Maqaalku waa qayb ka mid ah mowduuca cilmi-baarista "Hagaajinta iska caabbinta digirta ee cudurada iyo cayayaanka", fiiri dhammaan 5ta maqaal
Wakiilka sababa cudurka fangaska necrosis Sclerotinia sclerotiorum (Lib.) de Bary wuxuu adeegsadaa xeelado badan oo heer sare ah si uu u qaadsiiyo dhirta martida loo yahay ee kala duwan. Daraasaddani waxay soo jeedinaysaa isticmaalka diamine L-ornithine, oo ah amino acid aan borotiin ahayn oo kiciya isku-darka amino acids-ka kale ee muhiimka ah, sida istaraatiijiyad maamul oo kale si loo xoojiyo jawaabaha molecular, physiological iyo biochemical ee Phaseolus vulgaris L. ee caaryada cad ee uu keeno Pseudomonas sclerotiorum. Tijaabooyinka In vitro waxay muujiyeen in L-ornithine si weyn u joojisay koritaanka mycelial ee S. pyrenoidosa si ku xiran qiyaasta. Intaa waxaa dheer, waxay si weyn u yareyn kartaa darnaanta caaryada cad ee xaaladaha aqalka dhirta lagu koriyo. Intaa waxaa dheer, L-ornithine waxay kicisay koritaanka dhirta la daaweeyay, taasoo muujinaysa in fiirsashada la tijaabiyay ee L-ornithine aysan ahayn sun ku ah dhirta la daaweeyay. Intaa waxaa dheer, L-ornithine waxay xoojisay muujinta antioxidants-ka aan enzymatic ahayn (phenolics-ka milma iyo flavonoids) iyo antioxidants-ka enzymatic (catalase (CAT), peroxidase (POX), iyo polyphenol oxidase (PPO)), waxayna kordhisay muujinta saddex hiddo-side oo la xiriira antioxidant-ka (PvCAT1, PvSOD, iyo PvGR). Intaa waxaa dheer, falanqaynta silico waxay muujisay jiritaanka borotiin oxaloacetate acetylhydrolase (SsOAH) oo ku jira hiddo-wadaha S. sclerotiorum, kaas oo aad ugu ekaa borotiinnada oxaloacetate acetylhydrolase (SsOAH) ee Aspergillus fijiensis (AfOAH) iyo Penicillium sp. (PlOAH) marka loo eego falanqaynta shaqada, qaybaha la ilaaliyo, iyo topology-ga. Waxaa xiiso leh, ku darista L-ornithine ee maraqa baradhada dextrose (PDB) ayaa si weyn hoos ugu dhigtay muujinta hidda-wadaha SsOAH ee mycelia S. sclerotiorum. Sidoo kale, codsiga dibadda ee L-ornithine wuxuu si weyn hoos ugu dhigay muujinta hidda-wadaha SsOAH ee mycelia fangaska laga soo ururiyey dhirta la daweeyay. Ugu dambeyntii, codsiga L-ornithine wuxuu si weyn hoos ugu dhigay dheecaanka aashitada oxalic ee caleenta dhexe ee PDB iyo kuwa cudurka qaba labadaba. Gunaanadkii, L-ornithine waxay door muhiim ah ka ciyaartaa ilaalinta xaaladda redox iyo sidoo kale kor u qaadida jawaabta difaaca ee dhirta cudurka qabta. Natiijooyinka daraasaddan waxay gacan ka geysan karaan horumarinta habab cusub oo deegaanka u fiican si loo xakameeyo caaryada cad loona yareeyo saameynta ay ku leedahay wax soo saarka digirta iyo dalagyada kale.
Caariyaysi cad, oo ay keento fangaska necrotrophic Sclerotinia sclerotiorum (Lib.) de Bary, waa cudur ba'an oo yareeya wax soo saarka kaas oo khatar weyn ku ah wax soo saarka digirta caalamiga ah (Phaseolus vulgaris L.) (Bolton et al., 2006). Sclerotinia sclerotiorum waa mid ka mid ah jeermiska dhirta fangaska ee ugu adag in la xakameeyo, iyadoo leh tiro ballaaran oo ka badan 600 oo nooc oo dhir ah iyo awoodda ay si dhakhso ah ugu xoqdo unugyada martida loo yahay si aan gaar ahayn (Liang iyo Rollins, 2018). Xaaladaha aan fiicnayn, waxay martaa marxalad muhiim ah oo wareeggeeda nololeed ah, iyadoo ku sii nasanaysa muddo dheer qaab-dhismeedyo madow, adag, oo u eg iniin oo loo yaqaan 'sclerotia' ciidda ama sida koritaan cad, jilicsan oo ku jira mycelium ama jirida geedaha cudurka qaba (Schwartz et al., 2005). S. sclerotiorum wuxuu awood u leeyahay inuu sameeyo sclerotia, taasoo u oggolaanaysa inuu ku noolaado beeraha cudurka qaba muddo dheer iyo inuu sii jiro inta uu cudurku socdo (Schwartz et al., 2005). Sclerotia waxay qani ku tahay nafaqooyinka, waxay ku sii jiri kartaa ciidda muddo dheer, waxayna u adeegtaa sidii tallaalka ugu horreeya ee caabuqyada soo socda (Schwartz et al., 2005). Xaaladaha wanaagsan, sclerotia waxay soo baxdaa oo soo saartaa spores hawada ku jira oo ku dhici kara dhammaan qaybaha dhulka ka sarreeya ee geedka, oo ay ku jiraan laakiin aan ku xaddidnayn ubaxyada, jirridaha, ama ubaxyada (Schwartz et al., 2005).
Sclerotinia sclerotiorum waxay isticmaashaa xeelado badan oo heer sare ah si ay u qaadsiiso dhirta ay martida u tahay, taasoo ku lug leh taxane dhacdooyin isku xiran laga bilaabo biqilka sclerotial ilaa horumarinta calaamadaha. Bilowgii, S. sclerotiorum waxay soo saartaa unugyo la hakiyey (oo loo yaqaan ascospores) oo ka yimaada qaab-dhismeedka u eg boqoshaada oo loo yaqaan apothecia, kuwaas oo noqda kuwo hawada ku jira oo isu beddela sclerotia aan dhaqdhaqaaq lahayn oo ku dhaca haraaga dhirta ee cudurka qaba (Bolton et al., 2006). Fangaska ayaa markaa soo daaya oxalic acid, oo ah arrin fayras, si loo xakameeyo pH-ga derbiga unugyada dhirta, loo dhiirrigeliyo burburka enzymatic iyo duullaanka unugyada (Hegedus iyo Rimmer, 2005), loona xakameeyo burburka oksaydhka ee geedka martida loo yahay. Habkan aashitada ayaa daciifiya derbiga unugyada dhirta, isagoo siinaya jawi ku habboon hawlgalka caadiga ah iyo hufnaanta ee enzymes-ka burburiya derbiga unugyada fungal (CWDEs), taasoo u oggolaanaysa cudur-sidaha inuu ka gudbo caqabadda jireed oo uu dhex galo unugyada martida loo yahay (Marciano et al., 1983). Marka la geliyo, S. sclerotiorum wuxuu soo saaraa tiro ka mid ah CWDE-yada, sida polygalacturonase iyo cellulase, kuwaas oo fududeeya faafitaankooda unugyada cudurka qaba oo sababa necrosis unugyada. Horumarka nabarrada iyo gogosha hyphal waxay horseedaa astaamaha astaamaha caaryada cad (Hegedus iyo Rimmer, 2005). Dhanka kale, dhirta martida loo yahay waxay aqoonsadaan qaababka molecular-ka ee la xiriira pathogen-ka (PAMPs) iyada oo loo marayo soo-dhoweeyayaasha aqoonsiga qaabka (PRRs), taasoo kicinaysa taxane dhacdooyin calaamadayn ah oo ugu dambeyntii dhaqaajiya jawaabaha difaaca.
Iyadoo tobanaan sano ay socdeen dadaallo lagu xakameynayo cudurrada, yaraanta jeermiska u adkaysta ee ku filan ayaa weli ku jirta digirta, sida dalagyada kale ee ganacsiga, sababtoo ah iska caabbinta, badbaadada, iyo la qabsiga cudurka. Sidaa darteed, maaraynta cudurku waa mid aad u adag waxayna u baahan tahay istiraatiijiyad isku dhafan oo dhinacyo badan leh oo ay ku jiraan isku-darka dhaqamada, xakamaynta bayoolojiga, iyo kiimikooyinka fungicides (O'Sullivan et al., 2021). Xakamaynta kiimikada ee caaryada cad ayaa ah tan ugu waxtarka badan sababtoo ah fungicides-ka, marka si sax ah iyo waqtiga saxda ah loo isticmaalo, waxay si wax ku ool ah u xakamayn karaan faafitaanka cudurka, waxay yareyn karaan darnaanta caabuqa, waxayna yareyn karaan khasaaraha wax soo saarka. Si kastaba ha ahaatee, isticmaalka xad-dhaafka ah iyo ku tiirsanaanta badan ee fungicides-ka waxay horseedi kartaa soo ifbaxa noocyada u adkaysta ee S. sclerotiorum waxayna si xun u saameeyaan noolaha aan bartilmaameedsanayn, caafimaadka carrada, iyo tayada biyaha (Le Cointe et al., 2016; Ceresini et al., 2024). Sidaa darteed, helitaanka beddelaad deegaan u fiican ayaa noqotay mudnaanta koowaad.
