Lendzatjana iyincenye yesihloko selucwaningo “Kwenta ncono kumela kwema legumes kumagciwane kanye netilwane letibulalako”, buka tonkhe letindzatjana letingu 5 .
I-ejenti lebangela sifo sesitjalo se fungal iSclerotinia sclerotiorum (Lib.) de Bary isebentisa lisu lelinetigaba letiningi kwentela kutselela titjalo letehlukene letibambako. Lolucwaningo luphakamisa kusetjentiswa kwe diamine L-ornithine, i-amino acid lengasiyo iphrotheni lekhutsata kuhlanganiswa kwaletinye ti-amino esidi letibalulekile, njengelinye lisu lekulawula kwentela kutfutfukisa kuphendvula kwemamolekhyuli, kwemtimba kanye nekwemakhemikhali ekuphila kwe Phaseolus vulgaris L. kuya kusikhunta lesimhlophe lesibangelwa yi Pseudootio. Kuhlolwa kwe vitro kwakhombisa kutsi iL-ornithine ivimbele kakhulu kukhula kwe mycelial ye S. pyrenoidosa ngendlela lencike kumthamo. Ngetulu kwaloko, kungehlisa kakhulu bukhulu besikhunta lesimhlophe ngaphansi kwetimo te-greenhouse. Ngetulu kwaloko, iL-ornithine yakhutsata kukhula kwetitjalo letilashiwe, lokukhombisa kutsi lokuhloliwe kwe L-ornithine bekungasiyo i-phytotoxic kuletitjalo letilashiwe. Ngekwengeta, i-L-ornithine yenta ncono kuveta kwetintfo letingasiwo ema-enzyme (ema-phenolics laphelele lancibilikako kanye nema-flavonoids) kanye netintfo letilwa ne-enzyme (i-catalase (i-CAT), i-peroxidase (i-POX), kanye ne-polyphenol oxidase (PPO)), futsi yakhulisa kuveta emajini lamatsatfu lahlobene ne-e-enzyme, PGRvD, PvSOAT, kanye ne-PVSOT). Ngetulu kwaloko, ekuhlaziyweni kwe silico kwaveta kubakhona kweprotein letsatfwako ye oxaloacetate acetylhydrolase (SsOAH) ku S. (PlOAH) ngekwekuhlatiya lokusebenta, tindzawo letigcinwe, kanye ne-topology. Kuyajabulisa kutsi, kwengeteka kwe L-ornithine kumhluzi wemazambane (PDB) emkhatsini kwehlisa kakhulu kuveta kwelijini le SsOAH ku S. Ngendlela lefanako, kusetjentiswa kwe exogenous kwe L-ornithine kwehlisa kakhulu kuveta kwe SsOAH gene ku mycelia ye fungal lebutfwe etitjalweni letiphatsiwe. Ekugcineni, kufaka iL-ornithine kwehlisa kakhulu kukhishwa kwe oxalic acid kuwo omabili emacembe lasemkhatsini ePDB kanye nalawo latseleleke. Ekuphetseni, iL-ornithine idlala indzima lebalulekile ekugcineni simo se redox kanjalo nekutfutfukisa kuphendvula kwekuvikela kwetitjalo letingenwe ngulesifo. Imiphumela yalolucwaningo ingasita ekwakheni tindlela letinsha, letingalimati imvelo tekulawula sikhunta lesimhlophe kanye nekunciphisa umtselela waso ekukhiciteni emabhontjisi kanye naletinye tintjalo.
Sikhunta lesimhlophe, lesibangwa sikhunta lesibulalako se Sclerotinia sclerotiorum (Lib.) de Bary, sifo lesibhubhisako, lesinciphisa umkhicito lesibeka ingoti lenkhulu ekukhiciteni emabhontjisi (Phaseolus vulgaris L.) emhlabeni wonkhe (Bolton nalabanye, 2006). iSclerotinia sclerotiorum ngulenye yetintfo letilukhuni kakhulu tetitjalo letitsatfwa sikhunta letitsatfwa emhlabatsini kutsi tilawulelwe, ngeluhlu lolubanti lwetinhlobo tetitjalo letingetulu kwa 600 kanye nemandla ekusheshe ahlanganise ticubu tetitjalo ngendlela lengakacaci (Liang na Rollins, 2018). Ngaphansi kwetimo letingakalungi, idlula esigabeni lesibucayi semjikeleto wayo wekuphila, ihlala ingasebenti sikhatsi lesidze njengetintfo letimnyama, leticinile, letifana nembewu letibitwa nge ‘sclerotia’ emhlabatsini noma njengekukhula lokumhlophe, lokunebufuvu ku mycelium noma stem pith yetitjalo letingenwe ngulesifo (50). iS.sclerotiorum iyakhona kwenta iSclerotia, lokuyivumela kutsi iphile emasimini latseleleke sikhatsi lesidze futsi iphikelele ngesikhatsi sesifo (Schwartz nalabanye, 2005). Ema Sclerotia anotsile ngemanutrients, angaphikelela emhlabatsini sikhatsi lesidze, futsi asebenta njenge inoculum leyinhloko yekugula lokulandzelako (Schwartz et al., 2005). Ngaphansi kwetimo letikahle, iSclerotia ihluma bese ikhicita emagciwane lahamba emoyeni langatselela tonkhe tincenye letingetulu kwemhlabatsi talesitjalo, kufaka kepha kungapheleli etimbali, ticu, noma emapods (Schwartz et al., 2005).
iSclerotinia sclerotiorum isebentisa lisu lelinetigaba letiningi kwentela kutselela titjalo tayo letimukelako, lokufaka luchungechunge lwetintfo letihlangene kusuka ekuhlumeni kwe sclerotial kuya ekutfutfukeni kwetimphawu. Ekucaleni, iS. Le fungus ibese ikhipha i-oxalic acid, sici sekugula, kulawula i-pH yelubondza lwemaseli esitjalo, kukhutsata kubola kwe enzymatic kanye nekuhlaselwa kweticubu (Hegedus na Rimmer, 2005), kanye nekucindzetela kuqhuma kwe oxidative kwesitjalo lesiphetse. Lenchubo ye esidi yenta butsakatsaka lubondza lwemaseli esitjalo, kuniketa indzawo lekahle yekusebenta lokujwayelekile nalokusebentako kwema enzymes lacedza lubondza lwemaseli e fungal (CWDEs), kuvumela leligciwane kutsi lincobe sivimbo semtimba bese lingene eticubini letimukelako (Marciano et al., 1983). Uma seyingenile, iS.sclerotiorum ikhipha ema CWDE lamaningi, njenge polygalacturonase kanye ne cellulase, leyenta kutsi isabalale eticubini letingenwe ngulesifo futsi kubangele kufa kweticubu. Kuchubeka kwetilonda kanye nema hyphal mats kuholela etimphawu tetimphawu tesikhunta lesimhlophe (Hegedus na Rimmer, 2005). Ngaleso sikhatsi, titjalo letibambako tibona emaphethini emamolekhyuli lahlobene nemagciwane (PAMPs) ngekusebentisa emaphethini ekubona emaphethini (PRRs), lokuvusa luchungechunge lwetintfo letikhombisako letigcina tisebenta timphendvulo tekuvikela.
Noma nje emashumi eminyaka yemitamo yekulawula tifo, kushoda kwe germplasm leyanele lemelanako kuhlala emabhontjisi, njengaletinye tintjalo tekutsengisa, ngenca yekumelana, kuphila, kanye nekuvumelana ne pathogen. Ngakoke kulawulwa kwetifo kuyinsayeya kakhulu futsi kudzinga lisu lelihlanganisiwe, lelinetigaba letiningi lelifaka ekhatsi kuhlanganiswa kwetindlela temasiko, kulawula tilwane, kanye netibulala sikhunta ngemakhemikhali (O’Sullivan et al., 2021). Kulawula ngemakhemikhali kwesikhunta lesimhlophe ngiko lokusebenta kakhulu ngoba ema fungicides, uma asetjentiswa kahle futsi ngesikhatsi lesifanele, angalawula kahle kusakateka kwalesifo, anciphise bukhulu bekugula, futsi anciphise kulahleka kwesivuno. Nomakunjalo, kusebentisa kakhulu kanye nekwetsembela kakhulu kuma fungicides kungaholela ekuveleni kwetinhlobo letimelanako te S. sclerotiorum futsi kube nemtselela lomubi etilwaneni letingakahlosiwe, imphilo yemhlabatsi, kanye nelizinga lemanti (Le Cointe nalabanye, 2016; Ceresini nalabanye, 2024). Ngako-ke, kutfola letinye tindlela letingalimati imvelo sekuyinto lebaluleke kakhulu.
