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1 제출문 농림부장관귀하 본보고서를 결구상추의균핵병과밑둥썩음병방제를위한엽면살포제및종자처리 제미생물농약개발 과제의최종보고서로제출합니다 년 11 월 28 일 주관연구기관명 : 동아대학교 총괄연구책임자 : 문병주 세부연구책임자 : 정순재 세부연구책임자 : 이진우 연 구 원 : 김현주 연 구 원 : 박종영 연 구 원 : 백정우 연 구 원 : 이광열 연 구 원 : 전옥주

2 Ⅰ. 제목 결구상추의균핵병과밑둥썩음병방제를위한엽면살포제및종자처리제 미생물농약개발 Ⅱ. 연구개발의목적및필요성 1992년 UN 환경개발회의리우선언이후지구환경보호를위한노력과 WTO가창립되면서환경과무역을연계시킨그린라운드가진행되면서농업도환경친화적농업으로의전환이시급해졌다. 특히, 화학농약사용량의제한및잔류허용량강화로저독성, 저약량제품또는생물농약의개발이필요하게되었는데, 국내에서도최근친환경농업육성 5개년계획의수립으로화학농약사용량을 2005년도까지원제기준으로 30% 를감소시키고저농약재배로친환경농업재배면적을확대시키는친환경농업구현을계획하여실행하고있다 ( 농림부, 2001). 따라서, 현재국내에서는미생물농약에대한관심이크게일어나많은연구자와벤처기업등에서연구가진행되고있으며, 생물농약의개발과보급은필수가되어이는앞으로도계속가속화될전망이다. 현재전세계미생물농약시장은앞으로도고속성장할것으로예상되나, 국내에서개발되어등록, 시판되고있는미생물농약은극히미흡한상태이고이에이용되는균주의특허문제등많은문제가산적해있지만우리나라실정에맞는균주의선발과이를이용한미생물농약개발및정확한방제방법확립으로품질은물론이며수량을향상시키는친환경농업을일반화하는데본연구의목적이있다. 우리나라의결구상추시설재배는하우스내의다습조건으로인해밑둥썩음병 (Rhizoctonia solani) 이 1차발생하면균핵병 (Sclerotinia sclerotiorum) 과잿빛곰팡이병 (Botrytis cinerea) 이 2차발생하여그피해가더욱심해지고결구상추의수확량과품질을저하시켜농민들에게많은어려움을주고있다

3 특히, 결구상추는잎을생식하는건강채소이므로화학농약에의존할수가없고, 지금까지결구상추밑둥썩음병과균핵병방제용으로공시된화학약제가없어생물학적방제로서농작물을보호하고자한다. 이에, 본연구에서는항진균성길항세균인 Stenotrophomonas maltophilia BW-13 및 Pseudomonas aeruginosa LY-11 균주를이용하여결구상추밑둥썩음병방제용엽면살포제및종자처리제를개발하고, Bacillus amyloliquefaciens A-7 균주로는균핵병엽면살포제개발에이용하여환경친화적인병방제용미생물농약을개발하고이를실용화하고자한다

4 Ⅲ. 연구개발내용및범위 결구상추포장의토양과건전식물체로부터이미분리된길항균외항균활성이높은길항균의지속적분리 배지상에서길항효과검정 병원균과길항균의배양적특성검정및동정 제2, 3세부과제에서개발된제제의유묘와생육상내방제효과검정 제2, 3세부과제에서개발된제제의유묘와온실내방제효과검정 개발제제의포장적용시험 개발제제의자연발생농가실증시험 항균활성물질대량생산을위한최적조건확립및대량배양용배지선발 종자처리를위한전달매체개발 종자처리제의최적화실험 종자처리제제조기술확립및제제화 종자처리제의근권정착력및활성검정 종자처리제의안정성및경시적유효도검정 균핵병방제용길항균의대량배양을위한최적조건확립및대량배양용배지선발 엽면살포제를위한전달매체개발 엽면살포제의최적화실험 엽면살포제제조기술확립및제제화 엽면살포제의엽권정착력및활성검정 엽면살포제의안정성및경시적유효도검정 엽면살포제의우수제형최종선발 - 4 -

5 Ⅳ. 연구개발결과및활용에대한건의 제 1 절연구개발결과 제 1 연구목표 : 결구상추밑둥썩음병방제용미생물농약개발연구책임자 : 제 1 세부과제 ( 정순재 ) 제 2 세부과제 ( 이진우 ) 1. 유용미생물의탐색및동정 가. 밑둥썩음병발생및발병율조사 경상남도의령군부림면신반리의결구상추재배 플라스틱하우스에서 2003 년 10 월에서 11 월사이에발생한밑둥썩음병의평균이병주율은 5.3% 이었다. 나. 밑둥썩음병병원균의분리및동정 병든결구상추의잎아래밑둥부위에서분리하 여병원성이강한것으로확인된 PY-1 균주를공시병원균으로선발하고동정한결과, Rhizoctonia solani AG1 (IB) 로동정되었다. 다. 병원성검정 R. solani PY-1 균주의병원성검정을위한접종원및최적처리량과농도선발에서플러그포트의유묘검정에서는접종원균사조각부유액 1 ml (A 550 =0.8) 을, 생육실포트검정의성체식물에는균사조각부유액 10 ml (A 550=1.0) 을, 그리고플라스틱하우스토경재배에서는접종원인 WRSP( 밀기울 : 미강 : 톱밥 : PDB배지 =30 g: 10 g: 10g: 100 ml, w/w/w/v) 배지 40 ml 을선발하였다

6 마. 선발길항세균의동정 BW-13 균주와 LY-11 균주의생화학적, 형태적특성및 16S rdna sequence analysis 를통해동정한결과, Stenotrophomonas maltophilia BW-13 과 Pseudomonas aeruginosa LY-11 로각각최종동정, 명명되었다. 2. 밑둥썩음병방제용엽면살포제미생물농약제조기술확립 가. S. maltophilia BW-13 균주의최적배양조건확립 삼각플라스크배양 BW-13 균주는 NB배지에서 20시간배양째에 A 550=1.6 에도달하였고 48시간이되어도변화없이 OD값이 1.3으로생장이지속되었다. 생육온도는 35 에서가장세균밀도가높았으며, 증식최적 ph의범위는 6~8이었다. BW-13 균주의생육및길항력에가장효과적인탄소원과질소원은제빵개량제 (3.0%) 와 Yeast extract(0.5%) 로선발되었다. 그러나, 제빵개량제에는이미질소원으로대두유 12% 가함유되어있기때문에발효기배양에서는질소원을따로선발하지않기로하였다. 따라서, 기초배지에 3% 제빵개량제를첨가한제빵개량제배지가길항균증식과항생물질에의한병원균의균사생육저해가높아대량배양용배지로 1차선발되었다. 발효기배양대량배양용제빵개량제배지에 BW-13균주를배양 (450 rpm, 35, 2.0 기압, ph 6~7) 한후생균수를조사한결과, 배양 48시간과 72시간후에각각 , cfu/ml로현저히높았다. 하지만, 제빵개량제배지에우수저분자탄소원으로선발된 Sucrose(1%) 를첨가하여도세균밀도의증진효과는없었다. 대량배양용배지선발플라스크및발효기배양결과로기초배지 (K 2 HPO %, KH 2PO %, MgSO 4 7H 2O 0.01%) 에 3.0% 제빵개량제를첨가한제빵개량제배지를 S. maltophilia BW-13 균주의대량배양용배지로최종선발하였다. 나. Stenotrophomonas maltophilia BW-13 균주를이용한엽면살포제제형화 대량배 양용제빵개량제배지 4 L 에 BW-13 균주를접종하고 72 시간발효기배양 (450 rpm, 35, 2.0 기압, PH6~7) 한다음각종천연물고분자 ( 옥수수전분, 변성전분, 타피오카전분, 메주가 - 6 -

7 루 ) 을혼합하여 1 차제제 7 종과 2 차제제 3 종등총 10 종 ( 수화제 9 종, 액상수회제 1 종 ) 으 로제형화하였다. 3. S. maltophilia BW-13 균주를이용한엽면살포제방제효과 가. 생육실포트검정 BW-13 균주로종자코팅제개발을진행하던중엽면살포제로서의가능성이타진되어연구수행내용과는별도로엽면살포제를개발하고밑둥썩음병에대한방제효과를생육실포트검정하였다, 1차제제 7종중선발된 BW13-A제제와 2차제제 3종을추가하여생육실포트에서방제효과를비교검정한결과, BW13-A, BW13-I 및 BW13-H 제제가각각 76.6, 75.6, 71.8% 로서유의차없이방제가가높았고, 화학농약펜시쿠론의방제가 50.6% 보다훨씬효과적이어서생육실포트재배의밑둥썩음병방제용엽면살포제로 S. maltophilia BW-13 균주를이용하여제조한수화제형 BW13-A 및 BW13-I 제제를최종선발하였다. 나. 플라스틱하우스토경재배검정 생육실포트검정에서선발된 BW-13 균주의수화제 형 BW13-A와 BW13-I 제제를플라스틱하우스내토경재배한결구상추에서밑둥썩음병에대한방제효과를검정한결과, 수화제형 BW13-A 제제가방제가 75.7% 로가장우수하였으며, 다음으로는 69.8% 의 BW13-I 제제이었다. 화학농약펜시쿠론은 56.3% 로낮은방제가를보였다. 따라서, 토경실험결과밑둥썩음병방제용엽면살포제로서수화제형 BW13-A제제가 BW13-I 제제에비하여효과적인것으로확인하였다. 다. 엽면살포제수화제형 BW13-A 제제의저장안정성 밑둥썩음병방제용엽면살포제 수화제형 BW13-A 제제의온도차이에따른저장안정성을조사한결과, 초기세균수는제제 1 g 당 cfu/ml에서 7개월이경과하는동안 4 에서는 1.2x10 13 cfu/ml과실온에서는 1.6x10 13 cfu/ml으로매우높은수치의세균수가유지되었다. 따라서, 본미생물제제의안정성은매우높으며, 냉장보관혹은실온에서의보관에큰차이가없는것으로확인되었다