Polyamines (PAs), sida putrescine, spermidine, spermine, iyo cadaverine, waxay u adeegi karaan beddel rajo leh oo ka dhan ah jeermiska dhirta ee carrada ku dhaca, taasoo si buuxda ama qayb ahaan u yareynaysa isticmaalka sunta kiimikada ee khatarta ah (Nehela et al., 2024; Yi et al., 2024). Dhirta sare, PAs waxay ku lug leeyihiin habab badan oo jireed oo ay ku jiraan, laakiin aan ku xaddidnayn, kala-qaybinta unugyada, kala-soocidda, iyo ka jawaabista walbahaarka abiotic iyo biotic (Killiny iyo Nehela, 2020). Waxay u dhaqmi karaan sidii antioxidants, waxay ka caawin karaan inay nadiifiyaan noocyada oksijiinta ee falcelinta leh (ROS), waxay ilaaliyaan homeostasis-ka redox (Nehela iyo Killiny, 2023), waxay kicin karaan hiddo-wadaha la xiriira difaaca (Romero et al., 2018), waxay nidaamiyaan waddooyin kala duwan oo dheef-shiid kiimikaad (Nehela iyo Killiny, 2023), waxay hagaajiyaan phytohormones-ka endogenous (Nehela iyo Killiny, 2019), waxay dhisaan iska caabin nidaamsan (SAR), waxayna nidaamiyaan isdhexgalka dhirta iyo cudur-sidaha (Nehela iyo Killiny, 2020; Asija et al., 2022; Czerwoniec, 2022). Waa in la ogaadaa in farsamooyinka gaarka ah iyo doorka PAs ee difaaca dhirta ay kala duwan yihiin iyadoo ku xiran noocyada dhirta, jeermiska, iyo xaaladaha deegaanka. PA-ga ugu badan ee dhirta waxaa laga soo saaraa polyamine L-ornithine ee lagama maarmaanka ah (Killiny iyo Nehela, 2020).
L-ornithine wuxuu doorar badan ka ciyaaraa koritaanka iyo horumarka dhirta. Tusaale ahaan, daraasado hore ayaa muujiyay in bariiska (Oryza sativa), ornithine laga yaabo inay la xiriirto dib u warshadaynta nitrogen (Liu et al., 2018), wax soo saarka bariiska, tayada iyo caraf udgoonka (Lu et al., 2020), iyo jawaabta cadaadiska biyaha (Yang et al., 2000). Intaa waxaa dheer, codsiga dibadda ee L-ornithine si weyn ayuu u xoojiyay dulqaadka abaarta ee xididdo sonkor ah (Beta vulgaris) (Hussein et al., 2019) iyo yareynta cadaadiska milixda ee basasha (Allium Cepa) (Çavuşoǧlu iyo Çavuşoǧlu, 2021) iyo cashew (Anacardium occidentale) (da Rocha et al., 2012). Doorka suurtagalka ah ee L-ornithine ee difaaca walbahaarka abiotic-ga waxaa laga yaabaa inay sabab u tahay ku lug lahaanshaha ururinta proline ee dhirta la daweeyay. Tusaale ahaan, hiddo-sidayaasha la xiriira dheef-shiid kiimikaadka proline, sida hiddo-sidayaasha ornithine delta aminotransferase (delta-OAT) iyo hiddo-sidayaasha proline dehydrogenase (ProDH1 iyo ProDH2), ayaa hore loo soo sheegay inay ku lug leeyihiin difaaca Nicotiana benthamiana iyo Arabidopsis thaliana oo ka dhan ah noocyada aan martida loo ahayn ee Pseudomonas syringae (Senthil-Kumar iyo Mysore, 2012). Dhanka kale, fungal ornithine decarboxylase (ODC) ayaa loo baahan yahay si loo kobciyo cudurka (Singh et al., 2020). Bartilmaameedka ODC ee Fusarium oxysporum f. sp. lycopersici iyada oo loo marayo aamusinta hidda-wadaha ee martida loo yahay (HIGS) si weyn ayuu u xoojiyay iska caabbinta dhirta yaanyada ee Fusarium wilt (Singh et al., 2020). Si kastaba ha ahaatee, doorka suurtagalka ah ee codsiga ornithine ee dibadda ka ah ee ka dhanka ah walbahaarka biotic sida phytopathogens si fiican looma baran. Tan ugu muhiimsan, saameynta ornithine ee ku leedahay iska caabbinta cudurrada iyo ifafaalaha kiimikada iyo fiisiyoolajiyadda ee la xiriira ayaa inta badan aan la sahamin.
Fahmidda kakanaanta caabuqa S. sclerotiorum ee digiraha ayaa muhiim u ah horumarinta istaraatiijiyado xakameyn oo wax ku ool ah. Daraasaddan, waxaan ku talagalnay inaan aqoonsanno doorka suurtagalka ah ee diamine L-ornithine oo ah qodob muhiim ah oo lagu xoojinayo hababka difaaca iyo iska caabbinta dhirta digiraha ee caabuqa Sclerotinia sclerotiorum. Waxaan qiyaaseynaa in, marka laga soo tago kor u qaadista jawaabaha difaaca ee dhirta cudurka qabta, L-ornithine sidoo kale ay door muhiim ah ka ciyaarto ilaalinta xaaladda redox. Waxaan soo jeedineynaa in saameynta suurtagalka ah ee L-ornithine ay la xiriirto nidaaminta hababka difaaca antioxidant-ka enzymatic iyo kuwa aan enzymatic ahayn iyo faragelinta arrimaha cudurada fungal/virulent iyo borotiinada la xiriira. Shaqadan laba-geesoodka ah ee L-ornithine waxay ka dhigaysaa musharax rajo leh oo loogu talagalay istaraatiijiyad waara si loo yareeyo saameynta caaryada cad iyo in la xoojiyo iska caabbinta dalagyada digiraha caadiga ah ee cudurkan fangaska ee awoodda leh. Natiijooyinka daraasaddan hadda jirta waxay gacan ka geysan kartaa horumarinta habab cusub oo deegaanka u fiican si loo xakameeyo caaryada cad loona yareeyo saameynta ay ku leedahay wax soo saarka digiraha.
Daraasaddan, noocyo ganacsi oo u nugul digir caadi ah, Giza 3 (Phaseolus vulgaris L. cv. Giza 3), ayaa loo isticmaalay qalab tijaabo ah. Abuur caafimaad qaba waxaa si naxariis leh u bixiyay Waaxda Cilmi-baarista Legume, Machadka Cilmi-baarista Beeraha (FCRI), Xarunta Cilmi-baarista Beeraha (ARC), Masar. Shan abuur ayaa lagu beeray dheriyo balaastig ah (dhexroor gudaha ah 35 cm, qoto dheer 50 cm) oo lagu buuxiyay carro uu ku dhacay S. sclerotiorum xaaladaha aqalka dhirta lagu koriyo (25 ± 2 °C, qoyaan qaraabo ah 75 ± 1%, 8 saacadood iftiin/16 saacadood mugdi ah). 7-10 maalmood ka dib beerista (DPS), geedo yaryar ayaa la khafiifiyay si ay uga tagaan laba geed oo keliya oo leh koritaan isku mid ah iyo saddex caleemo oo si buuxda u fidsan dheri kasta. Dhammaan dhirta dheriga ku jirta waxaa la waraabin jiray labadii toddobaadba mar waxaana la bacrimin jiray bishiiba iyadoo lagu salaynayo heerka lagu taliyay ee nooca la bixiyay.
Si loo diyaariyo 500 mg/L oo ah L-ornithinediamine (oo sidoo kale loo yaqaan (+)-(S)-2,5-diaminopentanoic acid; Sigma-Aldrich, Darmstadt, Jarmalka), 50 mg ayaa markii ugu horreysay lagu milmay 100 mL oo biyo nadiif ah oo la sifeeyay. Xalka kaydka ah ayaa markaa la qasi jiray oo loo isticmaalay tijaabooyinkii xigay. Si kooban, lix taxane oo ah fiirsashada L-ornithine (12.5, 25, 50, 75, 100, iyo 125 mg/L) ayaa lagu tijaabiyay in vitro. Intaa waxaa dheer, biyo nadiif ah oo la sifeeyay ayaa loo isticmaalay sidii xakameyn taban (Mock) iyo budada cayayaan dilaha ganacsiga "Rizolex-T" 50% oo ah budada qoyan (toclofos-methyl 20% + thiram 30%; KZ-Kafr El Zayat Pesticides and Chemicals Company, Kafr El Zayat, Gharbia Governorate, Masar) ayaa loo isticmaalay sidii xakameyn togan. Daawada cayayaan dilaha ee ganacsiga ee "Rizolex-T" waxaa lagu tijaabiyay in vitro shan fiirsasho (2, 4, 6, 8 iyo 10 mg/L).
Tusaalooyin ka mid ah jirridaha digirta caadiga ah iyo qolofta oo muujinaya astaamaha caadiga ah ee caaryada cad (heerka caabuqa: 10-30%) ayaa laga soo ururiyay beeraha ganacsiga. Inkasta oo inta badan walxaha dhirta ee cudurka qaba lagu aqoonsaday noocyada/noocyada kala duwan (noocyada ganacsiga ee u nugul Giza 3), kuwa kale, gaar ahaan kuwa laga helay suuqyada maxalliga ah, waxay ahaayeen noocyo aan la garanayn. Alaabada cudurka qaba ee la soo ururiyay waxaa marka hore lagu nadiifiyay dusha sare 0.5% sodium hypochlorite xal muddo 3 daqiiqo ah, ka dibna dhowr jeer lagu raaciyay biyo nadiif ah oo la safeeyey oo lagu tirtiray warqad nadiif ah si looga saaro biyaha xad-dhaafka ah. Xubnaha cudurka qaba ayaa markaa loo jarjaray qaybo yaryar oo ka yimid unugyada dhexe (inta u dhaxaysa unugyada caafimaadka qaba iyo kuwa cudurka qaba), oo lagu beeray baradhada agar dextrose (PDA) oo lagu dhex beeray heerkul ah 25 ± 2 °C oo leh wareeg mugdi ah oo 12 saacadood ah/12 saacadood ah muddo 5 maalmood ah si loo kiciyo sameynta sclerotia. Habka mycelial cap ayaa sidoo kale loo isticmaalay in lagu nadiifiyo fangaska laga soocay dhaqamada isku dhafan ama kuwa wasakhaysan. Kala soocidda fangaska ee la nadiifiyey ayaa markii ugu horreysay la aqoonsaday iyadoo lagu saleynayo astaamaha dhaqankeeda ka dibna la xaqiijiyay inay tahay S. sclerotiorum iyadoo lagu saleynayo astaamaha yar yar. Ugu dambeyntii, dhammaan kala soocidda la nadiifiyey waxaa lagu tijaabiyey cudurro ku dhaca nooca digirta caadiga ah ee Giza 3 si loo waafajiyo ra'yiga Koch.
