Boosting from Below Ground

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By Deborah Komlos

For farmers, much more than just hope is riding on their seed — quite literally.

In preparation for planting, farmers load their seed with various substances such as chemical fertilizers, fungicides and pesticides to help generate a healthy and productive crop. An increasingly common part of this pre-planting regime, and one that’s showing success in augmenting crop yield, is pre-treatment with growth-promoting microbes.

A suite of beneficial soil microbes that is of particular interest to firms manufacturing agricultural inoculants is known as plant growth-promoting rhizobacteria (PGPR). Among the microbes included in the PGPR group is Rhizobium, a bacterium that develops a symbiotic relationship with legumes, including crops such as soybeans, beans, peanuts, peas, lentils and alfalfa. In this partnership, the bacteria colonize the plant roots and cause the formation of nodules, in which the bacteria live and transform gaseous nitrogen into a plant-usable form.

A Growing Market
Today’s rhizobial product market is effectively split in two, explains Hannah McIver, CEO – Marketing at Agribiotics Inc. (Cambridge, ON). One is the on-farm market, she says, where the farmer must apply a product to the seed at the farm.

The new area that has been developing in the last two years, McIver says, is the pre-treatment market, which is allowing the farmer to extend the life of bacteria on the seed through special additives, and is also allowing for compatibility of the inoculant with the various chemical treatments that become loaded onto the seed.

While the inoculant technology has been around for many years, the field is advancing in the mode of bacterial delivery, McIver says. “It could be granular or a movement toward liquids,” she explains. “Liquids are much easier to apply for the growers. So we’re really trying to access more market acres by making (the inoculant) easier to apply. But the one big drawback has been, most growers don’t want to be fussing around at the farm site with putting anything on the seed. So, of course, then you get into the new market area, which is the pre-treatment market.”

A further catalyst for the inoculant market, McIver says, is that growers have consolidated and become much larger, which has resulted in demand for more seed treatment and much earlier and faster planting.

Agribiotics’ product line includes PulseR, a powder peat inoculant that delivers 1.2 million rhizobia per seed and can be applied wet or dry. It is used for soybeans, peas, lentils and chickpeas. Another product, PulseR HP, provides higher performance, delivering 2.1 million rhizobia per seed for soybean. The latter is especially useful in harsh environmental conditions or on virgin land, where the land lacks residual soil rhizobia.

McIver says the product must exceed the minimum required levels of rhizobia per seed. These numbers are specified under the Fertilizers Act — which is administered by the Canadian Food Inspection Agency (CFIA) (Ottawa, ON) — and include, for instance, a minimum of 100,000 viable rhizobial cells on the seed of beans, peas and soybeans.

“As soon as (the bacteria) goes onto the seed, it begins to die off,” McIver says. “What you’re trying to get rid of is that on-seed die-off.” It’s also critical to build up the bacterial population in the soil, McIver says, especially if it is unknown what proportion of the rhizobial population has remained post-harvest.

Before chemical treatments are applied to inoculated seed, 50 per cent of the bacteria can be lost rather quickly, McIver says. With old liquid formulations, for instance, half the bacteria could be lost within four hours. Current formulations are providing a safeguard against relatively early loss by extending that window to 12 hours.

Getting a Jump Start
Aside from Rhizobium, other microbes are also taking root in the inoculant market.

At Saskatoon, Sask.-based Philom Bios Inc., Penicillium bilaii (referred to as P. bilaiae in scientific literature) has been incorporated into a wettable powder called JumpStart® that is being sold as a growth-promoting inoculant for use with wheat, canola, pea, lentil, bean and Brassica crops. The fungus helps plant growth by solubilizing less-available forms of soil phosphate and making them accessible to plant roots. This is especially useful in Prairie soils because their high levels of calcium phosphate tie up phosphate, says Mary Leggett, PhD, science advisor for Philom Bios.

“There are a lot of people working on phosphate inoculants, but we’re the first to commercialize it and sell it widespread to farmers,” Leggett says.

Use of the P. bilaii is patented by Agriculture and Agri-Food Canada (AAFC) (Ottawa, ON), which discovered and developed the microbe in Alberta through work at the AAFC Lethbridge Research Centre (Lethbridge, AB). The fungus had been isolated, Leggett explains, along with hundreds of bacteria and other fungi and all were tested for phosphate solubilizing characteristics.

