LIJO. Chudinov, V.A. Platonov, AV Alexandrova, S.N. Elansky
Ho sa tsoa bontšoa hore fungus ea ascomycete Ilyonectria crassa e khona ho tšoaetsa methapo ea methapo. Mosebetsi ona ke oa pele oa ho sekaseka litšobotsi tsa bioloji le ho hanyetsa li-fungicides tse ling tsa mofuta oa I. crassa o arohileng ho litapole. Ho latellana ha libaka tse ikhethileng tsa mofuta oa "litapole" ho tsamaisane le tse fumanoeng pejana bakeng sa fungus e arohaneng le metso ea daffodil, ginseng, aspen le beech, bulbs lily le makhasi a tulip. Kamoo ho bonahalang kateng, limela tse ngata tse hlaha le tsa serapeng e ka ba mehloli ea I. crassa. Mofuta o batlisisitsoeng oa likotoana tsa tamati le litapole, empa ha oa ka oa tšoaetsa litholoana tsohle tsa langa le le lej le tuber e tiileng ea litapole. Sena se bontša hore I. crassa ke pharasaete ea leqeba. Tlhatlhobo ea ho hana fludioxonil, difenoconazole le azoxystrobin setsing sa limatlafatsi e bonts'itse katleho ea meriana ena.
Index ea EC50 (mokokotlo oa fungicide, e liehang ho feta makhetlo a 2 sekhahla sa kholo ea maqhubu a kolone mabapi le taolo ea fungicidal) e ne e lekana le 0.4; 7.4 le 4 mg / l, ka ho latellana. Monyetla oa nts'etsopele ea lefu le bakoang ke I. crassa o lokela ho tsotelloa ha tlhahlobo ea phytopathological ea litapole tsa litapole le nts'etsopele ea mehato ea ts'ireletso ea limela.
Nts'etsopele ea likokoana-hloko tsa phytopathogenic e lebisa ho tahlehelo e phahameng maemong ohle a ho holisa le ho boloka litapole. Ha ho raloa mehato ea ts'ireletso, joalo ka molao, ho tsejoa likokoana-hloko tse tsebahalang joalo ka mefuta ea genera ea Alternaria, Fusarium, Phoma, Helminthosporium, Colletotrichum, Phytophthora, jj. Baeloji ea bona ha e ithutoe hantle, katleho ea fungicides e sebelisitsoeng litapole ha e tsejoe, mekhoa ea ho hlahloba ha e so ka e etsoa. Ka nts'etsopele ea bongata, li khona ho baka tšenyo e kholo lijalo tsa litapole. E 'ngoe ea likokoana-hloko tsena ke fungus ea ascomycete Ilyonectria crassa (Wollenw.) A. Cabral & Crous, e fumanoeng pele ke bangoli ba li-tubers tsa litapole (Chudinova et al., 2019).
Mosebetsi ona o hlahisa litholoana tsa tlhahlobo ea "crassa strain" e arotsoeng ke methapo ea litapole. Morphology ea likolone le meaho ea mycelial ea I. crassa, tatellano ea li-nucleotide ea libaka tse ikhethileng tsa DNA, bongata ba litapole le litamati le ho hanela li-fungicides tse ling tse tummeng li ile tsa ithutoa.
Lisebelisoa le mekhoa
Re sebelisitse mofuta oa I. crassa 18KSuPT2 o ikhethileng ho 2018 ho tsoa ho tuber e nang le tšoaetso ea litapole e holileng sebakeng sa Kostroma. Tuber e anngoe ke mofuta o bolileng o omileng o nang le lesoba le koahetsoeng ke mycelium e sootho. Ho sebelisoa nale e sa hlatsoeng e hlabang, mycelium ea fungal e ile ea fetisetsoa sejaneng sa Petri se nang le sesebelisoa sa agar (biri ea wort 10%, agar 1.5%, penicillin 1000 U / ml). Lipoleiti li ile tsa koaheloa lefifing ka 24 ° C.
