I-pine nematode iyi-endoparasite yokufuduka ehlala yodwa eyaziwa ngokubangela ukulahlekelwa okukhulu kwezomnotho ezindaweni zemvelo zehlathi likaphayini. Ucwaningo lwamanje lubuyekeza umsebenzi wokubulala ama-indole afakwe i-halogenated ngokumelene nama-pine nematode kanye nendlela yawo yokusebenza. Imisebenzi yokubulala ama-pine nematode ye-5-iodoindole kanye ne-avermectin (ukulawula okuhle) ngokumelene nama-pine nematode yayifana futhi iphezulu ngamanani aphansi (10 μg/mL). I-5-iodoindole yehlise ukuzala, umsebenzi wokuzala, ukufa kwama-embryonic kanye nama-larval, kanye nokuziphatha kwe-locomotor. Ukusebenzisana kwama-molecular kwama-ligands ane-glutamate-gated chloride channel receptors e-invertebrate kusekela umbono wokuthi i-5-iodoindole, njenge-avermectin, ibopha ngokuqinile endaweni esebenzayo ye-receptor. I-5-Iodoindole iphinde yabangela ukuguquguquka okuhlukahlukene kwe-phenotypic kuma-nematode, kufaka phakathi ukuwa/ukuncipha kwezitho okungavamile kanye nokwanda kwe-vacuolization. Le miphumela iphakamisa ukuthi ama-vacuole angadlala indima ekufeni okubangelwa yi-nematode methylation. Okubalulekile, i-5-iodoindole yayingesiyo ubuthi kuzo zombili izinhlobo zezitshalo (iklabishi kanye ne-radish). Ngakho-ke, lolu cwaningo lubonisa ukuthi ukusetshenziswa kwe-iodoindole ngaphansi kwezimo zemvelo kungalawula ukulimala kwe-pine wilt.
I-pine wood nematode (i-Bursaphelenchus xylophilus) ingeyama-pine wood nematode (i-PWN), ama-nematode afudukayo ahlala e-endoparasitic aziwa ngokubangela umonakalo omkhulu emvelweni ezindaweni zemvelo zamahlathi ephayini1. Isifo sokubuna kwephayini (i-PWD) esibangelwa yi-pine wood nematode siba yinkinga enkulu emazwenikazi amaningana, okuhlanganisa i-Asia neYurophu, kanti eNyakatho Melika, i-nematode ibhubhisa izinhlobo zephayini ezifakiwe1,2. Ukuncipha kwezihlahla zephayini kuyinkinga enkulu yezomnotho, futhi amathuba okusabalala kwayo emhlabeni wonke ayakhathaza3. Izinhlobo ezilandelayo zephayini zivame ukuhlaselwa yi-nematode: i-Pinus densiflora, i-Pinus sylvestris, i-Pinus thunbergii, i-Pinus koraiensis, i-Pinus thunbergii, i-Pinus thunbergii, kanye ne-Pinus radiata4. I-pine nematode yisifo esibi esingabulala izihlahla zephayini zingakapheli amasonto noma izinyanga zokutheleleka. Ngaphezu kwalokho, ukuqubuka kwe-pine nematode kuvamile ezindaweni ezahlukene zemvelo, ngakho-ke sekusungulwe uchungechunge lokutheleleka oluqhubekayo1.
I-Bursaphelenchus xylophilus iyi-nematode yezitshalo ezihlala zodwa etholakala emndenini omkhulu i-Aphelenchoidea kanye ne-clade 102.5. I-nematode idla isikhunta futhi izale ezicutshini zezihlahla zephayini, ikhule ibe yizigaba ezine ezahlukene zama-larval: L1, L2, L3, L4 kanye nomuntu omdala1,6. Ngaphansi kwezimo zokuntuleka kokudla, i-pine nematode idlulela esigabeni esikhethekile sama-larval - i-dauer, edla i-vector yayo - i-pine bark beetle (i-Monochamus alternatus) bese idluliselwa ezihlahleni zephayini ezinempilo. Kubantu abanempilo, ama-nematode ahamba ngokushesha ezicutshini zezitshalo futhi adle amaseli e-parenchymatous, okuholela ezinhlobonhlobo zezimpendulo zokungezwani komzimba, ukubuna kwephayini nokufa kungakapheli unyaka ngemuva kokutheleleka1,7,8.
