I-atikili le yingcenye yesihloko serhubhululo esithi “Ukwenza ngcono ukujamelana kwama-legumes emagciwaneni nezinambuzaneni”, qala woke ama-atikili amahlanu
I-ejenti ebanga isifo se-fungal plant necrosis Sclerotinia sclerotiorum (Lib.) de Bary isebenzisa iqhinga elineengaba ezinengi ukutheleleka iintjalo ezihlukahlukeneko. Irhubhululo leli liphakamisa ukusetjenziswa kwe-diamine L-ornithine, i-amino acid engekho ngamaphrotheyini ekhuthaza ukwakheka kwamanye ama-amino acid aqakathekileko, njengendlela ehlukileko yokulawula ukuthuthukisa iimpendulo zamamolekhyuli, zomzimba kanye ne-biochemical ze-Paseolus vulgaris L. esikhunjeni esimhlophe esibangelwa yi-Pseudomonasclerum sclerum. Iimhloliso ze-in vitro zitjengise bona i-L-ornithine ivimbele khulu ukukhula kwe-mycelial ye-S. pyrenoidosa ngendlela ethembele emthamo. Ngaphezu kwalokho, kungehlisa khulu ubudisi besikhunta esimhlophe ngaphasi kweemeko ze-greenhouse. Ukudlula lapho, i-L-ornithine yakhuthaza ukukhula kweentjalo eziphathweko, okutjengisa bona ukugxila okuhloliweko kwe-L-ornithine bekungasi yi-phytotoxic eentjalweni eziphathweko. Ukungezelela, i-L-ornithine ikhuphule ukuvezwa kwama-antioxidants angasiwo ama-enzymatic (ama-phenolics ancibilikako nama-flavonoids) kanye nama-antioxidants we-enzymatic (i-catalase (CAT), i-peroxidase (POX), kanye ne-polyphenol oxidase (PPO)), begodu yakhuphula ukuvezwa kwamajini amathathu ahlobene nama-antioxidant (PSO1, PVGRAT, kanye ne-PVGRAT). Ukudlula lapho, ukuhlaziywa kwe-silico kuveze ubukhona bephrotheyini ye-oxaloacetate acetylhydrolase (SsOAH) ku-S. sclerotiorum genome, ebegade ifana khulu namaphrotheyini we-oxaloacetate acetylhydrolase (SsOAH) we-Aspergillus fijiensis (AfOAH) kanye ne-SsOAH. (PlOAH) ngokuya ngokuhlaziywa okusebenzako, iindawo ezilondoloziweko, kanye nethopoloji. Ngokuthakazelisako, ukungezelelwa kwe-L-ornithine ku-potato dextrose broth (PDB) medium kwehlise khulu ukuvezwa kwejini le-SsOAH ku-S. sclerotiorum mycelia. Ngokufanako, ukusetjenziswa kwangaphandle kwe-L-ornithine kwehlise khulu ukuvezwa kwejini le-SsOAH ku-fungal mycelia ebuthelelwe eentjalweni eziphathweko. Ekugcineni, ukusetjenziswa kwe-L-ornithine kwehlise khulu ukukhiqizwa kwe-oxalic acid kuzo zombili i-PDB medium kanye namakari athelelekileko. Ekuphetheni, i-L-ornithine idlala indima eqakathekileko ekugcineni ubujamo be-redox kanye nokuthuthukisa ukuphendula kokuzivikela kweentjalo ezithelelekileko. Imiphumela yesifundo lesi ingasiza ekuthuthukiseni iindlela ezitjha, ezingalimazi ibhoduluko zokulawula isikhunta esimhlophe nokunciphisa umthelela waso ekukhiqizeni ubhontshisi nezinye iintjalo.
Isikhunta esimhlophe, esibangelwa yi-necrotrophic fungus Sclerotinia sclerotiorum (Lib.) de Bary, bulwelwe obubhubhisako, obunciphisa isivuno obubeka ingozi ekulu ekukhiqizweni kwebhontjisi (Phaseolus vulgaris L.) ephasini loke (u-Bolton et al., 2006). I-Sclerotinia sclerotiorum ngenye yamagciwane weentjalo ezithwala iinkhunta ezithwala ehlabathini ezibudisi ukulawula, inemihlobo ebanzi yeentjalo ezingaphezu kwama-600 kanye nekghono lokuqeda msinyana amathitjhu weentjalo ngendlela engakaqakatheki (u-Liang no-Rollins, 2018). Ngaphasi kweemeko ezingakalungi, idlula esigabeni esiqakathekileko somjikelezo wayo wokuphila, ihlale ingasebenzi isikhathi eside njengezakhiwo ezimnyama, eziqinileko, ezifana nembewu ezibizwa ngokuthi 'sclerotia' ehlabathini nofana njengemikhula emhlophe, e-fluffy ku-mycelium nofana isiqu seentjalo ezithelelekileko (Schwartz et al., 2005). I-S. sclerotiorum ikghona ukwakha i-sclerotia, okwenza bona iphile eendaweni ezithelelekileko isikhathi eside begodu iragele phambili ngesikhathi sokugula (Schwartz et al., 2005). I-Sclerotia inothile ngeemvundisi, ingahlala isikhathi eside ehlabathini, begodu isebenza njenge-inoculum yokuthoma yokutheleleka okulandelako (Schwartz et al., 2005). Ngaphasi kweemeko ezihle, i-sclerotia ihluma begodu ikhiqize ama-spore aphephetha emoyeni angathelela zoke iingcenye zesitjalo ezingaphezu kwehlabathi, kufaka hlangana kodwana kungapheleli emathuthumbo, iinqubu, nofana ama-pods (Schwartz et al., 2005).
I-Sclerotinia sclerotiorum isebenzisa iqhinga elineengaba ezinengi ukutheleleka iintjalo ezibambako, okufaka hlangana uchungechunge lwezehlakalo ezihlangeneko ukusuka ekumileni kwe-sclerotial ukuya ekuthuthukisweni kweempawu. Ekuthomeni, i-S. sclerotiorum ikhiqiza ama-spore alengako (abizwa ngokuthi ma-ascospores) avela ezakhiweni ezifana namakhowa ezibizwa ngokuthi yi-apothecia, eziba semoyeni bese zikhula zibe yi-non-motile sclerotia phezu kweentjalo ezithelelekileko (u-Bolton et al., 2006). I-fungus ibe seyikhipha i-oxalic acid, into ebangela ubungozi, ukulawula i-pH yamaseli wesitjalo, ukukhuthaza ukuwohloka kwe-enzymatic nokuhlasela kwezicubu (Hegedus no-Rimmer, 2005), nokugandelela ukuqhuma kwe-oxidative yesitjalo. Ikambiso yokwenza i-esidi yenza bona udonga lwamaseli wesitjalo lube buthakathaka, okwenza bona kube nendawo ehle yokusebenza okujayelekileko nokuphumelelako kwama-enzyme wokubulala udonga lwamaseli we-fungal (ama-CWDE), okwenza bona i-pathogen inqobe isithiyo esibonakalako begodu ingene emathitjhu we-host (u-Marciano et al., 1983). Nasele ingene ngaphakathi, i-S. sclerotiorum ikhipha ama-CWDE amanengi, afana ne-polygalacturonase ne-cellulase, okwenza bona irhatjheke eencutjhweni ezithelelekileko begodu ibangele ukufa kwezicubu. Ukuragela phambili kwezilonda nama-hyphal mats kurholela ekutheni kube namatshwayo wesikhunta esimhlophe (u-Hegedus no-Rimmer, 2005). Khatheso, iintjalo ezibambako zibona amaphetheni wamamolekhyuli ahlotjaniswa namagciwane (ama-PAMP) ngokusebenzisa ama-receptor wokubona amaphetheni (ama-PRR), okwenza bona kube nezehlakalo ezilandelana eziveza ukuphendula kokuzivikela.
Nanyana sele kuneminyaka ematjhumi yemizamo yokulawula amalwelwe, ukutlhayela kwe-germplasm ejamelana ngokwaneleko kusasele emabhontjisini, njengakwezinye iintjalo ezithengiswako, ngebanga lokujamelana, ukuphila, nokutjhuguluka kwe-pathogen. Ngalokho-ke ukulawulwa kwamalwele kuyinselele khulu begodu kutlhoga iqhinga elihlangeneko, elinemikhakha eminengi elifaka hlangana ukuhlanganiswa kwemikhuba yamasiko, ukulawulwa kwezinto eziphilako, kanye namakhemikhali wokubulala iinkhunta (O'Sullivan et al., 2021). Ukulawulwa kwamakhemikhali kwesikhunta esimhlophe kusebenza khulu ngombana i-fungicides, lokha nayisetjenziswa ngendlela efaneleko nangesikhathi esifaneleko, ingalawula kuhle ukurhatjheka kwesifo, yehlise ubudisi bokutheleleka, begodu yehlise ukulahlekelwa sivuno. Kodwana, ukusetjenziswa khulu nokuthembela khulu kuma-fungicides kungadosela ekuveleni kwemihlobo ejamelanako ye-S. sclerotiorum begodu kube nomthelela omumbi eenlwaneni ezingakaqalwa, ipilo yehlabathi, kanye nekhwalithi yamanzi (Le Cointe et al., 2016; Ceresini et al., 2024). Ngalokho-ke, ukuthola ezinye iindlela ezingalimazi ibhoduluko sekuyinto eqakathekileko.
