摘""要:云南怒江干熱河谷流域是小??Х龋–offea"arabica"L.)種植的理想?yún)^(qū)域,但是咖啡葉部病害的持續(xù)發(fā)生,對咖啡樹的健康、產(chǎn)量和品質(zhì)造成了嚴重影響,給當(dāng)?shù)剞r(nóng)業(yè)生產(chǎn)帶來一定損失。為了有效防控這些病害,本研究采集怒江干熱河谷流域不同咖啡種植地的典型病葉樣本,對咖啡葉部病害進行病原菌分離與鑒定。通過病原分離、純化、柯赫氏法則驗證、形態(tài)學(xué)觀察、ITS基因序列比對分析,鑒定到7個致病菌株,分別為Lu"11-2、Lu"9-1、Xin"5-2、Xin"7-5、Xin"8-2、Xin"15-2和Lu"23-1,其中Lu"11-2、Lu"9-1、Xin"5-2和Xin"7-5鑒定為鏈格孢菌(Alternaria"sp.),Xin"15-2和Lu"23-1菌株鑒定為炭疽菌(Colletotrichum"sp.),Xin"8-2菌株鑒定為擬盤多毛孢菌(Pestalotiopsis"sp.)。該地區(qū)的咖啡葉部病害主要致病菌有3類:鏈格孢菌、擬盤多毛孢菌和炭疽菌,其中鏈格孢菌、擬盤多毛孢菌為咖啡褐斑病的主要致病菌,炭疽菌為咖啡炭疽病的致病菌。研究結(jié)果不僅為深入了解云南怒江干熱河谷流域的咖啡葉部病害類型提供科學(xué)依據(jù),還為該地區(qū)咖啡病害的有效防控提供理論支持,對當(dāng)?shù)氐目Х确N植業(yè)具有重要的指導(dǎo)意義,也為其他地區(qū)的咖啡病害防控提供參考。
關(guān)鍵詞:小??Х?;葉部病害;鏈格孢菌;擬盤多毛孢菌;炭疽菌中圖分類號:S31""""""文獻標志碼:A
Isolation"and"Identification"of"Pathogens"Causing"Foliar"Diseases"in"Coffea"arabica"in"the"Nujiang"Dry-hot"Valley,"Yunnan,"China
WANG"Na,"YAN"Wei,"MA"Dongxiao,"YANG"Yang,"HU"Faguang,"FU"Xingfei,"LI"Guiping,"YU"Haohao,"LOU"Yuqiang*,"LI"Yanan*
Institute"of"Tropical"and"Subtropical"Cash"Crops,"Yunnan"Academy"of"Agricultural"Sciences,"Baoshan,"Yunnan"678000,"China
Abstract:"The"Nujiang"Dry-hot"Valley"region"in"Yunnan"is"an"ideal"area"for"the"cultivation"of"Arabica"coffee"(Coffea"arabica"L.)."However,"the"continuous"occurrence"of"coffee"leaf"diseases"has"a"significant"impact"on"the"health,"yield"and"quality"of"coffee"trees,"causing"considerable"losses"to"local"agricultural"production."To"effectively"manage"the"diseases,"this"study"focused"on"the"isolation"and"identification"of"the"pathogens"associated"with"coffee"leaf"diseases"in"the"Nujiang"Dry-hot"Valley"region."Typical"diseased"leaf"samples"were"collected"from"various"coffee"plantations,"and"through"pathogen"isolation,"purification,"verificationnbsp;using"Koch’s"postulates,"morphological"observation"and"ITS"gene"sequence"analysis."Seven"pathogenic"strains,"Lu"11-2,"Lu"9-1,"Xin"5-2,"Xin"7-5,"Xin"8-2,"Xin"15-2"and"Lu"23-1,"were"identified."Lu"11-2,"Lu"9-1,"Xin"5-2"and"Xin"7-5"were"identified"as"Alternaria"sp.,"Xin"15-2"and"Lu"23-1"were"identified"as"Colletotrichum"sp.,"and"Xin"8-2"was"identified"as"Pestalotiopsis"sp."The"primary"pathogens"causing"coffee"leaf"diseases"in"this"region"could"be"categorized"into"three"types,"Alternaria"sp.,"Pestalotiopsis"sp."and"Colletotrichum"sp.."Specifically,"Alternaria"sp."and"Pestalotiopsis"sp."were"the"primary"pathogens"responsible"for"coffee"leaf"spot"disease,"while"Colletotrichum"sp."was"the"causative"agent"of"coffee"anthracnose"disease."The"findings"of"this"study"would"not"only"provide"a"scientific"basis"for"a"deeper"understanding"of"the"types"of"coffee"leaf"diseases"in"the"Nujiang"Dry-hot"Valley"region"of"Yunnan"but"also"offer"theoretical"support"for"effective"disease"management"in"the"area."This"research"plays"a"significant"guiding"role"for"local"coffee"cultivation"and"serves"as"a"reference"for"disease"control"in"coffee-growing"regions"elsewhere.
