Methods of molecular identifications and lab. protocols
A simplified media for screening chitinase activity of Trichoderma
A SIMPLE MEDIUM FOR SCREENING CHITINASE ACTIVITY OF TRICHODERMA SPP.
Toshy Agrawal*1,2 and Anil S Kotasthane1
Dr Anil S Kotasthane: Senior Scientist & Associate Professor
Dr (Ms) Toshy Agrawal: Scientist
1Department of Biotechnology, Indira Gandhi Krishi Vishwavidyalaya, Krishak Nagar, Raipur 492 06, Chhattisgarh, India
2Barwale Knowledge & Study Centre, BT4, Phase II, Addl. MIDC Area, Jalna Aurangabad Road, Jalna 431 203, Maharashtra, India
Running title: Chitinase screening medium for Trichoderma spp.
* Corresponding author: Dr (Ms) Toshy Agrawal
Phone: +919860775610 email: email@example.com
We developed a simple medium for the screening of chitinase activity of the Trichoderma isolates. A pH indicator dye was incorporated into the medium for the assay of very clear, distinct and rapid chitinase activity. The medium is very sensitive, reliable, user- friendly, and economical to screen chitinase activity of large populations of Trichoderma.
Bromocresol purple, chitin, indicator dye
Fungal strain assigned to the genera Trichoderma are well known producer of chitinolytic enzymes and are used commercially as a source of these proteins. Additional interest in these enzymes is stimulated by the fact that chitinolytic strains of Trichoderma are among the most effective agents of biological control of plant diseases ((Chet, 1987; Harman, 1990; Harman et al., 1993a; Komatsu, 1976; Lo et al., 1996; Muthumeenakshi et al., 1994; Samuels, 1996; Tronsmo, 1991)). Research on chitinolytic enzymes from Trichoderma has flourished during past one and a half decades (Carsolio et al., 1994; Draborg et al. 1995; Fekete et al., 1996). To date, several laboratories around the world are applying these genes to a variety of biocontrol strategies and studying the mechanism of fungal antagonism and mycoparasitism.
There are several mechanisms involved in Trichoderma antagonism namely antibiosis whereby the antagonist fungus shows production of antibiotics; competition for nutrients; and mycoparasitism whereby Trichoderma directly attacks the plant pathogen by excreting lytic enzymes such as chitinases, ß-1,3 glucanases and proteases (Haran et al. 1996). Because the skeleton of filamentous fungi cell walls contains chitin, glucan and proteins, enzymes that hydrolyze these components have to be present in a successful antagonist in order to play a significant role in cell wall lysis of the pathogen (Lorito et al. 1994; Carsolio et al. 1999). Several distinct chitinolytic enzymes have been reported in T. harzianum (De la Cruz et al. 1992; Haran et al. 1996). These include endochitinases, exochitinases and 1,4-ß-N-acetylglucosaminidases, which are induced during growth of T. harzianum in liquid medium containing chitin as carbon source. Chitinase being the most important enzyme involved in mycoparasitic and subsequent biocontrol ability of the fungus Trichoderma, requires simple and yet effective means for screening.
In the present investigation two different chitin sources (Rhizoctonia cell wall derived and commercial chitin derived colloidal chitin) were amended in the medium used to assess / screen the chitinase activity of the Trichoderma isolates.
Trichoderma spp. was isolated from rhizosphere and non rhizosphere soil samples collected from different geographical locations of Chhattisgarh (Central India) using Potato Dextrose Agar (PDA) by following the procedure developed for serial dilution method. Rhizoctonia spp. was also isolated from the sick soil of the rice field.
