Chinyorwa ichi chikamu chenyaya yekutsvagisa inoti "Kuvandudza kusimba kwembeu kuzvirwere nezvipembenene", ona zvinyorwa zvese zvishanu
Chinhu chinokonzera chirwere chefungus plant necrosis Sclerotinia sclerotiorum (Lib.) de Bary inoshandisa nzira yakawanda yekurwisa zvirimwa zvakasiyana-siyana. Chidzidzo ichi chinokurudzira kushandiswa kwediamine L-ornithine, amino acid isiri protein inokurudzira kugadzirwa kwemamwe maamino acids akakosha, senzira yekuwedzera mhinduro dzemamorekuru, zvemuviri uye zvemakemikari ePhaseolus vulgaris L. kune mold chena inokonzerwa nePseudomonas sclerotiorum. Kuedza kwe in vitro kwakaratidza kuti L-ornithine yakadzivirira zvakanyanya kukura kwe mycelial kweS. pyrenoidosa nenzira inoenderana nedosi. Uyezve, inogona kuderedza zvakanyanya kuoma kwemold chena pasi pemamiriro egreenhouse. Uyezve, L-ornithine yakakurudzira kukura kwezvirimwa zvakarapwa, zvichiratidza kuti huwandu hwakaedzwa hweL-ornithine hwakanga husina chepfu kuzvirimwa zvakarapwa. Pamusoro pezvo, L-ornithine yakawedzera kuburitswa kwenon-enzyme antioxidants (total soluble phenolics and flavonoids) uye enzymatic antioxidants (catalase (CAT), peroxidase (POX), uye polyphenol oxidase (PPO)), uye yakawedzera kuburitswa kwemajini matatu ane chekuita neantioxidant (PvCAT1, PvSOD, uye PvGR). Uyezve, mukuongorora kwesilico kwakaratidza kuvapo kweprotein ye oxaloacetate acetylhydrolase (SsOAH) mu genome yeS. sclerotiorum, iyo yaive yakafanana zvikuru nemapuroteni e oxaloacetate acetylhydrolase (SsOAH) eAspergillus fijiensis (AfOAH) nePenicillium sp. (PlOAH) maererano nekuongorora kwebasa, nzvimbo dzakachengetwa, uye topology. Zvinofadza kuti kuwedzerwa kweL-ornithine kune potato dextrose broth (PDB) medium kwakaderedza zvakanyanya kuburitswa kweSsOAH gene muS. sclerotiorum mycelia. Saizvozvowo, kushandiswa kweL-ornithine kunze kwakaderedza zvakanyanya kuburitswa kweSsOAH gene mufungal mycelia yakaunganidzwa kubva mumiti yakarapwa. Pakupedzisira, kushandiswa kweL-ornithine kwakaderedza zvakanyanya kuburitswa kweoxalic acid mumashizha ese ari maviri ePDB nemashizha ane hutachiona. Mukupedzisa, L-ornithine inoita basa guru mukuchengetedza redox pamwe nekuwedzera simba rekudzivirira zvirimwa zvine hutachiona. Mhedzisiro yechidzidzo ichi inogona kubatsira mukugadzira nzira itsva, dzisingakuvadzi nharaunda dzekudzora mold chena uye kuderedza kukanganisa kwayo pakurima nyemba nezvimwe zvirimwa.
Chakuvhuvhu chena, chinokonzerwa nefungus inonzi necrotrophic fungus Sclerotinia sclerotiorum (Lib.) de Bary, chirwere chinoparadza, chinoderedza goho chinoisa njodzi huru kukugadzirwa kwebhinzi pasi rose (Phaseolus vulgaris L.) (Bolton et al., 2006). Sclerotinia sclerotiorum ndeimwe yezvirwere zvefungus zvinotapukira muvhu zvakanyanya kuoma kudzora, ine mhando dzakasiyana dzezvirimwa dzinopfuura 600 uye kugona kukurumisa matishu emhuka nenzira isiriyo chaiyo (Liang naRollins, 2018). Mumamiriro ezvinhu asina kunaka, inorarama kwenguva yakareba, ikasarara kwenguva yakareba semaumbirwo matema, akaomarara, akafanana nembeu anonzi 'sclerotia' muvhu kana semakuriro machena, akapfava mu mycelium kana stem pith yezvirimwa zvine hutachiona (Schwartz et al., 2005). S. sclerotiorum inokwanisa kugadzira sclerotia, iyo inoita kuti irarame muminda ine hutachiona kwenguva yakareba uye irambe iripo panguva yekurwara (Schwartz et al., 2005). Sclerotia ine zvinovaka muviri zvakawanda, inogona kuramba iri muvhu kwenguva yakareba, uye inoshanda senzira huru yekudzivirira hutachiona hunotevera (Schwartz et al., 2005). Mumamiriro ezvinhu akanaka, sclerotia inomera uye inoburitsa spores dziri mumhepo dzinogona kutapurira zvikamu zvese zviri pamusoro pevhu zvemuti, kusanganisira asi kwete kungova maruva, madzinde, kana pods (Schwartz et al., 2005).
Sclerotinia sclerotiorum inoshandisa nzira dzakasiyana-siyana dzekurwisa zvirimwa zvayo, dzinosanganisira zviitiko zvakasiyana-siyana kubva pakumera kwesclerotial kusvika pakukura kwezviratidzo. Pakutanga, S. sclerotiorum inoburitsa spores dzakamiswa (dzinonzi ascospores) kubva muzvimiro zvakaita sehowa zvinonzi apothecia, zvinozobuda mumhepo uye zvinokura kuita sclerotia isingafambi pamarara emiti ane hutachiona (Bolton et al., 2006). Howa iyi inobva yaburitsa oxalic acid, chinhu chinokanganisa hutachiona, kudzora pH yemadziro emasero emiti, kukurudzira kuora kwe enzyme uye kupinda kwematishu (Hegedus naRimmer, 2005), uye kudzvinyirira kuputika kwemuti wemuti. Maitiro aya ekusimbisa acid anoshayisa simba madziro emasero emiti, zvichipa nzvimbo yakanaka yekushanda kwakanaka kwema enzymes anoparadza madziro emasero efungus (CWDEs), zvichibvumira utachiona kukunda chipingamupinyi chemuviri uye kupinda mumatishu emuti wemuti (Marciano et al., 1983). Kana yapinda, S. sclerotiorum inoburitsa huwandu hweCWDEs, dzakadai sepolygalacturonase necellulase, izvo zvinoita kuti ipararire mumatishu ane utachiona uye zvinokonzera necrosis yematishu. Kufambira mberi kwemaronda nema hyphal mats kunotungamira kuzviratidzo zvemhando ye white mold (Hegedus naRimmer, 2005). Panguva iyi, zvirimwa zvinogamuchira mhuka zvinoziva pathogen-associated molecular patterns (PAMPs) kuburikidza nema pattern recognition receptors (PRRs), zvichikonzera zviitiko zvakawanda zvinozopedzisira zvaita kuti dziviriro ishande.
Pasinei nemakumi emakore ekuedza kudzora zvirwere, kushomeka kwembeu dzinorwisa zvipembenene zvakakwana kuchiripo mubhinzi, sezvinoitwa mune dzimwe zvirimwa zvekutengesa, nekuda kwekusagona kurwisa, kurarama, uye kugona kuchinja hutachiona. Saka kutonga zvirwere kwakaoma zvikuru uye kunoda nzira yakabatana, ine mativi akawanda inosanganisira musanganiswa wetsika, kudzora zvipenyu, uye mishonga inorwisa zvipembenene ine makemikari (O'Sullivan et al., 2021). Kudzora makemikari efungus chena ndiko kunonyanya kushanda nekuti mishonga inorwisa zvipembenene, kana ikashandiswa nemazvo uye panguva yakakodzera, inogona kudzora kupararira kwechirwere zvinobudirira, kuderedza kuoma kwehutachiona, uye kuderedza kurasikirwa kwegoho. Zvisinei, kushandisa zvakanyanya uye kuvimba zvakanyanya nemishonga inorwisa zvipembenene kunogona kutungamira mukubuda kwemhando dzinorwisa dzeS. sclerotiorum uye kukanganisa zvisikwa zvisiri zvekurwisa, hutano hwevhu, uye kunaka kwemvura (Le Cointe et al., 2016; Ceresini et al., 2024). Nokudaro, kuwana dzimwe nzira dzisingakuvadzi nharaunda kwave chinhu chakakosha.
