Some Plants Showing Antagonism to Five Plant Pathogenic Fungi

Fungi are among the main agents of plant diseases, being responsible for major losses in agriculture. The control of these microorganisms carried out using chemical compounds and numerous cases of resistance have already been observed, which makes it necessary to search for alternative methods of management of these pathogens. Therefore, the aim of this study, to evaluate the antifungal potential in plants. Twenty-four (24) plant extracts were tested for their antifungal potential against five plant pathogenic fungi: Sclerotinia sclerotiorum (Ss), Stromatinia cepivora (Sc), Fusarium oxysporum (Fox), Colletotrichum gloesporioides (Cg) and Verticillium dahlia (Vd). For the evaluation of the fungicidal potential, plant extracts were prepared by liquefying the plants in distilled water. The extracts were incorporated into PDA (Potato-Dextrose-Agar) culture medium to a final concentration of 35% and autoclaved. Then, PDA discs colonized by the aforementioned fungi were added to the center of each plate with the respective treatments (plant extracts). When all control treatments (PDA medium without plant extract) had colonized the entire Petri dish, the diameters of the fungal colonies were measured to calculate the Mycelial Growth Inhibition Index (MGI). Tests showed that all these plant extracts have some antifungal activity, ranging from 0 to 100% inhibition. In general, extracts of basil, lavender, guaco, rue, toxic cassava and black plum were the ones that stood out, with MGIs above 50%. New studies are being conducted to evaluate the activity of plant extracts without autoclaving, inhibition of sclerotia formation, to determine the minimum inhibitory concentration, as well as other parts of plants like roots and seeds, mixtures of plant extracts and in vivo antagonism tests.


Introduction
Plant diseases are among the most important phytosanitary problems, ahead of insects/arthropods and weeds. Among the phytopathogens, fungi stand out both in number and importance (Agrios, 2005). The control of these pathogens is basically performed with the use of chemical fungicides (Haq et al., 2020). Despite important strategies, it is known that such products are mostly costly and can affect the environment and living beings. In addition, many fungi have efficient survival mechanisms against adverse conditions (Massola Jr. & Krugner, 2011) and may also develop resistance to fungicide molecules used, as has often been observed in agriculture (Hobbelen et al., 2014). Note. *B = Branches, L = Leaves and F = Flowers.

In vitro Antagonism Evaluation
To evaluate the antifungal potential of crude plant extracts, a disc of PDA medium colonized by the pathogens mentioned was used and placed in the center of each Petri dish. The control treatment consisted of Petri dishes containing only PDA medium, without plant extract.
When the control treatment colonized the entire surface of the medium, the diameters of the fungal colonies were measured with the aid of a millimeter ruler. Assays were performed in triplicate. Radial mycelial growth data were used to calculate the Mycelial Growth Inhibition index (MGI) according to the formula: MGI = (D -T/D) × 100, where D is the diameter of the control colony, and T represents the mycelial growth, in cm, from treatment with plant extract (Menten et al., 1976).

Statistical Analysis
The experiment was conducted using a completely randomized design (CRD). MGI data were submitted to analysis of variance (ANOVA), followed by the Scott-Knott test, at 5% significance level, using the Sisvar 5.6 program (Ferreira, 2014).

Results
Considering the fungus Cg (Table 2, column), there was a variation in the MGI between 0 and 76.29% (Figure 1). The plant that stood out in the inhibition of growth of this pathogen was wild cassava (76.29%), with a significant difference from all others, which showed MGI below 34.16%. For Fox, the MGI ranged from 0 to 80%. In the inhibition of this plant pathogen, basil (80%) stood out, followed by sage (63.75%), lavender (60%), rue (53.33%), black plum (50.22%) and pomegranate (48.89%). As for Ss, the MGI ranged from 0 to 100%. In the inhibition of this fungus were highlighted the autoclaved extracts of basil, lavender and wild cassava (all with 100% of inhibition), followed by rue (70.41%) and guaco (62.50%). Horsetail extract inhibited only 7% of growth and the others were not antagonistic to this fungus. Against Sc, a variation was also observed in MGI between 0 and 100%. Five plant extracts formed an inhibition group without significant difference: lavender, were nt in antagonizing this pathogen. The alcoholic extract of this plant, dried and dissolved at 1% in water, inhibited 13% of Cg spore germination (Silva et al., 2009). Against Fox, no papers published in full in scientific journals were found that reported the use of autoclaved aqueous extract of basil, although the result here was significant: 80% inhibition. Camatti-Sartori et al. (2011) reported inhibition of 30.03 and 42.17%, with acetic extract at concentrations of 25 and 50%, respectively; and inhibition of 1.84 and 0% with ethanolic extract of this plant at 25 and 50% concentration, respectively. As regards Ss, 100% inhibition with the aqueous extract in question was observed here. Conversely, but using the same technique for a final concentration of 30%, Garcia et al. (2012) report an inhibition of only 25% of this pathogen. No scientific studies reporting the in vitro inhibition of Sc and Vd by basil extracts were accessed.