Ema Polyamines (ema PA), lafana ne putrescine, ispermidine, ispermine, kanye ne cadaverine, ingasebenta njengetindlela letitsembisako tekulwa nemagciwane etitjalo letitsatfwa emhlabatsini, ngaleyondlela kunciphise ngalokuphelele noma kancane kusetjentiswa kwemakhemikhali layingoti ekubulala sikhunta (Nehela nalabanye, 2024; Yi2 nalabanye, 2 . Etitjalweni letiphakeme, ema PA ayabandzakanyeka etinchubeni letiningi temtimba letifaka ekhatsi, kepha hhayi kuphela, kuhlukaniswa kwemaseli, kwehlukana, kanye nekuphendvula ekucindzetelekeni kwe abiotic kanye ne biotic (Killiny na Nehela, 2020). Tingasebenta njengetintfo letilwa nekugula, tisite kuhlanta tinhlobo te-oksijini lephendvulako (i-ROS), tigcine i-redox homeostasis (Nehela naKilliny, 2023), tikhutsate emajini lahlobene nekuvikela (Romero nalabanye, 2018), tilawule tindlela letehlukene tekugaya (i-Nehela naKilliny, 2023). Killiny, 2019), kusungula kumelana lokutfolakele lokuhlelekile (SAR), kanye nekulawula kusebentisana kwetitjalo-nemagciwane (Nehela na Killiny, 2020; Asija nalabanye, 2022; Czerwoniec, 2022). Kufanele kutsi kucashelwe kutsi tindlela letitsite kanye netindzima tema PAs ekuvikeleni titjalo tiyahlukahluka kuya ngetinhlobo tetitjalo, emagciwane, kanye netimo temvelo. iPA lenyenti kakhulu etitjalweni yentiwa ngetilwane kusuka ku polyamine L-ornithine lebalulekile (Killiny na Nehela, 2020).
iL-ornithine idlala tindzima letiningi ekukhuleni kwetitjalo kanye nekutfutfuka. Sibonelo, tifundvo letindlulileko tikhombise kutsi kulayisi (Oryza sativa), i-ornithine ingahle ihlobane nekuphindze kusetjentiswe initrogen (Liu nalabanye, 2018), umkhicito welilayisi, lizinga kanye neliphunga (Lu nalabanye, 2020), kanye nekuphendvula kwekucindzeteleka kwemanti (Yang nalabanye, 2000). Ngetulu kwaloko, kusetjentiswa kwe-exogenous kwe-L-ornithine kwenta ncono kakhulu kubeketela kwesomiso ku-sugar beet (Beta vulgaris) (Hussein nalabanye, 2019) kanye nekunciphisa kucindzeteleka kweluswayi ku-anyanisi (Allium Cepa) ( Rocha kanye nalabanye, 2012). Indzima lekhonakalako ye L-ornithine ekuvikeleni kucindzeteleka kwe abiotic kungenteka kutsi kungenca yekubandzakanyeka kwayo ekubutseni kwe proline etitjalweni letilashiwe. Sibonelo, emajini lahlobene nekugaya kwemtimba, njenge ornithine delta aminotransferase (delta-OAT) kanye nemajini e-proline dehydrogenase (ProDH1 na ProDH2), ngaphambilini kubikwe kutsi ayabandzakanyeka ekuvikeleni iNicotiana benthamiana kanye ne Arabidopsis thaliana ku Pse lengasiyo i-host Pse eMysore, nga-2012). Ngakulolunye luhlangotsi, i-ornithine decarboxylase yesikhunta (ODC) iyadzingeka ekukhuleni kwemagciwane (Singh et al., 2020). Kucondza i-ODC ye-Fusarium f. sp. lycopersici ngekuthulisa kwemajini labangelwa ngumsingatsi (HIGS) kwenta ncono kakhulu kumela kwetitjalo tetamatisi ku Fusarium wilt (Singh nalabanye, 2020). Nomakunjalo, indzima lekhonakalako yekusetjentiswa kwe exogenous ornithine ngekulwa nekucindzeteleka kwetilwane letifana nema phytopathogens akukafundvwa kahle. Lokubaluleke kakhulu, imiphumela ye ornithine ekumelaneni netifo kanye netintfo letihambisanako te biochemical kanye nemtimba tihlala tingahlolwa kakhulu.
Kuvisisa bunzima bekugula kwe S. sclerotiorum kwema legumes kubalulekile ekwakhiweni kwemasu ekulawula laphumelelako. Kulolucwaningo, besihlose kutfola indzima lekhonakalako ye diamine L-ornithine njengentfo lebalulekile ekwenteni ncono tindlela tekutivikela kanye nekumelana kwetitjalo te legume ekutselelekeni kwe Sclerotinia sclerotiorum. Sicabanga kutsi, ngetulu kwekutfutfukisa timphendvulo tekuvikela tetitjalo letingenwe ngulesifo, iL-ornithine iphindze idlale indzima lebalulekile ekugcineni simo se redox. Siphakamisa kutsi imiphumela lekhonakalako ye L-ornithine ihlobene nekulawulwa kwetindlela tekuvikela te antioxidant letine enzymatic naletingasiwo ema enzymatic kanye nekuphazamiseka kwetintfo tekugula/tekugula kwe fungal kanye nemaprotein lahlobene. Lokusebenta lokubili kwe L-ornithine kwenta kutsi kube ngummeleli lotsembisako welisu lelisimamako lekunciphisa umtselela wesikhunta lesimhlophe kanye nekutfutfukisa kumela kwetintjalo te legume letivamile kuleligciwane lelinemandla le fungal. Imiphumela yalolucwaningo lwanyalo ingasita ekwakhiweni kwetindlela letinsha, letingalimati imvelo tekulawula sikhunta lesimhlophe kanye nekunciphisa umtselela waso ekukhiciteni ema legume.
Kulolucwaningo, inhlobo yekutsengisa letsatsekako yebhonjisi levamile, iGiza 3 (Phaseolus vulgaris L. cv. Giza 3), yasetjentiswa njengentfo yekuhlola. Timbewu letiphilile taniketwa ngemusa Litiko Lelucwaningo Lwetinhlanyelo, Sikhungo Selucwaningo Lwetitjalo Temasimini (FCRI), Sikhungo Selucwaningo Lwekulima (ARC), eGibhithe. Timbewu letisihlanu tahlanyelwa etimbiteni tepulasitiki (bubanti bangekhatsi 35 cm, kujula 50 cm) lokugcwele umhlabatsi lone S.sclerotiorum ngaphansi kwetimo te greenhouse (25 ± 2 °C, umswakama lohlobene 75 ± 1%, 8 h kukhanya/16 h kumnyama). Emalangeni lasikhombisa kuya kulalishumi ngemuva kwekuhlwanyela (DPS), letitfombo betincishiswa kwentela kushiya titfombo letimbili kuphela letikhula ngendlela lefanako kanye nemacembe lamatsatfu lanwebekile ngalokuphelele ebhodweni ngalinye. Tonkhe titjalo letifakwe emabhodweni betiniselwa kanye ngemuva kwemaviki lamabili bese tivundiswa njalo ngenyanga ngesilinganiso lesinconyiwe seluhlobo loluniketiwe.
Kulungiselela 500 mg/L wekuhlanganiswa kwe L-ornithinediamine (lephindze yatiwe ngekutsi yi (+)-(S)-2,5-diaminopentanoic acid; Lesixazululo sesitoko sase sihlanjululwa futsi sasetjentiswa ekuhlolweni lokulandzelako. Kafishane, luchungechunge lwe-L-ornithine lolusitfupha (12.5, 25, 50, 75, 100, kanye na-125 mg / L) lwahlolwa nge-vitro. Ngekwengeta, emanti lahlungiwe lahlantekile asetjentiswa njengekulawula lokubi (Mock) kanye nesibulala sikhunta sekutsengisa “Rizolex-T” 50% wemphuphu lemanti (toclofos-methyl 20% + thiram 30%; njengekulawula lokuhle. Sibulali sikhunta sekutsengisa “Rizolex-T” sahlolwa nge vitro ngetigaba letisihlanu (2, 4, 6, 8 kanye na 10 mg/L).
