**Modern Seed Technology: Seed Coating Delivery Systems for Enhancing Seed and Crop Performance**

#### **Irfan Afzal <sup>1</sup> , Talha Javed <sup>1</sup> , Masoume Amirkhani <sup>2</sup> and Alan G. Taylor 2,\***


Received: 9 October 2020; Accepted: 3 November 2020; Published: 5 November 2020

**Abstract:** The objective of modern seed-coating technology is to uniformly apply a wide range of active components (ingredients) onto crop seeds at desired dosages so as to facilitate sowing and enhance crop performance. There are three major types of seed treating/coating equipment: dry powder applicator, rotary pan, and pelleting pan with the provisions to apply dry powders, liquids, or a combination of both. Additional terms for coatings produced from these types of equipment include dry coating, seed dressing, film coating, encrustments, and seed pelleting. The seed weight increases for these different coating methods ranges from <0.05% to >5000% (>100,000-fold range). Modern coating technology provides a delivery system for many other materials including biostimulants, nutrients, and plant protectants. This review summarizes seed coating technologies and their potential benefits to enhance seed performance, improve crop establishment, and provide early season pest management for sustainable agricultural systems.

**Keywords:** seed enhancement; seed treatment; seed dressing; seed coating; film coat; pellet; organic agriculture

#### **1. Introduction**

High seed quality is always demanded by farmers and may result in up to a 30% increase in crop yields [1,2]. Sowing high-quality seeds is essential, but their use does not guarantee successful stand establishment. The difference in time between sowing and stand establishment is a crucial period. Seeds may be exposed to a wide range of biotic and abiotic stresses resulting in decreased stand performance [3]. However, judicious use of chemical, biochemical, and biological seed treatments can protect and enhance establishment, growth and potential productivity [4]. In this review, seed treatments refer to materials that are active components, while seed dressings are the minimal coating that results after the application of seed treatments onto seeds. Seed treatments are most effective when they are objective oriented and crop specific to ensure optimal stand establishment and enhance yields under changing climatic conditions [5].

Seed treatments may be applied commercially by the seed industry or in some cases "on farm" for crop protection and enhanced seedling growth [2,6]. There is also a growing trend for the development and use of organically approved treatments for sustainable agriculture. Collectively, innovative seed coating technologies are needed as delivery systems for the application of active ingredients at effective dosages to crop seeds [7,8].

A brief history of seed treatments for plant protection illustrates the practical need for better delivery systems and improved ability to sow seeds [9]. Copper sulphate was found to be an effective seed treatment for bunt on cereals in the 1800s when applied as a soak. However, treating large quantities of seed required subsequent drying that made the process cumbersome and time consuming. The soaking process was replaced by the "heap" or "barn floor" method where a small amount of liquid was sprinkled over the seed and then mixed [9]. The soaking (also known as steeping) method is still in use for sugar beet seed using the method described by Halmer (2000) [10].

In 1866, a technique was developed to improve sowing of cotton seed using a paste of wheat flour to form a pellet [11]. During the mid-20th century, many coating technologies for improved agricultural productivity were developed and reviewed by Jeffs (1986) [9]. Seed coating technology continued to advance through the 1970s to 1990s and reviewed by Taylor and Harman (1990), Scott (1989) and Hill (1999) [7,12,13]. More recent reviews focus on seed enhancements and seed coating equipment in the 21st century by Taylor (2003), Pedrini et al. (2017), Halmer (2000), and Pedrini et al., (2020) [6,8,10,14].

Seed enhancements may be defined as post-harvest treatments that improve germination or seedling growth or facilitate the delivery of seeds and other materials required at time of sowing [15]. Seed coating is used for the application of biostimulants, plant nutrients, (including inoculants) and other products that will ameliorate biotic and abiotic stresses encountered after sowing [11,16].

The global market for seed coating materials (colorants, polymers, fillers and other additives) in 2019 was US \$1.8 billion and is forecasted to reach \$3.0 billion by 2025 [17]. The major group of active ingredients are chemical seed treatments estimated between \$3 to \$5 billion in 2020, and accounts for at least 2/3 of the total seed treatment market [18]. The biological seed treatment market includes a wide range of biologicals including biofertlizers, biopesticides and biostimulants [19]. The biological seed treatment market is estimated between \$1 to \$1.5 billion in 2020, and bioinoculants are the dominant group with about 70% of total [18].

The focus of this review is the use of selected seed coating components, including liquids and solid particulates, with designated seed coating equipment and technology for uniform delivery of treatments over seeds uniformly. Applications of selected seed treatment and coatings are presented as biostimulants, nutrients, and in management of abiotic and biotic stress. Seed coating technologies described may be applied to a wide range of crop seeds: grains, oilseed, vegetable, ornamentals, and other seed species [20].

There is considerable research and development by industry in the broader field of seed treatments, and much of this technology is proprietary. Many biological seeds treatments are being developed and marketed for pest management and as biostimulants. However, it is beyond the scope of this publication to critically review the merits and efficacy of these biologicals, though they are used commercially. Therefore, this review focuses on published papers and most are from refereed journals. This paper contains 112 references with 97 published papers or book chapters, 6 patents, 6 websites and 3 personal communications cited. Moreover, to provide relevancy to the seed coating industry, eight companies were acknowledged to provide valuable input in preparation of this review.

#### **2. Seed Treatment Active Components and Other Coating Materials**

A wide range of materials is used in seed treatments and coatings. These materials were categorized by their composition and origin as synthetic chemicals (SYN), natural products or derivatives from natural products (NP), biological agents (BIO) and minerals mined from the earth (MIN) (Table 1). Among these categories, particular materials may be used for organic use and labelling, and the US Organic Materials Review Institute (OMRI) [21] approved materials were noted as organic (*OR*). Seed treatment and coatings are further characterized by function, as active components, liquids or solid particulates.

**Table 1.** Seed treatment and coating materials grouped as active components, liquids and solid particulates. Each group of material is further classified by function and composition. Abbreviations for material source/origin: Synthetic Chemicals—SYN, Natural products or derivatives—NP, Biologicals—BIO, Mineral—MIN, substances may be Organically approved—*OR.*


#### *2.1. Active Components*

The purpose of active ingredients is aimed at protecting and enhancing seed and seedling performance in terms of germination, growth and development. The mode of action of the active ingredient dictates its role for protection and/or enhancement [16]. Active ingredients discussed in this paper include biostimulants, plant nutrients, protectants from abiotic and biotic stress, and inoculants (Table 1). Seed protectants are the most widely used group of ingredients for controlling pathogens and pests at the time of sowing. Fungicides, insecticides, nematicides, and bactericides are grouped as protectants [22]. Selected fungal and/or bacterial microorganisms are used commercially for plant protection, and as inoculants for nitrogen fixation [22,23]. Abiotic stresses due to saline soil conditions or drought stress may occur after sowing and selected biological and synthetic seed treatments may be applied in the seed coating to alleviate these stresses. Elicitors are being investigated as active components for pest management [24–26], and drought stress [27]. There is increased interest and demand for biostimulant- and nutrient-based seed treatments [8].

#### *2.2. Liquids*

Active components must be applied to seeds so that they adhere onto seeds throughout storage until planted. In addition, seeds treated with pesticides must easily be recognized as treated. Colorants are commonly used to indicate that seeds are treated and constitute about 60% of coating ingredient components, and in the case of seed pelleting are applied at the end of coating process [8]. Colorants also provide a visual of assessment of application uniformity, and cosmetic appearance. Water is the universal carrier of liquids that are atomized onto seeds during the coating process, and atomization is best achieved with low viscosity liquids. The proportion of water in the applied liquid is adjusted to maintain low solution viscosity. Adjuvants are used [20] as most chemical seed treatment active ingredients have limited water solubility, so surfactants are needed to produce aqueous seed treatment formulations. Surfactants may serve as an active component, and a seed coating technology with surfactants was documented to enhance germination and stand establishment when sown in water repellent soils [28].

Seed coating binders act as adhesives to adhere treatments to seeds. The binder provides the coating integrity during and after drying. They prevent cracking and dusting off during handling and sowing [2]. Commonly used binders (Table 1) for maintaining physical integrity of seeds are: polyvinyl alcohol [29], polyvinyl acetate [30], methyl cellulose [31], and carboxymethyl cellulose [32]. For organic seed coatings plant starches (maltodextrins) [33] and gum Arabic [34] are commonly used. Most binders are commonly referred to as polymers [35]. In preparing binders in water, solution viscosity must be low for complete atomization of the liquid onto seeds, based on the fourth author's experience preferably <100 centipoise (cP), or <0.1 pascal-second (Pa-s).

#### *2.3. Solid Particulates*

Solid particulates are the bulking materials used in seed coating technologies and form the physical coating after drying [7,30]. Solid particulates may also be binders. Solid particulate binders are applied as fine powders and become hydrolyzed as water is applied during the coating process. Fillers are also fine powders and can be mixed with the solid particulate binders to produce a seed-coating blend. Successful seed pelleting depends upon the optimization and selection of the most appropriate filler materials that do not interfere with germination [32]. *Agriculture* **2020**, *10*, x FOR PEER REVIEW 4 of 19 *2.3. Solid Particulates*  Solid particulates are the bulking materials used in seed coating technologies and form the

Filler materials are generally inexpensive, non-toxic, easily available, and produce a uniform coating surface texture that should not impede radicle emergence [6]. Several filler materials are used for seed pelleting including diatomaceous earth [36], limestone, gypsum [32], bentonite [34], vermiculite [37], talc [38], zeolite [32], silica sand [39] and barium sulphate [40] (Table 1). These fillers are generally mineral materials that are mined from the earth with minimal modification except for grinding to obtain a fine powder size used in seed coating. Particle size should pass through a 200-mesh sieve (<75 µm) for uniform distribution over the seed surface based on the fourth author's experience. physical coating after drying [7,30]. Solid particulates may also be binders. Solid particulate binders are applied as fine powders and become hydrolyzed as water is applied during the coating process. Fillers are also fine powders and can be mixed with the solid particulate binders to produce a seedcoating blend. Successful seed pelleting depends upon the optimization and selection of the most appropriate filler materials that do not interfere with germination [32]. Filler materials are generally inexpensive, non-toxic, easily available, and produce a uniform coating surface texture that should not impede radicle emergence [6]. Several filler materials are used for seed pelleting including diatomaceous earth [36], limestone, gypsum [32], bentonite [34], vermiculite [37], talc [38], zeolite [32], silica sand [39] and barium sulphate [40] (Table 1). These fillers are generally

#### **3. Seed Coating Equipment and Methods** mineral materials that are mined from the earth with minimal modification except for grinding to obtain a fine powder size used in seed coating. Particle size should pass through a 200-mesh sieve (<75 µm) for

The seed treatment and coating materials described in Section 2 provides an extensive list of potential ingredients. The next step in the seed coating process is when selected ingredients are applied with appropriate equipment to produce the final coated product. The selection of seed coating equipment and coating method is determined primarily by the dosage of actives, liquids and solid components applied per unit of seed. There are three major types of seed coating equipment used today: dry coating, rotary pan and pelleting pan (Figure 1). This coating equipment used singly or in some cases in tandem is paired with five coating methods: dry powder, seed dressing, film coating, encrusting and pelleting [6,8,10]. The overall goal of all coating equipment and methods is to achieve good application uniformity and adherence. Processes should not cause mechanical injury to seeds during coating [35]. uniform distribution over the seed surface based on the fourth author's experience. **3. Seed Coating Equipment and Methods**  The seed treatment and coating materials described in Section 2 provides an extensive list of potential ingredients. The next step in the seed coating process is when selected ingredients are applied with appropriate equipment to produce the final coated product. The selection of seed coating equipment and coating method is determined primarily by the dosage of actives, liquids and solid components applied per unit of seed. There are three major types of seed coating equipment used today: dry coating, rotary pan and pelleting pan (Figure 1). This coating equipment used singly or in some cases in tandem is paired with five coating methods: dry powder, seed dressing, film coating, encrusting and pelleting [6,8,10]. The overall goal of all coating equipment and methods is to achieve good application uniformity and adherence. Processes should not cause mechanical injury to seeds during coating [35].

**Figure 1.** The three major types of seed coating equipment: dry powder applicator, rotary coater and drum coater used to produce five seed coatings: dry coating, seed dressing, film coat, entrustment and seed pellet. **Figure 1.** The three major types of seed coating equipment: dry powder applicator, rotary coater and drum coater used to produce five seed coatings: dry coating, seed dressing, film coat, entrustment and seed pellet.

