Specie fungine endofite coltivabili che producono L-asparaginasi dalla regione arida del Sinai, Egitto
10.6092/issn.2531-7342/10063
Nel protettorato di Santa Caterina (Sinai meridionale, Egitto), fra il 2017 ed il 2019, venticinque specie di endofiti fungini sono state isolate in cultura pura ED e identificate da 23 specie di piante. Alternaria alternata ha fatto registrare il maggior numero di colonie (1382 su 4466 CFU) e la più alta frequenza di ritrovamento (22 su 23 specie vegetali totali). Con riferimento alla pianta ospite, il più alto numero di colonie è stato ottenuto da Euphorbia obovata mentre il maggior numero di specie fungine (9 su 25) sono state isolate in Artemisia herba-alba. Tutti gli solati sono stati testati per la produzione di L-asparaginasi e 7 ceppi appartenenti a 4 specie fungine hanno dato risultati positivi con un’attività enzimatica compresa fra 44.5 ± 1.66 e 152.58 ± 0.63 U ml-1. Lasiodiplodia theobromae isolata da Teucrium polium ha fatto registrare l’attività più elevata con 152.58 U ml-1. Questo studio dimostra che L. theobromae potrebbe rappresentare una valida fonte alternativa di L-asparaginasi quale potente antitumorale.
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Ahmed M. Abdel-Azeem
Italian Journal of Mycology
Saint Katherine Protectorate
Diversity of the culturable endophytic fungi producing L-asparaginase in arid Sinai, Egypt
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2019-11-27
2020-02-10
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Ital J Mycol
Ahmed M. Abdel-Azeem
2020/05/12
10.6092/issn.2531-7342/10063
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Botany Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt
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Saint Katherine Protectorate
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Zhao J, Shan T, Mou Y, Zhou L (2011) Plant-derived bioactive compounds produced by endophytic fungi. Mini Reviews in Medicinal Chemistry 11(2):159–168. https://doi.org/10.2174/138955711794519492
Diversity of the culturable endophytic fungi producing L-asparaginase in arid Sinai, Egypt
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49
Balbool and Abdel-Azeem                                                     Italian Journal of Mycology 49: 8-24 (2020)

DOI: 10.6092/issn.2531-7342/10063


Original Paper

           Diversity of the culturable endophytic fungi producing
                     L-asparaginase in arid Sinai, Egypt

                           Bassem A. Balbool1, Ahmed M. Abdel-Azeem2

1 Microbiology Department, Faculty of Biotechnology, October University for Modern Sciences and Arts, 6th October
city, Egypt
2 Botany Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt

Corresponding author e-mail: ahmed_abdelazeem@science.suez.edu.eg

Article info
Received 30/11/2019; accepted 28/04/2020
DOI: 10.6092/issn.2531-7342/10063

Abstract
Twenty-five endophytic fungal species were recovered and identified from a twenty-three plant species collected from
Saint Katherine Protectorate, South Sinai Egypt, during the period of 2017-2019` with a total count of 4,466 CFU.
Alternaria alternata recorded the highest count of 1382 CFU out of 4466 CFU and the highest frequency of occurrence
with 22 cases of isolation out of 23 plant species. In relation to host plant, Euphorbia obovata hosted the highest count
(366 CFU), while Artemisia herba-alba hosted the highest fungal species richness value with 9 species out of 25. Isolated
taxa were screened for L-asparaginase production where 7 strains belonging to 4 different species showed a positive result
with an enzymatic activity ranging from 44.5 ± 1.66 to 152.58 ± 0.63 U ml-1. Lasiodiplodia theobromae isolated from
Teucrium polium recorded the highest activity with 152.58 U ml-1. The present study suggests that L. theobromae has the
potential to be used as an alternative and reliable source of L-asparaginase for potent anticancer agents.

Keywords
endophytic fungi; L-asparaginase; Lasiodiplodia theobromae; Teucrium polium; Saint Katherine Protectorate

Introduction
Endobiontic fungi are microfungi that host plant tissue intercellularly and/or intracellularly without
any apparent pathological symptoms (Wilson, 1995; Das and Varma, 2009). To be able to sustain
steady symbiosis, plant-associated fungi develop chemical substances that enhance the development
of plants and help them to acclimatize to harsh environments (Gouda et al., 2018). As a treasure mine
of bioactive metabolites, endophytic fungi are considered a sustainable source of various natural
products such as antioxidants, quinones, saponins, alkaloids, steroids, phenolic acids, terpenoids, and
tannins that exhibit antimicrobial and anticancer properties (Verma et al., 2009; Abo Nahas, 2019).
        Recently, the term “bioprospecting” refers to the discovery and commercialization of natural
products from natural-biological sources (Beattie et al., 2011; Abdel-Azeem et al., 2012; Salem
and Abdel-Azeem, 2014). Fungi have been included in the interest of biotechnology because their
immense importance to the ecosystem components, especially to human (Strobel and Daisy, 2003). In
the last two decades scientific community worldwide has focused on the fungal plant-associated fungi
as a sustainable and ecofriendly source for production of bioactive metabolites that may be effective
candidates for the first-class drugs for treatment of human diseases (Bacon and White, 1994; Zhang
et al., 1999; Strobel and Daisy, 2003; Guo et al., 2008; Kumaran, 2008; Duan et al., 2009; Shweta et
al., 2010). It was indicated that endophytic fungi produce about 51% of new bioactive substances in
comparison with 38% from terricolous fungi (Schulz et al., 2002). Unique environments, diversity,


