Effect of biocontrol strain Hhs.015 on endophytic bacterial flora of apple trees

Abstract：【Objective】Apple Valsa canker is caused by Valsa mali Miyabe et Yamada which decomposes the apple tree bark and even the xylem, it is a serious fungal disease which brings significant damageto apple productivity in China. Biological control can be used on the plant surface or in vivo, regulatingthe balance and proportion of plant indigenous microflora and promoting plant growth to achieve thegoal of prevention and stimulation. Microorganisms are an important part of the plant microflora, whichare involved in the happenings, developing and declining of some life activities of the host in the level ofphysiology, pathology, pharmacology and many other life activities. Normal microbial flora is an important part of healthy plants. Saccharothrix yanglingensis Hhs.015 is effective in controlling apple Valsa canker both indoors and in field trials. This experiment aimed to explore the effects of Hhs.015 on themicro-ecology of apple tree twigs, which is helpful to evaluate its biosecurity.【Methods】A field trialwas carried out in a‘Fuji'apple orchard in Yangling, Shaanxi. 10 new sick spots on 10 randomly selected apple tree trunks were divided into two groups. The control was treated with the biocontrol agent Saccharothrix yanglingensis Hhs.015 broth, the contrast group was treated with water. Every 5 d the scarswere painted for a total of 4 times. Then 30 d after the last time, the tested samples at the boundary be-tween diseased and healthy areas of twigs were collected. DNA was extracted using the modified CTABmethod. The V4+V5 region of 16 S r DNA was amplified with a specific primer and sequenced by highthroughput sequencing. From raw pyrosequencing reads, primer and tag sequences were removed, the orig-inal tags were jointed by FLASH. After filtration treatment and mosaic deletion, the final valid data wereobtained. These sequences were clustered at 97% similarity using Uparse. To study the phylogenetic rela-tionship between OTUs, these sequences were multiple sequence aligned and a tree built to provide taxo-nomic identification. According to the relative abundance of top 10 genera of phylogenetic relationship in-formation and corresponding to the relative abundance of data, the samples were clustered. The top 35 spe-cies heat map were drawn based on the genus annotation and relative abundance, which is helpful to findthe differences between the samples of higher and lower species abundance.【Results】The quality of thesequencing data is ideal, the obtained sequence information could be used to build the OTUs and for thefollowing analysis. 223, 210, 220, 218, 232, 213 OTUs were obtained in F1, F2, F3, BA, BB and BC sam-ples, respectively. According to the results of OTUs species annotation, Cyanobacteria and Proteobacteriaare dominant in all 6 samples, especially Cyanobacteria, which accounted for more than 80% each. In ad-dition, there are also many sequences belonging to Acidobacteria, Actinobacteria and Gemmatimonadetes, etc. At the level of genus, the top ten are Saccharothrix, Pseudomonas, Gluconobacter, Novosphingobium, Lysobacter, Luteibacter, Thermomonas, Pseudoxanthomonas, Sphingomonas, and Brukholderia. Among thecontrol and the contrast groups, Burkholderia, Gluconobacter and Burkholderia are all present, but in theHhs.015 treated samples, the number of Burkholderia significantly decreased, Gluconobacter, Rhodococcus, Saccharothrix, Leuconostoc and Agrobacterium increased in the contrast group. The abundance of Saccharothrix remained at a high level in the sample taken after scraping the surface tree bark.【Conclusion】There were similar numbers of tags and OTUs in Valsa lesion and Valsa lesion applied with Hhs.015 broth. Cyanobacteria and Proteobacteria were dominant in both samples in the phylum level. However, theabundance of Gluconobacter and Actinobacteria in the treatment group were higher than the control groupwhich is consistent with the result of isolation. Pathogenic Rhodococcus fascians had also increased. Theabundance of Burkholderia and Luteibacter which may be associated with pathogenic species had a sub-stantial decline. The result showed that the impact of Hhs.015 broth on the Valsa lesion bacterial flora wascomplex. Hhs.015 favored the proliferation of Gluconobacter and Actinobacteria and inhibited the growthof harmful bacteria. The abundance of Saccharothrix remained at a high level in the bark scraped off theskin which shows that Hhs.015 could colonize in apple trees for some time and affect the bacterial flora ofthe cortex.