Promoting Agricultural Sustainability through the Use of Rhizosphere-Competent Fungi as an Alternative to Soil Fungicide

Objective 1: Delivery Methods.
Several delivery methods were tested in the course of finding one which was clearly superior. The fungus is commercially produced by growing it under highly controlled conditions on a matrix of small clay particles mixed with a food source. The grown-up fungal mass is matured so that it can be dried and stored. This material is the raw material for the different delivery systems.

The tested delivery systems are described below with the major conclusion for each.

Granules. Initial formulation. Clay granules covered with spores. Ineffective and expensive.

Apply by banding on top of soil. Not effective.

Apply in seed furrow. Requires specialized planter modification, marginally effective.

Planter box. Spore and clay dust mixed with graphite. Applied to seed by farmer. In use now. This formulation was used in most experiments and has good grower acceptance.

Seed treatment. Spores are collected and suspended in a sticker or film coat material that is applied to the seeds. Highly effective. The application can’t be done by farmer. Seed-company treatment is being developed, shelf life is the major challenge.

Cover crop. Planter box or seed treatment is applied to cover crop seed which then grows to distribute inoculum throughout the soil. Has potential to permanently establish T-22 in the field. Ineffective.

The main experiments comparing these treatments are described below. In all cases, we consider satisfactory colonization to be at least 10,000 colony forming units per gram of rhizosphere soil.

Granules. We initially used the granules for field experiments. While granule inoculum resulted in colonization, it was not consistently satisfactory. Furthermore, the cost of material was higher than anticipated (>$10/ac).

a. Placement of inoculum. Granules applied in the seed furrow consistently resulted in colonization, but it was not consistently above 10,000. (Table 1).

b. Type of inoculum. Granules were compared with the planter box seed treatment as well as a slurry seed treatment in a side-by-side trial without any fungicide (Fig. 1). The slurry seed treatment is most effective but is not yet in commercial production. Planter box resulted in more colonization than granules, and it was not significantly lower than the slurry treatment.

Recommendation:

• The granule formulation of T-22 in-furrow is not appropriate to inoculate sweet corn.
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Seed treatment. Treating seed with a slurry of spores places the spores in direct contact with the seed but this placement also means that the spores are in contact with fungicide and stickers on treated seed. Extended exposure to these materials may kill or inhibit the beneficial fungus. T-22 was applied to seeds as a film coat together with a variety of fungicides. Overall, this treatment resulted in satisfactory colonization, (4.4 treated vs. 2.4 background).

Fungicide compatibility. Treatments were several combinations of the fungicides Apron, Captan, Demosan and Imazilil (Fig. 2). T-22 was compatible with all the fungicides except Imazilil, which is used to protect against Penicillium. Since Penicillium is such an important seedling disease of sweet corn, we also investigated compatibility with one substitute for Imazilil, named Maxim. This fungicide was completely compatible with T-22 (Fig. 3).

Recommendation:

• Seed treated with T-22 by seed companies should be effective when it becomes available. Some technical aspects of the application process still need to be worked out by those companies.
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Planter Box. Planter box inoculum was shown to be superior in its first test in 1995 (Table 1). It was developed in May, 1995 and was only available for the last sowing that year. In 1996 and 1997, all field trials were done with this delivery system unless specifically designed to test something else.

Only the initial results are reported in this subsection. Colonization was universally high in 1995 and 1996, so we stopped making individual colonization measurements in grower trials.

Recommendation:

• Use T-22 in the planter box to obtain consistently high colonization by Trichoderma.
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Cover crops. In three field trials, we tested the ability of an uninoculated sweet corn crop to be colonized by Trichoderma when it followed inoculated winter rye that had been plowed down before sowing the sweet corn. At all three farms, no Trichoderma colonized the subsequent corn crop (Fig. 4.)