Emasampula eticu temabhonjisi lavamile kanye nema pods lakhombisa timphawu letijwayelekile tesikhunta lesimhlophe (lizinga lekuhlaselwa: 10–30%) kwabutfwa emapulazini ekutsengisa. Noma nje tintfo letiningi tetitjalo letingenwe ngulesifo takhonjwa ngetinhlobo/tinhlobo (tinhlobo tekutsengisa letitsatsekako Giza 3), letinye, ikakhulu leto letitfolakala etimakethe tendzawo, bekutinhlobo letingatiwa. Letintfo letibutfwe letingenwe ngulesifo tacala ngekubulawa nge 0.5% sodium hypochlorite solution imizuzu lemitsatfu, bese tiyahlanjululwa tikhatsi letiningi ngemanti lahlanjululwe lahlantekile bese tiyasulwa tomile ngeliphepha lekuhlunga lelingakagcini kwentela kususa emanti langetulu. Letitfo letingenwe ngulesifo tase tijutjwa tibe tincetu letincane kusuka eticubini letisemkhatsini (emkhatsini weticubu letiphilile naletingenwe ngulesifo), takhuliswa kumazambane dextrose agar (PDA) medium bese tiyafutfunyelwa ku 25 ± 2 °C nge 12 h kukhanya/12 h umjikeleto lomnyama emalanga langu 5 kwentela kuvuselela kwakheka kwe sclerotia . Indlela ye mycelial tip yaphindze yasetjentiswa kuhlanta ema fungal lahlukanisiwe kusuka kumasiko lahlanganisiwe noma langcolile. Le fungal isolate lehlanjululwe yacala kukhonjwa ngekuya ngetimphawu tayo temasiko e morphological kwase kucinisekiswa kutsi yi S. sclerotiorum ngekuya ngetimphawu letibonakala ngesibonakhulu. Ekugcineni, onkhe ema isolates lahlanjululwe ahlolwa kutsi angagula yini ku cultivar yebhontjisi levamile letsatsekako iGiza 3 kwentela kuhlangabetana ne postulates ya Koch.
Ngekwengeta, le S. sclerotiorum lehlasela kakhulu leyehlukile (leyehlukile #3) yaphindze yacinisekiswa ngekusebentisa kulandzelana kwesikhala lesibhalwe phansi sangekhatsi (ITS) njengoba kuchaziwe ngu White et al., 1990; Baturo-Ciesniewska et al., 2017. Kafishane, ema-isolates akhuliswa emhlutini we-dextrose wemazambane (PDB) futsi akhuliswa ku-25 ± 2 °C emalanga langu-5-7. iFungal mycelium yabese iyabutfwa, ihlungwe ngendvwangu yashizi, igezwe kabili ngemanti langakagcini, bese yomiswa ngeliphepha lekuhlunga lelingakagcini. I-DNA ye-Genomic yahlukaniswa ngekusebentisa iKhithi Yekulungiselela Lencane Yesikhunta / Yeligciwane Lesheshako-DNA TM (Kuramae-Izioka, 1997; Atallah nalabanye, 2022, 2024). Sigodzi sayo se-rDNA sabese siyakhuliswa ngekusebentisa umbhangqwana lotsite we-primer ITS1 / ITS4 ( Lemikhicito ye-PCR lehlanjululwe yetfulwa kutsi ilandzelane (iBeijing Aoke Dingsheng Biotechnology Co., Ltd.). Le ITS rDNA kulandzelana kwalandzelana ngetindlela letimbili kusetjentiswa indlela yekulandzelana kwe Sanger. Lokulandzelana kwemibuto lehlanganisiwe kwabese kucatsaniswa nemininingwane yakamuva ku GenBank kanye neSikhungo Savelonkhe Selwatiso Lwebuchwepheshe Betilwane (NCBI, http://www.ncbi.nlm.nih.gov/gene/) kusetjentiswa isoftware ye-BLASTn. Lokulandzelana kwemibuto kwacatsaniswa naletinye tinhlobo letingu 20 te S. Kuhlatiya kwekutigucukela kwemvelo kwentiwa kusetjentiswa indlela lenkhulu yekutsi kungenteka kanye nemodeli yekufaka esikhundleni se-nucleotide lebuyela emuva ngesikhatsi jikelele (Nei naKumar, 2000). Sihlahla lesinematfuba lamakhulu e-log siyakhonjiswa. Sihlahla sekucala sekufuna kwe heuristic sikhetwa ngekukhetsa sihlahla lesinematfuba lamakhulu e-log emkhatsini wesihlahla sekujoyina bomakhelwane (NJ) (Kumar et al., 2024) kanye nesihlahla se-parsimony (MP) lesikhulu. Lesihlahla se NJ sakhiwa kusetjentiswa i-matrix yebanga le-pairwise lebalwe kusetjentiswa imodeli jikelele yesikhatsi-lebuyela emuva (Nei na Kumar, 2000).
Umsebenti wekulwa nemagciwane we L-ornithine kanye nekubulala emagciwane “Rizolex-T” kwatsatfwa nge vitro ngendlela yekusakateka kwe agar. Indlela: Tsatsa linani lelifanele lesisombululo sesitoko se L-ornithine (500 mg/L) bese uyayihlanganisa kahle ne 10 ml ye PDA yekudla lokusemkhatsini kwentela kulungisa tisombululo letinekuhlanganiswa kwekugcina kwa 12.5, 25, 50, 75, 100 kanye na 125 mg/L, ngekulandzelana. Tintfo letisihlanu letihlanganisiwe tesibulala sikhunta “Rizolex-T” (2, 4, 6, 8 kanye na 10 mg/L) kanye nemanti lahlungiwe lahlantekile asetjentiswa njengekulawula. Ngemuva kwekutsi lendzawo seyicinile, ipulaki ye mycelial lesanda kulungiswa yelisiko le Sclerotinia sclerotiorum, 4 mm ngebubanti, yadluliselwa enkhabeni yesitja se Petri futsi yakhuliswa ku 25±2°C kwaze kwaba ngulapho le mycelium ivala sonkhe sitja se Petri sekulawula, ngemuva kwaloko kukhula kwe fungal kwabhalwa. Bala liphesenti lekuvimbela kukhula kwe-radial kwe-S. sclerotiorum usebentisa isilinganiso 1:
Lokuhlolwa kwaphindzaphindvwa kabili, ngekuphindzaphindza kwetilwane letisitfupha kwelicembu ngalinye lekulawula/lekuhlola kanye netimbita letisihlanu (titjalo letimbili ngembita) yekuphindzaphindza kwetilwane ngamunye. Kuphindzaphindza ngamunye kwetilwane kwahlaziywa kabili (kuphindzaphindza lokubili kwebuchwepheshe) kwentela kucinisekisa kuciniseka, kwetsembeka kanye nekukhicita kabusha kwemiphumela yekuhlola. Ngetulu kwaloko, kuhlatiya kwekubuyela emuva kwe-probit kwasetjentiswa kubala ihhafu-lephakeme yekuvimbela lokuhlushwa (IC50) kanye ne-IC99 (Prentice, 1976).
Kuhlola emandla eL-ornithine ngaphansi kwetimo te greenhouse, kuhlolwa kwembita lokubili lokulandzelanako kwentiwa. Kafishane, emabhodwe agcwaliswa ngemhlabatsi welibumba-wesihlabathi lohlanjululwe (3:1) bese agonywa ngelisiko lelisanda kulungiswa le S. sclerotiorum. Kwekucala, le isolate lehlaselako kakhulu ye S. sclerotiorum (isolate #3) yakhuliswa ngekutsi kusikwe i-sclerotium yinye ngesigamu, ibekwe ibuke phansi ku PDA bese iyafukamela ku 25°C ebumnyameni lobuhlala njalo (24 h) emalanga lamane kwentela kukhutsata kukhula kwe mycelial. Emapulagi lamane e-agar lanebukhulu lobungaba ngu 5 mm abese atsatfwa kusuka emnceleni loholako bese ayagonywa nge 100 g yekuhlanganiswa lokungakagcini kwakolweni kanye ne bran yelilayisi (1:1, v/v) bese onkhe emaflaski afutfunyelwa ku 25 ± 2 °C ngaphansi kwemjikeleto we 12 h wekukhanya/12 h we daysler kwentela 5. Lokucuketfwe kuwo onkhe emaflaski bekuhlanganiswa kahle kwentela kucinisekisa kufana ngaphambi kwekufaka umhlabatsi. Ngemuva kwaloko, 100 g wekuhlanganiswa kwe bran ye colonizing kwangetwa ebhodweni ngalinye kwentela kucinisekisa kutsi kuhlala kukhona emagciwane. Letimbita letigonyelwe betiniselwa kwentela kusebenta kukhula kwe fungal bese tibekwa etimeni te greenhouse emalanga lasikhombisa.
Timbewu letisihlanu tetinhlobo te Giza 3 tase tihlanyelwa ebhodweni ngalinye. Kuletimbita letiphatfwe nge L-ornithine kanye ne fungicide Rizolex-T, letinhlavu letibulala tifo tacala kufakwa emahora lamabili esixazululweni lesinemanti saletinhlanganisela letimbili letine IC99 yekugcina lecishe ibe ngu 250 mg/L kanye na 50 mg/L, ngekulandzelana, bese tiyamiswa emoyeni ngako lihora linye. Ngakulolunye luhlangotsi, letinhlavu betifakwa emantini lahlanjululwe lahlantekile njengekulawula lokubi. Ngemuva kwemalanga lalishumi, ngaphambi kwekunisela kwekucala, letitfombo betincishiswa, kwasala titfombo letimbili leticocekile kuphela ebhodweni ngalinye. Ngekwengeta, kwentela kucinisekisa kutseleleka nge S. sclerotiorum, ticu tesitjalo sebhontjisi esigabeni lesifanako sekutfutfuka (emalanga lalishumi) tajutjwa etindzaweni letimbili letehlukene kusetjentiswa i-scalpel lebulala tifo futsi cishe 0.5 g wemhlanganiso we colonizing bran wafakwa esilondzeni ngasinye sekugula kanye nekuvuselela umswakama lophakeme kwentela kutfutfuka kwesifo. Titjalo tekulawula betilimala ngendlela lefanako futsi linani lelilinganako (0.5 g) lekuhlanganiswa kwe bran lengakagcini, lengakakoloni yafakwa esilondzeni futsi yagcinwa ngaphansi kwemswakama lomkhulu kwentela kulingisa simondzawo sekutfutfuka kwetifo kanye nekucinisekisa kuhambisana emkhatsini wemacembu ekwelapha.