With regard to horsetail extract, in the present study only satisfactory antifungal activity was observed for Vd (74.36%). Using the same methodology, Rozwalka et al. (2008) observed 4.44% of inhibition growth of Cg from guava. Against Ss, Sc and Vd no works were found published in scientific journals that reported the use of autoclaved aqueous extract of this plant. Studying acetic and ethanolic extract, Camatti-Sartori et al. (2011) reported similar results, where they observed that two concentrations (25 and 50%) led to a low inhibition of Fusarium sp. (< 15.38%).
Carqueja extract at 35% showed low inhibition of the studied fungi (< 35.41%). In the case of Cg the inhibition was 13.75%. Studying different concentrations and with the same methodology, Milanesi et al. (2009) reported results close: 34.08% inhibition of this fungus. Against Fox no full scientific papers were found reporting the use of this plant to control the growth of this pathogen. For Ss, Pansera et al. (2012) observed that plant extracts obtained by infusion, hydroethanolic and ethanolic were not able to inhibit the fungus. Data on inhibition of Sc and Vd growth by compounds produced by this plant were not found.
With regard to lavender extract, excellent inhibition (> 60%) was observed for most fungi, except for Cg (16.16%), but no information about this fungus was available in literature with the present methodology. On the other hand, according to Domingues et al. (2009), L. augustifolia hexane extract exhibited good antagonism to C. acutatum (70%). Aqueous lavender extract completely inhibited the growth of Ss, Sc and Vd.
The aqueous extract of guaco was not antifungal regarding Cg. However, Bonett et al. (2012) observed that the 20% ethanolic extract of this plant was able to inhibit the fungus in a medium way. As for Fox, Ss, Sc and Vd, no information was found in the literature, although there was excellent antagonism of guaco toward the last three pathogens (100%).
Aloe, in the form of autoclaved extract, showed no fungicidal effect at the 35% concentration, except against Vd, which was low (5.12%). In contrast, Ranjitha et al. (2019) observed that this plant at a much lower concentration (7.5%) inhibited 50% of Cg growth. With different methodology, incorporation of plant filtrate into PDA medium after autoclaving, Mendy et al. (2019) reported that the 50% concentration was the most effective, inhibiting 100% of Cg growth, suggesting that heat sterilization may inactivate plant fungicidal compounds. The same was reported by Mendy et al. (2019) for Fusarium sp. Ss and Sc inhibition data were not accessed by extracts of this plant. Using another methodology, drying and incorporation of paper disk filtrate, Khaskheli et al. (2016) observed inhibition of Vd mycelial growth by approximately 24%.
The rue extract exhibited low Cg inhibition (34.16%). Using another methodology, dry plant material ground and diluted in boiling water, Celoto et al. (2008) report inhibition of 22.2% and 9.2% for extract with and without autoclaving at 20%, respectively, suggesting that this sterilization process may reduce the fungitoxic potential. In another study, the alcoholic extract dried and dissolved at 1% in water inhibited 6.6% of Cg spore germination (Silva et al., 2009). In the present study rue extract inhibited 53.33% Fox growth. Another species, F. solani, was inhibited by 77.6% with the autoclaved lyophilized extract of this plant (Oliva et al., 1999). The extract obtained from the milling and immersion in water, prepared by Garcia et al. (2016) at 30% concentration, inhibited 27.71% of the growth of Ss, a result lower than that observed here (70.41%). The aqueous extract of this plant inhibited 30 and 20% of growth of Sc at concentrations of 20 and 2%, according to Fuga et al. (2018), a result lower than that observed in the present study, of 70.83%. Autoclaved lyophilized extract, together with the culture medium, provided a 70.6% inhibition of Vd, according to Oliva et al. (1999). The result observed here was lower (33.33%).