Keywords:"Coffea"arabica"L.;"leaf"diseases;"Alternaria"sp.;"Pestalotiopsis"sp.;"Colletotrichum"sp.
DOI:"10.3969/j.issn.1000-2561.2025.02.011
咖啡作為全球經(jīng)濟作物的重要支柱,影響著許多國家的農(nóng)業(yè)經(jīng)濟和農(nóng)民生計[1-2]。小??Х龋–offea"arabica"L.)隸屬于茜草科咖啡屬,因其優(yōu)良的品質(zhì)和經(jīng)濟價值而受到廣泛關(guān)注[2]。其生長對氣候條件有較高的要求,尤其是溫暖濕潤的環(huán)境最為適宜[3],國際咖啡種植標準指出,咖啡適宜種植在海拔500~2000"m、溫度15~25"℃、年降雨量1500~2000"mm的具有火山灰質(zhì)土壤的地區(qū)[4]。云南怒江干熱河谷流域以其適宜的海拔、充足的陽光、適量的降水量和適宜的溫度,為小??Х鹊纳L提供了優(yōu)越的天然條件[5-6]。然而,干熱河谷獨特的氣候條件也帶來了病害發(fā)生及其擴散的復(fù)雜性和嚴重性。
云南怒江干熱河谷流域多以山地、坡地為主,生物多樣性豐富,咖啡葉部病害多發(fā),葉部病害的發(fā)生不僅對咖啡樹的健康造成嚴重威脅,還直接影響咖啡的產(chǎn)量和品質(zhì),導(dǎo)致咖啡產(chǎn)業(yè)的經(jīng)濟損失。常見的咖啡葉部病害主要有咖啡葉銹病、褐斑病、炭疽病、煤煙病等[7]。據(jù)報道,咖啡葉銹病由專性生物營養(yǎng)真菌Hemileia"vastatrix引起,是一種世界性的毀滅性病害,導(dǎo)致咖啡種植園的產(chǎn)量嚴重下降和經(jīng)濟受損[8-9]。褐斑病可由多種病原菌引起,通常表現(xiàn)為葉片上出現(xiàn)褐色斑點,這些斑點擴大并發(fā)展成具有深棕色邊緣的同心環(huán),最后壞死斑點蔓延到整片咖啡葉,導(dǎo)致咖啡葉卷曲和脫落,使幼樹大量死亡[10-11]。炭疽病主要感染咖啡的嫩葉、葉柄、枝條和果實,引發(fā)葉片脫落、枝條干枯及果實腐爛,嚴重損害咖啡的產(chǎn)量和品質(zhì)[12-14]。煤煙病則是由昆蟲分泌蜜露誘發(fā)的病害,導(dǎo)致咖啡葉面出現(xiàn)灰黑色的斑塊或霉層[7],這些葉部病害的發(fā)生均不同程度地影響了咖啡植株的正常生長發(fā)育。
研究咖啡葉部病害病原菌種類及其生物學(xué)特性,以及它們與環(huán)境因素的關(guān)系是深入了解和應(yīng)對咖啡病害的關(guān)鍵。本研究旨在對怒江干熱河谷流域的小??Х热~部病害進行系統(tǒng)地病原分離與鑒定,探究病原菌種類,明確致病菌種。以期為該地區(qū)咖啡病害的有效管理和防治提供科學(xué)依據(jù),并提出切實可行的防控建議和對策,從而改善咖啡質(zhì)量和提升咖啡生產(chǎn)效益。
1.1""材料
1.1.1""供試菌株""于2023年7月采自云南省保山市潞江鎮(zhèn)辛家山咖啡種植基地(海拔962.71"m,24°57′30″N,98°53′56″E)、瀘水市咖啡種植區(qū)域(海拔1079.6"m,25°48′25″N,98°51′3″E)的小??Х绕贩N鐵皮卡(Typica)病葉,分離純化獲得病原菌。
1.1.2""培養(yǎng)基""馬鈴薯葡萄糖瓊脂(PDA)培養(yǎng)基干粉購自廣東環(huán)凱微生物科技有限公司,按使用方法配制PDA培養(yǎng)基。