Polyamines (PAs), dzakadai seputrescine, spermidine, spermine, uye cadaverine, dzinogona kushanda senzira dzinovimbisa dzekudzivirira utachiona hunobva mumiti, nokudaro zvichideredza zvachose kana kuti zvishoma kushandiswa kwemishonga inouraya fungicides ine ngozi (Nehela et al., 2024; Yi et al., 2024). Mumiti yakakwirira, maPA anobatanidzwa mumabasa akawanda emuviri anosanganisira, asi kwete kungogumira pakupatsanurwa kwemasero, kusiyanisa, uye kupindura kumatambudziko ekusagadzikana uye biotic (Killiny naNehela, 2020). Zvinogona kushanda sema antioxidants, kubatsira kubvisa reactive oxygen species (ROS), kuchengetedza redox homeostasis (Nehela naKilliny, 2023), kukurudzira majini ane chekuita nekudzivirira (Romero et al., 2018), kudzora nzira dzakasiyana dze metabolic (Nehela naKilliny, 2023), kugadzirisa endogenous phytohormones (Nehela naKilliny, 2019), kusimbisa systemic acquired resistance (SAR), uye kudzora plant-pathogen interactions (Nehela naKilliny, 2020; Asija et al., 2022; Czerwoniec, 2022). Zvakakosha kuziva kuti maitiro chaiwo uye mabasa ePAs mukudzivirira zvirimwa anosiyana zvichienderana nemarudzi emiti, hutachiona, uye mamiriro ezvakatipoteredza. PA yakawanda mumiti inogadzirwa kubva ku polyamine L-ornithine yakakosha (Killiny naNehela, 2020).
L-ornithine ine mabasa akawanda mukukura nekukura kwezvirimwa. Semuenzaniso, zvidzidzo zvekare zvakaratidza kuti mumupunga (Oryza sativa), ornithine inogona kunge yakabatana nekudzokorodza nitrogen (Liu et al., 2018), goho remupunga, mhando uye hwema (Lu et al., 2020), uye mhinduro yekumanikidzwa nemvura (Yang et al., 2000). Uyezve, kushandiswa kweL-ornithine kunze kwenyika kwakasimudzira zvakanyanya kushivirira kusanaya kwemvura mushuga beet (Beta vulgaris) (Hussein et al., 2019) uye kwakaderedza kushushikana kwemunyu muhanyanisi (Allium Cepa) (Çavuşoǧlu naÇavuşoǧlu, 2021) uye cashew (Anacardium occidentale) zvirimwa (da Rocha et al., 2012). Basa rinogona kuitwa neL-ornithine mukudzivirira kushushikana kweabiotic rinogona kunge riri nekuda kwekubatanidzwa kwayo mukuunganidzwa kweproline muzvirimwa zvakarapwa. Semuenzaniso, majini ane chekuita neproline metabolism, akadai se ornithine delta aminotransferase (delta-OAT) uye majini e proline dehydrogenase (ProDH1 naProDH2), akambotaurwa kuti ane chekuita nekudzivirira Nicotiana benthamiana naArabidopsis thaliana kubva kuPseudomonas syringae strains dzisiri dzePseudomonas syringae (Senthil-Kumar naMysore, 2012). Kune rumwe rutivi, fungal ornithine decarboxylase (ODC) inodiwa kuti pave nekukura kwezvirwere (Singh et al., 2020). Kutarisa ODC yeFusarium oxysporum f. sp. lycopersici kuburikidza nehost-induced gene silencing (HIGS) kwakasimbisa zvakanyanya kuramba kwezvirimwa zvematomatisi kuFusarium wilt (Singh et al., 2020). Zvisinei, basa rinogona kuitwa nekushandiswa kwe exogenous ornithine kurwisa biotic stress dzakadai se phytopathogens harina kudzidzwa zvakanaka. Chinonyanya kukosha ndechekuti, mhedzisiro yeornithine pakuramba kwezvirwere pamwe nezviitiko zvemakemikari nemuviri zvinobatanidzwa hazvisati zvanyatsoonekwa.
Kunzwisisa kuoma kwehutachiona hweS. sclerotiorum hwembeu kwakakosha pakugadzira nzira dzinoshanda dzekudzora. Muchidzidzo chino, takanangana nekuona basa rinogona kuitwa nediamine L-ornithine sechinhu chakakosha mukusimudzira nzira dzekudzivirira uye kuramba kwembeu dzembeu kuhutachiona hweSclerotinia sclerotiorum. Tinofungidzira kuti, pamusoro pekusimudzira mhinduro dzekudzivirira dzembeu dzine utachiona, L-ornithine inoitawo basa guru mukuchengetedza redox. Tinokurudzira kuti mhedzisiro inogona kuitika yeL-ornithine ine chekuita nekudzora nzira dzekudzivirira dzinorwisa mabhakitiriya uye dzisiri dzemaenzyme uye kupindira kwezvinhu zvinokonzera fungus/hutachiona uye mapuroteni akabatana. Kushanda kweL-ornithine kaviri uku kunoita kuti ive mukana wekuwana nzira inogara kwenguva refu yekuderedza kukanganisa kwembeu chena uye kuwedzera kuramba kwembeu dzembeu dzakajairika kuhutachiona hwefungus hune simba. Mhedzisiro yechidzidzo ichi inogona kubatsira mukuvandudza nzira itsva, dzinochengetedza nharaunda dzekudzora mbeu chena uye kuderedza kukanganisa kwayo pakugadzirwa kwembeu.
Muchidzidzo ichi, mhando yembeu dzinowanikwa mumusika dzinoshandiswa, Giza 3 (Phaseolus vulgaris L. cv. Giza 3), yakashandiswa sechishandiso chekuyedza. Mbeu dzine hutano dzakapihwa neLegume Research Department, Field Crops Research Institute (FCRI), Agricultural Research Center (ARC), Egypt. Mbeu shanu dzakadyarwa muhari dzepurasitiki (dhayamita yemukati 35 cm, kudzika 50 cm) dzakazadzwa nevhu rine utachiona hweS. sclerotiorum pasi pemamiriro egreenhouse (25 ± 2 °C, hunyoro 75 ± 1%, chiedza chemaawa masere/maawa gumi nematanhatu erima). Pamazuva manomwe kusvika gumi mushure mekudyara (DPS), mbeu dzakachekwa kuti dzisiye mbeu mbiri chete dzakakura zvakaenzana uye mashizha matatu akakura zvakakwana muhari yega yega. Zvirimwa zvese zviri muhari zvakadiridzwa kamwe chete pamavhiki maviri ega ega uye zvakafemerwa pamwedzi zvichienderana nechiyero chinokurudzirwa chemhando yakapihwa.
Kugadzira huwandu hwe500 mg/L hweL-ornithinediamine (inozivikanwawo se (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Germany), 50 mg yakatanga kunyungudutswa mu100 mL yemvura yakacheneswa. Mushonga wemushonga wakazosanganiswa ndokushandiswa mukuyedza kwakatevera. Muchidimbu, huwandu hutanhatu hweL-ornithine (12.5, 25, 50, 75, 100, uye 125 mg/L) hwakaedzwa mu vitro. Pamusoro pezvo, mvura yakacheneswa yakashandiswa senzira yekudzivirira negative (Mock) uye mushonga wekutengesa we "Rizolex-T" 50% wettable powder (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Egypt) wakashandiswa senzira yekudzivirira negative. Mushonga wefungi wekutengesa "Rizolex-T" wakaedzwa mu vitro pahuwandu hushanu (2, 4, 6, 8 uye 10 mg/L).