Ukulawulwa kwezinto eziphilayo kwama-pine nematode sekuyisikhathi eside kuyinselele, njengoba izinyathelo zokuvalelwa zodwa zisukela ekhulwini lama-20. Amasu amanje okulawula ama-pine nematode ahilela kakhulu ukwelashwa ngamakhemikhali, okuhlanganisa ukubulala izinambuzane ngokhuni kanye nokufakelwa kwama-nematicides eziqwini zezihlahla. Ama-nematicides asetshenziswa kakhulu yi-avermectin kanye ne-avermectin benzoate, okungowomndeni we-avermectin. Lawa makhemikhali abizayo asebenza kahle kakhulu ezinhlotsheni eziningi zama-nematode futhi abhekwa njengaphephile emvelweni9. Kodwa-ke, ukusetshenziswa okuphindaphindiwe kwala ma-nematicides kulindeleke ukuthi kudale ingcindezi yokukhetha okuzoholela ekuveleni kwama-pine nematode amelana nawo, njengoba kuye kwabonakala ezinambuzaneni eziningana, njenge-Leptinotarsa decemlineata, i-Plutella xylostella kanye nama-nematode i-Trichostrongylus colubriformis kanye ne-Ostertagia circumcincta, okuye kancane kancane kwakhula ukumelana nama-avermectin10,11,12. Ngakho-ke, amaphethini okumelana kudingeka afundwe njalo futhi ama-nematicides ahlolwe njalo ukuze kutholakale ezinye izindlela, ezingabizi kakhulu nezinobungane nemvelo zokulawula i-PVD. Emashumini eminyaka amuva nje, abalobi abaningana baphakamise ukusetshenziswa kwezithako zezitshalo, amafutha abalulekile kanye ne-volatiles njengezinto zokulawula i-nematode13,14,15,16.
Muva nje sibonise umsebenzi wokubulala amaseli we-indole, i-molecule yesignali ephakathi kwamangqamuzana kanye ne-interkingdom, ku-Caenorhabditis elegans 17. I-Indole iyisignali ebanzi yangaphakathi kwamangqamuzana emvelweni yamagciwane, elawula imisebenzi eminingi ethinta i-physiology yamagciwane, ukwakheka kwe-spore, ukuzinza kwe-plasmid, ukumelana nemithi, ukwakheka kwe-biofilm, kanye ne-virulence 18, 19. Umsebenzi we-indole kanye ne-derivatives yayo ngokumelene namanye ama-nematode abangela izifo awukafundwa. Kulesi sifundo, siphenye umsebenzi wokubulala amaseli we-indole angu-34 ngokumelene nama-nematode ephayini futhi sachaza indlela yokusebenza kwe-5-iodoindole enamandla kakhulu sisebenzisa i-microscopy, izithombe ze-time-lapse, kanye nokuhlolwa kwe-molecular docking, futhi sahlola imiphumela yayo enobuthi ezitshalweni sisebenzisa i-seed germination assay.
Ukuhlushwa okuphezulu (>1.0 mM) kwe-indole kuye kwabikwa ngaphambilini ukuthi kunomphumela wokubulala ama-nematode17. Ngemva kokwelashwa kwe-B. xylophilus (izigaba zokuphila ezixubile) nge-indole noma izinhlobo ezingu-33 ezahlukene ze-indole ku-1 mM, ukufa kwe-B. xylophilus kwalinganiswa ngokubala ama-nematode aphilayo nafile emaqenjini okulawula nawokwelapha. Ama-indole amahlanu abonise umsebenzi obalulekile wokubulala ama-nematic; ukusinda kweqembu lokulawula elingaphathwanga kwaba ngu-95 ± 7% ngemva kwamahora angu-24. Kuma-indole angu-34 ahlolwe, i-5-iodoindole kanye ne-4-fluoroindole ku-1 mM kubangele ukufa okungu-100%, kanti i-5,6-difluoroindigo, i-methylindole-7-carboxylate, kanye ne-7-iodoindole kubangele ukufa okungu-50% (Ithebula 1).
