Nkhumba ya paini ndi endoparasite yosamukira yokhayokha yomwe imadziwika kuti imayambitsa kutayika kwakukulu kwachuma m'nkhalango ya paini. Kafukufukuyu akuwunikanso momwe ma indole okhala ndi halogenated amagwirira ntchito motsutsana ndi ma nematode a paini ndi momwe amagwirira ntchito. Zochita za 5-iodoindole ndi avermectin (kulamulira kwabwino) motsutsana ndi ma nematode a paini zinali zofanana komanso zapamwamba pamlingo wochepa (10 μg/mL). 5-iodoindole yachepetsa kubereka, kubereka, kufa kwa embryonic ndi larval, komanso khalidwe la locomotor. Kuyanjana kwa ma ligands ndi ma invertebrates-specific glutamate-gated chloride channel receptors kumathandizira lingaliro lakuti 5-iodoindole, monga avermectin, imamangirira mwamphamvu pamalo omwe receptor imagwira ntchito. 5-Iodoindole idayambitsanso kusintha kwa phenotypic mu ma nematode, kuphatikiza kugwa/kuchepa kwa ziwalo kosazolowereka komanso kuwonjezeka kwa vacuolization. Zotsatirazi zikusonyeza kuti ma vacuole amatha kukhala ndi gawo pakufa kwa nematode chifukwa cha methylation. Chofunika kwambiri n'chakuti, 5-iodoindole sinali poizoni kwa mitundu yonse ya zomera (kabichi ndi radish). Choncho, kafukufukuyu akusonyeza kuti kugwiritsa ntchito iodoindole m'malo okhala ndi chilengedwe kungathandize kuchepetsa kuvulala kwa pine wilt.
Nthambi ya mitengo ya paini (Bursaphelenchus xylophilus) ndi ya m'gulu la nthambi ya paini (PWN), yomwe imadziwika kuti imayambitsa kuwonongeka kwakukulu kwa chilengedwe cha nkhalango ya paini1. Matenda a kufota kwa pine (PWD) omwe amayamba chifukwa cha nthambi ya mitengo ya paini akukhala vuto lalikulu m'maiko angapo, kuphatikiza Asia ndi Europe, ndipo ku North America, nthambiyi imawononga mitundu ya mitengo ya paini yomwe yabwera1,2. Kuchepa kwa mitengo ya paini ndi vuto lalikulu lazachuma, ndipo chiyembekezo cha kufalikira kwake padziko lonse lapansi chikudetsa nkhawa3. Mitundu yotsatirayi ya mitengo ya paini imakhudzidwa kwambiri ndi nthambiyi: Pinus densiflora, Pinus sylvestris, Pinus thunbergii, Pinus koraiensis, Pinus thunbergii, Pinus thunbergii, ndi Pinus radiata4. Nthambi ya paini ndi matenda oopsa omwe amatha kupha mitengo ya paini mkati mwa milungu kapena miyezi ya matendawa. Kuphatikiza apo, kufalikira kwa nthambi ya paini kumakhala kofala m'malo osiyanasiyana achilengedwe, kotero maunyolo opatsirana nthawi zonse akhazikitsidwa1.
Bursaphelenchus xylophilus ndi nematode yochokera ku banja la Aphelenchoidea ndi clade 102.5. Nematode imadya bowa ndipo imaberekana m'mitengo ya mitengo ya paini, ndikupanga magawo anayi osiyanasiyana a mphutsi: L1, L2, L3, L4 ndi munthu wamkulu1,6. Pansi pa kusowa kwa chakudya, nematode ya paini imapita ku gawo lapadera la mphutsi - dauer, lomwe limawononga ventricle yake - kachilombo ka makungwa a paini (Monochamus alternatus) ndikusamutsidwira ku mitengo ya paini yathanzi. Mu eni ake athanzi, nematode amasuntha mwachangu kudzera mu minofu ya zomera ndikudyetsa maselo a parenchymatous, zomwe zimapangitsa kuti paini iwonongeke kwambiri, kufota ndi kufa mkati mwa chaka chimodzi kuchokera pamene kachilomboka kakufalikira1,7,8.
