Introduction

The genus Clonostachys comprises fungi with diverse lifestyles, including destructive mycoparasites that are used as biocontrol agents against plant pathogen fungi, as well as other species with other types of ecological associations (Schroer, 2001); few studies related to this genus have been published in Mexico, although Clonostachys rosea was recently cited to be parasiting avocado in Puebla (CóyotlPérez et al., 2022), along with C. chloroleuca, causing wilting in chickpea (Cicer arietinum) in Sinaloa and Baja California (^{Cota-Barreras et al., 2022}). Out of the mycoparasitic species, the most widely studied and used as a biological control agent is Clonostachys rosea (^{Funck and Dubey, 2022}), which acts against several plant pathogens such as the genera Alternaria, Botrytis, Bipolaris, Drechslera, Moniliophthora, Phytophthora, Rhizoctonia, Rhynchosporium and Sclerotinia (^{Sun et al., 2020}). One of the alternatives for the control of diseases caused by plant pathogens is the use of biocontrols, which are sustained in the ability of certain fungal groups to inhibit the growth of others, using hydrolitic enzymes such as chitnases and glucanases, giving them a distinctive advantage by letting them aim directly at the hyphae of other fungi, degrading cell walls more effectively (^{Moore et al., 2020}). Due to this, investigations on this group of fungi are important to discover new possibilities of biological control.

In Clonostachys, 11 mycoparasitic species have been reported (^{Schroers, 2001}).

To date there are no reports of Clonostachys as a mycoparasite in Veracruz, therefore the aim of this work was to evaluate the degree of antagonism in vitro of an isolated Clonostachys sp. strain against five strains of phytopathogenic fungi: Alternaria alternata, Colletotrichum musae, C. kahawae, Fusarium oxysporum and F. solani. Clonostachys was isolated (^{Senanayake et al. 2020}) as a mycoparasite of an ascomycete (Lachnum petridophyllum) living in the mesophilic forest of Veracruz. For the isolation, PDA medium (MCD LAB) was used, incubated for five days at 26 °C. The strain is located in the strain collection of the Centro de Micología Aplicada Research (CIMA) of the Universidad Veracruzana under the code CIMA-F-168. The strain was viewed under the Primo Star Iled compound microscope (Carl Zeiss, Oberkochen, Germany) and primary and secondary conidiophores were measured (^{Schroers et al., 2001}). Since the strain has morphological characteristics that do not correspond to any of the cited mycoparasitic species of Clonostachys, it is currently undergoing molecular identification.

Accrding to Comporota (1985), the first phase to select a strain with the potential for biocontrol is to evaluate its ability to invade other fungi. To corroborate this, five species of fungi related to diseases in previously studied crops were selected: Alternaria alternata (CIMAF-056; ^{Trigos et al., 2008}); Colletotrichum kahawae (CIMAF-078; isolated by A. Salinas from the Bola de Oro estate, in Coatepec, Veracruz); Colletotrichum musae (CIMAF-012; donated by COLPOS); Fusarium oxysporum (CIMA-F-068; ^{Adame et al., 2015}); F. solani (CIMA-F-127; ^{Lagunes et al., 2015}), all of which were deposited in the s CIMA-UV strain collection. The strains were planted in a PDA medium and incubated in a cultivation oven (BGE-71) at 26 °C for seven days.

The antagonistic activity was evaluated using dual cultures, placing two discs, 4 mm in diameter, in 90 mm Petri dishes with PDA medium. A PDA disc with Clonostachys sp. mycelial growth was placed on one side, and on the other, the fungus related to the cultures. The distance of separation between the discs was 50 mm and 15 mm from the edge of the dish. Five repetitions were carried out for each confrontation assay.

As a control, each one of the evaluated fungi were sown without the presence of Clonostachys sp., as well as the Clonostachys sp. strain without the presence of other fungi. Both the controls and the dual cultures were incubated at 26 ºC. Measurements of the growth (in millimeters) of both fungi in confrontation and the control were taken every 24 hours until 360 hours were completed (15 days).

The interaction between the fungi in vitro was observed and described, with particular attention to the production of reproductive structures, pigmentation and mycelium morphology. The degree of antagonism (Table 1) was determined using the scale by ^{Bell et al. (1980}).

Using the growth measurements, the percentage of colonization was determined, following the formula by ^{Camporota (1985}): C= DT/DE/100, where DT is the distance covered by Clonostachys sp. on the axis that separates the points of planting and DE is the distance between both (5 cm). The colonization was considered effective when the percentage was greater than 50% (^{Rollan et al., 1999}). The statistical analyses were carried out using the R software (R Core Team). The normality of the data was corroborated using the Shapiro-Wilks. Subsequently, a generalized linear model (GLM) analysis was carried out.

The strain under study displayed a mycoparasitic-like antagonistic behavior against the five fungal species evaluated, with Fusarium solani corresponding to

class 1; Alternaria alternata, Colletotrichum musae and C. kahawae, to class 2; Fusarium oxysporum, to class 3, following the scale proposed in Table 1. In none of the cases was an inhibition halo observed, so the interaction observed corresponds to the type of “overgrowth,” according to the classification by ^{Bertrand et al. (2013}). The percentage of colonization of Clonostachys sp. was 100% after 216 h against

A. alternata, C. kahawae and F. solani, whereas for the species C. musae and F. oxysporum, it was after 264 hours under in vitro conditions (Table 2). Although the colonization was effective, no significant differences were found in the percentage of colonization among the phytopathogens evaluated (p= 0.0073). Nevertheless, the time of invasion did have a significant effect on the percentage of colonization for each fungus, as indicated by the value of p < 0.0001 obtained in the GLM analysis.

