pine nematode ndi malo okhala kwaokha omwe amasamuka omwe amadziwika kuti amabweretsa kuwonongeka kwakukulu kwachuma m'nkhalango za pine. Kafukufuku wapano akuwunikira zochitika za nemacidal za ma halogenated indoles motsutsana ndi ma pine nematodes ndi momwe amagwirira ntchito. Zochita za nemacidal za 5-iodoindole ndi avermectin (zowongolera zabwino) motsutsana ndi ma pine nematodes zinali zofanana komanso zokwera kwambiri (10 μg/mL). 5-iodoindole idachepetsa kubereka, kubereka, kufa kwa embryonic ndi mphutsi, komanso machitidwe a locomotor. Kuyanjana kwa ma molekyulu a ligands okhala ndi invertebrate-specific glutamate-gated chloride channel receptors kumathandizira lingaliro lakuti 5-iodoindole, monga avermectin, imamangiriza zolimba ku malo omwe amalandila. 5-Iodoindole idapangitsanso kusinthika kwa phenotypic mu nematodes, kuphatikiza kugwa kwa chiwalo / kuchepera komanso kuchuluka kwa vacuolization. Zotsatirazi zikuwonetsa kuti ma vacuoles amatha kukhala ndi gawo pa kufa kwa nematode methylation-mediated. Chofunika kwambiri, 5-iodoindole sinali poizoni ku mitundu yonse ya zomera (kabichi ndi radish). Chifukwa chake, kafukufukuyu akuwonetsa kuti kugwiritsa ntchito iodoindole pansi pazachilengedwe kumatha kuwongolera kuvulala kwa pine wilt.
Pine wood nematode (Bursaphelenchus xylophilus) ndi ya pine wood nematodes (PWN), migratory endoparasitic nematodes yomwe imadziwika kuti imawononga kwambiri zachilengedwe ku nkhalango za pine. Pine wilt disease (PWD) yoyambitsidwa ndi pine wood nematode ikukhala vuto lalikulu m'makontinenti angapo, kuphatikiza Asia ndi Europe, ndipo ku North America, nematode imawononga mitundu ya paini1,2. Kutsika kwa mtengo wa pine ndi vuto lalikulu lazachuma, ndipo chiyembekezo cha kufalikira kwake padziko lonse lapansi chikudetsa nkhawa3. Mitundu ya paini yotsatirayi imakonda kugwidwa ndi nematode: Pinus densiflora, Pinus sylvestris, Pinus thunbergii, Pinus koraiensis, Pinus thunbergii, Pinus thunbergii, ndi Pinus radiata4. Pine nematode ndi matenda oopsa omwe amatha kupha mitengo ya paini mkati mwa milungu kapena miyezi ingapo itadwala. Kuonjezera apo, kuphulika kwa pine nematode kumakhala kofala m'madera osiyanasiyana a zachilengedwe, choncho maunyolo opitilira matenda akhazikitsidwa1.
Bursaphelenchus xylophilus ndi quarantine plant-parasitic nematode ya superfamily Aphelenchoidea and clade 102.5. Nematode imadya bowa ndipo imaberekana mumitengo ya paini, ikukula m'magulu anayi a mphutsi: L1, L2, L3, L4 ndi munthu wamkulu1,6. Pakakhala kuchepa kwa chakudya, pine nematode imapita kumalo apadera a larval - dauer, yomwe imasokoneza vector yake - kachilomboka ka pine bark (Monochamus alternatus) ndipo imasamutsidwa kumitengo yapaini yathanzi. M'magulu athanzi, nematode imasamuka mwachangu kudzera m'maselo a zomera ndikudya ma cell a parenchymatous, zomwe zimatsogolera ku zochitika zingapo za hypersensitivity, kufota kwa pine ndi kufa pakatha chaka chimodzi pambuyo pa matenda1,7,8.
