Getting “AM” fungi to go organic: greenhouse inoculation of sweet corn

Translating the yield boosting success of arbuscular mycorrhizal fungi to an organic greenhouse system.

By Molly Lohman, seasonal research technician

In many cases, farming organically requires reinventing the wheel; practices that worked in a conventional system often need to be redesigned to function in an organic context. Such is the case when inoculating greenhouse seedlings with arbuscular mycorrhizal (AM) fungi, soil fungi that form a symbiotic relationship with the roots of plants. While previous research has established a successful conventional greenhouse fertilization protocol for obtaining high colonization of roots by AM fungi, a comparable organic system has not been identified. Our current research at the Rodale Institute with USDA microbiologist Dr. David Douds strives to find an organic greenhouse system that will produce the same levels of colonization while increasing plant health. Most recently we tested our inoculation protocol in the field on sweet corn in response to the growing trend of starting early season sweet corn in the greenhouse.  Replicated both at the Institute and a local farm, this trial illustrates the potential benefits of outplanting seedlings pre-colonized by mycorrhiza.

Basic AM biology

Arbuscular mycorrhizal (AM) fungi form the most important mycorrhiza in agricultural ecosystems due to the fact that they colonize the majority of crop plants. Known as “obligate symbionts,” AM fungi must form an association with plant roots to survive; it is this association that begins a symbiosis between the fungi and the plant. In return for sugars from a plant, the thread-like structures of the mycorrhiza, or hyphae, act as extensions of a plant’s root system. While root hairs only extend 1-2 mm into to soil, the mycorrhiza’s hyphae explore a greater volume of soil and can extend up to 15 cm from the plant’s roots.

One of the most important benefits of this symbiotic relationship to a crop plant is increased nutrition. By extending further into the soil, the mycorrhizal fungus’ hyphae increase a plant’s access to immobile nutrients such as phosphorus, copper and zinc. AM fungi have also been shown to increase a plant’s disease resistance, enhance the plant’s ability to grow under drought conditions and improve the soil’s ability to retain water after periods of heavy rainfall or snowmelt. Because of these benefits, recent research has focused on how to bolster mycorrhiza populations in agroecosystems.

Applying mycorrhiza in agriculture

Many common management practices negatively impact native mycorrhiza populations. Using fungicides directly decreases mycorrhiza populations while using herbicides indirectly decreases populations by killing host plants. Frequent tilling also negatively affects native populations by destroying networks of hyphae in the soil. Adjusting management practices is one of the easiest ways to bolster native mycorrhiza populations. Reducing tillage, adding a winter cover crop and developing a diversified crop rotation all positively affect AM fungi populations.

In operations that are large or have lower returns per plant (such as grain) good management practices are the best way to increase and take advantage of mycorrhiza populations (to read more about managing AM fungi populations, see Douds’ article Improve your soil, increase your yields, and reduce your expenses with AM fungi"). On small-scale farms or when the return per plant is higher (such as working with vegetables), another way to take advantage of mycorrhizae is through the use of inoculum. Inoculation of plants with AM fungi prior to outplanting has been shown to increase or maintain crop yields while decreasing expenses by reducing the need for fertilization.

In a 2004 study on strawberries, Douds observed that plants inoculated with AM fungi before outplanting had a 17% higher overall yield compared to uninoculated control plants (Douds et al. 2008). While the fruits were the same size between inoculated and non-inoculated plants, the inoculated plants produced, on average, 3.6 more fruits per plant. In a separate study looking at green pepper yields, the use of an inoculum of various mycorrhiza species increased yields by 14 to 23% in compost-amended soils and, in one year, by 34% in soils using chemical fertilizers (Douds and Reider 2003). As Douds’ research shows, overall crop yield can benefit when seedlings are transplanted to the field already colonized by AM fungi.

The problem

As more and more farmers transition to organic, the next step is finding a way to ensure high mycorrhiza colonization of roots in an organic greenhouse setting. While Douds’ research has established a conventional protocol for establishing high root colonization in a conventional greenhouse setting, the unique nutrient sources of an organic system make it more difficult to obtain high colonization. Different from conventional systems, organic systems rely heavily upon compost as a principal soil amendment. And compost’s high phosphorus (P) levels restrict effective colonization. Plants that are P sufficient exude fewer hyphal branching signals, chemicals that stimulate branching of the hyphae to increase the probability of contact between the AM fungi and the host root. Additionally, plants that can absorb sufficient phosphorus without the aid of AM fungi, such as seedlings growing in the presence of high levels of compost, can prevent colonization of their roots by restricting the flow of plant sugars to the mycorrhiza. Although Douds has experimented with decreasing the amount of compost in potting media to encourage higher root colonization, his efforts were foiled when plant health declined rapidly without explanation.

