epiphytic or endophytic fungi spend a part of their lifecycle outside or inside leaf tissues , with no negative impact on the host ( 1 ) .
endophytic fungi , which have been reported in numerous plant species , are important components of the forest community and significantly contribute to the diversity of natural ecosystems ( 2 ) .
it has also been shown that some fungal endophytes can produce various bioactive chemicals and have potential applications in biocontrol and resistance ( 3 ) .
they play important roles in recycling nutrients in natural ecosystems ( 4 , 5 ) .
medicinal plants represent an important health and economic component of biodiversity ; therefore , it is essential to make a complete inventory of the medicinal components of each country s flora , in order to facilitate conservation and sustainable use ( 6 ) . according to mossa et al .
( 7 ) , the kingdom of saudi arabia possesses a wide range of flora , consisting of a large number of medicinal herbs , shrubs , and trees ; folk medicine has been practiced since time immemorial .
previous studies have reported the isolation and identification of endophytic fungi and bacteria from medicinal plants such as cressa cretica , achillea fragrantissima , and artemisia species ( 8 - 10 ) .
a few other studies have evaluated the antimicrobial activity of the fungal endophytes against plant pathogenic fungi such as neurospora sp . (
11 ) , magnaporthe grisea , corticium sasakii , botrytis cinerea , phytophthora infestans , puccinia recondita , blumeria graminis ( 12 ) , alternaria alternata , fusarium oxysporum , b. cinerea , and pythium ultimum ( 13 ) .
in the present investigation , we evaluated the diversity of endophytic fungi colonizing seven species of medicinal plants ( alhagi graecorum , c. cretica , citrullus colocynthis , tamarix nilotica , a. fragrantissima , artemisia sieberi , and neurospora retusa ) , collected from salt marshes of the al - gouf governorate in north saudi arabia . the molecular characterization and antimicrobial activity of these isolates
a total of 70 stem and roots samples of seven disease - free medicinal plants , namely a. graecorum , c. cretica , c. colocynthis , t. nilotica , a. fragrantissima , a. sieberi , and n. retusa , were randomly collected from the salt marshes of al - gouf governorate , kingdom of saudi arabia , during the period from july to september 2013 .
the stem and roots samples were cut into three segments measuring 0.5 to 1 cm .
( 14 ) , immersed in 70% ethanol for five seconds , in 4% sodium hypochlorite for 90 seconds , and then rinsed in sterile distilled water for 10 seconds .
the sterilized segments were placed in petri dishes containing pda medium ( himedia , mumbai , india ) ; these were sealed with parafilm , and incubated at 26  1c in 12-hours light/12-hours dark cycles for two to four weeks .
the petri dishes were monitored daily to check the growth of fungal endophytic colonies from the segments .
fungi growing out of the plant segments were isolated and identified using morphological characteristics , according to the established procedure ( 15 ) .  
the colonization frequency ( cf ) and the percentage of the dominant endophytic fungi were calculated ( 13 ) .
three plant pathogenic fungi obtained from the college of agriculture , king saud university ( f. oxysporum , f. solani , and alternaria ) and five human pathogenic bacteria obtained from the military hospital in riyadh city ( enterococcus faecalis , escherichia coli , staphylococcus aureus , pseudomonas aeruginosa , and klebsiella pneumoniae ) were used as target fungal and bacterial pathogens in this study .
a dual culture technique was adopted to investigate the antimicrobial activity of fungal endophytes isolated from a. graecorum , c. cretica , c. colocynthis , t. nilotica , a. fragrantissima , a. sieberi , and n. retusa against the selected fungal and bacterial pathogens . the conidial suspension and assay plates of plant pathogenic fungi
the inocula and assay plates for bacterial strains were prepared as described by pelaez et al .
endophytic fungi were grown on pda plates . five - day - old discs ( 5 mm in diameter ) of endophytes were placed on three points in petri plates containing pda medium . the target pathogens were inoculated in the center of the pda plates .
after incubation of the fungi at 25c for 10 days , and of the bacteria at 37c for 48 hours , the diameters ( in mm ) of the inhibition zones were measured .
the level of inhibition was calculated by subtracting the distance ( mm ) of fungal and bacterial growth in the direction of an antagonist colony from the fungal growth radius .
the widths of inhibition zones between the pathogen and the endophytes were grouped as follows : 10 mm ( + + + , strong inhibition ) , 2 to 10 mm ( + + , moderate inhibition ) , 1 mm ( + , weak inhibition ) , and less than 1 mm ( - , no activity determined ) ( 18 ) .
