Streptomyces ambofaciens S2-A Potential Biological Control Agent for Colletotrichum gleosporioides the Causal Agent for Anthracnose in Red Chilli Fruits

Anthracnose disease on chilli fruits has been known to be a serious problem faced by most of the chilli planters other than Chilli Mosaic Virus (CMV). Once the fruit had been infected by anthracnose the fruit is deem useless and need to be sorted out to reduce further infection on other fruits. Typical symptoms of anthracnose attack on chilli fruits are sunken necrotic tissues, with concentric rings of acervuli. The most widely spread Colletotrichum spp. found in the red chilli fruits are the Colletotrichum capsici and Colletotrichum gloeosporioides.


Introduction
Anthracnose disease on chilli fruits has been known to be a serious problem faced by most of the chilli planters other than Chilli Mosaic Virus (CMV). Once the fruit had been infected by anthracnose the fruit is deem useless and need to be sorted out to reduce further infection on other fruits. Typical symptoms of anthracnose attack on chilli fruits are sunken necrotic tissues, with concentric rings of acervuli. The most widely spread Colletotrichum spp. found in the red chilli fruits are the Colletotrichum capsici and Colletotrichum gloeosporioides.
Soil borne Streptomyces had been long known as the main producer of antibiotics in the world [1,2]. Scientists stated that at least 5000 documents relating to the production of bioactive compounds by actinomycetes were produced by the genus streptomycetes [3]. Jeffrey has stated that actinomycetes isolated from Sarawak farming soil were capable of inhibiting the growth of plant pathogens such as Fusarium palmivora, Pantoae dispersa and Ralstonia solanacearum [4]. Ndonde and Semu [5] in their study also noted that most of the Streptomyces spp. isolated from Tanzanian soils gave potential antimicrobial activities towards Clavibacter michiganesis sp. michiganensis (CMM IPO 542) and Xanthomonas vascatoria. Taechowisan et al. [6] showed that Streptomyces spp. SRM1 isolated from Nakorn Pathom, Thailand produced antagonistic activity on Colletotrichum musae on banana fruits. Shimizu et al. [7] and Yang et al. [8], stated that biocontrol agents have the potential to replace the currently widely used chemical pesticides. In agriculture practices, actinomycetes particularly the genus of streptomycetes has been used to control several plant diseases [9,10].
In this present study, isolation of Streptomyces spp. from peat soil were carried out and tested for their capability to produce antifungal substances towards anthracnose disease on chilli fruits.

Isolation and enumeration of streptomycetes
Soil samples were collected 15 cm below the surface of the soil under the canopy of selected plants at MARDI Research Station Pontian, Johor which was located at latitude 1°30'U and longitude 103°27'T. This research station was meant for the plantation of peat soil plants. The soil suspension was agitated with an orbital shaker at 250 rpm for approximately 1 h. Hundred and fifty microliter of the suspension was pipetted and spread onto Starch Casien Agar (SCA) [11] after 1 h of agitation. The plates were then incubated at 28 ± 2°C for 14 days before selection of emerging streptomycetes [5]. Selected streptomycetes were then subcultured onto fresh SCA plates and further incubated for 7 days before the pure colonies were used for the test and kept as stock in 20% (v/v) glycerol.

Abstract
Streptomycetes ambofaciens S2 was chosen to study its ability to control Colletotrichum gloeosporioides in chilli fruits. Soil samples were collected from Malaysia Agriculture Research Development Institute (MARDI) Pontain Research Station in Johor Darul Takzim, Malaysia. Streptomyces were later isolated from the soil samples and subjected to antifungal screening, metabolites characterization and in vivo testing of the potential microbes. In this study, 110 isolates of streptomycetes were successfully isolated from peat soil samples collected from Malaysia Agriculture Research Development Institute (MARDI) Pontain Research Station in Johor Darul Takzim, Malaysia. Screening for antifungal activity showed that 10 isolates of streptomycetes gave antifungal inhibition zone of 8-16 mm separately. Streptomyces ambofaciens S2 was later chosen for further testing based on the widest antifungal inhibition zone exhibited (16 mm). Characterization of S. ambofaciens S2 using both light microscope and scanning electron microscope showed that, S. ambofaciens S2 spores appeared to be rough while the spore chain arrangement was long and spiral. In vivo testing on S. ambofaciens S2, showed that C. gleosporioides infected chilli fruits sprayed with S. ambofacines S2 extract did not showed any sign of infection when compared with chilli fruits sprayed with ethyl acetate. Minimal inhibition concentration (MIC) performed on S. ambofaciens S2 against C. gleosporioides was observed to be 0.8125 mg/ml. The test conducted showed that S. ambofaciens S2 maybe an alternative biopesticide for control of C. gleosporioides. However, further tests should be in place to ascertain the viability and toxicity of the extract towards human health and environment.

