Eli nqaku liyinxalenye yesihloko sophando esithi “Ukuphucula ukumelana kweembotyi kwizifo ezibangela izifo kunye nezinambuzane”, jonga onke amanqaku ama-5
I-agent ebangela isifo sezityalo ezibangelwa yifungus necrosis I-Sclerotinia sclerotiorum (Lib.) de Bary isebenzisa icebo elinamanqanaba amaninzi ukosulela izityalo ezahlukeneyo ezihlala kuyo. Olu phononongo lucebisa ukusetyenziswa kwe-diamine L-ornithine, i-amino acid engeyiyo iproteni ekhuthaza ukwenziwa kwezinye ii-amino acid ezibalulekileyo, njengecebo lolawulo olulolunye ukuphucula iimpendulo ze-molecular, physiological kunye ne-biochemical ze-Phaseolus vulgaris L. kwi-white mold ebangelwa yi-Pseudomonas sclerotiorum. Uvavanyo lwe-in vitro lubonise ukuba i-L-ornithine ithintele kakhulu ukukhula kwe-mycelial ye-S. pyrenoidosa ngendlela exhomekeke kwidosi. Ngaphezu koko, inokunciphisa kakhulu ubunzima be-white mold phantsi kweemeko ze-greenhouse. Ngaphezu koko, i-L-ornithine ikhuthaze ukukhula kwezityalo ezinyangiweyo, nto leyo ebonisa ukuba ubuninzi be-L-ornithine obuvavanyiweyo abuzange bube yingozi kwizityalo ezinyangiweyo. Ukongeza, i-L-ornithine yonyuse ukubonakaliswa kwee-antioxidants ezingezizo ii-enzyme (ii-phenolics kunye nee-flavonoids ezinyibilikayo ngokupheleleyo) kunye nee-enzyme antioxidants (i-catalase (CAT), i-peroxidase (POX), kunye ne-polyphenol oxidase (PPO)), kwaye yonyusa ukubonakaliswa kweejini ezintathu ezinxulumene ne-antioxidant (i-PvCAT1, i-PvSOD, kunye ne-PvGR). Ngaphezu koko, kuhlalutyo lwe-silico kutyhile ubukho beproteni ye-oxaloacetate acetylhydrolase (SsOAH) kwi-genome ye-S. sclerotiorum, eyayifana kakhulu neeproteni ze-oxaloacetate acetylhydrolase (SsOAH) ze-Aspergillus fijiensis (AfOAH) kunye ne-Penicillium sp. (PlOAH) ngokubhekiselele kuhlalutyo olusebenzayo, iindawo ezigciniweyo, kunye ne-topology. Okunomdla kukuba, ukongezwa kwe-L-ornithine kwi-potato dextrose broth (PDB) medium kunciphisa kakhulu ukubonakaliswa kwe-SsOAH gene kwi-S. sclerotiorum mycelia. Ngokufanayo, ukusetyenziswa kwe-L-ornithine ngaphandle kwezityalo kunciphisa kakhulu ukubonakaliswa kwe-SSOAH gene kwi-fungus mycelia eqokelelwe kwizityalo ezinyangiweyo. Okokugqibela, ukusetyenziswa kwe-L-ornithine kunciphisa kakhulu ukukhutshwa kwe-oxalic acid kumagqabi aphakathi e-PDB kunye namagqabi asulelekileyo. Ukuqukumbela, i-L-ornithine idlala indima ebalulekileyo ekugcineni imeko ye-redox kunye nokuphucula impendulo yokuzikhusela kwizityalo ezisulelekileyo. Iziphumo zolu phononongo zinokunceda ekuphuhliseni iindlela ezintsha nezinobuhlobo nokusingqongileyo zokulawula ukungunda okumhlophe kunye nokunciphisa impembelelo yayo kwimveliso yeembotyi kunye nezinye izityalo.
Umngundo omhlophe, obangelwa yi-necrotrophic fungus iSclerotinia sclerotiorum (Lib.) de Bary, sisifo esitshabalalisayo nesinciphisa isivuno esibeka umngcipheko omkhulu kwimveliso yeembotyi zehlabathi (iPhaseolus vulgaris L.) (Bolton et al., 2006). I-Sclerotinia sclerotiorum yenye yezona pathogens zezityalo zefungus ezinzima kakhulu ukuzilawula, inoluhlu olubanzi lweentlobo zezityalo ezingaphezu kwama-600 kunye nokukwazi ukuqhekeza ngokukhawuleza izicubu zomsingathi ngendlela engacacanga (Liang and Rollins, 2018). Phantsi kweemeko ezingathandekiyo, idlula kwinqanaba elibalulekileyo lomjikelo wayo wobomi, ihlala ingasebenzi ixesha elide njengezakhiwo ezimnyama, eziqinileyo, ezifana nembewu ezibizwa ngokuba yi-'sclerotia' emhlabeni okanye njengezityalo ezimhlophe, ezithambileyo kwi-mycelium okanye isiqu sesityalo esinezifo (Schwartz et al., 2005). I-S. sclerotiorum inako ukwenza i-sclerotia, evumela ukuba iphile kwiindawo ezinesifo ixesha elide kwaye iqhubeke ngexesha lesifo (Schwartz et al., 2005). I-Sclerotia ityebile kwizakhamzimba, inokuhlala emhlabeni ixesha elide, kwaye isebenza njenge-inoculum ephambili yosulelo olulandelayo (Schwartz et al., 2005). Phantsi kweemeko ezilungileyo, i-sclerotia iyahluma kwaye ivelise ii-spores eziphuma emoyeni ezinokosulela zonke iindawo zesityalo ezingaphezulu komhlaba, kuquka kodwa kungaphelelanga apho kwiintyatyambo, iziqu, okanye ii-pods (Schwartz et al., 2005).
I-Sclerotinia sclerotiorum isebenzisa icebo elinamanqanaba amaninzi ukosulela izityalo ezihlala kuyo, elibandakanya uthotho lweziganeko ezidibeneyo ukusuka ekuhlumeni kwe-sclerotial ukuya ekuphuhlisweni kweempawu. Ekuqaleni, i-S. sclerotiorum ivelisa ii-spores ezixhonyiweyo (ezibizwa ngokuba yi-ascospores) ezivela kwizakhiwo ezifana neekhowa ezibizwa ngokuba yi-apothecia, eziba nomoya kwaye zikhule zibe yi-sclerotia enganyakaziyo kwinkunkuma yezityalo esulelekileyo (Bolton et al., 2006). Emva koko le fungus ikhupha i-oxalic acid, into ebangela ubukho bentsholongwane, ukulawula i-pH yodonga lweseli yesityalo, ukukhuthaza ukuwohloka kwe-enzymatic kunye nokuhlasela kwezicubu (Hegedus kunye noRimmer, 2005), kunye nokuthintela ukuqhuma kwe-oxidative kwesityalo esihlala kuyo. Le nkqubo yokwenza i-acid ibuthathaka udonga lweseli yesityalo, ibonelela ngendawo efanelekileyo yokusebenza okuqhelekileyo nokusebenza kakuhle kwee-enzymes eziwohlozayo zodonga lweseli yefungus (CWDEs), ivumela i-pathogen ukuba yoyise umqobo womzimba kwaye ingene kwizicubu ezihlala kuyo (Marciano et al., 1983). Xa sele ingenile, i-S. sclerotiorum ikhupha inani le-CWDE, ezifana ne-polygalacturonase kunye ne-cellulase, ezenza kube lula ukusasazeka kwayo kwizicubu ezisulelekileyo kwaye zibangele i-tissue necrosis. Ukuqhubela phambili kwezilonda kunye nee-hyphal mats kukhokelela kwiimpawu ezibonakalayo ze-white mold (Hegedus kunye noRimmer, 2005). Okwangoku, izityalo ezihlala kwindawo yazo ziyazibona iipatheni ze-molecular ezinxulumene ne-pathogen (ii-PAMP) ngokusebenzisa ii-receptors ze-pattern recognition (ii-PRR), nto leyo ebangela uthotho lweziganeko zesignali ezithi ekugqibeleni zisebenze iimpendulo zokuzikhusela.
Nangona kukho amashumi eminyaka emizamo yokulawula izifo, ukunqongophala kwe-germplasm efanelekileyo enganyangekiyo kusaqhubeka kwiimbotyi, njengakwezinye izityalo zorhwebo, ngenxa yokuxhathisa, ukusinda, kunye nokuziqhelanisa ne-pathogen. Ngoko ke, ulawulo lwezifo lunzima kakhulu kwaye lufuna icebo elidibeneyo nelineenkalo ezininzi eliquka indibaniselwano yeendlela zenkcubeko, ulawulo lwebhayoloji, kunye ne-fungicides yamakhemikhali (O'Sullivan et al., 2021). Ulawulo lwamakhemikhali lwe-white mold lolona lusebenzayo kuba i-fungicides, xa isetyenziswa ngokuchanekileyo nangexesha elifanelekileyo, inokulawula ngokufanelekileyo ukusasazeka kwesifo, inciphise ubunzima bosulelo, kwaye inciphise ilahleko yesivuno. Nangona kunjalo, ukusetyenziswa kakhulu kunye nokuxhomekeka kakhulu kwi-fungicides kunokukhokelela ekuveleni kweentlobo ezinganyangekiyo ze-S. sclerotiorum kwaye kuchaphazele kakubi izinto eziphilayo ezingajoliswanga, impilo yomhlaba, kunye nomgangatho wamanzi (Le Cointe et al., 2016; Ceresini et al., 2024). Ke ngoko, ukufumana ezinye iindlela ezinobuhlobo nokusingqongileyo kuye kwaba yinto ephambili.
