| Asparagus stem blight is a fungal disease caused by Phomopsis spp.,which is the main disease in the production of asparagus.It occurs in many asparagus producing areas in the world and is a typical asparagus stem disease.Asparagus stem blight can significantly reduce or even eliminate the yield of spears,which seriously affects the asparagus industry development.Hainan Province is located in the tropical region of China.The high temperature and high humidity tropical climate is conducive to the annual production of asparagus and to increase the spear yield;but it also makes the asparagus susceptible to the infection and damage of stem blight.In this paper,the asparagus stem blight disease investigation was carried out on the asparagus planting plots in Wenchang area of Hainan Province,and the pathogens were isolated from the disease samples,and the physical control,biological control technology and high-efficiency safety chemical control agent screening research were carried out in order to provide a basis for green control of stem blight diseases for asparagus production.The main results of this study are as follows:1.Isolation and identification of pathogens:The pathogenic stalks were collected from the asparagus planting plots at the Wenchang Experimental Base of the Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences.The pathogens were isolated by tissue separation.Eight purified strains were isolated,of which strain GZ05 was the most pathogenic.2.rDNA-ITS sequence analysis:The 8 isolates from the pathogens were amplified by universal primers ITS1 and ITS4.The ITS lengths of the 8 tested strains were all about 600 bp.The strains were compared by NCBI Blast.The homology of Phomopsis sp.is as high as 99%or more.The phylogenetic tree was constructed by MEGA6.0 software,and 8 test strains were clustered in the same branch as Phomopsis sp.According to the hyphal morphology and ITS sequence identification results,all the 8 tested strains were identified as Phomopsis sp.3.Biological characteristics of strain GZ05:The results of biological characteristics of GZ05 showed that the pH of the medium was 3～10,and the optimum pH was 7.0.There were differences in the growth rate of mycelium on medium containing different kinds of carbon and nitrogen sources.The optimum carbon source was sucrose,glucose or sorbic acid.The optimum nitrogen source was potassium nitrate.The optimum growth temperature was 28℃,and the lethal temperature was 55 ℃.4.Virulence determination of fungicides:11 fungicides with different dosage forms were used,and the inhibitory effect of 11 fungicides on GZ05 was determined by colony growth rate.The fungicides effect of different fungicides was found to be different.The most effective fungicidal was prochloraz aqueous emulsion,which had an EC50 of 0.015 mg/L,an EC75 value of 0.03171 mg/L,and an EC95 value of 0.09324 mg/L.5.Isolation and identification of antagonistic strains:26 strains of bacteria were isolated from asparagus stems,and 5 strains(BS-1,BS-5,BS-11,BS-17,BS-19)with strong antagonistic effect were screened by plate sputum method,among which BS-11 has the strongest antifungal effect.6.Physiological and biochemical characteristics of strain BS-11:The characteristics of colony morphology,Gram stain,spore staining and physiological and biochemical characteristics showed that strain BS-11 was Gram-positive and negative for spore staining.7.16S rDNA sequence analysis:5 strains of the selected strains were amplified by PCR using universal primers 27F and 1492R.After Blast alignment by NCBI,they had 99%highest homology with Bacillus pumilus(EU594558.1,KT003271.1),Bacillus safensis(MK517598,708899.1)and Bacterium(MK072997).MEGA6.0 software was used for cluster analysis and phylogenetic tree was constructed.The test strains were clustered on the same branch as B.pumilus,B.safensis and Bacterium,and they were initially identified as belonging to the genus Bacillus.8.GyrB gene sequence analysis:The gyrB gene was used to analyze the tested strains,and the genomic DNA of the test strain was used as a template to amplify the gyrB gene.The BLAST software homology comparison results showed that the tested strains and the reported B.safensis gyrase subunit B(gyrB)gene(KX011111.1)similarity of more than 99%.and it was identified as Bacillus safensis by combining morphological characteristics,physiological and biochemical characteristics,16S rDNA gene sequence and gyrB sequence.9.Field experiment:The field efficacy test was carried out by combining fungicides and biocontrol bacteria combined with insect nets.The results showed that the insect net,fungicide and biocontrol bacteria had different effects on stem blight of asparagus.Based on the results of three times of application,it was found that the field disease index and the disease index before the application were the most varied after the spraying of the fungicide and the bio-control fermentation broth in the test plot.The pre-application rate was 13.26.The control index was reduced to 4.09,and the control indexes of the three tests were 64.66%,72.87%,and 74.39%. |
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