Lesi sihloko siyingxenye yesihloko socwaningo esithi “Ukuthuthukisa ukumelana kwemifino ezifweni kanye nezinambuzane”, buka zonke izihloko ezi-5
I-ejenti eyimbangela yesifo sezitshalo ezibangelwa yi-fungus necrosis i-Sclerotinia sclerotiorum (Lib.) de Bary isebenzisa isu elinamazinga amaningi ukuthelela izitshalo ezahlukene eziphethwe yi-host. Lolu cwaningo luphakamisa ukusetshenziswa kwe-diamine L-ornithine, i-amino acid engeyona iphrotheni ekhuthaza ukwakheka kwamanye ama-amino acid abalulekile, njengesu lokuphatha elihlukile lokuthuthukisa izimpendulo zama-molecule, zomzimba kanye nezamakhemikhali ze-Phaseolus vulgaris L. ku-white mold ebangelwa yi-Pseudomonas sclerotiorum. Ukuhlolwa kwe-in vitro kubonise ukuthi i-L-ornithine ivimbele kakhulu ukukhula kwe-mycelial kwe-S. pyrenoidosa ngendlela encike kumthamo. Ngaphezu kwalokho, inganciphisa kakhulu ubunzima be-white mold ngaphansi kwezimo zokushisa. Ngaphezu kwalokho, i-L-ornithine yakhuthaza ukukhula kwezitshalo eziphathwe ngemithi, okubonisa ukuthi amazinga ahlolwe e-L-ornithine ayengewona ubuthi ezitshalweni eziphathwe ngemithi. Ngaphezu kwalokho, i-L-ornithine ithuthukise ukuvezwa kwama-antioxidant angewona ama-enzyme (ama-phenolics nama-flavonoid aphelele) kanye nama-antioxidant e-enzymatic (i-catalase (CAT), i-peroxidase (POX), kanye ne-polyphenol oxidase (PPO)), futhi yandisa ukuvezwa kwama-gene amathathu ahlobene nama-antioxidant (i-PvCAT1, i-PvSOD, kanye ne-PvGR). Ngaphezu kwalokho, ekuhlaziyweni kwe-silico kwembule ukuba khona kweprotheni ye-oxaloacetate acetylhydrolase (SsOAH) e-genome ye-S. sclerotiorum, eyayifana kakhulu namaprotheni e-oxaloacetate acetylhydrolase (SsOAH) e-Aspergillus fijiensis (AfOAH) kanye ne-Penicillium sp. (PlOAH) maqondana nokuhlaziywa kokusebenza, izindawo ezigciniwe, kanye ne-topology. Ngokuthakazelisayo, ukungezwa kwe-L-ornithine ku-potato dextrose broth (PDB) medium kunciphisa kakhulu ukuvezwa kwe-gene ye-SsOAH ku-S. sclerotiorum mycelia. Ngokufanayo, ukusetshenziswa kwe-L-ornithine ngaphandle kwe-L-ornithine kunciphisa kakhulu ukuvezwa kwe-SSOAH gene ku-fungal mycelia eqoqwe ezitshalweni eziphathwe ngemithi. Ekugcineni, ukusetshenziswa kwe-L-ornithine kunciphisa kakhulu ukukhishwa kwe-oxalic acid kokubili emaqabunga aphakathi ne-PDB kanye namaqabunga athelelekile. Ekuphetheni, i-L-ornithine idlala indima ebalulekile ekugcineni isimo se-redox kanye nokuthuthukisa impendulo yokuzivikela yezitshalo ezithelelekile. Imiphumela yalolu cwaningo ingasiza ekwakheni izindlela ezintsha nezinobungani nemvelo zokulawula isikhunta esimhlophe nokunciphisa umthelela waso ekukhiqizweni kobhontshisi nakwezinye izitshalo.
Isikhunta esimhlophe, esibangelwa yisikhunta se-necrotrophic i-Sclerotinia sclerotiorum (Lib.) de Bary, yisifo esibhubhisayo, esinciphisa isivuno esibeka usongo olukhulu ekukhiqizweni kwebhontshisi yomhlaba wonke (i-Phaseolus vulgaris L.) (Bolton et al., 2006). I-Sclerotinia sclerotiorum ingenye yezifo zezitshalo zesikhunta ezinzima kakhulu ukuzilawula, enezinhlobo eziningi zezitshalo ezingaphezu kuka-600 kanye nekhono lokusheshisa izicubu zomsingathi ngendlela engacacisiwe (Liang noRollins, 2018). Ngaphansi kwezimo ezingezinhle, idlula esigabeni esibucayi somjikelezo wayo wokuphila, ihlala ithule isikhathi eside njengezakhiwo ezimnyama, eziqinile, ezifana nembewu ezibizwa ngokuthi 'i-sclerotia' enhlabathini noma njengezimila ezimhlophe, ezithambile ku-mycelium noma isiqu sesitshalo esinegciwane (Schwartz et al., 2005). I-S. sclerotiorum iyakwazi ukwakha i-sclerotia, evumela ukuthi iphile emasimini athelelekile isikhathi eside futhi iqhubeke ngesikhathi sokugula (Schwartz et al., 2005). I-Sclerotia icebile ngezakhamzimba, ingahlala enhlabathini isikhathi eside, futhi isebenza njenge-inoculum eyinhloko yokutheleleka okulandelayo (Schwartz et al., 2005). Ngaphansi kwezimo ezinhle, i-sclerotia iyahluma futhi ikhiqize ama-spores aphuma emoyeni angatheleleka kuzo zonke izingxenye zesitshalo ezingenhla komhlaba, okuhlanganisa kodwa kungagcini ezimbalini, eziqwini, noma eziqongweni (Schwartz et al., 2005).
I-Sclerotinia sclerotiorum isebenzisa isu elinamazinga amaningi ukuthelela izitshalo ezihlala kuyo, okuhlanganisa uchungechunge lwezehlakalo ezihlanganisiwe kusukela ekuhlumeni kwe-sclerotial kuya ekukhuleni kwezimpawu. Ekuqaleni, i-S. sclerotiorum ikhiqiza ama-spores amisiwe (abizwa ngokuthi ama-ascospores) avela ezakhiweni ezifana namakhowe ezibizwa ngokuthi i-apothecia, eziba nomoya bese zikhula zibe yi-sclerotia enganyakazi emfucumfucwini yezitshalo ezithelelekile (Bolton et al., 2006). I-fungus bese ikhipha i-oxalic acid, i-virulence factor, ukulawula i-pH yodonga lweseli lesitshalo, ukukhuthaza ukuwohloka kwe-enzymatic kanye nokuhlasela kwezicubu (Hegedus noRimmer, 2005), kanye nokucindezela ukuqhuma kwe-oxidative kwesitshalo esihlala kuyo. Le nqubo yokwenza i-acid ibe buthaka udonga lweseli lesitshalo, ihlinzeka ngendawo enhle yokusebenza okuvamile nokusebenza kahle kwama-enzyme abhubhisa udonga lweseli lesikhunta (ama-CWDE), okuvumela i-pathogen ukuthi inqobe isithiyo esingokwenyama futhi ingene ezicutshini ezihlala kuyo (Marciano et al., 1983). Uma isingene, i-S. sclerotiorum ikhipha inani lama-CWDE, njenge-polygalacturonase kanye ne-cellulase, okwenza kube lula ukusabalala kwayo ezicutshini ezithelelekile futhi kubangele i-tissue necrosis. Ukuqhubeka kwezilonda kanye nama-hyphal mats kuholela ezimpawini eziphawulekayo zesikhunta esimhlophe (Hegedus noRimmer, 2005). Okwamanje, izitshalo ezibambayo zibona amaphethini amangqamuzana ahlobene ne-pathogen (ama-PAMP) ngokusebenzisa ama-receptor okuqaphela amaphethini (ama-PRR), okubangela uchungechunge lwezehlakalo zokubonisa ezigcina zivuselela izimpendulo zokuzivikela.
Naphezu kwemizamo yokulawula izifo emashumini eminyaka, ukushoda kwe-germplasm elwa nokumelana okwanele kusalokhu kubhontshisi, njengakwezinye izitshalo zezentengiselwano, ngenxa yokumelana, ukusinda, kanye nokuzivumelanisa nezimo kwegciwane. Ngakho-ke ukuphathwa kwezifo kuyinselele enkulu futhi kudinga isu elihlanganisiwe nelinezici eziningi elihlanganisa inhlanganisela yemikhuba yamasiko, ukulawulwa kwezinto eziphilayo, kanye ne-fungicides yamakhemikhali (O'Sullivan et al., 2021). Ukulawulwa kwamakhemikhali kwesikhunta esimhlophe kuphumelela kakhulu ngoba i-fungicides, uma isetshenziswa kahle nangesikhathi esifanele, ingalawula ngempumelelo ukusabalala kwesifo, inciphise ubukhali bokutheleleka, futhi inciphise ukulahlekelwa kwesivuno. Kodwa-ke, ukusetshenziswa ngokweqile kanye nokuthembela ngokweqile kuma-fungicides kungaholela ekuveleni kwezinhlobo ezilwa nokumelana ze-S. sclerotiorum futhi kuthinte kabi izinto eziphilayo ezingahlosiwe, impilo yomhlabathi, kanye nekhwalithi yamanzi (Le Cointe et al., 2016; Ceresini et al., 2024). Ngakho-ke, ukuthola ezinye izindlela ezinobungani nemvelo sekuyinto eza kuqala.
