A new chitinase-producer strain Streptomyces glauciniger WICC-A03: isolation and identification as a biocontrol agent for plants phytopathogenic fungi

This study discusses the isolation and identification of a new Streptomycetes highly active chitinase producer. Fifteen strains were isolated from Malaysian soil samples. The isolate WICC-A03 was found to be the most active chitinase producer. Its antifungal activity was evaluated against many phytopathogens. The identification of WICC-A03 using phenotypic and genotypic methods strongly indicated that strain WICC-A03 belonged to the genus Streptomyces and displayed similarity (91%) with Streptomyces glauciniger. Thus, it was given the suggested name S. glauciniger WICC-A03 with accession number: JX139754. WICC-A03 produces extracellular chitinase in a medium containing 1.5% colloidal chitin in submerged culture on 144 h. The produced enzyme was partially characterised and its molecular weight of 50 kDa was determined by using SDS-PAGE. This study indicates that WICC-A03 is a potential chitinase producer for biocontrol of plant pathogens. Further experiments are being carried out to optimise medium composition and cultivation conditions under lab and bioreactor scale.


Introduction
The search of new principles in the biocontrol of plant pathogens different from the classically used fungicides is of worldwide interest in recent years (McSpadden-Gardener & Fravel 2002). Such biological control of plant diseases is slow and gives few quick profits, but it can be long lasting, inexpensive and harmless to life (Zarandi et al. 2009). The chitin wastes, especially those derived from sea food-processing units, have caused remarkable environmental problems globally (Liu et al. 2003).
The production of inexpensive chitinolytic enzymes is of profound importance to the re-utilisation of shell fish wastes, which not only solves environmental problems, but also helps to extend the applications of microbial chitinases with added value in certain cases (Sezer et al. 2013). In the antagonistic activity of biocontrol agents, lysis of the host structure by secretion of extracellular lytic enzymes is a crucial mechanism (Kim et al. 2001). Other uses of chitinase include the treatment of chitin, biological research for the generation of fungal protoplasts and the biological control of many plant diseases. It is also used in crustacean chitin waste management (Suresh & Chandrasekaran 1999).
Chitinase is an industrially preferred choice mainly because it can degrade the chitin polymer in the cell wall of the fungal pathogen (Liu et al. 2003). The hydrolytic property of chitinases makes them attractive as environmentally safe biocontrol agents. To date, numerous reports have been published concerning chitinase-producing organisms used to inhibit the growth of phytopathogens. In most cases, the enzyme can either be used directly in the biological control of microorganisms, indirectly using purified proteins, or through gene manipulation (Oppenheim & Chet 1992).
Bacteria produce chitinase to digest chitin as it is their source of carbon and energy. Some chitinases produced by the chitinolytic bacteria such as Serratia marcescens MO-1 (Sezer et al. 2013) are potential agents for the biological control of plant diseases caused by various phytopathogenic fungi (Downing & Thompson 2000). Many chitinolytic bacteria produce only GH-18 chitinases, while some bacteria such as Streptomyces species produce both GH-18 and GH-19 chitinases (Watanabe et al. 2003). Therefore, Streptomyces microorganisms have been proposed to be a good source for chitinase fermentation and degradation of chitin (Christodoulou et al. 2001). As Streptomyces can use chitin as the sole carbon source, chitin can be used as an enrichment medium for the isolation of Streptomyces from soil (Saito et al. 1998). The aim of this study is to screen and molecularly identify chitinase antifungal activity-containing Streptomyces from Malaysian soil samples. The produced chitinase was characterised, and its antifungal activity was evaluated against phytopathogens isolated from domestically cultivated dragon fruits.

Results and discussion
2.1 Isolation of Streptomyces spp. producing chitinase Enrichment of chitinolytic microorganisms was performed with 13 samples derived from Malaysian soil. These samples were used to inoculate enrichment medium containing colloidal chitin as the only carbon and energy source. Cultures were incubated under aerobic conditions at 288C. The cultures were purified by plating on solidified enrichment medium. Within 24-96 h of incubation, the chitin-degrading organisms formed colonies in 1-3.0 mm in diameter and were surrounded by clear zones, indicating chitinase activity. The colonies served as inoculum for liquid cultures. The procedure of growing the organisms on agar plates and subsequently in liquid culture was repeated four times and this had led to the isolation of two different chitin-degrading strains.

Antibacterial, antifungal activity and antimicrobial susceptibility of strain WICC-A03
In this study, the potent Streptomyces isolate and crude chitinase preparation derived from it exhibited antibacterial and antifungal activity against phytopathogenic fungi, as shown in Table  S1. Most of the Gram-positive bacteria, Gram-negative bacteria and fungi were inhibited by the culture broth of WICC-A03 strain. These observations suggest that a good correlation exists between chitinolytic activity and the production of antimicrobial substances by Streptomycetes. In addition, the results are in agreement with those obtained by Yücel and Yamac (2010). In their study, the Streptomyces isolates exhibited antimicrobial activity against a panel of four bacteria, two yeasts and four filamentous fungi during the screening program in Turkey.
The antifungal activity ranged from 15 to 20 mm (inhibition zone diameter) was determined by agar well diffusion method, displaying the activity of WICC-A03 strain as a chitinase producer and inhibited the growth of the isolated pathogens. Antimicrobial susceptibility of the WICC-A03 strain is presented in Table S2.