8 4. 밑둥썩음병방제용종자처리미생물농약제조기술확립및방제효과검정 가. Pseudomonas aeruginosa LY-11 균주에의한우수코팅종자선발 종자처리미생 물농약개발용으로선발된 LY-11 균주를전달매체 (AF300, clay, zeosil, diatomaceous earth 325) 와함께종자에피막화하고 5종의코탕종자를제조하였다. 이들의출아율을조사한결과, C와 E 코팅종자의발아율이각각 78.9, 71.1% 로대체로높았으나, 비코팅종자의발아율 92.2% 보다는낮고, 반복실험에따른오차가많아효율적인새로운종자코팅방법이요구되었다. 나. P. aeruginosa LY-11 균주를이용한 alginate 종자코팅기술개발 LY-11 균주를 NB배지에 24시간진탕배양 (28 ) 한후세균부유액을 A 550 =1.0으로조정하여멸균된 sodium alginate solution과혼합한배양액을 10 ml용주사기를이용하여종자에피막시켜 0.1 M calcium chloride 용액에떨어뜨려서구형으로될때까지방치하여 alginate 코팅종자를제조하였다.. 다. Aalginate 종자코팅제의방제효과 유묘및플라스틱하우스포트검정 LY-11 균주를이용한 alginate 코팅제제는각각 모잘록병 ( 유묘검정 ) 에서 70.4%, 밑둥썩음병 ( 플라스틱하우스포트검정 ) 에서 85.4% 의방제가를 보여 P. aeruginosa LY-11 균주를이용하여제조한 alginate 코팅종자가모잘록병과밑둥 썩음병에모두효과적이었다. 라. alginate 코팅종자의근권및엽권정착력및안정성검정 근권및엽권정착력검정 P. aeruginosa LY-11R 균주를이용한 alginate 코팅종 자를파종하고유묘 ( 파종후 4주 ) 에서존재하는생균수를조사한결과, 밑둥부위에서잎중간부분 ( cfu/ml) 과밑둥에서뿌리부분 ( cfu/ml) 이가장높았으며뿌리의중간에서끝부위는 cfu/ml로약간감소하였다. 그러나잎의중간에서끝부위부분은밑둥부분에서뿌리부분보다낮았으며, 잎끝부위는 LY-11 균주가조사되지않았다. 중간 - 8 -

9 성체식물 ( 파종후 6주 ) 에서는전반적으로유묘와동일한경향이었으며, 성체식물 ( 파종후 8주 ) 에서는밑둥부위의잎부분과뿌리부분이각각 3.2x10 6, 1.8x10 5 cfu/ml로세균수가가장많았으며뿌리의중간부위까지는유사하였으나, 뿌리끝부분은약간감소하였다. 그러나잎에서는잎의밑둥부위을제외하고는 LY-11 균주가관찰되지못하였다. 저장온도에따른안정성검정 온도별 (4, 15, 25, 35, 40 ) 로밀봉하거나 밀봉하지않고 7일간보관한코팅종자의상태를조사한결과, 15 와 25 는종자가자연적으로발아되었으며, 35 와 40 에서는 alginate의건조현상이심하게일어나부적합하였다. 4 에서도밀봉하지않을경우일부건조및발아되는종자가관찰되므로최종적으로 4 에밀봉보관하는것이가장효과적이었다. 안정성검정에서는 4 에서 7일간밀봉보관 한 alginate 코팅종자내생균수는초기에 cfu/ml 였으나 1 일째는 cfu/ml, 7 일째는 cfu/ml 으로큰변화가없었으며, 50 일째조사한결과에도생균수가안정 적으로지속되었다

10 제 2 연구목표 : 결구상추균핵병방제용미생물농약개발연구책임자 : 제 1 세부과제 ( 정순재 ) 제 3 세부과제 ( 문병주 ) 1. 유용미생물의탐색및동정 가. 균핵병의발생및발병율조사 2003 년경상남도의령군부림면신반리의결구상추 재배농가에서발생된 1, 2월달의이병주율은각각 39.0%, 30.1% 로평균이병주율 21.9% 보다훨씬높았고, 3월은 24.2%, 4월과 5월은매우낮았다. 2차조사시기인 2003년 11월부터 2004년 4월까지의평균이병주율은 8.3% 로 2003년초에비하여현저히낮아졌다. 나. 균핵병병원균분리및동정병든결구상추로부터분리한총 140 여균주중병원성 이강한것으로확인된 YR-1 균주를공시하고형태적및배양적특성을조사하여기존의 보고와비교한결과, Sclerotinia sclerotiorum YR-1 로동정, 명명하였다. 다. 병원성검정 S. sclerotiorum YR-1 균주의병원성검정을위하여균사조각부유액을접종원으로하여접종원의최적처리량과농도을선발한결과, 접종농도 A 550 =0.4 또는 0.6에서 40 ml처리시 50% 의발병을나타내는 ED 50 값에가까워이를최적접종농도및처리량으로선발하였다. 라. 길항미생물의분리 건전결구상추의밑둥부위, 뿌리및근권토양으로부터분리된 702 균주중병원균 S. sclerotiorum YR-1에대해균사생육저지효과가큰 10균주를길항세균으로 1차선발하고이들일차길항세균에의한방제효과를생육실포트검정하여 3균주 (A-2, A-7, RH-4) 를 2차선발하고 2차선발균주와추가공시된 3균주의방제효과를비교하여최종선발하였는데, A-7 균주의방제가가 91.0% 로가장높았고, Pro-EB-15 균주가 90.1% 로다음으로높았으나서로유의차가없어방제가가다소높은 A-7 균주를균핵병방제용우수길항균으로최종선발하였다

11 마. 우수길항세균의동정 균핵병방제를위해최종선발된길항세균 A-7 균주의동 정을위하여생리학적, 생화학적특성및 16S rdna 와 gyrase A 유전자의염기서열을분 석한결과, Bacillus amyloliquefaciens A-7 동정, 명명되었다. 2. 균핵병방제용엽면살포제미생물농약제조기술확립 가. B. amyloliquefaciens A-7 균주의최적배양조건확립 삼각플라스크배양 A-7 균주는배양 14 시간째에최대값인 A 550=1.0 에도달하였으 며, 14시간이후에는점차감소하며 24시간이후에급격히감소하였다. 증식최적온도는 25 로확인되었으며, 생육최적 ph는 6과 7로확인되었다. A-7 균주의생육및길항력에가장효과적인탄소원과질소원은현미유 (3%) 와 Yeast extract(0.5%) 로선발되었다. 따라서, 기초배지에현미유 3.0%, Yeast extract 0.5% 를첨가한현미유배지를 B. amyloliquefaciens A-7균주의플라스크배양에서대량배양용배지로 1차선발하였다. 발효기배양 플라스크배양에서선발된대량배양용배지인현미유배지를발효기에 넣고, A-7 균주을발효기배양 (30, 350 rpm, 1.5 atm, ph 7) 하여생균수를조사한결과, 배양 72시간과 96시간배양에서각각 , cfu/ ml으로가장높게나타났다. 따라서, 발효기에서길항세균을배양하는기간은세균밀도가가장높은 72시간으로정하였다. 대량배양용배지선발이상플라스크및발효기배양결과로기초배지 (K 2 HPO %, MgSO 4 7H 2 O 0.05%, MnCl 2 4H 2 O %, CaCl 2 2H 2 O %, FeSO %) 에현미유 3.0%, Yeast extract 0.5% 를첨가한현미유배지를 B. amyloliquefaciens A-7 균주의대량배양용배지로최종선발하였다. 나. Bacillus amyloliquefaciens A-7 균주를이용한엽면살포제제형화 7 L 발효기에현미유배지 4 L 를넣고 A-7 균주를접종한배양액에전달매체인옥 수수전분, 타피오카전분, 변성전분, 메주가루, 올리브유, Fructose 등을혼합하여 1 차에서

12 4 차까지총수화제형 21 종을제조하였다. 제형화를달리하고자 5 차, 6 차에서는전달매체를 찰옥수수전분, 썬크리미, 썬텐더, 썬프리젤, 썬사이즈, 썬캡, 썬슈퍼젤, 썬배터, 제오 실, 변성전분등을이용하여수화제형 8 종과액상수화제 2 종을제조하였다. 3. B. amyloliquefaciens A-7 균주를이용한엽면살포제방제효과 가. 생육실포트검정 1 차에서 6 차까지제조한미생물제제의생육실포트검정결과방제 가가가장높았던 B. amyloliquefaciens A-7 균주를이용하여제조한액상수화제형 A7-2 제제및수화제형 A7A 제제를균핵병방제용엽면살포제의우수제형으로최종선발하였다. 나. 플라스틱하우스토경재배검정 플라스틱하우스포트검정에서최종선발된액상수화 제형 A7-2제제및수화제형 A7A제제와방제효과가높았던수화제형 A7-4 및 A7W제제 2 종류를추가로공시하여플라스틱하우스내토경재배한결구상추에서균핵병에대한방제효과를검정한결과, A7-2제제가살포 20일째의방제가가 80.5% 로가장우수하였으며, 다음으로는 A7A제제가 79.1% 의방제가를보였다. 그러나이들은서로간에유의차는없었으나대조구인베노밀처리구 75.2% 보다는유의적으로높았다. 따라서, A7-2제제와 A7A 제제는생육실포트검정에서와마찬가지로플라스틱하우스내토경재배에서도균핵병방제용으로효과적인것으로확인되었다. 다. 밑둥썩음병과균핵병의동시방제효과 최종선발된밑둥썩음병방제용엽면살포제인 수화제형 BW13-A제제와균핵병방제용엽면살포제인액상수화제형 A7-2제제를포트재배한결구상추에분무살포하여밑둥썩음병과균핵병을동시방제하기위해생육실포트검정한결과, A7-2 제제의단독처리가 2종류의병원균혼합접종에대해 81.3% 의방제가로 2종류제제의혼합처리 70.6% 보다는높았으나유의차는없었다. 그러나, BW13-A제제의단독처리는 2종류의병원균즉, 밑둥썩음병과균핵병의혼합접종에대해방제가가높지않았으므로 A7-2제제만으로도두가지병원균에대한방제는가능할것으로판단되었다