Iipolyamines (ii-PA), ezifana ne-putrescine, i-spermidine, i-spermine, kunye ne-cadaverine, zinokusebenza njengeendlela ezithembisayo zokulwa nezifo ezithwalwa zizityalo emhlabeni, ngaloo ndlela zinciphisa ngokupheleleyo okanye ngokuyinxenye ukusetyenziswa kwee-fungicides eziyingozi zeekhemikhali (Nehela et al., 2024; Yi et al., 2024). Kwizityalo eziphezulu, ii-PA zibandakanyeka kwiinkqubo ezininzi ze-physiological kuquka, kodwa kungaphelelanga apho, ukwahlulwa kweeseli, ukwahlukana, kunye nokuphendula kwiingcinezelo ze-abiotic kunye ne-biotic (Killiny kunye neNehela, 2020). Zingasebenza njengee-antioxidants, zincede ekususeni iintlobo zeoksijini ezisebenzayo (i-ROS), zigcine i-redox homeostasis (i-Nehela kunye ne-Killiny, 2023), zivuselele iijini ezinxulumene nokhuselo (uRomero et al., 2018), zilawule iindlela ezahlukeneyo ze-metabolic (i-Nehela kunye ne-Killiny, 2023), zilawule ii-phytohormones ze-endogenous (i-Nehela kunye ne-Killiny, 2019), zimise ukumelana ne-systemic acquired resistance (i-SAR), kwaye zilawule ukusebenzisana kwezityalo nezifo (i-Nehela kunye ne-Killiny, 2020; i-Asija et al., 2022; i-Czerwoniec, 2022). Kubalulekile ukuqaphela ukuba iindlela ezithile kunye neendima ze-PAs ekukhuseleni izityalo ziyahluka ngokuxhomekeke kwiintlobo zezityalo, iintsholongwane, kunye neemeko zokusingqongileyo. I-PA eninzi kakhulu kwizityalo yi-biosynthesized evela kwi-polyamine L-ornithine ebalulekileyo (i-Killiny kunye ne-Nehela, 2020).
I-L-ornithine idlala indima ezininzi ekukhuleni nasekuphuhlisweni kwezityalo. Umzekelo, izifundo zangaphambili zibonise ukuba kwirayisi (Oryza sativa), i-ornithine inokunxulunyaniswa nokurisayikilishwa kwe-nitrogen (Liu et al., 2018), isivuno serayisi, umgangatho kunye nevumba (Lu et al., 2020), kunye nokuphendula kuxinzelelo lwamanzi (Yang et al., 2000). Ngaphezu koko, ukusetyenziswa kwe-L-ornithine ngaphandle kwayo kuphucule kakhulu ukunyamezela imbalela kwi-beet yeswekile (Beta vulgaris) (Hussein et al., 2019) kwaye kunciphise uxinzelelo lwetyuwa kwi-anyanisi (Allium Cepa) (Çavuşoǧlu kunye ne-Çavuşoǧlu, 2021) kunye nezityalo ze-cashew (Anacardium occidentale) (da Rocha et al., 2012). Indima enokubakho ye-L-ornithine ekukhuseleni uxinzelelo lwe-abiotic inokuba ngenxa yokubandakanyeka kwayo ekuqokeleleni kwe-proline kwizityalo ezinyangiweyo. Umzekelo, iijini ezinxulumene ne-proline metabolism, ezifana ne-ornithine delta aminotransferase (delta-OAT) kunye ne-proline dehydrogenase (ProDH1 kunye ne-ProDH2) genes, ngaphambili kuye kwaxelwa ukuba zibandakanyeka ekukhuseleni i-Nicotiana benthamiana kunye ne-Arabidopsis thaliana kwiintlobo ze-Pseudomonas syringae ezingezizo ezibambekayo (Senthil-Kumar kunye neMysore, 2012). Kwelinye icala, i-fungal ornithine decarboxylase (ODC) iyadingeka ekukhuleni kwe-pathogen (Singh et al., 2020). Ukujolisa kwi-ODC ye-Fusarium oxysporum f. sp. lycopersici nge-host-induced gene silencing (HIGS) kuphucule kakhulu ukumelana kwezityalo zetamatato kwi-Fusarium wilt (Singh et al., 2020). Nangona kunjalo, indima enokubakho yokusetyenziswa kwe-ornithine yangaphandle ngokuchasene noxinzelelo lwe-biotic olufana ne-phytopathogens ayikafundwa kakuhle. Okubaluleke ngakumbi, imiphumo ye-ornithine ekuchaseni izifo kunye neziganeko ezinxulumene ne-biochemical kunye ne-physiological azikahlolwa kangako.
Ukuqonda ubunzima bosulelo lwe-S. sclerotiorum kwiimbotyi kubalulekile ekuphuhlisweni kwamaqhinga okulawula asebenzayo. Kolu phononongo, sijonge ukuchonga indima enokubakho ye-diamine L-ornithine njengento ebalulekileyo ekuphuculeni iindlela zokuzikhusela kunye nokuxhathisa izityalo ze-legume kusulelo lwe-Sclerotinia sclerotiorum. Sicinga ukuba, ukongeza ekuphuculeni iimpendulo zokuzikhusela zezityalo ezisulelekileyo, i-L-ornithine ikwadlala indima ebalulekileyo ekugcineni imeko ye-redox. Sicebisa ukuba iziphumo ezinokubakho ze-L-ornithine zinxulumene nokulawulwa kweendlela zokuzikhusela ze-enzyme kunye ne-non-enzymatic antioxidant kunye nokuphazamiseka kwezinto ze-fungus pathogenicity/virulence kunye neeproteni ezinxulumene nazo. Olu sebenzi lumbini lwe-L-ornithine lwenza ukuba ibe ngumviwa othembisayo wesicwangciso esizinzileyo sokunciphisa impembelelo ye-white mold kunye nokuphucula ukuxhathisa kwezityalo ze-legume eziqhelekileyo kule pathogen enamandla ye-fungus. Iziphumo zolu phononongo zinokunceda ekuphuhlisweni kweendlela ezintsha, ezinobuhlobo nokusingqongileyo zokulawula i-white mold kunye nokunciphisa impembelelo yayo kwimveliso ye-legume.
Kolu phononongo, uhlobo oluthile lweembotyi eziqhelekileyo oluthengiswayo, iGiza 3 (Phaseolus vulgaris L. cv. Giza 3), lusetyenziswe njengezinto zovavanyo. Imbewu enempilo inikwe ngobubele yiSebe loPhando lweZityalo zaseMasimini (FCRI), iZiko loPhando lwezoLimo (ARC), eJiphutha. Imbewu ezintlanu zityalwe kwiimbiza zeplastiki (ububanzi bangaphakathi obuyi-35 cm, ubunzulu obuyi-50 cm) ezizaliswe ngumhlaba one-S. sclerotiorum phantsi kweemeko zegreenhouse (25 ± 2 °C, umswakama ohambelanayo 75 ± 1%, ukukhanya kweeyure ezisi-8/16 iiyure ezimnyama). Emva kweentsuku ezisi-7-10 emva kokuhlwayela (i-DPS), izithole zancitshiswa ukuze kushiyeke izithole ezimbini kuphela ezikhula ngokulinganayo kunye namagqabi amathathu anwebe ngokupheleleyo kwimbiza nganye. Zonke izityalo ezikwimbiza zaziseliswa kanye kwiiveki ezimbini kwaye zichumiswe nyanga zonke ngesantya esicetyiswayo sohlobo olunikiweyo.
Ukulungiselela uxinaniso lwe-500 mg/L lwe-L-ornithinediamine (ekwaziwa ngokuba yi-(+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, eJamani), i-50 mg yaqala yanyibilikiswa kwi-100 mL yamanzi acocekileyo acocekileyo. Isisombululo sesitokhwe saxutywa saza sasetyenziswa kwiimvavanyo ezilandelayo. Ngokufutshane, uthotho oluthandathu lwamanqanaba e-L-ornithine (12.5, 25, 50, 75, 100, kunye ne-125 mg/L) lwavavanywa kwi-vitro. Ukongeza, amanzi acocekileyo acocekileyo asetyenziswa njenge-negative control (Mock) kwaye i-fungicide yorhwebo 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) yasetyenziswa njenge-positive control. I-fungicide yorhwebo ethi "Rizolex-T" ivavanywe kwi-vitro kumanqanaba amahlanu (2, 4, 6, 8 kunye ne-10 mg/L).
