microorganisms such as fungi , viruses and bacteria are recognized as biocontrol agents ( 1 ) .
nematodes are a large group of parasites which cause serious economic and hygienic problems in plants , animals and humans .
several programs are developed to minimize the unfavorable effects of gastrointestinal parasites ( 3 ) .
some of the programs attempted to reduce the infective larvae with antihelminthic compounds to reduce nematode infections in adult animals . even though using the antihelminthic compounds are the major tools to treat gastrointestinal parasites , there are limitations regarding their usage in drug residual in animal products such as milk and meat ( 4 ) .
these compounds can be also toxic for non - target organisms ( 5 ) and helminthes because of resistance to the antihelminthics ( 6 ) .
biological control seems to be a safe and efficient idea to decrease the population of infective nematodes larvae in gastrointestinal tract and pastures .
de souza maia filho et al . ( 8) evaluated ovicidal effect on toxacara canis ova by using several species of fungi including acremonium , aspergillus , bipolaris , fusarium , gliocladium , mucor and trichoderma isolated from local soil .
the results showed that trichoderma , fusarium 
 solani complex and acremonium have more ovicidal effect on t. canis ova ( 8) .
agriculture nematodes were controlled by nematocidal activity of fusarium and paecilomyces species isolated in india .
mishra and dwivedi indicated a reduction in the number of nematode farms such as root - knot meloidogyne incognita and m. javanica , and reniform rotylenchulus reniformiss using fusarium and paecilomyces isolates ( 9 ) .
showed that aspergillus alliaceus can be used as a potential biological controlling agent on root parasitic weed orobanche population ( 10 ) .
various types of fungi , mostly belonging to ascomycota , demonstrate antagonistic effects on eggs and parasitic nematodes in animal or human .
the secretion of nematode - antagonistic compounds in f. equiseti isolated from soybean cyst is recognized ( 11 ) .
these metabolites can influence the creation of nematicidal agents and promote the nematocidal activity of some bacteria in plant ( 12 ) .
showed that trichoderma 
 harzianum enhanced the production of nematicidal compounds in laboratory and developed biocontrol of meloidogyne 
 javanica with pseudomonas fluorescens in tomato ( 13 ) .
the current study mainly aimed to evaluate the in vitro activities of various saprophyte soil - fungi to reduce the infective larvae stage of parasitic nematode trichostrongylidae family .
two - hundred grams of feces from each sheep was incubated in room temperature for 10 days .
the third stage infective larvae of trichostrongylidae family containing ostertagia circumcincta , marshalgia marshali and haemonchos contortus , were collected by berman apparatus . to obtain larvae without fecal fungi and bacteria ,
they were washed 10 times in normal saline by centrifugation for two minutes at 500 rpm .
the larvae were kept at 4c in a solution including antibiotics penicillin - streptomycin ( sigma , germany ) and amphotericin b ( fungizone / brstol - myers squibb , paris ) to prevent bacteria and fungi growth .
fifteen isolates of filamentous fungi including penicillium sp . , a. flavus , cladosporium sp . ,
trichoderma sp . , f. equisetti , paecilomyces sp . , a. niger , fusarium sp . ,
acremonium sp . , alternaria sp . , derecselera sp . , trichotecium sp . and
the collected fungi were identified by a mycologist using special media and according to colony morphology characters and light microscope .
the fungi were cultured on sabouraud dextrose agar ( merck , germany ) at room temperature for 3 days .
the grown fungi were transferred on potato dextrose agar ( pda ) ( merck , germany ) for three days at room temperature .
after grow of fungi on pda , 7 mm diameter mycelium culture discs were cut and transferred to plates containing 2% water - agar medium with 1% gentamycin ( sigma , germany ) .
the larvae were washed 10 times with normal saline to remove antibiotics . on the 4th day
, 1 ml of larvae culture suspension containing 200 third stage nematode larvae from trichostrongylidae family l3 was separately added to the fungal cultures in 2% water - agar medium petri - dishes .
every day the live larvae were counted by light microscope ( 10x ) and the number of captured larvae was recorded in different days .
the control groups were use in each isolated fungi to compare data with the test groups .
the data were analyzed by chi - square test (  = 5% confidence index ) and descriptive analysis by spss software version 16 ( spss , inc , chicago , il , usa ) .
two - hundred grams of feces from each sheep was incubated in room temperature for 10 days .
the third stage infective larvae of trichostrongylidae family containing ostertagia circumcincta , marshalgia marshali and haemonchos contortus , were collected by berman apparatus . to obtain larvae without fecal fungi and bacteria ,
they were washed 10 times in normal saline by centrifugation for two minutes at 500 rpm .
the larvae were kept at 4c in a solution including antibiotics penicillin - streptomycin ( sigma , germany ) and amphotericin b ( fungizone / brstol - myers squibb , paris ) to prevent bacteria and fungi growth .
fifteen isolates of filamentous fungi including penicillium sp . , a. flavus , cladosporium sp . ,
trichoderma sp . , f. equisetti , paecilomyces sp . , a. niger , fusarium sp . , acremonium sp . , alternaria sp . , derecselera sp . , trichotecium sp . and
the collected fungi were identified by a mycologist using special media and according to colony morphology characters and light microscope .
the fungi were cultured on sabouraud dextrose agar ( merck , germany ) at room temperature for 3 days .
the grown fungi were transferred on potato dextrose agar ( pda ) ( merck , germany ) for three days at room temperature .
