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New help from ancient grains

Researchers at the University of Guelph are tapping into some ancient grains to see if their endophytes – microbes that live inside plants without causing disease – can help our modern crops. Manish Raizada and his lab have already found several endophytes that can control the fungal pathogen Fusarium graminearum in laboratory and greenhouse trials. Now they are testing these endophytes in field trials as a step toward possibly developing commercial biocontrol products.

Raizada is an associate professor in the university’s department of plant agriculture, and his research group has been studying endophytes since about 2007. They are adding to the growing body of endophyte research going on around the world, which is finding certain endophytes are able to promote plant growth by performing such functions as controlling plant pathogens, producing plant hormones and making nutrients available to the plant.

Two of Raizada’s PhD students have built a large collection of endophytes as a foundation for the lab’s further research, including the current anti-Fusarium endophyte work. To isolate endophytes from a plant, researchers sterilize the surface of the seeds, roots or shoots, and then culture the microbes from within the samples. The two students sampled mainly grain species and lines that have had to fight off pathogens without help from commercial fungicides.

“A former PhD student in my lab, David Johnston-Monje, isolated endophytes from 14 genotypes of corn from Central America, Mexico, Canada and the U.S. Those genotypes included three groups of corn: wild relatives of corn; Mexican landraces [traditional varieties] and a Canadian First Nations landrace from Quebec; and modern inbreds and hybrids,” Raizada explains. “The wild species obviously don’t grow with the use of fungicides, and farmers who are growing the traditional varieties are not usually using pesticides and fungicides. So we thought we might be able to capture endophytes that help combat fungal pathogens from those lines.”

PhD student Walaa Mousa isolated endophytes from finger millet. “Finger millet is an ancient Ethiopian crop. It is widely grown in Africa and South Asia. It is really valued by subsistence farmers because it is reported to be very resistant to a lot of pathogens,” Raizada says.

As a result of the work by Johnston-Monje and Mousa, Raizada’s lab now has a collection of over 250 cereal endophytes. Most are bacteria and a few are fungi.

Raizada and Mousa suspected finger millet might have anti-Fusarium endophytes. “Unlike a lot of cereal crops, finger millet is not susceptible to Fusarium graminearum. That is surprising given there is some evidence that Fusarium  pathogens evolved in Africa,” Raizada notes. “So we hypothesized: what if finger millet and its endophytes co-evolved with Fusarium, so there was a three-way co-evolution, and finger millet selected for endophytes that could combat Fusarium?”

Fusarium graminearum and its sexual stage, Gibberella zeae, cause tough-to-control diseases in many cereals, including Fusarium head blight in wheat and Gibberella ear rot in corn. These costly diseases reduce grain yield, grade and quality, and can produce mycotoxins, such as deoxynivalenol (DON), that limit the grain’s end-use.

Mousa tested all the endophytes in the lab’s collection by putting each one in a Petri dish with Fusarium graminearum. She found a handful of endophytes that could suppress the pathogen; some were from corn and some from finger millet, and most were bacteria.

Then she and Charles Shearer, who is now a Master’s student in Raizada’s lab, conducted greenhouse trials with five of the endophytes that controlled the pathogen in the lab. They applied the endophytes to wheat and corn as a seed coating or as a spray. The spray was applied on the corn silks at silking time and on the wheat heads at heading time. They used an Ontario corn hybrid and an Ontario wheat variety that are moderately susceptible to Fusarium graminearum. After the endophytes were applied, the plants were exposed to the pathogen.

In these replicated greenhouse trials, each of the five endophytes was able to control the pathogen, and one of the endophytes worked so well the treated plants didn’t show any symptoms at all of a Fusarium graminearum infection.

For Raizada, the most exciting results from the greenhouse trials were the endophytes’ remarkable effectiveness in reducing DON levels, which were analyzed by Victor Limay-Rios, a research associate at the university’s Ridgetown Campus. “At harvest, for whatever reason, all the samples had low DON levels, even the ones that hadn’t been treated with endophytes. Then Walaa stored the seeds for 14 months at room temperature; under those conditions, Fusarium is still active,” Raizada explains. “When Victor tested those stored seeds, he found that the DON mycotoxin levels were extremely high in the corn and wheat samples that had been exposed to Fusarium, but not treated with the endophytes. In contrast, the seeds that had been exposed to Fusarium and treated with the endophytes had very low DON levels, well below the acceptable level of DON mycotoxins, which is about 1 to 3 ppm, depending on which regulations and which conditions are involved.”