Iisampulu zeziqu zeembotyi eziqhelekileyo kunye neepods ezibonisa iimpawu eziqhelekileyo zesikhunta esimhlophe (izinga lokuhlasela: 10–30%) ziqokelelwe kwiifama zorhwebo. Nangona uninzi lwezixhobo zezityalo ezisulelekileyo zichongiwe ngokweentlobo/uhlobo (uhlobo lwentengiso olusesichengeni seGiza 3), ezinye, ingakumbi ezo zifunyenwe kwiimarike zasekuhlaleni, zazizezohlobo olungaziwayo. Izinto ezisulelekileyo eziqokelelweyo zaqala zacocwa ngesisombululo se-0.5% sodium hypochlorite imizuzu emi-3, emva koko zahlanjwa izihlandlo ezininzi ngamanzi acocekileyo acociweyo zaza zasulwa ngephepha lokucoca elicocekileyo ukuze kususwe amanzi angaphezulu. Izitho ezisulelekileyo zanqunyulwa zaba ziingceba ezincinci kwizicubu eziphakathi (phakathi kwezicubu eziphilileyo nezisulelekileyo), zakhuliswa kwi-potato dextrose agar (PDA) medium zaza zafakwa kwi-incubator kuma-25 ± 2 °C ngomjikelo wokukhanya weeyure ezili-12/12 iiyure zobumnyama kangangeentsuku ezi-5 ukukhuthaza ukwakheka kwe-sclerotia. Indlela ye-mycelial tip ikwasetyenziswa ukucoca ii-fungus isolates ezivela kwiinkcubeko ezixutyiweyo okanye ezingcolisiweyo. I-fungi ecociweyo yaqala yachongwa ngokusekelwe kwiimpawu zayo zenkcubeko yaza yaqinisekiswa ukuba yi-S. sclerotiorum ngokusekelwe kwiimpawu ezincinci. Ekugqibeleni, zonke ii-isolates ezicociweyo zavavanywa ukuba azinazo izifo kwi-common bean cultivar esengozini yeGiza 3 ukuze kuhlangatyezwane ne-Koch's postulates.
Ukongeza, eyona S. sclerotiorum isolate (isolate #3) iqinisekisiwe ngakumbi ngokusekelwe kwi-internal transcribed spacer (ITS) sequencing njengoko kuchaziwe nguWhite et al., 1990; Baturo-Ciesniewska et al., 2017. Ngamafutshane, ii-isolates zakhuliswa kwi-potato dextrose broth (PDB) zaza zafakwa kwi-incubator kwi-25 ± 2 °C kangangeentsuku ezi-5-7. I-Fungal mycelium yaqokelelwa emva koko, yahluzwa nge-cheesecloth, yahlanjwa kabini ngamanzi acocekileyo, yaza yomiswa ngephepha lesihluzi elicocekileyo. I-Genomic DNA yahlulwa kusetyenziswa i-Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Ummandla we-ITS rDNA wandiswa kusetyenziswa i-primer pair ethile ITS1/ITS4 (TCCGTAGGTGAACCTGGCG TCCTCGCTTTATTGATATGC; ubungakanani obulindelekileyo: 540 bp) (Baturo-Ciesniewska et al., 2017). Iimveliso ze-PCR ezicociweyo zingeniswe ukuze zilandelelaniswe (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). Ulandelelwano lwe-ITS rDNA lulandelelaniswe ngokulandelelana kusetyenziswa indlela ye-Sanger sequencing. Ulandelelwano lwemibuzo oluhlanganisiweyo emva koko luthelekiswa nedatha yamva nje kwiGenBank kunye neNational Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/) kusetyenziswa isoftware ye-BLASTn. Ulandelelwano lwemibuzo luthelekiswe nezinye iintlobo ezingama-20 ze-S. sclerotiorum/isolates ezifunyenwe kwidatha yamva nje kwi-NCBI GenBank (Itheyibhile Eyongezelelekileyo S1) kusetyenziswa i-ClustalW kwi-Molecular Evolutionary Genetics Analysis Package (MEGA-11; inguqulelo 11) (Kumar et al., 2024). Uhlalutyo lwe-Evolutionary lwenziwe kusetyenziswa indlela ye-maximum likelihood kunye ne-general time-reversible nucleotide substitution model (Nei and Kumar, 2000). Umthi one-log-likelihood ephezulu ubonisiwe. Umthi wokuqala wokukhangela i-heuristic ukhethwa ngokukhetha umthi one-log-likelihood ephezulu phakathi komthi we-neighbor-joining (NJ) (Kumar et al., 2024) kunye nomthi we-parsimony (MP) ophezulu. Umthi we-NJ wakhiwe kusetyenziswa i-pairwise distance matrix ebalwe kusetyenziswa i-general time-reversible model (Nei and Kumar, 2000).
Umsebenzi we-L-ornithine wokulwa neentsholongwane kunye ne-bactericide "Rizolex-T" uchongiwe kwi-vitro ngendlela ye-agar diffusion. Indlela: Thatha isixa esifanelekileyo sesisombululo se-stock se-L-ornithine (500 mg/L) kwaye usixube kakuhle ne-10 ml ye-PDA nutrient medium ukulungiselela izisombululo ezinezinga lokugqibela ze-12.5, 25, 50, 75, 100 kunye ne-125 mg/L, ngokulandelelana. Amazinga amahlanu e-fungicide "Rizolex-T" (2, 4, 6, 8 kunye ne-10 mg/L) kunye namanzi acocekileyo asetyenzisiweyo asetyenziswa njengolawulo. Emva kokuba i-medium iqinile, i-mycelial plug esandul' ukulungiswa ye-Sclerotinia sclerotiorum culture, ububanzi be-4 mm, yadluliselwa embindini wesitya sePetri yaza yakhuliswa kwi-25±2°C de i-mycelium igubungele sonke isitya sePetri solawulo, emva koko ukukhula kwefungus kwarekhodwa. Bala ipesenti yokuthintela ukukhula kwe-radial ye-S. sclerotiorum usebenzisa i-equation 1:
Olu vavanyo luphindwe kabini, kunye neekopi ezintandathu zebhayoloji kwiqela ngalinye lolawulo/lovavanyo kunye neembiza ezintlanu (izityalo ezimbini ngembiza nganye) kwikopi nganye yebhayoloji. Ikopi nganye yebhayoloji ihlalutywe kabini (iikopi ezimbini zobugcisa) ukuqinisekisa ukuchaneka, ukuthembeka kunye nokuphindaphinda kweziphumo zovavanyo. Ukongeza, uhlalutyo lokubuyela umva kweprobit lusetyenzisiwe ukubala uxinaniso lwe-half-maximal inhibitory concentration (IC50) kunye ne-IC99 (Prentice, 1976).
Ukuvavanya amandla e-L-ornithine phantsi kweemeko zegreenhouse, kwenziwe uvavanyo lweembiza ezimbini ezilandelelanayo. Ngokufutshane, iimbiza zazaliswa ngomhlaba wesanti yodongwe ocoliweyo (3:1) zaza zafakwa i-incubation nge-culture entsha ye-S. sclerotiorum. Okokuqala, eyona ndawo ihlaselayo ye-S. sclerotiorum (isolate #3) yakhuliswa ngokusika i-sclerotium enye phakathi, yabekwa ijonge phantsi kwi-PDA yaza yafakwa kwi-incubation kuma-25°C ebumnyameni obungaguqukiyo (iiyure ezingama-24) kangangeentsuku ezi-4 ukukhuthaza ukukhula kwe-mycelial. Iiplagi ezine ze-agar ezinobubanzi obuyi-5 mm zathathwa kumphetho ophambili zaza zafakwa i-incubation nge-100 g yomxube ongcolileyo wengqolowa kunye ne-rice bran (1:1, v/v) kwaye zonke iiflask zafakwa kwi-incubation kuma-25 ± 2 °C phantsi komjikelo wobumnyama weeyure ezili-12/iiyure ezili-12 kangangeentsuku ezi-5 ukukhuthaza ukwakheka kwe-sclerotia. Umxholo wazo zonke iiflask waxutywa kakuhle ukuqinisekisa ukulingana ngaphambi kokongeza umhlaba. Emva koko, i-100 g yomxube we-colonizing bran yongezwa kwimbiza nganye ukuqinisekisa ukuba kukho uxinano oluqhubekayo lwezifo ezibangelwa ziintsholongwane. Iimbiza ezifakwe inaliti zankcenkceshelwa ukuze kusebenze ukukhula kwefungus zaza zabekwa kwindawo eluhlaza kangangeentsuku ezisi-7.
Emva koko imbewu ezintlanu zohlobo lweGiza 3 zatyalwa kwimbiza nganye. Kwiimbiza eziphathwe nge-L-ornithine kunye ne-fungicide i-Rizolex-T, imbewu ecociweyo yaqala yacwiliswa iiyure ezimbini kwisisombululo samanzi sale mixube mibini kunye noxinzelelo lokugqibela lwe-IC99 olumalunga ne-250 mg/L kunye ne-50 mg/L, ngokulandelanayo, yaze yomiswa emoyeni iyure enye ngaphambi kokuba ityalwe. Kwelinye icala, imbewu yacwiliswa emanzini acociweyo angenazintsholongwane njengolawulo olubi. Emva kweentsuku ezili-10, ngaphambi kokunkcenkceshela kokuqala, izithole zancitshiswa, kwasala izithole ezimbini kuphela ezicocekileyo kwimbiza nganye. Ukongeza, ukuqinisekisa usulelo lwe-S. sclerotiorum, iziqu zezityalo zembotyi ezikwinqanaba elifanayo lokukhula (iintsuku ezili-10) zanqunyulwa kwiindawo ezimbini ezahlukeneyo kusetyenziswa i-scalpel ecociweyo kwaye malunga ne-0.5 g yomxube we-colonizing bran yafakwa kwinxeba ngalinye, kulandele ukufuma okuphezulu ukukhuthaza usulelo kunye nophuhliso lwezifo kuzo zonke izityalo ezigonyiweyo. Izityalo zokulawula zonzakele ngendlela efanayo kwaye inani elilinganayo (0.5 g) lomxube we-bran ongengowasekoloni wafakwa enxebeni waza wagcinwa phantsi komswakama ophezulu ukuze kuxelwe imeko-bume yophuhliso lwesifo nokuqinisekisa ukuba amaqela onyango ayahambelana.