ISSN 2531-7342 Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
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Balbool and Abdel-Azeem                                           Italian Journal of Mycology 49: 8-24 (2020)

DOI: 10.6092/issn.2531-7342/10063



ethnobotanical history and endemicity should be considered during selection and sampling processes
of endophytic fungi-hosting plants (Abdel-Azeem et al., 2019a).
       Leukemic cells require large amounts of asparagine for proliferation and growth. L-asparaginase
controls the tumor by lowering the serum’s asparagine (Egler et al., 2016). El-Nagga et al. (2014)
recommended L-asparaginase as a potential drug which can treat acute lymphoblastic leukemia as an
alternative to the classical chemotherapy protocols. L-asparaginase (EC 3.5.1.1) belongs to a group of
homologous amidohydrolases family, which catalysis the hydrolysis of the amino acid L-asparagine
to L-aspartate and ammonia (Dias et al., 2016). L-asparaginase discovered firstly from guinea pig
serum (Clementi, 1922) and its tumor activity were reported afterwards by recognizing the anti-
leukemic activity of the guinea pig serum on treated mice (Broome, 1963). First microbial source
of L-asparaginase was E. coli but L-asparaginase is broadly distributed among plants, animals and
microorganisms (Kumar et al., 2013a). Prokaryotic sources of L-asparaginase showed many side
effects like hypersensitivity which usually lead to anaphylaxis and hypersensitivity (Duval et al.,
2002). Based on aforementioned reasons, fungi are considered as an alternative and effective source
of L-asparaginase (Kumar and Sobha, 2012). More than 600,000 species of plant associated micro-
fungi are theorized to exist worldwide (Schmit and Mueller, 2007). In Egypt, endophytic fungi are a
potential fungal resources still unexplored (Abdel-Azeem, 2010).
       The mountainous region of southern Sinai exhibits greater biodiversity than the rest of Egypt, and
4,350 km2 of this area was declared a Protectorate in 1996 (Zalat et al., 2008). In South Sinai, Bedouin’s
traditional folk medicine depends upon approximately 170 plant species which inhabiting the area
(Fayed and Shaltout, 2004). No solid study on endophytic fungi producing L-asparaginase exists in
Egypt. In this study, we isolated endophytic fungi from 23 plant species growing on the mountainous
region in Saint Katherine Protectorate, in addition to analyzing the biodiversity and seasonal variation
in the numbers of these fungi and screening their potentiality to produce L-asparaginase.

Materials and Methods
Study area
The Protectorate of Saint Katherine covers about 4,350 km2 of Southern Sinai (Abdel-Azeem, 2009).
Saint Katherine is located at an elevation of 1,500 to 2,624 meters above sea level (m a.s.l.) which
includes the main mountains in the area. The area is situated between 28°30’ to 28°35’ North and
33°55’ to 34°30’ East (Abdel-Azeem et al., 2019a). The area is composed of igneous and metamorphic
rocks; chiefly granites are intensely dissected and rugged. The area is characterized by an extremely
arid climate with long, hot and rainless summers and cool winters. The mean annual precipitation in
the area is 45 mm but the high mountains receive more precipitation (100 mm y-1) as rain and snow. In
some parts of this place, floods resulting from connective rains have sometimes been observed during
winter and spring. The maximum air temperature ranged from 20.2 oC to 32.7 oC and the minimum
temperature ranged from 1.9 oC to 20.2 oC with the lowest temperature in December and January and
the highest temperature in July and August (Abdel-Azeem et al., 2019a).
      Within the Protectorate, more than 400 species of higher plants have been recorded, of which
19 species are endemic, 10 are extremely endangered and 53 are endangered (Guenther, 2005).
Approximately 170 plant species that inhabit south Sinai are used traditionally in folk medicine
(Fayed and Shaltout, 2004; Abdel-Azeem et al., 2012; 2019a).

Plant species sampling
Some dominant plant species (Table 1) belonging to thirteen families were collected from different
11 sites (for more data check supplementary material). Aerial parts of the plants were collected in
sterilized polyethylene bags and transferred to the laboratory, where they were subsequently plated


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DOI: 10.6092/issn.2531-7342/10063



out. Samples were collected under permission of the Saint Katherine Protectorate for scientific
purposes and no endangered species were involved in the study.

