Effect of transient exposure to carbaryl wettable powder on the gut microbial community of honey bees

Kathyleen Nogrado,이슬,전경미,이지훈 한국응용생명화학회 2019 Applied Biological Chemistry (Appl Biol Chem) Vol.62 No.-

Bees are important pollinators in agriculture. The bee population has recently begun to decline possibly due to pesticides. The bee gut microbiota strongly influences the health of bees. The gut microbiota of bees is composed of distinct members belonging to selective taxa. Chemicals like pesticides can alter the gut microbiota. The present study investigated the effect of carbaryl pesticides on gut microbiota of honey bees, which had come in contact with rapeseed plants (Brassica napus) sprayed with carbaryl wettable powder during the honey bee brood test under semifield condition. Molecular techniques (conventional and quantitative polymerase chain reaction (PCR), clone library method, and DNA sequencing) were employed to analyze changes in the microbial communities between the pesticide- exposed and unexposed bees. Phylogenetic analysis of 16S rRNA genes of the clones from both groups, showed differences in their respective compositions of core and non-core bacteria. Both groups contained carbohydratedegrading bacteria such as Gilliamella apicola and Lactobacillus. However, the unexposed bees harbored Alphaproteobacteria, which were absent in the exposed bees. Microorganisms found in honey bee guts such as Snodgrassella alvi and L. kullabergensis, however, were observed only in the exposed bees, but not in the unexposed bees. The difference between the two groups was distinctly recognized when copy numbers of 16S rRNA genes were compared by quantitative PCR. Results showed that the average gene copy number for the unexposed bees was higher than that for the exposed bees. This may indicate the toxic effect of pesticides on bees and gut microbiota.

Tetracycline‑resistant bacteria and ribosomal protection protein genes in soils from selected agricultural fields and livestock farms

Nogrado Kathyleen,Unno Tatsuya,Hur Hor-Gil,Lee Ji-Hoon 한국응용생명화학회 2021 Applied Biological Chemistry (Appl Biol Chem) Vol.64 No.3

Antibiotic resistance in soil environment has eminently been compared and studied between agricultural and pristine soils, and the role of concentrated animal feeding operations has markedly been recognized as one of the major sources of antibiotic resistance. This study described the tetracycline resistance in small-scale farms in pursuit of presenting its possible role and contribution to the persistence of antibiotic resistance in the environment. Results of the study would render additional information on the occurrence of the ribosomal protection protein (RPP) tet genes among the isolated bacteria from the selected agricultural soils. Four tetracycline resistance and RPP genes were determined in two different agricultural soil settings. Both the culture and molecular method were used to determine and measure tetracycline resistance in soils from arable land and animal house. Results revealed a significantly higher number of culturable antibiotic-resistant bacteria in animal houses than arable lands which was suggestive of higher antibiotic resistance in areas where there was direct administration of the antibiotics. However, quantification of the gene copy numbers in the agricultural soils indicated a different result. Higher gene copy number of tetO was determined in one animal house (IAH-3), while the two other tet genes tetQ and tetW were found to be higher in arable lands. Of the total 110 bacterial isolates, tetW gene was frequently detected, while tetO gene was absent in any of the culturable bacterial isolates. Principal component analysis of occurrence and gene copy number of RPP tet genes tetO, tetQ, and tetW also revealed highest abundance of RPP tet genes in the manure and arable soils. Another important highlight of this study was the similarity of the RPP tet genes detected in the isolated bacteria from the agricultural soils to the identified RPP tet genes among pathogenic bacteria. Some of the tetracycline-resistant bacterial isolates were also multidrug resistant as it displayed resistance to tetracycline, erythromycin, and streptomycin using disk diffusion testing.

