**1. Introduction**

Sclerotia are survival melanized structures of different sizes and shapes depending on the fungal species and host plant. They remain viable in soil for long periods of time and are resistant to chemicals, adverse conditions, and biological degradation [1]. *Sclerotinia sclerotiorum*, *Rhizoctonia solani*, *Athelia rolfsii*, *Stromatinia cepivora*, and *Microphomina phaseolina* are among the most harmful sclerotia-producing phytopathogenic fungi in agriculture [2–6]. These pathogens are hard to manage and cause expanding losses in horticultural crops worldwide due to their survival capabilities associated with successive production of the same crop in the field and the lack of safe and efficient soil fumigation methods.

Biological control products formulated with *Trichoderma* have grown as the best method to manage sclerotia-producing pathogens, once chemicals in soil are often inefficient, too expensive, and too environmentally harmful. All *Trichoderma* species are able to utilize the cell content of other fungi as a source of nutrients [7–9]. However, certain species are more adapted to parasitize sclerotia of pathogenic fungi [2–6, 10–12]. These species are able to successfully invade, parasitize, and kill sclerotia in soil, meaning that the capacity to survive and compete in this environment is an absolute requirement. Although *Trichoderma* is one of the most commonly found genera in soil, not all species of the genus are well adapted to survive and thrive in soil [13]. The species adapted to soil need to be equipped with structures such as chlamydospores to ensure their long-term survival.

*Trichoderma* is a hyper diverse genus, with more than 350 species already described [14]. Only a small fraction of these species were developed into commercial products. This raises the questions which *Trichoderma* species have potential to be commercialized? Only the limited number of species that is well adapted to the soil environment and frequently are the active ingredients of formulations. Or is there any potential in the unexploited species?

In this chapter, we adopted a more practical view of the use of *Trichoderma* to control sclerotia-producing fungal plant pathogens. More emphasis is given on the mechanisms employed by *Trichoderma*, the species used in commercial formulations and the practical use of *Trichoderma* to control sclerotia producers in the field in Brazil, where products containing these fungi are used by farmers to manage white mold disease in soybean.

### **2. Plant pathogens capable of producing sclerotia**

Sclerotia-producing fungi are very diverse, including saprotrophic, plant, animal and insect pathogens, mycoparasites, endophytes, insect symbionts, ecto- and ericoid-mycorrhizal fungi, and lichenicolous fungi [15]. However, they seem to be exclusively produced by fungi in the phyla Ascomycota and Basidiomycota that comprise the subkingdom Dikarya of the kingdom Fungi [15]. Sclerotia were documented in at least 85 fungal genera and 20 orders [15]. Outside of the kingdom fungi, sclerotia are relatively well studied in the slime molds or Myxomycetes [16], which belong to the kingdom Amoebozoa.

Sclerotia-producing fungi may be found in tropical and temperate regions, although sampling for these structures in natural environments is still scarce [15]. The structure and shape of fungal sclerotia are highly variable. While some are surrounded by much defined melanized ring encircling undifferentiated hyphae, others lack a distinct ring [17]. Some species, such as *Athelia rolfsii*, produce sclerotia with a round and smooth surface and dark color, whereas other fungi such as *Rhizoctonia solani* produce sclerotia with an irregular shape and lighter color. Some sclerotia are very small (sometimes smaller than 1 mm) such as the ones produced by *Macrophomina phaseolina*, *Stromatinia cepivora*, and *Verticillium dahliae*, whereas some fungal species, such as *Polyporus mylittae*, are able to produce giant sclerotia of up to 40 cm in diameter [18]. Fungi, such as *Claviceps purpurea*, *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Monilinia* spp., produce apothecia, which are sexual reproductive structures, directly on the sclerotia, whereas *Aspergillus flavus* produces cleistothecia inside the sclerotia [19].

