Cinye. Chudinov, V.A. Platonov, A.V. Alexandrova, S.N. Elansky
Kwanan nan aka nuna cewa asgus na naman gwari Ilyonectria crassa na iya cutar da tubers dankalin turawa. Wannan aikin shine farkon wanda zai binciki halayen halittu da kuma juriya ga wasu kayan gwari na I. mai kashe kwayar dankali. Jerin takamaiman yankuna na takamaiman nau'in "dankalin turawa" sun yi daidai da waɗanda aka samo a baya don fungi da aka keɓe daga tushen daffodil, ginseng, aspen da beech, kwararan fitila da kuma ganyen tulip. A bayyane yake, yawancin tsire-tsire na daji da na lambu na iya zama na I. crassa. Binciken da aka gudanar ya kamu da cutar tumatir da yankakken dankalin turawa, amma bai cutar da dukkan 'ya'yan itacen tumatir da tuber dankalin ba. Wannan yana nuna cewa I. crassa parasite ne na rauni. Kimantawa game da juriya ga fludioxonil, difenoconazole da azoxystrobin akan matsakaicin abinci mai gina jiki sun nuna ingancin waɗannan kwayoyi.
Alamar EC50 (narkar da kayan gwari, wanda ke raguwa da sau 2 adadin haɓakar radial na mulkin mallaka dangane da ikon da ba na kayan gwari ba) ya yi daidai da 0.4; 7.4 da 4 mg / l, bi da bi. Ya kamata a lura da yiwuwar ci gaban cutar da na sa ta hanyar karo. Sa'ilin da ake yin nazari kan pampoathological na tubers dankalin turawa da ci gaban matakan kariya ta shuka.
Ci gaban kwayoyin halittar phytopathogenic yana haifar da asara mai yawa a duk matakai na girma da adana dankali. Lokacin tsara matakan kariya, a matsayin doka, ana ɗaukar sanannun ƙwayoyin cuta, kamar su jinsunan Alternaria, Fusarium, Phoma, Helminthosporium, Colletotrichum, Phytophthora, da sauransu.Koda yake, a cikin recentan shekarun nan, ƙarin rahotanni sun bayyana game da bayyanar sabbin newananan ƙwayoyin cuta akan dankali. Ba a yi nazarin ilimin su ba sosai, ba a san tasirin maganin gwari da ake amfani da shi kan dankali dangane da su ba, ba a samar da hanyoyin bincike ba. Tare da ci gaba mai yawa, suna iya haifar da babbar illa ga amfanin gonar dankalin turawa. Ofaya daga cikin waɗannan ƙananan ƙwayoyin cuta shine naman gwari mai suna Ilyonectria crassa (Wollenw.) A. Cabral & Crous, waɗanda marubutan suka fara ganowa akan tubers dankalin turawa (Chudinova et al., 2019).
Wannan aikin yana gabatar da sakamakon binciken I. muguwar kwayar cuta wacce aka ware daga tubers dankalin turawa. Tsarin halittu na yankuna da tsarin gine-gine na I. crassa, jerin nucleotide na takamaiman yankuna na DNA, ƙwarewar dankali da tumatir, da juriya ga wasu sanannen kayan gwari.
kaya da matakai
Munyi amfani da nau'ikan na I. crassa 18KSuPT2 wanda aka ware shi a cikin shekarar 2018 daga cutar tuber dankalin turawa da aka shuka a yankin Kostroma. Aaƙasasshen bushe ya shafi tuber tare da ramin da aka rufe da mycelium mai ruwan kasa mai haske. Ta amfani da allura mai rarrabawa, an canza fungal mycelium a cikin abincin Petri tare da matsakaicin agar (giya wort 10%, agar 1.5%, penicillin 1000 U / ml). An saka faranti a cikin duhu a 24 ° C.
Anyi amfani da madubin hangen nesa mai suna Leica DM2500 tare da kyamarar dijital ICC50 HD da kuma Leica M80 binocular microscope tare da kyamarar dijital IC80HD (Leica Microsystems, Jamus) don daukar hoto, tantance girman da yanayin halittar spores da gabobin jiki.
Don keɓance DNA, func mycelium ya girma ne a matsakaicin matsar ruwan sha, sannan ya daskare a cikin sinadarin nitrogen, ya haɗu, aka saka shi a cikin CTAB buffer, aka tsarkake shi da chloroform, kuma aka wanke shi sau biyu tare da giya 2%.
