Mycosphere 7 (3): 236–261(2016) www.mycosphere.org ISSN 2077 7019 Article Doi 10.5943/mycosphere/7/3/1 Copyright © Guizhou Academy of Agricultural Sciences Biodiversity and molecular characterization of yeast and filamentous fungi in the air of citrus and grapevine plantations in Assiut area, Egypt Moubasher AH*, Abdel-Sater MA and Zeinab Soliman Department of Botany and Microbiology, Faculty of Science, Assiut University, P.O. Box 71516, Assiut, Egypt Moubasher AH, Abdel-Sater MA, Soliman Zeinab SM 2016 – Biodiversity and molecular characterization of yeast and filamentous fungi in the air of citrus and grapevine plantations in Assiut area, Egypt. Mycosphere 7(3), 236–261, Doi 10.5943/mycosphere/7/3/1 Abstract A total of 218 species and 3 varieties belonging to 83 genera of filamentous and yeast fungi were recovered from the air of both citrus and grapevine plantations. A relatively higher numbers of genera and species were recovered from the air of citrus plantations compared with those recovered from grapevine plantations. The peak of total propagules of fungi caught from the air of citrus plantations was shown in February on both media and from the air of grapevine in December and August on DYM and DRBC, respectively. Their troughs were shown in June and October on DYM and DRBC, respectively for both citrus and grapevine plantations. The widest spectrum of species recovered from the air of citrus plantations was registered in June on both media and from the air of grapevine plantations in February and in April on DYM and DRBC, respectively. The air of citrus plantations shared the air of grapevine plantations in some highly encountered filamentous fungi on both media (Cladosporium, Penicillium, Aspergillus and Alternaria) or on one medium (Cochliobolus, Fusarium, Myrothecium, Phoma and Pleospora). Eighty-four fungal species were isolated from the air of citrus only, while 46 species were isolated from the air of grapevine only.Yeast fungi showed their peak of total propagules from the air of citrus plantations in October and April and from the air of grapevine plantations in June and December on DYM and DRBC, respectively. Fifteen genera and 26 species of yeasts were collected. Two genera of yeasts were encountered in high frequency on one medium and moderate or low on the other medium in the air of both citrus and grapevine plantations and these were Cryptoccocus (4 species) and Rhodotorula (3 species). Key Words – Aerobiota – characterization – genotypic – phenotypic - seasonal fluctuation Introduction In the atmosphere many microbioparticles are present. These are fungal spores, pollen grains, insect parts. The study of aeromycology is important in plant pathology and in disease forecasting of plant diseases (Gregory 1973). Many fungal diseases of plants are spread by air. The deterioration of stored materials and the spoilage of foodstuffs are induced by growth of fungi which reach them from the air. Some fungal spores are regarded as important causes of allergic diseases such as bronchial asthma and allergic rhinitis. The fungal spores act passively as allergens acting on some individuals who have become sensitized (Moubasher 1993). Submitted 19 February 2016, Accepted 30 April 2016, Published online 13 May 2016 Corresponding Author: Moubasher AH – e-mail – [email protected] 236 Aerobiological studies conducted in relation to respiratory allergic diseases (Hasnain et al. 1984, 1985a,b) revealed that the total air spora of the Auckland region and many of its fungal components show a strong tendency towards an increase during warmer months and a gradual decline during winter. It is well known that fungi require certain optimum conditions for each phase of their growth (Gregory 1973). Aeromycological researchs from the Middle East area are scattered, in Egypt (Moubasher & Moustafa 1974, Abu-El-Souod 1974, Abdel-Hafez et al.1986, Abdul Wahid et al.1996, Ismail et al. 2002), in Kuwait (Moustafa 1975, Khan et al. 1999), in Qatar (Al-Subai 2002), in Saudi Arabia (Abdel-Hafez 1984, Hasnain et al. 2005), in Yemen (El-Essawy et al. 1992), in Turkey (Sarica et al. 2002; Asan et al. 2004, Özkara et al. 2007), in Iran (Hedayati et al. 2005, Nourian et al. 2007) and in Jordan (Shaheen 1992, Al-Qura’n 2008). Ben-Meir-Glueck (1952) isolated more than thirty different species from the air of orange groves and packing sheds and from the skins of fruits. These included Penicillium italicum and P. digitatum, which are the main incitants of citrus rot. Barkai-Gollan (1961) studied the air-borne fungi in citrus fruit packing houses and reported that P. digitatum and P. italicum predominated, whereas Fusarium, Trichoderma, Colletotrichum and Diplodia were encountered only occasiona- lly. Moubasher et al. (1971) found that the fungal aerospora in citrus plantations was considerably different from that in the soil with Cladosporium herbarum, Aspergillus niger, Curvularia sp., Alternaria alternata, Helminthosporium sativum and Fusarium were the most abundant in the air of citrus