Colloidal chitin was prepared from commercial chitin (HiMedia) as well as from Rhizoctonia cell wall and was amended in the chitinase assay medium as a sole carbon source. Colloidal chitin was prepared from commercial chitin by the method of Roberts and Selitrennikoff (1988) with a few modifications described herein. In the first step acid hydrolysis of commercial chitin was done by suspending 5.0 g of chitin in 60ml Conc. HCl by constant stirring using a magnetic stirrer at 4oC (refrigerator) overnight. For the acid hydrolysis of Rhizoctonia cell wall the vegetatively growing mycelia was harvested from the PDB by suction filtration through Whatman no. 1 filter paper using a buchner filtration apparatus connected to a vacuum pump. Blot dried mycelial mat (25 g) was ground to uniform consistency in small amount of concentrated HCl using a sintered glass tissue homogenizer. The grounded slurry was subjected to acid hydrolysis by adding concentrated HCl (to make up the final volume to 60 ml) and was kept at cooling condition as stated above.
Second step was the extraction of colloidal chitin by ethanol neutralization. To the resulting slurry (as obtained in step one), 2000 ml of ice-cold 95% ethanol was added and kept at 26oC for overnight. It was then centrifuge at 3000 rpm for 20 min at 4oC. The pellet was washed with sterile distilled water by centrifugation at 3000 rpm for 5 min at 4oC. The washing of the pellets was done till the smell of alcohol vanished. Colloidal chitin thus obtained was stored at 4oC until further use.
The final chitinase detection medium consisted of a basal medium comprising (all amounts are per liter) 4.5 g of colloidal chitin, 0.3 g of MgSO4.7H2O, 3.0 g of (NH4)SO4, 2.0 g of KH2PO4, 1.0 g of citric acid monohydrate, 15 g of agar, 0.15 g of bromocresol purple and 200 µl of Tween-80; pH was adjusted to 4.7 and then autoclaved at 121oC for 15 min. After cooling the medium was poured in to Petri plates and allowed to solidify. The fresh culture plugs of the isolates to be tested for chitinase activity was inoculated into the medium and incubated at 25±2 oC for 2-3 days and observed for the colored zone formation.
Chitinase activity was identified due to the formation purple colored zone. Color intensity and diameter of the purple colored zone were taken as the criteria to determine the chitinase activity after 3 days of incubation. In the present investigation screening was performed for about 400 isolates of Trichoderma (belonging to different species) collected from different geographical locations of Chhattisgarh. The isolates were grouped according to the diameter of the purple zone as A) Isolates showing no chitinase activity B) Isolates showing low chitinase activity C) Isolates showing medium chitinase activity D) Isolates showing high chitinase activity and E) Isolates showing highest chitinase activity.
The principle behind the formation of colored zone is that the media is supplemented with a pH indicator dye bromocresol purple which transforms the yellow color of the media (in acidic here pH 4.7) into purple color due to increase in pH. The pH increases because of the utilization of chitin by the Trichoderma and its breakdown into product N- acetyl glucosamine (chitin being a homopolysaccharide of N- acetyl glucosamine), which is basic in nature and thus the cause for corresponding shift in pH towards alkalinity. The enzyme activity on Rhizoctonia cell wall derived colloidal chitin was faster as compared to colloidal chitin derived from commercial chitin. Here Rhizoctonia acts as elicitor molecule for the expression and induction of of chitinolytic enzyme. The purpose of using colloidal chitin derived from Rhizoctonia is to test its usefulness as a source of chitin in order to determine chitinase activity.
Chitin agar plate has been used earlier for isolating chitinolytic microorganisms by observing clear zone around the colony of microorganisms (Cody, 1989; Wirth and Wolf, 1990). However, the clear zone produced could not determine and discriminate the intensity of chitin utilization by individual Trichoderma isolates. Several other reports used costly glycol chitin and indicators such as Calcoflour White M2R, Flourescein isothiocyanate, Rhodamine B etc. to screen hyperchitinase producing bacteria and fungus (Vaidya et al., 2003). However the medium developed herein could be very effectively used as a economical source for basic screening and categorization of large fungal populations for its chitinase activity. All the components of the medium are readily available and easily affordable by any basic laboratory working on plant pathogen interaction or bio-control. Moreover the medium is suitable for rapid and user friendly sensitive plate assays or semi-quantitative enzyme diffusion plate assay.
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posted by toshy agrawal on 04 Aug, 2009