Tsab xov xwm no yog ib feem ntawm lub ncauj lus tshawb fawb "Txhim kho qhov kev tiv thaiv ntawm cov taum pauv rau cov kab mob thiab cov kab tsuag", saib tag nrho 5 tsab xov xwm
Tus neeg ua rau muaj kab mob fungal plant necrosis Sclerotinia sclerotiorum (Lib.) de Bary siv ntau txoj kev los kis kab mob rau ntau hom nroj tsuag. Txoj kev tshawb fawb no qhia txog kev siv diamine L-ornithine, uas yog cov amino acid uas tsis yog protein uas txhawb nqa kev tsim cov amino acid tseem ceeb, ua lwm txoj hauv kev tswj hwm los txhim kho cov lus teb molecular, physiological thiab biochemical ntawm Phaseolus vulgaris L. rau pwm dawb los ntawm Pseudomonas sclerotiorum. Kev sim hauv vitro qhia tau tias L-ornithine inhibited mycelial kev loj hlob ntawm S. pyrenoidosa hauv txoj kev nyob ntawm koob tshuaj. Ntxiv mus, nws tuaj yeem txo qhov hnyav ntawm pwm dawb hauv qab cov xwm txheej greenhouse. Ntxiv mus, L-ornithine txhawb nqa kev loj hlob ntawm cov nroj tsuag kho, qhia tias cov concentration ntawm L-ornithine kuaj tsis yog phytotoxic rau cov nroj tsuag kho. Ntxiv mus, L-ornithine txhim kho qhov kev qhia tawm ntawm cov tshuaj antioxidants uas tsis yog enzymatic (tag nrho cov phenolics thiab flavonoids uas yaj tau tag nrho) thiab cov tshuaj antioxidants enzymatic (catalase (CAT), peroxidase (POX), thiab polyphenol oxidase (PPO)), thiab ua rau muaj kev qhia tawm ntawm peb lub noob caj noob ces uas cuam tshuam nrog antioxidant (PvCAT1, PvSOD, thiab PvGR). Ntxiv mus, kev tshuaj xyuas hauv silico tau qhia txog qhov muaj cov protein oxaloacetate acetylhydrolase (SsOAH) hauv S. sclerotiorum genome, uas zoo ib yam li cov protein oxaloacetate acetylhydrolase (SsOAH) ntawm Aspergillus fijiensis (AfOAH) thiab Penicillium sp. (PlOAH) hauv kev tshuaj xyuas kev ua haujlwm, cov chaw khaws cia, thiab topology. Qhov nthuav yog tias, kev ntxiv L-ornithine rau cov kua txiv qos dextrose (PDB) medium txo qis qhov kev qhia tawm ntawm SsOAH gene hauv S. sclerotiorum mycelia. Ib yam li ntawd, kev siv L-ornithine sab nraud ua rau txo qis qhov kev qhia tawm ntawm SsOAH gene hauv cov kab mob fungal mycelia sau los ntawm cov nroj tsuag kho. Thaum kawg, kev siv L-ornithine ua rau txo qis oxalic acid secretion hauv ob qho tib si PDB medium thiab cov nplooj uas muaj kab mob. Hauv kev xaus, L-ornithine ua lub luag haujlwm tseem ceeb hauv kev tswj hwm qhov xwm txheej redox nrog rau kev txhim kho kev tiv thaiv ntawm cov nroj tsuag uas muaj kab mob. Cov txiaj ntsig ntawm txoj kev tshawb fawb no yuav pab tsim cov txheej txheem tshiab, zoo rau ib puag ncig los tswj cov pwm dawb thiab txo nws qhov cuam tshuam rau kev tsim cov taum thiab lwm yam qoob loo.
Pwm dawb, uas yog los ntawm cov pwm necrotrophic Sclerotinia sclerotiorum (Lib.) de Bary, yog ib yam kab mob loj heev uas ua rau cov noob qoob loo tsis muaj zog thiab ua rau cov noob qoob loo tsis muaj zog (Phaseolus vulgaris L.) tsis muaj zog txaus (Bolton et al., 2006). Sclerotinia sclerotiorum yog ib yam kab mob fungal uas nyuaj tshaj plaws hauv av los tswj, muaj ntau hom nroj tsuag tshaj 600 hom thiab nws muaj peev xwm zom cov nqaij ntawm cov nroj tsuag sai sai (Liang thiab Rollins, 2018). Nyob rau hauv tej yam mob tsis zoo, nws yuav dhau mus ua ib theem tseem ceeb ntawm nws lub neej, nws yuav nyob tsis tau ntev ntev li cov noob dub, tawv, zoo li noob hu ua 'sclerotia' hauv av lossis ua cov noob dawb, mos mos hauv cov mycelium lossis cov qia ntawm cov nroj tsuag uas muaj kab mob (Schwartz et al., 2005). S. sclerotiorum muaj peev xwm tsim cov sclerotia, uas tso cai rau nws kom muaj sia nyob hauv cov teb uas muaj kab mob tau ntev thiab nyob tau thaum muaj kab mob (Schwartz et al., 2005). Sclerotia muaj ntau yam khoom noj khoom haus, tuaj yeem nyob tau hauv av tau ntev, thiab ua lub hauv paus tseem ceeb rau kev kis kab mob tom qab (Schwartz et al., 2005). Nyob rau hauv cov xwm txheej zoo, sclerotia germinate thiab tsim cov spores hauv huab cua uas tuaj yeem kis tau rau txhua qhov saum av ntawm cov nroj tsuag, suav nrog tab sis tsis txwv rau paj, qia, lossis pods (Schwartz et al., 2005).
Sclerotinia sclerotiorum siv ntau txoj kev npaj los kis tus kab mob rau nws cov nroj tsuag, uas muaj ntau yam xwm txheej sib koom ua ke los ntawm kev tawg paj mus rau kev loj hlob ntawm cov tsos mob. Thaum xub thawj, S. sclerotiorum tsim cov noob uas raug dai (hu ua ascospores) los ntawm cov qauv zoo li nceb hu ua apothecia, uas dhau los ua huab cua thiab loj hlob mus rau hauv cov sclerotia uas tsis txav tau ntawm cov nroj tsuag uas muaj kab mob (Bolton et al., 2006). Cov pwm tom qab ntawd tso tawm oxalic acid, uas yog ib qho virulence factor, los tswj cov pH ntawm phab ntsa ntawm cov nroj tsuag, txhawb kev puas tsuaj ntawm enzymatic thiab kev nkag mus rau cov ntaub so ntswg (Hegedus thiab Rimmer, 2005), thiab tswj cov nroj tsuag lub zog oxidative. Cov txheej txheem acidification no ua rau cov phab ntsa ntawm cov nroj tsuag tsis muaj zog, muab ib puag ncig zoo rau kev ua haujlwm ib txwm thiab ua haujlwm zoo ntawm cov enzymes rhuav tshem phab ntsa ntawm cov nroj tsuag (CWDEs), uas tso cai rau cov kab mob kom kov yeej qhov kev thaiv lub cev thiab nkag mus rau hauv cov ntaub so ntswg ntawm tus tswv tsev (Marciano et al., 1983). Thaum nkag mus rau hauv, S. sclerotiorum tso tawm ntau yam CWDEs, xws li polygalacturonase thiab cellulase, uas ua rau nws kis mus rau hauv cov ntaub so ntswg uas muaj kab mob thiab ua rau cov ntaub so ntswg necrosis. Kev nce qib ntawm cov qhov txhab thiab cov ntaub pua plag hyphal ua rau muaj cov tsos mob ntawm cov pwm dawb (Hegedus thiab Rimmer, 2005). Lub caij no, cov nroj tsuag hauv tsev lees paub cov qauv molecular cuam tshuam nrog cov kab mob (PAMPs) los ntawm cov qauv lees paub cov receptors (PRRs), ua rau muaj ntau yam kev tshwm sim uas thaum kawg ua rau muaj kev tiv thaiv teb.
Txawm hais tias muaj ntau xyoo ntawm kev siv zog tswj kab mob, qhov tsis txaus ntawm cov noob caj noob ces tiv taus txaus tseem nyob hauv taum pauv, zoo li lwm cov qoob loo lag luam, vim yog qhov tiv taus, kev ciaj sia, thiab kev hloov pauv ntawm cov kab mob. Yog li ntawd, kev tswj kab mob yog qhov nyuaj heev thiab xav tau kev sib koom ua ke, ntau yam kev coj ua uas suav nrog kev sib xyaw ua ke ntawm kev coj noj coj ua, kev tswj hwm kab mob, thiab tshuaj tua kab mob (O'Sullivan et al., 2021). Kev tswj hwm tshuaj ntawm cov pwm dawb yog qhov zoo tshaj plaws vim tias cov tshuaj tua kab mob, thaum siv kom raug thiab thaum lub sijhawm raug, tuaj yeem tswj tau qhov kis ntawm tus kab mob, txo qhov hnyav ntawm kev kis tus kab mob, thiab txo qhov poob ntawm cov qoob loo. Txawm li cas los xij, kev siv ntau dhau thiab kev vam khom ntau dhau rau cov tshuaj tua kab mob tuaj yeem ua rau muaj cov kab mob tiv taus ntawm S. sclerotiorum thiab cuam tshuam tsis zoo rau cov kab mob tsis yog lub hom phiaj, kev noj qab haus huv av, thiab dej zoo (Le Cointe et al., 2016; Ceresini et al., 2024). Yog li ntawd, kev nrhiav lwm txoj hauv kev zoo rau ib puag ncig tau dhau los ua qhov tseem ceeb tshaj plaws.
