Ipine nematode yi-quarantine endoparasite efudukayo eyaziwa ngokubangela ilahleko emandla yezoqoqosho kwi-ikhosistim yamahlathi epine. Uphononongo lwangoku luphonononga umsebenzi we-nemacidal we-halogenated indoles ngokuchasene ne-pine nematodes kunye nendlela yazo yokusebenza. Imisebenzi ye-nemacidal ye-5-iodoindole kunye ne-avermectin (ulawulo oluhle) ngokuchasene ne-pine nematodes yayifana kwaye iphezulu kwiindawo eziphantsi (10 μg / mL). I-5-iodoindole yanciphisa i-fecundity, umsebenzi wokuzala, ukufa kwe-embryonic kunye ne-larval, kunye nokuziphatha kwe-locomotor. Ukusebenzisana kwe-molecular ye-ligands kunye ne-invertebrate-specific glutamate-gated chloride channel receptors isekela ingcamango yokuba i-5-iodoindole, njenge-avermectin, ibophelela ngokuqinileyo kwindawo esebenzayo ye-receptor. I-5-Iodoindole iphinde ibangele ukuguqulwa kwe-phenotypic ezahlukahlukeneyo kwi-nematodes, kubandakanywa nokuwa kwe-organ engaqhelekanga / i-shrinkage kunye nokunyuka kwe-vacuolization. Ezi ziphumo zibonisa ukuba i-vacuoles inokudlala indima kwi-nematode methylation-mediated death. Okubalulekileyo, i-5-iodoindole yayingeyotyhefu kuzo zombini iintlobo zezityalo (ikhaphetshu kunye neradish). Ngaloo ndlela, olu phononongo lubonisa ukuba ukusetyenziswa kwe-iodoindole phantsi kweemeko zendalo kunokulawula ukulimala kwepine wilt.
Ipinewood nematode (iBursaphelenchus xylophilus) yeyomthi wepine nematodes (PWN), imigratory endoparasitic nematodes ezaziwa ngokwenza umonakalo omkhulu kwi-ikholoji kwindalo yamahlathi epine1. I-Pine wilt disease (PWD) ebangelwa yi-pine wood nematode iba yingxaki enkulu kumazwekazi amaninzi, kuquka i-Asia kunye ne-Europe, kwaye eMntla Melika, i-nematode itshabalalisa iintlobo zepine ezifakiweyo1,2. Ukwehla komthi wompayina yingxaki enkulu yezoqoqosho, kwaye ithemba lokusasazeka kwawo kwihlabathi liyaxhalabisa3. Ezi ndidi zilandelayo zompayina zidla ngokuhlaselwa yi nematode: iPinus densiflora, iPinus sylvestris, iPinus thunbergii, iPinus koraiensis, iPinus thunbergii, iPinus thunbergii, kunye nePinus radiata4. Ipine nematode sisifo esiyingozi esinokuthi sibulale imithi yompayina kwiiveki okanye iinyanga zosulelo. Ukongeza, ukuqhambuka kwepine nematode kuxhaphakile kwiintlobo ngeentlobo zendalo, ngoko ke amakhonkco osulelo oluzingileyo asekiwe1.
IBursaphelenchus xylophilus yinematode yesityalo ehlala yodwa ephuma kwi-superfamily i-Aphelenchoidea kunye ne-clade 102.5. I-nematode itya umngundo kwaye ivelise kwakhona kwizicubu zomthi zepineyini, ikhula ibe ngamanqanaba amane ahlukeneyo ombungu: L1, L2, L3, L4 kunye nomntu omdala1,6. Ngaphantsi kweemeko zokunqongophala kokutya, i-pine nematode idlula kwisigaba esikhethekileyo sombungu - i-dauer, ephazamisa i-vector yayo - i-pine bark beetle (i-Monochamus alternatus) kwaye idluliselwe kwimithi yepine enempilo. Kwimikhosi enempilo, i-nematodes ifuduka ngokukhawuleza ngeethishu zezityalo kwaye izondla kwiiseli ze-parenchymatous, ezikhokelela kwinani lokusabela kwe-hypersensitivity, i-pine wilting kunye nokufa kwisithuba sonyaka emva kosulelo1,7,8.