13 라. 미생물농약의자연발생농가실증시험 2004년경상남도의령군재배포장에서의방제효과 2004년 10월말경균핵병이자연발생하기시작한결구상추의경상남도의령군재배포장에서수화제형 A7A제제및액상수화제 A7-2제제의방제가를베노밀처리와비교하여검정한결과, A7-2 및 A7A제제 100배희석처리구와베노밀처리구간에는유의차가없었고무처리구의 60.0% 이병주율에비하여각각방제가가 84.7%, 84.3%, 87.0% 이었다. 한편 A7-2제제 500배희석처리에서도 70.3% 의방제가를보였다. 실용적인면에서수화제 A7A제제는약하지만약흔이발생되었으나액상수화제인 A7-2제제는이러한약흔의단점을보완하면서도 500배처리에서도방제가가높아산업화의가능성이높은제제로확인되었다. 2005년김해시대동면재배포장에서의방제효과 2005년 3월말경균핵병이자연발생하기시작한경남김해시대동면결구상추의재배포장의플라스틱하우스에서경남의령군의재배포장에서방제효과가확인된 A7-2제제를처리농도별로달리하여제제의방제효과를화학농약인베노밀처리와비교, 검정하였다. 그결과, 무처리의이병주율이 28.0% 로서 A7-2제제 100배처리가 76.7% 의방제가로베노밀처리의 75.0% 와유사하였으며, A7-2제제의 500배에서는 60.7% 로이보다는낮았다. 그러나, 2005년대동면재배지에서의무처리발병율이 2004년의령군에비하여현저히낮았음에도불구하고미생물농약뿐만아니라, 화학농약에의한방제효과도의령군에비하여약간낮은경향이었다. 4. A7-2 제제의엽권정착력검정및안정성검정 토경재배한결구상추에서의 A7-2제제의엽권정착력검정 Rifampicin 저항성 B. amyloliquefaciens A-7R 균주를이용하여제조한액상수화제형 A7-2R 제제를결구상추잎에 1회처리한후 21일동안 A-7 균주의생균수를근권토양, 밑둥부위및잎부위에서측정하였다. 그결과, 초기세균수는 cfu/ml 이었으며경시적으로약간감소하였으나처리 21일째에근권토양, 밑둥부위및잎부위에서는각각 2.8x10 9, 1.4x10 9, 6.8x10 9 cfu/ml로써 A-7균주의엽권정착력이장기간유지되는것으로확인되었다. 따라서, A7-2제제 1회처리후에도 A-7균주의엽권정착력이장기간유지되는것은 A7-2제제는

14 물론 A7A 제제의방제효과가균핵병발생포장에서도높았던원인일것으로생각되었다. 저장안정성검정 선발된미생물제제수화제형 A7A 제제와액상수화제형 A7-2 제 제를저장온도및저장기간에따른안정성을조사한결과, 2003년 10월의 A7A제제초기세균밀도는 cfu/ml이었으며, 1년후 2004년 12월의 4 와실온에서의세균밀도는각각 cfu/ ml와 cfu/ ml이었다. 이는 2005년 9월까지 1년 8개월간조사되었는데각각의세균밀도는 cfu/ ml와 cfu/ ml로초기보다는낮아졌으나여전히높은밀도의세균수가유지되고있었다. 이때실온과 4 보관에따른세균밀도차이는초기세균밀도와별차이가없었으나, 3개월째부터는실온보관에서 10 1 cfu/ml 정도낮아졌으나큰차이는없었다. 액상수화제 A7-2제제의경우, 2003년 10월의초기세균수가 cfu/ ml이었으며, 1년뒤인 2004년 12월의 4 와실온에서의세균밀도는각각 cfu/ ml와 cfu/ ml로 4 와실온보관에따른세균의밀도차이가다소낮아졌다. 연구가종료되는 2005년 9월에는각각 cfu/ ml와 cfu/ ml로초기보다는낮아졌으나여전히높은세균수가유지되고있었고 A7A제제보다는 A7-2제제가다소높은세균수로유지되는경향이었다. 결과적으로, 선발미생물제제 (A7A, A7-2) 의보관상안정성에는문제가없을것으로보여지며, 보관방법은실온보다는 4 보관이좋으나실온에보관하여도안정성에는문제가없을것으로생각된다. 또한, 4 에서 3개월과 8개월간 4 에서보관되어있었던 A7-2제제를 PDA배지상에서균핵병원균과대치배양한결과, 각각 5.0 mm, 5.3 mm의저지대를보였으며, 1년간보관한 A7-2제제를농도별로희석하여대치배양한결과 1,000배희석에서도 4.5 mm 이상의저지대를보여장기간의보관에도매우안정성이매우높음을재차확인하였다. 제 2 절 활용에대한건의 1. 본연구로부터탐색분리된항진균활성세균인 B. amyloliquefaciens A-7 균주는결구상 추균핵병방제용엽면살포제미생물농약의제제화에최초로이용되었으며, ( 주 ) 한국바이오

15 케미칼에기술이전예정에있다. 2. 결구상추밑둥썩음병방제용인종자처리제개발에이용된 Pseudomonas aeruginosa LY-11 균주와엽면살포제개발에이용된 Stenotrophomonas maltophilia BW-13은밑둥썩음병방제에처음으로이용되었으므로, 균주특허및미생물농약제조관련특허출원에활용된다. 3. Pseudomonas aeruginosa LY-11 균주로부터생성되는물질연구는본세균에의한항진균화합물의보고가지금까지알려진바가적어신규화합물일가능성이있고, 물질분리후물질특허출원과동시에유기합성및유도체합성을시도하여다양한약제개발및생산에활용될것이다. 4. 항진균활성물질중소분자량폴리펩타이드는농약용또는의약용항진균제로의개발에 이용될것이다. 5. 지금까지보고된연구또는국내외등록된특허와전혀다른 formulation 의구성성분으 로미생물제제가완성되어국내특허에출원예정에있다. 6. 종자코팅법의신기술개발로국내특허또는기술이전으로활용될수있다

16 SUMMARY Development of New Biofungicides and Seed Coating Technique for Sclerotinia rot and Bottom rot of Crisphead Lettuce Development of new biofungicides and seed coating technique using antagonistic bacteria, Bacillus amyloliquefaciens A-7, Stenotrophomonas maltophilia BW-13, and Pseudomonas aeruginosa LY-11 strain for Sclerotinia rot caused by Sclerotinia sclerotiorum and Bottom rot caused by Rhizoctonia solani on crisphead lettuce 1. Screening of useful antagonistic bacteria from soils 2. Identification of plant pathogenic fungus and antagonistic bacteria 3. In vitro and In vivo, antifungal activities of antagonistic bacteria, B. licheniformis N1 strain 4. Optimization of culture condition and mass production for Bacillus amyloliquefaciens A-7, Stenotrophomonas maltophilia BW-13, and Pseudomonas aeruginosa LY-11 strain. 5. Scale up of the fermentation process 6. Formulations of the biofungicide 7. Control effects of the biofungicides in a growth chamber, a plastic house, and the fields naturally occurred gray mold rot 8. Storage ability of biofungicide under the different conditions

17 1. Isolation and Identification of pathogen and antagonistic bacteria Occurrence of crisphead lettuce bottom rot The occurrence of crisphead lettuce bottom rot at Sinban-Ri, Burim-Myeon, Euryeong-Gun of Gyung-Sang Namdo was investigated from October of 2003 to November of the same year. The ratio of average number of diseased plants was 5.3%. The symptoms of the crisphead lettuce bottom rot include round water-soaking light brown or brown spots on the crown of the plant. When disease became severe, the symptoms were enlarged as irregular lesions and plant leaves became rotten and finally dead as brown blight tissue. The subsequent infection of the plant by sclerotinia and gray mold caused the dramatic yield loss on the crisphead lettuce. Identification of a causative pathogen The candidate causative fungi were isolated from the diseased plant tissues and tested for the virulence on the healthy crisphead lettuce. One of the isolate, PY-1, showed the original bottom rot symptom and were identified as Rhizoctonia. solani AG-1 (IB)

18 Pathogenicity test For the pathogenicity test, triturated mycelia-inoculum(a 550=1.0) of PY-1 isolate was selected as the most effective inoculum showing disease incidence of 51.1% for the mycelial inoculation at pot assay. Otherwise, WSRP media-inoculum (wheat bran, sawdust, rice bran, PDB media) of PY-1 isolate was effectual inoculum showing disease incidence of 61.6% for soil inoculation at the plastic house. 2. Development of biofungicide for bottom rot disease Optimum culture condition of antagonistic bacteria BW-13 The highest cell density of A 550=1.6 was obtained by 20 hr cultivation of S. maltophilia BW-13 and the density was maintained until 48 hr cultivation without

19 signicant decrease of the OD. The optimum temperature and ph range for the bacterial growth was 35 and ph 6~8, respectively. In flask cultivation experiment Measurement of bacterial density and assay of antagonistic activity of bacterial culture broth allowed us to select the best carbon source and nitrogen source for mass production of antifungal compounds from the BW-13 strain. Dough-conditioner and yeast extract were finally selected as the best carbon and nitrogen source, respectively. However, since dough-conditioner itself contained 12% of nitrogen compounds, we used dough-conditioner as a major nutrient for bacterial fermentation culture instead of addition of yeast extract. Establishment of mass-production of antifungal compounds Therefore, we used a basal salt medium with 3% dough-conditioner for fermentation broth. The bacterial density after 48 hr and 72 hr fermentation under the defined temperature and ph condition was exceptionally high upto approximately cfu/ml and cfu/ml, respectively. The addition of 1% of sucrose in the dough-conditioner media did not make a difference in the bacterial density. Formulation of BW-13 for foliar spray Various additives such as starches were supplemented into the 4 liter fermentation culture of S. maltophilia BW-13 grown under the defined condition. Among 10 different formulations tested, a liquid formulation using suncreamy as an additive exhibited the excellent disease control activity without the toxicity problem. Other formulations also showed good disease control activity but residual marks on the sparyed leaves

20 3. Biological control effect of biofungicides Pot assay at growth chamber Ten different formulations of BW-13 strain were evaluated for disease control activity against crisphead lettuce bottom rot in pots of plastic house. Formulations such as BW13-A, BW13-I and BW13-H exhibited disease control value of 76.6, 75.6, 71.8%, respectively. This control value was significantly higher than that of chemical fungicide Pencycuron with 50.6% of disease control value. Since BW-13H showed residual marks on treated leaves. liguid formulation BW13-A and BW13-I were finally selected as microbial pesticide to control the crisphead lettuce bottom rot and further tested in platic house. Pot assay at a plastic house The BW13-A and BW13-I exhibited disease control value of 75.7% and 69.8% in plastic house experiment, respectively, while chemical fungicide showed 56.3% disease control value. Therefore, BW13-A was finally selected as a microbial pesticide to control the crisphead lettuce bottom rot. Storage stability of BW13-A formulation on foliar leaves Storage of BW13-A at 4 and room temperature did not significantly affect the number of viable bacteria during 6 months. The number of bacteria was maintained at the level of cfu/ml (initial), 1.2x10 13 cfu/ml (4 ) and 1.6x10 13 cfu/ml (room temperature). Therefore, BW13-A formulation is remarkably stable irrespective of storage temperature. 4. Seed-coating technique for crisphead lettuce bottom rot and its biological control effects Establishment of seed-coating by Pseudomonas aeruginosa LY