Cov Polyamines (PAs), xws li putrescine, spermidine, spermine, thiab cadaverine, tuaj yeem ua haujlwm ua lwm txoj hauv kev zoo los tiv thaiv cov kab mob cog hauv av, yog li ntawd tag nrho lossis ib feem txo ​​​​kev siv cov tshuaj tua kab mob txaus ntshai (Nehela et al., 2024; Yi et al., 2024). Hauv cov nroj tsuag siab dua, PAs koom nrog ntau cov txheej txheem kev ua haujlwm suav nrog, tab sis tsis txwv rau, kev faib cell, kev sib txawv, thiab kev teb rau kev ntxhov siab abiotic thiab biotic (Killiny thiab Nehela, 2020). Lawv tuaj yeem ua haujlwm ua cov tshuaj antioxidants, pab tshem tawm cov tshuaj reactive oxygen (ROS), tswj cov redox homeostasis (Nehela thiab Killiny, 2023), ua rau cov noob caj noob ces tiv thaiv (Romero et al., 2018), tswj ntau txoj hauv kev metabolic (Nehela thiab Killiny, 2023), hloov kho endogenous phytohormones (Nehela thiab Killiny, 2019), tsim kom muaj kev tiv thaiv systemic (SAR), thiab tswj kev sib cuam tshuam ntawm cov nroj tsuag thiab kab mob (Nehela thiab Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). Nws yog ib qho tsim nyog sau cia tias cov txheej txheem tshwj xeeb thiab lub luag haujlwm ntawm PAs hauv kev tiv thaiv cov nroj tsuag sib txawv nyob ntawm cov nroj tsuag, cov kab mob, thiab cov xwm txheej ib puag ncig. PA ntau tshaj plaws hauv cov nroj tsuag yog biosynthesized los ntawm cov polyamine L-ornithine tseem ceeb (Killiny thiab Nehela, 2020).
L-ornithine ua ntau lub luag haujlwm hauv kev loj hlob thiab kev txhim kho ntawm cov nroj tsuag. Piv txwv li, cov kev tshawb fawb yav dhau los tau qhia tias hauv cov mov (Oryza sativa), ornithine tej zaum yuav cuam tshuam nrog kev siv nitrogen rov qab (Liu et al., 2018), cov qoob loo, qhov zoo thiab aroma (Lu et al., 2020), thiab kev teb rau kev ntxhov siab ntawm dej (Yang et al., 2000). Ntxiv mus, kev siv L-ornithine sab nraud ua rau muaj kev tiv thaiv kev qhuav hauv cov qab zib beet (Beta vulgaris) (Hussein et al., 2019) thiab txo kev ntxhov siab ntsev hauv dos (Allium Cepa) (Çavuşoǧlu thiab Çavuşoǧlu, 2021) thiab cashew (Anacardium occidentale) cov nroj tsuag (da Rocha et al., 2012). Lub luag haujlwm ntawm L-ornithine hauv kev tiv thaiv kev ntxhov siab abiotic tej zaum yuav yog vim nws koom nrog hauv kev sib sau ua ke ntawm proline hauv cov nroj tsuag kho. Piv txwv li, cov noob caj noob ces uas cuam tshuam nrog cov metabolism proline, xws li ornithine delta aminotransferase (delta-OAT) thiab proline dehydrogenase (ProDH1 thiab ProDH2) noob caj noob ces, tau tshaj tawm yav dhau los tias koom nrog kev tiv thaiv Nicotiana benthamiana thiab Arabidopsis thaliana tawm tsam cov kab mob Pseudomonas syringae uas tsis yog tus tswv tsev (Senthil-Kumar thiab Mysore, 2012). Ntawm qhov tod tes, fungal ornithine decarboxylase (ODC) yog qhov xav tau rau kev loj hlob ntawm cov kab mob (Singh et al., 2020). Kev tsom mus rau ODC ntawm Fusarium oxysporum f. sp. lycopersici ntawm tus tswv tsev-induced gene silencing (HIGS) tau txhim kho qhov kev tiv thaiv ntawm cov txiv lws suav rau Fusarium wilt (Singh et al., 2020). Txawm li cas los xij, lub luag haujlwm ntawm kev siv ornithine sab nraud tawm tsam kev ntxhov siab biotic xws li phytopathogens tsis tau kawm zoo. Qhov tseem ceeb tshaj, cov teebmeem ntawm ornithine rau kev tiv thaiv kab mob thiab cov xwm txheej biochemical thiab physiological tseem tsis tau tshawb nrhiav.
Kev nkag siab txog qhov nyuaj ntawm S. sclerotiorum kev kis tus kab mob ntawm cov taum pauv yog qhov tseem ceeb rau kev tsim cov tswv yim tswj hwm zoo. Hauv kev tshawb fawb no, peb lub hom phiaj yog txheeb xyuas lub luag haujlwm ntawm diamine L-ornithine ua lub hauv paus tseem ceeb hauv kev txhim kho cov txheej txheem tiv thaiv thiab kev tiv thaiv ntawm cov nroj tsuag legume rau Sclerotinia sclerotiorum kev kis tus kab mob. Peb xav tias, ntxiv rau kev txhim kho cov lus teb tiv thaiv ntawm cov nroj tsuag kis tus kab mob, L-ornithine kuj ua lub luag haujlwm tseem ceeb hauv kev tswj hwm qhov xwm txheej redox. Peb xav tias cov teebmeem ntawm L-ornithine muaj feem cuam tshuam nrog kev tswj hwm ntawm cov txheej txheem tiv thaiv antioxidant enzymatic thiab tsis-enzymatic thiab kev cuam tshuam nrog cov kab mob fungal pathogenicity / virulence thiab cov protein cuam tshuam. Qhov kev ua haujlwm ob npaug ntawm L-ornithine ua rau nws yog tus neeg sib tw cog lus rau lub tswv yim ruaj khov los txo qhov cuam tshuam ntawm pwm dawb thiab txhim kho kev tiv thaiv ntawm cov qoob loo legume rau cov kab mob fungal muaj zog no. Cov txiaj ntsig ntawm kev tshawb fawb tam sim no yuav pab tau hauv kev tsim cov txheej txheem tshiab, tus phooj ywg ib puag ncig los tswj cov pwm dawb thiab txo nws qhov cuam tshuam rau kev tsim cov taum pauv.
Hauv kev tshawb fawb no, ib hom taum pauv uas yoo kis tau los ntawm kev lag luam, Giza 3 (Phaseolus vulgaris L. cv. Giza 3), tau siv los ua cov khoom siv sim. Cov noob noj qab haus huv tau muab los ntawm Legume Research Department, Field Crops Research Institute (FCRI), Agricultural Research Center (ARC), Egypt. Tsib lub noob tau cog rau hauv cov lauj kaub yas (sab hauv txoj kab uas hla 35 cm, tob 50 cm) uas muaj cov av uas muaj kab mob S. sclerotiorum nyob rau hauv cov xwm txheej greenhouse (25 ± 2 °C, av noo 75 ± 1%, 8 teev kaj/16 teev tsaus ntuj). Thaum 7-10 hnub tom qab cog (DPS), cov noob tau raug txiav kom nyias nyias kom tsuas yog muaj ob lub noob nrog kev loj hlob zoo ib yam thiab peb nplooj nthuav dav hauv txhua lub lauj kaub. Tag nrho cov nroj tsuag hauv lauj kaub tau ywg dej ib zaug txhua ob lub lis piam thiab muab chiv txhua hli ntawm tus nqi pom zoo rau hom uas tau muab.
Yuav kom npaj tau 500 mg/L concentration ntawm L-ornithinediamine (tseem hu ua (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Lub Tebchaws Yelemees), 50 mg raug yaj ua ntej hauv 100 mL ntawm cov dej ntshiab uas tsis muaj kab mob. Tom qab ntawd cov kua tshuaj tau raug diluted thiab siv rau hauv kev sim tom qab. Hauv luv luv, rau series ntawm L-ornithine concentrations (12.5, 25, 50, 75, 100, thiab 125 mg/L) tau raug sim hauv vitro. Tsis tas li ntawd, cov dej ntshiab uas tsis muaj kab mob tau siv ua kev tswj tsis zoo (Mock) thiab cov tshuaj tua kab mob "Rizolex-T" 50% hmoov ntub (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Egypt) tau siv ua kev tswj zoo. Cov tshuaj tua kab mob "Rizolex-T" uas muag hauv khw tau raug sim hauv vitro ntawm tsib qhov concentration (2, 4, 6, 8 thiab 10 mg/L).