Ulawulo lwebhayoloji yepinematodes kudala ingumceli mngeni, ngamanyathelo okuvalela abantu asusela kwinkulungwane yama-20. Izicwangciso zangoku zokulawula ipine nematodes ikakhulu zibandakanya unyango lwekhemikhali, kubandakanywa ukufuthwa komthi kunye nokufakelwa kwe-nematicides kwiziqu zemithi. I-nematicides esetyenziswa kakhulu yi-avermectin kunye ne-avermectin benzoate, ezizezosapho lwe-avermectin. Ezi khemikhali zinexabiso eliphezulu zisebenza kakhulu kwiintlobo ezininzi ze-nematode kwaye zithathwa njengekhuselekile kokusingqongileyo9. Nangona kunjalo, ukusetyenziswa ngokuphindaphindiweyo kwezi nematicides kulindeleke ukuba kudale uxinzelelo lokukhetha oluya kuthi lukhokelele ekuveleni kwe-pine nematodes enganyangekiyo, njengoko kubonisiwe kwizinambuzane ezininzi zezinambuzane, ezifana neLeptinotarsa decemlineata, iPlutella xylostella kunye nematodes i-Trichostrongylus colubriformis kunye ne-Ostertagiaverctum ephuhliswe kancinci, i-12 ye-circumcinins, kunye ne-12 ye-circumcinins. Ke ngoko, iipateni zokuxhathisa kufuneka zifundwe rhoqo kwaye i-nematicides ihlolwe rhoqo ukufumana enye indlela, eyongayo kunye nemilinganiselo yokusingqongileyo yokulawula i-PVD. Kwiminyaka yakutshanje, inani lababhali licebise ukusetyenziswa kwezityalo zezityalo, i-oyile efunekayo kunye ne-volatile njenge-agent yokulawula i-nematode13,14,15,16.
Kutshanje sibonise umsebenzi we-nemacidal we-indole, i-intercellular kunye ne-interkingdom yokubonisa i-molecule, kwi-Caenorhabditis elegans 17. I-Indole iyisignali ye-intracellular ebanzi kwi-microbial ecology, elawula imisebenzi emininzi echaphazela i-microbial physiology, ukubunjwa kwe-spore, ukuzinza kwe-plasmid, ukumelana neziyobisi, ukubunjwa kwe-biofilm, kunye ne-virulence 18, 19. Umsebenzi we-indole kunye ne-derivatives yayo ngokuchasene nezinye i-nematodes ze-pathogenic ayizange ifundwe. Kolu phononongo, siphande umsebenzi we-nemacidal we-34 indoles ngokuchasene ne-pine nematodes kwaye sacacisa indlela yokusebenza kweyona nto inamandla ye-5-iodoindole kusetyenziswa i-microscopy, ukufota ixesha elidlulayo, kunye novavanyo lwe-molecular docking, kwaye yavavanya iziphumo zayo eziyityhefu kwizityalo zisebenzisa uvavanyo lokuntshula kwembewu.
Uxinzelelo oluphezulu (> 1.0 mM) lwe-indole luye lwabikwa ngaphambili ukuba lunempembelelo ye-nemacidal kwi-nematodes17. Ukulandela unyango lwe-B. xylophilus (izigaba zobomi obuxubeneyo) kunye ne-indole okanye i-33 eyahlukeneyo ye-indole derivatives kwi-1 mM, ukufa kwe-B. xylophilus kulinganiswa ngokubala i-nematodes ephilayo kunye efileyo kulawulo kunye namaqela aphathwayo. Ii-indoles ezintlanu zibonise umsebenzi obalulekileyo we-nemacidal; Ukusinda kweqela lolawulo elingaphathwanga kwaba ngama-95 ± 7% emva kweeyure ezingama-24. Kwii-indoles ze-34 ezivavanyiweyo, i-5-iodoindole kunye ne-4-fluoroindole kwi-1 mM yabangela ukufa kwe-100%, kanti i-5,6-difluoroindigo, i-methylindole-7-carboxylate, kunye ne-7-iodoindole ibangele malunga ne-50% yokufa (Itheyibhile 1).
