Fungal biodiversity of some substrates of the Sultanate of Oman

International symposium of Biotechnology & Conservation of Species from Arid Regions February 10-13, 2013, Muscat-Oman

Fungal biodiversity of some substrates of the Sultanate of Oman Saifeldien A. F. El-Nagerabi1, 2Abdulkadir E. Elshafie and 2 Ratiba Al Muaine 1

Department of biological Sciences and Chemistry ,College of Arts and Sciences ,University of Nizwa ,Birkat Al Mouz,Nizwa, Sultanate of Oman, 2 Department of Biology, College of Science, Sultan Qaboos University, PO Box 36 Al-Khod, Sultanate of Oman PC123.

ABSTRACT In this study the biodiversity, host-specificity of coprophilous, nematophagous and endophytic fungi of different substrates from semi-arid regions of Oman were evaluated. The distribution of coprophilous fungi among the different dung types was investigated. Forty five species belonging to 25 genera are reported. Most of the genera and species are new records for Oman. Twenty-one species are new records for the Arabian Peninsula and four are new records for Asia. Some dung types are new substrates. The species composition of Oman was comparable to those of East Africa. A survey of nematophagous fungi of 82 soil samples and leaf litter in the Sultanate of Oman yielded ten trapping fungi belonging to three genera. All isolates are new record for Oman. Arthrobotrys multiformis is the second record of the species world wise. All isolates tested trapped all the nematodes (500 nematodes/Petri dish) in 96 hours. The biodiversity of epndophytic fungi on Boswellia sacra, Ziziphus spinachristi and Z. hajanensis was studied. Forty three species and 3 varieties of fungi were recovered as new records to B. sacra, 45 fungal species, 18 sterile mycelia and 12 yeasts from Z. spina-christi, whereas 35 fungi, 11 sterile mycelia and 5 yeasts were recovered from Z. hajanensis. Its species preference, tissue-specificity and seasonal variation were investigated. Pure and herbarium cultures of all of the fungi were preserved as genetic resources and kept as SQU and Nizwa Fungal Culture Collection. Key Words Biodiversity, Boswellia sacra, coprophilous, culture collection, endophytic fungi, nematophagous and soil fungi, species-tissue- specificity, trapping efficiency, Oman, Ziziphus.

INTRODUCTION Oman has a variety of topographical features consisting of plains and mountains that are typical of harsh environmental conditions with rare erratic rainfall pattern typical of desert and semi-desert climates where the majority of fungi will be unable to survive these conditions. Mountains are important ecosystem attracting many interests of the world. They cover 24% of the earth surface and support 12% of the world population as an excellent source for water and inhabited by diverse flora and fauna [9]. In Oman, AlJabal AlAkhdar in the Western Hajer mountains (1500 m) and Dhofar Mountains are globally distinguished ecosystem of different climatic conditions and diverse vegetation. It has experienced rapid development which associated with noticeable climatic changes and vegetation deterioration. The problem is not limited to conspicuous flora and fauna, but extends to fungi and bacteria which depend on higher plants for their survival [34]. There are no extensive studies on the mycoflora of this region. Little is known about the coprophilous fungi of the Arabian Peninsula [1-5, 20-24, 119]. Very few studies have been carried out on the coprophilous fungi of Oman [50]. On the other hand, the taxonomy of nematophagous fungi has been under continuous revision and evaluation by many authors [15, 39, 43, 67, 85, 89, 97, 105, 108, 116, 124]. The most recent 1

