A recalcitrant soil inhabitant is the major threat for banana production

Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of the Fusarium wilt, also known as Panama disease (Figure 1). The hyphae of Foc accedes to the plant by the roots, once inside the plant, the fungus is incorporated into the xylem vessels and disseminates to the aerial tissue (^{Ploetz, 2015}). Yellowing of the leaves and bending of their petioles are typical symptoms of Panama disease (^{Pérez-Vicente et al., 2014}) (Figure 1). Browning of the pseudostem and necrosis of whole leaves are late disease symptoms. At this stage, Foc invades both xylem vessels and photosynthetic tissue. Three types of asexual spores are produced by Foc: macroconidia, microconidia and chlamydospores. Chlamydospores are spores produced when the plant decomposes, they are highly resistant to hostile conditions such as high radiation and temperature. Once chlamydospores are produced, they can persist in the soil for years in absence of the banana host, but they germinate and start its infectious cycle when banana plants are again placed in the fields (^{Ploetz, 2015}).

Figure 1: Symptoms of Fusarium wilt or Panama disease in a plantation of “plátano macho” from the coast of Oaxaca, southern Mexico (A). Growth of Fusarium oxysporum f sp. Cubense in potato dextrose agar, mycelium is initially white and later turns into red to purple (B). Trichoderma sp. Growing in potato dextrose agar, production of a ring of green conidia is characteristic of this fungus (C). 

Foc is a specialist pathogen of Musa plants, it is further classified into three races according to the host cultivar. Foc race 1 (FocR1) is pathogen of plants from the Gros Michel subgroup, which dominated the global market during XIX and early XX Centuries. Epidemic caused by Foc1 eliminated the production of Gros Michel. Recovery of banana production was achieved by the introduction cultivars from the FocR1-resistance Cavendish subgroup. Foc race 2 (FocR2) is pathogen of Bluggoe bananas. Foc race 4 in divided into tropical race 4 (FocTR4) and subtropical race 4 (FocSTR4), depending on the climate conditions where pathogen cause disease. They are pathogens of the bananas from the FocR1-resistant Cavendish subgroup, and shows increased virulence towards host of FocR1. Foc race 3 (FocR3) is rather pathogen of plants the genus Heliconia and it does not affect Musa plants (^{Ploetz, 2015}). 3Foc can invade weeds or species that coexist in the banana plantations (^{Pérez-Vicente et al., 2014}), which contributes to the persistency of this pathogen.

The outbreak of FocTR4 started in 1986 when FocR1-resistant Cavendish plants grown in Southeast Asia displayed Fusarium wilt, since then, FocTR4 has caused dramatic loses in producers in Southeast Asia, Australia, Africa (^{Ordonez et al., 2015}). FocTR4 has been recently reported in Israel (^{EPPO, 2018}), which entailed total destruction of the plants, however, due to the durability of the chlamydospores in the soil, the destruction of the plants does not eradicate the problem. The high similarity between the isolate from Israel and an isolate form the neighbor country Jordan is an evidence of the pathogen dispersion. The dispersion of FocTR4 has entailed a decrease in the global production and import restrictions in producers such as Philippines and Mozambique (^{FAO, 2017}). There are no reports of the presence in Latin America and the Caribbean, but the expansion of the pathogen by climatic events or infected rhizomes are factors that alert the danger of a future establishment of FocTR4 in Latin America. Furthermore, cultivars susceptible to FocR1 and FocR2 are still produced in Mexico (^{Nadal-Medina et al., 2009}). This scenario forces us to seek methods of management in the field, and thus ensure the supply of bananas and the welfare of the people involved in this production chain. The physical resistance of chlamydospores together with the ability to produce other two types of spores that contribute to preserve the fungus under unfavorable conditions, suggest that the methods of disease control must attack vegetative mycelium before the accession into the root. Therefore, native microbes already adapted to the soil that compete with this type of pathogens are reliable candidates of biological control agents.

The soil and rhizosphere are source of sustainable agents for protection of banana plants

The total amount of carbon in the soils is higher than the carbon pools in vegetation and the atmosphere (^{Tarnocai et al., 2009}). Atmospheric CO₂ is fixed in plants as unique source of carbon, and distinct organic compounds are produced by the plant’s metabolism, which together with the availability of organic nitrogen serves as nutrient sources that boosts the generation and selection of the microbial biodiversity in the soils. Fungal diversity in soil play important roles in modulating the soil carbon cycling, plant nutrition and pathology. Due to the eco-physiological relationship between plants and fungi, the fungal diversity positively correlates to the plant diversity (^{Tedersoo et al., 2014}). The fraction of the soil influenced by the roots is known as rhizosphere (^{Toal et al., 2000}). At the rhizosphere, microbial population is even higher than in plant-free portions of soil. The high microbial diversity implies that such microbes play distinct roles, either providing benefits to plants such as sources of organic nitrogen and phosphorus or causing detrimental effects as in the case of Foc and other pathogens. In both cases, the response of the fungus is induced after the sensing of roots exudates produced by the root metabolic activity (^{Bais et al., 2006}). Accumulation of root exudates in soil is a consequence of the metabolic flux of carbon. Organic compounds produced from the fixation of atmospheric CO₂ by the plants are incorporated in primary and secondary metabolism pathways that generates a combination of compounds secreted by the roots. Composition of the exudates is specific for every plant species, and they directly promotes the selection of specific phylotypes of the fungal community (^{Broeckling et al., 2008}). Hence, the biodiversity of the rhizosphere is plant-specific, including saprophytes, beneficial and pathogenic fungi. Foc is adapted to interact with diverse microbes at the rhizosphere of banana plants, and it is also able to sense the metabolism of the living roots necessary to start with its infective process. Survival of beneficial species such as endophytes depends on healthy banana roots; they might compete against Foc to directly colonize root tissue, and then, decrease the root surface prone to get infected. Competition for carbon or nitrogen sources between saprophytes and Foc at the rhizosphere can also affect their respective populations. Foc has to be quite adapted to the micro-environment of the rhizosphere however, introduction of adapted saprophytes in plantations of banana might be helpful to restrain the abundance of this pathogen.