Indlela yokunyanga: Izithole zeembotyi zinkcenkceshelwe nge-500 ml yesisombululo samanzi se-L-ornithine (250 mg/l) okanye i-fungicide i-Rizolex-T (50 mg/l) ngokunkcenkceshela umhlaba, emva koko unyango luphindwe kathathu kwisithuba seentsuku ezili-10. Ulawulo oluphathwe nge-placebo lunkcenkceshelwe nge-500 ml yamanzi acocekileyo acocekileyo. Zonke iindlela zonyango zenziwe phantsi kweemeko zegreenhouse (25 ± 2°C, 75 ± 1% yomswakama ohambelanayo, kunye nexesha le-photoperiod yeeyure ezi-8 zokukhanya/16 iiyure zobumnyama). Zonke iimbiza zinkcenkceshelwe rhoqo emva kweeveki ezimbini kwaye ziphathwe inyanga nenyanga ngesichumiso se-NPK esilinganayo (20-20-20, kunye ne-3.6% yesulfure kunye ne-TE microelements; iZain Seeds, eYiputa) kuxinzelelo lwe-3–4 g/l ngokutshiza ngefoliar ngokweengcebiso zohlobo oluthile kunye nemiyalelo yomenzi. Ngaphandle kokuba kuchazwe ngenye indlela, amagqabi avuthiweyo akhule ngokupheleleyo (amagqabi esi-2 nawesi-3 ukusuka phezulu) aqokelelwe kwi-replica nganye yebhayoloji emva kweeyure ezingama-72 emva konyango (hpt), afakwa kwi-homogenized, ahlanganiswe ndawonye aze agcinwe kwi--80 °C ukuze kuhlalutywe ngakumbi kuquka, kodwa kungaphelelanga apho, i-in situ histochemical localization ye-oxidative stress indicators, i-lipid peroxidation, i-enzymatic kunye ne-non-enzymatic antioxidants kunye nokubonakaliswa kwe-gene.
Ubunzulu bosulelo lwe-White mold buhlolwe ngeveki emva kweentsuku ezingama-21 emva kokugonyelwa (dpi) kusetyenziswa isikali esingu-1–9 (Itheyibhile Eyongezelelweyo S2) ngokusekelwe kwisikali sikaPetzoldt noDickson (1996) esiguqulwe nguTeran et al. (2006). Ngamafutshane, iziqu kunye namasebe ezityalo zembotyi ahlolwe ukuqala kwindawo yokugonyelwa ukuze kulandelwe ukuqhubela phambili kwezilonda ecaleni kwama-internode kunye nama-node. Umgama wesilonda ukusuka kwindawo yokugonyelwa ukuya kwindawo ekude ecaleni kwesiqu okanye isebe walinganiswa kwaye amanqaku angu-1–9 anikwa ngokusekelwe kwindawo yesilonda, apho (1) kwabonisa ukuba akukho ntsholongwane ibonakalayo kufutshane nendawo yokugonyelwa kwaye (2–9) kwabonisa ukunyuka kancinci kancinci kobukhulu besilonda kunye nokuqhubela phambili ecaleni kwama-node/ama-internode (Itheyibhile Eyongezelelweyo S2). Ubunzulu bosulelo lwe-White mold baguqulwa baba yipesenti kusetyenziswa ifomula yesi-2:
Ukongeza, indawo ephantsi kwe-disease progression curve (AUDPC) ibalwe kusetyenziswa ifomyula (Shaner and Finney, 1977), esandula ukulungiswa kwi-white rot of common bean (Chauhan et al., 2020) kusetyenziswa i-equation 3:
Apho uYi = ubunzima besifo ngexesha elithi ti, uYi+1 = ubunzima besifo kwixesha elizayo elithi ti+1, u-ti = ixesha lokulinganisa kokuqala (ngeentsuku), u-ti+1 = ixesha lokulinganisa okulandelayo (ngeentsuku), u-n = inani lilonke lamanqaku exesha okanye amanqaku okuqaphela. Iiparameter zokukhula kwesityalo seembotyi kuquka ukuphakama kwesityalo (cm), inani lamasebe ngesityalo ngasinye, kunye nenani lamagqabi ngesityalo ngasinye zirekhodwe veki nganye kangangeentsuku ezingama-21 kuzo zonke iikopi zebhayoloji.
Kwikopi nganye yebhayoloji, iisampulu zamagqabi (amagqabi esibini nawesithathu aphuhlileyo ukusuka phezulu) zaqokelelwa ngomhla wama-45 emva konyango (iintsuku ezili-15 emva konyango lokugqibela). Ikopi nganye yebhayoloji yayiquka iimbiza ezintlanu (izityalo ezimbini kwimbiza nganye). Malunga ne-500 mg yezicubu ezityumkileyo yasetyenziswa ekukhupheni iipigment zephotosynthetic (i-chlorophyll a, i-chlorophyll b kunye ne-carotenoids) kusetyenziswa i-80% ye-acetone kwi-4 °C ebumnyameni. Emva kweeyure ezingama-24, iisampulu zafakwa kwi-centrifuge kwaye i-supernatant yaqokelelwa ukuze kuchongwe umxholo we-chlorophyll a, i-chlorophyll b kunye ne-carotenoid ngokwemibala kusetyenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, eJapan) ngokwendlela ye (Lichtenthaler, 1987) ngokulinganisa ukufunxwa kwiiwavelengths ezintathu ezahlukeneyo (A470, A646 kunye ne-A663 nm). Ekugqibeleni, umxholo weepigment ze-photosynthetic ubalwe kusetyenziswa iifomyula ezilandelayo 4–6 ezichazwe nguLichtenthaler (1987).
Emva kweeyure ezingama-72 emva konyango (hpt), amagqabi (amagqabi esibini nawesithathu aphuhlileyo ukusuka phezulu) aqokelelwa kwi-replica nganye yebhayoloji ukuze kufumaneke indawo ye-hydrogen peroxide (H2O2) kunye ne-superoxide anion (O2•−). I-replica nganye yebhayoloji yayineembiza ezintlanu (izityalo ezimbini kwimbiza nganye). I-replica nganye yebhayoloji yahlalutywa ngokuphindwe kabini (ii-replica ezimbini zobugcisa) ukuqinisekisa ukuchaneka, ukuthembeka kunye nokuphindaphinda kwendlela. I-H2O2 kunye ne-O2•− zachongwa kusetyenziswa i-0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Germany) okanye i-nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Germany), ngokulandelanayo, kulandela iindlela ezichazwe nguRomero-Puertas et al. (2004) kunye no-Adam et al. (1989) ngotshintsho oluncinci. Ukuze i-H2O2 ifumaneke kwi-histochemical localization, amaphetshana afakwe kwi-vacuum ene-0.1% DAB kwi-10 mM Tris buffer (pH 7.8) aze afakwe kwi-incubator kubushushu begumbi ekukhanyeni imizuzu engama-60. Amaphetshana afakwe kwi-0.15% (v/v) TCA kwi-4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) aze abekwe ekukhanyeni de kube mnyama. Ngokufanayo, iivalvu zafakwa kwi-vacuum ene-10 mM potassium phosphate buffer (pH 7.8) equlethe i-0.1 w/v % HBT ukuze i-O2 •− ifumaneke kwi-histochemical localization. Amaphetshana afakwe kwi-incubator kubushushu begumbi imizuzu engama-20, emva koko afakwe kwi-vacuum njengoko kuchazwe ngasentla, aze akhanyiswe de kuvele amabala aluhlaza okwesibhakabhaka/violet amnyama. Ubungakanani bombala omdaka ophumayo (njengesalathisi se-H2O2) okanye oluhlaza okwesibhakabhaka (njengesalathisi se-O2•−) buvavanyiwe kusetyenziswa inguqulelo yeFiji yephakheji yokucubungula imifanekiso i-ImageJ (http://fiji.sc; ifikelelwe nge-7 Matshi 2024).
IMalondialdehyde (MDA; njengophawu lwe-lipid peroxidation) yachongwa ngokwendlela kaDu noBramlage (1992) ngotshintsho oluncinci. Amagqabi avela kwi-replica nganye yebhayoloji (igqabi lesibini nelesithathu eliphuhlileyo ukusuka phezulu) aqokelelwa emva kweeyure ezingama-72 emva konyango (hpt). I-replica nganye yebhayoloji yayiquka iimbiza ezintlanu (izityalo ezimbini kwimbiza nganye). I-replica nganye yebhayoloji yahlalutywa ngokuphindwe kabini (ii-replica ezimbini zobugcisa) ukuqinisekisa ukuchaneka, ukuthembeka kunye nokuphindaphinda kwendlela. Ngamafutshane, i-0.5 g yezicubu zamagqabi asemhlabeni yasetyenziselwa ukukhupha i-MDA nge-20% ye-trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) equlethe i-0.01% ye-butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). Umxholo we-MDA kwi-supernatant emva koko wamiselwa ngokwemibala ngokulinganisa ukufunxwa kwi-532 kunye ne-600 nm kusetyenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, eJapan) yaze yavezwa njenge-nmol g−1 FW.