Indlela yekwelapha: Titfombo temabhontjisi betiniselwa nge 500 ml yesisombululo lesinemanti se L-ornithine (250 mg/l) noma umutsi wekubulala sikhunta Rizolex-T (50 mg/l) ngekunisela umhlabatsi, bese kwelashwa kwaphindzaphindvwa katsatfu ngesikhatsi semalanga lalishumi. Letilawuli letiphatfwe nge placebo taniselwa ngemanti lahlanjululwe langu 500 ml. Konkhe kwelashwa kwentiwa ngaphansi kwetimo te greenhouse (25 ± 2°C, 75 ± 1% umswakama lohlobene, kanye nesikhatsi sekukhanya se 8 h kukhanya/16 h kumnyama). Onkhe emabhodo aniselwa njalo ngemuva kwemaviki lamabili bese alashwa njalo ngenyanga ngemanyolo eNPK lalinganiselako (20-20-20, nge 3.6% sulphur kanye ne TE microelements; Zain Seeds, Egypt) ngekuhlanganiswa kwa 3–4 g/l ngekufafata emacembe ngekuya kwetincomo tetinhlobo letitsite kanye nemkhiciti’. Ngaphandle uma kushiwo ngalenye indlela, emacembe lavutsiwe lanwetshiwe ngalokuphelele (emacembe esibili kanye nesitsatfu kusuka etulu) ahlanganiswa kusuka kukuphindzaphindza ngamunye kwetilwane nga 72 h ngemuva kwekwelashwa (hpt), ahlanganiswa, ahlanganiswa futsi agcinwa ku -80 °C kwentela kuhlatiya lokunyenti lokufaka ekhatsi, kepha hhayi kuphela, ku inkomba yekucindzeteleka kwe enoxidative yendzawo ye histochemical, kanye nema antioxidants langasiwo ema enzyme kanye nekuveta emajini.
Kucina kwekugula kwesikhunta lesimhlophe kwahlolwa njalo ngeliviki ngemuva kwemalanga langemashumi lamabili nakunye ngemuva kwekugonywa (dpi) kusetjentiswa sikali sa 1–9 (Lithebula Lelengetiwe S2) ngekuya ngesikali sa Petzoldt na Dickson (1996) lesiguculwe ngu Teran et al. (2006). Kafishane, ticu kanye nemagala etitjalo temabhontjisi kwahlolwa kucala endzaweni yekugonywa kwentela kulandzelela kuchubeka kwetilonda eceleni kwema internodes kanye nema nodes. Libanga lesilonda kusuka endzaweni yekugoma kuya endzaweni lekhashane kakhulu eceleni kwesicu noma ligatja labese lilinganiswa futsi emaphuzu langu 1–9 abelwa ngekuya ngendzawo yesilonda, lapho khona (1) kukhombisa kutsi kute kugula lokubonakalako edvute nendzawo yekugoma kanye (2–9) kukhombisa kukhula kancane kancane kwesayizi yesilonda a/interulong S2). Kucina kwekugula kwesikhunta lesimhlophe kwabese kuguculwa kwaba ngemaphesenti kusetjentiswa ifomula 2:
Ngekwengeta, indzawo lengaphansi kwendlela yekuchubeka kwesifo (AUDPC) yabalwa kusetjentiswa ifomula (Shaner na Finney, 1977), leyasandza kulungiswa ngekubola lokumhlophe kwebhontjisi lovamile (Chauhan et al., 2020) kusetjentiswa isilinganiso 3:
Lapho khona Yi = bukhulu besifo ngesikhatsi ti, Yi+1 = bukhulu besifo ngesikhatsi lesitako ti+1, ti = sikhatsi sekukala kwekucala (ngemalanga), ti+1 = sikhatsi sekukala lokulandzelako (ngemalanga), n = linani leliphelele lemaphuzu esikhatsi noma emaphuzu ekubuka. Emapharamitha ekukhula kwesitjalo sebhontjisi kufaka ekhatsi kuphakama kwesitjalo (cm), linani lemagala ngesitjalo ngasinye, kanye nelinani lemacembe ngesitjalo ngasinye kwabhalwa njalo ngeliviki emalanga lamashumi lamabili nakunye kutotonkhe letiphindzaphindziwe tetilwane.
Kukuphindzaphindza ngamunye kwetilwane, emasampula emacembe (emacembe esibili kanye nesitsatfu lakhule ngalokuphelele kusuka etulu) ahlanganiswa ngelilanga lemashumi lamane nesihlanu ngemuva kwekwelashwa (emalanga lalishumi nesihlanu ngemuva kwekwelashwa kwekugcina). Ngayinye yekuphindzaphindza kwetilwane beyinetimbita letisihlanu (titjalo letimbili ngembita). Cishe 500 mg weticubu letichoboshiwe tasetjentiswa kukhipha imibala lekhicitwa ngekukhanya (i-chlorophyll a, i-chlorophyll b kanye ne carotenoids) kusetjentiswa 80% we-acetone ku 4 °C ebumnyameni. Ngemuva kwemahora langemashumi lamabili nakune, lamasampula ahlanganiswa futsi lendzawo lesetulu yabutfwa kwentela kuncuma lokucuketfwe yi-chlorophyll a, i-chlorophyll b kanye ne-carotenoid ngendlela yekubala ngekusebentisa i-UV-160A spectrophotometer (Shimadzu Corporation, eJapan) ngendlela ye (Lichtebaring the35 leyehlukene, emagagasi (A470, A646 kanye na-A663 nm). Ekugcineni, lokucuketfwe kwemibala yekukhicita ngekukhanya kwabalwa kusetjentiswa lamafomula lalandzelako 4–6 lachazwe ngu Lichtenthaler (1987).
Ngesikhatsi se 72 h ngemuva kwekwelashwa (hpt), emacembe (emacembe esibili kanye nesitsatfu latfutfuke ngalokuphelele kusuka etulu) ahlanganiswa kusuka kukuphindzaphindza ngamunye kwetilwane kwentela kuhlala endzaweni ye histochemical ye hydrogen peroxide (H2O2) kanye ne superoxide anion (O2•−). Ngayinye yekuphindzaphindza kwetilwane beyinetimbita letisihlanu (titjalo letimbili ngembita). Ngayinye lephindzaphindvwako yetintfo letiphilako yahlatiya ngekuphindzaphindza (kuphindzaphindza lokubili kwebuchwepheshe) kwentela kucinisekisa kuciniseka, kwetsembeka kanye nekukhicita kabusha kwalendlela. I-H2O2 ne-O2•− kwatsatfwa ngekusebentisa 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Jalimane) noma i-nitroblue tetrazolium (NBT; (2004) kanye na-Adamu nalabanye. (1989) ngekuguculwa lokuncane. Kute kube nendzawo ye histochemical ye H2O2 endzaweni, emapheshana abefakwa nge vacuum nge 0.1% DAB ku 10 mM Tris buffer (pH 7.8) bese ayafutfunyelwa ngesikhatsi sekushisa ekamelweni ekukhanyeni 60 min. Emacembe abehlanjululwa ku 0.15% (v/v) TCA ku 4:1 (v/v) ethanol:chloroform (Imitsi ye Al-Gomhoria kanye neTimphahla Tekwelapha, eCairo, eGibhithe) bese abekwa ekukhanyeni kwaze kwaba mnyama. Ngendlela lefanako, emavalvu abefakwe nge-vacuum nge-10 mM ye-potassium phosphate buffer (pH 7.8) lecuketse 0.1 w/v % HBT yekubeka indzawo ye-O2•− endzaweni. Emapheshana abefutfunyelwa ekukhanyeni ngesikhatsi sekushisa kweligumbi emaminithi lamashumi lamabili, bese ahlanjululwa njengalokungenhla, bese akhanyiswa kwaze kwavela emabala lamnyama laluhlata sasibhakabhaka/violet. Kucina kwembala lophumelelako lonsundvu (njengenkhomba ye H2O2) noma loluhlata sasibhakabhaka (njengenkhomba ye O2•−) kwahlolwa kusetjentiswa inguqulo yase Fiji yephakheji yekucubungula titfombe ImageJ (http://fiji.sc; ifinyelelwe mhla tinge-7 Mashi 2024).
