I-pine nematode iyi-quarantine migratory endoparasite eyaziwa ngokudala ukulahlekelwa okukhulu kwezomnotho ku-ecosystem yehlathi likaphayini. Ucwaningo lwamanje lubuyekeza umsebenzi we-nematicidal we-halogenated indoles ngokumelene ne-pine nematodes kanye nendlela yabo yokusebenza. Imisebenzi ye-nematicidal ye-5-iodoindole ne-avermectin (ukulawula okuhle) ngokumelene ne-pine nematode yayifana futhi iphakeme ekugxilweni okuphansi (10 μg/mL). I-5-iodoindole yehlise i-fecundity, umsebenzi wokuzala, ukufa kwe-embryonic kanye nezibungu, kanye nokuziphatha kwe-locomotor. Ukusebenzisana kwamangqamuzana ama-ligands ane-invertebrate-specific glutamate-gated chloride channel receptors kusekela umbono wokuthi i-5-iodoindole, njenge-avermectin, ibophezela ngokuqinile kusayithi elisebenzayo le-receptor. I-5-Iodoindole iphinde yadala ukuwohloka okuhlukahlukene kwe-phenotypic kuma-nematodes, okuhlanganisa ukugoqeka/ukuncipha kwesitho esingavamile kanye nokukhuphuka kwe-vacuolization. Le miphumela iphakamisa ukuthi ama-vacuoles angase abambe iqhaza ekufeni kwe-nematode methylation-mediated. Okubalulekile, i-5-iodoindole yayingenabo ubuthi kuzo zombili izinhlobo zezitshalo (iklabishi nesithombo). Ngakho, lolu cwaningo lubonisa ukuthi ukusetshenziswa kwe-iodoindole ngaphansi kwezimo zemvelo kungalawula ukulimala kwe-pine wilt.
I-pine wood nematode (i-Bursaphelenchus xylophilus) iyingxenye ye-pine wood nematodes (PWN), i-endoparasitic nematode efudukayo eyaziwa ngokudala umonakalo omkhulu wemvelo endaweni yemvelo yehlathi likaphayini1. I-Pine wilt disease (PWD) ebangelwa i-pine wood nematode iba inkinga enkulu emazwenikazi amaningana, kuhlanganise ne-Asia ne-Europe, futhi eNyakatho Melika, i-nematode ibhubhisa izinhlobo zephayini ezifakiwe1,2. Ukwehla kwesihlahla sikaphayini kuyinkinga enkulu yezomnotho, futhi ithemba lokusabalala kwaso emhlabeni wonke liyakhathaza3. Lezi zinhlobo zikaphayini ezilandelayo zivame ukuhlaselwa i-nematode: i-Pinus densiflora, i-Pinus sylvestris, i-Pinus thunbergii, i-Pinus koraiensis, i-Pinus thunbergii, i-Pinus thunbergii, ne-Pinus radiata4. I-pine nematode yisifo esiyingozi esingabulala izihlahla zikaphayini phakathi namasonto noma izinyanga zokutheleleka. Ngaphezu kwalokho, ukuqubuka kwe-pine nematode kuvamile ezindaweni ezihlukahlukene zemvelo, ngakho-ke amaketanga okutheleleka aphikelelayo asesungulwe1.
I-Bursaphelenchus xylophilus iyi-quarantine plant-parasitic nematode eyingxenye yomndeni omkhulu i-Aphelenchoidea kanye ne-clade 102.5. I-nematode idla isikhunta bese izalana ezicutshini zokhuni zezihlahla zikaphayini, ikhula ibe izigaba ezine ezihlukene zezibungu: L1, L2, L3, L4 kanye nomuntu omdala1,6. Ngaphansi kwezimo zokushoda kokudla, i-pine nematode idlulela esigabeni se-larval esikhethekile - i-dauer, ephazamisa i-vector yayo - i-pine bark beetle (i-Monochamus alternatus) futhi idluliselwe ezihlahleni zikaphayini ezinempilo. Ezindaweni ezinempilo, ama-nematode ahamba ngokushesha ngezicubu zezitshalo futhi adla amangqamuzana e-parenchymatous, okuholela ekuphenduleni okuningi kwe-hypersensitivity, ukubola kukaphayini nokufa phakathi nonyaka ngemva kokutheleleka1,7,8.