Ama-polyamine (ama-PA), afana ne-putrescine, i-spermidine, i-spermine, kanye ne-cadaverine, angasebenza njengezinye iindlela ezithembisako zokulwisana namagciwane weentjalo ezithwala ehlabathini, ngaleyindlela ukwehlisa ngokupheleleko nofana ngokwengcenye ukusetjenziswa kwamakhemikhali ayingozi (Nehela et al., 2024; Yi et al., 2024). Eentjalweni eziphezulu, ama-PA abandakanyeka eenkambisweni ezinengi zomzimba ezifaka hlangana, kodwana kungapheleli lapho, ukwahlukana kwamaseli, ukuhlukaniswa, nokuphendula ekugandelelekeni kwe-abiotic ne-biotic (Killiny no-Nehela, 2020). Zingasebenza njengama-antioxidants, zisize ukukhipha imihlobo ye-oksijini esebenzako (ROS), zigcine i-redox homeostasis (Nehela no-Killiny, 2023), zikhuthaze amajini ahlobene nokuzivikela (Romero et al., 2018), zilawule iindlela ezihlukahlukeneko zokugaya ukudla (Nehela no-Killiny, 2023), ziguqule i-enhordogenous kanye nama-hetogenous (Nehela U-Killiny, 2019), wasungula ukujamelana okufunyenweko (i-SAR), begodu ulawula ukusebenzisana kweentjalo nama-pathogen (u-Nehela no-Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). Kuqakathekile ukutjheja bona iindlela ezithileko neendima zama-PA ekuvikeleni iintjalo ziyahluka ngokuya ngomhlobo weentjalo, amagciwane, kanye nobujamo bebhoduluko. I-PA enengi khulu eentjalweni yenziwa nge-biosynthesis kusuka ku-polyamine eqakathekileko L-ornithine (Killiny no-Nehela, 2020).
I-L-ornithine idlala iindima ezinengi ekukhuleni nekuthuthukeni kweentjalo. Ukwenza isibonelo, iimfundo zangaphambilini zitjengise bona elayisini (i-Oryza sativa), i-ornithine ingahlotjaniswa nokubuyiselwa kwe-nitrogen (Liu et al., 2018), isivuno serayisi, ikhwalithi nephunga (Lu et al., 2020), kanye nokuphendula kokugandeleleka kwamanzi (Yang et al., 2000). Ukudlula lapho, ukusetjenziswa kwangaphandle kwe-L-ornithine kukhuphule khulu ukubekezelela isomiso ku-sugar beet (Beta vulgaris) (Hussein et al., 2019) begodu kwehlisa ukugandeleleka kwetshwayi ku-anyanisi (Allium Cepa) (Çavuşoǧlu no-Çavuşoǧlu, 2021) kanye nesitjalo se-cashena (Awda) U-Rocha et al., 2012). Indima engaba khona ye-L-ornithine ekuvikeleni ukugandeleleka kwe-abiotic kungenzeka kungenxa yokuzibandakanya kwayo ekubutheleleni i-proline ezitjalweni eziphathweko. Ukwenza isibonelo, amajini ahlobene nokusebenza kwe-proline, afana ne-ornithine delta aminotransferase (delta-OAT) kanye nama-proline dehydrogenase (ProDH1 kanye ne-ProDH2) amajini, ngaphambilini abikwa bona abandakanyeka ekuvikeleni i-Nicotiana benthamiana kanye ne-Arabidopsis thaliana ngokujamelana ne-non-host Pseudomonas strains (Kumarh, no 2012). Ngakelinye ihlangothi, i-fungal ornithine decarboxylase (ODC) iyatlhogeka ekukhuleni kwe-pathogen (Singh et al., 2020). Ukuqonda i-ODC ye-Fusarium oxysporum f. sp. i-lycopersici nge-host-induced gene silencing (HIGS) ikhuphule khulu ukujamelana kweentjalo zamatamatisi ku-Fusarium wilt (Singh et al., 2020). Kodwana, indima engaba khona yokusetjenziswa kwe-ornithine yangaphandle malungana nokugandeleleka kwezinto eziphilako okufana nama-phytopathogens ayikafundwa kuhle. Okuqakatheke khulu, imiphumela ye-ornithine ekujamelaneni namalwelwe kanye nezinto ezihlobene namakhemikhali wezinto eziphilako nezemvelo zihlala zingakahlolwa.
Ukuzwisisa ubudisi bokutheleleka kwe-S. sclerotiorum kwemidumba kuqakathekile ekuthuthukiseni amaqhinga wokulawula asebenzako. Kulesi sifundo, besihlose ukukhomba indima engaba khona ye-diamine L-ornithine njengesici esiqakathekileko ekuthuthukiseni iindlela zokuzivikela nokujamelana kweentjalo ze-legume ekuthelelekeni kwe-Sclerotinia sclerotiorum. Sicabanga bona, ngaphezu kokuthuthukisa ukuphendula kokuzivikela kweentjalo ezithelelekileko, i-L-ornithine nayo idlala indima eqakathekileko ekugcineni ubujamo be-redox. Siphakamisa bona imiphumela engaba khona ye-L-ornithine ihlotjaniswa nokulawulwa kweendlela zokuzivikela ze-enzymatic nezingasizo ze-enzymatic kanye nokuphazamiseka kwe-fungal pathogenicity/virulence factors kanye namaphrotheyini ahlobene nawo. Ukusebenza kabili kwe-L-ornithine kwenza bona ibe yinto ethembisako yeqhinga elisimemeko lokunciphisa umthelela wesikhunta esimhlophe begodu kukhulise ukujamelana kweentjalo ze-legume ezijayelekileko kileli gciwane elinamandla le-fungal. Imiphumela yesifundo samanje ingasiza ekuthuthukisweni kweendlela ezitjha, ezingalimazi ibhoduluko zokulawula isikhunta esimhlophe nokunciphisa umthelela waso ekukhiqizeni imidumba.
Kulesi sifundo, umhlobo othengiswako we-common bean, i-Giza 3 (Phaseolus vulgaris L. cv. Giza 3), wasetjenziswa njengento yokuhlola. Imbewu enepilo yanikelwa ngomusa mNyango weRhubhululo le-Legume, i-Field Crops Research Institute (FCRI), i-Agricultural Research Center (ARC), e-Egypt. Imbewu emihlanu yatjalwa emabhodweni weplastiki (ububanzi bangaphakathi 35 cm, ukujula 50 cm) ezele ihlabathi ene-S. sclerotiorum ngaphasi kweemeko ze-greenhouse (25 ± 2 °C, umswakama ohlobene 75 ± 1%, 8 h ukukhanya/16 h ubumnyama). Emalangeni ama-7 ukuya keli-10 ngemva kokutjala (DPS), iintjalo zancipha ukobana kutjhiye iintjalo ezimbili kwaphela ezikhula ngokufanako kanye namakari amathathu avuleke ngokupheleleko embizeni ngayinye. Zoke iintjalo ezifakwe emabhodweni beziniselwa kanye ngemva kweemveke ezimbili begodu zivundiswe qobe nyanga ngezinga eliphakanyisiweko lomhlobo onikelweko.
Ukulungiselela i-500 mg/L ye-L-ornithinediamine (eyaziwa nangokuthi (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Germany), i-50 mg yathoma incibilikiswa ngaphakathi kwe-100 mL yamanzi ahlanjululweko. Isixazululo sesitokwe sabe sesihlanjululwa begodu sasetjenziswa emihlolweni elandelako. Kafitjhani, iinthombe ezisithandathu ze-L-ornithine (12.5, 25, 50, 75, 100, kanye ne-125 mg/L) zahlolwa nge-vitro. Ukungezelela, amanzi ahlanjululweko asetjenziswe njengokulawula okumbi (i-Mock) begodu i-fungicide yokuthengisa “Rizolex-T” 50% wettable powder (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governate, Egyp) yasetjenziswa njengokulawula okuhle. I-fungicide yokuthengisa “i-Rizolex-T” yahlolwa nge-vitro ngamazinga amahlanu (2, 4, 6, 8 kanye ne-10 mg/L).
Amasampula weziqu zebhontjisi ezijayelekileko kanye nama-pods atjengisa amatshwayo ajayelekileko wesikhunta esimhlophe (izinga lokutheleleka: 10-30%) abuthelelwe emaplasini wokuthengisa. Nanyana inengi lezinto zezitjalo ezithelelekileko zikhonjwe ngokwemihlobo/umhlobo (umhlobo wezokuthengisa othelelekako i-Giza 3), ezinye, khulukhulu lezo ezitholwe emakethe yendawo, beziyimihlobo engaziwako. Iinsetjenziswa ezithelelekeko ezibuthelelweko zathoma ngokubulawa amagciwani nge-0.5% ye-sodium hypochlorite solution imizuzu emi-3, bese zahlanzwa kanengi ngamanzi ahlanjululweko begodu zasulwa zomile ngephepha lokuhlunga elihlanzekileko ukususa amanzi amanengi. Izitho ezithelelekileko zabe seziqunywa zaba ziinquntu ezincani ukusuka esicutshini esiphakathi (hlangana nezicubu eziphilileko nezinesifo), zakhuliswa ku-potato dextrose agar (PDA) medium begodu zafukamela ku-25 ± 2 °C ngomjikelezo we-12 h wokukhanya/12 h wobumnyama amalanga ama-5 ukukhuthaza ukwakheka kwe-sclerotia. Indlela ye-mycelial tip nayo yasetjenziselwa ukuhlanza ama-fungal isolates avela emasikweni ahlanganisiweko nofana angcolileko. I-fungal isolate ehlanjululweko yathoma ukubonwa ngokuya ngobujamo bayo bezamasiko bese kwaqinisekiswa bona yi-S. sclerotiorum ngokuya ngeempawu ezibonakala ngesibonakhulu. Ekugcineni, woke ama-isolate ahlanjululweko ahlolwa ukutheleleka emhlobeni webhontjisi ovamileko we-Giza 3 ukuhlangabezana nama-postulate ka-Koch.