P. bilaii was chosen, she says, because it could grow at the cold Prairie temperatures and is a very efficient solubilizer, an ability that it kept over several subcultures, unlike some of the bacteria, which were quite unstable. “And it’s a very effective recolonizer, which was serendipity,” Leggett adds. “That’s been great because, so far, I haven’t found a crop that it can’t colonize the root cap.”

The firm also produces a combination product called TagTeam® that includes P. bilaii along with a superior strain of Rhizobium. The product is jointly patented between Philom Bios and AAFC.

As with a Rhizobium inoculant, the Penicillium product is applied to the seed to meet and exceed the minimum required count, which in this case is at least 100 viable spores when the seed goes into the ground. Because the Penicillium inoculant was new, data submitted to the CFIA were used to create the standard for this fungus as an inoculant, Leggett says.

Researching compatibility between its inoculants and commercial chemical seed treatments is a strong focus at Philom Bios. The firm tests all of the registered chemicals and then prepares compatibility information and detailed recommendations for the grower.

“It’s a huge amount of work because any time a formulation ingredient changes in the chemical, we have to re-do it. Any time we change our formulation, we have to re-do it,” Leggett says. “So, it’s something that we think is really important because there’s no point in applying a biological if it’s dead.”

The use of microbial inoculants is simply a way to maximize a natural process, Leggett says.

“I think people tend to think of biotechnology and they think just GM and that’s not true,” Leggett says. “There are a lot of us doing other things that are not modified . . . We’re really just helping Mother Nature along. These organisms come from the soil; it would be doing this to some extent in the soil anyway, in the plants anyway. What we’re doing is by putting enough on the seed, we’re giving it that better chance to do what it would do normally.”

Another Type of PGPR
At Brett-Young Seeds Ltd. (Winnipeg, MB), microbe-induced plant-growth promotion is also taking a novel role. The firm has produced a canola inoculant containing a sulphur-oxidizing PGPR. The product, which does not yet have a trade name, is awaiting registration in Canada.

Having an enhanced sulphur oxidation capability is crucial, says Manas Banerjee, PhD, director of Research and Development at Brett-Young Seeds. Amid the approximate 12 to 13 million acres of arable land for canola in Western Canada, Banerjee says about 25 to 30 per cent is sulphur-deficient, and canola is a high sulphur-demanding plant.

To remedy this deficiency, Banerjee says one option is applying sulphur fertilizer. But there are associated concerns. Such chemicals are expensive, he says, and the form in which sulphur is often applied — ammonium sulphate — is water-soluble. “So they apply this fertilizer, which is a chemical fertilizer and which is costly, and then rain comes in. So what happens, it directly solubilizes into the rainwater and goes down into the groundwater,” he explains. “So first of all, they are losing their nutrient . . . plus it is most probably going to pollute our groundwater as well.”

Applying elemental sulphur is another option, and one that Banerjee says is appealing because the oil and gas industry in Alberta produces piles of sulphur as a byproduct and these sites need cleaning up. The trouble is, the plant cannot take up elemental sulphur; soil microbes must first transform the sulphur into sulphate, and the process takes too long.

“So if a farmer applies elemental sulphur, it takes about 18 to 24 months before it gets available to the plant. The farmer doesn’t want to wait for two years,” Banerjee says.

And that’s where the PGPR steps in. Banerjee says that through its field trials, Brett-Young Seeds has found a yield increase of, on average, six to 10 per cent with its PGPR, which is applied to the seed in a peat-based powder.

Banerjee explains that the PGPR may be achieving its growth enhancement via six or seven possible mechanisms. Aside from the nutrient effect, the bacteria may also behave as a biocontrol agent, possibly producing antagonistic material to ward off harmful micro-organisms. Phytohormone production by the PGPR is another possibility, as is the formation of siderophores, compounds that chelate iron and make the element available to the plant.

Based on the firm’s findings and because the PGPR could have several possible modes of action, Banerjee says another advantage of the firm’s product is that it can even be used in non-sulphur-deficient soils.

While the results seem promising, Banerjee says registration is needed for the firm to be able to access the Canadian canola market, which is the largest in Western Canada. Product registration in Canada is much different than in the United States where it works on a “buyer beware” premise, Banerjee says. “So if your product is good, the marketplace will decide whether it will survive or not.”

Kate Billingsley, PhD, senior safety officer with the CFIA’s Fertilizer Section, mentions that in order for fertilizer and inoculant products to be registered, two years’ worth of field trials are required, and of these, 60 per cent must show significant positive results.