Ukulawulwa kwebhayoloji ye-pine nematode sekuyisikhathi eside kuyinselele, ngezinyathelo zokuvalela abantu kusukela ekhulwini lama-20. Amasu amanje okulawula i-pine nematode ikakhulukazi ahilela ukwelashwa kwamakhemikhali, okuhlanganisa ukuthuthwa kwezinkuni kanye nokufakwa kwe-nematicides eziqwini zezihlahla. Ama-nematicides asetshenziswa kakhulu yi-avermectin ne-avermectin benzoate, okungawomndeni we-avermectin. Lawa makhemikhali abizayo asebenza kahle kakhulu ezinhlotsheni eziningi ze-nematode futhi athathwa njengephephile emvelweni9. Kodwa-ke, ukusetshenziswa okuphindaphindiwe kwalawa ma-nematicides kulindeleke ukuthi kudale ingcindezi yokukhetha ezoholela ekuveleni kwezinambuzane zikaphayini ezingazweli, njengoba kuye kwaboniswa ezinambuzaneni ezimbalwa, njengeLeptinotarsa decemlineata, iPlutella xylostella kanye ne-nematodes i-Trichostrongylus colubriformis kanye ne-Ostertagiaver eziye 1 kancane kancane ukumelana ne-12 circumcinins. Ngakho-ke, amaphethini okumelana nawo adinga ukucutshungulwa njalo futhi ama-nematicides ahlolwe ngokuqhubekayo ukuze kutholwe ezinye izindlela, ezingabizi kakhulu futhi ezilungele imvelo zokulawula i-PVD. Emashumini eminyaka amuva nje, abalobi abaningi baye bahlongoza ukusetshenziswa kwezitshalo zezitshalo, amafutha abalulekile kanye nama-volatile njengama-ejenti okulawula i-nematode13,14,15,16.
Sisanda kukhombisa umsebenzi we-nematicidal we-indole, i-molecule ye-intercellular kanye ne-interkingdom ekhombisa, ku-Caenorhabditis elegans 17. I-Indole iyisiginali ye-intracellular esabalele ku-microbial ecology, elawula imisebenzi eminingi ethinta i-microbial physiology, ukwakheka kwe-spore, ukuzinza kwe-plasmid, ukumelana nezidakamizwa, ukwakheka kwe-biofilm, kanye ne-virulence 18, 19. Umsebenzi we-indole kanye nokuphuma kwawo ngokumelene namanye ama-nematode e-pathogenic awukafundwa. Kulolu cwaningo, siphenye umsebenzi we-nematicidal wama-indoles angu-34 ngokumelene ne-pine nematodes futhi sacacisa indlela yokusebenza ye-5-iodoindole enamandla kakhulu kusetshenziswa i-microscopy, ukuthwebula izithombe ezidlula isikhathi, nokuhlolwa kwe-docking yama-molecular, futhi sahlola umthelela wako wobuthi ezitshalweni zisebenzisa i-assay yokuhluma kwembewu.
Ukugxila okuphezulu (>1.0 mM) kwe-indole kubikwe ngaphambilini ukuthi kunomthelela we-nematicidal kuma-nematodes17. Ukulandela ukwelashwa kwe-B. xylophilus (izigaba zempilo ezixubile) ne-indole noma okuphuma kokunye okuhlukile kwe-indole okungu-33 ku-1 mM, ukufa kwe-B. xylophilus kukalwa ngokubala ama-nematode aphilayo nafile emaqenjini okulawula nalashwa. Ama-indoles amahlanu abonise umsebenzi obalulekile we-nemacidal; ukusinda kweqembu lokulawula elingaphathwanga kwaba ngama-95 ± 7% ngemva kwamahora angu-24. Kuma-indoles angu-34 ahloliwe, i-5-iodoindole ne-4-fluoroindole ku-1 mM ibangele ukufa okungu-100%, kuyilapho i-5,6-difluoroindigo, i-methylindole-7-carboxylate, ne-7-iodoindole ibangele cishe ukufa okungama-50% (Ithebula 1).