Ukuvavanya ii-antioxidants ezingezizo eze-enzyme kunye neze-enzymatic, amagqabi (amagqabi esibini nawesithathu aphuhlileyo ukusuka phezulu) aqokelelwa kwi-replica nganye yebhayoloji emva kweeyure ezingama-72 emva konyango (hpt). I-replica nganye yebhayoloji yayineembiza ezintlanu (izityalo ezimbini kwimbiza nganye). Isampuli nganye yebhayoloji yahlalutywa ngokuphindwe kabini (iisampuli ezimbini zobugcisa). Amagqabi amabini acolwa nge-nitrogen engamanzi kwaye asetyenziswa ngokuthe ngqo ekumiseleni ii-antioxidants ze-enzyme kunye ne-non-enzyme, ii-amino acids zizonke, umxholo we-proline, ukubonakaliswa kwezakhi zofuzo, kunye nokulinganiswa kwe-oxalate.
Iiphenolics ezinyibilikayo zifunyenwe kusetyenziswa i-Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) kunye notshintsho oluncinci kwindlela echazwe nguKahkonen et al. (1999). Ngamafutshane, malunga ne-0.1 g yezicubu zamagqabi ezihomogenized zakhutshwa nge-20 ml ye-80% methanol ebumnyameni iiyure ezingama-24 kwaye i-supernatant yaqokelelwa emva kwe-centrifugation. I-0.1 ml yesicatshulwa sesampulu yaxutywa ne-0.5 ml ye-Folin-Ciocalteu reagent (10%), yashukunyiswa imizuzwana engama-30 yaze yashiywa ebumnyameni imizuzu emi-5. Emva koko i-0.5 ml yesisombululo se-sodium carbonate se-20% (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, eCairo, eEgypt) yongezwa kwityhubhu nganye, yaxutywa kakuhle yaza yafakwa kubushushu begumbi ebumnyameni iyure e-1. Emva kokufunxwa, ukufunxwa komxube wempendulo kulinganiswe kwi-765 nm kusetyenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, eJapan). Uxinzelelo lwee-phenols ezinyibilikayo kwisampuli lufunyenwe kusetyenziswa i-gallic acid calibration curve (Fisher Scientific, eHampton, eNH, e-USA) kwaye luchazwa njengee-milligrams ze-gallic acid equivalent ngegram nganye yobunzima obutsha (mg GAE g-1 ubunzima obutsha).
Umxholo opheleleyo we-flavonoid enyibilikayo umiselwe ngokwendlela kaDjeridane et al. (2006) ngotshintsho oluncinci. Ngamafutshane, i-0.3 ml ye-methanol extract engentla ixutywe ne-0.3 ml yesisombululo se-aluminium chloride esingu-5% (AlCl3; Fisher Scientific, Hampton, NH, USA), yaxutywa ngamandla yaza yafakwa kubushushu begumbi imizuzu emi-5, yalandelwa kukongeza i-0.3 ml yesisombululo se-potassium acetate esingu-10% (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), yaxutywa kakuhle yaza yafakwa kubushushu begumbi imizuzu engama-30 ebumnyameni. Emva kokufunxwa, ukufunxwa komxube wempendulo kwalinganiswa kwi-430 nm kusetyenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, Japan). Uxinzelelo lwee-flavonoids ezinyibilikayo kwisampuli zamiselwa kusetyenziswa i-rutin calibration curve (TCI America, Portland, OR, USA) yaze yavezwa njengee-milligrams ze-rutin equivalent ngegram nganye yobunzima obutsha (mg RE g-1 ubunzima obutsha).
Ubungakanani be-amino acid epheleleyo yamagqabi eembotyi bufunyenwe kusetyenziswa i-reagent ye-ninhydrin eguquliweyo (Thermo Scientific Chemicals, Waltham, MA, USA) ngokusekelwe kwindlela ecetyiswe nguYokoyama noHiramatsu (2003) kwaye yaguqulwa nguSun et al. (2006). Ngamafutshane, i-0.1 g yezicubu zomhlaba yakhutshwa nge-pH 5.4 buffer, kwaye i-200 μL ye-supernatant yaphendulwa nge-200 μL ye-ninhydrin (2%) kunye ne-200 μL ye-pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), yafakwa kwibhafu yamanzi abilayo imizuzu engama-30, yaze yapholiswa yaze yalinganiswa kwi-580 nm kusetyenziswa i-UV-160A spectrophotometer (Shimadzu Corporation, Japan). Kwelinye icala, i-proline yamiselwa ngendlela yeBates (Bates et al., 1973). Iproline ikhutshwe nge-3% ye-sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) kwaye emva kokuyicoca, i-0.5 ml ye-supernatant ixutywe ne-1 ml ye-glacial acetic acid (Fisher Scientific, Hampton, NH, USA) kunye ne-ninhydrin reagent, efakwe kwi-90°C kangangemizuzu engama-45, yapholiswa yaza yalinganiswa kwi-520 nm kusetyenziswa i-spectrophotometer efanayo nengasentla. Ii-amino acids ezikhululekileyo kunye ne-proline kwi-leaf extracts zichongiwe kusetyenziswa i-glycine kunye ne-proline calibration curves (Sigma-Aldrich, St Louis, MO, USA), ngokulandelanayo, kwaye zibonakaliswe njenge-mg/g ubunzima obutsha.
Ukuze kufunyanwe umsebenzi we-enzymes ze-antioxidant, malunga ne-500 mg yezicubu ezihomogenized zakhutshwa nge-3 ml ye-50 mM Tris buffer (pH 7.8) equlethe i-1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) kunye ne-7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), efakwe kwi-centrifuge kwi-10,000 × g kangangemizuzu engama-20 phantsi kwefriji (4 °C), kwaye i-supernatant (i-crude enzyme extract) yaqokelelwa (El-Nagar et al., 2023; Osman et al., 2023). I-Catalase (CAT) emva koko yaphendulwa nge-2 ml ye-0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) kunye ne-100 μl yesisombululo se-269 mM H2O2 ukumisela umsebenzi wayo we-enzymatic ngokwendlela ye-Aebi (1984) ngotshintsho oluncinci (El-Nagar et al., 2023; Osman et al., 2023). Umsebenzi we-enzymatic we-peroxidase (POX) oxhomekeke kwi-Guaiacol wamiselwa kusetyenziswa indlela ye-Harrach et al. (2009). (2008) kunye notshintsho oluncinci (El-Nagar et al., 2023; Osman et al., 2023) kwaye umsebenzi we-enzymatic we-polyphenol oxidase (PPO) umiselwe emva kokusabela nge-2.2 ml ye-100 mM sodium phosphate buffer (pH 6.0), i-100 μl ye-guaiacol (iikhemikhali ze-TCI, ePortland, OR, e-USA) kunye ne-100 μl ye-12 mM H2O2. Le ndlela iguqulwe kancinci ukusuka (El-Nagar et al., 2023; Osman et al., 2023). Uvavanyo lwenziwe emva kokusabela nge-3 ml yesisombululo se-catechol (Thermo Scientific Chemicals, eWaltham, MA, e-USA) (0.01 M) esandula ukulungiswa kwi-0.1 M phosphate buffer (pH 6.0). Umsebenzi we-CAT ulinganiswe ngokujonga ukubola kwe-H2O2 kwi-240 nm (A240), umsebenzi we-POX ulinganiswe ngokujonga ukwanda kokufunxwa kwi-436 nm (A436), kwaye umsebenzi we-PPO ulinganiswe ngokurekhoda ukuguquguquka kokufunxwa kwi-495 nm (A495) rhoqo emva kwemizuzwana engama-30 imizuzu emi-3 kusetyenziswa i-UV-160A spectrophotometer (Shimadzu, eJapan).
I-RT-PCR yexesha langempela yasetyenziswa ukufumanisa amanqanaba e-transcript ye-genes ezintathu ezinxulumene ne-antioxidant, kuquka i-peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), i-superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), kunye ne-glutathione reductase (PvGR; GenBank Accession No. KY195009.1), kumagqabi eembotyi (amagqabi esibini nawesithathu aphuhlileyo ukusuka phezulu) emva kweeyure ezingama-72 emva konyango lokugqibela. Ngokufutshane, i-RNA yahlulwa kusetyenziswa i-Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) ngokwemigaqo yomenzi. Emva koko, i-cDNA yenziwa kusetyenziswa i-TOP script™ cDNA Synthesis Kit ngokwemiyalelo yomenzi. Ulandelelwano lwe-primer lwe-genes ezintathu ezingentla ludweliswe kwi-Supplementary Table S3. I-PvActin-3 (inombolo yokwamkelwa kweGenBank: XM_068616709.1) yasetyenziswa njenge-housekeeping gene kwaye ukubonakaliswa kwe-relative gene kwabalwa kusetyenziswa indlela ye-2-ΔΔCT (Livak kunye noSchmittgen, 2001). Ukuzinza kwe-Actin phantsi koxinzelelo lwe-biotic (unxibelelwano olungahambelaniyo phakathi kwe-common leaves kunye ne-anthracnose fungus Colletotrichum lindemuthianum) kunye noxinzelelo lwe-abiotic (imbalela, ityuwa, ubushushu obuphantsi) kwabonakaliswa (Borges et al., 2012).
