EJBR2018v8i1art42 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2018; 8 (2): 42-55 Diversity of inulinase-producing fungi associated with two Asteraceous plants, Pulicaria crispa (Forssk.) and Pluchea dioscoridis (L.) growing in an extreme arid environment Doaa M. A. Khalil 1 , Mohamed S. Massoud 1 , Mostafa Abdelrahman 1,2 *, Soad A. El-Zayat 1 , Magdi A. El-Sayed 1,3 * 1 Botany Department, Faculty of Sciences, Aswan University, Aswan 81528, Egypt 2 Graduate School of Life Sciences, Tohoku University, Sendai 9808577, Japan 3 Unit of Environmental Studies and Development, Aswan University, Aswan 81528, Egypt *Corresponding authors: Mostafa Abdelrahman, E-mail: meettoo2000@ige.tohoku.ac.jp; Magdi A. El-Sayed, E-mail: magdiel_sayed@aswu.edu.eg ABSTRACT Inulinases are potentially valuable enzymes catalyze the hydrolysis of plant’s inulin into high fructose syrups as sweetening ingredients for food industry and ethanol production. The high demands for inulinase enzymes have promoted interest in microbial inulinases as the most suitable approach for biosynthesis of fructose syrups from inulin. Arid land ecosystem represents a valuable bioresource for soil microbial diversity with unique biochemical and physiological properties. In the present study, we explored the fungi diversity associated with the rhizosphere and rhizoplane of two desert medicinal plants namely Pluchea dioscoridis and Pulicaria crispa growing in the South-Eastern desert of Aswan, Egypt. A total of 180 fungal isolates were screened based on their ability to grow on potato dextrose agar medium supplemented with 1% inulin. The isolated fungal colonies were morphologically identified according to cultural characteristics and spore-bearing structure. In addition, the inulinase activity of the isolated fungi was examined spectrophotometrically. Among these, Aspergillus terreus var. terreus 233, Botrytis cinerea, Aspergillus aegyptiacus, Cochliobolus australiensis 447 and Cochliobolus australiensis exhibited high inulinase activity ranging from 5.05 to 7.26 U/ml. This study provides a promising source of microbial inulinase, which can be scaled up for industrial applications. Keywords: Microbial inulinase; Arid land; Pluchea dioscoridis; Pulicaria crispa. 1. INTRODUCTION Fungi are a diverse group of microorganisms comprising seven known phyla, including Asco- mycota, Basidiomycota, Microsporidia, Glomero- mycota, Blastocladiomycota, Chytridiomycota and Neocallimastigomycota [1]. Fungi communities are essential soil components as both decomposers and plant symbionts, playing critical roles in the ecological and biogeochemical processes in natural environments [2-5]. Fungi communities can undergo Received: 15 January 2018; Revised submission: 09 March 2018; Accepted: 21 March 2018 Copyright: © The Author(s) 2018. European Journal of Biological Research © T.M.Karpiński 2018. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. DOI: http://dx.doi.org/10.5281/zenodo.1205649 43 | Khalil et al. Diversity of inulinase-producing fungi associated with two Asteraceous plants European Journal of Biological Research 2018; 8 (2): 42-55 transitory variations in its structure due to the selective pressure exerted by the surrounding environment [6, 7]. For example, it is well known that production and diffusion of root exudates are affected by plant health and development, and these exudates exercise a selective microbial stimulation/ inhibition that varies according to the unique rhizosphere nutrient pool [3, 5, 7, 8]. In this context, plant roots represent a favorable habitat for diverse fungi populations, including those colonize the soil zone around the root (Rhizosphere) as well as the root surface (Rhizoplane) [8-10]. Rhizosphere and rhizoplane areas are always affluent in various fungal populations with vigorous activities in close association with host plant [11]. Therefore, the co- evolution of plant roots and soil fungi plays a significant role in soil physical and biological processes that increase biodiversity and soil fertility in agricultural and natural systems [3, 4, 7]. Inulin is a widespread naturally occurring storage polysaccharides found in the roots of several plants belonging to Gramineae and Asteraceae families [12, 13]. Inulin composed of fructose unit chains with various length, linked by β-2,1-D- fructosyl-fructose bonds, and generally terminated by a single glucose unit connected by the α-D- glucopyranosyl bond [13, 14]. Inulinases (2,1-β-D- fructan fructanohydrolases) are potentially useful enzymes catalyze the conversion of plant inulin into high fructose syrups as sweetening ingredients for the food industry and ethanol