Oil formulations
One of the first steps for implementing the formulations with biocontrol fungi was to determine its compatibility with the microorganisms involved in the study. The percentage of T. asperellum TV190 spore germination in oil formulations without irradiation were 95.32, 96, and 97.66%, for the mineral oil (MO), vegetable oil (VO), and control, respectively (Figs. 1 and 2). It was observed that T. asperellum and emulsions seemed to be compatible, with non-significant differences (F = 0.018; df = 4; P = 0.99) between spore germination in formulations with mineral or vegetable oil. For the treatments with spores, exposed to UVR, a negative effect was evident on spore viability. Statistical differences were found in treatments with MO under UVB (F = 109; df: 32; P < 0.05) and UVC (F = 132.82; df: 32; P < 0.05) and VO under UVB (F = 176.3; df: 32; P < 0.05) and UVC (F = 132.39; df: 32; P < 0.05). In general, UVR decreased spore viability to 8–12% levels after 5 min of treatment on unformulated T. asperellum spores (NF), the effect being dependent on time exposure (Figs. 1 and 2).
However, in systems treated with oil formulations, partial protective effects were detected. After 5 min UVR exposure, viability decreased to 43–56% in systems with MO and to 56–63% in VO systems (Figs. 1 and 2). Once again, the negative effect was dependent on the exposure time. In treatments with lignosulphonate, non-significant differences were detected in the combination with VO under the same time of exposure to UVR (F = 0.16; df = 5; P = 0.71) (Fig. 2), while in the treatments with MO, differences were detected only when spores were exposed to UVB (F = 24.89; df = 5; P < 0.05) or UVC (F = 4.84; df = 5; P < 0.05) for 5 min (Fig. 1).
Results strongly suggested a protective effect of oil formulations and lignosulfonate to T. asperellum spores, when irradiated with UVR under laboratory conditions. Exposure time was a crucial parameter because spore viability was drastically reduced, when exposure increases from 1 to 5 min. A greater negative effect of UV-C radiation was also evident in spore viability. The UVR effect on fungal metabolism was related to DNA degradation in conidia and mycelium of Aspergillus nidulans (Braga et al. 2015). In addition, Seyedmousavi et al. (2014) reported that UVR affected proteolytic activity, cell growth, and carbohydrate synthesis in Candida albicans. Besides, it had also been reported that UV-B inhibited various fungal processes such as spore germination and hyphae elongation (Suthaparan et al. 2016) and affects negatively several fungi such as Botryris cinerea (Janisiewicz et al. 2016). Mutagenesis has been induced using UVR to obtain modifications in the genetic structure of two Trichoderma biocontrol agents, T. virens and T. asperellum (Alfiky 2019). If exposure time was longer, DNA damage will be great, producing a higher rate of mutations and reduced spore germination (Begum et al. 2009). UV-C radiation reduced the spore germination by more than 80% (Bell and Wheeler 1986). A decrease in spore viability by UVR depends on spore coloration, the medium in which it was evaluated, and time of exposure to radiation, the darker the spore, the greater its resistance to UV radiation, probably due to melanin that protects it from this radiation (Carzaniga et al. 2002). The photoprotective properties of melanin were considered to be important for the survival and longevity of spores (Bell and Wheeler 1986).
A protective effect of oil formulations against UVR has also been reported in other studies (Fernandes et al. 2015), observing mineral and vegetable oil protection on entomopathogenic fungi spores against UVR. Several oil-based formulations with T. asperellum have been developed to control cacao black pod disease caused by Phytophthora megakarya, in which the half-life of the conidia reached 22.5 and 5 weeks in aqueous and oil suspension, respectively (Mbarga et al. 2014). Oil and aqueous formulations have been proven to control frosty pod rot caused by Moniliophthora roreri on cocoa (Crozier et al. 2015), finding that an inverted corn oil formulation significantly enhanced cocoa yield, providing a promising model for optimizing Trichoderma-based biocontrol strategies. Finally, some vegetable oils are able to absorb UV radiation (Montenegro and Santagati 2019) suggesting the possibility to use them as UV blockers.
