In vitro inhibition of Fusarium solani by Trichoderma harzianum and biofertilizer Inibição in vitro de Fusarium solani por Trichocherma harzianum e biofertilizante Inhibición in vitro de Fusarium solani por Trichocherma harzianum y biofertilizante

Cassava root rot causes significant production losses. Difficulties of management, along with the lack of chemical fungicides officially registered by the Ministry of Agriculture, Livestock and Supply (MAPA), require alternative control methods. This study investigated the in vitro antagonistic activity of Trichoderma harzianum as well as a biological fertilizer MICROGEO on Fusarium solani. The phytophatogenic strains of F. solani, called F1 and F2 were isolated from rotted cassava tubers and T. harzianum, strain ESALQ 1306, from a biological fungicide. Continuous liquid composting of bovine ruminal content, water and MICROGEO produced the biological fertilizer. Dual culture method was used at the bioassay with T. harzianum. Sterilized (St) and unsterilized (USt) biological fertilizer were tested in different concentrations (% v/v) diluted in the culture media. Colony diameters were measured daily in order to establish the mycelial growth velocity index, inhibition percentage, aside from the sporulation rate and spore germination percentage. The mycelial growth of F. solani isolates was interrupted after hyphae encounter with T. harzianum, due to the occurrence of mycoparasitism, but without influence on the sporulation rate. Sterilized biological fertilizer induced no biocontrol, whereas the unsterilized product (concentration 2.5%) inhibited approximately 64% and 85% of the mycelial growth of isolates F1 and F2, respectively. Moreover, spore germination declined with increasing concentration. In conclusion, T. harzianum and the unsterilized biofertilizer showed in vitro antagonistic activity on F. solani.


Introduction
The capacity of adaptation to different cultivation conditions and the versatility of uses make cassava (Manihot esculenta Crantz) both socially and economically a highly relevant crop. The whole plant can be exploited, although the major interest is focused on the tuberous, starch-rich roots, which are worldwide an energy source in the diet of populations (Boas et al., 2016). From the economic point of view, it is the crop with the world's sixth highest production and Brazil is the fourth largest producer of cassava root (Conab, 2019).
Diseases such as cassava root rot, caused by fungi of the genus Fusarium, affect the plant health and are responsible for significant yield losses, hampering sales (Boas et al., 2016). Aside from affecting the quality of the harvested roots, some species of the genus are able to produce mycotoxins, which can be harmful to plants, animals and humans. These plant pathogens also colonize alternative host plants and survive in the soil for long periods under adverse conditions, foiling an effective plant health management (Kim & Vujanovic, 2016).
Although not very sustainable, chemical control by fungicide applications is one of the keys and most constantly applied measures of plant disease control (Kim & Vujanovic, 2016). However, the lack of chemical fungicides registered by the Ministry of Agriculture, Livestock and Food Supply (MAPA) to control this cassava disease requires alternative control methods (Mapa, 2021).
Biological control is an alternative for the use of agrochemicals in the management of agricultural diseases. It involves the use of living organisms, called biocontrol agents, and/or their metabolites against undesirable pathogens, in order to combat them or reduce their development and harmful effects on plants (Hridya et al., 2013). With regard to the interaction between fungi and/or bacteria with plant pathogenic fungi, e.g., Fusarium spp., biocontrol can occur through antibiosis (secretion of inhibitory extracellular metabolites), competition for nutrients and sites of infection and mycoparasitism or hyperparasitism. Other types of interactions that benefits the plant development at the expense of the pathogen are related to the induction of host resistance as well as growth promotion (Silva et al., 2016).
Species of the fungus Trichoderma are promising for the biocontrol of plant pathogens. These fast-growing soil microorganisms are suited for development under diverse conditions and have parasite and antagonistic behavior to many soilborne plant pathogenic fungi, e.g., Phytophtora sp., Fusarium spp., Sclerotinia sclerotiorum and Rizhoctonia solani, on different crops. In addition, they have shown to be avirulent plant symbionts and may induce plant resistance and growth (Qualhato et al, 2013).
Another promising alternative for the suppression of a disease is the manipulation physical, chemical and microbiological characteristics of the soil by means of management practices that enrich biodiversity and soil microbial activity, creating an environment deleterious to the plant pathogen (Barros et al., 2014). In this context, biological fertilizers serve not only as complement of mineral nutrition, but also to restore the microbial soil biodiversity (Bhardwaj et al., 2014;Zanuncio et al., 2020).
Biological fertilizers, also called biofertilizers, contain macro and micronutrients required for plant nutrition, as well as living beneficial microorganisms that can confer plant-hormonal and suppressive effects against different plant pathogens (Barros et al., 2020) in different crops, as described by Hridya et al. (2013) for Phytophthora palmivora on cassava and by Shen et al. (2013) for Fusarium oxysporum f. sp. cubense on banana trees. The fertilizers are applied on crop leaves or directly to the soil (Medeiros & Lopes, 2006), and their effect in disease suppression is related to their microbiological diversity, the soil microbiota and factors such as soil type, climatic conditions and management (Akter et al., 2013).
The purpose of this study was to determine the in vitro antagonistic activity of T. harzianum and biological fertilizer MICROGEO ® on F. solani, causal agent of cassava root rot.