Umphumela we-5-iodoindole ekwakhekeni kwe-vacuole kanye nokusetshenziswa kwe-pine wood nematode. (A) Umphumela we-avermectin kanye ne-5-iodoindole kuma-nematode amaduna amadala, (B) amaqanda e-L1 stage nematode kanye (C) ukusetshenziswa kwe-B. xylophilus, (i) ama-vacuole awabonwanga ngehora eli-0, ukwelashwa kwaholela ku-(ii) ama-vacuole, (iii) ukuqongelela kwama-vacuole amaningi, (iv) ukuvuvukala kwama-vacuole, (v) ukuhlanganiswa kwama-vacuole kanye (vi) ukwakheka kwama-vacuole amakhulu. Imicibisholo ebomvu ikhombisa ukuvuvukala kwama-vacuole, imicibisholo eluhlaza okwesibhakabhaka ikhombisa ukuhlanganiswa kwama-vacuole kanye nemicibisholo emnyama ikhombisa ama-vacuole amakhulu. Ibha yesikali = 50 μm.
Ngaphezu kwalokho, lolu cwaningo luchaze nenqubo elandelanayo yokufa okubangelwa yi-methane kuma-nematode ephayini (Isithombe 4C). Ukufa kwe-methanogenic uhlobo lokufa kwamaseli olungeyona i-apoptotic oluhlotshaniswa nokuqongelela kwama-vacuoles e-cytoplasmic avelele27. Amaphutha okwakheka abonwe kuma-nematode ephayini abonakala ehlobene eduze nendlela yokufa okubangelwa yi-methane. Ukuhlolwa kwe-microscopic ngezikhathi ezahlukene kubonise ukuthi ama-vacuoles amakhulu akheka ngemva kwamahora angama-20 okuchayeka ku-5-iodoindole (0.1 mM). Ama-vacuoles e-microscopic abonwe ngemva kwamahora ayi-8 okwelashwa, futhi inani lawo landa ngemva kwamahora ayi-12. Ama-vacuoles amakhulu amaningana abonwe ngemva kwamahora ayi-14. Ama-vacuoles amaningana ahlanganisiwe abonakala ngokucacile ngemva kwamahora ayi-12-16 okwelashwa, okubonisa ukuthi ukuhlanganiswa kwe-vacuole kuyisisekelo sendlela yokufa kwe-methanogenic. Ngemva kwamahora angama-20, ama-vacuoles amaningana amakhulu atholakala kulo lonke i-worm. Lokhu okubonwe kumelela umbiko wokuqala we-metuosis ku-C. elegans.
Ezimpethu eziphathwe nge-5-iodoindole, ukuhlangana kwe-vacuole kanye nokuqhekeka kwayo kwabonwa (Isithombe 5), njengoba kufakazelwa ukugoba kwezimpethu kanye nokukhululwa kwe-vacuole endaweni ezungezile. Ukuphazamiseka kwe-vacuole kwabonwa nasolwembeni lwegobolondo leqanda, oluvame ukugcinwa lungaphazanyiswa yi-L2 ngesikhathi sokuchamisela (Isithombe Esingeziwe S2). Lokhu okubonwayo kusekela ukubandakanyeka kokuqongelela koketshezi kanye nokwehluleka kwe-osmoregulatory, kanye nokulimala kweseli okuguquguqukayo (i-RCI), enkambisweni yokwakheka kwe-vacuole kanye ne-suppuration (Isithombe 5).
Sicabangela indima ye-iodine ekwakhekeni kwe-vacuole okubonwe, sihlole umsebenzi wokubulala i-sodium iodide (NaI) kanye ne-potassium iodide (KI). Kodwa-ke, ekugxilweni (0.1, 0.5 noma 1 mM), azizange zithinte ukusinda kwe-nematode noma ukwakheka kwe-vacuole (Isithombe Esingeziwe S5), yize i-1 mM KI yayinomphumela omncane wokubulala i-nematic. Ngakolunye uhlangothi, i-7-iodoindole (1 noma 2 mM), njenge-5-iodoindole, ibangele ama-vacuole amaningi kanye nokuguquguquka kwesakhiwo (Isithombe Esingeziwe S6). Ama-iodoindole amabili abonise izici ezifanayo ze-phenotypic kuma-nematode ephayini, kanti i-NaI kanye ne-KI azizange zibonise. Ngokuthakazelisayo, i-indole ayizange ibangele ukwakheka kwe-vacuole ku-B. xylophilus ekugxilweni okuvivinywe (idatha ayiboniswanga). Ngakho-ke, imiphumela iqinisekisile ukuthi i-indole-iodine complex inesibopho sokushintshashintsha kwe-vacuolization kanye ne-metabolism ye-B. xylophilus.