Umphumela we-5-iodoindole ekwakhekeni kwe-vacuole kanye ne-metabolism ye-pine wood nematode. (A) Umphumela we-avermectin kanye ne-5-iodoindole kuma-nematode wesilisa omdala, (B) amaqanda e-L1 esiteji se-nematode kanye (C) ne-metabolism ye-B. xylophilus, (i) ama-vacuoles awabonwanga ku-0 h, ukwelashwa kubangele (ii) ama-vacuoles, (iii) ukuqoqwa kwama-vacuoles amaningi, i-vacuoles ye-vacuoles (i-vacuoles) kanye (vi) nokwakhiwa kwama-vacuole amakhulu. Imicibisholo ebomvu ibonisa ukuvuvukala kwama-vacuoles, imicibisholo eluhlaza okwesibhakabhaka ibonisa ukuhlanganiswa kwama-vacuoles nemicibisholo emnyama ibonisa ama-vacuoles amakhulu. Ibha yesikali = 50 μm.
Ngaphezu kwalokho, lolu cwaningo luphinde lwachaza inqubo elandelanayo yokufa kwe-methane-induced in pine nematodes (Figure 4C). Ukufa kwe-Methanogenic kuwuhlobo lwe-non-apoptotic lokufa kweseli okuhlotshaniswa nokuqoqwa kwama-cytoplasmic vacuoles avelele27. Ukukhubazeka kwe-morphological okubonwa kuma-pine nematode kubonakala kuhlobene eduze nendlela yokufa okubangelwa i-methane. Ukuhlolwa kwe-Microscopic ngezikhathi ezihlukene kwabonisa ukuthi ama-vacuoles amakhulu akhiwa ngemva kwamahora angu-20 wokuchayeka ku-5-iodoindole (0.1 mM). Ama-vacuoles ama-Microscopic abonwa ngemuva kwamahora ayi-8 wokwelashwa, futhi inani lawo lenyuka ngemuva kwamahora ayi-12. Ama-vacuole amakhulu amaningana abonwa ngemuva kwamahora ayi-14. Ama-vacuole amaningana ahlanganisiwe abonakala ngokucacile ngemva kwamahora angu-12-16 okwelashwa, okubonisa ukuthi ukuhlanganiswa kwe-vacuole kuyisisekelo sendlela yokufa kwe-methanonic. Ngemva kwamahora angu-20, kwatholakala ama-vacuoles amakhulu amaningi kulo lonke isikelemu. Lokhu kubuka kumelela umbiko wokuqala we-metuosis ku-C. elegans.
Ezimpethuni ezilashwe nge-5-iodoindole, ukuhlanganiswa kwe-vacuole nokuqhekeka nakho kwabonwa (Umfanekiso wesi-5), njengoba kuboniswa ukugoba kwesikelemu kanye nokukhululwa kwe-vacuole endaweni ezungezile. Ukuphazamiseka kwe-Vacuole kuphinde kwabonwa kulwelwesi lwegobolondo leqanda, elivame ukugcinwa liphelele yi-L2 ngesikhathi sokuchanyuselwa (I-Supplementary Fig. S2). Lokhu kubheka kusekela ukubandakanyeka kokuqoqwa kwamanzi kanye nokuhluleka kwe-osmoregulatory, kanye nokulimala kweseli elibuyiselwayo (RCI), ohlelweni lokubunjwa kwe-vacuole kanye ne-suppuration (Fig. 5).
I-hypothesizing indima ye-iodine ekwakhekeni kwe-vacuole ephawuliwe, siphenye umsebenzi we-nematicidal we-sodium iodide (NaI) ne-potassium iodide (KI). Kodwa-ke, ekugxilweni (0.1, 0.5 noma 1 mM), akuzange kuthinte noma yikuphi ukusinda kwe-nematode noma ukwakheka kwe-vacuole (I-Supplementary Fig. S5), nakuba i-1 mM KI ibe nomthelela omncane we-nematicidal. Ngakolunye uhlangothi, i-7-iodoindole (1 noma 2 mM), njenge-5-iodoindole, yenze ama-vacuole amaningi kanye nokukhubazeka kwesakhiwo (I-Supplementary Fig. S6). Ama-iodoindoles amabili abonise izici ezifanayo ze-phenotypic kuma-pine nematodes, kuyilapho i-NaI ne-KI ayizange. Kuyathakazelisa ukuthi i-indole ayizange yenze ukwakheka kwe-vacuole ku-B. xylophilus ekugxilweni okuhloliwe (idatha engaboniswa). Ngakho-ke, imiphumela yaqinisekisa ukuthi inkimbinkimbi ye-indole-iodine inesibopho se-vacuolization kanye ne-metabolism ye-B. xylophilus.