Kulamulira tizilombo toyambitsa matenda a paini kwakhala kovuta kwa nthawi yayitali, ndipo njira zodzitetezera kuyambira m'zaka za m'ma 1900. Njira zamakono zothanirana ndi tizilombo toyambitsa matenda a paini makamaka zimakhudza mankhwala, kuphatikizapo kupopera ndi kuyika tizilombo toyambitsa matenda m'mitengo. Mankhwala ophera tizilombo omwe amagwiritsidwa ntchito kwambiri ndi avermectin ndi avermectin benzoate, omwe ali m'gulu la avermectin. Mankhwala okwera mtengo awa ndi othandiza kwambiri polimbana ndi mitundu yambiri ya tizilombo toyambitsa matenda ndipo amaonedwa kuti ndi otetezeka ku chilengedwe9. Komabe, kugwiritsa ntchito mankhwala ophera tizilombo mobwerezabwereza kukuyembekezeka kupanga mphamvu yosankha yomwe ingayambitse kubuka kwa tizilombo toyambitsa matenda a paini, monga momwe zasonyezedwera ndi tizilombo tina tambiri, monga Leptinotarsa decemlineata, Plutella xylostella ndi tizilombo toyambitsa matenda Trichostrongylus colubriformis ndi Ostertagia circumcincta, zomwe pang'onopang'ono zakhala zikukana avermectins10,11,12. Chifukwa chake, njira zothanirana ziyenera kuphunziridwa nthawi zonse ndipo mankhwala ophera tizilombo amafufuzidwa nthawi zonse kuti apeze njira zina, zotsika mtengo komanso zochezeka kuti athetse PVD. M'zaka makumi angapo zapitazi, olemba angapo apereka lingaliro loti agwiritse ntchito zotulutsa zomera, mafuta ofunikira ndi zinthu zowononga zomera monga mankhwala oletsa nematode13,14,15,16.
Posachedwapa tawonetsa momwe indole, molekyulu yolumikizirana pakati pa maselo ndi ma kingdom, imagwirira ntchito pochiza matenda a tizilombo toyambitsa matenda, mu Caenorhabditis elegans 17. Indole ndi chizindikiro chofala cha mkati mwa maselo mu chilengedwe cha tizilombo toyambitsa matenda, chomwe chimayang'anira ntchito zambiri zomwe zimakhudza thupi la tizilombo toyambitsa matenda, mapangidwe a spore, kukhazikika kwa plasmid, kukana mankhwala, mapangidwe a biofilm, ndi virulence 18, 19. Ntchito ya indole ndi zotumphukira zake motsutsana ndi nematode ena opatsirana sizinaphunziridwe. Mu kafukufukuyu, tafufuza momwe nematode 34 a indole amagwirira ntchito motsutsana ndi nematode a paini ndipo tafotokoza momwe 5-iodoindole yamphamvu kwambiri imagwirira ntchito pogwiritsa ntchito microscopy, time-lapse photography, ndi ma molecular docking experiments, ndipo tafufuza zotsatira zake zoopsa pa zomera pogwiritsa ntchito njira yoyesera kumera kwa mbewu.
Kuchuluka kwa indole (>1.0 mM) kwanenedwa kale kuti kumakhudza kupha tizilombo toyambitsa matenda m'thupi la nematode17. Pambuyo pochiza B. xylophilus (magawo osiyanasiyana a moyo) ndi indole kapena mitundu 33 yosiyanasiyana ya indole pa 1 mM, imfa ya B. xylophilus inayesedwa powerengera nematode amoyo ndi akufa m'magulu olamulira ndi ochiritsidwa. Indoles zisanu zinawonetsa ntchito yayikulu yopha tizilombo; kupulumuka kwa gulu lolamulira lomwe silinalandire chithandizo kunali 95 ± 7% patatha maola 24. Mwa indoles 34 zomwe zinayesedwa, 5-iodoindole ndi 4-fluoroindole pa 1 mM zinayambitsa imfa 100%, pomwe 5,6-difluoroindigo, methylindole-7-carboxylate, ndi 7-iodoindole zinayambitsa imfa pafupifupi 50% (Table 1).
