum’s lab is among several in the United States working to use these tools to clone and map resistance genes in soybeans. “We’re very close to identifying the genes. Once we know what the resistance genes are, then we can start asking questions about what genes downstream play a role in conferring resistance to the nematode.”

On the horizon is the completed genome sequence of the soybean cyst nematode. Soon, says Mitchum, we’ll have the whole molecular arsenal the nematode uses to attack soybeans.

And the payoff?

“If we can find multiple ways to disrupt this ability to make the feeding site, then we can pyramid these different approaches into soybeans to create the broad, durable resistance that soybean producers need.”

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Reader Comments

viridiana salazar wrote on February 18, 2011

this video and artical really interesting and really too look at i would love too get too lear and kn ow more about roundworms (:

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