Kuwongolera kwachilengedwe kwa pine nematodes kwakhala kovuta kwanthawi yayitali, ndi njira zodzipatula kuyambira zaka za zana la 20. Njira zamakono zothanirana ndi pine nematodes makamaka zimaphatikizapo chithandizo chamankhwala, kuphatikiza kufukiza nkhuni ndi kuyika kwa nematicides mumitengo yamitengo. Nematicides omwe amagwiritsidwa ntchito kwambiri ndi avermectin ndi avermectin benzoate, omwe ndi a banja la avermectin. Mankhwala okwera mtengowa ndi othandiza kwambiri ku mitundu yambiri ya nematode ndipo amawonedwa ngati otetezeka ku chilengedwe9. Komabe, kugwiritsa ntchito mobwerezabwereza kwa nematicides kumayembekezeredwa kupangitsa kukakamizidwa kwa kusankha komwe kungapangitse kuti pakhale kugonjetsedwa kwa pine nematodes, monga momwe zasonyezedwera tizirombo zingapo, monga Leptinotarsa decemlineata, Plutella xylostella ndi nematodes Trichostrongylus colubriformis ndi Ostertagia circumcinins, yomwe pang'onopang'ono yayamba kukana,12. Chifukwa chake, kukana kumayenera kuwerengedwa pafupipafupi ndikuwunika ma nematicides mosalekeza kuti apeze njira zina, zotsika mtengo komanso zosamalira zachilengedwe zowongolera PVD. M'zaka zaposachedwa, olemba angapo apereka lingaliro la kugwiritsa ntchito zopangira zamasamba, mafuta ofunikira komanso osasinthika ngati othandizira owongolera nematode13,14,15,16.
Posachedwapa tidawonetsa ntchito ya nematicidal ya indole, molekyulu yolumikizana ndi intercellular ndi interkingdom, mu Caenorhabditis elegans 17. Indole ndi chizindikiro chofala kwambiri cha intracellular mu chilengedwe cha tizilombo toyambitsa matenda, kulamulira ntchito zambiri zomwe zimakhudza thupi la tizilombo toyambitsa matenda, mapangidwe a spore, kukhazikika kwa plasmid, kukana mankhwala, kupanga biofilm, ndi virulence 18, 19. Ntchito ya indole ndi zotumphukira zake motsutsana ndi nematode zina za pathogenic sizinaphunzire. Mu kafukufukuyu, tidafufuza za 34 indoles zomwe zimachitika motsutsana ndi ma pine nematodes ndikuwonetsa momwe 5-iodoindole imagwirira ntchito pogwiritsa ntchito ma microscopy, kujambula kwanthawi yayitali, ndi kuyesa kwa ma cell docking, ndikuwunika momwe zimawonongera mbewu pogwiritsa ntchito kuyesa kwa kameredwe ka mbewu.
Kuchuluka kwambiri (> 1.0 mM) kwa indole kudanenedwa kale kuti kumakhala ndi nematodes17. Pambuyo pa chithandizo cha B. xylophilus (magawo osakanikirana a moyo) ndi indole kapena 33 zosiyana za indole pa 1 mM, imfa ya B. xylophilus inayesedwa powerengera nematode zamoyo ndi zakufa m'magulu olamulira ndi ochiritsidwa. Ma indoles asanu adawonetsa zochitika zazikulu za nemacidal; kupulumuka kwa gulu lolamulira lopanda chithandizo kunali 95 ± 7% pambuyo pa 24 h. Pa ma indoles a 34 omwe adayesedwa, 5-iodoindole ndi 4-fluoroindole pa 1 mM inachititsa kuti 100% ifa, pamene 5,6-difluoroindigo, methylindole-7-carboxylate, ndi 7-iodoindole inachititsa pafupifupi 50% kufa (Table 1).
