摘要
【目的】明确我国梨主产区引起黑斑病的链格孢属(Alternaria)真菌种类及其致病性,为制定有效防治措施提供理论依据。【方法】从我国梨主产区采集黑斑病病样进行组织分离,并利用形态学和分子生物学相结合的方法对获得的菌株进行种类鉴定和致病性验证。【结果】从我国14个省、自治区及直辖市的梨产区采集病样后,通过组织分离和纯化并依据其菌落形态特征,共获得405个链格孢属(Alternaria)菌株。对这些菌株的形态学观察和多基因(ITS、GAPDH、Alt a1、TEF 1、endoPG、及His 3)系统发育分析的结果显示,它们分别属于链格孢属(Alternaria)的6个种。其中属细极链格孢(Alternaria tenuissima)的有267个菌株、链格孢(A.alternata)115个菌株、乔木链格孢(A.arborescens)14个菌株、梨黑斑链格孢(A.gaisen)6个菌株、棉链格孢(A.gossypina)2个菌株、长柄链格孢(A.longipes)1个菌株。将这6个种的代表菌株在翠冠梨离体叶片上进行有伤接种的结果显示,它们均可致病,但其致病力之间存在差异。而在桃(Prunus persica)、猕猴桃(Actinidia chinensis)和柑橘(Citrus reticulate)离体叶片上进行有伤接种的结果显示,这些代表菌株均使桃和猕猴桃致病,但均不能使柑橘致病。【结论】引起我国梨黑斑病的病原菌有A.tenuissima、A.alternata、A.arborescens、A.gaisen、A.gossypina和A.longipes 6种链格孢属(Alternaria)真菌,其中细极链格孢(A.tenuissima)和链格孢(A.alternata)为优势种,分别占总分离菌株数的65.9%和28.4%。本研究是我国梨主产区引起黑斑病的链格孢属(Alternaria)真菌种类系统性鉴定的首次报道。
【Objective】Pear trees have been widely cultivated in China and the total yield and cultivation area of Chinese pear ranks first in the world.Pear belongs to a kind of perennial fruit tree and it is frequently infected by a large number of fungal diseases during growth and development.Black spot is one of the important diseases in pear production areas in China,which mainly damages leaves and fruits,resulting in serious precocious defoliation and fruit decay.Black spot has become a worldwide disease,especially in Japan,South Korea and the south of China.The disease has caused severe losses to the economic benefits and greatly affected the development of pear industry in major pear-cultivation areas in China.This study aimed to clarify the species of Alternaria spp.associated with black spot of pear in China based on multi-gene and morphological identification,as well as to determine the composition,distribution,morphological and pathogenic characteristics of the pathogens.The results of this study are expected to provide a better understanding on the etiology of the disease and scientific basis for its prevention and control.【Methods】The leaves and fruits infected by black spot collected from 14 provinces,municipalities or autonomous regions(including Anhui,Chongqing,Fujian,Gansu,Guizhou,Hubei,Jiangxi,Jilin,Shanxi,Shandong,Sichuan,Xinjiang,Yunnan and Zhejiang)were used as disease samples.The fungus pathogen was isolated by using the routine plant tissue isolation method.Total genomic DNA was extracted from pure cultures with a modified cetyltrimethyl ammonium bromide(CTAB)protocol,and subjected to polymerase chain reaction(PCR)amplification of partial regions of six loci including partial rDNA-ITS(ITS)region,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),translation elongation factor 1-alpha(TEF 1),endo polygalacturonase(endoPG),Alternaria major allergen gene(Alt a1)and histone 3(His 3).Phylogenetic trees were constructed by mrbayes-3.1.2.The best-fit models of nucleotide substitution for each partition were determined by using MrModeltest v.2.3.The sporulation phenotype and conidia morphology of representative strains were recorded under a microscope.The pathogenicity was determined on detached leaves,fruits and branches(wounded or unwounded)of pear inoculated with mycelial plugs of representative strains and the host ranges were determined on detached leaves of peach(Prunus persica),kiwifruit(Actinidia chinensis)and citrus(Citrus reticulate)inoculated with mycelial plugs of representative strains.