Zotsatira za 5-iodoindole pakupanga vacuole ndi kagayidwe kake ka nematode ya pine wood. (A) Zotsatira za avermectin ndi 5-iodoindole pa nematode amphongo akuluakulu, (B) mazira a nematode a L1 stage ndi (C) kagayidwe ka B. xylophilus, (i) vacuole sizinawonekere pa 0 ola, chithandizocho chinapangitsa (ii) vacuole, (iii) kusonkhanitsa vacuole zingapo, (iv) kutupa kwa vacuole, (v) kuphatikiza kwa vacuole ndi (vi) kupanga vacuole zazikulu. Mivi yofiira imasonyeza kutupa kwa vacuole, mivi yabuluu imasonyeza kuphatikiza kwa vacuole ndi mivi yakuda imasonyeza vacuole zazikulu. Scale bar = 50 μm.
Kuphatikiza apo, kafukufukuyu adafotokozanso njira yotsatizana ya imfa yoyambitsidwa ndi methane m'ma pine nematode (Chithunzi 4C). Imfa ya methanogenic ndi mtundu wosabadwa wa kufa kwa maselo womwe umagwirizanitsidwa ndi kusonkhanitsa kwa ma cytoplasmic vacuoles odziwika bwino27. Zolakwika za morphological zomwe zimawonedwa mu pine nematodes zikuwoneka kuti zikugwirizana kwambiri ndi momwe imfa yoyambitsidwa ndi methane imachitikira. Kufufuza kwa microscopic nthawi zosiyanasiyana kunawonetsa kuti ma giant vacuoles adapangidwa pambuyo pa maola 20 atakumana ndi 5-iodoindole (0.1 mM). Ma microscopic vacuoles adawonedwa pambuyo pa maola 8 a chithandizo, ndipo chiwerengero chawo chinawonjezeka pambuyo pa maola 12. Ma vacuoles angapo akuluakulu adawonedwa pambuyo pa maola 14. Ma fused vacuoles angapo adawonekera bwino pambuyo pa maola 12-16 a chithandizo, zomwe zikusonyeza kuti vacuole fusion ndiye maziko a methanogenic death mechanism. Pambuyo pa maola 20, ma giant vacuoles angapo adapezeka m'nyongolotsi yonse. Izi zikuyimira lipoti loyamba la metuosis mu C. elegans.
Mu nyongolotsi zothandizidwa ndi iodoindole 5, kusonkhana ndi kuphulika kwa vacuole kunawonedwanso (Chithunzi 5), monga momwe zasonyezedwera ndi kupindika kwa nyongolotsi ndi kutulutsa vacuole m'chilengedwe. Kusokonezeka kwa vacuole kunawonedwanso mu nembanemba ya dzira, yomwe nthawi zambiri imasungidwa bwino ndi L2 panthawi yoswana (Chithunzi Chowonjezera S2). Izi zikuthandizira kukhudzidwa kwa kuchulukana kwa madzi ndi kulephera kwa osmoregulatory, komanso kuvulala kwa maselo osinthika (RCI), pakupanga vacuole ndi suppuration (Chithunzi 5).
Poganizira za ntchito ya ayodini pakupanga vacuole yomwe yawonedwa, tinafufuza momwe sodium iodide (NaI) ndi potassium iodide (KI) zimagwirira ntchito popha tizilombo toyambitsa matenda. Komabe, pa kuchuluka kwa (0.1, 0.5 kapena 1 mM), sizinakhudze kupulumuka kwa nematode kapena kupanga vacuole (Chithunzi Chowonjezera S5), ngakhale kuti 1 mM KI inali ndi zotsatira zochepa zopha tizilombo toyambitsa matenda. Kumbali ina, 7-iodoindole (1 kapena 2 mM), monga 5-iodoindole, inayambitsa ma vacuole ambiri ndi kusintha kwa kapangidwe kake (Chithunzi Chowonjezera S6). Ma iodoindole awiriwa adawonetsa makhalidwe ofanana a phenotypic mu nematode za paini, pomwe NaI ndi KI sizinatero. Chosangalatsa n'chakuti, indole sinapangitse kupanga vacuole mu B. xylophilus pa kuchuluka komwe kunayesedwa (deta sinawonetsedwe). Chifukwa chake, zotsatira zake zatsimikizira kuti indole-iodine complex ndiyo imayambitsa vacuolization ndi kagayidwe ka B. xylophilus.