                    Table 1 - List of collected plant species and their Families dominant
                    in the study area
                   Family                           Species
                   Asteraceae                       Achillea fragrantissima Sch. Bip.
                                                    Artemisia judaica Täckh.
                                                    Artemisia herba-alba Asso.
                                                    Chiliadenus montanus (Vahl). Brullo
                                                    Tanacetum sinaicum Delil ex DC.
                   Boraginaceae                     Alkanna orientalis Boiss
                   Euphorbiaceae                    Euphorbia obovate Decne
                   Lamiaceae                        Ballota undulata Benth.
                                                    Origanum syriacum Boiss.
                                                    Phlomis aurea Decne.
                                                    Rosmarinus officinalis L.
                                                    Teucrium polium Decne.
                                                    Thymus decussatus Benth
                   Oleaceae                         Olea europaea L.
                   Peganaceae                       Peganum harmala L.
                   Plantaginaceae                   Plantago sinaica Decne.
                   Rhamnaceae                       Rhamnus frangula L.
                   Rosaceae                         Crataegus sinaica Boiss.
                   Rubiaceae                        Galium odoratum Scop.
                                                    G. sinaicum Boiss.
                   Scrophulariaceae                 Verbascum sinaiticum Benth.
                   Solanaceae                       Hyoscyamus muticus L.
                   Zygophyllaceae                   Fagonia mollis Delile

Isolation and phenotypic identification of plant-associated fungi
A total number of 2,760 of plates were used for recovering endophytic fungi (120 plate/plant) during
this study. Surface sterilization technique adopted by Abdel-Azeem and Salem (2012) was applied:
pant samples washed under running tap water for 10 min, then stem tissues cut down to pieces each
of a 2 cm in length, while leaves tissues cut into (2 x 2 cm) 4 cm2 segments and surface sterilized by
sodium hypochlorite and 70% ethanol and plated out on isolation media. For primary isolation, Potato
Dextrose Agar (PDA) and Malt Extract Agar medium (MEA) were supplemented with Rose Bengal
(1:1,500) and chloramphenicol (50 ppm) to suppress bacterial growth (Abdel-Azeem et al., 2016).
        Isolated taxa were identified morphologically to species level on standard media based on
the phenotypic characters and relevant identification keys [Raper and Thom (1949), Pitt (1980) for
Penicillium spp.; Raper and Fennell (1965), Klich (2002) for Aspergillus spp.; Booth (1971), Leslie
and Summerell (2006) for Fusarium spp.; Ellis (1971, 1976) for Dematiaceous hyphomycetes; Guarro
et al. (2012) for ascomycetes; Von Arx (1981), Domsch et al. (2007) for miscellaneous fungi; Cannon
(1986), Von Arx (1986), Asgari and Zare (2011), Doveri (2013) for Alternaria spp.].
        Authors of the fungal taxa names were abbreviated according to Kirk and Ansell (1992). All
name corrections, authors and taxonomic assignments of the recorded species in the present study
checked against the Index Fungorum Partnership (www.indexfungorum.org).



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Qualitative and quantitaive screening of fungal L-asparaginase
All of the recovered plant-associated fungi were screened for their capability to produce L-asparaginase
according to Gulati et al. (1997) with some modifications. Taxa were grown on malt extract agar
(MEA) at 28 °C for 15 days to enhance sporulation. Modified Czapek’s Dox broth (MCDB) composed
of Glucose, 2.0, L-asparagine 10.0; KH2PO4 1.52, KCL 0.52, MgSO4 7H2O 0.52, traces of Cu(NO3)2
3H2O, ZnSO4 7H20, FeSO4 7H2O (g l-1) (Gulati et al., 1997) was supplemented with bromothymol blue
as indicator and pH adjusted to 6.0, followed by sterilization for 20 min at 120 oC. Five millimeters
fungal mycelial plug was inoculated to 50 ml sterile falcon tube containing MCDB and were incubated
at 28 °C for 5 days. Color change from yellow to blue was recorded as an indication for the release of
ammonium due to L-asparaginase activity of positive taxa.
       Nine strains belongig to five species (showing blue color) were grown at MCDB without
bromothymol blue and incubated under static condition for 5 days at 28 °C. All the cultures were
filtered using Whatman filter papers No.1. One ml of culture filtrate was mixed with 0.1 ml of 40 mM
asparagine, 0.4 ml dH2O and 0.5 ml of 50 mM Tris-buffer (pH 7.0). Mixture was incubated at 37o C
for 30 min. Reaction was stopped by adding 1.5 M trichloroacetic acid, followed by centrifugation
at 10,000 rpm for 5 min at 4 °C. After having discarged the pellet, Pellets obtained discarded, 3.7 ml
of dH2O and 1 ml of Nessler’s reagent were vortexed with 0.1 ml of the supernatant and incubated at
room temperature for 10 min. Blank solution was prepared containing all the reagents except fungal
filtrate. Absorbance was measured at 450 nm using UV spectrophotometer (JEANWAY 6305, UK).
All readings were noted in triplicates and the amount of ammonia liberated from the crude sample
was calculated from the standard curve of ammonia previously created (Dias et al., 2016).


