The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Culturing Simpler and Bacterial Wilt Suppressive Microbial Communities from Tomato Rhizosphere

  • Roy, Nazish (Department of Applied Bioscience, Dong-A University) ;
  • Choi, Kihyuck (Department of Applied Bioscience, Dong-A University) ;
  • Khan, Raees (Department of Applied Bioscience, Dong-A University) ;
  • Lee, Seon-Woo (Department of Applied Bioscience, Dong-A University)
  • Received : 2019.07.02
  • Accepted : 2019.07.14
  • Published : 2019.08.01
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Abstract

Plant phenotype is affected by a community of associated microorganisms which requires dissection of the functional fraction. In this study, we aimed to culture the functionally active fraction of an upland soil microbiome, which can suppress tomato bacterial wilt. The microbiome fraction (MF) from the rhizosphere of Hawaii 7996 treated with an upland soil or forest soil MF was successively cultured in a designed modified M9 (MM9) medium partially mimicking the nutrient composition of tomato root exudates. Bacterial cells were harvested to amplify V3 and V4 regions of 16S rRNA gene for QIIME based sequence analysis and were also treated to Hawaii 7996 prior to Ralstonia solanacearum inoculation. The disease progress indicated that the upland MM9 $1^{st}$ transfer suppressed the bacterial wilt. Community analysis revealed that species richness was declined by successive cultivation of the MF. The upland MM9 $1^{st}$ transfer harbored population of phylum Proteobacteria (98.12%), Bacteriodetes (0.69%), Firmicutes (0.51%), Actinobacteria (0.08%), unidentified (0.54%), Cyanobacteria (0.01%), FBP (0.001%), OD1 (0.001%), Acidobacteria (0.005%). The family Enterobacteriaceae of Proteobacteria was the dominant member (86.76%) of the total population of which genus Enterobacter composed 86.76% making it a potential candidate to suppress bacterial wilt. The results suggest that this mixed culture approach is feasible to harvest microorganisms which may function as biocontrol agents.

Keywords

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Fig. 1. Disease progress of R. solanacearum strains SL341 on tomato. Thirty 21-days-old tomato plants (cv. Hawaii 7996) treated with bacterial cells harvested from inoculated MM9 culture media, control plants treated with 2.5 mM MES (pH 5.7) were inoculated by R. solanacearum SL341 at a final concentration of 1 × 107 cfu/g soil. Plants were rated for disease index through 14 days; vertical bars represent the standard error. Significant difference was observed by repeated measures ANOVA. P values are indicated by asterisks < 0.01 = **, < 0.001=***.

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Fig. 2. Bacterial community alpha diversity measured by observed species (A) and Shannon diversity index (B) from the rhizosphere MF of upland, forest MF treated Hawaii 7996 and their respective communities harvested from the cultures and subsequent cultures in MM9 media. The upland rhizosphere sample is abbreviated Upland_Rhi, forest rhizosphere sample as Forest_Rhi, bacterial mixed culture in MM9 inoculated with upland rhizosphere MF is abbreviated as UplandMM9, and bacterial mixed culture in MM9 transferred from UplandMM9 is abbreviated as UplandMM9_1st_Tra. The significance observed by Wilcoxon rank sum test are indicated by asterisks < 0.05 = *, < 0.01 = **.

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Fig. 3. Non-metric multidimensional scaling (NMDS) plot of Bray-Curtis dissimilarity on microbial communities harvested from the rhizosphere MF of upland and forest MF treated Hawaii 7996 and their respective communities harvested from the cultures and subsequent cultures in MM9 media. NMDS plot stress value equals 0.06. Permutational multivariate analysis of variance (PERMANOVA) showed significant difference (P = 0.001) with R2 value 0.5283. The upland rhizosphere sample is abbreviated Upland_Rhi, forest rhizosphere sample as Forest_Rhi, bacterial mixed culture in MM9 inoculated with upland rhizosphere MF is abbreviated as UplandMM9, and bacterial mixed culture in MM9 transferred from UplandMM9 is abbreviated as UplandMM9_1st_Tra.

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Fig. 4. Relative abundance (%) of bacterial OTUs at the phylum level from tomato rhizosphere, bacterial mixed culture in MM9 inoculated with rhizosphere MF (MM9) and bacterial mixed culture in MM9 transferred from MM9 (MM9 1st Tra). The x-axis represents the relative abundance (%) of each phylum. The y-axis represents the samples. The color box below the figure represents the phyla.

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Fig. 5. Disease progress of R. solanacearum strains SL341 on tomato in non-sterilized (A) and sterilized (B) nursery soil. Thirty 21-daysold tomato plants (cv. Hawaii 7996) treated with bacterial cells of Enterobacteriaceae strain, control plants treated with 2.5 mM MES (pH 5.7) were inoculated by R. solanacearum SL341 at a final density of 1 × 107 cfu/g soil. Plants were rated for disease index through 14 days, vertical bars represent the standard error of the mean (n = 30). Significant difference was observed by repeated measures ANOVA between control and plants treated with Enterobacteriaceae strain (P = 0.019471) in non-sterilized soil. No significant difference was observed by repeated measures ANOVA between control and plants treated with Enterobacteriaceae strain (P = 0.9615) in sterilized soil.

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