two microliters of potato dextrose broth ( pdb ) ( himedia , mumbai , india ) were poured into pda tubes and vortexed to disperse the spores , and the spores - pdb mixtures were poured into flasks containing 100 ml pdb .
the mycelium was harvested by filtration , frozen at -80c for 30 minutes , lyophilized , and stored at -80c .
the mycelium was ground in liquid nitrogen with a sterile mortar to obtain mycelium powder .
dna was extracted from 20 mg of mycelium powder using a dneasy plant mini kit .
the dna quantity and quality were checked by electrophoresis on a 0.8% agarose gel , and visualized with ethidium bromide under uv trans - illumination ( 13 ) .
the its region of the ribosomal dna was amplified by pcr with the primers its1-f ( 5-cttggtcatttagaggaagtaa-3 ) and its4 ( 5-tcctccgcttattgatatgc-3 ) ( 19 ) .
pcr amplifications were carried out in a final volume of 50 l , containing 2 l of dna , 0.5 mm of each primer , 150 mm of dntp , 1 u of taq dna polymerase ( promega ) , and pcr reaction buffer .
amplification was carried out in a thermal cycler with an initial denaturation of three minutes at 94c , followed by 35 cycles of one minute at 94c , one minute at 50c , one minute at 72c , and a final extension of 10 minutes at 72c .
the amplified products were checked by electrophoresis on a 1% agarose gel , and visualized with ethidium bromide under uv trans - illumination .
the pcr products were purified using an exosapit kit ( usb corporation , amersham place , uk , under license from ge healthcare ) , based on the manufacturer s instructions .
the purified products were sequenced in an automated dna sequencer ( abi prism 3700 ) using the bigdye deoxy terminator cycle - sequencing kit ( applied biosystems , darmstadt , germany ) , following the manufacturer s instructions .
sequences obtained in this study were compared with the previously deposited sequences in the genbank database , using blast , on the ncbi website ( http://www .
treecon ( 21 ) for windows ( version 1.3b , 1998 ) was used to construct a neighbor - joining tree using the jukes - cantor model .
a total of 70 stem and roots samples of seven disease - free medicinal plants , namely a. graecorum , c. cretica , c. colocynthis , t. nilotica , a. fragrantissima , a. sieberi , and n. retusa , were randomly collected from the salt marshes of al - gouf governorate , kingdom of saudi arabia , during the period from july to september 2013 .
the stem and roots samples were cut into three segments measuring 0.5 to 1 cm .
( 14 ) , immersed in 70% ethanol for five seconds , in 4% sodium hypochlorite for 90 seconds , and then rinsed in sterile distilled water for 10 seconds .
the sterilized segments were placed in petri dishes containing pda medium ( himedia , mumbai , india ) ; these were sealed with parafilm , and incubated at 26  1c in 12-hours light/12-hours dark cycles for two to four weeks .
the petri dishes were monitored daily to check the growth of fungal endophytic colonies from the segments .
fungi growing out of the plant segments were isolated and identified using morphological characteristics , according to the established procedure ( 15 ) .  
the colonization frequency ( cf ) and the percentage of the dominant endophytic fungi were calculated ( 13 ) .
three plant pathogenic fungi obtained from the college of agriculture , king saud university ( f. oxysporum , f. solani , and alternaria ) and five human pathogenic bacteria obtained from the military hospital in riyadh city ( enterococcus faecalis , escherichia coli , staphylococcus aureus , pseudomonas aeruginosa , and klebsiella pneumoniae ) were used as target fungal and bacterial pathogens in this study .
a dual culture technique was adopted to investigate the antimicrobial activity of fungal endophytes isolated from a. graecorum , c. cretica , c. colocynthis , t. nilotica , a. fragrantissima , a. sieberi , and n. retusa against the selected fungal and bacterial pathogens . the conidial suspension and assay plates of plant pathogenic fungi
the inocula and assay plates for bacterial strains were prepared as described by pelaez et al .
five - day - old discs ( 5 mm in diameter ) of endophytes were placed on three points in petri plates containing pda medium .
after incubation of the fungi at 25c for 10 days , and of the bacteria at 37c for 48 hours , the diameters ( in mm ) of the inhibition zones were measured .
the level of inhibition was calculated by subtracting the distance ( mm ) of fungal and bacterial growth in the direction of an antagonist colony from the fungal growth radius .
the widths of inhibition zones between the pathogen and the endophytes were grouped as follows : 10 mm ( + + + , strong inhibition ) , 2 to 10 mm ( + + , moderate inhibition ) , 1 mm ( + , weak inhibition ) , and less than 1 mm ( - , no activity determined ) ( 18 ) .
