IDYANI. Chudinov, V. A. Platonov, A. V. Alexandrova, S. N. Elansky
Zawoneka posachedwa kuti bowa wa ascomycete Ilyonectria crassa amatha kupatsira ma tubers a mbatata. Mu ntchitoyi, mawonekedwe achilengedwe komanso kukana kwa mankhwala ophera fungal a mtundu wa I. crassa olekanitsidwa ndi mbatata amawunikidwa koyamba. Kutsatizana kwa zigawo zamtundu wamtundu wa "mbatata" kumagwirizana ndi zomwe zidapezedwa kale kwa bowa olekanitsidwa ndi mizu ya narcissus, ginseng, aspen ndi beech, mababu a kakombo ndi masamba a tulip. Mwachiwonekere, zomera zambiri zakutchire ndi zamaluwa zimatha kukhala nkhokwe za I. crassa. Kufufuzidwa kupsyinjika kachilombo phwetekere ndi mbatata magawo, koma sanapatsire lonse phwetekere zipatso ndi wawo mbatata tuber. Izi zikuwonetsa kuti I. crassa ndi tizilombo toyambitsa matenda. Kuwunika kwa kukana kwa fludioxonil, difenoconazole ndi azoxystrobin pazakudya zopatsa thanzi kunawonetsa kuthandizira kwakukulu kwa mankhwalawa.
Chizindikiro cha EC50 (kuchuluka kwa fungicide, komwe kumachepetsa nthawi 2 kuchuluka kwa ma radial kukula kwa koloni ndi kuwongolera kopanda fungicide) kunali kofanana ndi 0.4; 7.4 ndi 4 mg / l, motero. Kuthekera kwa chitukuko cha matenda obwera chifukwa cha I. crassa kuyenera kuganiziridwa pakuwunika kwa phytopathological ma tubers a mbatata ndikukula kwa njira zotetezera zomera.
Kukula kwa tizilombo ta phytopathogenic kumabweretsa kutayika kwakukulu pamagawo onse akukula ndi kusunga mbatata. Pokonzekera njira zodzitetezera, monga lamulo, tizilombo toyambitsa matenda timaganiziridwa, monga mitundu ya genera Alternaria, Fusarium, Phoma, Helminthosporium, Colletotrichum, Phytophthora, etc. Komabe, m'zaka zaposachedwa, malipoti ochulukirapo awonekera za maonekedwe atsopano phytopathogenic tizilombo pa mbatata. Biology yawo sinaphunziridwe bwino, mphamvu ya fungicides yomwe imagwiritsidwa ntchito pa mbatata pokhudzana ndi iwo sikudziwika, njira zowunikira sizinapangidwe. Ndi chitukuko chachikulu, amatha kuwononga kwambiri mbewu ya mbatata. Chimodzi mwa tizilombo toyambitsa matenda ndi bowa wa ascomycete Ilyonectria crassa (Wollenw.) A. Cabral & Crous, omwe adapezeka koyamba ndi olemba pa ma tubers a mbatata (Chudinova et al., 2019).
Ntchitoyi akupereka zotsatira za kusanthula I. crassa kupsyinjika olekanitsidwa mbatata tubers. The morphology of colonies and mycelial structures of I. crassa, ma nucleotide sequence of the species-Special DNA regions, virulence to mbatata ndi tomato, and resistance to some popular fungicides adaphunzira.
Zida ndi njira
Tidagwiritsa ntchito mtundu wa I. crassa 18KSuPT2 wodzipatula mu 2018 kuchokera ku tuber ya mbatata yomwe ili ndi kachilombo yomwe idabzalidwa kudera la Kostroma. Tuber anakhudzidwa ndi youma zowola ndi patsekeke yokutidwa ndi kuwala bulauni mycelium. Pogwiritsa ntchito singano yosabala, mycelium ya mafangasi idasamutsidwa m'mbale ya Petri yokhala ndi sing'anga ya agar (mowa wowotcha 10%, agar 1.5%, penicillin 1000 U / ml). Mambale adakulungidwa mumdima pa 24 ° C.
Kamera yowala ya Leica DM2500 yokhala ndi kamera ya digito ya ICC50 HD ndi microscope ya Leica M80 yokhala ndi kamera ya digito ya IC80HD (Leica Microsystems, Germany) idagwiritsidwa ntchito kujambula, kuyesa kukula ndi mawonekedwe a spores ndi ziwalo za spore.