#### *3.1. Dry Powder Coating*

Dry powder application is a seed coating method used for mixing seeds with a dry powder. The older term for this application method is "planter box" treatment [6]. Dry powders, also known as dusts, [20] are used for fungal or bacterial treatments followed by drying (hydration/dehydration) and seeds can have a shorter shelf-life after application [6]. This technology can be conducted on-farm for the application of labeled treatments for the control of a pests [9]. *Agriculture* **2020**, *10*, x FOR PEER REVIEW 5 of 19 *3.1. Dry Powder Coating*  Dry powder application is a seed coating method used for mixing seeds with a dry powder. The older term for this application method is "planter box" treatment [6]. Dry powders, also known as

Dry powder application equipment and technology has evolved to allow for more precise loading of material onto seeds. As can be seen in Figure 1 [41,42] a rotating stainless-steel brush sifts a powder material through a metering screen (Figure 1). The equipment is calibrated on a weight basis to deliver powder to a given weight of seed. The seed is not shown in the illustration, but would be moving underneath the dry powder applicator via most delivery systems (auger, conveyor, seed tender, etc.) [https://www.ctapplicators.com] [43]. This equipment is used for stand-alone dry powder application, or for the application of finishing powders after seed dressing or film coating (described in Sections 3.2 and 3.3). Another dry powder feeder equipment uses a computer-controlled auger with hopper vibrator to deliver coating powders, finishing powders or dry powder actives to seed by volumetric or weight basis [44]. Dry powder carriers may act as lubricants to improve seed flowability by reducing seed-to-seed friction in the planter [6]. The most common dry powders are talc and graphite [45], and recent research revealed that soy-based protein is an environmentally friendly and cost-effective seed lubricant that improves flow and singulation during planting without creating dust [46]. Thus, the use of soy-based protein has the potential to reduce the risk of negative impact on pollinators and people. dusts, [20] are used for fungal or bacterial treatments followed by drying (hydration/dehydration) and seeds can have a shorter shelf-life after application [6]. This technology can be conducted onfarm for the application of labeled treatments for the control of a pests [9]. Dry powder application equipment and technology has evolved to allow for more precise loading of material onto seeds. As can be seen in Figure 1 [41,42] a rotating stainless-steel brush sifts a powder material through a metering screen (Figure 1). The equipment is calibrated on a weight basis to deliver powder to a given weight of seed. The seed is not shown in the illustration, but would be moving underneath the dry powder applicator via most delivery systems (auger, conveyor, seed tender, etc.) [https://www.ctapplicators.com] [43]. This equipment is used for stand-alone dry powder application, or for the application of finishing powders after seed dressing or film coating (described in Sections 3.2 and 3.3). Another dry powder feeder equipment uses a computer-controlled auger with hopper vibrator to deliver coating powders, finishing powders or dry powder actives to seed by volumetric or weight basis [44]. Dry powder carriers may act as lubricants to improve seed flowability by reducing seed-to-seed friction in the planter [6]. The most common dry powders are talc and graphite [45], and recent research revealed that soy-based protein is an environmentally friendly and cost-effective seed lubricant that improves flow and singulation during planting without creating dust [46]. Thus, the use of soy-based protein has the potential to reduce the risk of negative

The dosage of dry coating powders applied to seeds is limited by their adherence onto seeds, and ranges from 0.06 to 1.0% of seed weight (Figure 2). This loading rate is inversely proportional to seed size, and the amount of powder retained increases as seed size decreases due to the increase in seed surface area of smaller seeds [45]. impact on pollinators and people. The dosage of dry coating powders applied to seeds is limited by their adherence onto seeds, and ranges from 0.06 to 1.0% of seed weight (Figure 2). This loading rate is inversely proportional to seed size, and the amount of powder retained increases as seed size decreases due to the increase in seed surface area of smaller seeds [45].

**Figure 2.** Percent weight increase after dry coating, seed dressing, film coat, entrustment and seed pellet technologies. The grey shaded bar for seed dressing and film coat is addition of a finishing powder during the coating process. The percent weight increase shown on a log scale to aid comparison between technologies. **Figure 2.** Percent weight increase after dry coating, seed dressing, film coat, entrustment and seed pellet technologies. The grey shaded bar for seed dressing and film coat is addition of a finishing powder during the coating process. The percent weight increase shown on a log scale to aid comparison between technologies.

#### *3.2. Seed Dressing*

Seed dressing is the most widely used method for low dosages of active components onto seeds [33]. Although there are many types of equipment used for coating [9], the most commonly used device is the rotary coater (Figure 1). Liquids are applied onto a spinning disc and atomized onto seeds that are spinning inside a metal cylinder, then the freshly treated seeds are discharged. A wide range of active materials especially chemical plant protectants can be applied with this method.

The dosage of liquid seed treatment formulations typically ranges from <0.05 to 1.0% by weight (Figure 2). For higher loading rates of chemical seed treatment, in particular insecticides, finishing powders or fluency powders are added immediately after the liquid application to absorb excess liquid [45]. The dry finishing powders can be added into the rotary coater during operation or applied immediately downstream with the dry seed coating equipment (Figure 1).

#### *3.3. Film Coating*

Film coating originally developed for the pharmaceutical and confectionary industries was adapted as a seed coating method [6]. Film coating consists of producing a continuous thin layer over the seed surface. The rotary coater is the primary seed coating equipment used for film coating (Figure 1). Film coating polymers (liquid components) are formulated to dissolve/dispense active ingredient prior to application on seeds. Film coating resulted in 90% application recovery [7], with little modification of shape and size during this process [7,8]. Film coating has gained in use and is the most adaptable among all seed applied technologies. The performance of film-coated seed is evaluated on the basis of germination and dust control. Film coating improves flow-ability of seed during treating/processing and sowing operations. This value-added treatment is preferred over conventional methods due to excellent delivery of protectants on value seeds and have a cosmetic appearance [6].

The weight increases for film-coated seed, ranges from 2 to 5% of seed weight (Figure 2) [16]. Seed weight build-up greater than 5% requires other seed coating equipment with drying capability during coating, primarily a ventilated pan and fluidized bed seed coating facilitate concurrent treating and drying [10]. However, both side-ventilated or perforated pan and fluidized bed are used much less in commercial practice than the rotary pan technology. As described for seed treatment (Section 3.2), dry finishing powders can be added into the rotary coater, or with the dry seed coating equipment to increase loading from 5–8% (Figure 2).

The choice of film forming polymers is important for success in field sowing [5,13] and in the protection of the environment. Corn seeds coated with a proprietary film-forming polymer, PolySeed CF (Rigrantec, Porto Alegre, RS, Brazil), improved precision seed placement compared to graphite treated or non-coated seeds with significant reduction in dust formation and leaching of applied insecticides [47]. Further, the film coating polymer had good seed treatment adhesion resulting in less dust-off into the environment [47].

#### *3.4. Encrusting*

Encrusting is a seed coating method with the addition of liquids and solid particulates that results in a coated seed that is completely covered, but the original seed shape is retained [16]. Encrusted seeds can be referred to as mini-pellets [6] or sometimes as coated seeds. The primary coating methods to produce encrusted seed are the rotary coater or coating pan (Figure 1). The addition of large amounts of water during encrusting requires that the freshly coated seed be dried to back to its original seed moisture content prior to packaging and storing. The weight increase after encrusting can range from 8 to 500% (Figure 2).

Encrusted seeds have been shown to improve seedling emergence. Significantly higher germination of fescue seeds was measured when seeds were encrusted before storage compared to encrusting after storage or non-treated seeds [48]. The seed coating thickness or percent build-up may impact germination rate, and encrusted seed requires more time to germinate as compared to film-coated seed [49]. The amount of binder used in producing encrusted coatings changes mechanical properties including integrity, compressive strength and time to disintegrate after soaking [50].

#### *3.5. Pelleting and Agglomeration*

Seed pelleting is a continuation of the encrusting coating process resulting in even greater build-up so that the original size or shape of the coated crop seed is not visible [8,16]. The materials and techniques used for this purpose are proprietary [8], but common mineral materials cited in the literature and in patents are presented (Table 1). The binders may be liquid or formulated as dry powders (Table 1). Dry powder binders are mixed with filler materials to produce a coating blend [51], only requiring water applied during the coating process as the liquid. The percent weight increase after pelleting and drying ranges from 500 to >5000 percent (Figure 1). It is common that the percent weight increase is expressed as a ratio of seed weight to dried pellet weight, so a 500% weight increase is a 1:5 build-up of seed to coating.

The selection of liquids paired with fillers (Table 1) is essential to ensure that the pelleted seed will germinate unimpeded by the pellet matrix [16]. The pelleting seed industry has conducted tremendous research and development on optimizing commercial pelleting products for growers. The demand of pelleted seed continues to grow among growers so seeds can be planted with precision. Precise seed spacing achieved with pelleted seed reduces the need for thinning operations. Pelleted seeds are commonly used for growing transplants. Pelleting is frequently performed on high-value, small-seeded horticultural crops (e.g., onion, lettuce, carrot, tobacco, and tomato [6,32,34,36].

Material properties for successful pelleting include particle size distribution, porosity, water absorbing and holding capacity and lack of toxicity [32]. For tobacco seed pelleting, a combination of bentonite and talc [38] or pumice [52] was highly recommended. Similarly, diatomaceous earth and a combination of gypsum and calcium carbonate were found to be effective in broccoli [53] and lettuce [32], respectively. Calcium peroxide was added as a seed coating component [12] after sowing in a water-saturated soil with limited oxygen availability, the calcium peroxide releases oxygen gas to the germinating seed. Calcium peroxide applied in a seed pellet improved emergence and crop establishment of rice under submerged conditions [54].

Pelleting requires the most time and expertise compared to other coating technologies due to extensive application of active components, liquids, and solid particulates (Table 2). the pellet should not cause any restriction to germination when sown in the field. pellet integrity is dependent on the selection of material (fillers and binders) and appropriate technology [7].


**Table 2.** Comparison of amount of coating components and time needed for the dry coating, seed dressing, film coat, entrustment and seed pellet technologies. The (+) for seed dressing and film coating is the addition solid particulates as finishing powders. Relative comparisons are noted with number '+'.

The objective of all the described coating methods thus far is for each seed to be singulated during the coating process to avoid doubles or agglomerates (two or more seeds in one coated propagule). However, it may be needed in certain cases to have more than one seed in a pellet. Seed agglomeration is an alternative coating technology in which multiple seeds are pooled into a single delivery unit [36]. The purpose of this technology is to sow multiple seeds of the same seed lot, different varieties of the same crop or multiple seed species. Seed agglomerates may be produced with a pan coater or rotary coater (Figure 2). Other agglomeration technologies use extrusion equipment [14]

and molding technology [48]. Moreover, producing "seed balls" is a pelleting technique that utilizes materials, seeds and supporting additives in small amounts such as mineral fertilizer [55]. Both seed agglomeration technologies are used for improving handling and sowing of small-seeded species for arid land restoration [56,57].

#### *3.6. Comparison of Seed Treatment and Coating Technologies*

Five seed treatment and coating technologies were discussed in Sections 3.1–3.5, and now each technology can be compared to provide relative differences. The range of weight increase after treatment/coating is shown for the five methods and is expressed on a log scale to better visualize percent weight increase or build-up (Figure 2). The coating technologies cover from <0.05% to >5000% weight increase (>100,000-fold range) that accommodates all crop seed specific treatment and coating needs and applications. Additional comparisons of the five coating methods are illustrated with respect to weight increase after coating, and the relative amounts of active components, liquids and solid particulates applied, and the time required to treat or coat a batch of seeds (Table 2). All coating technologies can apply active components, but the potential amount per unit seed is limited by coating technology. No water or liquids are applied with the dry powder method, while with the other coating methods the amount of water/liquids increase is proportional to the percent weight increase (Figure 2). Solid particulates may be added with seed dressing and film coating as the amount of water increases resulting in "stickiness" during seed treating and inadvertent agglomeration. The solid particulates are termed drying powders [20], finishing powders or fluency agents that help absorb excess moisture applied during coating. As stated previously, these drying powders can also serve as seed lubricants to reduce friction as seed flows through the seed treater or planter [20]. There is a clear distinction in choice of seed coating technology with respect to the amount of water applied. Seed dressing and film coating as described do not require further drying after treatment, while encrusting and pelleting require post-coating drying to remove excess water and to dry seeds to their original seed moisture content. Finally, each seed dressing/coating method requires time, and longer processing times are needed as the amount of coating materials increases.

Many factors affect the final coated seed properties including the rotator and atomizing disc rpm, the solid particulate particle size, porosity, water holding capacity, and the binder adhesion properties [49]. The success of coating process and uniform distribution of active components requires time for mixing in the coating equipment [35] and for accurate adherence of binder and powder to seeds [34].

There are two types of seed treatment/coating equipment systems: batch treater and continuous flow treaters [20]. A batch treater matches a known amount of seed with seed treatment and coating material at one time, while the continuous flow treats a known amount of seed with seed treatment and coating material at a given flow rate [35]. Dry powder applicator or rotary coater may be either a batch or continuous flow based in equipment design, while most drum coater technology used for small-seeded vegetable crop seeds is performed on a batch basis. Each seed coating method (Figure 1) requires precise metering to deliver the target dosage onto seeds. Seed treatment equipment is needed to proportion an accurate amount of material to the seed. Computer technology is often used to monitor seed flow and seed treatment application, known as proportion control [35]. There are two stages of seed treatment application to achieve uniformity of application from seed to seed: primary and secondary application [35]. Primary application is the direct application of liquids onto seeds, for example the atomizer (atomizing disk) in the rotary coater disperses liquids directly onto seeds (Figure 1). Secondary application is the seed-to-seed transfer of the applied material during mixing while in the seed coating equipment [35]. Dosage can be expressed on a weight basis, for example g/100 kg seed or quantity per seed, for example mg ai/seed (ai—active ingredient) [35].