Cov qauv ntawm cov qia thiab cov noob taum uas pom cov tsos mob ntawm pwm dawb (tus nqi ntawm kev kis kab mob: 10–30%) tau sau los ntawm cov liaj teb ua lag luam. Txawm hais tias feem ntau ntawm cov khoom cog uas muaj kab mob tau txheeb xyuas los ntawm hom/ntau yam (hom lag luam uas yoo mob Giza 3), lwm yam, tshwj xeeb tshaj yog cov uas tau los ntawm cov khw hauv zos, yog cov hom tsis paub. Cov khoom uas muaj kab mob tau sau ua ntej tau muab tshuaj tua kab mob rau saum npoo av nrog 0.5% sodium hypochlorite kua rau 3 feeb, tom qab ntawd yaug ob peb zaug nrog dej ntshiab thiab so kom qhuav nrog daim ntawv lim dej kom tshem tawm cov dej ntau dhau. Cov noob uas muaj kab mob tom qab ntawd tau txiav ua tej daim me me los ntawm cov nqaij nruab nrab (ntawm cov nqaij noj qab haus huv thiab cov nqaij uas muaj kab mob), cog rau ntawm qos yaj ywm dextrose agar (PDA) nruab nrab thiab incubated ntawm 25 ± 2 °C nrog 12 teev lub teeb/12 teev tsaus ntuj rau 5 hnub los txhawb kev tsim sclerotia. Txoj kev mycelial tip kuj tau siv los ntxuav cov kab mob fungal los ntawm cov kab mob sib xyaw lossis cov kab mob. Cov kab mob fungal uas tau ntxuav tau txheeb xyuas thawj zaug raws li nws cov yam ntxwv ntawm kab lis kev cai thiab tom qab ntawd lees paub tias yog S. sclerotiorum raws li cov yam ntxwv me me. Thaum kawg, tag nrho cov kab mob uas tau lim lawm tau raug kuaj seb puas muaj kab mob rau ntawm cov noob taum Giza 3 uas yooj yim kis tau los ua kom tau raws li Koch cov lus pom zoo.
Ntxiv mus, tus kab mob S. sclerotiorum uas kis tau ntau tshaj plaws (isolate #3) tau raug lees paub ntxiv raws li kev txheeb xyuas sab hauv (ITS) raws li White et al., 1990 tau piav qhia; Baturo-Ciesniewska et al., 2017. Hauv luv luv, cov isolates tau cog rau hauv cov kua zaub qos yaj ywm dextrose (PDB) thiab muab tso rau ntawm 25 ± 2 °C rau 5-7 hnub. Tom qab ntawd, cov kab mob fungal mycelium tau sau, lim los ntawm cheesecloth, ntxuav ob zaug nrog dej huv, thiab ziab nrog daim ntawv lim dej huv. Genomic DNA tau raug rho tawm siv Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Thaj chaw ITS rDNA tau raug nthuav dav siv cov primer khub tshwj xeeb ITS1/ITS4 (TCCGTAGGTGAACCTGCGG TCCTCCGCTTATTGATATGC; qhov loj me xav tau: 540 bp) (Baturo-Ciesniewska et al., 2017). Cov khoom PCR purified tau xa mus rau sequencing (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). Cov kab ke ITS rDNA tau raug sequenced bidirectional siv txoj kev Sanger sequencing. Cov kab ke nug sib sau ua ke tau raug piv nrog cov ntaub ntawv tshiab kawg hauv GenBank thiab National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/) siv BLASTn software. Cov lus nug tau muab piv rau 20 lwm cov kab mob S. sclerotiorum / isolates uas tau txais los ntawm cov ntaub ntawv tshiab kawg hauv NCBI GenBank (Supplementary Table S1) siv ClustalW hauv Molecular Evolutionary Genetics Analysis Package (MEGA-11; version 11) (Kumar et al., 2024). Kev tshuaj xyuas kev hloov pauv tau ua tiav siv txoj kev nyiam siab tshaj plaws thiab cov qauv hloov pauv nucleotide lub sijhawm dav dav (Nei thiab Kumar, 2000). Tsob ntoo uas muaj qhov zoo li cav siab tshaj plaws tau pom. Tsob ntoo pib rau kev tshawb nrhiav heuristic yog xaiv los ntawm kev xaiv tsob ntoo uas muaj qhov zoo li cav siab dua ntawm tsob ntoo sib koom ua ke (NJ) (Kumar et al., 2024) thiab tsob ntoo parsimony siab tshaj plaws (MP). Tsob ntoo NJ tau tsim los ntawm kev siv cov matrix nrug sib npaug uas tau xam los ntawm kev siv cov qauv hloov pauv lub sijhawm dav dav (Nei thiab Kumar, 2000).
Cov tshuaj tua kab mob ntawm L-ornithine thiab cov tshuaj tua kab mob "Rizolex-T" tau txiav txim siab hauv vitro los ntawm txoj kev agar diffusion. Txoj Kev: Siv cov tshuaj L-ornithine (500 mg/L) uas tsim nyog thiab sib tov nrog 10 ml ntawm PDA nutrient medium los npaj cov tshuaj nrog qhov concentration kawg ntawm 12.5, 25, 50, 75, 100 thiab 125 mg/L, raws li. Tsib qhov concentration ntawm cov tshuaj fungicide "Rizolex-T" (2, 4, 6, 8 thiab 10 mg/L) thiab dej distilled sterile tau siv ua kev tswj hwm. Tom qab cov tshuaj tau khov lawm, ib qho mycelial plug tshiab ntawm Sclerotinia sclerotiorum kab lis kev cai, 4 hli hauv txoj kab uas hla, tau hloov mus rau hauv nruab nrab ntawm lub tais Petri thiab cog qoob loo ntawm 25 ± 2 ° C kom txog thaum cov mycelium npog tag nrho lub tais Petri tswj hwm, tom qab ntawd cov kab mob fungal tau sau tseg. Xam qhov feem pua ​​ntawm kev txwv tsis pub loj hlob ntawm S. sclerotiorum siv kab zauv 1:
Qhov kev sim tau rov ua dua ob zaug, nrog rau rau qhov kev rov ua dua ntawm cov kab mob rau txhua pawg tswj/kev sim thiab tsib lub lauj kaub (ob tsob nroj rau ib lub lauj kaub) rau txhua qhov kev rov ua dua ntawm cov kab mob. Txhua qhov kev rov ua dua ntawm cov kab mob tau raug soj ntsuam ob zaug (ob qhov kev rov ua dua ntawm kev siv tshuab) kom ntseeg tau tias qhov tseeb, kev ntseeg tau thiab kev rov ua dua ntawm cov txiaj ntsig ntawm kev sim. Tsis tas li ntawd, kev tshuaj xyuas kev rov qab los ntawm probit tau siv los xam qhov kev sib xyaw ua ke ntawm ib nrab-siab tshaj plaws ntawm kev txwv tsis pub muaj kab mob (IC50) thiab IC99 (Prentice, 1976).
Txhawm rau ntsuam xyuas lub peev xwm ntawm L-ornithine nyob rau hauv cov xwm txheej greenhouse, ob qhov kev sim ua ke tau ua tiav. Luv luv, cov lauj kaub tau ntim nrog cov av av nplaum-xuab zeb (3: 1) thiab txhaj tshuaj nrog cov kab lis kev cai tshiab ntawm S. sclerotiorum. Ua ntej, qhov kab mob S. sclerotiorum (tso tawm # 3) uas nkag mus rau hauv av ntau tshaj plaws tau cog los ntawm kev txiav ib lub sclerotium ua ob nrab, muab nws lub ntsej muag rau ntawm PDA thiab incubate ntawm 25 ° C hauv qhov tsaus ntuj tas li (24 teev) rau 4 hnub los txhawb kev loj hlob ntawm mycelial. Plaub lub 5 hli txoj kab uas hla agar plugs tom qab ntawd tau coj los ntawm ntug ua ntej thiab txhaj tshuaj nrog 100 g ntawm cov khoom sib xyaw ua ke ntawm cov nplej thiab mov bran (1: 1, v / v) thiab txhua lub raj mis tau incubated ntawm 25 ± 2 ° C nyob rau hauv 12 teev lub teeb / 12 teev tsaus ntuj voj voog rau 5 hnub los txhawb kev tsim sclerotia. Cov ntsiab lus ntawm txhua lub raj mis tau sib xyaw kom huv si kom ntseeg tau tias muaj kev sib xws ua ntej ntxiv av. Tom qab ntawd, muab 100 g ntawm cov hmoov nplej uas tseem tab tom loj hlob ntxiv rau hauv txhua lub lauj kaub kom ntseeg tau tias muaj cov kab mob sib xyaw tas li. Cov lauj kaub uas tau txhaj tshuaj lawm tau muab dej rau kom cov kab mob loj hlob thiab muab tso rau hauv cov tsev cog khoom rau 7 hnub.
Tsib noob ntawm Giza 3 hom tau raug cog rau hauv txhua lub lauj kaub. Rau cov lauj kaub kho nrog L-ornithine thiab fungicide Rizolex-T, cov noob sterilized tau muab tso rau hauv dej rau ob teev hauv cov kua dej ntawm ob lub tshuaj nrog qhov kawg IC99 concentration ntawm kwv yees li 250 mg / L thiab 50 mg / L, raws li, thiab tom qab ntawd cua-ziab rau ib teev ua ntej cog. Ntawm qhov tod tes, cov noob tau muab tso rau hauv dej distilled sterile ua qhov kev tswj tsis zoo. Tom qab 10 hnub, ua ntej thawj zaug ywg dej, cov noob tau thinned tawm, tawm tsuas yog ob lub noob zoo huv si hauv txhua lub lauj kaub. Tsis tas li ntawd, kom ntseeg tau tias muaj kev kis tus kab mob nrog S. sclerotiorum, cov qia ntawm cov nroj tsuag taum ntawm tib theem kev loj hlob (10 hnub) tau txiav ntawm ob qhov chaw sib txawv siv lub scalpel sterilized thiab kwv yees li 0.5 g ntawm cov khoom sib xyaw colonizing tau muab tso rau hauv txhua qhov txhab, ua raws li qhov av noo siab los txhawb kev kis tus kab mob thiab kev loj hlob ntawm kab mob hauv txhua cov nroj tsuag inoted. Cov nroj tsuag tswj tau raug mob zoo sib xws thiab muab cov hmoov nplej sib xyaw ua ke (0.5 g) uas tsis muaj kab mob sterile, tsis muaj kab mob tso rau hauv qhov txhab thiab khaws cia rau hauv qhov chaw noo noo kom zoo li qhov chaw rau kev loj hlob ntawm tus kab mob thiab xyuas kom meej tias muaj kev sib xws ntawm cov pab pawg kho mob.
