Patent Application: US-62912405-A

Abstract:
this invention refers to isolation and utilization of the fungus pochonia chlamydosporia var . chlamydosporia strain pcmr in biological control of nematodes , characterized in that it has a high virulence against meloidogyne spp . in in - vitro microbiologic tests , pot tests and field tests . in these tests , the strain pcmr , isolated in portugal , have a good performance in the ability to control meloidogyne spp . populations and shows a good capacity to produce high amounts of chlamydospores used to produce inoculum and to colonize plant roots that might be infected by meloidogyne spp . this strain can be utilized as part of biological control methods that effectively control root - knot - nematode populations belonging to genus meloidogyne and this invention refers also to production and utilization of nematicides based on pochonia chlamydosporia var . chlamydosporia strain pcmr and any organisms derived from this strain .

Description:
this strain of the fungus pochonia chlamydosporia has all the morphologic typical characteristics of the fungus pochonia chlamydosporia var . chlamydosporia , described by gams , 1988 . selection tests outcome , using the methods stated in iobc manual ( kerry & amp ; bourne , 2002 ): were utilized the 3 standard selection tests , the barley root colonization test in semi - sterile conditions , the chlamydospore production test in vitro , the egg parasitism test in water - agar petri dishes . b ) chlamydospore production test : 2 . 2 × 10 7 chlamydospores / g of barley used in the chlamydospore production medium . green pepper pot tests according to iobc methods ( kerry & amp ; bourne , 2002 ): the experiment was carry way in a growth chamber , at 25 ° c ., with appropriate light regime to green pepper growth , and with manual irrigation . soils used : 2 types : type 1 : non - autoclaved sandy soil ; type 2 : autoclaved sandy soil . fungus treatment : 3 strains tested and controlled ( non - inoculated soil ). all inoculations were performed with 5000 chlamydospores / g of soil . nematode treatment with meloidogyne incognita : were used 3 levels : level 1 : 6000 juveniles , level 2 : 2500 juveniles , level 3 : control ( non - inoculated ). each statistical unit , obtained by the combination of the different treatments stated was repeated 5 times . in pot tests the strain pcmr decreased significantly ( s & lt ; 0 . 05 ) the number of eggs / egg mass , in soil inoculated with 5000 chlamydospores / g of soil , compared to non - inoculated soil , in eggs collected from egg masses obtained from the green peppers grown in the pots with 2500 and 6000 juveniles of meloidogyne incognita . field trials , in 2 consecutive years , in conditions matching those utilized by commercial growers ( assay design and evaluation methods of assay parameters according to kerry & amp ; bourne , 2002 ) site : agricultural experimental station of patação , in direcção regional de agricultura do algarve , algarve , portugal . the plastic house soil was a meloidogyne javanica infested sandy soil , and had no indigenous population of p . chlamydosporia . plant density : 6 rows with 97 plants each , spaced 1 . 1 m between rows × 0 . 4 m in the row . 16 plots , each with 3 plant rows , 11 plants per row , in a total of 33 plants per plot . the plots were randomly distributed in the plastic house . treatment 2 : nematicide application + pcmr : methyl - bromide in pre - plantation + pcmr treatment ( same as treatment 3 ). treatment 3 : pcmr treatment : 2 - 3 inoculations , utilizing 5000 chlamydospores / g soil , inoculating 8 plants chosen in central row of the plot , and calculating the soil volume by multiplying soil area available for each plant × top 20 cm of soil . the inoculations started 2 - 3 weeks after planting . each treatment was repeated 4 times . all the evaluated parameters were determined 3 times in each sample . 1 st year ( treatment 3 compared with control ): soil colonization , final population recovered at the end of assay : 2 . 7 × 10 4 cfu / g of soil ( conf . int . t - student 95 %, between 1 . 5 and 3 . 8 × 10 4 cfu / g of soil ). root colonization : 1 . 3 × 10 4 cfu / g of root ( conf . int . t - student 95 %, between 0 . 7 e 1 . 9 × 10 4 cfu / g of root ) egg parasitism in meloidogyne eggs , tested in semi - selective media , from eggs extracted by mechanic methods from egg masses obtained from the field infected plants : average 45 % ( conf . int . t - student 95 %, between 39 and 51 %). meloidogyne population reduction : determination of the parameter : total viable juveniles plus eggs : average of 74 % ( conf . int . t - student 95 %, between 56 e 91 %). determination of parameter : number of viable eggs / egg mass : average of 64 % ( conf . int . t - student 95 %, between 49 e 79 %). 2 nd year ( treatment 3 compared with control ): soil colonization , final population recovered at the end of assay : 2 . 