Mienzaniso yemadzinde embeu nemapods anoratidza zviratidzo zvefungus chena (huwandu hwehutachiona: 10–30%) yakatorwa kubva kumapurazi ekutengeserana. Kunyangwe zvakawanda zvezvinhu zvezvirimwa zvine hutachiona zvakaonekwa nemarudzi/marudzi akasiyana (rudzi rweGiza 3 runogona kutengeswa), zvimwe, kunyanya izvo zvakatorwa kubva kumisika yemuno, zvaive zvemarudzi asingazivikanwe. Zvinhu zvakaunganidzwa zvine hutachiona zvakatanga zvacheneswa ne 0.5% sodium hypochlorite solution kwemaminitsi matatu, ndokuzosukurudzwa kakawanda nemvura yakacheneswa yakabviswa mvura yakachekwa ne sterile filter paper kubvisa mvura yakawandisa. Nhengo dzine hutachiona dzakazochekwa kuita zvidimbu zvidiki kubva pakati penyama (pakati penyama dzine hutano nedzine hutachiona), dzakarimwa pa potato dextrose agar (PDA) medium uye dzakaiswa pa25 ± 2 °C ne 12 h light/12 h dark cycle kwemazuva mashanu kukurudzira kuumbwa kwe sclerotia. Nzira ye mycelial tip yakashandiswawo kuchenesa fungus isolates kubva mu common kana contaminated cultures. Chinomera chefungus chakacheneswa chakatanga chaonekwa zvichibva pahunhu hwacho hwetsika uye ndokuzosimbiswa kuti iS. sclerotiorum zvichibva pahunhu hudiki. Pakupedzisira, zvese zvinomera zvakacheneswa zvakaongororwa kuti zvionekwe kuti zvine hutachiona hwakaita sei pamhando yeGiza 3 inobatwa nechirwere ichi kuti zvisangane neKoch's postulates.
Pamusoro pezvo, S. sclerotiorum isolate inonyanya kupinda (isolate #3) yakasimbiswa zvakare zvichibva pa internal transcribed spacer (ITS) sequencing sezvakatsanangurwa naWhite et al., 1990; Baturo-Ciesniewska et al., 2017. Muchidimbu, isolates dzakarimwa mu potato dextrose broth (PDB) ndokuchengetwa pa25 ± 2 °C kwemazuva 5-7. Fungal mycelium yakazounganidzwa, yakasefetwa kuburikidza ne cheesecloth, yakagezwa kaviri nemvura isina utachiona, uye yakaomeswa ne sterile filter paper. Genomic DNA yakabviswa uchishandisa Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Nzvimbo yeITS rDNA yakabva yawedzerwa uchishandisa primer pair chaiyo ITS1/ITS4 (TCCGTAGGTGAACCTGGCG TCCTCGCTTTATTGATATGC; saizi inotarisirwa: 540 bp) (Baturo-Ciesniewska et al., 2017). Zvigadzirwa zvePCR zvakacheneswa zvakaendeswa kuti zviongororwe (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). MaITS rDNA sequences akarongwa bidirectionally achishandisa nzira yeSanger sequencing. Ma query sequences akaunganidzwa akazoenzaniswa nedata razvino muGenBank neNational Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/) uchishandisa BLASTn software. Kutevedzana kwemibvunzo kwakaenzaniswa nedzimwe mhando makumi maviri dzeS. sclerotiorum strains/isolates dzakatorwa kubva mudata razvino muNCBI GenBank (Supplementary Table S1) uchishandisa ClustalW muMolecular Evolutionary Genetics Analysis Package (MEGA-11; version 11) (Kumar et al., 2024). Kuongororwa kwekushanduka-shanduka kwakaitwa uchishandisa nzira yepamusoro yekufungidzira uye general time-reversible nucleotide substitution model (Nei naKumar, 2000). Muti une mukana wepamusoro wekupinda unoratidzwa. Muti wekutanga wekutsvaga kweheuristic unosarudzwa nekusarudza muti une mukana wekupinda pakati pemuti wevakidzani (NJ) (Kumar et al., 2024) uye muti wepamusoro weparsimony (MP). Muti weNJ wakagadzirwa uchishandisa matrix yedaro repakati yakaverengerwa uchishandisa general time-reversible model (Nei naKumar, 2000).
Kushanda kweL-ornithine kurwisa mabhakitiriya uye mushonga we "Rizolex-T" kwakaongororwa mu vitro nenzira ye "agar diffusion". Nzira: Tora huwandu hwakakodzera hwemushonga weL-ornithine (500 mg/L) wosanganisa zvakakwana ne10 ml yePDA nutrient medium kuti ugadzire mhinduro dzine huwandu hwekupedzisira hwe12.5, 25, 50, 75, 100 uye 125 mg/L, zvichiteerana. Hunhu hushanu hwe "Rizolex-T" yefungicide (2, 4, 6, 8 uye 10 mg/L) nemvura yakacheneswa yakashandiswa senzira yekudzora. Mushure mekunge mushonga wacho waoma, mycelial plug yakagadzirwa itsva yeSclerotinia sclerotiorum culture, dhayamita ye4 mm, yakaendeswa pakati pePetri dish ndokurimwa pa25±2°C kusvika mycelium yafukidza dish yese yePetri control, mushure mezvo kukura kwefungus kwakanyorwa. Verenga huwandu hwekudzivirira kukura kweS. sclerotiorum uchishandisa equation 1:
Kuedza uku kwakadzokororwa kaviri, nemifananidzo mitanhatu yezvehupenyu yeboka rega rega rekudzora/kuyedza uye hari shanu (miti miviri pahari imwe neimwe) yemifananidzo yega yega yezvehupenyu. Mufananidzo wega wega wezvehupenyu wakaongororwa kaviri (mifananidzo miviri yezvehunyanzvi) kuti uve nechokwadi chekuti mhinduro dzekuedza dzakarurama, dzakavimbika uye dzinodzokororwa. Pamusoro pezvo, ongororo yekuongorora kweprobit regression yakashandiswa kuverenga huwandu hwehafu-maximal inhibitory concentration (IC50) uye IC99 (Prentice, 1976).
Kuti vaongorore kugona kweL-ornithine pasi pemamiriro ekunze ekudziya, vakaedza kaviri kakatevedzana muhari. Muchidimbu, hari dzakazadzwa nevhu revhu rakasanganiswa nevhu (3:1) uye dzakaiswa mushonga weS. sclerotiorum. Kutanga, S. sclerotiorum (isolate #3) yakanyanya kupararira yakakurirwa nekucheka sclerotium imwe nepakati, ndokuiisa yakatarisa pasi paPDA uye yakaiswa pa25°C murima risingaperi (24 h) kwemazuva mana kuti iwedzere kukura kwemycelial. Mapulagi mana e5 mm dhayamita agar akatorwa kubva kumucheto wepamberi ndokuiswa mushonga we100 g wegorosi nerayisi bran (1:1, v/v) uye maflask ese akaiswa pa25 ± 2 °C pasi pechiedza chemaawa gumi nemaviri/maawa gumi nemaviri erima kwemazuva mashanu kuti akurudzire kuumbwa kwesclerotia. Zviri mumaflask ese zvakasanganiswa zvakanaka kuti zvive nechokwadi chekuti homogeneity isati yawedzera ivhu. Zvadaro, 100 g yemusanganiswa we bran inoisa zvirimwa muhari yega yega kuti pave nechokwadi chekuti utachiona hunoramba huripo. Hari dzakabayiwa dzakadiridzwa kuti dzikurudzire kukura kwefungus uye dzakaiswa munzvimbo inodziya kwemazuva manomwe.
Mbeu shanu dzerudzi rweGiza 3 dzakazodyarwa muhari imwe neimwe. Pahari dzakarapwa neL-ornithine nemushonga wefungi weRizolex-T, mbeu dzakachekwa dzakatanga kunyikwa kwemaawa maviri mumvura ine huwandu hwekupedzisira hweIC99 hunosvika 250 mg/L uye 50 mg/L, zvichiteerana, uye dzozoomeswa nemhepo kweawa imwe dzisati dzadyarwa. Kune rumwe rutivi, mbeu dzakanyikwa mumvura yakachekwa isina utachiona senzira yekudzivirira. Mushure memazuva gumi, mbeu dzisati dzadirwa kwekutanga, mbeu dzakachekwa, zvichisiya mbeu mbiri chete dzakachena muhari imwe neimwe. Pamusoro pezvo, kuti ive nechokwadi chekuti S. sclerotiorum yabatwa nehutachiona, madzinde embeu ari padanho rimwe chete rekukura (mazuva gumi) akachekwa panzvimbo mbiri dzakasiyana uchishandisa scalpel yakachekwa uye inenge 0.5 g yemusanganiswa webran wakaiswa muronda rega rega, zvichiteverwa nehunyoro hwakawanda kuti zvikurudzire hutachiona uye kukura kwezvirwere muzvirimwa zvese zvakapinzwa. Miti yekudzivirira yakakuvara zvakafanana uye huwandu hwakaenzana (0.5 g) hwemusanganiswa webran usina kucheneswa, usina kuunganidzwa wakaiswa muronda ndokuchengetwa pasi pehunyoro hwakanyanya kuti utevedzere mamiriro ekukura kwechirwere uye kuve nechokwadi chekuti mapoka ekurapa anowirirana.