production [13-15]. Although inulinases were first isolated from plants, it is very difficult to separate plant inulinase in sufficient quantities for industrial and biochemical applications [12]. In addition, the acid hydrolysis of inulin to fructose displays several drawbacks, which resulted in difructose anhydrides as a final result with no sweetening properties [12, 16]. These drawbacks have forced interest in microbial inuli- nases as the most suitable method for biosynthesis of fructose syrups from inulin [13, 14, 17]. There are several fungal species can produce inulinase enzymes, among which, Aspergillus, Penicillium, and Khyveromyces species are the most common fungi that used for inulinase industrial production [18]. The increasing potential of inulinase appli- cations promoted the screening of new inulinase- producing fungi with high thermostable character. In the present study, we isolated, identified rhizosphere and rhizoplane fungi from two desert medicinal plants including Pluchea dioscoridis and Pulicaria crispa growing naturally in the South-Eastern desert of Aswan governorate, Egypt. The isolated fungi were screened based on their ability to grow on potato dextrose agar (PDA) medium supplemented with 1% inulin as the only carbon source, followed by quantitative analysis of their inulinase enzymatic activity using spectrophotometric analysis. The obtained results demonstrated the potential of the desert-adapted plants like a rich bioresource for various fungal stains with high inulinase activity, which can be used for industrial purpose. 2. MATERILAS AND METHODS 2.1. Study area This study was performed at Aswan University, Aswan governorate, Egypt (24° 5' 15" N 32° 53' 56" E). The rhizosphere and rhizoplane samples from P. dioscoridis and P. crispa were collected from three different locations including Aswan University campus (Location I, sites 1-6), Aswan airport road (location II, sites 7-8) and Aswan dam road (Location III, sites 9-10). The climatic conditions in this region ranged from very hot dry summer (30 to 50°C) to moderately cold dry winter (10 to 25°C) [3]. 2.2. Preparation of rhizosphere and rhizoplane samples In each study site, three replicates were established with one-meter distance from each other, at 20-35 cm depth from the surface. P. dioscoridis and P. crispa roots were carefully uprooted, and roots with adhering soil particles were placed in sterilized plastic bags and transported to the laboratory. The rhizosphere samples were collected according to Timonin [19], and Moubasher and Abdel-Hafez [20]. One gram of roots was lightly scraped to collect the firmly adhering soil particles to the root surface by using sterilized hairbrush and spatula. The collected rhizosphere-soil samples were subjected to serial dilution (10-4) using sterile distilled water (SDW). One ml of the rhizosphere soil suspension was transferred to PDA plates amended with chloramphenicol (500 mg l-1). The 44 | Khalil et al. Diversity of inulinase-producing fungi associated with two Asteraceous plants European Journal of Biological Research 2018; 8 (2): 42-55 plates were incubated in an incubation chamber at 28 ± 1°C for 7 days. The number of fungal colonies (cfu g-1 soil) formed on PDA dilution plates in each sample was calculated. The rhizoplane samples were collected from plant roots by washing one-gram of roots with SDW and cut into approximately 1 cm pieces. Root pieces were allowed to surface-dry under sterile conditions prior to placement into PDA plate. Five pieces of root were placed on PDA plates amended with chloramphenicol (500 mg l-1) and incubated at 28 ± 1°C for 7 days [21]. The number of fungal colonies (cfu g-1 root) formed on PDA dilution plates in each sample was calculated. 2.3. Identification of fungi All the isolated fungal colonies were morpho- logically identified according to cultural characte- ristics (color, texture, and pigmentation), and spores and spore-bearing structure using standard identi- fication manuals [22-28]. 2.4. Screening of inulinase-producing fungi All isolated strains from the rhizosphere and rhizoplane samples were screened for their ability to grow on Czapek’s agar medium supplemented with inulin as the only carbon source and were incubated at 28 ± 1°C for 7 days. A total of 180 isolates were able to grow on inulin-Czapek’s agar medium, and these strains were further used for inulinase assay. 