Ama-polyamine (ama-PA), njenge-putrescine, i-spermidine, i-spermine, kanye ne-cadaverine, angasebenza njengezindlela ezithembisayo zokulwa namagciwane ezitshalo athwalwa yinhlabathi, ngaleyo ndlela anciphise ngokuphelele noma kancane ukusetshenziswa kwama-fungicide amakhemikhali ayingozi (Nehela et al., 2024; Yi et al., 2024). Ezitshalweni eziphakeme, ama-PA ahilelekile ezinqubweni eziningi zomzimba okuhlanganisa, kodwa kungagcini lapho, ukuhlukaniswa kwamaseli, ukuhlukanisa, kanye nokusabela ekucindezelekeni okungenabungozi kanye ne-biotic (Killiny noNehela, 2020). Zingasebenza njengama-antioxidants, zisize ekususeni izinhlobo ze-oxygen ezisabelayo (i-ROS), zigcine i-redox homeostasis (i-Nehela ne-Killiny, 2023), zivuse izakhi zofuzo ezihlobene nokuzivikela (i-Romero et al., 2018), zilawule izindlela ezahlukene ze-metabolic (i-Nehela ne-Killiny, 2023), ziguqule ama-phytohormones angaphakathi (i-Nehela ne-Killiny, 2019), zisungule ukumelana okutholakala ohlelweni (i-SAR), futhi zilawule ukusebenzisana kwezitshalo nezifo (i-Nehela ne-Killiny, 2020; i-Asija et al., 2022; i-Czerwoniec, 2022). Kubalulekile ukuqaphela ukuthi izindlela ezithile kanye nezindima zama-PA ekuvikeleni izitshalo ziyahlukahluka kuye ngezinhlobo zezitshalo, izifo ezibangelwa amagciwane, kanye nezimo zemvelo. I-PA eningi kakhulu ezitshalweni yenziwe nge-biosynthesized kusuka ku-polyamine L-ornithine ebalulekile (i-Killiny ne-Nehela, 2020).
I-L-ornithine idlala indima eminingi ekukhuleni nasekuthuthukisweni kwezitshalo. Isibonelo, izifundo zangaphambilini zibonise ukuthi elayisini (Oryza sativa), i-ornithine ingahlotshaniswa nokuvuselelwa kwe-nitrogen (Liu et al., 2018), isivuno serayisi, ikhwalithi kanye nephunga (Lu et al., 2020), kanye nokusabela ekucindezelweni kwamanzi (Yang et al., 2000). Ngaphezu kwalokho, ukusetshenziswa kwe-L-ornithine ngaphandle kwandise kakhulu ukubekezelela isomiso ku-beetroot kashukela (Beta vulgaris) (Hussein et al., 2019) futhi kunciphisa ukucindezeleka kukasawoti ku-anyanisi (Allium Cepa) (Çavuşoǧlu no-Çavuşoǧlu, 2021) kanye nezitshalo ze-cashew (Anacardium occidentale) (da Rocha et al., 2012). Indima engaba khona ye-L-ornithine ekuvikeleni ukucindezeleka okungenabungozi ingase ibe ngenxa yokubandakanyeka kwayo ekuqongeleleni kwe-proline ezitshalweni eziphathwe kahle. Isibonelo, izakhi zofuzo ezihlobene nokuguqulwa kwe-proline, njengezakhi zofuzo ze-ornithine delta aminotransferase (delta-OAT) kanye ne-proline dehydrogenase (ProDH1 kanye ne-ProDH2), ziye zabikwa ngaphambilini ukuthi zihilelekile ekuvikeleni i-Nicotiana benthamiana kanye ne-Arabidopsis thaliana ngokumelene nezinhlobo ze-Pseudomonas syringae ezingezona ezibamba iqhaza (Senthil-Kumar kanye ne-Mysore, 2012). Ngakolunye uhlangothi, i-fungal ornithine decarboxylase (ODC) iyadingeka ekukhuleni kwe-pathogen (Singh et al., 2020). Ukuqondisa i-ODC ye-Fusarium oxysporum f. sp. lycopersici nge-host-induced gene silencing (HIGS) kuthuthukise kakhulu ukumelana kwezitshalo zikatamatisi ku-Fusarium wilt (Singh et al., 2020). Kodwa-ke, indima engaba khona yokusetshenziswa kwe-ornithine yangaphandle ngokumelene nokucindezeleka kwe-biotic njenge-phytopathogens ayikafundwa kahle. Okubaluleke kakhulu, imiphumela ye-ornithine ekumelaneni nezifo kanye nezimo ezihlobene namakhemikhali kanye nezomzimba azikahlolwa kangako.
Ukuqonda ubunzima bokutheleleka kwe-S. sclerotiorum kwezitshalo ezisawolintshi kubalulekile ekwakhiweni kwamasu okulawula asebenzayo. Kulolu cwaningo, sihlose ukuthola indima engaba khona ye-diamine L-ornithine njengento ebalulekile ekwandiseni izindlela zokuzivikela kanye nokumelana kwezitshalo ezisawolintshi ekuthelelekeni kwe-Sclerotinia sclerotiorum. Sicabanga ukuthi, ngaphezu kokuthuthukisa izimpendulo zokuzivikela zezitshalo ezithelelekile, i-L-ornithine nayo idlala indima ebalulekile ekugcineni isimo se-redox. Siphakamisa ukuthi imiphumela engaba khona ye-L-ornithine ihlobene nokulawulwa kwezindlela zokuzivikela ze-enzyme kanye ne-non-enzymatic antioxidant kanye nokuphazamiseka kwezinto ezibangela izifo/igciwane lesikhunta kanye namaprotheni ahlobene. Lokhu kusebenza okubili kwe-L-ornithine kwenza kube yinto ethembisayo yesu eliqhubekayo lokunciphisa umthelela wesikhunta esimhlophe nokuthuthukisa ukumelana kwezitshalo ezisawolintshi ezivamile kule pathogen enamandla yesikhunta. Imiphumela yalolu cwaningo ingasiza ekuthuthukisweni kwezindlela ezintsha, ezinobungani nemvelo zokulawula isikhunta esimhlophe nokunciphisa umthelela waso ekukhiqizweni kwezitshalo ezisawolintshi.
Kulolu cwaningo, uhlobo lwebhontshisi oluvamile olusengozini yokuhweba, i-Giza 3 (i-Phaseolus vulgaris L. cv. Giza 3), lusetshenziswe njengezinto zokuhlola. Imbewu enempilo yanikezwa ngomusa yi-Legume Research Department, i-Field Crops Research Institute (FCRI), i-Agricultural Research Center (ARC), eGibhithe. Imbewu ezinhlanu zahlwanyelwa ezitsheni zepulasitiki (ububanzi bangaphakathi obungu-35 cm, ukujula okungu-50 cm) ezigcwele inhlabathi etheleleke yi-S. sclerotiorum ngaphansi kwezimo ze-greenhouse (25 ± 2 °C, umswakama ohlobene ongu-75 ± 1%, ukukhanya kwamahora angu-8/amahora angu-16 amnyama). Ezinsukwini ezingu-7-10 ngemva kokuhlwanyela (i-DPS), izithombo zancishiswa ukuze kushiywe izithombo ezimbili kuphela ezinokukhula okufanayo namaqabunga amathathu anwetshwe ngokugcwele ebhodweni ngalinye. Zonke izitshalo ezifakwe ezitsheni zaniselwa kanye njalo emavikini amabili futhi zavundiswa njalo ngenyanga ngesilinganiso esinconywayo sohlobo olunikeziwe.
Ukuze kulungiswe ukuhlushwa okungu-500 mg/L kwe-L-ornithinediamine (eyaziwa nangokuthi (+)-(S)-2,5-diaminopentanoic acid; iSigma-Aldrich, eDarmstadt, eJalimane), ama-50 mg aqale ancibilikiswa ku-100 mL wamanzi acwecwe angenasici. Isixazululo sesitoko sabe sesincibilikiswa futhi sasetshenziswa ekuhlolweni okulandelayo. Kafushane, uchungechunge oluyisithupha lokuhlushwa kwe-L-ornithine (12.5, 25, 50, 75, 100, kanye no-125 mg/L) kwahlolwa ngaphandle kwemithi. Ngaphezu kwalokho, amanzi acwecwe angenasici asetshenziswa njenge-negative control (Mock) kanye ne-fungicide yezentengiselwano ethi “Rizolex-T” 50% wettable powder (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Egypt) yasetshenziswa njenge-positive control. I-fungicide yezentengiselwano ethi “Rizolex-T” ihlolwe ngaphandle kwe-vitro ngamanani amahlanu (2, 4, 6, 8 kanye no-10 mg/L).
