The peanut agroindustry chain (Arachis hypogaea) in Argentina is located mainly in Córdoba province and can be considered the enabler of local development. Currently, the peanut production system is under high pressure by seed-transported fungal diseases, including peanut smut, whose causal agent is Thecaphora frezzi, and peanut blight caused by Sclerotinia minor (INTA, 2018; ^{Rosso et al., 2018}). This has relevant implications for the long-term planning of the production system and important effects at the regional level. Although the use of synthetic fungicides has been the main tool for controlling this kind of diseases, it has proven to be ineffective, with controversial consequences for the environment, human health and the balance of edaphic microbiota (^{Andrés et al., 2016}). An alternative has emerged to address these limitations by using biocontrol as part of sustainable crop management in order to improve productivity by increasing the availability of nutrients and phytopathogen protection (^{Sherathia et al., 2016}). The products used for biological control are preparations of living or latent cells of microorganisms that, when applied to the soil or seed, favor the plant-rhizosphere relationship (^{Bashan et al., 2013}). So far, there are in vitro studies on peanut about the benefits of applying microorganisms (^{Ankati et al., 2018}; ^{Ganuza et al. 2017}). According to Bashan et al. (2013), the applied biologicals can directly or indirectly influence plant growth by controlling phytopathogens as well as producing effects that promote plant growth. Bacillus is a bacterial genus that is widely distributed in agri-systems (soil, water and plant) because of its ability to form endospores and produce antimicrobial and antifungal metabolites (phenazines, 2,4-diacetylphloroglucinol, cyclic lipopeptides) (^{Tejera-Hernández et al., 2011}, ^{Villarreal-Delgado et al., 2017}). Bacillus activates protection mechanisms in plants, including structural changes in the cell wall caused by lignin accumulation (^{Singh et al., 2016}) and the production of flavonoids, phytoalexins and auxins (^{Pretali et al., 2016}). In trials conducted by ^{Shifa et al. (2016}), the results showed that when B. subtilis was applied to peanut seed and the soil, the A. flavus population decreased significantly, as well as the infection and the aflatoxin content in grain; this showed the effect of B. subtilis as a biological control agent of fungi that can produce aflatoxins in peanut. Trichoderma is a widely distributed and highly adaptable fungus, of easy reproduction and antagonistic capacity (^{Sharma and Gothalwal, 2017}). Particularly in peanut, inoculation with Trichoderma promoted plant growth, according to Rojo et al. (2017). The proposed objective of this study was to evaluate the biocontrol effect on Aspergillus flavus, Fusarium sp., Sclerotinia minor and Thecaphora frezzi, and growth promotion in peanut by inoculating Trichoderma harzianum and Bacillus subtilis under controlled and field conditions.

The biological controls that were used, T. harzianum and B. subtilis, were isolated from peanut production plots in Córdoba and identified as CT306 and CT104 strains, respectively; they belong to the collection of Agri-food Quality Transfer Center, Faculty of Agronomy Sciences, National University of Córdoba (FCA UNC). The antagonistic effect of each microorganism was evaluated in vitro in previous studies. For the study, peanut seed of the Granoleico variety, 2016/17 harvest cycle, was provided by the company Aceitera General Deheza S.A., General. Deheza, Córdoba, Argentina. A trial under controlled conditions was used to evaluate the control efficiency of the studied biologicals on the following pathogens associated with seeds: A. flavus, Fusarium sp., S. minor and T. frezzi. The fungi were obtained from the culture collection of the Phytopathology Laboratory, FCA UNC, Argentina. To inoculate the peanut seeds, which were previously disinfected with 3% (v/v) sodium hypochlorite for 10 min and rinsed with sterile distilled water, the surface of the pure culture plate was washed with a 0.9% sterile saline solution. The concentration of the spore suspension for A. flavus and Fusarium sp. was 1x10⁶ conidia mL^-1 and 1x10⁵ sclerotia mL^-1 of S. minor. In the case of T. frezzi, the seeds were inoculated by applying a coat of teliospore suspension (1 g/25 mL of sterile saline solution). Then, using the pelletizing method, 100 seeds were infested with each one of the studied fungus. The T. harzianum inoculum was obtained in a malt extract medium for 7 days at 28 ºC and 12-h light cycles. The B. subtilis inoculum was prepared in an agar-potato-dextrose medium in a rotary stirrer for 48 h at 25 °C.