2.5. Inulinase assay A loop of the selected fungal isolates was inoculated separately in 5 ml fermentative medium consisted of inulin 10.0, NaNO3 3.0, K2PO4 1.0, MgSO4 0.5, KCl 0.5 and FeSO4 0.001 g/l. The inoculated-cultures were incubated at 30°C for 12 days. The fermented broth was centrifuged at 10000 rpm, for 20 minutes at 4°C, and the supernatant was filtered and used as the crude enzyme extract. Inulinase activity was carried out spectrophoto- metrically according to Singh et al. [29]. The reaction mixture consisted of one ml crude enzyme extract and 0.8 ml of 2% (w/v) inulin dissolved in 0.05 M sodium acetate buffer (pH 5.5). The reaction mixture was incubated at 37°C for 60 min, then 2 ml dinitrosalicylic acid (DNS) reagent was added to the reaction. The mixture was boiled for 10 min, immediately cooled on ice and absorbance was measured at 540 nm. One enzyme unit was defined as the amount of enzyme that produces 1 μmol of fructose from inulin per min under standard assay conditions. 3. RESULTS AND DISCUSSION Since the early 90s, the plant-soil fungi associations have been the subject of intensive research, which enable us to gain an insight into the critical role of soil fungal symbionts in plant ecology and physiology [30]. However, little is known about the plant-rhizosphere and rhizoplane fungi associations in the desert ecosystems where water availability is limited. In our recent studies, we were able to illustrate the significant roles of Trichoderma longibrachiatum isolated from desert soil in Egypt that confer beneficial agronomic traits to onion (Allium cepa) [3], and the eco- physiological role of Thermomyces endophyte CpE- mediated heat stress tolerance in cucumber, which will facilitate the cultivation of heat-tolerant cucum- ber (Cucumis sativus) [4]. This study extended the previous work to isolate prospective rhizosphere and rhizoplane fungi from two desert medicinal plants P. dioscoridis and P. crispa and their potential for inulinase enzyme production. 3.1. Fungal occurrence and diversity A total of 180 fungal isolates were isolated from the rhizosphere and rhizoplane of P. diosco- ridis, and P. crispa based on their ability to grow on PDA medium supplemented with 1% inulin as the only carbon source (Figs. 1-2, Supplementary Fig. S1). The morphological identification of all fungal isolates was confirmed based on the anamorph and teleomorph characters using a light microscope (Fig. 3). A total of 62 fungal isolates were obtained from rhizoplane of Pluchea dioscoridis, while 28 fungal isolates were isolated from rhizosphere of P. dioscoridis (Fig. 4A). In addition, five isolates were overlapped between P. dioscoridis rhizosphere and rhizoplane (Fig. 4A). 45 | Khalil et al. Diversity of inulinase-producing fungi associated with two Asteraceous plants European Journal of Biological Research 2018; 8 (2): 42-55 Figure 1. Heatmap clustering of the total number of fungi species isolated from Pluchea dioscoridis-rhizoplane (A) and P. dioscoridis-rhizosphere (B) at different sites (1-10). To construct heatmap color scale, the fungal counts were normalized using logarithm of base 10. TC, total count. 46 | Khalil et al. Diversity of inulinase-producing fungi associated with two Asteraceous plants European Journal of Biological Research 2018; 8 (2): 42-55 Figure 2. Heatmap clustering of the total number of fungi species isolated from Pulicaria crispa-rhizoplane (A) and P. crispa-rhizosphere (B) at different sites (1-10). To construct heatmap color scale, the fungal counts were normalized using logarithm of base 10. TC, total count. 47 | Khalil et al. Diversity of inulinase-producing fungi associated with two Asteraceous plants European Journal of Biological Research 2018; 8 (2): 42-55 Supplementary Figure S1. Total count of fungal isolates isolated from Pluchea dioscoridis-(A) and Pulicaria crispa- (B) rhizoplane and rhizosphere at studied sites. Similarly, the total number of fungal isolates isolated from Pulicaria crispa-rhizoplane and rhizo- sphere was 46 and 44, respectively, whereas nine fungal isolates were overlapped (Fig. 4B). The high number of fungi species obtained from the rhizo- plane and rhizosphere of desert medicinal plants indicates the significant role of root exudates in soil fungal diversity and dynamics, which are important for plant adaption to the arid land ecosystem [4]. Our data was in accordance with the early reports that showed a high diversity of fungal species were detected in the rhizosphere and rhizoplane of Hyoscyamus muticus and Hordelymus europaeus relative to other soil samples [31, 32]. In addition, Aspergillus niger, A. terreus and Aspergillus sp. were the most dominant fungi in the all sample sites (Figs. 1-2). Aspergillus spp. were highly abundant in the all sample sites represented by 13 identified species and three varieties, followed by Chaeto- mium, Emericella, and Phoma that were represented by six identified species (Figs. 1-2). 