Endophytic fungi of some medicinal plants in Egypt

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INTRODUCTION
Medicinal plants are plants that provide people with medicines to prevent disease, maintain health or cure ailment''.The World Health Organization (WHO) defines a medicinal plant as any plant, which in one or more of its organs، contains substances that can be used for therapeutic purposes or which are precursors for chemo-pharmaceutical semi-synthesis (Balicket al., 1996).An ethno-biological survey revealed that about 8,000 species of medicinal plants are used as food supplements, medicines, biocides and other phytochemicals.
The medicinal plants in Egypt represent a new promising resource as there is a relatively high representation of medicinal species in the native flora, that may induce the accumulation of a high concentration of secondary metabolites and there is a large market demand with higher economic value than other crops.All plant organs such as roots, rhizomes, flowers, leaves, fruits, seeds, as well as oils were used for medicaments in the form of powders, pills, suppositories, creams, pastes and ointments (Haggag, 1997).Endophytes refer to the microorganisms (mostly fungi and bacteria) colonizing the intercellular and intracellular regions of healthy plant tissues at a particular time, whose presence is unobtrusive and asymptomatic (Strobel, 2003and Schulz andBoyle, 2006).Endophytes form a symbiotic relationship with their plant host.It is believed that in many cases the microbes function as the biological defense for the plant against foreign phytopathogens.
The protection mechanism of the endophytes is exerted directly, by releasing metabolites to attack any antagonists or lyse affected cells and indirectly, by either inducing host defense mechanisms or promoting growth (Kloepper and Ryu, 2006).
Medicinal plants which are known to be used since centuries as an alternative source of medicine, are a valuable source for bioprospecting endophytes.Many studies recently found that endophytic fungi have the ability to protect the host from diseases and limit the damage caused by pathogenic microorganisms (Phongpaichit et al., 2006;Ganley et al., 2008;Sun andGuo, 2012 andVieira et al., 2012).Moreover, the production of chemical compounds by medicinal plants is believed to be associated with the endophytic fungi.
Wheat (Triticum aestivum L. em Thell.) is an important and a strategic cereal crop for the most of world's populations.It is the most staple food of about two billion people (36% of the world population).Worldwide, wheat provides nearly 55% of the carbohydrates and 20% of the food calories consumed globally (Breiman and Graur, 1995).
Wheat is mainly cultivated in Egypt, as it is one of world's leading crops and can affect the economy.Diseases are a major cause of yield loss in winter wheat.Wheatcrop is subjected to a number of diseases, which are responsible for reducing its overall production to a greatextent (Haggag, 2013).
Fusarium foot and root rot (FRR) represent major devastating disease of wheat worldwide.Generally, affected wheat plants with root rot disease may be stunted or less vigorous than healthy one.Plants may be yellow, wilted and die prematurely.Root system may be poor with roots and crown tissues discolored and deteriorated (Smiley andPatterson, 1996 andSmiley et al., 2005).
The present study was conducted to investigate the diversity of fungal endophytes of some medicinal plants grown in Egypt and their antagonistic activity against Fusarium oxsporum the most common pathogen of root rot of wheat plants.

Isolation of endophytic fungi The plant materials
Eight medicinal plants (basil, green onion, green pepper, mint, roselle, watercress, white radish and tagetes) were selected for the present investigation and collected from their natural habitats from El-Qalyubia (El-Qanatir el-Khairiya) and El-Fayoum governorates cultivated fields between April-October, 2012.The plant samples (healthy leaves, stems and roots)were placed in a plastic bag, stored in a refrigerator and then used for isolation of endophytic microorganisms within 72 h after sampling (Kim et al., 2007).

Surface sterilization of the plant materials
The collected plant material was first surface sterilized according to the following method with few modifications.Plant material was first cleaned by washing several times under running tap water to remove soil particles and adhered debris and then left to air-dried.Surface sterilization was performed by sequentially rinsing the plant material with 96% ethanol for 0.5 -1 minute, then with 2 -2.5% sodium hypochlorite solution for 1 -4 minute followed by 96% ethanol for 0.5 minute and finally with sterile distilled water for several times to remove the surface sterilization agents (Oberholzer-Tschütscher 1982;Sieber et al., 1988 andCrous et al., 1995).Finally, plant materials were then dried in between folds of sterile filter papers.