Zotsatira za 5-iodoindole pakupanga vacuole ndi metabolism ya pine wood nematode. (A) Zotsatira za avermectin ndi 5-iodoindole pa nematodes yamphongo wamkulu, (B) L1 siteji ya mazira a nematode ndi (C) metabolism ya B. xylophilus, (i) vacuoles sanawonedwe pa 0 h, chithandizo chinachititsa (ii) vacuoles, (iii) kudzikundikira kwa multiple vacuoles, vacuoles, vacuoles (vacuoles) ndi (vi) kupanga ma vacuoles akuluakulu. Mivi yofiira imasonyeza kutupa kwa vacuoles, mivi yabuluu imasonyeza kuphatikizika kwa vacuoles ndi mivi yakuda imasonyeza ma vacuoles akuluakulu. Scale bar = 50 μm.
Kuphatikiza apo, kafukufukuyu adafotokozanso njira yotsatizana ya kufa kwa methane mu pine nematodes (Chithunzi 4C). Imfa ya Methanogenic ndi mtundu wosakhala wa apoptotic wa cell kufa komwe kumalumikizidwa ndi kudzikundikira kwa ma cytoplasmic vacuoles27 otchuka. Zofooka za morphological zomwe zimawonedwa mu pine nematodes zikuwoneka kuti zimagwirizana kwambiri ndi momwe kufa kwa methane. Kuwunika kwapang'onopang'ono nthawi zosiyanasiyana kunawonetsa kuti ma vacuoles akulu adapangidwa pambuyo pa maola 20 atakumana ndi 5-iodoindole (0.1 mM). Ma vacuoles ang'onoang'ono adawonedwa pambuyo pa maola 8 a chithandizo, ndipo chiwerengero chawo chinawonjezeka pambuyo pa maola 12. Ma vacuoles akuluakulu angapo adawonedwa pambuyo pa maola 14. Ma vacuole angapo osakanikirana adawonekera bwino pambuyo pa 12-16 h ya chithandizo, kusonyeza kuti kuphatikizika kwa vacuole ndiko maziko a njira ya imfa ya methanogenic. Pambuyo pa maola 20, ma vacuoles akuluakulu angapo adapezeka mu nyongolotsi yonseyo. Zowonera izi zikuyimira lipoti loyamba la metuosis mu C. elegans.
Mu nyongolotsi zothandizidwa ndi 5-iodoindole, kuphatikizika kwa vacuole ndi kupasuka kunawonedwanso (mkuyu 5), monga zikuwonekera ndi kupindika kwa nyongolotsi ndi kutulutsidwa kwa vacuole m'chilengedwe. Kusokonezeka kwa Vacuole kunawonedwanso mu chipolopolo cha dzira, chomwe nthawi zambiri chimasungidwa bwino ndi L2 panthawi ya kuswa (Supplementary Fig. S2). Zowonera izi zimathandizira kukhudzidwa kwa kuchulukira kwamadzimadzi komanso kulephera kwa osmoregulatory, komanso kuvulala kwa cell reversible (RCI), popanga vacuole mapangidwe ndi suppuration (mkuyu 5).
Poyerekeza ndi gawo la ayodini pakupanga vacuole, tidafufuza ntchito ya nematicidal ya sodium iodide (NaI) ndi potassium iodide (KI). Komabe, pamagulu (0.1, 0.5 kapena 1 mM), sizinakhudze kupulumuka kwa nematode kapena mapangidwe a vacuole (Supplementary Fig. S5), ngakhale kuti 1 mM KI inali ndi zotsatira zochepa za nemacidal. Kumbali ina, 7-iodoindole (1 kapena 2 mM), monga 5-iodoindole, inachititsa ma vacuoles angapo ndi kuwonongeka kwapangidwe (Supplementary Fig. S6). Ma iodoindoles awiriwa adawonetsa mawonekedwe ofanana a phenotypic mu pine nematodes, pomwe NaI ndi KI sanatero. Chochititsa chidwi n'chakuti, indole sinapangitse mapangidwe a vacuole mu B. xylophilus pazitsulo zoyesedwa (deta yosasonyezedwa). Choncho, zotsatira zinatsimikizira kuti indole-iodine complex ndi yomwe imayambitsa vacuolization ndi metabolism ya B. xylophilus.