Impembelelo ye-5-iodoindole ekubunjweni kwe-vacuole kunye ne-metabolism ye-pine nematode yomthi. (A) Umphumo we-avermectin kunye ne-5-iodoindole kwi-nematodes yamadoda omdala, (B) i-L1 isigaba samaqanda e-nematode kunye (C) ne-metabolism ye-B. xylophilus, (i) i-vacuoles ayizange ibonwe kwi-0 h, unyango lubangele (ii) i-vacuoles, (iii) ukuqokelelwa kwe-vacuoles ezininzi, i-vacuoles, i-vacuoles, i-vacuoles (ii) kunye (vi) nokwenziwa kweevacuole ezinkulu. Iintolo ezibomvu zibonisa ukuvuvukala kwee-vacuoles, iintolo eziluhlaza okwesibhakabhaka zibonisa ukudibanisa kwee-vacuoles kunye neentolo ezimnyama zibonisa i-vacuoles enkulu. Isikali bar = 50 μm.
Ukongezelela, olu pho nonongo luphinde lwachaza inkqubo elandelelanayo yokufa kwe-methane-induced in pine nematodes (Figure 4C). Ukufa kwe-Methanogenic luhlobo lwe-non-apoptotic yokufa kweseli ehambelana nokuqokelelwa kwe-cytoplasmic vacuoles eveleleyo27. Iziphene ze-morphological ezibonwe kwi-pine nematodes zibonakala zihambelana ngokusondeleyo kwindlela yokufa okubangelwa yi-methane. Uvavanyo lweMicroscopic ngamaxesha ahlukeneyo lubonise ukuba ii-vacuoles ezinkulu zenziwe emva kwe-20 h yokuvezwa kwi-5-iodoindole (0.1 mM). I-Microscopic vacuoles yabonwa emva kwe-8 h yonyango, kwaye inani labo lenyuka emva kwe-12 h. Iivacuole ezininzi ezinkulu zabonwa emva kwe-14 h. Iivacuoles ezininzi ezidibeneyo zibonakala ngokucacileyo emva kwe-12-16 h yonyango, ebonisa ukuba i-vacuole fusion isisiseko sendlela yokufa kwe-methanogenic. Emva kweeyure ezingama-20, iivacuole ezininzi ezinkulu zafunyanwa kulo msundululu. Olu qwalaselo lubonisa ingxelo yokuqala ye-metuosis kwi-C. elegans.
Kwii-worms eziphathwa nge-5-iodoindole, i-vacuole aggregation kunye nokuqhekeka kwabonwa kwakhona (umzobo 5), njengoko kubonakaliswe kukugoba kweembungu kunye nokukhululwa kwe-vacuole kwindawo. Ukuphazamiseka kweVacuole kwabonwa kwi-eggshell membrane, ehlala igcinwe ikhuselekile yi-L2 ngexesha lokuqanduselwa (i-Supplementary Fig. S2). Olu qwalaselo luxhasa ukubandakanyeka kwe-fluid accumulation kunye nokungaphumeleli kwe-osmoregulatory, kunye nokulimala kwe-cell reversible (RCI), kwinkqubo yokwakhiwa kwe-vacuole kunye ne-suppuration (Umfanekiso 5).
I-hypothesizing indima ye-iodine ekubonweni kwe-vacuole formation, siye saphanda umsebenzi we-nemacidal we-sodium iodide (NaI) kunye ne-potassium iodide (KI). Nangona kunjalo, ekugxininiseni (i-0.1, i-0.5 okanye i-1 mM), ayizange ichaphazele okanye i-nematode iphile okanye i-vacuole formation (i-Supplementary Fig. S5), nangona i-1 mM KI ibe nefuthe elincinci le-nemacidal. Ngakolunye uhlangothi, i-7-iodoindole (1 okanye i-2 mM), njenge-5-iodoindole, yenza i-vacuoles ezininzi kunye nokuguqulwa kwesakhiwo (i-Supplementary Fig. S6). Ii-iodoindoles ezimbini zibonise iimpawu ezifanayo ze-phenotypic kwi-pine nematodes, kanti i-NaI kunye ne-KI ayizange. Kuyathakazelisa ukuba i-indole ayizange ibangele ukubunjwa kwe-vacuole kwi-B. xylophilus kwi-concentrations evavanyiweyo (idatha engaboniswa). Ngaloo ndlela, iziphumo ziqinisekisile ukuba i-indole-iodine eyinkimbinkimbi ijongene ne-vacuolization kunye ne-metabolism ye-B. xylophilus.
