isolation of different isolates of a. dactyloides was done with slight modification ( bandyopadhyay and singh , 2000 ) using the method described by duddington ( 1955 ) from different locations : varanasi , ghazipur , mirzapur , chunar ( uttar pradesh ) and ranchi ( jharkhand ) , india .
different isolates were purified and maintained on maize meal agar medium ( maize-20 g ; agar-20 g ; distilled water-1000 ml ) .
further single spore culture of each isolate was made by the method given by singh et al .
cultures of each isolate were maintained on maize meal agar medium by regular subculturing at an interval of 15 days .
the effect of urea , dap and muriate of potash on the spore germination of five isolates of a. dactyloides was studied by the method described by jackson ( 1958 ) for soil fungistasis .
soil samples were taken from vegetable research farm , banaras hindu university , varanasi , india and passed through a 2 mm mesh sieve .
urea , dap and muriate of potash were separately mixed in the soil at the rate of 1% , 0.5% and 0.1% .
50~60 g of soil of each concentration were taken in 90 mm petri dishes and soil was watered near full water holding capacity by addition of distilled water .
water agar blocks ( 10 mm size , 3 mm thickness ) were placed on whatman 's filter paper disc ( 20 mm size ) lying on the surface of soil at five places in the petri dishes and incubated at room temperature ( 25~30 ) for 24 h to allow the diffusates to reach in the agar blocks .
five agar blocks were taken for inoculation of spores of five isolates of a. dactyloides . for unamended soil ( control )
after incubation period , spore suspension of each isolate was made separately by harvesting the spores from 15 days old culuture .
a small drop of water containing 75~100 spores of each isolate were inoculated separately on each agar block and incubated for 24 h at room temperature for observations .
data on spore germination , direct trap formation and trap formation on spore germ tube was taken .
for each treatment three petri dishes were taken as replicates . in order to find out the effect of neem cake amendments in soil on the spore germination of five isolates of a. dactyloides and trapping of soil nematodes by the fungus ,
soil was collected from vegetable research farm , banaras hindu university , varanasi , india .
neem cake was thoroughly mixed in the soil at the rate of 0.5% and placed in pots for decomposition .
50~60 g soil from amended and unamended soils was placed into petri dishes and soil was wet to near full water holding capacity with distilled water .
five water agar blocks were placed directly on the surface of soil in petri dishes at different places and incubated at room temperature for 24 h. after incubation spores of different isolates of a. dactyloides were inoculated on agar block as per method described earlier and the inoculated petri dishes were further incubated for 24 or 48 hours for observations .
data on spore germination , direct trap formation and trap formation on germ tube were recorded .
the experiments on spore germination were conducted on 1 day , 15 day and 30 day after amendment .
isolation of different isolates of a. dactyloides was done with slight modification ( bandyopadhyay and singh , 2000 ) using the method described by duddington ( 1955 ) from different locations : varanasi , ghazipur , mirzapur , chunar ( uttar pradesh ) and ranchi ( jharkhand ) , india .
different isolates were purified and maintained on maize meal agar medium ( maize-20 g ; agar-20 g ; distilled water-1000 ml ) .
further single spore culture of each isolate was made by the method given by singh et al .
cultures of each isolate were maintained on maize meal agar medium by regular subculturing at an interval of 15 days .
the effect of urea , dap and muriate of potash on the spore germination of five isolates of a. dactyloides was studied by the method described by jackson ( 1958 ) for soil fungistasis .
soil samples were taken from vegetable research farm , banaras hindu university , varanasi , india and passed through a 2 mm mesh sieve .
urea , dap and muriate of potash were separately mixed in the soil at the rate of 1% , 0.5% and 0.1% .