Fungal inhabitants of the soil known as mycoparasites can take nutrients by the hyphae of another fungi, have the ability to destroy hyphae by mechanical force and production of cell wall degrading enzymes, produce antibiotics that inhibit growth of the host, furthermore, they can also protect plants by stimulation of the plant immune system (^{Brimner and Boland, 2003}). All this features have to be considered to select novel biological control agents in the soil of banana plants. In particular, several species of the genus Trichoderma -a ubiquitous soil fungus- fulfills the features that allow their implementation to protect plants against Fusarium wilt.

Soil Inhabitants from the genus Trichoderma as biological control agents

Species in the genus Trichoderma are mycoparasite of many fungal phytopathogens, some are endophytes and others also plays beneficial roles to the plants providing soil nutrients, plant growth promotion and induction of resistance to biotic and abiotic factors (^{Contreras-Cornejo et al., 2016}) (Figure 1). The first evidence of Trichoderma as mycoparasite was reported in 1932, and the first commercial formulation was registered in 1989, since then, over the 60% of the biopesticides are based on conidia or mycelia of Trichoderma spp. (^{Mukherjee et al., 2013}). Trichoderma harzianum has been used mainly to control leaf fungal pathogens (^{Samuelian, 2016}), however, its characteristics as a fungus adapted to the soil and rhizosphere, suggest that it could be a reliable alternative to prevent the Fusarium wilt of banana. Here, we describe and discuss the features of Trichoderma as a microorganism that offers multiple beneficial effects to plants.

Antifungal activity. Secondary metabolites are extensively produced by Trichoderma spp. (^{Reino et al., 2008}). Those compounds confer adaptive advantages, and contribute to the colonization of Trichoderma into very different ecological niches. It is well documented that diverse biosynthetic pathways in Trichoderma spp. produce metabolites such as peptabiols, polyketides and terpenes with antifungal activity (^{Keswani et al., 2014}). Production of antifungal metabolites by Trichoderma might restrict the growth of surrounding fungi, which facilitates the consumption of free nutrients and dispersion in the rhizosphere. Furthermore, direct antifungal activity might be required as part of the process of mycoparasitism. Likewise, Trichoderma can uptake and utilize nutrients directly from other fungi by mycoparasitism. Fungal recognition by Trichoderma leads the attachment to the hyphae of the host and formation of appressoria-like structures that exert a physical force over the host (^{Harman et al., 2004}). During mycoparasitism, the production of cell wall degrading enzymes such as chitinases, glucanases and proteases is necessary to utilize host macromolecules as carbon and nitrogen source (^{Gruber and Seidl-Seiboth, 2012}). The ability to sequester fungi by appressoria-like structures, production of antifungal compounds and activity of cell wall degrading enzymes are adaptations acquired by Trichoderma that place it as a potent fungal antagonist. Also its persistency in the soil could be potentiated by roots exudates produced by plants that took advantage of its antifungal character.

Iron uptake. Iron is an essential micronutrient, nevertheless, most of the iron present in the soil is insoluble. The absorption of available iron is mediated by the secretion of siderophores that chelate and carry Fe⁺³ into the cell, which is later reduced to Fe⁺². Harzianic acid is a Fe⁺³ siderophore produced by Trichoderma harzianum that promotes the growth of canola seedlings, possibly by facilitating the Fe⁺³ uptake to the roots, and also showing in vitro antifungal activity against phytopathogens such as Rhizoctonia solani (^{Vinale et al., 2009}). Over 10 different siderophores are produced by the Trichoderma spp. (^{Lehner et al., 2013}); which suggest the prominent role of Trichoderma in the mineral relocation between organism of the rhizosphere. During saprophytic growth, Foc and other phytopathogens might not have eff icient systems of iron uptake, hence, colonization of plant could facilitate the acquisition of this micronutrient. Fe⁺³ chelation in the rhizosphere might have a simultaneous effect as provider of Fe⁺³ to the plants and as inhibitor of Fe⁺³ uptake by the phytopathogens.