Intaa waxaa dheer, S. sclerotiorum isolate (isolate #3) oo ah nooca ugu weerarka badan ayaa si dheeraad ah loo xaqiijiyay iyadoo lagu saleynayo taxanaha spacer-ka gudaha ee la duubay (ITS) sida ay ku qeexeen White et al., 1990; Baturo-Ciesniewska et al., 2017. Si kooban, isolates-ka waxaa lagu beeray maraq baradho dextrose ah (PDB) waxaana lagu kululeeyay heerkul ah 25 ± 2 °C muddo 5-7 maalmood ah. Mycelium-ka fungal-ka ayaa markaa la soo ururiyay, laga shaandheeyay maro farmaajo ah, laba jeer lagu dhaqay biyo nadiif ah, ka dibna lagu qalajiyay warqad shaandho nadiif ah. DNA-da genomic-ga waxaa lagu go'doomiyay iyadoo la isticmaalayo Quick-DNA™ Fungal/Bacterial Miniprep Kit (Kuramae-Izioka, 1997; Atallah et al., 2022, 2024). Gobolka ITS rDNA ayaa markaa la ballaariyay iyadoo la adeegsanayo lammaanaha gaarka ah ee ITS1/ITS4 (TCCGTAGGTGAACCTGCGG TCCTCCGCTTATTGATATGC; cabbirka la filayo: 540 bp) (Baturo-Ciesniewska et al., 2017). Badeecadaha PCR ee la nadiifiyey waxaa loo gudbiyay si loo sameeyo isku xigxig (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). Taxanaha ITS rDNA waxaa loo kala saaray laba jiho iyadoo la adeegsanayo habka isku xigxigsiga Sanger. Taxanaha su'aalaha la isku daray ayaa markaa la barbar dhigay xogtii ugu dambeysay ee GenBank iyo Xarunta Qaranka ee Macluumaadka Bayoteknoolajiyadda (NCBI, http://www.ncbi.nlm.nih.gov/gene/) iyadoo la adeegsanayo software-ka BLASTn. Taxanaha su'aasha waxaa la barbar dhigay 20 nooc oo kale oo S. sclerotiorum ah/go'doomin ah oo laga soo qaatay xogtii ugu dambeysay ee NCBI GenBank (Jadwalka Dheeraadka ah S1) iyadoo la adeegsanayo ClustalW oo ku jira Xirmada Falanqaynta Hidde-sidayaasha Horumarka Molecular (MEGA-11; nooca 11) (Kumar et al., 2024). Falanqaynta horumarka waxaa lagu sameeyay iyadoo la adeegsanayo habka ugu macquulsan ugu badan iyo qaabka beddelka nucleotide-ka ee waqtiga-dib loo rogi karo (Nei iyo Kumar, 2000). Geedka leh suurtagalnimada ugu sarreysa ee looxa ayaa la muujiyay. Geedka bilowga ah ee raadinta heuristic waxaa lagu doortaa iyadoo la dooranayo geedka leh suurtagalnimada sare ee looxa ee u dhexeeya geedka deriska ku xiran (NJ) (Kumar et al., 2024) iyo geedka ugu badan ee looxa (MP). Geedka NJ waxaa lagu dhisay iyadoo la adeegsanayo matrix masaafada laba-geesoodka ah oo lagu xisaabiyay iyadoo la adeegsanayo qaabka guud ee waqtiga-dib loo rogi karo (Nei iyo Kumar, 2000).
Waxqabadka bakteeriyada dila ee L-ornithine iyo bakteeriyada disha "Rizolex-T" waxaa lagu go'aamiyay in vitro iyadoo la adeegsanayo habka faafinta agar. Habka: Qaado xaddiga ku habboon ee xalka kaydka ah ee L-ornithine (500 mg/L) oo si fiican ugu qas 10 ml oo ah daawada nafaqada ee PDA si aad u diyaariso xalal leh xaddiga ugu dambeeya ee 12.5, 25, 50, 75, 100 iyo 125 mg/L, siday u kala horreeyaan. Shan fiirsasho oo ah sunta disha "Rizolex-T" (2, 4, 6, 8 iyo 10 mg/L) iyo biyo nadiif ah oo la safeeyey ayaa loo isticmaalay xakamaynta. Ka dib markii daawada ay adkaato, fiilo mycelial ah oo cusub oo ah dhaqanka Sclerotinia sclerotiorum, dhexroorkeedu yahay 4 mm, ayaa loo wareejiyay bartamaha saxanka Petri waxaana lagu beeray heerkul ah 25±2°C ilaa mycelium uu daboolay saxanka xakamaynta Petri oo dhan, ka dibna koritaanka fangaska ayaa la duubay. Xisaabi boqolkiiba joojinta koritaanka radial ee S. sclerotiorum adoo isticmaalaya isla'egta 1:
Tijaabada waxaa lagu celceliyay laba jeer, iyadoo lix jeer la celceliyay bayooloji koox kasta oo xakameyn/tijaabo ah iyo shan dheri (laba dhir halkii dheri) oo loogu talagalay nuqul kasta oo bayooloji ah. Ku celcelin kasta oo bayooloji ah ayaa laba jeer la falanqeeyay (laba jeer oo farsamo ah) si loo hubiyo saxnaanta, isku halaynta iyo soo-saarista natiijooyinka tijaabada. Intaa waxaa dheer, falanqaynta dib-u-celinta probit ayaa loo isticmaalay in lagu xisaabiyo isku-darka nus-ugu sarreeya ee xakamaynta (IC50) iyo IC99 (Prentice, 1976).
Si loo qiimeeyo awoodda L-ornithine ee xaaladaha aqalka dhirta lagu koriyo, laba tijaabo oo isku xigta ayaa la sameeyay. Si kooban, dheriyada waxaa lagu buuxiyay ciid dhoobo-ciid ah oo la nadiifiyey (3:1) waxaana lagu tallaalay dhaqan cusub oo S. sclerotiorum ah. Marka hore, go'doominta ugu badan ee S. sclerotiorum (go'doominta #3) waxaa lagu beeray iyadoo la jarayo hal sclerotium kala bar, iyadoo la saarayo wejiga PDA oo lagu kululeynayo 25°C mugdi joogto ah (24 saacadood) muddo 4 maalmood ah si loo kiciyo koritaanka mycelium. Afar baloog oo agar ah oo dhexroorkoodu yahay 5 mm ayaa markaa laga soo qaaday geeska hore waxaana lagu tallaalay 100 g oo isku dar nadiif ah oo sarreen iyo bariis ah (1:1, v/v) dhammaan dhalooyinkana waxaa lagu kululeeyay 25 ± 2 °C iyadoo la raacayo wareeg mugdi ah oo 12 saacadood ah/12 saacadood ah muddo 5 maalmood ah si loo kiciyo sameynta sclerotia. Waxyaabaha ku jira dhammaan dhalooyinka si fiican ayaa loo isku qasay si loo hubiyo isku mid ahaanshaha ka hor inta aan lagu darin ciidda. Dabadeed, 100 g oo isku darka galleyda ee gumeysiga ah ayaa lagu daray dheri kasta si loo hubiyo inay si joogto ah u ururaan jeermisyada. Dheriyada la tallaalay ayaa la waraabiyay si loo kiciyo koritaanka fangaska waxaana la dhigay xaalado aqalka dhirta lagu koriyo ah muddo 7 maalmood ah.
Shan iniin oo ka mid ah noocyada kala duwan ee Giza 3 ayaa lagu beeray dheri kasta. Dheriyada lagu daweeyay L-ornithine iyo sunta cayayaanka ee Rizolex-T, iniinaha la jeermis dilay waxaa marka hore lagu qooyay laba saacadood xal biyood ah oo ka mid ah labada iskudhis iyadoo la adeegsanayo qiyaasta ugu dambeysa ee IC99 oo ah qiyaastii 250 mg/L iyo 50 mg/L, siday u kala horreeyaan, ka dibna hawo ayaa lagu qalajiyey hal saac ka hor inta aan la beerin. Dhanka kale, iniinaha waxaa lagu qooyay biyo nadiif ah oo la safeeyey si loo xakameeyo. 10 maalmood ka dib, ka hor waraabinta ugu horreysa, geedo yaryar ayaa la khafiifiyey, iyagoo ku haray laba geed oo nadiif ah dheri kasta. Intaa waxaa dheer, si loo hubiyo infekshinka S. sclerotiorum, jirridaha geedka digirta ee isla marxaladda koritaanka (10 maalmood) ayaa laga jaray laba meelood oo kala duwan iyadoo la isticmaalayo scalpel la jeermis dilay, qiyaastii 0.5 g oo isku darka bran-ka gumeysiga ah ayaa la geliyay nabar kasta, ka dibna waxaa ku xigay qoyaan sare si loo kiciyo caabuqa iyo horumarinta cudurrada dhammaan dhirta la tallaalay. Dhirta xakamaynta ayaa sidoo kale la dhaawacay, waxaana la geliyay tiro isku mid ah (0.5 g) oo ah isku darka bran-ka oo nadiif ah oo aan la gumeysan ayaa lagu daray nabarka, waxaana lagu hayaa qoyaan sare si loogu daydo deegaanka si loo horumariyo cudurrada loona hubiyo isku dheelitirnaanta kooxaha daaweynta.