two microliters of potato dextrose broth ( pdb ) ( himedia , mumbai , india ) were poured into pda tubes and vortexed to disperse the spores , and the spores - pdb mixtures were poured into flasks containing 100 ml pdb .
the mycelium was harvested by filtration , frozen at -80c for 30 minutes , lyophilized , and stored at -80c .
the mycelium was ground in liquid nitrogen with a sterile mortar to obtain mycelium powder .
dna was extracted from 20 mg of mycelium powder using a dneasy plant mini kit .
the dna quantity and quality were checked by electrophoresis on a 0.8% agarose gel , and visualized with ethidium bromide under uv trans - illumination ( 13 ) .
the its region of the ribosomal dna was amplified by pcr with the primers its1-f ( 5-cttggtcatttagaggaagtaa-3 ) and its4 ( 5-tcctccgcttattgatatgc-3 ) ( 19 ) .
pcr amplifications were carried out in a final volume of 50 l , containing 2 l of dna , 0.5 mm of each primer , 150 mm of dntp , 1 u of taq dna polymerase ( promega ) , and pcr reaction buffer .
amplification was carried out in a thermal cycler with an initial denaturation of three minutes at 94c , followed by 35 cycles of one minute at 94c , one minute at 50c , one minute at 72c , and a final extension of 10 minutes at 72c .
the amplified products were checked by electrophoresis on a 1% agarose gel , and visualized with ethidium bromide under uv trans - illumination .
the pcr products were purified using an exosapit kit ( usb corporation , amersham place , uk , under license from ge healthcare ) , based on the manufacturer s instructions .
the purified products were sequenced in an automated dna sequencer ( abi prism 3700 ) using the bigdye deoxy terminator cycle - sequencing kit ( applied biosystems , darmstadt , germany ) , following the manufacturer s instructions .
sequences obtained in this study were compared with the previously deposited sequences in the genbank database , using blast , on the ncbi website ( http://www .
treecon ( 21 ) for windows ( version 1.3b , 1998 ) was used to construct a neighbor - joining tree using the jukes - cantor model .
this is probably the rst report to describe the endophytic fungi that colonize the medicinal plants a. graecorum , c. cretica , c. colocynthis , and n. retusa in the northern desert of saudi arabia .
a total of 70 stem and root samples were derived from seven medicinal plants , and were screened for the presence of endophytic fungi .
275 isolates belonging to 23 species and 14 genera were obtained from stem and root segments of a. graecorum , c. cretica , c. colocynthis , t. nilotica , a. fragrantissima , a. sieberi , and n. retusa .
the isolates were identified as follows : one species of alternaria from 23 isolates , seven species of aspergillus from 68 isolates , one species of chaetomium from two isolates , one species of drechslera from two isolates , one species of emericella from one isolate , one species of eupenicillium from three isolates , four species of fusarium from 14 isolates , one species of gibberella from four isolates , one species of monilia from one isolate , one species of mucor from four isolates , one species of mycelia from 65 isolates , one species of penicillium from 81 isolates , one species of scopulariopsis from three isolates , and one species of ulocladium from four isolates .
the most commonly isolated species were p. chrysogenum with an overall colonization frequency of 98.57% , followed by mycelia sterilia with a colonization frequency of 92.86% ( table 2 ) .
a total of 263 endophytic fungal isolates were obtained from 70 stem and root fragments .
thirty five isolates were selected based on their antimicrobial activity , and identified based on the sequencing of the its region of rdna ( table 3 ) .
the sequence results corroborated with the morphological identification of the isolated fungal   endophytes ( figure 1 ) .
most of the isolates belonged to the taxa ascomycota ( 58.54% ) , deuteromycota ( 39.27% ) , or zygomycota ( 1.45% ) .
t. nilotica had the highest endophytic diversity ( relative frequency 27.27% ) , followed by c. cretica ( relative frequency 19.27% ) , and c. colocynthis had the lowest endophytic diversity ( relative frequency 5.82% ) among the seven medicinal plants used in this study .  
the tree was constructed by neighbor - joining algorithm , using the maximum composite likelihood model .
all endophytic fungi exhibited significant inhibition against a wide range of plant pathogenic fungi and human pathogenic bacteria .
the isolates , namely p. chrysogenum ( tuef2 ) , e. crustaceum ( tuef10 ) , a. sydowii ( tuef14 ) , f. brachygibbosum ( tuef26 ) , p. chrysogenum ( tuef30 ) , p. chrysogenum ( tuef31 ) , and e. crustaceum ( tuef32 ) showed strong inhibition towards plant pathogenic fungi .  
