海南白沙和儋州油梨叶部及果实炭疽病菌的鉴定

Identification of pathogen species of avocado(Persea americana Mill.)leaf and fruit anthracnose in Baisha and Danzhou,Hainan

【目的】明确海南省白沙县和儋州市油梨种植基地的油梨叶片和果实的病原菌种类,为该病害的准确鉴定和田间防治措施的制定提供理论依据。【方法】在海南省白沙县和儋州市的油梨种植基地调查炭疽病的发生情况并采集病叶和病果,通过组织分离法和单孢分离法获得纯化分离株,并对纯化菌株进行致病性测定,结合形态学和分子生物学对所得菌株进行病原菌种类鉴定。【结果】从采自白沙县的14份病叶种分离的得到3株菌株,儋州市的10份病叶中分离获得6株分离物以及自白沙市采集的5份病果中分离出的1株真菌,经致病性测定,菌株HNBSL01、HNDZL02和HNBSF03为致病菌,且均与田间症状一致。根据3种致病菌的菌落、分生孢子和附着胞的形态特征可初步判断引起油梨叶片和果实炭疽病的病原菌均为炭疽菌属(Colletotrichum sp.);多基因(ITS-ACT-TUB2-CHS-1-GAPHD-HIS3)联合分析构建系统发育树分析结果显示,病原菌HNBSL01与暹罗炭疽菌(C.siamense)的同源性为81%、HNDZL02与果生炭疽菌(C.fructicola)的同源性为100%、HNBSF03与长直孢炭疽菌(C.gigasporum)的相似性达100%。【结论】引起海南白沙县和儋州市油梨种植基地叶片和果实炭疽病的病原菌为暹罗炭疽菌(C.siamense)、果生炭疽菌(C.fructicola)和长直孢炭疽菌(C.gigasporum),其中,C.gigasporum为国内首次报道引起油梨果实炭疽病的病原菌。

【Objective】Avocado(Persea americana Mill.)is a tropical and subtropical evergreen fruit crop.As the demand for avocado increases year by year,the planting area has expanded.The incidence of anthracnose is increasing in the planting area.In 2021,the avocado planting base in Baisha and Danzhou of Hainan province found that the symptoms of anthracnose appeared on the leaves of large trees,and more than 50%trees were affected,which seriously affected the yield and quality of avocado.Danzhou Avocado Planting Base,found the dark brown disease spot on the fruit during the fruit harvest and storage,and a sticky orange-red conidiomata appeared,so that the heavy loss was caused.This experiment was conducted to describe and identify the pathogen causing leaf and fruit anthracnose of avocado,so as to provide a theoretical basis for the accurate identification of the disease and the development of control measures in the field.【Methods】The disease incidence of Baisha and Danzhou Avocado Planting Bases were investigated,and samples of anthracnose disease were collected.Tissue isolation method and single spore isolation method were used to isolate and purify the strains.To confirm the pathogenicity,the wounded and unwounded leaves and fruits of the avocad were inoculated by stem cake and conidial suspension inoculation method.The pathogen was re-isolated from the inoculated sites,and the morphological characteristics of the reisolated strains were observed and recorded.The purified strains were transferred onto PDA medium and incubated at 25℃and 12 h light/12 h dark.After 7 d,the morphology,color and growth rate of the colonies were recorded.After sporulation,the morphology and size of conidia and appressoria were observed and recorded under the optical microscope to clarify their morphological characteristics.Genomic DNA was extracted using the Fungal Genomic DNA Rapid Extraction Kit(OMEGA BIO-TEK).The six target gene sequences,ITS(ITS1/ITS4),TUB2(T1/βt2b),ACT(ACT-512F/ACT-783R),HIS3(CYLH3F/CYLH3R),CHS-1(CHS-79F/CHS354R)and GAPHD(GDF/GDR),were selected for PCR amplification.The products were detected by 1%agarose gel electrophoresis and purified,and then the sequence determination was done by Biotech Bioengineering(Shanghai)Co.SequenceMatrix software was used to perform sequence splicing in the order of ITS-ACT-TUB2-CHS-1-GAPHD-HIS3,and blast alignment.Phylogenetic tree was constructed using the Maximum Likelihood method with MEGA 7.0 software to clarify the taxonomic status of pathogens.【Results】Three strains(HNBSL01-03)were obtained from 14 diseased leaves and one strain(HNBSF03)was isolated from five diseased fruits from Baisha.Six isolates(HNDZL02-07)were obtained from 10 diseased leaves from Danzhou.After 10 days of inoculation,only the leaves inoculated with HNBSL01 and HNDZL02 and the fruits inoculated with HNBSF03 showed symptoms of infection.The symptoms that appeared on the inoculated leaves and fruits were similar to those collected from the field.The control was asymptomatic.The strains with the same morphology as HNBSL01,HNDZL02 and HNBSF03 were obtained by re-isolation and purification from the disease site.According to Koch's postulates,it was concluded that these three isolates were pathogens causing anthracnose on the leaves and fruits of avocado.After culturing on PDA medium at 25℃and 12 h light/12 h dark for 7 days,the colonies of strain HNBSL01 were white.Conidia with oil droplets were cylindrical,bluntly rounded at both ends or bluntly rounded at one end and acuminate at the other,the size was 14.11-16.97(15.61)μm×3.74-4.89(4.32)μm(n=100),with an aspect ratio of(3.47-3.77).Appressoria were light brown to brown,clavate,ellipsoidal,subglobose or spherical,entire,with a size of 7.7-10.24(9.03)μm×4.37-6.16(5.15)μm(n=100).The strain HNDZL02 was diaphanous green with white outer edges,conidia short cylindrical,bluntly rounded at both ends,without oil droplets,13.84-16.96(15.67)μm×5.38-6.52(5.97)μm(n=100)in size,and such an aspect ratio(2.57-2.60).Appressoria were light brown to brown,elliptical or irregularly shaped,entire or with obtusely serrate lobes,9.95-13.76(11.88)μm×4.82-6.22(5.53)μm(n=100)in size.The strain HNDZF03 colonies were inky gray to white on the front,bamboo green to onyx on the back,conidia without oil droplets were clavate,apical acuminate base obtuse round,13.77-17.65(15.49)μm×4.15-5.47(4.66)μm(n=100)in size,and an aspect ratio(3.23-3.32).Appressoria were brown to dark brown,ovoid to orbicular,entire,and 6.65-9.78(8.38)μm×4.95-6.91(6.25)μm in size(n=100).The results of phylogenetic tree constructed by multi-gene(ITS-ACT-TUB2-CHS-1-GAPHD-HIS3)association analysis showed that the pathogen HNBSL01 had 81%homology with Colletotrichum siamense,HNDZL02 had 100%homology with C.fructicola,and HNBSF03 was 100%similar to C.gigasporum.【Conclusion】The strains of anthracnose were isolated from the leaves and fruits of avocado in Baisha and Danzhou,Hainan,which belonged to C.siamense,C.fructicola and C.gigasporum.This is the first report of C.gigasporum causing anthracnose on avocado fruit in China.