Isolation and identification of Fusarium solani and Trichoderma harzianum
For the isolation of Fusarium, samples of cassava roots with rot symptoms were obtained from properties in the region of Paranavaí-PR. Fragments were cultured in Petri dishes containing Potato-Dextrose-Agar (PDA). Monosporic isolates were obtained to ensure pure cultures. The isolate morphology was differentiated by macroscopic (pigmentation) and microscopic (hyphae and conidia morphology under an optical microscope) observations of the colony. Fusarium isolates were submitted to molecular identification for F. solani confirmation. Genomic DNA was extracted according to the protocol proposed by Gontia-Mishra et al. (2014). The ITS region of the F. solani genome was amplified by Polymerase Chain
The T. harzianum fungus strain ESALQ 1306 was isolated by serial dilution of a biological fungicide in PDA culture medium.

Antagonist action of Trichoderma harzianum on Fusarium solani
The antagonistic action of T. harzianum on F. solani was evaluated in vitro, by the modified dual culture method proposed by Dennis & Webster (1971). Each Petri dish (150 mm diameter), containing PDA culture medium, received two Research, Society andDevelopment, v. 10, n. 3, e5210312994, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i3.12994 4 agar disks (8 mm diameter) with fungal mycelium, one F. solani and one T. harzianum colony disc, on opposite sides and 1.5 cm away from the edges of the plate. F. solani mycelial discs were taken from 10-days-old colonies and transferred to the Petri dish 96 h before T. harzianum, according to the methodology adapted from Carvalho et al. (2008). As controls, the fungi were grown separately, from a mycelium disc placed in the center of the plate. The Petri dishes were incubated in a BOD incubator at 25°C, with a photoperiod of 12 h for 9 days. Mycelial growth (mm) of the fungi was determined by measuring the colony diameters daily, to calculate the Mycelial Growth Velocity Index (MGVI), in mm day -1 , as proposed by Oliveira (1991).

Sporulation analysis of Fusarium solani
For the sporulation analysis of F. solani isolates, a methodology was adapted from Guzmán-de-Peña & Ruiz-Herrera (1997), counting the spores of three mycelial discs (8 mm diameter) collected from the central, intermediate and peripheral region of the colonies on the 10 th day of development. Samples from each colony were transferred to test tubes containing 10 mL of 0.05% Tween 80 solution and vortexed for 2 min. The counts were performed in a Neubauer chamber under light microscopy. Sporulation was expressed in number of spores cm -2 of colony.

Macroscopic and microscopic morphology of Fusarium solani
The macroscopic morphology was evaluated as proposed by Pitt & Hocking (1997) and the microscopy was analyzed by means of dual microcultivation of the fungi on microscope slides and later observation under optical microscope.

Determination of the microbial community of biological fertilizer
Continuous liquid composting of bovine ruminal content, water and the component MICROGEO ® produced the biological fertilizer used. The functional groups of microorganisms (population of total fungi, heterotrophic bacteria, cellulolytic, proteolytic, amilolytic, actinomycetes, free-living nitrogen-fixing and Pseudomonas fluorescens) in the broth were determined by serial dilution and inoculation on selective culture media.

Effect of the biological fertilizer on mycelial growth of Fusarium solani
The tests with the biological fertilizer were carried out in vitro, with the unsterilized (USt) and sterilized (St) product.
For sterilization, a fertilizer sample was submitted to: pH correction to 6.8-7.2; filtering through common filter paper; centrifugation at 4,000 rpm for 20 min; centrifugation of the supernatant at 10,000 rpm for 10 min and sterilization by vacuum pump filtration (0.22 μm Millipore membrane).
F. solani mycelium discs (8 mm diameter) were transferred to the center of the Petri dishes (90 mm diameter) containing the culture medium plus the biological fertilizer. Incubation conditions were 25 °C with a photoperiod of 12 h for 7 days. The mycelial diameter of the F. solani isolates was measured daily in the two perpendicular directions of the Petri dish to obtain the mean of the diameters (in mm) of each colony to calculate the Mycelial Growth Velocity Index (MGVI), according to Oliveira (1991). For the evaluation of mycelial growth inhibition, the values of colony diameters on the last day of incubation were used and the formula proposed by .

