Nkhaniyi ndi gawo la mutu wofufuza wakuti “Kukweza mphamvu ya nyemba ku matenda ndi tizilombo toononga”, onani nkhani zonse 5
Choyambitsa matenda a bowa ku zomera ndi necrosis Sclerotinia sclerotiorum (Lib.) de Bary amagwiritsa ntchito njira yosiyana siyana kuti afalitse zomera zosiyanasiyana. Kafukufukuyu akupereka lingaliro logwiritsa ntchito diamine L-ornithine, amino acid yosapangidwa ndi mapuloteni yomwe imalimbikitsa kupanga ma amino acid ena ofunikira, ngati njira ina yowongolera kuti iwonjezere mphamvu ya mamolekyulu, thupi, ndi zochita za biochemical za Phaseolus vulgaris L. ku nkhungu yoyera yomwe imayambitsidwa ndi Pseudomonas sclerotiorum. Kuyesa kwa in vitro kunawonetsa kuti L-ornithine inaletsa kwambiri kukula kwa mycelial kwa S. pyrenoidosa motengera mlingo. Kuphatikiza apo, ikhoza kuchepetsa kwambiri kuopsa kwa nkhungu yoyera pansi pa nyengo yobiriwira. Kuphatikiza apo, L-ornithine inalimbikitsa kukula kwa zomera zomwe zachiritsidwa, zomwe zikusonyeza kuti kuchuluka kwa L-ornithine komwe kunayesedwa sikunali poizoni ku zomera zomwe zachiritsidwa. Kuphatikiza apo, L-ornithine inawonjezera kufotokozedwa kwa ma antioxidants osapangidwa ndi enzymatic (ma phenolics ndi ma flavonoids onse osungunuka) ndi ma antioxidants a enzymatic (catalase (CAT), peroxidase (POX), ndi polyphenol oxidase (PPO)), ndikuwonjezera kufotokozedwa kwa majini atatu okhudzana ndi antioxidant (PvCAT1, PvSOD, ndi PvGR). Kuphatikiza apo, mu kusanthula kwa silico kunawonetsa kukhalapo kwa puloteni ya oxaloacetate acetylhydrolase (SsOAH) mu genome ya S. sclerotiorum, yomwe inali yofanana kwambiri ndi mapuloteni a oxaloacetate acetylhydrolase (SsOAH) a Aspergillus fijiensis (AfOAH) ndi Penicillium sp. (PlOAH) pankhani ya kusanthula kwantchito, madera osungidwa, ndi topology. Chosangalatsa ndichakuti, kuwonjezera kwa L-ornithine ku potato dextrose broth (PDB) medium kunachepetsa kwambiri kufotokozedwa kwa jini ya SsOAH mu S. sclerotiorum mycelia. Mofananamo, kugwiritsa ntchito L-ornithine kunja kunachepetsa kwambiri kufalikira kwa majini a SsOAH mu bowa wotchedwa mycelia womwe unasonkhanitsidwa kuchokera ku zomera zomwe zinapatsidwa chithandizo. Pomaliza, kugwiritsa ntchito L-ornithine kunachepetsa kwambiri kutulutsa kwa oxalic acid m'masamba a PDB apakatikati komanso omwe ali ndi kachilomboka. Pomaliza, L-ornithine imagwira ntchito yofunika kwambiri pakusunga redox komanso kukulitsa chitetezo cha zomera zomwe zakhudzidwa. Zotsatira za kafukufukuyu zingathandize kupanga njira zatsopano komanso zosamalira chilengedwe zowongolera nkhungu yoyera ndikuchepetsa mphamvu yake pakupanga nyemba ndi mbewu zina.
Nkhungu yoyera, yomwe imayambitsidwa ndi bowa wa necrotrophic Sclerotinia sclerotiorum (Lib.) de Bary, ndi matenda oopsa komanso ochepetsa zokolola omwe ali pachiwopsezo chachikulu pakupanga nyemba padziko lonse lapansi (Phaseolus vulgaris L.) (Bolton et al., 2006). Sclerotinia sclerotiorum ndi imodzi mwa matenda ovuta kwambiri oyambitsidwa ndi bowa m'nthaka, yokhala ndi mitundu yoposa 600 ya zomera komanso kuthekera kowononga minofu ya zomera mwachangu mwanjira yosadziwika bwino (Liang ndi Rollins, 2018). Mu mikhalidwe yoipa, imadutsa mu gawo lofunika kwambiri la moyo wake, kukhala chete kwa nthawi yayitali ngati zinthu zakuda, zolimba, zonga mbewu zotchedwa 'sclerotia' m'nthaka kapena ngati zomera zoyera, zofewa mu mycelium kapena tsinde la zomera zodwala (Schwartz et al., 2005). S. sclerotiorum imatha kupanga sclerotia, yomwe imalola kuti ipulumuke m'minda yodwala kwa nthawi yayitali komanso kupitirirabe panthawi ya matenda (Schwartz et al., 2005). Sclerotia ili ndi michere yambiri, imatha kukhalabe m'nthaka kwa nthawi yayitali, ndipo imakhala ngati njira yoyambira yochizira matenda ena (Schwartz et al., 2005). Mu nyengo yabwino, sclerotia imamera ndikupanga spores zomwe zimatha kufalikira mbali zonse za chomera pamwamba pa nthaka, kuphatikiza maluwa, tsinde, kapena nyemba (Schwartz et al., 2005).
Sclerotinia sclerotiorum imagwiritsa ntchito njira yosiyana siyana kuti ipatsire zomera zomwe zimalandira, zomwe zimaphatikizapo zochitika zosiyanasiyana kuyambira kumera kwa sclerotial mpaka kukula kwa zizindikiro. Poyamba, S. sclerotiorum imapanga spores zoyimitsidwa (zotchedwa ascospores) kuchokera kuzinthu zofanana ndi bowa zotchedwa apothecia, zomwe zimauluka mumlengalenga ndikukula kukhala sclerotia yosasuntha pa zinyalala za zomera zomwe zakhudzidwa (Bolton et al., 2006). Kenako bowa limatulutsa oxalic acid, chinthu chomwe chimayambitsa matenda, kuti chilamulire pH ya khoma la maselo a zomera, kulimbikitsa kuwonongeka kwa enzymatic ndi kulowa kwa minofu (Hegedus ndi Rimmer, 2005), ndikuletsa kuphulika kwa okosijeni kwa chomera. Njira yopangira asidiyi imafooketsa khoma la maselo a zomera, kupereka malo abwino ogwirira ntchito bwino komanso moyenera kwa ma enzymes owononga khoma la maselo a bowa (CWDEs), kulola tizilombo toyambitsa matenda kugonjetsa chotchinga chakuthupi ndikulowa m'thupi la chomera (Marciano et al., 1983). Akalowa, S. sclerotiorum imatulutsa ma CWDE angapo, monga polygalacturonase ndi cellulase, zomwe zimathandiza kufalikira kwake m'maselo omwe ali ndi kachilomboka ndikuyambitsa necrosis ya minofu. Kupita patsogolo kwa zilonda ndi ma hyphal mats kumabweretsa zizindikiro za nkhungu yoyera (Hegedus ndi Rimmer, 2005). Pakadali pano, zomera zomwe zimakhala nazo zimazindikira mapatani a maselo ogwirizana ndi tizilombo toyambitsa matenda (PAMPs) kudzera mu ma receptors ozindikira mawonekedwe (PRRs), zomwe zimayambitsa zochitika zingapo zodziwitsa zomwe pamapeto pake zimayambitsa mayankho a chitetezo.
Ngakhale kuti pakhala zaka zambiri zoyeserera zothana ndi matenda, kusowa kwa majeremusi okwanira olimbana ndi matenda kukupitirirabe mu nyemba, monga momwe zimakhalira ndi mbewu zina zamalonda, chifukwa cha kukana, kupulumuka, komanso kusinthasintha kwa tizilombo toyambitsa matenda. Chifukwa chake, kuthana ndi matenda ndi kovuta kwambiri ndipo kumafuna njira yolumikizana, yokhala ndi mbali zambiri zomwe zimaphatikizapo kuphatikiza miyambo yachikhalidwe, kuwongolera zachilengedwe, ndi mankhwala ophera fungicide (O'Sullivan et al., 2021). Kulamulira nkhungu yoyera ndi mankhwala ndikothandiza kwambiri chifukwa mankhwala ophera fungicide, akagwiritsidwa ntchito moyenera komanso panthawi yoyenera, amatha kuwongolera bwino kufalikira kwa matendawa, kuchepetsa kuopsa kwa matenda, ndikuchepetsa kutayika kwa zokolola. Komabe, kugwiritsa ntchito mopitirira muyeso komanso kudalira kwambiri mankhwala ophera fungicide kungayambitse kubuka kwa mitundu yolimbana ndi matenda a S. sclerotiorum ndikuwononga zinthu zomwe sizili m'gulu la zomera, thanzi la nthaka, komanso ubwino wa madzi (Le Cointe et al., 2016; Ceresini et al., 2024). Chifukwa chake, kupeza njira zina zotetezera chilengedwe kwakhala chinthu chofunikira kwambiri.