The isolation methods
After surface sterilization, two methods were used for isolation of endophytic microorganism.The first method, after proper drying, the surface-sterilized plant materials were cut into small pieces long 1 cm.(Kim et al., 2007) then three pieces per each plant tissue were placed over the surface of three isolation media (i) Malt yeast extract agar (Schulz et al., 1995) (ii) Potato dextrose agar (Rotem, 1994) and (iii) Tryptic soy Agar (Leavitt et al., 1955).The sterilized tissue pieces were pressed on to the surface of media to ensure the endophytic isolations only from internal tissues of the plant pieces.In the second method, the surface-sterilized plant materials were cut into small pieces then cut longitudinally with a sterile scalpel as the inner tissues were excised and then laid on plates of the isolation media mentioned before (Hung and Annapurna, 2004).
All the plates were analyzed in three replicates, incubated at 28 0 C for a about 2 weeks to promote the growth of endophytes and regularly observed.Total colony count was determined and any fungal growth was isolated, purified regularly, transferred onto slants of the isolation medium, accessioned by the plant and plant tissue, and incubated at 28 0 C when they appeared then stored at 4 0 C till further use.

Species richness
Expresses the total number of each fungal species isolated from each plant.

Relative frequency (%)
Relative frequency (RF) of each isolate was calculated as the number of the isolates of one species divided by the total number of isolates and express as percentage (Photita et al., 2001).

Tissues specifity
Expresses the total number of each fungal genus isolated from each organ tissue of all the medicinal plants.

Identification of endophytic fungi
All endophytic fungi isolated in this study were identified to the genus and species level depending on morphological methods.Identification of the genus Aspergillus was carried out using Raperand Fennelli, (1977) reference.Identification of fungal isolates belonging to Hyphomycetes, dematiaceous Hyphomycetes, general Mucorales, the genus Fusarium and the genus Penicillium was carried out in MYCOLOGICAL CENTER, Faculty of Science, Assiut University (AUMC).

Antagonistic activities of endophytic fungi against Fusarium oxysporum
Endophytic fungal isolates were screened for their in vitro antagonistic activity against Fusarium oxysporum by dual culture technique, which isolated from naturally infected wheat plants grown in Egyptfrom El-Qalyubia (El-Qanatir el-Khairiya) governorate.The endophytic isolates were streaked-inoculated to one side of petri-dishes containing potato dextrose agar medium (PDA) (El-Tarabily et al., 1997;Hassanein, 2010 andHassanein et al., 2010).The plates were then incubated at 28 0 C for 3 days to allow the production and diffusion of metabolites into the agar.Fusarium oxysporum(isolated from diseased wheat plants) agar disk from seven days old culture was placed into the opposite side of the inoculated streak plates.Pathogen mycelium disc was also placed on uninoculated potato dextrose agar separately as control.All the plates were incubated at 28 • C for 6 days and the plates were examined for inhibition of pathogens growth after 3, 4, 5 and 6 days.The reduction percentage in radial growth of the pathogen was calculated with each endophyte as described by Nouari et al. (2013) as follow: Percentage of inhibition (I %) = A -B / A X 100 Where : A -Radius of the pathogen in the control plate, B -Radius of pathogen in the dual culture plate.

RESULTS The study plants
During the study, endophytic fungi have been isolated from 8 different medicinal plants belonging to 6 families (Table 1).

Isolation of endophytic fungi
A total of 911 endophytic fungal colony forming units (CFU) were obtained from 8 medicinal plants during the study.

Identification of endophytic fungi from different plants
In the present study, 14 genera were obtained and identified as Acremonium, Alternaria, Aspergillus, Cladosporium, Cochliobolus, Epicoccum, Fusarium, Geosmithia, Macrophomina, Paecilomyces, Penicillium, Rhizopus, Scopulariopsis and Trichoderma (Table 2).Some fungal isolates could not be identified to the genus level.These cultures that not form spores were grouped as mycelia sterilia and divided into different morphological species according to their cultural characteristics.In this study, one morphospecies of mycelia sterilia were recorded and identified as dark septate sterile mycelium isolates.

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Thirty endophytic fungal isolates belonging to 14 genera and one mycelia sterilia group (4 isolates) were identified from 8 different medicinal plants.
Among the identified fungi, Aspergillus yielded the greatest fungal diversity with 7 different species being isolated from different plants tissues followed by Acremonium and Penicillium had 4 species for each of them.Mycelia sterilia group was represented by 4 isolates of dark septate sterile mycelium isolates obtained from 4 different plants (basil, mint, tagetes and watercress).One species was isolated and identified from the remaining 11 genera (Table 2).

Species richness of the endophytic fungi in different medicinal plants
The distribution of endophytic fungal species was investigated in the selected medicinal plants and their occurrence in different plant organs tissues also were investigated (Table 3).The diversity and abundance of the species varied according to the plant and the organs tissues tested as shown in Table (3).
Table (3) indicated that, the highest species count was observed with roselle plant (11 species) with the highest in roots and stems tissues (7 and 6 species, respectively) followed by tagetes plant (10 species) in stems, roots and leaves (7, 5 and 5 species, respectively).On the other hand, the same species count was recorded with basil, mint and watercress plants (7 species of each).