Phakathi kwama-indoles ahlolelwe umsebenzi we-nematicidal, i-5-iodoindole yayinenkomba ephakeme kakhulu ye-slip -5.89 kcal / mol, ilandelwa yi-7-iodoindole (-4.48 kcal / mol), 4-fluoroindole (-4.33), ne-indole (-4.03) (Figure 6). Ibhondi ye-hydrogen eqinile engumgogodla we-5-iodoindole ku-leucine 218 imisa ukubopha kwayo, kuyilapho zonke ezinye izinto eziphuma ku-indole zibophezela ku-serine 260 ngamabhondi e-hydrogen eseceleni. Phakathi kwamanye ama-iodoindoles amodelayo, i-2-iodoindole inenani elibophezelayo elingu -5.248 kcal/mol, elibangelwa isibopho sayo esikhulu se-hydrogen ne-leucine 218. Okunye okubophayo okwaziwayo kuhlanganisa i-3-iodoindole (-4.3 kcal/mol), 4-iodoindole (-4.0) kanye ne-6. kcal/mol) (Umfanekiso Owengeziwe S8). Ama-indoles amaningi ane-halogenated kanye ne-indole ngokwayo, ngaphandle kwe-5-iodoindole kanye ne-2-iodoindole, yakha isibopho ne-serine 260. Iqiniso lokuthi i-hydrogen bonding ne-leucine 218 libonisa ukusebenza kahle kwe-receptor-ligand, njengoba kuphawulwe ivermectin (Supplementary Fig. I-2-iodoindole, njenge-ivermectin, ibophezela ngokuqinile kusayithi elisebenzayo le-GluCL receptor nge-leucine 218 (Fig. 6 kanye ne-Supplementary Fig. S8). Siphakamisa ukuthi lokhu kubophezela kuyadingeka ukuze kugcinwe ukwakheka kwe-pore evulekile ye-GluCL complex futhi ngokubopha ngokuqinile endaweni esebenzayo ye-GluCL receptor, i-5-iodoindole, i-2-iodoindole, i-avermectin ne-ivermectin ngaleyo ndlela igcine isiteshi se-ion sivulekile futhi sivumele ukutholakala koketshezi.
Ukufakwa kwe-molecular ye-indole ne-halogenated indole ku-GluCL. Ukuma okubophezelayo kwe-(A) indole, (B) 4-fluoroindole, (C) 7-iodoindole, kanye (D) nama-ligand angu-5-iodoindole kusayithi elisebenzayo le-GluCL. Iphrotheni imelelwa iribhoni, futhi izibopho ze-hydrogen ezingemuva ziboniswa njengemigqa enamachashazi aphuzi. (A′), (B′), (C′), kanye (D′) abonisa ukusebenzisana kwemigqa ehambisanayo nezinsalela ze-amino acid ezizungezile, futhi amabhondi e-hydrogen e-side-chain aboniswa ngemicibisholo enamachashazi aphinki.
Kwenziwa izivivinyo zokuhlola umphumela onobuthi we-5-iodoindole ekuhlumeni kwembewu yeklabishi nesithombo. I-5-iodoindole (0.05 noma 0.1 mM) noma i-avermectin (10 μg/mL) ibe nomthelela omncane noma ingenawo nhlobo ekuhlumeni kokuqala kanye nokuvela kwesitshalo (Umfanekiso 7). Ngaphezu kwalokho, awukho umehluko obalulekile owatholakala phakathi kwezinga lokuhluma kwezilawuli ezingalashwa kanye nembewu ephathwa nge-5-iodoindole noma i-avermectin. Umthelela ekwelulweni kwe-taproot kanye nenani lezimpande ezisemaceleni ezakhiwe wawungabalulekile, nakuba i-1 mM (iphindwe ka-10 ukugxila kwayo okusebenzayo) ye-5-iodoindole yabambezela kancane ukukhula kwezimpande ezisemaceleni. Le miphumela ikhombisa ukuthi i-5-iodoindole ayinabo ubuthi kumaseli okutshala futhi ayiphazamisi izinqubo zokuthuthukiswa kwezitshalo ekugxiliseni okufundwe.
