Abstract
Colletotrichum gloeosporioides (Penz.) Sacc. is a fungus that causes anthracnose disease in tropical fruit plants, resulting in damages of the fruit plants and low yield and quality of fruits. The use of chemical fungicides is common for management of this disease, but it also results in the development of fungal resistance to the chemicals. Therefore, this study aims to in vitro evaluate the efficacy of 14 crude leaf extracts against C. gloeosporioides. The results showed that Piper sarmentosum leaf extracts, using 80% of ethanol, methanol, and chloroform as solvents, were found to have very high antifungal activities. Crude methanol extract of P. sarmentosum leaves could effectively inhibit the growth of fungal mycelium (100%), followed by crude chloroform extract (81.85%) and 80% ethanol extract (45.50%). Maximum inhibition of C. gloeosporioides spore germination could be obtained after application with crude methanol extract of P. sarmentosum leaves and crude chloroform extract of Mentha cordifolia leaves at 1.25 and 2.5%, respectively. In conclusion, crude extracts of P. sarmentosum leaves were found to be highly effective for inhibiting both C. gloeosporioides mycelium growth and spore germination, and they have a potential as the new natural fungicides for management of anthracnose disease.
1. Introduction
Colletotrichum gloeosporioides (Penz.) Sacc. is a causative agent for anthracnose disease in many tropical fruit trees such as mango and papaya. This disease is very harmful and can cause spoilage and rotting of fruit plants, resulting in low yield and poor quality of the fruits [1]. The use of chemical fungicides is the most common choice for management of anthracnose disease, but this also causes the development of fungal resistance [2]. In addition, continuous and inappropriate use of chemical fungicides to manage anthracnose disease is not considered to be the long-term solution because this can increase the investment expenses, the risk of having high levels of toxic residues, and also the concerns in human health and environmental settings [3]. Due to these reasons, there are several attempts to search for alternative measures to control the anthracnose disease effectively. Recent efforts have focused on the development of environmentally safe, long-lasting, and effective biocontrol methods for management of anthracnose diseases. The utilization of natural products, especially the plant extracts, has been shown to be effective against many plant pathogens and considered to be safe for consumers and environments [4]. A number of plant species have been reported to possess natural substances that are toxic to a variety of plant pathogenic fungi [5, 6]. The extracts derived from Curcuma longa (leaf and rhizome), Tagetes erecta (leaf), and Zingiber officinales (rhizome) were shown to have antifungal activities against fungal anthracnose by completely inhibiting conidial germination of C. gloeosporioides [7]. The aqueous leaf extracts of custard apple (Annona reticulate L.) and papaya could inhibit spore formation and germination of Rhizopus stolonifer and also conidial formation of C. gloeosporioides [8]. In addition, C. capsici mycelial growth and spore germination were found to be suppressed by crude leaf extracts of Piper betle L. using methanol, chloroform, and acetone as solvents [9]. Hence, in this study, the in vitro antifungal activities of 14 leaf extracts were evaluated against C. gloeosporioides, a causative agent of mango anthracnose disease.
2. Materials and Methods
2.1. Fungal Culture
C. gloeosporioides was isolated from the upper surface of infected mango and cultured using potato dextrose agar (PDA) medium at 25°C.
2.2. Plant Materials and Extractions
Leaves from 14 different plant species were collected locally or bought at local markets of Maha Sarakham province which is in the northeast region of Thailand (Table 1). Leaf samples were thoroughly washed using tap water, air-dried at room temperature for 3 to 4 h, and finally dried in a hot-air oven at 45–50°C for 1 to 2 days depending on the plant species. Dried leaf samples were ground using small grinder, then placed in polyethylene bags, and stored at 4°C until required. For each sample, 50 g of leaf powder were added to 150 mL of methanol (M), 80% ethanol (E), or chloroform (F) (thus ratio between leaf powder and solvent was 1 : 3). The mixtures were agitated for 72 h on rotary shaker (130 rpm). The obtained extracts were centrifuged at 8,000 rpm for 10 minutes, filtered through Whatman filter paper no. 1, and transferred to 250 mL round-bottom flasks. Finally, these 42 extracts were evaporated using rotary evaporator at 45°C. Concentrated extracts were allowed to dry in hot-air oven, weighed again, and kept at 4°C until required for antifungal assays.
