Advancements in organic production practices for peanut (Arachis hypogaea L.) have been made in the southeastern U.S., particularly for weed (Johnson et al., 2012; Wann et al., 2011b) and mid-season foliar disease control (Cantonwine et al., 2008; Wann et al., 2011a), but there are still serious obstacles to overcome. One of the key research needs is to improve plant stand without the use of synthetic seed treatments. Untreated peanut seed can result in 50% fewer established plants than seed treated with synthetic seed treatment (Melouk and Backman, 1995). Acceptable plant stands not only maximize yield potential but also minimize pest problems by reducing incidence of tomato spotted wilt virus (Branch et al., 2003), and competition by weeds (Place et al., 2010). Plant stands of around 11 to 12 plants/m of row maximized single row peanut production in conventional management in Georgia (Tubbs et al., 2011), and yields were equal with plant stands of 9 to 11 plants/m using various weed control regimes with organic management in Georgia (Wann et al., 2011b). Weed competition is a significant concern for organic growers since there are fewer options to control weeds in organic production systems than conventional systems.

There are several seedborne and soilborne pathogens that can attack peanut seed or seedlings, including Rhizopus spp., Penicillium spp., Fusarium spp., Aspergillus niger, and A. flavus (Sullivan, 1984). Rhizopus seed rot typically occurs during imbibition or just after germination, and is most common in cool-wet planting conditions (Melouk and Backman, 1995; Sullivan, 1984). Aspergillus crown rot, caused by A. niger, can attack the plant prior to emergence or after emergence before stems have hardened off significantly, and is more common in hot-dry conditions (Jackson and Bell, 1969; Melouk and Backman, 1995). A study conducted in North Carolina evaluating the efficacy of peanut seed or soil treated with organically acceptable seedling disease treatments, such as biocontrol organisms, activated charcoal, copper hydroxide, or hot water showed that seed treated with the biocontrol agent Bacillus subtilis showed the most promise among the other treatments tested (Ruark and Shew, 2010). Cantonwine et al. (2011) found that genotype and seed integrity were more important factors than B. subtilis seed treatment to improve plant stand and reduce Aspergillus crown rot, but the rate of the B. subltilis seed treatment used in the experiment was lower than that used by Ruark and Shew (2010).

The objectives of this research were to assess the direct toxicity of various seed treatments against Rhizopus and Aspergillus species, and relate these findings to the efficacy of these materials for improving plant stands and reducing postemergence plant mortality (damping-off) (Fig. 1). Effects on yield and time required for hand weeding were also investigated.

In the lab assay, three experimental trials with 26 replications total were used to evaluate treatment effects for each pathogen. A total of 10 trials were conducted, however four trials were excluded due to low pathogen pressure (<25% in untreated controls). Trends observed in these trials were consistent with the trials included in the analyses (data not shown). One trial was excluded due to high pressure (100% of untreated seed affected by Rhizopus), which was the result of adding too much inoculum. The trend observed in this trial differed from the trials included in the analysis and is discussed later. Two of the trials used to evaluate Rhizopus did not include all seed treatments. Therefore, only those treatments that were common to all trials were compared.

There were no interactions between seed treatment and trial, and seed treatment significantly affected incidence of Aspergillus and Rhizopus (P<0.01). Compared to the untreated control, Bacillus subtilis and S. lydicus seed treatments suppressed Aspergillus (P<0.01), with B. subtilis completely suppressing pathogen establishment (Table 1). The dry and cola copper sulfate treatments resulted in similar incidences of Aspergillus to the untreated control (P = 0.299 and 0.154, respectively), while incidence was higher than the untreated control when copper sulfate was applied with water (P<0.01) (Table 1). The effectiveness of B. subtilis against Aspergillus is in agreement with other studies (Kimura and Hirano, 1988; Zhang et al., 2008), while the lack of response by copper sulfate was unexpected, since similar copper containing compounds provided fungicidal activities against A. carbonarius in a lab study using grape-like media (Belli et al., 2006). Copper sulfate did suppress the incidence of Rhizopus, with similar levels of suppression to that of Dynasty (P = 0.161) (Table 1). However, the activity of copper sulfate was almost completely overcome when pathogen pressure was high (data not shown), which suggests limitations to this seed treatment. Compared to the untreated control, B. subtilis had no effect on Rhizopus (P = 0.926), while S. lydicus caused higher incidence (P = 0.021) (Table 1). Streptomyces lydicus has been shown to reduce Pythium and Rhizoctonia solani on pea and cotton seed (Yuan and Crawford, 1995). Rhizopus is not listed as target pathogens for S. lydicus (Cao et al., 2010), and may not have activity against this fungus.