Preparation of pathogens test strain
Colletotrichum gloeosporioides were isolated from infected red chili fruits using surface strerilization method. In this method, infected chili fruits were cut at the infected site and dipped into 2% sodium hypochloride for 2 min. After which, the infected sites were washed with sterile distilled water before being submerged into 75% ethanol for 30 sec. The pieces of cut chilli fruits were then left to dry in laminar flow for about 30 min before being placed onto fresh Potato Dextrose Agar plates (PDA). The plates were then incubated for about 21 days for the emerging of Colletotrichum spp. The emerging fungal colonies were picked and grown on fresh PDA for another 8 days to ensure the purity of the isolates. The pure isolates of Colletotrichum spp. were identified based on their conidia morphology using research microscope (Meiji MX 5000) and also molecular method using the ITS 1 and ITS 4 primers [12], before proceeding to be screened for the antigonistic activities.

Primary Screening for antimicrobial activity
Antimicrobial test was performed by using agar diffusion technique. In this technique, the test pathogens were inoculated on the middle of the petri dish surrounded by two plugs of the streptomycetes, one plug of PDA which act as negative control and one plug containing cycloheximide as positive control were placed in the petri dish ( Figure  1). All the plates were then incubated at 28 ± 2°C for 5 days. Positive results were determined with the formation of clear zones. All tests were conducted in a triplicate manner. Antifungal activity was evaluated using rating suggested by Baniasadi et al. [13] from the modification of rating by Lee and Hwang [14], whereby 0-4 mm= no inhibition, 5-9 mm=weak inhibition, 10-19 mm=moderate inhibition and ≥20 mm=strong inhibition.

Secondary screening
Selected streptomycetes isolates were grown in a 250 ml Erlenmeyer flask containing 100 ml of SCB at 28 ± 2°C for 3 days. Broth containing streptomycetes were later extracted using ethyl acetate. Equal volume of ethyl acetate was added to the 250 ml Erlenmeyer flask containing 100 ml of three days cultures. The flasks were later incubated at 28 ± 2°C with continuous agitation using an orbital shaker at 250 rpm for one hour. After one hour, the organic phase was pipetted into the 50 ml centrifuge tubes and was centrifuged at 4°C for 10 min at 8000 rpm. The supernatant obtained were then filtered using 0.45 µm nitrocellulose filter. Fifteen microliter of the filtrate which contained the crude antifungal extract was inoculated onto each sterile filter paper discs.
The discs were air dried in a laminar flow for about 30 min. Dried filter paper discs were then placed onto the prepared assay plates and further incubated at 28 ± 2°C for 5 days. The sizes of the clear zones formed were measured on the 5 th day of the incubation. Tests were conducted in triplicate manner.

Identification of selected actinomycetes
DNA extraction method as described by Vivantis was followed (http://www.vivantis.com) with little modification. Supernatant containing R1 buffer and lysozyme (50 mg/ml) was incubated at 37°C for 40 min. Another modification was the incubation time after the addition of R2 buffer and Proteinase K, where the time was prolonged to 40 min at 65°C. All other protocols were as stated by the manufacturer. DNA extracted was later used for Polymerase Chain Reaction (PCR).
PCR parameter and conditions used were as stated by Jeffrey [4]. The PCR products were later subjected to purification using Vivantis GF-1 Gel DNA recovery kit. Protocol used was as stated by the manufacturer (http://www.vivantis.com). The purified PCR products were sent for sequencing at First Base Laboratories Sdn. Bhd., Selangor using ABI PRISM ® 377 DNA Sequencer (Applied Biosytems). Results obtained from the sequencing were blast thru NCBI database [15].

Microscopic examination
Microscopic identification of selected Streptomycetes spp. were conducted using cover slip methods [16]. In this method, streptomycetes were streaked onto SCA. Sterilized cover slips were carefully stabbed onto the agar at the angle of 45° and the culture plates were incubated at 30°C for 6 days. Cover slip was then taken out from the agar and put onto prepared slide. Crystal Violet dye was used for the staining of the slide. Spore chain arrangement was then viewed using a light microscope (Meiji MX 5000 series). To view the spore morphology of selected streptomycetes, Scanning Electron Microscope (EL-30) was used.