Statistical analysis of the results
The bioassays were conducted in a completely randomized design with four replications. The results were subjected to analysis of variance. The means obtained from the bioassay with T. harzianum were compared by the t-test (p <0.05) and regression analysis and Tukey's test (p <0.05) were applied to the means of the bioassay with biological fertilizer.

Morphological and molecular identification of the plant pathogens
Monosporic fungus cultures allowed the identification of two Fusarium isolates, called F1 and F2, differentiated by morphological characteristics of the colonies in macroscopic (F1 with white-pigmented mycelium and F2 with pink-pigmented mycelium) and microscopic (F1 with sickle-shaped and F2 with oval conidia) characteristics. However, the identification of Fusarium species based only on the morphological characteristics of the fungus requires the skills of specialized taxonomists.
Thus, the molecular identification of the Fusarium isolates F1 and F2 confirmed the F. solani species, according to the observation of 354 bp fragments obtained from the amplification of the ITS region of the species.

Antagonist action of Trichoderma harzianum on Fusarium solani
The dual culture assay of T. harzianum with F. solani isolates demonstrated an interruption in the mycelial growth of plant pathogens at the moment their hyphae meet with those of T. harzianum, on the 7 th days after subculturing (DAS) ( Table   1). The biocontrol however was only perceptible on the 8 th DAS of the plant pathogens, due to the statistical difference in the colony diameters, compared to the controls. Before that, there was no inhibitory action of T. harzianum, since the MGVI (mm day -1 ) of isolates F1 and F2 did not differ from the controls ( Table 1).
One of the characteristics of Trichoderma sp. is the faster development, which is relevant for its performance as a biocontrol agent (Qualhato et al., 2013). The mean mycelial growth of T. harzianum was 40.77 mm day -1 , which is approximately four times the MGVI of isolate F1 (10.87 mm day -1 ) and three times the MGVI of isolate F2 (12.91 mm day -1 ) of F. solani. Although T. harzianum was transferred to the Petri dish 96 h after F. solani, it rapidly reached and surpassed the mycelial growth of the plant pathogens, aside from continuing to grow and sporulate on their colonies. Similar results were reported by Pereira (2009), in an analysis of the antagonism of ten Trichoderma isolates against Fusarium sp. isolates, causing vascular wilt of tomato. In their study, all Trichoderma sp. isolates grew faster than the tested plant pathogens.  The antagonism of Trichoderma sp. mainly involves the mechanisms of antibiosis (release of inhibitory extracellular compounds such as gliotoxins), parasitism (direct contact with production and release of enzymes that disintegrate the cell wall of the parasited fungus, e.g., chitinases) and competition for nutrients and space (Kim & Vujanovic, 2016). Despite the interruption in the mycelial growth of F. solani isolates F1 and F2 by the action of T. harzianum after mycelium contact, there was no statistical difference (Tukey test p <0.05) in their sporulation rate (mean spore number/cm² of colony) compared to their control colonies. Isolate F1 sporulation rate was 1.2547 x 10 7 spores/cm² (control F1: 1.2049 x 10 7 spores/cm²) and isolate F2 sporulation rate was 3.4066 x 10 7 spores/cm² (control F2: 3.1566 x 10 7 spores/cm²). The results suggest that T.
harzianum was not able to produce sporulation-inhibiting extracellular compounds, what differs from Santos (2008), who reported inhibition of sporulation of Fusarium oxysporum by metabolite production due to antibiosis.
The in vitro results demonstrated that T. harzianum is a fungus that could potentially be used for the biological control of Fusarium root rot. Apart from enriching the soil microbiota, they are antagonistic to Fusarium solani and other soil fungi due to their biocontrol mechanisms (Qualhato et al., 2013).
The microbial composition contained in the biological fertilizer is relevant for its performance in terms of disease suppression. Generally, phytosanitary problems associated with soil fungi such as Fusarium spp. are related to a decrease in the soil microbial diversity (Palmieri et al., 2017). Thus, the restructuring of the microbiota by the application of a biological fertilizer full of microorganisms, apart from accelerating the degradation of soil biological matter and providing plantaccessible nutrients, invigorates the control of diseases caused by soil plant pathogens, by creating a competitive and pathogenhostile environment (Bhardwaj et al., 2014). The suppressiveness to cassava root rot caused by F. solani can be improved by an increase of its biodiversity by using adequate cultivation and management system in the area, and the disease severity was highest in soils used for monoculture crops in the past (Barros et al., 2014). Analyzing the mechanisms of control, Bonilla et al.
(2012) stated that the suppression of diseases should not be attributed to the entire microbial population of the biofertilizer, but to the biocontrol activity and mechanisms used by specific groups of microorganisms.