Light microscopic examination for antifungal activity
Compared with the plate treated with sterile water (Figure S1(A)) and the Fusarium oxyporum has been exposed to crude enzyme after 48 h ( Figure S1(B)) had its cell wall disintegrated, protoplast leaked out and the mycelia cracked. The microscopic analysis shows that the main effect of the extracted enzyme is to degrade the cell wall. This is the target effect from chitinase, which is at the same time the most important antifungal proteins produced by some Streptomyces. These results corresponds with those of Dempsey et al. (1998) who demonstrated that the chitinase enzymes could inhibit fungal growth by hydrolysing the chitin presented in the fungal cell wall. Antifungal proteins such as chitinases are of great biotechnological interest because of their potential as seed preservative agents and for engineering plants to boost resistance to phytopathogenic fungi. In addition, Seidl (2008) mentioned that Streptomyces sp. degrades chitin with several chitinases acting synergistically.

Phenotypic identification of strain WICC-A03
Strain WICC-A03 exhibited different degrees of growth in a range of agar media and showed the morphology typical of Streptomyces (Locci 1989). The growth was abundant on most of the media tested, but was moderate on the others. The colour of the aerial mycelium varied from white to white grey. Therefore, the strain was assigned to the white series (Tables S3 and S4).
The physiological and biochemical properties of A03 strain are presented in Table S3. The morphology of the spore chains was of a Rectiflexibles type ( Figure S2(A)). The spore surface ornamentation was observed by transmission electron microscopy (TEM) and it revealed a smooth spore surface ( Figure S2(B)). According to the work of Shirling and Gottlieb (1966), laboratories with access to an electron microscope should include electron micrographs of the spore surface as one of the descriptive characterisations for each type of culture.
Analysis of the whole-cell hydrolysate of strain A03 indicated the presence of a chemotype I cell wall containing LL-DAP (Table S3). The presence of LL-DAP in the cell wall indicates that this strain is Streptomyces, as identified by Lechevalier and Lechevalier (1970) who established that the cell-wall composition analysis is one of the main chemotaxonomic characteristics of Streptomyces. The strain WICC-A03 was able to utilise different C-sources (Table S3). Taken from the work by Rosselló-Mor and Amann (2001), several additional tests relating to the use of arabinose, inositol, rhamnose, galactose and mannitol were used to identify new strains in their study.

Genotypic identification of strain WICC-A03
The nucleotide sequence of strain WICC-A03 was compared with the 16S rRNA gene sequences that have been reported in the GenBank database. The isolated DNA and PCR product size (1500 bp) are presented ( Figure S3). A phylogenetic tree was derived from the distance matrices using a neighbour-joining method ( Figure S4). A good congruence was found between the 16S rRNA sequences of the Streptomyces glauciniger and strain WICC-A03 and the use of genotypic and phenotypic techniques has given a better resolution in the species-level identification (Mizui et al. 2004). Thus, strain WICC-A03 was given the suggested name S. glauciniger WICC-A03 with accession number: JX139754.

Secondary structure prediction and restriction site analysis
The RNA secondary structure was predicted for the 16S rRNA of S. glauciniger WICC-A03 ( Figure S5) in this study. This prediction showed that the free energy of the structure was 2 35.2 kcal/mol; the threshold energy was 2 4.0 with its cluster factor and conserved factor set at 2 and compensated factor set at 4; and the conservativity was 0.8. The prediction of restriction sites in the strain A03 showed the restriction sites for various enzymes, such as Bsa BI, Sna BI, Eco RI, AgeI and BsaI ( Figure S6).

Chitinase production and characterisation
Prospective application of chitinases as biocontrol agent has been previously studied (Freeman et al. 2004) where strain WICC-A03 managed to produce up to 0.120 U/mL of chitinase in the production medium. The specific chitinase activities, using colloidal chitin as the substrate, recorded in the concentrated culture supernatant of this isolate was in the same range as those described for other crude bacterial chitinases (Wiwat et al. 1999). Meanwhile, a collection of 53 antibiotic-producing Streptomyces isolated from soils from Minnesota, Nebraska and Washington had been evaluated by Xiao et al. (2002) to identify their ability to inhibit plant pathogenic in vitro root rots on alfalfa and soybean.
Three protein bands were visible in this study with each recorded at about 63, 61 and 50 kDa in size. Among the protein bands detected, the prominent one was the 50 kDa band, as shown in Figure S7. The molecular weight of the chitinase obtained from this isolated strain is in agreement with those results put forth by Hoster et al. (2005). In their study, the microbial chitinases weighed from 20 to 120 kDa. In addition, the molecular weight of chitinase from Streptomyces has been reported to be between 30 (Tsujibo et al. 2000) and 68 kDa (Vetrivel et al. 2001).

Conclusion
A new chitinase producer S. glauciniger WICC-A03 was isolated from Malaysian soil samples and identified using phenotypic and genotypic methods. Its 16S rRNA has been deposited in GenBank under accession number: JX139754. WICC-A03 strain was able to produce chitinase in mineral medium containing colloidal chitin as a sole carbon and energy sources. The produced enzyme was partially characterised by SDS-PAGE, has the potential to control many pathogen species and can be used as biological control agent for plant diseases.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1 -S7 and Tables S1 -S4.