#### **4. E**ffi**cacy of Seed Treatments and Coatings**

#### *4.1. Biostimulants*

There has been considerable effort over many decades on applying chemicals to seeds to improve germination and seedling growth. The term "biostimulants" was adopted in the 21st century and provides a better definition and grouping of materials that serve to enhance plant performance. Biostimulants may be defined as natural compounds that trigger physiological and molecular processes modulating crop yield and quality. There are several categories of plant biostimulants and these materials are natural products or biologicals. A review of biostimulants applied as seed coatings is summarized by category (Table 3): beneficial bacteria and fungi [58–60], plant and animal-derived proteins, protein hydrolysates and amino acids [50,51,53,61], carbohydrate derivatives [62,63], seaweed [64] and herbal extracts [65]. There are no seed applied references for other biostimulant categories including vitamins, humic and fulvic acids. All these compounds may enhance plant metabolism when applied in small quantity, but their mode of action is only partially understood [66,67].

**Table 3.** Review of biostimulants applied as seed treatments on seed germination, seedling growth and other measured parameters.



**Table 3.** *Cont.*

\* Source of material: Natural products or derivatives—NP, Biologicals—BIO, substances may be Organically approved—*OR*.

The application of biostimulant components has not been widely integrated as seed treatments in agriculture. Biostimulants applied as seed treatments and coatings are more cost effective and provide great potential to enhance stand establishment compared to foliar and soil application methods [51,59]. The global market for biostimulants applied as seed treatments in 2015 and 2019 was USD 112 million and USD 181 million, respectively and is forecasted to reach USD 338 million by 2025 [68].

The studies summarized in Table 3, reports on the beneficial effects of biostimulants applied as seed treatments and coatings on germination enhancement and growth stimulation on several crop species. For example, Amirkhani et al. [51,53] reported that seed coating with plant-derived protein enhanced germination indices and seedling uniformity, as well as the vigor index of broccoli, compared to non-coated seeds under optimum conditions. Moreover, the co-application of plant-derived protein and a nutrient-rich micronized vermicompost as a dry seed-coating binder and biostimulant significantly enhanced plant biometric parameters in germination and greenhouse studies [51,53]. In another study, Qiu et al. [50] reported enhancement in the percent germination and germination rate in red clover, and root enhancement in ryegrass, in response to biostimulant seed coating. The above studies suggest that biostimulant seed treatment practices enhanced uptake of soil-media nitrogen. The application of nitrogen in the seed coating accounted for less than 5% of the total nitrogen taken up by the roots. Therefore, the biostimulant was not merely a nitrogen fertilizer, but acted as a biostimulant to enhance nutrient uptake [51,53].

#### *4.2. Nutrient Coating*

Adequate nutrient availability is very important starting at the early stages of plant growth. Seed coating with appropriate amounts of macro- and preferentially micro-nutrients can reduce nutrient losses by placement on the seed, and also reduce competition from weeds. However, germination and seedling growth can also be hindered by macronutrient coatings due to phytotoxicity. To prevent

such toxicity, direct contact of nutrients should be avoided with seeds by including the initial layer or boundary layer followed by the nutrient coating.

Several investigations conducted on plant nutrients applied as seed coatings were summarized by Scott (1989), Farooq (2012) and Masuthi (2009) [12,69,70]. Successful coating of phosphorus on oats improved early plant growth [71]. In rice seeds, boron (2 g/kg seed) was applied as seed coating and significantly increased grain yield and boron contents over a control [72]. Losses of nutrients by seed coating reduced the cost of production as compared to soil applications [69]. Conventional broadcasting of fertilizers exhibited higher cost and losses, while coating with an equivalent rate of nutrients significantly produced higher yield of cereal crops [69,73]. Slow release nutrient (N-P-K) coating on maize seeds resulted in improved emergence and yield attributes as compared to conventional compound fertilizer application in the field [74].

The effects of applied nutrients to a wide range of field and vegetable crop seeds with pre-defined quantity of nutrients are summarized, and plant improvements in germination, emergence, plant growth and yield were cited (Table 4). All fertilizers were synthetic chemicals, but several are available as organically approved. Zinc oxide [75,76] and zinc sulphate [70,77–80] are the most promising micronutrients used in seed coating of cereal crops and pulses. Wiatrak [81,82] evaluated the effect of polymer coating with manganese, copper and zinc on wheat and soybean crops and found a cost-effective technique for the enhancement of plant growth and ultimate yield of both crops [81,82]. In another study, coating with a range of micronutrients (Zn, B, K, Mo, Fe, Mg, Mn) increased productivity of cotton, chickpea, groundnut and pigeon pea with minimum expenditure and higher returns [83].


**Table 4.** Review of plant nutrients applied as seed treatments on seed germination, seedling growth, yield and other measured parameters.


#### **Table 4.** *Cont.*

\* Source of material: Synthetic Chemicals—SYN, Natural products or derivatives—NP, Biologicals—BIO, Mineral—MIN, substances may or may not be Organically approved—*OR*.

#### *4.3. Abiotic Stress*

Abiotic stresses may occur in the field and have a deleterious effect on germination and stand establishment. Abiotic stresses may be caused by drought stress or salinity stress. Both chemical and biological seed treatments and coatings have the potential to ameliorate deleterious effects of transient abiotic stress [4,85]. Superabsorbent polymers (SAPs) are hydrophilic polymers that can absorb over one hundred times their weight in water and have a long history of use in agriculture [86]. Seed coating technologies were developed to incorporate SAPs with filler materials to produce encrusted or pelleted seeds [87,88]. Hydro-absorbers and SAP improved germination potential by early and rapid completion of imbibition and active metabolism phases by improving water availability around the sown seed [49]. SAP supplies sufficient moisture and ensures oxygen availability to germinating seed under normal and stressful conditions [89]. SAP seed coatings were shown to increase germination and stand establishment at substantially lower application rates than soil-applied SAPs [90–92].

Salinity stress reduces soil water availability and results in an excess of sodium ions in the soil. Biological seed treatments may partially ameliorate the deleterious influence of salinity on plant growth. A commercial seed treatment formulation of *Trichoderma harziannum* was applied onto squash (*Cucurbita pepo*) seeds and studied in pot experiments in the greenhouse [93]. Pots were irrigated with 50 and 100 mM NaCl solutions and plant weight and leaf mineral content analyzed. The biological seed treatment increased plant growth at both salinity levels compared to the non-treated control. Moreover, the biological increased the leaf potassium to sodium ratio suggesting that one mechanism of a beneficial biological was altered mineral uptake. In another study, seed treatment with *T. harzianum* alleviated biotic, abiotic, and physiological stresses in germinating seeds and seedlings [94]. A recent

review on beneficial microbes applied as seed coatings stated several plant beneficial microbes (PBMs) enhanced drought or salinity tolerance [22].

#### *4.4. Plant Protectants and Inoculants*

Management of biotic stresses in agriculture is synonymous with plant protectants applied as seed treatments and coatings. These seed treatments may be fungicides, insecticides, bactericides, and nematicides [20]. In agriculture, control of these pests should be considered if damage exceeds an economic threshold [35]. Plant protectants are applied in anticipation that economic damage will occur from soil-borne or air-borne pathogens and/or pests. Therefore, seed treatments provide insurance from potential biotic stresses either singularly or in combination, as in the case with soil-borne pathogens and insect pests. A wide range of active components may serve as plant protectants including: synthetic chemicals, natural products, and biologicals (Table 1). Some of these plant protectants may be organically approved for use in crop protection. Based on the seed-treatment active component and its formulation, dosage and other attributes, these actives may be applied with specific paring of equipment. Methods include: dry powder applicator, rotary coater or drum coater to apply dry coating, seed dressing, film coat, and encrustment or pellet (Figure 1).

The literature on seed treatments as plant protectants is beyond the scope of this review. However, selected papers are highlighted on seed treatments and coatings as seed enhancements. Herbicide safeners are seed treatments that negate the potential herbicidal effect of selective herbicide chemistries on crop plants. Thus, herbicide safeners are tools for specialty crops and other plant species that lack chemical weed control options. The herbicide safener, fluxofenim was effective on field soil treated with the herbicide, metolachlor on switchgrass (*Panicum virgatum*) [95]. Biologicals also known as plant beneficial microbes (PBMs) [22] may provide inconsistent pest management under a wide range of field conditions encountered at time of sowing. Synthetic chemical seed treatments provide more reliable pest control for conventional agriculture but are prohibited for organic crop production. Biopesticides that are derived from natural products or microbes and are organically approved have potential for pest management comparable to synthetic chemical seed treatments. Spinosad, a biopesticide for foliar application, was investigated as an onion seed treatment at Cornell University [96]. Spinosad seed treatment was comparable in efficacy to chemical seed treatments in the control of onion maggot (*Delia antiqua*). An organic formulation of spinosad was also effective for control of onion maggot and seed-corn maggot (*Delia platura*) when used in combination with other seed treatments [97]. Collectively, seed-coating technology as described in this paper provides a delivery platform for many other active components for improved pest management that are environmentally friendly for sustained systems. In addition, new generation biochemical, bio-pesticides reduces the reliance on synthetic agrochemical seed treatments [97,98]. Greater efficacy of fungicides has been achieved with good treatment adhesion resulting in less dusting [98].

The use of plant extracts as seed treatments can improve seed quality and reduce infestation of microbial pathogens [99]. Such plant extracts have antibiotic and antimicrobial properties that help in alleviation of biotic and abiotic stresses during seed emergence in the soil [100]. Natural occurring plant extracts are readily available, less expensive, and have promising effects on germination, plant growth, and yield as compared to traditional chemical fungicide treatments [99,101].

Seed pelleting was effective in sowing sesame seed. Pelleting significantly enhanced plant height, lateral branches and number of capsules per plant as compared to non-pelleted seeds [102]. Damping-off disease incidence was significantly reduced by pelleting of sesame seeds with the plant growth promoting microbe (strain E681) [29]. Pelleting does not normally affect shelf-life. An investigation on the storage of pelleted seeds revealed that quality of tobacco seed after pelleting was maintained up to 720 days when properly stored in aluminum cans [103].

Microbial seed coating is a method of coating seeds with plant beneficial microorganisms such as plant growth promoting bacteria (PGPB), rhizobia, and fungi to increase crop growth and yield through improvement in nutrition and protection against diseases and pathogens [22,23]. Coating seeds with beneficial microbes is an efficient delivery system for application of beneficial microbes and is a promising tool for inoculation of different crop seeds with a reduced use of inoculum as compared to traditional seed treatments [7,12,37]. A typical inoculant formulation is based on the selection of the microorganism, a suitable carrier, and related additives [22,104]. Combination of carbon source materials with rhizobia not only aids in the survival of bacterial strains as a food source but also provides protection from the external environment [105]. In addition to seed coating as a carrier for food bases, pH can be adjusted for optimum growth of beneficial microbes [7]. Lime pelleting was shown to be helpful for rhizobia survival by neutralizing fertilizer acidity close to the seed [4]. Application of compatible rhizosphere microbes to chickpea seeds was effective to alleviate biotic stress through enhanced stand establishment, growth, and molecular attributes. Peat and biochar were effective for providing protection to the rhizobia by tightly absorbing it and preventing direct exposure to the external environment [106]. After seed coating with bacterial strains, rapid desiccation should be avoided, by selection of appropriate filler materials. Microbial survival on coated seeds may be attenuated, and generally old chemistry seed treatment fungicides including captain, thiram and carboxin are not recommended with Rhizobium inoculants [107]. Therefore, compatibility of new seed treatments should be tested to ensure efficacy of the biological.

#### *4.5. Other Coatings*

Different marker substances including visible dyes, fluorescent tracers and magnetic powders were incorporated into coatings to trace the seed in the supply chain and protect the true seeds from fake seeds in the market [8,52]. Color-coding is the most widely used marker system in coating processes for identification of a specific variety or seed treatment [23]. Colored seed is an indication of a seed coat treatment with appropriate fungicide or pesticide and is used to reduce the risk of livestock or human consumption [8]. Natural colorants can be used for storage of soybean seeds without loss of vigor [108]. Additionally, researchers have also evaluated the efficacy of fluorescein, rhodamine, and magnetic powder as anti-counterfeiting labels in tobacco seeds in order to enhance seed security in the supply chain [52]. Riboflavin is a natural fluorescent compound and was used for marking cucumber seeds for authentication [109]. Riboflavin was not phytotoxic after application nor after seed storage compared to non-treated seeds, and riboflavin fluorescence was not diminished after 10 months' storage [109].

#### **5. Conclusions and Future Prospects**

Seed coating technologies have many virtues including protecting seeds from pests and diseases at the time of sowing and improving flowability for precision seeding [15]. Improved stand establishment and seedling vigor under biotic and abiotic stresses can be achieved by using appropriate seed coating equipment, methods, and materials. The growing demand for coated seeds is documented with many small and large companies in the market. Despite the extensive information on natural or synthetic active components, coating methods and polymers, the seed industry in many developing countries is not adopting this technology. Farmers in these countries are not utilizing seed treatments due to lack of resources as compared to 100% adoption in developed countries [4]. Usually, economical treatments are preferred if cost is not exceeding USD 20 per planted hectare [7]. Therefore, the success of seed coating technology depends upon the selection of inexpensive and readily available coating agents with low cost. Collectively, cost effective, simple materials and methods are needed for use in third world countries.