This year, we hoped to identify a combination of potting media and fertilization regime that would produce organic seedlings that were both highly colonized and of satisfactory health. Our first trial looked at the effects of potting media and fertilization regime on colonization. We needed to balance the high colonization that had been observed in Douds’ conventional system with sufficient nutrient availability for good growth. 

AM colonization in organic media

As pictures of the treatments illustrate, there were clearly growth and health differences between the treatments for leeks, tomatoes and peppers. Additionally, analysis revealed significant differences in percent root length colonization (RLC). In general, the levels of RLC for the organic treatments were very low. Conversely, the organic plants showed significantly higher levels of P uptake than the conventional plants, suggesting that phosphorus was not limited enough in any of the organic treatments in order to facilitate colonization. Although the highest levels of colonization were observed in the conventional treatments, the plants were consistently smaller than the other treatments. While these results suggest changes for next year’s trial, such as further limiting P availability in the organic treatments, the results reflect the inherent difficulty of producing a highly colonized, healthy-looking plant.

Trying it out on sweet corn

In addition to getting an early start with peppers and tomatoes in the greenhouse, one growing trend in agriculture is to start sweet corn indoors to get a jump on early season production. This introduction of sweet corn into the greenhouse is a situation that lends itself to the use of mycorrhiza inoculum. However, different from other species previously used in Douds’ studies, sweet corn is kept in the greenhouse for a very short amount of time.  Therefore, with sweet corn, a new challenge arose of obtaining maximum root colonization in the shortest time possible. Instead of trying to increase colonization levels through potting media or fertilization regimes, we hoped to influence colonization by manipulating the potting media temperature. We hoped to “jump start” mycorrhiza spore germination by heating trays of potting media to elevate microbe activity and carbon dioxide generation. Due to the fact that mycorrhiza spores germinate in response to elevated carbon dioxide (CO2) levels, we assumed that the elevated CO2 levels would prompt germination and branching. Then, when corn was seeded into these flats one week later, there would be an existing network of hyphae that would be ready to colonize the corn’s roots sooner than if the soil had been kept at a cool temperature to prevent CO2 generation.

Our trial consisted of two treatments; one flat of potting media-inoculum mix was warmed in the greenhouse for one week prior to planting while one flat of the same mix was kept at 4ºC for one week. The root length colonization of the corn seedlings was analyzed after 7, 12 and 19 days. Both treatments produced what we considered to be satisfactory levels of colonization: approximately 10% RLC at 12 days and 19% RLC at 19 days. Although the difference was not statistically significant, the mean colonization of the seedlings planted into warmed soil was consistently higher than the cool soil seedlings over all three sampling periods. Given the small sample size of the trial, the experiment would need to be repeated with more replicates in order to see if the small difference between the two treatments over the three week period is indicative of a true difference between the treatments.

Although our two greenhouse trials didn’t provide us with any concrete method of increasing levels of colonization in an organic system, the trial that looked specifically at sweet corn revealed that our current method of inoculation using a 10% compost potting media did produce  colonized sweet corn seedlings. The next step was to test these colonized sweet corn seedlings to the field. In response to the growing trend of using sweet corn starts, we set up a field trial in hopes of answering whether mycorrhizal root colonization prior to outplanting would impact sweet corn yields in the same way that inoculation increased yields in other crops.

Taking it to the field

Our trial consisted of two sweet corn stands: one at Shenk’s Berry Farm in Lancaster County and one at the Rodale Institute. The same experimental procedure was followed at both locations although the dates of outplanting and harvest differed.  All seedlings were started in the greenhouse and were planted in either a standard 10% compost potting media or a mix of the potting media and mycorrhiza inoculum from our on-farm inoculation system (for instructions on how to prepare your own inoculum see Douds’ article On-farm production and utilization of AM fungus inoculum). Seedlings were sampled at outplanting and after 17 or 35 days in the field for height, weight, phosphorus content and percent root length colonization.  The corn was harvested when the silks were both brown and dry, at which point both the stalks and cobs were weighed and counted.