Umthelela we-5-iodoindole ekuhlumeni kwembewu. Ukumila, ukuhluma kanye nokumila kwezimpande kwe-lateral kwembewu ye-B. oleracea kanye ne-R. raphanistrum ku-Murashige ne-Skoog agar medium ene-avermectin noma ngaphandle kwayo noma i-5-iodoindole. Ukuhluma kwabhalwa ngemuva kwezinsuku ezi-3 zokufukamela ku-22°C.
Lolu cwaningo lubika izigameko eziningana zokubulawa kwe-nematode ngama-indoles. Okubalulekile, lona umbiko wokuqala we-iodoindole inducing methylation (inqubo ebangelwa ukunqwabelana kwama-vacuoles amancane ahlangana kancane kancane abe ama-vacuole amakhulu, agcine eholela ekuqhekekeni kolwelwesi nokufa) ezinalithini zikaphayini, ne-iodoindole ekhombisa izici ezibalulekile ze-nematicidal ezifana nalezo ze-avermectin ye-nematicide yokuhweba.
Ama-Indoles abikwe ngaphambilini ukuthi enze imisebenzi eminingi yokubonisa ama-prokaryotes nama-eukaryotes, okuhlanganisa ukuvimbela / ukwakheka kwe-biofilm, ukusinda kwamagciwane, kanye ne-pathogenicity19,32,33,34. Muva nje, imiphumela yokwelapha engaba khona yama-indoles ane-halogenated, i-indole alkaloid, kanye nokuphuma kwe-semisynthetic indole kuye kwaheha isithakazelo esibanzi socwaningo35,36,37. Isibonelo, ama-indoles ane-halogenated aboniswe ukubulala i-Escherichia coli eqhubekayo kanye namaseli e-Staphylococcus aureus37. Ngaphezu kwalokho, kuyintshisekelo yesayensi ukutadisha ukusebenza kahle kwama-indoles ane-halogenated ngokumelene nezinye izinhlobo zezilwane, isizukulwane, nemibuso, futhi lolu cwaningo luyisinyathelo sokufinyelela lo mgomo.
Lapha, siphakamisa indlela yokubulala i-5-iodoindole e-C. elegans esekelwe ekulimaleni kweseli okubuyiselekayo (RCI) kanye ne-methylation (Izibalo 4C kanye ne-5). Izinguquko ze-Edematous ezifana nokukhukhumeza kanye nokuwohloka kwe-vacuolar yizinkomba ze-RCI ne-methylation, ezibonakaliswa njengama-vacuoles amakhulu ku-cytoplasm48,49. I-RCI iphazamisa ukukhiqizwa kwamandla ngokunciphisa ukukhiqizwa kwe-ATP, okubangela ukwehluleka kwepompo ye-ATPase, noma ukuphazamisa ulwelwesi lwamaseli futhi kubangele ukugeleza okusheshayo kwe-Na +, Ca2 +, namanzi50,51,52. Ama-vacuoles e-Intracytoplasmic avela kumaseli ezilwane ngenxa yokuqoqwa koketshezi ku-cytoplasm ngenxa yokungena kwe-Ca2 + namanzi53. Kuyathakazelisa ukuthi le nqubo yokulimala kwamangqamuzana ibuyiselwa emuva uma umonakalo ungokwesikhashana futhi amangqamuzana aqala ukukhiqiza i-ATP isikhathi esithile, kodwa uma umonakalo uqhubeka noma uba mkhulu, amangqamuzana ayafa.54 Ukubuka kwethu kubonisa ukuthi i-nematodes ephathwa nge-5-iodoindole ayikwazi ukubuyisela i-biosynthesis evamile ngemva kokuchayeka ezimweni zokucindezeleka.