after grow of fungi on pda , 7 mm diameter mycelium culture discs were cut and transferred to plates containing 2% water - agar medium with 1% gentamycin ( sigma , germany ) .
the larvae were washed 10 times with normal saline to remove antibiotics . on the 4th day
, 1 ml of larvae culture suspension containing 200 third stage nematode larvae from trichostrongylidae family l3 was separately added to the fungal cultures in 2% water - agar medium petri - dishes . every day the live larvae were counted by light microscope ( 10x ) and the number of captured larvae
the control groups were use in each isolated fungi to compare data with the test groups .
the data were analyzed by chi - square test (  = 5% confidence index ) and descriptive analysis by spss software version 16 ( spss , inc , chicago , il , usa ) .
the table 1 shows the results of 15 isolates from 13 different tested fungi in this study .
these results indicated the highest level of efficiency among the isolates after seven days co - culture of nematodes larvae and fungi .
, trichothecium sp . and curvularia sp . had no significant effect on the reduction of live larvae population , compared with the control group , after seven days incubation ( p > 0.05 ) .
, a. flavus , paecilomyces sp . , a. niger , acremonium sp . , alternaria sp . and fusarium sp .
the killed larvae were introduced by being immobilized , elongation and appearance of several vacouele in body and cuticule of the larvae .
controlling nematodes in agriculture , livestock industries and human community is an important program in many countries . currently , chemical agents and anti - nematode drugs are utilized to control and treat nematode diseases .
the resistance of nematodes against anti - nematode drugs has increased since many years ago .
it seems that chemical control is not completely effective for this purpose . in recent years
, biologic control of nematodes is dramatically considered by many investigators ( 14 - 16 ) .
the current study results demonstrated that some saprophytic fungi spread in the environment can be effective in reducing the number of third stage larvae of trichostrongylidae family .
these results indicated great potential of using appropriate saprophyte fungi for biological control of trichostrongylidae family larvae in sheep .
two factors including adaptability to the environment and ability to produce extracellular enzymes which contribute in the process of infection are the major characteristics of selecting potentially suitable fungus to be employed as an agent for biocontrol ( 18 , 19 ) . in the current research , three fungi derecselera , trichothecium , curvularia species did not show nematophagous activity against trichostrongylidae family larvae in sheep . to employ fungi isolates as biological control ,
the in vitro efficacy of predacious fungal isolates on parasitic nematodes of cattle , cooperia punctata and haemonchus placei has been indicated by a previous study ( 20 ) .
in addition , the in vivo biological control of parasites is shown in bovine by nematode - trapping fungi ( 21 ) .
other researchers reported the capability of a. robusta isolates in the biological control of nematode parasites in bovine gastrointestinal .
it seems that d. flagrans has nematocidal activity ; hence , it is used to control ostertagia sp . , cooperia sp .
this fungus consists of one to three cells covered with adhesive material . in spite of the kind of trap , the entrapped nematode ineffectively struggle for relieve themselves ( 25 ) .
free - living nematodes of panagrellus sp . are used in many laboratories across the world .
this nematode is used as bait for the isolation and predation experiments of nematophagous fungi . in trapping tests by gomes et al .
. showed high susceptibility to predation by the entire genus monacrosporium isolates ( 26 ) .
also , duddingtonia flagrans can be as potential tool for biological controlling of nematodes by nematode - destroying ability .
the fungus creates sticky traps and seizes developing larval stages of parasitic nematodes in the feces of animals ( 27 ) . after feeding the grazing sheep with the spores of d. flagrans ,
biological control has become an important tool in maintaining an appropriate system of sheep products . regarding the time of infection , the fungus has various effects including decrease in the population of mature females , fecundity , and eggs parasitism ( 23 ) . in conclusion
, the studied fungal isolates could be applied as a suitable alternative to resolve the problems related to nematode infection .
compared with the chemical compounds , biological control agents need longer time and have lower ability to rapidly control of the nematodes infection .
in addition , more in vivo surveys are required to choose isolates which affect nematodes larvae in the environment .
 background : biological control of parasitic nematodes by microorganisms is a promising approach to control such parasites . 
 microorganisms such as fungi , viruses and bacteria are recognized as biocontrol agents of nematodes.objectives:the current study mainly aimed to evaluate the in vitro potential of various saprophyte soil - fungi in reducing the infective larvae stage of parasitic nematode trichostrongylidae family.materials and methods : sheep feces were employed to provide the required third stage larvae source for the experiments . 
 the nematode infective larvae of trichostrongylidae family including three species of ostertagia circumcincta , marshalgia marshali and heamonchos contortus were collected by berman apparatus . 
 fifteen isolates of filamentous fungi were tested in the current study . 
 one milliliter suspension containing 200 third stage larvae of trichostrongylidae family was separately added to the fungal cultures in 2% water - agar medium petri - dishes . every day the live larvae were counted with light microscope ( 10x ) and the number of captured larvae 
 was recorded on different days.results:significant differences were observed in the results of co - culture of nematodes larva and fungi after seven days . 
 the most effective fungi against the nematodes larvae were cladosporium sp . 
 , trichoderma sp . , fusarium equisetti , after seven days of incubation.conclusions:the studies on fungi could be applied as suitable tools in biocontrol of nematode infections . 
 however , additional surveys are required to select efficient with the ability to reduce the nematode larvae in the environment . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Preparation of Trichostrongylidae Family Larval Stage
3.2. Preparation of Fungi
3.3. Co-culture of Fungi and Larvae
3.4. Statistical Analysis
4. Results
5. Discussion