The Mexican-tea did not show antifungal activity regarding Cg with the methodology employed. Similarly, but using another methodology, low activity was observed by Celoto et al. (2008): 4.7% and 0.5% inhibition for extract with and without autoclaving at 20%, respectively. Using different concentrations of aqueous extracts obtained from dry and filtered material (without autoclaving), Ohunakin & Bolanle (2017) observed greater inhibition of Fox at 60% concentration. In the present study, there was no inhibition of autoclaved extract, indicating that it is possible that this process may have affected the properties of the botanical fungicide. No information on the antifungal effect of rue was accessed from Ss, Sc and Vd.
The aqueous extract of lemongrass showed low activity against most of the tested fungi. For Cg the inhibition was 15.42%. Moura et al. (2012) observed that the filtered aqueous crude extract inhibited the fungus more (35.1%) when compared to the autoclaved one (25%). A study developed by Silva and coworkers (2014), with the 20% extract and paper disc method, ie without autoclaving, indicated a 17.04% inhibition of this fungus. The infusion of lemongrass leaves in PDA medium (autoclaved) led to a median inhibition, being higher at a concentration of 20% when compared to previously reported 5 and 10% (Marcondes et al., 2014). Using the same methodology as in the present study, Rozwalka et al. (2008) observed a 26.66% inhibition of Cg growth, isolated from guava. Using another methodology described earlier, Celoto et al. (2008) report superior inhibition, 38% and 0.5% of this fungus, with and without autoclaving, respectively, of the 20% extract. No data were found on lemongrass extract to inhibit Fox, Ss, Sc and Vd.
Pomegranate extract exhibited low inhibition of the fungus Cg; however, the alcoholic extract dried and dissolved at 1% in water of this plant inhibited 72% of Cg spore germination (Silva et al., 2009). No information was found in the literature about the potential antagonist of this extract to Fox, Ss, Sc and Vd.
The sage plant in the form of autoclaved aqueous extract exhibited median antifungal potential for only two fungi. Regarding Cg, the inhibition was low (3.12%). The sage leaf infusion in PDA medium (autoclaved) led to a median inhibition of this fungus, being higher at 20%, when compared to previously reported 5 and 10% (Marcondes et al., 2014). The dried alcoholic extract, dissolved at 1% in water, inhibited 72% of Cg spore germination (Silva et al., 2009). The autoclaved extract in question was a good inhibitor of Fox growth (63.75%). No information on antagonistic sage extract was accessed for Fox in the literature; however, against F. solani, median inhibition was observed (Marcondes et al., 2014). The aqueous extract obtained by 50% hot water infusion was not effective in inhibiting Ss (Pansera et al., 2012), as observed in the present study. Also, information about the antagonistic sage extract was not accessed from Sc and Vd.
The Indian coleus extract showed, in general, low fungicidal potential. There was no inhibition of Cg with the extract of this plant. However, Figueiredo et al. (2016) observed 58.9% inhibition of C. musae. There are no reports in the literature of Fox, Ss, Sc and Vd inhibition by this plant.
The greatest antifungal potential of fennel extract was for Sc, although there is no report in the literature of the use of this plant as a fungicide. With regard to Cg, low inhibition (27.12%) was observed; using the same methodology, Rozwalka et al. (2008) observed 13.33% inhibition of the growth of this fungus of guava. The alcoholic extract of this plant, dried and dissolved at 1% in water, inhibited 85% of Cg spore germination (Silva et al., 2009). Fox inhibition by this plant was 40.27%. Using 10% alcohol extract, Bonapaz et al. (2019) observed 55% of mycelial growth inhibition. No information on the use of this plant to inhibit the fungus Ss and Vd was found in the literature.
