Microbial Communication Lab

Microbial biofilm, Microbial communication, Biological control, Pesticides

• Microbial cell to cell signaling and biofilm formation
• Biological control and integrated pest management
• Analysis of pesticide residues and metabolites produces by microbes

1. Microbial cell to cell signaling and biofilm formation

        Bacteria communicate to each other by secreting and sensing small molecules in response to cell density and coordinate their physiological processes. The known physiological processes include biofilm formation, motility, production of antimicrobial agents and expression of virulence factors. This phenomenon is known as “quorum sensing” and is crucial for a number of bacteria to successfully develop pathogenic, antagonistic or symbiotic relationships with their hosts. Thus, quorum sensing circuits represent an attractive target for the design of strategies to prevent bofilm formation, manipulate production of antimicrobial agents and attenuate infection.

         Research focuses of my group are on understanding microbial cell-to-cell signaling, and addressing the roles of the signaling in its physiological processes particular those for survival, pathogenesis and as biocontrol agents. Our main study organisms are Xanthomonas species, Bacillus subtilis group and Streptomyces species. The long term goal is to design strategies by interference of the signaling system against infections caused by microbes or to manipulate mechanisms for the management of plant disease by microbial agents.

2. Population diversity and integrated pest management

        The increase population of pesticide resistant pests in the field is partly due to long term exposure to and improper use of chemical pesticides. Integrated pest management (IPM) is an integrated approach of crop management. This approach with a main goal of significantly reducing or eliminating the use of pesticides while at the same time managing pest populations at an acceptable level, reducing human and environmental exposure to hazardous chemicals, and maintaining the balance of ecosystem. Biological control of plant pathogens by the purposeful utilization and introduction of antagonistic and live organisms in the field is emerging. Bacillus species and Streoptomyces species with properties of forming spores and production of varieties of antibiotics, enzymes, secondary metabolites, promoting animal/ plant growth or immune system are commonly used and investigated for the application in agriculture and industry. For control of bacterial diseases, there are only few suggested chemical pesticides available. The suggested pesticides include anitibiotics and copper compounds, while these compounds are known of high risk in causing pesticide resistance and environmental pollution. In addition, there is no known biocontol agent available. In our project, native microorganisms such as Bacillus subtilis group and Streptomyces species with antagonistic activity against X. axonopodis pv. citri, X. axonopodis pv. mangiferaindicae, X. oryzae pv. oryzae, anthracnose fungi and Botrytis species etc. will be screened and assessed. The potential application of native microorganisms as biocontrol agents for bacterial and fungal diseases will be examined. Information gained from this study will provide strategies by using biocontrol agents in disease management. In addition, fungicides and insecticides comparible with microorganism pesticides will be evaluated to serve as the basis for the application of abiotic and biotic pesticides in combination. In the long term, problems of pesticide residue in fruits and pesticide resistance will be resolved. The genetic identity based on rDNA sequences of Bacillus and Streptomyces strains from Taiwan and worldwide will be compared and the DNA fingerprints will be assayed for future patent and commercial use of the strains. Information gained from this study will provide strategies in integrated pest management of important plant diseases.

3. Diagnosis and identification of fungal pathogen

        Colletotrichum species are the causative agents of anthracnose on various important crops and the most notorious post-harvest pathogens greatly deteriorating the quality and shelf-life of agricultural products. Identification of Colletotrichum spp. by morphotaxonomic criteria is often frustrating and time consuming because these pathogens have wide host range, vary in morphological characteristics by environmental factors, and cross infect on one host. To cope with increasing demand of identification of this fungal pathogen, the application of heteroduplex mobility assay of ITS regions as a biochemical tool is explored. The aims of this project are to 1) survey and identify the Colletotrichum spp. that caused anthracnose disease in important exporting fruits by heteroduplex mobility assay of ITS regions; 2) verify the potential application of heteroduplex mobility assay in diagnosis and identification of Colletotrichum spp.; 3) investigate the application of heteroduplex pattern as an aid in identification. Information gained from this project will advance our knowledge in the species and genetic diversity of the Colletotrichum spp. in Taiwan. The heteroduplex pattern and reference isolates established in this study will provide a sensitive and inexpensive methodology in identification of large number of isolates. This methodology will be applicable in pathogen diagnosis and quarantine.