Phakathi kwee-indoles ezivavanyelwe umsebenzi we-nematicidal, i-5-iodoindole ine-index ephezulu ye-slip index -5.89 kcal / mol, ilandelwa yi-7-iodoindole (-4.48 kcal / mol), 4-fluoroindole (-4.33), kunye ne-indole (-4.03) (Umfanekiso 6). Umqolo owomeleleyo we-hydrogen bonding we-5-iodoindole ukuya kwi-leucine 218 uzinzisa ukubopha kwayo, ngelixa zonke ezinye izinto eziphuma kwi-indole zibophelela kwi-serine 260 ngekhonkco elisecaleni le-hydrogen bond. Phakathi kwamanye ama-iodoindoles angumzekelo, i-2-iodoindole inexabiso elibophezelayo le-5.248 kcal / mol, ngenxa yebhondi yayo ephambili ye-hydrogen kunye ne-leucine 218. Ezinye izibophelelo ezaziwayo ziquka i-3-iodoindole (-4.3 kcal / mol), 4-iodoindole (-4.2) kunye ne-6. kcal / mol) (Figure Supplementary S8). Uninzi lwee-indoles ze-halogenated kunye ne-indole ngokwayo, ngaphandle kwe-5-iodoindole kunye ne-2-iodoindole, yenza ibhondi kunye ne-serine 260. Inyaniso yokuba i-hydrogen bonding kunye ne-leucine 218 ibonisa ukubopha okusebenzayo kwe-receptor-ligand, njengoko kubonwa kwi-ivermectin (i-Supplementary Fig. I-2-iodoindole, njenge-ivermectin, ibophe ngokuqinileyo kwindawo esebenzayo ye-GluCL receptor nge-leucine 218 (umzobo we-6 kunye ne-Supplementary Fig. S8). Siphakamisa ukuba oku kubophezela kuyadingeka ukugcina i-pore evulekileyo ye-pore ye-GluCL eyinkimbinkimbi kwaye ngokuzibophelela ngokuqinileyo kwindawo esebenzayo ye-GluCL receptor, i-5-iodoindole, i-2-iodoindole, i-avermectin kunye ne-ivermectin ngaloo ndlela igcina i-ion channel ivulekile kwaye ivumela ukukhutshwa kwamanzi.
I-molecular docking ye-indole kunye ne-halogenated indole kwi-GluCL. Izibophelelo ezibophezelayo ze (A) indole, (B) 4-fluoroindole, (C) 7-iodoindole, kunye (D) i-5-iodoindole ligands kwindawo esebenzayo ye-GluCL. Iprotheni imelwe yiribhoni, kwaye i-backbone bonds ye-hydrogen iboniswa njengemigca enemibala ephuzi. (A′), (B′), (C′), kunye (D′) zibonisa intsebenziswano yee-ligands ezihambelanayo kunye neentsalela ze-amino acid ezijikelezileyo, kunye neebhondi ze-hydrogen ezisecaleni ziboniswa ngeentolo ezinamachaphaza apinki.
Iimvavanyo zenziwe ukuvavanya umphumo wetyhefu we-5-iodoindole ekuhlumeni kwekhaphetshu kunye nembewu yeradish. I-5-iodoindole (i-0.05 okanye i-0.1 mM) okanye i-avermectin (i-10 μg / mL) yayinempembelelo encinci okanye ayinayo impembelelo ekuhlumeni kokuqala kunye nokuvela kwe-plantlet (Umfanekiso 7). Ukongezelela, akukho mahluko abalulekileyo afunyenweyo phakathi kwezinga lokuntshula kokulawulwa okungaphendulwanga kunye nembewu ephathwa nge-5-iodoindole okanye i-avermectin. Impembelelo kwi-taproot elongation kunye nenani leengcambu ezisecaleni ezakhiweyo zazingabalulekanga, nangona i-1 mM (i-10 amaxesha e-concentration yayo esebenzayo) ye-5-iodoindole ilibazise kancinci ukuphuhliswa kweengcambu ezisecaleni. Ezi ziphumo zibonisa ukuba i-5-iodoindole ayinayo ityhefu kwiiseli zezityalo kwaye ayiphazamisi iinkqubo zophuhliso lwezityalo kwiindawo ezifundwayo.