Bigleaf mint extract exhibited low antifungal potential against Cg (13.98%). Similarly, Ribeiro & Bedendo (1999) report that the filtered extract of Mentha sp. (without autoclaving) and at 1000 ppm concentration inhibited the growth of this fungus only slightly (29.57%). Inhibition of aqueous plant extract was low (6.25%) for Fox. Camatti-Sartori et al. (2011) report inhibition of 26.41 and 45.49%, with acetic extract at concentrations of 25 and 50%, respectively; and inhibition of 0 and 2.74% with ethanolic extract of M. piperita at 25 and 50% concentration, respectively. No data were found on Ss, Sc and Vd inhibition by bigleaf mint extracts.
Autoclaved wild cassava extract inhibited almost all fungi. No information was found on Cg inhibition by this plant, although here it showed good inhibition (76.29%). Fox inhibition by autoclaved extract was low (10.74%); however, Ferreira et al. (2015) observed that only 10% root peel extract, without heat sterilization, led to inhibition of 53.17% of this passion fruit fungus. The extract of this plant showed low Ss inhibition (6.55%), according to a study by Garcia et al. (2016). In contrast, total inhibition of growth of this fungus was observed here, perhaps because it is a toxic cassava cultivar, which usually has more than 100 mg HCN/kg. No data on the antifungal potential of Sc and Vd of this plant were accessed, although they showed good inhibition of mycelial growth of 100 and 88.57%, respectively.
Citronella aqueous extract showed low inhibition of Cg (29.63%). The 20% aqueous extract and using the paper disc method, ie without autoclaving, led to a 59.22% inhibition of this fungus (Silva et al., 2014). Following the same pattern, the extract in question showed low fungitoxic activity against Fox (37.50%). The study carried out by Silva et al. (2014) indicated an inhibition of 48.28%. According to Monteiro et al. (2013), the 1% aqueous extract of this plant inhibited 90% mycelial growth of this fungus. On the other hand, this plant in the 2000 (0.2%) and 2500 ppm (0.25%) concentrations inhibited 94.44% of the Fox mycelial growth (Sreenivasa et al., jas.ccsenet.org Journal of Agricultural Science Vol. 12, No. 12; 2011). The aqueous extract of this plant inhibited 52% growth of Sc at a concentration of 20%, according to Fuga et al. (2018), a result lower than that observed in the present study, of 100% inhibition. No citronella Ss and Vd inhibition data were accessed, although there was good fungal inhibition in the present study (72.22%).
Rosemary exhibited low inhibition of the evaluated fungi. Cg was inhibited by 24.07%; however, using the same methodology as the present study, Rozwalka et al. (2008) observed 51.11% inhibition of growth of this Cg guava fungus. Similarly, 10% alcohol extract inhibited 45% growth of this fungus (Bonapaz et al., 2019), and the infusion of rosemary leaves, autoclaved in PDA medium, led to median inhibition of this fungus, being higher in the concentration of 20% when compared to previously reported 5 and 10% (Marcondes et al., 2014). Low inhibition was also observed for autoclaved aqueous extract against Fox (20.37%). A study carried out by Şesan et al. (2017) reports 58.6% growth inhibition of this fungus with 20% ethanolic extract. According to Monteiro et al. (2013), the 1% aqueous extract of this plant inhibited Fox mycelial growth by more than 80%. There was no inhibition of Ss with the aqueous extract of the plant. Ethanolic extract at 10, 15 and 20% inhibited 100% growth and sclerotia formation of this fungus (Goussous et al., 2013). On the other hand, autoclaved aqueous extract of this plant at a lower concentration (5%) inhibited 94.22% fungal growth (Montes-Belmont & Prados-Ligero, 2006).
Ginger Bush aqueous extract showed low antifungal activity. Testing aqueous extract at 7% of concentration against C. lindemuthianum, Pinto et al. (2009) observed an inhibition of 23.8%, slightly higher than that observed here (14.81%) for Cg, with 35% extract. No information was found in the literature on the investigation of fungicidal properties of this plant to the fungi Fox, Ss, Sc and Vd.
The plant commonly known as comfrey also showed no fungicidal activity, from the methodology used, except for a very low inhibition of Vd mycelial growth. The 1% alcoholic extract of this plant inhibited 67% of spore germination of Cg isolated from coffee (Silva et al., 2009). In contrast, studies carried out by Celoto et al. (2008) showed that neither aqueous nor hydroethanolic extracts inhibited papaya Cg conidia germination. No data were found in the scientific literature on the antifungal potential of this plant toward Fox, Ss, Sc and Vd.