Ekuqaleni senze uhlalutyo lwe-silico olupheleleyo lwe-oxaloacetate acetylhydrolase (OAH) proteins kwi-S. sclerotiorum sisebenzisa isixhobo se-BLAST se-protein-protein (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Ngokufutshane, sisebenzise i-OAH evela kwi-Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; GenBank accession number XP_040799428.1; 342 amino acids) kunye ne-Penicillium lagena (PlOAH; taxide: 94218; GenBank accession number XP_056833920.1; 316 amino acids) njenge-query sequences ukuze kudweliswe i-homologous protein kwi-S. sclerotiorum (taxide: 5180). I-BLASTp yenziwe ngokuchasene nedatha ye-genome ye-S. sclerotiorum efumaneka kutshanje kwiGenBank kwiwebhusayithi yeNational Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Ukongeza, i-gene ye-OAH eqikelelweyo evela kwi-S. sclerotiorum (SsOAH) kunye nohlalutyo lwe-evolutionary kunye nomthi we-phylogenetic we-AfOAH ovela kwi-A. fijiensis CBS 313.89 kunye ne-PlOAH evela kwi-P. lagena ziqikelelwe kusetyenziswa indlela ye-maximum likelihood kwi-MEGA11 (Tamura et al., 2021) kunye nemodeli esekwe kwi-JTT matrix (Jones et al., 1992). Umthi we-phylogenetic udibaniswe nohlalutyo lwe-multiple alignment lwe-protein sequences yazo zonke ii-genes ze-OAH eziqikelelweyo (SsOAH) ezivela kwi-S. sclerotiorum kunye nolandelelwano lwemibuzo kusetyenziswa i-Constraint-Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos kunye no-Agarwala, 2007). Ukongeza, ulandelelwano lwe-amino acid oluhambelanayo lwe-SsOAH oluvela kwi-S. sclerotiorum luhambelaniswe ne-query sequences (AfOAH kunye ne-PlOAH) (Larkin et al., 2007) kusetyenziswa i-ClustalW (http://www.genome.jp/tools-bin/clustalw), kwaye iindawo ezigciniweyo kulungelelwaniso ziboniswe kusetyenziswa isixhobo se-ESPRipt (inguqulelo 3.0; https://espript.ibcp.fr/ESPRipt/ESPRipt/index.php).
Ngaphezu koko, iidomeyini ezimele umsebenzi eziqikelelweyo kunye neendawo ezigciniweyo ze-S. sclerotiorum SsOAH zahlulwe ngokweentsapho ezahlukeneyo kusetyenziswa isixhobo se-InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Okokugqibela, imodeli yesakhiwo esinemilinganiselo emithathu (3D) ye-S. sclerotiorum SsOAH eqikelelweyo yenziwe kusetyenziswa iProtein Homology/Analogy Recognition Engine (Phyre2 server version 2.0; http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) (Kelley et al., 2015) kwaye yaqinisekiswa kusetyenziswa iseva ye-SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). Izakhiwo eziqikelelweyo ezinemilinganiselo emithathu (ifomathi yePDB) ziboniswe ngendlela yokusebenzisana kusetyenziswa iphakheji ye-UCSF-Chimera (inguqulelo 1.15; https://www.cgl.ucsf.edu/chimera/ ) (Pettersen et al., 2004).
I-PCR ye-fluorescence ye-quantitative real-time yasetyenziswa ukufumanisa inqanaba le-transcriptional le-oxaloacetate acetylhydrolase (SsOAH; inombolo ye-accession yeGenBank: XM_001590428.1) kwi-mycelia yeSclerotinia sclerotiorum. Ngokufutshane, i-S. sclerotiorum yafakwa kwiflaski equlethe i-PDB yaza yafakwa kwi-shaking incubator (imodeli: I2400, New Brunswick Scientific Co., Edison, NJ, USA) kwi-25 ± 2 °C kangangeeyure ezingama-24 kwi-150 rpm nakwi-darkness engaguqukiyo (iiyure ezingama-24) ukukhuthaza ukukhula kwe-mycelial. Emva koko, iiseli zanyangwa nge-L-ornithine kunye ne-fungicide i-Rizolex-T kumanqanaba okugqibela e-IC50 (malunga nama-40 kunye ne-3.2 mg/L, ngokulandelelana) zaze zakhuliswa ezinye iiyure ezingama-24 phantsi kweemeko ezifanayo. Emva kokufunxa, ii-cultures zafakwa kwi-centrifuge kwi-2500 rpm kangangemizuzu emi-5 kwaye i-supernatant (i-fungal mycelium) yaqokelelwa ukuze kuhlalutywe ukubonakaliswa kwezakhi zofuzo. Ngokufanayo, i-fungal mycelium yaqokelelwa kwiiyure eziyi-0, 24, 48, 72, 96, kunye ne-120 emva kosulelo kwizityalo ezisulelekileyo ezazenze i-white mold kunye ne-cottony mycelium kumphezulu wezicubu ezisulelekileyo. I-RNA yakhutshwa kwi-fungal mycelium yaza emva koko kwenziwa i-cDNA njengoko kuchaziwe apha ngasentla. I-primer sequences ye-SsOAH idweliswe kwi-Supplementary Table S3. I-SsActin (GenBank accession number: XM_001589919.1) yasetyenziswa njenge-housekeeping gene, kwaye ukubonakaliswa kwezakhi zofuzo okuhlobene kwabalwa kusetyenziswa indlela ye-2-ΔΔCT (Livak kunye noSchmittgen, 2001).
I-oxalic acid yafunyanwa kwi-potato dextrose broth (PDB) kunye neesampuli zezityalo eziqulethe i-fungus pathogen Sclerotinia sclerotiorum ngokwendlela kaXu noZhang (2000) kunye notshintsho oluncinci. Ngokufutshane, ii-S. sclerotiorum isolates zafakwa kwiiflasks ezine-PDB zaza zakhuliswa kwi-shaking incubator (model I2400, New Brunswick Scientific Co., Edison, NJ, USA) kwi-150 rpm kwi-25 ± 2 °C kangangeentsuku ezi-3-5 ebumnyameni obungapheliyo (iiyure ezingama-24) ukukhuthaza ukukhula kwe-mycelial. Emva kokufunxa, i-fungus culture yahluzwa kuqala ngephepha lesihluzo le-Whatman #1 yaza yafakwa kwi-centrifuge kwi-2500 rpm kangangemizuzu emi-5 ukususa i-mycelium eseleyo. I-supernatant yaqokelelwa yaza yagcinwa kwi-4°C ukuze kufunyanwe i-oxalate engaphezulu. Ukulungiselela iisampulu zezityalo, malunga ne-0.1 g yeziqwenga zezicubu zezityalo zakhutshwa kathathu ngamanzi acocekileyo (2 ml ngexesha ngalinye). Emva koko iisampulu zafakwa kwi-centrifuge kwi-2500 rpm imizuzu emi-5, i-supernatant yahluzwa ngephepha lesihluzo le-Whatman No. 1 yaza yaqokelelwa ukuze ihlalutywe ngakumbi.
Ukuze kuhlalutywe ubungakanani be-oxalic acid, umxube wempendulo ulungiselelwe kwityhubhu evaliweyo yeglasi ngolu hlobo lulandelayo: i-0.2 ml yesampulu (okanye isisombululo se-PDB culture filtrate okanye isisombululo esiqhelekileyo se-oxalic acid), i-0.11 ml ye-bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), i-0.198 ml ye-1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) kunye ne-0.176 ml ye-100 mM potassium dichromate (K2Cr2O7; TCI chemicals, Portland, OR, USA), emva koko isisombululo saxutywa kwi-4.8 ml ngamanzi acocekileyo, saxutywa ngamandla saza safakwa ngoko nangoko kwindawo yokuhlambela yamanzi engama-60 °C. Emva kwemizuzu eli-10, impendulo yamiswa ngokongeza i-0.5 ml yesisombululo se-sodium hydroxide (NaOH; 0.75 M). Ukufunxwa (A600) komxube we-reaction kulinganiswe kwi-600 nm kusetyenziswa i-UV-160 spectrophotometer (Shimadzu Corporation, eJapan). I-PDB kunye namanzi acociweyo asetyenziswe njengolawulo lokulinganisa i-culture filtrates kunye neesampuli zezityalo, ngokulandelelana. Amanqanaba e-oxalic acid kwi-culture filtrates, abonakaliswa njengee-micrograms ze-oxalic acid nge-milliliter ye-PDB medium (μg.mL−1), nakwi-leaf extracts, abonakaliswe njengee-micrograms ze-oxalic acid nge-gram yobunzima obutsha (μg.g−1 FW), amiselwe kusetyenziswa i-oxalic acid calibration curve (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
Kuyo yonke le sifundo, zonke iimvavanyo zenziwe ngendlela ecwangcisiweyo ngokupheleleyo (i-CRD) kunye neekopi ezintandathu zebhayoloji ngonyango ngalunye kunye neembiza ezintlanu ngekopi nganye yebhayoloji (izityalo ezimbini kwimbiza nganye) ngaphandle kokuba kuchazwe ngenye indlela. Iikopi ezimbini zebhayoloji zahlalutywa ngokuphindaphindiweyo (iikopi ezimbini zezobugcisa). Iikopi ezimbini zezobugcisa zasetyenziswa ukujonga ukuphinda kuphindaphindwe kovavanyo olufanayo kodwa azizange zisetyenziswe kuhlalutyo lwezibalo ukuze kuthintelwe iikopi ezingezizo. Idatha yahlalutywa ngokwezibalo kusetyenziswa uhlalutyo lokwahluka (i-ANOVA) olulandelwa luvavanyo lweTukey-Kramer olubonisa umahluko obalulekileyo (i-HSD) (p ≤ 0.05). Kwizilingo ze-in vitro, ixabiso le-IC50 kunye ne-IC99 libalwe kusetyenziswa imodeli ye-probit kwaye kwabalwa izithuba zokuzithemba ezingama-95%.