Nzira yekurapa: Mbeu dzebhinzi dzakadirwa mvura ne500 ml yeL-ornithine (250 mg/l) kana mushonga wefungicide Rizolex-T (50 mg/l) nekudiridza ivhu, uye kurapwa kwacho kwakadzokororwa katatu kwemazuva gumi. Mishonga yekurapa yakadiridzwa ne placebo yakadirwa mvura yakasanganiswa ne500 ml. Kurapa kwese kwakaitwa pasi pemamiriro ekunze egreenhouse (25 ± 2°C, 75 ± 1% hunyoro, uye nguva yechiedza kwemaawa masere/maawa gumi nematanhatu erima). Hari dzese dzakadirwa mavhiki maviri ega ega uye dzakarapwa mwedzi wega wega nefetereza yeNPK yakaringana (20-20-20, ne3.6% sulfur neTE microelements; Zain Seeds, Egypt) nehuwandu hwe3–4 g/l nekupfapfaidza mashizha zvichienderana nemazano emhando chaiyo uye mirairo yemugadziri. Kunze kwekunge zvataurwa neimwe nzira, mashizha akakura zvakakwana (mashizha echipiri neechitatu kubva kumusoro) akaunganidzwa kubva kune yega yega yakatorwa mumaawa makumi manomwe nemaviri mushure mekurapwa (hpt), akaiswa muhomogenized, akaiswa pamwe chete uye akachengetwa pa -80 °C kuti awedzere kuongororwa kusanganisira, asi kwete kungogumira pane, in situ histochemical localization ye oxidative stress indicators, lipid peroxidation, enzymatic uye non-enzyme antioxidants uye gene expression.
Kuwanda kwechirwere chevhudzi chena kwakaongororwa vhiki rega rega mazuva makumi maviri nerimwe mushure mekubayiwa (dpi) uchishandisa chiyero che1–9 (Tafura Yekuwedzera S2) zvichibva pachiyero chePetzoldt naDickson (1996) chakagadziriswa naTeran et al. (2006). Muchidimbu, madzinde nematavi embeu zvakaongororwa kutanga panzvimbo yekubayiwa kuti zvitevedzere kufambira mberi kwezvironda pamwe chete nemapfundo. Kureba kwechirwere kubva panzvimbo yekubayiwa kusvika panzvimbo yakanyanya pedyo nedzinde kana bazi kwakazoyerwa uye mamaki e1–9 akapihwa zvichibva panzvimbo yechironda, apo (1) yakaratidza kuti hapana hutachiona hunoonekwa pedyo nenzvimbo yekubayiwa uye (2–9) yakaratidza kuwedzera zvishoma nezvishoma kwehukuru hwechirwere uye kufambira mberi pamwe chete nemapfundo/mapfundo (Tafura Yekuwedzera S2). Kuwanda kwechirwere chevhudzi chena kwakazoshandurwa kuita chikamu uchishandisa fomura 2:
Pamusoro pezvo, nzvimbo iri pasi pechirwere cheprogression curve (AUDPC) yakaverengerwa uchishandisa fomura (Shaner naFinney, 1977), iyo yakagadziriswa nguva pfupi yadarika kuti ionekwe seyakaora bhinzi (Chauhan et al., 2020) vachishandisa equation 3:
Apo Yi = kuoma kwechirwere panguva ti, Yi+1 = kuoma kwechirwere panguva inotevera ti+1, ti = nguva yekuyera kwekutanga (mumazuva), ti+1 = nguva yekuyera kunotevera (mumazuva), n = huwandu hwenguva dzese kana nzvimbo dzekucherechedza. Zviyero zvekukura kwechirimwa chebhinzi zvinosanganisira kureba kwechirimwa (cm), huwandu hwematavi pachirimwa, uye huwandu hwemashizha pachirimwa chimwe nechimwe zvakanyorwa vhiki rega rega kwemazuva makumi maviri nerimwe mune zvese zvipenyu.
Mune imwe neimwe yakatorwa kubva muzvipenyu, sampuli dzemashizha (mashizha echipiri neechitatu akanyatsokura kubva kumusoro) dzakatorwa pazuva rechi45 mushure mekurapwa (mazuva gumi nemashanu mushure mekurapwa kwekupedzisira). Imwe neimwe yakatorwa kubva muzvipenyu yaisanganisira hari shanu (miti miviri pahari imwe neimwe). Inenge 500 mg yenyama yakapwanywa yakashandiswa kubvisa pigments dzephotosynthetic (chlorophyll a, chlorophyll b uye carotenoids) uchishandisa 80% acetone pa4 °C murima. Mushure memaawa makumi maviri nemana, sampuli dzakaiswa pakati uye supernatant yakatorwa kuti ionekwe kuti chlorophyll a, chlorophyll b uye carotenoid zviri mukati zvine ruvara seimetrically uchishandisa UV-160A spectrophotometer (Shimadzu Corporation, Japan) zvichienderana nenzira ya (Lichtenthaler, 1987) nekuyera absorbance pama wavelengths matatu akasiyana (A470, A646 uye A663 nm). Pakupedzisira, huwandu hwepigment yephotosynthetic hwakaverengerwa uchishandisa mafomula anotevera 4–6 akatsanangurwa naLichtenthaler (1987).
Mushure memaawa makumi manomwe nemaviri mushure mekurapwa (hpt), mashizha (mashizha echipiri neechitatu akanyatsokura kubva kumusoro) akaunganidzwa kubva kune rimwe nerimwe rezvechisikigo kuti hydrogen peroxide (H2O2) uye superoxide anion (O2•−) iwanikwe panzvimbo yehistochemical. Imwe neimwe replica yezvechisikigo yaisanganisira hari shanu (miti miviri pahari imwe neimwe). Replica imwe neimwe yezvechisikigo yakaongororwa kaviri (replica mbiri dzehunyanzvi) kuti ive nechokwadi chekuti nzira yacho yakarurama, yakavimbika uye inodzokororwa. H2O2 neO2•− zvakaongororwa uchishandisa 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Germany) kana nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Germany), zvichiteerana, zvichitevera nzira dzakatsanangurwa naRomero-Puertas et al. (2004) naAdam et al. (1989) nekuchinja kudiki. Kuti H2O2 ive panzvimbo chaiyo, mapepa akapinzwa ne 0.1% DAB mu 10 mM Tris buffer (pH 7.8) uye akazopinzwa patembiricha yemukamuri muchiedza kwemaminitsi makumi matanhatu. Mapepa akapinzwa ne 0.15% (v/v) TCA mu 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) uye ozoiswa pachiedza kusvika asviba. Saizvozvowo, mavharuvhu akapinzwa ne 10 mM potassium phosphate buffer (pH 7.8) ine 0.1 w/v % HBT kuti O2 •− ive panzvimbo chaiyo. Mapepa akapinzwa muchiedza patembiricha yemukamuri kwemaminitsi makumi maviri, ozopinzwa ne bleach sezvataurwa pamusoro apa, uye ozovhenekerwa kusvika madonhwe ebhuruu/violet asviba aonekwa. Kusimba kweruvara rwebrown rwunobva rwabuda (sechiratidzo cheH2O2) kana bhuruu-violet (sechiratidzo cheO2•−) kwakaongororwa uchishandisa vhezheni yeFiji yepakeji yekugadzirisa mifananidzo ImageJ (http://fiji.sc; yakasvikwa musi wa7 Kurume 2024).
Malondialdehyde (MDA; sechiratidzo che lipid peroxidation) yakaongororwa zvichienderana nenzira yaDu naBramlage (1992) nekuchinja kudiki. Mashizha kubva pamashizha ega ega ega ega ega ega (mashizha echipiri neechitatu akanyatsokura kubva kumusoro) akaunganidzwa maawa makumi manomwe nemaviri mushure mekurapwa (hpt). Makopi ega ega ega ega ega ega ega ega aisanganisira hari shanu (miti miviri pahari imwe neimwe). Makopi ega ega ega ega ega ega ega ega ega akaongororwa kaviri (makopi maviri ehunyanzvi) kuti ave nechokwadi chekuti nzira yacho yakarurama, yakavimbika uye inodzokororwa. Muchidimbu, 0.5 g yemashizha evhu akashandiswa pakuburitsa MDA ne20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) ine 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). Zviri muMDA mu supernatant zvakazoongororwa neruvara rwemavara nekuyera kunyudzwa kwemvura pa532 ne600 nm uchishandisa UV-160A spectrophotometer (Shimadzu Corporation, Japan) uye zvobva zvaratidzwa se nmol g−1 FW.