Promotion of plant growth. Mutual benefit between Trichoderma and plant species is favored by the physical contact between roots and this fungus, thus, the exchange of nutrients and signal molecules is facilitated. Moreover, the presence of Trichoderma at the root surface or inside the roots functions as a barrier that impedes the access of pathogens such as Foc (^{Contreras-Conejo et al., 2016}). In addition to colonizing the epidermis of cucumber roots, Trichoderma harzianum can penetrate and growth in intracellular spaces of outer cortical cells (^{Yedidia et al., 1999}). Roots colonized by Trichoderma adopt a barrier that protect them against subsequent pathogens invasion. Under hydroponic conditions, Trichoderma harzianum promoted an increased biomass production of cucumber plants as well as the germination rate of the seeds (^{Yedidia et al., 2001}). Several reports reviewed by ^{López-Bucio et al. (2015)} illustrate the positive effect of Trichoderma spp. as stimulator of growth of plant aerial tissue or roots. This stimulation of plant growth is consequence of changes in the hormonal balance (^{Guzmán-Guzmán et al., 2018}). Hormones produced by Trichoderma at the contact interface of the root can be efficiently recognized by the root cells, and consequently modulate cell proliferation and differentiation in the roots or get transported to induce a response in distant parts of the plant. Auxins, well-known phytohormones, are produced by Trichoderma virens (^{Contreras-Cornejo et al., 2009}). Auxins produced by Trichoderma virens are biologically active since auxin-dependent responses in the roots were also reported in the same research (^{Contreras-Cornejo et al., 2009}). Root branching induced by Trichoderma virens broaden the volume of soil explored, thus, increases the uptake of nutrients and water required to growth. Analysis of the Trichoderma atroviride genome identifies genes encoding biosynthetic enzymes of cytokinins, gibberellins, ethylene and abscisic acid, whose crosstalk mediates plant development (^{Guzmán-Guzmán et al., 2018}). The production of other fungal hormones has been demonstrated, but this finding suggests that the concentration of phytohormones in the root is directly altered by Trichoderma as a mechanism to potentiate endogenous signals of development. Besides the typical phytohormones, volatile compounds of low molecular weight are produced by Trichoderma atroviride, they enhanced the fresh weight and the production of secondary roots in the model plant Arabidopsis thaliana (^{Nieto-Jacobo et al., 2017}). All these results show that Trichoderma is endowed with biostimulant activity that complements its use as a biocontrol agent.

Induction of plant defense response. Conserved molecules among microbes, either phytopathogens or saprophytes can be recognized by the plant to induce defense responses (^{Boller and Felix, 2009}). Likewise, the effect produced by phytohormones, the attachment and penetration of Trichoderma spp. inside the roots facilitates identification of the chemical composition of the hyphae (for example, chitin oligomers and secreted proteins), that triggers a local defense response that might not be strong enough to kill the microbiota of the rhizosphere and Trichoderma itself, but can limit invasion of root phytopathogens such as Fusarium (^{Jogaiah et al., 2018}). The activity of cell wall degrading enzymes releases small fragments of cell wall constituents that can also be perceived by the plant and boost a defense response. The presence of Trichoderma in the root induces signaling mediated by of stress-related phytohormones such as salicylic acid and jasmonic acid (^{Salas-Marina et al., 2011}), and promotes establishment of preventive defense responses in distal parts of the plant, known as induced systemic resistant (ISR), that protect the plant against subsequent pathogen attacks, and it is also triggered by beneficial rhizobacteria (^{Mathys et al., 2012}; ^{Pieterse et al., 2014}). Enzymes related to biosynthesis of salicylic acid have been identified in Trichoderma atroviride (^{Guzmán-Guzmán et al., 2018}), which might be required to increase the salicylic acid pools during specific stage of the interaction. Promotion of the ISR by inoculation of Trichoderma hamatum in the roots of Arabidopsis thaliana suppressed necrotic lesions in leaves inoculated with the pathogen Botrytis cinerea (^{Mathys et al., 2012}). Same protective effect mediated by different Trichoderma spp. has been reported in soybean (^{Zhang et al., 2016}), tomato (^{Martínez-Medina et al., 2013}), and pearl millet (^{Siddaiah et al., 2017}). These evidences suggest that ISR has a general effect produced by Trichoderma spp. that enabled its selection as inhabitant of the rhizosphere. Beneficial microorganisms such as the species of Trichoderma also induce a priming effect. Priming consists on the induction of a physiological state of enhanced sensitivity to the presence of pathogens, and hyperactivation of induced defense responses when pathogens invade the plant (^{Martínez-Medina et al., 2016}). This effect is also promoted by Trichoderma spp. in different plants, it is effective against the fungus Botrytis cinerea (Martínez-Medina et al., 2013), the oomycete Plasmopara viticola (^{Perazzolli et al., 2011}) and the nematode Meloidogyne incognita (^{Martínez-Medina et al., 2017}).

The profitable ability of Trichoderma to induce general preventive defense responses should be explored in Trichoderma-banana interactions to develop methods to control the Fusarium wilt and other diseases.