Amasampula eziqu zebhontshisi ezivamile kanye nama-pod abonisa izimpawu ezivamile zesikhunta esimhlophe (izinga lokutheleleka: 10-30%) aqoqwe emapulazini ezentengiselwano. Nakuba iningi lezinto zezitshalo ezinegciwane lahlonzwa ngezinhlobo/izinhlobo (uhlobo lwezentengiselwano olusengozini lweGiza 3), ezinye, ikakhulukazi lezo ezitholakale ezimakethe zendawo, zazingezohlobo olungaziwa. Izinto ezinegciwane eziqoqwe zaqala zahlanzwa ngamanzi angcolile ngesisombululo se-sodium hypochlorite esingu-0.5% imizuzu emi-3, zabe sezihlanzwa kaningana ngamanzi ahlanzekile ahlanzekile futhi zasulwa ngephepha lokuhlunga elihlanzekile ukuze kususwe amanzi angaphezulu. Izitho ezinegciwane zabe sezinqunywa zibe yizicucu ezincane ezicutshini eziphakathi (phakathi kwezicubu ezinempilo nezinegciwane), zakhuliswa ku-potato dextrose agar (PDA) medium futhi zafakwa ku-25 ± 2 °C ngomjikelezo omnyama wamahora ayi-12/amahora ayi-12 izinsuku ezi-5 ukuze kukhuthazwe ukwakheka kwe-sclerotia. Indlela ye-mycelial tip yasetshenziswa futhi ukuhlanza ama-fungal isolates avela ezitshalweni ezixubile noma ezingcolisiwe. I-fungi ehlanziwe yaqala ukutholakala ngokusekelwe ezicini zayo zamasiko bese kuqinisekiswa ukuthi iyi-S. sclerotiorum ngokusekelwe ezicini ezincane kakhulu. Ekugcineni, zonke i-fungi ehlanziwe yahlolwa ukuthi ayinazo yini izifo ku-common bean cultivar Giza 3 ukuze kuhlangatshezwane ne-Koch's postulates.
Ngaphezu kwalokho, i-S. sclerotiorum isolate ehlasela kakhulu (isolate #3) yaqinisekiswa ngokususelwa ekulandeleni kwe-internal transcribed spacer (ITS) njengoba kuchaziwe nguWhite et al., 1990; Baturo-Ciesniewska et al., 2017. Kafushane, ama-isolates akhuliswa kumhluzi we-potato dextrose (PDB) futhi afakwa e-incubator ku-25 ± 2 °C izinsuku ezingu-5-7. I-fungal mycelium yabe isiqoqwa, yahlungwa nge-cheesecloth, yagezwa kabili ngamanzi ahlanzekile, futhi yomiswa ngephepha lokuhlunga elihlanzekile. I-Genomic DNA yahlukaniswa kusetshenziswa i-Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Isifunda se-ITS rDNA sabe sesikhuliswa kusetshenziswa i-primer pair ethize ITS1/ITS4 (TCCGTAGGTGAACCTGGCG TCCTCGCTTTATTGATATGC; usayizi olindelekile: 540 bp) (Baturo-Ciesniewska et al., 2017). Imikhiqizo ye-PCR ehlanziwe yathunyelwa ukuze ilandelelane (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). Ukulandelana kwe-ITS rDNA kwahlelwa ngokulandelana kusetshenziswa indlela ye-Sanger sequencing. Ukulandelana kwemibuzo okuhlanganisiwe kwabe sekuqhathaniswa nedatha yakamuva ku-GenBank kanye ne-National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/) kusetshenziswa isofthiwe ye-BLASTn. Uchungechunge lombuzo luqhathaniswe nezinye izinhlobo/izihlukanisi ze-S. sclerotiorum ezingu-20 ezithathwe kudatha yakamuva ku-NCBI GenBank (Ithebula Elingeziwe S1) kusetshenziswa i-ClustalW ku-Molecular Evolutionary Genetics Analysis Package (MEGA-11; inguqulo 11) (Kumar et al., 2024). Ukuhlaziywa kokuziphendukela kwemvelo kwenziwe kusetshenziswa indlela yokuba nokwenzeka okukhulu kanye nemodeli yokushintsha i-nucleotide eguquguqukayo yesikhathi (Nei noKumar, 2000). Isihlahla esinethuba eliphezulu kakhulu le-log liyabonakala. Isihlahla sokuqala sokusesha kwe-heuristic sikhethwa ngokukhetha isihlahla esinethuba eliphezulu le-log phakathi kwesihlahla esihlangana nomakhelwane (NJ) (Kumar et al., 2024) kanye nesihlahla esiphezulu se-parsimony (MP). Isihlahla se-NJ sakhiwe kusetshenziswa i-matrix yebanga eliphindwe kabili ebalwe kusetshenziswa imodeli ejwayelekile eguquguqukayo yesikhathi (Nei noKumar, 2000).
Umsebenzi we-L-ornithine wokulwa namagciwane kanye ne-“Rizolex-T” yatholakala ku-vitro ngendlela yokusabalalisa i-agar. Indlela: Thatha inani elifanele lesisombululo se-stock se-L-ornithine (500 mg/L) bese usixuba kahle ne-10 ml ye-PDA nutrient medium ukuze ulungiselele izixazululo ezinamazinga okugcina angu-12.5, 25, 50, 75, 100 kanye no-125 mg/L, ngokulandelana. Amazinga amahlanu e-fungicide “Rizolex-T” (2, 4, 6, 8 kanye no-10 mg/L) kanye namanzi acwengekile ahlanzekile asetshenziswa njengendlela yokulawula. Ngemva kokuba i-medium isiqinile, i-mycelial plug esanda kulungiswa ye-Sclerotinia sclerotiorum culture, engu-4 mm ububanzi, yadluliselwa enkabeni ye-Petri dish futhi yakhuliswa ku-25±2°C kuze kube yilapho i-mycelium imboza sonke i-control Petri dish, ngemva kwalokho kwaqoshwa ukukhula kwe-fungus. Bala iphesenti lokuvinjelwa kokukhula kwe-radial kwe-S. sclerotiorum usebenzisa i-equation 1:
Ukuhlolwa kwaphindwa kabili, kanye nokukopishwa kwebhayoloji okuyisithupha kweqembu ngalinye lokulawula/lokuhlola kanye nezimbiza ezinhlanu (izitshalo ezimbili ngembiza ngayinye) kokukopishwa kwebhayoloji ngakunye. Ukukopishwa ngakunye kwebhayoloji kwahlaziywa kabili (ukukopishwa kwebhayoloji okubili) ukuqinisekisa ukunemba, ukuthembeka kanye nokuphindaphindwa kwemiphumela yokuhlolwa. Ngaphezu kwalokho, ukuhlaziywa kokuhlehla kwe-probit kwasetshenziswa ukubala ukuhlushwa kwe-half-maximal inhibitory (IC50) kanye ne-IC99 (Prentice, 1976).
Ukuze kuhlolwe amandla e-L-ornithine ngaphansi kwezimo zokushisa, kwenziwa izivivinyo ezimbili ezilandelanayo zembiza. Kafushane, izimbiza zagcwaliswa ngenhlabathi yesihlabathi sobumba esihlanziwe (3:1) futhi zafakwa umjovo ngesimila esisha esilungisiwe se-S. sclerotiorum. Okokuqala, i-isolate ehlasela kakhulu ye-S. sclerotiorum (isolate #3) yakhuliswa ngokusika i-sclerotium eyodwa phakathi, yabekwa ibheke phansi ku-PDA futhi yafakwa ekushiseni okungama-25°C ebumnyameni obuhlala njalo (amahora angama-24) izinsuku ezi-4 ukuze kukhuthazwe ukukhula kwe-mycelial. Ama-plug e-agar amane anobubanzi obungama-5 mm athathwe onqenqemeni oluphambili futhi afakwa umjovo nge-100 g yengxube ehlanzekile kakolweni ne-rice bran (1:1, v/v) futhi wonke ama-flask afakwa ekushiseni okungama-25 ± 2 °C ngaphansi komjikelezo omnyama wamahora ayi-12/amahora ayi-12 izinsuku ezi-5 ukuze kukhuthazwe ukwakheka kwe-sclerotia. Okuqukethwe yizo zonke ama-flask kwaxutshwa kahle ukuqinisekisa ukuthi i-homogeneity ngaphambi kokufaka inhlabathi. Ngemuva kwalokho, kwafakwa amagremu ayi-100 engxube ye-bran ehlanganisa amagciwane ebhodweni ngalinye ukuqinisekisa ukuhlushwa okuqhubekayo kwamagciwane. Izimbiza ezigonyelwe zaniselwa ukuze kusebenze ukukhula kwesikhunta futhi zabekwa ezindaweni zokugcina izithombo izinsuku eziyi-7.
Imbewu ezinhlanu zohlobo lwe-Giza 3 zabe sezihlwanyelwa ebhodweni ngalinye. Ezitsheni eziphathwe nge-L-ornithine kanye ne-fungicide i-Rizolex-T, imbewu ehlanjululwe yaqala yacwiliswa amahora amabili esixazululweni samanzi salezi zinhlanganisela ezimbili nge-IC99 concentration yokugcina engaba ngu-250 mg/L kanye no-50 mg/L, ngokulandelana, bese yomiswa emoyeni ihora elilodwa ngaphambi kokuhlwanyela. Ngakolunye uhlangothi, imbewu yacwiliswa emanzini ahlanjululwe angenawo amagciwane njengendlela yokulawula engemihle. Ngemva kwezinsuku eziyi-10, ngaphambi kokunisela kokuqala, izithombo zancishiswa, kwasala izithombo ezimbili ezihlanzekile ebhodweni ngalinye. Ngaphezu kwalokho, ukuqinisekisa ukutheleleka nge-S. sclerotiorum, iziqu zezitshalo zobhontshisi esigabeni esifanayo sokukhula (izinsuku eziyi-10) zanqunywa ezindaweni ezimbili ezahlukene kusetshenziswa i-scalpel ehlanjululwe amagciwane futhi cishe u-0.5 g wengxube ye-bran ehlala emathunjini yafakwa enxebeni ngalinye, kulandelwe umswakama ophezulu ukukhuthaza ukutheleleka kanye nokukhula kwezifo kuzo zonke izitshalo ezifakwe umjovo. Izitshalo zokulawula zalimala ngendlela efanayo futhi inani elilinganayo (0.5 g) lengxube ye-bran engenamagciwane, engakafakwa ekoloni lafakwa enxebeni futhi lagcinwa ngaphansi komswakama ophezulu ukuze kufanekiselwe indawo yokukhula kwesifo nokuqinisekisa ukuvumelana phakathi kwamaqembu okwelapha.