An bayyana hanyar hakar DNA dalla-dalla a cikin labarin ta Kutuzova et al. (2017).
Don ƙayyade jinsin ta hanyar hanyoyin kwayoyi da kuma kwatanta su da sauran sanannun nau'ikan nau'ikan ruwa, an yi PCR tare da abubuwan share fage wanda ya ba da izinin haɓaka yankuna na takamaiman nau'ikan DNA: ITS1-5,8S-ITS2 (share fage ITS5 / ITS4, White et al., 1990), yankuna masu asali na b -tubulin (Bt2a / Bt2b, Glass, Donaldson, 1995) da kuma haɓakar fassara 1 factor (tef1α) (share fage EF1-728F / EF1-986R, Carbone da Kohn, 1999). An cire amplicons na tsayin da ake so daga gel ta amfani da kit ɗin Evrogen CleanUp. Yankunan da aka inganta an tsara su ta amfani da BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) akan Applied Biosystems 3730 xl mai sarrafa kansa mai amfani (Aiwatar da Biosystems, CA, Amurka) Anyi amfani da jerin nucleotide da aka samo don bincika wasa a cikin bayanan GenBank na Cibiyar Kula da Kayan Fasaha ta Amurka ta Amurka (NCBI). Anyi nazarin phylogenetic ta amfani da shirin MEGA 6 (Tamura et al., 2013).
An gudanar da ƙaddara game da ƙwayoyin cuta a kan dukkanin fruitsa greenan 'ya'yan itace masu girma-manyan tumatir (iri-iri Dubrava) da tubers na dankalin turawa (iri iri Gala) Bugu da kari, anyi amfani da yanyan itace da tubers iri daya don yin lahani ga 'ya'yan itatuwa da tubers da suka lalace. An sanya sassan tubers a ɗakuna masu ɗumi, waxanda suke da jita-jita na Petri tare da takardar tace ruwa a ƙasa. An sanya nunin faifai a kan takardar, wanda, bi da bi, an sanya sassan tubers ko 'ya'yan itatuwa. Hakanan an sanya duka tubers da 'ya'yan itatuwa a cikin kwantena tare da rigar matattarar takarda a ƙasan. A tsakiyar yanki (ko a saman daskararren tuber ko 'ya'yan itace), an sanya wani agar (5 × 5 mm) tare da fungal hyphae bayan kwanaki 5 da girma akan wort agar.
Kimantawa game da juriya na matsalolin fungal zuwa kayan gwari an gudanar da shi a cikin yanayin dakin gwaje-gwaje akan matsakaicin abinci mai gina jiki. Munyi nazarin saukin kamuwa da magungunan fungicidal Maxim, KS (mai amfani da sinadarin fludioxonil, 25 g / l), Quadris, KS (azoxystrobin 250 g / l), Scor, EC (difenoconazole 250 g / l) (Takardar Jiha ..., 2020). An gudanar da kimantawa a cikin jita-jita na Petri akan matsakaicin matsakaicin matsakaici tare da ƙari na magungunan da aka yi nazari a kan ƙarfin abu mai aiki na 0.1; daya; 1 ppm (mg / L) (don fludioxonil da difenoconazole), 10; goma; 1 ppm (don azoxystrobin) kuma a cikin kafofin watsa labarai ba tare da kayan gwari ba (sarrafawa). An saka kayan gwari a cikin narkewar kuma sanyaya zuwa matsakaicin 10 ° C, bayan haka an zuba matsakaicin cikin abincin Petri. An sanya katangar agar tare da func mycelium a cikin tsakiyar abincin Petri kuma an al'ada ta a zafin jiki na 100 ° C a cikin duhu. Bayan kwanaki 60 na yin incubation, an auna diamita na mulkin mallaka a cikin kwatankwacin daidaikun juna; Sakamakon matsakaita ga kowane yanki ya kasance matsakaita. An gudanar da gwaje-gwajen ne sau uku. Dangane da sakamakon binciken, an kirga EC24, daidai yake da narkar da kayan gwari, wanda ya rage ragin girman radial na mulkin dangi dangane da sarrafa kayan gwari.