Kuti zviongororwe ma antioxidants asiri e-enzyme uye enzymatic, mashizha (mashizha echipiri neechitatu akanyatsokura kubva kumusoro) akatorwa kubva kune rimwe nerimwe rezvechisikigo mushure memaawa makumi manomwe nemaviri mushure mekurapwa (hpt). Rimwe nerimwe rezvechisikigo raive nehari shanu (miti miviri pahari imwe neimwe). Muenzaniso wega wega wezvechisikigo wakaongororwa kaviri (mimwe mienzaniso miviri yehunyanzvi). Mashizha maviri akakuyiwa ne nitrogen yemvura uye akashandiswa zvakananga pakuona ma antioxidants e-enzyme uye asiri e-enzyme, maamino acid ese, huwandu hweproline, gene expression, uye oxalate quantification.
Ma phenolics ese anonyungudika akawanikwa achishandisa Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) nekuchinja kudiki kwenzira yakatsanangurwa naKahkonen et al. (1999). Muchidimbu, inenge 0.1 g yemashizha akafanana yakabviswa ne20 ml ye80% methanol murima kwemaawa makumi maviri nemana uye supernatant yakaunganidzwa mushure mekutenderera. 0.1 ml yemuenzaniso yakasanganiswa ne0.5 ml yeFolin-Ciocalteu reagent (10%), yakazununguswa kwemasekonzi makumi matatu ndokusiyiwa murima kwemaminitsi mashanu. Zvadaro 0.5 ml ye20% sodium carbonate solution (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) yakawedzerwa kuchubhu yega yega, yakasanganiswa zvakanaka uye yakachengetwa patembiricha yemumba murima kweawa imwe chete. Mushure mekuiswa musanganiswa, kunyudzwa kwemusanganiswa wereaction kwakayerwa pa765 nm uchishandisa UV-160A spectrophotometer (Shimadzu Corporation, Japan). Kuwanda kwetotal soluble phenols mu sample extracts kwakaongororwa uchishandisa gallic acid calibration curve (Fisher Scientific, Hampton, NH, USA) uye kwakatsanangurwa sema milligrams e gallic acid equivalent pa gram yehuremu hutsva (mg GAE g-1 fresh weight).
Huwandu hwese hwe flavonoid inonyungudika hwakaongororwa zvichienderana nenzira yaDjeridane et al. (2006) nekuchinja kudiki. Muchidimbu, 0.3 ml yemethanol extract yataurwa pamusoro apa yakasanganiswa ne 0.3 ml ye 5% aluminium chloride solution (AlCl3; Fisher Scientific, Hampton, NH, USA), yakasanganiswa zvine simba ndokuzoiswa patembiricha yemumba kwemaminitsi mashanu, zvichiteverwa nekuwedzera 0.3 ml ye 10% potassium acetate solution (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), yakasanganiswa zvakanaka ndokuiswa patembiricha yemumba kwemaminitsi makumi matatu murima. Mushure mekuiswa, kunyudzwa kwemusanganiswa we reaction kwakayerwa pa 430 nm uchishandisa UV-160A spectrophotometer (Shimadzu Corporation, Japan). Kuwanda kwe flavonoids inonyungudika mu sample extracts kwakaongororwa uchishandisa rutin calibration curve (TCI America, Portland, OR, USA) uye yakazoratidzwa se milligrams ye rutin yakaenzana pa gramu yehuremu hutsva (mg RE g-1 huremu hutsva).
Huwandu hweamino acid yemashizha ebhinzi hwakawanikwa uchishandisa modified ninhydrin reagent (Thermo Scientific Chemicals, Waltham, MA, USA) zvichibva panzira yakataurwa naYokoyama naHiramatsu (2003) uye yakagadziriswa naSun et al. (2006). Muchidimbu, 0.1 g yenyama yakagayiwa yakabviswa ne pH 5.4 buffer, uye 200 μL ye supernatant yakagadziriswa ne 200 μL ye ninhydrin (2%) uye 200 μL ye pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), yakaiswa mumvura inopisa kwemaminitsi makumi matatu, ndokuzotonhodzwa uye yakayerwa pa 580 nm uchishandisa UV-160A spectrophotometer (Shimadzu Corporation, Japan). Kune rumwe rutivi, proline yakayerwa nenzira yaBates (Bates et al., 1973). Proline yakatorwa ne3% sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) uye mushure mekutenderera, 0.5 ml ye supernatant yakasanganiswa ne 1 ml glacial acetic acid (Fisher Scientific, Hampton, NH, USA) uye ninhydrin reagent, yakaiswa pa90°C kwemaminitsi makumi mana nemashanu, yakatonhodzwa uye yakayerwa pa520 nm uchishandisa spectrophotometer yakafanana neyataurwa pamusoro apa. Total free amino acids uye proline mumashizha emashizha zvakaongororwa uchishandisa glycine uye proline calibration curves (Sigma-Aldrich, St Louis, MO, USA), zvichiteerana, uye zvakaratidzwa se mg/g huremu hutsva.
Kuti uone mashandiro eenzymes eantioxidant, inenge 500 mg ye homogenized tissue yakabviswa ne 3 ml ye 50 mM Tris buffer (pH 7.8) ine 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) uye 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), yakatenderedzwa pa centrifuge pa 10,000 × g kwemaminitsi makumi maviri pasi pefiriji (4 °C), uye supernatant (crude enzyme extract) yakaunganidzwa (El-Nagar et al., 2023; Osman et al., 2023). Catalase (CAT) yakazoshandiswa ne 2 ml ye 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) uye 100 μl ye 269 mM H2O2 solution kuti ione mashandiro ayo e enzyme zvichienderana nenzira yaAebi (1984) nekuchinja kudiki (El-Nagar et al., 2023; Osman et al., 2023). Basa re enzyme re Guaiacol-dependent peroxidase (POX) rakaonekwa uchishandisa nzira yaHarrach et al. (2009). (2008) nekuchinja kudiki (El-Nagar et al., 2023; Osman et al., 2023) uye mashandiro enzymatic e polyphenol oxidase (PPO) akaonekwa mushure mekuita ne 2.2 ml ye 100 mM sodium phosphate buffer (pH 6.0), 100 μl ye guaiacol (TCI chemicals, Portland, OR, USA) uye 100 μl ye 12 mM H2O2. Nzira iyi yakagadziriswa zvishoma kubva (El-Nagar et al., 2023; Osman et al., 2023). Kuyedzwa kwakaitwa mushure mekuita ne 3 ml ye catechol solution (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) yakagadzirwa patsva mu 0.1 M phosphate buffer (pH 6.0). Basa reCAT rakayerwa nekutarisa kuora kweH2O2 pa240 nm (A240), basa rePOX rakayerwa nekutarisa kuwedzera kwekunyudzwa kwesimba pa436 nm (A436), uye basa rePPO rakayerwa nekunyora kushanduka-shanduka kwekunyudzwa kwesimba pa495 nm (A495) masekondi makumi matatu ega ega kwemaminetsi matatu uchishandisa UV-160A spectrophotometer (Shimadzu, Japan).
RT-PCR yenguva chaiyo yakashandiswa kuona huwandu hwemajini matatu ane chekuita neantioxidant, anosanganisira peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), uye glutathione reductase (PvGR; GenBank Accession No. KY195009.1), mumashizha ebhinzi (mashizha echipiri neechitatu akanyatsokura kubva kumusoro) maawa makumi manomwe nemaviri mushure mekurapwa kwekupedzisira. Muchidimbu, RNA yakabviswa uchishandisa Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) zvichienderana neprotocol yemugadziri. Zvadaro, cDNA yakagadzirwa uchishandisa TOP script™ cDNA Synthesis Kit zvichienderana nemirairo yemugadziri. Ma primer sequences emajini matatu ari pamusoro akanyorwa muSupplementary Table S3. PvActin-3 (GenBank accession number: XM_068616709.1) yakashandiswa se housekeeping gene uye relative gene expression yakaverengerwa uchishandisa nzira ye 2-ΔΔCT (Livak naSchmittgen, 2001). Kugadzikana kweActin pasi pekumanikidzwa kwebiotic (kusawirirana pakati pembeu dzakajairika nefungus yeanthracnose Colletotrichum lindemuthianum) uye kushushikana kweabiotic (kusanaya kwemvura, munyu, tembiricha yakaderera) kwakaratidzwa (Borges et al., 2012).
