ʻAINA Chudinov, V.A. Platonov, A.V. Alexandrova, S.N. ʻO Elansky
Ua hōʻike ʻia nei i kēia manawa ka acommycete fungus Ilyonectria crassa hiki ke hoʻopili i nā tubers potato. ʻO kēia hana ka mea mua e kālailai i nā ʻano biology a me ke kūpaʻa ʻana i kekahi mau fungicides o ka I. crassa strain i hoʻokaʻawale ʻia mai kaʻuala. ʻO ke kaʻina o nā ʻano kikoʻī kikoʻī o ke kānana "ʻuala" i kūlike me nā mea i loaʻa ma mua no nā fungi i hoʻokaʻawale ʻia mai nā aʻa o daffodil, ginseng, aspen a me beech, nā līlī a me nā lau tulip. Me he mea lā, hiki i nā mea kanu hihiu a me nā māla ke lilo i waihona no I. crassa. ʻO nā kānana i noiʻi ʻia i hoʻopili ʻia i nā ʻāpana kōmato a me nā ʻuala, akā ʻaʻole i hoʻopili ʻia i nā hua kōmato āpau a me ka paila potato paʻa. Hōʻike kēia i ka I. crassa kahi maʻi palapū. ʻO ka loiloi o ke kūpaʻa ʻana i fludioxonil, difenoconazole a me azoxystrobin ma kahi ʻano mea momona i hōʻike i ka pono kiʻekiʻe o kēia mau lāʻau.
ʻO ka mea hōʻike EC50 (ke kikowaena o ka fungicide, kahi e lohi ai 2 mau manawa i ka helu o ka ulu radial o ke kolone e pili ana i ka non-fungicidal control) like ia me 0.4; 7.4 a me 4 mg / l. ʻO ke kūpono o ka ulu ʻana o ka maʻi i hoʻokumu ʻia e I. crassa e pono e noʻonoʻo ʻia i ka wā o ka loiloi ʻia o ka tubato uala a me ka hoʻomohala ʻana i nā pale pale mea kanu.
ʻO ka hoʻomohala ʻana o nā microorganism phytopathogenic e alakaʻi aku ai i nā pohō nui i nā wā āpau o ka ulu ʻana a mālama ʻana i kaʻuala. Ke hoʻolālā nei i nā hana pale, ma ke ʻano he rula, lawe ʻia i nā pathogens kaulana, e like me nā ʻano o ka genera Alternaria, Fusarium, Phoma, Helminthosporium, Colletotrichum, Phytophthora, a pēlā aku. Eia nō naʻe, i loko o nā makahiki i hala aku nei, ua ʻike ʻia ka nui o nā hōʻike e pili ana i ka hōʻike ʻana o nā microorganism phytopathogenic hou ma kaʻuala. ʻAʻole aʻo maikaʻi ʻia kā lākou biology, ʻaʻole hoʻohana ʻia ka hopena o nā fungicides ma nā ʻuala e pili ana iā lākou, ʻaʻole i hoʻomohala ʻia nā ʻano hana diagnostic. Me ka hoʻomohala nui, hiki iā lākou ke hōʻino nui i ka hua o kaʻuala. ʻO kekahi o kēia mau microorganism ka hualoaʻa ascomycete fungus Ilyonectria crassa (Wollenw.) A. Cabral & Crous, i ʻike mua ʻia e nā mea kākau ma nā kūmaka potato (Chudinova et al., 2019).
Hōʻike kēia hana i nā hopena o ke kālailai ʻana o ka I. crassa kānana i hoʻokaʻawale ʻia mai nā kūpe kaʻuala. ʻO ka morphology o nā kolone a me nā hale mycelial o I. crassa, nā nukleotide o nā ʻano DNA kikoʻī kikoʻī, ka virulence i kaʻuala a me nā ʻōmato, a me ke kūʻē ʻana i kekahi mau fungicides kaulana.