Ho ile ha sebelisoa microscope e bobebe ea Leica DM2500 e nang le kh'amera ea dijithale ea ICC50 HD le microscope ea Leica M80 e nang le kh'amera ea dijithale ea IC80HD (Leica Microsystems, Jeremane) e sebelisitsoeng ho nka lifoto, ho lekola boholo le morpholoji oa likokoana-hloko le litho tsa spore.
Ho arola DNA, mycelium ea fungal e ne e lengoa ka mokelikeli oa lierekisi tse metsi, ebe e hoamisoa ka naetrojene ea metsi, e homogenized, e kenngoe ka hara buffer ea CTAB, e hloekisitsoe ka chloroform, mme e hlatsuoa habeli ka joala ba 2%.
Mokhoa oa ho ntša DNA o hlalositsoe ka botlalo ho sengoloa ke Kutuzova et al. (2017).
Ho tseba mofuta ka mefuta ea limolek'hule le ho bapisoa le mefuta e meng e tsebahalang ea I. crassa, PCR e ile ea etsoa ka li-primers tse neng li lumella ntlafatso ea libaka tse ikhethileng tsa DNA: ITS1-5,8S-ITS2 (primers ITS5 / ITS4, White et al., 1990), liphatsa tsa lefutso -tubulin (Bt2a / Bt2b, Glass, Donaldson, 1995) le phetolelo elongation factor 1cy (tef1cy) (primers EF1-728F / EF1-986R, Carbone le Kohn, 1999). Li-amplicon tsa bolelele bo batlang li ne li ntšoa ka har'a gel ho sebelisa sesebelisoa sa Evrogen CleanUp. Libaka tse ntlafalitsoeng li ile tsa latellana ho sebelisoa BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) ho Applied Biosystems 3730 xl automencer sequencer (Applied Biosystems, CA, USA). Ho latellana ha li-nucleotide ho sebelisitsoe ho batla papali ho database ea GenBank ea Setsi sa Naha sa Naha sa Boitsebiso ba Biotechnology (NCBI). Tlhahlobo ea Phylogenetic e entsoe ho sebelisoa lenaneo la MEGA 6 (Tamura et al., 2013).
Boikemisetso ba virulence bo entsoe ka litholoana tse tala tse felletseng tsa langa le le lej tse nang le litholoana tse kholo (mefuta e fapaneng ea Dubrava) le li-tubers tsa litapole (mefuta e fapaneng ea Gala) Ntle le moo, ho etsisa tšenyo ea litholoana le methapo e senyehileng, re ne re sebelisa lilae tsa litholoana le tubers tse tšoanang. Likarolo tsa li-tubers li ile tsa beoa ka likamoreng tse mongobo, e leng lijana tsa Petri tse nang le pampiri e metsi e katlase. Slide e ile ea beoa pampiring, eo le eona ho ileng ha beoa likhae tsa li-tubers kapa litholoana. Li-tubers tse felletseng le litholoana le tsona li kentsoe ka har'a likhontheina tse nang le pampiri e metsi e katlase. Bohareng ba selae (kapa bokaholimo bo sa tsitsang ba tuber kapa litholoana), sengoathoana sa agar (5 × 5 mm) se nang le fungal hyphae se ile sa beoa kamora matsatsi a 5 a ho hola ho wort agar.
