Zucchini squash (Cucurbita pepo) is one of the main cucurbitaceae species (^{Zitter et al., 2004}). Its production is limited by biotic and abiotic agents during the whole cropping cycle, being the seedling stage one of the most critical. There are numerous pathogens that affect seed and seedlings and cause pre-emergence or post-emergence damping off. The most common causal agents of seedlings damping off in cucurbitaceae are Fusarium oxysporum (^{Vakalounakis and Chalkias, 2004}), Phytophthora capsici (^{Hausbeck and Lamour, 2004}), Rhizoctonia solani (^{Scheuerell et al., 2005}) and Pythium spp. (^{Deadman et al., 2002}; ^{Scheuerell et al., 2005}). In the past two decades, the use of synthetic pesticides has been considerably reduced because of the development of new control strategies such as biopesticides (^{Ongena and Jacques, 2007}). One of the species most used as bioplaguicide is Bacillus subtilis, because it produces antibiotics, lipoproteins and hydrolytic enzymes which confer antifungal activity, making it an excellent biological control agent of phytopathogens. Specifically, its activity has been reported in organisms which induce seedling damping off, and its beneficial effects on treated seedlings (^{Cavaglieri et al., 2005}; ^{Sharma and Sharma, 2008}). B. subtilis produces three families of cyclic polypeptides (surfactins, iturines and fengycins) involved in its antagonistic activity against bacteria, fungi, and oomycetes. These lipoproteins are also associated with a greater capacity to colonize the plant roots of the bacterial strain that produces them and are involved in the stimulation of the plant’s defense response (^{Ongena and Jacques, 2007}; ^{Villareal-Delgado et al., 2017}). In addition to cyclic polypeptides, B. subtilis produces antimicrobial compounds which increase its capacity to control phytopathogens (^{Hamdache et al., 2011}; ^{Wise et al., 2012}). Based on this, B. subtilis is a promising alternative for the biological control of phytopathogens (^{Falardeau et al., 2013}; ^{Khedher et al., 2015}). So far, the data published on the effectiveness of B. subtilis strain QST 713 for the combined control of F. oxysporum, P. capsici and R. solani are scarce. Therefore, the objective of this research was to morphologically identify the causal agents associated with damping off in zucchini squash seedlings and determine the effectiveness of B. subtilis strain QST 713 on the incidence of the disease produced by the combined inoculation of the isolated fungi.

During the 2019 spring-summer cycle, zucchini squash (Cucurbita pepo) seedlings with damping off symptoms were collected in Cuautla, Morelos. The fungi associated with the symptoms were isolated from root and stem cuts, as described by ^{Ayala-Armenta et al. (2020}). The morphological characterization was conducted under a compound microscope CX31RBSFA (Olympus^®, Japan) at 40X, where 50 reproductive structures of each isolate were measured. The molecular identification was done based on specialized taxonomic keys (^{Leslie and Summerell, 2006}; ^{Sneh et al., 1991}; ^{Erwin and Ribeiro, 1996}). To confirm the pathogenicity of the isolates, the Koch’s postulates were followed using healthy squash seedlings. The pathogenicity test was conducted twice selecting the most virulent isolate of each species.

