BIOTROPIA Vol. 28 No. 2, 2021: 141 - 148 DOI: 10.11598/btb.2021.28.2.1274 141 BIOCONTROL POTENTIAL OF ENDOPHYTIC Aspergillus spp. AGAINST Fusarium verticillioides RON PATRICK CUAGDAN CAMPOS* AND JAMES KENNARD SANZ JACOB Department of Biological Sciences, College of Arts and Sciences, Isabela State University, Echague 3309, Isabela, Philippines Received 15 June 2019/Accepted 8 September 2019 ABSTRACT The soil-borne fungus Fusarium verticillioides is the causal agent of ear, stalk and root rot of maize resulting in severe reduction in yields and quality of infected products. Endophytic fungi have been reported as potential candidates in controlling pathogens since they are considered strong plant mutualists that confer disease resilience to their host. The present study was carried out to determine the in vitro antagonistic activity and biocontrol potential of endophytic Aspergillus spp. associated with Plectranthus amboinicus leaves against F. verticillioides. Three fungal endophytes from the genus Aspergillus, namely; A. tamarii, A. terreus and A. niger were isolated and identified from the leaves of P. amboinicus,. The fungal isolates were tested for their antagonism against F. verticillioides in dual culture plates. Results indicated that the Aspergillus endophytes can restrict the growth of F. verticillioides through varying mechanisms of antagonism. A. niger inhibited F. verticillioides by 47.37%, followed by A. tamarii (41.02%) and A. terreus (27.91%). Dual culture observations revealed that A. tamarii and A. niger antagonized the growth of F. verticillioides via overgrowth mechanism while A. terreus employed antibiosis to restrict the pathogen. These varying degrees of antagonism by the Aspergillus endophytes exhibited their potential as biocontrol agents and source of bioactive compounds. Keywords: Aspergillus, biocontrol, endophytic fungi, Fusarium, rot INTRODUCTION Fusarium verticillioides is one of the most commonly reported soil-borne fungal pathogens infecting maize (Abbas et al. 1998; Bacon & Hinton 1996). It is the causal agent of ear, stalk and root rot of maize. This fungus also produces secondary metabolites such as fumonisins that accumulate in maize kernels, consequently causing severe reductions in yields and quality of the products (Leyva-Madrigal et al. 2017; Chu & Li 1994). In order to maintain the abundance and quality of agricultural products world-wide, these plant pathogens need to be controlled and managed appropriately (Pal & Gardener 2006). Currently, chemical fungicides are the most effective agents in preventing the infection of F. verticillioides. However, efforts to control postharvest diseases employing synthetic chemical control agents pose danger to the environment as they affect soil microorganism diversity (Cardoso et al. 2010). Endophytic fungi are microorganisms that inhabit internal plant tissues, usually involving metabolic interactions without apparent symptoms (Bacon & White 2000; Petrini 1991; Wilson 1995). Growing evidences from several studies indicate that endophytes are found in all plants and are extremely abundant and diverse (Arnold et al. 2000). Endophytic fungi are believed to be strong plant mutualists which can produce increased resilience against pests and plant pathogens (Carroll 1988). Since these microorganisms are systemically distributed throughout the host via metabolic translocation, they are noteworthy candidates for biological control (Rai et al. 2007). Studies have been done on the antagonistic mechanisms and actions, particularly the efficacy, of many endophytic fungi for their biocontrol potential against different plant pathogens (Rahman et al. 2009). Furthermore, considering their long-lasting effects and not requiring repeated periodic *Corresponding author, email: rpcampos023@gmail.com BIOTROPIA Vol. 28 No. 2, 2021 142 application, the use of fungi for biocontrol has more advantages than chemical fungicides (Okigbo & Ikediugwu 2000). Several studies recorded that Aspergillus species acted as biological control agents against fungal pathogens (Fusarium oxysporum, Pythium spp. and Sclerotinia sclerotiorum) through competition, mycelial lysis, mycoparasitism and antibiosis via the synthesis of volatile and/or non-volatile metabolites (Gomathi & Ambikapathy 2011; Daami-Remadi et al. 2006; Bhattacharyya & Jha 2011). Hence, the