Seasonal variation of air, soil and leaf surface fungi of broad bean and cellulolytic ability in Upper Egypt

Seventy-five species and 3 species varieties belonging to 21 fungal genera were collected from air, soil and leaf surface of broad bean plant on dicholran chloramphenicol malt extract agar (DCMA) and dichloran Rosebengal chloramphenicol agar (DRBC) at 28°C. The results obtained from leaf surface (phyllosphere and phylloplane), soil and atmosphere were basically similar in the two types of media and the most common fungi were: Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Cladosporium cladosporioides, Cladosporium sphaerospermum and Drechslera neergaardii. The monthly counts of these fungi on two types of media irregularly fluctuated giving maxima value at various months. A. flavus was the highest fungi that produced both exoand endo-ß-1,4-glucanases among the 9 tested isolates. Maximum production of two enzymes by A. flavus was 8 and 6 days after incubation at 30°C with culture medium containing glucose and cellulose as carbon sources and sodium nitrate as nitrogen source and initially adjusted to pH 6.


INTRODUCTION
Food legumes play an important and diverse role in the farming systems and in the diets of poor people around the world.They are ideal crops for simultaneously achieving three developmental goals in targeted population reducing poverty, improving human health and nutrition, and enhancing ecosystem resilience.Broad bean (Vicia faba L.) is one of the most important winter crops of high nutritive value in the world as well as in Egypt.Vicia faba, which has several common names (fava bean, faba bean, horse bean and tic bean), is a species of bean (Fabaceae) native to north Africa and southwest Asia (Elwakil et al., 2009).
World production of broad bean varied during the last 10 years.China came first in the production.It produced 13033750 tones while Egypt production was 155554 tones (FAO, 2009).
Cellulose, a major polysaccharide constituent of plant cell walls, is a 1,4 linked linear polymer of 8000~12000 E-mail: amanyattanew@yahoo.com.Gglucose units.Three major enzymes are involved in the degradation of cellulose to glucose which are endoglucanase , cellobiohydrolase (exo-1,4-d-glucanasem CBH) and β-glucosidase BG).EG acts in random fashion, cleaving linked bonds with in the cellulose molecule; CBH removes cellobiose units from the non reducing ends of the cellulose chain and BG degrades cellobiose and cellooligosaccharides to glucose (Saha, 2004).
The aim of the present investigation was to study the seasonal variations of fungus flora of leaf surface, airborne fungi and soil in Vicia faba field cultivated in Oena governorate and cellulolytic activity of some fungal isolates and the effect of some environmental and nutritional factors on cellulase production.

MATERIALS AND METHODS
During the growing season of broad bean crop which extended from December 2011 to April 2012, a broad bean field in South Valley University in Qena city in Upper Egypt was selected to study the mycoflora of leaf surface (phyllosphere and phylloplane) and soil, as well as the airborne fungi over the broad bean field.Samples were collected fortnightly and two media types were used: dicholran chloramphenicol malt extracts agar (DCMA) Andrews and Pitt (1986) and dichloran Rosebengal chloramphenicol agar (DRBC) at 28°C King et al. (1979).

Determination of phyllosphere fungi
The dilution plate method was used as employed by El-Said et al. (2006).

Determination of phylloplane fungi
The determination of phylloplane was as employed by El-Said et al. (2006).

Determination of airborne fungi
The "exposed plate" method was used to trap fungal spores over broad bean field during growing season.Six plates of 9 cm diameter were used for each exposure (3 plates for each type of medium).The plates were exposed at 10-11 a.m., for 15 min every 15 days (Abdel-Hafez et al., 1990).

Determination of soil fungi
The dilution-plate method as described by El-Said (1994) was used for estimation of soil fungi.The plates were incubated at 28°C for 5-10 days during which the developing fungi were counted, identified purely morphologically, based on macro-and microscopic characters (Raper and Fennell, 1965;Ellis, 1971Ellis, , 1976;;Domsch et al., 1980;Pitt, 1985) and calculated per g dry soil.