2.3. Screening of Leaf Extracts against C. Gloeosporioides Mycelial Growth
Forty two crude leaf extracts were in vitro tested for their efficacy against C. gloeosporioides mycelia growth using the poisoned food technique [10]. All crude leaf extracts were reconstituted to have the concentration of 5%. Then 1 mL of each extract was used for mixing with 19 mL of warm PDA and poured into 9 cm sterile Petri dish. After solidification, the plates were inoculated with the 6 mm agar piece containing a week old C. gloeosporioides mycelia. For each crude leaf extracts, the experiments were performed in three replicates. PDA plates mixed with carbendazim (commercial fungicide at 0.005%) and sterile distill water were served as positive and negative controls, respectively. The inoculated plates were incubated at 30°C, and the diameters of fungal colonies were measured every day for 5 days.
Inhibition of mycelial growth was calculated using the following formula [11]: : diameter of fungal colony grown on negative control plate, : diameter of fungal colony grown on plates containing crude leaf extracts.
2.4. Effect of Leaf Extracts Prepared at Different Concentrations on C. Gloeosporioides Mycelial Growth and Spore Germination
Twelve of the leaf extracts that were found to have high levels of activities against C. gloeosporioides mycelial growth were selected for further testing at lower concentrations. Various concentrations of selected crude leaf extracts were prepared (2.5, 1.25, 0.625, 0.3, 0.2, 0.1, and 0.05%) and in vitro tested against C. gloeosporioides mycelial growth (as described above) and spore germination. Inhibition of spore germination was examined by spreading 100 μL of C. gloeosporioides spore suspension (105 spores/mL) on PDA plates containing each leaf extracts. Carbendazim and sterile distill water were served as positive and negative controls, respectively. Plates were incubated at 30°C and monitored for 7 days.
2.5. Statistical Analysis
All data were subjected to analysis of variance (ANOVA) using the general linear models procedure (SAS Institute, Cary, NC, USA). The data of the percentages of mycelial inhibition were arcsine transformed before analysis. The means of % mycelial inhibition of all treatments were compared and determined using the LSD test at .
3. Results
3.1. Screening of 42 Crude Leaf Extracts against C. Gloeosporioides Mycelial Growth
Different solvents used for extraction could result in different levels of in vitro antifungal activities of the crude leaf extracts (5%) as measured by poisoned food technique. The antifungal activities of leaf extracts using 80% ethanol, methanol, and chloroform as solvents were found to range between 0.77–45.50%, 4.35–100% and 12.37–100%, respectively (Figure 1). Even though all crude leaf extracts exhibited certain levels of activities against C. gloeosporioides mycelia, the 80% ethanol extract of O. bacilicum and chloroform extracts of A. heterophyllus and C. siamea did not effectively prevent mycelial growth. C. siamea leaves that were extracted using 80% ethanol and methanol were found to have very low antifungal activities at 0.77 and 4.35%, respectively (Figure 1).
(a)
(b)
(c)
Crude leaf extracts using 80% ethanol as solvent were shown to have rather low antifungal activities (less than 50%), as shown by that P. sarmentosum, A. heterophyllus, and E. camaldulensis could prevent the growth of C. gloeosporiodes mycelia at 45.50, 42.75, and 33.85%, respectively (Figure 1(a)). Interestingly, crude methanol extracts of P. sarmentosum leaves exhibited the highest inhibition activities against C. gloeosporioides mycelialgrowth (100%), followed by E. camaldulensis (57.75%), O. sanctum (52.75%), and P. guajava (52.75%). However, the other methanol leaf extracts were found to have levels of antifungal activities less than 50% (Figure 1(b)). There were 5 chloroform extracts that were found to have more than 50% inhibition activities against C. gloeosporioides mycelial growth, including M. cordifolia (100%), P. sarmentosum (81.75%), E. camaldulensis (60.25%), M. paniculata (55.50%), and O. bacilicum (91.10%) (Figure 1(c)).