Phakathi kwama-indole ahlolwe umsebenzi wokubulala amagciwane, i-5-iodoindole yayine-slip index ephezulu kakhulu engu--5.89 kcal/mol, ilandelwa yi-7-iodoindole (-4.48 kcal/mol), i-4-fluoroindole (-4.33), kanye ne-indole (-4.03) (Isithombe 6). Ukubopha okuqinile kwe-hydrogen yomgogodla we-5-iodoindole ku-leucine 218 kuzinzisa ukubopha kwayo, kuyilapho zonke ezinye izinto ezitholakala ku-indole zibopha ku-serine 260 ngezibopho ze-hydrogen ze-side chain. Phakathi kwamanye ama-iodoindole amodeliwe, i-2-iodoindole inenani lokubopha elingu--5.248 kcal/mol, okubangelwa isibopho sayo esiyinhloko se-hydrogen ne-leucine 218. Ezinye izibopho ezaziwayo zifaka phakathi i-3-iodoindole (-4.3 kcal/mol), i-4-iodoindole (-4.0 kcal/mol), kanye ne-6-fluoroindole (-2.6 kcal/mol) (Isithombe Esingeziwe S8). Iningi lama-indole ane-halogenated kanye ne-indole uqobo, ngaphandle kwe-5-iodoindole kanye ne-2-iodoindole, akha isibopho ne-serine 260. Iqiniso lokuthi i-hydrogen bonding ne-leucine 218 libonisa ukubopha okusebenzayo kwe-receptor-ligand, njengoba kubonwe ku-ivermectin (Isithombe Esingeziwe S7), liqinisekisa ukuthi i-5-iodoindole kanye ne-2-iodoindole, njenge-ivermectin, zibopha ngokuqinile endaweni esebenzayo ye-GluCL receptor nge-leucine 218 (Isithombe 6 kanye nesithombe Esingeziwe S8). Siphakamisa ukuthi lokhu kubopha kuyadingeka ukuze kugcinwe isakhiwo se-pore evulekile se-GluCL complex nokuthi ngokubopha ngokuqinile endaweni esebenzayo ye-GluCL receptor, i-5-iodoindole, i-2-iodoindole, i-avermectin kanye ne-ivermectin ngaleyo ndlela kugcina isiteshi se-ion sivulekile futhi sivumele ukumuncwa koketshezi.
Ukufakwa kwama-molecule e-indole kanye ne-halogenated indole ku-GluCL. Ukuqondiswa kokubopha kwe-(A) indole, (B) 4-fluoroindole, (C) 7-iodoindole, kanye (D) 5-iodoindole ligands endaweni esebenzayo ye-GluCL. Iphrotheni imelelwe yiribhoni, kanti izibopho ze-hydrogen zomgogodla ziboniswa njengemigqa ephuzi enamachashazi. (A′), (B′), (C′), kanye (D′) zibonisa ukusebenzisana kwama-ligands ahambisanayo nezinsalela ze-amino acid ezizungezile, kanti izibopho ze-hydrogen ze-side-chain zikhonjiswa ngemicibisholo enamachashazi apinki.
Kwenziwe izivivinyo zokuhlola umphumela onobuthi we-5-iodoindole ekuhlumeni kwembewu yeklabishi kanye ne-radish. I-5-iodoindole (0.05 noma 0.1 mM) noma i-avermectin (10 μg/mL) ibe nomthelela omncane noma ingabi nawo nhlobo ekuhlumeni kokuqala kanye nokuvela kwezitshalo (Isithombe 7). Ngaphezu kwalokho, akukho mehluko obalulekile owatholakala phakathi kwesilinganiso sokuhluma kwezilawuli ezingalashwanga kanye nembewu ephathwe nge-5-iodoindole noma i-avermectin. Umphumela ekukhuleni kwezimpande ezithambile kanye nenani lezimpande eziseceleni ezakhiwe wawungabalulekile, yize i-1 mM (okuphindwe kayishumi ukuhlushwa kwayo okusebenzayo) ye-5-iodoindole yabambezela kancane ukuthuthukiswa kwezimpande eziseceleni. Le miphumela ikhombisa ukuthi i-5-iodoindole ayinabo ubuthi kumaseli ezitshalo futhi ayiphazamisi izinqubo zokuthuthukiswa kwezitshalo ekuhlungeni okufundwe.