Indlela Yokwelapha: Izithombo zobhontshisi zaniselwa nge-500 ml yesisombululo samanzi se-L-ornithine (250 mg/l) noma i-fungicide i-Rizolex-T (50 mg/l) ngokunisela inhlabathi, bese ukwelashwa kwaphindwa kathathu ngesikhawu sezinsuku eziyi-10. Izilawuli eziphathwe nge-placebo zaniselwa nge-500 ml yamanzi acwengekile angenamagciwane. Konke ukwelashwa kwenziwa ngaphansi kwezimo ze-greenhouse (25 ± 2°C, 75 ± 1% umswakama ohlobene, kanye nesikhathi sokukhanya samahora ayi-8/16 amahora amnyama). Zonke izimbiza zaniselwa njalo ngemva kwamasonto amabili futhi zaphathwa njalo ngenyanga ngomanyolo we-NPK olinganiselayo (20-20-20, nge-3.6% yesulfure kanye ne-TE microelements; i-Zain Seeds, eGibhithe) ekugxilweni okungu-3–4 g/l ngokufafaza ngamahlamvu ngokwezincomo zohlobo oluthile kanye nemiyalelo yomenzi. Ngaphandle kokuthi kushiwo ngenye indlela, amaqabunga avuthiwe anwetshiwe ngokuphelele (amaqabunga esibili nawesithathu ukusuka phezulu) aqoqwe kusuka kulokho okuphindaphindwayo kwezinto eziphilayo emahoreni angu-72 ngemuva kokwelashwa (hpt), ahlanganiswa abe munye, ahlanganiswe ndawonye futhi agcinwa ku--80 °C ukuze kuhlaziywe okwengeziwe okuhlanganisa, kodwa kungagcini lapho, kutholakala khona izinkomba zokucindezeleka kwe-oxidative endaweni ethile, i-lipid peroxidation, ama-antioxidants e-enzymatic kanye ne-non-enzymatic kanye nokubonakaliswa kwezakhi zofuzo.
Ukuqina kokutheleleka kwesikhunta esimhlophe kwahlolwa masonto onke ezinsukwini ezingama-21 ngemuva kokugonywa (dpi) kusetshenziswa isikali esingu-1–9 (Ithebula Elingeziwe S2) ngokusekelwe esikalini sikaPetzoldt noDickson (1996) esiguqulwe nguTeran et al. (2006). Kafushane, iziqu namagatsha ezitshalo zobhontshisi kwahlolwa kusukela endaweni yokugonywa ukuze kulandelwe ukuqhubeka kwezilonda eduze kwama-internode nama-node. Ibanga lesilonda kusukela endaweni yokugonywa kuya endaweni ekude kakhulu eduze kwesiqu noma igatsha labe selilinganiswa futhi kwabekwa amaphuzu angu-1–9 ngokusekelwe endaweni yesilonda, lapho (1) kwabonisa khona ukuthi akukho ukutheleleka okubonakalayo eduze nendawo yokugonywa futhi (2–9) kwabonisa ukwanda kancane kancane kosayizi wesilonda kanye nokuqhubekela phambili eduze kwama-node/ama-internode (Ithebula Elingeziwe S2). Ukuqina kokutheleleka kwesikhunta esimhlophe kwabe sekuguqulwa kwaba yiphesenti kusetshenziswa ifomula 2:
Ngaphezu kwalokho, indawo engaphansi kwejika lokuqhubeka kwesifo (AUDPC) ibalwe kusetshenziswa ifomula (Shaner noFinney, 1977), esanda kulungiswa ukubola okumhlophe kwebhontshisi evamile (Chauhan et al., 2020) kusetshenziswa isibalo 3:
Lapho u-Yi = ubukhali besifo ngesikhathi u-ti, u-Yi+1 = ubukhali besifo ngesikhathi esilandelayo u-ti+1, u-ti = isikhathi sokulinganisa kokuqala (ngezinsuku), u-ti+1 = isikhathi sokulinganisa okulandelayo (ngezinsuku), u-n = inani eliphelele lamaphuzu esikhathi noma amaphuzu okubuka. Imingcele yokukhula kwesitshalo sikabhontshisi ehlanganisa ukuphakama kwesitshalo (cm), inani lamagatsha ngesitshalo ngasinye, kanye nenani lamaqabunga ngesitshalo ngasinye kwaqoshwa masonto onke izinsuku ezingu-21 kuzo zonke izinhlobo zezinto eziphilayo.
Kulokho kuphindaphindwa kwezinto eziphilayo, amasampula amaqabunga (amaqabunga esibili nawesithathu akhule ngokugcwele kusukela phezulu) aqoqwa ngosuku lwama-45 ngemuva kokwelashwa (izinsuku eziyi-15 ngemuva kokwelashwa kokugcina). Ukuphindaphindwa ngakunye kwezinto eziphilayo kwakuhlanganisa izimbiza ezinhlanu (izitshalo ezimbili ngembiza ngayinye). Cishe ama-500 mg ezicubu ezichotshoziwe asetshenziswa ekukhipheni imibala ye-photosynthetic (i-chlorophyll a, i-chlorophyll b kanye ne-carotenoids) kusetshenziswa i-80% acetone ku-4 °C ebumnyameni. Ngemva kwamahora angama-24, amasampula afakwa ku-centrifuge futhi i-supernatant yaqoqwa ukuze kutholakale okuqukethwe kwe-chlorophyll a, i-chlorophyll b kanye ne-carotenoid ngombala kusetshenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, Japan) ngokwendlela ka (Lichtenthaler, 1987) ngokulinganisa ukumuncwa kwamaza amathathu ahlukene (A470, A646 kanye ne-A663 nm). Ekugcineni, okuqukethwe yi-pigment ye-photosynthetic kubalwe kusetshenziswa amafomula alandelayo 4–6 achazwe nguLichtenthaler (1987).
Ngemva kwamahora angu-72 ukwelashwa (hpt), amaqabunga (amaqabunga esibili nawesithathu akhule ngokugcwele kusukela phezulu) aqoqwa kusuka ku-replica ngayinye yezinto eziphilayo ukuze kutholakale indawo ye-hydrogen peroxide (H2O2) kanye ne-superoxide anion (O2•−). I-replica ngayinye yezinto eziphilayo yayinezimbiza ezinhlanu (izitshalo ezimbili ngembiza ngayinye). I-replica ngayinye yezinto eziphilayo yahlaziywa ngokuphindwe kabili (i-replica ezimbili zobuchwepheshe) ukuqinisekisa ukunemba, ukuthembeka kanye nokuphindaphindwa kwendlela. I-H2O2 kanye ne-O2•− zanqunywa kusetshenziswa i-0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Germany) noma i-nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Germany), ngokulandelana, kulandela izindlela ezichazwe nguRomero-Puertas et al. (2004) kanye no-Adam et al. (1989) ngokuguqulwa okuncane. Ukuze kutholakale indawo ye-H2O2 endaweni ye-histochemical, amapheshana afakwe nge-vacuum cleaner nge-0.1% DAB ku-10 mM Tris buffer (pH 7.8) abese efakwa ekushiseni kwegumbi ekukhanyeni imizuzu engama-60. Amapheshana afakwe nge-bleach ku-0.15% (v/v) TCA ku-4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) abese evezwa ekukhanyeni kuze kube mnyama. Ngokufanayo, ama-valve afakwe nge-vacuum cleaner nge-10 mM potassium phosphate buffer (pH 7.8) equkethe i-0.1 w/v % HBT ukuze kutholakale indawo ye-O2 •− endaweni ye-histochemical. Amapheshana afakwe ekukhanyeni ekushiseni kwegumbi imizuzu engama-20, abese efakwa nge-bleach njengoba kushiwo ngenhla, abese ekhanyiswa kuze kuvele amabala aluhlaza okwesibhakabhaka/aluhlaza okwesibhakabhaka. Ukuqina kombala onsundu ophumayo (njengenkomba ye-H2O2) noma oluhlaza okwesibhakabhaka (njengenkomba ye-O2•−) kuhlolwe kusetshenziswa inguqulo ye-Fiji yephakheji yokucubungula izithombe i-ImageJ (http://fiji.sc; ifinyelelwe ngomhlaka-7 Mashi 2024).
I-Malondialdehyde (MDA; njengophawu lwe-lipid peroxidation) yanqunywa ngokwendlela kaDu noBramlage (1992) ngokuguqulwa okuncane. Amaqabunga avela ku-replica ngayinye yezinto eziphilayo (amaqabunga esibili nawesithathu akhule ngokugcwele kusukela phezulu) aqoqwa amahora angama-72 ngemuva kokwelashwa (hpt). I-replica ngayinye yezinto eziphilayo yayihlanganisa izimbiza ezinhlanu (izitshalo ezimbili ngembiza ngayinye). I-replica ngayinye yezinto eziphilayo yahlaziywa ngokuphindwe kabili (i-replica ezimbili zobuchwepheshe) ukuqinisekisa ukunemba, ukuthembeka kanye nokuphindaphinda kwendlela. Ngamafuphi, i-0.5 g yezicubu zeqabunga elisemhlabathini yasetshenziswa ukukhipha i-MDA nge-20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) equkethe i-0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). Okuqukethwe kwe-MDA ku-supernatant kwabe sekunqunywa ngombala ngokukala ukumuncwa ku-532 kanye no-600 nm kusetshenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, Japan) bese kuvezwa njenge-nmol g−1 FW.