Pakati pa ma indoles omwe anayesedwa ntchito ya nematicidal, 5-iodoindole inali ndi chiwerengero chapamwamba kwambiri cha -5.89 kcal / mol, chotsatira 7-iodoindole (-4.48 kcal / mol), 4-fluoroindole (-4.33), ndi indole (-4.03) (Chithunzi 6). Kumangirira kolimba kwa hydrogen kwa 5-iodoindole ku leucine 218 kumakhazikika, pomwe zotumphukira zina zonse za indole zimamangiriza ku serine 260 kudzera pama chain chain hydrogen bond. Pakati pa ma iodoindoles ena, 2-iodoindole ili ndi mtengo womangirira wa -5.248 kcal / mol, womwe umakhala chifukwa cha mgwirizano wake waukulu wa hydrogen ndi leucine 218. Zomangira zina zodziwika zimaphatikizapo 3-iodoindole (-4.3 kcal / mol), 4-iodoindole (-4.0 kcal 6. kcal / mol) (Wowonjezera Chithunzi S8). Zambiri za halogenated indoles ndi indole zokha, kupatulapo 5-iodoindole ndi 2-iodoindole, zimapanga mgwirizano ndi serine 260. Mfundo yakuti hydrogen bonding ndi leucine 218 imasonyeza kuti imagwira bwino receptor-ligand kumanga, monga momwe taonera ivermectin (Supplementary Fig. 2-iodoindole, monga ivermectin, imangiriza mwamphamvu kumalo ogwira ntchito a GluCL receptor kudzera pa leucine 218 (Mkuyu 6 ndi Supplementary Fig. S8). Tikukulimbikitsani kuti kumangiriza uku kumafunika kusunga mawonekedwe otseguka a pore a GluCL complex komanso kuti pomangirira mwamphamvu kumalo omwe akugwiritsidwa ntchito a GluCL receptor, 5-iodoindole, 2-iodoindole, avermectin ndi ivermectin motero kusunga njira ya ion yotseguka ndikulola kuti madzi atengeke.
Kuyika kwa ma molekyulu a indole ndi halogenated indole kupita ku GluCL. Kumangirira kwa (A) indole, (B) 4-fluoroindole, (C) 7-iodoindole, ndi (D) 5-iodoindole ligands ku malo ogwira ntchito a GluCL. Puloteni imayimiridwa ndi riboni, ndipo zomangira za msana wa haidrojeni zimawonetsedwa ngati mizere yachikasu. (A′), (B′), (C′), ndi (D′) amawonetsa kugwirizana kwa ligand zofananira ndi zotsalira za amino acid zozungulira, ndipo zomangira zam'mbali za haidrojeni zimasonyezedwa ndi mivi ya madontho apinki.
Kuyesera kunachitika kuti aone momwe 5-iodoindole imawonongera pa kumera kwa kabichi ndi mbewu za radish. 5-iodoindole (0.05 kapena 0.1 mM) kapena avermectin (10 μg/mL) inali ndi zotsatira zochepa kapena zosakhudza kumera koyambirira ndi kumera kwa mbewu (Chithunzi 7). Kuphatikiza apo, palibe kusiyana kwakukulu komwe kunapezeka pakati pa kuchuluka kwa kumera kwa zowongolera zosasamalidwa ndi mbewu zothandizidwa ndi 5-iodoindole kapena avermectin. Zotsatira pa taproot elongation ndi chiwerengero cha ofananira nawo mizu anapanga analibechabe, ngakhale 1 mm (10 nthawi yogwira ndende) wa 5-iodoindole pang`ono anachedwa chitukuko cha ofananira nawo mizu. Zotsatira izi zikuwonetsa kuti 5-iodoindole ndi yopanda poizoni ku ma cell obzala ndipo samasokoneza njira zakukula kwa mbewu pazomwe amaphunzira.