The biocontrol of each pathogen with the microorganisms was conducted separately, for which pots 30 cm in diameter were filled with a soil:sand (3:1) substrate, previously sterilized in an autoclave (1 hour; 1 atm.). The pots were kept in a chamber at 25 °C and 12-h light photoperiod. The evaluated treatments were: a) the check (seed infected with the pathogen in sterile substrate); b) inoculation with T. harzianum (seed infected with the pathogen in a substrate inoculated with T. harzianum 1x10⁸ conidia mL^-1; 1 L m³ of substrate); c) inoculation with B. subtilis (seed infected with the pathogen and after 24 h inoculated with B. subtilis 2.5x10¹⁰ UFC using doses of 100 mL/100 kg of seeds, in sterile substrate); and d) inoculation with a combination of T. harzianum and B. subtilis (seed infected with the pathogen and after 24 h inoculated with B. subtilis and T. harzianum using the same doses and concentration of the previous treatments). The trial was arranged in a completely randomized design. Each unit of the experiment consisted of four pots per duplicates for each evaluated treatment. The trial was repeated twice. From sowing and up to 60 DAS (days after sowing), the signs and symptoms of each disease were observed, visually and with a stereoscope and an optical microscope, according to the description shown in Table 1. Based on the percentage of plants showing disease signs and symptoms, the following incidence categories were established: low level= 10% of plants; intermediate level= 25% of plants; high level= 50% of plants; no signs or symptoms= 0% of plants. In all cases where the disease was detected, isolation and morphological identification were conducted to confirm the pathogen.

In the field, the seeds were sown in the Peanut Module of the FCA UNC Field-School (31° 28 49.42” S and 64°00 36.04” W), Córdoba, Argentina. The evaluated treatments were: a) control (untreated seed); b) fungicide (pre-treatment with Metalaxil-M 1.0 g/Fludioxonil 2.5 g; 750 cc/100 kg seed); c) fungicide + T. harzianum (pre-treatment with fungicide + pre-inoculation with T. harzianum 1 x 10⁸ viable conidia mL^-1; 200 mL/100 kg seed + adhesive); d) fungicide + B. subtilis (pre-treatment with fungicide + pre-inoculation with B. subtilis (2.5 x 10 ¹⁰ UFC/L; 100 mL/100 kg seed); and e) fungicide + T. harzianum + B. subtilis (pre-treatment with fungicide + pre-inoculation with T. harzianum and B. subtilis at the same dose and concentration as in the previous treatments). Each treatment was sown rows 100 m long separated by a distance of 70 cm and a distribution of 14 seeds per linear meter. Each treatment was arranged in a completely randomized design with four replications. The data were collected by randomly sampling all the plants from the two central rows in 1 m². The evaluated variables were percentage of emerged plants at 28 DAS, plant growth measured as dry weight (DW) at 30 and 165 DAS (^{Pérez and Arguello, 1995}). At the end of the cycle (165 DAS), each replication of each treatment was manually harvested to determine the level of maturity, according to the husk scraping method (^{Pérez et al., 2004}), percentage of husks infected with T. frezzi (^{March and Marinelli, 2005}), husk and grain yield (qq/ha) and grain quality as the percentage of confectionery grains corresponding to 38/42, 40/50, 50/60 and 60/70 grains per ounce. The data were subjected to an analysis of variance (ANOVA), after validating the variance homogeneity assumption using Levene’s test (α = 0.05). The means were compared using the LSD test (p≤0.05) with the InfoStat statistical software (^{Di Rienzo et al., 2018}).

According to the results shown in Table 2, under controlled conditions the treatments with T. harzianum and B. subitilis, alone and combined, showed no Fusarium sp., while S. minor showed a low level of incidence when the antagonist was individually applied, and no disease symptoms were observed when the treatments were combined. The T. frezzi control was more effective when the biocontrols were combined (Table 2). However, when the biocontrols were applied alone, T. harzianum had better control than B. subtilis. These results coincide with the results of ^{Rojo et al. (2007}), who by applying T. harzianum ITEM 3636 on peanut seeds effectively controlled F. solani. The authors state that this biocontrol proved to be an efficient and competitive microorganism in the rhizosphere. The effect observed when Trichoderma is used could be related to complex mechanisms possibly associated with the degradation of the fungal walls, according to ^{Howell (2003}), ^{Harman et al. (2004}) and ^{Woo et al. (2006}).

Preliminary evaluations in which peanut was inoculated with B. subtilis showed effective control of fungi associated with seed (^{Illa et al., 2013}). In the results shown in Table 2, it must be highlighted that the growth of the A. flavus fungus was inhibited when B. subtillis was inoculated in peanut. Similar results were obtained by ^{Shifa et al. (2016}), who reported they inhibited the growth of A. flavus by 93-100% in dual crop trials and reduced the infection percentages in greenhouse and field trials. The authors highlight the importance of applying Bacillus to be able to reduce the presence of this fungus that can produce aflatoxins, which contributes to safe peanut production.

