Evaluation of Chitinase Enzyme Production in Some of the Fusarium Isolates Obtained from Globodera rostochiensis

The plant parasitic nematodes are among the most important agents causing losses in agricultural crops (Nicol et al. 2013). The golden cyst nematode, Globodera rostochiensis (Wollenweber, 1923; Behrens, 1975) is the most destructive nematode of potato in the world (Brodie, 1984). Chitin is the most abundant polymer in structure of nematode eggshell. Chitinases are widely distributed in fungi and play important roles in degradation of chitin. The aim of this study was to assay chitinase activity in 16 isolates of various species of Fusarium obtained from golden potato cyst nematode, Globodera rostochiensis. In the current study, 16 Fusarium isolates were recovered from infected eggs of the golden potato cyst nematode, G. rostochiensis. These isolates were identified based on morphological (Seifert, 1996) and molecular features including internal transcribed spacer (ITS) regions (ITS1, ITS2 and 5.8S gene) of ribosomal DNA. Total genomic DNA was extracted from lyophilized mycelia with a QIAGEN DNeasy Plant Mini Kit (Germany). ITS regions of ribosomal DNA were amplified with the ITS1 (forward primer) and ITS4 (reverse primer) primers. PCR amplifications were performed with total reaction volume of 25 μl using a Takara EmeraldAmp GT PCR Master Mix. PCR products were separated by electrophoresis technique using 1% agarose gel in 1X Tris-Borate-EDTA (TBE) buffer by adding 12 μl SYBR-safe 10,000X concentrate DNA stain to melted agarose before running the gel and finally visualized under ultraviolet illuminator. The ITS1, ITS2 and 5.8S sequences were obtained by sequencing both strands in opposite directions using the PCR amplification primers, ITS1 and ITS4 in genomics resource laboratory at Massachusetts University, USA. Colloidal chitin was prepared using the procedure of Tikhonov et al. (2002). For enzyme assay in liquid media, the 16 isolates were grown in minimal synthetic medium (MSM) containing colloidal chitin (1 g l-1) based on the method described by Zeilinger et al. (1999). The culture medium was filtered through Whatman paper No. 3 filter followed by filtration through 0.2-mm Millipore polydifluoropropilene membranes. The filtrate obtained was analyzed for chitinolytic activity. Chitinase activity was determined by measuring the release of reducing saccharides from colloidal chitin by the N-acetyl-glucosamine-dinitrosalicylate method according to the method described by Monreal and Reese (1969). Protein concentration was determined according to Bradford (1976) with bovine serum albumin (Sigma) as the standard. Chitinase specific activity was obtained by dividing the enzyme activity rate by total protein mass. Enzyme assay was done to determine the most promising isolates for biological control of G. rostochiensis in 24 and 96 h after fungal growth. The chitinase specific activity data was subjected to analysis of variance (ANOVA) using software SAS, version 9.0 (Statistical Analysis System Institute Inc., USA) in a CRD (completely randomized design) with three replicates. Two selected isolates were assayed using a chitinase assay, fluorimetric kit-CS1030 (Sigma). The assay included three substrates, 4-Methylumbelliferyl N,N′-diacetyl-β-D-chitobioside, 4-Methylumbelliferyl N-acetyl-β-Dglucosaminide and 4-Methylumbelliferyl β-D-N,N′,N′′-triacetylchitotriose for the detection of the chitinolytic isozymes Chitobiosidase, β-N-acetylglucosaminidase and Endochitinase. The experiment was a randomized complete block design (RCB) with three replicates. Chitinase activity was subjected to analysis of variance using software SAS, version 9.0. Enzyme activity was expressed over time using the Logistic-Peak, software Slide Write, version 2.0. Two isolates (the strongest and weakest ones) were selected to determine the optimum conditions for chitinase production. The measured conditions were pH, temperature and reaction time between enzyme and substrate in a N-acetyl-glucosaminedinitrosalicylate method. According to measurements taken by an enzyme activity assay kit, the fourth day (96 h) showed the highest activity for the two selected isolates. We then tested our two isolates at pHs of 3, 4, 5, 6, 7, 8 and 9, temperatures of 22°C, 25°C, 28°C, 31°C for differing periods of time (1 h, 6 h, and 24 h). The experiment was based on factorial experiment in a completely randomized design with three replicates. Analysis of variance chitinase activity was performed using SAS 9.0 software. Among 16 isolates, F12 (1/02 U/mg) and F15 (0/04 U/mg) had the maximum and minimum amount of specific activity, respectively. According to morphological features and sequencing of ITS regions (ITS1, ITS2 and 5.8S gene) of ribosomal DNA, these 16 isolates were classified in three species i.e. F. solani, F. oxysporum and F. equiseti. The optimum conditions to produce chitinase in isolates of F12 and F15 were pH=6, 96 hours after fungal growth in 25°C and the reaction between the enzyme and substrate during one hour achieved by the N-acetyl-glucosamine-dinitrosalicylate method. As a producer of various chitinase enzymes, the filamentous fungus, Fusarium spp. seems to be recommendable to biologically control Globodera rostochiensis.