Black plum extract showed low activity against Cg (33.33%); on the other hand, according to a study conducted by Srivastava et al. (2015), there was excellent antimycotic action of ethanolic extract to 10 and 15% of this plant, when compared to aqueous extract, to fungus C. capsici. Fox inhibition by aqueous extract was median (50.22%); however, according to More and colleagues (2017), the use of different extraction solvents provided inhibition of mycelial growth of this fungus ranging from 68.83 to 82.04%. The extract of this plant showed low inhibition of Ss (2.38%), according to a study by Garcia et al. (2016), similar to what was observed in the present study, where there was no inhibition of the fungus as an autoclaved extract. No report was found in the scientific literature on the use of this plant to inhibit the fungus Sc, which here was inhibited 60.95%. However, the autoclaved extract of the species S. aromaticum (clove) inhibited this pathogen by 85.44 % at a concentration of 5% (Montes-Belmont & Prados-Ligero, 2006). No report was found in the scientific literature regarding the use of this plant to inhibit the fungus Vd, which here was inhibited by 65.71%.
Banana leaf extract showed low mycelial growth inhibition of the evaluated fungi. For Cg, the inhibition was 13.89%. Evaluating the antifungal activity of banana tree leachate toward some postharvest fungi, Bele et al. (2018) observed that there was 100% inhibition of C. musae mycelial growth at the three concentrations tested (10, 15 and 20%). However, against F. verticillioides there was 100% inhibition at 20% concentration. Here, only 8.88% inhibition of Fox growth was observed using autoclaved leaf extract. No information was found in the literature on inhibition of Ss, Sc and Vd by extracts of this plant.
The antifungal activity of Brazilian joyweed extract was low, and scientific information was found in the literature only on its antiviral activity of this plant (Lagrota et al., 1987).
Another plant that showed low activity against the studied fungi was horseradish. MGI of Cg was only 10.55%. In contrast, Ranjitha et al. (2019) observed that this plant, at a much lower concentration (7.5%), inhibited Cg mycelial growth by 50%. On the other hand, the alcoholic extract at 1% inhibited 88.3% of Cg spore germination (Silva et al., 2009). Horseradish activity against Fox was even lower (2.22%). According to studies by Hlokwe et al. (2018), using the alcoholic extract of leaves, the greatest inhibition (35%) of this fungus was observed at a concentration of 6 g/ml. One mix of the dry extract from various parts of the plant at concentrations of 25 and 75% was able to inhibit Fox mycelial growth by 73.71 and 100%, respectively, according to studies carried out by Dwivedi and Sangeet (2015). Using different parts of the plant and seed oil, El-Mohamedy and Mohamed (2018) observed that the greatest inhibition of both Fox and Ss was with 50% leaf extract, 20% root extract and seed 3% oil. Ss sclerotia production was totally inhibited with 3% seed oil. No information was found in scientific articles on the use of this plant as an antagonist to fungi Sc and Vd.
Finally, the autoclaved aqueous of Mexican sunflower extract also exhibited low or no fungicidal activity. Inhibition of mycelial growth of the plant pathogenic fungi Cg was 11.11%; on the other hand, at a concentration of 1000 ppp (0.1%) inhibited 68.6% growth of this fungus (Bhuyan et al., 2015). A study carried out by Dissanayake and Jayasinghe (2013) showed that 20% extract inhibited 20% growth of Cg. According to Ngegba et al. (2018), the extract of the leaves of this plant at 100 and 40% provided an inhibition of 87.4 and 30.1% of Fox, respectively. At a lower concentration, 20%, Dissanayake and Jayasinghe (2013) observed an inhibition of approximately 10%. Here, no inhibition by extract of this plant was observed. No information was found in scientific papers published in widely circulated journals dealing with the antifungal potential of Mexican sunflower to Ss, Sc and Vd.

Conclusion
The aqueous extracts that stood out, inhibiting mycelial growth of some plant pathogenic fungi tested above 50%, were basil, lavender, guaco, rue, wild cassava, citronella and black plum. New studies are being conducted to evaluate the activity of plant extracts without autoclaving, inhibition of sclerotia formation, to determine the minimum inhibitory concentration, as well as other parts of plants like roots and seeds, mixtures of plant extracts and in vivo antagonism tests.