Biological Control Potential of Streptomyces sp . AR 10 Producing Albocycline Isolated from Soil around Ant Nest

Fifty actinomycetes were isolated from fifteen soil samples and were screened for their antagonism against fungal plant pathogens by dual culture assay, and one of the strain named AR10 was shown to be most effective in suppression of growth of plant pathogen. An antifungal compound of AR10 was extracted, and purified by TLC and HPLC. As a result of NMR and LC-MS analysis, the antifungal compound was identified as albocycline. AR10 suppressed Rhizoctonia damping-off of cucumber in infection control assay. The 16S rDNA sequence of AR10 shows high sequence similarity to those of genus Streptomyces, and the closest similarity was found in the sequence of S. lanatus NBRC 12787 with 98.7% similarity. However, the production of albocycline in Streptomyces closely related to AR10 in the phylogenetic tree has not been reported. Our finding suggests that AR10 can be a candidate for biological control agents.


Introduction
Food shortage is one of the most critical problems in the world, and to solve this problem, it is necessary to improve and protect the crop production (FAO, 2012).Phytopathogens affect the growths and productivities of crop plants, and there are wide varieties of phytopathogens in the world.For example, Rhizoctonia solani is a plant pathogen which infects crops such as rice, tomato and cucumber, and the incidence of disease caused by this pathogen is rising annually (Jaiswal, Elad, Graber, & Frenkel, 2007;Chuping, Xuehui, Huafei, Xiaoyu, & Zhiyi, 2014;Chellemi, 2002).Fusarium oxysporum also causes wilt disease in more than 100 kinds of plants such as banana, melon, and tomato (Chikh-Rouhou et al., 2013;Ploetz, 2006;Brzezinska, Jankiewicz, & Walczak, 2013).To control both pathogens and hence the diseases that caused, many chemical fungicides have been developed and used.However, excessive use of chemical fungicides has some problems, such as environmental pollution, deteriorating human health, and development of drug-resistant pathogens/insects (Staub & Sozzi, 1984;Ando, 1991;Clevo & Clem, 2001).Therefore, biological control agents which reduce the negative impacts on the environment and ecosystems have attracted much attention as an alternative to the use of chemical fungicide.
More than 70% of naturally occurring antibiotics have been isolated from different genus of actinomycetes (Prabavathy, Mathivanan, & Murugesan, 2006;Wang et al., 2013).Streptomycetes are known to produce much more antibiotics than actinomycetes in other genera, and are thought to have strong biocontrol activity against various phytopathogens.They also secrete extracellular hydrolytic enzymes such as chitinases and glucanases, which can degrade components in the cell walls of plant pathogens (Castillo et al., 2016;Sakdapetsiri et al., 2016).However, in spite of these advantages, reports of biological control application by actinomycetes are less than those from other bacteria such as Pseudomonas or Bacillus.Haeder et al. (2009) reported that leaf-cutting ants were using actinomycetes to control its own nest environment.Leaf-cutting ants live in symbiosis with fungi of the genus Leucoagaricus, and the symbiotic fungi serve as a major food source for the ants, and this symbiotic relationship is threatened by another pathogenic fungi.Leaf-cutting ants use actinomycetes to protect the symbiotic fungi from infections by pathogenic fungi (Haeder et al., 2009;Oh, Poulsen, Currie, & Clard, 2009;Poulsen et al., 2003).This symbiotic relationship of leaf cutting ants with actinomycetes are considered to be consistent with the concept of biological control application, and research on biological control using actinomycetes are considered to contribute to sustainable agriculture.
In this study, actinomycetes with strong antifungal activity against plant pathogens were isolated as candidates for biological control strain from the soil around ant nest.These actinomycetes were tested for infection control test against plant pathogens, and a strain with the strongest antifungal activity was identified and characterized.

Antifungal Activity of Actinomycetes
Antifungal activity of isolated fifty actinomycetes against plant pathogens were evaluated by dual culture assay.A single agar plug of 0.7-cm diameter, cut from the edge of actively growing fungal mycelium, was placed on the perimeters of the PDA plate at a distance of 1.5 cm, and then an actinomycete was inoculated on the opposite of the fungal mycelium.Plates were incubated for 3-4 days at 24 °C.The antifungal activity of the compound purified by HPLC was assayed against R. solani by disk diffusion test.The paper disks were placed at PDA plate and treated with 200 μg of purified compound.

Extraction of Antifungal Compound
The AR10 was cultivated on PDA medium at 24 °C.After 7 days of incubation, the colony was transferred into the 800 mL PDB medium and incubated at 1,000 rpm for 10 days.After incubation, 800 mL of ethyl acetate was added to the culture solution for extraction.The extraction was repeated three times, and the resulting organic layers were combined (ca.2.4 L) and evaporated.The dried residues were used for TLC analysis.

