129 1. Introduction Fusarium is an endophytic genus consisting of an ar- ray of species responsible for damping-off, root rot, and vascular wilt in a multitude of economically important plant species (Summerell et al., 2001). The importance of Fusarium spp. in the current context is that infection may sometimes occur in developing seeds (Tajehmiri et al., 2014). Because of this unusually wide range of symptoms, cell wall degrading enzymes (CWDEs) could have a range of important functions for the fungus in penetration, in heads, and in saprotrophic growth in dead tissue. This eco- logical habitat of the fungus implies that Fusarium could be a useful resource of extracellular enzymes (Kwon et al., 2007; Bakri et al., 2013). Extracellular enzymes are thought to be particularly important CWDEs in interactions between Fusarium spp. and their hosts (Ahmed et al., 2012). Much research work is still needed to fully understand the degradation process, and particularly the enzymes and other metabolites secret- ed by the fungi during infection. Information on these as- pects could help to elucidate the biochemical mechanisms of wheat infection by Fusarium diseases and consequently facilitate fungal strain selection for industrial applications. However, studies of enzyme production by a phytopatho- genic fungus are complicated by the presence of the plant, particularly by the presence of plant enzymes and micro- bial enzyme inhibitors that occur in plants. The most prac- tical way to study the production of enzymes by a fun- gus is therefore to study the production of its enzymes on artificial growth media that contain no plant or enzyme inhibitors. Solid-state fermentation (SSF) is considered an attrac- tive alternative method for the production of industrially demanded enzymes that employ microorganisms (Krish- na, 2005). SSF was defined by Pandey (2003) as a fermen- tation process involving solids in the absence (or near ab- sence) of free water. Among the microorganisms that are capable of growing on solid substrates, only filamentous fungi can grow to a significant extent in the absence of free water (Guimaraes et al., 2006). The present study was undertaken to assess the potenti- alities of several Fusarium spp. in the production of indus- trially relevant enzymes under SSF conditions. 2. Materials and Methods Isolation of Fusarium species Fusarium spp. have been isolated from infected wheat seeds showing disease symptoms, and 21 were screened out of 105 strains for their host-pathogen interactions (Alazem, 2007). Seeds were sterilized to remove all mi- crobial epiphytes by soaking them in 1:5 dilutions of Na- OCl (sodium hypochlorite) solution for 15 min. They were then rinsed in sterile distilled water and dipped in 70% ethanol for 10 min. Subsequently, the seeds were washed with distilled water, dried between sterilized filter paper, and incubated in 9-cm Petri dishes containing potato dex- trose agar (PDA, DIFCO, Detroit, MI. USA) for 10 days, at 23±1°C in the dark to allow mycelial growth. All strains were identified morphologically according to Nelson et al. Enzymatic activity of the endophytic Fusarium species strains isolated from wheat Y. Bakri (1), M. Jawhar, M.I. E. Arabi Department of Molecular Biology and Biotechnology, AECS, Damascus, Syria. Key words: Fusarium spp., hydrolytic enzymes, wheat. Abstract: Fusarium is a genus of fungi that cause some of the most important plant diseases affecting agricultural and horticultural crops. Members of the genus establish an endophytic role inside the tissue of plants and produce a wide range of biologically active metabolites and enzymes. In the present study, the enzymatic profiles of several dominant Fusarium spp. were determined under solid state fermentation and activities were detected for xylanase, lipase, amy- lase, polygalacturonase, filterpase, and carboxy-methyl cellulase. Each Fusarium spp. showed a wide range of enzyme activities and protein contents. The ability to produce these enzymes was distributed amongst the strains tested, however amylase and xylanase F. solani Sy7 was found in a high percentage of strains. This study provides additional information to support future research about the industrial potential of these enzyme-producing species. Adv. Hort. Sci., 2014 28(3): 129-132 (1) Corresponding author: ascientific@aec.org.sy Received for publication 12 June 2014 Accepted for publication 29 September 2014 130 (1983). The Fusarium spp. strains used in the study are listed in Table 1. The cultures were maintained on silica gel at 4°C until needed. Extraction of enzymes from solid-state cultures Enzyme production by the Fusarium spp. strains was carried out in 250 ml Erlenmeyer flasks containing 5 g of solid substrate and nutrients (based on 100 ml of liquid medium) plus distilled water to adjust the moisture content to 75%. Fresh fungal spores were used as