Nā meahana a me nā ʻano hana
Ua hoʻohana mākou i ka laha I. crassa 18KSuPT2 i hoʻokaʻawale ʻia i ka makahiki 2018 mai ka tuber uala i loaʻa i ka ʻāina ʻo Kostroma. Ua loli ʻia ka tuber e kahi ʻano popopo maloʻo me kahi lua i uhi ʻia me ka mycelium enaena māmā. I ka hoʻohana ʻana i kahi nila kuʻi ʻole, ua hoʻoneʻe ʻia ka mycelium fungal i loko o kahi ipu Petri me kahi ʻano agar (pia wort 10%, agar 1.5%, penicillin 1000 U / ml). Hoʻokomo ʻia nā pā i ka pouli ma 24 ° C.
Hoʻohana ʻia kahi microscope kukui Leica DM2500 me kahi kāmela uila ICC50 HD a me kahi microscope binocular Leica M80 me kahi kamera uila IC80HD (Leica Microsystems, Kelemānia) e paʻi kiʻi ai, loiloi i ka nui a me nā morphology o nā spores a me nā spore organ.
No ka hoʻokaʻawale ʻana iā DNA, ua ulu ka mycelium fungal i loko o ka wai pea, a laila paʻahau ʻia i ka nitrogen wai, homogenized, hoʻokomo ʻia i loko o ka buffer CTAB, hoʻomaʻemaʻe ʻia me ka chloroform, a holoi ʻelua ʻia me ka 2% ʻalekohola.
Hōʻike kikoʻī ʻia ka hana hoʻoiho DNA ma ka ʻatikala e Kutuzova et al. (2017).
No ka hoʻoholo ʻana i nā laha e nā hana moleʻaleʻa a hoʻohālikelike ʻia me nā ʻano I. crassa i ʻike ʻia, ua hana ʻia ʻo PCR me nā primers i ʻae ʻia e hoʻonui i nā wahi DNA kikoʻī kikoʻī: ITS1-5,8S-ITS2 (primers ITS5 / ITS4, White et al., 1990) -tubulin (Bt2a / Bt2b, Glass, Donaldson, 1995) a me ka unuhi elongation factor 1α (tef1α) (primers EF1-728F / EF1-986R, Carbone and Kohn, 1999). Ua kiʻi ʻia nā amplicons o ka lōʻihi i makemake ʻia mai ka gel e hoʻohana ana i ka pahu Evrogen CleanUp. Ua hoʻonohonoho ʻia nā wahi i hoʻonui ʻia me ka hoʻohana ʻana i ka BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) ma kahi Applied Biosystems 3730 xl automater sequencer (Applied Biosystems, CA, USA). Ua hoʻohana ʻia nā kaʻina nukleide hopena e ʻimi i kahi pāʻani i ka US National Center for Biotechnology Information (NCBI) GenBank database. Ua hana ʻia ka hoʻopili Phylogenetic e hoʻohana ana i ka papahana MEGA 6 (Tamura et al., 2013).
Hoʻoholo ʻia ka virulence ma luna o nā hua ʻōmaʻomaʻo piha o kahi ʻōmato hua nui (ʻano Dubrava) a me nā kūmaka potato (ʻano Gala. Hoʻohui ʻia, e simulate i ka poino i nā hua a me nā tubers i hōʻino ʻia, ua hoʻohana mākou i nā ʻāpana o nā hua like a me nā kūpe. Hoʻokomo ʻia nā ʻāpana o nā tubers i nā keʻena humuhū, ʻo ia nā ipu Petri me ka pepa kānana pulu ma lalo. Ua kau ʻia kahi pāheʻe ma luna o ka pepa, a ma luna, ua kau ʻia nā ʻāpana o nā kūmole a i ʻole nā huaʻai. Hoʻokomo ʻia nā tubers a me nā huaʻai i loko o nā pahu me ka pepa kānana pulu ma lalo. I waenakonu o ka ʻāpana (a i ʻole ma ka ʻaoʻao paʻa o ka tuber a hua paha), kau ʻia kahi ʻāpana agar (5 × 5 mm) me ka hyphae fungal ma hope o 5 mau lā o ka ulu ʻana ma ka agar wort.