Sakamako da tattaunawa
A kan jita-jita na petri tare da wort agar, naman gwari ya kafa yankuna tare da fararen fure mai ƙyalƙyali. Matsakaici a ƙarƙashin mycelium ya zama ja-launi. Lokacin da matsakaiciyar ta bushe, naman gwari ya samar da nau'ikan launuka iri biyu akan dunkulen wuri da kuma tarawa a cikin ƙananan sporodochia. Macroconidia suna da tsayi, suna zagaye, suna da septa daya zuwa uku, matsakaita tsayi 27.2 µm tare da kewayon dabi'u daga 23.2 zuwa 32.2 µm, nisa - har zuwa 4.9 µm (Fig. 1). Matsakaicin tsaran microconidia shine 14.3 µm tare da kewayon ƙimomi daga 10.3 zuwa 18.1 µm, faɗin ya kai 4.0 µm. Duk haruffan macro da micromorphological sun dace da yanayin bambancin jinsin Ilyonectria crassa (Cabral et al., 2012).
Jerin jerin takamaiman yankuna na DNA (ITS, b-tubulin, TEF 1α) gabaɗaya sun dace da jerin abubuwan I. muguwar haɗuwa da muka karanta a baya (Chudinova et al., 2019, Table 1). Don yin nazarin yaduwar I. crassa a wasu yankuna da kuma bincika bambance-bambancen al'adun da abin ya shafa, ana nazarin jerin DNA masu kama da juna a cikin bayanan GenBank (Table 1). Juyewar ya kasance 86 zuwa 100%. Tsarin dukkan yankuna uku na DNA na “dankalin turawa” I. crassa iri iri daya ne da jerin tsaran da aka ware daga bulb bul da kuma narcissus tushen a cikin Netherlands da kuma daga ginseng root a Kanada. Mun kasa gano wasu nau'ikan I. na haɗari tare da bincikar jerin abubuwa iri-iri a cikin ɗakunan bayanai na buɗe. Koyaya, nazarin abubuwan da aka ajiye na ITS da b-tubulin jerin sun nuna kasancewar I. jiri a jikin ganyen tulip a Burtaniya. Fungi mai irin wannan jerin an gano shi a cikin nazarin mycobiota na asalin aspen a cikin Kanada da tushen beech a Italiya, tubers dankalin turawa a Saudi Arabia (Table 1). Sakamakon wannan binciken ya nuna cewa I. crassa yana da rarraba a duniya kuma yana da ikon kamuwa da nau'ikan tsire-tsire iri-iri.
Lokacin da ake tantance cutar a cikin yanka tumatir da dankalin turawa a ranar 5, faɗin raunin ya kai cm 1.5. strainwayar da aka bincika ba ta cutar da fruita fruitan tumatir gaba ɗaya da tuber dankalin turawa. Koyaya, an lalata sassan jikin tumatir din. Don keɓance yiwuwar gurɓatarwa, wani keɓaɓɓen naman gwari daga mycelium da aka haɓaka a kan yanki tuber dankalin turawa an keɓe shi cikin tsarkakakken al'adu. Ya kasance daidai yake da matsalar iyaye. A bayyane, I. crassa shine mai cutar rauni.
Maganin shuki na tubers iri tare da kayan gwari yana rage ci gaban cututtuka akan tsire-tsire yayin lokacin girma. Don zaɓin magungunan gwari masu amfani, yana da mahimmanci a tantance wanne daga cikinsu ke da tasiri a kan I. сrassa. Aikin yayi nazarin abubuwa masu yaduwa masu yawa na kayan gwari - fludioxonil, azoxystrobin, difenoconazole. Fludioxonil yana cikin cakudawa da yawa da ake amfani da shi don ado iri da tubers iri kafin shuka. Fludioxonil (Maxim) ana amfani dashi don magance tubers iri kafin adanawa. Difenoconazole da azoxystrobin suma suna cikin wasu shirye-shiryen da ake amfani dasu don sarrafa kayan iri, haka nan kuma a cikin shirye-shiryen da aka shirya don sarrafa tsire-tsire masu tsire-tsire (kundin ƙasa ..., 2020).