TLC Analysis
Crude extracts were subjected to TLC (Silica gel 60 F 254 ; Merck Milipore) using toluene: ethyl acetate: acetic acid (16:4:1) as a solvent and observed under UV light at 254 nm.After TLC analysis, TLC plates were cut into 5 fractions and used for bioautography test.The cut TLC plates were placed on the perimeters of the PDA plate at a distance of 1.5 cm, and then R. solani was inoculated on the opposite of the TLC plate.PDA plates were incubated for 3-4 days at 24 °C.

Identification and Characterization of Antifungal Compound
Crude extract was applied to preparative TLC under the same condition described above (section 2.4).TLC fractions containing antifungal compounds were dissolved in methanol, and used for HPLC separation.HPLC-UV analysis was performed using a JASCO LC-2000 system equipped with an Inertsil ODS-3 column (4.6 mm i.d. by 150 mm; GL Science) at a flow rate of 1.0 mL/min.For separation, a mixture of 50% acetonitrile/water were used as mobile phase, and the antifungal compound was detected by absorbance at 254 nm.
LC-MS analysis was performed using a QTrap (AB SCIEX) system with an Inertsil ODS-3 column (2.1 mm i.d. by 150 mm; GL Science) at a flow rate of 0.2 mL/min.For separation, distilled water and acetonitrile were used as mobile phase, and a linear gradient of 30-80% acetonitrile was applied.The mass spectrometer analysis was performed using ESI+ mode, using [M+H] + = 309.5 amu as the qualifier ion.

Identification of Actinomycete AR10
Phylogenetic analysis based on 16S rDNA sequence and morphological observation was done as follows.Bacterial 16S rRNA genes were PCR-amplified with primers 9F (5'-GAGTTTGATCCTGGCTCAG) and 1510R (5'-GGCTACCTTGTTACGA).The 16S rRNA gene sequences determined were compared with those retrieved from DNA database of APORON DB-BA 11.0 (Techno Suruga Lab., Shizuoka, Japan) and GenBank/EMBL/DDBJ using a BLAST homology search, and phylogenetic tree was constructed to ascertain the phylogenetic position of the actinomycete strain AR10.Phylogenetic trees were generated by the neighbor-joining method.Gene sequencing and phylogenetic analysis were carried out at Techno Suruga Lab., Co., Ltd.(Shizuoka, Japan).

Infection Control Assay of Isolated Actinomycete Strain AR10 against R. solani
Cucumis sativus L. (cucumber) was used for infection control test.Seeds were soaked in 70% ethanol for 10 sec, sterilized with 1% NaClO for 10 min, and finally rinsed three times with sterile distilled water.Sterilized seeds were germinated under dark conditions for 3 days on water agar plate.The germinated seeds were then transferred to agripot containing water agar medium (agar; 0.8%), with 3 seeds in one pot.AR10 was cultured in PDB medium at 24 o C, 1,500 rpm (stirred culture) for 3 days.Mycelia of AR10 were collected by centrifugation and then resuspended by 0.01 g mycelia/100 μL of culture broth.Resulting mycelia solution was spread in the agripot just before transplant of germinated seeds.Five days after the transplant, the infection assay was carried out by placing 0.7-cm-diameter agar plugs of plant pathogens onto the center of agripots.Biocontrol effect was assessed after the plants were grown in growth chamber for 12 days at 24 o C, 12 h light/dark condition.

Results and Disscusion
This study was aimed to evaluate the antagonistic potential of actinomycetes isolated from the soil around the ant nests, and there was a great deal of antagonistic actinomycetes in the soil samples.The antifungal activities of actinomycetes against R. solani were investigated using in vitro dual culture assays.Of the fifty strains tested, seventeen strains were found to be moderately effective and five strains showed strong antifungal activities.The five strains were named AR1, AR2, AR3, AR4, AR10, and applied to assay for antifungal activity against eight phytopathogenic fungi.As shown in Table 1, AR10 was most effective in antagonistic suppression against phytopathogenic fungi in the dual culture assay.AR10 showed inhibitory effect on all plant pathogens except P. infestans.In particular, AR10 showed inhibitory effect on C. echinochloa and F. oxysporum, which suggested that the presence of antifungal compound(s) different from other actinomycetes.Note.* inhibition zone index; -, < 10 cm 2 ; +, 10-20 cm 2 ; ++, 20-30 cm 2 ; +++, > 30 cm 2 .
To extract and detect the antifungal substance from AR10, the extracts of the strain were fractionated by TLC analysis.Bioautography was used to assess the activity of the compounds present in the TLC (Figure 1).

Table 1 .
Antifungal activity of actinomycetes against plant pathogens on PDA