1. Microbial cell to cell signaling and biofilm formation

Phyllospere biofilm of antagonistic Bacillus species and its application in control of citrus canker disease

Citrus bacteria canker caused by Xanthomonas axonopodis pv. citri is the most devastating bacterial disease of citrus plants worldwide. Biofilm formation has been indicated to be important for various bacteria to successfully develop pathogenic relationships with their host. Thus strategies for disease control are developed by interference of biofilm formation. The objectives of this study are to understand the molecular mechanisms of biofilm formation on leaf surfaces of citrus plants by X. axonopodis pv. citri and the potential link to pathogenicity, and to investigate the efficacy of using antagonistic bacilli to interfere the biofilm formation by X. axonopodis pv. citri on surfaces of citrus leaves. To understand the mechanisms of biofilm formation by X. axonopodis pv. citri, the strain XW19was subjected to transposon mutagenesis. One mutant with mutation in a two-component response regulator and deficient in biofilm formation in polystyrene microplate was selected for further study. The two-component response regulator BfdR (for biofilm formation defective, regulator) mutant showed reduction in the development of biofilm and canker lesions as well as epiphytic growth on the leaf surfaces of citrus plants. BfdR was found to positively control the rpfF expression in XVM2 medium. The results indicated that biofilm formation and bacterial survival of X. axonopodis pv. citri on the leaf surfaces of citrus plants are associated with bacterial virulence and are controlled by BfdR through regulation of rpfF, which is required for the biosynthesis of diffusible signal factor [Published in PLoS ONE (2013) 8(4):e62824)].Application of antagonistic bacilli on citrus leaves resulted in interference of biofilm formation by X. axonopodis pv. citri which may be a feasible way for the disease control of citrus bacterial canker [Published in PLoS ONE (2012) 7(7):e42124].

• A novel two-component response regulator links rpf with biofilm formation and virulence of Xanthomonas axonopodis citri

• • The two-component response regulator BfdR in X. axonopodis pv. citri plays roles in biofilm formation, and virulence on leaf surfaces of citrus plants. (Publsihed in PLoS ONE (2013) 8(4):e62824)

     

• • BfdR positively controls the expression of rpfF in medium mimicking cytoplasmic fluids in planta.

• Biocontrol of citrus bacterial canker by antagonistic Bacillus species with indication of the interference of phyllosphere biofilms

• •  The colonization and biofilm formation of citrus canker bacteria (Red fluorescence) on leaf surfaces of citrus plants were reduced by the treatment with strains of B. subtilis group (TKS1-1 & WG6-14 – Green fluorescence). (Published in PLoS ONE (2012) 7(7):e42124)
     

• • The symptom development of citrus bacterial canker is attenuated by the treatment with B. subtilis TKS1-1 and B. amyloliquefaciens WG614. (Published in PLoS ONE (2012) 7(7):e42124)

2. Population diversity and integrated pest management

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a devastating disease resulting in significant crop losses in various citrus cultivars worldwide. A biocontrol agent has not been recommended for this disease. To explore the potential of native bacilli in Taiwan to control this disease, Bacillus species with a broad spectrum of antagonistic activity against various phytopathogens were isolated from plant potting mixes, organic compost and the rhizosphere soil. Seven strains TKS1-1, OF3-16, SP4-17, HSP1, WG6-14, TLB7-7, and WP8-12 showing superior antagonistic activity were chosen for biopesticide development. The genetic identity based on 16S rDNA sequences indicated that all seven native strains were close relatives of the B. subtilis group and appeared to be discrete from the B. cereus group. DNA polymorphisms in strains WG6-14, SP4-17, TKS1-1, and WP8-12, as revealed by repetitive sequence-based PCR with the BOXA1R primers were similar to each other, but different from those of the respective Bacillus type strains. Information gained from molecular typing of native strains would provide DNA fingerprints as an aid for patenting or commercializing the stains in the future [Published in PLoS ONE (2012) 7(7):e42124]. In addition, our previous study indicated that the application of a Bacillus strain TKS1-1 endospore formulation to the leaf surfaces of citrus reduced the incidence of citrus bacterial canker and could prevent development of the disease.