An yi nazarin saurin ci gaban I. crassa a kafofin watsa labarai (Siffa 2) tare da nau'ikan abubuwa daban-daban masu aiki: fludioxonil (EC50 = 0.4 ppm), azoxystrobin (EC50 = 4 ppm), da difenoconazole (EC50 = 7.4 ppm) (Table 2). Wadannan shirye-shiryen ana iya daukar su da matukar tasiri a kan I. jiri, tunda EC50 dinsu ya ragu sosai da shawarar da aka bayar na shirye-shiryen a cikin ruwan aiki wanda ake amfani dashi don magance tubers. Dangane da Kundin Kasuwa ... (2020), yawan fludioxonil a cikin ruwa don magance tubers dankalin turawa daga 500 zuwa 1000 ppm, azoxystrobin (a cikin ruwa don magance kasan furcin) - 3750-9375 ppm, difenoconazole (a cikin ruwa don magance ciyawar shuke shuke) - 187.5 - 625 ppm.
Tebur 1. Jerin kamannin jinsin takamaiman jerin nau'ikan nau'ikan nau'ikan 18KSuPT2 da Ilyonectria crassa iri dake cikin kundin bayanan Genbank.
Iri | Mai watsa shiri, shafin yanar gizo | Lambobin jerin da aka ajiye a cikin GenBank, yawan kamanni | mahada | ||
DAI | β-tubulin | TAFIYA 1α | |||
17KSPT1 da 18KSuPT2 | Tumakin dankalin turawa, yankin Kostroma | MH818326 | MH822872 | MK281307 | Chudinova et al., 2019, wannan aikin |
CBS 158/31 | Tushen Narcissus, Netherlands | JF735276 100 | JF735394 100 | JF735724 99.3 | Cabral et al., 2012 |
CBS 139/30 | Lily kwan fitila, Netherlands | JF735275 100 | JF735393 99.7 | JF735723 99.3 |
|
NSAC-SH-1 | Tushen Ginseng, Kanada | AY295311 99.4 | JF735395 100 | JF735 / 725 99.6 |
|
Saukewa: RHS235138 | Ganyen Tulip, Burtaniya | KJ475469 100 | KJ513266 100 | ND | Denton, 2014 |
MT294410 | Tushen Aspen, Kanada | MT294410 100 | ND | ND | Ramsfield et al., 2020 |
ER1937 | Beech, Italiya | KR019363 99.65 | ND | ND | Tizzani, Haegi, Motta. Kai tsaye sallama |
KAUF19 | Tumkin dankalin turawa, Saudi Arabia | HE649390 98.3 | ND | ND | Gashgari, Gherbawy, 2013 |
ND = ba a saka ba
Tebur 2. Resistance na Ilyonectria crassa zuwa fungicides
(abu mai aiki) | EC50, ppm | ||||
Ranar 3 | Ranar 5 | Ranar 7 | |||
Sarrafa | 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 | |||
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 |
A cikin aikinmu, I. ɓarna iri an keɓe daga tubers dankalin turawa a cikin yankunan Kostroma da Moscow (Chudinova et al., 2019). Wani kaso mai tsoka na nau'ikan fungal tare da tsarin sa na ITS wanda yayi daidai da na I. ya fada ne a cikin binciken mycobiota na tukunyar dankalin turawa a Saudi Arabia (Gashgari da Gherbawy, 2013). A bayyane yake, I. crassa ba shi da wuya a kan dankali kamar yadda ake iya gani. Gwaje-gwajen da muka yi sun nuna cewa naman gwari na iya cutar da yayan tumatir da suka lalace. An san shi ne daga wallafe-wallafen cewa I. crassa na iya haɓaka saprotrophically a cikin ƙasa (Moll et al., 2016), tare da shafar iri-iri, har ma da masu nesa da kai tsaye kamar su daffodils, lili, ginseng, aspen, da beech (Table 1). daya). A bayyane yake, yawancin tsire-tsire na daji da na lambu na iya zama na I. crassa. Abubuwan da ke sama sun nuna cewa yayin bunkasa matakan kariya, ya zama dole ayi la'akari da yiwuwar shafar tubers dankalin turawa da wannan naman gwari. Shirye-shirye masu yaduwa don maganin tubers dankalin turawa dauke da fludioxonil, azoxystrobin da difenoconazole sun nuna matukar tasirin gwari game da I. jiri.
Wannan aikin ya sami goyan bayan Gidauniyar Rasha don Nazarin Asali (Grant No. 20-016-00139).
An buga labarin a cikin mujallar "Bulletin Bulletin Plant", 2020, 103 (3)