There is limited information available on the shelf life of treated and coated seeds. Specifically, it would be helpful to know if seed treatment phytotoxicity increases with the loss of seed vigor in storage [110,111]. Is there a reduction in seed treatment efficacy after seed storage, particularly for seeds treated with biologicals [7]? Investigations are needed on how to better integrate seed coating technologies with weed management exploiting herbicide safeners [95], or herbicide seed treatments [112]. Additional research and development are needed for new biochemical, bio-pesticide

plant protectants [96,97] that can be used for organic or conventional crop production for sustainable agricultural systems. Lastly, the knowledge of seed treatment and coating technologies should be directed for reliable and consistent stand establishment under changing climatic conditions. To accomplish these goals will require the development of new active components, with complimentary coating equipment, and coating technologies. This can best be achieved by continued efforts from multidisciplinary teams of seed scientists, agronomists, chemists, pest management specialists and engineers. These achievements may be accomplished through a partnership of academia with industry for the development of cost-effective materials and methods for wide-scale adoption in developed and third-world countries.

**Author Contributions:** I.A. Conceptualization, writing and original draft preparation, T.J. Writing and original draft preparation. M.A. Writing, preparation of both figures and three tables, A.G.T. Conceptualization and editing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This material is based upon work that is supported by the United States Hatch Funds under Multi-state Project W-4168 under accession number 1007938 to the fourth author, and Pakistan Science Foundation under a research project PSF/NSLP-489 to the first author.

**Acknowledgments:** The authors thank Hilary Mayton for critically reviewing this manuscript, and helpful suggestions were provided by Simone Pedrini for developing the illustration in Figure 1. We thank valuable input from seed treatment and coating industries, and seed companies: ABM, Aginnovations, BASF, Beck's Hybrids, CTApplicators, Germains, Incotec and Syngenta.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


112. Kanampiu, F.K.; Kabambe, V.; Massawe, C.; Jasi, L.; Friesen, D.; Ransom, J.K.; Gressel, J. Multi-site, multi-season field tests demonstrate that herbicide seed-coating herbicide-resistance maize controls Striga spp. and increases yields in several African countries. *Crop Prot.* **2003**, *22*, 697–706. [CrossRef]

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Evaluating Genotypes and Seed Treatments to Increase Field Emergence of Low Phytic Acid Soybeans**

**Benjamin J. Averitt <sup>1</sup> , Gregory E. Welbaum <sup>2</sup> , Xiaoying Li <sup>2</sup> , Elizabeth Prenger <sup>3</sup> , Jun Qin <sup>4</sup> and Bo Zhang 2,\***

<sup>1</sup> Department of Crop and Soil Sciences, University of Georgia, Athens, GA 30602, USA; ben.averitt@uga.edu

<sup>2</sup> School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA 24060, USA; welbaum@vt.edu (G.E.W.); xiaoying@vt.edu (X.L.)


Received: 10 September 2020; Accepted: 29 October 2020; Published: 30 October 2020

**Abstract:** Low phytic acid (LPA) soybean [*Glycine max* (L.) Merr] genotypes reduce indigestible PA in soybean seeds in order to improve feeding efficiency of mono- and agastric animals, but often exhibit low field emergence, resulting in reduced yield. In this study, four LPA soybean varieties with two different genetic backgrounds were studied to assess their emergence and yield characters under 12 seed treatment combinations including two broad-spectrum, preplant fungicides (i.e., ApronMaxx (mefenoxam: (R,S)-2-[(2,6-dimethylphenyl)-methoxyacetylamino]-propionic acid methyl ester; fludioxonil: 4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile) and Rancona Summit (ipconazole: 2-[(4-chlorophenyl)methyl]-5-(1-methylethyl)-1-(1H-1,2,4-triazol-1-ylmethyl) cyclopentanol; metalaxyl: N-(methooxyacetyl)-N-(2,6-xylyl)-DL-alaninate)), osmotic priming, and MicroCel-E coating. Two normal-PA (NPA) varieties served as controls. Both irrigated and non-irrigated plots were planted in Blacksburg and Orange, Virginia, USA in 2014 and 2015. Results revealed that three seed treatments (fungicides Rancona Summit and ApronMaxx, as well as Priming + Rancona) significantly improved field emergence by 6.4–11.6% across all genotypes, compared with untreated seeds. Seed priming was negatively associated with emergence across LPA genotypes. Seed treatments did not increase the yield of any genotype. LPA genotypes containing *mips* or *lpa1*/*lpa2* mutations, produced satisfactory emergence similar to NPA under certain soil and environmental conditions due to the interaction of genotype and environment. Effective seed treatments applied to LPA soybeans along with the successful development of LPA germplasm by soybean breeding programs, will increase use of LPA varieties by commercial soybean growers, ultimately improving animal nutrition while easing environmental impact.

**Keywords:** field emergence; low phytic acid; seed treatment; soybean

#### **1. Introduction**

Grain soybean [*Glycine max* (L.) Merr] is one of the most important crops for animal feed in the United States due to its high protein content and wide adaptability. Seventy-five percent of phosphorus (P) in soybean seeds is in the form of phytic acid (PA), myo-inositol-1,2,3,4,5,6-hexakisphosphate, which is indigestible for agastric and monogastric animals such as swine, poultry, and most aquatic animals, leading to low feeding efficiency [1]. In addition, other essential minerals, such as calcium, iron, manganese, and zinc, are bound by phytic acid, forming insoluble phytate salts, that render them unavailable, resulting in nutrient deficiencies in monogastric animals [2]. Furthermore, these nondigestible phytate salts are excreted by animals and become an important source of P pollution detrimental to the environment causing massive algal blooms and fish death [3,4].

Although animal producers have long added synthetic phytase to animal feed to improve PA digestibility, a much more effective method would be the utilization of low-PA (LPA) seeds developed from mutant lines. Three mutant alleles have been reported to create soybean LPA varieties [5]. The first two, *lpa1* and *lpa2*, were both discovered in mutant line CX-1834. These alleles lower phytate by producing a truncated ABC transporter responsible for partitioning PA into seeds [6]. The third mutant allele, *mips1*, is responsible for the first step in PA biosynthesis, catalyzing the NADH-dependent conversion of glucose-6-phosphate to myo-inositol-3-phosphate [7]. However, these mutations not only reduce the seed phytic acid levels in soybean, but also affect the pathways associated with seed development, leading to reduced seed germinability and ultimately low emergence [8,9]. Recent studies showed that many transcriptional genes in biological processes, such as those related to phytic acid metabolism and seed dormancy were involved in this process and the expression diversification of antioxidation-related and hormone-related genes were reported to strongly contribute to variations of emergence rate of LPA soybean lines [8–10]. So far, the mechanism of seed emergence in LPA soybean lines remains unclear and requires further exploration.

Poor field emergence has greatly hindered the use of LPA germplasm in soybean breeding programs [10]. Many attempts have been made to improve emergence in the past few decades. Previous studies showed that soybean seeds produced in temperate environments exhibited higher field emergence than those from tropical/subtropical environments, which illustrates the importance of seed production environment on LPA cultivars for commercial production [11–13]. Maupin and Rainey (2011) reported some seeds derived from the LPA genotype (*mips1*) had field emergence (above 85%) similar to normal-PA (NPA) soybean lines, indicating the potential to develop high emerging LPA soybean lines from natural variations within some LPA mutants [12]. Recently, several new LPA soybean lines (such as 56CX-1273), which display rapid emergence and good agronomic performance, have been developed using traditional crossbreeding methods as well as transgenic technologies [2,14].

Seed treatments improve field emergence of a broad range of crops including soybean. Fungicide treatment is one of the most commonly used to increase soybean stand establishment because it protects seed/seedling from seed- and soil-borne diseases, such as seed rot and damping-off caused by *Phytophthora* spp. [15,16]. Seed priming increases soybean seed vigor, and consequently improves seedling emergence under normal or stressful conditions [17]. Priming involves a controlled hydration procedure followed by redrying applied preplant that allows initial metabolic processes required for seed germination to occur prior to planting resulting in faster germination and uniform field establishment [18]. Additionally, mineral nutrients have been applied preplant as seed coating treatments to improve seedling growth [19,20]. Micro-Cel E, a synthetic calcium silicate, produced by the hydrothermal reaction of diatomaceous silica and high purity lime, can supply plant-essential nutrients and has pesticidal properties. Micro-Cel can be applied as a seed coating and may improve soybean stand establishment.

However, no seed treatment consistently increases the field emergence of LPA soybean lines. The purpose of this study was to apply twelve combinations of four seed treatments: two fungicide treatments (ApronMaxx and Rancona Summit) reported to greatly improve soybean emergence previously [21,22], osmotic priming with potassium phosphate solution and seed coating using Micro-Cel E, to increase field emergence of LPA soybeans. The objective was to: (1) evaluate the seed and seedling vigor of four newly developed LPA soybean varieties (56CX-1283, MD 03-5453, V12-4557, and V12-BB144) with two different genetic backgrounds (i.e., 56CX-1283 and MD 03-5453 having both the *lpa1* and *lpa2* alleles, while V12-4557 and V12-BB144 have the *mips1* allele), and (2) establish a preplant seed enhancement treatment that can effectively improve field emergence and establishment of LPA soybeans.

#### **2. Materials and Methods**

#### *2.1. Plant Materials*

Six maturity group V soybean varieties were studied: four LPA and two NPA (Table 1). The four LPA genotypes were 56CX-1283, MD 03-5453, V12-4557 and V12-BB144. 56CX-1283 and MD 03-5453 were developed by the USDA-ARS-Purdue University and University of Maryland, respectively, and contain both the *lpa1* and *lpa2* alleles. V12-4557 and V12-BB144 were developed at Virginia Tech and have the *mips1* allele. Seeds of all six varieties were grown in the same field at the Virginia Tech Kentland Research Farm near Blacksburg, VA using identical agronomic practices the previous year. Seeds of all six genotypes were dried to 9% moisture (dwt basis) after harvest and stored in sealed paper bags maintained in dark in a room maintained at 21 ◦C until planted the following growing season. The LPA varieties' PA content ranged from 2132 to 4421 ppm. AG 5632 (Bayer, Pittsburgh, PA) and 5002T [23] are both NPA commercial varieties. Their PA content ranged from 5887 to 6116 ppm. MD 03-5453 and V12-4557 have a history of poor field emergence and were not tested in 2014 but were added into the study in 2015.

**Table 1.** The phytic acid (PA) content, genetic source of the low-PA (LPA) trait, and the years planted for each soybean genotype in this study.


#### *2.2. Field Plot Design and Trait Measurement*

The experimental design was a triplicated split plot generalized randomized complete block design (GRCBD) wherein the main plots were blocked by the two locations (VT Kentland Farm, Blacksburg and VT Northern Piedmont Research Station, Orange, VA) and split into irrigated and non-irrigated subplots. The Blacksburg location has Hayter loam fine-loamy, mixed, active, mesic Ultic Hapludalfs soil type. The Northern Piedmont site near Orange, VA has Davidson clay loam fine, kaolinitic, thermic Rhodic Kandiudults soil type [24]. Plots were irrigated shortly after planting and kept wet until emergence to simulate damp spring planting conditions typically encountered. All plots were planted using a small-plot mechanical seeder in the last week of May. Orange usually a little warmer than Blacksburg (about 4 ◦C higher in average in 2014 and 2015) and gets more precipitation (32 more mm in average in 2014 and 2015) than Blacksburg in late May. Each plot was planted in two 3.05 m-long rows spaced 0.82 m apart with 80 seeds per row at a density of 26 seeds per meter. Stand counts were taken at the V1 stage (one set of unfolded trifoliolate leaves) [25]. The plots were once-over destructively harvested in late October (Orange) and early November (Blacksburg). Grain weight and moisture content were recorded for each plot and converted to yield (kg ha−<sup>1</sup> ) at 13% moisture on dry weight basis. Phytic acid was measured by high-throughput indirect Fe colorimetry [26].

#### *2.3. Seed Treatments*

Twelve seed treatment combinations (Table 2) were tested in 2014: MicroCel-E (synthetic calcium silicate, CaSiO3; Manville, Denver, CO); osmotic priming; two fungicides, ApronMaxx (mefenoxam: (R,S)-2-[(2,6-dimethylphenyl)-methoxyacetylamino]-propionic acid methyl ester; fludioxonil: 4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile; Syngenta Crop Protection, Greensboro, NC) and Rancona Summit (ipconazole: 2-[(4-chlorophenyl)methyl]-5-(1-methylethyl)-1-(1H-1,2,4-triazol-1-ylmethyl) cyclopentanol; metalaxyl: N-(methooxyacetyl)-N-(2,6-xylyl)-DL-alaninate; Valent USA, Walnut Creek, CA, USA); all possible two and three way combinations; and an untreated control. The specific treatments were selected based on prior unpublished results of germination tests. MicroCel-E was ineffective in 2014, so it was excluded in the 2015 trials (Table 2).


**Table 2.** Seed treatments used in this study.

MicroCel-E was applied to seeds in seed coating. Polyvinyl acetate based-adhesive, (C4H6O2)<sup>n</sup> (Elmer's Glue-All, Elmer's Products, Westerville, OH, USA) was diluted 10 times with tap water and misted on seeds (2.5 mL/1000 seeds) in a rotating bowl. Powedered MicroCel-E was slowly added by hand to coat seeds with a thin layer (2.5 mg MicroCel-E/1000 seeds). Seeds were immediately dried at 32 ◦C in a forced-air dryer for 24 h.