Ukungezelela, i-S. sclerotiorum ehlasela khulu (i-isolate #3) yaqinisekiswa godu ngokuya ngokulandelana kwe-internal transcribed spacer (ITS) njengombana kuchaziwe ngu-White et al., 1990; U-Baturo-Ciesniewska et al., 2017. Kafitjhani, ama-isolate akhuliswa ngaphakathi komhluzi we-potato dextrose (PDB) begodu akhuliswa ku-25 ± 2 °C amalanga ama-5-7. I-mycelium ye-fungal yabuthelelwa, yahlungwa nge-cheesecloth, yahlanzwa kabili ngamanzi ahlanzekileko, begodu yomiswe ngephepha lokuhlunga elihlanzekileko. I-DNA ye-genomic yahlukaniswa kusetjenziswa i-Quick-DNATM Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Indawo ye-ITS rDNA yakhuliswa kusetjenziswa i-primer pair ethileko i-ITS1/ITS4 (TCCGTAGGTGAACCTGCGG TCCTCCGCTTATTGATATGC; ubukhulu obulindelweko: 540 bp) ( Baturo-Ciesniewska et al., 2017 ). Imikhiqizo ye-PCR ehlanjululweko yathunyelwa ukulandelana (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). Ukulandelana kwe-ITS rDNA kwalandelana ngokusebenzisa indlela yokulandelana ye-Sanger. Ukulandelana kwemibuzo ehlanganisiweko kwabe sekuqathaniswa nedatha yakamuva ku-GenBank kanye ne-National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/) kusetjenziswa isofthiwe ye-BLASTn. Ukulandelana kwemibuzo kwafaniswa nezinye iinhlobo/ama-isolate we-S. sclerotiorum ama-20 atholwe kudatha yakamuva ku-NCBI GenBank (Ithebula elingezelelweko S1) kusetjenziswa i-ClustalW ku-Molecular Evolutionary Genetics Analysis Package (MEGA-11; version 11) (Kumar et al., 2024). Ukuhlaziywa kokuziphendukela kwemvelo kwenziwa kusetjenziswa indlela ye-maximum likelihood kanye nemodeli yokufaka i-nucleotide ebuyiselwa emuva ngesikhathi (u-Nei no-Kumar, 2000). Umuthi onethuba eliphezulu lokuloga uyatjengiswa. Umuthi wokuthoma wokusesha kwe-heuristic ukhethwa ngokukhetha umuthi one-log-likelihood ephezulu hlangana nomuthi wokuhlanganisa umakhelwana (NJ) (Kumar et al., 2024) kanye nomuthi we-maximum parsimony (MP). Umuthi we-NJ wakhiwe kusetjenziswa i-pairwise distance matrix eyabalwa kusetjenziswa imodeli ejwayelekileko yesikhathi (u-Nei no-Kumar, 2000).
Umsebenzi wokulwa namagciwani we-L-ornithine kanye ne-bactericide “Rizolex-T” kwatholwa nge-vitro ngendlela ye-agar diffusion. Indlela: Thatha inani elifaneleko lesisombululo se-L-ornithine (500 mg/L) bese usihlanganisa kuhle ne-10 ml ye-PDA nutrient medium ukulungiselela iinsombululo ezinamazinga wokugcina we-12.5, 25, 50, 75, 100 kanye ne-125 mg/L, ngokulandelana. Amazinga amahlanu we-fungicide “Rizolex-T” (2, 4, 6, 8 kanye ne-10 mg/L) namanzi ahlanjululweko asetjenziswe njengokulawula. Ngemva kobana i-medium iqinile, i-mycelial plug esanda kulungiswa ye-Sclerotinia sclerotiorum culture, engu-4 mm ububanzi, yadluliselwa phakathi kwe-Petri dish begodu yakhuliswa ku-25±2°C bekwaba kulapho i-mycelium ivala yoke i-Petri dish yokulawula, ngemva kwalokho ukukhula kwe-fungal kwarekhodwa. Bala iphesenteji yokukhandela ukukhula kwe-S. sclerotiorum ngokusebenzisa i-equation 1:
Ukuhlolwa kwaphindwa kabili, ngokuphindaphinda okusithandathu kwezinto eziphilako kweqembu ngalinye lokulawula/lokuhlola kanye namabhodo amahlanu (izitjalo ezimbili ngembiza ngayinye) ekuphindaphindweni ngalinye kwezinto eziphilako. Ukuphindaphinda ngamunye kwezinto eziphilako kwahlaziywa kabili (ukuphindaphinda okubili kwezobuchwepheshe) ukuqinisekisa ukunemba, ukuthembeka nokukhiqizwa kabutjha kwemiphumela yokuhlola. Ukungezelela, ukuhlaziywa kwe-probit regression kwasetjenziselwa ukubala i-half-maximum inhibitory concentration (IC50) kanye ne-IC99 (Prentice, 1976).
Ukuhlola amandla we-L-ornithine ngaphasi kweemeko ze-greenhouse, kwenziwa iinhlahlubo ezimbili ezilandelanako. Kafitjhani, amabhodlelo azaliswa ngehlabathi elihlanjululweko (3:1) begodu afakwa isiko elisanda kulungiswa le-S. sclerotiorum. Kokuthoma, i-isolate ehlasela khulu ye-S. sclerotiorum (i-isolate #3) yakhuliswa ngokusika i-sclerotium yinye ngesigamu, yayibeka iqale phasi ku-PDA bese ifukamela ku-25°C ebumnyameni obungapheliko (24 h) amalanga ama-4 ukukhuthaza ukukhula kwe-mycelial. Amapulagi amane we-agar anobubanzi obuyi-5 mm athathwa emphethweni ophambili begodu afakwa nge-100 g yomxube ohlanzekileko wekoroyi ne-rice bran (1:1, v/v) begodu woke amaflaski afakwa ku-25 ± 2 °C ngaphasi komjikelezo we-12 h wokukhanya/12 h wobumnyama amalanga ama-5 ukuvuselela ukwakheka kwe-sclerotia. Okuqukethwe kiwo woke amaflaski kwahlanganiswa kuhle ukuqinisekisa ukufana ngaphambi kokufaka ihlabathi. Bese, i-100 g yengxube ye-bran yekoloni yefakwa embizeni ngayinye ukuqinisekisa ukugxila okuhlala njalo kwamagciwane. Iimbiza ezigonyweko zaniselwa ukukhula kwe-fungal begodu zafakwa ebujameni be-greenhouse amalanga ali-7.
Kwabe sekutjalwa imbewu emihlanu yomhlobo we-Giza 3 embizeni ngayinye. Emabhodweni aphathwe nge-L-ornithine kanye ne-fungicide i-Rizolex-T, imbewu ehlanjululweko yathoma ukuthambisa ama-awa amabili esixazululweni samanzi samakhompawundi amabili nge-IC99 yokugcina engaba ma-250 mg/L kanye nama-50 mg/L, ngokulandelana, bese yomiswa emoyeni i-awa elilodwa ngaphambi kokutjalwa. Ngakelinye ihlangothi, imbewu beyifakwe emanzini ahlanjululweko njengesilawuli esingakalungi. Ngemva kwamalanga ali-10, ngaphambi kokuthelelela kokuthoma, iintjalo zanciphiswa, kwasala iintjalo ezimbili ezihlanzekileko embizeni ngayinye. Ukungezelela, ukuqinisekisa ukutheleleka nge-S. sclerotiorum, iinqubu zesitjalo samabhontjisi ezisesigabeni esifanako sokukhula (amalanga ali-10) zasikwa eendaweni ezimbili ezihlukileko kusetjenziswa i-scalpel ehlanjululweko begodu pheze i-0.5 g yomxube we-bran ehlalako yafakwa esilondeni ngasinye, kwalandela umswakama ophakemeko ukukhuthaza ukutheleleka nokutheleleka kwazo zoke iintjalo ezithelelekako. Iintjalo zokulawula bezilimele ngokufanako begodu inani elifanako (0.5 g) lomxube we-bran ongakahlanzwa, ongakafakwa esilondani begodu wagcinwa ngaphasi komswakama omkhulu ukulingisa ibhoduluko lokuthuthukisa amalwelwe nokuqinisekisa ukunzinza hlangana namaqembu wokwelatjhwa.