Bacillus strains SP4-17, WP8-12, WG6-14 and TKS1-1, which showed the greatest antagonistic activity, were members of the B. subtilis group recognized as GRAS bacteria.

Strains SP4-17, TKS1-1, WG6-14 and WP8-12 showing the superior antagonistic activity against X. axonopodis pv. citri had the same BOXA1R-PCR fingerprint, which is different from their respective type strains.

3. Development of molecular techniques for diagnosis and identification of fungal Pathogen

        Colletotrichum spp. are the causal agents of anthracnose on various important crops and the most notorious post-harvest pathogens greatly deteriorating the quality and shelf-life of agricultural products. Identification of Colletotrichum spp. by morphotaxonomic criteria is often frustrating and time consuming because these pathogens have wide host range, vary in morphological characteristics by environmental factors, and cross infect on one host. To cope with increasing demand of identification of this fungal pathogen, the application of heteroduplex mobility assay of ITS regions as a biochemical tool was explored. The ITS regions of tested Colletotrichum isolates including C. gloeosporioides, C. acutatum, C. musae, C. graminicola , C. capsici, C. dematium, C. lindemuthianum, and Colletotrichum sp., were PCR amplified using the universal primers ITS1 and ITS4. The data from HMA of ITS regions suggested that tested Colletotrichum isolates could be divided into 5 distinctive species groups. The correlation of DNA distance and relative heteroduplex motility from reference isolates was calculated. The calculated formula was used to deduce DNA distances of tested sequences from relative heteroduplex motility. Phylogenetic analysis of ITS regions based on DNA distance inferred from relative heteroduplex mobility showed that tested Colletotrichum isolates was classified into five groups, namely CG, CA, CC, CM, and CL which is the same as that from DNA sequences. Analysis of the heteroduplex pattern (HP) with reference isolates C. gloeosporioides Cg1, C. gloeosporioides Cg16, C. musae Cm1, C. gloeosporioides Cg2, C. graminicola Cgr1 and C. lindemuthianum Cl1 indicated that 29 Colletotrichum isolates could be divided into 6 distinctive species groups. Isolates of C. gloeosporioides could be divided into two subgroups CG1 and CG2. In conclusion, HMA and HP of ITS regions is a relative rapid and simple method for species-specific identification of Colletotrichum spp. Because the uniqueness of HP when assaying with DAN from reference isolates, we suggest that the HP of ITS regions may be use as “DNA barcodes” for the identification of anthracnose fungi [published in J. Phytopath. 158(1):46-55. & New Biotech. (2011) 28(1):72-78.].

Pesticides,  Experiment in Pesticides,  Pesticide Residues Analyses and Food Safety, Agricultural Pesticides-Pharmacology and Application, Agricultural Pesticide Prescriptions, Microbial Communication,  Sustainable Agriculture and Ecology.