In field trials, the emergence of plants at 28 DAS (Table 3) in all the applied treatments was higher than that of the control (p≤0.05). The treatments that included biological treatments improved the percentage of emerged plants compared to that of the fungicide treatment, with no significant differences between the biological products used. The increase in emergence in the biological treatments could be due to the control of phytopathogenic fungi associated with peanut seed. In this regard, ^{Marani-Barbosa et al. (2013}) informed that by efficiently controlling A. flavus transported on the seed, they improved peanut seedling emergence in the field. Results in which a better effect was obtained by applying a combination of a biological and a fungicide, compared to the effect obtained when applied alone, have been already described for Bacillus (^{Illa et al., 2013}; ²⁰¹⁶) and Trichoderma (^{Vinale et al., 2008}). The plant growth measured at 30 DAS (Table 3) indicated that all the treatments with biological products were better than the control (p≤0.05). In addition, no significant differences were detected between the treatment with fungicide and the evaluated biocontrols. At 165 DAS, the positive effect of the applied biologicals, alone and combined, was observed and was statistically different from the fungicide treatment. So, the application of biologicals improved plant growth by 11.8% compared to the fungicide treatment, and by 27.4% compared to the control. The average value of mature husks was 29%, with no significant differences between the evaluated treatments (Table 3). Based on the obtained results, we can conclude that the maturity index behaved independently from the sanitary status of the husks and the application of the biocontrols.

Based on the results shown in Table 4, the husk and grain yields were higher in all the treatments compared to that of the control. In addition, the application of biologicals, alone and combined, increased grain yields by 45.2% compared to those of the control, and by 9.4% compared to the fungicide treatment. These results coincide with the results obtained by ^{Shifa et al. (2016}), who reported that when applying Bacillus and fungicide (Tebuconazole) to peanut seeds, there was an increase of around 40% in husk yield. The percentage of grains of confectionary quality (Table 4) did not show significant differences among the treatments applied to the seeds, but it did with the control. The yield increase appears to be a consequence of the growth promoter effect of the applied biologicals (^{Rojo et al., 2007}). In this regard, previous studies have shown that there is a positive correlation between the growth achieved by a peanut plant and its yield (^{Prassad et al., 2009}). The percentage of T. frezzi incidence on the husks was not significantly different between the control and the fungicide treatment (Table 4). This fact demonstrated that the synthetic product did not control the disease, in contrast with what ^{Astiz and Wojscko (2011)} observed in vitro, and ^{Buffoni and Marraro (2010}) in Colorado peanut in field trials. It must be highlighted that controlling this disease is relevant because of the important losses it causes and its potential spread to new crop areas (^{Ganuza et al., 2017}). The treatments with B. subtilis and T. harzianum that were applied, alone and combined, did not show significant differences (p≤0.05) between them, but they did when compared to the control and the fungicide treatment. The results obtained when biocontrols were applied show a decrease in T. frezzi incidence. This coincides with the results obtained by Ganuza et al. (2017), who evaluated peanut seeds inoculated with T. harzianum ITEM 3636, and controlled T. frezzi by 17% and 25%, depending on the evaluated year.

It is important to highlight that pre-treating seed with biologicals is an alternative for controlling this severe disease that reduces yields more than 30%, making peanut production economically non-viable (^{Paredes et al., 2017}). The results of this study show that there are benefits when applying B. subtilis and T. harzianum to peanut, from the phytosanitary perspective and for increasing yield. It should also be noted that numerous studies have shown that these microorganisms are safe and that there are no restrictions in the food codes when their presence is detected on grains. Inoculation with T. harzianum and B. subtilis under controlled conditions showed antifungal activity on the pathogens A. flavus, Fusarium sp., S. minor and T. frezzi in peanut plants at 60 DAS. The best control was achieved when the evaluated biologicals were combined. In field trials, the application of biological products, alone and combined, improved emergence at 28 DAS and plant growth at 165 DAS. This resulted in higher husk and grain yield, increase in the proportion of bigger grains, without affecting the maturity level. At the end of the cycle, the application of the evaluated biologicals reduced T. frezzi incidence by 14% compared with the control and the fungicide. The biocontrolling and stimulating effect of T. harzianum and B. subtilis on peanut growth suggests that their application will benefit the crop and can be considered a technological strategy to be added during sowing.