Garlic and soybean are examples among the economically important crops where *Trichoderma* has been used to manage sclerotia-producing pathogens, at least under experimental conditions. Garlic cultivation in Brazil is done in the provinces of Goiás, Minas Gerais, Santa Catarina, and Rio Grande do Sul and it reached 118,000 tons in 2018 [20]. High losses have been induced by the white rot disease caused by the fungus *Stromatinia cepivora*, which is a pathogen specific to plants of the family Alliaceae [21]. *Stromatinia cepivora* belongs in the Ascomycota phylum, Helotiales order, and Sclerotiniaceae family [22]. The white rot caused in plants of the Alliaceae family presents well characterized symptoms, such as yellowing and death of infected leaves, owing to the root system damage caused by the pathogen. The aerial part of infected plants is easily detached from the soil. In garlic bulbs, the symptoms are soft rot of the tissues and white mycelial on the structural axis of infected plants and production of black microsclerotia on the bulbs [23]. The microsclerotia (ranging from 0.2 to 0.5 mm diameter) can remain dormant and viable in soil for more than 20 years in the absence of suitable host plants [24].

Soybean is an agricultural commodity traded not only in Brazil but is also exported [21]. In this crop, the white mold caused by *S. sclerotiorum* is considered the second most important disease after soybean rust [24, 25]. *Sclerotinia sclerotiorum*, which also belongs in the family Sclerotiniaceae, promotes the white mold or sclerotinia stem mold, leading to losses that can reach 70% in productivity. It occurs in approximately 30% of the Brazilian soybean producing area, which is currently more than 35 million ha [26]. The pathogen infects more than 600 plant species and is distributed worldwide. The sclerotia (from 0.5 to 10 mm diameter) are able to survive up to 12 years in soil (50, 51).

*Rhizoctonia solani* and *Athelia rolfsii* (syn. *Sclerotium rolfsii*) are two sclerotiaproducing basidiomycetes from the families Ceratobasidiaceae and Atheliaceae, respectively, that cause rots in seeds, roots, stems, leaves, and fruits of a wide range of plant species (52, 53). These pathogens are commonly found in warm climates causing damping-offs, root rots, and wilts. The sclerotia are the only survival structures of these pathogens as they do not reproduce asexually and only rarely reproduce sexually.

These resistance structures are extremely difficult to destroy or inactivate by chemical methods, whereas physical methods such as solarization, inundation, and radiation are not feasible or too expensive to be adopted over large areas. In this context, biological control with antagonistic fungi of the genus *Trichoderma* is one of the best options available. The advantages of *Trichoderma* include its mycoparasitic capacity toward these structures, the excellent adaptation of certain species to the soil environment, the safety to humans and animals of the biocontrol species, and the relative ease of mass producing and formulating these agents.

### **3. Mechanisms employed by** *Trichoderma* **against sclerotia producers**

The mechanisms of activity present in *Trichoderma* include mycoparasitism, antibiosis, competition, induction of resistance, and plant growth promotion [27–30]. Mycoparasitism is the main mechanism employed against sclerotia in soil [31], although the benefits observed in the field probably result from a combination of all mechanisms acting in concert. Mycoparasitism is defined as the ability of organisms to actively parasitize fungi and live at their expense [31]. Mycotrophy, a more inclusive term, may be defined as the ability of organisms to feed on either dead (passive mycotrophy, i.e., saprophytism) or on living fungi (active mycotrophy, i.e., mycoparasitism) [9]. This ability to feed on fungi, either dead or alive, was shown to be the ancestral form of nutrition in all *Trichoderma* species [13]. Although there are technical differences between mycotrophy and mycoparasitism, the latter term is the only one traditionally employed for *Trichoderma* in the literature, even in cases where there is no evidence that these fungi killed the host or prey.

