摘要
【目的】明确引起琯溪蜜柚炭疽病的刺盘孢属种类及其致病力差异,为该病害的有效防控提供科学依据。【方法】采集福建省平和县13个乡镇琯溪蜜柚炭疽病典型病样进行组织分离,利用形态学和分子生物学等方法对分离菌株进行种类鉴定及致病性研究。【结果】共获得350株刺盘孢属真菌,从中选取培养性状有较大差异的58株刺盘孢属真菌进行形态学观察和多基因(ITS、ACT、TUB2、GAPDH及GS)系统发育分析,结果表明,其分属于胶胞炭疽菌复合种下的胶孢炭疽菌(Colletotrichum gloeosporioides)和果生炭疽菌(C.fructicola)、博宁炭疽菌复合种下的喀斯特炭疽菌(C.karstii)、平头炭疽菌复合种下的平头炭疽菌(C.truncatum)、C.magnum复合种下的短孢炭疽菌(C.brevisporum)以及C.orchidearum复合种下的兰花炭疽菌(C.cliviicola)。采用叶片和枝条有伤接种孢子悬浮液法,结果表明,除兰花炭疽菌(C.cliviicola)外的其他5种刺盘孢属真菌都可使琯溪蜜柚叶片和枝条致病,但致病力存在明显差异。病原菌菌丝生长速率、附着胞形成率与致病力相关性分析发现,相关系数分别为0.3733和0.3641,表明菌丝生长速率及附着胞形成率与致病力之间均呈弱相关性。【结论】琯溪蜜柚炭疽病的病原菌有胶孢炭疽菌、果生炭疽菌、喀斯特炭疽菌、短孢炭疽菌、平头炭疽菌(C.truncatum),其中胶孢炭疽菌(C.gloeosporioides)为病原优势种,果生炭疽菌、喀斯特炭疽菌、短孢炭疽菌和平头炭疽菌是新病原,证实了福建省琯溪蜜柚炭疽病病原菌具有多样性。不同病原菌的菌丝生长速率及附着胞形成率与致病力均呈弱相关性。
【Objective】Guanximiyou pomelo(Citrus grandis)is a famous and popular Citrus species for its sweet and excellent nutrients native to Pinghe county of Fujian province,China.In Guanximiyou pomelo,anthracnose caused by Colletotrichum spp.is a serious disease limiting its production.In 2018,the disease seriously affected over 60% of Guanximiyou pomelo trees in an orchard in Pinghe county.The Guanximiyou pomelo anthracnose mainly damages leaves,twigs and fruits.In the edge,tip or middle of damaged leaves develop brown spots,which form a“V”shape then withering and falling off after the diseased leaves die.The damaged twigs show spots from the petiole and the base of axillary bud to the bottom of twigs.On fruit,symptoms appear as green,irregular and sunken lesions in the young stage,that turn to brown rot and then falling off in mature stage.This study aimed to clarify the species of Colletotrichum spp.associated with Guanximiyou pomelo anthracnose and determine the pathogenic characteristics of the pathogens,so as to provide a better acknowledge for the diversity of the pathogen species and scientific basis for the prevention and control of the disease in Fujian province.【Methods】A survey of anthracnose disease was conducted in 13 Guanximiyou pomelo orchards from 2019 to 2021 in Pinghe county,Fujian province.The leaves,twigs and fruit with symptoms were collected from the pomelo trees and were used as disease samples.The Colletotrichum spp.were isolated by the plant tissue isolation method.4×4 mm diseased tissues were surface-sterilized with 70% ethanol for 30 s,0.1%mercuric chloride for 60 s,washed three times in sterile water and dried on sterilized filter paper,then placed onto potato dextrose agar(PDA)plates and incubated under 28℃ in the dark.The single mycelium was used for purifying strains,and pure cultures were stored in PDA at 4℃.The colony characteristics,conidia morphology and appressorium for representative strains of the identified Colletotrichum spp.were recorded.The Colletotrichum spp.genomic DNA was extracted using a fungus genomic DNA extraction kit,which was identified through partial rDNA-ITS(ITS),actin(ACT),beta-tubulin(TUB2),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),and glutamine synthetase(GS)region sequence.Phylogenetic tree based on the combined ITS-ACT-TUB2-GAPDH-GS sequences was constructed by maximum like-lihood method with MEGA 7.0.The conidia suspension of 58 representative Colletotrichum spp.were used in the pathogenicity tests to inoculate twigs and leaves of Guanximiyou pomelo according to Koch’s postulate.Correlation analysis of mycelial growth rate,appressorium formation rate and pathogenicity were determined by Pearson method.