【Results】A total of 405 strains of Alternaria,including 355 strains from leaves and 50 strains from fruits,were obtained.The obtained strains were identified by multiple genes,combined with morphological observation,and they were confirmed to be 6 species of Alternaria,including A.tenuissima(267 strains),A.alternata(115 strains),A.arborescens(14 strains),A.gaisen(6 strains),A.gossypina(2 strains)and A.longipes(1 strains).Phylogenetic analysis showed that specific species could not be identified only by ITS sequences.A.gaisen,A.arborescens and A.alternata could be identified by multiple genes(ITS,GAPDH,Alt a1,TEF 1 and endoPG).A.tenuissima and A.alternata could be identified by His 3 gene.Through PCR amplification,A.tenuissima produced a fragment of 550 bp,while A.alternata produced a fragment of 440 bp.However,SC-5,SC-10 and SC-16 strains were clustered together with A.longipes and A.gossypina,so there was no clear identification between A.longipes and A.gossypina.Combined with morphological identification,the conidia morphology of A.longipes and A.gossypina were different.The conidia of A.gossypina were dark brown and pear-shaped,while the conidia of A.longipes were gray and spindle-shaped.The results of pathogenicity test showed that the representative strains of A.alternata(SX-1),A.tenuissima(SX-2),A.arborescens(XJ-13),A.gaisen(CQ-35),A.longipes(SC-10)and A.gossypina(SC-5)could induce lesions on leaves and fruits of Pyrus pyrifolia cv.Cuiguan,but there were significant differences in their pathogenicity.However,under the same inoculation conditions,these representative strains could not induce lesions on pear branches.The pathogens isolated from the infected sites after inoculation were identical with the inoculated strains.These results showed that they were all pathogenic and responsible for black spot by fulfilling the Koch’s postulates.The results of the determination of host range showed that these representative strains could infect the leaves of peach(Prunus persica)and kiwifruit(Actinidia chinensis),but there were differences in their pathogenicity.None of these representative strains could cause symptom on leaves of citrus(Citrus reticulate).Analysis of the prevalence of 6 species of Alternaria revealed that A.tenuissima and A.alternata were dominant species,which occurred in all Pyrus spp.cultivated in 14 provinces,municipalities or autonomous regions,accounting for 65.9% and 28.4% of the total isolates,respectively.A.longipes and A.gossypina were only separated from the samples of Sichuan,A.arborescens was isolated from the samples of Xinjiang,Sichuan and Gansu,and A.gaisen was isolated from the samples of Chongqing,Hubei,and Anhui.【Conclusion】Based on the phylogenetic analysis and morphological characteristics of the 405 strains obtained in this study,the pathogens causing black spot of pear in China were associated with six species of Alternaria including A.tenuissima,A.alternata,A.arborescens,A.gaisen,A.gossypina and A.longipes.Among them,A.tenuissima and A.alternata were the dominant pathogens of the disease.This research is the first report on the identification of pathogens associated with black spot in the main pear producing areas in China.
作者
王文青
李扬
向均
洪霓
王国平
WANG Wenqing;LI Yang;XIANG Jun;HONG Ni;WANG Guoping(College of Plant Science and Technology,Huazhong Agricultural University/Key Lab of Crop Disease Monitoring and Safety Control in Hubei,Wuhan 430070,Hubei,China)
出处
《果树学报》
CAS
CSCD
北大核心
2020年第12期1922-1933,共12页
Journal of Fruit Science
基金
国家重点研发项目(2018YFD0201406)
国家梨产业技术体系(CARS-28-16)。
关键词
梨
黑斑病
链格孢
种类鉴定
致病性
Pear
Black spot
Alternaria
Species identification
Pathogenicity