8 × 10 4 cfu / g of soil ( conf . int . t - student 95 %, between 1 . 7 and 3 . 8 × 10 4 cfu / g of soil ). root colonization : 1 . 3 × 10 4 cfu / g of root ( conf . int . t - student 95 %, between 1 . 2 e 1 . 5 × 10 4 cfu / g of root ). egg parasitism in meloidogyne eggs , tested in semi - selective media , from eggs extracted by mechanic methods from egg masses obtained from the field infected plants : average 39 % ( conf . int . t - student 95 %, between 34 and 43 %). meloidogyne population reduction : determination of the parameter : total viable juveniles plus eggs : average of 65 % ( conf . int . t - student 95 %, between 60 e 71 %). determination of parameter : number of viable eggs / egg mass : average of 70 % ( conf . int . t - student 95 %, between 63 e 77 %). a — this strain can be utilized in mass production of chlamydospores , using fermentors of variable size , using the appropriate technology to produce large amounts of chlamydospores , namely solid state fermentors using an appropriate substratum . currently , several media can be used , namely media based in barley , rice , wheat or corn . these spores can then be extracted from liquid suspensions ( international patent request pti 04 / 000009 ), or through dry methods . b — its possible to obtain formulated products from extracted chlamydospores referred in a by adding to them inert substances . these products can be applied in soils , in plants , in seeds or other growing media for plants . these products can be utilized as part of a biological control method against root - knot - nematode ( meloidogyne spp .) which can be utilized in the majority of susceptible cultivated plants . control of a meloidogyne spp . population , composed mainly by meloidogyne javanica , in plastic house tomato crop , in algarve , portugal . the barley is milled and sieved through a 2 mm sieve and then washed through a 44 μm , collecting all the residue collected in 44 μm . sieve fine sand , through a 2 mm sieve , then wash it through a 44 μm , and then collect the particles between 2 mm and 44 μm . the production medium is obtained by mixing the collected fractions of milled barley and fine sand in 1 : 1 proportion , and let it dry to the appropriate moisture level . place 50 ml of this mixture in small 250 ml erlenmyer flasks ( fermentors ), close with cotton , seal with aluminium foil and then autoclave . 2 — inoculate each fermentor of 250 ml , in sterile conditions , with 4 agar plugs previously colonized with pcmr , wait 3 days at 25 ° c ., and then shake gently to spread inoculum and then let incubate for 1 month . from a sufficient number of 250 ml fermentors , extract all the colonized medium with chlamydospores , homogenise and wash it to the feeding tank of the extraction and separation apparatus . then operate the apparatus in order to correctly extract the chlamydospores ( international patent request pti 04 / 000009 ) and then collect the chlamydospores in appropriate vials , and keep them in the fridge at 4 ° c . prepare fine sand ( ø 90 to 700 mm ), sterilize it in the autoclave , dry it overnight at 90 ° c . in the oven . build up the inoculum by mixing the sand and chlamydospores , evenly at the 10 : 1 proportion , and then keep it at 4 ° c . until necessary . with soil moisture at field capacity , apply an aqueous mixture of inoculum to the plant by spreading it on the soil around the plant , at the rate of 5000 chlamydospores / g soil , on the top 20 cm of the soil . then slightly incorporate inoculum in soil surface and cover it with soil . apply the inoculum as much times as needed to achieve a recovered population from soil of 2 × 10 4 cfu / g soil . monitoring pochonia chlamydosporia var . chlamydosporia strain pcmr . perform soil analysis , with the available methods , to determine the population of pcmr in cfu / g soil . 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( 1996 ) the importance of rhizosphere interactions in the biological control of plant parasitic nematodes - a case study using verticillium chlamydosporium . pesticide science 47 : 69 - 75 . bourne j . m ., kerry , b . r ., and leij , f . a . a . m . de ( 1996 ) the importance of the host plant on the interaction between root - knot nematodes ( meloidogyne spp .) and the nematophagous fungus , verticillium chlamydosporium goddard . biocontrol science and technology 6 ( 4 ), 539 - 548 . leij , f . a . a . m . de , davies , k . g . and kerry , b . r . ( 1992 ) the use of ver - ticillium chlamydosporium goddard and pasteuria penetrans ( thorne ) sayre & amp ; starr alone and in combination to control meloidogyne incognita on tomato plants . fundamental and applied nematology 15 ( 3 ), 235 - 242 . leij , f . a . a . m . de , kerry , b . r . and dennehy , j . a . ( 1993 ) verticillium chlamy - dosporium as a biological control agent for meloidogyne incognita and m . hapla in pot and micro - plot tests . nematologica , 39 : 115 - 126 .