3.2. Effect of Leaf Extracts Prepared at Different Concentrations on C. Gloeosporioides Mycelial Growth and Spore Germination
Twelve crude leaf extracts (derived from 7 plant species), including the extracts of P. sarmentosum and E. camaldulensis in all solvents, the extract of A. heterophyllus in 80% ethanol, the extracts of O. sanctum and P. guajava in methanol, and the extracts of O. bacillicum and M. paniculata in chloroform were prepared at various concentrations (2.5, 1.25, 0.625, 0.3, 0.2, 0.1, and 0.05%) and determined for their efficacy against C. gloeosporioides mycelia growth and spore germination.
Although at lower concentrations these plant extracts exhibited lower antifungal activities, some plant extracts remained effective (Tables 2 and 3). In particular, when compared to carbendazim (commercial fungicide), the crude methanol extract of P. sarmentosum and chloroform extract of Mcordifolia at 2.5% could significantly inhibit the growth of C. gloeosporioides mycelium at 100 and 97.60%, respectively (Table 2); moreover, both of these plant extracts at 1.25% also completely prevented C. gloeosporioides spore germination (100%) (Table 3). However, the other crude leaf extracts at lower concentrations did not exhibit significant antifungal activity against C. gloeosporioides (Tables 2 and 3).
4. Discussion
In this study, 12 leaf extracts (obtained from 7 plant species) that were found to inhibit C. gloeosporioides mycelial growth very strongly at 5% were selected, then prepared at lower concentrations, and used for further evaluation against C. gloeosporioides mycelial growth and spore germination (Tables 2 and 3). At 2.5%, the crude methanol extract of P. sarmentosum and chloroform extract of M. cordifolia were shown to inhibit C. gloeosporioides mycelial growth, and at 1.25%, both of these could also completely prevent C. gloeosporioides spore germination.
From previous reports, there are a variety of plant extracts that were used to control fungal anthracnose. For instance, crude methanol, chloroform, and acetone extracts of Piper betle leaves at the concentration of 10 μg/mL could inhibit the growth of Colletotrichum capsici (responsible for anthracnose disease in pepper) mycelium at 85.25, 78.53, and 73.58%, respectively [9]. Also, at the same concentration, crude methanol, chloroform, and acetone extracts of these P. betle leaves were found to prevent C. capsici spore germination at 80.93, 74.09, and 72.91%, respectively [9]. Moreover, the leaf extracts of O. bacillicum and Allium sativum exhibited 100% inhibition of C. gloeosporioides (responsible for anthracnose in para rubber) mycelial growth when applying at 50 and 100% w/v, respectively, and both of these extracts could completely suppress spore germination when applying as minimal as 10% w/v [12]. Furthermore, the ethanol extracts of Ocimum gratissimum and Aframomum melegueta leaves were shown to inhibit the growth of Botryodiplodia theobromae mycelium (causative agent of banana anthracnose) at 72.1 and 68.2%, respectively [13].
Other plant pathogenic fungi could also be inhibited by plant extracts. For example, the ethanol extracts of O. gratissimum and A. melegueta leaves were also reported to prevent Fusarium oxysporum and Aspergillus niger spore germination at over 65% [13]. In addition, Rhizopus oryzae spore germination and mycelia growth were found to be suppressed by the leaf extracts of O. gratissimum [14].
This study showed that P. sarmentosum and M. cordifolia leaves had significant antifungal activity. The studies of phytochemical characteristics showed that bioactive compounds in Mentha sp. are sitosterol and β-sitosteryl-β-D-glucoside, and in Piper sp. are lignans, steroids, neolignans, alkaloids, propenylphenols, terpenes, piperolides, chalcones, flavanones, flavones, and amides bearing isobutyl, pyrrolidine, dihydropyridine, and piperidine moieties, all of which could exhibit high antimicrobial and antifungal properties [15–17]. The levels of plant bioactive compounds with antifungal activity could be influenced by many factors which include the age of plant, harvesting time point, extraction solvent, and method of extraction [18].
In conclusion, this study shows that crude leaf extracts of P. sarmentosum have strong antifungal activities against C. gloeosporioides. This may suggest their potential for future formulation into products for controlling anthracnose diseases of mango and other fruits. More extensive study of their phytochemical characteristics and in vivo efficacy remains to be determined.
Acknowledgments
This work was financially supported by the NRCT, Thailand. The authors also thank the Department of Biotechnology, Faculty of Technology, Mahasarakham University, Thailand, for laboratory equipments and facility.