50~60 g of soil of each concentration were taken in 90 mm petri dishes and soil was watered near full water holding capacity by addition of distilled water .
water agar blocks ( 10 mm size , 3 mm thickness ) were placed on whatman 's filter paper disc ( 20 mm size ) lying on the surface of soil at five places in the petri dishes and incubated at room temperature ( 25~30 ) for 24 h to allow the diffusates to reach in the agar blocks .
five agar blocks were taken for inoculation of spores of five isolates of a. dactyloides . for unamended soil ( control )
after incubation period , spore suspension of each isolate was made separately by harvesting the spores from 15 days old culuture .
a small drop of water containing 75~100 spores of each isolate were inoculated separately on each agar block and incubated for 24 h at room temperature for observations .
data on spore germination , direct trap formation and trap formation on spore germ tube was taken . for each treatment three petri dishes
in order to find out the effect of neem cake amendments in soil on the spore germination of five isolates of a. dactyloides and trapping of soil nematodes by the fungus , soil was collected from vegetable research farm , banaras hindu university , varanasi , india .
neem cake was thoroughly mixed in the soil at the rate of 0.5% and placed in pots for decomposition .
50~60 g soil from amended and unamended soils was placed into petri dishes and soil was wet to near full water holding capacity with distilled water .
five water agar blocks were placed directly on the surface of soil in petri dishes at different places and incubated at room temperature for 24 h. after incubation spores of different isolates of a. dactyloides were inoculated on agar block as per method described earlier and the inoculated petri dishes were further incubated for 24 or 48 hours for observations .
data on spore germination , direct trap formation and trap formation on germ tube were recorded .
the experiments on spore germination were conducted on 1 day , 15 day and 30 day after amendment .
in agar blocks placed over unamended soil ( control ) directly or on filter papers , the conidia of a. dactyloides largely germinated by direct traps ( fig .
there was no difference in the germination of spores by direct traps whether the spores placed on agar blocks directly on the soil or on the filter papers .
the percentage of spore germination in both the situations were almost similar ( table 1 ) .
however application of urea at the concentration of 1.0% , 0.5% and 0.1% completely inhibited the germination of spores of all the isolates of a. dactyloides ( table 2 ) .
similarly diammonium phosphate ( dap ) also checked the germination of spores or direct trap formation at 1% concentration .
however , in the soil amended with 0.5% dap , 61~83.3% spores germinated by direct trap while very low percentage of spores ( 0~1.7% ) germinated by germ tube ( fig .
the level of trap formation on germ tube was also very low in all the isolates . at 0.1% concentration of dap , the germination of spores by direct rings ranged between 66.7 to 92.2% irrespective of the isolates .
microconidia of isolates b also recorded direct ring formation at 0.1% concentration of dap in soil .
application of muriate of potash also showed adverse effect on the spore germination or ring formation at all the concentrations . in observations recorded after 24 h of incubation only scant germination of spores by germ tube
was recorded at 0.5% and 1% concentration of muriate of potash . however , at 0.1% concentration , the germination of spores by germ tube increased conspicuously and very low percentage of spores germinated by direct rings . when the same agar blocks were observed after 48 h , the germination of spores by direct trap increased conspicuously with the decreasing concentrations of this fertilizer .
soil freshly amended with neem cake at the rate of 0.5% completely checked the germination of spores by germ tube and direct trap ( table 3 ) .
after , 15 days of amendment , germination of spores only by germ tube was recorded in all the isolates , however , the percentage of germinated spores was very low in all the isolates .
irrespective of isolates , the germination rate of spores ranged between 1.6% and 2.6% in 24 h. the microconidia of isolate b , however , failed to germinate . when the observation was taken after 48 h , fairly higher percentage of spores germinated by direct rings , indicating that germination was delayed .
