Labarin wani ɓangare ne na batun bincike "Inganta juriyar wake ga ƙwayoyin cuta da kwari", duba duk labarai 5
Maganin da ke haifar da cutar fungal necrosis Sclerotinia sclerotiorum (Lib.) de Bary yana amfani da dabara mai matakai da yawa don kamuwa da tsire-tsire daban-daban masu masaukin baki. Wannan binciken ya ba da shawarar amfani da diamine L-ornithine, wani amino acid mara furotin wanda ke motsa haɗar wasu muhimman amino acid, a matsayin wata dabarar sarrafawa don haɓaka martanin kwayoyin halitta, na jiki da na biochemical na Phaseolus vulgaris L. ga farin mold wanda Pseudomonas sclerotiorum ya haifar. Gwaje-gwajen In vitro sun nuna cewa L-ornithine ya hana ci gaban mycelium na S. pyrenoidosa ta hanyar da ta dogara da kashi. Bugu da ƙari, yana iya rage tsananin farin mold a ƙarƙashin yanayin greenhouse. Bugu da ƙari, L-ornithine ya ƙarfafa ci gaban tsire-tsire da aka yi wa magani, yana nuna cewa yawan L-ornithine da aka gwada ba shi da guba ga tsire-tsire da aka yi wa magani. Bugu da ƙari, L-ornithine ya haɓaka bayyanar antioxidants marasa enzymatic (furotin da flavonoids masu narkewa gaba ɗaya) da antioxidants masu narkewa a cikin enzymatic (catalase (CAT), peroxidase (POX), da polyphenol oxidase (PPO)), kuma ya ƙara bayyanar kwayoyin halitta guda uku masu alaƙa da antioxidant (PvCAT1, PvSOD, da PvGR). Bugu da ƙari, a cikin binciken silico ya bayyana kasancewar furotin oxaloacetate acetylhydrolase (SsOAH) a cikin kwayar halittar S. sclerotiorum, wanda yayi kama sosai da furotin oxaloacetate acetylhydrolase (SsOAH) na Aspergillus fijiensis (AfOAH) da Penicillium sp. (PlOAH) dangane da nazarin aiki, yankunan da aka kiyaye, da yanayin ƙasa. Abin sha'awa, ƙara L-ornithine zuwa miyar dankalin turawa (PDB) ya rage yawan bayyanar kwayar halittar SsOAH a cikin mycelia na S. sclerotiorum. Hakazalika, amfani da L-ornithine daga waje ya rage yawan bayyanar kwayar halittar SsOAH a cikin mycelia na fungal da aka tattara daga tsire-tsire da aka yi wa magani. A ƙarshe, amfani da L-ornithine ya rage yawan fitar da sinadarin oxalic acid a cikin ganyayyakin PDB da na kamuwa da cuta. A ƙarshe, L-ornithine yana taka muhimmiyar rawa wajen kiyaye matsayin redox da kuma haɓaka martanin kariya daga tsire-tsire masu kamuwa da cuta. Sakamakon wannan binciken na iya taimakawa wajen haɓaka hanyoyin kirkire-kirkire, masu lafiya ga muhalli don sarrafa farin mold da rage tasirinsa akan samar da wake da sauran amfanin gona.
Farin mold, wanda naman gwari mai suna Sclerotinia sclerotiorum (Lib.) de Bary ke haifarwa, wata cuta ce mai matuƙar illa, mai rage yawan amfanin gona wadda ke haifar da babbar barazana ga samar da wake a duniya (Phaseolus vulgaris L.) (Bolton et al., 2006). Sclerotinia sclerotiorum yana ɗaya daga cikin cututtukan fungal da shukar ta shafa a ƙasa, tare da nau'ikan tsire-tsire sama da 600 da kuma ikon yin lalata da kyallen da ke cikinta cikin sauri ba tare da takamaiman yanayi ba (Liang da Rollins, 2018). A ƙarƙashin yanayi mara kyau, yana fuskantar wani muhimmin lokaci na zagayowar rayuwarsa, yana zama ba tare da ya huta ba na dogon lokaci a matsayin siffofi baƙi, masu tauri, masu kama da iri da ake kira 'sclerotia' a cikin ƙasa ko kuma a matsayin fararen, masu laushi a cikin mycelium ko tushen shuke-shuken da suka kamu da cutar (Schwartz et al., 2005). S. sclerotiorum yana da ikon samar da sclerotia, wanda ke ba shi damar rayuwa a cikin filayen da suka kamu da cutar na dogon lokaci kuma ya daɗe a lokacin cutar (Schwartz et al., 2005). Sclerotia tana da wadataccen abinci mai gina jiki, tana iya dawwama a cikin ƙasa na dogon lokaci, kuma tana aiki a matsayin babban allurar rigakafi don kamuwa da cuta daga baya (Schwartz et al., 2005). A ƙarƙashin yanayi mai kyau, sclerotia tana tsiro kuma tana samar da ƙwayoyin cuta masu iska waɗanda zasu iya kamuwa da dukkan sassan shukar da ke sama da ƙasa, gami da amma ba'a iyakance ga furanni, tushe, ko kwari ba (Schwartz et al., 2005).
Sclerotinia sclerotiorum tana amfani da dabara mai matakai da yawa don kamuwa da cututtukan tsire-tsire masu masaukin baki, wanda ya ƙunshi jerin abubuwan da suka faru tun daga tsirowar sclerotial zuwa ci gaban alamun cutar. Da farko, S. sclerotiorum yana samar da spores da aka dakatar (wanda ake kira ascospores) daga tsarin namomin kaza da ake kira apothecia, waɗanda ke zama a iska kuma suna girma zuwa sclerotia marasa motsi akan tarkacen tsire-tsire da suka kamu da cutar (Bolton et al., 2006). Sannan naman gwari yana fitar da oxalic acid, wani abu mai haifar da cututtuka, don sarrafa pH na bangon ƙwayoyin shuka, yana haɓaka lalacewar enzymatic da mamaye nama (Hegedus da Rimmer, 2005), da kuma danne fashewar iskar oxygen na shukar mai masaukin baki. Wannan tsarin acidification yana raunana bangon ƙwayoyin shuka, yana samar da yanayi mai kyau don aiki na yau da kullun da inganci na enzymes masu lalata bangon ƙwayoyin fungal (CWDEs), yana ba da damar ƙwayoyin cuta su shawo kan shingen zahiri kuma su shiga kyallen mai masaukin baki (Marciano et al., 1983). Da zarar an shiga, S. sclerotiorum yana fitar da wasu CWDEs, kamar polygalacturonase da cellulase, waɗanda ke sauƙaƙa yaɗuwarsu a cikin kyallen da suka kamu da cutar kuma suna haifar da necrosis na nama. Ci gaban raunuka da tabarmar hyphal yana haifar da alamun bayyanar farin mold (Hegedus da Rimmer, 2005). A halin yanzu, tsire-tsire masu masaukin baki suna gane tsarin kwayoyin halitta da ke da alaƙa da ƙwayoyin cuta (PAMPs) ta hanyar masu karɓar ganewar alamu (PRRs), suna haifar da jerin abubuwan da ke haifar da alamun da ke kunna martanin kariya.
Duk da shekarun da aka shafe ana ƙoƙarin shawo kan cututtuka, ƙarancin isasshen ƙwayoyin cuta masu juriya ga cututtuka ya kasance a cikin wake, kamar yadda yake a sauran amfanin gona na kasuwanci, saboda juriya, rayuwa, da kuma daidaitawar ƙwayoyin cuta. Saboda haka, kula da cututtuka yana da matuƙar ƙalubale kuma yana buƙatar dabarun haɗaka, masu fuskoki da yawa waɗanda suka haɗa da haɗakar ayyukan al'adu, kula da halittu, da magungunan kashe ƙwayoyin cuta masu guba (O'Sullivan et al., 2021). Kula da sinadarai na farin mold shine mafi inganci saboda magungunan kashe ƙwayoyin cuta, idan aka yi amfani da su daidai kuma a lokacin da ya dace, na iya sarrafa yaɗuwar cutar yadda ya kamata, rage tsananin kamuwa da cuta, da kuma rage asarar amfanin gona. Duk da haka, amfani da magungunan kashe ƙwayoyin cuta fiye da kima da kuma dogaro da magungunan kashe ƙwayoyin cuta fiye da kima na iya haifar da fitowar nau'ikan S. sclerotiorum masu juriya da kuma yin mummunan tasiri ga ƙwayoyin cuta marasa manufa, lafiyar ƙasa, da ingancin ruwa (Le Cointe et al., 2016; Ceresini et al., 2024). Saboda haka, nemo madadin da ya dace da muhalli ya zama babban fifiko.