Impembelelo ye-5-iodoindole ekuntshuleni kwembewu. Ukuntshula, ukuntshula kunye neengcambu ezisecaleni kwe-B. oleracea kunye ne-R. raphanistrum imbewu kwi-Murashige kunye ne-Skoog agar medium kunye okanye ngaphandle kwe-avermectin okanye i-5-iodoindole. Ukuntshula kushicilelwe emva kweentsuku ezi-3 zokufukamela kwi-22°C.
Olu phononongo lubika iimeko ezininzi zokubulawa kwe-nematode ngama-indoles. Okubalulekileyo, le yingxelo yokuqala ye-iodoindole inducing methylation (inkqubo ebangelwa kukuqokelelana kwe-vacuoles ezincinci ezidibanisa ngokuthe ngcembe kwii-vacuoles ezinkulu, ekugqibeleni zikhokelela ekuqhekekeni kwe-membrane kunye nokufa) kwiinaliti zepine, kunye ne-iodoindole ebonisa iimpawu ezibalulekileyo ze-nematicidal ezifana nezo zentengiso ye-nematicide avermectin.
I-Indoles iye yaxelwa ngaphambili ukuba yenze imisebenzi emininzi yokubonakalisa iimpawu kwiprokaryotes kunye ne-eukaryotes, kuquka i-biofilm inhibition / formation, ukusinda kwebhaktheriya, kunye ne-pathogenicity19,32,33,34. Kutshanje, iziphumo ezinokubakho zonyango ze-halogenated indoles, i-indole alkaloids, kunye ne-semisynthetic indole derivatives ziye zatsala umdla wophando olubanzi35,36,37. Ngokomzekelo, i-indoles ene-halogenated iboniswe ukubulala i-Escherichia coli eqhubekayo kunye neeseli ze-Staphylococcus aureus37. Ukongeza, kunomdla wesayensi ukufunda ukusebenza kwe-halogenated indoles ngokuchasene nezinye iintlobo, igenera, kunye nezikumkani, kwaye olu phononongo linyathelo lokufezekisa le njongo.
Apha, siphakamisa indlela yokubulala i-5-iodoindole-induced lethality kwi-C. elegans esekelwe kwi-reversible cell injury (RCI) kunye ne-methylation (Amanani 4C kunye ne-5). Utshintsho lwe-Edematous olufana ne-puffiness kunye ne-vacuolar degeneration zizibonakaliso ze-RCI kunye ne-methylation, ebonakaliswe njenge-vacuoles enkulu kwi-cytoplasm48,49. I-RCI iphazamisa ukuveliswa kwamandla ngokunciphisa ukuveliswa kwe-ATP, kubangela ukungaphumeleli kwepompo ye-ATPase, okanye ukuphazamisa i-membrane yeseli kunye nokubangela ukunyuka ngokukhawuleza kwe-Na +, i-Ca2 +, kunye namanzi50,51,52. I-Intracytoplasmic vacuoles ivela kwiiseli zezilwanyana ngenxa yokuqokelela kwamanzi kwi-cytoplasm ngenxa yokunyuka kwe-Ca2 + kunye namanzi53. Kuyathakazelisa ukuba le ndlela yokulimala kweeseli ibuyiselwa umva ukuba umonakalo ungowexeshana kwaye iiseli ziqala ukuvelisa i-ATP ixesha elithile, kodwa ukuba umonakalo uqhubeka okanye unzima, iiseli ziyafa.54 Ukuqwalasela kwethu kubonisa ukuba i-nematodes ephathwa nge-5-iodoindole ayikwazi ukubuyisela i-biosynthesis eqhelekileyo emva kokuvezwa kweemeko zoxinzelelo.