Mycoparasitism by *Trichoderma* involves a sequence of events, including host localization, recognition, direct contact, coiling, formation of hook-shaped structures with appressorium function, penetration, folding, and development of parallel hyphae [29, 31–37]. It involves a combination of invasive hyphae with secondary metabolites and hydrolytic enzymes in most cases [38]. The wide range of *Trichoderma* secondary metabolites includes epipolythiodioxopiperazines (ETPs), peptaibols, pyrones, butenolides, pyridines, azaphilones, steroids, anthraquinones, lactones, trichothecenes, and harzianic acid [28]. These compounds can interfere with the metabolic activities of other microorganisms by inhibiting growth and sporulation, reducing spore germination, and weakening the sclerotia. Many *Trichoderma* species are strong producers of cellulases, chitinases and β-1,3-glucanases, proteases, and lipases, which act in concert with metabolites in the mycoparasitic activity of these fungi [31, 39–45].

Three distinct strategies of mycoparasitism were described for *Trichoderma*, which were supported by transcriptomic analyses [7]. These strategies are 1) passive or weak mycoparasitism, where species such as *T. reesei* have no capacity to stop the growth of fungi, but secrete cell wall degrading enzymes that slowly dissolve the mycelium of the host or prey; 2) strong mycoparasitism occurs in *T. atroviride* that actively and aggressively grows over the host and parasitizes it swiftly and produces proteases and glucanases; 3) mycoparasitism by lytic enzymes and toxic metabolites, which is observed in *T. virens*, that first produces metabolites such as gliovirin and gliotoxin that kill the host and later, the mycoparasite moves in and further produces lytic enzymes to digest the mycelium. Probably, strategies 2 and 3 will be more effective against sclerotia producers. There is solid evidence in the literature showing that *Trichoderma* behaves markedly differently in interactions with different fungal species or Oomycetes [9]. These differences are seen at the phenotypic level and at the gene expression level, where distinct mycoparasitism strategies are employed depending on the host or prey [9].

In this study, scanning electron microscopy observations of *in vitro* and in soil interactions between *Trichoderma* spp. and *S. sclereotiorum* (*Tr.* × *Ssc.*) and with *Stromatinia cepivora* (*Tr.* × *Sce.*) were done with scanning electron microscopy (SEM). Colonization of *S. sclerotiorum* sclerotia and *S. cepivora* microsclerotia by aerial mycelium of *Trichoderma* spp. was easily seen with the naked eye 7 days after the inoculation (**Figure 1A** and **I**). SEM analysis revealed the *Trichoderma* aerial myceliacolonizing sclerotia of *Ssc.* and *Sce.* (**Figure 1B** and **J**). Conidia and conidiophores of *Trichoderma* spp. are produced on the surface of the sclerotia (**Figure 1C**, **D**, **M–O**). Cryo-fractures in both *Ssc.* sclerotia and *Sce*. microsclerotia evidenced the central medulla enclosed by the outer layer of rind cells free of *Trichoderma* colonization after 7 and 14 days after the incubation, respectively (**Figure 1B** and **K**). Aerial mycelia originated from the sclerotia were efficiently colonized by *Trichoderma* (**Figure 1E–H**, **M–P**). **Figure 1G** shows decaying hypha of *S. sclerotiorum*, indicating that cell wall-degrading enzymes acted on the pathogen.

#### **Figure 1.**

*Scanning electron microscopy (SEM) observations of the interactions between* Trichoderma *and Sclerotinia sclereotiorum (Tr. × Ssc.) and with Stromatinia cepivora (Tr. × Sce.). A. Photography of Ssc. Sclerotia colonized by Tr. After seven days under 17°C above sterile soil.* B–H *and J–P: Scanning electron micrographs. B and K. Cryo-fractured Ssc. Sclerotia and Sce. Microsclerotia evidencing the central medulla enclosed by the outer layer of rind cells, respectively. C–D. Trichoderma conidiophores producing conidia above Ssc. Sclerotia surface. E–H. Trichoderma-parasitizing Ssc. Hyphae. I. Photography of Sce. Microsclerotia colonized by Tr. After 14 days under 17°C above sterile soil. J–L. aerial mycelia of Sce. And Trichoderma above Sce. Microsclerotia. M–O. Trichoderma conidia and conidiophores above Sce. Microsclerotial surface. M–P.* Trichoderma *parasitizing Sce. Hyphae. The hyphae of Ssc. And Sce. were thicker than the hyphae of Trichoderma. a.m. = aerial mycelia; rin. = outer layer of rind cells in sclerotia; c.med. = central medulla enclosed by the rind cells in sclerotia; Tr.con. = Trichoderma conidia; Tr.cdf. = Trichoderma conidiophore; Tr. = Trichoderma; Ssc. = Sclerotinia sclerotiorum; Sce. = Stromatinia cepivora. Scale bars: A and I: 5 cm; B and J: 100 μm; C–H and K–P: 10 μm.*