Effect of Non-indigenous Bacterial Introductions on Rhizosphere Microbial Community

( Kathyleen Nogrado ),( Gwang-Su Ha ),( Hee-jong Yang ),( Ji-hoon Lee ) 한국환경농학회 2021 한국환경농학회지 Vol.40 No.3

BACKGROUND: Towards achievement of sustainable agriculture, using microbial inoculants may present promising alternatives without adverse environmental effects; however, there are challenging issues that should be addressed in terms of effectiveness and ecology. Viability and stability of the bacterial inoculants would be one of the major issues in effectiveness of microbial pesticide uses, and the changes within the indigenous microbial communities by the inoculants would be an important factor influencing soil ecology. Here we investigated the stability of the introduced bacterial strains in the soils planted with barley and its effect on the diversity shifts of the rhizosphere soil bacteria. METHODS AND RESULTS: Two different types of bacterial strains of Bacillus thuringiensis and Shewanella oneidensis MR-1 were inoculated to the soils planted with barley. To monitor the stability of the inoculated bacterial strains, genes specific to the strains (XRE and mtrA) were quantified by qPCR. In addition, bacterial community analyses were performed using v3-v4 regions of 16S rRNA gene sequences from the barley rhizosphere soils, which were analyzed using Illumina MiSeq system and Mothur. Alphaand beta-diversity analyses indicated that the inoculated rhizosphere soils were grouped apart from the uninoculated soil, and plant growth also may have affected the soil bacterial diversity. CONCLUSION: Regardless of the survival of the introduced non-native microbes, non-indigenous bacteria may influence the soil microbial community and diversity.

선정 된 농지 및 동물 집에서 채취 한 토양 시료에서 테트라 사이클린 내성 세균 및 tetO, tetQ, tetS 및 tetW의 리보솜 보호 단백질 (RPP) 코딩 유전자 검출

( Nogrado Kathyleen ),( Leeji-hoon ) 한국환경농학회 2019 한국환경농학회 학술대회집 Vol.2019 No.-

This study aims to determine the presence of commonly detected tetracycline genes in agricultural soil samples from three agricultural areas in Gimje, Iksan, and Jangsu. In this study both the methodologies of viable cell count and molecular approaches were employed to detect tetracycline-resistant genes and to estimate the prevalence of tetracycline-resistant bacteria as well. The highest and least total numbers of culturable tetracycline-resistant bacteria were 8.65 x 10⁴ CFU/gfromGAF-1(Gimje)and3.50x10² CFU/g from GAH-3 (Gimje), respectively. The highest and least prevalence of tetracycline resistance based on culture method were 88.18% from Jangsu (JAF-3) and 0.86% from Gimje (GAH-3), respectively. Of the total culturable tetracycline-resistant bacteria, 0.76% to 3.86% were found to be Gram-negative bacteria. Meanwhile, molecular testing showed that among the tested four genes, tetQ was the most prevalent in all collected samples based on the results of the conventional PCR. Further analysis using quantitative PCR was performed. This was done to determine the absolute quantification of the tetracycline-resistance genes expressed in average gene copy number per gram of soil (gene copy/g). The qPCR results showed that the average tetO gene was highest in Iksan (IAH-3) at 9.46 x 10⁵ gene copy/g, while tetQ was highest in Gimje agricultural field 1 (GAF-1) at 1.24 x 10⁵ gene copy/g. Further study to analyze and understand the distribution of antibiotic-resistant genes in animal houses and agricultural area remains to be performed.

김제, 익산, 장수 지역의 토양 시료에서 테트라사이클린 내성 박테리아 다중약물 내성발생 및 RPP-tet 유전자검출

노그라도캐시린 ( Kathyleen Nogrado ),이지훈 ( Ji-hoon Lee ) 한국환경농학회 2020 한국환경농학회 학술대회집 Vol.2020 No.-