Pakutanga takaita ongororo ye genome-wide mu silico proteins ye oxaloacetate acetylhydrolase (OAH) mu S. sclerotiorum tichishandisa protein-protein BLAST tool (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Muchidimbu, takashandisa OAH kubva kuAspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; GenBank accession number XP_040799428.1; 342 amino acids) uye Penicillium lagena (PlOAH; taxide: 94218; GenBank accession number XP_056833920.1; 316 amino acids) sequences dzekutsvaga mapuroteni akafanana mu S. sclerotiorum (taxide: 5180). BLASTp yakaitwa ichishandiswa pakutsvaga ruzivo rwe genome rweS. sclerotiorum rwakawanikwa munguva pfupi yapfuura muGenBank pawebsite yeNational Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Pamusoro pezvo, gene reOAH rakafanotaurwa kubva kuS. sclerotiorum (SsOAH) uye ongororo yekushanduka-shanduka uye muti wephylogenetic weAfOAH kubva kuA. fijiensis CBS 313.89 uye PlOAH kubva kuP. lagena zvakafungidzirwa uchishandisa nzira yekuwedzera mukana muMEGA11 (Tamura et al., 2021) uye muenzaniso weJTT matrix-based (Jones et al., 1992). Muti wephylogenetic wakabatanidzwa nekuongorora kwakawanda kweprotein sequences yemajini ese eOAH akafanotaurwa (SsOAH) kubva kuS. sclerotiorum uye query sequence uchishandisa Constraint-Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos naAgarwala, 2007). Pamusoro pezvo, ma amino acid akanakisisa anoenderana eSSOAH kubva kuS. sclerotiorum akabatana nema query sequences (AfOAH naPlOAH) (Larkin et al., 2007) vachishandisa ClustalW (http://www.genome.jp/tools-bin/clustalw), uye nzvimbo dzakachengetedzwa dziri mu alignment dzakaratidzwa uchishandisa ESPRipt tool (vhezheni 3.0; https://espript.ibcp.fr/ESPRipt/ESPRipt/index.php).
Uyezve, nzvimbo dzakafanotaurwa dzinomirira mashandiro uye nzvimbo dzakachengetedzwa dzeS. sclerotiorum SsOAH dzakapatsanurwa mumhuri dzakasiyana uchishandisa chishandiso cheInterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Pakupedzisira, kutevedzera chimiro chetatu-dimensional (3D) cheS. sclerotiorum SsOAH chakafanotaurwa kwakaitwa uchishandisa 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) uye zvakasimbiswa uchishandisa SWISS-MODEL server (https://swissmodel.expasy.org/) (Biasini et al., 2014). Maumbirwo akafanotaurwa emativi matatu (PDB format) akaoneswa zviri nyore achishandisa UCSF-Chimera package (vhezheni 1.15; https://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004).
PCR ye fluorescence yakashandiswa kuona huwandu hwe oxaloacetate acetylhydrolase (SsOAH; GenBank accession number: XM_001590428.1) mu mycelia yeSclerotinia sclerotiorum. Muchidimbu, S. sclerotiorum yakabayiwa muflask ine PDB ndokuiswa mu shaking incubator (model: I2400, New Brunswick Scientific Co., Edison, NJ, USA) pa25 ± 2 °C kwemaawa makumi maviri nemana pa150 rpm uye murima risingaperi (maawa makumi maviri nemana) kukurudzira kukura kwe mycelial. Mushure meizvozvo, masero akarapwa neL-ornithine nemushonga wefungicide Rizolex-T pahuwandu hwekupedzisira hweIC50 (inenge 40 ne3.2 mg/L, zvichiteerana) uye ndokuzorimwa kwemamwe maawa makumi maviri nemana pasi pemamiriro akafanana. Mushure mekuiswa mudumbu, mbeu dzakaiswa mu centrifuge pa 2500 rpm kwemaminitsi mashanu uye supernatant (fungal mycelium) yakaunganidzwa kuti iongororwe magene expression. Saizvozvowo, fungal mycelium yakaunganidzwa pa 0, 24, 48, 72, 96, uye 120 maawa mushure mekutapukirwa kubva kumiti ine utachiona yakanga yaumba mold chena uye cottony mycelium pamusoro pematishu ane utachiona. RNA yakabviswa kubva mu fungal mycelium uye cDNA yakazogadzirwa sezvakatsanangurwa pamusoro apa. Ma primer sequences eSsOAH akanyorwa muSupplementary Table S3. SsActin (GenBank accession number: XM_001589919.1) yakashandiswa se housekeeping gene, uye relative gene expression yakaverengerwa uchishandisa nzira ye 2-ΔΔCT (Livak naSchmittgen, 2001).
Oxalic acid yakawanikwa mumuto we potato dextrose (PDB) uye mumiti ine fungus pathogen Sclerotinia sclerotiorum zvichienderana nenzira yaXu naZhang (2000) nekuchinja kudiki. Muchidimbu, S. sclerotiorum isolates dzakabayiwa mumaflasks ane PDB ndokuzodyarwa mu shaken incubator (model I2400, New Brunswick Scientific Co., Edison, NJ, USA) pa 150 rpm pa 25 ± 2 °C kwemazuva 3-5 murima risingaperi (24 h) kuti ikurumidzise kukura kwe mycelial. Mushure mekuiswa mu incubation, fungi yakatanga kuseferwa kuburikidza neWhatman #1 filter paper uye yozoiswa centrifuge pa 2500 rpm kwemaminitsi mashanu kubvisa residual mycelium. Supernatant yakaunganidzwa ndokuchengetwa pa 4°C kuti iwedzere kutsanangurwa kwe oxalate. Pakugadzirira zvirimwa, inenge 0.1 g yezvidimbu zvemiti yakabviswa katatu nemvura yakasanganiswa (2 ml nguva imwe neimwe). Sampuli dzakazoiswa mu centrifuge pa 2500 rpm kwemaminitsi mashanu, supernatant yakaseferwa nepepa rekusefa reWhatman No. 1 ndokuunganidzwa kuti iongororwe zvakanyanya.
Kuti pave nekuongorora huwandu hwe oxalic acid, musanganiswa we reaction wakagadzirwa mugirazi rakavharirwa nenzira inotevera: 0.2 ml yesample (kana PDB culture filtrate kana oxalic acid standard solution), 0.11 ml ye bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), 0.198 ml ye 1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) uye 0.176 ml ye 100 mM potassium dichromate (K2Cr2O7; TCI chemicals, Portland, OR, USA), uye mhinduro yacho yakasanganiswa kusvika 4.8 ml nemvura yakasanganiswa, yakasanganiswa zvine simba uye pakarepo yakaiswa mubhavhu remvura re 60 °C. Mushure memaminitsi gumi, reaction yakamiswa nekuwedzera 0.5 ml ye sodium hydroxide solution (NaOH; 0.75 M). Kunyudzwa kwemvura (A600) kwemusanganiswa wereaction kwakayerwa pa600 nm uchishandisa UV-160 spectrophotometer (Shimadzu Corporation, Japan). PDB nemvura yakasanganiswa zvakashandiswa senzira dzekudzora huwandu hwema culture filtrates nema plant samples, zvichiteerana. Kuwanda kwe oxalic acid mu culture filtrates, kwakayerwa se micrograms ye oxalic acid pa milliliter ye PDB medium (μg.mL−1), uye mumashizha emashizha, kwakayerwa se micrograms ye oxalic acid pa gramu yehuremu hutsva (μg.g−1 FW), kwakayerwa uchishandisa oxalic acid calibration curve (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
Mukati mekudzidza kwose, kuyedza kwese kwakagadzirwa nenzira isina kurongeka zvachose (CRD) ine makopi matanhatu ezvehupenyu pakurapa uye hari shanu pakopi yezvehupenyu (miti miviri papoto imwe neimwe) kunze kwekunge zvataurwa neimwe nzira. Makopi maviri ezvehupenyu akaongororwa kaviri (makopi maviri ezvehunyanzvi). Makopi ezvehunyanzvi akashandiswa kutarisa kugona kudzokororwa kwekuyedza kumwe chete asi haana kushandiswa mukuongorora kwezviverengero kudzivirira makopi enhema. Data rakaongororwa nenhamba uchishandisa analysis of variance (ANOVA) richiteverwa neTukey-Kramer significant significant difference (HSD) test (p ≤ 0.05). Kune kuyedza kwe in vitro, ma IC50 ne IC99 values ​​​​zvakaverengerwa uchishandisa probit model uye 95% confidence intervals dzakaverengerwa.