Pakati pa ma indole omwe adayesedwa kuti aone ngati ali ndi mphamvu yopha tizilombo toyambitsa matenda, 5-iodoindole inali ndi chiŵerengero chapamwamba kwambiri cha -5.89 kcal/mol, kutsatiridwa ndi 7-iodoindole (-4.48 kcal/mol), 4-fluoroindole (-4.33), ndi indole (-4.03) (Chithunzi 6). Kugwirizana kwamphamvu kwa hydrogen kwa 5-iodoindole ndi leucine 218 kumalimbitsa mgwirizano wake, pomwe zinthu zina zonse zochokera ku indole zimamangirira ku serine 260 kudzera mu ma side chain hydrogen bonds. Pakati pa ma iodoindole ena opangidwa ndi chitsanzo, 2-iodoindole ili ndi mtengo womangirira wa -5.248 kcal/mol, womwe umachitika chifukwa cha mgwirizano wake waukulu wa hydrogen ndi leucine 218. Ma ma maning ena odziwika ndi awa: 3-iodoindole (-4.3 kcal/mol), 4-iodoindole (-4.0 kcal/mol), ndi 6-fluoroindole (-2.6 kcal/mol) (Chithunzi Chowonjezera S8). Ma indole ambiri okhala ndi halogenated ndi indole okha, kupatula 5-iodoindole ndi 2-iodoindole, amapanga mgwirizano ndi serine 260. Mfundo yakuti hydrogen bonding ndi leucine 218 imasonyeza kuti receptor-ligand imalumikizana bwino, monga momwe zawonedwera pa ivermectin (Chithunzi Chowonjezera S7), imatsimikizira kuti 5-iodoindole ndi 2-iodoindole, monga ivermectin, zimamangirira mwamphamvu pamalo ogwirira ntchito a GluCL receptor kudzera mu leucine 218 (Chithunzi 6 ndi Chithunzi Chowonjezera S8). Tikupereka lingaliro lakuti mgwirizanowu umafunika kuti pakhale mawonekedwe otseguka a GluCL complex ndipo pomangirira mwamphamvu pamalo ogwirira ntchito a GluCL receptor, 5-iodoindole, 2-iodoindole, avermectin ndi ivermectin motero zimasunga njira ya ion yotseguka ndikulola madzi kulowa.
Kuyika mamolekyu a indole ndi halogenated indole ku GluCL. Ma link a (A) indole, (B) 4-fluoroindole, (C) 7-iodoindole, ndi (D) 5-iodoindole ligands pamalo omwe GluCL ikugwira ntchito. Puloteniyi imayimiridwa ndi riboni, ndipo ma backbone hydrogen bonds amawonetsedwa ngati mizere yachikasu yokhala ndi madontho. (A′), (B′), (C′), ndi (D′) amawonetsa kuyanjana kwa ma ligands ofanana ndi ma amino acid residues ozungulira, ndipo ma side-chain hydrogen bonds amawonetsedwa ndi mivi ya pinki yokhala ndi madontho.
Kuyesera kunachitika kuti awone momwe 5-iodoindole imakhudzira kumera kwa mbewu za kabichi ndi radish. 5-iodoindole (0.05 kapena 0.1 mM) kapena avermectin (10 μg/mL) sizinakhudze kwambiri kumera koyamba ndi kumera kwa mbewu za zomera (Chithunzi 7). Kuphatikiza apo, palibe kusiyana kwakukulu komwe kunapezeka pakati pa kuchuluka kwa kumera kwa mbewu zomwe sizinachiritsidwe ndi mbewu zomwe zinapatsidwa 5-iodoindole kapena avermectin. Zotsatira zake pa kutalika kwa mizu ya taproot ndi kuchuluka kwa mizu ya mbali zomwe zinapangidwa sizinali zazikulu, ngakhale 1 mM (kuchulukitsa ka 10 kwake) ya 5-iodoindole inachedwetsa pang'ono kukula kwa mizu ya mbali. Zotsatirazi zikusonyeza kuti 5-iodoindole si poizoni kwa maselo a zomera ndipo sisokoneza njira zokulira kwa zomera pa kuchuluka komwe kunaphunziridwa.
