林业科学
林業科學
임업과학
SCIENTIA SILVAE SINICAE
2015年
4期
126-133
,共8页
余贤美%侯长明%王洁%王海荣%安淼%艾呈祥
餘賢美%侯長明%王潔%王海榮%安淼%艾呈祥
여현미%후장명%왕길%왕해영%안묘%애정상
牛心柿%柿树炭疽病%病原分离与鉴定%rDNA-ITS序列分析%离体接种试验
牛心柿%柿樹炭疽病%病原分離與鑒定%rDNA-ITS序列分析%離體接種試驗
우심시%시수탄저병%병원분리여감정%rDNA-ITS서렬분석%리체접충시험
beef heart persimmon%persimmon anthracnose%pathogen isolation and identification%rDNA-ITS sequence analysis%in vitro inoculation
【目的】对侵染牛心柿引起炭疽病的病原菌进行分离鉴定，分析其致病性，初步构建其侵染谱，为柿树病害的病原诊断和防治提供理论依据和技术支持。【方法】通过组织分离法和单孢分离法从感病牛心柿的果实、叶片和嫩梢中分离病原菌，根据菌落和孢子形态特征及其 rDNA-ITS 序列分析进行种类鉴定，采用以菌饼进行伤口和非伤口接种、孢子悬浮液进行喷雾接种的离体接种试验分析其致病性。【结果】从感病牛心柿果实、叶片和嫩梢中分离获得9个病原菌分离物，菌落及孢子形态特征显示均为炭疽菌。以真菌转录间隔区通用引物 ITS6和 ITS4为引物，以菌丝总 DNA 为模板，扩增获得病原菌 rDNA-ITS基因片段，经 rDNA-ITS序列分析和系统进化树的构建，将该病原物鉴定为柿树炭疽菌，将其 rDNA-ITS 基因序列提交到 GenBank 数据库(基因登录号 KF010811)。牛心柿炭疽病菌和次郎炭疽病菌对不同品种柿树的致病性相同，均无差异性，对果实的致病性比叶片强；接种7天后，叶片无病斑或病斑较小，菌饼无伤口接种几乎不发病，在叶片上不产生病斑，果实上病斑较小；不同的接种方式在果实和叶片上产生的病斑大小为：菌饼伤口接种＞孢子悬浮液接种＞菌饼无伤口接种；牛心柿炭疽病菌和次郎炭疽菌对桃、苹果、梨等果树的叶片和果实均不致病。【结论】柿炭疽病菌具有一定的专性寄生性，对除柿树外的其他果树不致病，且在遗传上有一定的保守性，因地理距离产生的变异很小，这些特性对于柿炭疽病的防治具有重要意义，在一定程度上可以避免或削弱因病菌的遗传变异而产生的抗药性，从而有利于炭疽病的防治。在柿树有伤口时最易侵染致病，雨天或高湿度的情况下经孢子侵染致病的可能性居其次。在田间管理时注意避免造成柿树伤口感染，或者避免在下雨天气进行修剪，可最大程度地减轻或消除柿树炭疽病菌的侵染以及柿炭疽病的发生和流行，从而在一定程度上解决该病流行而造成的减产问题。
【目的】對侵染牛心柿引起炭疽病的病原菌進行分離鑒定，分析其緻病性，初步構建其侵染譜，為柿樹病害的病原診斷和防治提供理論依據和技術支持。【方法】通過組織分離法和單孢分離法從感病牛心柿的果實、葉片和嫩梢中分離病原菌，根據菌落和孢子形態特徵及其 rDNA-ITS 序列分析進行種類鑒定，採用以菌餅進行傷口和非傷口接種、孢子懸浮液進行噴霧接種的離體接種試驗分析其緻病性。【結果】從感病牛心柿果實、葉片和嫩梢中分離穫得9箇病原菌分離物，菌落及孢子形態特徵顯示均為炭疽菌。以真菌轉錄間隔區通用引物 ITS6和 ITS4為引物，以菌絲總 DNA 為模闆，擴增穫得病原菌 rDNA-ITS基因片段，經 rDNA-ITS序列分析和繫統進化樹的構建，將該病原物鑒定為柿樹炭疽菌，將其 rDNA-ITS 基因序列提交到 GenBank 數據庫(基因登錄號 KF010811)。牛心柿炭疽病菌和次郎炭疽病菌對不同品種柿樹的緻病性相同，均無差異性，對果實的緻病性比葉片彊；接種7天後，葉片無病斑或病斑較小，菌餅無傷口接種幾乎不髮病，在葉片上不產生病斑，果實上病斑較小；不同的接種方式在果實和葉片上產生的病斑大小為：菌餅傷口接種＞孢子懸浮液接種＞菌餅無傷口接種；牛心柿炭疽病菌和次郎炭疽菌對桃、蘋果、梨等果樹的葉片和果實均不緻病。【結論】柿炭疽病菌具有一定的專性寄生性，對除柿樹外的其他果樹不緻病，且在遺傳上有一定的保守性，因地理距離產生的變異很小，這些特性對于柿炭疽病的防治具有重要意義，在一定程度上可以避免或削弱因病菌的遺傳變異而產生的抗藥性，從而有利于炭疽病的防治。在柿樹有傷口時最易侵染緻病，雨天或高濕度的情況下經孢子侵染緻病的可能性居其次。在田間管理時註意避免造成柿樹傷口感染，或者避免在下雨天氣進行脩剪，可最大程度地減輕或消除柿樹炭疽病菌的侵染以及柿炭疽病的髮生和流行，從而在一定程度上解決該病流行而造成的減產問題。
【목적】대침염우심시인기탄저병적병원균진행분리감정，분석기치병성，초보구건기침염보，위시수병해적병원진단화방치제공이론의거화기술지지。【방법】통과조직분리법화단포분리법종감병우심시적과실、협편화눈소중분리병원균，근거균락화포자형태특정급기 rDNA-ITS 서렬분석진행충류감정，채용이균병진행상구화비상구접충、포자현부액진행분무접충적리체접충시험분석기치병성。【결과】종감병우심시과실、협편화눈소중분리획득9개병원균분리물，균락급포자형태특정현시균위탄저균。이진균전록간격구통용인물 ITS6화 ITS4위인물，이균사총 DNA 위모판，확증획득병원균 rDNA-ITS기인편단，경 rDNA-ITS서렬분석화계통진화수적구건，장해병원물감정위시수탄저균，장기 rDNA-ITS 기인서렬제교도 GenBank 수거고(기인등록호 KF010811)。우심시탄저병균화차랑탄저병균대불동품충시수적치병성상동，균무차이성，대과실적치병성비협편강；접충7천후，협편무병반혹병반교소，균병무상구접충궤호불발병，재협편상불산생병반，과실상병반교소；불동적접충방식재과실화협편상산생적병반대소위：균병상구접충＞포자현부액접충＞균병무상구접충；우심시탄저병균화차랑탄저균대도、평과、리등과수적협편화과실균불치병。【결론】시탄저병균구유일정적전성기생성，대제시수외적기타과수불치병，차재유전상유일정적보수성，인지리거리산생적변이흔소，저사특성대우시탄저병적방치구유중요의의，재일정정도상가이피면혹삭약인병균적유전변이이산생적항약성，종이유리우탄저병적방치。재시수유상구시최역침염치병，우천혹고습도적정황하경포자침염치병적가능성거기차。재전간관리시주의피면조성시수상구감염，혹자피면재하우천기진행수전，가최대정도지감경혹소제시수탄저병균적침염이급시탄저병적발생화류행，종이재일정정도상해결해병류행이조성적감산문제。