21 Seed coating of BW-13 was performed with various coating materials including adhesives, various salt and gelling materials under various culture conditions. Bacterial culture condition did not affect the activity for seed coating and plant growth promotion activity. Although modified starch supergel was selected as a good coating material, seed germination was not satisfactory. Seed coating of crisphead lettuce seeds with Pseudomonas aruginosa LY-11 was performed using carrier AF300, clay, zeosil, diatomaceous earth 325 with various concentrations. Seed germination ratio was 78.9, 71.1% according to carriers but the germination ratio was lower than non-coated seeds. The seed coating process using the material might make a damage on crisphead lettuce seeds. Alginate coating-seed technique by P. aeruginosa LY-11 Seed coating of crisphead lettuce seeds with Pseudomonas aruginosa LY-11 was performed with more mild materials such as sodium alginate. The alginate seed coating method was easy and showed good seed germination. Therefore, alginate coating of the seeds was evaluated for disease control activity and plant growth promotion. Effect of alginate coating to control the crisphead lettuce bottom rot Suppression of seedling damping off by R. solani in crisphead lettuce was investigated with alginate coating seeds with the LY-11 strain and without the strain in plugpots by artificial inoculation of the pathogen. Disease severity from coating seeds with bacteria was 28.9% and that from uncoated seeds was 97.7%. Thus, disease control value by alginate seed coating with LY-11 strain was 70.4%. Similarly, disease control value of alginate seed coating with LY-11 strain against bottom rot was 85.4%, indicating the excellent seed coating effect of alginate with the strain LY

22 Colonization of LY-11 from alginate coated seeds on roots and leaves of crisphead lettuce Colonization of crisphead lettuce plant by LY-11 strain from the alginate coated seeds was estimated from various plant parts. The cfu/g of plant tissues were estimated as and in leaves and shoots of crisphead lettuce plant and was at the root tips. The density of bacteria in shoot tips or tip of the leave were not detectable. The similar colonization of the bacteria were observed from the 30-days old adult plants and the 50-days old adult plants. The cfu/g of plant tissues in adult plant root tips was maintained as 3.1x10 6 and 1.8 x Stability of formulations and coated seeds. Storage of alginate coated seeds of crisphead lettuce above 15 induced the seed germination and caused seed drying subsequently reducing seed germination ratio. Since unsealed storage of alginate coated seeds at 4 also caused seed drying, storage of the seeds at 4 by sealing was the best to maintain the seed viability. Storage of the seeds at the defined conditions maintained the stable bacterial viability upto 7 days from cfu/ml of bacteria (initial) to cfu/ml of bacteria (7 days). The number of viable bacteria was not decreased upto 50 days storage at the defined condition

23 1. Isolation and Identification of pathogen and antagonistic bacteria Occurrence of crisphead lettuce sclerotinia rot The crisphead lettuce sclerotinia rot at Sinban-Ri, Burim-Myeon, Euryeong-Gun of Gyung-Sang Namdo was significantly occurred from January of 2003 to May of the same year. The ratio of average number of diseased plants were 21.9%. The same disease was also occurred from November of 2003 to April of The symptoms of the crisphead lettuce sclerotinia rot include irregular water-soaking brown lesions on the crown of the plant and progressed into leaves. When disease became severe, the leaves became rotten and finally dead as brown blight tissue. Fungal mycelia and sclerotium were observed from the diseased tissues. The subsequent infection of the plant by gray mold caused the dramatic yield loss on the crisphead lettuce. Identification of a causative pathogen A total of 140 isolates of the candidate causative fungi were isolated from the diseased plant tissues and 80 of them were identified as Sclerotina sclerotiorum. One of the isolate YR-1 with the highest pathogenicity on crisphead lettuce plant was selected and described in detail morphologically and culturally in this report. Pathogenicity test The pathogenicity test of the isolated strain YR-1 was performed by the defined condition in this report. the most suitable inoculum quantity of YR-1 strain

24 was selected as the mycelial suspension of A 550=0.8, 40 ml showing disease incidence of 94% at the whole plant. 2. Development of biofungicide for Sclerotinia rot Optimum culture condition of antagonistic bacteria A-7 The highest cell density of OD 1.0 was obtained by 14 hr cultivation of B. amyloliquefaciens A-7 and the density was reduced dramatically 24 hr later. The optimum temperature and ph range for the bacterial growth was 25 and ph 6~7, respectively. In flask cultivation experiment In flask cultivation experiment, measurement of bacterial density and assay of antagonistic activity of bacterial culture broth allowed us to select the best carbon source and nitrogen source for mass production of antifungal compounds from the BW-13 strain. Using the Bacillus basal medium with 0.5% of yeast extract and NH 4NO 3, corn oil and yeast extract were finally selected as the best carbon and nitrogen source. Addition of other carbon sources for enhancing antifungal activity production in culture media did not significantly increase the antifungal

25 activity. Establishment of mass-production of antifungal compounds Therefore, we defined the optimum media for A-7 mass cultivation with the following ingredients: K 2 HPO %, MgSO 4 7H 2 O 0.05%, MnCl 2 4H 2 O, %, CaCl 2 2H 2 O %, FeSO %, corn oil 3.0% and yeast extract 0.5%. a basal salt medium with 3% dough-conditioner for fermentation broth. The A-7 bacterial density after 72 hr and 96 hr fermentation culture under the defined media, temperature and ph condition was exceptionally high up to approximately and cfu/ ml, respectively. The antifungal activity of the fermentation culture against S. sclerotiorum YR-1 was remarkably high with fungal growth inhibition zone of 15 mm. Formulation of A-7 for foliar spray Various additives and carriers such as starches, suncreamy, vegetable oils and cereal oils were supplemented into the 4 liter fermentation culture of B. amyloliquefaciens A-7 grown under the defined condition. Among 24 different formulations tested, a wettable powder formulation and a liquid formulation exhibited the excellent disease control activity without the toxicity problem. Some formulations also showed good disease control activity but residual marks on the sparyed leaves. 3. Biological control effect of biofungicides Pot assay at a growth chamber Twenty four different formulations of A-7 strain were generated through 6 round of formulation process and evaluated for disease control activity against crisphead lettuce sclerotinia rot in pots of a growth chamber. Formulations such as A7-2 and A7-A exhibited disease control value of 90.2 and 83.1%, respectively

26 This control value was not significantly lower than that of chemical fungicide Benomyl with 95.86% of disease control value. In conclusion, liquid formulation A7-2 and wettable powder formulation A7-A were finally selected as candidate microbial pesticides to control the crisphead lettuce sclerotinia rot and further tested in platic house. Pot assay at a plastic house The A7-2 and A7-A exhibited disease control value of 80.5% and 79.1% in plastic house experiment, respectively, while chemical fungicide Benomyl showed 75.2% disease control value. Both A7-2 and A7-A application showed significantly higher disease control value than Benomyl application. Synergy effect of A7-2 and BW-13A formulations for bottom rot and sclerotinia rot at pot assay in a plastic house Simultaneous application of A7-2 and BW-13A in crisphead lettuce plant grown in pots in plastic house showed lowere disease control value of 70.6% against crisphead lettuce bottom rot and sclerotinia rot compared to A7-2 single application (81.3%). However, single application of BW13-A did not show effective disease control activity against two diseases. Therefore, A7-2 alone can be applied to control both crisphead lettuce bottom rot and sclerotinia rot. Field trials in production condition. Trial 1 in Euryeong: Application of 100-fold diluted A7-2, A7-A and Benomyl in production condition of crisphead lettuce showed similar disease control value of 84.7%, 84.3% and 87.0%, respectively. Dilution of A7-2 to 500-fold exhibited 70.3% disease control value. The A7-2 formulation turned out to be excellent microbial pesticide. Trial 2 in Kimhae (Feb. 2004): Natural occurrence of sclerotinia rot of

27 crisphead lettuce in Kimhae was observed and different dilution effect of A7-2 was investigated in the production condition. Treatment of the 100-fold dilution of A7-2, 500-fold dilution of A7-2 and Benomyl showed 76.7%, 60.7% and 75% of sclerotinia rot control value. The A7-2 formulation was the desirable microbial pesticide at 100-fold dilution treatment. Stability of the selected microbial pesticide. The rifampicin resistant A7 mutant was generated by spontaneous mutation and named as A7R. The A7R strain was used to evaluate the bacterial viability in lettuce rhizosphere over time. The number of A7R in rhizosphere, on lettuce plant leave and in plant crown was stably maintained up to 2.8x10 9, 1.4x10 9, 6.8x10 9 cfu/ml, respectively until 21 days. Storage stability assay of BW13-A formulation on foliar leaves Storage of both A7-2 and A7-A at 4 and room temperature did not make significant difference on bacterial survival. Bacterial density of both liquid formulation A7-2 and wettable powder formulation A7-A stored at 4 and room temperature was stable over 1 year 8 month. In addition, antifungal activity against S. sclerotiorum was stably retained in both A7-2 and A7-A

28 CONTENTS Section 1. Purpose and significance of the research 1. Background of the research 2. Importance of the research Section 2. Contents and scope of the research 1. Final goal of the research 2. Contents and scope of the research 1. Research trend in foreign country 2. Research trend in Korea 3. Patents of biopesticides in Korea 4. Problems of technology 5. Future prospect 6. Validity of technology transfer