Biochemical characterization of Streptomyces ambofaciens S2
Biochemical characterizations of Streptomyces ambafaciens S2 was performed using BIOLOG Gen III microplate. Protocol used was as stated by the manufacturer (http://www.biolog.com).

Characterization of Streptomyces ambofaciens S2 cultural characteristic
Pure colonies of Streptomyces ambafaciens S2 was grown in different International Streptomyces Project (ISP) Media for 7 days at 28 ± 2°C. After 7 days, the morphology of Streptomyces ambafaciens S2 in different media were evaluated and recorded.

Characterization of Streptomyces ambofaciens S2 physiological characteristic
Physiological characterization includes the growth of streptomyces isolate at selected temperature, pH [17] and also the ability of the isolate to produce various enzymes (lipase, cellulase, protease, mannanase and xylanase).

Spore germination test
Spore germination test was conducted by preparing the spore concentration in a serial dilution manner (10 -6 -10 -1 ). The dilution of spores were later plated onto freshly prepared potato dextrose agar (PDA). Spores were incubated at 30°C for 24 hours before being observed using a microscope to determine the germination of spores.

In vivo testing for the effectiveness of Streptomyces ambofaciens S2 to control anthracnose
Chilli fruits were bought from the local market for the testing purposes. Chilli fruits were surface sterilized by dipping the fruits in a 2% sodium hypochloride solution for 2 min and 75% ethanol for 30 sec, the fruits were left to dry before being dipped into solution containing 1 × 10 5 C. gleosporioides spores. The inoculated chilli fruits were then incubated for 1 h in a moist container. After 1 h the chilli fruits were sprayed with 0.5 mg/ml of S. ambofaciens S2 crude extract. The chilli fruits were then incubated at 28 ± 2°C for 5 days. After 5 days the chilli fruits were evaluated for any infection by C. gleosporioides. Tests were conducted in triplicates manner. Negative control for the test is pure ethyl acetate while 50 mg/ml of cycloheximide was used as the positive control.

Minimal Inhibitory Concentration test
Minimal inhibitory concentration (MIC) of the selected Streptomyces sp. crude extract towards C. gleosporioides was investigated. The MIC test was conducted in a serially diluted two-fold of concentration ranging from 60 mg/ml, 30 mg/ml, 15 mg/ml, 7.5 mg/ ml, 3.25 mg/ml, 1.625 mg/ml, 0.8125 mg/ml, 0.4062 mg/ml, 0.2031 mg/ml, 0.1015 mg/ml and 0.0508 mg/ml. All tests were conducted in triplicate. A standard curve was plotted using cycloheximide to estimate the relative concentration of the crude extract towards a known antifungal compound.

Isolation and enumeration of streptomycetes
From the total of 110 isolates of streptomycetes isolated, observation on the aerial mycelium colour showed that 45.5% (50 isolates), 21.8% (24 isolates), 20% (22 isolates) and 12.7% (14 isolates) of the isolates were found to be white, grey, dark grey and red, respectively. A total of 38 isolates were observed to produce diffusible pigmentation on the agar plate. It was also observed that the colony forming unit per gram (cfu/g) of dry soil for actinomycetes range from 4.2 × 10 3 to 6.0 × 10 4 ( Table 1).

Identification of pathogen strains
Fungal Colletotrichum gleosporioides was identified according to the blast results obtained from the National Center for Biotechnology Information (NCBI) database.

Antifungal activity
Test results of the primary screening, revealed that 44 isolates (40%) of the streptomycetes gave antifungal activity towards C. gleosporioides. Upon secondary screening was done only 10 isolates of actinomycetes were selected based on the inhibition zone of 8 -15 mm ( Table 2). It was observed that highest antifungal activities of streptomycetes were obtained from rhizosphere of Stevia rebaudianaa plant while the lowest antifungal activities were obtained from rhizosphere of Ananas comosus plant. Streptomycetes isolate number S2 and S4 gave the highest antifungal activities towards C. gleosporioides with 15 mm in diameter of clear zone observed. Streptomyces sp. S2 was selected for further tests.

Molecular identification of Streptomyces sp. S2
From the blast result performed, it was confirmed that Streptomyces sp. S2 was from the genus of streptomycetes and species of ambofaciens. This isolate was given the name as Streptomyces ambafaciens S2.

