Introduction

The Echeveria genus belongs to the Crassulaceae family, which corresponds to a group of plants endemic to the American continent, with succulent leaves and few agricultural requirements. Out of this genus, Mexico has 83% of the diversity of species (^{Reyes-Santiago et al., 2011}a; Reyes-Santiago et al., 2011b; ^{Schoch et al., 2020}).

In this genus, Echeveria gibbiflora (tememetla, Echeveria, ornamental succulent) stands out as one of the native Mexican species with great potential in floriculture, due to its ability to thrive in adverse environments, the beauty of its leaves, the size of its inflorescence (1 m in length), number of batches (up to 12 per inflorescence), the attractiveness of its bright pink flowers and the continuous appearance of new buds (^{Reyes-Santiago et al., 2015}). These are characteristics that make it an excellent option for their commercialization, both in pots and also as cutting flowers (^{Leszczyñska-Borys, 2003}).

On the other hand, one of the factors that influence the quality of the Echeveria genus plants is the attack of diverse fungal diseases such as anthracnose induced by Colletotrichum destructivum (^{Yao et al., 2020}), powdery mildew by Erysiphe sp. (^{Shi et al., 2019}), stem rot by Fusarium oxysporum (Yao et al., 2021), vascular wilting by F. oxysporum f. sp. echeveriae (^{Ortu et al., 2015}), fungal leaf spot (Cladosporium tenuissimum) (^{Jo et al., 2018}), white mold (Sclerotinia sclerotiorum) (Terrones et al., 2024) and anthracnose induced by Colletotrichum truncatum (^{Apáez et al., 2025}). It is frequent that, for the management of diseases in urban areas, chemical pesticides are inadequately applied, which implies the release of toxic residues into the environment, making it a risk for human health and the environment (^{Meftaul et al., 2020}), whether directly by bioaccumulation, or indirectly by the food chain (^{Parra-Arroyo et al., 2022}). This leads to a need to generate sustainable strategies for the management of diseases in ornamental plants for urban areas that help reduce the resistance of the pathogens to chemical pesticides and contribute to the reduction of contamination of soils and aquifers (^{Reyes et al., 2015}). Biofungicides based on plant oils, plant extracts and antagonistic microorganisms suggest possible uses in the in vitro biological control of C. truncatum isolated from E. gibbiflora. Due to this, the aim of this study was to evaluate the sensitivity of C. truncatum that induces anthracnose in E. gibbiflora to 11 biofungicides as an alternative for the integrated management of the disease.

Materials and methods

Origin of the isolation. An isolation obtained from E. gibbiflora plants grown in the Research Center in Horticulture and Native Plants of UPAEP University, located in Atlixco, Puebla, Mexico, where symptoms of anthracnose was observed in leaves, with an incidence and severity of 45 and 40%, respectively. The fungus that induced the disease was isolated (isolate CtEg2) and characterized pathogenically, culturally, morphologically, morphometrically and phylogenetically in a previous study and identified as Colletotrichum truncatum (GenBank. ITS: PP406307; TUB2: PP616679; GAPDH: PP616680; ACT: PP616681) (^{Apáez et al., 2025}).

Preparation of modified culture media and planting of C. truncatum . Using the poisoned culture medium method (^{Barroso et al., 2021}; ^{Guerrero et al., 2007}), the sensitivity of the C. truncatum isolation was evaluated with 11 biofungicides (Table 1). All biofungicides were tested with six concentrations, 0 (control), 1, 10, 100, 500 and 1000 mg L ^-1. A potato dextrose agar (PDA) culture medium was used, modified with the biofungicides poured into Petri dishes (100 × 15 mm). Subsequently, a 5 mm PDA disc with an active, seven-day old C. truncatum growth was planted, and the dishes were incubated in the dark at 25 ± 1 °C (^{Iturbide et al., 2017}).