Ukuze kuhlolwe ama-antioxidants angewona ama-enzyme nama-enzyme, amaqabunga (amaqabunga esibili nawesithathu akhule ngokugcwele kusukela phezulu) aqoqwa kusuka ku-replica ngayinye yezinto eziphilayo emahoreni angu-72 ngemuva kokwelashwa (hpt). I-replica ngayinye yezinto eziphilayo yayinezimbiza ezinhlanu (izitshalo ezimbili ngembiza ngayinye). Isampula ngayinye yezinto eziphilayo yahlaziywa ngokuphindwe kabili (amasampula amabili obuchwepheshe). Amaqabunga amabili agaywa nge-nitrogen ewuketshezi futhi asetshenziswa ngqo ekunqumeni ama-antioxidants e-enzyme nama-non-enzyme, ama-amino acid aphelele, okuqukethwe yi-proline, ukubonakaliswa kwezakhi zofuzo, kanye nokulinganiswa kwe-oxalate.
Ama-phenolic aphelele ancibilikayo atholakale kusetshenziswa i-reagent ye-Folin-Ciocalteu (Sigma-Aldrich, St. Louis, MO, USA) ngokuguqulwa okuncane kwendlela echazwe nguKahkonen et al. (1999). Kafushane, cishe u-0.1 g wezicubu zamaqabunga ezihlanganisiwe wakhishwa nge-20 ml ye-80% methanol ebumnyameni amahora angama-24 kwathi i-supernatant yaqoqwa ngemuva kokuxutshwa. I-0.1 ml yesampula ekhishwe yaxutshwa ne-0.5 ml ye-Folin-Ciocalteu reagent (10%), yanyakaziswa imizuzwana engama-30 yashiywa ebumnyameni imizuzu emi-5. Kwabe sekufakwa i-0.5 ml yesisombululo se-sodium carbonate esingu-20% (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, eCairo, eGibhithe) epayipini ngalinye, yaxutshwa kahle futhi yafakwa ekushiseni kwegumbi ebumnyameni ihora eli-1. Ngemva kokufukamela, ukumuncwa kwengxube yokusabela kwalinganiswa ku-765 nm kusetshenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, Japan). Ukuhlushwa kwama-phenol ancibilikayo aphelele ezikhishweni zesampula kwanqunywa kusetshenziswa i-gallic acid calibration curve (Fisher Scientific, Hampton, NH, USA) futhi kwavezwa njengama-milligrams e-gallic acid equivalent per gram of fresh weight (mg GAE g-1 fresh weight).
Okuqukethwe okuphelele kwe-flavonoid encibilikayo kunqunywe ngokwendlela kaDjeridane et al. (2006) ngokuguqulwa okuncane. Kafushane, i-0.3 ml ye-methanol extract engenhla ixutshwe ne-0.3 ml yesisombululo se-aluminium chloride esingu-5% (AlCl3; Fisher Scientific, Hampton, NH, USA), yavuselelwa ngamandla yabe isifakwa ekushiseni kwegumbi imizuzu emi-5, kwalandelwa ukwengezwa kwe-0.3 ml yesisombululo se-potassium acetate esingu-10% (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), yaxutshwa kahle yafakwa ekushiseni kwegumbi imizuzu engama-30 ebumnyameni. Ngemva kokufukulwa, ukumuncwa kwengxube yokusabela kwalinganiswa ku-430 nm kusetshenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, Japan). Ukuhlushwa kwe-flavonoids encibilikayo iyonke ezikhishweni zesampula kwanqunywa kusetshenziswa i-rutin calibration curve (TCI America, Portland, OR, USA) bese kuvezwa njengama-milligrams e-rutin equivalent per gram of fresh weight (mg RE g-1 fresh weight).
Okuqukethwe kwe-amino acid yamahhala okuphelele kwamaqabunga ebhontshisi kunqunywe kusetshenziswa i-reagent ye-ninhydrin eguquliwe (i-Thermo Scientific Chemicals, i-Waltham, i-MA, i-USA) ngokusekelwe endleleni ephakanyiswe nguYokoyama noHiramatsu (2003) futhi kwaguqulwa nguSun et al. (2006). Kafushane, i-0.1 g yezicubu ezigayiwe yakhishwa nge-pH 5.4 buffer, kwathi i-200 μL ye-supernatant yaphendulwa nge-200 μL ye-ninhydrin (2%) kanye ne-200 μL ye-pyridine (10%; i-Spectrum Chemical, i-New Brunswick, i-NJ, i-USA), yafakwa emanzini abilayo imizuzu engama-30, yabe isipholiswa futhi yalinganiswa ku-580 nm kusetshenziswa i-UV-160A spectrophotometer (i-Shimadzu Corporation, i-Japan). Ngakolunye uhlangothi, i-proline yanqunywa ngendlela kaBates (i-Bates et al., 1973). I-Proline ikhishwe nge-3% ye-sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) kwathi ngemva kokuyicubungula, i-0.5 ml ye-supernatant yaxutshwa ne-1 ml ye-glacial acetic acid (Fisher Scientific, Hampton, NH, USA) kanye ne-ninhydrin reagent, yafakwa ku-90°C imizuzu engama-45, yapholiswa futhi yalinganiswa ku-520 nm kusetshenziswa i-spectrophotometer efanayo naleyo engenhla. Ama-amino acid amahhala kanye ne-proline ephelele kuma-extracts amaqabunga kunqunywe kusetshenziswa ama-glycine kanye nama-proline calibration curves (Sigma-Aldrich, St Louis, MO, USA), ngokulandelana, futhi kwavezwa njengesisindo esisha se-mg/g.
Ukuze kutholakale umsebenzi we-enzyme wama-enzyme e-antioxidant, cishe u-500 mg wezicubu ezihlanganisiwe wakhishwa nge-3 ml ye-50 mM Tris buffer (pH 7.8) equkethe i-1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) kanye ne-7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), efakwe ku-centrifuge ku-10,000 × g imizuzu engama-20 ngaphansi kwesiqandisi (4 °C), kwathi i-supernatant (i-crude enzyme extract) yaqoqwa (El-Nagar et al., 2023; Osman et al., 2023). I-Catalase (CAT) yabe isiphendulwa nge-2 ml ye-0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) kanye ne-100 μl yesisombululo se-269 mM H2O2 ukuze kutholakale umsebenzi wayo we-enzymatic ngokwendlela ye-Aebi (1984) ngokuguqulwa okuncane (El-Nagar et al., 2023; Osman et al., 2023). Umsebenzi we-enzymatic we-peroxidase (POX) oxhomeke ku-Guaiacol wanqunywa kusetshenziswa indlela ka-Harrach et al. (2009). (2008) ngokuguqulwa okuncane (El-Nagar et al., 2023; Osman et al., 2023) kanye nomsebenzi we-enzymatic we-polyphenol oxidase (PPO) wanqunywa ngemva kokusabela nge-2.2 ml ye-100 mM sodium phosphate buffer (pH 6.0), i-100 μl ye-guaiacol (amakhemikhali e-TCI, ePortland, OR, e-USA) kanye ne-100 μl ye-12 mM H2O2. Indlela yashintshwa kancane kusukela ku-(El-Nagar et al., 2023; Osman et al., 2023). Ukuhlolwa kwenziwa ngemva kokusabela nge-3 ml yesisombululo se-catechol (Thermo Scientific Chemicals, Waltham, MA, e-USA) (0.01 M) esisanda kulungiswa ku-0.1 M phosphate buffer (pH 6.0). Umsebenzi we-CAT ulinganiswe ngokuqapha ukubola kwe-H2O2 ku-240 nm (A240), umsebenzi we-POX ulinganiswe ngokuqapha ukwanda kokumuncwa ku-436 nm (A436), kanti umsebenzi we-PPO ulinganiswe ngokurekhoda ukuguquguquka kokumuncwa ku-495 nm (A495) njalo ngemizuzwana engama-30 imizuzu emi-3 kusetshenziswa i-UV-160A spectrophotometer (Shimadzu, Japan).
I-RT-PCR yesikhathi sangempela yasetshenziswa ukuthola amazinga okubhalwa kwezakhi zofuzo ezintathu ezihlobene ne-antioxidant, okuhlanganisa i-peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), i-superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), kanye ne-glutathione reductase (PvGR; GenBank Accession No. KY195009.1), emaqabungeni ebhontshisi (amaqabunga esibili nawesithathu akhule ngokugcwele kusukela phezulu) emahoreni angu-72 ngemva kokwelashwa kokugcina. Kafushane nje, i-RNA yahlukaniswa kusetshenziswa i-Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) ngokwephrothokholi yomenzi. Ngemuva kwalokho, i-cDNA yahlanganiswa kusetshenziswa i-TOP script™ cDNA Synthesis Kit ngokwemiyalelo yomenzi. Ukulandelana kokuqala kwezakhi zofuzo ezintathu ezingenhla kubhalwe kuThebula Elingeziwe S3. I-PvActin-3 (inombolo yokungena kwe-GenBank: XM_068616709.1) yasetshenziswa njenge-housekeeping gene kanti ukubonakaliswa kwe-gene okuhlobene kwabalwa kusetshenziswa indlela ye-2-ΔΔCT (Livak noSchmittgen, 2001). Ukuzinza kwe-Actin ngaphansi kokucindezeleka kwe-biotic (ukuxhumana okungahambisani phakathi kwemifino evamile kanye ne-anthracnose fungus Colletotrichum lindemuthianum) kanye nokucindezeleka kwe-abiotic (isomiso, usawoti, izinga lokushisa eliphansi) kwaboniswa (Borges et al., 2012).