Tekolo ea ho hanyetsa mefuta ea fungal ho fungicides e entsoe maemong a laboratori molemong oa phepo ea agar. Re ithutile ho hlaseloa habonolo ke lithethefatsi tsa fungicidal Maxim, KS (sesebelisoa se sebetsang sa fludioxonil, 25 g / l), Quadris, KS (azoxystrobin 250 g / l), Scor, EC (difenoconazole 250 g / l) (Catalog ea Naha ..., 2020). Tlhatlhobo e entsoe ka lijana tsa Petri ho sesebelisoa sa wort-agar le ho eketsoa ha lithethefatsi tse ithutoang maemong a ntho e sebetsang 0.1; ngoe; 1 ppm (mg / L) (bakeng sa fludioxonil le difenoconazole), 10; leshome; 1 ppm (bakeng sa azoxystrobin) le mecheng ea litaba ntle le fungicide (taolo). Fungicide e ile ea eketsoa molemong oa ho qhibilihisoa le ho pholisoa ho isa selemong sa 10 ° C, kamora moo seaplane seo se ile sa tšeloa lijaneng tsa Petri. Sebaka sa agar se nang le fungus mycelium se ile sa beoa bohareng ba sejana sa Petri mme sa holisoa mochesong oa 100 ° C lefifing. Kamora matsatsi a 60 a poloko, bophara ba likolone bo ne bo lekantsoe ka likarolo tse peli tse lumellanang; liphetho tsa litekanyo tsa kolone ka 'ngoe li ne li lekantsoe. Liteko li entsoe ka makhetlo a mararo. Ho ipapisitsoe le liphetho tsa litlhahlobo, ho ile ha baloa EC24, e lekanang le mohopolo oa fungicide, o fokotseng sekhahla sa kholo ea radial ea kolone mabapi le taolo ea fungicidal.
Liphetho le puisano
Lijaneng tsa petri tse nang le wort agar, fungus e thehile likolone tse tšoeu tsa flocculent mycelium. Mecha e ka tlas'a mycelium e ile ea fetoha bosootho bo bofubelu. Ha seaplane se omella, fungus e thehile spores ea mefuta e 'meli ka li-conidiophores tse le' ngoe le tse kopantsoeng ka sporodochia e nyane. Macroconidia li bolelele, li-cylindrical, li na le septa e le 'ngoe ho isa ho tse tharo, bolelele ba bolelele ba 27.2 withm bo nang le litekanyetso tse fapaneng ho tloha 23.2 ho isa ho 32.2 µm, bophara - ho fihlela 4.9 µm (Setšoantšo sa 1). Bolelele ba karolelano ea microconidia ke 14.3 withm e nang le litekanyetso tse fapaneng ho tloha ho 10.3 ho isa ho 18.1 µm, bophara bo fihla ho 4.0 µm. Litlhaku tsohle tsa macro le micromorphological li lekana le mefuta-futa ea mefuta ea Ilyonectria crassa (Cabral et al., 2012).
Tatellano ea libaka tse ikhethileng tsa DNA (ITS, b-tubulin, TEF 1cy) li tsamaellane ka ho felletseng le tatellano ea mefuta ea I. crassa eo re ithutileng eona pejana (Chudinova et al., 2019, Tafole 1). Bakeng sa ho ithuta ho ata ha I.crassa libakeng tse ling le ho sekaseka mefuta e mengata ea litso tse amehileng, ho ile ha hlahlojoa tatellano e ts'oanang ea DNA ho database ea GenBank (Lethathamo la 1). Ho kopana ho ne ho le 86 ho ea ho 100%. Ho latellana ha libaka tsohle tse tharo tsa DNA tsa "litapole" I. khatello ea crassa e ne e ts'oana le tatellano ea mefuta e arohaneng le tleloubu ea lily le metso ea narcissus Netherlands le motso oa ginseng naheng ea Canada. Re hlotsoe ho fumana mefuta e meng ea I. crassa e nang le tatellano e meraro e hlahlobisitsoeng ka tatellano e fumanehang. Leha ho le joalo, tlhahlobo ea tatellano ea ITS le b-tubulin e kentsoeng e bonts'a boteng ba I. crassa makhasi a tulip UK. Li-fungus tse nang le tatellano e ts'oanang ea ITS li fumanoe tlhahlobisong ea mycobiota ea metso ea aspen naheng ea Canada le metso ea beech naheng ea Italy, litapole tsa litapole ho Saudi Arabia (Letlapa la 1). Liphetho tsa phuputso ena li bonts'a hore I. crassa e na le kabo ea lefats'e mme e khona ho tšoaetsa mefuta e fapaneng ea limela.