Before seeds were incubated in osmoticum for priming, they were surface sanitized with 30% bleach (8.25%, sodium hypoclorite, NaOCl) solution for 4 min then rinsed in double distilled water (ddH2O). A single layer of seeds was osmotically primed in 3% monopotassium phosphate (KH2PO4) solution in ddH2O on two thicknesses of germination blotter paper (Anchor Paper Co., St. Paul, MN, USA, 9.5 × 9.5 cm) saturated with 20 mL of solution. Seeds were sealed in square clear plastic boxes (10.1 × 10.1 × 3.5 cm OD). Seeds were placed in an incubator at 16 ◦C for 72 h in dark and force-air dried to their original moisture content.

Seeds were briefly soaked with two aqueous broad-spectrum fungicides, ApronMaxx and Rancona Summit according to label instructions. Twelve mL ApronMaxx was mixed with 10 mL red dye and 78 mL water, and 26 mL Rancona was mixed with 10 mL dye and 64 mL water, respectively. Seeds were treated by applying 2.25 mL of fungicide solution per 1000 seeds in a rotating drum. The seeds were force-air dried to their original moisture content after treated. For treatments with both fungicides and MicroCel-E, solutions were modified to contain either 8 mL Rancona, 7.5 mL dye, and 34.5 mL water; or 6 mL ApronMaxx, 7.5 mL dye, and 36.5 mL water. Three mL of fungicide solutions were applied per 1000 seeds. Once treated, the seeds were dried in a 32 ◦C dryer for 24 h. All untreated controls were also dried as previously described, so moisture contents of all treatments ranged from 7.5 to 9.5% (dwt basis, 17 h 103 ◦C).

#### *2.4. Statistical Analysis*

Correlation analysis of linear lines was calculated using JMP 11 software (SAS Inc, Raleigh, NC, USA) and R software package "corrplot" [27]. A split-split plot analysis of variance was performed using R software with packages "lattice" [28], "car" [29], and "agricolae" [30]. Analysis of variance effects included treatment, genotype, location, irrigation, and replication. The AOV function was used

to perform an ANOVA using the following formula for each year, 2014 and 2015, separately. Means of significant *F*-test were separated by Tukey's Honestly Significant Difference (HSD<sup>α</sup> <sup>=</sup> 0.05).

#### **3. Results**

#### *3.1. E*ff*ects of Genetic and Environmental Factors on Field Emergence*

In 2014, an ANOVA revealed significant variation among treatments, varieties, and irrigation regimes. Significant interactions included treatment × genotype, treatment × location, line × location, treatment × irrigation, line × irrigation, treatment × line × location, treatment × location × irrigation, and line × location × irrigation (*p* < 0.05). In 2015, an ANOVA revealed significant variation among treatments and genotype. Significant interactions occurred for treatment × line, treatment × location, line × location, line × irrigation, and treatment × line × irrigation (*p* < 0.05). Field emergence data were averaged separately for each irrigation regime, location in 2014 and 2015 for all soybean genotypes (Table 3). The average field emergence of NPA AG 5632 was 82.6% in 2014, higher than LPA varieties 56CX-1283 (72.1%) and V12-BB144 (70.3%), both of which had significantly higher emergence than NPA genotype 5002T (68.4%). In 2015, 79.5% of 56CX-1283 emerged, followed by NPA genotypes AG-5632 and 5002T without significant differences. For LPA genotype V12-4557, 71.9% of seeds emerged not significantly different from 5002T. Both V12-BB144 (61.8%) and LPA genotype MD 03-5453 (46.7%) were significantly lower than all other genotypes grown in 2015. Across treatments in both years, LPA genotype 56CX-1283 seeds emerged to similar percentages compared to NPA varieties, while the LPA genotype V12-BB144 had variable performance relative to the NPA varieties in Northern Piedmont (Table 3). MD03-5453 and V12-4557 had lower field emergence than the NPA varieties in 2015 with MD03-5453 being the lowest.

For all varieties, field emergence was 4.2% higher in 2014 compared to 2015, probably partly due to the introduction of MD 03-5453 into the study. An overall trend of higher mean emergence in non-irrigated trials compared to irrigated trials occurred across years and locations, indicated that excessive water may have been applied, irrigation increased disease pressure, or some genotypes were sensitive to moist soils. Field emergence percentage varied by location from 77.9% in Orange in 2014 to 68.8% in Blacksburg for 2014. The 2015 mean emergence in Blacksburg was 75.2%, and 63.0% for Orange (Table 3).

#### *3.2. General E*ff*ects of Seed Treatments on Field Emergence*

Seeds treated by Rancona Summit displayed the highest emergence across locations and irrigation regimes in 2014 with an average of 82.1% (Table 4). Rancona Summit was followed in descending order by ApronMaxx (81.9%), the control (80.2%), MicroCel-E + ApronMaxx (78.4%), MicroCel-E + Rancona Summit (76.8%), and Priming + Rancona Summit (76.3%). The untreated control was not significantly different from any seed treatment. Untreated seeds emerged to higher percentages than MicroCel-E, Priming + MicroCel-E + Rancona Summit, Priming, Priming + MicroCel-E, Priming + MicroCel-E + ApronMaxx, and Priming + ApronMaxx.

Emergence data were collected on fewer treatments in 2015 after ineffective treatments were identified in 2014. Untreated seed emergence was 67.4%, significantly lower compared with the three most effective seed treatments. Rancona Summit (79.0%) and ApronMaxx (76.6%) producing the highest emergence (Table 4). The Priming + Rancona Summit treatment performed better compared to the control in 2015 with emergence of 73.8%. Untreated control emergence was significantly higher than Priming and Priming + ApronMaxx treatments.


**Table 3.**Field emergence of two normal-PA (NPA) and four LPA soybean varieties grown at Blacksburg and Orange in 2014 and 2015.

1 BB = Blacksburg; O = Orange. 2 I = Irrigated; N = Non-irrigated. 3 Values within a column with values for a single year (2014 only; 2015 only or within a set of columns with values for a single year (2014 BB and O; 2015 BB and O) followed by the same letter are not significantly different based on Tukey's HSD (α = 0.05). \* Indicates a significant difference (*p* ≤ 0.05) in emergence between irrigation regimes for the corresponding genotype.


**Table 4.** Average field emergence and Tukey's separation of means for 12 seed treatment combinations in 2014 and 2015.

<sup>1</sup> BB = Blacksburg; O = Orange; <sup>2</sup> I = Irrigated; N = Non-irrigated; <sup>3</sup> M = MicroCel-E; A = ApronMaxx; R = Rancona; P = Priming; <sup>4</sup> Values within a column with values for a single year (2014 only; 2015 only or within a set of columns with values for a single year (2014 BB & O; 2015 BB & O) followed by the same letter are not significantly different based on Tukey's HSD (α = 0.05). \* Indicates a significant difference (*p* ≤ 0.05) in emergence between irrigation regimes for the corresponding.

#### *3.3. E*ff*ects of Seed Treatments on Field Emergence by PA Phenotype*

Analysis of the two NPA and four LPA soybean varieties and six treatments showed patterns of field emergence among phytic acid types in 2015 (Figure 1). The untreated control treatment average field emergence for the two *mips1* genotypes was 70.5%, while untreated seed emergence for the two *lpa1*/*lpa2* genotypes was 59.3%. Seeds treated with Rancona Summit, ApronMaxx, and Priming + Rancona had slightly higher average field emergence compared to untreated seeds. Priming and Priming + ApronMaxx tended to decrease emergence relative to untreated seeds. The overall mean of untreated emergence of the LPA varieties was 64.0%, while emergence of the two NPA varieties was 72.2%. This confirms that the LPA varieties may have lower inherent emergence than NPA varieties, although this could be largely due to the inclusion of the low-emerging genotype MD 03-5453 and the likelihood of greater disease incidence in wet soils. ApronMaxx and Rancona fungicide treatments, however, improved the emergence of LPA varieties by between 12.9% and 14.1%. This improvement suggests that fungicide treatments have the potential, when optimized, to improve LPA seed emergence to essentially the same percentages as NPA varieties.

#### *3.4. E*ff*ect of Seed Treatments on Field Emergence of LPA and NPA Genotypes*

The application of some seed treatments to the six different soybean LPA and NPA genotypes significantly improved field emergence, but effects were specific to each line (Table 5). The treatment × genotype combinations produced significant variations in both 2014 and 2015. In 2014, no treatment × genotype combination significantly improved emergence over untreated seeds of the same genotype. ApronMaxx, Rancona Summit, MicoCel-E, MicroCel-E + ApronMaxx, MicroCel-E + Rancona Summit, and Priming + Rancona Summit treatments increased emergence relative to untreated controls for at least one of four genotypes grown in 2014 (Table 5). For LPA genotypes, nearly all treatments decreased emergence relative to the control by 2–37%. The exceptions to this decrease was line V12-BB144, which ApronMaxx, Rancona Summit, and MicroCel-E + ApronMaxx treatments slightly increased (0.2–1.4%) or had no effect on emergence. MicroCel-E, Priming, Priming + ApronMaxx, Priming + MicroCel-E, Priming + MicroCel-E + ApronMaxx, and Priming + MicroCel-E + Rancona Summit each had a significant decrease in emergence relative to the control for at least one of four genotypes (Table 5).

In 2015, several treatment × genotype combinations improved emergence compared to untreated seeds of the same genotype. ApronMaxx and Rancona Summit treatments increased emergence over untreated seeds of LPA line MD 03-5453, and the highest emergence was 69.5% (Table 5). Rancona Summit as well as Priming + Rancona Summit increased emergence for NPA line 5002T compared to the control. ApronMaxx, Rancona Summit, and Priming + Rancona Summit treatments increased emergence for most genotypes relative to untreated, but the increases were not significant except as described previously. Both Priming and Priming + ApronMaxx treatments decreased emergence for five out of six genotypes from 0.7–27% compared to the control (Table 5).

### *3.5. E*ff*ect of Treatments, Genotypes, and Treatment* × *Line Interactions on Yield*

No seed treatment significantly increased yield of any genotype in either year (Table 6). However, significant differences in yield existed among genotypes in both years. In 2014, NPA line AG-5632 had the highest mean yield across all treatments at 5145 kg ha−<sup>1</sup> , followed by LPA line 56CX-1283 (4849 kg ha−<sup>1</sup> ), NPA line 5002T (4768 kg ha−<sup>1</sup> ), and V12-BB144 (4479 kg ha−<sup>1</sup> ). Only 56CX-1283 and 5002T were not significantly different. In 2015, the yield of 56CX-1283 and AG-5632 were not significantly different. Genotypes 5002T, V12-BB144, and V12-4557 yielded significantly less than either 56CX-1283 or AG 5632. The lowest yielding line was MD 03-5453 at 1211 kg ha−<sup>1</sup> , significantly lower than all other genotypes. No seed treatment increased yield compared to the control for each respective genotype. Seed treatment differences existed among genotypes, but not for treatments applied to the same genotype.

**Figure 1.** Field emergence for six seed treatments across different soybean varieties grown in 2015. Emergence rates of four low phytic acid (LPA) and two normal phytic acid (NPA) soybean varieties (**A**) as well as two LPA varieties with *lpa1* and *lpa2* alleles and two other LPA varieties with the *mips1* allele (**B**) were calculated. White horizontal lines at the center of each box show median values. The bounds of each black box show the quartiles, and the upper and lower bars show the maximum and minimum values, respectively. This image was drawn using ggplot2 package. Numbers above the violin plot indicate the means ± standard deviations. PA: Priming + Rancona; PR: Priming + ApronMaxx.


**Table 5.**Effects of seed treatments on field emergence in NPA and LPA soybeans and Tukey's separation of means in 2014 and 2015.

1 C = control; A = ApronMaxx; R = Rancona Summit; M = MicroCel-E; P = Priming; 2 NPA = normal phytic acid; LPA = low phytic acid; 3 Values followed by the same letter within bordered columns or rows are not significantly different based on Tukey's HSD (α=0.05). \* Indicates a treatment is significantly different from the control treatment for the corresponding genotype.


**Table 6.**Effects of seed treatments on yield of six soybean lines grown at Blacksburg and Orange and Tukey's separation of means in 2014 and 2015.

1 C = control; A = ApronMaxx; R = Rancona Summit; M = MicroCel-E; P = Priming 2 NPA = normal phytic acid; LPA = low phytic acid 3 Values followed by the same letter within bordered columns or rows are not significantly different based on Tukey's HSD (α=0.05).

#### **4. Discussion**

A major use of grain soybean is animal feed because of its high protein content. However, high levels of PA in soybean seeds may lead to animal mineral and protein malnutrition. In addition, phytic acid phosphorus excreted by monogastric animals such as poultry, swine, and fish can become a pollutant. These problems have provided plant geneticists with an incentive to develop LPA soybean varieties [31]. However, PA is also important for the growth and development of soybean seedlings because it is a primary storage reserve of phosphate in seeds. Phosphate is an essential component of adenosine triphosphate (ATP) that provides energy necessary for seedling growth and development. Thus, phosphorus is essential for the general health and vigor of developing seedlings. Reducing seed phytate by re-engineering synthesis pathway often has the unintended consequence of reducing seedling vigor and harming crop establishment [32]. Unfortunately, LPA soybeans often exhibit lower field emergence, making them problematic to grow particularly during stressful growing conditions.