ʻO ka loiloi ʻana i ke kūpaʻa ʻana o nā maʻi fungal i nā fungicides i lawe ʻia i loko o nā keʻena hoʻokolohua ma ka agar nutrient medium. Ua aʻo mākou i ka maʻalahi i nā lāʻau fungicidal Maxim, KS (mea hana fludioxonil, 25 g / l), Quadris, KS (azoxystrobin 250 g / l), Scor, EC (difenoconazole 250 g / l) (State katala ..., 2020). Lawe ʻia ka loiloi i nā kīʻaha Petri ma kahi medium wort-agar me ka hoʻohui ʻana o nā lāʻau i aʻo ʻia me ka noʻonoʻo o ka mea hana o 0.1; ekahi; 1 ppm (mg / L) (no fludioxonil a me difenoconazole), 10; umi; 1 ppm (no ka azoxystrobin) a ma ka pāpaho me ka ʻole o ka fungicide (kaohi). Hoʻohui ʻia ka fungicide i hoʻoheheʻe ʻia a hoʻoluʻolu i ka 10 ° C waena, a ma hope iho ua ninini ʻia ka mea i loko o nā ipu Petri. Ua hoʻokau ʻia kahi āpala agar me ka mycelium fungal i waenakonu o kahi ipu Petri a mahi ʻia ma kahi mahana o 100 ° C i ka pouli. Ma hope o 60 mau lā o ka hoʻouluulu ʻana, ana ʻia nā anawaena o nā kolone i nā ʻaoʻao kaulike ʻelua. ua hoʻohālikelike ʻia nā hopena ana no kēlā me kēia kolone. Hana ʻia nā hoʻokolohua i ka pālua. Ma muli o nā hopena o nā kālailai, ua helu ʻia ka EC24, e like me ka nui o ka fungicide, ʻo ia ka hapalua o ka ulu radial o ke kolone e pili ana i ka mana fungicidal.
Nā hopena a me nā kūkā kamaʻilio
Ma nā ipu petri me ka agar wort, ua hoʻokumu ʻia ka fungus i nā kolone me ka mycelium keʻokeʻo keʻokeʻo. Ua huli ka mea waena ma lalo o ka mycelium i ʻulaʻula. Ke maloʻo ka mea waena, hana ka fungus i nā spores o nā ʻano ʻelua ma ka conidiophores hoʻokahi a hōʻuluʻulu ʻia i ka sporodochia liʻiliʻi. Hoʻolālā ʻia nā Macroconidia, paukūʻolokaʻa, me hoʻokahi a i ʻekolu septa, ka awelika o ka lōʻihi 27.2 µm me kahi pae o nā waiwai mai 23.2 a 32.2 µm, ākea - a i 4.9 µm (Fig 1). ʻO ka lōʻihi awelika o ka microconidia he 14.3 µm me kahi pae o nā waiwai mai 10.3 a 18.1 µm, a hiki i ka 4.0 µm ka laulā. Kūpono nā makona a me nā micromorphological a pau i ka laulā o ka loli o ka laha Ilyonectria crassa (Cabral et al., 2012).
ʻO ke kaʻina o nā ʻano DNA kikoʻī kikoʻī (ITS, b-tubulin, TEF 1α) i kūlike loa me nā kaʻina o nā ʻano I. crassa a mākou i aʻo ai ma mua (Chudinova et al., 2019, Papa 1). No ke aʻo ʻana i ka laha ʻana o I. crassa i nā wahi ʻē aʻe a no ke kālailai ʻana i ke ʻano o nā moʻomeheu i hoʻopilikia ʻia, ua kālailai ʻia nā kaʻina DNA analogous ma ka waihona GenBank (Papa 1). ʻO 86 ka 100 ka mea i hoʻopili ʻia. ʻO nā kaʻina o nā ʻāpana DNA ʻekolu o ka “ʻuala” I. mānoanoa ʻo Crassa i nā kaʻina o nā ʻano i hoʻokaʻawale ʻia mai ka līlī a me nā aʻa narcissus ma Netherlands a mai ke aʻa ginseng ma Kanada. ʻAʻole i loaʻa iā mākou nā ʻano crassa I. ʻē aʻe me ʻekolu mau ʻano o nā kaʻina i kālailai ʻia i nā ʻikepili wehe. Eia naʻe, ua hōʻike ʻia ka anamanaʻo o ka waiho ʻia ʻana o ITS a me nā kaʻina b-tubulin i ka loaʻa ʻana o I. crassa ma nā lau tulip ma UK. ʻO nā fungi me kahi kaʻina ITS like i ʻike ʻia i ke kālailai ʻana i ka mycobiota o nā aʻa aspen ma Kanada a me nā aʻa beech ma ʻItalia, a me nā kōpuʻu potato ma Saudi Arabia (Papa 1). Hōʻike nā hopena o kēia noi ua hāʻawi ʻia ʻo I. crassa i ka mahele ākea a hiki ke hoʻololi i nā ʻano mea kanu.