      Enzyme activity was calculated using the following equation:


      Enzyme activity (units ml-1) =



      Fungal taxa selected for production of L-asparaginase were molecularly identified. The CTAB
extraction procedure (Arenz and Blanchette, 2011) was applied to extract genomic DNA. PCR
amplification of the internal transcribed spacer (ITS) region of the genomic rDNA was carried out by
using the primer combination of ITS1F/ITS2 (White et al., 1990) according to Abdel-Azeem et al.
(2019b). By using Geneious 9.0 (Kearse et al., 2012) software a consensus sequence was assembled
and compared to those deposited in GenBank using BLASTn for identification.

Data Analysis
The data presented in tables and figures were checked for normality and outlier detection.
Fungal diversity was studied using Shannon diversity and Margalef richness indices according to
Muthukrishnan (2012). Three variables (isolation media, seasonal variation and plant species) were
analysed using multivariate test and analysis of variance (ANOVA) using the SPSS ver. 23 for Mac
OS. To determine differences among groups ANOVA was followed by Duncan’s multiple range test
(DMRTs) at p < 0.05.




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DOI: 10.6092/issn.2531-7342/10063



Results
Overview and biodiversity of the recorded plants associated fungi
In this study twenty five fungal species were isolated; the isolated species were belonging to 16
genera, 10 families, 8 orders, 5 classes and three phyla. Taxa were distributed as 22 ascosporic
(anamorphic and teleomorphic), two zygosporic species and one basidiosporic taxon (for more
data check supplementary materials). Taxa with uncertain taxonomic position (Incertae sedis) were
distributed among orders and families (for more data check supplementary materials).
       The prevailing genera were Aspergillus (4 species; 16% of the total species number), Fusarium
and Alternaria (3 species; 12%) Penicillium and Chaetomium (2 species; 8%) and the remaining taxa
were represented only by one species each.
       Family Aspergillaceae contained the highest number of species (7 species) belonged to 3
genera among all families. The species genus ratio (S/G) per family showed that family Necteriaceae
recorded 3 species within one genus by recording a ratio of 3 followed by Aspergillaceae (2.3),
Chaetomiaceae (2), Pleosporaceae (1.7), while the other families recorded a ratio of 1 (for more data
check supplementary materials).
       Data concerning frequency classes showed that Alternaria alternata and Aspergillus niger
recorded the highest frequency of occurrence while, Aspergillus flavus and Penicillium chrysogenum
showed medium occurrence.The other species showed low to rare frequency of occurrence (Table 2).
       A total count of 4,466 CFU was recorded during this study from the collected 23 plant species.
Among the isolated taxa, A. alternata recorded the highest total count with 1,382 CFU followed by A.
niger (1225) and P. chrysogenum with 757 CFU respectively. Other taxa showed total count ranging
from 8 to 300 CFU/plant (Table 2).
       Comparing the total colony count of recovered fungi per plant, Euphorbia obovata came first
by recorded the highest count with 366 CFU, followed by Thymus decussates, Tanacetum sinaicum,
Verbascum sinaiticum, Galium odoratum with 341, 295, 279, 272 CFU/plant respectively. The other
plant species showed a total count ranging between 116 to 216 CFU (Table 3).
       Regarding the hosting plant’ species richness, Artemisia herba-alba showed the highest richness
value by hosting 9 species, followed by Achillea fragrantissima, Ballota undulata, Rosmarintus
officinalis with 8 fungal species, Thymus decussates recorded 7 species and other plant species showed
richness value ranging from 1 up to 6 (for more data check supplementary materials).
       Seasonally, spring recorded the highest number of taxa with 21, followed by summer, autumn
and winter with 20, 15 and 12 respectively.
       Regarding the diversity of the isolated endophytic fungi in relation to hosting plant species,
Artemisia herba-alba recorded the highest values of Margalef richness index and Shannon diversity
index with values of 3.64 and 2.20, respectively, followed by Achillea fragrantissima, Ballota
undulata ,Rosmarintus officinalis, Tanacetum sinaicum with values of 3.37 for Margalef index and
2.08 for Shannon index; other plant species exhibited a range of 2.49 to 1.44 for Margalef index and
1.95 to 0.69 for Shannon index (for more data check supplementary materials).
       The diversity of the isolated fungal species analyzed statistically using Multi-way ANOVA
analysis showed high significance values in relation to plant species, with F-ratio ranging from
105.15 to 4.84, while the seasonal variation showed a variation of significance from high to no
significance values with F-ratio ranging from 31.31 to 0.46; thetype of isolation media showed a
range of significance from high to no significance results with F-ratio from 26.3 to 0.04 (Table 5).
       Regarding the biodiversity of recorded taxa over seasons, A. alternata recorded the highest
number of cases of isolation over the four seasons with 73, followed by A. niger with 67 cases of
isolation (Table 4).