Screening of fungal isolates for cellulase production
Nine species (the most common species) belonging to 5 genera were screened for their abilities to produce exo-and endo-β-1,4glucanase (C1 and Cx enzymes, respectively).Isolates were cultured on Eggins and Pugh medium (1962).Cultures were incubated at 28°C for 7 days.Using sterile cork borer, 10 mm diameter, discs  were cut to inoculate 50 ml sterile liquid medium (in 250 ml Erlenmeyer flasks) of Eggins and Pugh medium (1962) for exoglucanase production and Prasad and Verma medium (1979) for endo-glucanase.The cultures were incubated at 28°C for 7 days.The cultures were filtered and the filtrates were used to detect the activity of the enzymes as follows:

Detection of exo-β-1, 4-glucanase (C1 enzyme)
Using a sterile cork borer, 3 cavities (10 mm diameter) were made in plates containing solid Eggins and Pugh medium (1962).A 0.1 ml of culture filtrate was dropped in each of these cavities followed by incubation at 28°C for 24 h, then the plates were flooded with chloroiodide of zinc solution and the uncolored zone gave a measure of cellulolytic power of isolates.

Detection of endo-β-1.4-glucanase (Cx enzyme)
Ten millimeters cavities were cut in plates containing solid medium of Dingle et al. (1953), filtrate obtained from 7 days old fungal cultures grown on Prasad and Verma (1979) medium was dropped in each cavity.After 24 h of incubation at 28°C, plates were flooded with chloroiodide of zinc solution and the clear zones around cavities were measured.

Factors affecting cellulase production
The effect of different ecological and nutritional factors on production of cellulase enzymes (C1 and Cx) by Aspergillus flavus was shown.Since this species was found to be highly active in cellulase production so this species was used for this study.The previous isolate was grown on liquid medium (Deacon, 1985).Fifty milliliters of the medium were dispensed into each 250 ml Erlenmeyer flask and each flask was inoculated with an agar mycelial disc (10 mm diameter) of the mould obtained from 7 days old fungal cultures growing on the solid basal medium.Experiments were done to indicate the best conditions which produce a good deal of the enzyme as well as of the best expense.

Effect of temperature and time course
The inoculated flasks were incubated at 20, 30 and 40°C for 14 days and harvested at 48 h intervals.Culture fluid were filtered and centrifuged at 5000 r.p.m. for 10 min, the clear supernatants were assayed for enzyme activity.

Effect of pH values
The test isolate (A.flavus) was grown on the basal medium of Deacon (1985).The initial pH of the medium was adjusted with 0.1 N NaOH or 0.1 N HCL to different values ranging from 2 to 14.After inoculation with A. flavus, cultures were incubated at 30°C for 8 days for C1 and Cx, respectively.At the end of incubation period the cultures were filtered, centrifuged at 5000 r.p.m. for 10 min and the clear supernatants were assayed for cellulase activity.

Effect of different carbon sources
The basal medium (Deacon, 1985) with pH 8 (the best pH for cellulase production) was supplemented with 1% of one of the following carbon sources: glucose, fructose, lactose, sucrose, cellulose, starch and carboxymethyl cellulose.The flasks were inoculated with A. flavus and incubated at 30°C (the best temperature of C1 and Cx enzymes production) for 8 days (the best incubation periods for C1 and Cx enzymes, respectively) and the cultures were filtered.After centrifugation the filtrate was used to detect the cellulase activity.

Effect of different nitrogen sources
To determine the effect of nitrogen source on cellulase production, sodium nitrate in the basal medium was replaced by the same amount of various nitrogen compounds such as: sodium nitrite, potassium nitrate, yeast extract, ammonium sulphate, ammonium nitrate and peptone in addition to sodium nitrate as a control.Cultures were incubated at 30°C for 8 days and the cultures were filtered, centrifuged and the filtrates were used for detection of cellulase activity.

Assay for cellulase activity (C1 and Cx enzymes)
The method described by Nelson (1944) and modified by Naguib (1964) was employed.The amount of reducing sugars produced was estimated by determining the optical density (absorption spectrum) at 700 nm wave length with a spectrophotometer model (Spectronic ® Genesys TM 2PC USA).A standard curve was plotted using aqueous solution of D-glucose.