We tested whether the placement of the inoculated rye residue affects the colonization of sweet corn. We expect the highest inoculum near the crown of the rye plant. Thus no-till places the corn seed with the highest inoculum, chisel plowing mixes the inoculum more, and moldboard plowing places the inoculum 4 to 6 inches below the seed. None of these tillage methods resulted in colonization above background (Fig. 5). We interpret this, and the results of the grower experiments, to mean that there was little or no effective inoculum present on the rye in the spring.

Investigations into other potential cover crops is discussed under Objective 3.

Recommendation:

• Do not expect Trichoderma to colonize crops that follow the crop that was inoculated with T-22.
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T-22 had a positive economic impact by increasing yields. The effect of Trichoderma was pronounced where untreated plots had low yields (Fig. 6). The yields were low at these sites because of intentional management practices to get a particular market, such as early planting, premature harvest, or Organic nitrogen management. None were low due to poor management. In 1995 and 1997, both dry growing seasons, the response to Trichoderma was seen where control yields were below 5 to 6 tons, whereas in 1996 the threshold was around 4 tons. In dry years, root development, which is often increased by Trichoderma, is more limiting to growth. The wet season in 1996 allowed even crops with minimal root systems to grow well.

The economic return was substantial (Table 4). The average return paid for the treatment many times over, on sweet corn and on other crops. Therefore the economic risk of using T-22 is minimal. An additional benefit is that the maximum benefit is when it is most needed by the farmer. When conditions arise during the season that compromise the yield and result in a low-income year, the response to T-22 is greatest. This effect could help manage economic risk in addition to having an overall positive return.

Recommendations:

1. Using Trichoderma (T-22) is profitable. The cost is so low that one additional dozen per acre will more than pay for the use, so the risk is small. The return averages 15 to 30 times the cost.

2. Consider T-22 to be biological insurance against unpredictable crop loss. T-22 preserves the crop’s yield potential under certain adverse conditions.
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Objective 3. Additional cover crops
Cover crops may differ in their ability to support a population of Trichoderma that is sufficient for colonizing a subsequent crop. The colonization of Trichoderma on several species used as cover crops was examined in greenhouse trials. Six cover crops of regional significance were selected: Annual ryegrass, canola, red clover, grain rye, hairy vetch and winter wheat. The experiment was done twice with comparable results.

Colonization was best and growth most vigorous in annual ryegrass (Fig. 7). This cover crop is killed in the winter so it would not be suitable for over wintering Trichoderma, but could potentially increase the population for later in the same growing season. Clover was reasonably well colonized, but the root mass was very small compared to the others, so the total inoculum provided may be insufficient. Vetch did not have sufficiently high inoculum to be considered a likely carrier. Rye was modestly colonized, but proved to be a poor vehicle in field tests described in Objective 1. Canola was also modestly colonized, but its winter root growth and exuded carbohydrates are different, so there is a possibility that it is better than rye for overwintering the fungus. These results suggest wheat as the most likely cover crop to use for carrying over and multiplying Trichoderma inoculum, with canola the likely second. We are not especially optimistic about any of them in light of the field results.

To distribute inoculum throughout the soil, the fungus needs to grow as fast as the roots. In the summer, that is always the case, but the hyphae of Trichoderma slow down when the temperature is below 60°F, and it becomes dormant when the temperature falls much further. Overwintering crops have a lot of root growth when the soil is too cold for active Trichoderma growth. The lack of fungal growth when the cover crop is doing most of the critical expansion through the soil will limit the usefulness of this technique. It is possible that a summer cover crop can be useful for inoculating a later summer crop, but that was not considered relevant for sweet corn in the Northeast. In the future, cold tolerant strains of Trichoderma are likely to be developed. These should be tested on overwintering cover crops and also on fall-sown crops such as winter grains.

Recommendation:

• Colonized cover crops are not an effective way to inoculate sweet corn with Trichoderma. Many cover crops are themselves well-colonized by T-22.
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a. Common garden. In order to identify specific traits of soils that are important to colonization, a variety of soils were tested in a common-garden experiment. Soils were collected from 12 locations that were to be planted to corn. The locations were selected to represent a diversity of relevant soil types. The soils were put in 12-liter rhizotrons that were placed in trenches to form a garden containing randomized and replicated soil types planted with inoculated and uninoculated seed of supersweet corn. For each soil, 5 rhizotrons were planted with inoculated seed, and 5 with uninoculated seed, for a total of 120 each year. This experiment was conducted in all three seasons, with preliminary data collected in 1995.