Habka Daaweynta: Geedaha digirta waxaa lagu waraabiyay 500 ml oo xal biyood ah oo L-ornithine ah (250 mg/l) ama sunta cayayaanka disha ee Rizolex-T (50 mg/l) iyadoo la waraabinayo ciidda, ka dibna daawaynta waxaa lagu celceliyay saddex jeer muddo 10 maalmood ah. Xakamaynta lagu daweeyay placebo waxaa lagu waraabiyay 500 ml oo biyo nadiif ah oo la sifeeyay. Dhammaan daawaynta waxaa lagu sameeyay xaaladaha aqalka dhirta lagu koriyo (25 ± 2°C, 75 ± 1% qoyaan qaraabo ah, iyo muddo sawireed oo ah 8 saacadood iftiin/16 saacadood mugdi ah). Dhammaan dheriyada waxaa la waraabiyay labadii toddobaadba mar waxaana lagu daaweeyay bille iyadoo la isticmaalayo bacrimin NPK oo dheellitiran (20-20-20, oo leh 3.6% sulfur iyo microelements TE; Zain Seeds, Masar) oo leh isku-darka 3-4 g/l iyadoo la isticmaalayo buufin caleenta ah sida ku xusan talooyinka noocyada gaarka ah iyo tilmaamaha soo saaraha. Haddii aan si kale loo sheegin, caleenta bislaaday ee si buuxda u fidsan (caleemaha 2aad iyo 3aad ee kor) ayaa laga soo ururiyay nuqul kasta oo bayooloji ah 72 saacadood ka dib daaweynta (hpt), la isku daray, la isku daray oo lagu kaydiyay -80 °C si loo sameeyo falanqayn dheeraad ah oo ay ku jiraan, laakiin aan ku xaddidnayn, in situ histochemical logu soo bandhigo tilmaamayaasha cadaadiska oksaydhka, lipid peroxidation, antioxidants enzymatic iyo non-enzymatic iyo muujinta hidda-wadaha.
Xoogga caabuqa caaryada cad waxaa la qiimeeyay 21 maalmood ka dib tallaalka (dpi) toddobaadle iyadoo la adeegsanayo miisaan ah 1-9 (Jadwalka Dheeraadka ah S2) iyadoo lagu saleynayo cabbirka Petzoldt iyo Dickson (1996) oo uu wax ka beddelay Teran et al. (2006). Si kooban, jirridaha iyo laamaha dhirta digirta ayaa la baaray laga bilaabo meesha tallaalka si loo raaco horumarka nabarrada ku teedsan internodes-ka iyo qanjidhada. Masaafada dhaawaca laga bilaabo meesha tallaalka ilaa meesha ugu fog ee ku teedsan jirridda ama laanta ayaa markaa la cabbiray ka dibna dhibco ah 1-9 ayaa loo qoondeeyay iyadoo lagu saleynayo goobta nabarku ku yaal, halkaas oo (1) ay muujisay in aan la arkin caabuq u dhow meesha tallaalka iyo (2-9) ay muujisay koror tartiib tartiib ah oo ku yimid cabbirka nabarka iyo horumarka qanjidhada/internodes-ka (Jadwalka Dheeraadka ah S2). Xoogga caabuqa caaryada cad ayaa markaa loo beddelay boqolkiiba iyadoo la isticmaalayo qaacidada 2:
Intaa waxaa dheer, aagga hoos yimaada qalooca horumarka cudurka (AUDPC) waxaa lagu xisaabiyay qaacidada (Shaner iyo Finney, 1977), kaas oo dhawaan loo habeeyey qudhunka cad ee digirta caadiga ah (Chauhan et al., 2020) iyadoo la adeegsanayo isla'egta 3:
Halka Yi = darnaanta cudurka waqtiga ti, Yi+1 = darnaanta cudurka waqtiga xiga ti+1, ti = waqtiga cabbirka koowaad (maalmo), ti+1 = waqtiga cabbirka xiga (maalmo), n = wadarta tirada dhibcaha waqtiga ama dhibcaha la socodka. Xuduudaha koritaanka dhirta digirta oo ay ku jiraan dhererka dhirta (cm), tirada laamaha geed kasta, iyo tirada caleenta geed kasta ayaa la diiwaangeliyey toddobaadle muddo 21 maalmood ah dhammaan noocyada bayoolojiga ah.
Nuqul kasta oo bayooloji ah, muunado caleen ah (caleemo labaad iyo saddexaad oo si buuxda loo sameeyay oo laga soo qaaday xagga sare) ayaa la soo ururiyay maalintii 45aad ka dib daawaynta (15 maalmood ka dib daawaynta ugu dambeysay). Nuqul kasta oo bayooloji ah wuxuu ka koobnaa shan dheri (laba dhir dheri kasta). Qiyaastii 500 mg oo ah unugyada la jajabiyey ayaa loo isticmaalay soo saarista midabada sawir-samaynta (chlorophyll a, chlorophyll b iyo carotenoids) iyadoo la isticmaalayo 80% acetone heerkul ah 4 °C mugdiga. 24 saacadood ka dib, muunado ayaa la centrifugedeeyay ka dibna waxa ka sarreeya ayaa la soo ururiyay si loo go'aamiyo waxyaabaha ku jira chlorophyll a, chlorophyll b iyo carotenoid iyadoo la adeegsanayo spectrophotometer UV-160A (Shimadzu Corporation, Japan) iyadoo la raacayo habka (Lichtenthaler, 1987) iyadoo la cabbirayo nuugista saddex mowjadood oo kala duwan (A470, A646 iyo A663 nm). Ugu dambeyntii, nuxurka midabada sawir-samaynta waxaa lagu xisaabiyay iyadoo la adeegsanayo qaacidooyinka soo socda 4-6 oo uu sharraxay Lichtenthaler (1987).
72 saacadood kadib daaweynta (hpt), caleenta (caleemaha labaad iyo saddexaad ee si buuxda u koray ee laga soo qaaday xagga sare) ayaa laga soo ururiyay nuqul kasta oo bayooloji ah si loogu helo in situ histochemical localisation ee hydrogen peroxide (H2O2) iyo superoxide anion (O2•−). Nooc kasta oo bayooloji ah wuxuu ka koobnaa shan dheri (laba dhir dheri kasta). Nooc kasta oo bayooloji ah waxaa lagu falanqeeyay nuqul (laba nuqul oo farsamo ah) si loo hubiyo saxnaanta, isku halaynta iyo dib-u-soo-saarka habka. H2O2 iyo O2•− waxaa lagu go'aamiyay iyadoo la isticmaalayo 0.1% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, Darmstadt, Jarmalka) ama nitroblue tetrazolium (NBT; Sigma-Aldrich, Darmstadt, Jarmalka), siday u kala horreeyaan, iyadoo la raacayo hababka ay qeexeen Romero-Puertas et al. (2004) iyo Adam et al. (1989) iyadoo wax laga beddelay. Si loo helo meelaynta histochemical ee H2O2 ee goobta, waraaqaha waxaa lagu shubay faakiyuum 0.1% DAB oo ku jirta 10 mM Tris buffer (pH 7.8) ka dibna lagu shubay heerkulka qolka iftiinka muddo 60 daqiiqo ah. Warqadaha waxaa lagu shubay 0.15% (v/v) TCA oo ku jirta 4:1 (v/v) ethanol:chloroform (Al-Gomhoria Pharmaceuticals and Medical Sahayda, Qaahira, Masar) ka dibna waxaa loo bandhigay iftiin ilaa ay madoobaadaan. Sidoo kale, waalka waxaa lagu shubay faakiyuum 10 mM potassium phosphate buffer (pH 7.8) oo ka kooban 0.1 w/v % HBT si loo helo meelaynta histochemical ee O2•− in situ. Warqadaha waxaa lagu shubay iftiinka heerkulka qolka muddo 20 daqiiqo ah, ka dibna waxaa la dhalaaliyay sida kor ku xusan, ka dibna waxaa la iftiimiyay ilaa ay ka soo muuqdaan dhibco buluug/violet madow. Xoogga midabka bunniga ah ee ka dhashay (sida tilmaame H2O2) ama buluug-guduud (sida tilmaame O2•−) ayaa lagu qiimeeyay iyadoo la adeegsanayo nooca Fiji ee xirmada farsamaynta sawirka ImageJ (http://fiji.sc; la galay 7 Maarso 2024).
Malondialdehyde (MDA; oo ah calaamad muujinaysa lipid peroxidation) ayaa lagu go'aamiyay habka Du iyo Bramlage (1992) iyadoo wax laga beddelayo si yar. Caleenta laga soo qaaday nuqul kasta oo bayooloji ah (caleemo labaad iyo saddexaad oo si buuxda loo sameeyay oo ka yimid xagga sare) ayaa la ururiyay 72 saacadood ka dib daaweynta (hpt). Nooc kasta oo bayooloji ah waxaa ku jiray shan dheri (laba dhir ah dherigiiba). Nooc kasta oo bayooloji ah waxaa lagu falanqeeyay nuqul (laba nuqul oo farsamo ah) si loo hubiyo saxnaanta, isku halaynta iyo soo-saarka habka. Si kooban, 0.5 g oo ah unug caleen dhulka ah ayaa loo isticmaalay soo saarista MDA iyadoo la isticmaalayo 20% trichloroacetic acid (TCA; MilliporeSigma, Burlington, MA, USA) oo ka kooban 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich, St. Louis, MO, USA). Waxyaabaha ku jira MDA ee ku jira dusha sare waxaa lagu go'aamiyay midab ahaan iyadoo la cabbirayo nuugista 532 iyo 600 nm iyadoo la adeegsanayo cabbiraadda UV-160A spectrophotometer (Shimadzu Corporation, Japan) ka dibna loo muujiyey nmol g−1 FW.
Si loo qiimeeyo antioxidants-ka aan enzymatic-ka ahayn iyo enzymatic-ka, caleenta (caleemaha labaad iyo saddexaad ee si buuxda loo soo saaray ee ka soo baxay xagga sare) ayaa laga soo ururiyay nuqul kasta oo bayooloji ah 72 saacadood ka dib daawaynta (hpt). Nooc kasta oo bayooloji ah wuxuu ka koobnaa shan dheri (laba dhir dherigiiba). Muunad bayooloji ah kasta waxaa lagu falanqeeyay nuqul (laba muunad farsamo). Laba caleemood waxaa lagu shiiday nitrogen dareere ah waxaana si toos ah loogu isticmaalay go'aaminta antioxidants-ka enzymatic-ka ah iyo kuwa aan enzymatic-ka ahayn, wadarta amino acids-ka, nuxurka proline-ka, muujinta hidda-wadaha, iyo cabbirka oxalate.