Txoj Kev Kho Mob: Cov noob taum tau muab dej nrog 500 ml ntawm cov kua dej ntawm L-ornithine (250 mg/l) lossis cov tshuaj tua kab mob Rizolex-T (50 mg/l) los ntawm kev ywg dej rau hauv av, tom qab ntawd kev kho mob tau rov ua dua peb zaug ntawm lub sijhawm 10 hnub. Cov neeg tswj hwm uas tau kho placebo tau muab dej nrog 500 ml ntawm cov dej ntshiab huv. Txhua txoj kev kho mob tau ua tiav hauv cov xwm txheej tsev cog khoom (25 ± 2°C, 75 ± 1% qhov av noo, thiab lub sijhawm photoperiod ntawm 8 teev lub teeb/16 teev tsaus ntuj). Txhua lub lauj kaub tau muab dej ob lub lis piam thiab kho txhua hli nrog cov chiv NPK sib npaug (20-20-20, nrog 3.6% sulfur thiab TE microelements; Zain Seeds, Egypt) ntawm qhov concentration ntawm 3-4 g/l los ntawm kev txau nplooj raws li cov lus pom zoo rau hom tshwj xeeb thiab cov lus qhia ntawm cov chaw tsim khoom. Tshwj tsis yog hais lwm yam, cov nplooj loj hlob tag nrho (cov nplooj thib 2 thiab thib 3 los ntawm sab saum toj) tau sau los ntawm txhua qhov kev rov ua dua ntawm lub cev ntawm 72 teev tom qab kev kho mob (hpt), homogenized, pooled thiab khaws cia ntawm -80 ° C rau kev tshuaj xyuas ntxiv suav nrog, tab sis tsis txwv rau, in situ histochemical localization ntawm oxidative stress indicators, lipid peroxidation, enzymatic thiab non-enzymatic antioxidants thiab gene expression.
Qhov hnyav ntawm kev kis tus kab mob pwm dawb tau raug soj ntsuam txhua lub lim tiam 21 hnub tom qab txhaj tshuaj (dpi) siv qhov ntsuas ntawm 1-9 (Supplementary Table S2) raws li Petzoldt thiab Dickson scale (1996) hloov kho los ntawm Teran et al. (2006). Hauv luv luv, cov qia thiab ceg ntawm cov noob taum tau raug tshuaj xyuas pib ntawm qhov chaw txhaj tshuaj kom ua raws li kev nce qib ntawm cov qhov txhab raws cov internodes thiab nodes. Qhov deb ntawm qhov txhab los ntawm qhov chaw txhaj tshuaj mus rau qhov deb tshaj plaws raws li cov qia lossis ceg tau raug ntsuas thiab qhov qhab nia ntawm 1-9 tau muab raws li qhov chaw ntawm qhov txhab, qhov twg (1) qhia tias tsis muaj kev kis tus kab mob pom ze ntawm qhov chaw txhaj tshuaj thiab (2-9) qhia txog kev nce qib maj mam ntawm qhov loj me ntawm qhov txhab thiab kev nce qib raws cov nodes / internodes (Supplementary Table S2). Qhov hnyav ntawm kev kis tus kab mob pwm dawb tau hloov mus rau feem pua ​​​​siv cov mis 2:
Ntxiv mus, thaj chaw nyob hauv qab kab mob nce qib (AUDPC) tau suav los ntawm kev siv cov mis (Shaner thiab Finney, 1977), uas nyuam qhuav tau hloov kho rau cov kab mob dawb ntawm cov taum pauv (Chauhan et al., 2020) siv kab zauv 3:
Qhov twg Yi = qhov mob hnyav ntawm lub sijhawm ti, Yi + 1 = qhov mob hnyav ntawm lub sijhawm tom ntej ti + 1, ti = lub sijhawm ntsuas thawj zaug (hauv hnub), ti + 1 = lub sijhawm ntsuas tom ntej (hauv hnub), n = tag nrho cov sijhawm lossis cov ntsiab lus soj ntsuam. Cov txheej txheem kev loj hlob ntawm cov noob taum suav nrog qhov siab ntawm cov nroj tsuag (cm), tus lej ntawm cov ceg ntoo ib tsob nroj, thiab tus lej ntawm nplooj ib tsob nroj tau raug kaw txhua lub lim tiam rau 21 hnub hauv txhua qhov kev rov ua dua ntawm cov tsiaj txhu.
Hauv txhua qhov kev rov ua dua ntawm cov tsiaj txhu, cov qauv nplooj (cov nplooj thib ob thiab thib peb uas loj hlob tag nrho los ntawm sab saum toj) tau sau rau hnub 45 tom qab kev kho mob (15 hnub tom qab kev kho mob zaum kawg). Txhua qhov kev rov ua dua ntawm cov tsiaj txhu muaj tsib lub lauj kaub (ob tsob nroj ib lub lauj kaub). Kwv yees li 500 mg ntawm cov ntaub so ntswg raug siv rau kev rho tawm cov xim photosynthetic (chlorophyll a, chlorophyll b thiab carotenoids) siv 80% acetone ntawm 4 ° C hauv qhov tsaus ntuj. Tom qab 24 teev, cov qauv tau centrifuged thiab cov supernatant tau sau rau kev txiav txim siab ntawm chlorophyll a, chlorophyll b thiab carotenoid cov ntsiab lus colorimetrically siv UV-160A spectrophotometer (Shimadzu Corporation, Nyiv) raws li txoj kev ntawm (Lichtenthaler, 1987) los ntawm kev ntsuas qhov absorbance ntawm peb qhov sib txawv wavelengths (A470, A646 thiab A663 nm). Thaum kawg, cov ntsiab lus ntawm cov xim photosynthetic tau suav siv cov qauv hauv qab no 4-6 piav qhia los ntawm Lichtenthaler (1987).
Tom qab 72 teev kho (hpt), nplooj (cov nplooj thib ob thiab thib peb uas loj hlob tag nrho los ntawm sab saum toj) tau sau los ntawm txhua qhov kev hloov pauv ntawm cov kab mob rau qhov chaw nyob hauv qhov chaw ntawm hydrogen peroxide (H2O2) thiab superoxide anion (O2•−). Txhua qhov kev hloov pauv ntawm cov kab mob muaj tsib lub lauj kaub (ob tsob nroj rau ib lub lauj kaub). Txhua qhov kev hloov pauv ntawm cov kab mob tau raug tshuaj xyuas ob zaug (ob qhov kev hloov pauv ntawm kev siv tshuab) kom ntseeg tau tias qhov tseeb, kev ntseeg tau thiab kev rov ua dua ntawm txoj kev. H2O2 thiab O2•− tau txiav txim siab siv 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Lub Tebchaws Yelemees) lossis nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Lub Tebchaws Yelemees), raws li cov txheej txheem piav qhia los ntawm Romero-Puertas et al. (2004) thiab Adam et al. (1989) nrog kev hloov kho me me. Rau qhov chaw histochemical ntawm H2O2 hauv situ, cov ntawv tau raug infiltrated nrog 0.1% DAB hauv 10 mM Tris buffer (pH 7.8) thiab tom qab ntawd incubated ntawm chav tsev kub hauv lub teeb rau 60 feeb. Cov ntawv tau bleached hauv 0.15% (v/v) TCA hauv 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals thiab Medical Supplies, Cairo, Egypt) thiab tom qab ntawd raug rau lub teeb kom txog thaum lawv tsaus ntuj. Ib yam li ntawd, cov valves tau raug infiltrated nrog 10 mM potassium phosphate buffer (pH 7.8) uas muaj 0.1 w/v% HBT rau qhov chaw histochemical ntawm O2•− hauv situ. Cov ntawv tau raug incubated hauv lub teeb ntawm chav tsev kub rau 20 feeb, tom qab ntawd bleached raws li saum toj no, thiab tom qab ntawd illuminated kom txog thaum tsaus xiav / violet me ntsis tshwm sim. Qhov xim av (ua ib qho cim qhia H2O2) lossis xim xiav-violet (ua ib qho cim qhia O2•−) tau raug soj ntsuam siv Fiji version ntawm pob khoom ua duab ImageJ (http://fiji.sc; nkag mus rau 7 Lub Peb Hlis 2024).