The Basics
Though demonstrating appropriate field results is critical to obtaining product registration, achieving consistent positive plant growth effects in the field is not easy. Jim Germida, PhD, director of the Saskatchewan Centre for Soil Research at the University of Saskatchewan (U of S) (Saskatoon, SK), certainly knows so, having worked for over 20 years with bacteria and fungi as plant growth-promoting micro-organisms.

“In the growth chamber, pretty much 95 or 100 per cent of the time you’re going to get these things to work,” explains Germida, also professor and head of U of S’s department of Soil Science. “When you go to the field, your success is not that great, the increases are not that great, the field variability, the environmental pressures and conditions that everything comes under cause problems.

“And the other problem that we’ve run into is, as a research group at a university, we don’t necessarily have the best delivery systems, the formulations, the commercial processes that companies who market different kinds of microbial inoculants might have,” he explains. “And so, I think that’s been one of the hurdles that we’ve run into when we’ve done our studies.”

Part of Germida’s current research is trying to understand microbial biodiversity in the rhizosphere of different plant species under different management conditions. “The idea being that part of the success of any inoculant is going to depend on how it competes with the native indigenous microflora,” he says.

One project is examining the use of endophytes. “It’s a fact that there is a demonstrable population of endophytes that one can find for almost any plant . . . the work that we’ve done shows that there’s a subset of bacteria that colonize the rhizosphere that are in fact colonizing the interior of the root,” Germida says, mentioning that others have shown this as well. “The practical application and utilization of these endophytes as bacterial or as microbial inoculants to actually promote crop growth out in the field, I don’t think there’s been a lot of success stories per se because I don’t think there’s been a lot of energy and resources directed into that area.”

Because many of the microbes will do multiple things depending on the selected lab conditions, Germida believes a first step to producing successful inoculants is to thoroughly understand the microbes. “So, truly understanding how they work, truly understanding how competitive they can be and how they can colonize root systems and perhaps even enter those root systems and carry out their activities as endophytes, and then developing very good delivery systems will be the key,” he says.

More to Consider
A research scientist with the Soils and Crops Research and Development Centre (Ste-Foy, QC) of AAFC, Danielle Prévost, PhD also emphasizes the importance of much more fundamental research on PGPR.

Echoing Germida’s comments, Prévost says that results with PGPR under field conditions can be quite variable. “Depending on the soil we use, we can see differences,” she says. “Some strains are very good in a type of soil, but they can be deleterious to the plant in another type of soil.

“So more is needed to be known about the root interaction because you know that the bacteria are there, but exudate from roots influence the growth of these bacteria or their interaction with the plant,” Prévost says.

Prévost is investigating the effectiveness of strains of Bradyrhizobium japonicum on the growth and mineral uptake of corn. Another of her projects is aiming to improve legume productivity under cold growing conditions by screening and finding rhizobia from Canadian soils that are adapted to the cold.

Prévost says an improved understanding of the plant-microbial relationship requires more observation during the early stages of the symbiosis.

Usually plant growth is measured at the mature stages, and includes tests such as nitrogen and phosphorus content, Prévost says. But she adds that there are other questions, such as influence of PGPR on seedling emergence. “It is one thing to know under in vitro conditions, but it is another thing to prove with the plant,” she says. Physiological work is also imperative, Prévost adds, as is looking at the plant-microbe molecular exchange and what signal molecules are moving between the two symbionts.

Nutrient stresses at different stages of growth should be considered as well. “Because, for example, for corn, sometimes we can see an effect with the young plant. But if we wait after maturity, we cannot see any more difference. So is there really an advantage?” Prévost says. “But maybe it is an advantage if the plant is stronger at the beginning of the growth, maybe the plant will not get diseased.”

For ongoing research projects, even when a product does make it to market, “your work’s not finished,” Leggett points out. “The amount that we spend on those compatibility tests, the amount that we spend on quality assurance . . . Plus looking into new areas or new crops. So, just because you’ve developed it and started to market it, your research doesn’t end — it’s a continuous process.”

McIver sees the future inoculants market as one that will involve product positioning.

In the Canadian soybean market, for instance, only 50 to 60 per cent of the 2.7 to 2.8 million acres is inoculated, McIver says. It’s a market that “somebody’s going to access,” she says, and one that will have high value to the grower. “It’s going to be who does it and when does it occur and that’s going to change the inoculant dynamic. And what we’re all jostling for is, of course, a position in that.”