Ha ho khethoa tšoaetso ea likokoana-hloko ka likhae tsa langa le le lej le litapole ka letsatsi la bohlano, bophara ba leqeba bo fihlile ho cm 5. Mofuta o batlisisitsoeng ha oa ka oa tšoaetsa litholoana tsohle tsa langa le le lej le tuber e tiileng ea litapole. Leha ho le joalo, li-sepals li ile tsa ameha ka langa le le lej. Ho khetholla monyetla oa tšilafalo, fungus e itšehla thajana ho mycelium e hlahisitsoeng ka selae sa litapole e arotsoe ka setso se hloekileng. E ne e ts'oana hantle le khatello ea batsoali. Kamoo ho bonahalang kateng, I. crassa ke pharasaete ea leqeba.
Kalafo ea pele ho jala ea li-tubers tsa peo e nang le fungicides e fokotsa nts'etsopele ea mafu ho limela nakong ea ho hola. Bakeng sa khetho ea li-fungicides tse sebetsang, ho bohlokoa ho lekola hore na ke efe ea tsona e sebetsang khahlano le I. сrassa. Mosebetsi o ithutile lintho tse atileng tse sebetsang tsa fungicides - fludioxonil, azoxystrobin, difenoconazole. Fludioxonil e kenyellelitsoe mefuteng e 'maloa e sebelisetsoang ho apara peo le methapo ea peo pele o lema. Fludioxonil (Maxim) e boetse e sebelisoa ho phekola methapo ea peo pele e bolokoa. Difenoconazole le azoxystrobin le tsona li kenyelelitsoe litokisetsong tse 'maloa tse sebelisetsoang ho sebetsana le lisebelisoa tsa peo, le litokisetsong tse reretsoeng ho sebetsana le limela tsa limela (K'hathaloko ea Naha ..., 2020).
Sekhahla sa kholo ea I. crassa se ithutiloe mecheng ea litaba (Setšoantšo sa 2) ka likhakanyo tse fapaneng tsa lintho tse sebetsang: fludioxonil (EC50 = 0.4 ppm), azoxystrobin (EC50 = 4 ppm), le difenoconazole (EC50 = 7.4 ppm) (Tafole 2). Litokisetso tsena li ka nkuoa li sebetsa haholo khahlano le I. crassa, kaha EC50 ea tsona e tlase haholo ho feta mohopolo o khothalelitsoeng oa ho itokisetsa mokelikeli o sebetsang o sebelisetsoang ho phekola methapo. Ho latela State Catalog ... (2020), mokokotlo oa fludioxonil ka mokelikeli bakeng sa ho phekola methapo ea litapole e tsoa ho 500 ho isa ho 1000 ppm, azoxystrobin (ka mokelikeli oa ho phekola tlase ea foro) - 3750-9375 ppm, difenoconazole (ka mokelikeli bakeng sa ho phekola limela tsa limela) - 187.5– 625 ppm.