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      Table 2 - The total count (TC), number of cases of isolation (NCI out of 23 plants species),
      frequency (F) and frequency classes (FC) of recovered taxa during this study
      Taxa                                                                TC NCI %F FC
      Alternaria alternata (Fr.) Keissl.                                  1382 22     95.7 H
      Aspergillus niger Tiegh                                             1225 20     87.0 H
      Penicillium chrysogenum Thom                                        757 16      69.6 M
      Aspergillus flavus Link                                             300 12      52.2 M
      Penicillium brevicompactum Dierckx                                  174 8       34.8 L
      Alternaria atra (Preuss) Woudenb. & Crous                           100 5       21.7 R
      Cladosporium cladosporioides (Fresen.) G.A. de Vries                68     7    30.4 L
      Rhizopus stolonifer (Ehrenb.) Vuill.                                67     4    17.4 R
      Chaetomium globosum Kunze                                           60     3    13.0 R
      Aspergillus terreus Thom                                            44     6    26.1 L
      Trichoderma viride Pers                                             35     6    26.1 L
      Talaromyces stipitatus C.R. Benj.                                   33     1    4.3 R
      Sarocladium strictum (W. Gams) Summerb.                             29     1    4.3 R
      Fusarium oxysporum sensu Smith & Swingle                            28     1    4.3 R
      Syncephalastrum racemosum Cohn ex J. Schröt.                        25     1    4.3 R
      Cochliobolus bicolor A.R. Paul & Parbery                            23     3    13.0 R
      Exserohilum monoceras (Drechler) KJ. Leonard & Suggs                20     1    4.3 R
      Lasiodiplodia theobromae (Pat.) Griffon & Maubl.                    15     1    4.3 R
      Fusarium solani (Mart.) Sacc.                                       15     1    4.3 R
      Aspergillus nidulans (Eidam) G. Winter                              14     1    4.3 R
      Chaetomium madrasense Natarajan                                     13     1    4.3 R
      Alternaria solani (Ellis & G. Martin) L.R. Jones & Grout            11     1    4.3 R
      Nigrospora oryzae (Berk. & Broome) Petch                            11     1    4.3 R
      Phanerodontia chrysosporium (Burds.) Hjortstam & Ryvarden           9      1    4.3 R
      Fusarium cylindricum (Mont.) Sacc.                                  8      1    4.3 R
      Frequency class: H, the species observed is “High” when recovered from 18 or more plants; M, species
      observed is “Medium” when recovered from (17-12) plants; L, the species observed is “Low” when
      recovered from (9-3) plants; R, species frequency is “Rare” when recovered from (5-0) plants.

          Table 3 - The total count of endophytic fungi recorded per plant species
          Plant species                  Total count     Plant species                  Total count
          Euphorbia obovata              366             Alkanna orientallis            162
          Thymus decussates              341             Fagonia mollis                 162
          Tanacetum sinaicum             295             Plantago sinaica               162
          Verbascum sinaiticum           279             Artemisia herba-alba           161
          Galium odoratum                272             Gallium siriacum               156
          Rosmarintus officinalis        216             Crataegus sinaica              141
          Tecrium polium                 214             Rhammus frangula               139
          Peganum harmala                189             Hyoscyamus muticus             136
          Phlomis aurea                  187             Ballota undulate               132
          Olea europaea                  184             Origanum syriacum              121
          Artemisia Judaica              169             Chiliadenus montanus           116
          Achillea fragrantissima        166
          Mean                           194.17          Minimum                        116.00
          Standard Error                 14.56           Maximum                        366.00
          Standard Deviation             69.81           Sum                            4466.00

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       As an overview on the dominating recorded fungal endophytic taxa over the nine collection
sites, Alternaria alternata showed the highest frequency over 7 different sites, while Aspergillus
terreus showed a high frequency over 4 different sites (for more data check supplementary materials).
       Distribution of endophytic fungi hosting plants collected from different elevation wadis showed
that low elevation wadis (Abu Sayla and Itlah) recorded the highest mean of content per plant species
with 2.14 species/plant followed by mid elevation wadis (Shyraj, Farsh Sefafa, Arbaein and Fara`)
with 1.78 fungal species/plant and high elevation wadis (Sefsafa, Ahmar and Abas) with 1.6 fungal
species/plant (for more data check supplementary materials).
       The combined effect of plant species, isolation medium and seasonal variation show a range
of significance from high to no significance with F-ratio ranging from 16.42 to 0, 21.13 to 1.29
respectively, while the combined effect of seasonal variation and media types showed a variation of
significance values from medium to no significance with F-ratio ranging from 0.04 to 4.31 (Table 5).
       Regarding the effect of three variables combined, Table 5 showed a high significance with some
fungal endophytic species with F-ratio ranging from 12.1 to 1.75 and showed a range of significance
from low to no significance with the other endophytic fungal species with F-ratio ranging from 0.04
to 1.41.