Zotsatira za 5-iodoindole pa kumera kwa mbewu. Kumera, kumera ndi mizu ya mbali ya mbewu za B. oleracea ndi R. raphanistrum pa Murashige ndi Skoog agar medium yokhala ndi kapena yopanda avermectin kapena 5-iodoindole. Kumera kunalembedwa patatha masiku atatu kuchokera pamene mbewu zinamera pa 22°C.
Kafukufukuyu akufotokoza za milandu ingapo ya kuphedwa kwa nematode ndi indoles. Chofunika kwambiri, iyi ndi lipoti loyamba la iodoindole yomwe imayambitsa methylation (njira yomwe imachitika chifukwa cha kuchuluka kwa ma vacuoles ang'onoang'ono omwe pang'onopang'ono amaphatikizana kukhala ma vacuoles akuluakulu, zomwe pamapeto pake zimapangitsa kuti nembanemba iwonongeke ndi kufa) mu singano za paini, ndipo iodoindole ikuwonetsa mphamvu zazikulu zopha nematode zofanana ndi zomwe zimagwiritsidwa ntchito ndi nematicide avermectin.
Ma Indoles adanenedwa kale kuti ali ndi ntchito zambiri zodziwitsa ma prokaryotes ndi ma eukaryotes, kuphatikizapo kuletsa/kupanga kwa biofilm, kupulumuka kwa mabakiteriya, komanso matenda19,32,33,34. Posachedwapa, zotsatira zochiritsira za ma indoles a halogenated, ma indole alkaloids, ndi ma indole derivatives a semisynthetic zakopa chidwi chachikulu cha kafukufuku35,36,37. Mwachitsanzo, ma indoles a halogenated awonetsedwa kuti amapha maselo a Escherichia coli ndi Staphylococcus aureus omwe amapitilira37. Kuphatikiza apo, ndikofunikira kwasayansi kuphunzira momwe ma indoles a halogenated amagwirira ntchito motsutsana ndi mitundu ina, mitundu, ndi mafumu, ndipo kafukufukuyu ndi sitepe yokwaniritsira cholinga ichi.
Pano, tikupereka njira yopangira 5-iodoindole-induced letality mu C. elegans kutengera kuvulala kwa maselo komwe kumasinthidwa (RCI) ndi methylation (Zithunzi 4C ndi 5). Kusintha kwa edematous monga kutupa 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 pampu ya ATPase, kapena kusokoneza nembanemba ya maselo ndikuyambitsa kuchuluka kwa Na+, Ca2+, ndi water50,51,52 mwachangu. Ma vacuole a intracytoplasmic amabuka m'maselo a nyama chifukwa cha kuchuluka kwa madzi mu cytoplasm chifukwa cha kuchuluka kwa Ca2+ ndi water53. Chochititsa chidwi n'chakuti, njira imeneyi yowonongera maselo imatha kusinthidwa ngati kuwonongekako kuli kwakanthawi ndipo maselo ayamba kupanga ATP kwa nthawi inayake, koma ngati kuwonongekako kukupitirira kapena kukulirakulira, maselowo amafa.54 Zomwe tawona zikusonyeza kuti ma nematode omwe amathandizidwa ndi 5-iodoindole sangathe kubwezeretsa biosynthesis yabwinobwino atatha kuvutika ndi mavuto.