Mphamvu ya 5-iodoindole pa kumera kwa mbeu. Kumera, kumera ndi mizu ya m'mbali mwa njere za B. oleracea ndi R. raphanistrum pa Murashige ndi Skoog agar medium yokhala kapena popanda avermectin kapena 5-iodoindole. Kumera kumalembedwa pambuyo pa masiku atatu a incubation pa 22 ° C.
Kafukufukuyu akuwonetsa milandu ingapo ya kupha nematode ndi ma indoles. Chofunika kwambiri, ili ndi lipoti loyamba la iodoindole inducing methylation (njira yoyambitsidwa ndi kudzikundikira kwa ma vacuoles ang'onoang'ono omwe amalumikizana pang'onopang'ono kukhala ma vacuoles akuluakulu, pamapeto pake amatsogolera kuphulika kwa membrane ndi imfa) mu singano za pine, ndi iodoindole kusonyeza katundu wochuluka wa nematicidal wofanana ndi wa malonda a nematicide avermectin.
Ma Indoles adanenedwa kale kuti ali ndi ntchito zambiri zowonetsera mu prokaryotes ndi eukaryotes, kuphatikizapo biofilm inhibition / mapangidwe, kupulumuka kwa mabakiteriya, ndi pathogenicity19,32,33,34. Posachedwapa, zotsatira zochiritsira za ma halogenated indoles, indole alkaloids, ndi semisynthetic indole zotumphukira zakopa chidwi chofufuza35,36,37. Mwachitsanzo, ma indoles a halogenated awonetsedwa kuti amapha Escherichia coli yosalekeza ndi ma cell a Staphylococcus aureus37. Kuphatikiza apo, ndizosangalatsa asayansi kuphunzira momwe ma halogenated indoles amagwirira ntchito motsutsana ndi mitundu ina, mibadwo, ndi maufumu, ndipo kafukufukuyu ndi sitepe loti akwaniritse cholingachi.
Pano, tikupangira njira yopangira 5-iodoindole-induced lethality mu C. elegans yochokera ku reversible cell injury (RCI) ndi methylation (Zithunzi 4C ndi 5). Kusintha kwa edematous monga kudzitukumula ndi kuwonongeka kwa vacuolar ndi zizindikiro za RCI ndi methylation, zomwe zimawonetsedwa ngati ma vacuoles akuluakulu mu cytoplasm48,49. RCI imasokoneza kupanga mphamvu mwa kuchepetsa kupanga kwa ATP, kuchititsa kulephera kwa mpope wa ATPase, kapena kusokoneza ma membrane a selo ndi kuchititsa kuti Na +, Ca2 +, ndi madzi50,51,52 azithamanga mofulumira. Ma vacuoles a intracytoplasmic amatuluka m'maselo a nyama chifukwa cha kuchuluka kwa madzi mu cytoplasm chifukwa cha kuchuluka kwa Ca2 + ndi madzi53. Chochititsa chidwi n'chakuti, njira iyi ya kuwonongeka kwa maselo imasinthidwa ngati kuwonongeka kuli kwakanthawi ndipo maselo amayamba kupanga ATP kwa nthawi inayake, koma ngati kuwonongeka kukupitirira kapena kuwonjezereka, maselo amafa.54 Zomwe taziwona zimasonyeza kuti nematodes omwe amachiritsidwa ndi 5-iodoindole sangathe kubwezeretsa biosynthesis yachibadwa pambuyo pokumana ndi zovuta.