Qualitative and quantitative screening of L-asparaginase
L-asparaginase producing taxa shifted the pH from acidic (yellow) to the alkaline (blue) conditions
(Fig. 1a). Out of the screened twenty-five taxa one strain of L. theobromae and two strains for each
of F. oxysporum, C. cladosporioides, A. flavus showed positive L-asparaginase activities (Fig. 1b).
       Quantitatively, activity of L-asparaginase have been assayed spectrophotometrically for the
positive recorded taxa and results showed a range of activity between 44.5 ± 1.66 to 152.58 ± 0.63 U
ml-1 (Fig. 2). L-asparaginase activity results analyzed statistically using ANOVA one-way analysis of
variance and showed F-ratio of 1382.2 and p < 0.001 (Fig. 1). Lasiodiplodia theobromae recorded the
highest activity results with 152.58 ± 0.63 U ml-1 (Figs. 1c and 2).
       All the positive L-asparaginase species were sequenced and deposited at the NCBI under
accession numbers of: MH752509 A. flavus SCUF2020, MH752510 F. oxysporum SCUF2021,
MH752511 C. cladosporioides SCUF2022 and MK537303 L. theobromae SCUF-TP2016.


Discussion
Screening of bioactive natural products secreted by endophytic fungi got attention by Egyptian
scientists lately (Abdel-Azeem, 2010; Abdel-Azeem and Salem 2012; Abdel-Azeem et al., 2012;
Salem and Abdel-Azeem 2014; Abdel-Azeem et al., 2016; 2018; 2019a) as they directed their
researches on endemic and medicinal plants grow over areas of great biodiversity e.g. Saint Katherine
Protectorate. From the collected twenty-three plant species a 25 fungal species with a total count of
4,466 CFU were isolated.
      During the presented study, twenty-five fungal species have been isolated with a 4,466 colonies
from twenty-three collected plant species. Abdel-Azeem et al. (2016) recovered a total count of 1490
CFU from 27 plants in Saint Katherine Protectorate with a mean value of 178.64 CFU, while each
plant showed a mean value of 194.17 CFU.
      All of the twenty-five taxa were recorded previously as plant-associated fungi with no new
records from Saint Katherine Protectorate (Abdel-Azeem, 2009; Abdel-Azeem et al., 2016; 2018;
2019a; Abo Nahas, 2019).




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DOI: 10.6092/issn.2531-7342/10063



            Table 4 - Number of cases of isolation of plant-asscoiated fungi over the four seasons
             Fungal species                   NCI                                            Total
                                                                                             NCI
                                              Spring Summer Autumn Winter
             Alternaria alternata             16         18         17            22         73
             Aspergillus niger                15         17         16            19         67
             Penicillium chrysogenum          12         13         9             13         47
             Aspergillus flavus               8          9          8             10         35
             Aspergillus terreus              4          5          4             4          17
             Penicillium brevicompactum       5          4          3             4          16
             Alternaria atra                  4          3          3             3          13
             Cladosporium cladosporioides 4              3          1             4          12
             Trichoderma viride               3          1          2             3          9
             Rhizopus stolonifer              1          3          2             3          9
             Cochliobolus bicolor             2          1          1             2          6
             Chaetomium globosum              2          2          0             1          5
             Fusarium oxysporum               1          1          1             1          4
             Fusarium solani                  1          1          1             1          4
             Talaromyces stipitatus           1          1          1             1          4
             Exserohilum monoceras            1          1          1             0          3
             Lasiodiplodia theobromae         1          1          1             0          3
             Nigrospora oryzae                1          1          1             0          3
             Syncephalastrum racemosum        1          1          1             0          3
             Sarocladium strictum             1          1          0             0          2
             Alternaria solani                1          1          0             0          2
             Aspergillus nidulans             1          1          0             0          2
             Chaetomium madrasense            1          0          0             0          1
             Phanerodontia chrysosporium 1               0          0             0          1
             Fusarium cylindricum             0          1          0             0          1
             Yeast                            0          0          0             0          0
             No. of Taxa/Season                    21         20          15           12




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Balbool and Abdel-Azeem                                                      Italian Journal of Mycology 49: 8-24 (2020)