48 | Khalil et al. Diversity of inulinase-producing fungi associated with two Asteraceous plants European Journal of Biological Research 2018; 8 (2): 42-55 Chaetomium elatum Emericella violaceae Cochliobolus lunatus Rhizopus oryzae Figure 3. Morphology of some isolated fungi. Figure 4. Venn diagram of the total number of fungal isolates isolated from Pluchea dioscoridis- (A) and Pulicaria crispa- (B) rhizoplane and rhizosphere. Our data was in accordance with the study by Lima et al. [33] demonstrating that Aspergillus spp. were highly abundant in the rhizosphere and rhizoplane of P. crispa and P. dioscoridis. Aspergillus spp. have a wide range of optimum growth temperature ranging from 25 to 40°C and minimum growth temperature around 10°C compared with other fungi [34]. The dynamic range of Aspergillus growth temperature is an important physiological character, which enables Aspergillus spp. to survive under different environ- mental conditions. Among all recovered species during this work, there were twenty three identified species and one variety belonging to 14 terrestrial fungal genera were recovered from Pluchea dios- 49 | Khalil et al. Diversity of inulinase-producing fungi associated with two Asteraceous plants European Journal of Biological Research 2018; 8 (2): 42-55 coridis in rhizosphere samples. In addition, 31 identified species and 2 varieties appertaining to 17 genera were recovered from Pulicaria crispa. While, 27 species and two varieties belonging to 13 genera were isolated and identified from Pluchea dioscoridis, whereas thirty one and three varieties appertaining to 15 genera were recovered from Pulicaria crispa in rhizoplane samples. Out of all recorded species in this investigation, 12 identified species were isolated from rhizoplane only of the two plants were; Aspergillus carneus, A. flavus var. columnaris, A. fumigatus, A. parasiticus, Botryo- trichum piluliferm, Chaetomium cochliodes, Phoma euprena, Phoma sp., P. pomorum, Emericella quadrillineata, Memoniella echinata and Pleospora herbarum. In Pakistan, Qureshi et al. [35] isolated Fusarium solani, Cochliobolus australiensis, Mac- rophomina phaseolina and Rhizoctoinia solani from rhizoplane of 65 plants belonging to 58 genera and 19 families. The following 14 fungal species; Aspergillus egyptiacus, Botrytis cinerea, Cepha- liophora tropica, Cladosporium cladosporioides, C. macrocarpum, Chaetomium bostrychodes, C. glo- bosum, Cochliobolus sativus, Emericella rugulosa, Gibberella intricans, Mucor fuscus, Nectaria haematocoa, Phoma acteae and Phycomyces sp. were isolated only from rhizosphere of the two tested plants. Most of the fungal genera that were recorded in this investigation were repeatedly reported for rhizosphere of different wild and cultivated plants in the South-Eastern desert of Egypt and different parts of the world by [29, 32, 36-39]. In the rhizoplane of Pluchea dioscoridis, there were four species found to be dominant are; Aspergillus ustus, Aspergillus flavus var. colum- naris, Aspergillus flavus and Aspergillus fumigatus, Phoma viride, Fusarium sp. and Chaetomium cochloides were showed an intermediate frequency while remaining isolated were found in low frequencies whereas, in rhizoplane of Pulcaria crispa, A. tamarii, A. awamori, A. flavus and Aspergillus ustus, Cochliobolus australiensis and Emericella violacea were found to be dominant. Mucor hiemalis and Rhizopus oryzae were showed an intermediate frequency while, remaining isolates were found in low frequency, these genera were represented by few species that did not exceed five species for each genus. Acremonium, Botryotrichum, Fusarium and Microascus which were recovered from Pluchea dioscoridis in moderate and low incidence, were completely missed in Pulicaria crispa. Whereas, Cladosporium, Cunninghamella, Memnoniella, Pleospora, Rhizopus, Syncephlastrum and Trichoderma which were represented by two or one identified species and present in moderate to low incidence, were recovered from Pulicaria crispa and completely absent in Pluchea dioscoridis. All fungal genera and species which were recovered during this work were previously recovered from soil, rhizosphere, rhizoplane and endophytic fungi at different parts of the world and on different habitats by many investigators [35, 38, 40-45]. 