**5. Interaction with other beneficial microorganisms**

The combination of more than one biocontrol agent is thought to be advantageous, but it depends on the individual strains compatibility [51]. Six out of the 34 registered products in Brazil are formulated with one or two *Trichoderma* and *Bacillus amyloliquefaciens* strains (**Table 1**). However, it is not known whether these microorganisms are compatible or not or if there is any synergism in their combination. Bacterial genera such as *Bacillus* and *Pseudomonas* are potential biocontrol agents of soil-borne pathogens due to the secretion of antibiotics and lytic enzymes in the rhizosphere of plants. Therefore, they are potential agents to be combined with *Trichoderma*, especially when they do not inhibit each other [51–55]. However, the compatibility of combinations needs to be evaluated with *in vitro* and *in planta* assays [56].

Interactions between *Trichoderma* and mycorrhyzae are sometimes antagonistic, such as with the ectomycorrhyzal basidiomycetous genus *Laccaria* spp., where there was clear inhibition of growth, colonization, and spore germination on both partners [57–59]. However, sometimes these interactions are synergistic, such as with *Glomus* spp. Although there was an increase in plant biomass in the interaction, microscopical observations clearly showed that *Trichoderma* was parasitizing this endomycorrhyzal fungus [60]. *Trichoderma* can parasitize the hyphae of the endomycorrhyzal fungus *Glomus irregulare* and gain entry into potato roots [61]. On the other hand, endomycorrhyzal species of *Rhizophaga* use *Trichoderma* to penetrate into the roots of nonhost Brassicaceae, resulting in increased plant productivity [62].

The compatibility and synergism in interactions between *Trichoderma* and other beneficial microorganisms is so specific that they vary according to the strain of each partner and the host plant. Therefore, determining the outcome of these interactions is crucial for the successful field applications.

#### **6. Field trials and uses in agriculture**

Biological control of plant diseases is a reality in the agricultural world, since the abuse and inappropriate use of chemicals have led to major problems to the environment and human health. In view of sustainable agriculture, the use of chemical molecules is becoming unfeasible due to their high cost and toxicity. The use of products based on *Trichoderma* proved to be effective, especially against root pathogens able to produce resistance structures such as sclerotia [63–65]. The application of these microorganisms aiming to manage different plant diseases can be performed on seeds before planting, *via* foliar spraying, in the substrate, in the planting furrow or even in organic matter that will be incorporated before transplanting seedlings [66]. The form of application of products formulated with these microorganisms depends on the target to be controlled, the host crop, the environmental conditions, and the manufacturer's recommendations.

In Brazil, the control of white mold in soybeans is done by a combination of biological and chemical methods. This system will be used here as a case study to exemplify the application of *Trichoderma* in the field. White mold is the second most important soybean disease in Brazil and causes between 20 and 30% of losses on average, but under some conditions may reach 70–100% [26, 30, 67]. Approximately 10 million ha of soil is infested by this pathogen in Brazil out of total 35.9 million ha devoted to soybean cultivation in the country [67]. Approximately 5 million ha of soybean is currently treated with *Trichoderma*-based products in Brazil [67]. A common recommendation for white mold management is one spray application of *Trichoderma* at the vegetative stage V2 and another application at V4-V6 with concentrations varying from 109 to 1011 CFU/ha, depending on the commercial product adopted. Spray application of *Trichoderma* should be done on overcast days with high soil humidity and mild temperatures. Since the levels of resistance in commercial cultivars are not satisfactory, the combination of other practices is desirable. No-tillage planting with mulch produced by *Urochloa ruziziensis* (Syn. *Brachiaria*) is highly recommended as it stimulates the sexual germination of sclerotia and at the same time functions a barrier for the spread of ascospores produced in apothecia [67, 68]. This mulch will also provide conducive conditions for the colonization of sclerotia and apothecia by *Trichoderma* [68, 69]. Another practice that must be adopted is the application of fungicides at the reproductive stage R1 and another application 15 days later. The most commonly used fungicides are fluazinam, thiophanate-methyl, procymidone, carbendazim, and trifloxystrobin [26]. Monitoring is essential for finetuning these recommendations to specific locations and environmental conditions. Some farmers may adopt the biological seed treatment with *Trichoderma* on top of the standard chemical seed treatment, as an additional measure to control damping-off, which is caused by many soil-borne pathogens, including *S. sclerotiorum*.

To verify the efficacy of the biological treatments, some of the manufactures of *Trichoderma* provide Petri plates with culture medium to farmers. Sclerotia should be collected from soil at the end of the cycle of soybean, but before harvest and plated to determine the percentage of sclerotia colonized by *Trichoderma*. When the


*The experiment was done in the field with four replicates per treatment. Trichoderma was applied at a concentration of 109 CFU/ha at the stages V4 and V6 and the fungicides at the stages R1 and R2. The area under the disease progress curve (AUDPC) was determined by integrating multiple measurements of disease severity; disease incidence was determined by measuring the percentage of infected plants per treatment; the number of sclerotia per ha was determined by separating them from seeds with sieves and weighing; TGW is the total grain weight and yield was determined by weighing. Means followed by different uppercase letters in the same columns are statistically different by the Scott-Knott test (p* ≤ *0.05).*

#### **Table 3.**

*Combination of Trichoderma and fungicides to control Sclerotinia stem rot in soybean.*

level of colonization of sclerotia in the plates is above 50%, the level of control is considered satisfactory. Although this level of control cannot be directly correlated with success, they serve to show that *Trichoderma* is present in the treated area at a relatively high rate.

Control of white mold is also dependent on the density of sclerotia in soil. Best results are obtained when the densities are 1–10 sclerotia/kg of soil [30]. Field experiments have shown that *Trichoderma* spp. as an exclusive control method is not sufficient to reduce the severity of white mold (**Table 3**). Fungicides normally have to be deployed to complement the activity of *Trichoderma*. However, in this experiment, where 1 year only was evaluated, two *Trichoderma* strains used alone were able to maintain the productivity at high levels even in the absence of fungicides. The use of *Trichoderma* in multiple years is expected to promote the build-up of inoculum in the soil and consequently decrease the levels of sclerotia to acceptable levels [70]. In the long run, the objectives are to maintain the inoculum in equilibrium and increase the plant growth and productivity.

#### **7. Conclusion**

In Brazil, 34 *Trichoderma*-based products are currently registered and most of them are recommended to control soil-borne pathogens that produce sclerotia as resistance structures. Relatively few species of the genus *Trichoderma* were developed into commercial formulations, despite the high number of publications that have shown the potential of many other species. *Trichoderma* spp. are widely used in Brazil to control white mold in soybean and its use is expected to increase in the near future as only 50% of the infected area is currently treated with these biocontrol agents. Besides providing partial control of white mold, these fungi can also increase plant growth and productivity coupled with a reduction in the use of chemical fungicides.