present study was carried out to determine the in vitro antagonistic activity and biocontrol potential of endophytic Aspergillus spp. associated with Plectranthus. amboinicus against F. verticillioides. MATERIALS AND METHODS Collection of Plant Material Mature and healthy leaf samples of Plectranthus amboinicus were collected in May 2018 from Echague, Isabela (16.6701° N, 121.7171° E). The plant materials were authenticated based on taxonomic characters through the assistance of an experienced botanist. Samples were transported in sterile polypropylene bags and processed within 6 hours of collection. Isolation of Endophytic Fungi Plant samples were surface sterilized and the endophytic fungi were isolated using the method described by Kusari et al. (2009) with minor modifications. Leaves of P. amboinicus were washed and rinsed with running tap water and cut into 10 mm (length) by 5 mm (width) segments. Each segment was then surface sterilized by sequential immersion in 75% ethanol for 2 minutes, 1% Sodium hypochlorite (NaOCl) for 3 minutes, and then once again in 75% ethanol for 1 minute. The leaf segments were finally rinsed three times in sterile distilled water to remove excess sterilant and blot dried in sterile filter paper. The leaf segments were later inoculated onto Potato Dextrose Agar (PDA) plates supplemented with streptomycin (1 ml/L) to suppress bacterial growth. Four (4) leaf segments were equidistantly placed on each amended PDA plate. The plates were then sealed with parafilm and incubated at 28 °C until the growth of endophytic fungi was detected. The hyphal tip of each endophytic fungi growing out from the leaf segments were separately transferred into new amended PDA plates and routinely maintained. Identification of Endophytic Fungi The endophytic fungi were identified according to their macroscopic and microscopic characteristics, particulalrly the fruiting structures and spore. Colony morphology of the endophytes was observed on Coconut Water Agar (CWA), Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA). To ascertain the identification of species, a microscopic examination of morphological structures was conducted using the agar block technique. The identification of fungi was done using the dichotomous keys and descriptions provided by Quimio and Hanlin (1999) and Samson et al. (2014). Source of Test Fungus Pure cultures of the pathogenic fungus F. verticillioides were obtained from the maintained cultures of Mycology Laboratory, College of Arts and Sciences, ISU-Echague, Isabela. The cultures were transferred into sterilized Potato Dextrose Agar (PDA) plates and incubated at ±30 °C to allow growth of the mycelia for seven (7) days. In vitro Antagonistic Activity The in vitro biocontrol potential and antagonistic activity of the endophytic fungi were tested against the pathogenic fungus F. verticillioides using the dual culture method described by Matroudi et al. (2009) and John et al. (2010). Agar plugs of each endophytic fungi and pathogen were obtained from the edge of 7-day-old pure cultures using sterile cork borer. The plugs were aseptically transferred 20 mm apart respectively on the center of 90 mm MEA plates. Control plates were inoculated with the pathogenic fungi alone. Plates were incubated at 37 °C for 15 days. The interactions exhibited by the co-cultures were monitored daily and the diameter growth of both endophyte and pathogen were recorded at 5, 10 and 15 days, respectively. All control and test plates were Biocontrol potential of endophytic Aspergillus spp. from Mexican thyme – Campos and Jacob 143 conducted in triplicates. Percentage inhibition was calculated as compared to control (Gaspar et al. 2004). The growth inhibition was calculated by using the formula: Statistical Analysis Each of the tests was carried out using Completely Randomized Design (CRD) with three replicates for each treatment. All means were treated statistically using one-way Analysis of Variance (ANOVA) and compared by Tukey’s Honest Significant Difference test at P < 0.05 using IBM™ SPSS v25. RESULTS AND DISCUSSION Identification and Characterization of Endophytic Fungi Three fungal isolates belonging to Aspergillus species were selected and their morphology and growth characteristics on CWA, PDA and MEA were recorded for the determination of their biocontrol potential (Table 1). The colony color of A. tamarii endophytes ranged from yellow to olive green which turned dark green with age (Fig. 1A). The margin and form are both filamentous and elevation is slightly raised. Production of colorless exudates and brown-black sclerotia was observed in various plates after extended incubation. The colonies of A. terreus showed cinnamon-brown color with floccose white mycelia which eventually turns brown to yellow-brown consisting of a dense felt of conidiophores (Fig. 1B). Reverse morphology was brownish to orange in color, indicating the secretion of metabolites into the medium. Sclerotia was absent. Meanwhile, A. niger has a distinct black- brown colony (Fig. 1C). It was both filamentous on margin and form and has umbonate elevation. The colonies initially grow with felt- like yellow to white hyphae, turning black with the formation of conidia. Formation of black sclerotia and black exudate beads was also observed. The microscopic structures of the endophytes were observed in a microscope through the agar block method. The vesicles of A. tamarii have sub-globose shape, while A. terreus and A. niger have pyriform and globose shaped vesicles, respectively (Fig. 1D-1F). The asexual conidia of A. terreus are smooth and hyaline, while A. tamarii conidia are finely roughened compared to the conidia of A. niger which are echinulate (Fig. 1G-1I). The endophytic fungi also exhibited some common morphological structures, such as biseriate conidial heads and septate hyaline hyphae. The obtained microscopic descriptions of the Aspergillus endophytes coincide with the keys and descriptions provided by Samson et al. (2002). Table 1 Macroscopic and microscopic characteristics of the Aspergillus endophytes Macroscopic Characteristics Microscopic Characteristics Endophytic Fungi Culture Media Colony Color Reverse Color Colony Density Shape of Vesicle Texture of Conidia Seriation A. tamarii CWA Brown- green White Abundant Sub- globose Smooth/Finely roughened Biseriate PDA Parrot green White Luxuriant MEA Yellow Light yellow Abundant A. terreus CWA Beige Tan Sparse Pyriform Smooth Biseriate PDA Cinnamon Brown Sparse MEA Cream yellow Yellow orange Abundant A. niger CWA Grey White Luxuriant Globose Echinulate Biseriate PDA Black Cream Abundant MEA Brown- black Black Luxuriant BIOTROPIA Vol. 28 No. 2, 2021 144 Figure 1 Colony morphology of (A) A. tamarii; (B) A. terreus; and (C) A. niger; Conidiophore of (D) A. tamarii; (E) A. terreus; and (F) A. niger; Conidia of (G) A. tamarii; (H) A. terreus; and (I) A. niger In vitro Antagonistic Activity of Endophytic Fungi Results of this study showed that the endophytes can variably restrict the growth of F. verticillioides (Table 2). Among the three fungi, A. niger produced the largest inhibition on the mycelial growth of F. verticillioides by 47.37%, followed by A. tamarii (41.02%) and A. terreus (27.91%). F. verticillioides recorded a mean radial growth diameter of 69.91 mm on the control plate. In comparison, the pathogenic fungi produced radial growth of 49.85 mm, 60.92 mm and 44.48 mm in the dual culture with A. tamarii, A. terreus and A. niger, respectively. The smaller radial growth on the dual culture plates indicated the presence of antagonism between the pathogen and the endophytes. C B A D E F G H I Biocontrol potential of endophytic Aspergillus spp. from Mexican thyme – Campos and Jacob 145 Table 2 Radial mycelial growth and percent inhibition of F. verticillioides by three endophytic strains of Aspergillus after 14 days of incubation Fungal antagonists F. verticillioides Radial Mycelial Growth (mm) Percent Inhibition (%) A. tamarii 49.85±10.64a 28.69% A. terreus 60.92±11.51b 12.86% A. niger 44.48±4.39a 36.38% Control 69.91±16.31b - Notes: Values are means of three replications. Means in the same column with different superscript indicate significant difference at P ≤ 0.05. All the fungal antagonists inhibited the mycelial growth of F. verticillioides through different antagonistic mechanisms (Fig. 2). Observations on the dual culture plates of A. tamarii and F. verticillioides indicated that the endophyte antagonized the pathogen through overgrowth mechanism. The overgrowth is achieved when a fungus exhibits a higher growth rate, tolerance against metabolites produced, and a higher capacity of antibiotic production (Mathiavanan