Unleash™ Mechanism of ActionNitrogen (N), potassium (K), and phosphorous (P) are the three primary...

Unleash™ White Paper – May 15, 2017 1

Unleash™ Mechanism of Action

White Paper Prepared for AquaBella Organic Solutions By Tracy Letain, PhD, Microbiologist

Issued: May 15, 2017

Abstract The purpose of this report is to describe how Unleash™ works to increase soil fertility including the benefits gained from using Unleash™.


1.0 Introduction Since agriculture’s beginnings thousands of years ago, growers have struggled to find ways to increase crop yield. The advent of industrial agriculture over the last two centuries has resulted in dramatic developments in intensive farming techniques including the use of modern plant cultivars, mechanization, additives such as fertilizers and pesticides, crop rotation and irrigation. While these techniques have resulted in enormous increases in crop yield and range, they also have their costs, limits, and detrimental consequences such as soil depletion, erosion, and widespread outbreaks of plant disease.

Research has clearly demonstrated the importance of a healthy microbial community in the soil, particularly in the soil surrounding plant roots, called the rhizosphere, for plant growth. This has led to the development of a new area for agricultural advancement and investment in the use of natural microbial soil inoculants that can result in significant improvements in crop yield and plant and soil health. The use of natural microbial inoculants can benefit growers in several ways including 1) the ability to assist the plant in nutrient uptake; 2) the displacement of harmful soil life that may be present; and 3) to help mitigate/minimize plant reaction to stress such as drought or disease.

Rhizosphere microbes consist of a select group of beneficial bacteria and fungi capable of promoting healthy plant growth and soil fertility. These “good” microbes not only aid in nutrient uptake, but also displace harmful soil life that may be present. While common in natural settings, their populations are often very low in agricultural, urban and residential settings. Unleash™ contains a highly concentrated mixture of many of these beneficial rhizosphere microorganisms that can colonize plant roots and root hairs. As the name implies, Unleash™ is designed to work optimally on plant roots. Unleash™ microbes are capable of several activities that can facilitate various applications for both agricultural and personal use (for a list of common rhizosphere microbes, see Table 1), including:

1. Mobilization/release of nutrients already in the soil/water;

2. Degradation of compounds detrimental to plant growth;

3. Displacement/destruction of organisms detrimental to plant growth;

4. Production of nutrients beneficial to plant growth;

5. Alteration of the growth matrix to conditions more favorable to plant growth.


TABLE 1:

List of Common Rhizosphere Microbes with Beneficial Activities Similar to Activities Found in Unleash™ Microbes

*Fungal organism. All other organisms are bacterial.


2.0 Roots and Plant Growth Figure 1 Root hairs and rhizosphere microbes.

Figure 1A Tree root covered with root hairs

Figure 1B Root hairs (orange) colonized with microbes (yellow)

• Figure 1A taken from link http://www.treedictionary.com/DICT2003/R/root_hairs.html • Figure 1B taken from the American Society for Microbiology link

http://www.microbelibrary.org/ASMOnly/Details.asp?ID=535

The most active area involved in nutrient uptake for a plant is the root, and the area of soil immediately surrounding the roots, called the rhizosphere (Figure 1). The rhizosphere is quite small, including a few millimeters or at most centimeters of soil around the plant root area. The rhizosphere is of particular importance as it contains a microbial population whose actions differ significantly from bulk soil (Figure 1). This occurs because plant roots release small quantities of oxygen, organic compounds, and other compounds, which can collectively serve as food and energy sources for microbes. In return, these rhizosphere microbes assist the plant in nutrient uptake, using several different mechanisms including:

1. Changing the chemical form of the nutrient to make it available for plant use;

2. Mobilizing nutrients absorbed/adsorbed onto soil particles.

3. These mechanisms help provide the nutrients required for good plant root development. In particular, nutrients are required for the root hairs to develop. These root hairs have a large surface area relative to the rest of the root, and are responsible for much of the water and nutrient uptake into a plant (figure 1).


4. Unleash™ contains beneficial bacteria that aid in the colonization of plant roots by arbuscular mycorrhizae (AM), a group of beneficial fungi that play a key role in the uptake of nutrients in plants. AM form a symbiotic association with plant roots, increasing the root surface area allowing the plant to extend the soil volume from which it receives water and nutrients, as well protecting against abiotic stresses such as soil compaction, salinity and drought (figure 2).