Kupatula DNA, fungal mycelium idakula mu sing'anga yamadzimadzi, kenako kuzizira mu nayitrogeni yamadzimadzi, yopangidwa ndi homogenized, yolumikizidwa mu CTAB buffer, yoyeretsedwa ndi chloroform, ndikutsukidwa kawiri ndi mowa 2%.
Njira yochotsera DNA ikufotokozedwa mwatsatanetsatane m'nkhani ya Kutuzova et al. (2017).
Kuti mudziwe zamoyo mwa njira zamamolekyu ndikuyerekeza ndi mitundu ina yodziwika bwino ya I. crassa, PCR idapangidwa ndi zoyambira zomwe zimalola kukulitsa madera amtundu wa DNA: ITS1-5,8S-ITS2 (zoyambira ITS5 / ITS4, White et al., 1990), zigawo za majini b -tubulin (Bt2a / Bt2b, Glass, Donaldson, 1995) ndi kumasulira elongation factor 1α (tef1α) (zoyambira EF1-728F / EF1-986R, Carbone ndi Kohn, 1999). Ma Amplicons a utali wofunidwa adachotsedwa mu gel osakaniza pogwiritsa ntchito zida za Evrogen CleanUp. Madera okulitsa adatsatidwa pogwiritsa ntchito BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) pa Applied Biosystems 3730 xl automated sequencer (Applied Biosystems, CA, USA). Zotsatira za ma nucleotide zidagwiritsidwa ntchito posaka machesi mu US National Center for Biotechnology Information (NCBI) GenBank database. Kusanthula kwa Phylogenetic kunachitika pogwiritsa ntchito pulogalamu ya MEGA 6 (Tamura et al., 2013).
Kutsimikiza kwa virulence kunkachitika pa zipatso zobiriwira zazikulu za phwetekere (zosiyanasiyana Dubrava) ndi ma tubers a mbatata (zosiyanasiyana Gala). Komanso, kuti yesezera kuwonongeka kwa kuonongeka zipatso ndi tubers, tinkagwiritsa ntchito magawo a yemweyo zipatso ndi tubers. Magawo a tubers adayikidwa m'zipinda zonyowa, zomwe zinali mbale za Petri zokhala ndi pepala lonyowa losefera pansi. Chojambula chinayikidwa pamapepala, pomwe, nawonso, magawo a tubers kapena zipatso adayikidwa. Onse tubers ndi zipatso anaikidwanso muli ndi chonyowa fyuluta pepala pansi. Pakatikati pa kagawo (kapena pamwamba pa tuber kapena chipatso), chidutswa cha agar (5 × 5 mm) chokhala ndi fungal hyphae chinayikidwa patatha masiku asanu akumera pa wort agar.
Kuwunika kukana kwa mitundu ya mafangasi ku fungicides kunachitika mu labotale pa sing'anga yazakudya za agar. Tinaphunzira chiwopsezo cha fungicidal mankhwala Maxim, KS (yogwira pophika fludioxonil, 25 g / l), Quadris, KS (azoxystrobin 250 g / l), Scor, EC (difenoconazole 250 g / l) (State m'buku ..., 2020 ). Kuunikira kunachitika muzakudya za Petri pa wort-agar sing'anga ndi kuwonjezera kwa mankhwala omwe amaphunzira pamagulu azinthu zogwira ntchito 0.1; 1; 10 ppm (mg / L) (ya fludioxonil ndi difenoconazole), 1; khumi; 10 ppm (ya azoxystrobin) komanso mu media popanda fungicide (kuwongolera). Fungicide idawonjezeredwa ku sing'anga yomwe idasungunuka ndikukhazikika mpaka 100 ° C, kenako sing'angayo idatsanuliridwa mu mbale za Petri. Chida cha agar chokhala ndi fungal mycelium chinayikidwa pakati pa mbale ya Petri ndikutukuka pa kutentha kwa 60 ° C mumdima. Pambuyo pa masiku 24 a incubation, ma diameter a maderawo adayesedwa m'njira ziwiri zosiyana; zotsatira za kuyeza kwa koloni iliyonse zidawerengedwa. Zoyesererazo zidachitika mwapatatu. Kutengera zotsatira za kusanthula, EC7 idawerengedwa, yofanana ndi kuchuluka kwa fungicide, yomwe idachepetsa ndi theka la kukula kwa koloni pokhudzana ndi kuwongolera kopanda fungicide.