International symposium of Biotechnology & Conservation of Species from Arid Regions February 10-13, 2013, Muscat-Oman revision was carried out by Scholler et al. (1999) [114] who proposed a new genus concept considering biochemical, morphological, and molecular features (rDNA sequence data). Nonetheless, nematophagous fungi have been poorly studied in the arid and desert habitat [45, 51, 53, 95]. We hope that this survey and others will help to provide a better understanding of the correlation among the distribution, biodiversity and morphological variation within the arid habitat strains and differences with known temperate and tropical species. On the other hand ,Worldwide, few attempts were carried on the biological control of nematodes by nematophagus fungi [50]. Epiphytic or endophytic fungi are microorganisms which spend at least part of their life cycle on or inside leaf tissues without negative impact [10, 40, 46, 61, 62, 70, 72, 86, 115]. Numerous and diverse fungi were isolated from the tissues of most parts of terrestrial and aquatic plants specially the leaves as epiphytes or endophytes [70]. However, certain endophytic fungi might promote growth and improve the ecological adaptability of the host by enhancing plant tolerance to environmental stress and resistance to phytopathogens and/or herbivores [16, 36, 85, 117, 120]. Therefore, the alteration of beneficial endophytes could lead to the development of new and devastating disease to the host plant [19]. Generally endophytes among poorly understood group of fungi [63]. In Oman there is no single study on the epiphytic fungi of wild and cultivated crops. Of these plants, Ziziphus also known as “Sedra” is an important genus of the family Rhamnaceae found growing extensively in arid and semi-arid regions and represented by 135-170 species [17, 86-88]. Of these, only Z. spina-christi (L.) Wild and Z. hajanensis are the most common species inhabiting AlJabal AlAkhdar and is indigenous to Oman [87]. It is excellent source of food, fodder and fuel [88]. Recently the anti-inflammatory analgesic and antispasmodic activities were reported in rodents [25, 26]. On the other hand, Boswellia sacra Flueck. (Frankincense, Olibanum) synonyms (B. carteri, B. undulata, B. crenata) belong to Burceraceae family which includes several species growing in the Arabian Peninsula, India and East Africa [31, 68]. B. sacra from Dhofar Mountains are currently and ecologically relevant species showing symptoms of decline due to anthropic factors and possibly global warming [85, 102]. It provides several good services as timber, fodder, nectar and gum which are useful for traditional medicine, religious ritual and income regeneration [60]. Frankincense (gum olibanum) is the most important product of several traditional and industrial uses as a raw material in many industries such as pharmacology, flavouring, beverage, liqueurs, cosmetics, detergents, creams and perfumery [60, 82, 83]. Anticancer, anti-inflammatory, immunomodulatory, antimicrobial and antiviral activities of several Boswellia species in addition to be a rich source of non-volatile triterpenoic constituents have been reported [6, 11, 30, 92-94]. Worldwide, many researchers collecting and isolating fungi from unexplored sites, habitats, substrates, particularly in extreme environmental conditions [71]. However, of all world plants, it seems that only a few species have had their complete complement of endophytes and epiphytes studies [70, 115]. The variations of endophytes/epiphytes are due in part to generic differences among plants and the variations in environmental conditions [46]. In Oman, the few research carried up to now focused on coprophilous fungi [50, 65], mycotoxins and mycotoxigenic moulds [52, 55], nematophagous fungi [51, 53], and some plant diseases [7, 56]. There are few studies on some plant diseases of cultivated crops in different areas of Oman, nonetheless, there is no single study on the biodiversity of the fungal flora of the wild and cultivated plants of Dhofar and AlJabal AlAkhdar. It is evident that endophytes are among poorly understood group of fungi [63]. It is quite promising to explore interesting endophytic fungal species among the myriad plants including the main two species of the genus Ziziphus namely Z. spina-christi, and Z. hajanensis and Boswellia sacra which have never been explored so far. 2