DOI: 10.6092/issn.2531-7342/10063




 Table 5 - The multivariate analysis (ANOVA) for the effect of plant species, isolation media and seasonal
 variation on taxa composition.
                                                                  Variables
                                                                                                          Plants*
                                                    Plants*                     Plants*      Media*
                         Plants        Media                      Season                                  Media*
  Fungal species                                    Media
                                                                                Season       Season       Season
                                                    F-ratio with degree of significance
                         F-ratio       F-ratio      F-ratio       F-ratio       F-ratio      F-ratio      F-ratio
  A. alternata           30.41***      13.46*** 4.41***           5.33**        11.52***     0.99n.s      1.17n.s
  A. atra                36.22***      0.18n.s      0.21n.s       2.98*         4.31***      2.13n.s      1.68***
  A. solani              14.12***      5.72*        5.72***       5.41***       5.41***      1.98n.s      1.98***
  A. flavus              41.47***      3.99*        3.75***       19.02*** 9.63***           0.93n.s      0.81n.s
  A. nidulans            20.79***      0n.s         0n.s          14***         14***        1.13n.s      1.13n.s
  A. niger               59.4***       26.3***      4.36***       7.35***       8.5***       3.19*        1.41*
  A. terreus             11.69***      0n.s         0.83n.s       2.76*         4.22***      0.21n.s      0.45n.s
  C. globosum            37.31***      12.73*** 4.15***           31.31*** 21.13*** 3.48*                 2.35***
  C. madrasense          14.97***      2.22n.s      2.22***       14.97*** 14.97*** 2.22n.s               2.22***
  C. cladosporioides 17***             0.07   n.s
                                                    10.81***      9.56***       5.67***      1.17   n.s
                                                                                                          2.01***
  C. bicolor             15.6***       2.5n.s       1.76*         0.46n.s       1.99***      2.64*        1.75***
  E. monoceras           27.18***      0.27n.s      0.27n.s       7.43***       7.43***      0.45n.s      0.45n.s
  F. cylindricum         8.15***       2.04n.s      2.04**        8.15***       8.15***      2.04n.s      2.04***
  F. oxysporum           29.19***      0.15   n.s
                                                    0.15   n.s
                                                                  1.29 n.s
                                                                                1.29   n.s
                                                                                             2.23   n.s
                                                                                                          2.23***
  F. solani              17.12***      0.68n.s      0.68n.s       1.5n.s        1.5**        0.89n.s      0.89n.s
  L. theobromae          13.02***      2.83n.s      2.83***       5.61***       5.61***      0.37n.s      0.37n.s
  N. oryzae              11.29***      2.33n.s      2.33***       2.33n.s       2.33***      4.32**       4.32***
  P. brevicompactum 105.15*** 2.58n.s               5.34***       1.76n.s       5.74***      2.74*        12.1***
  P. chrysogenum         55.42***      17.04*** 16.42***          0.73n.s       12.05*** 0.56n.s          1.86***
  P. chrysosporium       12.06***      0.15n.s      0.15n.s       12.06*** 12.06*** 0.15n.s               0.15n.s
  R. stolonifer          36.98***      0.21n.s      4.65***       11.1***       8.77***      2.89*        1.47**
  S. strictum            32.52***      0.04n.s      0.04n.s       15.2***       15.2***      0.04n.s      0.04n.s
  S. racemosum           26.68***      0.04n.s      0.04n.s       3.12*         3.12***      0.16n.s      0.16n.s
  T. stiptatus           33.29***      3.7n.s       3.7***        2.39n.s       2.39***      0.52n.s      0.52n.s
  T. viride              4.84***       1.1n.s       0.21n.s       1.46n.s       2.89***      0.08n.s      0.13n.s
 *** p < 0.0001, ** p < 0.01, * p < 0.01, n.s not significant




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Balbool and Abdel-Azeem                                           Italian Journal of Mycology 49: 8-24 (2020)

DOI: 10.6092/issn.2531-7342/10063




                Figure 1 - Positive and negative colour changes using bromothymol blue indicator
                (a). Positive L-asparaginase morphotypes (b). Ranges of OD values with Duncan
                grouping (c)

       Regarding the occurrence levels of the isolated endophytic species; A. alternata and A. niger
showed high occurrence, A. flavus and P. chrysogenum showed medium occurrence, and the other
species showed low to rare occurrence. Our results are in agreement with Salem and Abdel-Azeem
(2014) and Abdel-Azeem et al. (2016, 2018, 2019a). They reported that A. alternata and A. niger were
recovered with 25% of frequency and 250 cases of isolation and with 2.8% of frequency and 28 cases
of isolation respectively. As an overview for the biodiversity of endophytic fungi among the collected
plant species; Artemisia herba-alba showed the highest richness value of nine species, followed with,
Achillea fragrantissima, Ballota undulata, Rosmarintus officinalis, Tanacetum sinaicum. Our data
was compatible with Abdel-Azeem (2009) and Abdel-Azeem and Salem (2012).
       On studying the diversity of the recorded endophytic fungi among the collected plant species
using Shannon and Margalef indeces, Artemisia herba-alba recorded the highest diversity indeces,
while the other plant species showed a varied diversity indeces. These diversity levels are similar to
those reported by Li et al. (2016) who examined the plant-associated fungi isolated from Zanxthoylum
bungeanum.