Multi-drug resistance is another urgent concern raised with antibiotic resistance because it narrows the treatment option, extends ailment which might lead to death if get worsen. Incidentally, antibiotics consumed in large amounts by the agricultural sector has found its way to the environment like soil. This in turn affects functions and causes selective pressure to antibiotic resistance among microbial soil residents. This study determined multi-drug resistance by disk diffusion testing and detected four ribosomal protection (RPP)-tet genes by PCR among tetracycline-resistant bacterial isolates obtained from soils in three agricultural areas. A total of 110 tetracycline-resistant bacterial isolates were obtained and tested. Using disk diffusion, it was determined that 17/110 bacterial isolates were multi-drug resistant (resistant to erythromycin, streptomycin, and tetracycline) and the majority was isolated from Gimje. Disk diffusion test also revealed different pattern of high resistance to individual antimicrobial: Gimje to tetracycline, Iksan to streptomycin, and Jangsu to erythromycin. This pattern could be reflective of which antibiotic is consumed in high amount in that area. On the other hand, PCR testing revealed that all four RPF-tet genes were detected m the culturable bacteria except tetO. PCR testing also revealed that a very small percentage (1.82%) carries 3 RPP tet genes, a slightly higher percentage (12.73%) carries two RPP-tet genes, and most bacterial isolates (31.82%) carries only one BPP-tet gene. The results or the two tests revealed that there were bacterial isolates that were phenotypically resistant but did not carry any of the four EPP-tet genes and also there were bacterial isolates phenotypically susceptible to tetracycline but carries one of the four RPP- tet genes. The former is explained by the presence of other tetracycline resistance gene determinant while the latter indicates that risk is not eliminated as the gene could still be transferred and expressed by other bacterial host. In 2017,a report on the rate of consumption of antibiotics in South Korea showed that tetracycline antibiotics have not been used since 2009; the results of this study could give information on the effect of the intervention applied concerning antibiotic resistance and agricultural health status in terms of the presence of antibiotic-resistant bacteria and antibiotic resistance genes.

염내성 세균에 의한 보리의 염 스트레스 내성 촉진

이슬 ( Seul Lee ),( Anamika Khanal ),( Kathyleen Nogrado ),송형근 ( Hyung-geun Song ),조유성 ( Yu-sung Cho ),이지훈 ( Ji-hoon Lee ) 한국환경농학회 2021 한국환경농학회지 Vol.40 No.4

BACKGROUND: Salinity is one of the major limiting factors in agriculture that affect the growth and productivity of crops. It is economically difficult to artificially purify the soil affected by salt. Therefore, the use of plant growth-promoting bacteria (PGPB) in an effort to reduce stress caused by salt is emerging as a cost-effective and environment-friendly method. In this study, the purpose was to isolate the salt-tolerant bacteria from the rhizosphere soil and identify their ability to promote plant growth under salt stress condition. METHODS AND RESULTS: The isolates KST-1, KST-2, AST-3, and AST-4 that showed plant growth-promoting activity for barley in salt conditions were close to Bacillus cereus (KST-1, KST-2, and AST-4) and Bacillus thuringiensis (AST-3) and showed high salt tolerance up to 7% of additional NaCl to the media. When inoculated to barley, the strains had only minor effect on the length of the barley. However, the concentrations of chlorophyll in the barley leaves were found to be higher from the bacteria-inoculated pots than those from the uninoculated control. In particular, the chlorophyll concentration in Bacillus cereus AST-4 experiment was 5.45 times higher than that of the uninoculated control under the same experimental condition. CONCLUSION(S): The isolated salt-tolerant bacteria were found to influence on chlorophyll concentration of the barley. As represented by the strain AST-4, microbes may suggest a cost-effective and environmentally benign method to alleviate salt stress of crops cultivated in salt-accumulated soils such as reclaimed lands.