Polyamines (PAs), kamar putrescine, spermidine, spermine, da cadaverine, na iya zama madadin da zai yi kyau ga cututtukan tsire-tsire da ke ɗauke da ƙasa, ta haka ne rage amfani da magungunan kashe ƙwayoyin cuta masu haɗari gaba ɗaya ko kuma wani ɓangare (Nehela et al., 2024; Yi et al., 2024). A cikin manyan tsire-tsire, PAs suna shiga cikin ayyukan jiki da yawa, ciki har da, amma ba'a iyakance ga, rarraba ƙwayoyin halitta, bambance-bambance, da kuma amsawa ga damuwa mai hana ƙwayoyin cuta da ƙwayoyin cuta ba (Killiny da Nehela, 2020). Suna iya aiki a matsayin antioxidants, suna taimakawa wajen wargaza nau'ikan iskar oxygen masu amsawa (ROS), suna kula da yanayin redox (Nehela da Killiny, 2023), suna haifar da kwayoyin halitta masu alaƙa da tsaro (Romero et al., 2018), suna daidaita hanyoyin rayuwa daban-daban (Nehela da Killiny, 2023), suna daidaita phytohormones na ciki (Nehela da Killiny, 2019), suna kafa juriya ta tsarin da aka samu (SAR), da kuma daidaita hulɗar tsirrai da ƙwayoyin cuta (Nehela da Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). Ya kamata a lura cewa takamaiman hanyoyin da rawar da PA ke takawa wajen kare tsirrai sun bambanta dangane da nau'in shuka, ƙwayoyin cuta, da yanayin muhalli. Mafi yawan PA a cikin tsire-tsire ana samar da shi ne daga mahimmin polyamine L-ornithine (Killiny da Nehela, 2020).
L-ornithine yana taka rawa da yawa a cikin girma da ci gaban shuke-shuke. Misali, binciken da aka yi a baya ya nuna cewa a cikin shinkafa (Oryza sativa), ornithine na iya kasancewa da alaƙa da sake amfani da nitrogen (Liu et al., 2018), yawan shinkafa, inganci da ƙamshi (Lu et al., 2020), da kuma amsawar damuwa ta ruwa (Yang et al., 2000). Bugu da ƙari, amfani da L-ornithine na waje yana ƙara yawan haƙurin fari a cikin beet na sukari (Beta vulgaris) (Hussein et al., 2019) da kuma rage matsin lamba na gishiri a cikin shuke-shuken albasa (Allium Cepa) (Çavuşoǧlu da Çavuşoǧlu, 2021) da cashew (Anacardium occidentale) (da Rocha et al., 2012). Matsayin da L-ornithine ke da shi a cikin kariyar damuwa na iya kasancewa saboda shigarsa cikin tarin proline a cikin shuke-shuken da aka yi wa magani. Misali, an riga an ruwaito cewa kwayoyin halittar da ke da alaƙa da metabolism na proline, kamar ornithine delta aminotransferase (delta-OAT) da proline dehydrogenase (ProDH1 da ProDH2), suna da hannu a cikin kare Nicotiana benthamiana da Arabidopsis thaliana akan nau'ikan Pseudomonas syringae marasa masaukin baki (Senthil-Kumar da Mysore, 2012). A gefe guda kuma, ana buƙatar fungal ornithine decarboxylase (ODC) don haɓakar ƙwayoyin cuta (Singh et al., 2020). Yin niyya ga ODC na Fusarium oxysporum f. sp. lycopersici ta hanyar rufe kwayar halittar da aka haifar (HIGS) ya ƙara juriyar tsire-tsire na tumatir ga Fusarium wilt (Singh et al., 2020). Duk da haka, ba a yi nazari sosai kan rawar da aikace-aikacen ornithine na waje ke takawa kan matsalolin biotic kamar phytopathogens ba. Mafi mahimmanci, tasirin ornithine akan juriyar cututtuka da kuma abubuwan da ke tattare da sinadarai da kuma abubuwan da ke tattare da su ba a bincika su sosai ba.
Fahimtar sarkakiyar kamuwa da cutar S. sclerotiorum a cikin legumes yana da mahimmanci don haɓaka dabarun sarrafawa masu inganci. A cikin wannan binciken, mun yi nufin gano yuwuwar rawar da diamine L-ornithine ke takawa a matsayin muhimmin abu wajen haɓaka hanyoyin kariya da juriyar shuke-shuken legume ga kamuwa da cutar Sclerotinia sclerotiorum. Mun yi hasashen cewa, baya ga haɓaka martanin kariya na shuke-shuken da suka kamu da cutar, L-ornithine kuma yana taka muhimmiyar rawa wajen kiyaye matsayin redox. Muna ba da shawarar cewa tasirin L-ornithine yana da alaƙa da daidaita hanyoyin kariya na enzymatic da waɗanda ba enzymatic ba da kuma tsangwama ga abubuwan da ke haifar da cututtukan fungal/ƙwayar cuta da sunadarai masu alaƙa. Wannan aiki biyu na L-ornithine ya sa ya zama ɗan takara mai kyau don dabarun dorewa don rage tasirin farin mold da haɓaka juriyar amfanin gona na legume ga wannan ƙwayar fungal mai ƙarfi. Sakamakon wannan binciken na iya taimakawa wajen haɓaka hanyoyin kirkire-kirkire, masu aminci ga muhalli don sarrafa farin mold da rage tasirinsa akan samar da legume.
A cikin wannan binciken, an yi amfani da nau'in wake na yau da kullun da ke da sauƙin kamuwa da shi a kasuwa, Giza 3 (Phaseolus vulgaris L. cv. Giza 3), a matsayin kayan gwaji. Sashen Bincike na Legume, Cibiyar Bincike ta Gonaki (FCRI), Cibiyar Bincike ta Noma (ARC), Masar, ta samar da iri mai lafiya. An shuka iri biyar a cikin tukwane na filastik (diamita ta ciki 35 cm, zurfin 50 cm) cike da ƙasa mai kamuwa da cutar S. sclerotiorum a ƙarƙashin yanayin greenhouse (25 ± 2 °C, ɗanɗano mai kyau 75 ± 1%, haske awanni 8/sa'o'i 16 duhu). Bayan kwana 7-10 bayan shuka (DPS), an rage girman iri don barin iri biyu kawai tare da girma iri ɗaya da ganye uku masu faɗi a cikin kowace tukunya. Ana shayar da duk tsire-tsire da aka dasa a cikin tukunya sau ɗaya a kowane mako biyu kuma a ba su takin wata-wata bisa ga shawarar da aka bayar ga nau'in da aka bayar.
Domin shirya yawan L-ornithinediamine mai nauyin 500 mg/L (wanda kuma aka sani da (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Jamus), an fara narkar da 50 mg a cikin 100 ml na ruwan da aka tace. Daga nan aka narkar da ruwan da aka tace sannan aka yi amfani da shi a gwaje-gwajen da suka biyo baya. A takaice, an gwada jerin nau'ikan L-ornithine guda shida (12.5, 25, 50, 75, 100, da 125 mg/L) a cikin vitro. Bugu da ƙari, an yi amfani da ruwan da aka tace mai tsafta azaman maganin hana kumburi (Mock) kuma an yi amfani da maganin kashe kwari na kasuwanci "Rizolex-T" foda mai laushi 50% (toclofos-methyl 20% + thiram 30%; Kamfanin KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Masar) azaman maganin hana kumburi. An gwada maganin kashe ƙwayoyin cuta na "Rizolex-T" na kasuwanci a cikin vitro a cikin taro biyar (2, 4, 6, 8 da 10 mg/L).
