By Carolyn King
University of Guelph researchers are shedding light on white mould to help breeders develop resistant soybean varieties. Photo by Evelyn Valera Rojas, University of Guelph
December 1, 2014 - The results of a recently completed project are shedding light on how soybean plants respond to white mould at the cellular and molecular levels. That information is an important step towards more efficient breeding of soybean varieties with resistance to this yield-limiting disease.
White mould, also called sclerotinia stem rot, is a soil-borne disease caused by the fungal pathogen Sclerotinia sclerotiorum. The fungus overwinters in the soil as resting bodies called sclerotia, which can survive for up to seven years in the soil. In the spring, the sclerotia produce apothecia, which are little golf tee-shaped mushrooms. The apothecia release millions of tiny spores into the air. The spores can colonize soybean flowers and use them as an energy source to infect the plant’s stem. Each step of the pathogen’s development depends on having the right set of environmental conditions.
As the third most important soybean disease in Canada in terms of the crop damage, white mould is a key focus of Dr. Istvan Rajcan, who leads the soybean breeding program at the University of Guelph. He says, “White mould is considered one of the most damaging diseases in soybeans in the northern parts of the United States and Canada. It affects the crop more frequently in Quebec and Manitoba, and in some years in Ontario as well.”
Sclerotinia sclerotiorum is a widespread pathogen that affects more than 400 plant species, including many crops in addition to soybeans, such as canola, dry beans and sunflowers. “The losses due to white mould in the U.S. according to data from 2009 were estimated at $500 million,” notes Rajcan.
He worked on the project with PhD student Evelyn Valera Rojas and Dr. Greg Boland, a plant pathologist and Professor Emeritus at the University of Guelph. The project was funded by the Canadian Agricultural Adaptation Program in conjunction with the Grain Farmers of Ontario (GFO), and Rojas received a Highly Qualified Personnel (HQP) scholarship from the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) to do this project.
At present, no soybean varieties are completely resistant to white mould. “It’s a very complex disease that is conditioned by multiple genes with small effects. There is no soybean variety that would have all those genes to give it complete immunity or full resistance. The best that has been accomplished so far is partial resistance,” explains Rajcan.
This genetic complexity is one of the major challenges in breeding resistant soybean varieties. It’s much easier to develop disease-resistant varieties if only one or two genes are needed to confer resistance to a disease. Trying to pyramid all of the many genes needed for complete white mould resistance into a single soybean variety would be extremely difficult.
Another key challenge for breeders is the weather factor. “Breeders want the disease to be present so they can see which plants are resistant [and select those plants as the parents for the next generation in their breeding program]. However, white mould will not develop unless certain weather conditions and soil conditions are met. So we are hampered by our inability to control the weather conditions [in the field] for a meaningful evaluation of our genetic material,” notes Rajcan.
“My group and other research groups have tried a number of different approaches, like irrigating plots, misting them, and so on, to try to create conditions that favour white mould. Sometimes even when you think you are providing the perfect conditions, you still don’t get the disease.”
One way to enhance the process of developing resistant varieties is to create “markers.” Breeders use markers for screening the thousands of plants in their breeding programs – which helps identify those individual plants with the desired characteristics. For instance, a molecular marker is a specific sequence of DNA that is associated with a particular trait. Researchers use molecular markers to quickly screen the DNA of their breeding material in the lab, rather than having to take weeks or months to grow seeds into plants and test them for the trait.
As a step towards developing such markers for white mould resistance, the project aimed to characterize in detail the soybean plant’s physiological, anatomical and molecular responses to the pathogen as it infects and spreads in the plant.
“Characterizing the disease in detail allows us to understand what processes are ongoing in the course of plant infection and disease development,” says Rajcan. “Unless we understand the genetic and physiological basis of disease development – or lack thereof in terms of resistance – we will not understand how we can address the issue of developing new resistant varieties.”
To examine the plant’s physiological responses to the disease, Rojas inoculated a susceptible soybean variety, OAC Shire, and a partially resistant variety, OAC Salem, with the pathogen. Then she measured a number of physiological characteristics at the cellular level. For example, she looked at whether the susceptible plants accumulated more starch or less starch, whether their photosynthesis was slower or faster, and whether the pathogen’s mycelia (the thread-like filaments that make up the vegetative part of the fungus) developed faster or slower, in comparison to the partially resistant plants.
The molecular characterization involved comparing the inoculated susceptible and partially resistant plants to see which genes were more expressed and less expressed as the disease developed.
Rajcan outlines a few of the highlights from the project’s results. “Starch was accumulated more in the infected cells of the susceptible plant versus the partially resistant plant. We see that as a sign that the susceptible plant is feeling stressed out. It is accumulating more energy in the starch granules because it is trying to fight off the pathogen, but that fight is taking a toll on its energy level. Therefore, the plant may wilt and die because it doesn’t allocate its resources properly.
“Also the susceptible plants had a higher stomatal conductance. That means the infected plants were transpiring faster – losing water faster – which resulted in their wilting.”
For the molecular response, Rojas looked at all the genes that were expressed in the diseased versus the healthy tissues. Some genes were down-regulated, meaning their expression was reduced, and others were up-regulated, in response to infection. Rajcan notes, “Each of these genes belongs to a group of genes that is made up of genes with similar functions. Based on the function of the genes that changed their expression, we identified those that have been reported previously as being associated with disease resistance. In these groups, there are specific genes that we will now be able to focus on in a follow-up study.”
So the next step for Rajcan’s research group is to isolate and test a few key genes that have been identified by Rojas.
“We’re planning to take the knowledge we gained from this project, which was based on only two soybean varieties, and to assess a whole range of other soybean cultivars that were not related to these two cultivars to see if we get the same physiological and molecular patterns of response to the pathogen,” says Rajcan.
“If we do see those same patterns, then that would give us the chance to validate the findings from this study and to develop physiological markers or molecular markers for more efficient selection of partially resistant soybeans to white mould.”
In particular, the researchers hope to narrow down the dozens of genes identified in the project to the most important ones for use as molecular markers. And that advance could help breeders to
develop more white mould-resistant soybean varieties more quickly.