I-methylation phenotype eyenziwa yi-5-iodoindole kwi-B. xylophilus ingaba ngenxa yobukho be-iodine kunye nokusabalalisa kwayo i-molecular, ekubeni i-7-iodoindole yayinempembelelo engaphantsi kwe-inhibitory kwi-B. xylophilus kune-5-iodoindole (Itheyibhile 1 kunye ne-Supplementary Figure S6). Ezi ziphumo zihambelana ngokuyinxenye kunye nezifundo zaseMaltese et al. (2014), owabika ukuba ukufuduswa kwe-pyridyl nitrogen moiety kwi-indole ukusuka kwi-para- ukuya kwi-meta-position yaphelisa i-vacuolization, inhibition yokukhula, kunye ne-cytotoxicity kwiiseli ze-U251, ebonisa ukuba ukusebenzisana kwe-molecule enendawo ethile esebenzayo kwiprotheni ibaluleke kakhulu27,44,45. Ukusebenzisana phakathi kwe-indole okanye i-halogenated indoles kunye ne-GluCL receptors eziqatshelwe kolu cwaningo nazo zixhasa le ngcamango, njengoko i-5- kunye ne-2-iodoindole ifunyenwe ukuba ibophe i-GluCL receptors ngamandla ngakumbi kunezinye i-indoles ezihlolwe (Umfanekiso 6 kunye ne-Supplementary Figure S8). I-iodine kwindawo yesibini okanye yesihlanu ye-indole yafunyanwa ukuba ibophe kwi-leucine 218 ye-receptor ye-GluCL nge-backbone bond hydrogen, kanti ezinye ii-indoles ze-halogenated kunye ne-indole ngokwayo zenza i-side-chain chain bond hydrogen kunye ne-serine 260 (Umfanekiso 6). Ngoko ke sicinga ukuba indawo ye-halogen idlala indima ebalulekileyo ekufakweni kwe-vacuolar degeneration, ngelixa i-tight binding ye-5-iodoindole igcina i-ion channel ivulekile, ngaloo ndlela ivumela ukungena ngokukhawuleza kwamanzi kunye nokuphuka kwe-vacuole. Nangona kunjalo, indlela ecacileyo yesenzo se-5-iodoindole isahlala isaziwa.
Ngaphambi kokusetyenziswa okusebenzayo kwe-5-iodoindole, umphumo wayo oyityhefu kwizityalo kufuneka uhlalutywe. Iimvavanyo zethu zokuhluma kwembewu zibonise ukuba i-5-iodoindole yayingenayo impembelelo embi ekuhlumeni kwembewu okanye iinkqubo zophuhliso ezilandelayo kwiindawo ezifundwayo (Umfanekiso 7). Ngaloo ndlela, olu phononongo lubonelela ngesiseko sokusetyenziswa kwe-5-iodoindole kwindawo yendalo ukulawula ubungozi bepine nematodes kwimithi yepine.
Iingxelo zangaphambili zibonise ukuba unyango olusekwe kwi-indole lumele indlela enokubakho yokujongana nengxaki yokunganyangeki kwe-antibiotics kunye nokuqhubekeka komhlaza55. Ukongezelela, i-indoles ine-antibacterial, i-anticancer, i-antioxidant, i-anti-inflammatory, i-antidiabetic, i-antiviral, i-antiproliferative kunye ne-antituberculosis imisebenzi kwaye ingasebenza njengesiseko esithembisayo sokuphuhliswa kweziyobisi56,57. Olu phononongo lucebisa okokuqala ngqa ukusetyenziswa okunokwenzeka kwe-iodine njenge-antiparasitic kunye ne-anthelmintic agent.
I-Avermectin yafunyanwa kumashumi amathathu eminyaka eyadlulayo kwaye yaphumelela ibhaso leNobel ngo-2015, kwaye ukusetyenziswa kwayo njenge-anthelmintic kusaqhuba. Nangona kunjalo, ngenxa yophuhliso olukhawulezayo lokuchasana ne-avermectins kwi-nematodes kunye nezinambuzane zezinambuzane, enye indlela, indleko ephantsi, kunye neqhinga lokusingqongileyo liyafuneka ukulawula ukusuleleka kwe-PWN kwimithi yepineyini. Olu phononongo lukwaxela indlela i-5-iodoindole ebulala ngayo i-pine nematodes kwaye i-5-iodoindole inetyhefu ephantsi kwiiseli zezityalo, ezivula amathuba amahle kwisicelo sayo sorhwebo kwixesha elizayo.