1.2""方法
1.2.1""病原菌分離與純化""采用組織分離法分離病原菌[15],將采摘收集到的病葉置于超凈工作臺,用滅菌剪刀將病健交界處的組織剪成5"mm×"5"mm的小塊,用滅菌鑷子將組織塊置于75%醫(yī)用酒精中浸泡1"min,后用滅菌離子水漂洗1"min,重復(fù)漂洗3次,取出置于滅菌吸水紙上吸干水分,然后將其接種到PDA平板上,每皿接種4塊,于28"℃培養(yǎng)箱中倒置培養(yǎng)。每天觀察病原菌的生長情況,待組織周圍長出菌絲后,挑取菌絲尖端進行純化,重復(fù)純化3次左右,待病原菌生長一致即可保存菌株,備用。
1.2.2""致病性測定""采用離體接菌的方法,采集健康無病的鐵皮卡咖啡葉片,先用滅菌棉蘸取75%醫(yī)用酒精擦拭咖啡葉面消毒,再用滅菌水擦拭一遍葉片,將葉片置于保鮮盒內(nèi)。用無菌接種針刺傷葉脈一側(cè)的葉片,另一側(cè)不刺傷。將經(jīng)純化的病原菌菌餅接種到葉面,以菌絲面接觸葉面,以PDA培養(yǎng)基作對照,每個菌株接種3片葉,3次重復(fù)。葉柄處用濕棉花包住,28"℃保濕條件下培養(yǎng)7"d,并于接種后3、5、7"d對葉片進行拍照,使用Image"J軟件測量葉片病斑面積。待葉片發(fā)病后進行柯赫氏法則驗證,并將發(fā)病組織進行分離純化。
1.2.3""病原菌形態(tài)學(xué)觀察""將純化好的病原菌接種到PDA平板上,每個菌株接3皿,3次重復(fù),置于28"℃培養(yǎng)箱中倒置培養(yǎng)7"d,于接種后3、5、7"d觀察病原菌的菌落生長情況,測量菌落直徑并計算生長速率。采用德國Leica"DM5000B顯微鏡觀察菌絲和分生孢子形態(tài),對病原菌進行形態(tài)鑒定,并隨機選取50個分生孢子測量大小。
1.2.4""病原菌分子鑒定""將上述病原菌送樣,委托昆明擎科生物有限公司進行DNA測序,測序引物:ITS1(5¢-TCCGTAGGTGAACCTGCGG-"3¢),ITS4(5¢-TCCTCCGCTTATTGATATGC-3¢)。將測序結(jié)果上傳至NCBI進行BLAST同源分析,下載與分離病原菌親緣關(guān)系相近的菌株序列,利用MEGA"11.0軟件構(gòu)建系統(tǒng)發(fā)育樹。
1.3""數(shù)據(jù)處理
利用IBM"SPSS"Statistics"25軟件進行數(shù)據(jù)統(tǒng)計與分析,采用GraphPad"Prism"v8.0軟件制圖。
2.1""咖啡葉部病害的田間癥狀
咖啡葉部病害一年四季均可發(fā)生,葉部病害的發(fā)生既會導(dǎo)致咖啡生長受阻,又會導(dǎo)致產(chǎn)量降低,從而影響咖啡品質(zhì)。發(fā)現(xiàn)的咖啡葉部病害田間癥狀表現(xiàn)如圖1,發(fā)病初期葉片出現(xiàn)小黃點,然后慢慢擴大為圓形或近圓形的褐色病斑,病斑邊界明顯,中心為灰白色,周圍有黃色暈圈(圖1A,圖1B);發(fā)病后期病斑連片形成大病斑,呈灰白色,病斑邊緣有黑色點狀物(圖1C,圖1D)。
2.2""咖啡葉部病害病原菌的致病性
采用組織分離法對采集到的病葉病原進行分離純化,得到45個菌株,根據(jù)菌落形態(tài)特征和生長情況從中選取9株進行離體葉片回接試驗,每個菌株接種3片葉,3次重復(fù),接種時分別做刺