Relative frequencies of theendophytic fungi isolated from each plant
The endophytic fungal species isolated from the medicinal plants were classified into 14 genera and one mycelia sterilia group (Table 4).
Aspergillus species had the highest relative frequency values (66.63 %) and considered the most dominant genus isolated followed by Acremonium, dark septate sterile mycelium isolates, Penicillium, Alternaria and Paecilomyces which considered as moderate in their dominance and gave relative frequency values of 7.46 %, 5.27 %, 5.16 %, 4.61 % and 4.17 %, respectively.Finally, Scopulariopsis and Fusarium species gave the lowest relative frequency values (2.74 % and 1.54 %, respectively).
All the medicinal plants were found to harbor various endophytic fungi.Among the 8 medicinal plants tagetes, basil and roselle yielded the greatest fungal diversity with 8, 6 and 6, respectively different genera being isolated from different tissues.
The highest number of fungal genera (8) was obtained from tagetes plant as they belong to 7 different genera and one morphospecies of mycelia sterilia.Among these fungal genera, Aspergillus was the most frequent (40.70 %) followed by Alternaria (20.93 %) and Dark septate sterile mycelium isolate (9.30 %).
The common endophytic fungi had a wide distribution in the medicinal plants with a high species abundance, for example Aspergillus were found in all the plants except green pepper which had a relative high occurrence in 7 of these plants (RF= 66.63 %), especially in watercress and white radish (RF= 84.80 % and 83.97 %,respectively).The genera Alternaria and Penicillium were isolated from 5 medicinal plants with a major occurrence in tagetes (RF= 20.93 %) and watercress (RF= 10.40 %) respectively, followed by dark septate sterile mycelium isolates and isolated from 4 plants with a major occurrence in basil (RF= 16.11 %).The genus Acremonium was obtained from 3 plants with a major occurrence in green onion and basil plants (RF= 23.73 % and 20.56 %, respectively) (Table 4).

Tissues specificity of the endophytic fungi
The occurrence of the endophytic fungi in roots, stems and leaves was investigated for plants under investigation.
Acremonium, Alternaria, Aspergillus, dark septate sterile mycelium isolates, Paecilomyces and Penicillium were the most frequent fungiand had a special presence in all tissues of these plants (Table 5).Cladosporium and Scopulariopsis could not found in roots of any of 8 medicinal plants while Fusarium could not found in stems of these plants.Cochliobolus, Epicoccum and Macrophomina colonized only medicinal plants roots while Geosmithia, Rhizopus and Trichoderma colonized their stems only.
Some endophytic fungi could colonize tissue more than the others.For example, Aspergillus, Paecilomyces and Penicillium were obtained from medicinal plants roots than from stems and leaves while, Acremonium species were detected in the stems than in the roots and leaves.Finally, Alternaria and dark septate sterile mycelium isolates were found in the leaves than roots and stems.

In vitro antagonistic activity of the endophytic fungi against Fusarium oxysporum
The in vitro studies showed that among the thirty endophytic fungal species tested, only 7 produced inhibition of radial growth of Fusarium oxysporum under the conditions of this study compared to untreated control as shown in Table (6) and Figure (1) and there was a significant difference in the percentage of inhibition of pathogen radial growth by the tested endophytic antagonists.Alternaria alternata and Cochliobolus lunatus inhibited the growth of F. oxysporum and gave a percentage of inhibition of 71.43 % for both, followed by Fusarium oxysporum and Cladosporium cladosporioides with percentage of 68.57% and 60%, respectively.On the other hand, Aspergillus punicus, Epicoccum purpurascens and Aspergillus versicolor gave the lowest percentage of inhibition of F. oxysporum (54.28 %, 48.57 % and 40 %, respectively).