Ma polyamines (PAs), monga putrescine, spermidine, spermine, ndi cadaverine, angagwiritsidwe ntchito ngati njira zina zabwino zopewera matenda ochokera ku zomera zomwe zimafalikira m'nthaka, motero amachepetsa kwathunthu kapena pang'ono kugwiritsa ntchito mankhwala ophera fungicides oopsa (Nehela et al., 2024; Yi et al., 2024). Mu zomera zapamwamba, ma PA amachita nawo zinthu zambiri zokhudzana ndi thupi kuphatikizapo, koma osati kokha, kugawa maselo, kusiyanitsa, ndi kuyankha ku zovuta za abiotic ndi biotic (Killiny ndi Nehela, 2020). Zitha kugwira ntchito ngati ma antioxidants, kuthandiza kuchotsa mitundu ya okosijeni yogwira ntchito (ROS), kusunga redox homeostasis (Nehela ndi Killiny, 2023), kuyambitsa majini okhudzana ndi chitetezo (Romero et al., 2018), kulamulira njira zosiyanasiyana za kagayidwe kachakudya (Nehela ndi Killiny, 2023), kusintha ma phytohormones amkati (Nehela ndi Killiny, 2019), kukhazikitsa kukana kwa systemic acquired resistance (SAR), ndikuwongolera kuyanjana kwa zomera ndi tizilombo toyambitsa matenda (Nehela ndi Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). Ndikofunikira kudziwa kuti njira ndi ntchito za ma PA pakuteteza zomera zimasiyana kutengera mitundu ya zomera, tizilombo toyambitsa matenda, ndi momwe zinthu zilili. PA yochuluka kwambiri m'zomera imapangidwa kuchokera ku polyamine L-ornithine yofunika (Killiny ndi Nehela, 2020).
L-ornithine imagwira ntchito zambiri pakukula ndi chitukuko cha zomera. Mwachitsanzo, kafukufuku wakale wasonyeza kuti mu mpunga (Oryza sativa), ornithine ikhoza kugwirizanitsidwa ndi kubwezeretsanso kwa nayitrogeni (Liu et al., 2018), kukolola mpunga, ubwino ndi fungo (Lu et al., 2020), komanso kuyankha kupsinjika kwa madzi (Yang et al., 2000). Kuphatikiza apo, kugwiritsa ntchito L-ornithine kunja kwa dzikolo kunathandizira kwambiri kupirira chilala mu shuga beet (Beta vulgaris) (Hussein et al., 2019) ndikuchepetsa kupsinjika kwa mchere mu anyezi (Allium Cepa) (Çavuşoǧlu ndi Çavuşoǧlu, 2021) ndi zomera za cashew (Anacardium occidentale) (da Rocha et al., 2012). Udindo womwe L-ornithine ingathe kukhala nawo pa kuteteza kupsinjika kwa abiotic ukhoza kukhala chifukwa cha kutenga nawo mbali pakusonkhanitsa proline m'zomera zomwe zachiritsidwa. Mwachitsanzo, majini okhudzana ndi kagayidwe ka proline, monga majini a ornithine delta aminotransferase (delta-OAT) ndi majini a proline dehydrogenase (ProDH1 ndi ProDH2), adanenedwa kale kuti amatenga nawo mbali poteteza Nicotiana benthamiana ndi Arabidopsis thaliana motsutsana ndi mitundu ya Pseudomonas syringae yomwe siili m'gulu la tizilombo toyambitsa matenda (Senthil-Kumar ndi Mysore, 2012). Kumbali ina, bowa ornithine decarboxylase (ODC) imafunika kuti tizilombo toyambitsa matenda tikule (Singh et al., 2020). Kuyang'ana ODC ya Fusarium oxysporum f. sp. lycopersici kudzera mu host-induced gene silencing (HIGS) kunawonjezera kwambiri kukana kwa zomera za phwetekere ku Fusarium wilt (Singh et al., 2020). Komabe, ntchito yomwe ingagwiritsidwe ntchito kunja kwa ornithine motsutsana ndi kupsinjika kwa biotic monga phytopathogens sinaphunziridwe bwino. Chofunika kwambiri, zotsatira za ornithine pa kukana matenda komanso zochitika zokhudzana ndi biochemical ndi physiological sizikudziwikabe.
Kumvetsetsa kuopsa kwa matenda a S. sclerotiorum a nyemba ndikofunikira pakupanga njira zowongolera bwino. Mu kafukufukuyu, cholinga chathu chinali kuzindikira ntchito yomwe diamine L-ornithine ingakhale nayo ngati chinthu chofunikira kwambiri pakukweza njira zodzitetezera komanso kukana kwa zomera za nyemba ku matenda a Sclerotinia sclerotiorum. Tikuganiza kuti, kuwonjezera pa kukulitsa mayankho a chitetezo cha zomera zomwe zakhudzidwa, L-ornithine imagwiranso ntchito yofunika kwambiri pakusunga redox. Tikupereka lingaliro lakuti zotsatira zomwe L-ornithine ingakhale nazo zikugwirizana ndi kuwongolera njira zodzitetezera za enzymatic ndi non-enzyme komanso kusokoneza zinthu zoyambitsa matenda/zowononga bowa ndi mapuloteni ogwirizana nawo. Kagwiridwe kake ka L-ornithine kawiri kamapangitsa kuti ikhale njira yabwino yopezera njira yokhazikika yochepetsera mphamvu ya nkhungu yoyera ndikuwonjezera kukana kwa mbewu za nyemba wamba ku matenda amphamvu a bowa. Zotsatira za kafukufukuyu zingathandize pakupanga njira zatsopano komanso zosamalira chilengedwe zowongolera nkhungu yoyera ndikuchepetsa mphamvu yake pakupanga nyemba.
Mu kafukufukuyu, mtundu wa nyemba wamba womwe ungathe kugulitsidwa, Giza 3 (Phaseolus vulgaris L. cv. Giza 3), unagwiritsidwa ntchito ngati zinthu zoyesera. Mbewu zathanzi zinaperekedwa mokoma mtima ndi Dipatimenti Yofufuza za Nyemba, Field Crops Research Institute (FCRI), Agricultural Research Center (ARC), Egypt. Mbewu zisanu zinabzalidwa m'miphika ya pulasitiki (mkati mwake 35 cm, kuya 50 cm) yodzazidwa ndi nthaka yodwala S. sclerotiorum pansi pa kutentha (25 ± 2 °C, chinyezi cha 75 ± 1%, kuwala kwa maola 8/maola 16 amdima). Patatha masiku 7-10 kuchokera pamene mbewu zinabzalidwa (DPS), mbewuzo zinachepetsedwa kuti zisiye mbewu ziwiri zokha zomwe zikumera mofanana ndi masamba atatu okulirapo mumphika uliwonse. Mbewu zonse zomwe zinali m'miphika zinathiriridwa kamodzi pa milungu iwiri iliyonse ndipo zinkapatsidwa feteleza pamwezi pamlingo woyenera wa mtundu womwe waperekedwa.
Pofuna kukonza kuchuluka kwa 500 mg/L kwa L-ornithinediamine (yomwe imadziwikanso kuti (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Germany), 50 mg idasungunuka koyamba mu 100 mL ya madzi osakanizidwa. Kenako yankho la stock linachepetsedwa ndikugwiritsidwa ntchito mu zoyeserera zotsatira. Mwachidule, magulu asanu ndi limodzi a L-ornithine (12.5, 25, 50, 75, 100, ndi 125 mg/L) adayesedwa mu vitro. Kuphatikiza apo, madzi osakanizidwa osakanizidwa adagwiritsidwa ntchito ngati negative control (Mock) ndipo fungicide yogulitsa "Rizolex-T" 50% wettable powder (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Egypt) idagwiritsidwa ntchito ngati positive control. Fungivi yogulitsa "Rizolex-T" idayesedwa mu vitro pamlingo wachisanu (2, 4, 6, 8 ndi 10 mg/L).