Determining the percentage of inhibition. The diameter of the colony was measured every 24 h for 12 days (time taken for the control Petri dishes to fill with fungal mycelium) in two perpendicular directions using a digital caliper (Truper^®, Mexico) and using the data obtained, the percentage of growth inhibition (PGI) was calculated using Abbott’s formula (1925): PGI= (𝐷𝑐−𝐷𝑡) 𝑥 100 where: PGI: Percentage of growth inhibition; Dc: 𝐷𝑐 Diameter of the control; Dt: Diameter of the treatment (^{García et al., 2021}).

Experimental design. It was a factorial experiment, arranged in a totally randomized design, 11 × 6 [11 biofungicides with six concentrations (0, 1, 10, 100, 500 and 1000 mg L ^-1) each] with 66 treatments, four repetitions and 264 experimental units, each consisting of one Petri dish measuring 100 × 15 mm, and two repetitions of the experiment were performed.

Data analysis. The data obtained underwent normality tests, using the Shapiro-Wilk test. Likewise, the Levene test was used for the variance homogeneity test and the level of significance used was alfa=0.05. A concentration-response analysis was performed to evaluate the biological effectiveness of 11 different biofungicides in terms of percentages of inhibition. The data were adjusted to a log-logistical model to determine the effective concentration for an inhibition of 50% (EC50) for each biofungicide. An analysis of variance was carried out, along with Tukey’s test to carry out multiple comparisons between the percentage of mycelial inhibition of the 11 biofungicides, with a level of significance of 5%. All statistical tests were performed in the program R, version 4.3.2.

Results

The effective concentration for 50% inhibition (EC50) was different for each biofungicide. For the M. alternifolia oil, it was 109.23 mg L ^-1, whereas for the R. sachalinensis extract, it was 168.76 mg L ^-1. In the case of B. amyloliquefaciens QST 713, the estimation of EC50 was 61.07 mg L ^-1 and in the L. digitata oil, EC50 was 19.95 mg L ^-1. Regarding the T. vulgaris and M. spicata essential oils a EC50 of 112.47 mg L ^-1 was observed. In the cases of the L. tridentata and R. communis extracts, a EC50 of 78.51 mg L ^-1 was identified; a EC50 of 71.55 mg L ^-1 was found for the S. aromaticum, P. nigrum,

R. communis and R. graveolens extracts. The L. tridentata extract obtained a value of

99.47 mg L ^-1, the EC50 of the L. graveolens, C. verum and R. communis extracts presented a value of 27.57 mg L ^-1, and the M. tenuiflora and Q. robur extract displayed a EC50 of

60.15 mg L ^-1. Finally, the EC50 was determined for the fatty-acid-rich potassium salts with a value of 295.70 mg L ^-1 (Table 1, Figure 1).

The estimated values of the 50% effective concentration (standard error and t and p values) indicated that the estimation is highly significant (p < 0.001) for all biofungicides, except for the T. vulgaris and M. spicata essential oils, as the values suggest that the estimation is significant (p < 0.05) (Table 1).

The analysis of variance displayed significant differences between the percentages of inhibition of the mycelial growth of biofungicides (p-value < 2e-16***). Tukey’s test was used to carry out multiple comparisons between the percentages of inhibition of the mycelial growth of the 11 biofungicides, with a level of significance of 5%. The residual variability is relatively low (MSerror = 3.94), which suggests that the model is well adjusted to the data observed. The coefficient of variation was 3.65%, which indicates that the variability of the data is relatively low in comparison to the mean observed (53.46%); Tukey’s means comparison test displayed statistically different group.

Figure 1 Concentration response and EC50 of 11 biofungicides against C. truncatum isolated from E. gibbiflora. A) e M. alternifolia oil; B) R. sachalinensis extract; C) B. amyloliquefaciens QST 713; D) L. digitata oil; E) ac T. vulgaris and M. spicata essential oils; F) L. tridentata and R. communis extracts; G) cinnamaldehyde, S. aromaticum, P. nigrum, R. communis and R. graveolens extracts; H) L. tridentata extract; I) L. graveolens, C. verum and R. communis extracts; J) M. tenuiflora and Q. robur extract; K) fatty-acid-rich potassium salts. 