Cultural characterization and microscopic identification of Streptomyces sp. S2
Microscopic identification of Streptomyces ambofaciens S2 using SEM showed that the spore surface was rough (Figure 1) while the spore chain arrangement was observed to be long and straight ( Figure  2) when viewed using research microscope. Physiological test showed that S. ambofaciens S2 grew on 4% NaCl, pH 5 and 11 and temperature of 42°C (Table 3). From the observation on different ISP media plates, it was observed that S. ambofaciens S2 produces only grey, white or yellow aerial mycelium when grown on different agar media (Table 4).

In vivo testing
Chilli fruits that were sprayed with 0.5 mg/ml of crude extract from S. ambofaciens S2 were observed to be free from the inhibition of C. gleosporioides after 5 days. Meanwhile, chilli fruits which were sprayed with the ethyl acetate (negative control) were observed to be fully colonized by the fungi after 5 days of incubation (Figure 3).

MIC test for Streptomyces ambofaciens S2
The MIC test conducted showed that S. ambofacines S2 has the MIC of 0.8125 mg/ml (0.3 cm of clear zone) towards C. gleosporioides The MIC produced by S. ambofaciens S2 was approximately 3.0 mg/ml of cycloheximide

Discussion
The ability to use effective microorganisms or its secondary metabolite to control plant pathogens had open up a doorway to a more sustainable and environmentally friendly solution to pests and Soil sample collected from the rhizospere of:   It has been widely known that soil rhizosphere representing a unique biological niche with a diverse of microorganisms living in it [19]. A total of 110 isolates of streptomycetes were isolated from 5 soil samples collected from this study. It was observed that the majority of the streptomycetes showed the aerial mycelium of white followed by grey. The results obtained, however did not correlate with study done by Jeffrey [4] and Ndonde and Semu [5], where grey aerial mycelium streptomycetes were noted as the most frequently and predominantly isolated streptomycetes from both Sarawak and Tanzania soil, respectively. The differences of population may be due to the pH and soil moisture content of the soil.
The population density of streptomycetes (cfu/g) in this study    was much lower compared to study by Jeffrey [4]. He showed that the range for streptomycetes isolated from an agriculture site at Semonggok, Sarawak was 9.8 × 10 6 -8.0 × 10 7 cfu/g [4]. The low number of actinomycetes population in this study may be due to the condition of the rhizosphere itself whereby the actinomycetes were isolated from acidic soil. According to Suzuki et al. [20], soil with low acid level will suppress the population of streptomycetes. This is because streptomycetes can be widely found in soil with pH value of 5.0 to 8.0 while other genera of actinomycetes could be normally isolated from the neutral or alkaline soil [20].
Highest antifungal activity was observed from the rhizosphere of Stevia rebaudianaa plant while the lowest antifungal activity was observed from the rhizosphere of Ananas comosus plant. This could be due to the reason that diversity of a microbial community in specific environment was dependent upon the type of plant species. Only 9.1% (10 potential isolates) of the total isolates gave showed potential for as a biocontrol agent in the secondary screening. Khamna et al. [21] stated that the negative results obtained from other actinomycetes may suggest that the bioactive compounds produced are not meant for the tested fungi but for other untested microorganisms [4]. Another reason may be due to the antifungal compounds produced was not strong enough to inhibit the Colletotrichum sp. used in the study. Third reason for the low antifungal activity may be related to the cultivation condition of S. ambofaciens S2. According to Porter [22], different cultivation condition might trigger the production of different antimicrobial metabolite.
The ability of Streptomyces ambofacines S2 to inhibit the infection of anthracnose disease on chilli fruits during the in vivo trial, may suggest to the alternative application of the antifungal compounds for post-harvest usage other than pre-harvest usage.

Conclusion
Streptomyces ambofacines S2 showed the ability of producing antifungal activity towards C. gleosporioides was able to grew on 4% NaCl, pH 5 and 11 and temperature of 42°C. Apart from this S. ambofaciens also showed the ability to utilize a high number of carbon sources such as Gentiobiose, D-Turanose, Stachyose, D-Raffinose, D-Melibiose, A-Acetyl-β-D-Mannosamine, α-D-Glucose, D-Manose, D-fructose and D-Galactose. The ability to inhibit the growth of chilli anthracnose showed that S. ambofacines S2 could be used for the control of anthracnose on field or postharvest.