International symposium of Biotechnology & Conservation of Species from Arid Regions February 10-13, 2013, Muscat-Oman OBJECTIVES This study aimed at obtaining information on the mycobiota of coprophilous fungi associated with different dung types in an arid environment and to compare their distribution among different sites and dung types in Oman. We intended also to compare the mycobiota of Oman with the nearest studied area such as East Africa. Also nematophagous fungi were studied aiming at their efficiency as biocontrol to nematodes. It is quite promising to explore interesting and diverse endophytic fungal species from plants grown in mountainous areas. In the present investigation, we examined the diversity of the endophytic fungi colonizing two species of the genus Ziziphus (Z. spina Christi, Z. hajanensis) and B. sacra plant collected from Aljabal Alakhdar and Dhofar Mountains. The fungal communities in the leaves and stems tissues were evaluated for their biodiversity, species-tissue-specificity and seasonal variation. MATERIALS AND METHODS Dungs samples for isolation of coprophilous fungi The dung samples from cattle, camel, goat, oryx, donkey, gazelle, ibex and tahr which ranged between fresh to semi fresh, were collected, stored at room temperature (24 ± 2oC) and were examined within three weeks of collection. Each sample was soaked in distilled water for a few minutes and placed in a sterile crystalline dish containing sterilized moistened absorbent cotton lined with wet filter papers. The dishes were covered with small glass plates, incubated at room temperature 24 ± 2oC and placed near a window. The samples were examined every four days for up to three months. Fungi that subsequently developed on the dung were examined and identified. Soil samples for isolation of nematophagous fungi Soil samples were randomly collected from the rhizosphere of plants that were infected by nematodes. The sprinkled-plate technique described by Rubner (1996) [108] was used for the isolation of the nematophagous fungi in which one gram of soil was sprinkled onto each of four Petri dishes containing water agar (WA).One to two hundred nematodes (Panagarellus redivivus, Linne Goodey) were added to the cultures. The cultures were incubated for 1-2 months at room temperature (23ºC).Trapped nematodes were then transferred to half-strength Corn Meal agar (CMA) which was prepared by adding 8.5 g CMA [Oxoid] to 12.5 g additional agar [Oxoid] and 1 liter water). Using sterile human hair fixed onto a fine needle, single spores of nematophagous fungi were isolated from the erect conidiophores, cultured on CMA and challenged with nematodes. The efficiency of the fungi was tested by adding about 500 nematodes to each Petri dish. The trapping efficiency of the fungi was compared. Endophytic fungi Sampling site This study was carried out in AlJabal AlAkhdar and Dhofar Mountains of Sultanate of Oma. Oman which is located at the South of the Arabian Gulf, bordered by Yemen on the South, the Arabian Sea on the Southeast, Iran on the Northeast, the United Arab Emirates on the Northwest, and Saudi Arabia on the West. It lies between latitude of 21°00, N-29°00, N and longitude of 51°00, E - 59°40, E. The climate is hot-dry in the interior, hot-humid in the coastal area and humid in the South with summer monsoon rain. The average temperature is about 26°C with annual precipitation of less than 100 mm [8]. AlJabal AlAkhdar in the Western Hajer mountain range above 1500 m with average temperature on the plateau of 18.5°C which is much lower than the surrounding region (29°C) and of 46% relative humidity. Dhofar Mountains range are located in a coastal region known as "Nejd" covered 3