Greenhouse assays
The ability of T. asperellum formulations (granular and liquid) to control R. solani was evaluated by determining the number of infected plants and necrotic spot size (NSS) on corn leaves. Results showed statistical differences in treatments with T. asperellum (F = 1875.892; df = 29; P < 0.05) producing a decrease in the number of infected plants after being treated with T. asperellum (Fig. 3). The number of infected plants was similar (70%) in treatments with oil formulations (MO and VO) and with granules (G), both without T. asperellum, and in the treatment with R. solani alone (R) (F = 13.63; df = 8; P = 0.2). After applying T. asperellum spores to oil formulations (MOT and VOT) or to granules (GTR), the percentage of infected plants decreased to 20, 29, and 19%, respectively. The number of infected plants was reduced by 72% (MOT), 59% (VOT), and 73% (GTR).
Necrotic spot size (NSS) produced by R. solani on corn seedlings was another evaluated parameter (Fig. 4). Treatments without T. asperellum resulted in spot sizes larger than 20 mm (MO = 26.8 mm, VO = 28.31 mm, GR = 20.33 mm, R = 27.53 mm). In contrast, by using T. asperellum without formulation, NSS was 12.23 mm. When T. asperellum was included in oil formulations, spot size decreased to 2.67 mm (MOT) and 3.6 mm (VOT), with significant differences in relation to other treatments (H = 113.972; df = 9; P < 0.05), but not with each other (F = 0.222; df = 1; P = 0.64). The decrease in spot size caused by T. asperellum was 55.58%, when applied alone, compared to 90.04 and 87.29%, when applied with MO or VO, respectively. In GTR formulation, spot size decreased to 1.36 mm (93.32%).
In greenhouse assays, R. solani incidence was evaluated when liquid and granular formulations were applied. However, it is not possible to determine if the effect observed is due to the protection they exert against the UVR, since it was not possible to determine how much radiation these spores received. Obtained results were similar to those of Battan (2004) who used an oil formulation of T. harzianum to evaluate its biocontrol effect on Rhizopus stolonifer, Botrytis cinerea, and Penicillium expansum, fungi that affected apple, peach, pear, and strawberry. Although it was not studied in this work, one of the objectives of granular formulation was to serve as a substrate for fungal sporulation to increase spore number. The substrate used for granular formulation (DCG) has been characterized by the manufacturer containing 65.06% carbohydrates, 13.82% protein, 10.97% water content, 5.37% crude fiber, 4.19% of ash, and 0.59% of fats. It was also rich in minerals such as phosphorus, magnesium, iron, and zinc. DCG could be an excellent substrate for sporulation of several filamentous fungi. In this sense, a granulated formulation with DCG using the entomopathogenic fungus Nomurea rileyi spores was used to increase inoculum over 600 times and to protect spores from UVR (Pavone et al. 2009), probably because spores were immersed within a granule matrix, where UVR cannot reach them. Protection exerted by granule against UVR could be important for maintaining inoculum viability in the field.
Extruded granular formulations, containing rice flour, gluten, and biomass of Gliocladium virens and Trichoderma spp. among other components reduced eggplant damping-off caused by Rhizoctonia solani (Lewis and Larkin 1997). Formulations prepared with several components like talc and lignite were produced for seed treatment and control of tomato damping-off caused by Pythium aphanidermatum, in which active colonization of T. harzianum in the rhizosphere was observed (Jayaraj et al. 2006). Microencapsulation has been proposed to prolong shelf life and enhance application efficiency of Trichoderma (Cumagun 2014) focusing on seed treatment using solid matrix priming, liquid coating, and double coating.
It will be important to test these formulations under field conditions to obtain conclusive results that will allow their use as commercial products. Formulation compatibility with other control measures such as insecticides and herbicides commonly used in field should also be evaluated. Due to its mode of action and good performance under in vitro and greenhouse conditions, protecting spore against UVR and plants from R. solani, oil and granular formulation seems to have great potential to be incorporated in Integrated Pest Management Programs.