This study included MD 03-5453 and 56CX-1283 expressing *lpa1*/*lpa2* homologs responsible for a low phytic acid phenotype (Table 1). In combination, *lpa1*/*lpa2* lower the PA content to about 25% of NPA genotypes while the remaining 75% phosphorus is inorganic [1,33]. This study also included V12-4557 and V12-BB144 genotypes, expressing *mips1*, another allele responsible for LPA soybeans (Table 1). Compared with *lpa* mutants, *mips1* mutants have higher seed PA content where it usually accounts for 50% of total phosphorus. However, *mips1* mutants increase feed efficiency for mono-and a-gastric animals with the added benefit of a modified, beneficial sugar profile. Since they have higher PA than *lpa* mutants, germination would be predicted to be similar to wild-type soybeans.

The emergence data for LPA genotypes were inconsistent between genotypes and years compared to NPA genotypes. The *mips1* LPA genotype V12-BB144 showed higher emergence than NPA 5002T in 2014 but significantly lower emergence in 2015, while emergence of *mips1* LPA genotype V12-4557 was essentially the same as 5002T in 2015, the only year it was grown. LPA genotype MD 03-5453, containing *lpa1*/*lpa2,* emerged to lower percentages than all others in 2015. However, except for MD 03-5453 and V12-BB144 in 2015, the other two LPA genotypes exhibited average field emergence of around 70% or greater. This suggests that LPA genotypes containing *mips* or *lpa1*/*lpa2* mutations, can produce satisfactory emergence if seeds are carefully produced and stored properly prior to planting. Other studies have shown that LPA genotypes, *lpa1*/*lpa2* as well as *lpa1*/*lpa2* with GmIPK2 silenced, produced satisfactory germination or field emergence [14,34]. Maupin and Rainey (2011) reported average emergence of between 74–84% for varieties with *mips* or *lpa1*/*lpa2* mutations tested across 12 unique environments [12]. Anderson and Fehr (2008) reported up to 81.0% field emergence for *lpa1*/*lpa2* mutants from various seed sources [11].

Final grain yield was only loosely correlated with field emergence. Grain yield was not significantly affected by seed treatments. Soybean plants compensate by producing more pods per plant at wider spacings, so when emergence is slightly reduced, as was the case for most treatments in this study, yield was not affected [35].

Inconsistencies in emergence data among genotypes, treatments, and years were influenced by several important seed quality factors irrespective of the genetic-controlling phytate accumulation. This study was conducted at a cooler location at 650 m elevation (Blacksburg) and a warmer climate (Orange) at lower elevation in the Virginia Piedmont with vastly different soil types. Edaphic differences between locations such as soil microbes, soil texture, water holding capacity, etc., likely contributed to variation in emergence among genotypes and treatments complicating the conclusions about the role of seed phytate on emergence.

Environmental factors regulating seed fill can negatively impact seed vigor expression when seeds are grown for propagation. High temperatures, for example, during seed development decreased seed weight, caused shriveling, and decreased seed quality of soybean [36] and reduced soybean seed vigor in the absence of mechanical injury and seedborne diseases [37]. Drought stress on the parent soybean plant had little effect on seed quality although yields were reduced [38,39]. To mitigate maternal environmental effects on quality, seeds of all six genotypes used in this study were produced in the same season and location.

Seed vigor, another important determinant of emergence particularly under stressful field conditions, is affected by a number of factors such as: seed maturity at harvest, physical seed damage during harvest and transport, and improper storage. McDonald (1985) reviewed losses in seed vigor from maturation to planting in soybean as well as identifying seed quality tests that detected physical seed damage [40]. Although the seeds tested in the current study were grown at the same location to minimize differences in seed vigor, tailoring the time of harvest for highest seed vigor was not a focus. Delayed harvest may reduce soybean seed vigor [41]. Maximum seed quality and vigor often correlates with maximum dry weight accumulation [42]. However, physiological maturity can be better detected morphologically in some seeds. For example, maximum seed dry weight was not the best indicator of physiological maturity in common bean as pod color change [43]. Bean seeds with low quality produced fewer nodules, less nodule weight, and less nitrogen fixation that resulted in less plant growth and yield [44]. Vigor tests are more sensitive measures of seed quality than the standard germination tests or field stand counts, which are often used to assess germination of low phytate genotypes. Vigor tests in future studies could yield additional valuable information about the poor emergence sometimes observed in LPA soybean genotypes.

Improper post-harvest handling compromises seed quality. Open storage in combination with high relative humidity and high temperature can quickly result in a loss of seed vigor. All genotypes were grown and stored under identical conditions, so differences were most likely due to seed genotypes and not environment. Chauhan (1985) found the growing points of the embryonic axis in soybean were most prone to aging than other seed tissues [45]. This illustrates that seed tissues do not age simultaneously, and cotyledons may be healthy even after embryonic axis is damaged resulting in poor emergence. In this study all seeds were adjusted to the same moisture content after harvest and stored in paper bags at a room temperature. Seeds may have aged under these conditions, but all genotypes were exposed to the same aging conditions.

Hoy and Gamble (1985, 1987) found that soybean seed size had no effect on specific growth rate or seedling weight from planted seeds possessing no mechanical injury [46,47]. No improvement in speed of field emergence or final yield was detected when soybean seeds were separated into varying seed density classes [48]. Thus, in this study, seeds were not sized before field planting due to the poor correlation between seed size and seed vigor.

Seed treatments may benefit field emergence and were investigated as a strategy for improving establishment of LPA soybean genotypes. While there is no consensus about the exact reason for low emergence by LPA soybean genotypes, there are likely causes. Because some fungicide treatments improved LPA emergence, disease pressure before emergence is likely higher for LPA than NPA genotypes. Soil-borne pathogens are possibly the main cause of poor emergence in some seed lots planted in wet soils. Cellular leakage occurs in all seeds during imbibition because of cell membrane damage that occurs during desiccation that must be repaired. Cells repair membrane damage during hydration, and the duration of this process depends on seed quality. Electrolyte leakage is widely used vigor test to assess soybean seed quality [49]. Aged seeds leak more solutes and electrolytes than newly harvested undamaged high vigor seeds. Evidence suggests that some LPA genotypes naturally leak more compounds that attract seed/seedling pathogens because 75% of their phosphorus is inorganic [1,33]. The loss of inorganic P from the cytoplasm of LPA varieties due to imbibitional leakage could increase disease since leaked P can attract soil-borne pathogens to the emerging seedling explaining the benefits of fungicide treatments [50]. In addition, Douglass, et al. (1993) found a negative correlation between seed sugar content of differing sweet corn genotypes and emergence in cold soils [51] while this correlation is still unclear for soybean.

The lower emergence in irrigated plots supports the hypothesis that LPA are more prone to fungal attack since moist soils would create favorable conditions for disease development possibly leading to greater seed/seedling mortality. Fungicide was the most effective seed treatment in this

study. Both fungicides significantly increased the field emergence of LPA genotype MD 03-5453, supported the hypothesis higher pre-emergence disease pressure could be a major cause of the low field emergence of LPA soybeans.

Osmotic priming is a common preplant controlled hydration treatment often applied to high value flower and vegetable seeds. Benefits of priming include faster germination, advancing seed maturity, leaching of inhibitors, and removal of dormancy. However, priming also reduces the storage life of seeds [18]. Priming treatments are less often applied to lower value agronomic seeds because the cost of application may outweigh benefits. Osmotic priming was used to increase germination rate so that seedlings would establish before diseases could infect vulnerable young plants [52]. Surprisingly, osmotic priming did not improve establishment and reduced field emergence similar to hydroprimed soybeans [53]. In the current study, seeds were primed in potassium phosphate solution which was not removed by washing at the end of treatment. These salts combined with the leakage of electrolytes that occurred during the controlled hydration priming treatment, described above, likely increased susceptibility to pathogenic attack as nutrients surrounded seeds and aided the proliferation of plant pathogens. Similarly, MicroCel-E, a calcium silicate processed from diatomaceous earth with a low salt index that contains small amounts of plant nutrients including phosphate, was applied as a seed coating. Ideally the nutrients would stimulate early seedling growth and the antipathogenic properties of diatomaceous earth may provide protection from insect and fungal predation. However, MicroCel-E consistently failed to improve emergence unless it was combined with a fungicide. The antifungal properties of diatomaceous earth were likely ineffective against seedling pathogens and insect predation. The fertilizer may have attracted and stimulated microbial growth unless fungicides Rancona Summit or ApronMaxx were present.

Emergence results were variable in this study, making it difficult to draw simple conclusions about treatments or genotypes. This is because of the complexity of factors interacting to affect field emergence. Edaphic stressors in the field commonly reduce emergence compared to results obtained from standardized laboratory germination tests conducted under near ideal conditions. In some plots, LPA seeds with *lpa1*/*lpa2* and *mips1* alleles had satisfactory field emergence compared to NPA. In other trials emergence of LPA genotypes was less than NPA likely because of conditions favoring seedling disease due to greater metabolite leakage from LPA seeds because of the altered phosphate and sugar metabolism which increased mortality. Osmotic priming and diatomaceous earth coating were ineffective. In some plots, seed fungicide treatments improved emergence of certain genotypes likely by protecting seeds/seedlings from pathogens that reduce emergence.

**Author Contributions:** Conceptualization, B.J.A., G.E.W. and B.Z.; methodology, G.E.W. and B.Z.; software, B.J.A., E.P. and J.Q.; formal analysis, B.J.A., E.P.; investigation, B.J.A.; resources, B.Z.; data curation, B.J.A.; writing—original draft preparation, B.J.A.; writing—review and editing, G.E.W., X.L., E.P. and J.Q., and B.Z.; visualization, B.J.A.; supervision, B.Z.; project administration, G.E.W. and B.Z.; funding acquisition, B.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by United Soybean Board.

**Acknowledgments:** This work was conducted under US multi-state project, W-468. Thanks to Luciana Rosso, Tom Pridgen, Steve Gulick, and Andy Jensen for technical support. Thanks to Hwasoo Shin for helping to make the graph.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article* **Tomato Seed Coat Permeability: Optimal Seed Treatment Chemical Properties for Targeting the Embryo with Implications for Internal Seed-Borne Pathogen Control**

**Hilary Mayton 1,† , Masoume Amirkhani 1,† , Daibin Yang <sup>2</sup> , Stephen Donovan <sup>3</sup> and Alan G. Taylor 1,\***


**Abstract:** Seed treatments are frequently applied for the management of early-season pests, including seed-borne pathogens. However, to be effective against internal pathogens, the active ingredient must be able to penetrate the seed coat. Tomato seeds were the focus of this study, and the objectives were to (1) evaluate three coumarin fluorescent tracers in terms of uptake and (2) quantify seed coat permeability in relation to lipophilicity to better elucidate chemical movement in seed tissue. Uptake in seeds treated with coumarin 1, 120, and 151 was assessed by fluorescence microscopy. For quantitative studies, a series of 11 *n*-alkyl piperonyl amides with log *Kow* in the range of 0.02–5.66 were applied, and two portions, namely, the embryo, and the endosperm + seed coat, were analyzed by high-performance liquid chromatography (HPLC). Coumarin 120 with the lowest log *Kow* of 1.3 displayed greater seed uptake than coumarin 1 with a log *Kow* of 2.9. In contrast, the optimal log *Kow* for embryo uptake ranged from 2.9 to 3.3 derived from the amide series. Therefore, heterogeneous coumarin tracers were not suitable to determine optimal log *Kow* for uptake. Three tomato varieties were investigated with the amide series, and the maximum percent recovered in the embryonic tissue ranged from only 1.2% to 5%. These data suggest that the application of active ingredients as seed treatments could result in suboptimal concentrations in the embryo being efficacious.

**Keywords:** tissue lipophilicity; systemic uptake; coumarin; piperonyl amides

## **1. Introduction**

Seed-borne pathogens are responsible for the initiation of numerous plant diseases and are one of the primary mechanisms for the global spread of plant pathogens [1–4]. Internal infection of seeds and colonization of the embryo and endosperm are most often associated with infection of the mother plant via the xylem, stigma, or non-vascular tissue [4–6]. Seedborne pathogens have been observed in the seed embryo, storage tissue (endosperm and perisperm), and seed coat or testa [4,7]. Disinfection techniques can be used to remove and clean contaminants from the seed surface; however, plant pathogenic organisms located within the seed endosperm and embryo are much more difficult to control. Tomatoes are an important high-value vegetable crop and are susceptible to multiple pathogens. Tomato seeds can harbor fungal, bacterial, and viral pathogens [8–10]. Several systemic conventional pesticide seed treatments are available for fungal pathogens of tomato, but options are more limited for organic production and control of bacterial pathogens [3,11,12].