International symposium of Biotechnology & Conservation of Species from Arid Regions February 10-13, 2013, Muscat-Oman by drought deciduous broadleaf anogeissus forest typical for the cloud oasis of the Dhofar Governorate in Oman [69, 79, 90]. The site is 500 m above the sea level and about 25 km away from the coast with 113-115 mm precipitation at the coast and the temperature of between 24-26°C, while they are 252 mm and 21°C near the mountain crest of 880 m elevation. The wet season which is known as "Khareef" in Arabic is in summer from midJune through mid-September where moist air from Indian Ocean is pushed against the coastal mountains range, leading to orographic clouds and drizzle [69]. During the rest of the year, desert climate prevails, beside, "Khareef" rain, precipitation is rare and erratic, mainly from cyclones occurring about once in each three years [28]. Plant materials Duplicate samples of healthy green leaves from two species of Ziziphus (Z. spinachristi, Z. hajanensis), and stems of Boswellia sacra were collected from AlJabal AlAkhdar and Dhofar Mountains, Oman, respectively. The selected plant was identified at the Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa, and Department of Biology, College of Science, Sultan Qaboos University. Samples were collected at different times and season of between April 2008 through October 20011. The samples were collected in sterile polyethylene bags and stored in refrigerator at 5°C. Isolation of endophytic fungi The tissues of the selected plant parts were cut into small pieces of approximately 1 cm in length and washed with several changes of sterile distilled water. The pieces were surface disinfected with 70% ethanol for I min followed by 5% sodium hypochlorite for 5 min [63, 85]. The disinfected pieces were aseptically inoculated on Potato Dextrose Agar (PDA, Potato, 200g; dextrose, 20g; agar 15g; distilled water, 1L) supplemented with 0.05 mg/ml chloramphenicol to suppress the bacterial growth, and enable the mycelia development on the plant tissues. The inoculated plates were incubated at the ambient temperature (27-29°C) for 7-10 days until the mycelium is apparent on the growth media. The developing fungal colonies were then inoculated on Petri dishes containing Malt Extract Agar (MEA) for preparation of pure colonies, identification and preservation as dry herbarium materials. Duplicates of the isolated fungi were deposited at the herbarium of Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa, and Department of Biology, College of Science, Sultan Qaboos University, Oman. Identification of endophytic fungi The fungal isolates were identified using macroscopic features based upon colony morphology on the growth media and microscopic observations of mycelia, asexual conidia and sexual spores according to different taxonomic books, numerous monographs and taxonomic papers published by many authors [12-14, 47, 48, 96, 101, 103, 118, 121].The incidence of the isolated fungi was reported. Data analysis The number of cases isolation (NCI) of each fungal species was calculated according to modified formula of Gazis & Charerril (2010) [63] as the number of the samples from which the fungus was isolated, whereas the occurrence remarks (OR) as a total number of the samples from which a given species was isolated compared to the total number of samples. The number of the samples from which a given species was isolated divided by the total number of the collected samples was used to calculate the incidence percentages of the fungal species in each genus.

4

International symposium of Biotechnology & Conservation of Species from Arid Regions February 10-13, 2013, Muscat-Oman RESULTS AND DISCUSSION Distribution of coprophilous fungi among the dung of animals Table 1 shows a list of coprophilous fungi, their incidence of occurrence, distribution (Map1) and herbarium numbers. The table shows a total of 45 species belonging to 25 genera. All the genera and species except Coprotus dhofarensis, Thecotheus harasisus and Chaetomidium khodense [32, 65] are new records for Oman. The genera are distributed taxonomically as follows: Discomycetes (8 genera, 19 species; 42.2% of the species); Loculoascomycetes (3 genera, 6 species; 13.3% of the species); Pyrenomycetes (9 genera, 15 species; 33.3% of the species); Plectomycetes (2 genera, 2 species; 4.4% of the species); Myxomycetes, Basidiomycetes, and Zygomycetes (one genus, one species; 2.2% each of the species). Species of Discomycetes and Pyrenomycetes are the most common (75.5%). Five species were recorded for Coprotus, Sporomiella (4 species), Ascobolus (3 species), Iodophanus (2 species), one species for each of the remaining genera (Saccobolus, Lasiobolus, Chaetomium, Lophotrichus, and Podospora). Most of the fungal species were very uncommon and were found in less than 5% of dung examined (Table 2). The species that were extremely common (31%) were Sporormiella minima and Iodophanus carneus. Other common fungal species found in 21-30% of the samples were Ascobolus hawaiensis, Coprotus disculus, Coprotus granuliformis, Coprotus leucopocillum and Chaetomium globosum. Table 2 shows the distribution of the fungal species among the dung types of animals. Twenty seven species (17 genera) were found on camel dung; 24 species (15 genera) were found on goat dung; 16 species (14 genera) were on cattle dung and 1-7 species on the dung of other animals such as oryx, donkey, gazelle, ibex and tahr. Species of Discomycetes (19 species) were the most common species found in this study. Of these 19 species, 5 were found on cattle dung, 10 on camel dung ,12 on goat dung, and 6 on oryx dung. The most common group were species belonging to Pyrenomycetes (15 species). Of these 7 species were found on cattle dung, 9 on camel dung and 5 on goat dung. Some fungi were found only on one dung type. Of the 16 fungal species found on cattle dung, 7 were restricted to cow dung and were not found on other dung types. Three species out of 24 species, and 7 species out of 27 species were restricted to goat and camel dung respectively ( Table 2). Some fungi were found on more than one dung type. Thirteen species out of the total 45 species were found on both camel and goat dung, 6 on camel and cattle dung, 5 on cattle and goat dung and 3 on dung of cattle, goats and camels. Some species are new to the Arabian Peninsula and/or to Asia whereas some dung types are new substrates (Table 1). In this study, 8 genera of Discomycetes were found on different dung types in this study. The same genera were reported, among 11 genera, in East Africa [76]. Some of these genera are cosmopolitan and were also found in temperate and tropical habitats. Thirty nine genera of Pyrenomycetes and nine genera of Loculoascomycetes were reported in East Africa [76]. In our survey we found only 9 genera of Pyrenomycetes, two of which, namely Chaetomidium and Zopfiella, were not reported in East Africa. Of the nine Loculascomycetes recorded in East Africa [76] three genera namely Delitschia, Sporormiella and Faurelina were also found in Oman. Our study has shown that 75.5% of our species were Discomycetes and Pyrenomycetes as reported in similar results [75, 76]. If we compare the number of species in some of our genera with the number of species of similar genera in East Africa we find Coprotus (4 5