inoculums and 1 ml spore suspension (containing around 106 spores/ml) was added to sterilized medium and incubated at 30°C. Flasks were removed after cultivation and the enzyme was extracted by adding distilled water containing 0.1% Triton x 100 to make the in-flask volume equivalent to 100 ml. Flask contents were stirred for 1.5 h on a magnetic stir- rer. The clear supernatant was obtained by centrifugation (5000 x g for 15 min) followed by filtration (Whatman no. 1. paper). Carboxy methyl-cellulase and filter paperase (Fpase) activity Extra cellular enzymes were extracted by filtering the culture through Whatman No. 1 filter paper. The CMCase and Fpase activity were measured using the methods de- scribed by Refaz et al. (2013). One unit of enzyme activity (IU) was defined as the amount of enzyme that released 1 μmol of glucose per ml per minute. Amylolytic activity Α-amylase activity was determined as described by Okolo et al. (2001). The reaction mixture consisted of 1.25 ml of 1% soluble starch, 0.25 of 0.1 M acetate buf- fer (pH5.0), 0.25 ml distilled water and 0.25 ml of crude enzyme extract. After 10 min of incubation at 50°C, the liberated reducing sugars (glucose equivalent) were esti- mated by the dinitrosalicylic acid method of Miller (1959). One unit (IU) of α-amylase is defined as the amount of enzyme that releases 1 μmol of glucose equivalent per min under the assay conditions. Lipolytic activity Lipase activity was determined using 1 ml sunflower oil, 5 ml of 50 mM phosphate buffer pH 7.0 and 1 ml en- zyme solution. The assay was carried out according to the method of Park et al. (1988). One unit of lipase activity was defined as the amount of enzyme liberating 1μ mol of fatty acid per min under the experimental conditions. Pectinolytic activity Assay of polygalacturonase (PGase) activity was car- ried out according to Marcia et al. (1999). PG activity was determined by measuring the release of reducing groups using the dinitrosalicylic acid reagent (DNS) assay (Mill- er, 1959). The reaction mixture containing 0.8 ml of 1% citric pectin with 67% of metoxilation in 0.2M citrate- phosphate, pH 6.0 buffer and 0.2 ml of culture superna- Table 1 - Enzyme activity (U/g) of the 21 strains of Fusarium species used in this study Strain Xylanase carboxy-methyl cellulase Fpase Polygalacturonase Amylase Lipase F.culmorum SY1 20.3 3.31 2.46 23.52 45.5 61.28 2 96.36 6.67 3.64 38.72 55.36 0.30 3 163.69 3.89 2.03 70.96 54.6 0.90 6 131.93 2.78 2.03 80.4 51.13 117.04 9 12.16 1.76 2.03 15.68 52 65.04 12 115.92 2.32 2.03 74.24 66.8 81.28 13 90.64 2.87 2.03 23.92 47.4 0.50 14 19.52 2.09 2.03 41.04 51.4 0.10 F.solani SY7 757.2 5.27 2.03 67.92 118.35 81.28 11 112.16 2.66 2.03 22.72 40.4 0.70 20 234.96 1.43 2.03 61.04 39.25 0.90 35 125.6 2.39 2.03 43.76 41.7 57.52 F.verticillioides SY15 61.92 1.79 2.03 32 44.8 0.40 16 16.56 3.01 2.03 31.44 54.3 0.20 19 108.56 5.74 2.03 97.92 70.95 87.6 27 129.92 8.92 2.03 35.52 76.13 73.76 29 138.72 2.47 2.03 80 38.7 60.4 31 151.92 2.14 2.03 41.52 43.5 54.8 F.equiseti SY41 93.2 2.41 2.03 29.52 33.6 0.60 42 84.64 4.92 2.03 20.56 45.1 0.40 43 122.24 0 2.03 68 58.8 82.48 LSD= Least Significant Difference at P < 0.05. 131 tant, was incubated at 40ºC for 10 min. One unit of enzy- matic activity (U) was defined as 1 µmol of galacturonic acid release per minute. Xylanolytic activity Xylanase activity was measured with the optimized method described by Bailey et al. (1992), using 1% birch- wood xylan as substrate. The solution of xylan and the en- zyme at appropriate dilution were incubated at 55°C for 5 min and the reducing sugars were determined by the dini- trosalicylic acid method described by Miller (1959), with xylose as standard. The released xylose was measured spectrophotometrically at 540 nm. One unit of xylanase is defined as the amount of enzyme required to release 1 µmol of reducing sugar as xylose equivalent per min under the above assay conditions. Protein determination Total proteins were determined for their importance when purifying an enzyme, since purity depends on the re- moval of unwanted proteins, and can be assessed by relat- ing the activity to total protein present. The protein content in the enzyme preparation was determined according to the method of Lowry et al. (1951). Statistical analysis All the experiments were performed in triplicate and the means were analyzed statistically with the analysis of variance (Anonymous, 1988) using the STAT-ITCF com- puter package to test for differences in enzyme production among Fusarium spp. strains. 