Malondialdehyde (MDA; ua ib qho cim ntawm lipid peroxidation) tau txiav txim siab raws li txoj kev ntawm Du thiab Bramlage (1992) nrog kev hloov kho me ntsis. Nplooj ntawm txhua qhov kev rov ua dua ntawm cov tsiaj txhu (qhov thib ob thiab thib peb nplooj uas tsim tag nrho los ntawm sab saum toj) tau sau 72 teev tom qab kev kho mob (hpt). Txhua qhov kev rov ua dua ntawm cov tsiaj txhu suav nrog tsib lub lauj kaub (ob tsob nroj hauv ib lub lauj kaub). Txhua qhov kev rov ua dua ntawm cov tsiaj txhu tau raug soj ntsuam ua ob zaug (ob qhov kev rov ua dua ntawm kev siv tshuab) kom ntseeg tau tias qhov tseeb, kev ntseeg siab thiab kev rov ua dua ntawm txoj kev. Hauv luv luv, 0.5 g ntawm cov nplooj hauv av tau siv rau MDA rho tawm nrog 20% ​​trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) uas muaj 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). Cov ntsiab lus MDA hauv cov supernatant ces tau txiav txim siab xim los ntawm kev ntsuas qhov absorbance ntawm 532 thiab 600 nm siv UV-160A spectrophotometer (Shimadzu Corporation, Nyiv) thiab tom qab ntawd qhia ua nmol g−1 FW.
Rau kev ntsuam xyuas cov tshuaj antioxidants uas tsis yog enzymatic thiab enzymatic, nplooj (nplooj thib ob thiab thib peb uas loj hlob tag nrho los ntawm sab saum toj) tau sau los ntawm txhua qhov kev rov ua dua ntawm lub cev ntawm 72 teev tom qab kev kho mob (hpt). Txhua qhov kev rov ua dua ntawm lub cev muaj tsib lub lauj kaub (ob tsob nroj ib lub lauj kaub). Txhua qhov qauv ntawm lub cev tau raug tshuaj xyuas ua ob daim (ob qho qauv kev siv tshuab). Ob nplooj tau raug zom nrog cov kua nitrogen thiab siv ncaj qha rau kev txiav txim siab ntawm cov tshuaj antioxidants enzymatic thiab tsis yog enzymatic, tag nrho cov amino acids, cov ntsiab lus proline, kev qhia txog gene, thiab kev ntsuas oxalate.
Tag nrho cov phenolics uas yaj tau raug txiav txim siab siv Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) nrog kev hloov kho me ntsis ntawm txoj kev piav qhia los ntawm Kahkonen et al. (1999). Hauv luv luv, kwv yees li 0.1 g ntawm cov nplooj ntoos homogenized tau rho tawm nrog 20 ml 80% methanol hauv qhov tsaus ntuj rau 24 teev thiab cov supernatant tau sau tom qab centrifugation. 0.1 ml ntawm cov qauv rho tawm tau sib xyaw nrog 0.5 ml Folin-Ciocalteu reagent (10%), co rau 30 vib nas this thiab tso rau hauv qhov tsaus ntuj rau 5 feeb. Tom qab ntawd 0.5 ml ntawm 20% sodium carbonate kua (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) tau ntxiv rau txhua lub raj, sib xyaw kom huv si thiab incubated ntawm chav tsev kub hauv qhov tsaus ntuj rau 1 teev. Tom qab kev sib xyaw ua ke, qhov nqus ntawm cov tshuaj sib xyaw tau ntsuas ntawm 765 nm siv lub tshuab ntsuas UV-160A (Shimadzu Corporation, Nyiv). Qhov concentration ntawm tag nrho cov phenols soluble hauv cov qauv rho tawm tau txiav txim siab siv cov kab ntsuas gallic acid (Fisher Scientific, Hampton, NH, Tebchaws Meskas) thiab qhia ua milligrams ntawm gallic acid sib npaug rau ib gram ntawm qhov hnyav tshiab (mg GAE g-1 qhov hnyav tshiab).
Tag nrho cov flavonoid uas yaj tau raug txiav txim siab raws li txoj kev ntawm Djeridane et al. (2006) nrog kev hloov kho me ntsis. Hauv luv luv, 0.3 ml ntawm cov tshuaj methanol saum toj no tau sib xyaw nrog 0.3 ml ntawm 5% txhuas chloride kua (AlCl3; Fisher Scientific, Hampton, NH, USA), do kom zoo thiab tom qab ntawd incubated ntawm chav tsev kub rau 5 feeb, ua raws li ntxiv 0.3 ml ntawm 10% potassium acetate kua (Al-Gomhoria Pharmaceuticals thiab Medical Supplies, Cairo, Egypt), sib xyaw kom huv si thiab incubated ntawm chav tsev kub rau 30 feeb hauv qhov tsaus ntuj. Tom qab incubation, qhov absorbance ntawm cov tshuaj sib tov tau ntsuas ntawm 430 nm siv UV-160A spectrophotometer (Shimadzu Corporation, Nyiv). Qhov concentration ntawm tag nrho cov flavonoids uas yaj tau hauv cov qauv rho tawm tau txiav txim siab siv cov kab rutin calibration (TCI America, Portland, OR, USA) thiab tom qab ntawd qhia ua milligrams ntawm rutin sib npaug rau ib gram ntawm qhov hnyav tshiab (mg RE g-1 qhov hnyav tshiab).
Tag nrho cov amino acid dawb ntawm cov nplooj taum tau txiav txim siab siv cov tshuaj ninhydrin reagent hloov kho (Thermo Scientific Chemicals, Waltham, MA, USA) raws li txoj kev uas Yokoyama thiab Hiramatsu (2003) tau hais thiab hloov kho los ntawm Sun et al. (2006). Hauv luv luv, 0.1 g ntawm cov ntaub so ntswg hauv av tau rho tawm nrog pH 5.4 buffer, thiab 200 μL ntawm supernatant tau cuam tshuam nrog 200 μL ntawm ninhydrin (2%) thiab 200 μL ntawm pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), incubated hauv lub dab dej kub rau 30 feeb, tom qab ntawd txias thiab ntsuas ntawm 580 nm siv UV-160A spectrophotometer (Shimadzu Corporation, Nyiv). Ntawm qhov tod tes, proline tau txiav txim siab los ntawm txoj kev Bates (Bates et al., 1973). Proline tau rho tawm nrog 3% sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) thiab tom qab centrifugation, 0.5 ml ntawm supernatant tau sib xyaw nrog 1 ml glacial acetic acid (Fisher Scientific, Hampton, NH, USA) thiab ninhydrin reagent, incubated ntawm 90 ° C rau 45 feeb, txias thiab ntsuas ntawm 520 nm siv tib lub spectrophotometer li saum toj no. Tag nrho cov amino acids dawb thiab proline hauv cov nplooj extracts tau txiav txim siab siv glycine thiab proline calibration curves (Sigma-Aldrich, St Louis, MO, USA), feem, thiab qhia ua mg / g qhov hnyav tshiab.
Txhawm rau txiav txim siab qhov kev ua haujlwm enzymatic ntawm cov enzymes antioxidant, kwv yees li 500 mg ntawm cov ntaub so ntswg homogenized tau rho tawm nrog 3 ml ntawm 50 mM Tris buffer (pH 7.8) uas muaj 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) thiab 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), centrifuged ntawm 10,000 × g rau 20 feeb hauv qab lub tub yees (4 ° C), thiab cov supernatant (crude enzyme extract) tau sau (El-Nagar et al., 2023; Osman et al., 2023). Catalase (CAT) ces tau reacted nrog 2 ml ntawm 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) thiab 100 μl ntawm 269 mM H2O2 kua los txiav txim siab nws cov enzymatic ua haujlwm raws li txoj kev ntawm Aebi (1984) nrog me ntsis kev hloov kho (El-Nagar et al., 2023; Osman et al., 2023). Guaiacol-dependent peroxidase (POX) enzymatic ua haujlwm tau txiav txim siab siv txoj kev ntawm Harrach et al. (2009). (2008) nrog kev hloov kho me me (El-Nagar et al., 2023; Osman et al., 2023) thiab qhov kev ua haujlwm enzymatic ntawm polyphenol oxidase (PPO) tau txiav txim siab tom qab tshuaj tiv thaiv nrog 2.2 ml ntawm 100 mM sodium phosphate buffer (pH 6.0), 100 μl ntawm guaiacol (TCI tshuaj, Portland, OR, USA) thiab 100 μl ntawm 12 mM H2O2. Txoj kev tau hloov kho me ntsis los ntawm (El-Nagar et al., 2023; Osman et al., 2023). Qhov kev ntsuam xyuas tau ua tom qab tshuaj tiv thaiv nrog 3 ml ntawm catechol kua (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) tshiab npaj hauv 0.1 M phosphate buffer (pH 6.0). Kev ua haujlwm CAT tau ntsuas los ntawm kev saib xyuas qhov kev lwj ntawm H2O2 ntawm 240 nm (A240), kev ua haujlwm POX tau ntsuas los ntawm kev saib xyuas qhov nce ntawm kev nqus ntawm 436 nm (A436), thiab kev ua haujlwm PPO tau ntsuas los ntawm kev sau cov kev hloov pauv ntawm kev nqus ntawm 495 nm (A495) txhua 30 s rau 3 feeb siv UV-160A spectrophotometer (Shimadzu, Nyiv).