et al. 2000). A noticeable change in the morphology of A. tamarii in all co-culture plates was also observed wherein the isolates became highly floccose and conidia were rarely present (Fig. 2A). This suggested that the A. tamarii isolates were adapting and responding to the presence of F. verticillioides. Aspergillus species are known to grow via the formation of a floccose mycelium, producing aerial hyphae that are capable of enhanced oxygen absorption and increased rates of respiration (Rahardjo et al. 2005). Isolates of A. niger outgrew those of F. verticillioides implying that the antagonism involved is overgrowth mechanism. Macroscopic observation also revealed that the A. niger has a mutually intermingled growth with F. verticillioides without any zone of inhibition, indicating the failure of the production of antibiotics either by the pathogen or by the antagonist. A. terreus zone of inhibition was clearly observed in which there was a conspicuous space between the antagonist and the test fungus (Fig. 2E). The inhibition zone is observed in all co-culture plates of A. terreus and F. verticillioides and mycelial growth of both fungi is either stunted or severely decreased in the region. The formation of a zone of inhibition is an indication of the production of antibiotic substances either by the pathogen against antagonistic fungi or vice versa (Gomathi & Ambikapathy 2011). The capacity of Aspergillus species to inhibit Fusarium isolates has been reported by several authors. A. niger initiated lysis of F. oxysporum mycelium through antibiosis (Patibandn & Sen 2007; Dwivedi & Enespa 2013). A. niger, A. tamarii and A. terreus successfully controled the growth of Fusarium sambucinum and Phytophthora erythroseptica (Abdallah et al. 2015). A. niger was also reported as one of the best antagonists for several soil-borne, seed-borne and foliar plant pathogens (Kamil et al. 2009; Ahmed & Upadhyay 2009). Moreover, certain atoxigenic strains of Aspergillus have been reported to competitively exclude aflatoxin-producing strains during crop infection and thereby reduce aflatoxin contamination. One of these, AF36, has been registered as a biological control for the competitive exclusion of aflatoxin producing fungi from cottonseed (Cotty 2018). Aspergillus species have diverse adaptations and responses to cellular stress which allows them to be resilient in the presence of other organisms (Abdallah et al. 2015). These include the deployment of biophysically diverse compatible solutes and functionally diverse protein‐stabilization proteins; hyperaccumula- tion of melanin in the cell wall; oxidative stress responses; ability to resist high temperatures; production of extracellular polymeric substances (EPS) and formation of biofilms; and the ability to compete with other microbes. BIOTROPIA Vol. 28 No. 2, 2021 146 Figure 2 Antagonism of F. verticillioides with A. tamarii (A, D), A. terreus (B, E) and A. niger (C, F); Microscopic hyphal interactions of A. tamarii and F. verticillioides (G) and A. niger (H) with F. verticillioides CONCLUSION The three Aspergillus endophytes, namely A. tamarii, A. terreus and A niger isolated from the foliar segments of Plectranthus amboinicus restricted the growth of Fusarium verticillioides and the mechanisms involved overgrowth and antibiosis. The capacity of these endophytes to restrict the growth of F. verticillioides implied that these organisms can be exploited as possible alternatives to chemical control agents. ACKNOWLEDGEMENTS The authors acknowledge the provision of materials and facilities by the Department of Biological Sciences of Isabela State University, Echague, Philippines. We also gratefully acknowledge Dr Helen C. Ramos and Dr Florenda B. Temanel for their assistance in checking this manuscript. C B A F E D Endophyte Endophyte Endophyte Endophyte Endophyte Endophyte Pathogen Pathogen Pathogen Pathogen Pathogen Pathogen G H F. verticillioides hyphae A. tamarii hyphae A. niger hyphae F. verticillioides hyphae Biocontrol potential of endophytic Aspergillus spp. from Mexican thyme – Campos and Jacob 147 REFERENCES Abbas HK, Mirocha CJ, Meronuk RA, Pokorny JD, Gould SL, Kommedahl T. 1988. Mycotoxins and Fusarium spp. associated with infected ears of corn in Minnesota. Appl Environ Microbiol 54:1930-3. 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