I-methylation phenotype eyenziwa yi-5-iodoindole ku-B. xylophilus ingase ibe ngenxa yokuba khona kwe-iodine nokusatshalaliswa kwayo kwamangqamuzana, njengoba i-7-iodoindole ibe nomthelela omncane ovimbelayo ku-B. xylophilus kune-5-iodoindole (Ithebula 1 kanye Nomfanekiso Owengeziwe S6). Le miphumela ihambisana kancane nezifundo zaseMaltese et al. (2014), owabika ukuthi ukudluliswa kwe-pyridyl nitrogen moiety ku-indole kusuka ku-para- kuya ku-meta-position kuqede i-vacuolization, ukuvimbela ukukhula, kanye ne-cytotoxicity kumaseli e-U251, okuphakamisa ukuthi ukusebenzisana kwe-molecule enendawo ethile esebenzayo kuphrotheni kubalulekile27,44,45. Ukusebenzisana phakathi kwama-indole ama-indole noma ama-halogenated nama-GluCL receptors abonwe kulolu cwaningo nakho kuyawusekela lo mbono, njengoba i-5- kanye ne-2-iodoindole kutholakale ukuthi ibophezela kuma-GluCL receptors ngokuqinile kunamanye ama-indoles ahloliwe (Umfanekiso 6 kanye Nomfanekiso Owengeziwe S8). I-iodine endaweni yesibili noma yesihlanu ye-indole itholwe ibophezela ku-leucine 218 yesamukeli se-GluCL ngamabhondi e-hydrogen e-backbone, kanti amanye ama-indoles ane-halogenated kanye ne-indole ngokwayo enza amabhondi e-hydrogen aseceleni abuthakathaka ne-serine 260 (Umfanekiso 6). Ngakho-ke sicabangela ukuthi ukwenziwa kwasendaweni kwe-halogen kudlala indima ebalulekile ekungenisweni kokuwohloka kwe-vacuolar, kuyilapho ukubopha okuqinile kwe-5-iodoindole kugcina isiteshi se-ion sivulekile, ngaleyo ndlela kuvumela ukugeleza koketshezi olusheshayo nokugqashuka kwe-vacuole. Nokho, indlela enemininingwane yokusebenza kwe-5-iodoindole isazonqunywa.
Ngaphambi kokusetshenziswa okusebenzayo kwe-5-iodoindole, umphumela wayo onobuthi ezitshalweni kufanele uhlaziywe. Ukuhlolwa kwethu kokuhluma kwembewu kubonise ukuthi i-5-iodoindole yayingenawo umthelela omubi ekuhlumeni kwembewu noma ezinqubweni zokukhula ezalandela ekugxilweni okufundwe (Umfanekiso 7). Ngakho, lolu cwaningo lunikeza isisekelo sokusebenzisa i-5-iodoindole endaweni ezungezile ukuze kulawulwe ubungozi be-pine nematodes ezihlahleni zikaphayini.
Imibiko yangaphambilini ikhombisile ukuthi ukwelashwa okusekelwe kwi-indole kumelela indlela engaba khona yokubhekana nenkinga yokumelana nama-antibiotic kanye nokuqhubekela phambili komdlavuza55. Ngaphezu kwalokho, ama-indoles ane-antibacterial, anticancer, antioxidant, anti-inflammatory, antidiabetic, antiviral, antiproliferative and antituberculosis imisebenzi futhi angase asebenze njengesisekelo esithembisayo sokuthuthukiswa kwezidakamizwa56,57. Lolu cwaningo luphakamisa okokuqala ukusetshenziswa okungenzeka kwe-iodine njenge-antiparasitic kanye ne-anthelmintic.
I-Avermectin yatholwa emashumini amathathu eminyaka edlule futhi yawina uMklomelo KaNobel ngo-2015, futhi ukusetshenziswa kwayo njenge-anthelmintic kusaqhubeka. Kodwa-ke, ngenxa yokuthuthukiswa okusheshayo kokumelana nama-avermectins kuma-nematodes nezinambuzane zezinambuzane, elinye isu, elingabizi kakhulu, nelinobungani bemvelo liyadingeka ukulawula ukutheleleka kwe-PWN ezihlahleni zikaphayini. Lolu cwaningo luphinde lubike indlela i-5-iodoindole ebulala ngayo i-pine nematode nokuthi i-5-iodoindole inobuthi obuphansi kumaseli okutshala, okuvula amathuba amahle okusetshenziswa kwayo kwezohwebo esikhathini esizayo.