To evaluate B. subtilis against the isolated fungi, seeds of zucchini squash Grey var. were used. The fungicide treatment on the seeds was eliminated as described by ^{Márquez-Licona et al. (2018}). Seven days after planting, the emerging seedlings were transplanted to 500 mL polyurethane cups containing Peat Moss and agrolite sterile substrate (75:25 v/v). The evaluated treatments consisted of three concentrations of B. subtilis QST 713 strain (Serenade ASO^®) (2, 4 and 6×10⁷ UFC mL^-1) and methyl thiophanate + propamocarb hydrochloride, which were applied preventively and curatively (Table 1). The incidence of damping off caused by the combined infection of the isolated fungi and under greenhouse conditions was determined using zucchini squash seedlings (24 °C and 70% HR). The treatments were evaluated in duplicate. The viability of the commercial product was determined by placing an aliquot of the product in four Petri dishes containing PDA medium. The Petri dishes were incubated in darkness at 36 °C for 48 h. The viability evaluation was made in duplicate. Inoculum was increased for each pathogen in specific culture mediums (SNA, V8 Agar and PDA). The inoculum load of each of the pathogens applied per plant was adjusted at a concentration of 4×10⁵ UFC using a hemocytometer (Marienfeld^®). The inoculation of the pathogens was made three days after transplanting. The biological product based on B. subtilis was applied twice to zucchini squash seedlings, the first to the transplanted seedlings, and the second six days after transplanting; in both cases, 5 mL of the suspension were applied at the seedlings neck. The preventive chemical treatment was applied when the seedlings were transplanted, and the curative treatment 72 h after the pathogens were inoculated, for which 5 mL of the solution were applied to the seedlings neck. To determine the effectiveness of the treatments on the disease, the damping off incidence in the seedlings was measured every 24 h, starting at the time when the pathogens were inoculated. The statistical analysis included evaluations at three, six, nine and 12 days. The experimental unit consisted of 100 seedlings, and the obtained data were analyzed using a randomized complete block design with four replications per treatment. The data were subjected to an analysis of variance and comparison of multiple means using the Tukey’s honest significant difference method at a significance level of 5%, and SAS for Windows v. 9.4 (SAS^® Systems). The phytotoxicity evaluation of the treatments was made using the percentage scale proposed by the European Weed Research Society (https://www.ewrs.org/). To confirm the presence of the pathogens that were inoculated in the diseased plants, re-isolations were made in PDA culture medium. At the end of the experiment, the surviving plants were kept under observation for another 14 days.

One-hundred isolates were obtained from zucchini squash seedlings with damping off symptoms collected in Cuautla, Morelos, where Phytophthora capsici, Rhizoctonia solani and Fusarium oxysporum (Figure 1) were identified in 62, 26 and 12%, respectively. The pathogenicity tests confirmed that the isolated organisms were pathogenic. The plants inoculated with P. capsici showed damping off symptoms four days after inoculation, whereas the plants which were independently inoculated with F. oxysporum and R. solani showed disease symptoms six days after inoculation. In this research, the symptoms occurred earlier than those reported by ^{Chehri et al. (2010}), who observed damping off symptoms seven days after inoculating P. capsici and F. oxysporum in cucurbitaceae. SNA, PDA and V8-Agar juice culture mediums increased the amount of inoculum of F. oxysporum, R. solani and P. capsici, respectively. The B. subtilis bacterium strain QST 713 (Serenade ASO^®) colonized the Petri dish 48 h after the strain was sown and incubated at 36 °C, thus confirming the viability of the commercial product. The first damping off symptoms in zucchini squash plants appeared 72 h after inoculation. The syndrome coincides with the result described by ^{Koike et al. (2007}).

Figure 1  Fusarium oxysporum colonies in SNA culture medium (A), Phytophthora capsici in V8 Agar (B) and Rhizoctonia solani in PDA medium (C) isolated from zucchini squash seedlings (Cucurbita pepo) with damping off symptoms. Appearance of the colonies 12 days after sowing in selective media. 

The disease incidence was directly proportional to the time elapsed after inoculation. Twelve days after the pathogens were inoculated into the control plants, the disease incidence reached 100%, whereas the absolute control plants remained healthy. The average disease incidence in the evaluated treatments is shown in Table 2. Three days after inoculation (dai) (first evaluation), the control inoculated with methyl thiophanate + propamocarb hydrochloride (preventive) and the treatments based on B. subtilis 2×10⁷ and 4×10⁷ UFC mL^-1 showed a disease incidence of 2 and 2.5%, respectively, being statistically different to the rest of the evaluated treatments, which remained healthy. In the second evaluation (six dai), the inoculated control reached 60% average incidence, while the treatments based on B. subtilis 2×10⁷ and 4×10⁷ UFC mL^-1 showed 31.8 and 33.8% disease incidence, respectively. The treatments based on B. subtilis 6×10⁷ UFC mL^-1 and methyl thiophanate + propamocarb hydrochloride (curative) were statistically equal, while the treatment with methyl thiophanate + propamocarb hydrochloride (preventive) and the absolute control were statistically equal, with 0% disease incidence. In the third evaluation (nine dai), the previous tendency was continuous and there was only an increase in the percent of the disease incidence. The inoculated control reached 75% average incidence, while the treatments based on B. subtilis 2×10⁷ and 4×10⁷ UFC mL^-1 showed 33.8 and 38.8% disease incidence, respectively. The treatments based on B. subtilis 6×10⁷ UFC mL^-1 and methyl thiophanate + propamocarb hydrochloride (curative) were statistically equal, while the treatment with methyl thiophanate + propamocarb hydrochloride (preventive) and the absolute control were also statistically equal, with 0% disease incidence. Finally, in the fourth evaluation (12 dai), the inoculated control reached 100% average incidence, while the treatment based on B. subtilis 2×10⁷ UFC mL^-1 reached 41.3% incidence, and the treatment based on B. subtilis 4×10⁷ UFC mL^-1 reached 38.8% incidence; however, both treatments (2×10⁷ and 4×10⁷ UFC mL^-1) were statistically equal. In the treatments based on B. subtilis 6×10⁷ UFC mL^-1 and methyl thiophanate + propamocarb hydrochloride (curative) the disease incidence increased 2% compared to the percent obtained in the third evaluation, being statistically equal. Finally, the plants treated with methyl thiophanate + propamocarb hydrochloride (preventive) remained healthy, this is, with 0% disease incidence.