Identification of Key Functional Genes Related with Nitrogen Cycle in the Terrestrial Habitat

Anamika Khanal,Junkoo Yeo(여준구),Kathyleen Nogrado,Seul Lee(이슬),Ahyeon Cho(조아현),Yoonjin Song(송윤진),Miji Kwon(권미지),Ji-Hoon Lee(이지훈) 한국토양비료학회 2018 한국토양비료학회 학술발표회 초록집 Vol.2018 No.5

살코스키 시약과 Indole-3-acetic Acid의 반응 중 트립토판의 간섭

이슬 ( Seul Lee ),어나미까커날 ( Anamika Khanal ),여준구 ( Junkoo Yeo ),노그라도캐시린 ( Kathyleen Nogrado ),조아현 ( Ahyeon Cho ),송윤진 ( Yoonjin Song ),권미지 ( Miji Kwon ),이지훈 ( Ji-hoon Lee ) 한국환경농학회 2018 한국환경농학회 학술대회집 Vol.2018 No.-

Indole-3-acetic acid (IAA) is the most common auxin hormone produced in the plant and is very important because it regulates various aspects of plant growth and development. Tryptophan is the main precursor for IAA while IAA can be synthesized by two pathways i.e., tryptophan-dependent pathway and tryptophan-independent pathway. There have been many studies using Salkowski regent to measure the IAA production by bacteria. In this study, we have found that there was the interference of tryptophan on the colorimetric estimations of indole-3-acetic acid (IAA) by using Salkowski reagent (35% HClO4 and 10 mM FeCl3), which developed a pink color complex of tris-(indole-3-acetate) iron(III) with IAA. Moreover, we detected the effect of different concentration of tryptophan (100, 200, and 300 μM) on different concentration of IAA (100, 200, and 300 μM). The absorbance on IAA for the tested concentrations was found to be increased with the increasing concentration of tryptophan. It seems that both the IAA and tryptophan show the maximum absorbance at the similar wavelengths. It was also observed that the difference between the predicted and measured concentrations of IAA became larger as the predicted IAA concentration decreased. From this study, it is expected that tryptophan would interfere with accurate measurements of IAA, and therefore care should be taken for the measurement of IAA generated by the precursor, tryptophan.

실시간 중합효소연쇄반응을 이용하여 논 토양 내 질소순환 관련 기능 유전자의 평가

어나미까커날 ( Anamika Khanal ),이슬 ( Seul Lee ),여준구 ( Junkoo Yeo ),노그라도캐시린 ( Kathyleen Nogrado ),조아현 ( Ahyeon Cho ),송윤진 ( Yoonjin Song ),권미지 ( Miji Kwon ),이지훈 ( Ji-hoon Lee ) 한국환경농학회 2018 한국환경농학회 학술대회집 Vol.2018 No.-

The nitrogen cycle and the microbes responsible for mediating this process have an important role in natural ecosystems, terrestrial habitats and also have a major impact on climate change. In this study, we use molecular techniques such as conventional PCR, clone library construction, sequencing, phylogenetic analysis and real-time PCR to explore microbial communities in a rice paddy soil by detecting and quantifying some of the key functional genes that are involved in the process of nitrogen cycle i.e., nitrogen fixation (nifH), hydrazine synthase (hzsA), nitrous oxide reductase (nosZ), copper-containing (nirK), cytochrome cd1- containing nitrite reductase (nirS), nitrite oxidoreductase (nxrB), and ammonium monoxygenase (amoA). The sequence assessment from the clone library targeting these different genes i.e., nifH, hzsA, nosZ, nirK, nirS, nxrB, and amoA showed a high diversity and dominance of bacterial community. Besides that, Real-time PCR using SYBR green dye and some old and recently developed primers specific for each individual gene revealed the high abundance of nxrB gene and low abundance of hzsA gene. This finding will be very helpful in exploring and exploiting microorganism in terrestrial habitat.

Identification of Nickel-resistant Bacteria from Metal Contaminated Soil and Potential Mechanism by Genome Sequencing

Seul Lee(이슬),Anamika Khanal,Junkoo Yeo(여준구),Kathyleen Nogrado,Ahyeon Cho(조아현),Yoonjin Song(송윤진),Miji Kwon(권미지),Ji-Hoon Lee(이지훈) 한국토양비료학회 2018 한국토양비료학회 학술발표회 초록집 Vol.2018 No.5