Wadarta phenolics-ka milma waxaa lagu go'aamiyay iyadoo la adeegsanayo reagent Folin-Ciocalteu (Sigma-Aldrich, St. Louis, MO, USA) iyadoo wax laga beddelay habka uu sharraxay Kahkonen et al. (1999). Si kooban, qiyaastii 0.1 g oo ah unug caleen isku dhafan ayaa laga soo saaray 20 ml 80% methanol mugdiga muddo 24 saacadood ah ka dibna dheecaanka ayaa la ururiyay ka dib markii la centrifugation. 0.1 ml oo ka mid ah soosaarka muunadda ayaa lagu qasay reagent Folin-Ciocalteu 0.5 ml (10%), la ruxay 30 ilbiriqsi ka dibna mugdiga ayaa lagu daayay muddo 5 daqiiqo ah. Kadib 0.5 ml oo ah 20% xal sodium carbonate ah (Na2CO3; Al-Gomhoria Pharmaceuticals and Medical Supplies Company, Cairo, Masar) ayaa lagu daray tuubo kasta, si fiican ayaa loo qasay oo heerkulka qolka mugdiga ah muddo 1 saac ah. Ka dib markii la beero, nuugista isku darka falcelinta waxaa lagu cabiray 765 nm iyadoo la adeegsanayo spectrophotometer UV-160A (Shimadzu Corporation, Japan). Isugeynta phenols-ka milma ee ku jira soosaarka muunadda waxaa lagu go'aamiyay iyadoo la adeegsanayo qalooca cabbiraadda gallic acid (Fisher Scientific, Hampton, NH, USA) waxaana lagu muujiyay milligrams oo ah gallic acid u dhiganta garaamkiiba miisaanka cusub (mg GAE g-1 miisaan cusub).
Wadarta guud ee flavonoid-ka milmi kara waxaa lagu go'aamiyay habka Djeridane et al. (2006) iyadoo wax laga beddelayo si yar. Si kooban, 0.3 ml oo ka mid ah soosaarka methanol ee kor ku xusan ayaa lagu qasay 0.3 ml oo ah 5% xal aluminium chloride ah (AlCl3; Fisher Scientific, Hampton, NH, USA), si xoog leh ayaa loo walaaqay ka dibna lagu kululeeyay heerkulka qolka muddo 5 daqiiqo ah, ka dibna waxaa lagu daray 0.3 ml oo ah 10% xal potassium acetate ah (Al-Gomhoria Pharmaceuticals and Medical Sahayda, Qaahira, Masar), si fiican ayaa loogu qasay oo lagu kululeeyay heerkulka qolka muddo 30 daqiiqo ah mugdiga. Ka dib markii la kululeeyo, nuugista isku darka falcelinta waxaa lagu cabiray 430 nm iyadoo la isticmaalayo spectrophotometer UV-160A (Shimadzu Corporation, Japan). Isugeynta flavonoid-yada milmi kara ee laga soo saaray muunadda waxaa lagu go'aamiyay iyadoo la isticmaalayo qalooca cabbiraadda rutin (TCI America, Portland, OR, USA) ka dibna waxaa lagu muujiyay milligrams oo u dhigma rutin garaamkiiba miisaan cusub (mg RE g-1 miisaan cusub).
Wadarta guud ee amino acid-ka bilaashka ah ee caleenta digirta waxaa lagu go'aamiyay iyadoo la adeegsanayo reagent ninhydrin oo wax laga beddelay (Thermo Scientific Chemicals, Waltham, MA, USA) iyadoo lagu saleynayo habka ay soo jeediyeen Yokoyama iyo Hiramatsu (2003) waxaana wax ka beddelay Sun et al. (2006). Si kooban, 0.1 g oo unugyo dhulka ah ayaa laga soo saaray pH 5.4 buffer, 200 μL oo ka mid ah supernatant-ka waxaa lagu falceliyay 200 μL oo ninhydrin ah (2%) iyo 200 μL oo pyridine ah (10%; Spectrum Chemical, New Brunswick, NJ, USA), oo lagu dhex geliyay biyo karkaraya muddo 30 daqiiqo ah, ka dibna lagu qaboojiyey oo lagu cabbiray 580 nm iyadoo la isticmaalayo spectrophotometer UV-160A (Shimadzu Corporation, Japan). Dhanka kale, proline-ka waxaa lagu go'aamiyay habka Bates (Bates et al., 1973). Proline waxaa laga soo saaray 3% sulfosalicylic acid (Thermo Scientific Chemicals, Waltham, MA, USA) ka dib markii la centrifugation-ka la sameeyay, 0.5 ml oo ka mid ah dheecaanka sare waxaa lagu qasay 1 ml oo ah glacial acetic acid (Fisher Scientific, Hampton, NH, USA) iyo reagent ninhydrin, oo lagu kululeeyay 90°C muddo 45 daqiiqo ah, la qaboojiyey oo lagu cabiray 520 nm iyadoo la isticmaalayo isla spectrophotometer-ka sida kor ku xusan. Amino acids-ka iyo proline-ka bilaashka ah ee ku jira soosaarka caleenta waxaa lagu go'aamiyay iyadoo la isticmaalayo qalooca glycine iyo proline (Sigma-Aldrich, St Louis, MO, USA), siday u kala horreeyaan, waxaana lagu muujiyey miisaan cusub oo mg/g ah.
Si loo go'aamiyo dhaqdhaqaaqa enzymatic ee ensaymes-ka antioxidant-ka, qiyaastii 500 mg oo unugyo isku-dhafan ah ayaa laga soo saaray 3 ml oo ah 50 mM Tris buffer (pH 7.8) oo ka kooban 1 mM EDTA-Na2 (Sigma-Aldrich, St. Louis, MO, USA) iyo 7.5% polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA), oo lagu centrifuged 10,000 × g muddo 20 daqiiqo ah oo qaboojiyaha ah (4 °C), ka dibna waxaa la soo ururiyay supernatant (soo saarista ensaymka cayriin) (El-Nagar et al., 2023; Osman et al., 2023). Dabadeed Catalase (CAT) waxaa lagu falceliyay 2 ml oo ah 0.1 M sodium phosphate buffer (pH 6.5; Sigma-Aldrich, St. Louis, MO, USA) iyo 100 μl oo ah xalka 269 mM H2O2 si loo go'aamiyo dhaqdhaqaaqiisa enzymatic iyadoo la raacayo habka Aebi (1984) iyadoo wax laga beddelay (El-Nagar et al., 2023; Osman et al., 2023). Hawlaha enzymatic ee ku xiran peroxidase (POX) ee Guaiacol-ku-tiirsan waxaa lagu go'aamiyay habka Harrach et al. (2009). (2008) oo leh wax-ka-beddello yar yar (El-Nagar et al., 2023; Osman et al., 2023) iyo dhaqdhaqaaqa enzymatic ee polyphenol oxidase (PPO) ayaa la go'aamiyay ka dib markii falcelin lagu sameeyay 2.2 ml oo ah 100 mM sodium phosphate buffer (pH 6.0), 100 μl oo guaiacol ah (kiimikada TCI, Portland, OR, USA) iyo 100 μl oo ah 12 mM H2O2. Habka waxaa wax yar laga beddelay (El-Nagar et al., 2023; Osman et al., 2023). Baaritaanka waxaa la sameeyay ka dib falcelin lagu sameeyay 3 ml oo xal catechol ah (Thermo Scientific Chemicals, Waltham, MA, USA) (0.01 M) oo si cusub loogu diyaariyey 0.1 M phosphate buffer (pH 6.0). Dhaqdhaqaaqa CAT waxaa lagu cabiray iyadoo la kormeerayo kala-goynta H2O2 240 nm (A240), dhaqdhaqaaqa POX waxaa lagu cabiray iyadoo la kormeerayo kororka nuugista 436 nm (A436), dhaqdhaqaaqa PPO waxaa lagu cabiray iyadoo la duubayo isbeddellada nuugista 495 nm (A495) 30 ilbiriqsi kasta muddo 3 daqiiqo ah iyadoo la isticmaalayo spectrophotometer UV-160A (Shimadzu, Japan).
RT-PCR-ka waqtiga-dhabta ah ayaa loo isticmaalay in lagu ogaado heerarka qoraalka ee saddex hiddo-side oo la xiriira antioxidant-ka, oo ay ku jiraan peroxisomal catalase (PvCAT1; GenBank Accession No. KF033307.1), superoxide dismutase (PvSOD; GenBank Accession No. XM_068639556.1), iyo glutathione reductase (PvGR; GenBank Accession No. KY195009.1), caleenta digirta (caleemaha labaad iyo saddexaad ee si buuxda loo horumariyay ee ka soo baxa xagga sare) 72 saacadood ka dib daawaynta ugu dambeysay. Si kooban, RNA waxaa lagu go'doomiyay iyadoo la isticmaalayo Simply P Total RNA Extraction Kit (Cat. No. BSC52S1; BioFlux, Biori Technology, Shiinaha) sida ku cad hab-raaca soo saaraha. Kadib, cDNA waxaa lagu sameeyay iyadoo la adeegsanayo TOP script™ cDNA Synthesis Kit sida ku cad tilmaamaha soo saaraha. Taxanaha asaasiga ah ee saddexda hiddo-side ee kor ku xusan waxaa lagu taxay Jadwalka Dheeraadka ah ee S3. PvActin-3 (Lambarka gelitaanka GenBank: XM_068616709.1) ayaa loo isticmaalay sidii hidda-wadaha guriga lagu hayo, muujinta hidda-wadaha qaraabada ahna waxaa lagu xisaabiyay habka 2-ΔΔCT (Livak iyo Schmittgen, 2001). Xasiloonida actin ee hoos timaada cadaadiska bayoolojiga (isdhexgalka aan iswaafaqsanayn ee u dhexeeya digirta caadiga ah iyo fangaska anthracnose Colletotrichum lindemuthianum) iyo cadaadiska abiotic (abaarta, milixda, heerkulka hooseeya) ayaa la muujiyay (Borges et al., 2012).