An tattara samfuran tushen wake da kwasfa da ke nuna alamun fararen mold (yawan kamuwa: 10-30%) daga gonakin kasuwanci. Duk da cewa an gano yawancin kayan shuka da suka kamu da cutar ta hanyar nau'in/nau'in (irin Giza 3 na kasuwanci mai saurin kamuwa), wasu, musamman waɗanda aka samo daga kasuwannin gida, nau'ikan da ba a san su ba ne. An fara tsaftace kayan da suka kamu da cutar da maganin sodium hypochlorite 0.5% na tsawon mintuna 3, sannan a wanke sau da yawa da ruwan da aka tace sannan a goge su da takarda mai tsafta don cire ruwan da ya wuce kima. Daga nan aka yanke gabobin da suka kamu da cutar zuwa ƙananan guntu daga tsakiyar nama (tsakanin kyallen lafiya da masu kamuwa da cutar), aka noma su a kan matsakaiciyar dankalin turawa dextrose agar (PDA) sannan a saka su a zafin 25 ± 2 °C tare da haske na awanni 12/zagayen duhu na awanni 12 na tsawon kwanaki 5 don ƙarfafa samuwar sclerotia. An kuma yi amfani da hanyar mycelial tip don tsarkake ƙwayoyin fungal daga al'adu masu gauraye ko masu gurbata. An fara gano sinadarin fungal da aka tsarkake bisa ga halayensa na al'ada sannan aka tabbatar da cewa S. sclerotiorum ne bisa ga siffofin ƙananan halittu. A ƙarshe, an gwada duk wani sinadarin da aka tsarkake don gano cutar da ke cikin nau'in wake mai sauƙin kamuwa da cutar Giza 3 don cika ka'idojin Koch.
Bugu da ƙari, an ƙara tabbatar da cewa mafi girman S. sclerotiorum isolate (isolate #3) ya samo asali ne daga jerin spacer na ciki (ITS) kamar yadda White et al., 1990 suka bayyana; Baturo-Ciesniewska et al., 2017. A taƙaice, an noma isolates a cikin ruwan dankalin turawa (PDB) kuma an saka su a zafin 25 ± 2 °C na tsawon kwanaki 5-7. Daga nan aka tattara mycelium na fungal, aka tace ta hanyar cheesecloth, aka wanke sau biyu da ruwa mai tsafta, sannan aka busar da shi da takardar tacewa mai tsafta. An ware DNA ta genomic ta amfani da Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Daga nan aka ƙara girman yankin ITS rDNA ta amfani da takamaiman nau'in primer ITS1/ITS4 (TCCGTAGGTGAACCTGCGG TCCTCCGCTTATTGATATGC; girman da ake tsammani: 540 bp) (Baturo-Ciesniewska et al., 2017). An gabatar da samfuran PCR da aka tsarkake don jerin abubuwa (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). An tsara jerin ITS rDNA ta hanyoyi biyu ta amfani da hanyar jerin abubuwa ta Sanger. Daga nan aka kwatanta jerin tambayoyin da aka haɗa da sabbin bayanai a GenBank da Cibiyar Bayar da Bayanai kan Fasahar Halittu ta Ƙasa (NCBI, http://www.ncbi.nlm.nih.gov/gene/) ta amfani da software na BLASTn. An kwatanta jerin tambayoyin da wasu nau'ikan S. sclerotiorum/isolates guda 20 da aka samo daga sabbin bayanai a cikin NCBI GenBank (Ƙarin Tebur S1) ta amfani da ClustalW a cikin Kunshin Nazarin Halittar Juyin Halittar Kwayoyin Halitta (MEGA-11; sigar 11) (Kumar et al., 2024). An gudanar da nazarin juyin halitta ta amfani da hanyar mafi girman yuwuwar da kuma samfurin maye gurbin nucleotide na lokaci-lokaci (Nei da Kumar, 2000). An nuna bishiyar da ke da mafi girman yuwuwar yin amfani da ...
An gano tasirin maganin kashe ƙwayoyin cuta na L-ornithine da kuma maganin kashe ƙwayoyin cuta na "Rizolex-T" a cikin vitro ta hanyar hanyar yaduwar agar. Hanya: A ɗauki adadin da ya dace na maganin L-ornithine (500 mg/L) sannan a gauraya shi sosai da 10 ml na maganin gina jiki na PDA don shirya mafita tare da yawan ƙarshe na 12.5, 25, 50, 75, 100 da 125 mg/L, bi da bi. An yi amfani da yawan maganin kashe ƙwayoyin cuta guda biyar na "Rizolex-T" (2, 4, 6, 8 da 10 mg/L) da ruwan da aka tace a matsayin magani. Bayan maganin ya taurare, an mayar da wani sabon toshe na al'adar Sclerotinia sclerotiorum, diamita na mm 4, zuwa tsakiyar abincin Petri kuma an shuka shi a 25±2°C har sai mycelium ya rufe dukkan abincin Petri, bayan haka an yi rikodin girman naman gwari. Lissafa kashi na hana ci gaban radial na S. sclerotiorum ta amfani da lissafi na 1:
An maimaita gwajin sau biyu, tare da kwafi shida na halitta ga kowane rukuni na sarrafawa/gwaji da kuma tukwane biyar (tsirrai biyu a kowace tukunya) ga kowane kwafi na halitta. An yi nazarin kowace kwafi na halitta sau biyu (kwafi biyu na fasaha) don tabbatar da daidaito, aminci da sake haifar da sakamakon gwajin. Bugu da ƙari, an yi amfani da nazarin komawar probit don ƙididdige yawan hanawa rabin-mafi girma (IC50) da IC99 (Prentice, 1976).
Domin kimanta yuwuwar L-ornithine a ƙarƙashin yanayin greenhouse, an gudanar da gwaje-gwaje guda biyu a jere a cikin tukunya. A takaice, an cika tukwane da ƙasa mai yashi da aka yi wa fenti (3:1) sannan aka yi wa S. sclerotiorum allurar rigakafi. Da farko, an noma mafi girman ƙwayar S. sclerotiorum (isolate #3) ta hanyar yanke sclerotium ɗaya a rabi, a sanya shi a kan PDA sannan a saka shi a zafin jiki na 25°C a cikin duhu mai ɗorewa (awanni 24) na tsawon kwanaki 4 don ƙarfafa ci gaban mycelium. Daga nan aka ɗauki matosai huɗu na agar mai diamita 5 mm daga gefen gaba aka yi wa allurar allurar 100 g na cakuda alkama da shinkafa mai tsabta (1:1, v/v) kuma an saka dukkan kwalba a zafin jiki na 25 ± 2°C a ƙarƙashin haske na awanni 12/zagayen duhu na awanni 12 na tsawon kwanaki 5 don ƙarfafa samuwar sclerotia. An haɗa abubuwan da ke cikin dukkan kwalba sosai don tabbatar da daidaito kafin a ƙara ƙasa. Sannan, an ƙara gram 100 na cakuda bran mai cike da ƙura a kowace tukunya don tabbatar da yawan ƙwayoyin cuta akai-akai. An shayar da tukwanen da aka yi wa allurar don kunna haɓakar fungal sannan aka sanya su a cikin yanayin greenhouse na tsawon kwanaki 7.
An shuka iri biyar na nau'in Giza 3 a kowace tukunya. Ga tukwanen da aka yi wa magani da L-ornithine da kuma maganin kashe kwari na Rizolex-T, an fara jiƙa iri da aka yi wa magani na tsawon awanni biyu a cikin ruwan magani na mahaɗan biyu tare da jimlar IC99 na ƙarshe na kimanin 250 mg/L da 50 mg/L, bi da bi, sannan a busar da su ta iska na tsawon awa ɗaya kafin a shuka. A gefe guda kuma, an jiƙa iri a cikin ruwan da aka tace a matsayin maganin da ba shi da kyau. Bayan kwana 10, kafin a fara shayar da su, an rage girman iri, an bar iri biyu kawai masu tsabta a kowace tukunya. Bugu da ƙari, don tabbatar da kamuwa da cuta tare da S. sclerotiorum, an yanke tushen shukar wake a matakin ci gaba ɗaya (kwanaki 10) a wurare biyu daban-daban ta amfani da scalpel da aka yi wa magani kuma an sanya kimanin 0.5 g na cakuda bran mai kama da nama a cikin kowane rauni, sannan a biyo baya da zafi mai yawa don ƙarfafa kamuwa da cuta da ci gaban cututtuka a duk tsire-tsire da aka yi wa allura. An kuma ji wa cibiyoyin da ke kula da cutar rauni iri ɗaya, kuma an saka daidai adadin (0.5 g) na cakuda bran da ba a shafa ba, wanda ba a shafa ba a cikin raunin, sannan a kula da shi a ƙarƙashin zafi mai yawa don kwaikwayon yanayin ci gaban cututtuka da kuma tabbatar da daidaito tsakanin ƙungiyoyin jiyya.