iMalondialdehyde (MDA; njengemaka yekukhicita emafutsa) yatsatfwa ngendlela ya Du na Bramlage (1992) ngekuguculwa kancane. Emacembe lasuka ku replicate ngayinye yetintfo letiphilako (emacembe esibili kanye nesitsatfu latfutfuke ngalokuphelele kusuka etulu) ahlanganiswa 72 h ngemuva kwekwelashwa (hpt). Ngayinye yekuphindzaphindza kwetilwane beyifaka emabhodwe lasihlanu (titjalo letimbili ngembita). Ngayinye lephindzaphindvwako yetintfo letiphilako yahlatiya ngekuphindzaphindza (kuphindzaphindza lokubili kwebuchwepheshe) kwentela kucinisekisa kuciniseka, kwetsembeka kanye nekukhicita kabusha kwalendlela. Kafishane, 0.5 g weticubu temacembe laphansi tasetjentiswa kukhipha iMDA nge 20% we trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) lecuketse 0.01% we hydroxytoluene (BHT; Sigma-Aldrich, St. Lokucuketfwe yi MDA kule supernatant kwabese kuncunywa ngekukala ngekukala lokumuncwako ku 532 na 600 nm kusetjentiswa i-UV-160A spectrophotometer (Shimadzu Corporation, eJapan) bese kuvetwa njenge nmol g−1 FW.
Kuhlolwa kwema antioxidants langasiyo enzymatic kanye ne enzymatic, emacembe (emacembe esibili kanye nesitsatfu latfutfuke ngalokuphelele kusuka etulu) ahlanganiswa kusuka ku replicate ngayinye yetintfo letiphilako nga 72 h ngemuva kwekwelashwa (hpt). Ngayinye yekuphindzaphindza kwetilwane beyinetimbita letisihlanu (titjalo letimbili ngembita). Sampula ngayinye yetintfo letiphilako yahlaziywa ngekuphindzaphindza (emasampula lamabili ebuchwepheshe). Emacembe lamabili abegayiwe nge nitrogen lengemanti bese asetjentiswa ngco kwentela kutfola ema antioxidants lane enzymatic nalawo langasiwo ema enzymatic, ema amino acid laphelele, lokucuketfwe yi proline, kuveta emajini, kanye nekulinganisa kwe oxalate.
Ema phenolics laphelele lancibilikako lancunywa kusetjentiswa iFolin-Ciocalteu reagent (Sigma-Aldrich, St. (1999). Kafishane, cishe 0.1 g weticubu temacembe lahlanganisiwe takhishwa nge 20 ml 80% methanol ebumnyameni emahora lamashumi lamabili nakune bese le supernatant yabutfwa ngemuva kwekuhlanganiswa. 0.1 ml wesampula lekhishwako yahlanganiswa ne 0.5 ml ye Folin-Ciocalteu reagent (10%), yanyakatiswa 30 s yashiywa ebumnyameni 5 min. Ngemuva kwaloko 0.5 ml we 20% we sodium carbonate sisombululo (Na2CO3; Inkampani Yemitsi Nekuphakela Yekwelapha ye Al-Gomhoria, eCairo, eGibhithe) yafakwa kulelishubhu ngalinye, yahlanganiswa kahle futsi yafutfunyelwa ngesikhatsi sekushisa ekamelweni ebumnyameni sikhatsi lesingu 1 h. Ngemuva kwekufukamela, kutsatfwa kwemhlanganiso wekuphendvula kwakalwa nga 765 nm kusetjentiswa i-UV-160A spectrophotometer (iShimadzu Corporation, eJapan). Kugcwala kwema phenols laphelele lancibilikako kuletintfo letikhishwako tesampula kwatsatfwa ngekusebentisa indlela yekulinganisa i-gallic acid (Fisher Scientific, Hampton, NH, USA) futsi kwavetwa njengema milligrams e-asidi ye-gallic lelinganako ngegramu yesisindvo lesisha (mg GAE g-1 sisindvo lesisha).
Linani leliphelele le flavonoid lencibilikako lancunywa ngendlela ya Djeridane et al. (2006) ngekuguculwa kancane. Kafishane, 0.3 ml we methanol lekhishiwe ngenhla yahlanganiswa ne 0.3 ml ye 5% yesisombululo se aluminium chloride (AlCl3; (Al-Gomhoria Imitsi kanye Netinsita Tekwelapha, Cairo, Egypt), kuhlanganiswe kahle futsi kufutfunyelwe ngesikhatsi sekushisa ekamelweni 30 min ebumnyameni. Ngemuva kwekufukamela, kutsatfwa kwemhlanganiso wekuphendvula kwakalwa ku 430 nm kusetjentiswa i-UV-160A spectrophotometer (iShimadzu Corporation, eJapan). Lokucuketfwe kwema flavonoids laphelele lancibilikako etincenyeni tesampula kwatsatfwa ngekusebentisa indlela yekulinganisa ye rutin (TCI America, Portland, NOMA, USA) bese kuvetwa njengema milligrams e rutin lalinganako nge gram yesisindvo lesisha (mg RE g-1 sisindvo lesisha).
Linani leliphelele le amino acid lekhululekile lemacembe emabhontjisi latsatfwa ngekusebentisa i-ninhydrin reagent leguculwe (Emakhemikhali esayensi lashisako, Waltham, MA, USA) ngekuya ngendlela lephakanyiswe ngu Yokoyama na Hiramatsu (2003) futsi yaguculwa ngu Sun et al. (2006). Kafishane, 0.1 g weticubu letiphansi takhishwa nge pH 5.4 buffer, bese 200 μL we supernatant waphendvulwa nge 200 μL we ninhydrin (2%) kanye na 200 μL we pyridine (10%; kulinganiswa ku 580 nm kusetjentiswa i-UV-160A (Inkampani yaShimadzu, eJapan). Ngakulolunye luhlangotsi, i-proline yancunywa yindlela yaBates (Bates nalabanye, 1973). I-Proline yakhishwa nge-3% ye-sulfosalicylic acid (Emakhemikhali Esayensi Yekushisa, Waltham, MA, USA) futsi ngemuva kwekuhlanganiswa, 0.5 ml we-supernatant yahlanganiswa ne-1 ml ye-asidi ye-acetic ye-glacial (Fisher Scientific, Hampton, NH, USA) kanye ne-ninhydrin reagent lelinganisiwe ku-min5 ku 520 nm kusetjentiswa i-spectrophotometer lefanako njengaleyo lengetulu. Linani selilonkhe lema amino acid lakhululekile kanye ne proline kuletintfo letikhishwako temacembe kwatsatfwa kusetjentiswa emagoba ekulinganisa eglycine kanye ne proline (Sigma-Aldrich, St Louis, MO, eMelika), ngekulandzelana, futsi kwavetwa njenge mg/g sisindvo lesisha.
Kute kutfolakale umsebenti we-enzymatic we-enzymes yekulwa nekugula, cishe 500 mg weticubu letifanako takhishwa nge-3 ml ye-50 mM Tris buffer (pH 7.8) lecuketse 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA), i-centrifuged ku-10,000 × g imizuzu lengu-20 ngaphansi kwekubandza (4 °C), futsi le-supernatant (i-enzyme lengakahlutwa) yabutfwa (El-Nagar nalabanye, 2023; Osman nalabanye, 2023). I-Catalase (CAT) yabese isabela nge-2 ml ye-0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. 2023; Osman nalabanye, 2023). Umsebenti we-enzymatic we-peroxidase (POX) loncike ku-Guaiacol wancunywa kusetjentiswa indlela yaHarrach et al. (2009). (2008) ngekuguculwa lokuncane (El-Nagar nalabanye, 2023; Osman nalabanye, 2023) kanye nemsebenti we-enzymatic we-polyphenol oxidase (PPO) wancunywa ngemuva kwekuphendvula nge-2.2 ml ye-100 mM ye-sodium phosphate buffer (pH 6.0l), EPortland, NOMA, eMelika) kanye ne-100 μl ye-12 mM H2O2. Indlela yashintjwa kancane kusuka ku (El-Nagar nalabanye, 2023; Osman nalabanye, 2023). Lokuhlolwa kwentiwa ngemuva kwekuphendvula nge 3 ml yesisombululo se catechol (Emakhemikhali Esayensi Yekushisa, Waltham, MA, USA) (0.01 M) lesanda kulungiswa ku 0.1 M phosphate buffer (pH 6.0). Umsebenti we-CAT walinganiswa ngekugadza kubola kwe-H2O2 ku-240 nm (A240), umsebenti we-POX walinganiswa ngekugadza kukhula kwekumunwa ku-436 nm (A436), kanye nemsebenti we-PPO walinganiswa ngekubhala kugucugucuka kwekumuncwa ku-495 nm njalo nge-U305) silinganiso sekukhanya (Shimadzu, eJapan).