傷和不刺傷處理。結(jié)果如圖2所示,接種4"d后,有7個菌株出現(xiàn)明顯的發(fā)病癥狀,Xin"1-3和Xin"12-1菌株未發(fā)病。測量發(fā)病菌株的病斑面積及統(tǒng)計發(fā)病率(表1),發(fā)現(xiàn)Xin"5-2菌株的病斑面積最大,為(1.47±0.08)cm2,且發(fā)病率最高,為100%;Xin"15-2菌株次之,病斑面積為(0.79±0.23)cm2;而菌株Lu"11-2的病斑面積最小,僅為(0.13±"0.05)cm2,且發(fā)病率最低,為52.78"%。對發(fā)病的7個葉片進行病原分離純化,得到與原接種菌株形態(tài)特征和培養(yǎng)性狀一致的病原菌,符合柯赫氏法則,說明接種菌株是致病菌。
2.3""咖啡葉部病害病原菌的形態(tài)學(xué)特征
2.3.1""病原菌菌落形態(tài)特征及生長速率""將符合柯赫氏法則的病原菌菌株接種到PDA培養(yǎng)基上,培養(yǎng)7"d后觀察菌落形態(tài)特征及生長情況。結(jié)果發(fā)現(xiàn)這些菌株在某些特征上存在共性,同時也表現(xiàn)出一定的差異性。所有菌株的菌落均為同心圓形態(tài),邊緣菌絲為白色且菌絲形態(tài)相似;在菌落顏色和生長速率方面有差異,如Lu"11-2、Lu"9-1、Xin"5-2和Xin"7-5菌株的菌落顏色由內(nèi)到外逐漸變淡,由深褐色到淺褐色再到白色,而Xin"8-2、Xin"15-2和Lu"23-1菌株的菌絲則呈現(xiàn)灰白色或白色。此外,7個菌株的菌落生長速率略有差異,其中Xin"15-2菌株的菌落生長速率顯著高于其他菌株,其5"d的菌落直徑達(7.82±0.31)cm,平均生長速率為(1.65±0.09)cm/d,而Lu"9-1菌株生長速率較慢,5"d的菌落直徑為(4.10±0.15)cm,平均生長速率為(0.74±0.04)cm/d(圖3,表2)。
2.3.2""病原菌的分生孢子及菌絲形態(tài)特征""7個菌株的分生孢子及菌絲形態(tài)如圖4,Lu"11-2菌株的分生孢子呈卵形,有隔,橫隔1~3個,縱隔0~1個,分生孢子平均大小為(24.60±4.08)μm×(11.39±"2.27)μm,長寬比為2.18±0.17,菌絲直徑為(3.78±0.80)μm;Lu"9-1菌株的分生孢子呈梨形,有隔,橫隔1~2個,縱隔0~1個,分生孢子平均大小為(24.87±6.07)μm×(11.17±1.21)μm,長寬比為2.23±0.46,菌絲直徑為(4.10±0.77)μm;Xin"5-2的分生孢子呈卵形或梨形,有隔,橫隔1~3個,縱隔0~2個,分生孢子平均大小為(26.44±2.71)μm×"(12.51±3.27)μm,長寬比為2.24±0.53,菌絲直徑為(5.31±0.83)μm;Xin"7-5的分生孢子呈倒梨形,有隔,橫隔1~2個,縱隔0~2個,分生孢子平均大小(31.51±6.20)μm×(14.04±2.97)μm,長寬比為2.29±0.41,菌絲直徑為(3.61±0.87)μm;Xin"8-2的分生孢子共5個細胞,4分隔,基部細胞顏色較淡,倒三角形或倒圓錐形,中間3個細胞顏色偏深,翁形,頂端細胞顏色偏淡,圓錐形或三角形,頂端具1根附屬絲,基部具1~3根附屬絲,分生孢子平均大小為(27.43±2.87)μm×(6.52±0.77)μm,長寬比為4.26±0.60,菌絲直徑為(3.52±0.77)μm;Xin"15-2的分生孢子呈長圓柱形,單胞,分生孢子平均大小為(15.95±4.70)μm×(4.94±1.03)μm,長寬比為3.41±1.31,菌絲直徑為(5.31±0.83)μm;Lu"23-1的分生孢子呈長圓柱形,單胞,分生孢子平均大小為(18.38±2.88)μm×(5.04±0.81)μm,長寬比為3.69±0.56,菌絲直徑為(3.70±0.67)μm。