Hanyar Magani: An shayar da 'ya'yan wake da ruwan L-ornithine (250 mg/l) ko maganin kashe kwari na Rizolex-T (50 mg/l) ta hanyar ban ruwa a ƙasa, sannan aka maimaita maganin sau uku a tazara ta kwanaki 10. An yi ban ruwa da ruwan da aka yi wa magani da placebo da ruwa mai tsafta na 500 ml. An yi duk maganin a ƙarƙashin yanayin greenhouse (25 ± 2°C, ɗanɗano 75 ± 1%, da kuma hasken awanni 8/haske 16 a duhu). An shayar da dukkan tukwane sau biyu a mako kuma an yi wa magani kowane wata da takin NPK mai daidaito (20-20-20, tare da 3.6% sulfur da microelements na TE; Zain Seeds, Egypt) a yawan 3-4 g/l ta hanyar fesa ganye bisa ga shawarwarin da aka bayar na takamaiman nau'in da umarnin masana'anta. Sai dai idan an faɗi akasin haka, an tattara ganyen da suka girma gaba ɗaya (ganye na 2 da na 3 daga sama) daga kowace kwafin halitta a cikin awanni 72 bayan magani (hpt), an haɗa su daidai, an tattara su kuma an adana su a -80 °C don ƙarin bincike, gami da, amma ba'a iyakance ga, inda aka gano alamun damuwa na oxidative a cikin wurin ba, lipid peroxidation, antioxidants enzymatic da non-enzymatic da bayyanar kwayoyin halitta.
An tantance ƙarfin kamuwa da cutar farin mold a kowane mako bayan allurar (dpi) ta amfani da sikelin 1-9 (Tebur na Ƙarin S2) bisa ga sikelin Petzoldt da Dickson (1996) wanda Teran et al. (2006) suka gyara. A takaice, an duba tushen da rassan shuke-shuken wake tun daga lokacin allurar don bin diddigin ci gaban raunuka a kan ƙananan ƙwayoyin cuta da ƙananan ƙwayoyin cuta. Daga nan aka auna nisan raunin daga wurin allurar zuwa mafi nisa a kan tushe ko reshe kuma aka sanya maki 1-9 bisa ga wurin raunin, inda (1) ya nuna babu wani kamuwa da cuta da ake gani kusa da wurin allurar kuma (2-9) ya nuna ƙaruwa a hankali a girman raunuka da ci gaba tare da ƙananan ƙwayoyin cuta/ƙwayoyin cuta (Tebur na Ƙarin S2). Daga nan aka canza ƙarfin kamuwa da cutar farin mold zuwa kashi ta amfani da dabara ta 2:
Bugu da ƙari, an ƙididdige yankin da ke ƙarƙashin lanƙwasa ci gaban cutar (AUDPC) ta amfani da dabarar (Shaner da Finney, 1977), wanda aka daidaita kwanan nan don farin ruɓewar wake (Chauhan et al., 2020) ta amfani da lissafi na 3:
Inda Yi = tsananin cutar a lokacin ti, Yi+1 = tsananin cutar a lokaci na gaba ti+1, ti = lokacin aunawa na farko (a cikin kwanaki), ti+1 = lokacin aunawa na gaba (a cikin kwanaki), n = jimlar adadin lokutan lokaci ko wuraren lura. An rubuta ma'aunin girma na shukar wake gami da tsayin shuka (cm), adadin rassan kowace shuka, da adadin ganyen kowace shuka a kowane mako na tsawon kwanaki 21 a cikin dukkan kwafi na halitta.
A cikin kowace kwafin halitta, an tattara samfuran ganye (ganye na biyu da na uku da suka cika girma daga sama) a rana ta 45 bayan magani (kwanaki 15 bayan magani na ƙarshe). Kowace kwafin halitta ta ƙunshi tukwane biyar (shuke-shuke biyu a kowace tukunya). An yi amfani da kimanin 500 MG na nama da aka niƙa don cire pigments na photosynthetic (chlorophyll a, chlorophyll b da carotenoids) ta amfani da 80% acetone a 4 °C a cikin duhu. Bayan awanni 24, an cire samfuran kuma an tattara supernatant don tantance abubuwan da ke cikin chlorophyll a, chlorophyll b da carotenoid ta hanyar amfani da na'urar auna UV-160A spectrophotometer (Shimadzu Corporation, Japan) bisa ga hanyar (Lichtenthaler, 1987) ta hanyar auna sha a tsawon tsayi daban-daban guda uku (A470, A646 da A663 nm). A ƙarshe, an ƙididdige abubuwan da ke cikin pigments na photosynthetic ta amfani da waɗannan dabarun 4-6 da Lichtenthaler ya bayyana (1987).
Bayan awanni 72 bayan magani (hpt), an tattara ganyen (na biyu da na uku ganyen da aka haɓaka gaba ɗaya daga sama) daga kowace kwafin halitta don gano wurin da hydrogen peroxide (H2O2) da superoxide anion (O2•−) ke zaune a ciki. Kowace kwafin halitta ta ƙunshi tukwane biyar (tsirrai biyu a kowace tukunya). An yi nazarin kowace kwafin halitta a cikin kwafi (kwafi biyu na fasaha) don tabbatar da daidaito, aminci da sake haifuwar hanyar. An tantance H2O2 da O2•− ta amfani da 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Jamus) ko nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Jamus), bi da bi, bin hanyoyin da Romero-Puertas et al. (2004) da Adam et al. (1989) suka bayyana tare da ƙananan gyare-gyare. Don gano wurin da H2O2 ke a wurin da aka yi amfani da shi a cikin histochemical, an saka takardar da ke cikin injin tsabtace iska da 0.1% DAB a cikin 10 mM Tris buffer (pH 7.8) sannan a saka ta a zafin ɗaki a cikin haske na tsawon minti 60. An saka takardar da ke cikin injin tsabtace iska da 0.15% (v/v) TCA a cikin 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) sannan a fallasa ta ga haske har sai sun yi duhu. Hakazalika, an saka bawuloli da ke cikin injin tsabtace iska da 10 mM potassium phosphate buffer (pH 7.8) wanda ke ɗauke da 0.1 w/v% HBT don gano wurin da aka yi amfani da shi a cikin injin tsabtace iska da O2•− a wurin da aka yi amfani da shi a cikin injin tsabtace iska. An saka takardar da ke cikin haske a zafin ɗaki na tsawon minti 20, sannan a yi ta a cikin injin tsabtace iska kamar yadda aka ambata a sama, sannan a haskaka har sai tabo masu duhu shuɗi/violet sun bayyana. An tantance ƙarfin launin launin ruwan kasa (a matsayin alamar H2O2) ko shuɗi-violet (a matsayin alamar O2•−) ta amfani da sigar Fiji ta kunshin sarrafa hoto ImageJ (http://fiji.sc; an duba a ranar 7 ga Maris 2024).
An tantance Malondialdehyde (MDA; a matsayin alamar lipid peroxidation) bisa ga hanyar Du da Bramlage (1992) tare da ɗan gyare-gyare. An tattara ganyen daga kowace kwafin halitta (ganye na biyu da na uku da aka haɓaka gaba ɗaya daga sama) awanni 72 bayan magani (hpt). Kowace kwafin halitta ta haɗa da tukwane biyar (tsirrai biyu a kowace tukunya). An yi nazarin kowace kwafin halitta a cikin kwafi biyu (kwafi biyu na fasaha) don tabbatar da daidaito, aminci da sake haifuwar hanyar. A taƙaice, an yi amfani da 0.5 g na nama na ganyen ƙasa don cire MDA tare da 20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, Amurka) wanda ke ɗauke da 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, Amurka). An tantance abubuwan da ke cikin MDA a cikin ruwan da ke cikinsa ta hanyar launi ta hanyar auna sha a 532 da 600 nm ta amfani da na'urar auna haske ta UV-160A (Shimadzu Corporation, Japan) sannan aka bayyana shi a matsayin nmol g−1 FW.
Don tantance sinadaran antioxidants marasa enzymatic da enzymatic, an tattara ganyen (ganyayyaki na biyu da na uku da suka fito daga sama) daga kowace kwayar halitta a cikin awanni 72 bayan magani (hpt). Kowace kwayar halitta ta ƙunshi tukwane biyar (tsirrai biyu a kowace tukunya). An yi nazarin kowane samfurin halitta a cikin kwafi biyu (samfuran fasaha guda biyu). An niƙa ganyen biyu da ruwa mai nitrogen kuma an yi amfani da su kai tsaye don tantance sinadaran antioxidants masu enzymatic da waɗanda ba enzymatic ba, jimlar amino acid, abubuwan da ke cikin proline, bayyanar kwayoyin halitta, da kuma ƙididdige oxalate.