Marudzi mana akatorwa kubva muminda yakasiyana yesoya muEl Ghabiya Province, Egypt. PaPDA medium, marudzi ese akatorwa akaburitsa mycelium chena yakakora iyo yakakurumidza kuchinja kuita donje jena (Mufananidzo 1A) uye yozoshanduka kuita beige kana brown padanho resclerotium. MaSclerotia anowanzo kuve akakora, matema, akaita sedenderedzwa kana asina kurongeka muchimiro, akareba 5.2 kusvika 7.7 mm uye dhayamita 3.4 kusvika 5.3 mm (Mufananidzo 1B). Kunyangwe marudzi mana akatorwa akagadzira marginal pattern ye sclerotia pamucheto we culture medium mushure memazuva 10-12 ekuiswa pa25 ± 2 °C (Mufananidzo 1A), huwandu hwe sclerotia paplate imwe neimwe hwakasiyana zvakanyanya pakati pawo (P < 0.001), ne isolate 3 ine nhamba yepamusoro ye sclerotia (32.33 ± 1.53 sclerotia paplate imwe neimwe; Mufananidzo 1C). Saizvozvowo, isolate #3 yakagadzira oxalic acid yakawanda muPDB kupfuura mamwe masora (3.33 ± 0.49 μg.mL−1; Mufananidzo 1D). Isolate #3 yakaratidza hunhu hwe morphological uye microscopic hwe phytopathogenic fungus Sclerotinia sclerotiorum. Semuenzaniso, paPDA, makoroni eisolate #3 akakura nekukurumidza, aive machena (Mufananidzo 1A), reverse beige kana light salmon yellow-brown, uye zvaida mazuva 6-7 pa25 ± 2°C kuti afukidze zvakakwana pamusoro peplate ine dhayamita ye9 cm. Zvichibva pane hunhu hwe morphological uye microscopic hwataurwa pamusoro apa, isolate #3 yakaonekwa seSclerotinia sclerotiorum.
Mufananidzo 1. Hunhu uye hutachiona hwezvipembenene zveS. sclerotiorum kubva muzvirimwa zvembeu zvakajairika. (A) Kukura kwezvipembenene zvina zveS. sclerotiorum paPDA medium, (B) sclerotia yezvipembenene zvina zveS. sclerotiorum, (C) huwandu hwesclerotia (pandiro imwe neimwe), (D) oxalic acid secretion paPDB medium (μg.mL−1), uye (E) kuoma kwechirwere (%) chezvipembenene zvina zveS. sclerotiorum pazvirimwa zvembeu zvinotengeswa zvinokurumidza kubatwa neGiza 3 pasi pemamiriro egreenhouse. Makoshero anomiririra avhareji ± SD yezvipembenene zvishanu zvebhayoloji (n = 5). Mabhii akasiyana anoratidza mutsauko wakakosha pakati pekurapa (p < 0.05). (F–H) Zviratidzo zvebvudzi jena zvakaonekwa pamadzinde epamusoro pevhu ne silique, zvichiteerana, mazuva gumi mushure mekubayiwa ne isolate #3 (dpi). (I) Kuongororwa kwekushanduka-shanduka kwenzvimbo yemukati yakadhindwa (ITS) yeS. sclerotiorum isolate #3 kwakaitwa uchishandisa nzira yekuwedzera mukana uye kwakaenzaniswa ne20 reference isolates/strains dzakawanikwa kubva kuNational Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). Nhamba dziri pamusoro pemitsara yekuunganidza dzinoratidza nzvimbo inofukidzwa nedunhu (%), uye nhamba dziri pasi pemitsara yekuunganidza dzinoratidza kureba kwebazi.
Uyezve, kusimbisa kuti chirwere ichi chine utachiona, maS. sclerotiorum isolates mana akawanikwa akashandiswa kubaya mbeu yeGiza 3 inobatwa nechirwere ichi pasi pemamiriro ekunze egreenhouse, izvo zvinoenderana neKoch's postulates (Mufananidzo 1E). Kunyangwe maS. 1E ese akawanikwa aine fungus isolates aikonzera chirwere uye aigona kutapukira bhinzi yegirini (cv. Giza 3), zvichikonzera zviratidzo zvechivhu chena pazvikamu zvese zviri pamusoro pevhu (Mufananidzo 1F), kunyanya pamadzinde (Mufananidzo 1G) nemapods (Mufananidzo 1H) pamazuva gumi mushure mekubaya (dpi), isolate 3 ndiyo yaive isolate yakanyanya mukuyedza kwakazvimirira. Isolate 3 yaive nechirwere chakanyanya (%) pazvirimwa zvebhinzi (24.0 ± 4.0, 58.0 ± 2.0, uye 76.7 ± 3.1 pamazuva 7, 14, uye 21 mushure mekutapukirwa, zvichiteerana; Mufananidzo 1F).
Kuzivikanwa kweS. sclerotiorum isolate inonyanya kupinda mumiti #3 kwakasimbiswa zvakare zvichibva pa internal transcribed spacer (ITS) sequencing (Mufananidzo 1I). Kuongorora kwephylogenetic pakati peisolate #3 ne20 reference isolates/strains kwakaratidza kufanana kwakanyanya (>99%) pakati pawo. Zvakakosha kuziva kuti S. sclerotiorum isolate #3 (533 bp) yakafanana zvakanyanya neAmerican S. sclerotiorum isolate LPM36 yakabviswa kubva kumbeu dzepea dzakaoma (GenBank accession number MK896659.1; 540 bp) uye Chinese S. sclerotiorum isolate YKY211 (GenBank accession number OR206374.1; 548 bp), izvo zvinokonzera kuora kweviolet (Matthiola incana), zvese zvakarongwa zvakasiyana pamusoro pedendrogram (Mufananidzo 1I). Kutevedzana kutsva kwacho kwaiswa mudhatabhesi reNCBI uye kwainzi “Sclerotinia sclerotiorum – isolate YN-25” (GenBank accession number PV202792). Zvinogona kuonekwa kuti isolate 3 ndiyo isolate inonyanya kupinda; saka, isolate iyi yakasarudzwa kuti iongororwe mune zvese zvakazotevera.
Kushanda kwe diamine L-ornithine (Sigma-Aldrich, Darmstadt, Germany) pahuwandu hwakasiyana (12.5, 25, 50, 75, 100 uye 125 mg/L) pakurwisa S. sclerotiorum isolate 3 kwakaongororwa mu vitro. Zvakakosha kuziva kuti L-ornithine yakaita basa rekurwisa mabhakitiriya uye zvishoma nezvishoma yakadzivisa kukura kweS. sclerotiorum hyphae nenzira inoenderana nedosi (Mufananidzo 2A, B). Pahuwandu hwakanyanya hwakaedzwa (125 mg/L), L-ornithine yakaratidza huwandu hwakanyanya hwekudzivirira kukura kwemycelial (99.62 ± 0.27%; Mufananidzo 2B), hwakaenzana nemushonga wekutengesa weRizolex-T (huwandu hwekudzivirira 99.45 ± 0.39%; Mufananidzo 2C) pahuwandu hwakanyanya hwakaedzwa (10 mg/L), zvichiratidza kushanda kwakafanana.
Mufananidzo 2. Basa reL-ornithine rinorwisa mabhakitiriya muropa reSclerotinia sclerotiorum. (A) Kuenzanisa basa rekurwisa mabhakitiriya rehuwandu hwakasiyana hweL-ornithine kurwisa S. sclerotiorum nemushonga wefungi wekutengesa Rizolex-T (10 mg/L). (B, C) Kudzora (%) kwekukura kweS. sclerotiorum mycelial mushure mekurapwa nehuwandu hwakasiyana hweL-ornithine (12.5, 25, 50, 75, 100 uye 125 mg/L) kana Rizolex-T (2, 4, 6, 8 uye 10 mg/L), zvichiteerana. Makoshero anomiririra avhareji ± SD yezvinyorwa zvishanu zvebhayoloji (n = 5). Mabhii akasiyana anoratidza mutsauko wehuwandu pakati pekurapa (p < 0.05). (D, E) Kuongororwa kweprobit model regression yeL-ornithine nemushonga wefungi wekutengesa Rizolex-T, zvichiteerana. Mutsetse wekuregera weprobit model unoratidzwa semutsetse webhuruu wakasimba, uye nguva yekuvimba (95%) inoratidzwa semutsetse mutsvuku wakachekwa.