Ke hoʻoholo nei i ka pathogenicity ma nā ʻāpana o ke kōmato a me kaʻuala ma ka lā 5, ua hiki ke anawaena o ka liona i 1.5 cm. ʻAʻole i hoʻopili ʻia ke ʻano o ka ʻimi Tomato i ka hua kōmato āpau a me ka paipu potato paʻa. Eia naʻe, ua hoʻopili ʻia nā tomba i ka ʻōmato. E kāpae i ka hiki o ka hoʻohaumia, kahi kaʻawale fungal hoʻokaʻawale ʻia mai ka mycelium i hoʻomohala ʻia ma kahi ʻoki uala i hoʻokaʻawale ʻia i kahi moʻomeheu maʻemaʻe. Kūlike loa ia i ke kānana a nā mākua. ʻOiai, ʻo I. crassa kahi maʻi palapū.
ʻO ka mālama mua ʻana i nā tubers hua me nā fungicides e hōʻemi ana i ka ulu ʻana o nā maʻi i nā mea kanu i ka wā ulu. No ke koho ʻana o nā fungicides maikaʻi, he mea nui e nānā i kahi o lākou e kūʻē iā I. сrassa. Ua aʻo ka hana i nā mea hana ākea o fungicides - fludioxonil, azoxystrobin, difenoconazole. Hoʻokomo ʻia ʻo Fludioxonil i loko o nā hui i hoʻohana ʻia no ke kāhiko ʻana i nā ʻanoʻano a me nā tubers hua ma mua o ke kanu ʻana. Hoʻohana ʻia ʻo Fludioxonil (Maxim) e mālama i nā tubers hua ma mua o ka waiho ʻana. Hoʻokomo pū ʻia ʻo Difenoconazole a me azoxystrobin i kekahi mau hoʻomākaukau i hoʻohana ʻia no ka hana ʻana i nā mea kanu, a me nā hoʻomākaukau i manaʻo ʻia no ka hana ʻana i nā mea kanu (State catalog ..., 2020).
Ua aʻo ʻia ka helu ulu o I. crassa ma ka pāpaho (Fig 2) me nā ʻano like ʻole o nā mea hana: fludioxonil (EC50 = 0.4 ppm), azoxystrobin (EC50 = 4 ppm), a me difenoconazole (EC50 = 7.4 ppm) (Papa 2). Hiki ke noʻonoʻo pono ʻia kēia mau hoʻomākaukau kūʻēʻē kūʻē iā I. crassa, ʻoiai ʻo kā lākou EC50 ʻoi aku ka haʻahaʻa ma mua o ka paukū i manaʻo ʻia o ka hoʻomākaukau i ka wai hana i hoʻohana ʻia no ka mālama ʻana i nā tubers Wahi a ka State Catalog ... (2020), ka hoʻopaʻapaʻa ʻana o fludioxonil i ka wai no ka mālama ʻana i nā ʻōpala potato mai 500 a 1000 ppm, azoxystrobin (i ka wai no ka mālama ʻana i ka lalo o ka ʻaihue) - 3750-9375 ppm, difenoconazole (i ka wai no ka mālama ʻana i nā mea kanu mea kanu) - 187.5– 625 ppm.
Pakuhi 1. Ka like o nā kaʻina o nā ʻāpana kikoʻī o nā kānana 18KSuPT2 a me nā ʻano o Ilyonectria crassa i loaʻa i ka waihona pūnaewele Genbank.
