DLA. Chudinov, V.A. Platonov, A. V. U-Alexandrova, S.N. Elansky
Kusanda kuboniswa ukuthi i-ascomycete fungus Ilyonectria crassa iyakwazi ukuthelela izilimo eziyizigaxa zamazambane. Kulo msebenzi, izici zebhayoloji kanye nokumelana kwamanye ama-fungicides e-I. crassa strain ehlukanisiwe namazambane kuyahlaziywa okokuqala. Ukulandelana kwezifunda ezithize zezinhlobo ezithile zohlobo “lwamazambane” kwaqondana nalezo ezitholwe ngaphambili zesikhunta ezihlukaniswe nezimpande ze-narcissus, i-ginseng, i-aspen ne-beech, ama-lily bulbs namaqabunga e-tulip. Ngokusobala, izitshalo eziningi zasendle nezingadini zingaba izinqolobane ze-I. crassa. I-strain ephenyiwe enegciwane le-tomato kanye nezingcezu zamazambane, kodwa ayizange ithele izithelo zonke zikatamatisi kanye ne-tuber yamazambane eqinile. Lokhu kubonisa ukuthi i-I. crassa iyi-parasite yamanxeba. Ukuhlolwa kokumelana ne-fludioxonil, i-difenoconazole ne-azoxystrobin endaweni yezakhi kubonise ukusebenza kahle okuphezulu kwale mithi.
Isikhombi se-EC50 (i-concentration ye-fungicide, enciphisa izikhathi ezingu-2 izinga lokukhula kwe-radial ye-coloni ehlobene nokulawula okungenayo isikhunta) yayilingana no-0.4; 7.4 no-4 mg / l, ngokulandelana. Amathuba okuthuthukiswa kwesifo esibangelwa yi-I. crassa kufanele kucatshangelwe lapho ukuhlolwa kwe-phytopathological yezilimo eziyizigaxa zamazambane kanye nokuthuthukiswa kwezinyathelo zokuvikela izitshalo.
Ukuthuthukiswa kwama-microorganisms e-phytopathogenic kuholela ekulahlekelweni okuphezulu kuzo zonke izigaba zokukhula nokugcina amazambane. Lapho uhlela izinyathelo zokuzivikela, njengomthetho, amagciwane aziwa kakhulu ayacatshangelwa, njengezinhlobo ze-genera Alternaria, Fusarium, Phoma, Helminthosporium, Colletotrichum, Phytophthora, njll Nokho, eminyakeni yamuva nje, kuye kwavela imibiko eyengeziwe. mayelana nokuvela kwama-microorganisms amasha e-phytopathogenic kumazambane. I-biology yabo ayifundwanga kahle, ukusebenza kahle kwama-fungicides asetshenziswa kumazambane maqondana nawo akwaziwa, izindlela zokuxilonga azikasungulwa. Ngokuthuthuka okukhulu, bayakwazi ukubangela umonakalo omkhulu esitshalweni samazambane. Enye yalezi zinambuzane isikhunta se-ascomycete i-Ilyonectria crassa (Wollenw.) A. Cabral & Crous, esatholwa okokuqala ngababhali ngezigaxa zamazambane (Chudinova et al., 2019).
Lo msebenzi wethula imiphumela yokuhlaziywa kwe-I. crassa strain ehlukanisiwe nezilimo eziyizigaxa zamazambane. I-morphology yamakholoni kanye nezakhiwo ze-mycelial ze-I. crassa, ukulandelana kwe-nucleotide yezifunda ze-DNA eziqondene nezinhlobo ezithile, ubungako kumazambane notamatisi, kanye nokumelana kwamanye ama-fungicides adumile kwacwaningwa.
Izimpahla nezindlela
Sisebenzise uhlobo lwe-I. crassa 18KSuPT2 olwahlukaniswa ngo-2018 kubhatata elinegciwane elitshalwe esifundeni sase-Kostroma. I-tuber yathinteka ukubola okomile okunomgodi ombozwe nge-mycelium ensundu ngokukhanyayo. Kusetshenziswa inaliti yokuhlanza eyinyumba, i-mycelium yefungal yadluliselwa esitsheni sePetri esine-agar medium (ubhiya wort 10%, i-agar 1.5%, i-penicillin 1000 U / ml). Amapuleti afakwa ebumnyameni ngo-24 ° C.