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33 목 차 제 1 장연구개발과제의개요 38 제 1 절연구개발의목적과필요성 연구의배경 연구개발의필요성 40 제 2 절연구개발내용및범위 최종연구목표 연구개발의내용및범위 45 제 2 장국내외기술개발현황 46 제 1 절국내외관련기술의현황과문제점 외국에서의개발현황 국내에서의개발현황 국내의특허관련동향 관련기술문제점 55 5 앞으로의전망 기술도입의타당성 57 제 3 장연구개발수행내용및결과 58 제 1 연구목표 결구상추밑둥썩음병방제용미생물농약개발 제 1 절서론 58 제 2 절유용미생물의탐색및동정 연구수행방법 연구결과 69 가. 밑둥썩음병발생과병원균분리및동정 69 나. 병원성검정 73 다. 우수길항균선발

34 라. 길항미생물의혼합처리에의한방제효과증진검정 82 마. 선발길항세균의동정 85 제 3 절밑둥썩음병방제용엽면살포제미생물농약제조기술확립 연구수행방법 연구결과 93 가. S. maltophilia BW-13 균주의최적배양조건확립 93 1) 삼각플라스크배양 93 2) 발효기배양 100 나. 엽면살포제의제형화 101 1) 1차제형화 ( 수화제형, BW13-A~G) 101 2) 2차제형화 ( 액상수화제형 BW13-H 및수화제형 BW13-I, J) 102 제 4 절밑둥썩음병방제용종자처리미생물농약제조기술확립 연구수행방법 연구결과 105 가. S. maltophilia BW-13 균주에의한종자처리효과 105 나. P. aeruginosa LY-11 균주에의한우수코팅종자선발 112 다. P. aeruginosa LY-11 균주를이용한 alginate 종자코팅기술개발 116 제 5 절미생물농약의방제효과검정및우수제형선발 연구수행방법 연구결과 119 가. S. maltophilia BW-13 균주를이용한엽면살포제의방제효과 119 1) 생육실포트재배에서의밑둥썩음병방제효과 119 나. P. aeruginosa LY-11 균주를이용한 alginate 종자코팅제의방제효과 122 1) 모잘록병방제효과 122 2) 생육실포트재배에서의밑둥썩음병방제효과

35 제 6 절플라스틱하우스내토경재배에서의방제효과검정 연구수행방법 연구결과 126 제 7 절 Alginate 코팅종자의작물에서의근권및엽권정착력검정 연구수행방법 연구결과 129 가. 종자처리제의근권및엽권정착력검정 129 제 8 절엽면살포제와 alginate 코팅종자의안정성검정 연구수행방법 연구결과 133 가. BW13-A 엽면살포제의저장안정성 133 나. Alginate 코팅종자의저장온도에따른안정성검정 134 제 2 연구목표 결구상추균핵병방제용미생물농약개발 제 1 절서론 137 제 2 절유용미생물의탐색및동정 연구수행방법 연구결과 145 가. 균핵병발생과병원균분리및동정 145 나. 병원성검정 152 다. 길항미생물의분리 155 라. 길항미생물의혼합처리에의한방제효과증진검정 162 마. 우수길항세균의동정 164 제 3 절길항세균의대량배양기술확립 연구수행방법

36 2. 연구결과 171 가. B. amyloliquefaciens A-7 균주의최적배양조건확립 171 1) 삼각플라스크배양 171 2) 발효기배양 178 제 4 절균핵병방제용엽면살포제미생물농약제조기술확립 연구수행방법 연구결과 181 가. 길항세균 A-7 균주를이용한엽면살포제의제형화 181 1) 전달매체선발 181 2) 제형화 181 제 5 절미생물농약의방제효과검정및우수제형선발 연구수행방법 연구결과 185 가. B. amyloliquefaciens A-7 균주를이용한균핵병방제용엽면살포제의방제효과 185 1) 생육실포트검정에서의방제효과 185 가 ) 1차제제 ( 수화제형 A7A, A7B, A7C, A7D, A7E, A7F) 185 나 ) 2차제제 ( 수화제형 A7G, A7H, A7I, A7J, A7K, A7L) 186 다 ) 3차제제 ( 수화제형 A7M, A7V, A7O, A7P, A7Q) 187 라 ) 4차제제 ( 수화제형 A7R, A7S, A7T, A7U) 188 마 ) 5차제제 ( 수화제형 (A7-1, A7-3, A7-4, A7-5, A7-7, A7-8, A7V와액상수화제형 A7-2, A 바 ) 6차제제 ( 수화제형 A7W 제제 ) 193 제 6 절플라스틱하우스내토경재배에서의방제효과검정 연구수행방법 연구결과

37 가. 균핵병방제효과검정 196 나. 밑둥썩음병과균핵병의동시방제효과 197 제 7 절미생물농약의자연발생농가실증시험 연구수행방법 연구결과 200 가. 2004년결구상추의경상남도의령군재배포장에서의방제효과 200 나. 2005년김해시대동면재배포장에서의방제효과 202 제 8 절선발미생물농약의작물에서의엽권정착력및저장안정성검정 연구수행방법 연구결과 205 가. 토경재배한결구상추에서의 A7-2제제의엽권정착력 ( 활성 ) 검정 205 나. 저장안정성검정 206 제 4 장목표달성도및관련분야에의기여도 210 제 1 절연구개발목표의달성도 210 제 2 절관련분야의기술발전에의기여도및기대효과 221 제 5 장연구개발결과의활용계획 223 제 6 장해외과학기술정보 225 제 7 장참고문헌

38 제 1 장 연구개발과제의개요

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44 항진균성길항세균인 Stenotrophomonas maltophilia BW-13 및 Pseudomonas aruginosa LY-11 균주를이용하여결구상추밑둥썩음병방제용엽면살포제및종자처리제를개발하고, Bacillus amyloliquefaciens A-7 균주로는균핵병방제용엽면살포제의환경친화적인미생물농약을개발하고이를실용화하고자한다

45 결구상추포장의토양과건전식물체로부터이미분리된길항균외항균활성이높은길항균의지속적분리 배지상에서길항효과검정 병원균과길항균의배양적특성검정및동정 제2, 3세부과제에서개발된제제의유묘와생육상내방제효과검정 제2, 3세부과제에서개발된제제의유묘와온실내방제효과검정 개발제제의포장적용시험 개발제제의자연발생농가실증시험 항균활성물질대량생산을위한최적조건확립및대량배양용배지선발 종자처리를위한전달매체개발 종자처리제의최적화실험 종자처리제제조기술확립및제제화 종자처리제의근권정착력및활성검정 종자처리제의안정성및경시적유효도검정 균핵병방제용길항균의대량배양을위한최적조건확립및대량배양용배지선발 엽면살포제를위한전달매체개발 엽면살포제의최적화실험 엽면살포제제조기술확립및제제화 엽면살포제의엽권정착력및활성검정 엽면살포제의안정성및경시적유효도검정 엽면살포제의우수제형최종선발

46 제 2 장 국내외기술개발현황

47 미생물이용균주대상병해상품명등록국 ( 년 ) Agrobacterium radiobacter strain 84 Crown gall Galltrol Bakuterozu Dygall USA('79) Japan('89) Canada 세균 A. radiobacter K1026 Bacillus subtilis Pseudomonas cepacia Crown gall Nogall Australia Seedling root diseases Infection seed-born Quantum 4000 GUS 2000 USA USA Seedling root Blue circle USA 곰팡이 Pseudomonas fluorescens EG-1053 Streptomyces griseovirides Gliocladium virens GL-21 Pythium ligandam T.harzianum Rifaistrain KRL-AG 2 T. harzianum /polysporum T. lignorum T. viridae Damping-off Dagger G USA('88) Damping-off(Fusar ium, Alternaria etc.) Dampint-off (Rhizoctonia, Pythium) Sugar beet disease Damping-off (Pythium) Wood-decaying fungus Southern blight Sore shin(tobacco) Verticillium in mushroom Plum silver leaf disease Mycostop WRC-GL-21-W RC-AP-1 Polygandron F-Stop BinabTM T Trichoderma (spore) BINAB T SEPPIC BINAB USA USA('90) Czechoslovaki a USA USA Japan('62) France UK

48 방제미생물상품명대상병원균

49 항생물질생산균주대상병 ( 작물 ) Blasticidin-S Streptomyces griseochsomogenes Blast(Rice) 등록년도 ( 한국 ) '58('66) Kasugamycin St. kasugaensis St. kasugapinus Blast(Rice) '64('69) Validamycin St. hygroscopicus var.limoneus Sheath blight(rice) '70('76) Polyoxin Streptomycin St. cacaoi var. asoensis St. griseus Sheath blight(rice) Alternaria leaf spot(apple) Powdery mildew (Apple, Pear, Cucumber) Black rot(pear) Brown spot(tobacco) Gray mold(red pepper) Scab(Pump) Canker(Apple) Canker(Citrus) Bacteria shot hole(peach) Late blight(potato) '64('71) '44('81)

50 작물 대상병 미생물종류 보고년도

51 - 51 -

52 - 52 -

53 대상식물병균주연구팀제품명

54 파리목해충에대해방제효과를가지는바실러스슈린지에스균주를이용한미생물살충제 ( 제 호 ) 와바실러스리케니포미스 N1 및이를포함하는식물병원성진균방제용미생물제제 ( 제 호 ) 등의 2건이미생물농약의제형화로특허등록되었다

55 - 55 -

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58 제 3 장연구개발수행내용및결과 세부과제별연구기관 : 동아대학교연구책임자 : 제 1 세부과제 ( 정순재 ) 제 2 세부과제 ( 이진우 )

59 - 59 -

60 - 60 -

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63 - 63 -

64 공시병원균 PY-1 균주의효과적인병원성검정을위해 2종류의접종원을준비하여결구상추에처리하고이들의발병도를조사하여가장적합한접종원종류와처리농도를선발하였다. 먼저균사조각부유액 (triturated mycelia-inoculum) 접종원은 PDA배지에전배양한 PY-1 균주를 yeast extract glucose broth (YGB, glucose 15.0 g, K 2HPO g, KH 2 PO g, NH 4 Cl 0.54 g, yeast extract 0.50 g, MgSO g, distilled water 1 L) 에 6일간진탕배양 (150 rpm, 25 ) 한후균사를걸러내어탈이온수로 5회세척하고분쇄기 (Waring, USA) 로 15초간균사를절단하여완충용액 (ph 7.0) 으로농도 (A 550 = 0.6, 0.8, 1.0) 를조정하여접종원으로사용하였다. 다음은 WRSP배지접종원을이용하였는데, 3000 ml용삼각플라스크에 WRSP배지 ( 밀기울 : 미강 : 톱밥 : PDB배지 =30 g: 10 g: 10g: 100 ml, w/w/w/v) 를잘혼합하여고압살균 (121, 20분 ) 하고여기에 PY-1 병원균의균사절편 (9 mm) 을 10개이식한후 25 의항온기에 3주간배양하여흰색의균사가삼각플라스크내에완전히퍼지면살균수 1 L를첨가하여분쇄기로 15 초간갈아접종원으로이용하였다. 이때, 균사조각부유액접종원은포트재배한 30일된결구상추잎에주당 40 ml를농도별로분무살포하였으며, WRSP배지접종원은포트내토양 800 g 당 20, 30, 40 ml의처