2.4""咖啡葉部病害病原菌的分子鑒定
將7個菌株的ITS序列測序結(jié)果進行整理,分別得到不同大小的目標片段,Lu"11-2菌株的ITS片段大小為539"bp,Lu"9-1的為539"bp,Xin"5-2的為580"bp,Xin"7-5為575"bp,Xin"8-2為575"bp,Xin"15-2為583"bp,Lu"23-1為587"bp。將7個菌株的ITS序列分別在NCBI上進行BLAST比對,并下載同源性較高的代表菌株序列,以AB496639.1"Methanobacterium"alcaliphilum作外群聯(lián)合構(gòu)建系統(tǒng)發(fā)育進化樹(圖5)。Lu"11-2、Lu"9-1、Xin"5-2、Xin"7-5菌株與鏈格孢菌(Alternaria"sp.)以100%的支持率聚為一支,其中Lu"11-2和Lu"9-1鑒定為交鏈格孢(A."alternata),Xin"5-2鑒定為極細鏈格孢(A."tenuissima),Xin"7-5鑒定為鏈格孢屬(Alternaria"sp.);Xin"15-2和Lu"23-1菌株與炭疽菌(Colletotrichum"sp.)聚為一支,其中Xin"15-2鑒定為果生炭疽菌(C."fructicola),Lu"23-1則鑒定為博寧炭疽菌(C."boninense);菌株Xin"8-2與擬盤多毛孢菌(Pestalotiopsis"sp.)聚為一支,能明顯與其他種區(qū)分。結(jié)合各菌株的形態(tài)學(xué)特征,將菌株Lu"11-2、Lu"9-1、Xin"5-2、Xin"7-5鑒定為鏈格孢菌,Xin"15-2和Lu"23-1則鑒定為炭疽菌,Xin"8-2鑒定為擬盤多毛孢菌。
在怒江干熱河谷流域的咖啡種植區(qū),咖啡葉部病害發(fā)生頻繁且多樣,為明確該區(qū)域咖啡葉部病害的種類及其病原,采集不同咖啡種植地的典型病葉樣本,利用形態(tài)學(xué)和分子生物學(xué)方法,確定了咖啡葉部的病原菌分別是鏈格孢菌(Alternaria"sp.)、擬盤多毛孢菌(Pestalotiopsis"sp.)和炭疽菌(Colletotrichum"sp.)。
鏈格孢菌(Alternaria"sp.)是一種分布廣泛的植物病原真菌,具有廣泛的寄主范圍,能夠侵染多種植物,造成嚴重的經(jīng)濟損失。已有研究發(fā)現(xiàn),交鏈格孢(A."alternata)可以引起蘋果葉斑病,導(dǎo)致蘋果葉片上形成棕色或褐色病斑,嚴重時導(dǎo)致葉片干枯脫落[16-17];交鏈格孢也是繡球花葉斑病的致病菌之一,在繡球花葉片上的癥狀表現(xiàn)為深褐色病斑,對植物的觀賞價值造成嚴重影響[18];覃茜等[19]從蘇鐵葉片上分離出引起褐斑病的病原真菌巴斯基鏈格孢(A."burnsii),并證實該病原菌能引起多種蘇鐵褐斑??;此外,鏈格孢菌也是龍牙百合褐斑病的主要致病菌[20-21]。2024年,ZHANG等[10]報道了由交鏈格孢引起的咖啡葉斑病。本研究對采集到的咖啡病葉進行病原菌分離鑒定,鑒定到4個菌株屬于鏈格孢菌,與ZHANG等[10]的研究結(jié)果相似,表明鏈格孢菌是咖啡褐斑病的致病菌之一,且咖啡褐斑病是怒江干熱河谷流域咖啡種植區(qū)域的主要葉部病害之一。