#### **4. Species of** *Trichoderma* **employed against sclerotia-producing fungi**

In 2014, there were 177 *Trichoderma*-based fungicides commercially available in the world [46]. These products contained mainly *Trichoderma asperellum, T. hamatum, T. harzianum*, and *T. viride* as active ingredients and were recommended mainly for seed and soil treatments [46]. In Brazil, there are currently 34 formulated products with *Trichoderma* as active ingredients registered in the Ministry of Agriculture, Livestock and Food Supply (MAPA) (**Table 1**) [39, 47, 48]. These 34 products are based on four species: *T. harzianum*, *T. asperellum*, *T. koningiopsis*, and *T. stromaticum*.





Most products are indicated for sclerotia-producing pathogens, for example, 26 products are recommended for *S. sclerotiorum*, 23 for *R. solani*, three for *Asclepias rolfsii*, and two for *Macrophomina phaseolina*. Most products are formulated with one strain and only six are combinations of strains. Some of the products are recommended to manage Oomycetes, nematodes, or for *Moniliophthora perniciosa*, the causative agent of cacao witches' broom disease (**Table 1**). No products are available for *S. cepivora*, even though potential *Trichoderma* strains are described in the literature [49].

Most commercial products based on *Trichoderma* are recommended for soil applications. Soil environments have few variations in temperature and humidity than the aerial parts of plants and these biocontrol agents show more potential in more stable niches. In 2020, there were more than 350 described species of *Trichoderma* in the world [14] and although only a limited number of species (approximately 30) appear to be well adapted to soil environments, the number of species used in commercial products is certainly under-represented. Additionally, it is possible that some of the species listed in **Table 1** are not identified correctly at the species level, as shown for members of the *T. harzianum* species complex [50].

Many species of *Trichoderma* other than the ones listed in **Table 1** were shown to have potential in the inactivation of sclerotia. *In vitro* assays performed by the first author of this chapter demonstrated the potential of different species of *Trichoderma* and eight undescribed species to colonize sclerotia of two pathogenic fungi (**Table 2**). Some of these strains were able to colonize up to 100% of the sclerotia of S*. cepivora* in soil (**Table 2**). The sclerotia of *S. sclerotiorum* appear to be more resistant to colonization than the ones produced by *S. cepivora*. Some of the novel strains were superior in comparison with a commercial product based on *T. harzianum*. These data underscore the potential of other than the species that are traditionally commercialized and novel *Trichoderma* species to be developed into commercial products to control sclerotia-producing plant pathogens. However, this potential has yet to be confirmed in field trials.



*Sclerotia of the pathogens were placed on the soil surface and sprayed with a suspension of* Trichoderma *conidia. The boxes containing the sclerotia were incubated at 17°C for* S. cepivora *and at 25°C for* S. sclerotiorum*. The evaluations were done at 7 and 14 days after the inoculations. The data are averages of two experiments with five replicates per experiment. \* Species identification was done by sequencing the tef-1 fragment. \* Not determined.*

#### **Table 2.**

*Colonization of sclerotia of* Stromatinia cepivora *and* Sclerotinia sclerotiorum *by strains of* Trichoderma*.*