An tantance jimillar sinadaran phenolic da ke narkewa ta amfani da sinadarin Folin-Ciocalteu (Sigma-Aldrich, St. Louis, MO, Amurka) tare da ɗan gyare-gyare kaɗan na hanyar da Kahkonen et al. (1999) suka bayyana. A takaice, an cire kimanin 0.1 g na kyallen ganyen da aka haɗa da 20 ml 80% methanol a cikin duhu na tsawon awanni 24 kuma an tattara ruwan da ke cikinsa bayan an yi amfani da shi. An haɗa 0.1 ml na samfurin da aka cire da sinadarin Folin-Ciocalteu 0.5 ml (10%), an girgiza shi na tsawon daƙiƙa 30 sannan aka bar shi a cikin duhu na tsawon mintuna 5. Sannan an ƙara 0.5 ml na maganin sodium carbonate 20% (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Egypt) a kowace bututu, an gauraya sosai sannan aka sanya shi a zafin ɗaki a cikin duhu na tsawon awanni 1. Bayan an gama, an auna yadda cakudawar amsawar ke sha a 765 nm ta amfani da na'urar auna haske ta UV-160A (Shimadzu Corporation, Japan). An tantance yawan phenols masu narkewa a cikin samfurin da aka cire ta amfani da lanƙwasa na daidaita sinadarin gallic acid (Fisher Scientific, Hampton, NH, Amurka) kuma an bayyana su a matsayin milligrams na sinadarin gallic acid daidai da kowace gram na sabon nauyin (mg na nauyin g-1 na sabon nauyin GAE).
An ƙayyade jimillar sinadarin flavonoid mai narkewa bisa ga hanyar Djeridane et al. (2006) tare da ɗan gyare-gyare kaɗan. A taƙaice, an haɗa 0.3 ml na ruwan methanol da ke sama da 0.3 ml na ruwan aluminum chloride 5% (AlCl3; Fisher Scientific, Hampton, NH, Amurka), an juya shi da ƙarfi sannan a saka shi a zafin ɗaki na tsawon minti 5, sannan a ƙara 0.3 ml na ruwan potassium acetate 10% (Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt), an haɗa shi sosai kuma an saka shi a zafin ɗaki na tsawon minti 30 a cikin duhu. Bayan an saka shi, an auna shan ruwan haɗin a 430 nm ta amfani da na'urar auna UV-160A (Shimadzu Corporation, Japan). An tantance yawan flavonoids masu narkewa a cikin samfurin cirewa ta amfani da lanƙwasa na daidaita rutin (TCI America, Portland, OR, Amurka) sannan a bayyana shi a matsayin milligrams na daidai da rutin a kowace gram na nauyin sabo (mg RE g-1 sabon nauyi).
An tantance jimlar amino acid kyauta na ganyen wake ta amfani da wani sinadarin ninhydrin da aka gyara (Thermo Scientific Chemicals, Waltham, MA, Amurka) bisa ga hanyar da Yokoyama da Hiramatsu (2003) suka gabatar kuma Sun et al. (2006) suka gyara. A takaice, an cire 0.1 g na nama da aka niƙa da pH 5.4 buffer, kuma an mayar da 200 μL na supernatant da 200 μL na ninhydrin (2%) da 200 μL na pyridine (10%; Spectrum Chemical, New Brunswick, NJ, Amurka), an saka shi a cikin ruwan zafi na tsawon minti 30, sannan aka sanyaya aka auna shi a 580 nm ta amfani da na'urar auna haske ta UV-160A (Shimadzu Corporation, Japan). A gefe guda kuma, an tantance proline ta hanyar hanyar Bates (Bates et al., 1973). An cire Proline da kashi 3% na sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, Amurka) kuma bayan an yi amfani da centrifugation, an haɗa 0.5 ml na supernatant da glacial acetic acid 1 ml (Fisher Scientific, Hampton, NH, Amurka) da kuma ninhydrin reagent, an saka su a zafin 90°C na tsawon minti 45, aka sanyaya su sannan aka auna su a 520 nm ta amfani da irin wannan spectrophotometer kamar yadda aka ambata a sama. An tantance jimlar amino acid da proline kyauta a cikin cirewar ganye ta amfani da lanƙwasa na daidaita glycine da proline (Sigma-Aldrich, St Louis, MO, Amurka), bi da bi, kuma an bayyana su a matsayin sabon nauyin mg/g.
Domin tantance aikin enzymatic na enzymes masu hana tsufa, an cire kimanin 500 MG na nama mai kama da juna tare da 3 ml na 50 mM Tris buffer (pH 7.8) wanda ke ɗauke da 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, Amurka) da 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, Amurka), an sanya shi a centrifuge a 10,000 × g na tsawon minti 20 a ƙarƙashin firiji (4 °C), kuma an tattara supernatant (ɗanyen enzyme da aka cire) (El-Nagar et al., 2023; Osman et al., 2023). Daga nan aka mayar da martani ga Catalase (CAT) da 2 ml na sodium phosphate buffer mai nauyin 0.1 M (pH 6.5; Sigma-Aldrich, St. Louis, MO, Amurka) da 100 μl na maganin H2O2 na 269 mM don tantance aikin enzymatic ɗinsa bisa ga hanyar Aebi (1984) tare da ɗan gyare-gyare (El-Nagar et al., 2023; Osman et al., 2023). An tantance aikin enzymatic peroxidase (POX) na Guaiacol-dependent (POX) ta amfani da hanyar Harrach et al. (2009). (2008) tare da ƙananan gyare-gyare (El-Nagar et al., 2023; Osman et al., 2023) da kuma aikin enzymatic na polyphenol oxidase (PPO) an tantance shi bayan an yi maganin tare da 2.2 ml na 100 mM sodium phosphate buffer (pH 6.0), 100 μl na guaiacol (TCI chemicals, Portland, OR, USA) da 100 μl na 12 mM H2O2. An ɗan gyara hanyar daga (El-Nagar et al., 2023; Osman et al., 2023). An yi gwajin bayan an yi maganin tare da 3 ml na maganin catechol (Thermo Scientific Chemicals, Waltham, MA, Amurka) (0.01 M) wanda aka shirya sabo a cikin 0.1 M phosphate buffer (pH 6.0). An auna aikin CAT ta hanyar sa ido kan rugujewar H2O2 a 240 nm (A240), an auna aikin POX ta hanyar sa ido kan karuwar sha a 436 nm (A436), kuma an auna aikin PPO ta hanyar yin rikodin canjin sha a 495 nm (A495) kowace daƙiƙa 30 na tsawon minti 3 ta amfani da na'urar auna UV-160A (Shimadzu, Japan).
An yi amfani da RT-PCR na ainihin lokaci don gano matakan tantance kwayoyin halitta guda uku masu alaƙa da antioxidant, waɗanda suka haɗa da peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), da glutathione reductase (PvGR; GenBank Accession No. KY195009.1), a cikin ganyen wake (ganye na biyu da na uku da aka haɓaka gaba ɗaya daga sama) awanni 72 bayan magani na ƙarshe. A takaice, an ware RNA ta amfani da Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, China) bisa ga ka'idar masana'anta. Sannan, an haɗa cDNA ta amfani da TOP script™ cDNA Synthesis Kit bisa ga umarnin masana'anta. Jerin farko na kwayoyin halitta guda uku da ke sama an jera su a cikin Tebur na Ƙarin S3. An yi amfani da PvActin-3 (lambar shiga GenBank: XM_068616709.1) a matsayin kwayar halittar kula da gida kuma an ƙididdige yanayin kwayar halittar da ke da alaƙa ta amfani da hanyar 2-ΔΔCT (Livak da Schmittgen, 2001). An nuna kwanciyar hankali na Actin a ƙarƙashin damuwa ta biotic (mu'amala mara jituwa tsakanin wake da naman gwari na anthracnose Colletotrichum lindemuthianum) da damuwa ta abiotic (fari, gishiri, ƙarancin zafin jiki) (Borges et al., 2012).