65 리량별로토양표면과아랫잎밑둥부위에접종하였다. 접종한결구상추를생육실 (25, 상대습도 90%) 에보관하면서매일발병도 (%) 를조사하였다. 이때각처리구는포트당 1주씩 3 포트, 3반복으로 3회조사되었으며이를평균하여주당발병도로표시하였다. 발병도 (%) 는농약등록시험기준과방법에따라병반면적율을조사하여다음과같이발병도로환산하였다 ( 한국농약공업협회, 2001). 발병도 (%) = ( 발병수 계수 ) 4 엽수 ml

66 , PY-1 (A 550 =1.0) (Kim, 2004), = 무처리의발병도 - 처리구의발병도 무처리구의발병도 x

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69 2003년 10월에서 11월사이에경상남도의령군부림면신반리의결구상추재배플라스틱하우스 6동에발생한밑둥썩음병의발병율을조사한결과, 평균이병주율은 5.3% 이었다 (Table 1, Fig. 1A). 밑둥썩음병의병징은발병초기에아랫잎밑둥부위에수침상의원형내지타원형의담갈색또는갈색점무늬가나타나고, 진전되면윗잎쪽으로병반이부정형으로크게확대되어결국잎이무르게썩고누렇게변하며종이처럼말라죽는증상이관찰되었다 (Fig. 1B, 1C, 1D). 한편, 밑둥썩음병의발생후에 2차적으로균핵병과잿빛곰팡이병이유발되어작물에더큰피해를주었는데, 기온이다소높고비가많이오는장마철이나가을비가계속되는시기에수확기가가까워지면서병반이확대되고그표면에검고편평한균핵이발생하는등그피해정도가심각해졌다. crisphead Plastic house a Disease severity index (%) b Ⅰ Ⅱ 6.6 Ⅲ 6.8 Ⅳ 8.5 Ⅴ 8.0 Ⅵ 2.1 mean 5.3 Disease severity index

70 B D 병든결구상추의아랫잎밑둥부위에서분리된 30 균주를결구상추의윗잎과아랫잎에각각접종하여병원성검정을실시하였다. 그결과대부분의균주가잎윗부분과아래밑둥부분에서강한병원성을보였으며, 잎윗부분과아래부분에서모두병원성이강한 PY-1 균주를공시

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73 Fig. 4)

74 Disease incidence (%) OD 0.6 OD 0.8 OD ml 30 ml 40 ml Non incubation periods (days) Fig. 4. Selection of effective inoclumn concentration of Rhizoctonia solani PY-1 isolate for the pathogenicity on crisphead lettuce at pots in a growth chamber. Disease severity index (%) was obtained by measuring the percentage of infected leaf area on each plant by R. solani PY-1 isolate. A 550 = 0.6 (- -), 0.8 (- -) and 1.0 (- -) of triturated mycelia-inoculum ; 20ml (- -), 30ml (- -), and 40ml (- -) of WRSP inoculum ; - -, non-treatment. R. solani PY-1균주의균사조각부유액의처리량에따른모잘록병의병원성검정을실시한결과, 플러그포트재배에서 1 플러그당 1 ml(a 550 =0.8) 을처리한구가 7일째에 50% 의이병주율을나타내어가장적정한접종원의처리량으로선발되었다 (Table 2)

75 Table 2. Pathogenicity and selection of suitable inoculum volume of R. solani AG-1 on crisphead lettuce at plugpots experiment ml /plant b Disease severity index (%) a 1 day 2 days 3 days 4 days 5 days 6 days 7 days Control a.8) 감염초기에잎아래부분에원형내지타원형의암갈색점무늬가형성되고진전되면서부정형으로확대되었다. 병이심해지면줄기의지제부가잘록해지고썩어결국유묘가쓰러지고심하게감염된잎은흑색으로변하여썩어말라죽었다 (Fig 5)

76 Fig. 5. Symptoms of damping-off caused by Rhizoctonia solani PY-1 isolate for the pathogenicity on crisphead lettuce at plugpots in a growth chamber. A, spot rot on stem; B, spot rot on leaves; C, irregular spot rot on leaves; D, rotten leaves; E, fallen seedling; E, decay of seedlings., 702. R. solani PY-1 PDA,

77 Antagonistic bacteria Inhibition zone R-13 + a R R RH-1 + RH-4 ++ CAA-2 ++ BW

78 RH-1 RH-4 R-13 R-26 R-39 CAA-2 BW

79 100 Seedlings a a Control value (%) b b cd b e cd Adult plants c de e e c d 20 0 R-13 + Pa R-26 + Pa R-39 + Pa RH-1 + Pa e RH-4 + Pa CAA-2 + Pa Treatments d BW-13+Pa Pathogen (Pa) only g g Control

80 - 80 -

81 Table 4. Inhibitory effect of 14 antagonisitic bacteria on mycelial growth of R. solani PY-1 on PDA medium Isolates BW13 KT-25 LY-11 N1 Pro-EB-15 CT11 CT13 Inhibition zone (mm) 4.5 b 0.0 d 7.0 a 2.8 c 2.8 c 0.0 d 0.0 d 이들 2균주를포함한총 11 균주를결구상추종자에처리한후유묘의생장촉진효과를검정한결과, LY-11 균주가 BW-13 균주보다유근의길이, 묘생체중및건조중의촉진효과가약간우수하였다. 따라서, 종자처리미생물농약제조에이용할길항세균으로 LY-11 균주를최종선발하게되었다 (Table 5). 이상의결과로, BW-13 균주는밑둥썩음병방제용엽면살포제개발에, LY-11 균주 는종자처리미생물농약개발에이용할우수길항균으로각각최종선발되었다

82 Table 5. Growth of coated crisphead seeds with the each 11 antagonistic bacteria Isolates Length of young root ( cm ) Dry weight (g) Seedling Fresh weight (g) R c 11.4 e 3.0 c R a 16.5 a 3.5 b R c 12.4 d 3.8 a S ab 15.5 b 2.6 de LY a 16.6 a 3.9 a BW c 12.8 cd 2.6 de Pro-EB a 15.5 h 2.4 df RH ab 14.7 b 3.9 a RH ab 15.1 b 2.3 f RH c 12.2 de 3.5 b RH d 10.4 f 2.3 f Control 12.8 ab 13.5 c 2.7 d

83 Treatments Control value (%) 7 days after inoculation R-13 + Pa 86.7 ab R-26 + Pa 75.3 b BW-13 + Pa R-13+R-26 + Pa R-13+R-26+BW13 + Pa Pathogen (Pa) only Control 89.4 ab 90.1 ab 96.6 a 0.0 d a

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86 Characteristics BW-13 Stenotrophomonas maltophilia a Gram strain No. of flagella >1 >1 Endospore formation Cell diameter > 1.0 μm Anaerobic growth + + Growth in NaCl 2% + + 5% + + 7% + Growth at Growth at ph ph ph Activity of Catalase + + Protease + + Chitinase ± b Starch Hydrolysis + + Nitrate reduction + + Utilization of Citrate + + Glucose + + Sorbitol + Oxidase reaction + + Plant pathogenicity

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89 1 GGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGAAGGGAGC 61 TTGCTCCTGGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGG 121 GGATAACGTCCGGAAACGGGCGCTAATACCGCATACGTCCTGAGGGAGAAAGTGGGGGAT 181 CTTCGGACCTCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAAAGG 241 CCTACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAG 301 ACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCC 361 TGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGG 421 GAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAGAATAAGCACCGG 481 CTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTG 541 GGCGTAAAGCGCGCGTAGGTGGTTCAGCAAGTTGGATGTGAAATCCCCGGGCTCAACCTG 601 GGAACTGCATCCAAAACTACTGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGT 661 AGCGGTGAAATGCGTATATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTG 721 ATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCC 781 ACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAAC 841 GCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAATGAATTGACGGG 901 GGCCCGCACAAGCGGTGAAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTG 961 CCCTTGACATGCTAAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAG 1021 GTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGC 1081 GCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGA 1141 CAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACA 1201 CACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATA 1261 AAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAG 1321 TAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC 1381 ACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCGCAAGGGGGACGGTTACC 1441 ACGGAGTGATTCATGACTGGGGTG

90 - 90 -

91 Casein, Casamino acid, (NH 4) 2SO 4, Beef extract, Yeast extract) 과

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93 2 Optical density (550nm) BW Incubation periods (hours)

94 Optical density(550nm) a a 24 b b 48 c d d c d e f d g d Temperature Optical density(550nm) a a a a ab ab 24 b 48 b c e ph

95 Optical density(at 550nm)) A B C D E F G H I J K Low molecular weight carbon sources

96 Optical density(at 550nm)) A B C D E F G H High molecular weight carbon sources

97 Inhibition zone(mm) A B C D E F G H I J K L Low molecular weight carbon sources 7 6 Inhibition zone(mm) A B C D E F High molecular weight carbon sources

98 길항세균 BW-13균주의생육에미치는질소원영향을조사하기위해서탄소원제빵개량제 (3.0%) 가첨가된기초배지에저분자질소원 (Casein, Casamino acid, (NH 4 ) 2 SO 4, Beef extract, Yeast extract) 과고분자질소원 (Soy flake, Soy meal, Biji) 을각각 0.5% 로첨가하고여기에 BW-13균주를접종하여진탕배양 5일동안 OD값을측정하였다. 그결과 Yeast extract와비지가루 (Biji) 배지에서 OD 값이 5.9와 5.6 순으로높았다 (Table 8). Table 8. Nitate sources Optical density (550nm) 24 hr 48 hr 72 hr 96 hr 120 hr Casein 4.5a 4.8a 5.0a 5.0ab 5.0bc Casamino acid 1.8e 2.0e 2.2e 2.7c 3.2d (NH4)2SO4 0.1f 0.0f 0.1f 0.1e 0.1f Beef extract 4.0b 4.3ab 4.6ab 4.5b 4.6c Yeast extract 4.0b 4.2ab 4.9a 5.2a 5.9a Soy flake 3.1cd 3.3cd 3.8cd 4.5b 4.8bc Soy meal 2.5d 2.9d 3.1d 3.1c 2.8d Biji powder 3.4bc 3.8bc 3.9bc 4.7ab 5.6ab NB 1.5e 1.8f 1.9f 1.8e 0.2f Basal media 0.0f 0.1e 0.1e 0.1d 1.8e 배지가저지대 5.6 mm으로우수질소원이었다