Zitsanzo za mitengo ya nyemba ndi nyemba zomwe zimasonyeza zizindikiro za nkhungu yoyera (chiwerengero cha kufalikira: 10–30%) zinasonkhanitsidwa kuchokera ku minda yamalonda. Ngakhale kuti zomera zambiri zomwe zakhudzidwa zinazindikirika ndi mitundu/mitundu (mtundu wa Giza 3 womwe ungagulitsidwe), zina, makamaka zomwe zinapezeka m'misika yakomweko, zinali za mitundu yosadziwika. Zinthu zomwe zakhudzidwazo zinasonkhanitsidwa poyamba zinatsukidwa pamwamba ndi yankho la 0.5% sodium hypochlorite kwa mphindi zitatu, kenako zinatsukidwa kangapo ndi madzi oyeretsedwa ndikupukutidwa ndi pepala loyeretsedwa kuti zichotse madzi ochulukirapo. Ziwalo zomwe zakhudzidwazo zinadulidwa mzidutswa tating'onoting'ono kuchokera ku minofu yapakati (pakati pa minofu yathanzi ndi minofu yomwe yakhudzidwa), zinalimidwa pa potato dextrose agar (PDA) medium ndikuyikidwa pa 25 ± 2 °C ndi kuwala kwa maola 12/maola 12 kwa masiku 5 kuti zilimbikitse mapangidwe a sclerotia. Njira ya mycelial tip inagwiritsidwanso ntchito kuyeretsa bowa kuchokera ku zomera zosakanikirana kapena zoipitsidwa. Chotupa cha bowa choyeretsedwacho chinayamba kuzindikirika kutengera mawonekedwe ake achikhalidwe kenako chinatsimikiziridwa kuti ndi S. sclerotiorum kutengera mawonekedwe ake a microscopic. Pomaliza, chotupa chonse choyeretsedwacho chinayesedwa kuti chipeze matenda pa mtundu wa nyemba wamba womwe ungathe kukhudzidwa ndi Giza 3 kuti ukwaniritse zizindikiro za Koch.
Kuphatikiza apo, S. sclerotiorum isolate yoopsa kwambiri (isolate #3) idatsimikiziridwanso kutengera kusanthula kwa internal transcribed spacer (ITS) monga momwe White et al., 1990; Baturo-Ciesniewska et al., 2017 adafotokozera. Mwachidule, isolates adalimidwa mu potato dextrose broth (PDB) ndikuyikidwa pa 25 ± 2 °C kwa masiku 5-7. Kenako fungal mycelium idasonkhanitsidwa, kusefedwa kudzera mu cheesecloth, kutsukidwa kawiri ndi madzi osabala, ndikuumitsidwa ndi pepala losefera losabala. Genomic DNA idachotsedwa pogwiritsa ntchito Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Kenako dera la ITS rDNA linakulitsidwa pogwiritsa ntchito primer pair yeniyeni ITS1/ITS4 (TCCGTAGGTGAACCTGGCG TCCTCGCTTTATTGATATGC; kukula komwe kukuyembekezeka: 540 bp) (Baturo-Ciesniewska et al., 2017). Zogulitsa za PCR zoyeretsedwa zinatumizidwa kuti zigwiritsidwe ntchito potsatira njira (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). Ma sequence a ITS rDNA adasankhidwa motsatira njira ya Sanger sequencing. Ma query sequence omwe adasonkhanitsidwa adayerekezeredwa ndi deta yaposachedwa mu GenBank ndi National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/gene/) pogwiritsa ntchito pulogalamu ya BLASTn. Mndandanda wa mafunso unayerekezeredwa ndi mitundu/zosiyana 20 zina za S. sclerotiorum zomwe zapezeka kuchokera ku deta yaposachedwa mu NCBI GenBank (Supplementary Table S1) pogwiritsa ntchito ClustalW mu Molecular Evolutionary Genetics Analysis Package (MEGA-11; version 11) (Kumar et al., 2024). Kusanthula kwa kusinthaku kunachitika pogwiritsa ntchito njira ya maximum likelihood ndi general time-reversible nucleotide substitution model (Nei and Kumar, 2000). Mtengo wokhala ndi log-likelihood yapamwamba kwambiri wawonetsedwa. Mtengo woyamba wa heuristic search umasankhidwa posankha mtengo wokhala ndi log-likelihood yayikulu pakati pa mtengo wolumikizana ndi anansi (NJ) (Kumar et al., 2024) ndi mtengo wa maximum parsimony (MP). Mtengo wa NJ unapangidwa pogwiritsa ntchito pairwise distance matrix yowerengedwa pogwiritsa ntchito general time-reversible model (Nei and Kumar, 2000).
Mphamvu ya mabakiteriya ya L-ornithine ndi mankhwala ophera tizilombo toyambitsa matenda a "Rizolex-T" zinapezeka mu vitro pogwiritsa ntchito njira yofalitsira agar. Njira: Tengani kuchuluka koyenera kwa yankho la L-ornithine (500 mg/L) ndikusakaniza bwino ndi 10 ml ya PDA nutrient medium kuti mukonze mayankho okhala ndi kuchuluka komaliza kwa 12.5, 25, 50, 75, 100 ndi 125 mg/L, motsatana. Kuchuluka kasanu kwa mankhwala ophera tizilombo a "Rizolex-T" (2, 4, 6, 8 ndi 10 mg/L) ndi madzi oyeretsedwa oyeretsedwa kunagwiritsidwa ntchito ngati njira yowongolera. Pambuyo poti mankhwalawo akhazikika, cholumikizira chatsopano cha mycelial cha Sclerotinia sclerotiorum culture, cha mainchesi 4 mm, chinasamutsidwira pakati pa mbale ya Petri ndikuchikulitsa pa 25±2°C mpaka mycelium itaphimba mbale yonse ya Petri, pambuyo pake kukula kwa bowa kunalembedwa. Werengerani kuchuluka kwa kuletsa kukula kwa radial kwa S. sclerotiorum pogwiritsa ntchito equation 1:
Kuyeseraku kunabwerezedwa kawiri, ndi makopi asanu ndi limodzi a zamoyo pa gulu lililonse lowongolera/loyesera ndi miphika isanu (zomera ziwiri pa mphika uliwonse) papepala lililonse la zamoyo. Kubwereza kulikonse kwa zamoyo kunasanthulidwa kawiri (makopi awiri aukadaulo) kuti zitsimikizire kulondola, kudalirika, ndi kuberekanso kwa zotsatira za kuyesaku. Kuphatikiza apo, kusanthula kwa probit regression kunagwiritsidwa ntchito kuwerengera kuchuluka kwa theka la maximum inhibitory concentration (IC50) ndi IC99 (Prentice, 1976).
Kuti aone momwe L-ornithine ingathandizire pa kutentha kwa dziko lapansi, mayeso awiri otsatizana a mphika adachitika. Mwachidule, miphika idadzazidwa ndi dothi losakanizidwa ndi dothi (3:1) ndipo idayikidwa katemera ndi chikhalidwe chatsopano cha S. sclerotiorum. Choyamba, cholowa kwambiri cha S. sclerotiorum (chosalowa #3) chidalimidwa mwa kudula sclerotium imodzi pakati, ndikuyiyika moyang'anizana ndi PDA ndikuyikidwa pa 25°C mumdima wosalekeza (maola 24) kwa masiku 4 kuti ilimbikitse kukula kwa mycelial. Kenako mapulagi anayi a agar a 5 mm m'mimba mwake adatengedwa kuchokera m'mphepete mwa kutsogolo ndikuyikidwa katemera ndi 100 g ya chisakanizo chosabala cha tirigu ndi mpunga (1:1, v/v) ndipo ma flask onse adayikidwa pa 25 ± 2 °C pansi pa kuwala kwa maola 12/maola 12 amdima kwa masiku 5 kuti alimbikitse kupanga sclerotia. Zomwe zili m'ma flask onse zidasakanizidwa bwino kuti zitsimikizire kuti zimagwirizana bwino musanawonjezere dothi. Kenako, 100 g ya chisakanizo cha chimanga chomwe chimalowa m'malo mwa mbewu chinawonjezeredwa ku mphika uliwonse kuti zitsimikizire kuti tizilombo toyambitsa matenda timakhala ndi tizilombo tosiyanasiyana. Miphika yophimbidwa ndi katemera inathiriridwa kuti iyambe kukula kwa bowa ndipo inayikidwa m'malo obiriwira kwa masiku 7.
Mbewu zisanu za mtundu wa Giza 3 zinabzalidwa mumphika uliwonse. Pa miphika yomwe inapatsidwa L-ornithine ndi Rizolex-T yophera fungicide, mbewu zophikidwa poyamba zinanyowa kwa maola awiri mu yankho lamadzi la mankhwala awiriwa ndi kuchuluka komaliza kwa IC99 kwa pafupifupi 250 mg/L ndi 50 mg/L, motsatana, kenako n’kuumitsa mpweya kwa ola limodzi musanabzale. Kumbali ina, mbewuzo zinanyowa m’madzi osungunuka ngati njira yopewera kufalikira kwa matenda. Patatha masiku 10, mbewuzo zisanathiridwe madzi oyamba, zinachepetsedwa, n’kusiya mbewu ziwiri zokha zoyera mumphika uliwonse. Kuphatikiza apo, kuti zitsimikizire kuti S. sclerotiorum yafalikira, nthambi za nyemba zomwe zili pagawo lomwelo la kukula (masiku 10) zinadulidwa m’malo awiri osiyana pogwiritsa ntchito scalpel yophikidwa ndipo pafupifupi 0.5 g ya chisakanizo cha nthambi ya m’mimba inayikidwa pa bala lililonse, kutsatiridwa ndi chinyezi chambiri kuti zilimbikitse matenda ndi kukula kwa matenda m’zomera zonse zopatsidwa katemera. Zomera zowongolera zinavulala mofananamo ndipo kuchuluka kofanana (0.5 g) kwa ufa wosabala, wosadulidwa unayikidwa pabala ndikusungidwa pansi pa chinyezi chambiri kuti zifanane ndi malo omwe matenda amakulira ndikuwonetsetsa kuti magulu ochizira ali bwino.