Markii hore waxaan sameynay falanqayn hidde-side ah oo ku saabsan borotiinnada oxaloacetate acetylhydrolase (OAH) ee S. sclerotiorum annagoo adeegsanayna qalabka BLAST-protein-ka ee BLAST (BLASTp 2.15.0+) (Altschul et al., 1997, 2005). Si kooban, waxaan u isticmaalnay OAH laga soo bilaabo Aspergillus fijiensis CBS 313.89 (AfOAH; taxide: 1191702; lambarka gelitaanka GenBank XP_040799428.1; 342 amino acids) iyo Penicillium lagena (PlOAH; taxide: 94218; lambarka gelitaanka GenBank XP_056833920.1; 316 amino acids) oo ah taxane su'aalo ah si loo sawiro borotiinka isku midka ah ee S. sclerotiorum (taxide: 5180). BLASTp waxaa lagu sameeyay iyadoo la barbar dhigayo xogta hidde-sideyaasha S. sclerotiorum ee ugu dambeysay ee laga heli karo GenBank bogga internetka ee Xarunta Qaranka ee Macluumaadka Bayoteknoolajiyadda (NCBI), http://www.ncbi.nlm.nih.gov/gene/.
Intaa waxaa dheer, hidda-wadaha OAH ee la saadaaliyay ee ka yimid S. sclerotiorum (SsOAH) iyo falanqaynta isbeddelka iyo geedka phylogenetic-ga ee AfOAH ee ka yimid A. fijiensis CBS 313.89 iyo PlOAH ee ka yimid P. lagena ayaa lagu go'aamiyay iyadoo la adeegsanayo habka ugu macquulsan ee MEGA11 (Tamura et al., 2021) iyo qaabka ku salaysan matrix-ka JTT (Jones et al., 1992). Geedka phylogenetic-ga waxaa lagu daray falanqaynta isku-dubaridka badan ee taxanaha borotiinka ee dhammaan hidda-wadaha OAH ee la saadaaliyay (SsOAH) ee ka yimid S. sclerotiorum iyo taxanaha weydiinta iyadoo la adeegsanayo Qalabka Isku-dubaridka Ku Salaysan Xaddidaadda (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos iyo Agarwala, 2007). Intaa waxaa dheer, taxanaha amino acid ee ugu habboon ee SsOAH ee ka yimid S. sclerotiorum waxaa la waafajiyay taxanaha weydiinta (AfOAH iyo PlOAH) (Larkin et al., 2007) iyadoo la adeegsanayo ClustalW (http://www.genome.jp/tools-bin/clustalw), gobollada la ilaaliyay ee isku-xidhkana waxaa lagu sawiray iyadoo la adeegsanayo qalabka ESPript (nooca 3.0; https://espript.ibcp.fr/ESPript/ESPript/index.php).
Intaa waxaa dheer, meelaha la saadaaliyay ee matalaya shaqada iyo goobaha la ilaaliyay ee S. sclerotiorum SsOAH waxaa si is-dhexgal ah loogu kala saaray qoysas kala duwan iyadoo la adeegsanayo qalabka InterPro (https://www.ebi.ac.uk/interpro/) (Blum et al., 2021). Ugu dambeyntii, qaab-dhismeedka saddex-geesoodka ah (3D) ee S. sclerotiorum SsOAH ee la saadaaliyay waxaa lagu sameeyay iyadoo la adeegsanayo 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) waxaana la xaqiijiyay iyadoo la adeegsanayo server-ka SWISS-MODEL (https://swissmodel.expasy.org/) (Biasini et al., 2014). Qaab-dhismeedka saddexda-cabbir ee la saadaaliyay (qaabka PDB) waxaa si is-dhexgal ah loogu sawiray iyadoo la adeegsanayo xirmada UCSF-Chimera (nooca 1.15; https://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004).
PCR-ka iftiinka waqtiga-dhabta ah ee tirada leh ayaa loo isticmaalay in lagu go'aamiyo heerka qoraalka ee oxaloacetate acetylhydrolase (SsOAH; lambarka gelitaanka GenBank: XM_001590428.1) ee mycelia ee Sclerotinia sclerotiorum. Si kooban, S. sclerotiorum waxaa lagu tallaalay dhalo ay ku jirto PDB waxaana la geliyay qalab gariiraya (nooca: I2400, New Brunswick Scientific Co., Edison, NJ, USA) heerkul ah 25 ± 2 °C muddo 24 saacadood ah 150 rpm iyo mugdi joogto ah (24 saacadood) si loo kiciyo koritaanka mycelium. Intaa ka dib, unugyada waxaa lagu daaweeyay L-ornithine iyo sunta cayayaanka ee Rizolex-T oo ah fiirsashada ugu dambeysa ee IC50 (qiyaastii 40 iyo 3.2 mg/L, siday u kala horreeyaan) ka dibna waxaa la beeray 24 saacadood oo kale xaalado isku mid ah. Ka dib markii la beero, dhaqamada waxaa lagu centrifuge gareeyay 2500 rpm muddo 5 daqiiqo ah, ka dibna waxaa la ururiyay dusha sare ee jirka (mycelium fungal) si loo falanqeeyo muujinta hidda-wadaha. Sidoo kale, mycelium fungal waxaa la ururiyay 0, 24, 48, 72, 96, iyo 120 saacadood ka dib caabuqa laga soo qaaday dhirta cudurka qabta ee samaysatay caaryada cad iyo mycelium cudbi ah oo dusha sare ee unugyada cudurka qaba. RNA waxaa laga soo saaray mycelium fungal ka dibna cDNA ayaa la sameeyay sida kor lagu sharaxay. Taxanaha asaasiga ah ee SsOAH waxaa lagu taxay Jadwalka Dheeraadka ah S3. SsActin (Lambarka gelitaanka GenBank: XM_001589919.1) waxaa loo isticmaalay hidda-wadaha daryeelka guriga, muujinta hidda-wadaha qaraabada ahna waxaa lagu xisaabiyay habka 2-ΔΔCT (Livak iyo Schmittgen, 2001).
Asiidhka Oxalic waxaa lagu go'aamiyay maraq baradho dextrose ah (PDB) iyo muunado dhir ah oo ay ku jiraan cudur-sidaha fangaska Sclerotinia sclerotiorum iyadoo la raacayo habka Xu iyo Zhang (2000) iyadoo wax laga beddelay. Si kooban, S. sclerotiorum isolates ayaa lagu tallaalay dhalooyin ay ku jiraan PDB ka dibna lagu beeray qalab gariiraya (nooca I2400, New Brunswick Scientific Co., Edison, NJ, USA) 150 rpm heerkul ah 25 ± 2 °C muddo 3-5 maalmood ah mugdi joogto ah (24 saacadood) si loo kiciyo koritaanka mycelium. Ka dib markii la beero, dhaqanka fangaska waxaa marka hore lagu sifeeyay warqadda shaandhada Whatman #1 ka dibna lagu dhex geliyay 2500 rpm muddo 5 daqiiqo ah si looga saaro mycelium-ka haray. Dusha sare waxaa la ururiyay oo lagu kaydiyay 4°C si loo helo go'aanno dheeraad ah oo ku saabsan oxalate. Diyaarinta muunadaha dhirta, qiyaastii 0.1 g oo ah jajabyada unugyada dhirta ayaa saddex jeer lagu soo saaray biyo la safeeyey (2 ml mar kasta). Shaybaarrada ayaa markaa lagu dhex shubay 2500 rpm muddo 5 daqiiqo ah, dareeraha ka soo baxa biyaha ayaa sifeeyay iyadoo la mariyay warqadda shaandhada Whatman No. 1 ka dibna la ururiyey si loo sameeyo falanqayn dheeraad ah.
Falanqaynta tirada ee asiidhka oxalic, isku darka falcelinta waxaa lagu diyaariyey tuubo galaas ah oo xiran sida soo socota: 0.2 ml oo muunad ah (ama xalka PDB culture filtrate ama xalka caadiga ah ee asiidhka oxalic), 0.11 ml oo ah bromophenol blue (BPB, 1 mM; Fisher Chemical, Pittsburgh, PA, USA), 0.198 ml oo ah 1 M sulfuric acid (H2SO4; Al-Gomhoria Pharmaceuticals and Medical Sahayda, Cairo, Masar) iyo 0.176 ml oo ah 100 mM potassium dichromate (K2Cr2O7; kiimikooyinka TCI, Portland, OR, USA), ka dibna xalka waxaa lagu qasi jiray 4.8 ml biyo la sifeeyay, si xooggan ayaa loo isku qasay oo isla markiiba lagu riday qubeys biyo ah oo 60 °C ah. 10 daqiiqo ka dib, falcelinta waxaa la joojiyay iyadoo lagu daray 0.5 ml oo ah xalka sodium hydroxide (NaOH; 0.75 M). Nuugista (A600) ee isku darka falgalka waxaa lagu cabiray 600 nm iyadoo la adeegsanayo cabbiraadda UV-160 (Shimadzu Corporation, Japan). PDB iyo biyaha la sifeeyay ayaa loo isticmaalay sidii xakamaynta cabbirka shaandhaynta dhaqanka iyo muunadaha dhirta, siday u kala horreeyaan. Fiirsashada aashitada oxalic ee ku jirta shaandhaynta dhaqanka, oo lagu muujiyay micrograms oo ah aashitada oxalic halkii millilitir ee dhexdhexaadka PDB (μg.mL−1), iyo soosaarka caleenta, oo lagu muujiyay micrograms oo ah aashitada oxalic halkii garaam oo miisaan cusub ah (μg.g−1 FW), waxaa lagu go'aamiyay iyadoo la isticmaalayo qalooca cabbiraadda aashitada oxalic (Thermo Fisher Scientific Chemicals, Waltham, MA, USA).