Da farko mun yi nazarin silico mai faɗi na furotin oxaloacetate acetylhydrolase (OAH) a cikin S. sclerotiorum ta amfani da kayan aikin BLAST-protein BLAST (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). A takaice, mun yi amfani da OAH daga Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; lambar shiga GenBank XP_040799428.1; amino acid 342) da Penicillium lagena (PlOAH; taxide: 94218; lambar shiga GenBank XP_056833920.1; amino acid 316) a matsayin jerin tambayoyi don taswirar furotin mai kama da juna a cikin S. sclerotiorum (taxide: 5180). An yi gwajin BLASTp akan bayanan kwayoyin halittar S. sclerotiorum da aka samu kwanan nan a GenBank a gidan yanar gizon Cibiyar Bayar da Bayanai kan Fasahar Halittu ta Ƙasa (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Bugu da ƙari, an yi hasashen kwayar halittar OAH da aka annabta daga S. sclerotiorum (SsOAH) da kuma nazarin juyin halitta da kuma bishiyar phylogenetic ta AfOAH daga A. fijiensis CBS 313.89 da PlOAH daga P. lagena ta amfani da hanyar mafi girman yuwuwar a cikin MEGA11 (Tamura et al., 2021) da kuma samfurin JTT matrix (Jones et al., 1992). An haɗa bishiyar phylogenetic tare da nazarin daidaitawa da yawa na jerin furotin na duk kwayoyin halittar OAH da aka annabta (SsOAH) daga S. sclerotiorum da jerin tambayoyi ta amfani da Kayan Aikin Daidaita Daidaito (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos da Agarwala, 2007). Bugu da ƙari, mafi kyawun jerin amino acid masu dacewa na SsOAH daga S. sclerotiorum an daidaita su da jerin tambayoyi (AfOAH da PlOAH) (Larkin et al., 2007) ta amfani da ClustalW (http://www.genome.jp/tools-bin/clustalw), kuma an hango yankunan da aka kiyaye a cikin jeri ta amfani da kayan aikin ESPript (sigar 3.0; https://espript.ibcp.fr/ESPript/ESPript/index.php).
Bugu da ƙari, an rarraba yankunan wakilcin aiki da aka annabta da wuraren da aka kiyaye na S. sclerotiorum SsOAH zuwa iyalai daban-daban ta amfani da kayan aikin InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). A ƙarshe, an yi ƙirar tsarin girma uku (3D) na S. sclerotiorum SsOAH da aka annabta ta amfani da 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) kuma an tabbatar da su ta amfani da sabar SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). An yi amfani da fakitin UCSF-Chimera (sigar 1.15; https://www.cgl.ucsf.edu/chimera/) wajen nuna tsarin da aka annabta mai girma uku (tsarin PDB).
An yi amfani da PCR mai haske na ainihin lokaci don tantance matakin rubutun oxaloacetate acetylhydrolase (SsOAH; lambar shiga GenBank: XM_001590428.1) a cikin mycelia na Sclerotinia sclerotiorum. A takaice, an yi wa S. sclerotiorum allura a cikin kwalba mai ɗauke da PDB kuma aka sanya shi a cikin incubator mai girgiza (samfurin: I2400, New Brunswick Scientific Co., Edison, NJ, Amurka) a 25 ± 2 °C na tsawon awanni 24 a 150 rpm da kuma cikin duhu mai ɗorewa (awanni 24) don haɓaka haɓakar mycelium. Bayan haka, an yi wa ƙwayoyin halittar magani da L-ornithine da kuma maganin kashe ƙwayoyin cuta Rizolex-T a matakin ƙarshe na IC50 (kimanin 40 da 3.2 mg/L, bi da bi) sannan aka noma su na tsawon wasu awanni 24 a ƙarƙashin irin wannan yanayi. Bayan an dasa shi, an yi amfani da shuke-shuken a cikin 2500 rpm na tsawon mintuna 5 sannan aka tattara saman (mycelium na fungal) don nazarin bayyanar kwayoyin halitta. Hakazalika, an tattara mycelium na fungal a awanni 0, 24, 48, 72, 96, da 120 bayan kamuwa da cuta daga tsire-tsire masu kamuwa da cuta waɗanda suka samar da farin mold da mycelium na auduga a saman kyallen da suka kamu da cutar. An cire RNA daga mycelium na fungal sannan aka haɗa cDNA kamar yadda aka bayyana a sama. An jera jerin farko na SsOAH a cikin Tebur na Ƙarin S3. An yi amfani da SsActin (lambar shiga GenBank: XM_001589919.1) a matsayin kwayar halittar kula da gida, kuma an ƙididdige bayyanar kwayar halitta ta hanyar amfani da hanyar 2-ΔΔCT (Livak da Schmittgen, 2001).
An gano sinadarin Oxalic acid a cikin ruwan dankalin turawa (PDB) da samfuran shuka waɗanda ke ɗauke da cutar fungal Sclerotinia sclerotiorum bisa ga hanyar Xu da Zhang (2000) tare da ɗan gyare-gyare kaɗan. A taƙaice, an yi wa S. sclerotiorum isolates allura a cikin kwalba da ke ɗauke da PDB sannan aka shuka su a cikin injin girgiza (samfurin I2400, New Brunswick Scientific Co., Edison, NJ, Amurka) a 150 rpm a 25 ± 2 °C na tsawon kwanaki 3-5 a cikin duhu mai ɗorewa (awanni 24) don haɓaka haɓakar mycelium. Bayan haɗuwa, an fara tace al'adar fungal ta hanyar takarda tace Whatman #1 sannan aka sanya centrifuge a 2500 rpm na tsawon mintuna 5 don cire sauran mycelium. An tattara ruwan kuma an adana shi a 4°C don ƙarin tantance adadin oxalate. Don shirya samfuran shuka, an cire kusan 0.1 g na gutsuttsuran ƙwayoyin shuka sau uku da ruwan da aka tace (2 ml kowane lokaci). An sanya samfuran a cikin injin centrifuge a 2500 rpm na tsawon mintuna 5, an tace ruwan da ke cikinsa ta hanyar tace takarda ta Whatman No. 1 sannan aka tattara su don ƙarin bincike.
Don yin nazarin adadi na oxalic acid, an shirya cakudawar amsawar a cikin bututun gilashi mai rufewa kamar haka: 0.2 ml na samfurin (ko PDB culture filtrate ko oxalic acid standard solution), 0.11 ml na bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, Amurka), 0.198 ml na 1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Supplies, Cairo, Egypt) da 0.176 ml na 100 mM potassium dichromate (K2Cr2O7; TCI chemicals, Portland, OR, USA), sannan aka narkar da maganin zuwa 4.8 ml da ruwan da aka tace, aka gauraya sosai sannan aka sanya shi nan da nan a cikin ruwan wanka mai zafin digiri 60. Bayan mintuna 10, an dakatar da amsawar ta hanyar ƙara 0.5 ml na sodium hydroxide solution (NaOH; 0.75 M). An auna shan ruwa (A600) na cakuda amsawar a 600 nm ta amfani da na'urar auna haske ta UV-160 (Shimadzu Corporation, Japan). An yi amfani da PDB da ruwan da aka tace a matsayin iko don auna yawan tacewar al'adu da samfuran shuka, bi da bi. Yawan sinadarin oxalic a cikin tacewar al'adu, wanda aka bayyana a matsayin micrograms na oxalic acid a kowace milliliter na matsakaicin PDB (μg.mL−1), da kuma a cikin cirewar ganye, wanda aka bayyana a matsayin micrograms na oxalic acid a kowace gram na sabon nauyi (μg.g−1 FW), an tantance ta ta amfani da lanƙwasa na daidaita acid oxalic (Thermo Fisher Scientific Chemicals, Waltham, MA, Amurka).
A cikin binciken, an tsara dukkan gwaje-gwajen a cikin tsari mai tsari gaba ɗaya (CRD) tare da kwafi shida na halitta a kowace magani da tukwane biyar a kowace kwafi na halitta (tsirrai biyu a kowace tukunya) sai dai idan an faɗi akasin haka. An yi nazarin kwafi na halitta a cikin kwafi (kwafi biyu na fasaha). An yi amfani da kwafi na fasaha don duba yuwuwar sake haifuwa na wannan gwajin amma ba a yi amfani da su a cikin binciken ƙididdiga don guje wa kwafi na ƙarya ba. An yi nazarin bayanai ta hanyar nazarin bambance-bambance (ANOVA) sannan aka biyo baya gwajin bambanci mai mahimmanci (HSD) na Tukey-Kramer (p ≤ 0.05). Don gwaje-gwajen in vitro, an ƙididdige ƙimar IC50 da IC99 ta amfani da samfurin probit kuma an ƙididdige tazara na amincewa 95%.