Njira Yochiritsira: Mbeu za nyemba zinathiriridwa ndi 500 ml ya madzi a L-ornithine (250 mg/l) kapena Rizolex-T (50 mg/l) pothirira nthaka, kenako mankhwalawa anabwerezedwa katatu pakatha masiku 10. Oyang'anira omwe adathiridwa ndi placebo adathiriridwa ndi 500 ml ya madzi oyeretsedwa. Mankhwala onsewa adachitika pansi pa kutentha (25 ± 2°C, chinyezi cha 75 ± 1%, komanso nthawi ya kuwala kwa maola 8/maola 16 amdima). Miphika yonse idathiriridwa milungu iwiri iliyonse ndipo pamwezi idathiriridwa ndi feteleza wa NPK woyenerera (20-20-20, ndi 3.6% sulfure ndi TE microelements; Zain Seeds, Egypt) pamlingo wa 3-4 g/l pothirira masamba motsatira malangizo a mtundu winawake ndi malangizo a wopanga. Pokhapokha ngati tanena mwanjira ina, masamba okhwima mokwanira (masamba achiwiri ndi achitatu kuchokera pamwamba) adasonkhanitsidwa kuchokera ku chofanana chilichonse chamoyo patatha maola 72 chithandizo (hpt), kusinthidwa kukhala homogenized, kusungidwa pamodzi ndikusungidwa pa -80 °C kuti ziwunikidwe zina kuphatikiza, koma osati kokha, in situ histochemical localization of oxidative stress indicators, lipid peroxidation, enzymatic and non-enzyme antioxidants and gene expression.
Kuchuluka kwa matenda a nkhungu yoyera kunayesedwa sabata iliyonse masiku 21 pambuyo pa kubayidwa (dpi) pogwiritsa ntchito sikelo ya 1–9 (Table Supplementary S2) kutengera sikelo ya Petzoldt ndi Dickson (1996) yosinthidwa ndi Teran et al. (2006). Mwachidule, tsinde ndi nthambi za zomera za nyemba zinayesedwa kuyambira pomwe kubayidwa kuti zitsatire kupita patsogolo kwa zilonda m'mbali mwa ma internode ndi ma node. Mtunda wa chotupacho kuchokera pomwe kubayidwa mpaka patali kwambiri m'mbali mwa tsinde kapena nthambi unayesedwa ndipo chigoli cha 1–9 chinaperekedwa kutengera komwe chotupacho chinali, pomwe (1) chinasonyeza kuti palibe matenda owoneka pafupi ndi pomwe kubayidwa ndipo (2–9) chinasonyeza kuwonjezeka pang'onopang'ono kwa kukula kwa chotupacho ndi kupita patsogolo m'mbali mwa ma node/ma internode (Table Supplementary S2). Kuchuluka kwa matenda a nkhungu yoyera kunasinthidwa kukhala peresenti pogwiritsa ntchito fomula 2:
Kuphatikiza apo, dera lomwe lili pansi pa curve yopita patsogolo kwa matenda (AUDPC) linawerengedwa pogwiritsa ntchito njira (Shaner ndi Finney, 1977), yomwe idasinthidwa posachedwapa kuti igwirizane ndi kuvunda koyera kwa nyemba wamba (Chauhan et al., 2020) pogwiritsa ntchito equation 3:
Kumene Yi = kuopsa kwa matenda panthawi ya ti, Yi+1 = kuopsa kwa matenda nthawi yotsatira ti+1, ti = nthawi yoyezera koyamba (m'masiku), ti+1 = nthawi yoyezera yotsatira (m'masiku), n = chiwerengero chonse cha nthawi kapena malo owonera. Zigawo za kukula kwa mbewu za nyemba kuphatikizapo kutalika kwa chomera (cm), chiwerengero cha nthambi pa chomera, ndi chiwerengero cha masamba pa chomera chilichonse zinalembedwa sabata iliyonse kwa masiku 21 m'magawo onse a zamoyo.
Mu chitsanzo chilichonse cha zamoyo, zitsanzo za masamba (masamba achiwiri ndi achitatu omwe adakula bwino kuchokera pamwamba) zinasonkhanitsidwa pa tsiku la 45 pambuyo pa chithandizo (masiku 15 pambuyo pa chithandizo chomaliza). Chitsanzo chilichonse cha zamoyo chinali ndi miphika isanu (zomera ziwiri pa mphika uliwonse). Pafupifupi 500 mg ya minofu yophwanyika idagwiritsidwa ntchito pochotsa utoto wa photosynthetic (chlorophyll a, chlorophyll b ndi carotenoids) pogwiritsa ntchito 80% acetone pa 4 °C mumdima. Pambuyo pa maola 24, zitsanzozo zinayikidwa pakati ndipo supernatant inasonkhanitsidwa kuti idziwe zomwe zili mu chlorophyll a, chlorophyll b ndi carotenoid pogwiritsa ntchito utoto pogwiritsa ntchito UV-160A spectrophotometer (Shimadzu Corporation, Japan) malinga ndi njira ya (Lichtenthaler, 1987) poyesa kuyamwa kwa madzi pa mafunde atatu osiyanasiyana (A470, A646 ndi A663 nm). Pomaliza, kuchuluka kwa utoto wa photosynthetic kunawerengedwa pogwiritsa ntchito njira zotsatirazi 4-6 zomwe zinafotokozedwa ndi Lichtenthaler (1987).
Patatha maola 72 kuchokera pamene mankhwala aperekedwa (hpt), masamba (masamba achiwiri ndi achitatu okhwima bwino kuchokera pamwamba) anasonkhanitsidwa kuchokera ku replica iliyonse ya zamoyo kuti apeze malo a hydrogen peroxide (H2O2) ndi superoxide anion (O2•−). Replica iliyonse ya zamoyo inali ndi miphika isanu (zomera ziwiri pa mphika uliwonse). Replica iliyonse ya zamoyo inasanthulidwa kawiri (replicas ziwiri zaukadaulo) kuti zitsimikizire kulondola, kudalirika, ndi kuberekana kwa njirayo. H2O2 ndi O2•− zinapezeka pogwiritsa ntchito 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Germany) kapena nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Germany), motsatana, potsatira njira zomwe Romero-Puertas et al. (2004) ndi Adam et al. (1989) adafotokoza ndi zosintha zazing'ono. Kuti tipeze malo a H2O2 mu histochemical, timapepala ta vacuum tinalowetsedwa ndi 0.1% DAB mu 10 mM Tris buffer (pH 7.8) kenako tinaikidwa pa kutentha kwa chipinda mu kuwala kwa mphindi 60. Timapepala ta vacuum tinayeretsedwa mu 0.15% (v/v) TCA mu 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) kenako tinaikidwa pa kuwala mpaka titada. Mofananamo, ma valve analowetsedwa ndi 10 mM potassium phosphate buffer (pH 7.8) yokhala ndi 0.1 w/v % HBT kuti tipeze malo a O2 •− mu histochemical. Timapepala ta vacuum tinalowetsedwa pa kuwala pa kutentha kwa chipinda kwa mphindi 20, kenako tinayeretsedwa monga tafotokozera pamwambapa, kenako tinawalitsidwa mpaka madontho akuda abuluu/violet atawonekera. Mphamvu ya mtundu wa bulauni womwe watuluka (monga chizindikiro cha H2O2) kapena buluu-violet (monga chizindikiro cha O2•−) idayesedwa pogwiritsa ntchito mtundu wa Fiji wa phukusi lokonza zithunzi la ImageJ (http://fiji.sc; idapezeka pa 7 Marichi 2024).
Malondialdehyde (MDA; monga chizindikiro cha lipid peroxidation) idapezeka motsatira njira ya Du ndi Bramlage (1992) ndi kusintha pang'ono. Masamba ochokera ku replicate iliyonse yachilengedwe (masamba achiwiri ndi achitatu okhwima bwino kuchokera pamwamba) adasonkhanitsidwa maola 72 atatha kuchiritsidwa (hpt). Replicate iliyonse yachilengedwe idaphatikizapo miphika isanu (zomera ziwiri pa mphika uliwonse). Replicate iliyonse yachilengedwe idasanthulidwa kawiri (replicate ziwiri zaukadaulo) kuti zitsimikizire kulondola, kudalirika, komanso kuberekanso kwa njirayo. Mwachidule, 0.5 g ya minofu ya masamba odulidwa idagwiritsidwa ntchito pochotsa MDA ndi 20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) yokhala ndi 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). Kuchuluka kwa MDA mu supernatant kunatsimikiziridwa ndi mtundu poyesa kuyamwa kwa 532 ndi 600 nm pogwiritsa ntchito spectrophotometer ya UV-160A (Shimadzu Corporation, Japan) kenako nkufotokozedwa ngati nmol g−1 FW.