The treatment based on M. alternifolia oil was significantly different to the rest of the treatments, with the highest percentage of inhibition of the mycelial growth with a value of 93.6%, while the biofungicide made with fatty-acid-rich potassium salts was significantly different to the rest of the treatments, since it presented the lowest percentage of inhibition of the mycelial growth, with a value of 7.63%. The biofungicides composed of R. sachalinensis extract, B. amyloliquefaciens QST 713, L. digitata oil, T. vulgaris and

M. spicata essential oils and the L. tridentata extract displayed percentages of inhibition above 50%, with values of 78.85, 69.5, 65.4, 64.55 and 63.4 %, respectively (Figure 2).

Figure 2 Percentage of inhibition of the mycelial growth of C. truncatum isolated from E. gibbiflora, evaluated in vitro with 11 biofungicides. The mean values followed by the same letters are statistically similar (Tukey α = 0.05) according to Tukey’s honest significant difference test. T1= M. alternifolia oil; T2= R. sachalinensis extract; T3= B. amyloliquefaciens QST 713; T4= L. digitata oil; T5= T. vulgaris and M. spicata essential oils; T6= L. tridentata extract; T7= cinnamaldehyde, S. aromaticum extracts, P. nigrum, R. communis y R. graveolens; T8= L. tridentata and R. communis extracts; T9= L. graveolens, C. verum and R. communis extracts; T10= M. tenuiflora and Q. robur extract; and T11= fatty-acid-rich potassium salts. 

The percentage of inhibition of the mycelial growth of C. truncatum was calculated for each one of the six concentrations (0, 1, 10, 100, 500 and 1000 mg L ^-1) of the 11 biofungicides evaluated. In concentration of 0 mg L ^-1, the percentage of inhibition was 0% in all biofungicides. When 1 mg L ^-1 was evaluated, the percentage of inhibition varied from 0.5 to 90.0%; when the concentration of 10 mg L ^-1 was tested, the percentage of inhibition was maintained in a range of 4.8 to 92.0%, whereas when 100 mg L ^-1 was tested, a range of 6.0 to 93.8% of inhibition was identified. In the concentration of 500 mg L ^-1, the percentage of inhibition values found were between 8.3 and 94.8%. Finally, when a concentration of 1000 mg L ^-1 was used, the percentage of inhibition was found to range between 11.5 and 97.5. In all concentrations used, the fatty-acid-based potassium salt treatment presented the lowest percentage values, whereas the M. alternifolia oil- based treatment displayed the highest values (Table 2). Additionally, in all the biofungicides evaluated, the percentage of inhibition grew as the concentration increased. For example, in the case of M. alternifolia oil, when concentrations of 1, 10, 100, 500 and 1000 mg L ^-1 were evaluated, percentages of inhibition were identified in the C. truncatum mycelial growth of 90.0, 92.0, 93.8, 94.8 and 97.5 respectively. The biofungicides composed of L. tridentata plant extract, T. vulgaris and M. spicata oils, L. digitata plant oil, B. amyloliquefaciens QST 71 antagonist, R. sachalinensis plant extract and de M. alternifolia oil presented percentages of mycelial growth inhibition higher than 50% (Table 2).

Discussion

Calculating the EC50 of each product helped identify that seven biofungicides (64%) were effective at concentrations lower than 100 mg L ^-1 and 4 of them (36%), at concentrations between 101 and 300 mg L ^-1, whereas the most efficient biofungicides in the control of C. truncatum were the M. alternifolia oil, the R. sachalinensis extract and the B. amyloliquefaciens QST 713, effective in the control at relatively low concentrations of 109.23, 168.76 and 61.07 mg L ^-1, respectively.

According to ^{Quyen and Quoc (2024}), the essential oils of the M. alternifolia extract contain 45 compounds, of which the main compound is terpinen-4-ol (44.55%), associated to antioxidant and antimicrobial activity. Likewise, the action mechanism of this essential oil in Penicillium expansum has been documented to be on the plasmid membrane of the fungus, although whether this is due to the alteration in the structure of the membrane or a change in its permeability is unknown. However, the exposure of the fungus for 4 hours to the vapor phase of the M. alternifolia essential oil caused a loss in DNA, protein and glucose, as well as lipid damage (^{da Rocha et al., 2019}).