Indlela yokwelapha: Iintjalo zamabhontjisi zathelelwa nge-500 ml yesisombululo samanzi se-L-ornithine (250 mg/l) nofana i-fungicide i-Rizolex-T (50 mg/l) ngokuthelelela ihlabathi, bese ukwelapha kwaphindwa kathathu ngesikhatjhana samalanga ali-10. Iinlawuli eziphathwe nge-placebo zathelelelwa ngamanzi ahlanjululweko angama-500 ml. Zoke iindlela zokwelapha zenziwa ngaphasi kweemeko ze-greenhouse (25 ± 2°C, 75 ± 1% umswakama, kanye nesikhathi sokukhanya se-8 h ukukhanya/16 h ubumnyama). Zoke iimbiza beziniselwa qobe ngemva kwamaviki amabili begodu ziphathwe qobe nyanga ngesivundisi esilinganiselweko se-NPK (20-20-20, esine-3.6% yesulfure kanye nama-microelements we-TE; i-Zain Seeds, e-Egypt) ngezinga elingu-3-4 g/l ngokufafaza ngamakari ngokuya ngeencomo zomhlobo othileko kanye nemilayo yomkhiqizi. Ngaphandle kobana kutjhiwo ngenye indlela, amakari avuthiweko anwebileko (amakari wesibili nowesithathu ukusuka phezulu) abuthelelwe ekuphindaphindweni ngalinye kwezinto eziphilako ngemva kwama-72 h ngemva kokwelatjhwa (hpt), ahlanganiswa, ahlanganiswa begodu agcinwa ku--80 °C ukuhlaziywa okuragela phambili okufaka hlangana, kodwana kungapheleli lapho, ukutholakala kwe-histochemical ye-in situ yeenkomba zokugandeleleka kwe-oxidative, i-peroxidative peromatic, kanye ne-enzymatic enzyme ama-antioxidant angasiwo ama-enzayimu kanye nokuvezwa kwezakhi zofuzo.
Ubukhulu bokutheleleka kwesikhunta esimhlophe bebuhlolwa qobe veke ngemva kwamalanga ama-21 ngemva kokugonywa (dpi) kusetjenziswa isikali esingu-1 ukuya ku-9 (Ithebula elingezelelweko S2) elisekelwe esikalini se-Petzoldt no-Dickson (1996) esitjhugululwe ngu-Teran et al. (2006). Kafitjhani, iinqubu namagatja weentjalo zebhontjisi ahlolwa ukuthoma ngesikhathi sokugonywa ukulandela ukuragela phambili kwezilonda eduze nama-internode nama-node. Ibanga lesilonda ukusuka endaweni yokugonywa ukuya endaweni ekude khulu eceleni kwesiqu nofana igatja lalinganiswa begodu kwanikelwa isikolo esingu-1-9 ngokuya ngendawo yesilonda, lapho (1) kutjengisa bona akukho ukutheleleka okubonakalako eduze nendawo yokugonywa begodu (2-9) kutjengisa ukwanda kancanikancani kobukhulu besilonda nokuragela phambili ngama-node/ama-node (Ithebula S2). Ubukhulu bokutheleleka kwesikhunta esimhlophe kwatjhugululwa kwaba maphesenti kusetjenziswa ifomula 2:
Ukungezelela, indawo engaphasi kwejika lokuragela phambili kwesifo (AUDPC) yabalwa kusetjenziswa ifomula (u-Shaner no-Finney, 1977), esanda kutjhugululwa ukubola okumhlophe kwebhontjisi evamileko (u-Chauhan et al., 2020) kusetjenziswa i-equation 3:
Lapho i-Yi = ubukhulu besifo ngesikhathi se-ti, i-Yi+1 = ubukhulu besifo ngesikhathi esilandelako ti+1, i-ti = isikhathi sokulinganisa kokuthoma (ngamalanga), i-ti+1 = isikhathi sokulinganisa okulandelako (ngamalanga), n = inani elipheleleko lamaphuzu wesikhathi nofana amaphuzu wokutjheja. Amapharamitha wokukhula kwesitjalo samabhontjisi afaka hlangana ukuphakama kwesitjalo (cm), inani lamagatja ngesitjalo ngasinye, kanye nenani lamakari ngesitjalo ngasinye atlolwa qobe veke amalanga ama-21 kiwo woke amaphindaphindo wezinto eziphilako.
Ekuphindaphindweni ngalinye kwezinto eziphilako, amasampula wamakari (amakari wesibili newesithathu akhule ngokuzeleko ukusuka phezulu) abuthelelwa ngelanga lama-45 ngemva kokwelatjhwa (amalanga ali-15 ngemva kokwelatjhwa kokugcina). Iphindaphindo ngayinye yezinto eziphilako beyinamabhodo amahlanu (iintjalo ezimbili ngembiza ngayinye). I-500 mg yezicubu eziphukileko yasetjenziselwa ukukhipha ama-pigment we-photosynthetic (i-chlorophyll a, i-chlorophyll b kanye nama-carotenoids) kusetjenziswa i-80% ye-acetone ku-4 °C ebumnyameni. Ngemva kwama-awa ama-24, amasampula ahlanganiswa begodu i-supernatant yabuthelelwa ukuthola i-chlorophyll a, i-chlorophyll b kanye nokuqukethwe kwe-carotenoid ngokusebenzisa i-UV-160A spectrophotometer (i-Shimadzu Corporation, Japan) ngokuya ngendlela ye-(Lichtenthaler, 1987) ngokulinganisa amagagasi amathathu ahlukileko (A470, A646 kanye no-A663 nm). Ekugcineni, okuqukethwe kwemibala ye-photosynthetic kwabalwa kusetjenziswa amafomula alandelako 4-6 achazwe ngu-Lichtenthaler (1987).
Ngemva kwama-awa ama-72 ngemva kokwelatjhwa (hpt), amakari (amakari wesibili newesithathu akhule ngokuzeleko ukusuka phezulu) abuthelelwa kiyo yoke i-biological replication ukuthola indawo ye-histochemical ye-hydrogen peroxide (H2O2) kanye ne-superoxide anion (O2•−). Iphindaphindo ngayinye yezinto eziphilako beyinamabhodo amahlanu (iintjalo ezimbili ngembiza ngayinye). Ukuphindaphinda ngamunye kwezinto eziphilako kwahlaziywa kabili (ukuphindaphinda okubili kwezobuchwepheshe) ukuqinisekisa ukunemba, ukuthembeka nokukhiqizwa kabutjha kwendlela. I-H2O2 ne-O2•− zatholwa kusetjenziswa i-0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Germany) nofana i-nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Germany), ngokulandelana, ngokulandela iindlela ezichazwe ngu-Romero-Puertas et al. (2004) no-Adam et al. (1989) ngokutjhugulula okuncani. Ukuthola indawo ye-histochemical ye-H2O2 endaweni, amaphetjhana afakwa nge-vacuum nge-0.1% DAB ku-10 mM Tris buffer (pH 7.8) bese afakwa emazingeni womtjhiso wegumbi ekukhanyeni isikhathi esingaba mizuzu ema-60. Amaphetjhana ahlanjululwe nge-0.15% (v/v) TCA ku-4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) bese avezwa ekukhanyeni bekwaba mnyama. Ngokufanako, amavalvu bekangene nge-vacuum nge-10 mM ye-potassium phosphate buffer (pH 7.8) ene-0.1 w/v % HBT yokutholakala kwe-histochemical ye-O2•− in situ. Amaphetjhana afukanywa emkhanyweni emazingeni womtjhiso wegumbi isikhathi esingaba mizuzu ema-20, bese ahlanjululwa njengaphezulu, bese akhanyiswa bekwaba kulapho kuvela khona amabala ahlaza okwesibhakabhaka/abomvu. Ukuqina kombala onsundu (njengesibonisi se-H2O2) nofana ohlaza satjani (njengesibonisi se-O2•−) kwahlolwa kusetjenziswa inguqulo ye-Fiji yephakheji yokucubungula iinthombe i-ImageJ (http://fiji.sc; ifinyelelwe mhlazi-7 kuNtaka 2024).
I-Malondialdehyde (i-MDA; njengetshwayo lokutjhuguluka kwamafutha) yatholakala ngokuya ngendlela ka-Du no-Bramlage (1992) ngokutjhugulula okuncani. Amakari avela ekuphindaphindweni kwezinto eziphilako (amakari wesibili newesithathu akhule ngokuzeleko avela phezulu) abuthelelwa ngemva kwama-awa ama-72 ngemva kokwelatjhwa (hpt). Ukuphindaphinda ngamunye kwezinto eziphilako bekufaka hlangana amabhodo amahlanu (iintjalo ezimbili ngembiza ngayinye). Ukuphindaphinda ngamunye kwezinto eziphilako kwahlaziywa kabili (ukuphindaphinda okubili kwezobuchwepheshe) ukuqinisekisa ukunemba, ukuthembeka nokukhiqizwa kabutjha kwendlela. Kafitjhani, i-0.5 g yezicubu zamakari agayiweko yasetjenziselwa ukukhipha i-MDA nge-20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) ene-0.01% ye-butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). Okuqukethwe kwe-MDA ngaphakathi kwe-supernatant kwabe sekuhlolwa ngokulinganisa ukumuncwa ku-532 no-600 nm kusetjenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, Japan) bese kuvezwa njenge-nmol g−1 FW.