DISCUSSION
Plants-associated microorganisms are known to be beneficial, neutral or pathogenic, but others are still poorly understood.Endophytes which are free living organisms able to colonize plant tissues through stomata, wounds or cracks, when a chance is available (Hardoim et al., 2008 andRodriguez et al., 2009).This relationship is complex and involve many factors.Some studies have shown that endophyte colonization can be stimulated by host plant species, geographic location, seasonality and different tissues of the same plant (Porras-Alfaro and Bayman, 2011).
From our results, it was found that some fungal pathogens are commonly isolated as endophytes and several researches have reported that many of these species can be pathogenic for some hosts and nonpathogenic for others (Malcolm et al., 2013) that depending on environmental conditions and on the host (Sieber, 2007 andMalcolm et al., 2013).
The present study also found that fungal endophytes showed significant differences in their presence, absence and abundance in the study plants.Some plants harbored more endophytic fungi than others (as roselle and tagetes).The endophytic fungi had different relative frequencies in different host plants as Aspergillus not only found in most of plantsbut also had the highest relative frequencies within each of them.On the other hand, some other endophytic fungi were detected in only single plant (Geosmithia from roselle and Trichoderma from tagetes).These results were consistent with the previous reports of Arnold et al. (2001) and Bettucci et al. (2004).
The endophytic fungi have been found in different tissues of a single plant which is a reflection of tissue specificity (Collado et al., 2001;Frohlich et al., 2000 andGanley andNewcombe, 2006).The current results showed that there is a difference between 1 2 3 4 various plants tissues and that might be due to their degree of exposure to air, sun, wind, rain, moisture and aeration conditions and availability of nutrients (Andreote et al., 2014).
Although the production of wheat has increased but several diseases have greatly affected its yield and quality.Many Fusarium pathogens cause the same essentially symptoms on different crops such as cortical decay of roots, root rot, wilting, yellowing and premature death on the diseased plants (Summerell et al., 2001 andSaremi, 2005).Several Fusarium species are dangerous as they known to produce mycotoxins as deoxynivalenol (DON) and nivalenol (NIV) (Bewick et al., 2008) and these toxins can also transport from the roots to the other upper plant parts (Covarelli et al., 2012).
Fusarium oxysporumis the most important Fusarium species isolated from diseased wheat plants, Mehta (2014) reported that both F. oxysporum and F. solaniare secondary pathogens of wheat plants.Also, Saremi et al., (2011) showed that F. oxysporum can cause vascular and damping-off diseases on different crops.
Non-pathogenic endophytic microorganisms have a specific interest as biological control agents (Alabouvette et al., 2006 andBerg, 2009).They are well fully adapted to their host plant as they colonize an ecological niche similar to that of plant pathogens (Berg et al., 2005) and can produce secondary metabolites which required for their survival against host defense responses (Strobel andDaisy, 2003 andBrader et al., 2014).
In the present study, all the endophytic fungi isolated from medicinal plants were examined for their antagonistic activity against Fusarium oxysporum.Out of these endophytes, only 7 endophytic fungi showed a significant difference in the pathogen radial growth inhibition.
Mycelial growth inhibition of the target pathogen in dual culture method revealed that the highest suppression rate was with a wider inhibition zone.A microbial biological control agent may be showed several mechanisms against plant pathogens during their antagonistic activity.There are about 3 primary mechanisms by which endophytes can promote host resistance to pathogens: by the production of antimicrobial substances toxic to pathogens, by the competition for the same ecological niche and nutrients used by the pathogen and by the production of enzymes that attack the cell wall components of the pathogens, stimulating pathogen cell death (Mandyam and Jumpponen, 2005;Ganley et al., 2008 andShittu et al., 2009).Abdel-Sater (2001) showed that the culture filtrates of Alternaria alternate and Cochliobolus lunatus caused high inhibition of Pleospora herbarum that caused spot diseases on onions leaves.Hellwig et al. (2002) demonstrated that antimicrobial compounds like altersetin from Alternaria sp., showed significant antibacterial activities.
The antagonistic effect expressed by the Cladosporium cladosporioides in dual culture method also reported by Bisht et al. (2016) who isolated Cladosporium cladosporioides from of Cupressus torulosa which were found to be shown strong antagonistic activity against phytopathogens Macrophomina phaseolina and Fusarium solani.
The dual culture technique reveals that 2 Aspergillus species, Aspergillus punicus and Aspergillus versicolor have an antagonist effect against Fusarium oxysporum.Previous reports by Bosah et al. (2010) showed that Aspergillus species were identified as effective antagonists against the known destructive pathogens to most agricultural crop plants.
The successful in pathogens suppressions in dual culture methods provide useful information on the effective use of these isolates as biological control agents against wheat diseases.Therefore, they may be used as biofertilizer and biocontrol agents for crops production in agricultural environment.

Table 1 :
List of medicinal plants studied with their English, latin and family names

Table 2 :
List of endophytic fungal genera speciesand mycelia sterilia isolated from medicinal plants

Table 3 :
The distribution and count (CFU) of the endophytic fungal species in different organs tissues of medicinal plants

Table 4 :
Relative frequencies of the endophytic fungi isolated from medicinal plants

Table 5 :
The endophytic fungi in different organs tissues of medicinal plants

Table 6 :
In vitro antagonistic activity of the endophytic fungi against Fusarium oxysporum Radius of the pathogen in the control plate, B -Radius of pathogen in the dual culture plate.