plantations. Fifteen isolates from air sampled at the vineyard were identified as A. niger (88.2%) and 2 isolates were A. carbonarius (11.8%) (Díaz et al. 2009). In the aerospora of tomato plantations Cladosporium sp. was found to be the most dominant type followed by species of Alternaria, aspergilli, Nigrospora, Torula and Curvularia (Lohare et al. 2009). There are several reports on the occurrence of yeasts in the air (Di Menna 1955, Turner 1966, Gregory 1973). Al-Doory (1967) found that species of Cryptococcus, Rhodotorula, Sporobolomyces, and Debaryomyces were the most dominant species from the air in San Antonia, Texas, U.S.A. Rhodotorula mucilaginosa and Cryptococcus albidus were the most dominant species followed by Debaryomyces hansenii isolated from the air of El-Minia city, Egypt while Rhodotorula rubra, R. aurantiaca, Kluyveromyces marxianus, Torulaspora delbrueckii, Saccharomyces kluyveri, and Hansenula polymorpha were of less frequency (Haridy 1992). The present work was designed to investigate the diversity of filamentous and yeast (for the first time in this laboratory) fungi in the air of two economically–important plants (citrus and grapevine). Also, seasonal fluctuations of these fungi were carried out bimonthly during April 2008 to February 2009. Materials & Methods Sampling location This study was carried out in Sahel-Saleem city at approximately 25 km south-east of Assuit city. Sampling was conducted bimonthly over a twelve - month period from April 2008- February 2009. Three different plantations of citrus in the suburbs of Sahel-Saleem city and three of grapevine in El-Khawaled village (about 6 Km to the east border of the river Nile), in the northeast of Sahel-Saleem city were selected. Isolation of air -borne fungi Five replicate plates of 9 cm diameter of each of two media (DYM and DRBC) were exposed for five minutes at a height of 60 cm above the ground level during the same hours of the day (10 am-2 pm) at each of the six sites. The plates were then sealed and brought back to the laboratory and incubated at 25ºC for 7-15 days, during which, the developing colonies were counted, isolated and identified. The meteorological data during the period of study were as follows: the maximum temperatures varied from 25ºC–46ºC, the relative humidity from 36-86℅. A total of 36 exposures (3 farms of each of citrus and grapevine) were carried out bimonthly, with collectively 360 exposed plates. 237 Media used for isolation The exposure plate method relied on two isolation media "yeast extract malt extract agar supplemented with dichloran (DYM) and dichloran rose bengal chloramphenicol agar (DRBC)" were used in this study: 1- Dichloran yeast extract malt extract agar (DYM): Yeast extract malt extract agar (Wickerham 1951) of the following composition was employed: (g/l) yeast extract 3.0, malt extract 3.0, peptone 5.0, glucose 10.0, agar 20.0. Chloramphenicol (250 μg/ml) was used as a bacteriostatic agent. This medium was modified in the present work after preliminary survey by addition of 1 ml/l of 2 mg of dichloran dissolved in 10 ml ethanol which restricts the growth of mucoraceous fungi without affecting the other species. 2- Dichloran rose bengal chloramphenicol agar (DRBC), (King et al. 1979): (g/l) peptone 5.0, KH2 PO4 1.0, Mg SO4 0.5, glucose 10.0, agar 15.0, to which rose bengal (25 μg/ml) and chloramphenicol (100 μg/ml) as bacteriostatic agents (Smith & Dawson 1944, Al- Doory 1980) and dichloran (20 μg/ml) were used. Identification of filamentous fungi The following references were used for the identification of fungal genera and species (purely morphologically, based on macroscopic and microscopic features): Booth (1971), Ellis (1971, 1976), Pitt (1979); Domsch et al. (2007); Moubasher (1993), de Hoog et al. (2000), Schroers (2001), Samson & Frisvad (2004), Zare & Gams (2004), Leslie & Summerell (2006), Crous et al. (2007), Samson & Varga (2007), Simmons (2007), and De Seifert et al. (2011). Identification of yeasts Morphological characters Formation of pseudomycelium and true mycelium (Wickerham 1951) and the ability to form ascospores on three sporulation media (corn meal agar, potato glucose agar and yeast extract malt extract agar at 25°C) (Barnett et al. 2000) were tested. Physiological characters Fermentation of 14 different sugars (D-glucose, D-galactose, maltose, Me-α-D-glucoside, sucrose, trehalose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin, starch and D-xylose) and oxidative utilization of 36 carbon compounds were performed according to Barnett et al. (2000). Assimilation of 9 nitrogen compounds (potassium nitrate, sodium nitrite, ethylamine-HCl, L-lysine-HCl, creatine, creatinine, D-glucosamine, imidazole, or D-tryptophan) was determined (Suh et al. 2008). Hydrolysis of urea, growth at high osmotic pressure, growth at different temperatures, growth in the presence of cycloheximide, diazonium blue B (DBB) test and production of extracellular starch-like compounds were tested. Identification keys of Barnett et al.