Ukuhlolwa kwama-antioxidants angasiwo we-enzyme kanye nama-enzyme, amakari (amakari wesibili newesithathu akhule ngokuzeleko ukusuka phezulu) abuthelelwa ekuphindaphindweni kwezinto eziphilako nge-72 h ngemva kokwelatjhwa (hpt). Iphindaphindo ngayinye yezinto eziphilako beyinamabhodo amahlanu (iintjalo ezimbili ngembiza ngayinye). Isampula ngayinye yebhayoloji yahlaziywa kabili (amasampula amabili wezobuchwepheshe). Amakari amabili asilwa nge-liquid nitrogen begodu asetjenziselwa ukunquma ama-antioxidants we-enzymatic ne-non-enzymatic, ama-amino acid woke, okuqukethwe kwe-proline, ukuvezwa kwezakhi zofuzo, kanye nokulinganisa kwe-oxalate.
Amaphenolikhi ancibilikako woke ahlolwa kusetjenziswa i-Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) ngokutjhugululwa okuncani kwendlela echazwe ngu-Kahkonen et al. (1999). Kafitjhani, pheze i-0.1 g yezicubu zamakari ezihlanganisiweko zakhishwa nge-20 ml ye-80% ye-methanol ebumnyameni ama-awa ama-24 begodu okuphezulu kwabuthelelwa ngemva kokukhipha. I-0.1 ml yesampula extract yahlanganiswa ne-0.5 ml ye-Folin-Ciocalteu reagent (10%), yanyakaziswa ama-30 s begodu yatjhiywa ebumnyameni imizuzu emi-5. Bese i-0.5 ml yesixazululo se-20% ye-sodium carbonate (Na2CO3; i-Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) yafakwa etjhubeni ngalinye, yahlanganiswa kuhle begodu yafukanywa emazingeni womtjhiso wegumbi ebumnyameni i-1 h. Ngemva kokufukamela, ukumuncwa komxube wokuphendula kwalinganiswa ku-765 nm kusetjenziswa i-UV-160A spectrophotometer (i-Shimadzu Corporation, Japan). Ukugxila kwama-phenoli ancibilikako woke eensetjenzisweni zesampula kwatholwa kusetjenziswa i-gallic acid calibration curve (Fisher Scientific, Hampton, NH, USA) begodu kwavezwa njengama-milligrams we-gallic acid elingana ngegremu yesisindo esitjha (mg GAE g-1 isisindo esitjha).
Okuqukethwe okupheleleko kwe-flavonoid okuncibilikako kwatholakala ngokuya ngendlela ka-Djeridane et al. (2006) ngokutjhugulula okuncani. Kafitjhani, i-0.3 ml ye-methanol extract engenhla yahlanganiswa ne-0.3 ml ye-5% yesixazululo se-aluminium chloride (AlCl3; Fisher Scientific, Hampton, NH, USA), yavuselelwa ngamandla bese yafukanywa emazingeni womtjhiso wegumbi imizuzu emi-5, kwalandela ukufakwa kwe-0.3 ml ye-0.3 ml yesixazululo se-potasiteum 0% ye-potasite. (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), ihlanganiswe kuhle begodu yafukamela emazingeni womtjhiso wegumbi imizuzu ema-30 ebumnyameni. Ngemva kokufukamela, ukumuncwa komxube wokuphendula kwalinganiswa ku-430 nm kusetjenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, Japan). Ukugxila kwama-flavonoids apheleleko ancibilikako eensetjenzisweni zesampula kwatholakala kusetjenziswa i-rut calibration curve (TCI America, Portland, OR, USA) bese kuvezwa njengama-milligrams we-rutin okulingana ngegremu yesisindo esitjha (mg RE g-1 isisindo esitjha).
Inani elipheleleko le-amino acid yamahlamvu wamabhontjisi latholwa kusetjenziswa i-ninhydrin reagent (i-Thermo Scientific Chemicals, Waltham, MA, USA) ngokuya ngendlela ephakanyiswe ngu-Yokoyama no-Hiramatsu (2003) begodu yatjhugululwa ngu-Sun et al. (2006). Kafitjhani, i-0.1 g yezicubu eziphasi yakhutjhwa nge-pH 5.4 buffer, begodu i-200 μL ye-supernatant yahlanganiswa ne-200 μL ye-ninhydrin (2%) kanye ne-200 μL ye-pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), yafakwa emanzini abilayo iminithi, bese yalinganiswa ku-580 nm kusetjenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, Japan). Ngakelinye ihlangothi, i-proline beyinqunywa ngendlela ka-Bates (u-Bates et al., 1973). I-Proline yakhishwa nge-3% ye-sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) begodu ngemva kokukhipha, i-0.5 ml ye-supernatant yahlanganiswa ne-1 ml ye-glacial acetic acid (Fisher Scientific, Hampton, NH, USA) kanye ne-ninhydrin reagent, yafakwa ku-90°C, begodu yapholiswa kulinganiswa ku-520 nm kusetjenziswa i-spectrophotometer efanako naleyo engenhla. Ama-amino acid wamahhala woke kanye ne-proline emaqabungeni atholakala ngokusebenzisa i-glycine ne-proline calibration curves (Sigma-Aldrich, St Louis, MO, USA), ngokulandelana, begodu ivezwa njenge-mg/g yesisindo esitjha.
Ukuthola umsebenzi wama-enzyme we-antioxidant, pheze i-500 mg yezicubu ezihlangeneko zakhishwa nge-3 ml ye-50 mM Tris buffer (pH 7.8) ene-1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) kanye ne-7.5% polylypyrrolyone (VPP; PH; I-Sigma-Aldrich, St. Louis, MO, USA), i-centrifuged ku-10,000 × g imizuzu ema-20 ngaphasi kwefriji (4 °C), begodu i-supernatant (i-enzyme extract engakalungiswa) yaqoqwa (El-Nagar et al., 2023; Osman et al., 2023). I-Catalase (i-CAT) yabe isiphendulwa nge-2 ml ye-0.1 M ye-sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) kanye ne-100 μl ye-269 mM H2O2 solution ukuthola umsebenzi wayo we-enzymatic ngokuya ngendlela ka-Aebi (1984) nge-Ellight-Nagar et al. 2023; Osman et al., 2023). Umsebenzi we-enzymatic we-Guaiacol-dependent peroxidase (POX) watholakala kusetjenziswa indlela ka-Harrach et al. (2009). (2008). I-Portland, OR, USA) kanye ne-100 μl ye-12 mM H2O2. Indlela leyo yatjhugululwa kancani ukusuka ku-(El-Nagar et al., 2023; Osman et al., 2023). Ukuhlolwa kwenziwa ngemva kokuphendula nge-3 ml yesisombululo se-catechol (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) esanda kulungiswa ku-0.1 M phosphate buffer (pH 6.0). Umsebenzi we-CAT walinganiswa ngokutjheja ukubola kwe-H2O2 ku-240 nm (A240), umsebenzi we-POX walinganiswa ngokutjheja ukwanda kokumuncwa ku-436 nm (A436), begodu umsebenzi we-PPO walinganiswa ngokurekhoda ukutjhuguluka kokumuncwa ku-495 nm (A495) qobe ngemizuzu ema-30 kusetjenziswa i-UV-phorometer (Shimadzu, Japan).
I-RT-PCR yesikhathi sangempela yasetjenziselwa ukuthola amazinga wokutloliswa kwamajini amathathu ahlobene nama-antioxidant, kufaka hlangana i-peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), i-superoxide dismutase (PvSOD; i-GenBank Accession No. XM_0686395), kanye ne-glutase reductase (GenBank; Inombolo yokungena KY195009.1), emakarini webhontjisi (amaqabunga wesibili newesithathu akhule ngokuzeleko ukusuka phezulu) ngemva kwama-awa ama-72 ngemva kokwelatjhwa kokugcina. Kafitjhani, i-RNA yahlukaniswa kusetjenziswa i-Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) ngokuya ngephrothokholi yomkhiqizi. Bese, i-cDNA yenziwa kusetjenziswa i-TOP script™ cDNA Synthesis Kit ngokuya kwemiyalo yomkhiqizi. Ukulandelana kwama-primer wamajini amathathu angaphezulu kufakwe kuThebuli yokuNgezelela i-S3. I-PvActin-3 (inomboro yokungena ye-GenBank: XM_068616709.1) yasetjenziswa njengejini lokugcina indlu begodu ukuvezwa kwejini okuhlobeneko kwabalwa kusetjenziswa indlela ye-2-ΔΔCT (u-Livak no-Schmittgen, 2001). Ukuzinza kwe-actin ngaphasi kokugandeleleka kwe-biotic (ukusebenzisana okungahambisaniko hlangana nama-legumes avamileko kanye ne-anthracnose fungus i-Colletotrichum lindemuthianum) kanye nokugandeleleka kwe-abiotic (isomiso, itswayi, amazinga womtjhiso aphasi) kwatjengiswa (Borges et al., 2012).