Ekuqaleni senze ukuhlaziywa kwe-silico okubanzi kwe-genome kwamaprotheni e-oxaloacetate acetylhydrolase (OAH) ku-S. sclerotiorum sisebenzisa ithuluzi le-BLAST le-protein-protein (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Kafushane, sisebenzise i-OAH evela ku-Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; inombolo yokungena ye-GenBank XP_040799428.1; ama-amino acid angu-342) kanye ne-Penicillium lagena (PlOAH; taxide: 94218; inombolo yokungena ye-GenBank XP_056833920.1; ama-amino acid angu-316) njengochungechunge lwemibuzo ukuze kudwetshwe iphrotheni efana ne-S. sclerotiorum (taxide: 5180). I-BLASTp yenziwe ngokumelene nedatha ye-genome ye-S. sclerotiorum etholakala muva nje ku-GenBank kuwebhusayithi ye-National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Ngaphezu kwalokho, i-gene ye-OAH ebikezelwe evela 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 kwacatshangwa kusetshenziswa indlela ye-maximum likelihood ku-MEGA11 (Tamura et al., 2021) kanye nemodeli esekelwe ku-JTT matrix (Jones et al., 1992). Umuthi we-phylogenetic wahlanganiswa nokuhlaziywa kwe-multiple alignment kwe-protein sequences yazo zonke izakhi zofuzo ze-OAH ezibikezelwe (SsOAH) ezivela ku-S. sclerotiorum kanye nochungechunge lwemibuzo kusetshenziswa i-Constraint-Based Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos no-Agarwala, 2007). Ngaphezu kwalokho, ukulandelana okuhle kakhulu kwe-amino acid kwe-SsOAH okuvela ku-S. sclerotiorum kwahambisana nokulandelana kwemibuzo (AfOAH kanye ne-PlOAH) (Larkin et al., 2007) kusetshenziswa i-ClustalW (http://www.genome.jp/tools-bin/clustalw), kanti izindawo ezigciniwe ekulinganisweni zabonakala kusetshenziswa ithuluzi le-ESPRipt (inguqulo 3.0; https://espript.ibcp.fr/ESPRipt/ESPRipt/index.php).
Ngaphezu kwalokho, izizinda ezimele umsebenzi ezibikezelwe kanye nezindawo ezilondoloziwe ze-S. sclerotiorum SsOAH zahlukaniswa ngemindeni ehlukene kusetshenziswa ithuluzi le-InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Okokugcina, ukumodelwa kwesakhiwo esinezinhlangothi ezintathu (3D) se-S. sclerotiorum SsOAH esibikezelwe kwenziwa kusetshenziswa i-Protein Homology/Analogy Recognition Engine (iseva ye-Phyre2 inguqulo 2.0; http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) (Kelley et al., 2015) futhi kwaqinisekiswa kusetshenziswa iseva ye-SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). Izakhiwo ezibikezelwe ezinobukhulu obuthathu (ifomethi ye-PDB) zaboniswa ngokuxhumana kusetshenziswa iphakheji ye-UCSF-Chimera (inguqulo 1.15; https://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004).
I-PCR ye-fluorescence yesikhathi sangempela elinganiselwe yasetshenziswa ukunquma izinga lokubhalwa kwe-oxaloacetate acetylhydrolase (SsOAH; inombolo yokungena ye-GenBank: XM_001590428.1) ku-mycelia ye-Sclerotinia sclerotiorum. Kafushane, i-S. sclerotiorum yafakwa ebhodleleni eliqukethe i-PDB yafakwa ku-incubator enyakazayo (imodeli: I2400, New Brunswick Scientific Co., Edison, NJ, USA) ku-25 ± 2 °C amahora angama-24 ngo-150 rpm kanye nasebumnyameni obuhlala njalo (amahora angama-24) ukuze kukhuthazwe ukukhula kwe-mycelial. Ngemva kwalokho, amaseli aphathwa nge-L-ornithine kanye ne-fungicide i-Rizolex-T ekugxilweni kokugcina kwe-IC50 (cishe ama-40 no-3.2 mg/L, ngokulandelana) bese kukhuliswa amanye amahora angama-24 ngaphansi kwezimo ezifanayo. Ngemva kokufukamela, ama-culture afakwa ku-centrifuge ngo-2500 rpm imizuzu emi-5 kanti i-supernatant (i-fungal mycelium) yaqoqwa ukuze kuhlaziywe ukubonakaliswa kwezakhi zofuzo. Ngokufanayo, i-fungal mycelium yaqoqwa emahoreni angu-0, 24, 48, 72, 96, kanye namahora angu-120 ngemva kokutheleleka ezitshalweni ezithelelekile ezazakhe isikhunta esimhlophe kanye ne-cottony mycelium ebusweni bezicubu ezithelelekile. I-RNA yakhishwa ku-fungal mycelium kwabe sekwenziwa i-cDNA njengoba kuchaziwe ngenhla. Ukulandelana kwe-primer kwe-SsOAH kubhalwe ku-Supplementary Table S3. I-SsActin (inombolo yokungena kwe-GenBank: XM_001589919.1) yasetshenziswa njengezakhi zofuzo zokunakekela indlu, futhi ukubonakaliswa kwezakhi zofuzo okuhlobene kwabalwa kusetshenziswa indlela ye-2-ΔΔCT (Livak and Schmittgen, 2001).
I-oxalic acid yatholakala kumhluzi we-potato dextrose (PDB) kanye namasampula ezitshalo aqukethe i-fungus pathogen i-Sclerotinia sclerotiorum ngokwendlela ka-Xu no-Zhang (2000) ngokuguqulwa okuncane. Kafushane nje, ama-isolates e-S. sclerotiorum afakwa emaflaskini aqukethe i-PDB abese ekhuliswa ku-shaking incubator (imodeli I2400, i-New Brunswick Scientific Co., i-Edison, NJ, e-USA) ngesivinini esingu-150 rpm ku-25 ± 2 °C izinsuku ezingu-3-5 ebumnyameni obuhlala njalo (amahora angu-24) ukuze kukhuthazwe ukukhula kwe-mycelial. Ngemva kokufukamela, isiko le-fungus lahlungwa kuqala ngephepha lokuhlunga le-Whatman #1 bese lifakwa ku-centrifuge ku-2500 rpm imizuzu emi-5 ukuze kususwe i-mycelium esele. I-supernatant yaqoqwa futhi yagcinwa ku-4°C ukuze kutholakale i-oxalate eyengeziwe. Ukuze kulungiselelwe amasampula ezitshalo, cishe u-0.1 g wezingcezu zezicubu zezitshalo zakhishwa kathathu ngamanzi acwengekile (2 ml isikhathi ngasinye). Amasampula abe esefakwa ku-centrifuge ku-2500 rpm imizuzu emi-5, i-supernatant yahlungwa ngephepha lokuhlunga le-Whatman No. 1 futhi yaqoqwa ukuze ihlaziywe kabanzi.
Ukuze kuhlaziywe inani le-oxalic acid, ingxube yokusabela yalungiswa ngepayipi elivalwe ngengilazi ngokulandelana okulandelayo: 0.2 ml yesampula (noma i-PDB culture filtrate noma isixazululo esijwayelekile se-oxalic acid), 0.11 ml ye-bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), 0.198 ml ye-1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) kanye no-0.176 ml we-100 mM potassium dichromate (K2Cr2O7; TCI chemicals, Portland, OR, USA), bese ikhambi lixutshwe ku-4.8 ml ngamanzi acwecwe, lixutshwe ngamandla bese lifakwa ngokushesha ebhavini lamanzi elingu-60 °C. Ngemva kwemizuzu eyi-10, ukusabela kwamiswa ngokungeza u-0.5 ml wekhambi le-sodium hydroxide (NaOH; 0.75 M). Ukumuncwa (A600) kwengxube yokusabela kukalwe ku-600 nm kusetshenziswa i-UV-160 spectrophotometer (Shimadzu Corporation, Japan). I-PDB kanye namanzi acwengekile asetshenziswa njengezilawuli zokulinganisa ama-culture filtrates namasampula ezitshalo, ngokulandelana. Ukuhlushwa kwe-oxalic acid kuma-culture filtrates, okuvezwe njengama-microgram e-oxalic acid nge-milliliter ye-PDB medium (μg.mL−1), kanye nasezikhutsheni zamaqabunga, okuvezwe njengama-microgram e-oxalic acid nge-gram yesisindo esisha (μg.g−1 FW), kunqunywe kusetshenziswa i-oxalic acid calibration curve (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
Kuyo yonke ucwaningo, zonke izivivinyo zaklanywa ngendlela engahleliwe ngokuphelele (i-CRD) enezikopi eziyisithupha zezinto eziphilayo ngokwelashwa ngakunye kanye nezimbiza ezinhlanu ngekhophi ngayinye yezinto eziphilayo (izitshalo ezimbili ngembiza ngayinye) ngaphandle kokuthi kuchazwe ngenye indlela. Izikopi ezimbili zezinto eziphilayo zahlaziywa ngokuphindwe kabili (izikopi ezimbili zobuchwepheshe). Izikopi ezimbili zobuchwepheshe zasetshenziswa ukuhlola ukuphindaphindwa kokuhlolwa okufanayo kodwa azisetshenziswanga ekuhlaziyweni kwezibalo ukuze kugwenywe izikopi ezingamanga. Idatha yahlaziywa ngokwezibalo kusetshenziswa ukuhlaziywa kokuhlukahluka (i-ANOVA) kulandelwa ukuhlolwa kwe-Tukey-Kramer okubalulekile ngokweqiniso (i-HSD) (p ≤ 0.05). Kokuhlolwa kwe-in vitro, amanani e-IC50 kanye ne-IC99 abalwa kusetshenziswa imodeli ye-probit kanye nezikhawu zokuzethemba ezingama-95%.