Tafole ea 1. Ho tšoana ka tatellano ea mefuta e ikhethileng ea mefuta ea mofuta oa 18KSuPT2 le Ilyonectria crassa strains tse fumanehang ho database ea Genbank
Mathata | Semela sa moamoheli, sebaka sa mantle | Linomoro tsa tatellano e kentsoeng ho GenBank, liperesente tsa ho tšoana | kgokahanyo | ||
ITS | β-tubulin | TEF 1A | |||
17KSPT1 le 18KSuPT2 | Tuber ea litapole, sebaka sa Kostroma | MH818326 | MH822872 | MK281307 | Chudinova et al., 2019, mosebetsi ona |
EA-158/31 | Metso ea Narcissus, Netherlands | JF735276 100 | JF735394 100 | JF735724 99.3 | Cabral et al., 2012 |
EA-139/30 | Tleloubu ea Lily, Netherlands | JF735275 100 | JF735393 99.7 | JF735723 99.3 |
|
EA-1-NSAC | Motso oa Ginseng, Canada | EA-295311 99.4 | JF735395 100 | EA-735/725 99.6 |
|
RHS235138 | Lekhasi la Tulip, UK | KJ475469 100 | KJ513266 100 | ND | Denton, Denton, 2014 |
MT294410 | Metso ea Aspen, Canada | MT294410 100 | ND | ND | Ramsfield et al., 2020 |
ER1937 | Beech, Italy | EA-019363-KR 99.65 | ND | ND | Tizzani, Haegi, Motta. Tlhahiso e tobileng |
KAUF19 | Tuber ea litapole, Saudi Arabia | HE649390 98.3 | ND | ND | Gashgari, Gherbawy, 2013 |
ND = ha e kenngoe
Tafole ea 2. Khanyetso ea Ilyonectria crassa ho fungicides
(ntho e sebetsang) | EC50, ppm | ||||
Letsatsi la 3 | Letsatsi la 5 | Letsatsi la 7 | |||
Melao | 17 ± 2 | 33 ± 5 | 47 ± 3 | ||
Quadris, KS (fsoxystrobin) | 18 ± 1 | 34 ± 2 | 48 ± 2 | ||
11 ± 1 | 11 ± 1 | 12 ± 1 | |||
11 ± 1 | 11 ± 1 | 12 ± 1 | |||
Boholo, KS (fludioxonil) | 16 ± 1 | 28 ± 2 | 48 ± 2 | ||
7 ± 1 | 13 ± 3 | 19 ± 4 | |||
5 ± 1 | 12 ± 1 | 17 ± 5 | |||
Skor, EC (difenoconazole) | 18 ± 1 | 35 ± 2 | 48 ± 1 | ||
11 ± 1 | 24 ± 3 | 35 ± 4 | |||
11 ± 1 | 13 ± 1 | 17 ± 3 |
Mosebetsing oa rona, mefuta ea crassa e ile ea aroloa ho methapo ea litapole libakeng tsa Kostroma le Moscow (Chudinova et al., 2019). Karolo e phahameng ea mefuta ea fungal e nang le tatellano ea ITS e ts'oanang le I. crassa e senotsoe ha ho hlahlojoa mycobiota ea litapole tubers ho la Saudi Arabia (Gashgari le Gherbawy, 2013). Kamoo ho bonahalang kateng, I. crassa ha se ntho e sa tloaelehang litapole kamoo ho ka bonahalang. Liteko tsa rona li bonts'itse hore fungus e ka tšoaetsa litholoana tsa langa le le lej tse senyehileng. Hoa tsebahala ho tsoa lingoliloeng hore I. crassa o khona ho nts'etsapele mobung saprotrophically (Moll et al., 2016), hape le ho ama limela tse fapaneng, esita le tse hole haholo joalo ka tekanyetso joalo ka daffodils, lilies, ginseng, aspen, le beech (Tafole ea 1). ngoe). Kamoo ho bonahalang kateng, limela tse ngata tse hlaha le tsa serapeng e ka ba mehloli ea I. crassa. Lintlha tse kaholimo li bonts'a hore ha ho etsoa mehato ea ts'ireletso, ho hlokahala hore ho nahanoe ka monyetla oa ho ama methapo ea litapole ka fungus ena. Litokisetso tse atileng tsa kalafo ea methapo ea litapole e nang le fludioxonil, azoxystrobin le difenoconazole li bonts'itse katleho e kholo ea fungicidal khahlano le I. crassa.
Mosebetsi ona o ne o tšehelitsoe ke Russian Foundation for Basic Research (Grant No. 20-016-00139).
Sengoloa se phatlalalitsoe koranteng "Bulletin ea Ts'ireletso ea Limela", 2020, 103 (3)