Ekuthomeni senze ukuhlaziywa kwe-genome-wide in silico yamaphrotheyini we-oxaloacetate acetylhydrolase (OAH) ku-S. sclerotiorum sisebenzisa ithulusi le-protein-protein BLAST (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Kafitjhani, sisebenzise i-OAH evela ku-Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; GenBank accession number XP_040799428.1; 342 amino acids) kanye ne-Penicillium lagena (PlOAH; taxide: 942181; GenBank accession number; XP_056833920.1; 316 amino acids) njengokulandelana kwemibuzo ukudweba iphrotheyini efanako ku-S. sclerotiorum (taxide: 5180). I-BLASTp yenziwa ngokujamelana nedatha ye-S. sclerotiorum genome esanda kutholakala ku-GenBank kuwebhusayithi ye-National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Ukungezelela, isakhi se-OAH esibikezelweko esivela ku-S. sclerotiorum (SsOAH) kanye nokuhlaziywa kokuziphendukela kwemvelo kanye nomuthi we-phylogenetic we-AfOAH ovela ku-A. fijiensis CBS 313.89 kanye ne-PlOAH evela ku-P. lagena kwaphethwa ngokusebenzisa indlela yokuba khona okuphezulu ku-MEGA11 (Tamura et al., 2021) kanye nemodeli esekelwe ku-JTT matriones (JTT et al. al., 1992). Umuthi we-phylogenetic wahlanganiswa nokuhlaziywa kokulandelana kwamaphrotheyini wawo woke amajini we-OAH abikezelweko (SsOAH) avela ku-S. sclerotiorum kanye nokulandelana kwemibuzo kusetjenziswa i-Constraint-Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools//cobalt_cobalt) Agarwala, 2007). Ukungezelela, ukulandelana kwe-amino acid ehambelana kuhle khulu kwe-SsOAH evela ku-S. sclerotiorum kwahambisana nokulandelana kwemibuzo (AfOAH ne-PlOAH) (Larkin et al., 2007) kusetjenziswa i-ClustalW (http://www.genome.jp/tools-bin/clustalw), begodu iindawo ezilondoloziweko zalungiswa ngokusebenzisa i-ESP ithuluzi (inguqulo 3.0; https://espript.ibcp.fr/ESPript/ESPript/index.php).
Ukudlula lapho, iindawo ezijameleko ezisebenzako ezibikezelweko kanye neendawo ezilondoloziweko ze-S. sclerotiorum SsOAH zahlukaniswa ngokusebenzisana emindenini ehlukileko kusetjenziswa ithuluzi le-InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Ekugcineni, ukumodela isakhiwo esinemikhakha emithathu (3D) ye-SsOAH ebikezelweko ye-S. sclerotiorum kwenziwa kusetjenziswa i-Protein Homology/Analogy Recognition Engine (Phyre2 server version 2.0; http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cid=index) (Kelley et al., 2015) begodu yaqinisekiswa kusetjenziswa iseva ye-SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). Izakhiwo ezibikezelweko ezinemikhakha emithathu (ifomethi ye-PDB) zabonwa ngokusebenzisana kusetjenziswa iphakheji ye-UCSF-Chimera (inguqulo 1.15; https://www.cgl.ucsf.edu/chimera/ ) (Pettersen et al., 2004).
I-PCR ye-Quantitative real-time fluorescence yasetjenziselwa ukuthola izinga lokutloliswa kwe-oxaloacetate acetylhydrolase (SsOAH; inomboro yokungena ye-GenBank: XM_001590428.1) ku-mycelia ye-Sclerotinia sclerotiorum. Kafitjhani, i-S. sclerotiorum yafakwa ngaphakathi kweflaski ene-PDB begodu yafakwa ngaphakathi kwe-incubator (imodeli: I2400, New Brunswick Scientific Co., Edison, NJ, USA) ku-25 ± 2 °C ama-awa ama-24 nge-150 rpm begodu ebumnyameni obuhlala njalo (ama-awa ama-24) ukuvuselela ukukhula kwe-mycelial. Ngemva kwalokho, amaseli alatjhwa nge-L-ornithine kanye ne-fungicide i-Rizolex-T emazingeni wokugcina we-IC50 (cishe ama-40 kanye nama-3.2 mg/L, ngokulandelana) bese akhuliswa amanye ama-awa ama-24 ngaphasi kweemeko ezifanako. Ngemva kokufukamela, amasiko ahlanganiswa nge-2500 rpm imizuzu emi-5 begodu i-supernatant (i-fungal mycelium) yabuthelelwa ukuhlaziywa kokuvezwa kwezakhi zofuzo. Ngokufanako, i-fungal mycelium yabuthelelwa nge-0, 24, 48, 72, 96, kanye ne-120 h ngemva kokutheleleka ukusuka eentjalweni ezithelelekileko ezakhiwe isikhunta esimhlophe kanye ne-mycelium yekoteni phezu kwezicubu ezithelelekileko. I-RNA yakhishwa ku-fungal mycelium bese i-cDNA yenziwa njengombana kuchaziwe ngehla. Ukulandelana kwe-primer ye-SsOAH kufakwe kuThebuli yokuNgezelela i-S3. I-SsActin (inomboro yokungena ye-GenBank: XM_001589919.1) yasetjenziswa njengejini yokugcina indlu, begodu ukuvezwa kwejini okuhlobeneko kwabalwa kusetjenziswa indlela ye-2-ΔΔCT (u-Livak no-Schmittgen, 2001).
I-oxalic acid yatholwa emhluzweni we-potato dextrose (PDB) kanye namasampula weentjalo ane-fungal pathogen Sclerotinia sclerotiorum ngokuya ngendlela ka-Xu no-Zhang (2000) ngokutjhugululwa okuncani. Kafitjhani, i-S. sclerotiorum isolates yafakwa ngaphakathi kwamaflaski ane-PDB bese yakhuliswa ngaphakathi kwe-incubator (imodeli I2400, New Brunswick Scientific Co., Edison, NJ, USA) nge-150 rpm ku-25 ± 2 °C amalanga ama-3-5 ebumnyameni obuhlala njalo (24 h) ukukhuthaza ukukhula kwe-mycelial. Ngemva kokufukamela, isiko le-fungal lathoma ngokuhlungwa ngephepha lokuhlunga le-Whatman #1 bese lahlungwa nge-2500 rpm imizuzu emi-5 ukususa i-mycelium eseleko. I-supernatant yaqoqwa begodu yagcinwa ku-4°C ukobana kuragele phambili nokutholakala kwe-oxalate. Ukulungiselela amasampula weentjalo, pheze i-0.1 g yeengcezu zezicubu zesitjalo zakhutjhwa kathathu ngamanzi ahlanjululweko (2 ml isikhathi ngasinye). Amasampula abese atjhugululwa nge-2500 rpm isikhathi esingaba mizuzu emi-5, i-pernatant yahlungwa ngomile ngephepha lokuhlunga le-Whatman No. 1 bese yabuthelelwa ukuhlaziywa okungeziweko.
Ukuhlaziywa kwe-oxalic acid, ingxube yokusabela yalungiswa ngetjhubhu yengilazi evaliweko ngokulandelana okulandelako: 0.2 ml yesampula (nofana i-PDB culture filtrate nofana isisombululo se-oxalic acid ejwayelekileko), 0.11 ml ye-bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburghburgh, PA, PA, Ml 1919), Ml. i-sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) kanye ne-0.176 ml ye-100 mM potassium dichromate (K2Cr2O7; amakhemikhali we-TCI, i-Portland, OR, USA), bese isisombululo sahlanjululwa saba 48 mM ngamanzi ahlanjululweko begodu sahlanganiswa ngamandla. msinyana ifakwe ebhadini lamanzi elingu-60 °C. Ngemva kwemizuzu eli-10, ukusabela kwamiswa ngokufaka i-0.5 ml yesixazululo se-sodium hydroxide (NaOH; 0.75 M). Ukumuncwa (A600) komxube wokuphendula kwalinganiswa ku-600 nm kusetjenziswa i-UV-160 spectrophotometer (Shimadzu Corporation, Japan). I-PDB namanzi ahlanjululweko asetjenziswe njengezilawuli zokulinganisa ama-filtrate wamasiko namasampula weentjalo, ngokulandelana. Ukugxila kwe-oxalic acid kuma-culture filtrate, okuvezwe njengama-micrograms we-oxalic acid nge-mililitha ye-PDB medium (μg.mL−1), begodu nasemaqabunga akhiqizwako, okuvezwe njengama-micrograms we-oxalic acid nge-gram yesisindo esitjha (μg.g−1 FW), kwatholakala kusetjenziswa i-oxalic acid cuve (The Firmors Amakhemikhali wezeSayensi, Waltham, MA, USA).
Kuso soke isifundo, zoke iimhloliso zakhiwe ngendlela engakahleleki ngokupheleleko (i-CRD) eneziphindaphindo ezisithandathu zezinto eziphilako ngokwelapha kanye namabhodo amahlanu ngokuphindaphinda kwezinto eziphilako (izitjalo ezimbili ngembiza ngayinye) ngaphandle kobana kutjhiwo ngenye indlela. Ukuphindaphinda kwezinto eziphilako kwahlaziywa ngokuphindaphindiweko (ukuphindaphinda okubili kwezobuchwepheshe). Ukuphindaphinda kwezobuchwepheshe kwasetjenziselwa ukuhlola ukukhiqizwa kwesilingo esifanako kodwana azange kusetjenziswe ekuhlaziyweni kwezibalo ukubalekela ukuphindaphinda okungakalungi. Idatha yahlaziywa ngokwezibalo kusetjenziswa ukuhlaziywa kokuhluka (ANOVA) kulandelwe yi-Tukey-Kramer honestly significant difference (HSD) test (p ≤ 0.05). Ekuhlolweni kwe-in vitro, amanani we-IC50 ne-IC99 abalwa kusetjenziswa imodeli ye-probit begodu ama-95% wokuqiniseka abalwa.