Kwaqoqwa ama-isolate amane avela emasimini ahlukene kabhontshisi e-El Ghabiya Province, eGibhithe. Ku-PDA medium, wonke ama-isolate akhiqiza i-mycelium emhlophe ekhilimu eyasheshe yaphenduka yaba mhlophe njengekotini (Isithombe 1A) bese iba beige noma nsundu esigabeni se-sclerotium. Ama-Sclerotia avame ukuba makhulu, amnyama, ayindilinga noma angajwayelekile ngesimo, angama-5.2 kuya ku-7.7 mm ubude kanye nobubanzi obungu-3.4 kuya ku-5.3 mm (Isithombe 1B). Nakuba ama-isolate amane athuthukise iphethini engezansi ye-sclerotia emaphethelweni e-culture medium ngemva kwezinsuku eziyi-10-12 zokufukamela ku-25 ± 2 °C (Isithombe 1A), inani le-sclerotia ngepuleti ngalinye lalihluke kakhulu phakathi kwawo (P < 0.001), lapho i-isolate 3 inenani eliphakeme kakhulu le-sclerotia (32.33 ± 1.53 sclerotia ngepuleti ngalinye; Isithombe 1C). Ngokufanayo, i-isolate #3 ikhiqize i-oxalic acid eningi ku-PDB kunezinye i-isolate (3.33 ± 0.49 μg.mL−1; Isithombe 1D). I-Isolate #3 ibonise izici ezijwayelekile ze-morphological kanye ne-microscopic ze-phytopathogenic fungus i-Sclerotinia sclerotiorum. Isibonelo, ku-PDA, amakholoni e-isolate #3 akhula ngokushesha, ayemhlophe okhilimu (Isithombe 1A), e-beige ephendukayo noma e-salmon ekhanyayo ephuzi-nsundu, futhi kwadingeka izinsuku ezingu-6-7 ku-25 ± 2°C ukuze amboze ngokuphelele ubuso bepuleti elingu-9 cm ububanzi. Ngokusekelwe ezicini ze-morphological kanye ne-microscopic ezingenhla, i-isolate #3 yatholakala njenge-Sclerotinia sclerotiorum.
Isithombe 1. Izici kanye nokugula kwe-S. sclerotiorum isolates ezivela ezitshalweni ezivamile zemidumba. (A) Ukukhula kwe-mycelial kwe-S. sclerotiorum isolates ezine ku-PDA medium, (B) i-sclerotia ye-S. sclerotiorum isolates ezine, (C) inani le-sclerotia (ngepuleti ngalinye), (D) ukukhiqizwa kwe-oxalic acid ku-PDB medium (μg.mL−1), kanye (E) ubunzima besifo (%) se-S. sclerotiorum isolates ezine ku-legume cultivar ethengiswayo esengozini yokukhula ngaphansi kwezimo zokushisa. Amanani amelela isilinganiso ± SD sezinhlobo ezinhlanu zebhayoloji (n = 5). Izinhlamvu ezahlukene zibonisa umehluko obalulekile ngokwezibalo phakathi kokwelashwa (p < 0.05). (F–H) Izimpawu ezijwayelekile zesikhunta esimhlophe zivele eziqwini ezingenhla komhlaba kanye ne-silique, ngokulandelana, ezinsukwini eziyi-10 ngemuva kokugonywa nge-isolate #3 (dpi). (I) Ukuhlaziywa kokuziphendukela kwemvelo kwesifunda se-internal transcribed spacer (ITS) se-S. sclerotiorum isolate #3 kwenziwe kusetshenziswa indlela ye-maximum likelihood futhi kwaqhathaniswa nama-reference isolates/strains angu-20 atholwe ku-National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). Izinombolo ezingaphezu kwemigqa ye-clustering zibonisa ukumbozwa kwesifunda (%), kanti izinombolo ezingaphansi kwemigqa ye-clustering zibonisa ubude begatsha.
Ngaphezu kwalokho, ukuqinisekisa ukuthi lokhu kubangelwa yizifo, kwasetshenziswa ama-isolate amane e-S. sclerotiorum atholakale ukugoma uhlobo lwebhontshisi olusengozini yokuhweba i-Giza 3 ngaphansi kwezimo zokushisa, okuhambisana nama-postulates kaKoch (Isithombe 1E). Nakuba wonke ama-isolate e-fungus atholakale ayeyi-pathogenic futhi angatheleleka kubhontshisi oluhlaza (cv. Giza 3), okubangela izimpawu zesikhunta esimhlophe ezivamile kuzo zonke izingxenye ezingenhla komhlabathi (Isithombe 1F), ikakhulukazi eziqwini (Isithombe 1G) kanye nama-pod (Isithombe 1H) ezinsukwini eziyi-10 ngemva kokugoma (dpi), i-isolate 3 yayiyi-isolate enamandla kakhulu ekuhlolweni okubili okuzimele. I-Isolate 3 yayinobunzima obukhulu besifo (%) ezitshalweni zebhontshisi (24.0 ± 4.0, 58.0 ± 2.0, kanye no-76.7 ± 3.1 ezinsukwini eziyi-7, 14, kanye nezinsuku ezingama-21 ngemva kokungenwa yizifo, ngokulandelana; Isibalo 1F).
Ukuhlonza i-S. sclerotiorum isolate #3 ehlasela kakhulu kwaqinisekiswa ngokususelwa ekulandeleni kwe-internal transcribed spacer (ITS) (Isithombe 1I). Ukuhlaziywa kwe-phylogenetic phakathi kwe-isolate #3 kanye ne-20 reference isolates/strains kubonise ukufana okuphezulu (>99%) phakathi kwazo. Kubalulekile ukuqaphela ukuthi i-S. sclerotiorum isolate #3 (533 bp) inokufana okuphezulu ne-American S. sclerotiorum isolate LPM36 ehlukaniswe nembewu ye-pea eyomile (inombolo ye-GenBank accession MK896659.1; 540 bp) kanye ne-Chinese S. sclerotiorum isolate YKY211 (inombolo ye-GenBank accession OR206374.1; 548 bp), okubangela ukubola kwe-stem violet (Matthiola incana), konke okuqoqwe ngokwahlukana phezulu kwe-dendrogram (Isithombe 1I). Uchungechunge olusha lufakwe kusizindalwazi se-NCBI futhi luqanjwe ngokuthi “i-Sclerotinia sclerotiorum – isolate YN-25” (inombolo yokungena ye-GenBank i-PV202792). Kungabonakala ukuthi isolate 3 iyona isolate ehlasela kakhulu; ngakho-ke, le isolate yakhethwa ukuze ifundwe kuzo zonke izivivinyo ezalandela.
Umsebenzi we-diamine L-ornithine (Sigma-Aldrich, Darmstadt, eJalimane) ngamanani ahlukene (12.5, 25, 50, 75, 100 kanye no-125 mg/L) ngokumelene ne-S. sclerotiorum isolate 3 uhlolwe ngaphakathi kwe-vitro. Kuyaphawuleka ukuthi i-L-ornithine ibe nomphumela wokulwa namagciwane futhi yavimbela kancane kancane ukukhula kwe-radial kwe-S. sclerotiorum hyphae ngendlela encike kumthamo (Isithombe 2A, B). Ekuhlungeni okuphezulu okuvivinywe (125 mg/L), i-L-ornithine ibonise izinga eliphezulu kakhulu lokuvinjelwa kokukhula kwe-mycelial (99.62 ± 0.27%; Isithombe 2B), elilingana ne-fungicide yezentengiselwano i-Rizolex-T (izinga lokuvinjelwa 99.45 ± 0.39%; Isithombe 2C) ekuhlungeni okuphezulu okuvivinywe (10 mg/L), okubonisa ukusebenza okufanayo.
Umfanekiso 2. Umsebenzi we-in vitro wokulwa namagciwane we-L-ornithine ngokumelene ne-Sclerotinia sclerotiorum. (A) Ukuqhathaniswa komsebenzi wokulwa namagciwane wokuhlushwa okuhlukene kwe-L-ornithine ngokumelene ne-S. sclerotiorum ne-fungicide yezentengiselwano i-Rizolex-T (10 mg/L). (B, C) Izinga lokuvinjelwa (%) kokukhula kwe-mycelial ye-S. sclerotiorum ngemuva kokwelashwa ngokuhlushwa okuhlukile kwe-L-ornithine (12.5, 25, 50, 75, 100 kanye no-125 mg/L) noma i-Rizolex-T (2, 4, 6, 8 kanye no-10 mg/L), ngokulandelana. Amanani amelela isilinganiso ± SD seziphindaphindo ezinhlanu zebhayoloji (n = 5). Izinhlamvu ezahlukene zibonisa umehluko wezibalo phakathi kokwelashwa (p < 0.05). (D, E) Ukuhlaziywa kokuhlehliswa kwemodeli ye-Probit kwe-L-ornithine kanye ne-fungicide yezentengiselwano i-Rizolex-T, ngokulandelana. Umugqa wokuhlehla wemodeli ye-probit uboniswa njengomugqa oluhlaza okwesibhakabhaka oqinile, kanti isikhawu sokuzethemba (95%) siboniswa njengomugqa obomvu onezingqimba.