RESULTS AND DISCUSSION
The monthly total counts of phyllosphere and phylloplane surface fungi of broad bean on plates of DCMA and DRBC irregularly fluctuated giving peaks during March and March, respectively.Thirty-four species and 3 species varieties belonging to 15 genera were collected from phyllosphere (10 genera and 20 species + 2 varieties) and phylloplane (12 genera and 21 species +1 var.) of broad bean leaves on DCMA and DRBC at 28°C (Table 1).The most common fungi of two substrates on the two types of media were: Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Cladosporium cladosporioides, Cladosporium sphaerospermum and Drechslera neergaardii.The monthly counts of the above fungal species were widely varied and fluctuated irregularly giving maxima during different months (Figures 1 and 2 2006) found that the most common fungi isolated from 60 samples of leaf surface of broad bean on DCMA and DRBC at 28°C were: Alternaria petroselini, A. citri, Aspergillus flavus, A. niger, C. cladosporioides and C. sphaerospermum.Also, 25 species and one species variety belong to 17 genera from leaf surface of broad bean on dichloranchloram-phenicol-malt extract agar (DCMA) at 28°C and the most common species were A. alternata, C. cladosporioides and C. sphaerospermum.All fungal species recovered from leaf surface of broad bean on the two types of media were previously isolated but with different inciden-ces from leaf surface of several plants growing or cultiva-ted in many parts of the world (Abdel-Hafez et al., 1990, 1995;Abdel-Sater, 1993;El-Kholl et al., 1994;Murace and Cellitin, 2005;Moharram et al., 2010;Saleem et al., 2010Saleem et al., , 2012)).
Forty species and 1 variety representing 14 genera were collected from the air above broad bean field on plates of DCMA (14 genera and 29 species) and DRBC (10 and 32+1var.) at 28°C (Table 2).The monthly counts of fungi on DCMA and DRBC in the atmosphere over broad bean field irregularly fluctuated and widely varied between 50-12 and 100-200 colonies/6 plates in 2 expo-sures of 10 min each giving peaks during February and February, respectively (Figure 3).The most common fun-gi on the two types of media were: A. citri, Alternaria petroselini, Alternaria pluriseptata, A. flavus, A. melleus, A. niger, C. sphaerospermum, Curvularia richardiae, D. neergaardii and P. chrysogenum.The monthly counts of these fungi were widely varied and irregularly fluctuated giving maxima during various months (Figure 3).Some species were prevalent on one type of media such as: Emericella nidulans and Mucor circinelloides on DRBC.Abdel-Hafez et al. (1990) 1995) isolated fifty species and 3 varieties representing 26 genera were collected from the air above sugarcane field and the most common fungi were: A. alternata, A. flavus, A. niger, A. terreus, P. chrysogenum and P. oxalicum.Patel (2008) carried out aeromyco-logical studies on tomato (Lycopersicon esculentum Mill.and Solanum melongana L.) fields at Nashik (M.S.) during two seasons.Spores of Cladosporium, Alternaria, Curvularia, Helminthosporium, Aspergillus, Penicillium, Fusarium and Periconia were found in maximum percentage in the total air-spora.More spore catch was found in the month of January, March and August, September.During the period of investigations, 66 types of fungal spores were observed.The maximum numbers of spores were found in the second season.Low temperature, high relative humidity and moderate and alternate spell of rain show effect on release and dissemination of air borne fungal spores.Chavan (2012) studied the occurrence of Ascomycetes fungal spores over a paddy field and observed that the spores belonging to groups Deuteromcotina contributed 66.61%, Basidiomycotina 8.89%, Ascomycotina 24.56% and other types 0.35% of the total airspora.The most prevalent genera were Curvularia, Fusarium, Helminthosporium, Phoma, Nigrospora, Alternaria and Cladosporium.Several of the above species were also frequently isolated from the air of some Egyptian localities (Abdel-Hafez, 1989;Abdel-Sater, 1990;Abdel-Hafez et al., 1990, 1993, 1995;Patel, 2008;Chavan, 2012).
Thirty-one species and 1 variety belonging to 12 genera were collected from soil of broad bean field on plates of DCMA (9 genera and 24 species + 1 var.) and DRBC (11 and 24 + 1 var.) at 28°C (Table 3).The monthly counts of fungi on DCMA and DRBC in soil of broad bean field irregularly fluctuated and giving peaks during different months Table 1.Total counts of phyllosphere (per g fresh leaves) and phylloplane (120 leaf segments) fungi, number of cases of isolation (NCI), occurrence remarks (OR) and relative importance value (RIV) of the fungal genera and species on dicholran chloramphenicol malt extract agar (DCMA) and dichloran Rosebengal chloramphenicol agar (DRBC) at 28°C.*OR = Occurrence remarks: H = high occurrence from 6-10 cases, M = moderate occurrence from 3-5cases, L = low occurrence 2 cases and R = rare occurrence 1 case.