Regarding the control of some Colletotrichum species, ^{do Nascimento et al. (2019}) found that with a concentration of 25 μL of M. alternifolia essential oil, the germination of spores from Colletotrichum gloeosporioides isolated from bell pepper (Capsicum annuum) plants becomes inhibited by 100%. In turn, ^{da Costa et al. (2023}) reported that the EC100 on the mycelial growth of C. musae in in vitro plantations achieved this with a concentration of 2.15 μg mL ^-1 of M. alternifolia essential oil, whereas treatments with banana fruits with aqueous emulsions of the essential oil reduce the severity of anthracnose induced by C. musae by more than 80% without any phytotoxic effects. These data coincide with the results presented in this investigation, unlike a study by ^{Kiranmayee et al. (2020}) that evaluated the effect of eight essential oils on the in vitro growth of C. gloeosporioides isolated from pommegranate (Punica granatum) stems and roots, and showed that M. alternifolia essential oil had a lower effect on concentrations of 0.5, 1.0 and 2.0%, in comparison with thyme (T. vulgaris) and clove (S. aromaticum) oils, which were the most effective biofungicides for the inhibition of mycelial growth, which suggests that, in order to evaluate the fungicidal effect of M. alternifolia, several factors must be taken into account, such as the species of the pathogen, the host from which it was isolated, the concentration of the biofungicide and the environment.

On the other hand, M. alternifolia has been proven to have an effect on the growth of other phytopathogenic fungal species, as indicated in an investigation by ^{Rani and Tripathi (2022}), where it was reported that with a dose of 0.02% of M. alternifolia essential oil, inhibited the germination of Botrytis cinerea spores isolated from chili pepper (C. annuum) fruits by 100%. Likewise, the authors indicate that the application of this oil as a fumigant and in submersion treatments for fruits increases the shelf life of the chili pepper. The sensitivity of the Alternaria alternata fungus to the essential oil of M. alternifolia was also evaluated, with 11 concentrations that ranged between 0.0125 and 1.0%, and found that the concentration in which full inhibition was reached was 0.2%. Meanwhile, evaluating in vivo by treatments of the cowpea bean (Vigna unguiculata) seed with the essential oil did not reduce the percentage of incidence of the pathogen (^{de Figuerêdo et al., 2019}), which indicates that, alongside the in vitro tests of sensitivity of

C. truncatum to the M. alternifolia essential oil, it is necessary to carry out in vivo tests on E. gibbiflora plants to determine the optimum dose on the field.

In addition to the antifungal effect, there are reports indicating that M. alternifolia has an inhibiting effect on the pathogenic bacteria of humans and some fungi that produce mycotoxins. ^{Quyen and Quoc (2024}) calculated the EC50 of the M. alternifolia essential oils for the inhibition of the growth of Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 25923), Salmonella enterica (ATCC 13076) and Escherichia coli (ATCC 25922) and reported that the 50% inhibition of bacterial growth was at a concentration of 360 mg mL ^-1. Another similar study, carried out by ^{Zhang et al. (2018}) shows that the M. alternifolia essential oil has an antimicrobial effect, not only on the growth of bacteria such as E. coli, S. aureus and Pseudomonas aeruginosa, but also of fungi such as Penicillium italicum and P. digitatum with a EC50 of 48.35 μg mL ^-1. This is an advantage in the use of the M. alternifolia essential oil since E. gibbiflora is commonly used in plants, where these plants can come into contact with children and pets. Therefore, should there be any contamination of the water or organic residues used for the cultivation of this plant, the oil could contribute to minimizing the risks of contamination, although further study on this is necessary.