Pau ʻole | Mea kanu hoʻokipa, kahua o excretion | Waiho ʻia nā helu hoʻonohonoho i GenBank, ka pākēneka o nā like | loulou | ||
KANAKA | .-tubulin | TEF 1α | |||
17KSPT1 a me 18KSuPT2 | Uī ʻū, ʻāpana ʻo Kostroma | MH818326 | MH822872 | MK281307 | Chudinova et al., 2019, kēia hana |
CBS 158/31 | Nā aʻa Narcissus, Netherlands | ʻO JF735276 100 | ʻO JF735394 100 | ʻO JF735724 99.3 | ʻO Cabral et al., 2012 |
CBS 139/30 | ʻO Lily u kukui, Netherlands | ʻO JF735275 100 | ʻO JF735393 99.7 | ʻO JF735723 99.3 |
|
NSAC-SH-1 | Aʻa Ginseng, Kanada | AY295311 99.4 | ʻO JF735395 100 | JF735 / 725 99.6 |
|
RHS235138 | Lau tulip, UK | KJ475469 100 | KJ513266 100 | ʻO ND | Denton, Denton, 2014 |
MT294410 | Nā aʻa Aspen, Kanada | MT294410 100 | ʻO ND | ʻO ND | ʻO Ramsfield et al., 2020 |
ER1937 | ʻO Beech, ʻItalia | KR019363 99.65 | ʻO ND | ʻO ND | ʻO Tizzani, Haegi, Motta. Waiho pololei |
KAUF19 | ʻUala potato, Saudi ʻAlapia | HE649390 98.3 | ʻO ND | ʻO ND | ʻO Gashgari, Gherbawy, 2013 |
ND = ʻaʻole i waiho ʻia
Pakuhi 2. Ke kūʻē ʻana o Ilyonectria crassa i nā fungicides
(ʻeleu hana) | EC50, ppm | ||||
ʻO 3 lā | ʻO 5 lā | ʻO 7 lā | |||
Nā mana | 17 ± 2 | 33 ± 5 | 47 ± 3 | ||
ʻO Quadris, KS (fsoxystrobin) | 18 ± 1 | 34 ± 2 | 48 ± 2 | ||
11 ± 1 | 11 ± 1 | 12 ± 1 | |||
11 ± 1 | 11 ± 1 | 12 ± 1 | |||
ʻO Maxim, 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 |
I kā mākou hana, ua hoʻokaʻawale ʻia ʻo I. crassa strains mai nā kōpuʻu ʻuala ma nā ʻāina ʻo Kostroma a me Moscow (Chudinova et al., 2019). Ua hōʻike ʻia kahi mahele kiʻekiʻe o nā maʻi fungal me nā kaʻina ITS e like me I. crassa i ke kālailai ʻana i ka mycobiota o nā kūmaka potato ma Saudi Arabia (Gashgari and Gherbawy, 2013). ʻIke, ʻaʻole liʻiliʻi ʻo I. crassa ma kaʻuala e like me ka mea. Ua hōʻike kā mākou mau hoʻokolohua hiki i ka fungus ke hoʻopili i nā hua kōmato i hōʻino ʻia. ʻIke ʻia mai nā puke i hiki i ka I. crassa ke hoʻomohala i ka lepo saprotrophically (Moll et al., 2016), a me ka loli ʻana i nā ʻano mea kanu like ʻole, a ʻo nā mea ʻauhau hoʻi e like me nā daffodil, nā līlia, nā ginseng, aspen, a me nā beech (Papa 1). ekahi). Me he mea lā, hiki i nā mea kanu hihiu a me nā māla ke lilo i waihona no I. crassa Hōʻike ka mea i luna aʻe nei i ka hoʻomohala ʻana i nā ana palekana, pono pono e noʻonoʻo i ka hiki ke hoʻopili i nā kōpelu uala me kēia fungus. ʻO nā hoʻomākaukau ākea no ka mālama ʻana i nā kūmaka potato me ka fludioxonil, azoxystrobin a me difenoconazole i hōʻike i ka hopena fungicidal kiʻekiʻe e kūʻē iā I. crassa.
Ua kākoʻo ʻia kēia hana e ka Russian Foundation for Basic Research (Grant No. 20-016-00139).
Ua paʻi ʻia kēia ʻatikala ma ka puke pai "Bulletin Protection Protection", 2020, 103 (3)