In corn, all five endophytes reduced the amount of DON to well below 0.1 ppm. In wheat, two of the endophytes reduced the DON levels to below 0.1 ppm; the other three significantly decreased DON levels but not to such a low level.

Field trials underway
In June 2015, Raizada’s lab received approval from the Canadian government to do field trials with the five endophytes. Shearer is heading up these two-year trials, which are taking place at the Ridgetown Campus. He is collaborating with Limay-Rios and Art Schaafsma, a professor at Ridgetown who is a leading expert in Fusarium.

In a field setting, endophyte applications face a couple of key challenges. One is that they may be outcompeted by microbes in the environment, so they might not even be able to colonize the plant. The other issue is how varying weather conditions might affect the endophytes.

The field trials are comparing the five endophytes in seed treatments and in-crop sprays on corn and wheat. Although a seed treatment would likely be the easiest for growers, Raizada thinks it might not be the most effective option because the endophytes might be outcompeted by soil microbes. However, the researchers are trying various ways to try to get around that problem.

Shearer is comparing different timings for the spray applications: at the same time as another product, like nitrogen fertilizer, is applied; or at the time of silking or heading. In addition, he’s testing the endophytes individually and as a cocktail of all the endophytes together.

The trials are comparing moderately susceptible and very susceptible cultivars of corn and wheat. The researchers will be assessing Fusarium graminearum symptoms on the ears and heads and measuring DON levels in the seeds. They will also be sampling different tissues from the treated plants to determine which tissues in the plant are being colonized by the endophytes. And they’ll be watching to see if perhaps the endophytes have other growth-promoting effects on the plants, such as controlling other pathogens or enhancing root growth.

In 2016, the researchers will complete the field trials and analyze the results.

Down the road
Once they’ve analyzed the field trial data, the researchers will decide on their next steps. For instance, they might investigate whether the endophytes work well in combination with a fungicide. “Of course, we’re hoping an endophyte alone will work really well, but let’s say that either a fungicide alone or an endophyte alone does not provide effective control of the pathogen. Maybe the two together might,” Raizada says.

If one or more of the endophytes seem to have commercial potential for controlling Fusarium graminearum, then Raizada’s lab will collaborate with a company, and that company will undertake the necessary tests regarding human and ecosystem safety and so on, to develop commercial biocontrol products.

Looking at the bigger picture, Raizada sees exciting times ahead for endophytes in agriculture. According to Raizada, when his lab first started working on endophytes, agricultural input companies were showing only moderate interest in such research. But since then, companies like Monsanto and Syngenta have been investing more and more into microbial products like biocontrol products and biofertilizers. “The large seed companies are now looking at endophytes and other microbes as a new frontier. Within a few years, I think growers will increasingly see microbe-based products coated onto their seeds or available as sprays.”

He adds, “Where I see the best opportunity is with microbes that have multiple functions. Perhaps a microbe that has anti-Fusarium activity is also able to combat other pathogens and also has some other activity. For example, we are intensively studying microbes that can stimulate root growth when the soil is waterlogged in the spring. If the soil is waterlogged in the spring, the roots don’t grow, and then if you have a hot period, the plants don’t do well because they never developed a good root system.”

Multi-functional endophytes are a key research area for Raizada’s lab. The researchers have screened the endophytes in their collection for several functions such as phosphorus solubilization and root growth stimulation, in addition to Fusarium control, and they’ve already found some with multiple functions.

Raizada is also looking forward to many interesting discoveries about the intriguing world of endophytes. For example, his lab has studied in great detail the relationship between a fungal endophyte species and yew trees, and has developed a step-by-step picture of how this endophyte helps the tree fight pathogens. “When a tree branches, it creates cracks, and yew trees hyperbranch; they are always branching and always creating bark cracks. This endophyte swarms to the crack, that wound site, and then it releases a fungicide in fatty bodies. So it’s no different than if you have a cut and you apply a Band-Aid with antibiotics in it. The fatty barrier is similar to a plastic Band-Aid barrier, and it’s laced with a fungicide. It is amazing.”

He adds, “I think every endophyte has a fascinating story. And any individual plant has hundreds of species of endophytes. So I think there will be many years ahead of interesting discoveries to be made and some really fascinating biotech applications."


September 6, 2016
By Carolyn King


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