International symposium of Biotechnology & Conservation of Species from Arid Regions February 10-13, 2013, Muscat-Oman vs. 9), Sporormiella (4 vs. 28), Ascobolus (3 vs. 7), Saccobolus (3 vs. 9), Lasiobolus (3 vs. 6) and Chaetomium (3 vs. 32). The number of the species is fewer in our study than for East Africa. This difference in number of species between Oman and East Africa could be due to the difference in the number of samples studied and the variation in the environmental conditions [104]. The most abundant fungi we found in our study were Sporormiella minima, Iodophanus carneus, Coprotus disculus, C. granuliformis, C. leucopocillum and Chaetomium globosum. The majority of these species are common and have been reported in East Africa [33, 73, 74]. Some fungi in this study were found only on one dung type. The fungal species composition has been shown to be dependent upon the type of stomachs, digestive processes, feeding habits and food preferences of the animals [44, 98]. However, in this study, we did not attempt to make quantitative assessment of the diversity and species richness. Nonetheless, we are aiming to survey the coprophilous mycobiota of a harsh environment that has not been previously studied and to be compared with the nearest well studied area such as East Africa. Diversity of nematophagous fungi Table 3 shows the place of isolation, source, incidence and trapping devices of the ten nematophagous fungi isolated from only 82 soil and litter samples from different places in Oman. Like the other Gulf countries, Oman has one of the world's harshest environments, but still it has its unique fungal biodiversity distributed all over the places (Table 3). More collections will reveal more mycobiota diversity, some of which might be unique. In this study, Arthrobotrys oudemansii, A. multiformis, and A. javanica were found to be morphologically more variable than so far known in the literature (Fig.1,2,3). Moreover, A.oudemansii was found to have shorter and wider conidia than the type species and all other described isolates. A.multiformis was found to be rare and is the second record to be described worldwide [42] and re-examined by [108]. It differs from the described species in having larger conidia, fewer septa and longer secondary conidia. A. javanica conidia are slightly longer, narrower, and not constricted at the septa when compared with described isolates. Significant differences were found in the trapping efficiency of the fungi studied. A.thaumasia was the most efficient trapping fungus followed by A.oligospora and D.brochopaga (Fig.4). Each fungus was capable in trapping 500 nematodes in 96 hours. These fungi which are adapted to harsh environmental conditions of high temperature, aridity and high salinity are potential biological agents for control of nematodes.