3.2. Inulinase activity of fungal isolates associated with Asteraceous plants A total of 180 fungal isolates were able to grow on PDA medium supplemented with 1% inulin. Out of which, 23 fungal isolates (12.77%) exhibited high inulinase activity ranging from 5.05 to 7.26 U/ml (Figs. 5-6). Whereas 67 fungal isolates (37.22%) displayed moderate inulinase activity, ran- ging from 3.01 to 4.99 U/ml (Figs. 5-6). Aspergillus terreus var. terreus 233 isolated from P. dioscoridis- rhizoplane, and Botrytis cinerea isolated from P. dioscoridis-rhizosphere exhibited the highest inulinase activity ranging from 6.28 to 6.41 U/ml (Fig. 5A-B). On the other hand, Chaetomium cochliods isolated from P. dioscoridis-rhizoplane, and Emericla nidulans var. nidulans isolated from P. dioscoridis-rhizosphere displayed the lowest inulinase activity, ranging from 0.17 to 0.34 U/ml (Fig. 5A-B). Similarly, Aspergillus aegyptiacus iso- lated from P. crispa-rhizoplane, and Cochliobolus australiensis 447 isolated from P. crispa-rhizosphe- re exhibited highest inulinase activity ranging from 7.10 to 7.26 U/ml (Fig. 6A-B). However, Mucor hiemalis isolated from P. crispa rhizoplane and rhizosphere displayed the lowest inulinase activity of 0.85 U/ml (Fig. 5A-B). In general, our results indicated that the isolated fungal species exhibited significant differences in inulinase activities, and several Aspergillus spp. isolated in this study showed high inulinase activity in comparison with other fungi species. The high inulinase activity of Aspergillus spp. seems to be a physiological characteristic of this species to enable them to extract nutrition under severe environmental condi- 50 | Khalil et al. Diversity of inulinase-producing fungi associated with two Asteraceous plants European Journal of Biological Research 2018; 8 (2): 42-55 tions. Similar studies have also indicated the high inulinase activity derived from Aspergillus spp. [46, 47]. The histogram analysis of inulinase activity of fungal isolates isolated from P. dioscoridis- rhizosphere and rhizoplane exhibited a right- skewed/asymmetrical distribution due to the high number of fungi showed low inulinase activity (Fig. 7). Figure 5. Inulinase activity (U/ml) of fungi species isolated from Pluchea dioscoridis-rhizoplane (A) and rhizosphere (B). 51 | Khalil et al. Diversity of inulinase-producing fungi associated with two Asteraceous plants European Journal of Biological Research 2018; 8 (2): 42-55 Figure 6. Inulinase activity (U/ml) of fungi species isolated from Pulicaria crispa-rhizoplane (A) 0020 and rhizosphere (B). On the other hand, the histogram analysis of inulinase activity of fungal isolates isolated from P. crispa-rhizoplane and rhizosphere exhibited nor- mal distribution due to the high number of fungi showed moderate inulinase activity (Fig. 7). The high inulinase activity for Aspergillus terreus var. terreus 233, Botrytis cinerea, Aspergillus aegy- ptiacus and Cochliobolus australiensis 447 observed in this study was in accordance with previous reports [41, 48, 49]. For examples, Coitinho et al. [49] demonstrated the high efficiency and thermo- stability of inulinase enzyme purified from Asper- 52 | Khalil et al. Diversity of inulinase-producing fungi associated with two Asteraceous plants European Journal of Biological Research 2018; 8 (2): 42-55 gillus terreus using sugarcane bagasse as a substrate. B. cinerea xylanase activity has been reported as an essential component for their virulence effect [50]; however, this is the first report about B. cinerea inulinase activity, which might be a starting point for further in-depth studies about its role in the plant-pathogen interaction. Souza-Motta et al. [41] also demonstrated the ability of filamentous fungi isolated from rhizosphere to hydrolyze inulin. Figure 7. Histogram analysis of inulinase activity on the X-axis and total fungal isolates on the Y-axis isolated from Pluchea dioscoridis- and Pulicaria crispa-rhizoplane and -rhizosphere. 4. CONCLUSION In conclusion, we were able to isolate and identify 180 fungal isolated from the rhizosphere and rhizoplane of two desert medicinal plants P. dioscoridis and P. crispa in the South-Eastern desert of Aswan governorate, Egypt. We also exa- mined the inulinase activity of the isolated fungi, revealing high ability of several fungal isolates including Aspergillus terreus var. terreus 233, Botrytis cinerea, Aspergillus aegyptiacus, Cochlio- bolus australiensis 447 and Cochliobolus austra- liensis. These fungal isolates could be a potential bio-resource for microbial inulinase production. Optimization experiments are needed for exploring the best conditions for increasing the enzyme productivity by these isolates for potential commercialization. AUTHOR’S CONTRIBUTION MA, SE: Project supervisors, research design, wrote and revised the manuscript; DKh, MS: experimental work, MAR: statistical analysis, Figures and wrote the first draft. 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