Pofuna kuwunika ma antioxidants omwe si a enzymatic ndi enzymatic, masamba (masamba achiwiri ndi achitatu omwe adakula bwino kuchokera pamwamba) adasonkhanitsidwa kuchokera ku replicate iliyonse yachilengedwe patatha maola 72 atalandira chithandizo (hpt). Replicate iliyonse yachilengedwe inali ndi miphika isanu (zomera ziwiri pa mphika uliwonse). Chitsanzo chilichonse chachilengedwe chidawunikidwa kawiri (zitsanzo ziwiri zaukadaulo). Masamba awiri adaphwanyidwa ndi nayitrogeni yamadzimadzi ndipo adagwiritsidwa ntchito mwachindunji kuti adziwe ma antioxidants a enzymatic ndi omwe si a enzymatic, ma amino acid onse, kuchuluka kwa proline, mawonekedwe a majini, ndi kuchuluka kwa oxalate.
Ma phenolic onse osungunuka adapezeka pogwiritsa ntchito reagent ya Folin-Ciocalteu (Sigma-Aldrich, St. Louis, MO, USA) ndi kusintha pang'ono kwa njira yomwe idafotokozedwa ndi Kahkonen et al. (1999). Mwachidule, pafupifupi 0.1 g ya minofu ya masamba ofanana idachotsedwa ndi 20 ml ya 80% methanol mumdima kwa maola 24 ndipo supernatant idasonkhanitsidwa pambuyo pa centrifugation. 0.1 ml ya chotsitsa chachitsanzo idasakanizidwa ndi 0.5 ml ya reagent ya Folin-Ciocalteu (10%), idagwedezeka kwa masekondi 30 ndikusiyidwa mumdima kwa mphindi 5. Kenako 0.5 ml ya yankho la 20% sodium carbonate (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) idawonjezedwa ku chubu chilichonse, kusakaniza bwino ndikuyikidwa kutentha kwa chipinda mumdima kwa ola limodzi. Pambuyo poikamo, kuyamwa kwa chisakanizo cha reaction kunayesedwa pa 765 nm pogwiritsa ntchito UV-160A spectrophotometer (Shimadzu Corporation, Japan). Kuchuluka kwa ma phenols onse osungunuka mu zitsanzo kunadziwika pogwiritsa ntchito gallic acid calibration curve (Fisher Scientific, Hampton, NH, USA) ndipo kunafotokozedwa ngati ma milligrams a gallic acid ofanana pa gramu imodzi ya kulemera kwatsopano (mg GAE g-1 kulemera kwatsopano).
Kuchuluka kwa flavonoid yonse yosungunuka kunapezeka motsatira njira ya Djeridane et al. (2006) ndi kusintha pang'ono. Mwachidule, 0.3 ml ya methanol extract yomwe ili pamwambapa inasakanizidwa ndi 0.3 ml ya 5% aluminiyamu chloride solution (AlCl3; Fisher Scientific, Hampton, NH, USA), inasakanizidwa mwamphamvu kenako inayikidwa kutentha kwa chipinda kwa mphindi 5, kenako inawonjezeredwa 0.3 ml ya 10% potassium acetate solution (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), inasakanizidwa bwino ndikuyikidwa kutentha kwa chipinda kwa mphindi 30 mumdima. Pambuyo poikamo, kuyamwa kwa reaction mix kunayesedwa pa 430 nm pogwiritsa ntchito UV-160A spectrophotometer (Shimadzu Corporation, Japan). Kuchuluka kwa flavonoids yonse yosungunuka mu zitsanzo kunapezeka pogwiritsa ntchito rutin calibration curve (TCI America, Portland, OR, USA) kenako inafotokozedwa ngati ma milligrams a rutin ofanana pa gramu imodzi ya kulemera kwatsopano (mg RE g-1 kulemera kwatsopano).
Kuchuluka kwa amino acid yonse yaulere m'masamba a nyemba kunapezeka pogwiritsa ntchito reagent yosinthidwa ya ninhydrin (Thermo Scientific Chemicals, Waltham, MA, USA) kutengera njira yomwe Yokoyama ndi Hiramatsu (2003) adapereka ndipo Sun et al. (2006) adasintha. Mwachidule, 0.1 g ya minofu yophwanyika idachotsedwa ndi pH 5.4 buffer, ndipo 200 μL ya supernatant idapangidwa ndi 200 μL ya ninhydrin (2%) ndi 200 μL ya pyridine (10%; Spectrum Chemical, New Brunswick, NJ, USA), yomwe idayikidwa m'madzi otentha kwa mphindi 30, kenako idaziziritsidwa ndikuyesedwa pa 580 nm pogwiritsa ntchito UV-160A spectrophotometer (Shimadzu Corporation, Japan). Kumbali ina, proline idapezeka pogwiritsa ntchito njira ya Bates (Bates et al., 1973). Proline inachotsedwa ndi 3% sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) ndipo pambuyo pa centrifugation, 0.5 ml ya supernatant inasakanizidwa ndi 1 ml glacial acetic acid (Fisher Scientific, Hampton, NH, USA) ndi ninhydrin reagent, yosungidwa pa 90°C kwa mphindi 45, yoziziritsidwa ndikuyesedwa pa 520 nm pogwiritsa ntchito spectrophotometer yomweyi monga pamwambapa. Ma amino acid onse aulere ndi proline mu masamba ochotsedwa adapezeka pogwiritsa ntchito glycine ndi proline calibration curves (Sigma-Aldrich, St Louis, MO, USA), motsatana, ndipo adawonetsedwa ngati mg/g kulemera kwatsopano.
Kuti mudziwe momwe ma enzyme oletsa antioxidant amagwirira ntchito, pafupifupi 500 mg ya minofu yofanana idachotsedwa ndi 3 ml ya 50 mM Tris buffer (pH 7.8) yokhala ndi 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) ndi 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), yomwe idayikidwa mu centrifuge pa 10,000 × g kwa mphindi 20 pansi pa firiji (4 °C), ndipo supernatant (crude enzyme extract) idasonkhanitsidwa (El-Nagar et al., 2023; Osman et al., 2023). Kenako Catalase (CAT) inachitidwa ndi 2 ml ya 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) ndi 100 μl ya yankho la 269 mM H2O2 kuti adziwe momwe imagwirira ntchito malinga ndi njira ya Aebi (1984) ndi kusintha pang'ono (El-Nagar et al., 2023; Osman et al., 2023). Ntchito ya enzymatic ya Guaiacol-dependent peroxidase (POX) inadziwika pogwiritsa ntchito njira ya Harrach et al. (2009). (2008) ndi zosintha zazing'ono (El-Nagar et al., 2023; Osman et al., 2023) ndipo ntchito ya enzymatic ya polyphenol oxidase (PPO) idadziwika pambuyo pochita ndi 2.2 ml ya 100 mM sodium phosphate buffer (pH 6.0), 100 μl ya guaiacol (TCI chemicals, Portland, OR, USA) ndi 100 μl ya 12 mM H2O2. Njirayi idasinthidwa pang'ono kuchokera ku (El-Nagar et al., 2023; Osman et al., 2023). Kuyesaku kunachitika pambuyo pochita ndi 3 ml ya catechol solution (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) yokonzedwa mwatsopano mu 0.1 M phosphate buffer (pH 6.0). Ntchito ya CAT inayesedwa poyang'anira kuwonongeka kwa H2O2 pa 240 nm (A240), ntchito ya POX inayesedwa poyang'anira kuwonjezeka kwa kuyamwa kwa kuwala pa 436 nm (A436), ndipo ntchito ya PPO inayesedwa polemba kusinthasintha kwa kuyamwa kwa kuwala pa 495 nm (A495) masekondi 30 aliwonse kwa mphindi zitatu pogwiritsa ntchito spectrophotometer ya UV-160A (Shimadzu, Japan).
RT-PCR ya nthawi yeniyeni idagwiritsidwa ntchito kuzindikira milingo yolembedwa ya majini atatu okhudzana ndi antioxidant, kuphatikiza peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), ndi glutathione reductase (PvGR; GenBank Accession No. KY195009.1), m'masamba a nyemba (masamba achiwiri ndi achitatu omwe adakula bwino kuchokera pamwamba) maola 72 pambuyo pa chithandizo chomaliza. Mwachidule, RNA idapezeka pogwiritsa ntchito Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) malinga ndi njira ya wopanga. Kenako, cDNA idapangidwa pogwiritsa ntchito TOP script™ cDNA Synthesis Kit malinga ndi malangizo a wopanga. Ma primer sequences a majini atatu omwe ali pamwambapa alembedwa mu Supplementary Table S3. PvActin-3 (GenBank accession number: XM_068616709.1) idagwiritsidwa ntchito ngati jini yosamalira nyumba ndipo kufotokozera kwa majini ogwirizana kunawerengedwa pogwiritsa ntchito njira ya 2-ΔΔCT (Livak ndi Schmittgen, 2001). Kukhazikika kwa Actin pansi pa biotic stress (kusagwirizana pakati pa nyemba zodziwika bwino ndi bowa wa anthracnose Colletotrichum lindemuthianum) ndi abiotic stress (chilala, mchere, kutentha kochepa) zidawonetsedwa (Borges et al., 2012).