The data were analyzed by first describing the different soils with principal components. The principal component analysis was performed on the soil descriptors to get a manageable set of predictors. The first three principal components explained 41, 21 and 15% of the variation in soils using 13 descriptors (pH, NO3, P, K, Mg, Ca, Fe, Mn, Al, Zn, Organic Matter, clay %, soluble salts).

The soils were distributed fairly uniformly across the observed range of each principal component, and there were no obvious clusters of soils when the components were plotted against each other (Fig. 8). This indicates that the soils were a good representation of the variation of interest.

The effect of each principal component on colonization was then tested. In all three years of this trial, calcium has been a major part of the principal component that is correlated with colonization. A regression analysis showed that Principal Component 2 was correlated with colonization (Fig. 9). This component was related to alkaline calcareous (or well-limed) soils. Two other principal components were not significantly correlated with colonization; PC1 represented Mg and soil texture, and PC3 was dominated by P and K.

Two traits were expected to be significant factors in colonization, and were analyzed individually. Soil clay content has been found to be positively related to colonization by beneficial fungi in the past. It was not a predictor of colonization by T-22. This strain of Trichoderma is more closely associated with roots than other strains, and may therefore be less dependent on clay particles for a niche in which to grow. High soil salinity can inhibit growth of fungi, but soluble salts were again not correlated with colonization.

Recommendation:

• T-22 will colonize sweet corn on a great diversity of soils. The only Northeastern soils where there would be a concern are those very low in Ca and acidic. Liming in accord with normal recommendations for sweet corn production should eliminate even the small chance of inadequate colonization.
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b. Field trials. Also from the common-garden experiment, we examined whether different types of soil management alter the ability of Trichoderma to colonize. Of specific interest is whether organic matter management that promotes growth of a diverse soil microflora will make it more difficult for Trichoderma to invade. This experiment was done using soil from the three systems at the Rodale Farming Systems Trial which have been managed continuously for 18 years with conventional fertilization, animal manure or legumes to supply nitrogen. The latter two provide more food for soil fungi. The results indicate that the animal based system is as easily colonized as the conventional one (Fig. 10). The lower colonization in the legume-based system did not recur in 1996.

Recommendation:

• Stable microbial communities will not prevent colonization by T-22. This organism may be effectively used by growers using organic management.
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c. Grower trials. The performance of Trichoderma on different soils was tested by planting strip trials at 12 different farms representing the full range of soil types used for sweet corn production in New York. At each site, fields were sowed with strips of 4 or 6 rows, alternating treated and untreated seed. Six blocks, each containing a pair of treated and untreated corn, were selected and the effect of Trichoderma was calculated as the mean of the effects in each pair.

Colonization was consistently good on a variety of soils (Fig. 11). The soils included gravel, sand, and muck as well as several loams. The last planting was on gravel, which had a low population. We found earlier that excessively well-drained soils, such as gravel and sand, support lower populations of Trichoderma. That pattern was mainly evident in the wild populations this year.

The population of wild Trichoderma was higher in later plantings. While we cannot distinguish wild strains from strain 22 in the assay, other tests have shown that strain 22 displaces the wild ones on inoculated roots. Even when the wild strains reach high populations, they are less effective at protecting from disease or increasing growth. They also do not colonize the roots as quickly when the seed germinates. These are the traits that were bred into strain 22 that sets it apart from the wild strains.

Recommendation:

• Sweet corn is colonized by T-22 on a wide variety of soil types. Soil type does not need to be a consideration in deciding whether to use T-22.
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Objective 5. Stresses.
a. Cold. T-22 grows sparingly below 60°F and not at all below 50°F. This may limit colonization immediately upon germination. Supersweet corn also does not germinate at low temperatures, so the germination of both may only be delayed by cold. Field experiments varying the cold stress were performed by sowing three to four different times in each of three years, and recording the mean soil temperature over the subsequent three days. Colonization was assayed at the four-leaf stage, about 20 d after emergence. The time to emergence varied widely because of the temperature differences. In 1997, early colonization was also assayed at emergence.