99 Nitrogen sources (0.5%) Inhibition zone ( mm ) BW-13 Casein 3.3d Casamino acid 4.3c (NH4)2SO4 2.9e Beef extract 2.0e Yeast extract 8.8a Soy flake 2.1e Soy meal 4.0c Biji powder 5.6b

100 Time cfu/ml 24 hr hr hr media 24 hr 48 hr 72 hr Dough Conditioner Dough Conditioner+Sucrose

101 이상플라스크및발효기배양결과로기초배지 BW-13 균주의대량 배양용배지로최종선발하였다. Table 12와같이 Table 12. Formulation of biofungicides using commercial additives and carriers Formulation name Sources BW13-A Corn starch 40%, Modified starch 10% BW13-B Modified starch 40% BW13-C Corn starch 40% BW13-D Tapioca starch 40%, Modified starch 10% BW13-E Tapioca starch 40% BW13-F Corn starch 40%, Meju flour 5% BW13-G Modified starch 40%, Meju flour 5%

102 Fig 21. Powder type formulation BW13-A using commercial additive and carrier Formulation of biofungicides using commercial additives and carriers Formulation name Sources BW13-H Suncreamy 8% BW13-I Sunsize 8% BW13-J Sun supergel 8%

103 LY

104 우수코팅종자선발 앞서의밑둥썩음병방제용엽면살포제이용으로선발된 S. maltophilia BW-13 균주를이용한종자처리제개발대신에종자처리제로선발된 종자코팅용드럼 ( 회전기 ) 에결구상추종자 20 g을넣고 LY-11 균주배양액 (297 ml) 과접착제썬슈퍼젤 (3 g, 1%) 을혼합한접착제배양액을분당 1회씩스프레이로총 5~10회정도살포한다음접착제배양액이골고루입혀지면전달매체 (carrier) 인 AF300( 생석회형흡습제, 왕표화학 ) 을 Table 18의코팅종자별성분에따라 1회당처리량별로손으로종자에흩뿌려피막화하였다. 경우에따라수분이과도하게살포되어피막화하는데어려움이있을경우드럼이회전하는동안드라이어로수분을재빨리증발시킨다. 이때, 전달매체 AF300의 1차코팅이완료되면접착제배양액을계속살포해주면서 2차코팅물질인 clay, zeosil을혼합하여소량씩손으로으깨어주면서흩뿌려준다. 이어 3차코팅물질인 zeosil과 AF325( 규조토, diatomaceous earth 325, 왕표화학 ) 을처리량별로피막화한다음, 적절한습도만을유지한

105 채드럼회전으로자연건조시키고일정한크기의공모양으로피막화가된후 4 에보관하면서모든실험에이용하였다. 세균부유액과각종의전달매체로코팅된종자는상토를넣은플러그포트에파종하고생육실 (25, 상대습도 71%) 에서 12일간배양하여종자의출아율 (%), 유근길이, 주근의길이를측정하여이들중가장효과적인코팅종자를선발하였다. LY-11 LY

106 LY-11 균주로 LY-11 LY-11 Treatments Emergency Length of root rate (%) (cm) 10 6 cfu/ml T50 (days) Noncontaminated R ab 8.5a 4.2a 2.8 LY b 8.0a 5.0a 2.6 soil BW a 8.4a 5.6a 2.6 R bc 6.4b 2.2b 3.9 contaminated soil LY b 6.7b 2.0b 3.8 BW b 7.0b 2.9b 3.7 Pathogen only 26.7c 4.8c Control 77.8ab 8.1a

107 Treatments Shaking time BW-13 Emergency rate (%) Result of experiment Length of root (cm) cfu/ml T50 (days) 10 min. 92.0a 6.3a 0.2bc min. 92.0a 6.4a 1.3b hr. 94.0a 6.7a 4.3a hr. 96.0a 6.8a 4.7a hr. 94.0a 6.5a 4.9a hr. 88.0a 6.5a 4.8a hr. 16.0b 5.3b 5.0a 4.8 Control 90.0a 6.5a

108 Treatments Soaking Result of experiment Emergency Length of time T50 (days) rate (%) root (cm) cfu/ml 30 min. 86.0a 6.1a 0.2b 2.9 BW-13 1 hr. 88.0a 6.3a 1.0b hr. 92.0a 6.2a 4.3a hr. 88.0a 5.9a 4.2a hr. 88.0a 6.0a 4.5a hr. 12.0b 4.7b 4.3a 4.9 Control 90.0a 6.5a Result of experiment Treatments Dry Time Emergency rate (%) Length of root (cm) cfu/ml T50 (days) 5 min. 90.0a 6.4a 6.4a min. 88.0a 6.8a 6.9a 2.6 BW min. 88.0a 6.7a 6.7a min. 90.0a 6.6a 6.5a hr. 94.0a 6.8a 6.7a hr. 92.0a 6.4a 6.7a 2.7 Control 90.0a 6.5a

109 처리구에서는유근길이가가장길었다 Treatments mineral salts Emergency rate (%) Length of root (cm) T50 (%) (cfu/ml) K 3PO 4 100mM 92.0a 6.3ab a 50mM 94.0a 6.4ab a 200mM 88.0a 6.0ab a KH 2PO 4 100mM 94.0a 6.5ab a 50mM 96.0a 7.1a a 200mM 90.0a 6.4ab a KNO 3 100mM 88.0a 6.0ab a 50mM 92.0a 6.3ab a BW mM 84.0a 5.4ab a 50mM 20.0c 5.9ab - 4.6a NaCl 100mM 0.0d mM 0.0d mM 96.0a 6.5ab a Ca(NO 3) 2 4H 2O 100mM 92.0a 6.1ab a 200mM 86.0a 5.7ab a 0.5% 94.0a 6.4ab a PEG % 86.0a 6.2ab a 2.0% 74.0b 6.0ab a Only BW a 6.3ab a

110 Methyl cellulose 400은농도가높을수록효과적이었으나, arabic gum은정반대의경향이었다. 따라서가장유근길이가높았던 1.5% methyl cellulose 400를접착제로선발되었다 (Table 19). 하지만, 다음의 LY-11 균주를이용한종자코팅제개발에서는접착제재료는환경친화적이면서길항미생물의생육을돕는무독한물질을선발하고자노력하였으며, 최종적으로변성전분의종류인슈퍼겔을접착제로선발하여이용하였다. Young root length (cm) 0.5% 1.0% 1.5% Methyl cellulose ab 3.44a 3.60a Tween c 0.80cd 0.26d Carboxymethyl cellulose 0.25c 0.20d 0.19d Polyvinyl alcohol 0.69c 0.90cd 0.9cd Arabic gum 2.25a 2.13b 1.91b

111 6 young root length (cm)) Kaolin Clay Calcium stearate diatomaceous earth 325 AF-300 Celite Carrier Zeolite Calcium carbonate Talc Dolomite

112 우수코팅종자선발 71.1% 로대체로높았으나, 비코팅종자의발아율 92.2% 보다는낮았다. 이는드럼내에서종자가전달매체 (carrier) 와접착제 (sticker) 등에의해혼합되는동안종자가다소손상을입는것으로예측되었다. Table 20. Amount of coating materials for seed coating using by adhesive and carriers (unit : g) coating materials (carrier) Type of coating seeds 1st 2nd 3rd AF300 clay zeosil AF325 zeosil AF325 A B C D E

113

114 Type of coated seeds A B C Emergency rate (%) 12 days after sowing 51.1bc 61.1ab 78.9ab D 22.2c E 71.1ab uncoated seeds 92.2a

115 12 days after sowing Type of coated seeds Young stem length( mm ) Young root length ( mm ) A 22.3bc 48.0a B 15.7c 47.4ab C 28.2ab 33.2c D 33.0a 35.4c E 26.2ab 38.5bc uncoated seeds 25.9ab 38.3c A B C D E F

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117

118 1 포트재배에서의밑둥썩음병방제효과 BW-13 균주를이용하여제조한 1차개발미생물제제와 2차개발미생물제제의밑둥썩음병방제효과를생육실포트재배에서검정하였다. 먼저, 미생물제제 100배희석액 (BW13A~G, BW13H~J) 과화학농약펜시쿠론 (Pencycuron, 품목명 : 몬세렌액상수화제 ) 1000배희석액을분무기로포트재배한 8주된결구상추잎의앞, 뒷면에골고루각각 100 ml 씩살포하고, 살포 24시간후에밑둥썩음병균 R. solani PY-1 균주의균사조각부유액접종원 (A 500 =1.0) 40 ml을잎의앞, 뒷면에골고루살포하여, 생육실 ( 상대습도 90%, 온도 24~25 ) 에보관하였다. 접종 7일과 10일후발병도를조사하고방제가로환산하였다. 균사조각부유액은 300 ml의 YGB (yeast glucose broth) 배지에 PDA배지에서전배양한 PY-1 균주의균사절편 15개를접종하여 7일간진탕배양 (25, 160 rpm) 한후균사를걸러내어탈이온수로 5회정도세척하였다. 이를분쇄기 (Warning, USA) 로 15초간균사를마쇄한후농도를 A 550=1.0이되도록완충용액으로조정하여이용하였다. 방제가는다음과같이환산하였다. = x

119 1 방제효과 LY-11 균주를이용한 alginate 코팅종자와비코팅종자를플러그포트에 1개씩파종하고발아한지 3일된어린유묘에밑둥썩음병원균 R. solani PY-1 접종원균사조각부유액 (A 550 =0.8) 1 ml을접종하고이를 25, 상대습도 90% 의생육실에서 4일간보관한뒤하우스로옮겨접종 7일후에이병주율을조사하여방제가로환산하였다. 균사조각부유액은앞서의생육실포트재배에서의밑둥썩음병방제효과검정과동일하였다. 포트재배에서의 방제효과 (A 550=1.0) 25, 상대습도 90% 의 방제가로환산하였다 가 ) 1 차제제