Effect of the biological fertilizer on Fusarium solani development
The sterilized biological fertilizer had no significant effect on the MGVI of F. solani isolate F1 when cultured in TSA medium ( Figure 1a). However, the MGVI of isolate F2 decreased slightly (Figure 1b), with a mean inhibition percentage of only 5.74% at the highest concentration.
For the unsterilized biological fertilizer (USt), the effect on fungal mycelial growth of both F. solani isolates was similar. There was a marked decrease in MGVI until a product concentration of 2.5% (USt5) (Figures 2a and 2b), which did not differ from the higher concentrations by the Tukey test (p <0.05).
The unsterilized biological fertilizer at a concentration of 2.5% (USt5) promoted inhibitions of approximately 64% in mycelial growth of isolate F1 ( Figure 3a) and 85% inhibition of isolate F2 ( Figure 3b). As observed for the MGVI of the fungi (Figures 2a and 2b), at concentrations above 2.5% of the unsterilized product, its effect on the inhibition percentage did not differ statistically by the Tukey test (p <0.05).
Similar results were reported by Tratch and Bettiol (1997) regarding the effect of a biofertilizer produced by anaerobic fermentation of cattle manure enriched with biological compounds on the mycelial growth of F. oxysporum f. sp. phaseoli. In this study, they observed that the biocontrol of the plant pathogen was effective at a product concentration of 10% and higher, not differing at the highest concentrations tested.
For foliar applications, biofertilizers are generally sprayed at concentrations ranging from 0.1 to 5.0% of the product diluted in water. Due to their nutritional and microbial composition, the application of very high concentrations may interfere at the plant development, causing physiological stress, apart from favoring vegetative plant growth and delaying the reproductive stage (Medeiros & Lopes, 2006). The technical recommendations of the manufacturer of this biological fertilizer suggest product doses of 3 to 5% diluted in water. The behavior of the microbial population present in the biological fertilizer was essential for the in vitro biological control of F. solani. Since the microbial community is rich and diverse, mechanisms of microbial action can occur simultaneously, e.g., parasitism, competition for nutrients, and production of antimicrobial compounds (Kupper et al., 2009).
The results of MGVI ( Figure 2) and inhibition percentage of the plant pathogens ( Figure 3) indicate that in the presence of plant pathogenic fungi, the microorganisms present in the biological fertilizer were able to produce antimicrobial metabolites by the mechanism of biocontrol, called antibiosis. A relevant fact for the confirmation of this mechanism was the noninhibition of F. solani under the effect of the sterilized product.
The in vitro and in vivo suppressive effect of biofertilizers on various plant pathogens was described by a number of researchers, in crops such as citrus (Kupper et al., 2009), cucumber (Akter et al., 2013) and banana (Shen et al., 2013). In an experiment of Shen et al. (2013) with Fusarium wilt (Fusarium oxysporum f. sp. cubense) of banana, different biological compounds were tested, as well as a biofertilizer based on pig slurry and amino acids inoculated with an antagonistic Bacillus amyloliquefaciens strain. The biofertilizer action reduced the disease incidence to 20%, compared to the other treatments.
The results confirm those of Kupper et al. (2009), who analyzed the effect of different concentrations of two biofertilizers based on cattle manure, one produced under anaerobic (Bio1) and the other under aerobic conditions (Bio2), on the spore germination percentage of Colletotrichum acutatum, causal agent of preharvest drop of citrus fruit. The authors observed inhibition rates exceeding 50% of the spore germination in response to the products at concentrations above 10%, particularly for Bio2, with highest inhibition percentages.
The harmful effect of biofertilizers on spore germination of various plant pathogenic fungi was also mentioned by Tratch & Bettiol (1997). For the fungus Botrytis cinerea, concentrations above 20% inhibited spore germination completely, while for Alternaria solani, a concentration of 10% was sufficient.
In view of the advantages of the use of biofertilizers to maintain the chemical, physical and microbiological characteristics of the soil and its responses in relation to plant pathogen suppression and the development of agricultural crops, their inclusion in sustainable agricultural production systems is becoming increasingly viable (Bhardwaj et al., 2014). (1.25%), USt5 (2.5%), USt6 (5.0%), USt7 (10.0%), USt8 (20.0%), and USt9 (40.0%).

Conclusion
Antagonistic activity of T. harzianum on F. solani was confirmed in vitro and biocontrol occurred by mycoparasitism after the mycelial encounter of the fungi.
Microorganisms presented in the unsterilized biofertilizer MICROGEO ® , starting from concentration 2.5% (USt5), caused mycelial growth inhibition in vitro of F. solani isolates and induced a reduction in the germination spores.
The proposed management alternatives proved to be relevant for the in vitro biological control of Fusarium solani, causal agent of the cassava root rot disease. Future research would be important to verify the in vivo biological control of F.

solani.
Scientific and Technological Development of the State of Paraná (Fundação Araucária) for funding and scholarships in an undergraduate research program.