3. Results All 21 strains of endophytic Fusarium spp. tested were able to produce one or the other extracellular enzymes (Table 1); some of the strains were able to produce all six enzymes tested. The profiles of extracellular enzymatic activities varied among Fusarium spp., but some general features were noted. Significant differences (P<0.05) in the mean yield values were detected among strains, with high values being consistently higher in the strain F. so- lani7 for xylanase and amylase activities with mean values of 757.2 and 118.35 U/g, respectively. Some authors have suggested that the absence of catabolic repression in SSF systems is due to several factors collectively, including the slow and low processes of diffusion in solid state cultures due to low water activity (Krishna, 2005). On the other hand, CMCase and Fpase activity were detected in the lowest quantity, in terms of total units per culture (Table 1). Whereas, F. verticillioides strain SY19, showed the highest PGase activity with a mean value 97.92 U/g. Hoondal et al. (2002) reported that degradation of host tissue by phytopathogens generally begins with the produc- tion of pectinolytic enzymes, which are the major enzymes involved in plant attack. The effect of different carbon sources on pectinase synthesis by fungi in SSF have been studied and it is generally agreed that the optimum medium for the enhanced production of extracellular pectinase con- tains pectic materials as an inducer (Yadav et al., 2005). Additionally, the results show that Fusarium spp. strains varied in lipase activity (Table 1), which could be related to a differential capacity to infect wheat. Pritsch et al. (2000) suggested that lipases might have participated, to a certain extent, in prior degradation of the cuticle. On the other hand, protein contents also showed significant (P<0.05) differenc- es among Fusarium spp. (Fig. 1). The increase in protein content in some strains - F. solani Sy7, F. culmorumSy2 and F. verticillioides Sy27 - that produced high enzymatic activ- ity suggests the growth of fungi contributing to the fungal protein by utilizing available nutrients in the substrate, mak- ing it possible to metabolize the available protein in the sub- strate. Consumption of substrate protein the production of biomass protein are reported by Scopes (1993). 4. Discussion and Conclusions The Fusarium spp. strains studied here produced signif- icant levels of enzyme activity in vitro. The results might indicate that this activity influences the aggressiveness of the strains of Fusarium spp. towards wheat plants. Kang and Buchenauer (2000) showed that Fusarium culmorum infects the wheat ovary usually through the junctions be- tween the epidermal cell walls. These junctions may be a more preferable site for entry of the pathogen, allowing a quicker establishment of infection. However, Priest (1984) showed that there are several possible regulatory mecha- nisms in enzyme production including induction. The ac- tion of such enzymes gives rise to the possibility that the “genetic recombination” of the endophyte with the host which occur in evolutionary time. This may be the reason why some endophytes Fusarium spp. can produce some photochemicals originally characteristic of the host. Promputtha et al. (2007) provided phylogenetic evi- dence indicating that the endophytes produced the same degrading enzymes as their saprobic counterparts. How- ever, the capability of studied endophytes, such as the Fusarium spp. in this work, to produce different enzymes should have an important role as saprobes since they do not decompose the host living tissue. These degrading en- Fig. 1 - Total protein of Fusarium spp. strains under SSF. 132 zymes are important factors which affect the lifestyle of these species to become pathogens on wheat plants. In- deed, Fusarium species strains produced in vitro enzymes, a good indication that they may also do so under natural conditions. Schwarz et al. (2002) reported that Fusarium can produce various CWDEs in vitro and analyzed their regulation, suggesting that the initial infection depends of the secretion of these enzymes. The present study demonstrates that Fusarium spp. are able to produce quite a good source of different types of in- dustrially important enzymes. Knowledge of the types and amounts of enzymes produced by these species would be useful for the selection of strains best suited for industrial requirements. There are currently no reported studies on xylanase, lipase, amylase, polygalacturonase, filterpase, and carboxy-methyl cellulase enzymes to the authors’ knowledge and thus there is a need for further in-depth studies on these isolated bioactive Fusarium spp. strains. Acknowledgements The authors thank the Director General of AECS and the Head of the Molecular Biology and Biotechnology Department for their continuous support throughout this work. Thanks are also extended to Dr. B. Al-Safadi for critical reading of the manuscript. References AHMED R.N., AL-HINDI R., MAOHMED A.S., 2012 - Char- acterization of polygalacturonases from fruit spoilage Fu- sarium oxysporum and Aspergillus tubingensis. - Afri. J. Biotech., 11: 8527-8536. 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