Mtundu wa methylation womwe umayamba chifukwa cha 5-iodoindole mu B. xylophilus ukhoza kukhala chifukwa cha kukhalapo kwa ayodini ndi kufalikira kwake kwa mamolekyu, popeza 7-iodoindole inali ndi mphamvu yochepa yoletsa B. xylophilus kuposa 5-iodoindole (Table 1 ndi Supplementary Picture S6). Zotsatirazi zikugwirizana pang'ono ndi maphunziro a Maltese et al. (2014), omwe adanenanso kuti kusamutsidwa kwa pyridyl nitrogen moiety mu indole kuchokera ku para- kupita ku meta-position kunathetsa vacuolization, kuletsa kukula, ndi cytotoxicity mu maselo a U251, zomwe zikusonyeza kuti kuyanjana kwa molekyulu ndi malo enaake ogwirira ntchito mu puloteni ndikofunikira27,44,45. Kuyanjana pakati pa indole kapena halogenated indoles ndi GluCL receptors zomwe zawonedwa mu kafukufukuyu kumathandiziranso lingaliro ili, chifukwa 5- ndi 2-iodoindole zidapezeka kuti zimamangirira ku GluCL receptors mwamphamvu kwambiri kuposa ma indole ena omwe adawunikidwa (Chithunzi 6 ndi Supplementary Picture S8). Iodini yomwe ili pamalo achiwiri kapena achisanu a indole inapezeka kuti imagwirizana ndi leucine 218 ya GluCL receptor kudzera mu backbone hydrogen bonds, pomwe ma halogen ena a indole ndi indole okha amapanga ma hydrogen bonds ofooka a side-chain ndi serine 260 (Chithunzi 6). Chifukwa chake tikuganiza kuti malo a halogen amatenga gawo lofunikira pakuyambitsa kuwonongeka kwa vacuolar, pomwe kumangirira kolimba kwa 5-iodoindole kumasunga njira ya ion yotseguka, motero kulola kulowa kwamadzimadzi mwachangu ndi kuphulika kwa vacuole. Komabe, njira yodziwira bwino ya 5-iodoindole idakalipo.
Musanagwiritse ntchito 5-iodoindole, muyenera kusanthula mphamvu yake ya poizoni pa zomera. Kuyesera kwathu kumera kwa mbewu kunasonyeza kuti 5-iodoindole sinali ndi zotsatirapo zoyipa pa kumera kwa mbewu kapena njira zina zokulira pambuyo pake pa kuchuluka komwe kunaphunziridwa (Chithunzi 7). Chifukwa chake, kafukufukuyu amapereka maziko ogwiritsira ntchito 5-iodoindole m'chilengedwe kuti athetse kuopsa kwa pine nematodes ku mitengo ya paini.
Malipoti am'mbuyomu asonyeza kuti mankhwala ochokera ku indole akuyimira njira yothetsera vuto la kukana maantibayotiki komanso kukula kwa khansa55. Kuphatikiza apo, indoles ili ndi zochita zotsutsana ndi mabakiteriya, zotsutsana ndi khansa, zotsutsana ndi antioxidant, zotsutsana ndi kutupa, zotsutsana ndi matenda a shuga, zotsutsana ndi mavairasi, zotsutsana ndi kufalikira kwa matenda komanso zotsutsana ndi chifuwa chachikulu ndipo zitha kukhala maziko abwino opangira mankhwala56,57. Kafukufukuyu akuwonetsa koyamba kugwiritsa ntchito ayodini ngati mankhwala oletsa tizilombo toyambitsa matenda komanso oletsa kufalikira kwa matenda.
Avermectin idapezeka zaka makumi atatu zapitazo ndipo idapambana Mphoto ya Nobel mu 2015, ndipo kugwiritsidwa ntchito kwake ngati mankhwala oletsa kufalikira kwa matenda kukupitirirabe. Komabe, chifukwa cha kukula mwachangu kwa kukana kwa avermectins mu nematodes ndi tizilombo toononga, njira ina, yotsika mtengo, komanso yosamalira chilengedwe ikufunika kuti ithetse matenda a PWN m'mitengo ya paini. Kafukufukuyu akufotokozanso momwe 5-iodoindole imaphera nematodes a paini ndipo 5-iodoindole ili ndi poizoni wochepa ku maselo a zomera, zomwe zimatsegula mwayi wabwino wogwiritsidwa ntchito mtsogolo m'malonda.