Iintlobo ezine eziqokelelweyo ziqokelelwe kwiintsimi ezahlukeneyo zesoya kwiPhondo lase-El Ghabiya, e-Egypt. Kwi-PDA medium, zonke ezi ntlobo zivelise i-mycelium emhlophe ekhrim eyakhawuleza yajika yaba mhlophe njengekotoni (Umfanekiso 1A) yaze yaba yi-beige okanye mdaka kwinqanaba le-sclerotium. Ii-Sclerotia zihlala zixinene, zimnyama, zingqukuva okanye zingaqhelekanga ngesimo, ubude obuyi-5.2 ukuya kwi-7.7 mm kunye nobubanzi obuyi-3.4 ukuya kwi-5.3 mm (Umfanekiso 1B). Nangona iintlobo ezine eziqokelelweyo zaphuhlisa ipateni engaphantsi ye-sclerotia kumda we-culture medium emva kweentsuku ezili-10-12 zokufunxwa kwi-25 ± 2 °C (Umfanekiso 1A), inani le-sclerotia kwipleyiti nganye lahluke kakhulu phakathi kwazo (P < 0.001), apho i-isolate 3 inenani eliphezulu le-sclerotia (32.33 ± 1.53 sclerotia kwipleyiti nganye; Umzobo 1C). Ngokufanayo, i-isolate #3 ivelise i-oxalic acid engaphezulu kwi-PDB kunezinye ii-isolate (3.33 ± 0.49 μg.mL−1; Umfanekiso 1D). I-Isolate #3 ibonise iimpawu eziqhelekileyo ze-morphological kunye ne-microscopic ze-phytopathogenic fungus i-Sclerotinia sclerotiorum. Umzekelo, kwi-PDA, iikholoni ze-isolate #3 zakhula ngokukhawuleza, zazimhlophe okhrim (Umfanekiso 1A), zibe beige okanye zibe mdaka nge-salmon ekhanyayo, kwaye zafuna iintsuku ezi-6-7 kwi-25 ± 2°C ukugubungela ngokupheleleyo umphezulu wepleyiti enobubanzi obuyi-9 cm. Ngokusekelwe kwiimpawu ze-morphological kunye ne-microscopic ezingentla, i-isolate #3 ichongiwe njenge-Sclerotinia sclerotiorum.
Umfanekiso 1. Iimpawu kunye nesifo se-S. sclerotiorum isolates ezivela kwizityalo ze-common legume. (A) Ukukhula kwe-Mycelial kwe-S. sclerotiorum isolates ezine kwi-PDA medium, (B) i-sclerotia ye-S. sclerotiorum isolates ezine, (C) inani le-sclerotia (ngepleyiti nganye), (D) ukukhutshwa kwe-oxalic acid kwi-PDB medium (μg.mL−1), kunye (E) ubunzima besifo (%) se-S. sclerotiorum isolates ezine kwi-secutable commercial legume cultivar Giza 3 phantsi kweemeko ze-greenhouse. Amaxabiso amele i-avareji ± SD ye-biological replicates ezintlanu (n = 5). Oonobumba abahlukeneyo babonisa umahluko obalulekileyo phakathi konyango (p < 0.05). (F–H) Iimpawu ze-white mold eziqhelekileyo zavela kwiziqu ezingaphezulu komhlaba kunye ne-silique, ngokulandelanayo, kwiintsuku ezili-10 emva kokugonywa nge-isolate #3 (dpi). (I) Uhlalutyo lokuvela kwezinto kwingingqi ye-internal transcribed spacer (ITS) ye-S. sclerotiorum isolate #3 lwenziwe kusetyenziswa indlela ye-maximum likelihood kwaye luthelekiswa ne-20 reference isolates/strains ezifunyenwe kwi-National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). Amanani angaphezulu kwemigca ye-clustering abonisa indawo egutyungelwe yingingqi (%), kwaye amanani angaphantsi kwemigca ye-clustering abonisa ubude besebe.
Ngaphezu koko, ukuqinisekisa ukuba isifo sibangelwa yintsholongwane, kusetyenziswe ii-isolates ezine ze-S. sclerotiorum ukugonya i-Giza 3 yembotyi ethengiswayo phantsi kweemeko ze-greenhouse, nto leyo ehambelana nee-postulates zikaKoch (Umfanekiso 1E). Nangona zonke ii-isolates ze-fungus ezifunyenweyo bezibangela isifo kwaye zinokosulela imbotyi eluhlaza (cv. Giza 3), nto leyo ebangela iimpawu zesikhunta esimhlophe eziqhelekileyo kuzo zonke iindawo ezingaphezulu komhlaba (Umfanekiso 1F), ingakumbi kwiziqu (Umfanekiso 1G) kunye nee-pods (Umfanekiso 1H) kwiintsuku ezili-10 emva kokugonya (dpi), i-isolate 3 yayiyeyona isolate inamandla kakhulu kwiimvavanyo ezimbini ezizimeleyo. I-Isolate 3 yayinobunzima obukhulu besifo (%) kwizityalo zembotyi (24.0 ± 4.0, 58.0 ± 2.0, kunye ne-76.7 ± 3.1 kwiintsuku ezisi-7, 14, kunye neentsuku ezingama-21 emva kokusuleleka, ngokulandelelana; Umfanekiso 1F).
Ukuchongwa kwe-S. sclerotiorum isolate #3 ehlaselayo kakhulu kuqinisekiswe ngakumbi ngokusekelwe kulandelelwano lwangaphakathi olubhalwe phantsi (ITS) (Umzobo 1I). Uhlalutyo lwe-phylogenetic phakathi kwe-isolate #3 kunye ne-20 reference isolates/strains lubonise ukufana okuphezulu (>99%) phakathi kwazo. Kubalulekile ukuqaphela ukuba i-S. sclerotiorum isolate #3 (533 bp) ifana kakhulu ne-American S. sclerotiorum isolate LPM36 ehlukaniswe kwimbewu ye-pea eyomileyo (inombolo ye-GenBank accession MK896659.1; 540 bp) kunye ne-Chinese S. sclerotiorum isolate YKY211 (inombolo ye-GenBank accession OR206374.1; 548 bp), ebangela ukubola kwe-stem violet (Matthiola incana), zonke ezihlelwe ngokwahlukeneyo phezulu kwe-dendrogram (Umfanekiso 1I). Olu luhlu lutsha lufakwe kwisiseko sedatha se-NCBI kwaye lubizwa ngokuba yi-“Sclerotinia sclerotiorum – isolate YN-25” (inombolo yokungena yeGenBank PV202792). Kuyabonakala ukuba isolate 3 yeyona isolate ihlaselayo; ke ngoko, le isolate ikhethwe ukuba ifundwe kuzo zonke iimvavanyo ezilandelayo.
Umsebenzi we-diamine L-ornithine (Sigma-Aldrich, Darmstadt, eJamani) kwiingqinisiso ezahlukeneyo (12.5, 25, 50, 75, 100 kunye ne-125 mg/L) ngokuchasene ne-S. sclerotiorum isolate 3 uphandwe kwi-vitro. Kuyaphawuleka ukuba i-L-ornithine yavelisa isiphumo sokulwa ne-bacterial kwaye yathintela kancinci ukukhula kwe-radial kwe-S. sclerotiorum hyphae ngendlela exhomekeke kwidosi (Umfanekiso 2A, B). Kwingqinisiso ephezulu evavanyiweyo (125 mg/L), i-L-ornithine ibonise izinga eliphezulu lokuthintelwa kokukhula kwe-mycelial (99.62 ± 0.27%; Umfanekiso 2B), elilingana ne-fungicide yorhwebo i-Rizolex-T (izinga lokuthintelwa 99.45 ± 0.39%; Umfanekiso 2C) kwingqinisiso ephezulu evavanyiweyo (10 mg/L), ebonisa ukusebenza okufanayo.
Umfanekiso 2. Umsebenzi we-in vitro antibacterial we-L-ornithine ngokuchasene ne-Sclerotinia sclerotiorum. (A) Uthelekiso lomsebenzi we-antibacterial we-concentrations ezahlukeneyo ze-L-ornithine ngokuchasene ne-S. sclerotiorum kunye ne-fungicide yorhwebo i-Rizolex-T (10 mg/L). (B, C) Izinga lokuthintelwa (%) kokukhula kwe-mycelial ye-S. sclerotiorum emva konyango nge-concentrations ezahlukeneyo ze-L-ornithine (12.5, 25, 50, 75, 100 kunye ne-125 mg/L) okanye i-Rizolex-T (2, 4, 6, 8 kunye ne-10 mg/L), ngokwahlukeneyo. Amaxabiso amele i-mean ± SD yee-replicates ezintlanu zebhayoloji (n = 5). Oonobumba abahlukeneyo babonisa umahluko wezibalo phakathi konyango (p < 0.05). (D, E) Uhlalutyo lohlengahlengiso lwemodeli ye-Probit ye-L-ornithine kunye ne-fungicide yorhwebo i-Rizolex-T, ngokwahlukeneyo. Umgca wokubuyela umva wemodeli yeprobit uboniswa njengomgca oluhlaza okwesibhakabhaka oqinileyo, kwaye isithuba sokuzithemba (95%) siboniswa njengomgca obomvu onemigca.