擬盤多毛孢菌(Pestalotiopsis"sp.)是一類廣泛分布于自然界中的微生物,它們與多種植物病害有關(guān),既可以作為病原菌導(dǎo)致植物病害,也可以作為內(nèi)生菌或腐生菌存在于植物體內(nèi)。擬盤多毛孢作為內(nèi)生菌大多被研究者用來探究真菌的次級代謝產(chǎn)物等,如紅樹林內(nèi)生擬盤多毛孢(P."microspora)可以通過生物合成途徑產(chǎn)生抗腫瘤的化合物[22];耐鋁樹種千年桐的內(nèi)生鏈格孢可吸收環(huán)境中的Al3+,提高宿主植物對鋁脅迫的耐受性[23]。2020年,安小麗等[24]從貴州省茶樹葉片上分離出茶擬盤多毛孢(P."theae),該病原菌可引起茶葉斑?。焕罹У萚25]報道了Neopestalotiopsis"sonneratae會引起檳榔葉斑病,初期檳榔葉片出現(xiàn)黑褐色小病斑,隨著病斑的擴展,最終導(dǎo)致葉片干枯倒垂;袁榕等[26]在華中五味子上鑒定出5種不同的擬盤多毛孢屬真菌,并且這些菌株具有較強的致病性;還有其他研究表明,擬盤多毛孢還可引起草莓根腐病[27]、番石榴環(huán)斑病[28]、松樹赤枯病[29]等。本研究對咖啡褐斑病進行病原菌分離鑒定,鑒定到1個菌株屬于擬盤多毛孢菌,并且符合柯赫氏法則,說明擬盤多毛孢菌是咖啡褐斑病的致病菌之一。
炭疽菌(Colletotrichum"sp.)作為一種重要的植物病原菌,引起了全球的廣泛關(guān)注,它能夠侵染多種農(nóng)作物和經(jīng)濟作物,導(dǎo)致炭疽病的發(fā)生,對農(nóng)業(yè)生產(chǎn)造成嚴重損失[30]。近年來,隨著對炭疽菌的深入研究,研究者們在炭疽菌的病原鑒定、生物學(xué)特性以及病害防治方面取得了一系列進展。炭疽菌的研究涵蓋了多個作物,包括碭山酥梨、咖啡、油茶等。研究發(fā)現(xiàn),不同地區(qū)的炭疽菌種類存在差異,同一作物可能受到多種炭疽菌的侵染,如,碭山酥梨炭疽病的主要病原菌為果生炭疽菌(C."fructicola)[31];而油茶炭疽病的病原菌則包括果生炭疽菌和膠孢炭疽菌(C."gloeosporioides)[32];寧夏地區(qū)葡萄炭疽病的主要病原菌為毀滅炭疽菌(C."destructivum)、果生炭疽菌和暹羅炭疽菌(C."siamense)[33];然而,導(dǎo)致重慶多花黃精炭疽病的病原菌有5種,分別為松針炭疽菌(C.nbsp;fioriniae)、博寧炭疽菌(C."boninense)、膠孢炭疽菌(C."gloeosporioides)、果生炭疽菌和冬麥炭疽菌(C.liriopes)[34]。國內(nèi)外已經(jīng)鑒定出多種可侵染咖啡的炭疽菌,CRISTóBAL-"MARTíNEZ等[35]在發(fā)病的咖啡植株上鑒定到5種炭疽菌,2019年,CAO等[36]在海南咖啡種植區(qū)鑒定到8種炭疽菌,這8種炭疽菌中也包含了本研究中鑒定到的果生炭疽菌和博寧炭疽菌。這些研究表明咖啡炭疽病在全球分布廣泛且危害嚴重。
本研究對怒江干熱河谷流域的小??Х热~部病害進行病原菌分離鑒定,鑒定到小??Х热~部病害的主要致病菌為鏈格孢菌(Alternaria"sp.)、擬盤多毛孢菌(Pestalotiopsis"sp.)和炭疽菌(Colletotrichum"sp.)。研究結(jié)果可為該地區(qū)咖啡病害的防控提供理論支持,對農(nóng)業(yè)生產(chǎn)具有重要意義。隨著研究的不斷深入,期待未來能夠開發(fā)出更多高效、環(huán)保的病害防控技術(shù),為咖啡產(chǎn)業(yè)的可持續(xù)發(fā)展做出貢獻。
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