Testing biological seed treatment compatibility at Insight Plant Health
Legumes like pulse crops and soybeans form symbiotic associations with soil bacteria called rhizobia. These rhizobia make nodules on the plant’s roots where they fix atmospheric nitrogen and supply it to the plant as ammonia or amino acids in exchange for carbon and energy from the plant. Because supplying nitrogen-fixing rhizobia to the seed or soil is generally far cheaper than supplying the equivalent amount of nitrogen fertilizer, most farmers either inoculate their legume seeds with commercial rhizobia products prior to planting or apply the rhizobia in furrow with a granular inoculant product.
What affects inoculant viability on seed?
The rhizobia applied to seed have to be alive at planting to infect the plant roots, induce nodulation, and begin fixing nitrogen for the plant. In most cases, companies (and some government regulatory agencies, where such rules exist) recommend having at least 100,000 colony forming units (CFUs) of rhizobia per seed at planting for large-seeded legumes like soybeans and peas. There are several factors that affect the viability of seed-applied rhizobia prior to planting, including time on seed, temperature, humidity, and compatibility with other seed treatment products. The number of viable cells on seed declines over time, so even at ideal temperature and humidity, there will be a limit to how long you can treat a seed before planting it. This decline in viability is accelerated as the temperature and/or relative humidity rise. The time on bare seed (i.e. without additional treatments) also varies greatly by rhizobia genus and species. For example, Bradyrhizobium japonicum, a soybean symbiont, can survive for weeks or months on seed if kept cool and dry, whereas Rhizobium leguminosarum, a pea and lentil symbiont, can only survive a few days under the same conditions.
For most Bradyrhizobium products applied to soybeans and at least one Rhizobium product for peas/lentils, inoculant manufacturers include an extender, a liquid co-packed product that is applied together with the inoculant. As their name suggests, extenders help to extend the life of the bacteria on seed.
Every year, new crop protection seed treatments are registered and sold for pulse crops and soybeans to protect against plant diseases and insects. Each of these new seed treatments must be tested for compatibility with inoculants on the crops they’re intended for to ensure that the rhizobia applied to the same seeds are still alive at planting. Inoculant companies publish compatibility guides (like this, this, or this) that show how their products interact with various seed treatments based on either their own internal testing or contract testing through external labs like Insight Plant Health. This is how we conduct seed treatment compatibility testing here at Insight Plant Health:
Seed treatment compatibility testing
The first step to testing seed treatment compatibility is to treat the seed. The inoculant and seed treatment combination can be treated in any of 3 ways:
Tank mixed, where the two products are mixed together and then applied to seed
Simultaneously, where the products are applied to seed at the same time without prior mixing
Sequentially, where one product (usually the seed treatment) is applied, held until dry and then the inoculant is applied over top
In general, the compatibility between products is better when applied sequentially, followed by simultaneous, and is least compatible when tank mixed. We treat our seed in a Hege 11 seed treater made by Wintersteiger, which is a lab scale version of a commercial spinning disk treater.
Hege 11 lab scale seed treater
Hege 11 in action - treating soybeans
Once the seed is treated, it is left to air dry in the open for about 10 minutes before packaging. For the most part, we package the seed in ziploc bags, but at the request of customers, we have used paper or combination paper and plastic layered bags. There’s generally not much difference in rhizobial survival between bag types but it’s the type of thing that generates a lot of discussion in seed treatment compatibility circles. For North American testing, most of the storage is conducted at 4°C or 10°C, but customers sometimes request higher temperature testing.
At treatment time zero and regular intervals after that, the rhizobia are recovered from the seed in a dilution buffer. The dilution buffer is another contested aspect of the testing with some labs using dilute saline, others phosphate buffer or peptone water, and others still dilute surfactant buffers. We use a dilute Tween buffer because it’s easy to make and works well in our hands. We recover the bacteria by counting out 20 seeds into 20 mL of buffer and incubating them for 15 minutes with several rounds of vigorous shaking. The 20 seeds in 20 mL allows for easy math later in the assay, as the CFU/seed is the same as the CFU/mL at this step in the protocol.
Treated seeds in extraction buffer before and after extraction
After the bacteria are thoroughly rinsed from the seeds, they are diluted in series and then plated onto a selective medium for colony counting. For most testing, we use yeast extract mannitol agar with brilliant green, Congo red, vancomycin, or tetracycline or a combination of those additives. We spot plate most of our samples because it saves on the number of plates, but where there is a lot of background contamination on the seeds or we are testing against a biological seed treatment that makes counting the rhizobia harder, we’ll use spread plating to make the counting easier.
Spotted Bradyrhizobium colonies ready for counting
Once all of the counts are in, we tabulate and graph the results for our customers and provide a report with a planting window that tells them how long before planting the treatments can be applied to seed.
Example graph of an inoculant alone, inoculant + extender, inoculant with a seed treatment, and inoculant with a seed treatment + extender. The black line represents the 100,000 CFU/seed cutoff
In both cases, the extender improves the counts - moving the time on seed from about 80 days (yellow line) to 105 days (green line) in the presence of the chemical seed treatment.
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