Culturing Simpler and Bacterial Wilt Suppressive Microbial Communities from Tomato Rhizosphere

Nazish Roy,Kihyuck Choi,Raees Khan,Seon-Woo Lee 한국식물병리학회 2019 Plant Pathology Journal Vol.35 No.4

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 1st transfer suppressed the bacterial wilt. Community analysis revealed that species richness was declined by successive cultivation of the MF. The upland MM9 1st 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.

Culturing Simpler and Bacterial Wilt Suppressive Microbial Communities from Tomato Rhizosphere

Roy, Nazish,Choi, Kihyuck,Khan, Raees,Lee, Seon-Woo The Korean Society of Plant Pathology 2019 Plant Pathology Journal Vol.35 No.4

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.

Dynamics of gut microbiome upon pollination in bumblebee (Bombus terrestris)

Choi Heeyun,Roy Nazish,Kim Jin-Myung,Hyung Joo Yoon,Kyeong Yong Lee,이광식,최기혁 한국응용곤충학회 2023 Journal of Asia-Pacific Entomology Vol.26 No.1

Bumblebees are crucial buzz pollinators of poricidal plants and commercial crops. The population of these crucial pollinators is globally declining. The need to focus on factors contributing to bee health such as gut microbiota is imperative. We evaluated the effect of source on gut microbiota composition in adult worker Bombus terrestris from two environments using multiplexed 16S rRNA amplicon sequencing on the Illumina MiSeq platform. Indoor reared adult worker bees were segregated into non-pollination (NP) and pollination (P) groups. The NP group bumblebees were raised and kept in the insectary for the entire experiment. P group bumblebees worked in a tomato-planted greenhouse for 15 days as pollinators. Our results show that members of Proteobacteria and Firmicutes were significantly different between the two groups. Bifidobacterium bombi (Actinobacteriota) and Candidatus_Schmidhempelia (Proteobacteria) were found to be important gut colonizer exclusive to NP and P group bees, respectively. DESeq2 analysis showed differentially abundant OTUs belonging to Lactobacillus spp. in each group. But for the differentially abundant detected OTUs, resolution till genus level was obtained which impairs our knowledge of the species associated with each group. Our data show that the gut microbiota between the groups differed when indoor- reared adult worker bees were exposed to different environments. The distinct gut microbiota between the two groups may also have been influenced by the different diets fed to bees upon segregation. Further studies can give important insights into the role of gut microbiota on bee health when indoor reared bees are employed for pollination.

Specific and Sensitive Primers Developed by Comparative Genomics to Detect Bacterial Pathogens in Grains

백광열,이현희,손근주,이평안,Nazish Roy,서영수,이선우 한국식물병리학회 2018 Plant Pathology Journal Vol.34 No.2

Accurate and rapid detection of bacterial plant pathogen is the first step toward disease management and prevention of pathogen spread. Bacterial plant pathogens Clavibacter michiganensis subsp. nebraskensis (Cmn), Pantoea stewartii subsp. stewartii (Pss), and Rathayibacter tritici (Rt) cause Goss’s bacterial wilt and blight of maize, Stewart’s wilt of maize and spike blight of wheat and barley, respectively. The bacterial diseases are not globally distributed and not present in Korea. This study adopted comparative genomics approach and aimed to develop specific primer pairs to detect these three bacterial pathogens. Genome comparison among target pathogens and their closely related bacterial species generated 15-20 candidate primer pairs per bacterial pathogen. The primer pairs were assessed by a conventional PCR for specificity against 33 species of Clavibacter, Pantoea, Rathayibacter, Pectobacterium, Curtobacterium. The investigation for specificity and sensitivity of the primer pairs allowed final selection of one or two primer pairs per bacterial pathogens. In our assay condition, a detection limit of Pss and Cmn was 2 pg/μl of genomic DNA per PCR reaction, while the detection limit for Rt primers was higher. The selected primers could also detect bacterial cells up to 8.8 × 103 cfu to 7.84 × 104 cfu per gram of grain seeds artificially infected with corresponding bacterial pathogens. The primer pairs and PCR assay developed in this study provide an accurate and rapid detection method for three bacterial pathogens of grains, which can be used to investigate bacteria contamination in grain seeds and to ultimately prevent pathogen dissemination over countries.

Diverse soil microbiota differentially influence bacterial wilt occurrence in tomato

Pyeong An Lee,Kihyuck Choi,Jinhee Choi,Nazish Roy,Raees Khan,Seon-Woo Lee 한국농약과학회 2018 한국농약과학회 학술발표대회 논문집 Vol.2018 No.10

Analysis of soil microbiome modulating bacterial wilt resistance in tomato

Kihyuck Choi,Pyeong An Lee,Nazish Roy,Jinhee Choi,Seon-Woo Lee 한국농약과학회 2018 한국농약과학회 학술발표대회 논문집 Vol.2018 No.10

Specific and Sensitive Primers Developed by Comparative Genomics to Detect Bacterial Pathogens in Grains

Baek, Kwang Yeol,Lee, Hyun-Hee,Son, Geun Ju,Lee, Pyeong An,Roy, Nazish,Seo, Young-Su,Lee, Seon-Woo The Korean Society of Plant Pathology 2018 Plant Pathology Journal Vol.34 No.2