【Results】A total of 350 strains with the similar morphology to Colletotrichum spp.were isolated from the collected samples infected by the Guanximiyou pomelo anthracnose disease.C.gloeosporioides species complexes,C.boninense species complexes,C.truncatum species complexes,C.magnum species complexes and C.orchidearum species complexes were identified based on morphological characteristics and ITS sequencing.Colony observation showed that there were significant differences in aerial and substrate mycelia’s color among the five Colletotrichum species complexes.C.gloeosporioides species complexes,C.boninense species complexes,C.truncatum species complexes,C.magnum species complexes and C.orchidearum species complexes had thirteen,thirty,one,nine,five different colony characteristics,respectively.Conidia of C.truncatum,and C.orchidearum species complexes were significant different from the other three Colletotrichum species complexes.Conidia of C.orchidearum species complexes was curve and rounded at both ends,which was sickle and acuminate at both ends by C.truncatum species complexes,and the other three Colletotrichum species complexes were cylindrical,rounded at both ends or top end rounded,base end raised.To classify the taxonomic status of the Colletotrichum spp.,the multi-genes(ITS,ACT,TUB2,GAPDH and GS)were used to build phylogenetic tree of 58 Colletotrichum isolates with different colony characteristics.Among the 58 Colletotrichum isolates,one was identified to be C.gloeosporioides,twelve were C.fructicola,thirty were C.karstii,nine were C.brevisporum,five were C.cliviicola and one was C.truncatum.Pathogenicity tests revealed that the above five Colletotrichum spp.induced lesions on both twigs and leaves of Guanximiyou pomelo,with C.cliviicola being the sole non-pathogenic exception.The strains isolated from infected sites were identical to the strains inoculated.The pathogenicity of C.gloeosporioides was slightly stronger than C.fructicola,but significantly stronger than the other three species.BZMYTJ20 demonstrated significantly stronger pathogenicity than JFMYTJ53,while DXMYTJ5 had no pathogenicity in leaf infection assays,although they all belonged to C.karstii.The mycelial growth rates of those pathogens ranged from 6.27 to 13.53 mm·d^(-1).C.gloeosporioides was the fastest,followed by C.fructicola,C.brevisporum and C.truncatum,while C.karstii was the slowest.The appressorium formation rate of those pathogens ranged from 33.65% to 82.52%;C.gloeosporioides was the highest,followed by C.brevisporum,C.fructicola and C.karstii,and C.truncatum was the lowest.By analyzing the correlation between mycelium growth rate and appressorium formation rate and pathogenicity,the correlation coefficient r=0.3733 and 0.3641,so it was clear there were some positive correlation between mycelial growth rate,appressorium formation rate and pathogenicity.【Conclusion】Based on colony and morphological characteristics,phylogenetic analysis of the multiple genes(ITS,ACT,TUB2,GAPDH and GS)and pathogenicity,the pathogens of Guanximiyou pomelo anthracnose disease in Fujian were identified as 5 species,including C.gloeosporioides,C.fruticola,C.karstii,C.brevisporum and C.truncatum,among which C.gloeosporioides was the dominant.C.fruticola,C.karstii,C.brevisporum and C.truncatum were first identified as the Guanximiyou pomelo anthracnose pathogen in Fujian province,which confirmed that the pathogens of Guanximiyou pomelo anthracnose tended to be diversified and differentiated in Fujan province.There were great differences in the growth rate of mycelium,appressorium formation rate and pathogenicity among different Colletotrichum species,and there was some positive correlation between mycelial growth rate,appressorium formation rate and pathogenicity.This study can provide theoretical data for the diversity research and sustainable prevention and control of Guanximiyou pomelo anthracnose in Fujan province.
作者
戴瑞卿
曾天宝
陈淑妹
潘永红
何云燕
赖宝春
DAI Ruiqing;ZENG Tianbao;CHEN Shumei;PAN Yonghong;HE Yunyan;LAI Baochun(Zhangzhou Institute of Agricultural Sciences,Zhangzhou 363005,Fujian,China;Zhangzhou Plant Protection Station,Zhangzhou 363000,Fujian,China;Zhangzhou Agriculture and Rural Bureau,Zhangzhou 363000,Fujian,China)
出处
《果树学报》
北大核心
2025年第8期1835-1850,共16页
Journal of Fruit Science
基金
福建省科技计划项目(2022N0044)。
关键词
琯溪蜜柚
炭疽病
刺盘孢属真菌
致病性
Guanximiyou pomelo
Anthracnose
Colletotrichum spp.
Pathogenicity