Daraasadda oo dhan, dhammaan tijaabooyinka waxaa lagu sameeyay naqshad gebi ahaanba aan kala sooc lahayn (CRD) oo leh lix nuqul oo bayooloji ah halkii daaweyn iyo shan dheri oo nuqul bayooloji ah halkii (laba dhir dheri) haddii aan si kale loo sheegin. Ku celcelinta bayoolojiga waxaa lagu falanqeeyay laba nuqul (laba nuqul oo farsamo ah). Ku celcelinta farsamada ayaa loo isticmaalay in lagu hubiyo dib-u-soo-saarka tijaabada isku midka ah laakiin looma isticmaalin falanqaynta tirakoobka si looga fogaado ku celcelinta been abuurka ah. Xogta waxaa lagu falanqeeyay tirakoob ahaan iyadoo la adeegsanayo falanqaynta kala duwanaanshaha (ANOVA) oo ay ku xigto tijaabada Tukey-Kramer ee kala duwanaanshaha dhabta ah ee muhiimka ah (HSD) (p ≤ 0.05). Tijaabooyinka In Vitro, qiimayaasha IC50 iyo IC99 waxaa lagu xisaabiyay iyadoo la adeegsanayo qaabka probit waxaana la xisaabiyay 95% kala-goysyada kalsoonida.
Wadarta afar go'doomin ayaa laga soo ururiyay beero kala duwan oo soy ah oo ku yaal Gobolka El Ghabiya, Masar. Qaybta PDA, dhammaan go'doominnada waxay soo saareen mycelium cad oo kareem ah oo si dhakhso ah u noqday cad suuf ah (Jaantuska 1A) ka dibna beige ama bunni marxaladda sclerotium. Sclerotia badanaa waa cufan yihiin, madow, wareegsan ama aan caadi ahayn, dhererkoodu waa 5.2 ilaa 7.7 mm iyo dhexroorkeedu yahay 3.4 ilaa 5.3 mm (Jaantuska 1B). In kasta oo afar go'doomin ay sameeyeen qaab yar oo sclerotia ah oo ku yaal geeska warshadda dhaqanka ka dib 10-12 maalmood oo lagu beeray heerkul ah 25 ± 2 °C (Jaantuska 1A), tirada sclerotia halkii saxan aad bay uga duwanayd dhexdooda (P < 0.001), iyadoo go'doominnada 3 ay leeyihiin tirada ugu badan ee sclerotia (32.33 ± 1.53 sclerotia halkii saxan; Sawirka 1C). Sidoo kale, isolate #3 waxay soo saartay asiidh oxalic ah oo ka badan PDB marka loo eego isolates-yada kale (3.33 ± 0.49 μg.mL−1; Jaantuska 1D). Isolate #3 waxay muujisay astaamo caadi ah oo qaab-dhismeed iyo mikroskoob ah oo ku saabsan fangaska phytopathogenic Sclerotinia sclerotiorum. Tusaale ahaan, PDA, kolonyada isolate #3 si degdeg ah ayay u koreen, waxay ahaayeen caddaan kareem ah (Jaantuska 1A), beige rogan ama salmon khafiif ah oo huruud-bunni ah, waxayna u baahdeen 6-7 maalmood heerkul ah 25 ± 2°C si ay si buuxda u daboolaan dusha sare ee saxan dhexroorkiisu yahay 9 cm. Iyada oo lagu saleynayo astaamaha qaab-dhismeedka iyo mikroskoobka ee kor ku xusan, isolate #3 waxaa loo aqoonsaday Sclerotinia sclerotiorum.
Jaantuska 1. Astaamaha iyo astaamaha cudurka ee go'doominta S. sclerotiorum ee dalagyada digirta caadiga ah. (A) Kobaca Mycelial ee afar go'doomin S. sclerotiorum ah oo ku jira dhexdhexaadka PDA, (B) sclerotia ee afar go'doomin S. sclerotiorum ah, (C) tirada sclerotia (saxankiiba), (D) dheecaanka aashitada oxalic ee dhexdhexaadka PDB (μg.mL−1), iyo (E) darnaanta cudurka (%) ee afar go'doomin S. sclerotiorum ah oo ku jira nooca go'doominta ganacsiga ee u nugul Giza 3 xaaladaha aqalka dhirta lagu koriyo. Qiimayaashu waxay matalaan celceliska ± SD ee shan nuqul oo bayooloji ah (n = 5). Xarfo kala duwan waxay muujinayaan kala duwanaansho tirakoob ahaan muhiim ah oo u dhexeeya daawaynta (p < 0.05). (F–H) Calaamadaha caaryada cad ee caadiga ah waxay ka soo muuqdeen jirridda dhulka korkiisa iyo siliques, siday u kala horreeyaan, 10 maalmood ka dib tallaalka go'doomin #3 (dpi). (I) Falanqaynta isbeddelka ee gobolka gudaha ee loo yaqaan 'spacer' (ITS) ee S. sclerotiorum isolate #3 ayaa la sameeyay iyadoo la adeegsanayo habka ugu macquulsan waxaana la barbar dhigay 20 nooc oo tixraac ah/noocyo ah oo laga helay xogta Xarunta Qaranka ee Macluumaadka Bayoteknoolajiyadda (NCBI) (https://www.ncbi.nlm.nih.gov/). Tirooyinka ka sarreeya xariiqyada kooxaynta waxay muujinayaan daboolida gobolka (%), tirooyinka ka hooseeya xariiqyada kooxayntana waxay muujinayaan dhererka laanta.
Intaa waxaa dheer, si loo xaqiijiyo cudurka, afar nooc oo S. sclerotiorum ah oo la helay ayaa loo isticmaalay in lagu tallaalo nooca digirta ganacsiga ee u nugul Giza 3 xaaladaha aqalka dhirta lagu koriyo, taas oo la jaanqaadaysa shuruudaha Koch (Jaantuska 1E). Inkasta oo dhammaan noocyada fangaska ee la helay ay ahaayeen kuwo cudur keena oo ay ku dhici karaan digirta cagaaran (cv. Giza 3), taasoo keentay calaamadaha caaryada cad ee caadiga ah dhammaan qaybaha dhulka korkiisa (Jaantuska 1F), gaar ahaan jirridda (Jaantuska 1G) iyo qolofta (Jaantuska 1H) 10 maalmood ka dib tallaalka (dpi), isolate 3 wuxuu ahaa kan ugu daran laba tijaabo oo madax-bannaan. Isolate 3 wuxuu lahaa darnaanta cudurka ugu sarreysa (%) ee dhirta digirta (24.0 ± 4.0, 58.0 ± 2.0, iyo 76.7 ± 3.1 7, 14, iyo 21 maalmood ka dib caabuqa, siday u kala horreeyaan; Sawirka 1F).
Aqoonsiga S. sclerotiorum isolate #3 ee ugu duullaanka badan ayaa si dheeraad ah loo xaqiijiyay iyadoo lagu saleynayo isku xigxiga spacer-ka gudaha ee la qoray (ITS) (Jaantuska 1I). Falanqaynta phylogenetic-ga ee u dhaxaysa isolate #3 iyo 20 nooc/noocyo tixraac ah ayaa muujisay isku ekaanshaha sare (>99%) dhexdooda. Waxaa xusid mudan in S. sclerotiorum isolate #3 (533 bp) ay leedahay isku ekaanshaha sare ee S. sclerotiorum isolate LPM36 ee laga soocay abuurka digirta qalalan (GenBank accession number MK896659.1; 540 bp) iyo S. sclerotiorum isolate YKY211 (GenBank accession number OR206374.1; 548 bp), kaas oo sababa qudhunka stem-ka ee violet (Matthiola incana), kuwaas oo dhammaantood si gaar ah loogu kala saaray xagga sare ee dendrogram-ka (Jaantuska 1I). Taxanaha cusub ayaa lagu shubay xogta NCBI waxaana loogu magac daray "Sclerotinia sclerotiorum - isolate YN-25" (lambarka gelitaanka GenBank PV202792). Waxaa la arki karaa in isolate 3 uu yahay isolate-ka ugu duullaanka badan; sidaas darteed, isolate-kan waxaa loo doortay daraasad dhammaan tijaabooyinka xigay.
Waxqabadka bakteeriyada diamine L-ornithine (Sigma-Aldrich, Darmstadt, Jarmalka) ee ku urursan tirooyin kala duwan (12.5, 25, 50, 75, 100 iyo 125 mg/L) ee ka dhanka ah S. sclerotiorum isolate 3 ayaa lagu baaray in vitro. Waxaa xusid mudan in L-ornithine ay samaysay saameyn bakteeriyada dila oo si tartiib tartiib ah u joojisay koritaanka radial ee S. sclerotiorum hyphae si ku xiran qiyaasta (Jaantuska 2A, B). Heerka ugu sarreeya ee la tijaabiyay (125 mg/L), L-ornithine waxay muujisay heerka ugu sarreeya ee joojinta koritaanka mycelial (99.62 ± 0.27%; Sawirka 2B), kaas oo u dhigma Rizolex-T ee ganacsiga ee sunta disha (heerka joojinta 99.45 ± 0.39%; Sawirka 2C) ee ku urursan heerka ugu sarreeya ee la tijaabiyay (10 mg/L), taasoo muujinaysa waxtar la mid ah.