Colonization of roots by T-22 was strong at the 4-leaf stage regardless of the soil temperature (Fig. 12). Wild strains had low colonization in cold soil. They were also were suppressed in later sowings, presumably due to competition with other soil microorganisms. Early colonization was affected by low temperature, confirming the slow growth of T-22 in cool soil.

These data are the first strong field evidence of the remarkable rhizosphere competence of T-22. No other biological on the market has comparable ability to colonize roots under field conditions.

In greenhouse experiments, cold had the same proportional effect at all doses, there was no interaction that would indicate a specific reversal of cold injury by Trichoderma colonization (Fig. 13). For applying cold treatments, seeds were sown in pots with each treatment consisting of 12 pots of 3 plants. The seeds were allowed to imbibe for 1 day at 20 to 25 °C. They were then transferred to a growth chamber on a 5/10 °C diurnal temperature cycle. After the cold treatment the pots were returned to the greenhouse and grown for three weeks.

If the cold injury were specifically reversed an interaction would be expected, with colonized plants being less sensitive to intermediate stresses that cause only lesions that can be reversed by the metabolic action of the fungus. Nevertheless, Trichoderma-colonized seedlings grew better after cold stress. Even though Trichoderma does not grow well at <15 °C, a growth enhancement resulted even after substantial cold treatment.

b. Oxidation. Treatment of the seeds with dilute hypochlorite to cause oxidation injury resulted in considerably reduced vigor. Subsequent colonization with Trichoderma completely restored the vigor of these seedlings (Fig. ). Hypochlorite and Trichoderma significantly changed the size distributions compared to untreated seedlings (Fig. 14). Trichoderma changed seedling-size distribution of hypochlorite-treated seeds so that it was the same as seedlings that had been treated only with Trichoderma. The effect of both hypochlorite and Trichoderma treatment on seedling vigor occurred mainly in the moderately vigorous seedlings. In contrast, the strongest seedlings in each group performed similarly.

Soil treated with the allelopathic compound benzoxazolinone (BOA) resulted in truncated roots with the same biomass as roots grown in the absence of BOA. Trichoderma increased the biomass growth (dry-weight gain) by 52% with no BOA effect (Fig. 15a).
Allelopathic stress did have a dramatic effect on root architecture. Trichoderma increased the elongation growth resulting in a large effect of BOA on the root system length (Fig. 15b). The radicle apex died quickly in the BOA-treated soil, with compensatory growth in branch roots . These branch-root apices also died relatively quickly, resulting in early higher-order branching as adventitious roots were initiated. The root systems had almost no radicle and a short, highly branched root system whether or not they were colonized by Trichoderma.

d. Fungicides.
Seed-applied fungicides, particularly Imazilil, represent a stress that can potentially diminish the effectiveness of the beneficial fungus. Seed-applied fungicides are often used in conjunction with T-22 to control seed rots, which are not affected by T-22. The effect of fungicide seed treatment on colonization and establishment was tested with the granule formulation in 1995. We used a 2x2 factorial with fungicide and T-22 granules, repeated in 5 different plantings.

There were two clear conclusions:

1. T-22 colonization was unaffected by the fungicides.

2. Fungicides were sometimes necessary for establishment, and T-22 did not substitute.

The increase in colonization with granules was adequate, but it was greater with the planter box formulation (Table 5). Stand establishment was not affected by T-22, but fungicides are sometimes necessary for a satisfactory stand (Table 6). The main conclusions form these experiments should also apply to the Planter Box formulation.

Recommendations:

1. T-22 can be used with fungicide-treated seed without reducing colonization.

2. If seed-applied fungicides are necessary for stand establishment, they need to be used regardless of whether T-22 is also used.
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