1a ) ranged between 3.8 to 8.9% , while constricting rings on germ tubes ( fig .
after one month of amendment , 91~94% of spores germinated by direct rings in 24 h. microconidia of isolate
the level of germination of spores by germ tube was very low ranging from 1.34 to 3.9% only . from the observations it is evident that after 24 h of amendment of neem cake there was neither spore germination nor nematode trapping
. however after 15 days of incubation of amended soil , few nematodes were attracted on agar blocks incubated for 24 h without any capturing . when the same blocks were observed after 48 h of incubation ,
this clearly indicated that even after 15 days of incubation of amended soil the toxic substances of neem cake had its effect although it was reduced .
however after 30 days of amendment , fairly higher number of nematodes were attracted and captured . in the corresponding controls ,
i.e. , unamended soil after 1 , 15 and 30 days the germination of spores by direct trap were almost similar as given in table 1 .
in general , soils amended with higher concentrations of fertilizers reduced spore germination of all the isolates of a. dactyloides ( table 2 ) . inhibition of germination of spores by urea at the concentrations used may be attributed to direct toxicity of urea on the spores . however , dap at 0.5% and 0.1% concentrations caused little inhibition on the germination of spores of almost all the isolates .
muriate of potash also reduced germination of spores as well as direct ring formation on conidia . from the observations it is very clear that application of fertilizers would reduce the population of nematode trapping fungi in the soil , which might be playing important role in reducing the population of nematodes . in soils ,
top - dressed with the fertilizers , the concentration of these fertilizers might be fairly high on the soil surface , which would be detrimental to sensitive fungi like a. dactyloides .
it is also possible that excessive use of these fertilizers may slowly reduce the population of sensitive nematode trapping fungi in the soil .
inhibition of germination of conidia of a. dactyloides in soil duly amended with 0.5% neem cake may be attributed to the toxic principle present in the neem cake .
( 1980 ) on the spore germination of some pathogenic fungi by neem plant extract .
the toxicity of neem cake was also reported on second stage juveniles of heterodera cajani by singh and singh ( 1997 ) . however , in observations after 15 days , the toxic effect of neem cake was considerably reduced which was evident from the observations on germination or trap formation after 48 hours of spore inoculation . this change in toxicity
may be attributed to biodegradation of neem toxic factor in soil , which was completely eliminated after 30 days of application of neem cake .
it was interesting to note that a large number of soil nematode migrated to the top of the agar block containing the spores of a. dactyloides , which may be attributed to attraction of nematode to fungal traps ( table 3 ) .
further most of the nematodes were captured and killed on the agar block ( fig .
1d ) and the developing hypha from the spore cell produced few to many traps on the hyphae ( fig .
it is concluded that mass culture of a. dactyloides should be applied only after 15 days of neem cake application to make the biocontrol agent more effective .
it has been observed that the population of saprophytic nematodes increases several folds after neem cake amendment ( data not shown ) , which can serve as pabulum for the fungus and increase the population of a. dactyloides .
 application of fertilizers such as urea , diammonium phosphate ( dap ) and muriate of potash in soil adversely affected the spore germination of arthrobotrys dactyloides . 
 amendment of soil with urea at the concentrations of 1.0% , 0.5% and 0.1% completely inhibited spore germination and direct trap formation on the conidium , whereas muriate of potash delayed and reduced the spore germination even at the lowest concentration . 
 dap also inhibited spore germination at 1.0% concentration , while at lower concentration the percentage of spore germination was reduced . 
 application of neem cake at the concentration of 0.5% also inhibited spore germination after 24 h of amendment . 
 the inhibitory effect of neem cake was reduced after 15 days of amendment , while after 30 days after amendment the inhibitory effect was completely lost and the spore germinated by direct trap as in unamended soil . 
 nematodes were not attracted to ungerminated spores after 24 h of amendment . 
 after 15 days of amendment nematodes were attracted to agar blocks containing fewer germinated spores after 24 h of incubation but after 48 h of incubation large number of nematodes were attracted and trapped by the germinated spores with direct traps . 
 after 30 days of amendment , larger number of nematodes were attracted and trapped by direct traps . 
Materials and Methods
Fungal isolates
Effect of urea, DAP and muriate of potash on spore germination and trap formation
Effect of neem cake on spore germination and trap formation
Results
Discussion