Umphumela we-5-iodoindole ekuhlumeni kwembewu. Ukuhluma, ukuhluma kanye nokuhluma kwezimpande kwembewu ye-B. oleracea kanye ne-R. raphanistrum ku-Murashige kanye ne-Skoog agar medium ene-avermectin noma i-5-iodoindole noma engenayo. Ukuhluma kwaqoshwa ngemva kwezinsuku ezi-3 zokufukamela ku-22°C.
Lolu cwaningo lubika amacala amaningana okubulawa kwezinambuzane ezincelisayo ngama-indoles. Okubalulekile, lo ngumbiko wokuqala we-iodoindole ebangela i-methylation (inqubo ebangelwa ukuqongelela kwama-vacuole amancane ahlangana kancane kancane abe ama-vacuole amakhulu, ekugcineni aholela ekuqhekekeni kwe-membrane nokufa) ezinalitini zephayini, lapho i-iodoindole ikhombisa izakhiwo ezibalulekile zokubulala izinambuzane ezifana neze-avermectin ye-nematicide yezentengiselwano.
Ama-Indole aseke abikwa ngaphambilini ukuthi anemisebenzi eminingi yokubonisa izimpawu kuma-prokaryotes nama-eukaryotes, okuhlanganisa ukuvimbela/ukwakheka kwe-biofilm, ukusinda kwamagciwane, kanye nesifo se-pathogenicity19,32,33,34. Muva nje, imiphumela yokwelapha engaba khona yama-indole akhiwe nge-halogenated, ama-alkaloid e-indole, kanye ne-semisynthetic indole derivatives idonsele isithakazelo esikhulu socwaningo35,36,37. Isibonelo, ama-indole akhiwe nge-halogenated aboniswe ukuthi abulala amaseli e-Escherichia coli aqhubekayo kanye namaseli e-Staphylococcus aureus37. Ngaphezu kwalokho, kuyathakazelisa ngokwesayensi ukutadisha ukusebenza kahle kwama-indole akhiwe nge-halogenated ngokumelene nezinye izinhlobo, izizukulwane, kanye nemibuso, futhi lolu cwaningo luyisinyathelo sokufeza lo mgomo.
Lapha, siphakamisa indlela yokubulala okubangelwa yi-5-iodoindole kuma-elegans e-C. ngokusekelwe ekulimaleni kweseli okuguqukayo (RCI) kanye ne-methylation (Izibalo 4C no-5). Izinguquko ezibangelwa ukudumba njengokuvuvukala kanye nokuwohloka kwe-vacuolar ziyizinkomba ze-RCI kanye ne-methylation, ezibonakala njenge-vacuoles ezinkulu ku-cytoplasm48,49. I-RCI iphazamisa ukukhiqizwa kwamandla ngokunciphisa ukukhiqizwa kwe-ATP, okubangela ukwehluleka kwephampu ye-ATPase, noma ukuphazamisa ulwelwesi lwamaseli futhi kubangele ukungena okusheshayo kwe-Na+, Ca2+, kanye ne-water50,51,52. Ama-vacuoles e-intracytoplasmic avela kumaseli ezilwane ngenxa yokuqongelela koketshezi ku-cytoplasm ngenxa yokungena kwe-Ca2+ kanye ne-water53. Ngokuthakazelisayo, le ndlela yokulimala kwamangqamuzana iyaguqulwa uma umonakalo ungowesikhashana futhi amangqamuzana eqala ukukhiqiza i-ATP isikhathi esithile, kodwa uma umonakalo uqhubeka noma uba mubi kakhulu, amangqamuzana ayafa.54 Okubonwe yithi kukhombisa ukuthi ama-nematode aphathwe nge-5-iodoindole awakwazi ukubuyisela i-biosynthesis evamile ngemva kokuchayeka ezimweni zokucindezeleka.