Kusetshenziswe isibonakhulu esikhanyayo se-Leica DM2500 esinekhamera yedijithali ye-ICC50 HD kanye nesibonakhulu se-binocular Leica M80 enekhamera yedijithali ye-IC80HD (Leica Microsystems, Germany) kusetshenziswe ukuthwebula, ukuhlola usayizi kanye nokumila kwezinhlamvu nezitho zembewu.
Ukuze kuhlukaniswe i-DNA, i-mycelium yesikhunta yayitshalwe endaweni eyi-liquid pea medium, bese iqandiswa ku-nitrogen ewuketshezi, ifakwe i-homogenized, ifakwe ku-CTAB buffer, ihlanzwe nge-chloroform, futhi igezwe kabili ngotshwala obungama-2%.
Indlela yokukhipha i-DNA ichazwe ngokuningiliziwe esihlokweni sikaKutuzova et al. (2017).
Ukuze kunqunywe uhlobo ngezindlela zamangqamuzana futhi kuqhathaniswe nezinye izinhlobo ezaziwayo ze-I. crassa, i-PCR yenziwa ngeziqalo ezivumela ukukhuliswa kwezifunda ze-DNA eziqondene nezinhlobo ezithile: ITS1-5,8S-ITS2 (primers ITS5 / ITS4, White et al., 1990 ), izifunda zofuzo b -tubulin (Bt2a / Bt2b, Glass, Donaldson, 1995) kanye nesici sokuhumusha se-elongation factor 1α (tef1α) (ama-primers EF1-728F / EF1-986R, Carbone and Kohn, 1999). Ama-amplicon obude obufunekayo akhishwe kujeli kusetshenziswa ikhithi ye-Evrogen CleanUp. Izifunda ezikhulisiwe zilandelaniswe kusetshenziswa i-BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) ku-Applied Biosystems 3730 xl sequencer automated (Applied Biosystems, CA, USA). Umphumela wokulandelana kwe-nucleotide wasetshenziselwa ukucinga okufanayo kusizindalwazi se-US National Center for Biotechnology Information (NCBI) GenBank. Ukuhlaziywa kwe-Phylogenetic kwenziwa kusetshenziswa uhlelo lwe-MEGA 6 (Tamura et al., 2013).
Ukunqunywa kwe-virulence kwenziwa ngezithelo eziluhlaza ezigcwele utamatisi omkhulu-fruited (izinhlobonhlobo ze-Dubrava) nezilimo eziyizigaxa zamazambane (izinhlobonhlobo ze-Gala). Ngaphezu kwalokho, ukulingisa umonakalo ezithelweni ezilimele nezilimo eziyizigaxa, sasisebenzisa izingcezu zezithelo ezifanayo nezilimo eziyizigaxa. Izingcezu zezilimo eziyizigaxa zazifakwa emakamelweni anomswakama, okwakuyizitsha zePetri ezinephepha lokuhlunga elimanzi ngaphansi. Kwafakwa isilayidi ephepheni, lapho-ke, kwabekwa izingcezu zezilimo eziyizigaxa noma izithelo. Izilimo eziyizigaxa eziphelele nezithelo nazo zafakwa ezitsheni ezinephepha lokuhlunga elimanzi phansi. Phakathi nendawo yocezu (noma endaweni engashintshile ye-tuber noma isithelo), ucezu lwe-agar (5 × 5 mm) olune-fungal hyphae lubekwe emva kwezinsuku ezi-5 zokukhula ku-wort agar.