I-RT-PCR yesikhatsi sangempela yasetjentiswa kutfola emazinga ekubhala emajini lamatsatfu lahlobene ne-antioxidant, kufaka ekhatsi i-peroxisomal catalase (PvCAT1; Kungena No. KY195009.1), emacembe emabhontjisi (emacembe esibili kanye nesitsatfu lakhule ngalokuphelele kusuka etulu) 72 h ngemuva kwekwelashwa kwekugcina. Kafishane, i-RNA yahlukaniswa kusetjentiswa iKhithi Yekukhipha I-RNA Lephelele Ye-Simply P (Cat. No. BSC52S1; BioFlux, Buchwepheshe Be-Biori, eShayina) ngekuya kwendlela yekusebenta yemkhiciti. Ngemuva kwaloko, i-cDNA yahlanganiswa kusetjentiswa i-TOP script TM i-cDNA Synthesis Kit ngekuya kweticondziso temkhiciti. Lokulandzelana kwe-primer kwalamajini lamatsatfu langetulu kubhalwe kuLithebula Lelengetiwe S3. I-PvActin-3 (inombolo yekungena yeGenBank: XM_068616709.1) yasetjentiswa njengelifutsi lekugcina indlu futsi kuveta lifutsi lelihlobene kwabalwa kusetjentiswa indlela ye-2-ΔΔCT (Livak naSchmittgen, 2001). Kusimama kwe Actin ngaphansi kwekucindzeteleka kwetilwane (kusebentisana lokungahambisani emkhatsini wema legumes lavamile kanye nesikhunta se anthracnose Colletotrichum lindemuthianum) kanye nekucindzeteleka kwetilwane letiphilako (somiso, luswayi, kushisa lokuphansi) kwakhonjiswa (Borges et al., 2012).
Sicale senta kuhlatiya kwe-genome-wide ku-silico kwemaprotheni e-oxaloacetate acetylhydrolase (OAH) ku-S. Kafishane, sasebentisa i-OAH lesuka ku-Aspergillus fijiensis CBS 313.89 (i-AfOAH; i-taxide: 1191702; inombolo yekungena ye-GenBank XP_040799428.1; XP_056833920.1; 316 ema-amino esidi) njengekulandzelana kwemibuto kwenta imephu yephrotheni lefanako ku S. I-BLASTp yentiwa ngekumelene nemininingwane ye-genome ye-S.
Ngetulu kwaloko, lifutsi le-OAH lelibiketelwe kusuka ku-S. imodeli (Jones nalabanye, 1992). Lesihlahla se phylogenetic sahlanganiswa nekuhlatiya kwekuhambisana lokunyenti kwekulandzelana kwemaprotheyini awo onkhe emajini e-OAH labiketelwe (SsOAH) kusuka ku S. sclerotiorum kanye nekulandzelana kwemibuto kusetjentiswa Lithuluzi Lekuhambisana Lelisekelwe Ekuvimbeleni (COBALT; kanye ne-Agarwala, ngemnyaka wa-2007). Ngetulu kwaloko, kulandzelana kwe-amino acid lokuhambisanako lokuhle kakhulu kwe-SsOAH kusuka ku-S. Lithuluzi le-ESPript (inguqulo 3.0; https:
Ngetulu kwaloko, tindzawo letimele letisebentako letibiketelwe kanye netindzawo letigcinwe te-S. Ekugcineni, kumodela kwesakhiwo se-S. sclerotiorum SsOAH lebikezelwe kwentiwa ngekusebentisa Injini Yekubona I-Protein Homology/Analogy (i-Phyre2 server version 2.0; 2015) futsi yacinisekiswa kusetjentiswa iseva ye-SWISS-MODEL (Biasini nalabanye, 2014). Letakhiwo letibikezelwe letinetigaba letintsatfu (ifomethi ye-PDB) tabonwa ngekusebentisana kusetjentiswa iphakheji ye-UCSF-Chimera (inguqulo 1.15; https://www.
I-PCR ye-fluorescence yesikhatsi sangempela yesilinganiso yasetjentiswa kutfola lizinga lekubhala le-oxaloacetate acetylhydrolase (SsOAH; inombolo yekungena ye-GenBank: XM_001590428.1) ku-mycelia ye-Sclerotinia sclerotiorum. Kafishane, iS. Ngemuva kwaloko, lamaseli alashwa nge L-ornithine kanye ne fungicide Rizolex-T ngesikhatsi sekugcina se IC50 (cishe 40 na 3.2 mg/L, ngekulandzelana) bese ayakhuliswa lamanye emahora lamashumi lamabili nakune ngaphansi kwetimo letifanako. Ngemuva kwekufukamela, lamasiko abekwa endzaweni lesetulu ku 2500 rpm ngemaminithi lasihlanu bese le supernatant (i mycelium ye fungal) yabutfwa kwentela kuhlatiya kuveta kwemajini. Ngendlela lefanako, imycelium ye fungal yabutfwa nga 0, 24, 48, 72, 96, kanye na 120 h ngemuva kwekutseleleka kusuka etitjalweni letingenwe ngulesifo lebetakhe sikhunta lesimhlophe kanye ne mycelium yekotini etulu kweticubu letingenwe ngulesifo. iRNA yakhishwa ku mycelium ye fungal bese icDNA yahlanganiswa njengoba kuchaziwe ngenhla. Lokulandzelana kwe-primer ye-SsOAH kubhalwe kuThebula Lelengetiwe S3. I-SsActin (inombolo yekungena ye-GenBank: XM_001589919.1) yasetjentiswa njengelifutsi lekugcina indlu, futsi kuveta lifutsi lelihlobene kwabalwa kusetjentiswa indlela ye-2-ΔΔCT (Livak naSchmittgen, 2001).
iOxalic acid yatsatfwa kumhluzi we dextrose wemazambane (PDB) kanye nemasampula etitjalo lacuketse sifo sesikhunta se Sclerotinia sclerotiorum ngendlela ya Xu na Zhang (2000) ngekuguculwa kancane. Kafishane, iS. Ngemuva kwekufukamela, lelisiko le fungal lacala kuhlungwa ngephepha lekuhlunga le Whatman #1 bese liyafakwa ku 2500 rpm ngemaminithi langu 5 kwentela kususa imycelium lesele. Le supernatant yabutfwa yagcinwa ku 4°C kwentela kutfola lokunyenti kwesilinganiso se oxalate. Kulungiselela emasampula etitjalo, cishe 0.1 g wetincenye teticubu tetitjalo takhishwa katsatfu ngemanti lahlungiwe (2 ml sikhatsi ngasinye). Lamasampula abese aya centrifuged ku 2500 rpm nge 5 min, le supernatant yase yomile ihlungwa nge Whatman No. 1 iphepha lekuhlunga bese iyabutfwa kwentela kuhlatiya kabanti.
Kuhlatiya kwesilinganiso se-oxalic esidi, le ngcucuko yekuphendvula yalungiswa ngeshubhu levaliwe ngengilazi ngekulandzelana lokulandzelako: 0.2 ml wesampula (noma i-PDB isiko lekuhlunga noma sisombululo lesijwayelekile se-oxalic esidi), 0.11 ml we-bromophenol leluhlata sasibhakabhaka (BPB, 1 mM; esidi (H2SO4; Al-Gomhoria Imitsi Netimphahla Tekwelapha, eCairo, eGibhithe) kanye na 0.176 ml we 100 mM we potassium dichromate (K2Cr2O7; emakhemikhali e TCI, ePortland, NOMA, eMelika), bese lesisombululo sahlanjululwa ngendlela lenemandla, nge 4.8 ml. endzaweni yekugeza ngemanti langu 60 °C. Ngemuva kwemizuzu lengu-10, lokusabela kwamiswa ngekwengeta 0.5 ml wesisombululo se-sodium hydroxide (NaOH; 0.75 M). Kumuncwa (A600) kwekuhlanganiswa kwekuphendvula kwakalwa ku 600 nm kusetjentiswa i-UV-160 spectrophotometer (Inkampani yaShimadzu, eJapan). iPDB kanye nemanti lahlungiwe asetjentiswa njengekulawula kwekulinganisa emasiko lahlungiwe kanye nemasampula etitjalo, ngekulandzelana. Lokucuketfwe kwe oxalic esidi kuletintfo letihlungiwe temasiko, lokuvetwa njengema micrograms e oxalic esidi nge mililitha ye PDB lesemkhatsini (μg.mL−1), kanye nasetintfweni letikhishwako temacembe, lokuvetwa njengema micrograms e oxalic esidi nge gram yesisindvo lesisha (μg.g−1 FW), kwatsatfwa ngekusebentisa i-oxalic acid calibration ( MA, eMelika).