The methylation phenotype yopangidwa ndi 5-iodoindole mu B. xylophilus ikhoza kukhala chifukwa cha kukhalapo kwa ayodini ndi kugawa kwa maselo, popeza 7-iodoindole inali ndi zotsatira zochepa zoletsa B. xylophilus kuposa 5-iodoindole (Table 1 ndi Supplementary Figure S6). Zotsatira izi zimagwirizana pang'ono ndi maphunziro a Malta et al. (2014), yemwe adanenanso kuti kusuntha kwa pyridyl nitrogen moiety mu indole kuchokera ku para- kupita ku meta-position kunathetsa vacuolization, kulepheretsa kukula, ndi cytotoxicity m'maselo a U251, kutanthauza kuti kuyanjana kwa molekyulu ndi malo enieni ogwira ntchito mu mapuloteni ndi ofunika27,44,45. Kuyanjana pakati pa indole kapena halogenated indoles ndi ma GluCL receptors omwe adawonedwa mu phunziroli amathandiziranso lingaliroli, popeza 5- ndi 2-iodoindole adapezeka kuti amamanga ma GluCL receptors mwamphamvu kuposa ma indoles ena omwe adawunikidwa (Chithunzi 6 ndi Supplementary Figure S8). Iodine pamalo achiwiri kapena achisanu a indole adapezeka kuti amamanga leucine 218 ya cholandilira cha GluCL kudzera m'maboma a hydrogen, pomwe ma indoles ena okhala ndi halogenated ndi indole yokha amapanga zomangira zofooka za hydrogen chain ndi serine 260 (Chithunzi 6). Chifukwa chake timaganiza kuti kukhazikika kwa halogen kumagwira ntchito yofunika kwambiri pakulowetsa vacuolar degeneration, pomwe kumangirira kolimba kwa 5-iodoindole kumapangitsa kuti njira ya ion ikhale yotseguka, motero imalola kuchulukira kwamadzimadzi mwachangu komanso kuphulika kwa vacuole. Komabe, ndondomeko yatsatanetsatane ya 5-iodoindole iyenera kutsimikiziridwa.
Musanagwiritse ntchito 5-iodoindole, zotsatira zake zapoizoni pa zomera ziyenera kufufuzidwa. Kuyesa kwathu kwa kameredwe ka mbeu kunawonetsa kuti 5-iodoindole inalibe vuto lililonse pa kameredwe ka mbewu kapena njira zachitukuko zotsatizana ndi zomwe zaphunziridwa (Chithunzi 7). Choncho, phunziroli limapereka maziko ogwiritsira ntchito 5-iodoindole m'chilengedwe kuti athetse kuwonongeka kwa pine nematodes ku mitengo ya pine.
Malipoti am'mbuyomu awonetsa kuti chithandizo chochokera ku indole chimayimira njira yomwe ingathe kuthana ndi vuto la kukana maantibayotiki komanso kukula kwa khansa55. Kuonjezera apo, ma indoles ali ndi antibacterial, anticancer, antioxidant, anti-inflammatory, antidiabetic, antiviral, antiproliferative ndi antituberculosis ntchito ndipo angakhale ngati maziko odalirika a chitukuko cha mankhwala56,57. Kafukufukuyu akuwonetsa kwa nthawi yoyamba kugwiritsa ntchito ayodini ngati antiparasitic ndi anthelmintic.
Avermectin adapezeka zaka makumi atatu zapitazo ndipo adapambana Mphotho ya Nobel mu 2015, ndipo kugwiritsidwa ntchito kwake ngati anthelmintic kukupitilirabe. Komabe, chifukwa cha kukula kofulumira kwa kukana kwa avermectins mu nematodes ndi tizilombo towononga tizilombo, njira ina, yotsika mtengo, komanso yosamalira zachilengedwe ndiyofunika kuti muteteze matenda a PWN mumitengo ya pine. Kafukufukuyu akuwonetsanso momwe 5-iodoindole imaphera pine nematodes komanso kuti 5-iodoindole ili ndi kawopsedwe kakang'ono kubzala ma cell, zomwe zimatsegula chiyembekezo chabwino cha ntchito yake yamtsogolo.