Ngaphezu kwalokho, kwenziwa ukuhlaziywa kokuhlehla kwe-probit futhi ama-plot ahambisanayo aboniswe kuThebula 1 kanye neZithombe 2D,E. Kafushane, inani le-slope elamukelekayo (y = 2.92x - 4.67) kanye nezibalo ezibalulekile ezihlobene (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 kanye no-p < 0.0001; Umfanekiso 2D) we-L-ornithine libonise umsebenzi othuthukisiwe we-antifungal ngokumelene ne-S. sclerotiorum uma kuqhathaniswa ne-fungicide yezentengiselwano i-Rizolex-T (y = 1.96x - 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 kanye no-p < 0.0001) (Ithebula 1).
Ithebula 1. Amanani okuhlushwa kwesigamu esiphezulu sokuvimbela (IC50) kanye ne-IC99 (mg/l) ye-L-ornithine kanye ne-fungicide yezentengiselwano "i-Rizolex-T" ngokumelene ne-S. sclerotiorum.
Sekukonke, i-L-ornithine (250 mg/L) inciphise kakhulu ukukhula kanye nobunzima besikhunta esimhlophe ezitshalweni zikabhontshisi ezivamile eziphathwe kahle uma kuqhathaniswa nezitshalo ezinegciwane le-S. sclerotiorum ezingaphathwanga kahle (ukulawula; Isithombe 3A). Kafushane, yize ubunzima besifo sezitshalo ezilawula izifo ezingaphathwanga kahle bukhuphuka kancane kancane (52.67 ± 1.53, 83.21 ± 2.61, kanye no-92.33 ± 3.06%), i-L-ornithine inciphise kakhulu ubunzima besifo (%) kulo lonke ukuhlolwa (8.97 ± 0.15, 18.00 ± 1.00, kanye no-26.36 ± 3.07) ezinsukwini ezingu-7, 14, kanye no-21 ngemuva kokwelashwa (dpt), ngokulandelana (Isithombe 3A). Ngokufanayo, lapho izitshalo zobhontshisi ezinegciwane le-S. sclerotiorum zelashwa nge-250 mg/L L-ornithine, indawo engaphansi kwejika lokuqhubeka kwesifo (AUDPC) yehle isuka ku-1274.33 ± 33.13 kulawulo olungelashwanga yaya ku-281.03 ± 7.95, eyayiphansi kancane kuneyomuthi we-fungicide we-Rizolex-T ongu-50 mg/L (183.61 ± 7.71; Umfanekiso 3B). Umkhuba ofanayo wabonwa ekuhlolweni kwesibili.
Isithombe 3. Umphumela wokusetshenziswa kwe-L-ornithine ngaphandle ekuthuthukisweni kokubola okumhlophe kwebhontshisi evamile okubangelwa yi-Sclerotinia sclerotiorum ngaphansi kwezimo zokushisa. (A) Ijika lokuqhubeka kwesifo lesikhunta esimhlophe sebhontshisi evamile ngemva kokwelashwa nge-250 mg/L L-ornithine. (B) Indawo engaphansi kwejika lokuqhubeka kwesifo (AUDPC) lesikhunta esimhlophe sebhontshisi evamile ngemva kokwelashwa nge-L-ornithine. Amanani amelela isilinganiso ± SD sezinhlobo ezinhlanu zebhayoloji (n = 5). Izinhlamvu ezahlukene zibonisa umehluko obalulekile ngokwezibalo phakathi kokwelashwa (p < 0.05).
Ukusetshenziswa kwe-250 mg/L L-ornithine ngaphandle kwandise kancane kancane ukuphakama kwesitshalo (Isithombe 4A), inani lamagatsha ngesitshalo ngasinye (Isithombe 4B), kanye nenani lamaqabunga ngesitshalo ngasinye (Isithombe 4C) ngemva kwezinsuku ezingu-42. Ngenkathi i-Rizolex-T (50 mg/L) yesikhunta esithengiswayo ibe nomthelela omkhulu kuzo zonke izilinganiso zokudla okunempilo ezifundwe, ukusetshenziswa kwe-250 mg/L L-ornithine ngaphandle kwaba nomphumela wesibili omkhulu uma kuqhathaniswa nezilawuli ezingalashwanga (Izithombe 4A–C). Ngakolunye uhlangothi, ukwelashwa kwe-L-ornithine akuzange kube nomthelela omkhulu kokuqukethwe kwe-photosynthetic pigments chlorophyll a (Isithombe 4D) kanye ne-chlorophyll b (Isithombe 4E), kodwa kwandise kancane okuqukethwe kwe-carotenoid iyonke (0.56 ± 0.03 mg/g fr wt) uma kuqhathaniswa nokulawula okungekuhle (0.44 ± 0.02 mg/g fr wt) kanye nokulawula okuhle (0.46 ± 0.02 mg/g fr wt; Isithombe 4F). Sekukonke, le miphumela ikhombisa ukuthi i-L-ornithine ayinabo ubuthi be-phytotoxic kuma-legume aphathwe futhi ingakhuthaza nokukhula kwawo.
Isithombe 4. Umphumela wokusetshenziswa kwe-L-ornithine yangaphandle ezicini zokukhula kanye nemibala ye-photosynthetic yamaqabunga ebhontshisi atheleleke nge-Sclerotinia sclerotiorum ngaphansi kwezimo ze-greenhouse. (A) Ukuphakama kwesitshalo (cm), (B) Inani lamagatsha ngesitshalo ngasinye, (C) Inani lamaqabunga ngesitshalo ngasinye, (D) Okuqukethwe kwe-Chlorophyll a (mg g-1 fr wt), (E) Okuqukethwe kwe-Chlorophyll b (mg g-1 fr wt), (F) Okuqukethwe kwe-carotenoid okuphelele (mg g-1 fr wt). Amanani yi-average ± SD yama-replicates amahlanu ebhayoloji (n = 5). Izinhlamvu ezahlukene zibonisa umehluko obalulekile ngokwezibalo phakathi kokwelashwa (p < 0.05).
Indawo ye-histochemical ye-in situ yezinhlobo ze-oxygen ezisabelayo (i-ROS; evezwa njenge-hydrogen peroxide [H2O2]) kanye nama-free radicals (evezwa njenge-superoxide anions [O2•−]) kwembule ukuthi ukusetshenziswa kwe-L-ornithine (250 mg/L) ngaphandle kwe-exogenous kunciphise kakhulu ukuqongelela kwe-H2O2 (96.05 ± 5.33 nmol.g−1 FW; Umfanekiso 5A) kanye ne-O2•− (32.69 ± 8.56 nmol.g−1 FW; Umfanekiso 5B) uma kuqhathaniswa nokuqongelela kwezitshalo zombili ezithelelekile ezingalashwanga (173.31 ± 12.06 kanye no-149.35 ± 7.94 nmol.g−1 FW, ngokulandelana) kanye nezitshalo eziphathwe nge-50 mg/L ye-fungicide yezentengiselwano i-Rizolex-T (170.12 ± 9.50 kanye no-157.00 ± 7.81 nmol.g−1 fr wt, ngokulandelana) emahoreni angu-72. Amazinga aphezulu e-H2O2 kanye ne-O2•− aqongelelwe ngaphansi kwe-hpt (Isithombe 5A, B). Ngokufanayo, ukuhlolwa kwe-malondialdehyde (MDA) okusekelwe ku-TCA kubonise ukuthi izitshalo zobhontshisi ezinegciwane le-S. sclerotiorum ziqongelele amazinga aphezulu e-MDA (113.48 ± 10.02 nmol.g fr wt) emaqabunga azo (Isithombe 5C). Kodwa-ke, ukusetshenziswa kwe-L-ornithine ngaphandle kwe-L-ornithine kunciphisa kakhulu i-lipid peroxidation njengoba kufakazelwa ukwehla kokuqukethwe kwe-MDA ezitshalweni eziphathwe kahle (33.08 ± 4.00 nmol.g fr wt).
Isithombe 5. Umphumela wokusetshenziswa kwe-L-ornithine yangaphandle ezimpawini ezinkulu zokucindezeleka kwe-oxidative kanye nezindlela zokuvikela i-antioxidant ezingezona eze-enzyme emaqabungeni ebhontshisi atheleleke nge-S. sclerotiorum emahoreni angu-72 ngemuva kokutheleleka ngaphansi kwezimo 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) i-malondialdehyde (MDA; nmol g−1 FW) ku-72 hpt, (D) ama-phenol aphelele ancibilikayo (mg GAE g−1 FW) ku-72 hpt, (E) ama-flavonoid aphelele ancibilikayo (mg RE g−1 FW) ku-72 hpt, (F) ama-amino acid aphelele amahhala (mg g−1 FW) ku-72 hpt, kanye (G) okuqukethwe kwe-proline (mg g−1 FW) ku-72 hpt. Amanani amelela isilinganiso ± ukuphambuka okujwayelekile (isilinganiso ± SD) samakhophi ayi-5 ezinto eziphilayo (n = 5). Izinhlamvu ezahlukene zibonisa umehluko obalulekile ngokwezibalo phakathi kokwelashwa (p < 0.05).