In the field experiments, plant stands were not improved on any sample date with B. subtilis compared to the untreated check (Figs. 2 and 3). There was also equal or greater levels of plant mortality after emergence compared to untreated seed (Figs. 4 and 5), although final totals of both stand counts and dead plants were equal to the untreated in all three field experiments (including the Blackshank location in 2010 – data not shown). Other studies have suggested that B. subtilis has the potential to aid in germination and emergence for improved stand establishment (Cantonwine et al., 2011; Ruark and Shew, 2010; Turner and Backman, 1991). The most favorable responses to B. subtilis have been under stressed conditions, such as seed that were mechanically shelled (Cantonwine et al., 2011), or unfavorable planting conditions from limited water, poor rotation, or cool soils (Turner and Backman, 1991). Yet, in the previous research from South Georgia, only one out of 15 genotypes in each year of the trial had a positive plant stand response that was solely related to B. subtilis seed treatment (Cantonwine et al., 2011). Warm soil temperatures at the 10 cm depth in the Lang-Rigdon-2010 experiment (25 C) and very hot and dry conditions at the Ponder-2011 experiment (36 C) may have caused greater pressure from A. niger to overburden the B. subtilis treatment, or poor establishment conditions for B. subtilis where it could not suppress the pathogen.

The only seed treatment that provided some level of efficacy in stand establishment was copper sulfate. When applied dry to the seed, there was an 18% increase in plant stand compared to the untreated seed at 4 wk after planting (Fig. 2), and half as many plants that died after emergence by week 4 at Lang-Rigdon in 2010 (Fig. 4). When copper sulfate was applied after the seed were sprayed with cola, the number of plants that died after emergence was reduced even further, with 79% fewer dead plants than the untreated seed. This is presumably due to greater adherence of the material to the seed. However, emergence of seedlings from the soil was severely delayed compared to all other treatments in both years, and never fully recovered in 2011 at the Ponder location, resulting in a plant stand that was nearly 50% of the untreated seed (Fig. 3). Although care was taken when wetting the seed prior to application of the copper sulfate, it is possible that there was some damage to the seed testae from this process. Such damage is detrimental to seed health, and is one reason the seed industry does not use liquid seed treatments. It is also possible that the additional sugars from the cola attracted facultative pathogens leading to slowed or limited emergence. There was no evidence in the laboratory study of phytotoxicity from a higher rate adherence in the treatments including cola.

Since the lab assays showed good activity from B. subtilis and no benefit from copper against A. niger, yet the field trials displayed no effect from B. subtilis and a positive response where copper sulfate was applied, this would indicate that there were additional variables affecting seedling response to the treatments in this research. It is possible that Rhizopus or other pathogens sensitive to copper sulfate contributed to seedling deterioration during germination, although the hot and relatively dry soil environment in these field studies would have been expected to be very suitable for A. niger. It can also be speculated that there may be some supplemental micronutrient benefit from the copper as well, aiding in growth and a resulting healthier seedling that had greater resistance to damping-off. Peanut seed soaked in low concentrations (10⁻⁴M) of copper sulfate, and other inorganic salt solutions, were shown to reduce Aspergillus collar rot (crown rot) in greenhouse and field studies in India (Dasgupta et al., 2000). The authors attributed this effect to enhanced defense responses of the plant rather than direct toxicity to A. niger due to the low concentration of the material. Copper is one of the 13 essential mineral elements needed for plant growth, and copper sulfate is a common micronutrient fertilizer with a nutrient content of 25–35% copper (Brady, 1974). In previous research, copper has increased seed germination for several plant species (Ouzounidou, 1995) and aided in root and shoot growth in alfalfa (Medicago sativa L.) (Peralta et al., 2001), although it can also inhibit germination and growth at high concentrations. The phenomena that caused higher plant stands and reduced mortality with the inclusion of copper in this study was not confirmed; however, it does open opportunities for supplemental research on the effect of copper on germination and stand establishment, and also in reducing damping-off.

Despite variations in plant stand in 2010 at both locations, there were no resulting yield differences (Table 2). However, there were yield differences at the 2011 Ponder location, and this was highly correlated with plant stand (Pearson coefficient  =  0.86, P < 0.0001). Plant stand and yield were also highly correlated in another organically managed trial in Georgia (Wann et al., 2011a), which proves the importance of establishing an optimum plant stand in organic production. Although there were no yield differences in 2010, there was a difference in the amount of time required for hand weeding the plots (Table 3). There was likewise a strong correlation between plant stand and time spent hand weeding in the Lang-Rigdon 2010 experiment for all four stand count dates (Pearson coefficient ranging from −0.39 to −0.51, P < 0.025 for all dates). Therefore, whether plant stand affects yield or amount of time spent hand weeding, either way affects net revenue for the organic peanut farmer, such that improved plant stands result in increased income.

Research from the lab assay experiment showed promising results for multiple biological control materials depending on the seed or seedling pathogen in question. However, these effects were not consistent with field results. Based on field results, copper sulfate appeared to have more consistent potential as an organically acceptable material to assist with stand establishment in an organic peanut production system compared to the other materials investigated. The fungicidal activity of B. subtilis against Aspergillus species in vitro did not translate to the field in this study. These data also suggest that copper sulfate may provide an indirect mode of action against the development of Aspergillus crown rot. More research related to this effect would be valuable. Evaluating other particle sizes in combination with an organically acceptable sticking agent that does not damage the seed testae could result in even better activity of copper sulfate on seed and seedling disease.