Accurate and rapid detection of bacterial plant pathogen is the first step toward disease management and prevention of pathogen spread. Bacterial plant pathogens Clavibacter michiganensis subsp. nebraskensis (Cmn), Pantoea stewartii subsp. stewartii (Pss), and Rathayibacter tritici (Rt) cause Goss's bacterial wilt and blight of maize, Stewart's wilt of maize and spike blight of wheat and barley, respectively. The bacterial diseases are not globally distributed and not present in Korea. This study adopted comparative genomics approach and aimed to develop specific primer pairs to detect these three bacterial pathogens. Genome comparison among target pathogens and their closely related bacterial species generated 15-20 candidate primer pairs per bacterial pathogen. The primer pairs were assessed by a conventional PCR for specificity against 33 species of Clavibacter, Pantoea, Rathayibacter, Pectobacterium, Curtobacterium. The investigation for specificity and sensitivity of the primer pairs allowed final selection of one or two primer pairs per bacterial pathogens. In our assay condition, a detection limit of Pss and Cmn was $2pg/{\mu}l$ of genomic DNA per PCR reaction, while the detection limit for Rt primers was higher. The selected primers could also detect bacterial cells up to $8.8{\times}10^3cfu$ to $7.84{\times}10^4cfu$ per gram of grain seeds artificially infected with corresponding bacterial pathogens. The primer pairs and PCR assay developed in this study provide an accurate and rapid detection method for three bacterial pathogens of grains, which can be used to investigate bacteria contamination in grain seeds and to ultimately prevent pathogen dissemination over countries.

Identification of a Gene Involved in the Negative Regulation of Pyomelanin Production in Ralstonia solanacearum

( Shabir Ahmad ),( Seung Yeup Lee ),( Raees Khan ),( Hyun Gi Kong ),( Geun Ju Son ),( Nazish Roy ),( Kihyuck Choi ),( Seon-woo Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.9

Ralstonia solanacearum causes bacterial wilt in a wide variety of host plant species and produces a melanin-like blackish-brown pigment in stationary phase when grown in minimal medium supplemented with tyrosine. To study melanin production regulation in R. solanacearum, five mutants exhibiting overproduction of melanin-like pigments were selected from a transposon (Tn) insertion mutant library of R. solanacearum SL341. Most of the mutants, except one (SL341T), were not complemented by the original gene or overproduced melanins. SL341T showed Tn insertion in a gene containing a conserved domain of eukaryotic transcription factor. The gene was annotated as a hypothetical protein, given its weak similarity to any known proteins. Upon complementation with its original gene, the mutant strains reverted to their wild-type phenotype. SL341T produced 3-folds more melanin at 72 h post-incubation compared with wild-type SL341 when grown in minimal medium supplemented with tyrosine. The chemical analysis of SL341T cultural filtrate revealed the accumulation of a higher amount of homogentisate, a major precursor of pyomelanin, and a lower amount of dihydroxyphenylalanine, an intermediate of eumelanin, compared with SL341. The expression study showed a relatively higher expression of hppD (encoding hydroxyphenylpyruvate dioxygenase) and lower expression of hmgA (encoding homogentisate dioxygenase) and nagL (encoding maleylacetoacetate isomerase) in SL341T than in SL341. SL341 showed a significantly higher expression of tyrosinase gene compared with SL341T at 48 h post-incubation. These results indicated that R. solanacearum produced both pyomelanin and eumelanin, and the novel hypothetical protein is involved in the negative regulation of melanin production.

Rhizosphere microbiome structure alters to enable wilt resistance in tomato

Kwak, Min-Jung,Kong, Hyun Gi,Choi, Kihyuck,Kwon, Soon-Kyeong,Song, Ju Yeon,Lee, Jidam,Lee, Pyeong An,Choi, Soo Yeon,Seo, Minseok,Lee, Hyoung Ju,Jung, Eun Joo,Park, Hyein,Roy, Nazish,Kim, Heebal,Lee, M Nature Pub. Co 2018 Nature biotechnology Vol.36 No.11

<P> Tomato variety Hawaii 7996 is resistant to the soil-borne pathogen Ralstonia solanacearum, whereas the Moneymaker variety is susceptible to the pathogen. To evaluate whether plant-associated microorganisms have a role in disease resistance, we analyzed the rhizosphere microbiomes of both varieties in a mesocosm experiment. Microbiome structures differed between the two cultivars. Transplantation of rhizosphere microbiota from resistant plants suppressed disease symptoms in susceptible plants. Comparative analyses of rhizosphere metagenomes from resistant and susceptible plants enabled the identification and assembly of a flavobacterial genome that was far more abundant in the resistant plant rhizosphere microbiome than in that of the susceptible plant. We cultivated this flavobacterium, named TRM1, and found that it could suppress R. solanacearum-disease development in a susceptible plant in pot experiments. Our findings reveal a role for native microbiota in protecting plants from microbial pathogens, and our approach charts a path toward the development of probiotics to ameliorate plant diseases. </P>