Ukongeza, kwenziwe uhlalutyo lwe-probit regression kwaye iiploti ezihambelanayo ziboniswe kwiTheyibhile 1 kunye neMifanekiso 2D,E. Ngamafutshane, ixabiso le-slope elamkelekileyo (y = 2.92x - 4.67) kunye nezibalo ezibalulekileyo ezinxulumene nazo (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 kunye no-p < 0.0001; Umfanekiso 2D) we-L-ornithine ubonise umsebenzi ophuculweyo wokulwa ne-antifungal ngokuchasene ne-S. sclerotiorum xa kuthelekiswa ne-fungicide yorhwebo i-Rizolex-T (y = 1.96x - 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 kunye no-p < 0.0001) (Itheyibhile 1).
Itheyibhile 1. Amaxabiso e-half-maximum inhibitory concentration (IC50) kunye ne-IC99 (mg/l) ye-L-ornithine kunye ne-fungicide yorhwebo "iRizolex-T" ngokuchasene ne-S. sclerotiorum.
Lilonke, i-L-ornithine (250 mg/L) inciphise kakhulu uphuhliso kunye nobunzima bomngundo omhlophe kwizityalo zembotyi eziqhelekileyo ezinyangiweyo xa kuthelekiswa nezityalo ezine-S. sclerotiorum ezinganyangwanga (ulawulo; Umfanekiso 3A). Ngamafutshane, nangona ubunzima besifo sezityalo ezilawula izifo ezinganyangwanga zanda kancinci kancinci (52.67 ± 1.53, 83.21 ± 2.61, kunye ne-92.33 ± 3.06%), i-L-ornithine inciphise kakhulu ubunzima besifo (%) kulo lonke uvavanyo (8.97 ± 0.15, 18.00 ± 1.00, kunye ne-26.36 ± 3.07) kwiintsuku ezisi-7, 14, kunye ne-21 emva konyango (dpt), ngokulandelanayo (Umfanekiso 3A). Ngokufanayo, xa izityalo zeembotyi ezine-S. sclerotiorum zinyangwa nge-250 mg/L L-ornithine, indawo ephantsi kwe-disease progression curve (AUDPC) yehla ukusuka kwi-1274.33 ± 33.13 kulawulo olunganyangwanga ukuya kwi-281.03 ± 7.95, eyayingaphantsi kancinci kune-positive control 50 mg/L Rizolex-T fungicide (183.61 ± 7.71; Umfanekiso 3B). Le ndlela ifanayo ibonwe kuvavanyo lwesibini.
Umzobo 3. Isiphumo sokusetyenziswa kwe-L-ornithine ngaphandle ekuphuhlisweni kokubola okumhlophe kwembotyi eqhelekileyo okubangelwa yiSclerotinia sclerotiorum phantsi kweemeko zegreenhouse. (A) Ijika lokuqhubekeka kwesifo lesikhunta esimhlophe sembotyi eqhelekileyo emva konyango nge-250 mg/L L-ornithine. (B) Indawo engaphantsi kwejika lokuqhubekeka kwesifo (AUDPC) sesikhunta esimhlophe sembotyi eqhelekileyo emva konyango nge-L-ornithine. Amaxabiso amele i-avareji ± SD yeekopi ezintlanu zebhayoloji (n = 5). Oonobumba abahlukeneyo babonisa umahluko obalulekileyo phakathi konyango (p < 0.05).
Ukusetyenziswa ngaphandle kwe-250 mg/L L-ornithine kwandise kancinci ubude besityalo (Umzobo 4A), inani lamasebe ngesityalo ngasinye (Umzobo 4B), kunye nenani lamagqabi ngesityalo ngasinye (Umzobo 4C) emva kweentsuku ezingama-42. Ngelixa i-fungicide yorhwebo i-Rizolex-T (50 mg/L) ibe nefuthe elikhulu kuzo zonke iiparameter zesondlo ezifundwe, ukusetyenziswa ngaphandle kwe-250 mg/L L-ornithine kube nefuthe lesibini elikhulu xa kuthelekiswa nolawulo olunganyangwanga (Umzobo 4A–C). Kwelinye icala, unyango lwe-L-ornithine aluzange lube nefuthe libalulekileyo kumxholo weepigment ze-photosynthetic chlorophyll a (Umzobo 4D) kunye ne-chlorophyll b (Umzobo 4E), kodwa lwandise kancinci umxholo we-carotenoid iyonke (0.56 ± 0.03 mg/g fr wt) xa kuthelekiswa nolawulo olubi (0.44 ± 0.02 mg/g fr wt) kunye nolawulo oluhle (0.46 ± 0.02 mg/g fr wt; Umzobo 4F). Ngokubanzi, ezi ziphumo zibonisa ukuba i-L-ornithine ayinabungozi kwizityalo ezivunyiweyo kwaye inokukhuthaza nokukhula kwazo.
Umzobo 4. Isiphumo sokusetyenziswa kwe-L-ornithine yangaphandle kwiimpawu zokukhula kunye nemibala ye-photosynthetic yamagqabi eembotyi asuleleke yiSclerotinia sclerotiorum phantsi kweemeko zegreenhouse. (A) Ukuphakama kwesityalo (cm), (B) Inani lamasebe ngesityalo ngasinye, (C) Inani lamagqabi ngesityalo ngasinye, (D) Umxholo weChlorophyll a (mg g-1 fr wt), (E) Umxholo weChlorophyll b (mg g-1 fr wt), (F) Umxholo we-carotenoid iyonke (mg g-1 fr wt). Amaxabiso yi-avareji ± SD yee-replicates ezintlanu zebhayoloji (n = 5). Oonobumba abahlukeneyo babonisa umahluko obalulekileyo phakathi konyango (p < 0.05).
Indawo ye-histochemical kwi-in situ histochemical species ye-reactive oxygen (ROS; evezwa njenge-hydrogen peroxide [H2O2]) kunye ne-free radicals (evezwa njenge-superoxide anions [O2•−]) ityhile ukuba ukusetyenziswa kwe-L-ornithine (250 mg/L) ngaphandle kwayo kunciphise kakhulu ukuqokelelwa kwe-H2O2 (96.05 ± 5.33 nmol.g−1 FW; Umfanekiso 5A) kunye ne-O2•− (32.69 ± 8.56 nmol.g−1 FW; Umfanekiso 5B) xa kuthelekiswa nokuqokelelwa kwezityalo ezinganyangwanga (173.31 ± 12.06 kunye ne-149.35 ± 7.94 nmol.g−1 FW, ngokulandelelana) kunye nezityalo ezinyangwa nge-50 mg/L ye-fungicide yorhwebo i-Rizolex-T (170.12 ± 9.50 kunye ne-157.00 ± 7.81 nmol.g−1 fr wt, ngokulandelelana) ngeeyure ezingama-72. Amanqanaba aphezulu e-H2O2 kunye ne-O2•− aqokelelene phantsi kwe-hpt (Umzobo 5A, B). Ngokufanayo, uvavanyo lwe-malondialdehyde (MDA) olusekelwe kwi-TCA lubonise ukuba izityalo zeembotyi ezine-S. sclerotiorum ziqokelelene amanqanaba aphezulu e-MDA (113.48 ± 10.02 nmol.g fr wt) kumagqabi azo (Umzobo 5C). Nangona kunjalo, ukusetyenziswa kwe-L-ornithine ngaphandle kwe-L-ornithine kunciphisa kakhulu i-lipid peroxidation njengoko kuboniswe kukwehla komxholo we-MDA kwizityalo ezinyangiweyo (33.08 ± 4.00 nmol.g fr wt).
Umzobo 5. Isiphumo sokusetyenziswa kwe-L-ornithine yangaphandle kwiimpawu eziphambili zoxinzelelo lwe-oxidative kunye neendlela zokuzikhusela ze-antioxidant ezingezizo i-enzyme kumagqabi eembotyi asuleleke yi-S. sclerotiorum kwiiyure ezingama-72 emva kosulelo phantsi kweemeko zegreenhouse. (A) I-hydrogen peroxide (H2O2; nmol g−1 FW) kwi-72 hpt, (B) i-superoxide anion (O2•−; nmol g−1 FW) kwi-72 hpt, (C) i-malondialdehyde (MDA; nmol g−1 FW) kwi-72 hpt, (D) ii-phenols ezinyibilikayo zizonke (mg GAE g−1 FW) kwi-72 hpt, (E) ii-flavonoids ezinyibilikayo zizonke (mg RE g−1 FW) kwi-72 hpt, (F) ii-amino acids zizonke zasimahla (mg g−1 FW) kwi-72 hpt, kunye (G) nomxholo we-proline (mg g−1 FW) kwi-72 hpt. Amaxabiso amele i-mean ± standard deviation (i-mean ± SD) yee-5 biological replicates (n = 5). Oonobumba abahlukeneyo babonisa umahluko obalulekileyo ngokwezibalo phakathi konyango (p < 0.05).