Cov RT-PCR tiag tiag tau siv los ntes cov theem sau ntawv ntawm peb lub noob caj noob ces uas cuam tshuam nrog antioxidant, suav nrog peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), thiab glutathione reductase (PvGR; GenBank Accession No. KY195009.1), hauv nplooj taum (cov nplooj thib ob thiab thib peb uas loj hlob tag nrho los ntawm sab saum toj) 72 teev tom qab kev kho mob kawg. Hauv luv luv, RNA tau raug rho tawm siv Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, Tuam Tshoj) raws li cov txheej txheem ntawm cov chaw tsim khoom. Tom qab ntawd, cDNA tau synthesized siv TOP script™ cDNA Synthesis Kit raws li cov lus qhia ntawm cov chaw tsim khoom. Cov kab ke primer ntawm peb lub noob caj noob ces saum toj no tau teev nyob rau hauv Supplementary Table S3. PvActin-3 (GenBank accession number: XM_068616709.1) tau siv ua tus gene housekeeping thiab qhov kev qhia txog gene tau suav los ntawm kev siv txoj kev 2-ΔΔCT (Livak thiab Schmittgen, 2001). Kev ruaj khov ntawm Actin nyob rau hauv kev ntxhov siab biotic (kev sib cuam tshuam tsis sib xws ntawm cov legumes thiab cov pwm anthracnose Colletotrichum lindemuthianum) thiab kev ntxhov siab abiotic (kev qhuav, salinity, qhov kub qis) tau pom (Borges et al., 2012).
Peb pib ua qhov kev tshuaj xyuas genome-wide hauv silico ntawm oxaloacetate acetylhydrolase (OAH) cov protein hauv S. sclerotiorum siv cov cuab yeej protein-protein BLAST (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Hauv luv luv, peb siv OAH los ntawm Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; GenBank accession number XP_040799428.1; 342 amino acids) thiab Penicillium lagena (PlOAH; taxide: 94218; GenBank accession number XP_056833920.1; 316 amino acids) ua cov lus nug kab lus los kos duab cov protein homologous hauv S. sclerotiorum (taxide: 5180). BLASTp tau ua tiav tawm tsam cov ntaub ntawv genome S. sclerotiorum tshiab tshaj plaws hauv GenBank ntawm lub vev xaib National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Ntxiv rau, cov noob caj noob ces OAH uas tau kwv yees los ntawm S. sclerotiorum (SsOAH) thiab kev tshuaj xyuas kev hloov pauv thiab tsob ntoo phylogenetic ntawm AfOAH los ntawm A. fijiensis CBS 313.89 thiab PlOAH los ntawm P. lagena tau xav txog siv txoj kev xav tau siab tshaj plaws hauv MEGA11 (Tamura et al., 2021) thiab JTT matrix-based model (Jones et al., 1992). Tsob ntoo phylogenetic tau muab tso ua ke nrog kev tshuaj xyuas ntau yam kev sib phim ntawm cov protein sequences ntawm txhua cov noob caj noob ces OAH uas tau kwv yees (SsOAH) los ntawm S. sclerotiorum thiab cov lus nug sequence siv Constraint-Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos thiab Agarwala, 2007). Ntxiv rau, cov kab lus amino acid zoo tshaj plaws ntawm SsOAH los ntawm S. sclerotiorum tau sib phim nrog cov kab lus nug (AfOAH thiab PlOAH) (Larkin et al., 2007) siv ClustalW (http://www.genome.jp/tools-bin/clustalw), thiab cov cheeb tsam khaws cia hauv qhov sib phim tau pom siv cov cuab yeej ESPript (version 3.0; https://espript.ibcp.fr/ESPript/ESPript/index.php).
Ntxiv mus, cov chaw sawv cev ua haujlwm kwv yees thiab cov chaw khaws cia ntawm S. sclerotiorum SsOAH tau raug faib ua pawg sib txawv siv cov cuab yeej InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Thaum kawg, kev tsim qauv peb-seem (3D) ntawm S. sclerotiorum SsOAH uas kwv yees tau ua tiav siv Protein 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) thiab tau lees paub siv SWISS-MODEL server (https://swissmodel.expasy.org/) (Biasini et al., 2014). Cov qauv peb-seem uas kwv yees tau (hom ntawv PDB) tau pom tau los ntawm kev siv pob UCSF-Chimera (version 1.15; https://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004).
Kev siv PCR fluorescence real-time los txiav txim siab txog qib transcriptional ntawm oxaloacetate acetylhydrolase (SsOAH; GenBank accession number: XM_001590428.1) hauv cov mycelia ntawm Sclerotinia sclerotiorum. Hauv luv luv, S. sclerotiorum tau txhaj tshuaj rau hauv lub raj mis uas muaj PDB thiab muab tso rau hauv lub tshuab co (qauv: I2400, New Brunswick Scientific Co., Edison, NJ, USA) ntawm 25 ± 2 °C rau 24 teev ntawm 150 rpm thiab hauv qhov tsaus ntuj tas li (24 teev) los txhawb kev loj hlob ntawm mycelial. Tom qab ntawd, cov hlwb tau kho nrog L-ornithine thiab fungicide Rizolex-T ntawm qhov kawg IC50 concentration (kwv yees li 40 thiab 3.2 mg / L, feem) thiab tom qab ntawd cog rau lwm 24 teev nyob rau hauv tib qho xwm txheej. Tom qab kev sib xyaw ua ke, cov kab lis kev cai tau centrifuged ntawm 2500 rpm rau 5 feeb thiab cov supernatant (fungal mycelium) tau sau rau kev tshuaj xyuas kev qhia txog noob caj noob ces. Ib yam li ntawd, fungal mycelium tau sau ntawm 0, 24, 48, 72, 96, thiab 120 teev tom qab kev kis tus kab mob los ntawm cov nroj tsuag uas muaj kab mob uas tau tsim cov pwm dawb thiab paj rwb mycelium ntawm qhov chaw ntawm cov ntaub so ntswg uas muaj kab mob. RNA tau rho tawm los ntawm fungal mycelium thiab tom qab ntawd cDNA tau synthesized raws li tau piav qhia saum toj no. Cov kab ke primer rau SsOAH tau teev nyob rau hauv Supplementary Table S3. SsActin (GenBank accession number: XM_001589919.1) tau siv ua housekeeping gene, thiab kev qhia txog noob caj noob ces tau suav siv txoj kev 2-ΔΔCT (Livak thiab Schmittgen, 2001).
Oxalic acid tau txiav txim siab hauv cov kua txiv qos yaj ywm dextrose (PDB) thiab cov qauv cog uas muaj cov kab mob fungal Sclerotinia sclerotiorum raws li txoj kev ntawm Xu thiab Zhang (2000) nrog kev hloov kho me ntsis. Hauv luv luv, S. sclerotiorum isolates tau txhaj tshuaj rau hauv cov fwj uas muaj PDB thiab tom qab ntawd cog rau hauv lub tshuab co (qauv I2400, New Brunswick Scientific Co., Edison, NJ, USA) ntawm 150 rpm ntawm 25 ± 2 ° C rau 3-5 hnub hauv qhov tsaus ntuj tas li (24 teev) los txhawb kev loj hlob ntawm mycelial. Tom qab kev cog qoob loo, cov kab lis kev cai fungal tau lim thawj zaug los ntawm daim ntawv lim Whatman # 1 thiab tom qab ntawd centrifuged ntawm 2500 rpm rau 5 feeb kom tshem tawm cov mycelium seem. Lub supernatant tau sau thiab khaws cia ntawm 4 ° C rau kev txiav txim siab ntxiv ntawm oxalate. Rau kev npaj cov qauv cog, kwv yees li 0.1 g ntawm cov ntaub so ntswg cog tau rho tawm peb zaug nrog dej distilled (2 ml txhua lub sijhawm). Cov qauv kuaj tau centrifuged ntawm 2500 rpm rau 5 feeb, cov supernatant tau lim qhuav los ntawm Whatman No. 1 lim ntawv thiab sau rau kev tshuaj xyuas ntxiv.
Rau kev tshuaj xyuas kom muaj nuj nqis ntawm oxalic acid, cov tshuaj sib tov tau npaj rau hauv lub raj iav stoppered hauv qab no: 0.2 ml ntawm cov qauv (lossis PDB kab lis kev cai filtrate lossis oxalic acid txheem kua), 0.11 ml ntawm bromophenol xiav (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, Tebchaws Meskas), 0.198 ml ntawm 1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals thiab Medical Supplies, Cairo, Egypt) thiab 0.176 ml ntawm 100 mM potassium dichromate (K2Cr2O7; TCI tshuaj, Portland, OR, Tebchaws Meskas), thiab tom qab ntawd cov tshuaj tau diluted rau 4.8 ml nrog dej distilled, sib xyaw ua ke thiab tam sim ntawd muab tso rau hauv 60 ° C dej da dej. Tom qab 10 feeb, cov tshuaj tau nres los ntawm kev ntxiv 0.5 ml ntawm sodium hydroxide kua (NaOH; 0.75 M). Qhov absorbance (A600) ntawm cov tshuaj sib xyaw tau ntsuas ntawm 600 nm siv lub UV-160 spectrophotometer (Shimadzu Corporation, Nyiv). PDB thiab dej distilled tau siv los ua kev tswj hwm rau kev ntsuas ntawm cov kab lis kev cai lim dej thiab cov qauv cog, raws li. Cov oxalic acid concentration hauv cov kab lis kev cai lim dej, qhia ua micrograms ntawm oxalic acid ib milliliter ntawm PDB medium (μg.mL−1), thiab hauv cov nplooj extracts, qhia ua micrograms ntawm oxalic acid ib gram ntawm qhov hnyav tshiab (μg.g−1 FW), tau txiav txim siab siv cov kab oxalic acid calibration (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
Thoob plaws hauv kev tshawb fawb, txhua qhov kev sim tau tsim los ntawm kev tsim qauv randomized tag nrho (CRD) nrog rau rau qhov kev rov ua dua ntawm cov kab mob hauv ib qho kev kho mob thiab tsib lub lauj kaub rau ib qho kev rov ua dua ntawm cov kab mob hauv ib qho (ob tsob nroj rau ib lub lauj kaub) tshwj tsis yog hais lwm yam. Cov kev rov ua dua ntawm cov kab mob hauv ib qho tau soj ntsuam ua ob zaug (ob qhov kev rov ua dua ntawm kev siv tshuab). Cov kev rov ua dua ntawm kev siv tshuab tau siv los xyuas qhov rov ua dua ntawm tib qho kev sim tab sis tsis tau siv rau hauv kev tshuaj xyuas cov ntaub ntawv kom tsis txhob muaj kev rov ua dua cuav. Cov ntaub ntawv tau tshuaj xyuas cov ntaub ntawv siv kev tshuaj xyuas ntawm qhov sib txawv (ANOVA) ua raws li Tukey-Kramer qhov sib txawv tseem ceeb (HSD) xeem (p ≤ 0.05). Rau kev sim hauv vitro, IC50 thiab IC99 tus nqi tau suav siv tus qauv probit thiab 95% kev ntseeg siab tau suav.