Inani elipheleleko lama-isolate amane laqoqwa emasimini ahlukileko wesoya e-El Ghabiya Governorate, e-Egypt. Emkhakheni we-PDA, woke ama-isolate akhiqiza i-mycelium emhlophe ekhrimu eyaphenduka msinyana yaba mhlophe njengekotini (Umfanekiso 1A) bese yaba yi-beige nofana ensundu esigabeni se-sclerotium. I-Sclerotia ivamise ukuba minyene, imnyama, ibe yindilinga nofana ingakajayeleki, ubude obungu-5.2 ukuya ku-7.7 mm kanye nobubanzi obungu-3.4 ukuya ku-5.3 mm (Umfanekiso 1B). Nanyana ama-isolate amane athuthukisa iphetheni encani ye-sclerotia emphethweni wendawo yokukhula ngemva kwamalanga ali-10 ukuya keli-12 wokufukamela ku-25 ± 2 °C (Umfanekiso 1A), inani lama-sclerotia ngepuleti ngalinye belihluke khulu hlangana nawo (P < 0.001), lapho i-isolate 3 inenani eliphezulu lama-sclerotia (23 ± 33.000). i-sclerotia ngepuleti ngayinye; Umfanekiso 1C). Ngokufanako, i-isolate #3 yakhiqiza i-oxalic acid enengi ku-PDB ukudlula ezinye i-isolate (3.33 ± 0.49 μg.mL−1; Fig. 1D). I-Isolate #3 itjengise ubujamo obujayelekileko nobuncani be-fungus ye-phytopathogenic Sclerotinia sclerotiorum. Ukwenza isibonelo, ku-PDA, amakoloni we-isolate #3 akhula msinyana, bekamhlophe (Umfanekiso 1A), be-beige obubuyela emuva nofana i-salmon ephuzi-onsundu, begodu bekutlhogeka amalanga ama-6-7 ku-25 ± 2°C ukuvala ngokupheleleko ubuso bepuleti elingu-9 cm ububanzi. Ngokuya ngeemfanelo ezingenhla ze-morphological kanye ne-microscopic, i-isolate #3 yabonwa njenge-Sclerotinia sclerotiorum.
Umfanekiso 1. Ubunjalo nokutheleleka kwe-S. sclerotiorum ehlukaniswe eentjalweni ezijayelekileko ze-legume. (A) Ukukhula kwe-mycelial yama-S. sclerotiorum amane ahlukanisiweko ku-PDA medium, (B) ama-sclerotia wama-S. sclerotiorum amane ahlukanisiweko, (C) inani lama-sclerotia (ngepuleti ngalinye), (D) ukukhiqizwa kwe-oxalic acid ku-PDB medium (μg.mL−1), kanye (E) nobukhulu besifo (%) sama-S. sclerotiorum amane ahlukanisiweko emilenzeni yokuthengisa. umhlobo we-Giza 3 ngaphasi kwemibandela ye-greenhouse. Amanani ajamele i-mean ± SD yamaphindaphindo amahlanu wezinto eziphilako (n = 5). Iinhlamvu ezihlukileko zitjengisa umehluko oqakathekileko hlangana nokwelatjhwa (p <0.05). (F–H) Amatshwayo ajwayelekileko wesikhunta esimhlophe avela eenhlathini ezingaphezu kwehlabathi kanye nama-siliques, ngokulandelana, ngemva kwamalanga ali-10 wokugonywa nge-isolate #3 (dpi). (I) Ukuhlaziywa kokuziphendukela kwemvelo kwe-internal transcribed spacer (ITS) yesifunda se-S. sclerotiorum isolate #3 kwenziwa kusetjenziswa indlela ye-maximum likelihood begodu kuqathaniswa nama-20 we-reference isolates/strains atholakala ku-National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.h.v./). Inomboro engaphezulu kwemigqa yokuhlanganisa itjengisa ukuhlanganiswa kwesifunda (%), begodu amanani angaphasi kwemigqa yokuhlanganisa atjengisa ubude begatja.
Ukudlula lapho, ukuqinisekisa ukutheleleka, ama-S. sclerotiorum amane atholakalako asetjenziselwa ukugoma umhlobo wamabhontjisi wokuthengisa othengiswako we-Giza 3 ngaphasi kwemibandela ye-greenhouse, okuhambisana nama-postulates ka-Koch (Umfanekiso 1E). Nanyana woke ama-fungal isolates atholakeleko bekanamalwelwe begodu angathelela ibhontjisi eluhlaza (cv. Giza 3), okwenza bona kube namatshwayo wesikhunta esimhlophe kizo zoke iingcenye ezingaphezulu kwehlabathi (Umfanekiso 1F), khulukhulu eentweni (Umfanekiso 1G) kanye nama-pods (Umfanekiso 1H) emalangeni ali-10 ngemva kokugonywa (Umfanekiso 1H), i-isolate beyihlasela khulu ukuhlolwa okuzijameleko okubili. I-Isolate 3 beyinesifo esiphezulu khulu (%) eentjalweni zebhontjisi (24.0 ± 4.0, 58.0 ± 2.0, kanye ne-76.7 ± 3.1 emalangeni ama-7, 14, kanye nama-21 ngemva kokutheleleka, ngokulandelana; Umfanekiso 1F).
Ukukhonjwa kwe-S. sclerotiorum isolate #3 ehlasela khulu kwaqinisekiswa godu ngokuya ngokulandelana kwe-internal transcribed spacer (ITS) (Umfanekiso 1I). Ukuhlaziywa kwe-phylogenetic hlangana ne-isolate #3 kanye nama-20 we-reference isolates/strains kutjengise ukufana okukhulu (>99%) hlangana nazo. Kuqakathekile ukutjheja bona i-S. sclerotiorum isolate #3 (533 bp) inokufana okukhulu ne-American S. sclerotiorum isolate LPM36 ehlukaniswe embewini ye-pea eyomileko (inomboro yokungena ye-GenBank MK896659.1; 540 bp) kanye nenomboro yokungena ye-S. sclerotiorum ye-YYY OR206374.1; 548 bp), okubangela ukubola kwesiqu se-violet (Matthiola incana), zoke zihlanganiswe ngokwehlukana phezulu kwe-dendrogram (Umfanekiso 1I). Ukulandelana okutjha kufakwe ku-database ye-NCBI begodu kwabizwa ngokuthi "Sclerotinia sclerotiorum - isolate YN-25" (inomboro yokungena ye-GenBank PV202792). Kungabonakala bona i-isolate 3 yiyona ehlasela khulu; ngalokho-ke, i-isolate le ikhethelwe ukufundwa kizo zoke iimhloliso ezilandelako.
Ukusebenza kwe-antibacterial ye-diamine L-ornithine (Sigma-Aldrich, Darmstadt, Germany) emazingeni ahlukileko (12.5, 25, 50, 75, 100 kanye ne-125 mg/L) ngokujamelana ne-S. sclerotiorum isolate 3 kwaphenywa nge-in vitro. Kuyaphawuleka bona i-L-ornithine yenza umphumela wokulwa namagciwani begodu kancanikancani yavimbela ukukhula kwe-S. sclerotiorum hyphae ngendlela ethembele emthamo (Umfanekiso 2A, B). Ebujameni obuphezulu obuhloliweko (125 mg/L), i-L-ornithine itjengise izinga eliphezulu lokukhandela ukukhula kwe-mycelial (99.62 ± 0.27%; Umfanekiso 2B), ebelilingana ne-fungicide yokuthengisa i-Rizolex-T (izinga lokukhandela 99.45 ± 0.39%; Umfanekiso 2C) ekuhlolweni okuphezulu (10 mg/L), umfanekiso 2C) ekuhlolweni okuphezulu (10 mg/L), umfanekiso 2C) ekuhlolweni okuphezulu (10 mg/L), umfanekiso 2C okutjengisa ukusebenza okufanako.
Umfanekiso 2. Umsebenzi wokulwa namagciwani we-L-ornithine ngokujamelana ne-Sclerotinia sclerotiorum. (A) Ukumadanisa umsebenzi wokulwisana namagciwani wamazinga ahlukileko we-L-ornithine ngokujamelana ne-S. sclerotiorum nge-fungicide yokuthengisa i-Rizolex-T (10 mg/L). (B, C) Izinga lokuvimbela (%) lokukhula kwe-S. sclerotiorum mycelial ngemva kokwelatjhwa ngamazinga ahlukileko we-L-ornithine (12.5, 25, 50, 75, 100 kanye ne-125 mg/L) nofana i-Rizolex-T (2, 4, 6, 8 kanye ne-10 mg/L), ngokulandelana. Amanani ajamele i-mean ± SD yamaphindaphindo amahlanu wezinto eziphilako (n = 5). Iinhlamvu ezihlukileko zitjengisa umehluko weembalo hlangana nokwelatjhwa (p <0.05). (D, E) Ukuhlaziywa kwe-Probit model regression ye-L-ornithine kanye ne-fungicide yokuthengisa i-Rizolex-T, ngokulandelana. Umugqa wokubuyela emuva wemodeli ye-probit utjengiswa njengomugqa ohlaza okwesibhakabhaka, begodu isikhala sokuqiniseka (95%) sitjengiswa njengomugqa obomvu ophukileko.