I-methylation phenotype ebangelwa yi-5-iodoindole ku-B. xylophilus ingase ibangelwe ukuba khona kwe-iodine kanye nokusatshalaliswa kwayo kwama-molecule, njengoba i-7-iodoindole yayinomphumela omncane wokuvimbela ku-B. xylophilus kune-5-iodoindole (Ithebula 1 kanye neSithombe Esingeziwe S6). Le miphumela ihambisana kancane nezifundo zikaMaltese et al. (2014), ababike ukuthi ukudluliselwa kwe-pyridyl nitrogen moiety ku-indole kusuka ku-para- kuya endaweni ye-meta-position kwaqeda i-vacuolization, ukuvinjelwa kokukhula, kanye ne-cytotoxicity kumaseli e-U251, okuphakamisa ukuthi ukusebenzisana kwe-molecule nendawo ethile esebenzayo kuprotheni kubalulekile27,44,45. Ukusebenzisana phakathi kwe-indole noma i-halogenated indoles kanye nama-receptor e-GluCL abonwe kulolu cwaningo nakho kusekela lo mbono, njengoba i-5- kanye ne-2-iodoindole kutholakale ukuthi kubopha kuma-receptor e-GluCL ngamandla kakhulu kunezinye i-indoles ezihlolwe (Isithombe 6 kanye neSithombe Esingeziwe S8). I-iodine esesikhundleni sesibili noma sesihlanu se-indole itholakale ibopha ku-leucine 218 ye-GluCL receptor ngezibopho ze-hydrogen zomgogodla, kuyilapho ezinye i-indole ze-halogenated kanye ne-indole ngokwazo zakha izibopho ze-hydrogen ze-side-chain ezibuthakathaka ne-serine 260 (Isithombe 6). Ngakho-ke sicabanga ukuthi indawo ye-halogen idlala indima ebalulekile ekuqalisweni kokuwohloka kwe-vacuolar, kanti ukubopha okuqinile kwe-5-iodoindole kugcina isiteshi se-ion sivulekile, ngaleyo ndlela kuvumele ukungena koketshezi okusheshayo kanye nokuqhuma kwe-vacuole. Kodwa-ke, indlela eningiliziwe yokusebenza kwe-5-iodoindole isazonqunywa.
Ngaphambi kokusebenzisa i-5-iodoindole ngokoqobo, kufanele kuhlaziywe umphumela wayo onobuthi ezitshalweni. Ukuhlolwa kwethu kokuhluma kwembewu kubonise ukuthi i-5-iodoindole ayinawo umphumela omubi ekuhlumeni kwembewu noma ezinqubweni zokuthuthukiswa ezalandela ekugxilweni okufundwe (Isithombe 7). Ngakho-ke, lolu cwaningo luhlinzeka ngesisekelo sokusetshenziswa kwe-5-iodoindole endaweni yemvelo ukulawula ubungozi be-pine nematodes ezihlahleni zephayini.
Imibiko yangaphambilini ikhombisile ukuthi ukwelashwa okusekelwe ku-indole kumelela indlela engaba khona yokubhekana nenkinga yokumelana nama-antibiotic kanye nokuqhubeka komdlavuza55. Ngaphezu kwalokho, ama-indole anemisebenzi yokulwa namagciwane, yokulwa nomdlavuza, i-antioxidant, i-anti-inflammatory, i-antidiabetic, i-antiviral, i-antiproliferative kanye ne-anti-tuberculosis futhi angasebenza njengesisekelo esithembisayo sokuthuthukiswa kwemithi56,57. Lolu cwaningo luphakamisa okokuqala ukusetshenziswa okungenzeka kwe-iodine njenge-ejenti yokulwa nezilokazane kanye ne-anthelmintic.
I-Avermectin yatholakala eminyakeni engamashumi amathathu edlule futhi yawina umklomelo weNobel ngo-2015, futhi ukusetshenziswa kwayo njenge-anthelmintic kusaqhubeka. Kodwa-ke, ngenxa yentuthuko esheshayo yokumelana nama-avermectin kuma-nematode nezinambuzane, kudingeka isu elihlukile, elingabizi kakhulu, nelinobungani nemvelo ukulawula ukutheleleka kwe-PWN ezihlahleni zikaphayini. Lolu cwaningo luphinde lubike indlela i-5-iodoindole ebulala ngayo ama-nematode ephayini nokuthi i-5-iodoindole inobuthi obuphansi kumaseli ezitshalo, okuvula amathuba amahle okusetshenziswa kwayo kwezentengiselwano esikhathini esizayo.