Ukuhlolwa kokumelana nezinhlobo zesikhunta kuma-fungicides kwenziwa ezimeni zaselabhorethri endaweni yezakhi ze-agar. Sifunde ukuthambekela kwemithi ebulala isikhunta i-Maxim, KS (isithako esisebenzayo i-fludioxonil, 25 g / l), i-Quadris, i-KS (i-azoxystrobin 250 g / l), i-Scor, i-EC (i-difenoconazole 250 g / l) (ikhathalogu yombuso ..., 2020 ). Ukuhlolwa kwenziwa ezitsheni zePetri ku-wort-agar medium kanye nokwengezwa kwezidakamizwa ezihloliwe ekugxilweni kwento esebenzayo engu-0.1; 1; 10 ppm (mg / L) (ye-fludioxonil ne-difenoconazole), 1; ishumi; 10 ppm (ye-azoxystrobin) nakumaphephandaba ngaphandle kwesikhunta (ukulawula). I-fungicide yengezwe endaweni encibilikisiwe futhi ipholiswe ku-100 ° C, emva kwalokho okuphakathi kuthelwa ezitsheni zePetri. I-agar block ene-fungal mycelium yafakwa phakathi nesitsha sePetri futhi yakhuliswa ekushiseni okungama-60 ° C ebumnyameni. Ngemva kwezinsuku ezingu-24 zokufukamela, ububanzi bamakholoni balinganiswa ngezindlela ezimbili ezihambisanayo; imiphumela yokulinganisa yekholoni ngayinye yalinganiselwa. Izivivinyo zenziwe ngokuphindwe kathathu. Ngokusekelwe emiphumeleni yokuhlaziya, i-EC7 ibalwe, ilingana nokugxiliswa kwesibulala-sikhunta, okunciphise isilinganiso sokukhula kwe-radial sekoloni ngokuhlobene nokulawula ngaphandle kokubulala isikhunta.
Imiphumela nengxoxo
Ezitsheni ze-petri ezine-wort agar, ukhunta kwakheka amakoloni nge-white flocculent mycelium. Indawo ephakathi ngaphansi kwe-mycelium yaphenduka yaba nsundu ngokubomvu. Lapho okuphakathi komile, ukhunta kwakheka izinhlamvu zezinhlobo ezimbili ku-conidiophores eyodwa futhi ehlanganisiwe ku-sporodochia encane. Ama-Macroconidia anwetshiwe, ayi-cylindrical, ane-septa eyodwa kuya kwezintathu, ubude obuyisilinganiso ngu-27.2 µm nobubanzi bamanani kusuka ku-23.2 kuya ku-32.2 µm, ububanzi - kufika ku-4.9 µm (Fig. 1). Ubude obumaphakathi be-microconidia bungu-14.3 µm nobubanzi bamanani ukusuka ku-10.3 ukuya ku-18.1 µm, ububanzi bufika ku-4.0 µm. Zonke izinhlamvu ze-macro- kanye ne-micromorphological zilingana nohlu lokuhlukahluka kwezinhlobo ze-Ilyonectria crassa (Cabral et al., 2012).
Ukulandelana kwezifunda ze-DNA eziqondene nezinhlobo ezithile (ITS, b-tubulin, TEF 1α) kwaqondana ngokuphelele nokulandelana kwezinhlobo ze-I. crassa esizifunde ngaphambili (Chudinova et al., 2019, Ithebula 1). Ukuze kufundwe ukusabalala kwe-I. crassa kwezinye izifunda futhi kuhlaziywe i-spectrum yamasiko athintekile, ukulandelana okufanayo kwe-DNA kusizindalwazi se-GenBank kwahlaziywa (Ithebula 1). Ukugqagqana kwaba ngu-86 kuya ku-100%. Ukulandelana kwazo zontathu izifunda ze-DNA "yezambane" I. crassa strain kwakufana nokulandelana kwezinhlobo ezihlukanisiwe ne-lily bulb kanye nezimpande ze-daffodil eNetherlands kanye nempande ye-ginseng eCanada. Asikwazanga ukuthola ezinye izinhlobo ze-I. crassa ezinokulandelana okulinganayo okuhlaziywe okuthathu kuzigcinilwazi ezivuliwe. Kodwa-ke, ukuhlaziywa kokulandelana kwe-ITS ne-b-tubulin efakiwe kubonise ukuba khona kwe-I. crassa emaqabunga e-tulip e-UK. Ukhunta olunokulandelana kwe-ITS okufanayo kwabonakala lapho kuhlaziywa i-mycobiota yezimpande ze-aspen eCanada kanye nezimpande ze-beech e-Italy, nezilimo eziyizigaxa zamazambane e-Saudi Arabia (Ithebula 1). Imiphumela yalolu cwaningo ibonisa ukuthi i-I. crassa inokusabalalisa emhlabeni wonke futhi iyakwazi ukuthelela izinhlobo zezitshalo ezihlukahlukene.