Ukungezelela, ukuhlaziywa kwe-probit regression kwenziwa begodu amaplothi ahambelana nawo atjengiswa kuThebuli 1 kanye neMifanekiso 2D,E. Kafitjhani, inani elivumelekileko lomthambeka (y = 2.92x − 4.67) kanye neembalo eziqakathekileko ezihlobene (u-Cox & Snell R2 = 0.3709, u-Nagelkerke R2 = 0.4998 kanye ne-p < 0.0001; Umfanekiso 2D) we-L-ornithine utjengise ukusebenza okuthuthukisiweko kwe-antifungal S sclerum nayiqathaniswa ne-srocerum. i-fungicide Rizolex-T (y = 1.96x − 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 kanye p < 0.0001) (Ithebula 1).
Ithebula 1. Amanani we-half-maximum inhibitory concentration (IC50) kanye ne-IC99 (mg/l) ye-L-ornithine kanye ne-fungicide yokuthengisa “Rizolex-T” ngokujamelana ne-S. sclerotiorum.
Ngokupheleleko, i-L-ornithine (250 mg/L) yehlise khulu ukuthuthukiswa nokuqina kwesikhunta esimhlophe eentjalweni zebhontjisi ezijayelekileko eziphathweko naziqathaniswa neentjalo ezingakalatjhwako ezitheleleke nge-S. sclerotiorum (ukulawula; Umfanekiso 3A). Kafitjhani, nanyana ubudisi besifo sezitjalo zokulawula ezithelelekileko ezingalatjhwako bukhula kancanikancani (52.67 ± 1.53, 83.21 ± 2.61, kanye no-92.33 ± 3.06%), i-L-ornithine yehlisa khulu ubudisi bokugula (%) kiyo yoke isilingo (8.97 ± 0.15, 80 ± .000, 26.36 ± 3.07) emalangeni ama-7, 14, kanye nama-21 ngemva kokwelatjhwa (dpt), ngokulandelana (Umfanekiso 3A). Ngokufanako, lokha iintjalo zamabhontjisi ezitheleleke nge-S. sclerotiorum naziphathwa nge-250 mg/L L-ornithine, indawo engaphasi kwe-disease progression curve (AUDPC) yehla ukusuka ku-1274.33 ± 33.13 esilawulweni esingakaphathwa ukuya ku-281.03 ± 7.95, ebegade iphasi kancani kunalokho okuhle kwe-50. mg/L Rizolex-T fungicide (183.61 ± 7.71; Umfanekiso 3B). Umkhuba ofanako wabonwa ekuhlolweni kwesibili.
Umfanekiso 3. Umphumela wokusetjenziswa kwangaphandle kwe-L-ornithine ekuthuthukisweni kokubola okumhlophe kwebhontjisi evamileko okubangelwa yi-Sclerotinia sclerotiorum ngaphasi kweemeko ze-greenhouse. (A) Ijika lokuragela phambili kwesifo sesikhunta esimhlophe sebhontjisi ngemva kokwelatjhwa nge-250 mg/L L-ornithine. (B) Indawo engaphasi kwe-disease progression curve (AUDPC) yesikhunta esimhlophe se-common bean ngemva kokwelatjhwa nge-L-ornithine. Amanani ajamele i-mean ± SD yamaphindaphindo amahlanu wezinto eziphilako (n = 5). Iinhlamvu ezihlukileko zitjengisa umehluko oqakathekileko hlangana nokwelatjhwa (p <0.05).
Ukusetjenziswa kwangaphandle kwe-250 mg/L L-ornithine kwandisa kancanikancani ukuphakama kwesitjalo (Umfanekiso 4A), inani lamagatja ngesitjalo ngasinye (Umfanekiso 4B), kanye nenani lamakari ngesitjalo ngasinye (Umfanekiso 4C) ngemva kwamalanga ama-42. Nanyana i-fungicide yokuthengisa i-Rizolex-T (50 mg/L) inomthelela omkhulu kiwo woke amapharamitha wezondleko afundwako, ukusetjenziswa kwangaphandle kwe-250 mg/L L-ornithine kwaba nomphumela wesibili omkhulu nayiqathaniswa nezilawuli ezingakaphathwa (Iinthombe 4A–C). Ngakelinye ihlangothi, ukwelatjhwa nge-L-ornithine bekunganamthelela omkhulu ekuqukethwe kwama-pigment we-photosynthetic chlorophyll a (Umfanekiso 4D) kanye ne-chlorophyll b (Umfanekiso 4E), kodwana kwakhuphula kancani okuqukethwe kwe-carotenoid (0.56 ± 0.03 mg/g fr wt) nayiqathaniswa nokulawula okumbi (0.44 ± 0.02 mg/g fr wt ukulawula (0.46 ± 0.02 mg/g fr wt; Umfanekiso 4F). Ngokupheleleko, imiphumela le itjengisa bona i-L-ornithine ayisiyo i-phytotoxic eentjalweni eziphathweko begodu ingakhuthaza ukukhula kwazo.
Umfanekiso 4. Umphumela wokusetjenziswa kwe-L-ornithine yangaphandle eemfanelweni zokukhula kanye nama-pigments we-photosynthetic wamaqabunga webhontjisi atheleleke nge-Sclerotinia sclerotiorum ngaphasi kweemeko ze-greenhouse. (A) Ukuphakama kwesitjalo (cm), (B) Inani lamagatja ngesitjalo ngasinye, (C) Inani lamakari ngesitjalo ngasinye, (D) Okuqukethwe yi-Chlorophyll a (mg g-1 fr wt), (E) Okuqukethwe kwe-Chlorophyll b (mg g-1 fr wt), (F) Okuqukethwe kwe-carotenoid okupheleleko (mg g-1 fr wt). Amanani aphakathi ± SD weemphindaphindo ezihlanu zezinto eziphilako (n = 5). Iinhlamvu ezihlukileko zitjengisa umehluko oqakathekileko hlangana nokwelatjhwa (p <0.05).
Ukutholakala kwe-histochemical ye-reactive oxygen species (ROS; evezwe njenge-hydrogen peroxide [H2O2]) kanye nama-free radicals (evezwe njenge-superoxide anions [O2•−]) kuveze bona ukusetjenziswa kwangaphandle kwe-L-ornithine (250 mg/L) kwehlise khulu ukubuthelela kwe-HO (250 ± nmol.g−1 FW; Umfanekiso 5A) kanye ne-O2•− (32.69 ± 8.56 nmol.g−1 FW; Umfanekiso 5B) nayiqathaniswa nokubuthelela kweentjalo ezithelelekileko ezingalatjhwako (173.31 ± 12.06 kanye ne-149.35 ± 7.94 nmol.g−1) ngokulandelana ngesitjalo esiphathweko mg/L ye-fungicide yokuthengisa i-Rizolex-T (170.12 ± 9.50 kanye ne-157.00 ± 7.81 nmol.g−1 fr wt, ngokulandelana) nge-72 h. Amazinga aphezulu we-H2O2 ne-O2•− abuthelele ngaphasi kwe-hpt (Umfanekiso 5A, B). Ngokufanako, i-TCA-based malondialdehyde (MDA) assay itjengise bona iintjalo zebhontjisi ezitheleleke nge-S. sclerotiorum zibuthelele amazinga aphezulu we-MDA (113.48 ± 10.02 nmol.g fr wt) emakarini wazo (Umfanekiso 5C). Kodwana, ukusetjenziswa kwangaphandle kwe-L-ornithine kwehlise khulu i-lipid peroxidation njengombana kufakazelwa kukwehla kokuqukethwe kwe-MDA eentjalweni eziphathweko (33.08 ± 4.00 nmol.g fr wt).
Umfanekiso 5. Umphumela wokusetjenziswa kwe-L-ornithine yangaphandle emamakeni amakhulu wokugandeleleka kwe-oxidative kanye neendlela zokuzivikela ezingezizo ze-enzymatic emaqabungeni wamabhontjisi atheleleke nge-S. sclerotiorum ngemva kwama-awa ama-72 ngemva kokutheleleka ngaphasi kweemeko ze-greenhouse. (A) I-Hydrogen peroxide (H2O2; nmol g−1 FW) ku-72 hpt, (B) i-superoxide anion (O2•−; nmol g−1 FW) ku-72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) ku-72 hpt, (D) i-phenol epheleleko encibilikako (GAE g−1) ku-72 hpt hpt, (E) ama-flavonoids ancibilikako (mg RE g−1 FW) ku-72 hpt, (F) ama-amino acid wamahhala woke (mg g−1 FW) ku-72 hpt, kanye (G) nokuqukethwe kwe-proline (mg g−1 FW) ku-72 hpt. Amanani ajamele isilinganiso ± ukuphambuka okujayelekileko (isilinganiso ± SD) sokuphindaphinda kwezinto eziphilako ezi-5 (n = 5). Iinhlamvu ezihlukileko zitjengisa umehluko oqakathekileko hlangana nokwelatjhwa (p <0.05).