120 Formulations 19.2(72.4)a 20.2(85.2)a 42.5(44.0)bcd 75.9(14.9)e 60.0(18.7)e 75.9(14.9)e 39.2(48.4)bc 59.1(33.6)cd 62.5(17.7)e 78.4(4.5)fg 70.9(6.6)fg 88.(0.9)g 71.7(5.5)fg 84.2(5.6)fg 31.9(52.6)bc 37.7(50.4)bc 75.9(0.0)g 89.2(0.0g 0.0(100)a 0.0(100)a

121 나 ) 2차제제 after 6 days Formulations disease severity index (%) a disease control value (%) b BW13-H + Pa b BW13-I + Pa b BW13-J + Pa c BW13-A + Pa b Pencycuron + Pa d e Control a Disease severity

122 LY-11 균주를이용한 alginate 코팅종자와비코팅종자를플러그포트에 1개씩파종하여발아한지 3일된어린유묘에밑둥썩음병원균 R. solani PY-1 접종원의균사조각부유액 (A 550=0.8) 1 ml을접종하고이를 22 의생육실에서 4일간보관한뒤하우스로옮겨이병주율을조사하고방제가를환산한결과,

123 Treatment disease severity Disease control index (%) b value (%) Coated seed + Pa a b Uncoated seed + Pa c Coated seed a Uncoated seed a Disease severity

124 (A 550=1.0), 병원균처리토양에서의비코팅종자는 100% 의발병도를보인반면, 코팅종자는 14.6% 로서방제가는 85.4% 이었다 (Table 26, Fig 30). Treatment Disease severity (%) b Coated seed + Pa a 14.6b d 85.4b Uncoated seed + Pa 100.0a 0.0a Coated seed 0.0c 100.0c Uncoated seed 0.0c 100.0c

125

126

127 Formulations 20 days after treatment disease control value (%) BW13-A +Pa b 75.7b BW13-I + Pa 69.8c Pencycuron 56.3d Pathogen (Pa) only 0.0e 100.0a b

128

129 cfu/ml 이었, 1.3 cfu/ml으로가장높았으며뿌리의중간에서끝부위는 (1-3) 에는약간감소하였으나, 1.1 cfu/ml 정도로유지되었다. 그러나잎중간에서끝부위부분 (1-2 ) 은 9.8 cfu/ml로밑둥에서뿌리부분보다낮았으며, 잎끝부위 (2-3 ) 에서는 LY-11 균주가조사되지않았다

130 , 2.1 cfu/ml 로가장높았으며뿌리끝부분에서는 3.1x cfu/ml 으로유지되었으며, 잎중간부분 (1 ~2 ) 에서로 9.6x 10 3 으로유지되었으나잎끝부분 에서는관찰되지않았다. Section cfu/g of tissue Log (cfu/g of tissue) seedlings Adults 2'-3' - - Leaf 1'-2' 9.8 x ' 6.8 x x Root x x '-3' - - Leaf 1'-2' 9.6 x ' 7.3 x x Root x x

131 3.2 x 10 6, 1.8 x 10 5 cfu/ml로세균수가가장많았으며뿌리의중간부위까지는유사하였으나 (2-5), 뿌리끝부분 (6-11) 은약간감소하였지만최소 1.8 x 10 4 cfu/ml 정도로유지되었다. 그러나잎에서는잎의밑둥부위 (0-1 ) 을제외하고는 LY-11 균주가관찰되지못하였다

132 Section cfu/g of tissue Log (cfu/g of tissue) Adults Leaf Root - 5'-6' - 4'-5' - - 2'-3' ' 3.2 x x x x x x x

133

134

135 cfu/ml 였으나 cfu/ml 로조사되었다. 1 일 간격으로 7 일이지나도 cfu/ml 으로큰변화가없었으며, 50 일째조사한결과에도 생균수가안정적으로지속되는것을확인하였다 (Table 31)

136 Stored period (Day) cfu/4 seeds a

137 세부과제별연구기관 : 동아대학교연구책임자 : 제 1 세부과제 ( 정순재 ) 제 3 세부과제 ( 문병주 )

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141 발병도 (%) 는농약등록시험기준과방법에따라병반면적율을조사하여다음과같이발병도로환산하였다 ( 한국농약공업협회, 2001). 발병도 (%) = ( 발병수 계수 ) 4 엽수

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146 Plastic house Disease severity index (%) January February March April May A b 38.2 a B C Average (%) 39.0a 30.1b 24.2b 10.4c 5.7c

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148 C

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152 Disease severity index (%) a ml/pot 1 day 2 days 3 days 4 days 5 days 6 days 7 days Control

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155 A A RH RH RH RH R R R S

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159 Treatment Disease control value (%) 9 days after BJ a d BJ de Pro-EC c Pro-EC de Pro-EC e Pro-EB ab Pro-EB b Pathogen ( 0.0f Control 100.0a

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161 Disease disease control value 7 days after inoculumn Treatment disease severity index (%) a (%) A b A ab RH b Pro-EC b Pro-EB ab Pro-EB b Pathogen ( c Control a

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163 Treatment Control value (%) a A-2 + Pa 75.0 ab A-7 + Pa 80.0 ab RH-4 + Pa 80.0 ab (A-2+A-7) + Pa 80.0 ab (A-7+RH-4) + Pa 90.0 a (A-2+RH-4) + Pa 75.0 ab (A-2+A-7+RH-4) + Pa 60.0 b Pathogen (Pa) only 0.0 c Control a

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172 1.2 Optical density (550nm) Incubation periods A7 Optical density (550nm) a 24 b bc bc c d e Temp.( )

173 Optical density (550nm) d ab a a c ph

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185 1) 생육실 A-7 균주를이용하여제조한 1차미생물제제 (A7A~A7F), 2차미생물제제 (A7G~A7L), 3차미생물제제 (A7M~A7Q), 4차미생물제제 (A7R~A7U), 5차미생물제제 (A7-1~A7-8 및 A7V), 6차미생물제제 (A7W) 의 100배희석액과화학농약베노밀 1000배희석액을생육실 결구상추잎의앞, 뒷면에 100 ml씩골고루살포하고, 24시간후에 S. sclerotiorum YR-1균주의접종원인균사조각부유액 (A 550 =0.6) 40ml을잎의앞, 뒷면에골고루접종하여상대습도 90%, 온도 24~25 의생육실에보관하였다. 7일후에발병도를조사하여방제가로환산하였다. 균사조각부유액의조제는 YR-1균주를 PDB배지에서 3일간진탕배양 (25, 160 rpm) 후균사를걸러내어탈이온수로 5회정도세척하였다. 이를분쇄기 (Warning, USA) 로 15초간균사를절단한후그농도를 A 550=0.6이되도록완충용액으로조정하였다

186 Formulations Disease severity index (%) a Disease control value (%) b A7A + Pa c b A7B + Pa d A7C + Pa c A7D + Pa e A7E + Pa f A7F + Pa g Benomyl a Pathogen (Pa) only h Control a

187 Formulations Disease severity Disease control index (%) a value (%) b A7G + Pa c c A7H + Pa cd A7I + Pa b A7J + Pa de A7K + Pa e A7L + Pa de Benomyl a Pathogen (Pa) only f Control a

188 Formulations Disease Disease control severity(%) a value (%) b A7M + Pa c d A7N + Pa e A7O + Pa d A7P + Pa f A7Q + Pa c A7A + Pa b Benomyl b Pathogen (Pa) nly g Control a

189 Formulations Disease severity index (%) a Disease control value (%) b A7R + Pa c cd A7S+ Pa c A7T+ Pa ef A7U+ Pa e Benomyl b Pathogen (Pa) only g Control a

190 Formulations Disease severity Disease control index (%) a value (%) b A7-1 + Pa c ef A7-2 + Pa abc A7-3 + Pa g A7-4 + Pa ad A7-5 + Pa e A7-6 + Pa d A7-7 + Pa cd A7-8 + Pa fg A7V + Pa bcd Benomyl ab Pathogen (Pa) only h Control a

191

192 Formulations Disease severity Disease control index (%) a value (%) b A7-2 + Pa c bc A7V + Pa cd A7-4 + Pa cd A7-6 + Pa e A7-7 + Pa d Benomyl ab Pathogen (Pa) only f Control a

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194 Formulations Disease severity Disease control index (%) a value (%) b A7A + Pa c c A7-2 + Pa c A7-4 + Pa cd A7W + Pa cd Benomyl b Pathogen (Pa) only f Control a

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197 20 days after treatment Formulations disease control value (%) b A7A + Pa c 79.1b A7-2 + Pa 80.5b A7-4 + Pa 70.2c A7W + Pa 69.6d Benomyl 75.2c Pathogen (Pa) only 0.0f Control 100.0a

198 Formulations Pathogens Seven days after inoculation Disease severity Disease control index (%) value (%) A7-2 SS+RS a BW13-A SS+RS b (A7-2 + BW13-A) SS+RS ab (A7-2 + BW13-A) SS ab (A7-2 + BW13-A) RS ab Benomyl SS a Pencycuron RS a Pathogen SS+RS c Pathogen SS c Pathogen RS c

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201 Disease control value Eleven days after treatment Formulations c Disease severity index (%) a (%) b A7A (100) a A7-2 (100) a Benomyl (1,000) a A7-2 (500) b Control d

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205 x x10 9 x10 9 Days after treatment No. of bacteria (cfu/ ml) Rhizoshere soil Bottom of lettuce Leaves x x x x x x x x x x x x x x x x x x

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207 Table 40. Bacterial cell density in A7A formulation according to storage periods under various temperature conditions ( ) Cell density (cfu/ ml ) Periods (Date) Cell density (cfu/ ml ) A7A formulation A7-2 formulation 4 RT 4 RT 2003년 11월 월 년 1월 월 월 월 월 월 월 월 월 월 월 월 년 1월 월

208 Cell density (cfu/ ml ) Periods (Date) Cell density (cfu/ ml ) A7A formulation A7-2 formulation 4 RT 4 RT 2005 년 3 월 월 월 월 월 월 월

209 CONT CONT SS 100 SS 1000 MAR AUG 500 A B