Seed treatments are applied worldwide for crop protection against pests and plant pathogens [13,14]; however, the systemic uptake and distribution of active ingredients of pesticide seed treatments in seed tissue have not been as well defined as root and leaf

**Citation:** Mayton, H.; Amirkhani, M.; Yang, D.; Donovan, S.; Taylor, A.G. Tomato Seed Coat Permeability: Optimal Seed Treatment Chemical Properties for Targeting the Embryo with Implications for Internal Seed-Borne Pathogen Control. *Agriculture* **2021**, *11*, 199. https:// doi.org/10.3390/agriculture11030199

Academic Editor: Rentao Song

Received: 13 February 2021 Accepted: 23 February 2021 Published: 28 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

transport [15,16]. The long-term efficacy of seed treatments and control of seed-borne pathogens are dependent on seed coat permeability, as the active ingredients must be able to penetrate the seed coat and diffuse to the embryo. There have been several studies focused on the physiochemical barriers that prevent or allow a chemical to permeate the seed coats of several plant species [17–19]. Taylor and Salanenka (2012) developed a system to classify seed coat permeability based on the passage of ionic and non-ionic compounds through the seed coat of ten plant species from seven plant families [18]. Tomato seeds have selective permeability defined as only non-ionic compounds diffusing through the seed coat, while ionic compounds are blocked [17,18].

Seed uptake research on potential chemical pesticides applied as seed treatments is problematic due to the potential human risk of exposure to agrochemicals and/or radioactively labeled compounds. Fluorescent tracers provide an alternative approach, and coumarins are one group that includes several fluorescent, non-ionic tracers differing in chemical properties and that allows for a more comprehensive analysis of seed coat permeability characteristics [16]. Therefore, coumarin compounds were used both for qualitative uptake [16,17,19] and, using a single coumarin compound, for quantitative uptake research [20]. One objective of this research is to use three coumarin compounds with different chemical properties for tomato seed uptake to assess optimum log *Kow*.

A key chemical property that affects the uptake of an organic compound in a seed is the log *Kow*, also known as the log *P* [20–23]. A compound's lipophilicity is measured as the log *Kow* and is the ratio of its chemical concentration in octanol (o) to its concentration in the aqueous (w) phase expressed on a log10 scale [24]. A series of fluorescent piperonyl amides were synthesized, and a novel combinatorial pharmacokinetic technique was developed to provide a range of compounds with log *Kow* from 0.2 to 5.8. This series of fluorescent piperonyl amides was used to explore seed coat permeability and systemic uptake in soybean and corn seeds [23]. This same approach was adopted for tomato seed in this study.

Understanding the chemical/physical properties associated with the uptake of active ingredients in tomato seed tissue will aid in the development of new products for the control of internal seed-borne pathogens. The key objectives of this study were to evaluate the movement of selected coumarin compounds in uptake by fluorescence imaging and assess the role of log *Kow* in seed tissue permeability using a homologous series of 11 fluorescent piperonyl amides quantified by high-performance liquid chromatography (HPLC).

#### **2. Materials and Methods**

#### *2.1. Fluorescence Microscopy of Coumarin 1, 120, and 151 in Tomato Seeds*

The first study was on the uptake of selected coumarin tracers in tomato seeds imaged by fluorescence microscopy. Tomato seeds of the variety "Hypeel 696" were provided by Seminis, Oxnard, CA, and coumarin 1, 120, and 151 were purchased from TCI America, Portland, OR. The chemical and other properties of these three coumarin compounds are shown in Table 1. Tomato seeds were treated with 3 µmoles of each coumarin per gram of seed, which was 0.833, 0.631, and 0.825 mg coumarin 1, 120, or 151, respectively, per gram of seed. Each coumarin compound was mixed with 3.8 mg L650 seed treatment binder (Incotec, Salinas, Canada), 250 µL deionized water, and mixed in a 50 mL centrifuge tube using a vortex mixer (Scientific Industries, Inc., Model 2-Genie No. G560, New York, NY, USA). Ten non-treated and treated tomato seeds of each tracer were sown in 20% moisture content silica sand (#1 Q-ROK, 0.15–0.84 mm, New England Silica, Inc., South Windsor, CT, USA) and maintained in a germinator at 20 ◦C for 40 h in the dark. Imbibed seeds were then removed and washed with deionized water, and then the seeds were dissected with scalpel blades and imaged under an Olympus microscope (SZX12, Tokyo, Japan), imaging camera (Infinity 3- 3URC, Lumenera Corp., Ottawa, ON, Canada), and Infinity Analyze (Revision 6.5.2, Teledyne Lumenera, Ottawa, ON, Canada). Seed tissue was illuminated

with long UV light, UV lamp (Model 9-circular illuminator, Stocker & Yale, Salem, NH, USA). Non-treated seeds were used as the control.


**Table 1.** Physical/chemical properties of coumarin 120, 151, and 1.

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\* Log *Kow* and water solubility data obtained from Chemicalize, ChemAxon's cheminformatic tool. Excitation/emission, molar absorbance coefficient, and quantum yield information were obtained from Aazam (2010) [25] and Taniguchi and Lindsey (2018) [26].

#### *2.2. Chemicals and Synthesis of N-alkyl Piperonyl Amides*

An experimental series of *n*-alkyl piperonyl amides developed by S. Donovan and B. Black [23] was used in this study. There were 11 custom synthesized homologous piperonyl amides with carbons ranging from 1 to 14, with molecular weights of 189.2 to 361.5 g/mol. The methods and materials are described in Yang et al. (2018B) [23]. Briefly, 3.0 g of piperonylic acid was added to 5 mL of thionyl chloride. The solution was then refluxed for 30 min after which 5 mL pyridine, 25 mL toluene, and 18.1 mM amines were added and the solution was refluxed for 1 h. After cooling to ambient temperature, ethyl acetate (50 mL) was added and the solution was washed with saturated NaCl, 5% NaOH, and 5% HCl. The solution was then dried (using anhydrous sodium sulfate), filtered, and concentrated using a rotary evaporator. Recrystallization was achieved by refluxing 50 mL of methylcyclohexane until a solution was attained. The C1, C2, C3, and C4 *n*-alkyl piperonyl amides were made by adding a small amount of methylene chloride until the desired solution was completed. Lastly, all solutions were cooled and vacuum filtered before use. Each piperonyl amide (0.56 mM) solution consisted of 70% acetone + 30% water.

A short octadecyl-poly (vinyl alcohol) column was used to determine the log of the octanol-water partition coefficient for each compound by HPLC [27,28]. The HPLC-log *Kow* of the *n*-alkyl piperonyl amides series is shown in relation to the number of carbon groups and water solubility determined by Chemicalize, ChemAxon's cheminformatics tool (Figure 1).

**Figure 1.** The log *Kow* of the piperonyl amide fluorescent tracers with corresponding number of carbon atoms and water solubility, log S.

#### *2.3. Sample Preparation for High-Performance Liquid Chromatography (HPLC) Analysis* 2.3.1. Coating Tomato Seeds with Amides

Tomato seeds "Florida 47" and "Hypeel 696" were donated by Seminis, Oxnard, CA, and "OH88119" was provided by The Ohio State University, Columbus, OH. A seed coating formulation was developed to apply high loading rates of the fluorescent tracer series as a single seed treatment. A thin adsorbent seed coating was first applied to single seeds to facilitate the high loading rates of the fluorescent tracer series in a single seed treatment. General methods and materials are described in Yang et al. (2018B) [23]. Twenty grams of diatomaceous earth (DE) was dispersed in 80 g of 4% polyvinyl alcohol (PVA) aqueous solution to prepare a 20% DE suspension concentrate. One gram of tomato seeds and 1.5 g of 20% DE suspension concentrate were stirred until a layer of dry DE was coated on the surface of each seed. The coated seeds were allowed to dry in a gentle air stream. A 1.2 mL solution of amides (approximately 6 µL for each seed) was loaded gradually onto 200 tomato seeds with a micropipette. The resulting dosage was 1 µmole of each amide per gram of seed—applied to each tomato variety. Now considering the molecular weights of the 11 amides, which ranged from 179.2 to 361.5 [23], the seed treatment dosage ranged from 0.179 to 0.361 mg per gram of seed. The seeds were again dried with a gentle air stream.

#### 2.3.2. Incubation and Harvest of Treated Seeds in Growth Chamber

Seeds treated with the piperonyl amide series were imbibed as described in Section 2.1. Seed tissue was separated after imbibition, just prior to visible germination. Seeds were removed and washed (to remove seed treatment) with sterile distilled water, cut with a razor blade, and the embryo was removed. Embryos of 50 seeds were pooled to comprise one replicate. The endosperm and seed coat of 50 seeds were also pooled together as one sample. Three replicates were evaluated for each treatment. Ten seeds were pooled together as one sample or replicate. The covering layers consisted of the endosperm and seed coat, while the internal tissues were comprised of the embryo.

#### 2.3.3. Harvesting Tomato Seed Tissue for HPLC Analysis

For each embryo sample, 1.5 mL of acetonitrile (MeCN) was added and the embryos were homogenized with a glass rod. For each sample containing the endosperm and seed coat, the samples were frozen with liquid nitrogen and then homogenized in a mortar after which 1.5 mL of MeCN was added [23]. The homogenized samples were vortexed for 2 min. The extract was transferred into a tube containing 20 mg of PSA, 5 mg of GCB, and 50 mg of MgSO4, then shaken for 1 min, and was then passed through a 0.22 µm syringe filter. The recovery is shown in Table S1 in the supporting information of Yang et al. (2018B) [23].

The tomato embryo and internal tissue samples were extracted by the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method as described for soybean and corn seeds [23]. Ten tomato embryos or ten tomato endosperm + seed coats were placed into a frozen mortar and frozen with liquid nitrogen, and ground into a fine powder. The powder was transferred into a 50 mL centrifuge tube with a screw cap, and 8 mL of MeCN was added and the mixture was shaken for 2 min using a Vortex mixer at room temperature. Following this, a mixture of 2.5 g of MgSO<sup>4</sup> and 1.0 g of NaCl was added. The tube was immediately shaken vigorously for 1 min to prevent the formation of MgSO<sup>4</sup> agglomerates and centrifuged at 3500 rpm for 5 min. Then, 3.0 mL of the supernatant was subjected to dispersive solid-phase extraction (SPE) using a mixture of 8 mg GCB, 50 mg PSA, and 100 mg MgSO4. The mixture was shaken vigorously for 1 min using a Vortex mixer. Finally, the extract was filtered through a 0.22 µm syringe filter. In developing the HPLC method, the percent recoveries were determined for the eleven amides from soybean embryo + testa (seed coat), and corn endosperm + embryo, and pericarp + testa. The recovery at ≤3.82 log *Kow* for both seed tissues was > 82% for soybean and > 85% for corn [23].

#### 2.3.4. HPLC Analysis of Tomato Seed Tissue

The amides content was determined using an Agilent 1100 HPLC equipped with a 1200 fluorescence detector (FLD) using an ODS-3 column (GL Sciences Inc., 5 µm, 4.6 mm × 75 mm column). The wavelengths of FLD were set at 292 nm (excitation) and 340 nm (emission). The mobile phase used was 0 min 30% MeCN + 70% water, 22 min 40% MeCN + 60% water, 25 min 80% MeCN + 20% water, 40 min 90% MeCN + 10% water. The temperature of the column was 30 ◦C. The injected volume was 20 µL. The retention time in minutes for each amide derivative was 3.63 (C1), 5.94 (C2), 10.38 (C3), 15.43 (C4), 18.59 (C5), 21.04 (C6), 23.15 (C7), 24.96 (C8), 27.70 (C10), 31.08 (C12), and 35.58 (C14). ℃

#### 2.3.5. Tomato Seed Coat Permeability Data Calculation

Percent uptake in relation to the maximal log *Kow* (relative amount) ܍ܝܛܛܑܜ ܖܑ ܍܌ܑܕ܉ ܐ܋܉܍ ܗ ܖܗܑܜ܉ܚܜܖ܍܋ܖܗ۱ ܍ܝܛܛܑܜ ܍ܕ܉ܛ ܍ܐܜ ܖܑ ܗܔ ܔ܉ܕܑܠ܉ܕ ܍ܐܜ ܜ܉ ܍܌ܑܕ܉ ܍ܐܜ ܗ ܖܗܑܜ܉ܚܜܖ܍܋ܖܗ۱ ൈ %

= Concentration of each amide in tissue Concentration of the amide at the maximal log *Kow* in the same tissue × 100%

܌܍܍ܛ ܉ ܡ܊ ܌܍܊ܚܗܛ܊܉ ܍܌ܑܕ܉ ܐ܋܉܍ ܗ ܜܖܝܗܕۯ

**2021**, , x FOR PEER REVIEW 5 of 12

Percent uptake based on amount applied (uptake efficiency) ܍܌ܑܕ܉ ܐ܋܉܍ ܗ ܜܖܝܗܕ܉ ܌܍ܑܔܘܘ ൈ %

= Amount of each amide absorbed by a seed Applied amount of each amide × 100%

Percent in embryo of total seed uptake ܗܡܚ܊ܕ܍ ܍ܐܜ ܖܑ ܍܌ܑܕ܉ ܐ܋܉܍ ܗ ܜܖܝܗܕۯ

Amount of each amide in the embryo

= Sum amount of each amide in the covering <sup>+</sup> internal tissues × 100% ܛ܍ܝܛܛܑܜ ܔ܉ܖܚ܍ܜܖܑ ା ܖܑܚ܍ܞܗ܋ ܍ܐܜ ܖܑ ܍܌ܑܕ܉ ܐ܋܉܍ ܗ ܜܖܝܗܕ܉ ܕܝ܁

#### **3. Results**

#### *3.1. Fluorescence Microscopy of Coumarin 1, 120, and 151 in Tomato Seeds*

Assessment of coumarin 1, 120, and 151 uptake was conducted by visual fluorescence observation of the applied seed treatment tracers in tomato seed tissue. Coumarin tracers evaluated in this study were all non-ionic and therefore were expected to permeate the seed coat and move to the embryo [17]. Results showed that coumarin 120, with the greatest water solubility and the lowest log *Kow* (Table 1), was readily taken up in the embryonic tissue, whereas coumarin 1 and coumarin 151 were only partially taken up in the embryo (Figure 2). The low level of fluorescence in the non-treated control was attributed to autofluorescence in the embryonic tissues.