Kulo lonkhe lolucwaningo, konkhe kuhlola kwakhiwe ngendlela lekhetsiwe ngalokuphelele (CRD) ngekuphindzaphindza lokusitfupha kwetilwane ngekwelashwa kanye netimbita letisihlanu ngekuphindzaphindza kwetilwane (titjalo letimbili ngembita) ngaphandle uma kushiwo ngalenye indlela. Tiphindvwe letiphindzaphindvwako tetilwane tahlaziywa ngekuphindzaphindza (kuphindzaphindza lokubili kwebuchwepheshe). Kuphindzaphindza kwebuchwepheshe kwasetjentiswa kuhlola kukhicita kabusha kwekuhlola lokufanako kepha akuzange kusetjentiswe ekuhlaziyeni kwetibalo kwentela kugwema kuphindzaphindza lokungemanga. Idatha yahlaziywa ngekwetibalo kusetjentiswa kuhlatiya kwekuhluka (ANOVA) kulandzelwe nguTukey-Kramer ngekwetsembeka umehluko lomkhulu (HSD) test (p ≤ 0.05). Kulokuhlolwa kwe vitro, emanani eIC50 kanye ne IC99 abalwa kusetjentiswa imodeli ye probit kanye netikhatsi tekwetsemba letingu 95% tabalwa.
Inani letinhlobo letine letihlukanisiwe tahlanganiswa emasimini lehlukene esoya eEl Ghabiya Governorate, eGibhithe. Ku PDA medium, tonkhe letihlukanisiwe takhicita imycelium lemhlophe lekhilimu lesheshe yaphenduka ibe mhlophe njenge cotton (Umfanekiso 1A) bese ibe beige noma insundvu esigabeni se sclerotium. Ema Sclerotia avame kuba mnyama, amnyama, ayindingilizi noma angajwayelekile ngesimo, 5.2 kuya ku 7.7 mm ngebudze kanye na 3.4 kuya ku 5.3 mm ngebubanti (Umfanekiso 1B). Noma nje ema-isolate lamane atfutfukisa iphethini lesemnceleni ye-sclerotia emnceleni wendzawo yekulima ngemuva kwemalanga langu-10–12 ekufukamela ku-25 ± 2 °C (Fig. 1A), linani le-sclerotia ngelipuleti ngalinye belihluke kakhulu emkhatsini wawo (P <0.001). 1.53 ipuleti ngalinye; Umfanekiso 1C). Ngendlela lefanako, leyehlukile #3 yakhicita i-oxalic esidi lenyenti ku-PDB kunaletinye letehlukaniswe (3.33 ± 0.49 μg.mL−1; Fig. 1D). Isolate #3 yakhombisa timphawu letijwayelekile tekwakheka kanye nesibonakhulu sesikhunta lesibulala titjalo iSclerotinia sclerotiorum. Sibonelo, ku PDA, emakoloni e-isolate #3 akhula ngekushesha, abemhlophe njengekhilimu (Umfanekiso 1A), abe beige emuva noma i-salmon lekhanyako lephuti-nsundvu, futsi bekudzingeka emalanga langu 6-7 ku 25 ± 2°C kwentela kumbonya ngalokuphelele indzawo yepuleti lelinebukhulu lobungaba ngu 9 cm. Kususelwa kuletimphawu letingenhla tekwakheka kanye nesibonakhulu, i-isolate #3 yakhonjwa njenge Sclerotinia sclerotiorum.
Umfanekiso 1. Timphawu kanye nekugula kwe S. sclerotiorum lehlukaniswe etitjalweni te legume letivamile. (A) Kukhula kwe-mycelial kwetintfo letine te-S. legume yekutsengisa letsatsekako Giza 3 ngaphansi kwetimo te greenhouse. Emanani amelela kusho ± SD kwekuphindzaphindza lokusihlanu kwetilwane (n = 5). Tinhlavugama letehlukene tikhombisa umehluko lomkhulu ngekwetibalo emkhatsini wekwelashwa (p <0.05). (F–H) Timphawu tesikhunta lesimhlophe letijwayelekile tavela eticumbini letingetulu kwemhlabatsi kanye nema siliques, ngekulandzelana, emalanga lalishumi ngemuva kwekugonywa nge isolate #3 (dpi). (I) Kuhlatiya kwekutigucukela kwemvelo kwesigodzi se-spacer lebhalwe phansi lengekhatsi (ITS) se-S. sclerotiorum isolate #3 kwentiwa kusetjentiswa indlela lenkhulu yekutsi kungenteka futsi kwacatsaniswa netinhlobo letingu-20 tekubhekisa/tinhlobo letitfolakala ku-database yeSikhungo Savelonkhe Selwatiso Lwetebucwepheshe Betilwane (NCBI). Tinombolo letingetulu kwemigca yekuhlanganisa tikhombisa kuhlanganiswa kwesigodzi (%), bese tinombolo letingephansi kwemigca yekuhlanganisa tikhombisa budze beligatja.
Ngetulu kwaloko, kucinisekisa kugula, ema S. sclerotiorum isolates lamane latfolakalako asetjentiswa kugoma le cultivar yebhontjisi yekutsengisa letsatsekako iGiza 3 ngaphansi kwetimo te greenhouse, lokuhambisana ne postulates ya Koch (Fig. 1E). Noma tonkhe letintfo letitfolakele letihlukaniswe ngesikhunta betinetifo futsi tingatseleleka ebhonjisini leliluhlata (cv. Giza 3), kubangela timphawu tesikhunta lesimhlophe lesijwayelekile kutotonkhe tincenye letingetulu kwemhlabatsi (Fig. 1F), ikakhulu eticumbini (Fig. 1G) kanye ne post inoculation (Fig. 1H) ngesikhatsi lesiningi se 10. isolate lenelulaka ekuhlolweni lokubili lokutimele. Isolate 3 beyinesifo lesikhulu kakhulu (%) etitjalweni temabhonjisi (24.0 ± 4.0, 58.0 ± 2.0, kanye na 76.7 ± 3.1 ngemalanga langu 7, 14, kanye na 21 ngemuva kwekutseleleka, ngekulandzelana; Umfanekiso 1F).
Kukhonjwa kwe S. sclerotiorum isolate #3 lehlasela kakhulu kwaphindze kwacinisekiswa ngekusebentisa kulandzelana kwesikhala lesibhalwe phansi sangekhatsi (ITS) (Fig. 1I). Kuhlatiya kwe Phylogenetic emkhatsini we isolate #3 kanye ne 20 yekubhekisa isolates/tinhlobo kwakhombisa kufana lokusetulu (>99%) emkhatsini wato. Kufanele kutsi kucashelwe kutsi iS. OR206374.1; 548 bp), lokubangela kubola kwesicu se violet (Matthiola incana), konkhe loku kuhlanganiswa ngekwehlukana ngetulu kwe dendrogram (Umfanekiso 1I). Lokulandzelana lokusha sekufakwe ku-NCBI futsi kwabitwa ngekutsi “Sclerotinia sclerotiorum – kuhlukanisa i-YN-25” (inombolo yekungena ye-GenBank PV202792). Kungabonakala kutsi isolate 3 nguyona isolate lehlasela kakhulu; ngako-ke, le isolate yakhetfwa kutsi ifundvwe kuto tonkhe tilingo letalandzela.
Umsebenti wekulwa nemagciwane we diamine L-ornithine (Sigma-Aldrich, Darmstadt, eJalimane) ngetigaba letehlukene (12.5, 25, 50, 75, 100 kanye na 125 mg/L) ngekumelene ne S. Kuyaphawuleka kutsi iL-ornithine yaveta umphumela wekulwa nemagciwane futsi kancane kancane yavimbela kukhula kwe radial kwe S. sclerotiorum hyphae ngendlela lencike kumthamo (Umfanekiso 2A, B). Ngesikhatsi sekuhlanganiswa lokusetulu lokuhloliwe (125 mg/L), i-L-ornithine yakhombisa lizinga leliphakeme lekuvimbela kukhula kwe-mycelial (99.62 ± 0.27%; Umfanekiso 2B), lebelilingana ne-fungicide yekutsengisa iRizolex-T) (lizinga lekuvimbela lelisetulu kakhulu Umfanekiso 395 the Umfanekiso 2B). (10 mg / L), lokukhombisa kusebenta lokufanako.
Umfanekiso 2. Umsebenti wekulwa nemagciwane we-L-ornithine ngekumelene ne-Sclerotinia sclerotiorum. (A) Kucatsanisa umsebenti wekulwa nemagciwane wekuhlanganiswa lokuhlukene kwe L-ornithine ngekumelene ne S. (B, C) Lizinga lekuvimbela (%) lekukhula kwe mycelial ye S. Emanani amelela kusho ± SD kwekuphindzaphindza lokusihlanu kwetilwane (n = 5). Tinhlavugama letehlukene tikhombisa umehluko wetibalo emkhatsini wekwelashwa (p <0.05). (D, E) Kuhlatiya kwekubuyela emuva kwemodeli ye-Probit ye-L-ornithine kanye ne-Rizolex-T yekutsengisa sikhunta, ngekulandzelana. Umugca wekuhlehla kwemodeli ye probit ukhonjiswa njengemugca loluhlata sasibhakabhaka locinile, bese sikhatsi sekwetsemba (95%) sikhonjiswa njengemugca lobovu lonemadashi.