1. Yun-Chien Wu, Chien-Wen Yu, Jo-Yu Chiu, Yu-Hsuan Chiang, Nobutaka Mitsuda, Xu-Chen Yen, Tzu-Pi Huang, Tzu-Fang Chang, Cen-Jie Yen, Woei-Jiun Guo. 2023. The AT-hook protein AHL29 promotes Bacillus subtilis colonization by suppressing SWEET2-mediated sugar retrieval in Arabidopsis roots. Plant Cell Environ (Published Online https://doi.org/10.1111/pce.1477)
2. Bo-Lin Ho, Jhun-Chen Chen, Tzu-Pi Huang*, Su-Chiung Fang*. 2022. Protocorm-like-body extract of Phalaenopsis aphrodite combats watermelon fruit blotch disease. Plant Sci. 13:1054586. (doi: 10.3389/fpls.2022.1054586)
3. Ting-Zhi Liao, Yu-Hsuan Chen, Jyh-Nong Tsai, Chieh Chao, Tzu-Pi Huang, Cheng-Fang Hong, Zong-Chi Wu, Isheng J. Tsai, Hsin-Han Lee, Ned B. Klopfenstein, Mee-Sook Kim, Jane Stewart, Ndeme Atibalentja, Fred E. Brooks, Philip G. Cannon, A. Mohd Farid, Tsutomu Hattori, Hoi-Shan Kwan, Regent Yau Ching Lam, Yuko Ota, Norio Sahashi, Robert L. Schlub, Louise S. Shuey, Alvin M. C. Tang, and Chia-Lin Chung^*. Translocation of fungicides and their efficacy in controlling Phellinus noxius, the cause of brown root rot disease. Plant Dis. (Published Online https://apsjournals.apsnet.org/doi/10.1094/PDIS-06-22-1285-RE)
4. Tzu-Pi Huang*, Jenn-Wen Huang, Chuan-Shun Lin, Chung-Lun Lu, Chien-Ya Kao, Wen-Di Huang, and Wen-Hsin Chung. 2022. Multiple functions of Bacillus biocontrol agents for agricultural production. FFTC APBB 263 (Aug. 29, 2022., https://apbb.fftc.org.tw/article/263) 
5. Ying-Ru Liang, Fang-Chin Liao, Tzu-Pi Huang*. 2022. Deciphering the influence of Bacillus subtilis strain Ydj3 colonization on the vitamin C contents and rhizosphere microbiomes of sweet peppers. PLoS ONE 17(2): e0264276.
6. Yu-Hsuan Chen, Pei-Chun Lee, and Tzu-Pi Huang*. Biological control of collar rot on passion fruits via induction of apoptosis in the collar rot pathogen by Bacillus subtilis. Phytopathology 111(4): 627-638.
7. Chia-Jung Yang, Tzu-Pi Huang*, and Jenn-Wen Huang*. 2021. Field sanitation and foliar application of Streptomyces padanus PMS-702 for the control of rice sheath blight. Plant Pathol. J. 37(1): 57-71.
8. Li-Hsuan Ho, Yung-I Lee, Shu-Ying Hsieh, I-Shiuan Lin, Yun-Chien Wu, Han- Yu Ko, Patrick A. Klemens, H. Ekkehard Neuhaus, Yi-Min Chen, Tzu-Pi Huang, Chih-Hsin Yeh, Woei-Jiun Guo, 2021. GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae). Plant, Cell Environ. 44:20-33
9. 黃姿碧。2021。農業也要益菌多全方位保護農漁牧-益生菌在農業上的多元應用。豐年雜誌，71卷8期: 20-25頁。110年8月號。
10. Kai-Wei Lin, Yu-Shen Liang, Chia-Yu Hsiao, Fei Wang, Tzu-Pi Huang and Yi-Hsien Lin* (2021) Application of fermentation broth of Bacillus amyloliquefaciens PMB05 to control bacterial canker disease on lemon. J. Plant Med., 63 (4), 17-26.
11. 蔡羽琳、黃振文、黃姿碧、陳郁璇、馮啟倫、林傳順。(2020)。飼糧中添加枯草桿菌對生長肥育豬生長性能、屠體肉質及糞便氣味之影響。中國畜牧學會會誌，49(增刊)：359。
12. Ting-Hsin Ho, Chiao-Yu Chuang, Jing-Lin Zheng, Hong-Hua Chen, Yu-Shen Liang, Tzu-Pi Huang, and Yi-Hsien Lin. 2020. Bacillus amyloliquefaciens strain PMB05 intensifies plant immune responses to confer resistance against bacterial wilt of tomato. 110(12):1877-1885
13. Yu-HsuanChen and Tzu-Pi Huang.* First report of anthracnose caused by Colletotrichum capsici on passion fruit in Taiwan. Plant Dis. 102(12):2648. https://doi.org/10.1094/PDIS-03-18-0462-PDN
14. Mei-Ling Chen, Tzu-Pi Huang, Tai-Wei Chen, Hsin-Hua Chan, Bing-Fang Hwang *. 2018. Interactions of genes and sodium intake on the development of hypertension: a cohort-based case-control study. J. Environ. Res. Public Health 15(6): 1110.
15. Yu-Hsuan Chen, Yia-Han Lin, Ying Yeh, and Tzu-Pi Huang*. 2018. Induction of apoptosis in the anthracnose fungi by Bacillus subtilis. Phytopathology 108(10): 141. (Abstract)
16. Ying-Ru Liang, Fang-Chin Liao, and Tzu-Pi Huang*. 2018. Influence of applying microbial agents on the quality of sweet pepper. Phytopathology 108(10): 142. (Abstract)
17. 梁瑩如、黃郁容、黃姿碧。2018。淺談現代農藥製劑發展趨勢。農業世界， 416:39-45。