Objective]This study aims to isolate and identify a pathogen which infects the persimmon plants and causes persimmon anthracnose,then investigate the pathogenicity so as to analyze its infect spectrum,and provide the theoretical basis and technical assistance for the pathogenic diagnosis and disease control of persimmon anthracnose in persimmon productivity.[Methods]The pathogen was isolated from anthracnose lesions on beef heart persimmon fruits,leaves and twigs in the persimmon orchards in Zhujiapo town,Linju county,Zhuge town,Yishui county and Wanjishan experimental station of Shandong Institute of Pomology with tissue separation and single spore separation, and identified by morphological characteristics and rDNA-ITS sequence analysis. The pathogenicity was investigated via in vitro inoculation test by disk-wound inoculation,disk-no-wound inoculation and spore-suspension inoculation.[Results]Nine isolates were obtained from persimmon fruits,leaves and twigs via tissue separation and single spore separation. The analysis of morphological characteristics showed that the 9 isolates belong to genus Colletotrichum. Then rDNA-ITS sequence analysis, via PCR amplification with the universal primers of ITS6 and ITS4 ,showed that the 9 isolates obtained were in full accord with the morphological observation,and the nucleotide sequence of rDNA-ITS gene (GenBank accession No. KF010811)shared 100%, 100% and 99. 8% identity with that of anthracnose pathogens on Jiro persimmon ( JQ957543 ) ,‘Wuheshi’persimmon in Zhejiang ( AY787483 ) and New Zealand isolate ( GQ329690 ) respectively. The phylogenetic analysis based on ITS sequences showed that beef heart persimmon isolate was located at the same branch of phylogenetic tree with the isolates of Jiro persimmon (JQ957543),‘Wuheshi’persimmon in Zhejiang (AY787483,AY791890) and New Zealand persimmon ( GQ329687 , GQ329688 and GQ329690 ) , which indicated that the pathogen causing the anthracnose disease of beef heart persimmon could be identified as Colletotrichum horii. The in vitro inoculation test showed that there was equal pathogenicity on different persimmon cultivars between‘Niuxinshi ’ isolate and‘Cilang ’ isolate of C. horii,and they have no pathogenicity on peach,apple and pear. On the 7th day after inoculation,the lesions on persimmon fruits were much larger than that on leaves, there were no symptoms on leaves via disk-no-wound inoculation,and lesions were small via spore suspension inoculation. The lesion size on fruits and leaves via different inoculation was inthe following order of:disk-wound inoculation > spore-suspension inoculation > disk-no-wound inoculation. [Conclusions]C. horii possessed a certain degree of obligate parasitic and genetic conservatism,with no pathogenicity to other fruit trees besides persimmon and little variation due to the geographic distance. These characteristics were significant for the control of persimmon anthracnose,which could play an important role in avoiding and weakening the resistance to chemicals due to the genetic variation of the pathogen,and thus facilitate the control of persimmon anthracnose,and guarantee quality of fruits and increase persimmon productivity. The wound infection by the pathogen will occurred most easily,followed by spore infecton in raining days or under the high humidity condition,which indicated that the pathogen infection, the occurrence and popularity of persimmon anthracnose will be avoided or eliminated largely by reducing persimmon wound in the field management and avoiding pruning in raining days. Thus,the measures would to some extent solve the problem of lost production in persimmon due to the popularity of persimmon anthracnose.