Poyamba tinachita kafukufuku wa silico wa ma protein a oxaloacetate acetylhydrolase (OAH) mu S. sclerotiorum pogwiritsa ntchito chida cha protein-protein BLAST (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Mwachidule, tinagwiritsa ntchito OAH kuchokera ku Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; GenBank accession number XP_040799428.1; 342 amino acids) ndi Penicillium lagena (PlOAH; taxide: 94218; GenBank accession number XP_056833920.1; 316 amino acids) ngati njira zofufuzira kuti tipeze ma protein ofanana mu S. sclerotiorum (taxide: 5180). BLASTp idachitidwa motsutsana ndi deta ya genome ya S. sclerotiorum yomwe yapezeka posachedwa mu GenBank patsamba la National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Kuphatikiza apo, jini ya OAH yoloseredwa kuchokera ku S. sclerotiorum (SsOAH) ndi kusanthula kwa chisinthiko ndi mtengo wa phylogenetic wa AfOAH kuchokera ku A. fijiensis CBS 313.89 ndi PlOAH kuchokera ku P. lagena zinaganiziridwa pogwiritsa ntchito njira ya maximum likelihood mu MEGA11 (Tamura et al., 2021) ndi chitsanzo cha JTT matrix (Jones et al., 1992). Mtengo wa phylogenetic unaphatikizidwa ndi kusanthula kwa ma protein a ma protein onse a OAH omwe analoseredwa (SsOAH) ochokera ku S. sclerotiorum ndi ma query sequence pogwiritsa ntchito Constraint-Based Alignment Tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos ndi Agarwala, 2007). Kuphatikiza apo, ma amino acid ofananira bwino a SsOAH ochokera ku S. sclerotiorum adagwirizana ndi ma query sequences (AfOAH ndi PlOAH) (Larkin et al., 2007) pogwiritsa ntchito ClustalW (http://www.genome.jp/tools-bin/clustalw), ndipo madera osungidwa mu alignment adawonetsedwa pogwiritsa ntchito chida cha ESPRipt (mtundu 3.0; https://espript.ibcp.fr/ESPRipt/ESPRipt/index.php).
Kuphatikiza apo, madera oimira ntchito omwe adanenedweratu komanso malo osungidwa a S. sclerotiorum SsOAH adagawidwa m'mabanja osiyanasiyana pogwiritsa ntchito chida cha InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Pomaliza, chitsanzo cha kapangidwe ka magawo atatu (3D) ka S. sclerotiorum SsOAH chomwe chidanenedweratu chidachitika pogwiritsa ntchito 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) ndipo adatsimikiziridwa pogwiritsa ntchito seva ya SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). Kapangidwe ka magawo atatu komwe kananenedweratu (mtundu wa PDB) kanawonetsedwa pogwiritsa ntchito phukusi la UCSF-Chimera (mtundu 1.15; https://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004).
PCR yowunikira nthawi yeniyeni yowunikira idagwiritsidwa ntchito kudziwa kuchuluka kwa oxaloacetate acetylhydrolase (SsOAH; nambala yolowera ya GenBank: XM_001590428.1) mu mycelia ya Sclerotinia sclerotiorum. Mwachidule, S. sclerotiorum idabayidwa mu botolo lomwe lili ndi PDB ndikuyikidwa mu chosungira chogwedeza (chitsanzo: I2400, New Brunswick Scientific Co., Edison, NJ, USA) pa 25 ± 2 °C kwa maola 24 pa 150 rpm komanso mumdima wosalekeza (maola 24) kuti ilimbikitse kukula kwa mycelial. Pambuyo pake, maselo adachiritsidwa ndi L-ornithine ndi Rizolex-T yophera fungicide pamlingo womaliza wa IC50 (pafupifupi 40 ndi 3.2 mg/L, motsatana) kenako adakulitsidwa kwa maola ena 24 pansi pa mikhalidwe yomweyi. Pambuyo poikamo, zomerazo zinayikidwa mu centrifuge pa 2500 rpm kwa mphindi 5 ndipo supernatant (fungal mycelium) inasonkhanitsidwa kuti iwonetse momwe majini amaonekera. Mofananamo, fungal mycelium inasonkhanitsidwa pa maola 0, 24, 48, 72, 96, ndi 120 pambuyo pa matenda kuchokera ku zomera zomwe zinayambitsa nkhungu yoyera ndi cottony mycelium pamwamba pa minofu yodwala. RNA inachotsedwa mu fungal mycelium kenako cDNA inapangidwa monga tafotokozera pamwambapa. Ma primer sequences a SsOAH alembedwa mu Supplementary Table S3. SsActin (GenBank accession number: XM_001589919.1) inagwiritsidwa ntchito ngati housekeeping gene, ndipo relative gene expression inawerengedwa pogwiritsa ntchito njira ya 2-ΔΔCT (Livak ndi Schmittgen, 2001).
Oxalic acid inapezeka mu potato dextrose broth (PDB) ndi zitsanzo za zomera zomwe zili ndi bowa la Sclerotinia sclerotiorum motsatira njira ya Xu ndi Zhang (2000) ndi kusintha pang'ono. Mwachidule, S. sclerotiorum isolates inayikidwa mu ma flasks okhala ndi PDB kenako inalimidwa mu shaking incubator (model I2400, New Brunswick Scientific Co., Edison, NJ, USA) pa 150 rpm pa 25 ± 2 °C kwa masiku 3-5 mumdima wosalekeza (maola 24) kuti ilimbikitse kukula kwa mycelial. Pambuyo pa incubation, bowa anayamba kusefedwa kudzera mu pepala losefera la Whatman #1 kenako nkuyikidwa centrifuge pa 2500 rpm kwa mphindi 5 kuti achotse mycelium yotsala. Supernatant inasonkhanitsidwa ndikusungidwa pa 4°C kuti idziwe kuchuluka kwa oxalate. Pokonzekera zitsanzo za zomera, pafupifupi 0.1 g ya zidutswa za minofu ya zomera zinachotsedwa katatu ndi madzi osungunuka (2 ml nthawi iliyonse). Kenako zitsanzozo zinayikidwa mu centrifuge pa 2500 rpm kwa mphindi 5, supernatant inasefedwa pogwiritsa ntchito pepala losefera la Whatman No. 1 ndipo inasonkhanitsidwa kuti iwunikidwenso.
Kuti mufufuze kuchuluka kwa oxalic acid, chisakanizo cha reaction chinakonzedwa mu chubu chotsekedwa ndi galasi motere: 0.2 ml ya chitsanzo (kapena PDB culture filtrate kapena oxalic acid standard solution), 0.11 ml ya bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), 0.198 ml ya 1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) ndi 0.176 ml ya 100 mM potassium dichromate (K2Cr2O7; TCI chemicals, Portland, OR, USA), kenako yankho linachepetsedwa kufika pa 4.8 ml ndi madzi osungunuka, kusakaniza mwamphamvu ndikuyikidwa nthawi yomweyo mu bafa la madzi la 60 °C. Pambuyo pa mphindi 10, reaction inayimitsidwa powonjezera 0.5 ml ya sodium hydroxide solution (NaOH; 0.75 M). Kuchuluka kwa kuyamwa kwa madzi (A600) kwa osakaniza kwa madzi kunayesedwa pa 600 nm pogwiritsa ntchito UV-160 spectrophotometer (Shimadzu Corporation, Japan). PDB ndi madzi osungunuka anagwiritsidwa ntchito ngati zowongolera kuchuluka kwa zosakaniza za zomera ndi zomera, motsatana. Kuchuluka kwa oxalic acid mu zosakaniza za zomera, zomwe zimafotokozedwa ngati ma microgram a oxalic acid pa milliliter ya PDB medium (μg.mL−1), ndi m'masamba otulutsa, zomwe zimafotokozedwa ngati ma microgram a oxalic acid pa gramu imodzi ya kulemera kwatsopano (μg.g−1 FW), zinadziwika pogwiritsa ntchito njira yowerengera ya oxalic acid (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
Mu kafukufukuyu, zoyeserera zonse zidapangidwa mwadongosolo losasinthika (CRD) ndi ma replica asanu ndi limodzi achilengedwe pa chithandizo chilichonse ndi miphika isanu pa replica yachilengedwe (zomera ziwiri pa mphika uliwonse) pokhapokha ngati zanenedwa mwanjira ina. Ma replica achilengedwe adasanthulidwa kawiri (ma replica awiri aukadaulo). Ma replica aukadaulo adagwiritsidwa ntchito kuwona kubwerezabwereza kwa kuyesera komweko koma sanagwiritsidwe ntchito mu kusanthula kwa ziwerengero kuti apewe ma replica onyenga. Deta idasanthulidwa mwa ziwerengero pogwiritsa ntchito kusanthula kwa kusiyana (ANOVA) kutsatiridwa ndi mayeso a Tukey-Kramer osonyeza kusiyana kwakukulu (HSD) (p ≤ 0.05). Pa zoyeserera za mu vitro, ma values ​​​​a IC50 ndi IC99 adawerengedwa pogwiritsa ntchito chitsanzo cha probit ndipo magawo odalirika a 95% adawerengedwa.