ൈ %

**Figure 2.** Tomato "Hypeel 696" seed coat permeability of three different coumarin tracers: (**a**) nontreated, (**b**) coumarin 1, (**c**) coumarin 151, and (**d**) coumarin 120.

#### *3.2. Tomato Seed Coat Permeability*

3.2.1. Maximal Uptake of Piperonyl Amides in Relation to log *Kow*

The maximum (100%) relative amount of *n*-alkyl piperonyl amide recovered in tomato seed tissue was in the range of 2.88–3.39 log *Kow* in embryonic seed tissue and 3.39–4.18 log *Kow* in endosperm + seed coat tissue (Figures 3 and 4). Amide diffusion to the embryo was limited when log *Kow* exceeded 4.18 (Figure 3). The maximal uptake in relation to log *Kow* was achieved at lower log *Kow* values for Hypeel 696 in both types of seed tissue compared with OH88119 and Florida 47, which had very similar uptake profiles (Figures 3 and 4).

**2021**, , x FOR PEER REVIEW 6 of 12

**2021**, , x FOR PEER REVIEW 6 of 12

**Figure 3.** *N*-alkyl piperonyl amide uptake in tomato embryo in relation to maximal log *Kow* of 100%. Means with standard error bars are shown.

**Figure 4.** *N*-alkyl piperonyl amide uptake of tomato seed coat + endosperm in relation to maximal log *Kow* of 100%. Means with standard error bars are shown.

3.2.2. Uptake Efficiency (%) of Piperonyl Amides in Seed Tissue in Relation to Amount Applied

The uptake efficiency, based on the total amount recovered in the embryo compared with the amount applied, showed that maximum uptake associated with log *Kow* for the embryo occurred at 2.88 for Hypeel 696 and 3.39 for OH99119 and Florida 47 (Figure 5). However, even at the maximal log *Kow*, the percent uptake was only 5.0% for Florida 47, 4.3% for Hypeel 696, and 1.2% for OH99119. In contrast, uptake efficiency for the entire seed was much greater than the embryo, and ranged from 27% to 36% for the three varieties (Figure 6).

**Figure 5.** Uptake efficiency of piperonyl amides in the embryo, measured as percent compound applied of *n*-alkyl piperonyl amides. Means with standard error bars are shown.

**Figure 6.** Uptake efficiency of piperonyl amides in the seed, measured as percent compound applied of *n*-alkyl piperonyl amides. Means with standard error bars are shown.

3.2.3. Percent of Piperonyl Amides in the Embryo Compared with the Entire Seed

The percent of the lipophilic amide series in the tomato embryo declined with log *Kow* from 0.02 to 4.18 for Hypeel 696 and OH8819 (Figure 7). In contrast, Florida 47 revealed a slight increase in the percent embryo distribution from log *Kow* 0.02 to 2.88–3.18, followed by a decrease to 4.78. **2021**, , x FOR PEER REVIEW 8 of 12

**Figure 7.** Percent of the absorbed *n*-alkyl piperonyl amides in seed embryo compared with the entire seed uptake of three tomato varieties. Means with standard error bars are shown.

#### **4. Discussion**

Fluorescent tracers were used in many previous studies in our lab to examine seed coat permeability in vegetable and field crop seed species. Application of single tracer compounds was used in these qualitative studies to determine seed coat permeability characteristics, resulting in three categories: (1) permeable, (2) selectively permeable, and (3) non-permeable [17]. A dual fluorescent tracer method was later developed to investigate corn pericarp/testa permeability of 27 maize lines [19]. This method could be readily adopted to determine the seed coat permeability category of other seed species. Collectively, both tomato [17] and corn [19] have selective permeability as only non-ionic compounds diffused through the seed coat, while ionic compounds were blocked. A single coumarin compound, coumarin 120, was used in quantitative uptake studies, and a linear increase in seed uptake was measured for corn seed treatment dosage in the range of 0.01 to 1.0 mg coumarin 120 applied per gram of seed [20]. In this study, coumarin 120 and each amide in the series were applied at 3 and 1 µmole per gram of seed, respectively. These seed treatment dosages convert to a range of 0.83 to 0.17 mg per gram of seed, which was in the linear uptake range of corn [20].

The objective of the first investigation in this study was the evaluation of the uptake of three coumarin fluorescent tracers in an attempt to develop a simple method to assess the optimum log *Kow* for the penetration of neutral compounds through the tomato seed coats. The major advantage was the use of readily available chemical compounds, and these tracers were previously documented with systemic uptake in seeds and seedlings [16]. In addition, fluorescence microscopy could be used for rapid assessment for comparisons without the need for chemical extraction and chemical analyses. Unfortunately, fluorescence intensity was more related to water solubility than log *Kow* (Table 1 and Figure 2). Moreover, limited conclusions can be drawn using only three tracer compounds. These inconclusive results were attributed to the use of heterogeneous compounds with different physical/chemical properties (Table 1). In addition, there are other properties unique to each coumarin compound including polarizability, topological polar surface area (TPSA), polar surface area (PSA), calculated molar refractivity (CMR), the number of hydrogen bond donors and acceptors, and pK<sup>a</sup> (Chemicalize, ChemAxon's cheminformatics tool), and these properties may play a role in seed uptake. Moreover, fluorescence microscopy images may produce false-positive images with the confounding effect of auto-fluorescence from internal seed structure constituents.

There is great value in understanding the chemical/physical properties of active ingredients, and this information can guide a directed chemical synthesis program giving optimal uptake. Alternatively, potential uptake of an existing active ingredient with known or predicted log *Kow* can be assessed through knowledge of the optimal lipophilicity for seed coat permeability. For this second objective, a combinatorial pharmacodynamic technique was employed using a homogeneous series of 11 *n*-alkyl piperonyl amides that varied in log *Kow* from 0.02 to 5.66. The mixture of amides was applied as a seed treatment, and tomato seeds were imbibed and dissected into two portions, the embryo, and the endosperm + seed coat. The relative amounts of the amides in these two fractions were quantified by HPLC and plotted as a function of log *Kow*. This allowed a clear understanding of the role of lipophilicity as it relates to uptake through the tomato seed coat and endosperm and the resulting transport into the embryo by neutral compounds. This knowledge of the optimal physical properties is an invaluable guide in the targeted control of internal seed-borne pathogens.

The overall uptake profile for the tomato embryo and endosperm + seed coat of all three varieties revealed a Gaussian distribution (Figure 3) that is similar to root uptake in plants [21–23]. This similar Gaussian distribution for both tomato roots and seeds was expected based on the composition of the barrier layers. Suberin is found in the endodermis and exodermis of tomato roots [29] and also the inner layer of the tomato seed coat [30]. In contrast, the Gaussian distribution pattern in root uptake in corn and soybean was not revealed for corn or soybean seed uptake [23].

The maximal uptake for the tomato embryo tissue of the three varieties ranged from 2.88 to 3.39 log *Kow* (Figure 3), while the maximal uptake ranged from 3.39 to 3.88 log *Kow* for the endosperm + seed coat (Figure 4). Thus, a slight shift to lower log *Kow* for the embryo tissue in comparison with the other seed tissues was revealed. In the case of corn, the maximal uptake was 3.39 log *Kow* for both the endosperm + embryo and pericarp/testa using a similar method with the 11 *n*-alkyl piperonyl amides [20]. Therefore, both tomato and corn have similar maximal uptake profiles. Unfortunately, the tomato embryo readily detached from the endosperm during dissection of the fully imbibed seed, which did not allow the measurement of the sum of endosperm with embryo, so a comparison of the effect of the endosperm on shifting the maximal log *Kow* could not be directly made between corn and tomato.

The uptake efficiency was calculated as the percent of each amide taken up in relation to the amount applied. The maximum uptake efficiency of the entire seed of the three varieties ranged from 27% to 36% (Figure 6), while the maximum uptake efficiency of corn was 43% [20]. Therefore, tomato had lower seed coat permeability than corn, which may be attributed to seed coat composition. The inner layer of the tomato seed coat is known as the semipermeable layer [31] and was shown to be composed of suberin [30], while corn has a semipermeable cutinized or suberized membrane that is located below the inner integument [32].

The maximum uptake efficiency of the embryo in relation to the amount applied of the three varieties ranged from 1.2% to 5.0% (Figure 5). Another calculation based only on the absorbed *n*-alkyl piperonyl amides uptake revealed that less than 30% of an amide was measured in the embryo compared with the entire seed (Figure 7). These data demonstrate that most of the piperonyl compounds were unable to reach the embryo. However, fluorescence images revealed the greatest fluorescence intensity in the embryo compared with the endosperm or seed coat (Figure 2). Therefore, fluorescence imaging that provides excellent qualitative data on the presence or absence of a tracer in the seed tissue was not related to quantitative results from our analytical method.

The pathway by which the applied seed treatment moved to the embryo was not investigated in this study. In the dicot seed *Sedum acre*, movement between the seed compartments was attributed to symplastic movement with cell-to-cell movement through the plasmodesmata [33]. We assume that movement from the endosperm to embryo in tomato seed is by the same symplastic pathway.

Three tomato varieties were investigated with the 11 *n*-alkyl piperonyl amides. The maximal log *Kow* for both the embryo and endosperm + seed was shifted to a slightly lower value for Hypeel 696 compared with the two other varieties (Figures 3 and 4). Florida 47 had the greatest accumulation in the embryo with 5.0% (Figure 5), while Hypeel 696 had the greatest accumulation in the entire seed with 36% (Figure 6). After an amide was absorbed, Florida 47 had a greater distribution in the embryo than the other two varieties (Figure 7). These varietal differences may be attributed to differences in seed coat composition and/or structural properties. Varietal differences in tomato seed coat permeability were related to the efficacy of jasmonic acid seed treatments used as an elicitor of defense against western flower thrips [34]. The thickness and compactness of the inner tomato seed coat layer composed of suberin [28] may be responsible for varietal differences. Further, the composition of the seed coat and embryo may differ and thus can affect both permeability and affinity for a compound. Further study is needed to investigate varietal differences in seed uptake, as retention of an active ingredient in the seed coat could result in a suboptimal concentration in the embryo being efficacious.

#### **5. Conclusions**

This study quantitively described the relationship between the log *Kow* and the permeation capacity of a chemical through the seed coat to the embryo of tomato seeds. The relatively hard and thick tomato testa attenuated the movement of the seed treatment to the embryo tissue. Less than 5% of the applied compound was measured in the embryo, while most resided in the seed coat + endosperm. For the control of internal seed-borne pathogens, seed treatment with log *Kow* in the range of 2.9 to 3.8 log *Kow* is suggested as these chemicals were found to most effectively reach the tomato embryo tissues.

The piperonyl amide method uses a combinatorial pharmacodynamic technique to probe the uptake and transport of xenobiotic compounds in seeds. This is in contrast with the use of heterogeneous compounds that differ in a multitude of physical properties, isolation efficiencies, and detection sensitivities. When using heterogeneous compounds, often the experimental method involves a separate experiment for each compound to generate an uptake and/or transport parameter. Combining the ensemble of data from the piperonyl amide method resulted in a trend that identified the optimum chemical properties for uptake and accumulation in specific seed tissues. Moreover, the combinatorial pharmacodynamic method used eleven piperonyl amides combined into a single experiment, with significant benefits with regard to time, cost, and many experimental variables being eliminated. Further, very subtle absorption and transport trends were quantified in different crop seeds [23], and also in plants and insects [Donovan and Black, unpublished]. Thus, the method can be broadly adapted for agricultural research, and provides detailed physical property space information at a level of precision that is not available using other techniques.

**Author Contributions:** Conceptualization, A.G.T. and S.D.; methodology, H.M., M.A. and D.Y.; software, H.M. and M.A.; validation, A.G.T., H.M. and M.A.; formal analysis, H.M. and M.A.; investigation, H.M., M.A. and D.Y.; resources, A.G.T.; data curation, A.G.T., S.D., H.M., M.A. and D.Y.; writing—original draft preparation, H.M. and M.A.; writing—review and editing, A.G.T., S.D., H.M., M.A. and D.Y.; visualization, A.G.T. and M.A.; supervision, A.G.T.; project administration, A.G.T.; funding acquisition, A.G.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Specialty Crop Research Initiative (grant no. 2015-51181- 24312) from the USDA National Institute of Food and Agriculture. D. Yang was partially supported by the China Scholarship Council (grant No. 201503250009). This material is based upon work that is supported by the National Institute of Food and Agriculture, US Department of Agriculture, Multi-state Project W-4168, under accession #1007938.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Authors would like to thank Lailiang Cheng, Cornell University, for providing access to his HPLC for this project.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