Lapho kunqunywa i-pathogenicity ezincekwini zikatamatisi namazambane ngosuku lwe-5, ububanzi be-lesion bufinyelele ku-1.5 cm.Kulokhu, uhlobo oluphenyiwe aluzange luthelele sonke isithelo sikatamatisi kanye ne-tuber yamazambane eqinile. Kodwa-ke, ama-sepals athintekile kutamatisi. Ukuze kukhishwe amathuba okutheleleka, isikhunta esihlukanisa isikhunta esivela ku-mycelium esakhiwe ocezu lwe-tuber yamazambane sahlukaniswa saba isiko elihlanzekile. Kwakufana ngokuphelele nokucindezeleka kwabazali. Ngokusobala, i-I. crassa iyi-parasite yamanxeba.
Ukwelashwa kwangaphambi kokutshala kwezilimo eziyizigaxa zembewu ngama-fungicides kunciphisa ukukhula kwezifo ezitshalweni ngesikhathi sokukhula. Ukuze kukhethwe ama-fungicides asebenzayo, kubalulekile ukuhlola ukuthi yimaphi kuwo asebenzayo ngokumelene ne-I. сrassa. Umsebenzi wafunda izinto ezisebenzayo ezisabalele ze-fungicides - i-fludioxonil, i-azoxystrobin, i-difenoconazole. I-Fludioxonil yakhiwe ngezingxube eziningana ezisetshenziselwa ukugqoka imbewu nezilimo eziyizigaxa zembewu ngaphambi kokutshala. I-Fludioxonil (Maxim) iphinde isetshenziselwe ukwelapha izilimo eziyizigaxa zembewu ngaphambi kokugcinwa. I-Difenoconazole ne-azoxystrobin nayo ifakiwe enanini lamalungiselelo asetshenziselwa ukucubungula imbewu, kanye namalungiselelo ahloselwe ukucubungula izitshalo zemifino (ikhathalogi yoMbuso ..., 2020).
Izinga lokukhula kwe-I. crassa lafundwa emithonjeni yezindaba (Umdwebo 2) ngokugxila okuhlukile kwezithako ezisebenzayo: i-fludioxonil (EC50 = 0.4 ppm), i-azoxystrobin (EC50 = 4 ppm), ne-difenoconazole (EC50 = 7.4 ppm) (Ithebula 2 ). Lawa malungiselelo angabhekwa asebenza kahle kakhulu ngokumelene ne-I. crassa, njengoba i-EC50 yawo iphansi kakhulu kunokugxilisa okunconywayo kokulungiswa oketshezini olusebenzayo olusetshenziselwa ukwelapha izilimo eziyizigaxa. Ngokwe-State Catalogue ... (2020), ukugcwala kwe-fludioxonil oketshezini lokwelapha izilimo eziyizigaxa zamazambane kusuka ku-500 kuye ku-1000 ppm, i-azoxystrobin (oketshezini lokwelapha ngaphansi komsele) - 3750-9375 ppm, i-difenoconazole (oketshezini lokwelapha izitshalo ezimilayo) - 187.5– 625 ppm.