Jaantuska 2. Waxqabadka bakteeriyada dila ee gudaha jirka ee L-ornithine ee ka dhanka ah Sclerotinia sclerotiorum. (A) Isbarbardhigga dhaqdhaqaaqa bakteeriyada dila ee fiirsashada kala duwan ee L-ornithine ee ka dhanka ah S. sclerotiorum iyo fungicider-ka ganacsiga ee Rizolex-T (10 mg/L). (B, C) Heerka joojinta (%) ee koritaanka mycelial ee S. sclerotiorum ka dib daaweynta fiirsashada kala duwan ee L-ornithine (12.5, 25, 50, 75, 100 iyo 125 mg/L) ama Rizolex-T (2, 4, 6, 8 iyo 10 mg/L), siday u kala horreeyaan. Qiimayaashu waxay matalaan celceliska ± SD ee shan nuqul oo bayooloji ah (n = 5). Xarfaha kala duwan waxay tilmaamayaan kala duwanaanshaha tirakoobka ee u dhexeeya daawaynta (p < 0.05). (D, E) Falanqaynta dib-u-celinta qaabka Probit ee L-ornithine iyo fungicider-ka ganacsiga ee Rizolex-T, siday u kala horreeyaan. Khadka dib-u-celinta qaabka probit-ka waxaa lagu muujiyay xariiq buluug adag, muddada kalsoonidana (95%) waxaa lagu muujiyay xariiq cas oo jajaban.
Intaa waxaa dheer, falanqaynta dib-u-celinta probit-ka ayaa la sameeyay, jaantusyada u dhigmana waxaa lagu muujiyay Shaxda 1aad iyo Jaantusyada 2D,E. Si kooban, qiimaha la aqbali karo ee leexashada (y = 2.92x − 4.67) iyo tirakoobyada muhiimka ah ee la xiriira (Cox & Snell R2 = 0.3709, Nagelkerke R2 = 0.4998 iyo p < 0.0001; Jaantuska 2D) ee L-ornithine waxay muujiyeen firfircooni antifungal ah oo la xoojiyay oo ka dhan ah S. sclerotiorum marka la barbar dhigo fungicides-ka ganacsiga ee Rizolex-T (y = 1.96x − 0.99, Cox & Snell R2 = 0.1242, Nagelkerke R2 = 0.1708 iyo p < 0.0001) (Jadwalka 1).
Shaxda 1. Qiimaha fiirsashada ugu badan ee ugu badan ee xakamaynta (IC50) iyo IC99 (mg/l) ee L-ornithine iyo fungicider-ka ganacsiga "Rizolex-T" ee ka dhanka ah S. sclerotiorum.
Guud ahaan, L-ornithine (250 mg/L) waxay si weyn u yareysay koritaanka iyo darnaanta caaryada cad ee dhirta digirta caadiga ah ee la daweeyay marka la barbar dhigo dhirta ay S. sclerotiorum-ku ku dhacday ee aan la daweyn (xakamaynta; Jaantuska 3A). Si kooban, inkastoo darnaanta cudurka ee dhirta xakamaynta cudurka ee aan la daweyn ay si tartiib tartiib ah u korodhay (52.67 ± 1.53, 83.21 ± 2.61, iyo 92.33 ± 3.06%), L-ornithine waxay si weyn u yareysay darnaanta cudurka (%) intii lagu jiray tijaabada (8.97 ± 0.15, 18.00 ± 1.00, iyo 26.36 ± 3.07) 7, 14, iyo 21 maalmood kadib daaweynta (dpt), siday u kala horreeyaan (Jaantuska 3A). Sidoo kale, markii dhirta digirta ee uu ku dhacay cudurka S. sclerotiorum lagu daweeyay 250 mg/L L-ornithine, aagga hoos yimaada qalooca horumarka cudurka (AUDPC) ayaa hoos uga dhacay 1274.33 ± 33.13 ee xakamaynta aan la daweyn ilaa 281.03 ± 7.95, taasoo wax yar ka hooseysay kan xakamaynta togan 50 mg/L Rizolex-T fungicidal (183.61 ± 7.71; Jaantuska 3B). Isbeddel isku mid ah ayaa lagu arkay tijaabadii labaad.
Jaantuska 3. Saamaynta isticmaalka dibadda ee L-ornithine ee horumarinta qudhunka cad ee digirta caadiga ah ee ay keento Sclerotinia sclerotiorum xaaladaha aqalka dhirta lagu koriyo. (A) Qallooca horumarka cudurka ee caaryada cad ee digirta caadiga ah ka dib markii lagu daaweeyay 250 mg/L L-ornithine. (B) Aagga hoos yimaada qalooca horumarka cudurka (AUDPC) ee caaryada cad ee digirta caadiga ah ka dib markii lagu daaweeyay L-ornithine. Qiimayaashu waxay matalaan celceliska ± SD ee shan nuqul oo bayooloji ah (n = 5). Xarfaha kala duwan waxay muujinayaan kala duwanaansho tirakoob ahaan muhiim ah oo u dhexeeya daawaynta (p < 0.05).
Codsashada dibadda ee 250 mg/L L-ornithine ayaa si tartiib tartiib ah u kordhisay dhererka dhirta (Jaantuska 4A), tirada laamaha geedkiiba (Jaantuska 4B), iyo tirada caleenta geedkiiba (Jaantuska 4C) ka dib 42 maalmood. Iyadoo daawada cayayaan dilaha ee ganacsiga ee Rizolex-T (50 mg/L) ay saameyn weyn ku yeelatay dhammaan xuduudaha nafaqada ee la bartay, codsiga dibadda ee 250 mg/L L-ornithine wuxuu lahaa saameynta labaad ee ugu weyn marka la barbar dhigo kantaroolka aan la daaweyn (Jaantusyada 4A–C). Dhanka kale, daawaynta L-ornithine ma lahayn saameyn muhiim ah oo ku saabsan waxa ku jira midabada sawir-samaynta chlorophyll a (Jaantuska 4D) iyo chlorophyll b (Jaantuska 4E), laakiin si yar ayay u kordhisay wadarta guud ee carotenoid-ka (0.56 ± 0.03 mg/g fr wt) marka la barbar dhigo xakamaynta taban (0.44 ± 0.02 mg/g fr wt) iyo xakamaynta togan (0.46 ± 0.02 mg/g fr wt; Sawirka 4F). Guud ahaan, natiijooyinkani waxay muujinayaan in L-ornithine uusan sun ku ahayn digirta la daweeyay xitaa waxay kicin kartaa koritaankooda.
Jaantuska 4. Saamaynta codsiga L-ornithine ee dibadda ku yimaada sifooyinka koritaanka iyo midabada sawir-samaynta ee caleenta digirta ee uu ku dhacay Sclerotinia sclerotiorum xaaladaha aqalka dhirta lagu koriyo. (A) Dhererka dhirta (cm), (B) Tirada laamaha geedkiiba, (C) Tirada caleenta geedkiiba, (D) Chlorophyll a (mg g-1 fr wt), (E) Chlorophyll b (mg g-1 fr wt), (F) Wadarta guud ee kaartoonotiga (mg g-1 fr wt). Qiimayaashu waa celceliska ± SD ee shan nuqul oo bayooloji ah (n = 5). Xarfaha kala duwan waxay muujinayaan kala duwanaansho tirakoob ahaan muhiim ah oo u dhexeeya daawaynta (p < 0.05).
In situ histochemical genetation ee noocyada oksijiinta falgalka ah (ROS; oo loo muujiyey hydrogen peroxide [H2O2]) iyo xagjirrada xorta ah (oo loo muujiyey anions superoxide [O2•−]) ayaa shaaca ka qaaday in isticmaalka dibadda ee L-ornithine (250 mg/L) uu si weyn u yareeyay ururinta H2O2 (96.05 ± 5.33 nmol.g−1 FW; Jaantuska 5A) iyo O2•− (32.69 ± 8.56 nmol.g−1 FW; Jaantuska 5B) marka la barbar dhigo ururinta labada dhir ee cudurka qaba ee aan la daaweyn (173.31 ± 12.06 iyo 149.35 ± 7.94 nmol.g−1 FW, siday u kala horreeyaan) iyo dhirta lagu daaweeyay 50 mg/L oo ah sunta cayayaanka ee ganacsiga ee Rizolex-T (170.12 ± 9.50 iyo 157.00 ± 7.81 nmol.g−1 fr wt, siday u kala horreeyaan) 72 saacadood. Heerar sare oo H2O2 iyo O2•− ah oo lagu ururiyay hpt (Jaantuska 5A, B). Sidoo kale, tijaabada malondialdehyde (MDA) ee ku salaysan TCA waxay muujisay in dhirta digirta ee uu ku dhacay S. sclerotiorum ay ku urureen heerar sare oo MDA ah (113.48 ± 10.02 nmol.g fr wt) caleemahooda (Jaantuska 5C). Si kastaba ha ahaatee, codsiga dibadda ee L-ornithine si weyn ayuu u yareeyay lipid peroxidation sida lagu caddeeyay hoos u dhaca ku yimid maadada MDA ee dhirta la daweeyay (33.08 ± 4.00 nmol.g fr wt).
Jaantuska 5. Saamaynta codsiga L-ornithine ee dibadda ku jira ee calaamadaha waaweyn ee walbahaarka oksaydhka iyo hababka difaaca antioxidant-ka ee aan enzymatic ahayn ee caleenta digirta ee uu ku dhacay S. sclerotiorum 72 saacadood ka dib caabuqa marka lagu jiro xaaladaha aqalka dhirta lagu koriyo. (A) Haydarojiin peroxide (H2O2; nmol g−1 FW) 72 hpt, (B) anion superoxide (O2•−; nmol g−1 FW) 72 hpt, (C) malondialdehyde (MDA; nmol g−1 FW) 72 hpt, (D) phenols-ka milma (mg GAE g−1 FW) 72 hpt, (E) flavonoids-ka milma (mg RE g−1 FW) 72 hpt, (F) amino acids-ka bilaashka ah ee guud (mg g−1 FW) 72 hpt, iyo (G) nuxurka proline (mg g−1 FW) 72 hpt. Qiimayaashu waxay matalaan celceliska ± heerka leexashada (celceliska ± SD) ee 5 nuqul oo bayooloji ah (n = 5). Xarfo kala duwan ayaa tilmaamaya kala duwanaansho tirakoob ahaan muhiim ah oo u dhexeeya daawaynta (p < 0.05).