| Jujube shrunken-fruit disease?JSFD?is one of the most destructive jujube diseases in China.In order to find the most effective methods to control Alternaria alternata on jujube,the infection characteristics of the JSFD pathogen and fungicide resistance were investigated.Agrobacterium tumefaciens-mediated transformation was carried out for GFP-labelling of strain CN193.Infection experiments of jujube were performed by spray inoculation on jujube blossoms and stab-wound inoculation of young jujube fruits with A.alternata CN193::gfp.Potential host plants of A.alternata were explored through the infection of strain CN193::gfp,and based on the screening results of potential hosts of A.alternata,substitutional host of the pathogen was studied as well to solve the problem of seasonal restrictions for the research of JSFD.The resistant isolates of A.alternata strains CN191,CN192,CN193 and CN194,to iprodione or benzoyl propiconazole were detected to rapidly find fungicides with no cross resistance,and finally to determine the most effective control methods for JSFD.The main results are as follows:GFP-tagged strain CN193,designated as CN193::gfp,was obtained successfully by Agrobacterium tumefaciens-mediated transformation through transferring gfp gene plasmids into CN193.The green fluorescence of the labelled strain was stable after five subcultures,and A.alternata CN193::gfp maintained the same pathogenicity as the wild type strain CN193.Conidial spore suspension of A.alternata CN193::gfp was spray inoculated to jujube blossoms and PDA agar disks with mycelia of A.alternata CN193::gfp was stabbing inoculated to young jujube fruits to observe the symptoms.The inoculated pathogen were isolated again by growing them on PDA agar plates with hygromycin B and observed under fluorescence microscope.A.alternata CN193::gfp could be recovered from the diseased fruits by spray inoculation of jujube blossoms,the diseased fruits on the non-inoculated neighbouring branches close to inoculated branches by flower spraying,the diseased fruits by inoculation of needle stabbing,and the diseased fruits on the non-inoculated neighbouring branches close to inoculated branches by needle stabbing,demonstrating that the pathogen was capable of infecting jujube plant through blossoms or young fruits.Lycopersicon esculentum,Nicotiana tabacum,Malus domestica cv.Red Fuji,Pyrus bretschneideri,Populus tomentosa and P.canadensis from the surrounding area of jujube plantations were selected for inoculating with and without stab wound to the fruits and/or leaves with the spore suspension of CN193::gfp.The marginal tissues with healthy and diseased parts were transferred onto potato dextrose agar media with supplement of hygromycin B at 10?g·mL-1,and the infected tissues and the hyphae isolated from each of the inoculated material were observed under fluorescence microscope.All inoculated materials with stabbing wound,except for N.tabacum plant,developed diseased symptoms after 7 d of inoculation.The disease rates of all the above materials inoculated with spore suspension were 45.8%of tomato fruits,79.2%of apple fruits,75.0%of apple leaves,56.3%of pear fruits,52.1%of pear leaves,66.7%of P.tomentosa leaves and 54.2%of P.canadensis leaves.Through non-wounded inoculation only tomatoes fruits showed symptoms,and all the other treated materials and the controls did not present any visible disease development.Strong fluorecence could be observed in the infected tissues of tomato fruits,and in the hyphae of isolates from all infected materials,including fruits of L.esculentum,fruits and leaves of M.domestica cv.Red Fuji,fruits and leaves of P.bretschneideri,and leaves of P.tomentosa and P.canadensis.The pathogen of JSFD was able to successfully infect the tomatoes,apples,pears,P.tomentosa,and P.canadensis,and it could cause the same symptoms as tomato melasma,apple brown spot,pear black spot,and poplar leaf blight,inferring that L.esculentum,M.domestica cv.Red Fuji,P.bretschneideri,P.tomentosa and P.canadensis are potential hosts of JSFD,expanding the host range of A.alternata,the primary infection pathogen of JSFD.Conidial suspension of strain CN193 was spray inoculated to the blossoms and unripened fruits of L.esculentum with and without stab wound,and the leaves and blossoms of N.tabacum.The inoculated materials were incubated in a humid chamber for disease development,and the causal agents were isolated from the infected tomato tissues.All the isolated strains,including the pathogenic A.alternata strain ZN-XR from naturally infected tomato fruits,were tested for their pathogenicities on jujube fruits.The identification of strain ZN-XR,the pathogen of tomato fruit black spot was performed based on the characteristics of colony morphology and ITS sequencing.Results showed that the symptoms of the detached tomato fruits could be observed 5 d after stabbing or non-wound inoculation.The fruits of living tomato plants were diseased as well after spray inoculation of blossoms or stabbing inoculation of unripened fruits with conidial spore suspension,with similar symptoms to JSFD.No disease symptoms developed either on living tobacco plants inoculated by spraying blossoms or on detached tobacco leaves with or without stabbing wound inoculation with spore suspension.The isolates obtained from diseased detached fruits,fruits with shrunken symptoms and flowers in living tomato plants,could infect jujube fruits,and there was no significant difference compared with the incidence of the control inoculated with the original strain CN193.The strain ZN-XR from naturally infected tomato fruits was the same as strain CN193 in colony morphology,sporulation pattern,morphological characteristics,and showed 100%homology in ITS sequence with strain CN193,and it is identified as A.alternata.Therefore tomato is suitable for a substitutional host of A.alternata,the pathogen of JSFD,and can be used for rapid screening of fungicides to control JSFD.To investigate the resistance of A.alternata,to iprodione and benzoyl propiconazole,four strains,i.e.CN191,CN192,CN193 and CN194,were used to test for their sensitivities to both fungicides with mycelial growth rate.The most sensitive strain to the two fungicides was screened,and the obtained resistant strain to one fungicide was tested for its resistance to another fungicide.In 2016 and 2017,field control experiments were performed using two fungicides,which were previously applied or never applied before,and the disease incidences were recorded at the early stage and peak period of disease development.Results showed that CN194 is most sensitive strain to both fungicides,and after growing it on potato dextrose agar supplemented with stepwise increase of iprodione,a stable iprodione-resistant mutant strain,designated as CN194y,was obtained.No cross resistance to benzoyl propiconazole in strain CN194y was found.The control effect of benzoyl propiconazole was 81.3%at the end of August and 51.3%at mid-September,respectively,much better than that of iprodione in jujube plantations,in which two fungicides were previously sprayed.However,the control effect of iprodione reached92.9%and 68.4%on two investigation dates,respectively,much higher than that of benzoyl propiconazole in the jujube fields,in which two fungicides were never applied previously.The sensitivity of A.alternata to iprodione and benzoyl propiconazole is different in different regions,but the sensitive trend is the same.It is inclined for A.alternata to appear resistant mutants to iprodione,but not to benzoyl propiconazole,and there is no cross resistance for A.alternata to the above mentioned two fungicides.In conclusion,it is recommended that two fungicides had better be applied alternately in jujube fields for effective control of JSFD. |
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