Konke ukuhlola kuvunywe iKomidi Lezimiso Zokuziphatha laseYunivesithi yaseYeungnam, eGyeongsan, Korea, futhi izindlela zenziwa ngokuvumelana neziqondiso zeKomidi Lokuziphatha laseYunivesithi yaseYeungnam.
Ukuhlolwa kokufukanyelwa kweqanda kwenziwa kusetshenziswa izinqubo ezimisiwe43. Ukuhlola amazinga okuchanyuselwa (HR), ama-nematode amadala anosuku olu-1 (cishe abesifazane abayi-100 nabesilisa abayi-100) adluliselwa ezitsheni ze-Petri eziqukethe ukhunta futhi zavunyelwa ukukhula amahora angama-24. Amaqanda abe esehlukaniswa futhi aphathwa nge-5-iodoindole (0.05 mM kanye no-0.1 mM) noma i-avermectin (10 μg/ml) njengendlela yokumiswa emanzini oyinyumba acwecwe. Lokhu kumiswa (500 μl; cishe amaqanda ayi-100) kudluliselwe emithonjeni yepuleti le-tissue elinemithombo engama-24 futhi yafakwa ku-22 °C. Izibalo ze-L2 zenziwa ngemva kwamahora angu-24 ekufukanyelwa kodwa zazibhekwa njengezifile uma amaseli enganyakazi lapho ekhuthazwa ngocingo oluhle lweplatinamu. Lokhu kuhlolwa kwenziwa ngezigaba ezimbili, ngasinye sinezimpinda eziyisithupha. Idatha evela kokubili ukuhlolwa yahlanganiswa futhi yethulwa. Iphesenti lika-HR libalwa ngale ndlela elandelayo:
Ukufa kwezibungu kwahlolwa kusetshenziswa izinqubo ezakhiwe ngaphambilini. Amaqanda eNematode aqoqwa futhi imibungu yavunyelaniswa ngokuchanyuselwa emanzini acwebile angenalutho ukuze kukhiqizwe izibungu zesiteji se-L2. Izibungu ezivumelanisiwe (cishe ama-nematode angu-500) zalashwa nge-5-iodoindole (0.05 mM no-0.1 mM) noma i-avermectin (10 μg/ml) futhi zakhuliswa kumapuleti e-B. cinerea Petri. Ngemva kwamahora angu-48 ekufukamela ku-22 °C, ama-nematode aqoqwa emanzini acwecwe angenalutho futhi ahlolelwa ukuba khona kwezigaba ze-L2, L3, ne-L4. Ukuba khona kwezigaba ze-L3 ne-L4 kubonise ukuguqulwa kwezibungu, kuyilapho ukuba khona kwesiteji se-L2 kubonise ukuthi akukho nguquko. Izithombe zitholwe kusetshenziswa i-iRiS™ Digital Cell Imaging System. Lokhu kuhlolwa kwenziwa ngezigaba ezimbili, ngasinye sinezimpinda eziyisithupha. Idatha evela kokubili ukuhlolwa yahlanganiswa futhi yethulwa.
Ubuthi be-5-iodoindole ne-avermectin kumbewu buhlolwe kusetshenziswa izivivinyo zokuhluma kumapuleti e-Murashige kanye ne-Skoog agar. Imbewu ye-oleracea engu-62 B. oleracea ne-R. raphanistrum yaqale yacwiliswa emanzini angenalutho acwengekile usuku olulodwa, yagezwa nge-1 ml 100% sterilized 5% ye-sodium ethanol, i-3 ml ye-sodium ethengiswayo. i-hypochlorite) imizuzu engu-15, futhi igeze kahlanu ngo-1 ml wamanzi oyinyumba. Imbewu evalwe inzalo yabe isicindezelwa kumapuleti e-agar okumila aqukethe u-0.86 g/l (0.2X) i-Murashige ne-Skoog medium kanye no-0.7% we-bacteriological agar ene-5-iodoindole noma i-avermectin noma engenawo. Amapuleti abe esefukanyelwa ku-22 °C, futhi izithombe zathathwa ngemva kwezinsuku ezi-3 zokufukamela. Lolu cwaningo lwenziwe ngezigaba ezimbili, ngasinye sazo saba nezimpinda eziyisithupha.
Isikhathi sokuthumela: Feb-26-2025