Tag nrho plaub hom kab mob tau sau los ntawm ntau hom teb taum pauv hauv El Ghabiya Governorate, tebchaws Iziv. Ntawm PDA medium, txhua hom kab mob tau tsim cov mycelium dawb creamy uas sai sai hloov mus ua paj rwb dawb (Daim Duab 1A) thiab tom qab ntawd beige lossis xim av ntawm theem sclerotium. Sclerotia feem ntau yog ntom, dub, kheej kheej lossis tsis sib luag, ntev 5.2 txog 7.7 hli thiab txoj kab uas hla 3.4 txog 5.3 hli (Daim Duab 1B). Txawm hais tias plaub hom kab mob tau tsim cov qauv ntawm sclerotia ntawm ntug ntawm cov kab lis kev cai tom qab 10-12 hnub ntawm kev cog qoob loo ntawm 25 ± 2 ° C (Daim Duab 1A), tus lej ntawm sclerotia ib phaj sib txawv ntawm lawv (P < 0.001), nrog rau isolate 3 muaj tus lej ntau tshaj plaws ntawm sclerotia (32.33 ± 1.53 sclerotia ib phaj; Daim Duab 1C). Ib yam li ntawd, isolate #3 tsim tau ntau oxalic acid hauv PDB dua li lwm cov isolates (3.33 ± 0.49 μg.mL−1; Daim Duab 1D). Isolate #3 qhia txog cov yam ntxwv morphological thiab microscopic ntawm phytopathogenic fungus Sclerotinia sclerotiorum. Piv txwv li, ntawm PDA, cov pawg ntawm isolate #3 loj hlob sai, yog creamy dawb (Daim Duab 1A), reverse beige lossis lub teeb salmon daj-xim av, thiab xav tau 6-7 hnub ntawm 25 ± 2 ° C kom npog tag nrho ntawm qhov chaw ntawm 9 cm txoj kab uas hla phaj. Raws li cov yam ntxwv morphological thiab microscopic saum toj no, isolate #3 tau txheeb xyuas tias yog Sclerotinia sclerotiorum.
Daim Duab 1. Cov yam ntxwv thiab pathogenicity ntawm S. sclerotiorum isolates los ntawm cov qoob loo legume feem ntau. (A) Mycelial kev loj hlob ntawm plaub S. sclerotiorum isolates ntawm PDA medium, (B) sclerotia ntawm plaub S. sclerotiorum isolates, (C) tus lej ntawm sclerotia (ib phaj), (D) oxalic acid secretion ntawm PDB medium (μg.mL−1), thiab (E) qhov mob hnyav (%) ntawm plaub S. sclerotiorum isolates ntawm cov lag luam legume cultivar Giza 3 nyob rau hauv cov xwm txheej greenhouse. Cov nqi sawv cev rau qhov nruab nrab ± SD ntawm tsib biological replicates (n = 5). Cov ntawv sib txawv qhia txog qhov sib txawv tseem ceeb ntawm kev kho mob (p < 0.05). (F-H) Cov tsos mob pwm dawb feem ntau tshwm sim ntawm cov qia saum av thiab siliques, feem, 10 hnub tom qab inoculation nrog isolate #3 (dpi). (I) Kev tshuaj xyuas kev hloov pauv ntawm thaj chaw sab hauv transcribed spacer (ITS) ntawm S. sclerotiorum isolate #3 tau ua tiav siv txoj kev xav tau siab tshaj plaws thiab piv nrog 20 qhov kev sib cais / hom kab mob tau los ntawm National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). Cov lej saum toj no cov kab sib sau ua ke qhia txog thaj chaw npog (%), thiab cov lej hauv qab cov kab sib sau ua ke qhia txog qhov ntev ntawm ceg.
Ntxiv mus, txhawm rau kom paub meej tias muaj kab mob, plaub hom kab mob S. sclerotiorum tau txais los txhaj tshuaj rau cov noob taum Giza 3 uas yooj yim kis tau hauv qab cov huab cua sov, uas yog sib xws nrog Koch's postulates (Daim Duab 1E). Txawm hais tias tag nrho cov kab mob fungal tau txais yog cov kab mob thiab tuaj yeem kis rau cov taum ntsuab (cv. Giza 3), ua rau muaj cov tsos mob pwm dawb rau txhua qhov saum av (Daim Duab 1F), tshwj xeeb tshaj yog rau cov qia (Daim Duab 1G) thiab cov noob (Daim Duab 1H) ntawm 10 hnub tom qab txhaj tshuaj (dpi), isolate 3 yog isolate hnyav tshaj plaws hauv ob qhov kev sim ywj pheej. Isolate 3 muaj qhov mob hnyav tshaj plaws (%) ntawm cov nroj tsuag taum (24.0 ± 4.0, 58.0 ± 2.0, thiab 76.7 ± 3.1 ntawm 7, 14, thiab 21 hnub tom qab kis kab mob, feem; Daim Duab 1F).
Qhov kev txheeb xyuas ntawm tus kab mob S. sclerotiorum isolate #3 uas kis tau ntau tshaj plaws tau raug lees paub ntxiv raws li kev sib cais sab hauv (ITS) sequencing (Daim Duab 1I). Kev tshuaj xyuas phylogenetic ntawm isolate #3 thiab 20 tus kab mob/hom kab mob qhia tau tias muaj kev sib xws siab (>99%) ntawm lawv. Nws yog ib qho tsim nyog sau cia tias S. sclerotiorum isolate #3 (533 bp) muaj kev sib xws siab rau American S. sclerotiorum isolate LPM36 uas cais los ntawm cov noob taum qhuav (GenBank accession number MK896659.1; 540 bp) thiab Suav S. sclerotiorum isolate YKY211 (GenBank accession number OR206374.1; 548 bp), uas ua rau violet (Matthiola incana) qia lwj, tag nrho cov no tau muab faib ua pawg sib cais rau sab saum toj ntawm dendrogram (Daim Duab 1I). Cov kab ke tshiab tau muab tso rau hauv NCBI database thiab hu ua "Sclerotinia sclerotiorum - isolate YN-25" (GenBank accession number PV202792). Nws tuaj yeem pom tias isolate 3 yog qhov invasive tshaj plaws; yog li ntawd, qhov isolate no tau raug xaiv los kawm hauv txhua qhov kev sim tom qab.
Cov tshuaj diamine L-ornithine (Sigma-Aldrich, Darmstadt, Lub Tebchaws Yelemees) ntawm ntau qhov sib txawv (12.5, 25, 50, 75, 100 thiab 125 mg/L) tawm tsam S. sclerotiorum isolate 3 tau raug tshawb xyuas hauv vitro. Nws yog ib qho tseem ceeb uas L-ornithine muaj cov nyhuv tiv thaiv kab mob thiab maj mam txwv tsis pub S. sclerotiorum hyphae loj hlob hauv txoj kev uas nyob ntawm qhov koob tshuaj (Daim Duab 2A, B). Ntawm qhov siab tshaj plaws uas tau sim (125 mg/L), L-ornithine tau qhia txog qhov siab tshaj plaws ntawm kev txwv tsis pub loj hlob ntawm mycelial (99.62 ± 0.27%; Daim Duab 2B), uas sib npaug rau cov tshuaj tua kab mob Rizolex-T (qhov txwv tsis pub 99.45 ± 0.39%; Daim Duab 2C) ntawm qhov siab tshaj plaws uas tau sim (10 mg/L), qhia txog qhov ua tau zoo sib xws.
Daim Duab 2. Kev ua haujlwm tiv thaiv kab mob hauv vitro ntawm L-ornithine tawm tsam Sclerotinia sclerotiorum. (A) Kev sib piv ntawm kev ua haujlwm tiv thaiv kab mob ntawm ntau yam kev sib txawv ntawm L-ornithine tawm tsam S. sclerotiorum nrog cov tshuaj tua kab mob Rizolex-T (10 mg / L). (B, C) Tus nqi txwv (%) ntawm S. sclerotiorum mycelial kev loj hlob tom qab kev kho mob nrog ntau yam kev sib txawv ntawm L-ornithine (12.5, 25, 50, 75, 100 thiab 125 mg / L) lossis Rizolex-T (2, 4, 6, 8 thiab 10 mg / L), feem. Cov nqi sawv cev rau qhov nruab nrab ± SD ntawm tsib qhov kev rov ua dua ntawm cov kab mob (n = 5). Cov ntawv sib txawv qhia txog qhov sib txawv ntawm kev kho mob (p <0.05). (D, E) Kev tshuaj xyuas qauv rov qab ntawm L-ornithine thiab cov tshuaj tua kab mob Rizolex-T, feem. Cov kab rov qab ntawm tus qauv probit tau qhia ua kab xiav khov kho, thiab qhov nruab nrab ntawm kev ntseeg siab (95%) tau qhia ua kab liab uas muaj kab txaij.