thirty one isolates in this work showed promising growth - inhibitory activity against at least one of the test human pathogenic bacteria , but no endophyte had antimicrobial activity against all five pathogenic microbes .
the numbers of fungal isolates displaying antimicrobial activity against s. aureus , e. faecalis , e. coli , p. aeruginosa , and k. pneumonia were 10 , 13 , 6 , 18 , and 19 , respectively .
the isolates p. chrysogenum ( tuef27 ) , p. chrysogenum ( tuef23 ) , f. oxysporum ( tuef35 ) , and f. nygamai ( tuef22 ) displayed the highest inhibition against the human pathogenic bacteria s. aureus , e. coli , p. aeruginosa , and k. pneumonia , respectively .
the isolates a. sydowii ( tuef6 ) , p. chrysogenum ( tuef23 ) , and e. crustaceum ( tuef32 ) showed strong activity against e. faecalis ( table 5 ) .
inhibition zone , ( - ) no activity determined ; + , > 2 mm ; + + , 2 - 10 mm ; + + + , < 10 mm .
all endophytic fungi exhibited significant inhibition against a wide range of plant pathogenic fungi and human pathogenic bacteria .
the isolates , namely p. chrysogenum ( tuef2 ) , e. crustaceum ( tuef10 ) , a. sydowii ( tuef14 ) , f. brachygibbosum ( tuef26 ) , p. chrysogenum ( tuef30 ) , p. chrysogenum ( tuef31 ) , and e. crustaceum ( tuef32 ) showed strong inhibition towards plant pathogenic fungi .  
thirty one isolates in this work showed promising growth - inhibitory activity against at least one of the test human pathogenic bacteria , but no endophyte had antimicrobial activity against all five pathogenic microbes .
the numbers of fungal isolates displaying antimicrobial activity against s. aureus , e. faecalis , e. coli , p. aeruginosa , and k. pneumonia were 10 , 13 , 6 , 18 , and 19 , respectively .
the isolates p. chrysogenum ( tuef27 ) , p. chrysogenum ( tuef23 ) , f. oxysporum ( tuef35 ) , and f. nygamai ( tuef22 ) displayed the highest inhibition against the human pathogenic bacteria s. aureus , e. coli , p. aeruginosa , and k. pneumonia , respectively .
the isolates a. sydowii ( tuef6 ) , p. chrysogenum ( tuef23 ) , and e. crustaceum ( tuef32 ) showed strong activity against e. faecalis ( table 5 ) .
inhibition zone , ( - ) no activity determined ; + , > 2 mm ; + + , 2 - 10 mm ; + + + , < 10 mm .
275 isolates , belonging to 23 species and 14 genera , were recovered from the stem and root segments of seven medicinal plants collected from salt marshes in north saudi arabia .
penicillium chrysogenum was the most frequently isolated species with a colonization frequency of 98.57% , followed by mycelia sterilia with a colonization frequency of 92.86% .
high colonization rates , ranging from 81 - 89% , were reported in palms in brunei and australia ( 22 ) , and up to 95 - 98% in leaf fragments of guarea guidonia ( meliaceae ) in puerto rico ( 23 ) .
( 85.3% in stems ) and colletotrichum gloeosporioides ( 30% in leaves ) endophytically colonized suaeda fruticosa in england .
( 25 ) found that the salt marsh fungus camarosporium roumeguerii , occurring on plants belonging to chenopodiaceae , produced a dark green pigment .
were always isolated from plants in deserts and saline lands ( 26 , 27 ) . a possible explanation for the relatively low overall colonization rate noted in this study could be the high salinity of salt marshes at the collection site , and the desert nature of saudi arabia
.   molecular analysis of fungal rdna sequences provides a powerful technique for assessing fungal diversity at the genus level .
most fungal isolates described in this study belong to ascomycota ( 58.54% ) , deuteromycota ( 39.27% ) , and zygomycota ( 1.45% ) , confirming the previous reports by huang et al .
( 10 ) and other reports on the fungal endophytes of t. nilotica , a. fragrantissima , and artemisia plants from various locations ( 9 , 28 ) .
the its sequences of the isolated species submitted to embl - ebi were compared with the previously deposited sequences using blast .
isolates used for the sequencing analysis , their codes , and genbank accession numbers are listed in table 3 .
most of the isolates belonged to ascomycota , which confirms petrini s findings ( 29 ) that fungal endophytes are mainly ascomycetes .
khan et al . ( 30 ) reported that all the endophytic fungi , collected from calotropis procera and situated at different locations within karachi university campus , belonged to ascomycetes .