The results of the effectiveness of B. subtilis obtained in this study agree with the results reported by other researchers. It has been consistently demonstrated that B. subtilis is effective to control F. oxysporum (^{Mejía-Bautista et al., 2016}), Fusarium sp. (^{Illa et al., 2020}), P. capsici (^{Khalaf and Raizada, 2018}) and R. solani (^{Brewer and Larkin, 2005}) in different hosts, including cucurbitaceae. Similarly, the results obtained using methyl thiophanate are consistent with the results of previous studies that document the effectiveness of methyl benzimidazole carbamate to control F. oxysporum in several hosts (^{Nel et al., 2007}; ^{Tarekegn et al., 2007}). Methyl thiophanate is also effective to reduce R. solani infection in different hosts (^{Hancock, 1993}; ^{Kumar et al., 2003}; ^{Olaya et al., 1994}). Propamocarb hydrochloride is a widely used active ingredient for the control of oomycetes, including Phytophthora spp. in cucurbitaceae (^{Deadman et al., 2002}).

After the 12 days of the experiment, no increase in the percentage of diseased plants in the treatments was observed. None of the evaluated treatments caused phytotoxicity to zucchini squash seedlings. In the re-isolation of the phytopathogenic fungi which were recovered from the inoculated control, the frequency obtained was of 50, 35 and 15% for P. capsici, R. solani and F. oxysporum; these data coincided with the frequency of seedlings collected in the field. This suggests that the dumping off symptoms observed in the combined inoculation were mainly caused by P. capsici infection. The first symptoms, caused by the combined inoculation, appeared in a shorter period than that observed when the pathogens were individually inoculated (^{Fernández-Herrera et al., 2007}), thus indicating that when F. oxysporum, P. capsici and R. solani were combined, the inoculation was more aggressive, because the incubation period was of only 72 h. In this study, a higher radical development of plants treated with B. subtilis was qualitatively observed. Although none of the treatments based on B. subtilis completely protected the zucchini squash seedlings from the combined infection of F. oxysporum, P. capsici and R. solani, the following tendency was observed: the higher the concentration of bacterium, the lower the disease incidence. The treatment based on B. subtilis at 6×10⁷ UFC mL^-1 was statistically equal to the curative chemical treatment, which demonstrates its potential for the biological management of the disease. The preventive chemical treatment was statistically equal to the absolute control, thereby demonstrating the effectiveness of the chemical control. However, as part of the current trend in reducing the use of synthetic molecules in agricultural production, the preventive use of biological products is the ecologically viable response to manage the disease. The use of higher concentrations of B. subtilis is suggested in further studies.

The products evaluated helped consistently to reduce the incidence of zucchini squash seedlings death. The biological product based on B. subtilis at a concentration of 6×10⁷ UFC mL^-1 protected more than 80% of the zucchini squash seedlings which were inoculated with F. oxysporum, P. capsici and R. solani; its effectiveness was statistically equal to thatof the curative treatment based on methyl thiophanate + propamocarb hydrochloride. The preventive treatment based on methyl thiophanate + propamocarb hydrochloride protected 100% of the seedlings from the inoculation of the three pathogens.