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Balbool and Abdel-Azeem                                              Italian Journal of Mycology 49: 8-24 (2020)

DOI: 10.6092/issn.2531-7342/10063




                      Figure 2 - L-asparaginase activities for the nine positive screened plant-
                      associated fungi


      On studying the effect of seasonal variation, spring recorded the highest number of taxa,
followed by summer, autumn and winter. Our results is compatible with Kim et al. (2013) and Salem
and Abdel-Azeem (2014) who suggested that the seasonal variation affected the biodiversity of
endophytic fungi in Saint Katherine Protectorate.
      Regarding the effect of elevation variation on the biodiversity of isolated endophytic taxa.
Low elevation wadis recorded the highest mean of endophytic taxa per plant species followed by
mid elevation wadis and high elevation wadis. Our results in agreement with Terhonen et al. (2011),
Kim et al. (2013), Salem and Abdel-Azeem (2014) who suggested that the fungal diversity would
increase with decreasing elevation altitude. In contrast Higgins et al. (2007) reported a high diversity
of plant-associated fungi over high elevations. Furthermore, Terhonen et al. (2011) suggested that the
elevation influences the biodiversity of plant associated fungi for its effets on climate.
      Statistical analyses showed that plant species strongly influence the diversity of plant-associated
fungi. This sociability may be referred to the chemical constituents of the plants. The ability of some
of these species to live under water stress and various chemical compounds has been proven by Salem
and Abdel-Azeem (2014) and Abdel-Azeem et al. (2018, 2019a) on endophytic fungi in Saint Katherine
Protectorate. Furthermore, Tan and Zahou (2001) concluded that it is possible to isolate hundreds of
endophytic species from a single plant, and among them, at least one generally shows host specificity.
Schulz et al. (2002), Jia et al. (2016) and Khare et al. (2018) suggested that there is a close relationship
between the host plant and associated fungi which already proven by Zhao et al. (2011).
      Four species with seven morphotypes showed the anti-leukemic enzyme (L-asparaginase)
production. Our result are in accordance with previous studies on the plants-associated fungi producing
L-asparaginase carried by Kour et al. (2007) and Yadav and Sarkar (2014) on F. oxysporum, Ali et al.
(1993) and Kumar et al. (2013) on C. cladosporiodes, Mishra (2006) and Rahiman et al. (2014) on A.
niger and Nagarajan et al. (2014) on L. theobromae.
      The positive taxa showed an enzymatic activity ranging from 44.5 U ml-1 to 152.58 U ml-1 where
L. theobromae recorded the highest activity with 152.58 U ml-1 which is higher than what previously
reported from A. terrus, R. miehi, A. oryzae and L. theobromae (Sarquis et al., 2004; Balbool et al.,
2018).

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Balbool and Abdel-Azeem                                            Italian Journal of Mycology 49: 8-24 (2020)

DOI: 10.6092/issn.2531-7342/10063



       During this work L. theobromae hosted by T. polium showed a positive result to produce
L-asparaginase. Plant extract of T. polium are used widely as antioxidants, antibacterial, antifungal
and anticancer (Rajabalian, 2008; Kandouz et al., 2010; Khan et al., 2011; Movahedi et al., 2014;
Emami Zeydi, 2016). It was previously suggested that both the plants-associated fungi and their host
plants produce active biometabolites with similar or higher activity (Zhao et al., 2011; Jia et al. 2016;
Khare et al. 2018).
       Our results showed that L. theobromae produces L-asparaginase extracellularly with a high
enzymatic activity, while Narayana et al. (2008) reported that most of the L-asparaginase with
microbial sources is intracellular. Extracellular L-asparaginase interacts minimally with cellular
constituents and easier to extract; hence, the induction of extracellular L-asparaginases with high
activity would be more advantageous for their expoliation as anticancer agents.
       L-asparaginase was reported to be used clinically as antileukemic drug (Pieters et al., 2011; El-
Nagga et al., 2014; Egler et al., 2016), and industrially as biosensor (Kumar et al., 2013b), and in the
biosynsthesis of amino acids (Shrivastava et al., 2016).
       In general, this study shed the light on the effect of different parameters like plant species,
seasonal variations, elevation and isolation media on the diversity of endophytic fungi in Saint
Katherine Protectorate. In addition, it gives an example for the ability of Egyptian fungi to be a gold
mine for production of bioactive compounds in a sustainable way.

Acknowledgements
We are deeply appreciated the kind help of Prof. Abdel Ghafar M. Abo El Saud (Botany Department, Faculty of
Science, University of Suez Canal) during data analyses. We extend our thanks to Prof. Robert Blanchette and
Dr. Benjamin Held (Plant pathology Department, University of Minnesota, USA) for their unlimited support
during molecular analyses of taxa.

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Balbool and Abdel-Azeem                                        Italian Journal of Mycology 49: 8-24 (2020)

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