Mayesero onse adavomerezedwa ndi Komiti ya Makhalidwe Abwino ya Yunivesite ya Yeungnam, Gyeongsan, Korea, ndipo njirazo zidachitika motsatira malangizo a Komiti ya Makhalidwe Abwino ya Yunivesite ya Yeungnam.
Kuyesa kuyika mazira m'mazira kunachitika pogwiritsa ntchito njira zokhazikitsidwa43. Pofuna kuwona kuchuluka kwa kuswana (HR), nematode akuluakulu a tsiku limodzi (pafupifupi akazi 100 ndi amuna 100) adasamutsidwira ku mbale za Petri zomwe zili ndi bowa ndipo adaloledwa kukula kwa maola 24. Kenako mazira adachotsedwa ndikuchiritsidwa ndi 5-iodoindole (0.05 mM ndi 0.1 mM) kapena avermectin (10 μg/ml) ngati kuyika m'madzi osungunuka. Kuyika kumeneku (500 μl; pafupifupi mazira 100) kudasamutsidwira ku zitsime za mbale yopangira minofu ya zitsime 24 ndikuyikidwa pa 22 °C. Kuwerengera kwa L2 kudachitika patatha maola 24 akuyika koma kumaonedwa kuti kwafa ngati maselo sanasunthe akalimbikitsidwa ndi waya wabwino wa platinamu. Kuyesaku kunachitika m'magawo awiri, chilichonse ndi kubwereza kasanu ndi kamodzi. Deta kuchokera ku kuyesa konseku idaphatikizidwa ndikuperekedwa. Chiwerengero cha HR chimawerengedwa motere:
Imfa ya mphutsi inayesedwa pogwiritsa ntchito njira zomwe zidapangidwa kale. Mazira a Nematode anasonkhanitsidwa ndipo mazira anagwirizanitsidwa mwa kuswana m'madzi oyeretsedwa kuti apange mphutsi za L2. Mphutsi zolumikizidwa (pafupifupi 500 nematodes) zinapatsidwa mankhwala a 5-iodoindole (0.05 mM ndi 0.1 mM) kapena avermectin (10 μg/ml) ndipo zinaleredwa pa mbale za B. cinerea Petri. Pambuyo pa maola 48 a kusungidwa pa 22 °C, nematodes anasonkhanitsidwa m'madzi oyeretsedwa ndipo anafufuzidwa kuti awone ngati pali magawo a L2, L3, ndi L4. Kupezeka kwa magawo a L3 ndi L4 kunasonyeza kusintha kwa mphutsi, pomwe kupezeka kwa gawo la L2 kunasonyeza kuti palibe kusintha. Zithunzi zinapezedwa pogwiritsa ntchito iRiS™ Digital Cell Imaging System. Kuyesera kumeneku kunachitika m'magawo awiri, chilichonse ndi kubwereza kasanu ndi kamodzi. Deta kuchokera ku zoyeserera zonse ziwiri idaphatikizidwa ndikuwonetsedwa.
Kuopsa kwa 5-iodoindole ndi avermectin ku mbewu kunayesedwa pogwiritsa ntchito mayeso omera pa mbale za Murashige ndi Skoog agar.62 Mbewu za B. oleracea ndi R. raphanistrum zinayamba kunyowa m'madzi oyeretsedwa kwa tsiku limodzi, kutsukidwa ndi 1 ml ya 100% ethanol, kutsukidwa ndi 1 ml ya 50% bleach yogulitsa (3% sodium hypochlorite) kwa mphindi 15, ndikutsukidwa kasanu ndi madzi oyeretsedwa a 1 ml. Mbewu zoyeretsedwazo zinakanikizidwa pa mbale za germinate agar zomwe zili ndi 0.86 g/l (0.2X) Murashige ndi Skoog medium ndi 0.7% bacteriological agar yokhala ndi kapena yopanda 5-iodoindole kapena avermectin. Kenako mbalezo zinayikidwa mu incubation pa 22 °C, ndipo zithunzi zinajambulidwa patatha masiku atatu kuchokera mu incubation. Kuyesera kumeneku kunachitika m'magawo awiri, iliyonse yomwe inali ndi kubwereza kasanu ndi kamodzi.
Nthawi yotumizira: Feb-26-2025