Figure 2 Plant roots and arbuscular mycorrhizae Figure 2A Depiction of the plant root-mycorrhizal network

Figure 2B Figure 2B shows a pine tree seedling and the mycorrhizae-plant root system. The visible tan and white lines are the plant root network, and the “cloudy” grayish surrounding area are the mycorrhizae. (Image taken from http://mpgranch.com)


3.0 Nutrients and Plant Growth Continuous farming of a plot of land depletes nutrients from the soil essential for plant growth. To minimize the amount of time required for soil recovery, nutrient-containing fertilizers are added to the soil. While this allows the land to be continuously farmed, it is both expensive and inefficient, with much of the fertilizer being absorbed by the soil or migrating into the local water table, making it unavailable for uptake into the plant.

Nitrogen (N), potassium (K), and phosphorous (P) are the three primary plant nutrients needed in large quantities by plants. The addition of these nutrients to the soil and uptake by the plants presents difficulties specific to each nutrient.

Nitrogen (N)

Plants require larger quantities of N than any other nutrient. While N is in abundant supply in the environment, most of this N is in a form unavailable for plant use. N deficiency in plants can result in restricted growth of tops, roots and especially lateral shoots. Plants can also become spindly with a general chlorosis of the entire plant to a lighter color and ultimately a yellowing of older leaves, which may proceed toward younger leaves. This occurs because N is required for formation of chlorophyll and the various proteins involved in photosynthesis, which is responsible for the green color seen in plant leaves.

Figure 2 Conversion of nitrogen in the environment by bacteria.

Figure 2 taken from Zumft, W. G. 1997. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61:533-616.


N conversion from one chemical form to another chemical form in the environment is most commonly carried out by bacterial microorganisms found naturally in the soil (Figure 2). The conversion of atmospheric N2 gas to ammonium by bacteria, called nitrogen fixation, takes place in the soil and in specialized nodules found in the roots of legume plants, such as alfalfa. This process historically was commonly used to replenish the nitrogen in the soil by growing crops that have root nodules containing bacteria capable of N fixation where needed. Unfortunately, not all plants are able to support nitrogen fixation, so other processes must be used such as the addition of N-containing fertilizer to nutrient-depleted soil.

The most common forms of N in the environment available to plants are the nitrate (NO3-), and ammonium (NH4+) ions. Although nitrate is the form of N most easily taken up by many agricultural plants such as peas and tomatoes, there are significant downfalls to its use: (1) it is highly mobile in most soils, migrating easily from the soil to the water table, particularly in saturated soils; (2) it is a major contaminant in drinking water, with relatively low levels of nitrate contamination resulting in a water body being no longer usable in municipal systems. Because of this, N is generally added to the soil in the form of the ammonium ion, which then is converted by biological or by chemical oxidation into nitrate by a process called nitrification. In fact, nitrification is so common that nitrate contamination of the water table often occurs in agricultural soils where only ammonium is added. The more effectively a plant is able to use N, the less N-containing fertilizer is needed, thus reducing the chance that nitrate may migrate to the local water table.

Rhizosphere bacteria are capable of many other N conversion processes. One of the most important is denitrification, where nitrate is converted to dinitrogen gas. Unlike some of the other conversion processes discussed in this paper, denitrification can only be carried out by bacteria. For agricultural operations that see a significant amount of nitrate runoff, denitrification is the primary process responsible for nitrate degradation.

Potassium (K)

Plants require more potassium (K) than any other nutrient except for N. Unlike most nutrients, K plays no structural role in the plant – it is not a major component of the leaves, roots, or any other plant structure. Instead, it is found in the water-containing parts of the plant, playing a major role in root growth, stalk strength, water and nutrient transport, disease control, plant pH, photosynthesis regulation, and sugar and starch transport, as well as other plant functions.

Plants grown with an insufficient supply of K are much more susceptible to water and temperature stress, and will result in reduced yield potential and quality long before any


physical symptoms occur. Even short periods of K deficiency during critical developmental stages can cause serious losses. Plants grown with a sufficient amount of K, on the other hand, will result in improved shelf life of fruits and vegetables, and improved feed values of grain and forage crops, as well as the fiber value of cotton crops.

Although there is a high amount of K in the soil, less than 2% of it is available for plant use, with 90-98% found in soil minerals such as mica, which are unavailable for use. Because such a high level of K is required for all agricultural crops, additional K must be added to agricultural soils. The most common K source for fertilizer is from salt mined from seawater beds. However, K present in manure is also commonly used as a fertilizer source.

The mechanism by which microbes help with K uptake by plants is not known, although agricultural soils with depleted amounts of rhizosphere microbes have been shown to have reduced levels of K uptake. Microbes may play a role by altering the rhizosphere environment pH and oxidation level such that K is either more readily obtainable or less likely to leach out. Microbes may also increase K uptake by increasing the surface area of the roots and root hairs, and by extending the soil volume that can be reached by the plant roots (figure 2). There is also a hypothesis that a healthy rhizosphere bacterial and mycorrhizal community will negatively impact non-mycorrhizae fungi, which can complete with the plant for K.