Zoyesera zonse zidavomerezedwa ndi Ethics Committee ya Yeungnam University, Gyeongsan, Korea, ndipo njirazo zidachitika motsatira malangizo a Ethics Committee of Yeungnam University.
Kuyesa kwa mazira kunkachitika pogwiritsa ntchito njira zokhazikitsidwa43. Kuti awone kuchuluka kwa hatching (HR), nematodes wamkulu wa tsiku limodzi (pafupifupi zazikazi 100 ndi amuna 100) adasamutsidwa ku mbale za Petri zomwe zimakhala ndi bowa ndikuloledwa kukula kwa 24 h. Mazira adasiyanitsidwa ndikuthandizidwa ndi 5-iodoindole (0.05 mM ndi 0.1 mM) kapena avermectin (10 μg/ml) ngati kuyimitsidwa m'madzi osabala osungunuka. Zoyimitsidwa izi (500 μl; pafupifupi mazira 100) zidasamutsidwa ku zitsime za mbale ya 24-zitsime zamtundu wa minofu ndikukulungidwa pa 22 ° C. Ziwerengero za L2 zinapangidwa pambuyo pa maola 24 a incubation koma zinkaonedwa kuti zafa ngati maselo sanasunthe pamene amalimbikitsidwa ndi waya wabwino wa platinamu. Kuyesera uku kunachitika mu magawo awiri, iliyonse ndi kubwereza kasanu ndi kamodzi. Deta yochokera muzoyesera zonsezo idaphatikizidwa ndikuwonetsedwa. Peresenti ya HR imawerengedwa motere:
Kufa kwa larval kunayesedwa pogwiritsa ntchito njira zomwe zidapangidwa kale. Mazira a nematode anasonkhanitsidwa ndipo miluza inalumikizidwa ndi kuswa m'madzi osabala osungunuka kuti apange mphutsi za L2. Mphutsi zosakanikirana (pafupifupi 500 nematodes) zinkagwiritsidwa ntchito ndi 5-iodoindole (0.05 mM ndi 0.1 mM) kapena avermectin (10 μg / ml) ndikuleredwa pa mbale za B. cinerea Petri. Pambuyo pa maola 48 a incubation pa 22 ° C, nematodes anasonkhanitsidwa m'madzi osabala osungunula ndikuwunikiridwa ngati pali magawo a L2, L3, ndi L4. Kukhalapo kwa magawo a L3 ndi L4 kumasonyeza kusintha kwa mphutsi, pamene kukhalapo kwa gawo la L2 sikunasonyeze kusintha. Zithunzi zidapezedwa pogwiritsa ntchito iRiS™ Digital Cell Imaging System. Kuyesera uku kunachitika mu magawo awiri, iliyonse ndi kubwereza kasanu ndi kamodzi. Deta yochokera muzoyesera zonsezo idaphatikizidwa ndikuwonetsedwa.
Kuopsa kwa 5-iodoindole ndi avermectin ku mbewu kunayesedwa pogwiritsa ntchito kuyesa kumera pa mbale za Murashige ndi Skoog agar. hypochlorite) kwa mphindi 15, ndikutsuka kasanu ndi madzi osabala 1 ml. Mbeu zosawilitsidwazo zinakanikizidwa pa mbale za kumera za agar zomwe zinali ndi 0.86 g/l (0.2X) Murashige ndi Skoog medium ndi 0.7% bacteriological agar yokhala ndi kapena popanda 5-iodoindole kapena avermectin. Kenako mbalezo zidakulungidwa pa 22 ° C, ndipo zithunzi zidatengedwa pambuyo pa masiku atatu atakulitsidwa. Kuyesera uku kunachitika mu magawo awiri, omwe anali ndi kubwereza kasanu ndi kamodzi.
Nthawi yotumiza: Feb-26-2025