18. Lo, S. F., T. H. Ho, Y. L. Liu, M. J. Jiang, K. T. Hsieh, K. T. Chen, L. C. Yu, M. H. Lee, C. Y. Chen, P. Huang, M. Kojima, H. Sakakibara, L. J. Chen, S. M. Yu. 2017. Ectopic expression of specific GA2 oxidase mutants promotes yield and stress tolerance in rice. Plant Biotech. J. 15(7):850-864.
19. Tuan, S. J., F. T. Hu, H. Y. Chang, P. W. Chang, Y. H. Chen, P. Huang.^* 2016. Xylella fastidiosa transmission and life history of two Cicadellinae sharpshooters, Kolla paulula and Bothrogonia ferruginea (Hemiptera: Cicadellidae), in Taiwan. J. Econ. Entomol. 109(3) 1034-1040.
20. Chang, H. Y., P. Huang, and S. J. Tuan. 2014. Transmission efficiency of Pierce’s disease by Bothrogonia ferruginea (F.) (Hemiptera: Cicadellidae) in Taiwan. J. Agri. Forest. 63:205-215.
21. Huang, T. P.^*, M. Lu, and Y. H. Chen. 2013. A novel two-component response regulator links rpf with biofilm formation and virulence of Xanthomonas axonopodis pv. citri. PLoS ONE 8(4):e62824
22. Huang, T. P.^* Regulation of biofilm formation and pathogenicity of citrus canker bacteria and its application in disease management. Plant Pathol. Bull. 22(1):1-11.
23. Huang, T. P.*, D. D. S. Tzeng, A. C. L. Wong, C. H. Chen, K. M. Lu, Y. H. Lee, W. D. Huang, B. F. Hwang, and K. C. Tzeng. 2012. DNA polymorphisms and biocontrol of Bacillus antagonistic to citrus bacterial canker with indication of the interference of phyllospere biofilm. PLoS ONE 7(7):e42124
24. Hwang, B. F., I. P. Liu, P. Huang. 2012. Gene-environment interaction between interleukin-4 promoter and molds in childhood asthma. Ann. Epidemiol. 22(4):250-256.
25. Hwang, B. F., I. P. Liu, P. Huang. 2011. Molds, parental atopy and pediatric incident asthma. Indoor air 21(6)472-478
26. Huang, T. P. , H. Chen, Y. H. Lee, B. F. Hwang, D. S. Tzeng, K. C. 2011. Genetic diversity of antagonistic Bacillus subtilis against citrus canker bacteria. Phytopathology 101:S76.
27. Huang, W. D., Y. H. Chiu, T. H. Wang, P. Huang, D. S. Tzeng. 2011. The amplification culture of endospore formulation of Bacillus subtilis biofungicide and its use in disease management. Phytopathology 101:S76.
28. Tzeng, D. S., W. D. Huang, Y. R. Liang, C. Y. Chen, P. Huang, T. L. Lee, W. R. Lai. 2011. The use of arthrospore formulation of antagonistic Streptomyces for the control of diseases caused by Phytophthora species. Phytopathology 101:S180.
29. Huang, T. P., Y Yeh and D. D.-S. Tzeng. 2011. Barcode-like heteroduplex DNA pattern as an aid for rapid identification of anthracnose fungi. New Biotech. 28(1):72-78. Huang, T. P., Y Yeh and D. D.-S. Tzeng. 2010. Heteroduplex mobility assay for identification and phylogenetic analysis of anthracnose fungi. Phytopath. 158(1):46-55.
30. Huang, T. P. and K. C. Tzeng. 2009. Biofilm formation and motility by strains of Xanthomonas axonopodis citri causing differential symptoms on citrus leaves. Phytopathology 99(6 Suppl.):S56
31. Huang, T. P., Yeh and D. D.-S. Tzeng. 2008. Identification of anthracnose fungi by heteroduplex mobility assay and heteroduplex pattern. Phytopathology 98(6 Suppl.):S9.
32. Huang, T. P.* and A. C. L. Wong. 2007. Extracellular fatty acids stimulate flagella-independent translocation by Stenotrophomonas maltophilia. Microbiol. 158(8-9): 702-711.
33. Huang, T. P. and A. C. L. Wong. 2007. cAMP receptor protein regulated cell-cell communication system mediates expression of a FecA homologue in Stenotrophomonas maltophilia. Environ. Microbiol. 73(15): 5034-5040.
34. Huang, T. P., E. B. Somers, and A. C. L. Wong. 2006. Differential biofilm formation and motility associated with lipopolysaccharide/exopolysaccharide-coupled biosynthetic genes in Stenotrophomonas maltophilia. Bacteriol. 188(8):3116–3120.
35. Shimizu, K. J. K. Hicks, T. P. Huang, and N. P. Keller. 2003. Pka, Ras and RGS protein interactions regulate activity of AflR, a Zn(II)2Cys6 transcription factor in Aspergillus nidulans. Genetics 165(3):1095-1104.