Zonke iimvavanyo zivunyiwe yiKomiti yezokuziphatha yeYunivesithi yaseYeungnam, eGyeongsan, eKorea, kwaye iindlela zenziwa ngokuhambelana nezikhokelo zeKomiti yezokuziphatha yeYunivesithi yaseYeungnam.
Uvavanyo lokufukamela iqanda lwenziwa kusetyenziswa iinkqubo ezimiselweyo43. Ukuvavanya amazinga okuqandusela (HR), i-nematodes endala yosuku olu-1 (malunga ne-100 yamabhinqa kunye ne-100 yamadoda) yatshintshelwa kwizitya ze-Petri eziqulethe i-fungus kwaye zavunyelwa ukuba zikhule i-24 h. Amaqanda abekwe yedwa kwaye aphathwa nge-5-iodoindole (0.05 mM kunye ne-0.1 mM) okanye i-avermectin (10 μg / ml) njengokunqunyanyiswa kumanzi angcolileyo adibeneyo. Oku kunqunyanyiswa (ama-500 μl; malunga namaqanda ali-100) aye akhutshelwa kumaqula epleyiti yokukhulisa inyama enamaqula angama-24 kwaye aqanduselwa kuma-22 °C. Izibalo ze-L2 zenziwa emva kwe-24 h yokufukanyelwa kodwa zithathwa njengefile ukuba iiseli azizange zihambe xa zikhuthazwa ngocingo oluhle lweplatinam. Olu lingelo lwenziwe ngokwamanqanaba amabini, ngalinye liphindaphindwa kathandathu. Idatha evela kuzo zombini iimfuniselo yadityaniswa kwaye yaboniswa. Ipesenti yeHR ibalwa ngolu hlobo lulandelayo:
Ukufa kombungu kwavavanywa kusetyenziswa iinkqubo eziphuhliswe ngaphambili. Amaqanda e-Nematode aqokelelwa kwaye iimbumba zadityaniswa ngokuqanduselwa kumanzi acwengileyo acoliweyo ukwenza imibungu yenqanaba le-L2. I-larvae edibeneyo (malunga ne-500 nematodes) iphathwe nge-5-iodoindole (0.05 mM kunye ne-0.1 mM) okanye i-avermectin (10 μg / ml) kwaye ikhuliswe kwi-B. cinerea Petri plates. Emva kweyure ezingama-48 zokufukanyelwa kwi-22 °C, iinematode zaqokelelwa kumanzi acwengileyo adiyiliweyo kwaye zivavanyelwe ubukho benqanaba le-L2, L3, kunye ne-L4. Ubukho benqanaba le-L3 kunye ne-L4 lubonise ukuguqulwa kombungu, ngelixa ubukho benqanaba le-L2 bubonisa ukuba akukho tshintsho. Imifanekiso yafunyanwa kusetyenziswa i-iRiS™ Digital Cell imaging System. Olu lingelo lwenziwe ngokwamanqanaba amabini, ngalinye liphindaphindwa kathandathu. Idatha evela kuzo zombini iimfuniselo yadityaniswa kwaye yaboniswa.
Ityhefu ye-5-iodoindole kunye ne-avermectin kwimbewu yavavanywa kusetyenziswa iimvavanyo zokuhluma kwi-Murashige kunye ne-Skoog agar plates. I-62 B. oleracea kunye nembewu ye-R. raphanistrum yaqala ukucwina emanzini angcolileyo ahlambulukileyo ngosuku olunye, ihlanjwe nge-1 ml 100% ye-ethanol ye-sodium, i-100% ye-sodium e-3% ye-sodium, i-3% i-hypochlorite) i-15 min, kwaye ihlanjwe kahlanu nge-1 ml yamanzi angcolileyo. Iimbewu ezizintsholongwane ziye zacinezelwa kwiipleyiti ze-agar zokuntshula ezine-0.86 g/l (0.2X) Murashige kunye ne-Skoog medium kunye ne-0.7% ye-bacteriological agar kunye okanye ngaphandle kwe-5-iodoindole okanye i-avermectin. Iipleyiti emva koko zafakwa kwi-22 °C, kwaye imifanekiso yathathwa emva kweentsuku ezi-3 zokufukamela. Olu lingelo lwenziwe ngezigaba ezibini, ngalinye libe nokuphindaphinda amathandathu.
Ixesha lokuposa: Feb-26-2025