Ithebula 1. Ukufana kokulandelana kwezinhlobo ezithile zezinhlobo ze-strain 18KSuPT2 kanye nezinhlobo ze-Ilyonectria crassa ezitholakala kusizindalwazi se-Genbank
Ukucindezela | Isitshalo sokusingatha, indawo yokukhishwa | Izinombolo zokulandelana ezifakwe ku-GenBank, iphesenti lokufana | Izikhombo | ||
ITS | β-tubulin | TEF 1a | |||
17KSPT1 kanye ne-18KSuPT2 | I-Potato tuber, esifundeni saseKostroma | I-MH818326 | I-MH822872 | MK281307 | Chudinova et al., 2019, lo msebenzi |
I-CBS 158/31 | Izimpande zeNarcissus, eNetherlands | JF735276 100 | JF735394 100 | JF735724 99.3 | UCabral et al., 2012 |
I-CBS 139/30 | I-Lily bulb, eNetherlands | JF735275 100 | JF735393 99.7 | JF735723 99.3 |
|
I-NSAC-SH-1 | Izimpande ze-ginseng, eCanada | I-AY295311 99.4 | JF735395 100 | JF735 / 725 99.6 |
|
I-RHS235138 | Iqabunga le-Tulip, e-UK | I-KJ475469 100 | I-KJ513266 100 | ND | Denton, Denton, 2014 |
MT294410 | Izimpande ze-Aspen, eCanada | MT294410 100 | ND | ND | Ramsfield et al., 2020 |
ER1937 | Beech, Italy | KR019363 99.65 | ND | ND | Tizzani, Haegi, Motta. Ukuhambisa okuqondile |
KAUF19 | I-Potato tuber, eSaudi Arabia | HE649390 98.3 | ND | ND | Gashgari, Gherbawy, 2013 |
ND = ayifakwanga
Ithebula 2. Ukumelana ne-Ilyonectria crassa kuma-fungicides
(into esebenzayo) | EC50, ppm | ||||
Usuku lwe-3 | Usuku lwe-5 | Usuku lwe-7 | |||
Izilawuli | I-17 ± 2 | I-33 ± 5 | I-47 ± 3 | ||
I-Quadris, KS (fsoxystrobin) | I-18 ± 1 | I-34 ± 2 | I-48 ± 2 | ||
I-11 ± 1 | I-11 ± 1 | I-12 ± 1 | |||
I-11 ± 1 | I-11 ± 1 | I-12 ± 1 | |||
UMaxim, KS (fludioxonil) | I-16 ± 1 | I-28 ± 2 | I-48 ± 2 | ||
I-7 ± 1 | I-13 ± 3 | I-19 ± 4 | |||
I-5 ± 1 | I-12 ± 1 | I-17 ± 5 | |||
I-Skor, EC (difenoconazole) | I-18 ± 1 | I-35 ± 2 | I-48 ± 1 | ||
I-11 ± 1 | I-24 ± 3 | I-35 ± 4 | |||
I-11 ± 1 | I-13 ± 1 | I-17 ± 3 |
Emsebenzini wethu, izinhlobo ze-I. crassa zahlukaniswa nezigaxa zamazambane esifundeni saseKostroma naseMoscow (Chudinova et al., 2019). Ingxenye ephezulu yohlobo lwesikhunta olunokulandelana kwe-ITS efana ne-I. crassa yavezwa ekuhlaziyweni kwe-mycobiota yezigaxa zamazambane e-Saudi Arabia (Gashgari ne-Gherbawy, 2013). Ngokusobala, i-I. crassa ayitholakali kumazambane njengoba kungase kubonakale. Ekuhloleni kwethu, kwaboniswa ukuthi isikhunta singangena izithelo ezilimele zikatamatisi. Kuyaziwa kusukela ezincwadini ukuthi i-I. crassa iyakwazi ukuthuthukisa i-saprotrophically emhlabathini (Moll et al., 2016), kanye nokuthinta izitshalo ezihlukahlukene, ngisho nezikude kakhulu njenge-daffodils, iminduze, i-ginseng, i-aspen, kanye ne-beech (Ithebula 1) XNUMX). Ngokusobala, izitshalo eziningi zasendle nezingadini zingaba izinqolobane ze-I. crassa. Okungenhla kubonisa ukuthi lapho uthuthukisa izindlela zokuvikela, kuyadingeka ukucabangela ukuthi kungenzeka ukuthinta izilimo eziyizigaxa zamazambane ngalesi sikhunta. Amalungiselelo asabalele okwelashwa kwezilimo eziyizigaxa zamazambane eziqukethe i-fludioxonil, i-azoxystrobin kanye ne-difenoconazole abonise ukusebenza kahle kwesikhunta ngokumelene ne-I. crassa.
Lo msebenzi usekelwe yi-Russian Foundation for Basic Research (Grant No. 20-016-00139).
Indatshana yashicilelwa kujenali ethi "Plant Protection Bulletin", 2020, 103 (3)