Weighing and recording cob weights in the field. 
(L to R: Molly Lohman, Dave Douds, Christine Ziegler-Ulsh)

At the time of outplanting, seedlings from the two treatments in both locations were not significantly different in height, weight or phosphorus uptake. As expected, no colonization was observed on non-inoculated plants while inoculated seedlings averaged 11% RLC at Shenk’s and 45% RLC at Rodale. The higher colonization of the Rodale seedlings at outplanting can be attributed to the fact that these seedlings remained in the greenhouse nine days longer than the plants at Shenk’s Berry Farm.

Percent root length colonization as a function of time in the field.
The higher RLC of the mycorrhiza inoculated plants (M) compared
 to the non-inoculated plants (NM) illustrates that the 
increased colonization as a result of inoculation 
persists for over one month in the field.

When the seedlings were sampled after 17 (Rodale) or 35 (Shenk’s) days in the field there was still a significant difference between the RLC of the inoculated and non-inoculated plants at both locations. After 17 days in the field Rodale’s inoculated plants averaged 0.268 higher RLC than the non-inoculated plants while even after 35 days of field growth Shenk’s inoculated plants averaged 0.201 higher RLC than the non-inoculated plants. Therefore, not only did the inoculum work by producing colonized seedlings, but this increase in colonization persisted even after an extended period of field growth in the presence of healthy native mycorrhiza populations.

At Shenk’s, there was also a significant difference between the average plant height and dry weight of the two treatments. On average, the inoculated plants at Shenk’s were 9.75 g heavier and 16.7 cm taller than the non-inoculated after 35 days of growth in the field. These results suggest that early growth in the field can be enhanced by outplanting colonized seedlings that can immediately take advantage of mycorrhiza’s benefits. Although the treatments were not significantly different at Rodale, the lack of difference is most likely due to the fact that samples were taken after 17 days of field growth, only half as long as Shenk’s at sampling. It is likely that 17 days after outplanting the seedlings were still in the process of establishing themselves in the field and experienced relatively slow growth. Early on seedlings will grow slowly, but once established their growth rate increases.

By the time of harvest, differences between the treatments had dissipated at both locations.  There was no difference in shoot weight, cob weight or production of marketable ears between treatments. The only significant difference between the treatments was observed at Rodale; the non-inoculated plants produced 0.11 more ears per plant than the inoculated plants. Both surprising and unexpected, we are still unsure of how to explain this difference given the slight early-growth advantage of inoculated seedlings. Due to the fact that our results also indicate a strong block effect in the Rodale trial, it is possible that the observed difference in cob production can be attributed to fertility differences throughout the plots or other landscape effects, a common setback in agricultural research.

What our results do tell us is that parts of our protocol leave room for improvement. The difference in average mature shoot weight between the Rodale plants and Shenk plants (40.5 and 103.5 g fresh weight, respectively) illustrates the negative impacts of the extra week in the greenhouse that the plants received at Rodale. This lost week of growth in the field could be vital considering the relatively short growing season of the cultivar, 75 days. In addition to suffering an extended period in the greenhouse, the Rodale plants also endured five weeks without rain and intense weed competition. Although the Rodale plots were cultivated multiple times throughout the experiment, it was obvious that some portions of the field experienced more weed competition than others. Therefore, next year’s trial will put greater emphasis on ensuring an appropriate outplanting date and identifying a better weed control method that will hopefully decrease the variability of weed competition we observed throughout the field.

Although we did not see changes in sweet corn yield in response to inoculation, our setbacks at the Rodale site illustrate that we need to refine our management of sweet corn both in the greenhouse and in the field. This lack of any significant treatment effect at the time of harvest may indicate that some setbacks to growth and production cannot be overcome by mycorrhiza inoculation including late outplanting and severe drought. However, the significantly larger inoculated plants at Shenk’s Berry Farm one month after outplanting suggest that inoculation of sweet corn is worth exploring further. Given the slight advantage of the inoculated plants after one month in the field, it is possible that we could see an effect of inoculation on yield in future replicates of this trial. The fact that inoculated seedlings have higher levels of root colonization than their non-inoculated counterparts for over one month after outplanting means that inoculated plants have a better chance of taking advantage of mycorrhiza’s benefits when the right conditions arise. As Douds has observed in previous studies, inoculation can lead to significant yield gains in some years and have no effect on yield in other years.

What’s up next

We still have more ongoing mycorrhiza trials in the field involving leeks, field corn and tomatoes. Although the summer has been dry and hot, the weather has kept late blight at bay and therefore this season may be the first year we are able to collect data from our infamous “Tarp Trial.” Be sure to keep posted as we collect data from this plot where Dr. Douds is currently trying to kill off all native mycorrhiza populations in order to observe the growth response of crops in the absence of mycorrhiza.