Zonke izivivinyo zavunyelwa yiKomidi Lokuziphatha laseYeungnam University, eGyeongsan, eKorea, futhi izindlela zenziwa ngokuhambisana neziqondiso zeKomidi Lokuziphatha laseYeungnam University.
Ukuhlolwa kokufukamela amaqanda kwenziwa kusetshenziswa izinqubo ezimisiwe43. Ukuhlola amazinga okufukamela (HR), ama-nematode amadala anosuku olulodwa (cishe abesifazane abayi-100 kanye nabesilisa abayi-100) adluliselwa ezitsheni zePetri eziqukethe isikhunta futhi avunyelwa ukukhula amahora angama-24. Amaqanda abe esehlukaniswa futhi aphathwa nge-5-iodoindole (0.05 mM kanye ne-0.1 mM) noma i-avermectin (10 μg/ml) njengokumiswa emanzini acwengekile angenamagciwane. Lokhu kumiswa (500 μl; amaqanda angaba yi-100) kwadluliselwa emithonjeni yepuleti lokukhulisa izicubu elinamanzi angu-24 futhi kwafakwa ku-22 °C. Izibalo ze-L2 zenziwa ngemva kwamahora angama-24 okufukamela kodwa kwabhekwa njengokufile uma amaseli engashukumi lapho eshukunyiswa ngentambo yeplatinum encane. Lokhu kuhlolwa kwenziwa ngezigaba ezimbili, ngasinye siphindaphindwa kasithupha. Idatha evela kuzo zombili izivivinyo yahlanganiswa futhi yethulwa. Iphesenti le-HR libalwa kanje:
Ukufa kwezibungu kwahlolwa kusetshenziswa izinqubo ezithuthukisiwe ngaphambilini. Amaqanda e-Nematode aqoqwa futhi ama-embryo avumelaniswa ngokuchanyuselwa emanzini acwebezelwe angenasici ukuze kukhiqizwe izibungu zesigaba se-L2. Izibungu ezivumelanisiwe (cishe ama-nematode angu-500) zaphathwa nge-5-iodoindole (0.05 mM kanye ne-0.1 mM) noma i-avermectin (10 μg/ml) futhi zakhuliswa kuma-plate e-B. cinerea Petri. Ngemva kwamahora angu-48 okufukamela ku-22 °C, ama-nematode aqoqwa emanzini acwebezelwe angenasici futhi ahlolwa ukuthi akhona yini amazinga esigaba se-L2, L3, kanye ne-L4. Ukuba khona kwezigaba ze-L3 kanye ne-L4 kwabonisa ukuguqulwa kwezibungu, kanti ukuba khona kwesigaba se-L2 kwakubonisa ukuthi akukho kuguqulwa. Izithombe zatholakala kusetshenziswa i-iRiS™ Digital Cell Imaging System. Lokhu kuhlolwa kwenziwa ngezigaba ezimbili, ngasinye siphindaphindwa kasithupha. Idatha evela kuzo zombili izivivinyo yahlanganiswa futhi yethulwa.
Ubuthi be-5-iodoindole kanye ne-avermectin embewini buhlolwe kusetshenziswa izivivinyo zokuhluma kumapuleti e-agar e-Murashige kanye ne-Skoog.62 Imbewu ye-B. oleracea kanye ne-R. raphanistrum yaqala yacwiliswa emanzini acwengekile angenazibi usuku olulodwa, yagezwa nge-1 ml ye-ethanol engu-100%, yahlanzwa nge-1 ml ye-bleach yokuhweba engu-50% (3% ye-sodium hypochlorite) imizuzu eyi-15, yagezwa kahlanu ngamanzi ahlanzekile angu-1 ml. Imbewu ehlanziwe yabe isicindezelwa kumapuleti e-agar okuhluma aqukethe i-0.86 g/l (0.2X) ye-Murashige kanye ne-Skoog medium kanye ne-0.7% ye-bacteriological agar ene-5-iodoindole noma i-avermectin noma engenayo. Amapuleti abe esefakwa e-incubation ku-22 °C, futhi izithombe zathathwa ngemva kwezinsuku ezi-3 zokufuya. Lokhu kuhlola kwenziwa ngezigaba ezimbili, ngasinye sazo saba nokuphindaphinda okuyisithupha.
Isikhathi sokuthunyelwe: Feb-26-2025