Zotsatira ndi zokambirana
Pazakudya za petri zokhala ndi wort agar, bowawo adapanga magulu okhala ndi white flocculent mycelium. Sing'anga pansi pa mycelium inasanduka wofiira-bulauni. Mbalameyo ikauma, bowawo amapanga timbewu tamitundu iwiri pamtundu umodzi komanso wophatikizana wa sporodochia yaying'ono. Macroconidia ndi elongated, cylindrical, ndi septa imodzi kwa atatu, pafupifupi kutalika 27.2 µm ndi osiyanasiyana makhalidwe 23.2 kuti 32.2 µm, m'lifupi mpaka 4.9 µm (mkuyu. 1). Pafupifupi kutalika kwa microconidia ndi 14.3 µm ndi mitundu yosiyanasiyana kuchokera ku 10.3 mpaka 18.1 µm, m'lifupi mwake ndi 4.0 µm. Mitundu yonse ya macro- ndi micromorphological imagwirizana ndi mitundu yosiyanasiyana ya mitundu ya Ilyonectria crassa (Cabral et al., 2012).
Kutsatizana kwa zigawo zamtundu wa DNA (ITS, b-tubulin, TEF 1α) zimagwirizana kwathunthu ndi machitidwe a I. crassa strains omwe tidaphunzira kale (Chudinova et al., 2019, Table 1). Pofuna kuphunzira kufalikira kwa I. crassa m'madera ena ndikuwunika kuchuluka kwa zikhalidwe zomwe zimakhudzidwa, ma DNA ofanana nawo mu database ya GenBank adawunikidwa (Table 1). Kuphatikizikako kunali 86 mpaka 100%. Mayendedwe a zigawo zonse zitatu za DNA za "mbatata" I. crassa strain zinali zofanana ndi zotsatizana za mitundu yosiyana ndi bulb ya kakombo ndi mizu ya daffodil ku Netherlands komanso kuchokera ku mizu ya ginseng ku Canada. Sitinathe kupeza mitundu ina ya I. crassa yokhala ndi matsatidwe atatu ofananira nawo m'mankhokwe otseguka. Komabe, kuwunika kwamayendedwe a ITS ndi b-tubulin omwe adayikidwa adawonetsa kupezeka kwa I. crassa pamasamba a tulip ku UK. Bowa omwe ali ndi machitidwe a ITS ofanana adadziwika posanthula mycobiota ya mizu ya aspen ku Canada ndi mizu ya beech ku Italy, ndi ma tubers a mbatata ku Saudi Arabia (Table 1). Zotsatira za kafukufukuyu zikuwonetsa kuti I. crassa imagawidwa padziko lonse lapansi ndipo imatha kupatsira mitundu yosiyanasiyana ya zomera.
Pozindikira za pathogenicity pa magawo a phwetekere ndi mbatata pa tsiku la 5, kutalika kwa chotupacho kunafika masentimita 1.5. Pankhaniyi, kupsyinjika komwe kunkafufuzidwa sikunawononge zipatso zonse za phwetekere ndi tuber ya mbatata. Komabe, ma sepals adakhudzidwa pa phwetekere. Kupatula kuthekera kwa kuipitsidwa, fungal kudzipatula ku mycelium yopangidwa pa kagawo ka tuber ya mbatata idapatulidwa kukhala chikhalidwe choyera. Zinali zofanana kwambiri ndi zovuta za makolo. Mwachiwonekere, I. crassa ndi tizilombo toyambitsa matenda.
Kubzala kusanachitike ma tubers ambewu ndi fungicides kumachepetsa kukula kwa matenda pazitsamba nthawi yakukula. Posankha mankhwala opha fungicides ogwira mtima, ndikofunikira kuwunika kuti ndi ati omwe ali othandiza motsutsana ndi I. сrassa. Ntchitoyi idaphunzira zamitundu yogwira ntchito ya fungicides - fludioxonil, azoxystrobin, difenoconazole. Fludioxonil amapangidwa muzosakaniza zingapo zomwe zimagwiritsidwa ntchito kuvala njere ndi ma tubers asanabzalidwe. Fludioxonil (Maxim) amagwiritsidwanso ntchito pochiza ma tubers ambewu asanasungidwe. Difenoconazole ndi azoxystrobin zimaphatikizidwanso muzokonzekera zingapo zomwe zimagwiritsidwa ntchito pokonza mbewu, komanso pokonzekera kukonza zomera zamasamba (Buku la State ..., 2020).