( 13 ) during their research on c. procera from the taif region of saudi arabia .
some of the fungi isolated in this study ( a. alternata , g. moniliformis , and some species of m. sterilia ) are well - known plant pathogens .
it is known that an endophyte in one plant may act as a pathogen in another plant , depending on the balance between the pathogenicity and endophytism of the microorganism in different hosts .
the fungi isolated during this study have previously been isolated as endophytes from a wide range of different host plants such as pinus tabulaeformis ( 31 ) , avicennia marina ( 32 ) , ginseng leaves ( 33 ) , thymus decussatus ( 34 ) , and c. procera ( 13 ) .
the results clearly showed that the endophytic fungi have stronger inhibition against plant pathogenic fungi , compared to human pathogenic bacteria .
this could be because the endophytic fungi and plant pathogenic fungi naturally exist in the same habitat .
the antibacterial activity of the endophytic isolates from medicinal plants shown in this work against human pathogenic bacteria indicates the probable role of endophytes in the medicinal activity of these plants , partially agreeing with jahanpour et al .
( 6 ) , who demonstrated that the ethanolic extract of p. harmala seed and p. granatum peel exhibited potential activity against all mycobacterium tuberculosis isolates with a mean inhibitory zone of 18.7 and 18.8 mm , at a 200 mg / ml concentration .
several groups ( 11 , 12 , 18 ) have previously reported the antifungal activities of plant endophytic fungi and actinomycetes .
( 35 ) evaluated the antibacterial activity of nine endophytic fungi isolated from leaves of luehea divaricata against phytopathogens and the human pathogenic bacteria e. coli and s. aureus .
( 36 ) isolated 28 fungal endophytes from agarwood and found that 13 ( 46.4% ) showed antimicrobial activity against at least one of the test human pathogenic bacteria .
the diameters of the inhibition zones of ynas07 , ynas14 , hnas04 , hnas05 , hnas08 , and hnas11 were equal to or higher than 14.0 mm against s. aureus , e. coli , bacillus subtilis , b. subtilis , a. fumigatus , and b. subtilis , respectively .
production of active metabolites by endophytes may be related to the characters of the host plants and to a genetic recombination of the endophyte with the host that may have occurred in evolutionary time .
this is probably the first study that demonstrates the diversity of molecular identification of endophytic fungi from the stems and roots of medicinal plants , collected from salt marshes in al - gouf governorate , saudi arabia .
the successful colonization of these plants by such fungi suggests that they can be utilized in future applications , such as delivery of degradative enzymes for controlling certain plant diseases .
meanwhile , the use of endophytes as producers of bioactive agents will help in the conservation of medicinal plants and the maintenance of environmental biodiversity .
 background : endophytic fungi , which have been reported in numerous plant species , are important components of the forest community and contribute significantly to the diversity of natural ecosystems.objectives:the current study aimed to evaluate and characterize , at the molecular level , the diversity and antimicrobial activities of endophytic fungi from medicinal plants in saudi arabia.materials and methods : fungi growing on plant segments were isolated and identified based on morphological and molecular characteristics . 
 the isolates were grouped into 35 distinct operational taxonomic units , based on the sequence of the internal transcribed spacer regions in the rrna gene . the colonization frequency and the dominant fungi percentage of these endophytic fungi were calculated . 
 a dual culture technique was adopted to investigate the antifungal activity of these endophytes.results:tamarix nilotica showed the highest endophytic diversity with a relative frequency of 27.27% , followed by cressa cretica with a relative frequency of 19.27% . 
 the most frequently isolated species was penicillium chrysogenum with an overall colonization rate of 98.57% . 
 seven out of 35 endophytic fungi exhibited strong antifungal activity to all plant fungal pathogens tested . 
 p. chrysogenum , fusarium oxysporum , and f. nygamai exhibited the highest inhibition against the human pathogenic bacteria staphylococcus aureus , escherichia coli , pseudomonas aeruginosa , and klebsiella pneumoniae . 
 aspergillus sydowii , p. chrysogenum , and eupenicillium crustaceum showed strong antimicrobial activity against enterococcus faecalis.conclusions:the antimicrobial activity of these endophytic microorganisms could be exploited in biotechnology , medicine , and agriculture . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Collection of Plant Samples
3.2. Isolation of Fungal Endophytes
3.3. Antimicrobial Activity of Endophytic Fungi
3.4. DNA Extraction, Amplification, and Sequencing
3.5. ITS Sequence and Phylogenetic Analysis
4. Results
4.1. Antimicrobial Activity of Endophytic Fungi
5. Discussion