Mitundu inayi yonse inasonkhanitsidwa kuchokera m'minda yosiyanasiyana ya soya ku El Ghabiya Governorate, Egypt. Pa PDA medium, mitundu yonseyi inapanga mycelium yoyera yokoma yomwe inasanduka yoyera ngati thonje (Chithunzi 1A) kenako beige kapena bulauni pagawo la sclerotium. Mitundu ya sclerotia nthawi zambiri imakhala yolimba, yakuda, yozungulira kapena yosakhazikika, kutalika kwa 5.2 mpaka 7.7 mm ndi 3.4 mpaka 5.3 mm m'mimba mwake (Chithunzi 1B). Ngakhale mitundu inayi inapanga mawonekedwe a sclerotia m'mphepete mwa njira yokulira pambuyo pa masiku 10-12 a incubation pa 25 ± 2 °C (Chithunzi 1A), chiwerengero cha sclerotia pa mbale chinali chosiyana kwambiri pakati pawo (P < 0.001), pomwe mtundu wa isolate 3 unali ndi chiwerengero chachikulu cha sclerotia (32.33 ± 1.53 sclerotia pa mbale; Chithunzi 1C). Mofananamo, isolate #3 inapanga oxalic acid yambiri mu PDB kuposa isolate zina (3.33 ± 0.49 μg.mL−1; Chithunzi 1D). Isolate #3 inawonetsa mawonekedwe achilengedwe komanso a microscopic a bowa wa phytopathogenic Sclerotinia sclerotiorum. Mwachitsanzo, pa PDA, madera a isolate #3 anakula mofulumira, anali oyera bwino (Chithunzi 1A), beige kapena salmon wopepuka wachikasu-bulauni, ndipo amafunika masiku 6-7 pa 25 ± 2°C kuti aphimbe bwino pamwamba pa mbale ya mainchesi 9 cm. Kutengera mawonekedwe a morphological ndi microscopic omwe ali pamwambapa, isolate #3 idadziwika kuti Sclerotinia sclerotiorum.
Chithunzi 1. Makhalidwe ndi matenda a S. sclerotiorum isolates kuchokera ku mbewu za nyemba wamba. (A) Kukula kwa mycelial kwa S. sclerotiorum isolates zinayi pa PDA medium, (B) sclerotia ya S. sclerotiorum isolates zinayi, (C) kuchuluka kwa sclerotia (pa mbale iliyonse), (D) oxalic acid secretion pa PDB medium (μg.mL−1), ndi (E) kuopsa kwa matenda (%) kwa S. sclerotiorum isolates zinayi pa Giza 3 yamalonda yomwe imakhudzidwa ndi nyengo yobiriwira. Ma Values ​​​​akuyimira avereji ± SD ya mitundu isanu ya zamoyo (n = 5). Zilembo zosiyanasiyana zimasonyeza kusiyana kwakukulu pakati pa mankhwala (p < 0.05). (F–H) Zizindikiro zoyera za bowa zimawonekera pamitengo ya pamwamba pa nthaka ndi silique, motsatana, masiku 10 pambuyo pa kubayidwa ndi isolate #3 (dpi). (I) Kusanthula kwa kusintha kwa dera la internal transcribed spacer (ITS) la S. sclerotiorum isolate #3 kunachitika pogwiritsa ntchito njira ya maximum likelihood ndipo kuyerekezeredwa ndi ma reference isolates/strains 20 omwe apezeka kuchokera ku database ya National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). Manambala omwe ali pamwamba pa mizere ya clustering akuwonetsa chigawo (%), ndipo manambala omwe ali pansi pa mizere ya clustering akuwonetsa kutalika kwa nthambi.
Kuphatikiza apo, kuti atsimikizire kufalikira kwa matendawa, mitundu inayi ya S. sclerotiorum yomwe yapezeka idagwiritsidwa ntchito poika katemera wa mbewu ya nyemba yogulitsa yomwe imakhudzidwa ndi kufalikira kwa zomera, zomwe zikugwirizana ndi zomwe Koch's postulates adalemba (Chithunzi 1E). Ngakhale mitundu yonse ya bowa yomwe idapezeka inali yowopsa ndipo imatha kufalikira ku nyemba zobiriwira (cv. Giza 3), zomwe zimayambitsa zizindikiro zoyera za nkhungu m'malo onse pamwamba pa nthaka (Chithunzi 1F), makamaka pa tsinde (Chithunzi 1G) ndi m'mapodi (Chithunzi 1H) patatha masiku 10 kuchokera pamene katemera adachitika (dpi), mtundu 3 unali woopsa kwambiri pa mayeso awiri odziyimira pawokha. Mtundu 3 unali ndi matenda oopsa kwambiri (%) pa zomera za nyemba (24.0 ± 4.0, 58.0 ± 2.0, ndi 76.7 ± 3.1 pa masiku 7, 14, ndi 21 pambuyo pa matenda, motsatana; Chithunzi 1F).
Kuzindikira kwa S. sclerotiorum isolate #3 yomwe imalowa kwambiri kunatsimikiziridwanso kutengera kusanthula kwa mkati mwa spacer (ITS) (Chithunzi 1I). Kusanthula kwa phylogenetic pakati pa isolate #3 ndi 20 reference isolates/strains kunawonetsa kufanana kwakukulu (>99%) pakati pawo. Ndikofunikira kudziwa kuti S. sclerotiorum isolate #3 (533 bp) ili ndi kufanana kwakukulu ndi American S. sclerotiorum isolate LPM36 yochotsedwa ku mbewu zouma za nandolo (GenBank accession number MK896659.1; 540 bp) ndi Chinese S. sclerotiorum isolate YKY211 (GenBank accession number OR206374.1; 548 bp), zomwe zimayambitsa kuvunda kwa tsinde la violet (Matthiola incana), zonse zomwe zili m'magulu padera pamwamba pa dendrogram (Chithunzi 1I). Mndandanda watsopanowu waikidwa mu database ya NCBI ndipo watchedwa “Sclerotinia sclerotiorum – isolate YN-25” (GenBank accession number PV202792). Zikuoneka kuti isolate 3 ndiye isolate yoopsa kwambiri; chifukwa chake, isolate iyi idasankhidwa kuti iphunziridwe mu zoyeserera zonse zotsatira.
Ntchito yolimbana ndi mabakiteriya ya diamine L-ornithine (Sigma-Aldrich, Darmstadt, Germany) pamlingo wosiyanasiyana (12.5, 25, 50, 75, 100 ndi 125 mg/L) motsutsana ndi S. sclerotiorum isolate 3 idafufuzidwa mu vitro. Ndikofunikira kudziwa kuti L-ornithine inali ndi mphamvu yolimbana ndi mabakiteriya ndipo pang'onopang'ono inaletsa kukula kwa radial kwa S. sclerotiorum hyphae motengera mlingo (Chithunzi 2A, B). Pamlingo wapamwamba kwambiri woyesedwa (125 mg/L), L-ornithine inawonetsa kuchuluka kwakukulu kwa mycelial growth inhibition (99.62 ± 0.27%; Chithunzi 2B), komwe kunali kofanana ndi Rizolex-T yogulitsa fungicide (kuchuluka kwa kuletsa 99.45 ± 0.39%; Chithunzi 2C) pamlingo wapamwamba kwambiri woyesedwa (10 mg/L), zomwe zikusonyeza kuti inali yofanana.
Chithunzi 2. Ntchito ya L-ornithine yolimbana ndi matenda a Sclerotinia sclerotiorum mu vitro. (A) Kuyerekeza ntchito ya antibacterial ya kuchuluka kosiyanasiyana kwa L-ornithine motsutsana ndi S. sclerotiorum ndi Rizolex-T (10 mg/L). (B, C) Kuchuluka kwa kuletsa (%) kwa kukula kwa mycelial ya S. sclerotiorum pambuyo pa chithandizo ndi kuchuluka kosiyana kwa L-ornithine (12.5, 25, 50, 75, 100 ndi 125 mg/L) kapena Rizolex-T (2, 4, 6, 8 ndi 10 mg/L), motsatana. Ma Values ​​​​akuyimira avereji ± SD ya ma replicates asanu achilengedwe (n = 5). Zilembo zosiyanasiyana zimasonyeza kusiyana kwa ziwerengero pakati pa chithandizo (p < 0.05). (D, E) Kusanthula kwa probit model regression kwa L-ornithine ndi fungicide yogulitsa Rizolex-T, motsatana. Mzere wowongolera chitsanzo cha probit umawonetsedwa ngati mzere wabuluu wolimba, ndipo nthawi yodzidalira (95%) imawonetsedwa ngati mzere wofiira wodulidwa.