An tattara jimillar ƙwayoyin cuta guda huɗu daga gonakin waken soya daban-daban a cikin El Ghabiya, Masar. A kan hanyar PDA, duk ƙwayoyin cuta suna samar da farin mycelium mai kauri wanda ya yi sauri ya zama fari mai kama da auduga (Hoto na 1A) sannan kuma ya zama launin ruwan kasa ko launin ruwan kasa a matakin sclerotium. Sclerotia yawanci suna da kauri, baƙi, mai siffar zagaye ko mara tsari, tsawon mm 5.2 zuwa 7.7 da diamita na mm 3.4 zuwa 5.3 (Hoto na 1B). Duk da cewa ƙwayoyin cuta guda huɗu sun sami tsarin sclerotia a gefen hanyar al'ada bayan kwanaki 10-12 na lokacin da aka yi amfani da su a zafin 25 ± 2 °C (Hoto na 1A), adadin ƙwayoyin cuta a kowace faranti ya bambanta sosai a tsakaninsu (P < 0.001), tare da ƙwayoyin cuta guda 3 suna da mafi girman adadin ƙwayoyin cuta (32.33 ± 1.53 sclerotia a kowace faranti; Hoto na 1C). Hakazalika, isolate #3 ya samar da ƙarin sinadarin oxalic acid a cikin PDB fiye da sauran isolates (3.33 ± 0.49 μg.mL−1; Hoto na 1D). Isolate #3 ya nuna halaye na yau da kullun na siffar jiki da ƙananan ƙwayoyin cuta na naman gwari na Sclerotinia sclerotiorum. Misali, akan PDA, ƙwayoyin isolate #3 sun girma da sauri, sun kasance fari mai tsami (Hoto na 1A), beige ko launin ruwan kasa mai haske na salmon, kuma sun buƙaci kwanaki 6-7 a zafin 25 ± 2°C don rufe saman farantin diamita na santimita 9 gaba ɗaya. Dangane da halayen siffar jiki da ƙananan ƙwayoyin cuta da ke sama, an gano isolate #3 a matsayin Sclerotinia sclerotiorum.
Hoto na 1. Halaye da kuma illolin da ke tattare da ƙwayoyin cuta na S. sclerotiorum daga amfanin gona na legume. (A) Girman ƙwayoyin cuta na S. sclerotiorum guda huɗu a kan matsakaiciyar PDA, (B) sclerotia na ƙwayoyin cuta na S. sclerotiorum guda huɗu, (C) adadin ƙwayoyin cuta na S (kowace faranti), (D) fitar da sinadarin oxalic acid akan matsakaiciyar PDB (μg.mL−1), da kuma (E) tsananin cutar (%) na ƙwayoyin cuta na S. sclerotiorum guda huɗu a kan nau'in legume na kasuwanci mai saurin kamuwa da cutar Giza 3 a ƙarƙashin yanayin greenhouse. Ƙimar tana wakiltar matsakaicin ± SD na kwafi biyar na halitta (n = 5). Haruffa daban-daban suna nuna bambance-bambance masu mahimmanci tsakanin jiyya (p < 0.05). (F–H) Alamomin fararen mold na yau da kullun sun bayyana a kan tushe da siliques na sama, bi da bi, kwanaki 10 bayan allurar rigakafi tare da isolate #3 (dpi). (I) An yi nazarin juyin halitta na yankin spacer na ciki (ITS) na S. sclerotiorum isolate #3 ta amfani da hanyar mafi girman yuwuwar kuma an kwatanta shi da nau'ikan da aka samo daga bayanan Cibiyar Bayar da Bayanai kan Fasahar Halittu ta Ƙasa (NCBI) (https://www.ncbi.nlm.nih.gov/). Lambobin da ke sama da layukan tarawa suna nuna yankin da aka rufe (%), kuma lambobin da ke ƙasa da layukan tarawa suna nuna tsawon reshen.
Bugu da ƙari, don tabbatar da cutar, an yi amfani da ƙwayoyin cuta guda huɗu da aka samu daga S. sclerotiorum don yin allurar rigakafin wake na kasuwanci mai saurin kamuwa da cutar Giza 3 a ƙarƙashin yanayin kore, wanda ya yi daidai da ka'idojin Koch (Hoto na 1E). Duk da cewa duk ƙwayoyin cuta na fungal da aka samu suna da illa kuma suna iya kamuwa da wake kore (cv. Giza 3), suna haifar da alamun farin mold na yau da kullun a duk sassan da ke sama da ƙasa (Hoto na 1F), musamman a kan tushe (Hoto na 1G) da kwasfa (Hoto na 1H) a cikin kwanaki 10 bayan allurar (dpi), isolate 3 shine mafi tsananin cutar a cikin gwaje-gwaje guda biyu masu zaman kansu. Isolate 3 yana da mafi girman tsananin cutar (%) akan tsire-tsire na wake (24.0 ± 4.0, 58.0 ± 2.0, da 76.7 ± 3.1 a cikin kwanaki 7, 14, da 21 bayan kamuwa da cuta, bi da bi; Hoto na 1F).
An ƙara tabbatar da gano mafi girman S. sclerotiorum isolate #3 bisa ga jerin abubuwan da aka rubuta a ciki (ITS) (Hoto na 1I). Binciken phylogenetic tsakanin isolate #3 da nau'ikan da aka ambata 20 ya nuna babban kamanceceniya (>99%) a tsakaninsu. Ya kamata a lura cewa isolate S. sclerotiorum isolate #3 (533 bp) yana da babban kamanceceniya da isolate S. sclerotiorum isolate LPM36 na Amurka da aka ware daga busassun tsaba na wake (lambar shiga GenBank MK896659.1; 540 bp) da kuma isolate S. sclerotiorum na China YKY211 (lambar shiga GenBank OR206374.1; 548 bp), wanda ke haifar da ruɓewar tushen violet (Matthiola incana), waɗanda duk an haɗa su daban-daban a saman dendrogram (Hoto na 1I). An ajiye sabon jerin a cikin bayanan NCBI kuma an sanya masa suna "Sclerotinia sclerotiorum - isolate YN-25" (lambar shiga GenBank PV202792). Za a iya ganin cewa isolate 3 shine mafi girman keɓewa; saboda haka, an zaɓi wannan keɓewa don nazari a duk gwaje-gwajen da suka biyo baya.
An binciki aikin maganin kashe ƙwayoyin cuta na diamine L-ornithine (Sigma-Aldrich, Darmstadt, Jamus) a cikin adadi daban-daban (12.5, 25, 50, 75, 100 da 125 mg/L) akan S. sclerotiorum isolate 3 a cikin vitro. Abin lura ne cewa L-ornithine yana da tasirin kashe ƙwayoyin cuta kuma a hankali yana hana ci gaban radial na S. sclerotiorum hyphae ta hanyar da ta dogara da kashi (Hoto na 2A, B). A mafi girman yawan da aka gwada (125 mg/L), L-ornithine ya nuna mafi girman adadin hana ci gaban mycelium (99.62 ± 0.27%; Hoto na 2B), wanda yayi daidai da Rizolex-T na maganin kashe ƙwayoyin cuta na kasuwanci (ƙimar hanawa 99.45 ± 0.39%; Hoto na 2C) a mafi girman yawan da aka gwada (10 mg/L), yana nuna irin wannan tasiri.
Hoto na 2. Ayyukan ƙwayoyin cuta na L-ornithine a cikin vitro akan Sclerotinia sclerotiorum. (A) Kwatanta ayyukan ƙwayoyin cuta na yawan L-ornithine daban-daban akan S. sclerotiorum tare da maganin kashe ƙwayoyin cuta na kasuwanci Rizolex-T (10 mg/L). (B, C) Yawan hanawa (%) na girman S. sclerotiorum mycelium bayan magani tare da yawan L-ornithine daban-daban (12.5, 25, 50, 75, 100 da 125 mg/L) ko Rizolex-T (2, 4, 6, 8 da 10 mg/L), bi da bi. Ƙimar tana wakiltar matsakaicin ± SD na kwafi biyar na halittu (n = 5). Haruffa daban-daban suna nuna bambance-bambancen ƙididdiga tsakanin jiyya (p < 0.05). (D, E) Binciken koma-baya na samfurin Probit na L-ornithine da maganin kashe ƙwayoyin cuta na kasuwanci Rizolex-T, bi da bi. An nuna layin komawa ga tsarin probit a matsayin layin shuɗi mai ƙarfi, kuma an nuna tazara ta amincewa (95%) a matsayin layin ja mai lanƙwasa.