K is highly mobile in the soil, similar to the nitrate ion. K is not by itself a water contaminant, unlike the N nitrate ion. However, high salinity in the water table due to K and other ions associated with fertilizer use in agricultural areas is a major problem where this is a concern, such as the Central Valley of California. Decreased fertilizer use can help minimize these high salinity problems.

Phosphorous (P)

Plants require a relatively large amount of phosphorous, with only N and K required in higher quantities. P is involved in numerous plant functions, particularly any functions involved with photosynthesis, as P is required for all processes involved with energy production. It is also required for root development, ripening, and flower development. Plants grown in P-depleted soil will result in stunted growth/delayed maturation, bluish green/purple leaves (especially older leaves), and small acidic fruit formation. Plants grown in P-depleted conditions are often depleted of other trace minerals as well such as iron and zinc.


Virtually all P in fertilizer is added to soils in the form of the phosphate anion (PO4-3). Animal manure is also a common source of fertilizer phosphate. Once added, phosphate rapidly precipitates, with only 10-30% of the fertilizer phosphate being available for plants. This precipitated phosphate is commonly referred to as “fixed” phosphate. Phosphate precipitates with various other minerals in the soil, with calcium, aluminum, carbonate, and iron all playing major roles at different soil pHs. Of these precipitates, the phosphate-calcium complex (which tends to occur the most in alkaline, or high pH soils) is the easiest to make available for plant use, so phosphate availability tends to be of minor concern in more alkaline soils. While some of the fixed phosphate becomes usable in subsequent years, most of it remains unavailable.

Phosphate is a major threat to water quality, particularly in surface waters. This is because high phosphate levels in water can result in excessive algae growth. High levels of algae reduce water clarity and can lead to decreases in available dissolved oxygen as the algae decays, conditions that can be detrimental to fish populations. Because phosphate rapidly precipitates in soil, there isn’t as much concern that it will leach into the water table, unlike nitrate and potassium. In fact, when fertilizer-associated phosphate is found in the water table, it is usually due to soil erosion or runoff. Good soil conservation practices are the best defense against unwanted phosphate losses.

Microbes play a direct role in phosphate uptake by mobilizing precipitated phosphate for plant use. Since phosphate availability is highly pH-dependent, local pH changes in the rhizosphere due to microbial activity can have a significant effect on the availability of this nutrient. Bacteria are especially good at recovery of precipitated phosphate, as active bacterial populations require phosphate for growth. Arbuscular mycorrhizae are known to play a significant role in phosphate uptake by increasing the root surface area, allowing the plant to extend the soil volume that can be reached by the plant roots (figure 2). In fact, an estimated 80% of the phosphate taken up by a mycorrhizal-colonized plant is supplied by the fungus.

Secondary Nutrients

Plants need several other soil nutrients in addition to the primary nutrients discussed above (N, K, and P). These include the secondary nutrients calcium (Ca), magnesium (Mg), and sulfur (S). While there are sufficient quantities of these secondary nutrients in moderate and high pH soils to support plant growth, lime is commonly added to more acidic soils, which increases the soil pH as well as supplying additional Ca and Mg. Rhizosphere organisms can increase availability of Ca and Mg by mobilizing


precipitated Ca and Mg. Rhizosphere microbes are central to S uptake, converting S into a form plants can use, serving a similar role for S as they do for N.

Micronutrients

Elements essential for plant growth that are only needed in trace quantities are called micronutrients. Plant micronutrients include boron (B), copper (Cu), iron (Fe), chloride (Cl), manganese (Mn), molybdenum (Mo), and zinc (Zn). Rhizosphere bacteria may be able to play a role in the availability of most of these elements; for example, several rhizosphere bacteria can chelate and mobilize several different metals in soils. However, as only trace quantities of these elements are necessary for growth, plants may be capable of obtaining sufficient quantities without rhizosphere microbial activity.

Plant Disease Resistance

Unleash™ contains rhizosphere bacteria and fungi that are known to increase resistance by plants to various diseases. They do this by preventing the growth of various pathogenic organisms in the plant rhizosphere, where many plant diseases are caused. Microorganisms can do this using multiple mechanisms including (1) Competing for growth in the same environmental niche such as mycorrhizae can do in the plant root zone against pathogenic fungi; (2) Limiting available nutrients such as iron to plant soil pathogens; (3) Producing compounds such as antibiotics and antifungals that actively block pathogen growth.


5.0 Conclusions and Recommendations

Figure 3 Effect of Unleash™ treatment on Huntington Farm vegetables.

Figure 3A

Figure 3B

Figure 3A depicts cauliflower leaves from treated (right) vs. untreated (left) plants. Figure 3B depicts broccoli heads from treated (right) vs. untreated (left) plants. The Huntington Farms owners found treated plants for both types of vegetables to have better color, healthier leaves and stalks, and highly uniform vegetable maturation compared to untreated plants. They also found the treated cauliflower had superior shelf life and yield.