Pamusoro pezvo, ongororo yeprobit regression yakaitwa uye maplots anoenderana anoratidzwa muTable 1 neMifananidzo 2D,E. Muchidimbu, kukosha kwekutsvedza kunogamuchirwa (y = 2.92x - 4.67) uye nhamba dzakabatana dzakakosha (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 uye p < 0.0001; Mufananidzo 2D) weL-ornithine yakaratidza kuwedzera kwekushanda kwehutachiona kurwisa S. sclerotiorum zvichienzaniswa neRizolex-T yefungicide yekutengeserana (y = 1.96x - 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 uye p < 0.0001) (Table 1).
Tafura 1. Hunhu hwehafu yepamusoro yekudzivirira (IC50) uye IC99 (mg/l) yeL-ornithine uye mushonga wekuuraya fungi wekutengesa "Rizolex-T" kurwisa S. sclerotiorum.
Kazhinji, L-ornithine (250 mg/L) yakaderedza zvakanyanya kukura nekuoma kwebvudzi jena pamiti yebhinzi yakajairika yakarapwa zvichienzaniswa nemiti ine hutachiona hweS. sclerotiorum isina kurapwa (control; Mufananidzo 3A). Muchidimbu, kunyangwe kuoma kwechirwere chemiti yekudzivirira hutachiona isina kurapwa kwakawedzera zvishoma nezvishoma (52.67 ± 1.53, 83.21 ± 2.61, uye 92.33 ± 3.06%), L-ornithine yakaderedza zvakanyanya kuoma kwechirwere (%) mukati mekuedza (8.97 ± 0.15, 18.00 ± 1.00, uye 26.36 ± 3.07) pamazuva manomwe, gumi nemana, uye makumi maviri nerimwe mushure mekurapwa (dpt), zvichiteerana (Mufananidzo 3A). Saizvozvowo, apo zvirimwa zvebhinzi zvine utachiona hweS. sclerotiorum zvakarapwa ne250 mg/L L-ornithine, nzvimbo iri pasi pechirwere cheprogression curve (AUDPC) yakaderera kubva pa1274.33 ± 33.13 muchirongwa chisina kurapwa kusvika pa281.03 ± 7.95, iyo yaive yakaderera zvishoma pane ye positive control 50 mg/L Rizolex-T fungicide (183.61 ± 7.71; Mufananidzo 3B). Maitiro akafanana akaonekwa mukuyedza kwechipiri.
Mufananidzo 3. Mhedzisiro yekushandiswa kweL-ornithine kunze kwemhuka pakukura kwekuora kwembeu chena kunokonzerwa neSclerotinia sclerotiorum pasi pemamiriro egreenhouse. (A) Kukura kwechirwere chemold chembeu chena mushure mekurapwa ne250 mg/L L-ornithine. (B) Nzvimbo iri pasi pechirwere chemold (AUDPC) chembeu chena mushure mekurapwa neL-ornithine. Mavalues ​​​​anomiririra avhareji ± SD yemhando shanu dzezvipenyu (n = 5). Mabhii akasiyana anoratidza mutsauko wakakosha pakati pekurapwa (p < 0.05).
Kushandiswa kwe250 mg/L L-ornithine kunze kwenyika kwakawedzera zvishoma nezvishoma kureba kwemuti (Mufananidzo 4A), huwandu hwematavi pamuti (Mufananidzo 4B), uye huwandu hwemashizha pamuti (Mufananidzo 4C) mushure memazuva 42. Kunyange zvazvo mushonga wekutengesa weRizolex-T (50 mg/L) wakakanganisa zvakanyanya pahutano hwese hwakadzidzwa, kushandiswa kwe250 mg/L L-ornithine kunze kwenyika kwakawedzerawo mhedzisiro yechipiri yakakura zvichienzaniswa nemidziyo isina kurapwa (Mufananidzo 4A–C). Kune rumwe rutivi, kurapwa kweL-ornithine hakuna kukanganisa zvakanyanya huwandu hwepigment ye photosynthetic chlorophyll a (Mufananidzo 4D) uye chlorophyll b (Mufananidzo 4E), asi kwakawedzera zvishoma huwandu hwe carotenoid (0.56 ± 0.03 mg/g fr wt) zvichienzaniswa ne negative control (0.44 ± 0.02 mg/g fr wt) uye positive control (0.46 ± 0.02 mg/g fr wt; Mufananidzo 4F). Zvose izvi zvinoratidza kuti L-ornithine haina chepfu kune mbesa dzakarapwa uye inogona kutokurudzira kukura kwadzo.
Mufananidzo 4. Mhedzisiro yekushandiswa kweL-ornithine yekunze pahunhu hwekukura uye pigments dzemashizha ebhinzi ane chirwere cheSclerotinia sclerotiorum pasi pemamiriro egreenhouse. (A) Kureba kwemuti (cm), (B) Huwandu hwematavi pamuti, (C) Huwandu hwemashizha pamuti, (D) Chlorophyll a content (mg g-1 fr wt), (E) Chlorophyll b content (mg g-1 fr wt), (F) Total carotenoid content (mg g-1 fr wt). Values ​​​​ndiyo avhareji ± SD yezvinyorwa zvishanu zvebhayoloji (n = 5). Mabhii akasiyana anoratidza mutsauko wakakosha pakati pekurapa (p < 0.05).
Kuwanikwa kwemakemikari emhando dzeokisijeni dzinoshanda (ROS; dzinoratidzwa sehydrogen peroxide [H2O2]) uye ma free radicals (anoratidzwa se superoxide anions [O2•−]) kwakaratidza kuti kushandiswa kweL-ornithine (250 mg/L) kwakaderedza zvakanyanya kuunganidzwa kweH2O2 (96.05 ± 5.33 nmol.g−1 FW; Mufananidzo 5A) uye O2•− (32.69 ± 8.56 nmol.g−1 FW; Mufananidzo 5B) zvichienzaniswa nekuunganidzwa kwezvirimwa zvese zvine hutachiona zvisina kurapwa (173.31 ± 12.06 uye 149.35 ± 7.94 nmol.g−1 FW, zvichiteerana) uye zvirimwa zvakarapwa ne50 mg/L yeRizolex-T inouraya fungi (170.12 ± 9.50 uye 157.00 ± 7.81 nmol.g−1 fr wt, zvakateerana) pamaawa makumi manomwe nemaviri. H2O2 neO2 •− zvakaunganidzwa pasi pe hpt (Mufananidzo 5A, B). Saizvozvowo, bvunzo yemalondialdehyde (MDA) yakavakirwa paTCA yakaratidza kuti zvirimwa zvebhinzi zvine utachiona hweS. sclerotiorum zvakaunganidza huwandu hwakakwira hweMDA (113.48 ± 10.02 nmol.g fr wt) mumashizha azvo (Mufananidzo 5C). Zvisinei, kushandiswa kweL-ornithine kunze kwenyika kwakaderedza zvakanyanya lipid peroxidation sezvakaratidzwa nekudzikira kwehuwandu hweMDA muzvirimwa zvakarapwa (33.08 ± 4.00 nmol.g fr wt).
Mufananidzo 5. Mhedzisiro yekushandiswa kweL-ornithine yekunze pazviratidzo zvikuru zvekushushikana kwe oxidative uye nzira dzekudzivirira antioxidant dzisiri dze enzyme mumashizha ebhinzi ane S. sclerotiorum mumaawa makumi manomwe nemaviri mushure mehutachiona pasi pemamiriro egreenhouse. (A) Hydrogen peroxide (H2O2; nmol g−1 FW) pa 72 hpt, (B) superoxide anion (O2•−; nmol g−1 FW) pa 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) pa 72 hpt, (D) total soluble phenols (mg GAE g−1 FW) pa 72 hpt, (E) total soluble flavonoids (mg RE g−1 FW) pa 72 hpt, (F) total free amino acids (mg g−1 FW) pa 72 hpt, uye (G) proline content (mg g−1 FW) pa 72 hpt. Mavalues ​​​​anomiririra avhareji ± standard deviation (avhareji ± SD) ye5 biological replicates (n = 5). Mabhii akasiyana anoratidza mutsauko wakakosha pakati pekurapa (p < 0.05).