Ntxiv rau, kev tshuaj xyuas probit regression tau ua tiav thiab cov phiaj xwm sib xws tau qhia hauv Rooj 1 thiab Cov Duab 2D, E. Hauv luv luv, tus nqi nqes hav uas lees txais tau (y = 2.92x − 4.67) thiab cov ntaub ntawv tseem ceeb cuam tshuam (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 thiab p < 0.0001; Daim Duab 2D) ntawm L-ornithine qhia txog kev ua haujlwm antifungal zoo dua tiv thaiv S. sclerotiorum piv rau cov tshuaj fungicide Rizolex-T (y = 1.96x − 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 thiab p < 0.0001) (Rooj 1).
Rooj 1. Cov nqi ntawm ib nrab ntawm qhov siab tshaj plaws inhibitory concentration (IC50) thiab IC99 (mg / l) ntawm L-ornithine thiab cov tshuaj tua kab mob "Rizolex-T" tawm tsam S. sclerotiorum.
Zuag qhia tag nrho, L-ornithine (250 mg/L) txo qhov kev loj hlob thiab qhov hnyav ntawm cov pwm dawb ntawm cov nroj tsuag taum pauv uas tau kho piv rau cov nroj tsuag uas tsis tau kho S. sclerotiorum-kis kab mob (tswj; Daim Duab 3A). Hauv luv luv, txawm hais tias qhov hnyav ntawm cov kab mob ntawm cov nroj tsuag tswj uas tsis tau kho mob maj mam nce ntxiv (52.67 ± 1.53, 83.21 ± 2.61, thiab 92.33 ± 3.06%), L-ornithine txo qhov hnyav ntawm cov kab mob (%) thoob plaws hauv kev sim (8.97 ± 0.15, 18.00 ± 1.00, thiab 26.36 ± 3.07) ntawm 7, 14, thiab 21 hnub tom qab kev kho mob (dpt), feem (Daim Duab 3A). Ib yam li ntawd, thaum cov noob taum uas muaj kab mob S. sclerotiorum raug kho nrog 250 mg/L L-ornithine, thaj chaw nyob hauv qab kab mob nce qib (AUDPC) tau txo qis los ntawm 1274.33 ± 33.13 hauv kev tswj tsis kho mus rau 281.03 ± 7.95, uas qis dua me ntsis ntawm kev tswj zoo 50 mg/L Rizolex-T fungicide (183.61 ± 7.71; Daim Duab 3B). Tib yam sib xws tau pom hauv qhov kev sim thib ob.
Daim Duab 3. Cov nyhuv ntawm kev siv L-ornithine sab nraud rau kev loj hlob ntawm cov kab dawb ntawm cov taum pauv uas tshwm sim los ntawm Sclerotinia sclerotiorum nyob rau hauv cov xwm txheej greenhouse. (A) Kab nkhaus kev loj hlob ntawm cov pwm dawb ntawm cov taum pauv tom qab kho nrog 250 mg / L L-ornithine. (B) Thaj chaw nyob rau hauv kab nkhaus kev loj hlob ntawm cov kab mob (AUDPC) ntawm cov pwm dawb ntawm cov taum pauv tom qab kho nrog L-ornithine. Cov nqi sawv cev rau qhov nruab nrab ± SD ntawm tsib qhov kev rov ua dua ntawm cov kab mob (n = 5). Cov ntawv sib txawv qhia txog qhov sib txawv tseem ceeb ntawm kev kho mob (p < 0.05).
Kev siv 250 mg/L L-ornithine sab nraud maj mam ua rau cov nroj tsuag siab dua (Daim Duab 4A), tus naj npawb ntawm cov ceg ntoo ib tsob (Daim Duab 4B), thiab tus naj npawb ntawm cov nplooj ib tsob (Daim Duab 4C) tom qab 42 hnub. Thaum cov tshuaj tua kab mob Rizolex-T (50 mg/L) muaj qhov cuam tshuam loj tshaj plaws rau txhua yam kev noj haus uas tau kawm, kev siv 250 mg/L L-ornithine sab nraud muaj qhov cuam tshuam loj tshaj plaws thib ob piv rau cov kev tswj tsis tau kho (Daim Duab 4A–C). Ntawm qhov tod tes, kev kho L-ornithine tsis muaj qhov cuam tshuam loj rau cov ntsiab lus ntawm cov xim photosynthetic chlorophyll a (Daim Duab 4D) thiab chlorophyll b (Daim Duab 4E), tab sis me ntsis ua rau tag nrho cov carotenoid ntsiab lus (0.56 ± 0.03 mg/g fr wt) piv rau cov kev tswj tsis zoo (0.44 ± 0.02 mg/g fr wt) thiab cov kev tswj zoo (0.46 ± 0.02 mg/g fr wt; Daim Duab 4F). Zuag qhia tag nrho, cov txiaj ntsig no qhia tau tias L-ornithine tsis yog phytotoxic rau cov legumes uas tau kho thiab tej zaum yuav txhawb lawv txoj kev loj hlob.
Daim Duab 4. Cov nyhuv ntawm kev siv L-ornithine sab nraud rau cov yam ntxwv kev loj hlob thiab cov xim photosynthetic ntawm cov nplooj taum uas muaj kab mob Sclerotinia sclerotiorum nyob rau hauv cov xwm txheej greenhouse. (A) Qhov siab ntawm cov nroj tsuag (cm), (B) Tus naj npawb ntawm cov ceg ntoo ib tsob nroj, (C) Tus naj npawb ntawm nplooj ib tsob nroj, (D) Chlorophyll a cov ntsiab lus (mg g-1 fr wt), (E) Chlorophyll b cov ntsiab lus (mg g-1 fr wt), (F) Tag nrho cov ntsiab lus carotenoid (mg g-1 fr wt). Cov nqi yog qhov nruab nrab ± SD ntawm tsib qhov kev rov ua dua ntawm cov kab mob (n = 5). Cov ntawv sib txawv qhia txog qhov sib txawv tseem ceeb ntawm kev kho mob (p < 0.05).
Kev tshawb nrhiav qhov chaw nyob ntawm cov tshuaj reactive oxygen (ROS; qhia ua hydrogen peroxide [H2O2]) thiab cov free radicals (qhia ua superoxide anions [O2•−]) tau qhia tias kev siv L-ornithine (250 mg/L) los ntawm sab nraud tau txo qhov kev sib sau ua ke ntawm H2O2 (96.05 ± 5.33 nmol.g−1 FW; Daim Duab 5A) thiab O2•− (32.69 ± 8.56 nmol.g−1 FW; Daim Duab 5B) piv rau kev sib sau ua ke ntawm ob qho tib si cov nroj tsuag uas tsis tau kho (173.31 ± 12.06 thiab 149.35 ± 7.94 nmol.g−1 FW, feem) thiab cov nroj tsuag uas tau kho nrog 50 mg/L ntawm cov tshuaj tua kab mob Rizolex-T (170.12 ± 9.50 thiab 157.00 ± 7.81 nmol.g−1 fr wt, feem) ntawm 72 teev. Cov H2O2 thiab O2•− ntau ntau tau sib sau ua ke hauv qab hpt (Daim Duab 5A, B). Ib yam li ntawd, TCA-based malondialdehyde (MDA) assay qhia tau tias cov nroj tsuag taum uas muaj kab mob S. sclerotiorum tau sib sau ua ke ntau dua ntawm MDA (113.48 ± 10.02 nmol.g fr wt) hauv lawv cov nplooj (Daim Duab 5C). Txawm li cas los xij, kev siv L-ornithine sab nraud tau txo qis lipid peroxidation raws li pom los ntawm kev txo qis hauv MDA cov ntsiab lus hauv cov nroj tsuag kho (33.08 ± 4.00 nmol.g fr wt).
Daim Duab 5. Cov nyhuv ntawm kev siv L-ornithine sab nraud rau cov cim tseem ceeb ntawm kev ntxhov siab oxidative thiab cov txheej txheem tiv thaiv antioxidant tsis-enzymatic hauv cov nplooj taum uas muaj kab mob S. sclerotiorum ntawm 72 teev tom qab kev kis kab mob hauv cov xwm txheej greenhouse. (A) Hydrogen peroxide (H2O2; nmol g−1 FW) ntawm 72 hpt, (B) superoxide anion (O2•−; nmol g−1 FW) ntawm 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) ntawm 72 hpt, (D) tag nrho cov phenols soluble (mg GAE g−1 FW) ntawm 72 hpt, (E) tag nrho cov flavonoids soluble (mg RE g−1 FW) ntawm 72 hpt, (F) tag nrho cov amino acids dawb (mg g−1 FW) ntawm 72 hpt, thiab (G) cov ntsiab lus proline (mg g−1 FW) ntawm 72 hpt. Cov nqi sawv cev rau qhov nruab nrab ± tus qauv sib txawv (mean ± SD) ntawm 5 qhov kev hloov pauv ntawm cov kab mob (n = 5). Cov ntawv sib txawv qhia txog qhov sib txawv tseem ceeb ntawm kev kho mob (p < 0.05).