The R. sachalinensis extract was the biofungicide with the second highest inhibition in the growth of C. truncatum. According to ^{Margaritopoulou et al. (2020}), in a study on the control of powdery mildew induced by Podosphaera xanthii in zucchini, the R. sachalinensis extract works as an elicitor or inducer of the plant defense mechanisms, since it triggers several reactions, such as the formation of callose, the accumulation of hydrogen peroxide and the increase in the production of salicylic, p-coumaric and caffeic acids, which significantly reduced the germination of conidia and the severity of the disease. ^{Santos-Esteban et al. (2021}) reported that the R. sachalinensis extract inhibited the growth of Fusarium oxysporum isolated from the bean plant (Phaseolus vulgaris) by 100%, whereas ^{Baysal-Gurel and Bika (2021}) reported the foliar applications of this same extract at a concentration of 0.5 mL L ^-1 reduced the severity of powdery mildew (Podosphaera physocarpi) on the ornamental tree ninebark (Physocarpus opulifolius) grown in a shade house.

On the other hand, B. amyloliquefaciens QST 713 had a significantly greater effect than the rest of the treatments after the M. alternifolia essential oil and the R. sachalinensis extract. B. amyloliquefaciens QST 713 has been reported to have an effect on the regulation of genes related to the activation of transcription factors of the WRKY family and NAC, related to the defense mechanisms against stress factors and LRR-RLK receptors, which intervene in the recognition of the pathogen and the transduction of signals, through transcriptomic studies on peach fruits treated with B. amyloliquefaciens QST 713 for the control of Monilinia fructicola, where it is also pointed out that the biofungicide inhibited the in vitro growth of the fungus in the culture and reduced the severity of the symptoms of the disease (Tsalgatidou et al., 2014). In turn, ^{Pérez-Moreno et al. (2015}) found that B. amyloliquefaciens QST 713 inhibited the in vitro culture growth of Sclerotinia minor isolated from symptomatic lettuce (Lactuca sativa) plants with an average radial mycelial growth of 1.9 cm. Another study, carried out by Solano- Báez et al. (2021) indicates that B. amyloliquefaciens QST 713, at a concentration of 6×10⁷ UFC mL ^-1 reduced by 81.7% the incidence of drowning of zucchini plants produced by a complex made up of Phytophthora capsici, Rhizoctonia solani and F. oxysporum.

Finally, the biopesticides used to control fungal diseases have diverse advantages, since they have multiple action mechanisms due to the amount of phytochemicals they contain, leading them to generate no resistance, being ecological, easy to produce, effective and inexpensive, they produce no waste and have a low toxicity on organisms that are not their target, including humans, making risks on human health reduced (^{Deresa and Diriba, 2023}; ^{Suteu et al., 2020}). It is worth mentioning that for the management of plant diseases in gardens, parks and public areas, where E. gibbiflora is appreciated, it is crucial to use fungicides that generate no residuality, are eco-friendly and low-risk for humans and fauna, due to the constant contact of users with this type of environments.

Due to the above, this investigation represents a contribution for the integrated management of diseases in E. gibbiflora in urban areas. However, ^{Lecomte et al. (2016}) indicate that once the biofungicide with efficiency in the control of phytopathogens in ornamental plants is identified, it is necessary to standardize the doses, formulation and application method, making it a guideline to continue with research.

Conclusions

C. truncatum, which induces anthracnose in E. gibbiflora leaves is more sensitive to biofungicides composed of M. alternifolia oil (109.23 mg L ^-1), R. sachalinensis extract (168.76 mg L ^-1) and B. amyloliquefaciens QST 713 (61.07 mg L ^-1) in in vitro conditions. E L. digitata oil (19.95 mg L ^-1), T. vulgaris and M. spicata essential oils (112.47 mg L ^-1) and the L. tridentata and R. communis extract (78.51 mg L ^-1) have an inhibiting effect on the growth of the fungus of over 50% (which vary between 63.4 and 93.6%) and represent an alternative for the integrated management of the disease. The logistic regression has a good adjustment and helps model percentages with significant biological predictions, in addition to helping calculate the EC50 of biofungicides.