Ngekwengeta, kuhlatiya kwe probit regression kwentiwa futsi tindzawo letihambisanako tikhonjiswe ku Lithebula 1 kanye ne Tibalo 2D,E. Kafishane, linani lekuthambeka lelemukelekako (y = 2.92x − 4.67) kanye netibalo letibalulekile letihambisanako (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 kanye ne p <0.0001; Umfanekiso 2D) we L-ornithine kukhombisa kusebenta lokukhulisiwe lokumelene ne Sfungal. umutsi wekubulala sikhunta Rizolex-T (y = 1.96x − 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 kanye ne p <0.0001) (Lithebula 1).
Lithebula 1. Emanani esigamu-lesikhulu sekuvimbela (IC50) kanye ne IC99 (mg/l) ye L-ornithine kanye nesibulali sikhunta sekutsengisa “Rizolex-T” ngekumelene ne S.
Kuko konkhe, iL-ornithine (250 mg/L) yehlisa kakhulu kutfutfuka kanye nebukhulu besikhunta lesimhlophe etitjalweni temabhontjisi letivamile letilashiwe uma kucatsaniswa netitjalo letingakaphatfwa letine S. sclerotiorum (kulawula; Umfanekiso 3A). Kafishane, noma bukhulu besifo setitjalo tekulawula letingenwe ngulesifo letingakalashwa bukhula kancane kancane (52.67 ± 1.53, 83.21 ± 2.61, kanye na 92.33 ± 3.06%), iL-ornithine yehlisa kakhulu bukhulu besifo (%) kulo lonkhe lokuhlolwa (8.9 ± 0.0. 1.00, kanye 26.36 ± 3.07) ku 7, 14, kanye 21 emalanga ngemuva kwekwelashwa (dpt), ngokulandelana (Umfanekiso 3A). Ngendlela lefanako, ngesikhatsi titjalo temabhontjisi letingenwe yi S. sclerotiorum tilashwa nge 250 mg/L L-ornithine, indzawo lengaphansi kwe curve yekuchubeka kwesifo (AUDPC) yehla kusuka ku 1274.33 ± 33.13 ekulawuleni lokungakaphatfwa kuya ku 281.03 ± 7.0L yekulawula lokuhle ngaphansi kwaloko s/0. Rizolex-T umutsi wekubulala sikhunta (183,61 ± 7,71; Umfanekiso 3B). Umkhuba lofanako wabonakala ekuhlolweni kwesibili.
Fig. 3. Umphumela wekusetjentiswa kwe L-ornithine ngephandle ekwakhiweni kwekubola lokumhlophe kwebhontjisi lovamile lokubangelwa yi Sclerotinia sclerotiorum ngaphansi kwetimo tendlu leluhlata. (A) Sifo sekuchubeka kwe curve yesikhunta lesimhlophe sebhontjisi lovamile ngemuva kwekwelashwa nge 250 mg/L L-ornithine. (B) Indzawo lengaphansi kwekuchubeka kwesifo (AUDPC) yesikhunta lesimhlophe sebhontjisi lovamile ngemuva kwekwelashwa nge L-ornithine. Emanani amelela kusho ± SD kwekuphindzaphindza lokusihlanu kwetilwane (n = 5). Tinhlavugama letehlukene tikhombisa umehluko lomkhulu ngekwetibalo emkhatsini wekwelashwa (p <0.05).
Kusetjentiswa kwe Exogenous kwe 250 mg/L L-ornithine kancane kancane kwakhulisa budze besitjalo (Fig. 4A), linani lemagala ngesitjalo ngasinye (Fig. 4B), kanye nelinani lemacembe ngesitjalo ngasinye (Fig. 4C) ngemuva kwemalanga langemashumi lamane nakubili. Ngesikhatsi umutsi wekubulala sikhunta wekutsengisa iRizolex-T (50 mg/L) waba nemtselela lomkhulu kuwo onkhe emapharamitha ekudla lafundvwako, kusetjentiswa kwe exogenous kwe 250 mg/L L-ornithine kwaba nemtselela wesibili lomkhulu uma kucatsaniswa nekulawula lokungakaphatfwa (Figs. 4A–C). Ngakulolunye luhlangotsi, kwelashwa kwe L-ornithine bekute umphumela lomkhulu kuloko lokucuketfwe yimibala yekukhicita i-chlorophyll a (Umfanekiso 4D) kanye ne-chlorophyll b (Umfanekiso 4E), kepha kwakhulisa kancane lokucuketfwe kwe-carotenoid lephelele (0.56 ± 0.03 mg2 ±0r w4 fr wt kanye nekulawula lokubi (0.0. kulawula lokuhle (0.46 ± 0.02 mg / g fr wt; Umfanekiso 4F). Kuko konkhe, lemiphumela ikhombisa kutsi iL-ornithine ayisiyo iphytotoxic kuma legumes laphatsiwe futsi ingaze ivuselele kukhula kwawo.
Fig. 4. Umphumela wekusetjentiswa kwe L-ornithine lengaphandle etimphawu tekukhula kanye nemibala yekukhicita emacembe emabhontjisi latseleleke nge Sclerotinia sclerotiorum ngaphansi kwetimo tendlu leluhlata. (A) Kuphakama kwesitjalo (cm), (B) Linani lemagala ngesitjalo ngasinye, (C) Linani lemacembe ngesitjalo ngasinye, (D) Lokucuketfwe yi Chlorophyll a (mg g-1 fr wt), (E) Lokucuketfwe yi Chlorophyll b (mg g-1 fr wt), (F) Kucuketfwe kwe carotenoid lokuphelele (mg g-1 fr). Emanani asho ± SD yekuphindzaphindza lokusihlanu kwetilwane (n = 5). Tinhlavugama letehlukene tikhombisa umehluko lomkhulu ngekwetibalo emkhatsini wekwelashwa (p <0.05).
In situ histochemical indzawo yetinhlobo te-oksijini lephendvulako (ROS; levetwa njenge-hydrogen peroxide [H2O2]) kanye nema-radicals lakhululekile (lavetwa njenge-anions ye-superoxide [O2•−]) yaveta kutsi kusetjentiswa kwe-exogenous kwe-L-ornithine (250 mg/L) kunciphise kakhulu kugcwala kwe-H. nmol.g−1 FW; Umfanekiso 5A) kanye ne-O2•− (32.69 ± 8.56 nmol.g−1 FW; Umfanekiso 5B) uma kucatsaniswa nekugcwala kwato totimbili titjalo letingenwe ngulesifo letingakaphatfwa (173.31 ± 12.06 kanye na-149.35 ± 7.94 nmol) lehlolwe nge-F−1. mg/L yesibulali sikhunta sekutsengisa iRizolex-T (170.12 ± 9.50 kanye na 157.00 ± 7.81 nmol.g−1 fr wt, ngekulandzelana) ngesikhatsi se 72 h. Emazinga laphakeme e-H2O2 kanye ne-O2•− labutselelwe ngaphansi kwe-hpt (Fig. 5A, B). Ngendlela lefanako, kuhlolwa kwe malondialdehyde (MDA) lokusekelwe ku TCA kwakhombisa kutsi titjalo temabhontjisi letingenwe yi S. Nomakunjalo, kusetjentiswa kwe L-ornithine ngekwemvelo kwehlisa kakhulu kukhishwa kwemafutsa njengoba kufakazelwa kwehla kwe MDA etitjalweni letiphatsiwe (33.08 ± 4.00 nmol.g fr wt).
Fig. 5. Umphumela wekusetjentiswa kwe exogenous L-ornithine kumamaki lamakhulu ekucindzeteleka kwe oxidative kanye netindlela tekuvikela letingekho enzymatic emacembe emabhontjisi latseleleke nge S. sclerotiorum nga 72 h ngemuva kwekutseleleka ngaphansi kwetimo te greenhouse. (A) I-hydrogen peroxide (H2O2; nmol g-1 FW) ku-72 hpt, (B) i-anion ye-superoxide (O2•-; nmol g-1 FW) ku-72 hpt, (C) i-malondialdehyde (MDA; nmol g-1 FW) ku-72 hpt, (D) i-phenol lephelele lencibilikako. (E) ema-flavonoids lancibilikako laphelele (mg RE g-1 FW) ku-72 hpt, (F) ema-amino esidi lakhululekile laphelele (mg g-1 FW) ku-72 hpt, kanye (G) nekucuketfwe kwe-proline (mg g-1 FW) ku-72 hpt. Emanani amelela kusho ± kuphambuka lokujwayelekile (kusho ± SD) kwe-5 yekuphindzaphindza kwetilwane (n = 5). Tinhlavugama letehlukene tikhombisa umehluko lomkhulu ngekwetibalo emkhatsini wekwelashwa (p <0.05).