Bugu da ƙari, an yi nazarin koma-baya na probit kuma an nuna zane-zane masu dacewa a cikin Jadawali na 1 da Hotuna na 2D,E. A taƙaice, ƙimar gangara mai karɓuwa (y = 2.92x − 4.67) da ƙididdiga masu mahimmanci masu alaƙa (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 da p < 0.0001; Hoto na 2D) na L-ornithine ya nuna ingantaccen aikin antifungal akan S. sclerotiorum idan aka kwatanta da maganin kashe ƙwayoyin cuta na kasuwanci Rizolex-T (y = 1.96x − 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 da p < 0.0001) (Tebur na 1).
Tebur 1. Ƙimar rabin ƙarfin hanawa (IC50) da IC99 (mg/l) na L-ornithine da maganin kashe ƙwayoyin cuta na kasuwanci "Rizolex-T" akan S. sclerotiorum.
Gabaɗaya, L-ornithine (250 mg/L) ya rage ci gaba da tsananin farin mold a kan tsire-tsire na wake da aka yi wa magani idan aka kwatanta da tsire-tsire masu kamuwa da cutar S. sclerotiorum (ikon sarrafawa; Hoto na 3A). A taƙaice, kodayake tsananin cutar na tsire-tsire masu kamuwa da cutar da ba a yi musu magani ba ya ƙaru a hankali (52.67 ± 1.53, 83.21 ± 2.61, da 92.33 ± 3.06%), L-ornithine ya rage tsananin cutar (%) sosai a duk lokacin gwajin (8.97 ± 0.15, 18.00 ± 1.00, da 26.36 ± 3.07) a kwanaki 7, 14, da 21 bayan magani (dpt), bi da bi (Hoto na 3A). Hakazalika, lokacin da aka yi wa shuke-shuken wake da S. sclerotiorum ya kamu da cutar magani da 250 mg/L L-ornithine, yankin da ke ƙarƙashin lanƙwasa ci gaban cutar (AUDPC) ya ragu daga 1274.33 ± 33.13 a cikin maganin da ba a yi masa magani ba zuwa 281.03 ± 7.95, wanda ya ɗan yi ƙasa da na maganin kashe ƙwayoyin cuta na Rizolex-T mai inganci 50 mg/L (183.61 ± 7.71; Hoto na 3B). An lura da irin wannan yanayin a gwajin na biyu.
Hoto na 3. Tasirin amfani da L-ornithine na waje akan ci gaban farin ruɓewar wake na yau da kullun wanda Sclerotinia sclerotiorum ke haifarwa a ƙarƙashin yanayin greenhouse. (A) Lanƙwasa ci gaban cuta na farin mold na wake na yau da kullun bayan an yi masa magani da 250 mg/L L-ornithine. (B) Yankin da ke ƙarƙashin lanƙwasa ci gaban cutar (AUDPC) na farin mold na wake na yau da kullun bayan an yi masa magani da L-ornithine. Ƙimar tana wakiltar matsakaicin ± SD na kwafi biyar na halitta (n = 5). Haruffa daban-daban suna nuna bambance-bambance masu mahimmanci tsakanin magunguna (p < 0.05).
Shafawa ta waje ta 250 mg/L L-ornithine a hankali ta ƙara tsayin shuka a hankali (Hoto na 4A), adadin rassan kowace shuka (Hoto na 4B), da adadin ganyen kowace shuka (Hoto na 4C) bayan kwana 42. Duk da cewa maganin kashe kwari na kasuwanci na Rizolex-T (50 mg/L) yana da mafi girman tasiri akan duk sigogin abinci mai gina jiki da aka yi nazari a kansu, shawa ta waje ta 250 mg/L L-ornithine tana da mafi girman tasiri na biyu idan aka kwatanta da magungunan da ba a yi musu magani ba (Hoto na 4A–C). A gefe guda kuma, maganin L-ornithine bai yi wani tasiri mai mahimmanci ba kan abubuwan da ke cikin pigments na photosynthetic chlorophyll a (Hoto na 4D) da chlorophyll b (Hoto na 4E), amma ya ɗan ƙara yawan carotenoid (0.56 ± 0.03 mg/g fr wt) idan aka kwatanta da rashin kulawar da ba ta dace ba (0.44 ± 0.02 mg/g fr wt) da kuma tabbataccen iko (0.46 ± 0.02 mg/g fr wt; Hoto na 4F). Gabaɗaya, waɗannan sakamakon sun nuna cewa L-ornithine ba shi da guba ga wake da aka yi wa magani kuma yana iya ƙarfafa ci gaban su.
Hoto na 4. Tasirin amfani da L-ornithine na waje akan halayen girma da kuma launukan photosynthetic na ganyen wake da suka kamu da cutar Sclerotinia sclerotiorum a ƙarƙashin yanayin greenhouse. (A) Tsawon shuka (cm), (B) Adadin rassan kowace shuka, (C) Adadin ganye a kowace shuka, (D) Chlorophyll a abun ciki (mg g-1 fr wt), (E) Chlorophyll b abun ciki (mg g-1 fr wt), (F) Jimlar abun ciki na carotenoid (mg g-1 fr wt). Ƙimar su ne matsakaicin ± SD na kwafi biyar na halitta (n = 5). Haruffa daban-daban suna nuna bambance-bambance masu mahimmanci tsakanin jiyya (p < 0.05).
A cikin yanayin da ake ciki, an gano nau'ikan iskar oxygen masu amsawa (ROS; an bayyana su a matsayin hydrogen peroxide [H2O2]) da kuma free radicals (wanda aka bayyana a matsayin superoxide anions [O2•−]) cewa amfani da L-ornithine (250 mg/L) a waje ya rage yawan tarin H2O2 (96.05 ± 5.33 nmol.g−1 FW; Hoto na 5A) da O2•− (32.69 ± 8.56 nmol.g−1 FW; Hoto na 5B) idan aka kwatanta da tarin tsire-tsire biyu da suka kamu da cutar (173.31 ± 12.06 da 149.35 ± 7.94 nmol.g−1 FW, bi da bi) da kuma tsire-tsire da aka yi wa magani da 50 mg/L na maganin kashe ƙwayoyin cuta na kasuwanci Rizolex-T (170.12 ± 9.50 da 157.00 ± 7.81 nmol.g−1 fr wt, bi da bi) a cikin awanni 72. Yawan H2O2 da O2•− da aka tara a ƙarƙashin hpt (Hoto na 5A, B). Hakazalika, gwajin malondialdehyde (MDA) da aka yi a TCA ya nuna cewa tsire-tsire masu kamuwa da wake da S. sclerotiorum suka kamu da su sun tara matakan MDA mafi girma (113.48 ± 10.02 nmol.g fr wt) a cikin ganyayyakinsu (Hoto na 5C). Duk da haka, amfani da L-ornithine na waje ya rage yawan lipid peroxidation kamar yadda aka nuna ta hanyar raguwar abun ciki na MDA a cikin tsire-tsire da aka yi wa magani (33.08 ± 4.00 nmol.g fr wt).
Hoto na 5. Tasirin amfani da L-ornithine na waje akan manyan alamomin damuwa na oxidative da hanyoyin kariya daga antioxidant marasa enzymatic a cikin ganyen wake da suka kamu da S. sclerotiorum a cikin awanni 72 bayan kamuwa da cuta a ƙarƙashin yanayin greenhouse. (A) Hydrogen peroxide (H2O2; nmol g−1 FW) a 72 hpt, (B) anion superoxide (O2•−; nmol g−1 FW) a 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) a 72 hpt, (D) jimlar phenols masu narkewa (mg GAE g−1 FW) a 72 hpt, (E) jimlar flavonoids masu narkewa (mg RE g−1 FW) a 72 hpt, (F) jimlar amino acid kyauta (mg g−1 FW) a 72 hpt, da (G) yawan proline (mg g−1 FW) a 72 hpt. Ƙima suna wakiltar matsakaicin ± karkacewar daidaito (matsakaicin ± SD) na kwafi 5 na halittu (n = 5). Haruffa daban-daban suna nuna bambance-bambance masu mahimmanci a kididdiga tsakanin jiyya (p < 0.05).