Kuphatikiza apo, kusanthula kwa probit regression kunachitika ndipo ma plots ofanana akuwonetsedwa mu Table 1 ndi Zithunzi 2D,E. Mwachidule, mtengo wovomerezeka wa slope (y = 2.92x - 4.67) ndi ziwerengero zofunika zogwirizana nazo (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 ndi p < 0.0001; Chithunzi 2D) cha L-ornithine chinasonyeza kuti pali ntchito yowonjezereka yolimbana ndi S. sclerotiorum poyerekeza ndi Rizolex-T yophera fungicide (y = 1.96x - 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 ndi p < 0.0001) (Table 1).
Gome 1. Mtengo wa theka lapamwamba kwambiri la kuletsa (IC50) ndi IC99 (mg/l) ya L-ornithine ndi mankhwala ophera fungicide ogulitsa "Rizolex-T" motsutsana ndi S. sclerotiorum.
Ponseponse, L-ornithine (250 mg/L) inachepetsa kwambiri kukula ndi kuopsa kwa nkhungu yoyera pa zomera za nyemba zomwe zachiritsidwa poyerekeza ndi zomera zomwe sizinalandire chithandizo cha S. sclerotiorum (kulamulira; Chithunzi 3A). Mwachidule, ngakhale kuti kuopsa kwa matenda a zomera zomwe sizinalandire chithandizo kunawonjezeka pang'onopang'ono (52.67 ± 1.53, 83.21 ± 2.61, ndi 92.33 ± 3.06%), L-ornithine inachepetsa kwambiri kuopsa kwa matendawa (%) panthawi yonse yoyesera (8.97 ± 0.15, 18.00 ± 1.00, ndi 26.36 ± 3.07) pa masiku 7, 14, ndi 21 pambuyo pa chithandizo (dpt), motsatana (Chithunzi 3A). Mofananamo, pamene nyemba zomwe zili ndi kachilombo ka S. sclerotiorum zinapatsidwa mankhwala a 250 mg/L L-ornithine, dera lomwe lili pansi pa curve ya matenda (AUDPC) linachepa kuchoka pa 1274.33 ± 33.13 mu njira yoletsa matenda mpaka 281.03 ± 7.95, zomwe zinali zochepa pang'ono kuposa mankhwala a 50 mg/L Rizolex-T fungicide (183.61 ± 7.71; Chithunzi 3B). Chizolowezi chomwecho chinawonedwa mu kuyesa kwachiwiri.
Chithunzi 3. Zotsatira za kugwiritsa ntchito L-ornithine kunja kwa thupi pakukula kwa kuvunda koyera kwa nyemba wamba komwe kumachitika chifukwa cha Sclerotinia sclerotiorum pansi pa nyengo yobiriwira. (A) Kuzungulira kwa matenda a nkhungu yoyera ya nyemba wamba mutalandira chithandizo ndi 250 mg/L L-ornithine. (B) Malo omwe ali pansi pa kuzungulira kwa matenda (AUDPC) a nkhungu yoyera ya nyemba wamba mutalandira chithandizo ndi L-ornithine. Ma Values ​​​​akuyimira apakati ± SD ya ma replica asanu achilengedwe (n = 5). Zilembo zosiyanasiyana zimasonyeza kusiyana kwakukulu pakati pa chithandizo (p < 0.05).
Kugwiritsa ntchito kunja kwa 250 mg/L L-ornithine pang'onopang'ono kunawonjezera kutalika kwa chomera (Chithunzi 4A), kuchuluka kwa nthambi pa chomera (Chithunzi 4B), ndi kuchuluka kwa masamba pa chomera (Chithunzi 4C) patatha masiku 42. Ngakhale kuti Rizolex-T (50 mg/L) yopha tizilombo toyambitsa matenda yogulitsa inali ndi zotsatira zabwino kwambiri pa zakudya zonse zomwe zaphunziridwa, kugwiritsa ntchito kunja kwa 250 mg/L L-ornithine kunali ndi zotsatira zachiwiri zazikulu poyerekeza ndi mankhwala oletsa omwe sanachiritsidwe (Chithunzi 4A–C). Kumbali ina, chithandizo cha L-ornithine sichinakhudze kwambiri kuchuluka kwa utoto wa photosynthetic chlorophyll a (Chithunzi 4D) ndi chlorophyll b (Chithunzi 4E), koma chinawonjezera pang'ono kuchuluka kwa carotenoid (0.56 ± 0.03 mg/g fr wt) poyerekeza ndi negative control (0.44 ± 0.02 mg/g fr wt) ndi positive control (0.46 ± 0.02 mg/g fr wt; Chithunzi 4F). Ponseponse, zotsatirazi zikusonyeza kuti L-ornithine si poizoni ku nyemba zomwe zachiritsidwa ndipo ingayambitse kukula kwawo.
Chithunzi 4. Zotsatira za kugwiritsidwa ntchito kwa L-ornithine yakunja pa kukula ndi utoto wa photosynthesis wa masamba a nyemba omwe ali ndi kachilombo ka Sclerotinia sclerotiorum pansi pa kutentha. (A) Kutalika kwa chomera (cm), (B) Chiwerengero cha nthambi pa chomera chilichonse, (C) Chiwerengero cha masamba pa chomera chilichonse, (D) Chlorophyll yomwe ili ndi (mg g-1 fr wt), (E) Chlorophyll yomwe ili ndi (mg g-1 fr wt), (F) Chiwerengero chonse cha carotenoid (mg g-1 fr wt). Ma Values ​​​​ndi apakati ± SD a ma replicates asanu achilengedwe (n = 5). Zilembo zosiyanasiyana zimasonyeza kusiyana kwakukulu pakati pa mankhwala (p < 0.05).
Kupezeka kwa histochemical kwa mitundu ya okosijeni yogwira ntchito (ROS; yowonetsedwa ngati hydrogen peroxide [H2O2]) ndi ma free radicals (owonetsedwa ngati ma superoxide anions [O2•−]) kunawonetsa kuti kugwiritsa ntchito kwa L-ornithine (250 mg/L) kunja kunachepetsa kwambiri kuchuluka kwa H2O2 (96.05 ± 5.33 nmol.g−1 FW; Chithunzi 5A) ndi O2•− (32.69 ± 8.56 nmol.g−1 FW; Chithunzi 5B) poyerekeza ndi kuchuluka kwa zomera zonse ziwiri zomwe sizinalandire chithandizo (173.31 ± 12.06 ndi 149.35 ± 7.94 nmol.g−1 FW, motsatana) ndi zomera zomwe zalandira 50 mg/L ya Rizolex-T yophera fungicide (170.12 ± 9.50 ndi 157.00 ± 7.81 nmol.g−1 fr wt, motsatana) pa maola 72. Kuchuluka kwa H2O2 ndi O2 •− komwe kunasonkhanitsidwa pansi pa hpt (Chithunzi 5A, B). Mofananamo, mayeso a malondialdehyde (MDA) ochokera ku TCA adawonetsa kuti mbewu za nyemba zomwe zili ndi matenda a S. sclerotiorum zinasonkhanitsa milingo yambiri ya MDA (113.48 ± 10.02 nmol.g fr wt) m'masamba awo (Chithunzi 5C). Komabe, kugwiritsa ntchito L-ornithine kunja kunachepetsa kwambiri lipid peroxidation monga momwe zasonyezedwera ndi kuchepa kwa kuchuluka kwa MDA m'zomera zochiritsidwa (33.08 ± 4.00 nmol.g fr wt).
Chithunzi 5. Zotsatira za kugwiritsidwa ntchito kwa L-ornithine yakunja pa zizindikiro zazikulu za kupsinjika kwa okosijeni ndi njira zodzitetezera zosakhala za enzymatic antioxidant m'masamba a nyemba omwe ali ndi kachilombo ka S. sclerotiorum pa maola 72 pambuyo pa matenda pansi pa nyengo yofunda. (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) phenols zonse zosungunuka (mg GAE g−1 FW) pa 72 hpt, (E) flavonoids zonse zosungunuka (mg RE g−1 FW) pa 72 hpt, (F) ma amino acid onse aulere (mg g−1 FW) pa 72 hpt, ndi (G) kuchuluka kwa proline (mg g−1 FW) pa 72 hpt. Ma values ​​​​akuyimira pakati ± standard deviation (avereji ± SD) ya ma replicates 5 achilengedwe (n = 5). Zilembo zosiyanasiyana zimasonyeza kusiyana kwakukulu pakati pa chithandizo (p < 0.05).