On the other hand, the evaluated fungi display different growth rates and particular characteristics for instance, in the case of Alternaria alternata (Figure 1), after 168 hours, it produced a bright yellow pigment in the culture medium, which intensified into an orange color. According to ^{Scott and Stoltz (1980)}, the diffuse yellow pigments in the medium are related to the production of toxins such as altertoxin II in A. alternata.

Figure 1 In vitro antagonism of Clonostachys sp. (Csp.) against Alternaria alternata (Aa). A: After 24 hours of evaluation. B. 120 hours of evaluation. C. 216 hours of evaluation. D. 360 hours of evaluation. 

After144 hours in C. kahawae, a brown pigment was observed in the center of the colony, which intensified as the antagonist grew on the fungus (Figure 2). Likewise, during the interaction between Clonostachys sp. agains C. musae, the growth of the antagonist was observed on the acervuli produced by C. musae (Figure 2). This process is highly important, since it can contribute significantly to the disruption of the cycle of the pathogen, since acervuli form at the end of the infection cycle, breaking the cuticle of the plant to emerge and continue with asexual reproduction (^{da Silva et al., 2020}).

Figure 2 In vitro antagonism of Clonostachys sp. (Csp.) against Colletotrichum kahawae (Ck) and Colletotrichum musae (Cm). A-D) Colletotrichum kahawae (Ck). A: 24 hours after evaluation. B: 192 hours of evaluation. C: 360 hours of evaluation. D: reverse Petri dish at 360 hours of evaluation. E-H) Colletotrichum musae (Cm) against Clonostachys sp. (Csp.). E: 24 hours of evaluation. F: 192 hours of evaluation. G: 360 hours of evaluation. H: Conidiophores of Clonostachys sp. growing on acervuli of C. musae. 

Although the Clonostachys genus has been the object of study in relation to its antagonistic activity, there are few investigations centered on its interaction with the species of the Colletotrichum genus. ^{Peters et al. (2020}) isolated Clonostachys rosea as the endophyte of the açai plant (Euterpe oleracea) and proved its ability to inhibit the growth of Colletotrichum gloeosporioides, the causal agent of anthracnose. This study presents, for the first time, the antagonistc evaluation against two species of the Colletotrichum genus that had not been previously evaluated.

In the case of the interaction between F. solani and Clonostachys sp. after 168 hours, the aerial mycelium of Clonostachys sp. changed its morphology, forming a convex colony with a fimbriated edge, unlike the control and trials with other fungi. Masses of conidia with an aqueous consistency were observed on the edge after 240 horas (Figure 3).

Some of the characteristics between F. oxysporum and Clonostachys sp. was that, after 168 hours, they both colonized half of the Petri dish, with their edges touching. During this period, the F. oxysporum pigments intensified, changing from a lilac color at the beginning of the assay to a dark purple color in the middle with bright edges at the end of the experiment, keeping the same color as the control (Figure 3).

Several studies conducted with the same genera tested in this trial showed that the in vitro and in vivo antagonistic activity of 10 strains of Clonostachys and F. circinatum in Pinus radiata seedlings (^{Moraga et al., 2011}) was variable. The strains inhibited F. circinatum by up to 23% in in vitro, but not in vivo, in which the percentage of survival of the seedlings infected by the fungus increased. It has also been proven that the endochitinase enzymes participate in the degragation process of cell walls of species such as Fusarium culmorum (^{Mamarabadi et al., 2008}).

The interactions observed betweem Clonostachys sp. and the fungi under study displayed distinctive charateristics, such as the production of masses of conidia and a different mycelial morphology during the trial, particularly in F. solani. In addition,

Figure 3 In vitro antagonism of Clonostachys sp. against Fusarium spp. A-D) Fusarium solani (Fs) against Clonostachys sp. (Csp.) A: after 24 hours of evaluation. B: at 96 hours of evaluation. C: at 360 hours of evaluation. D: masses of aqueous conidiophores at the margin of Clonostachys sp. E-H) Fusarium oxysporum (Fo) against Clonostachys sp. (Csp.) E: 24 hours of evaluation. F: 144 hours of evaluation. G: 192 hours of evaluation. H: 360 hours of evaluation. 

a variation was recorded in the speed of invasión of each fungus evaluated. These differences can be attributed to factors such as transcriptomic regulation and the ability of Clonostachys to discriminate between the species it will mycoparasitize, as seen in the case of C. rosea (^{Nygren et al., 2018}).

Dual trials to verify the antagonistic effect of species at an in vitro level constitute the basis to seek better candidates for the biological control of fungi that cause diseases in economically important crops. Given the marked noticeable activity of the Clonostachys sp. strain against all the fungi evaluated, it emerges as a promising candidate for future research to evaluate its in vivo effectiveness.