Kukula kwa I. crassa kunaphunziridwa pa zofalitsa (mkuyu 2) ndi mitundu yosiyanasiyana ya zinthu zogwira ntchito: fludioxonil (EC50 = 0.4 ppm), azoxystrobin (EC50 = 4 ppm), ndi difenoconazole (EC50 = 7.4 ppm) (Table 2). ). Zokonzekerazi zitha kuonedwa kuti ndizothandiza kwambiri motsutsana ndi I. crassa, popeza EC50 yawo ndi yotsika kwambiri kuposa momwe zimakhalira zokonzekera mumadzimadzi omwe amagwiritsidwa ntchito pochiza ma tubers. Malinga ndi State Catalogue ... (2020), kuchuluka kwa fludioxonil mumadzimadzi opangira ma tubers a mbatata kumachokera ku 500 mpaka 1000 ppm, azoxystrobin (mumadzi opangira pansi pa ngalande) - 3750-9375 ppm, difenoconazole. (mumadzimadzi ochizira zomera zamasamba) - 187.5– 625 ppm.
Gulu 1. Kufanana kwa mndandanda wamtundu wamtundu wamtundu 18KSuPT2 ndi mitundu ya Ilyonectria crassa yomwe ikupezeka munkhokwe ya Genbank
Kupsyinjika | Host chomera, malo excretion | Nambala zotsatizana zomwe zasungidwa ku GenBank, kuchuluka kwa zofanana | kugwirizana | ||
ITS | β-tubulin | TEF1 pa | |||
17KSPT1 ndi 18KSuPT2 | Mbatata tuber, Kostroma dera | MH818326 | MH822872 | MK281307 | Chudinova et al., 2019, ntchitoyi |
CBS 158/31 | Mizu ya Narcissus, Netherlands | JF735276 100 | JF735394 100 | JF735724 99.3 | Cabral et al., 2012 |
CBS 139/30 | Lily bulb, Netherlands | JF735275 100 | JF735393 99.7 | JF735723 99.3 |
|
NSAC-SH-1 | Muzu wa Ginseng, Canada | AY295311 99.4 | JF735395 100 | JF735/725 99.6 |
|
RHS235138 | Tulip Leaf, UK | KJ475469 100 | KJ513266 100 | ND | Denton, Denton, 2014 |
MT294410 | Mizu ya Aspen, Canada | MT294410 100 | ND | ND | Ramsfield et al., 2020 |
ER1937 | Beech, Italy | KR019363 99.65 | ND | ND | Tizzani, Haegi, Motta. Kugonjera mwachindunji |
KAUF19 | Potato tuber, Saudi Arabia | HE649390 98.3 | ND | ND | Gashgari, Gherbawy, 2013 |
ND = sichinasungidwe
Table 2. Kukana kwa Ilyonectria crassa ku mankhwala ophera fungal
(chinthu chogwira ntchito) | EC50, pa | ||||
Tsiku la 3 | Tsiku la 5 | Tsiku la 7 | |||
Kulamulira | 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 |
Mu ntchito yathu, mitundu ya I. crassa idasiyanitsidwa ndi ma tubers a mbatata kumadera a Kostroma ndi Moscow (Chudinova et al., 2019). Kuchuluka kwa mitundu ya mafangasi okhala ndi machitidwe a ITS ofanana ndi I. crassa adawululidwa pakuwunika kwa mycobiota wa ma tubers a mbatata ku Saudi Arabia (Gashgari ndi Gherbawy, 2013). Mwachiwonekere, I. crassa sichiri chosowa pa mbatata monga momwe zingawonekere. M'zoyeserera zathu, zidawonetsedwa kuti bowa amatha kupatsira zipatso zowonongeka za phwetekere. Amadziwika kuchokera m'mabuku omwe I. crassa amatha kupanga saprotrophically m'nthaka (Moll et al., 2016), komanso kukhudza zomera zosiyanasiyana, ngakhale zamtundu wa taxonomically monga daffodils, maluwa, ginseng, aspen, ndi beech (Table 1) XNUMX). Mwachiwonekere, zomera zambiri zakutchire ndi zamaluwa zimatha kukhala nkhokwe za I. crassa. Zomwe zili pamwambazi zikuwonetsa kuti popanga njira zodzitetezera, ndikofunikira kuganizira za kuthekera kwa kuwonongeka kwa ma tubers a mbatata ndi bowa. Kukonzekera kofala kwa machubu a mbatata okhala ndi fludioxonil, azoxystrobin ndi difenoconazole kwawonetsa mphamvu yayikulu ya fungicidal motsutsana ndi I. crassa.
Ntchitoyi inathandizidwa ndi Russian Foundation for Basic Research (Grant No. 20-016-00139).
Nkhaniyi idasindikizidwa mu nyuzipepala "Plant Protection Bulletin", 2020, 103 (3)