Cellulolytic activities of some fungal isolates
Nine species (most common species) belonging to 5 genera were screened for their abilities to produce C 1 and C x enzymes on solid media and proved to be active to utilize cellulose but with different degrees (Table 4).Five isolates (55.6% of total isolates) showed high activity in production of C 1 enzyme only and these were: A. citri, A. petroselini, A. flavus, A. fumigatus and P. chrysogenum.
On the other hand, one fungal isolate exhibited high activity on production of C X enzyme only and this was A. flavus.Three and two isolates (33.0 and 22.2% of total isolates) were found to be of moderate production of C 1 and C X enzymes, respectively, while one and six isolates (11.1 and 66.6% of total isolates) were of weak cellulolytic acti-vity.Most of the above fungal isolates were reported as cellulase producers, but with variable capabilities by several workers (Abraha and Gashe, 1992;Abdel-Hafez et al., 1995;Moharram et al., 1995Moharram et al., , 2004;;Berlin et al.,2005;Rashid et al., 2009;Sohila et al., 2009;Saleem et al. 2010Saleem et al. ,2013)).
A. flavus was the highest fungi in the production of endo and exo-ß-1,4glucanase in this investigation so it was chosen for further studies to achieve the most favorable environmental and nutritional conditions for C 1 and C x enzymes production.
Maximum production of exo and endo--1,4-glucanase by A. flavus was obtained after 8 and 6 days of incubition at 30°C with culture media containing glucose and cellulose as a carbon sources and sodium nitrate as nitrogen source and the culture medium was initially adjusted to pH 6 (Figures 5 and 6).These findings are almost in agreement with those reported by El-Said et al. (2006).They found that F. oxysporum was the highest fungi in producing endo--1,4-glucanses among the 70 tested isolates obtained from 60 samples leaves of Vicia faba.
Reducing sugar (µg/ml) Maximum production of endo--1,4-glucanses by F. oxysporum was achieved after 8 days of incubation at 30°C with culture medium containing carboxymethyl cellulose as carbon and peptone as nitrogen source and initially adjusted to pH 6. Immanuel et al. (2007) studied the effect of environmental factors on production of cellulase enzyme by A. fumigatus and A. niger.They reported that the optimum pH for cellulase production was 6 to 7 and optimum temperature was about 40°C.El Said and Saleem (2008) found that maximum production of endoß-1,4 glucanase by Cheatomium globosum was achieved 6 days after incubation at 30°C with incorporation of maltose as carbon source and NH 4 NO 3 as nitrogen source in the culture medium which is initially adjusted to pH 6.Recently Saleem et al. (2013) found that maximum pro-duction of exo-and endo-ß-1,4 glucanase by Mucor circinelloides and A. flavus was achieved 6 days after incubation at 30°C with incorporation of fructose or sucrose as a sole carbon source and potassium nitrate or sodium nitrate as a sole nitrogen source, respectively in the basal medium initially adjusted to pH 6.

Figure 1 .
Figure 1.Monthly counts (per g fresh leaves) of common phyllosphere fungi of Vicia faba on DCMA and DRBC at 28°C.

Figure 2 .
Figure 2. Monthly counts (per 120 leaf segments) of common phylloplane fungi of Vicia faba on DCMA and DRBC at 28°C.

Figure 4 .
Figure 4. Monthly counts (per 1 g soil) of common soil fungi on DCMA and DRBC at 28°C.
observed that the most preva-lent fungi in the air over lentil field were: A. alternata, A. flavus, A.

Table 2 .
Total counts (TC, calculated per 30 plates in 10 exposures of 15min), number of cases of isolation (NCI, out of 10) and occurrence remarks (OR) of various fungal genera and species recovered from air of Vicia faba field on dicholran chloramphenicol malt extract agar (DCMA) and dichloran Rosebengal chloramphenicol agar (DRBC) at 28°C.

Table 3 .
Total counts (TC, calculated per 1 g soil), number of cases of isolation (NCI, out of 10) and occurrence remarks (OR) of various fungal genera and species recovered from soil of Vicia faba field on dicholran chloramphenicol malt extract agar (DCMA) and dichloran Rose bengal chloramphenicol agar (DRBC) at 28°C.

Table 4 .
Degree of cellulolytic activities (calculated as diameter of clear zone in mm) of the most common fungal isolates.