6

International symposium of Biotechnology & Conservation of Species from Arid Regions February 10-13, 2013, Muscat-Oman

MAP 1: Dung samples collection areas

7

International symposium of Biotechnology & Conservation of Species from Arid Regions February 10-13, 2013, Muscat-Oman Table 1. Incidence of occurrence and distribution of coprophilous fungi in Oman Occurrence*

Fungi

DistributionS

Herbarium SQU** FMR***

18 19

Discomycetes Ascobolus hawaiiensis Brumm. Ascobolus immersus Pers. Ascobolus stictoideus Speg. Saccobolus citrinus Boud. & Torrend Saccobolus minimus Velen. Saccobolus versicolor (P.Karst) P.Karst Coprotus aurora (P.Crouan & H.Crouan) K.S.Thind & Waraitch Coprotus disculus Kimbr., Luck-Allen & Cain Coprotus dhofarensis Gené, ElShafie & Guarro Coprotus granuliformis (P.Crouan & H.Crouan) Kimbr. Coprotus leucopocillum Kimbr., Luck-Allen & Cain Lasiobolus intermedius J.L.Bezerra & Kimbr. Lasiobolus microsporus J.L.Bezerra & Kimbr. Lasiobolus trichoboloides S.R.Khan & J.L.Bezerra Lasiobolidium orbiculoides Malloch & Benny Iodophanus carneus (Pers.) Korf Iodophanus verrucosporus (P.W.Graff) Kimbr., Luck-Allen &Cain Ascodesmis nigricans Tiegh. Thecotheus harasisus Gené, ElShafie & Guarro

20 21 22 23 24 25

Loculoascomycetes Sporormiella australis (Speg.) S.I.Ahmed & Cain Sporormiella intermedia (Auersw.) S.I.Ahmed & Cain Sporormiella minimoides S.I Ahmed & Cain Sporormiella minima (Auersw.) S.I.Ahmed & Cain Delitschia marchalii Berl. & Voglino Faurelina indica Arx, Mukerji & N. Singh

B B B F A B

DFZ3 D K2 IJK1K2P2UWXYZ1S A A

31 32 33 30 25 10

3691

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Pyrenomycetes Arnium arizonense (Griffiths) N.Lundq. & J.C.Krug Chaetomidium khodense Cano, Guarro & ElShafie Chaetomium bostrychodes Zopf Chaetomium globosum Kunze Chaetomium murorum Corda Lophotrichus ampullus R.K.Benj. Lophotrichus sp. 1 Lophotrichus sp. 2 Podospora anserina (Ces. ex Rabh.) G.Winter Podospora prethopodalis Cain Podospora setosa (G.Winter) Niessl Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. Kernia nitida (Sacc.) Nieuwl. Zopfiella erostrata (Griffiths) Udagawa & Furuya Cercophora sp.

B A B E B B B B B B D A B B B

K2Z4 A IK2P2P3 IJK1K2NP2U K1L K1K2J V J A A AIJK2Y A BZ5 BDFV A

21 14 40 52 15 19 53 54 24 22 62 29 6 5 23

3688 3692 3689 3695 3690 3702

414 2

Plectomycetes Gymnoascus dankaliensis (Castell.) Arx Neosartorya fischeri (Wehmer) Malloch & Cain

B B

K2 AK2

18 17

3694

43

Myxomycetes Physarum sp.

B

K1K2MUU1Z3Z4

55

-

C

UZ3Z4

57

-

B

TZ2Z4

56

-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

44 45

Basidiomycetes Coprinus sp. Zygomycetes Pilobolus kleinii Tiegh.

E C B C B B B

BCDE AK2Z2Z3Z4H A ADC K1 K2 CK1K2

40 43 39 41 42 50 3

3682 3700 3698 3699 3684

E A E E B B B B F B

AGJK1P2TZ2Z4P1RR1 Z4 BDK1M JK1K2P2Z5Q FG FG FG V IJK2K3LMNP1P2TWY K2Z2

1 51 2 4 36 37 45 48 28 44

3685 3921 3696 3686 3701 3703 -

B A

DEFZ5 AG

35 34

4159

*Occurrence remarks:A = extremely rare (found in