Unleash™ significantly promotes healthy plant growth using several different mechanisms that result in increased nutrient availability and plant pathogen resistance. Unleash™ treatment has been shown to have this effect on many different plants grown in a variety of soils and climates. Some specific examples of Unleash™ results include (1) Huntington Farms treated cauliflower plants showed a 50% increase in yield, improved shelf life, and larger, healthier leaves compared to untreated plants (Figure 3A). Treated broccoli plants were also healthier and showed more uniform growth than untreated plants (Figure 3B); (2) treated grapevines were found to mature six months more rapidly, with bigger leaves, more Ca in the petioles, and a large amount of fruit production compared to untreated grapevines (Laura Zahtila Vinyards); (3) treated mâche (lamb lettuce) leaves had better color, texture, thickness, and shelf life than untreated plants, even in the absence of added fertilizer (Epic Roots); (4) treated green beans showed a 23% increase in yield compared to untreated green beans (Sarabian Farms); (5) treated grapevines had a 10% increase in grape yield and larger, healthier leaves with better color than untreated grapevines (Vineyard of Pasterick); (6) Rocking B Farms (Sonoma, CA) tested Unleash™ on a wide variety of plants including basil, mulberry trees, peach trees, raspberries, roses, thyme, tomatoes, starburst squash,


summer squash and zucchini. In all cases, treated plants were noticeably healthier, with increased fruit/vegetable yields compared to previous seasons; (7) field studies were done in Vietnam testing Unleash™ on a wide variety of plants including bananas, tomatoes, broccoli, carrots, yams, garlic, green onions, soybeans, watermelon, sugarcane, barley, and rice. In all cases increased yield was seen compared to untreated fields, with the increases ranging from 16% to 53%; (8) treated medical marijuana plants showed a faster maturation time and a 25% increase in yield compared to untreated plants; (9) treated eggplant and pepper plants were healthier, matured faster, and showed increased yield compared to untreated plants (Vic Dintino).

Some of the mechanisms used by rhizosphere microbes such as those found in Unleash™ to improve plant growth and yield are summarized below. Examples of specific rhizosphere microorganisms listed in Table 1 associated with the described processes are also indicated.

• Unleash™ microorganisms can increase nitrogen (N) availability by changing the chemical form of N. For example, when N is limited they can fix N2 gas into NH4-ammonium (e.g. Azotobacter vinlandii, Table 1). They can also change the more common N fertilizer form, NH4-ammonium, into the more mobile N form preferred for uptake by many common agricultural plants, NO3-nitrate (e.g. Nitrosomonas europaea, Table 1).

• Unleash™ can increase potassium (K) availability by altering the rhizosphere environment pH and oxidation level such that K is either more readily obtainable or less mobile.

• Unleash™ can increase phosphate availability by mobilizing precipitated “fixed” phosphate for plant use (e.g. Pseudomonas fluorescens, Table 1). Phosphate availability is highly pH-dependent, so local pH changes in the rhizosphere due to microbial activity will have a significant effect on the availability of this nutrient for plants.

• Unleash™ can act on secondary nutrients as well. Unleash™ microorganisms can change the chemical form of sulfur (S) into a form plants can use. They can mobilize precipitated calcium (Ca) and magnesium (Mg). In fact, an abundance of Ca was seen in treated grape petioles from Laura Zahtila Vineyards compared to untreated plants, indicating Unleash™ treatment had increased Ca availability in rhizosphere soil.

• Unleash™ treatment ensures that a healthy rhizosphere microbial community exists, which can suppress the growth of harmful soil life that would otherwise compete with the plant for nutrients. Unleash™ suppresses harmful soil organisms by (1) competing for nutrients; and (2) synthesizing compounds that prevent plant pathogen growth (e.g. Bacillus subtilis, Table 1).


• Unleash™ can also help ensure the colonization of plant roots by arbuscular

mycorrhizae (AM) – a type of beneficial fungi that play a key role in the uptake of nutrients and water in plants (e.g. Glomus intraradices, Table 1). AM form a symbiotic association with plant roots, increasing the root surface area allowing the plant to extend the soil volume from which it receives water and nutrients.

All of these activities can result in healthy root growth during plant development, which will also increase nutrient uptake and make the plant more stress resistant. By allowing plants to more effectively use the nutrients in the fertilizer and soil, Unleash™ maximizes the probability that plants will get sufficient nitrate, phosphate, and other nutrients throughout critical growth stages, ensuring high yield and harvest quality.

Unleash™ Mechanism of ActionNitrogen (N), potassium (K), and phosphorous (P) are the three primary...

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