*corresponding author

1. 黃姿碧*、黃振文、林傳順、呂仲倫、黃文的、鍾文鑫。2021。健康微生物菌叢創造農畜水產的安全與綠金。科技部全球事務與科學發展中心台灣亮點研究電子報。2021年5月。(https://trh.gase.most.ntnu.edu.tw/tw/article/content/207)
2. Tzu-Pi Huang*, Jenn-Wen Huang, Chuan-Shun Lin, Chung-Lun Lu, Wen-Di Huang, and Wen-Hsin Chung. 2021. Probiotic microbiomes for agriculture safety and green gold creation. GASE Newsletter – Taiwan Research Highlight. 2021.5 (https://trh.gase.most.ntnu.edu.tw/en/article/content/207)
3. Huang, T. P. and A. C. L. Wong. 2004. Involvement of lipopolysaccharide in biofilm formation by a septic tank system clogger Stenotrophomonas maltophilia. FRI Newsletter 16(4)2-3.

1. Huang, T. P. *, Chen, Y. H., Huang, W. D., Lin, C. S., Kao, C. Y., Lu, C. L., Yang, S. S., Chung, W. H., and Huang, J. W. 2020. Application of probiotic microbiomes in agriculture. Proceedings of the Workshop on Novel Crop Protection Technologies in Accord with Pesticide Reduction Policy. Chen, C. C., Lin, C. P., Tsai, J. N., Lin, M. J., Huang, C. W., and Hsieh, T. F. Ed. Special Publication of TARI No. 229. Taiwan Agricultural Research Institute, Executive Yuan, Taiwan. 121-133 pp. ISBN 978-986-5449-11-7.
2. Huang, T. P. * and Chen, Y. H. Bacillus subtilis-based products for plant health care. Handbook of Eco-Friendly Products for Plant Health Care. Huang, J. W., Hseih, T. F., Hsieh, F. C., and Lo, C. T. Ed. Wu-Nan Book Inc. 55-78 pp. ISBN 978-957-7635-51-8.
3. Der-Syh Tzeng, Huang, T. P. et al. 2009. Maximum Residue Limits of Taiwanese Agricultural Products in Major Exported Countries (Regions). Version 7. 362 PP. ISBN:957-01-9724-2. Agriculture and Food Agency, Council of Agriculture, Executive Yuan, R.O.C. and National Chung-Hsing University.
4. Der-Syh Tzeng, Huang, T. P. et al. 2008. Maximum Residue Limits of Taiwanese Agricultural Products in Major Exported Countries (Regions). Version 6. 475 PP. ISBN:957-01-9724-2. Agriculture and Food Agency, Council of Agriculture, Executive Yuan, R.O.C. and National Chung-Hsing University.
5. Biofilm formation and cell-cell signaling in Stenotrophomonas maltophilia. 2007. University of Wisconsin-Madison, USA. ( D dissertation)
6. Application of rDNA internal transcribed spacer (ITS) sequence characteristics as an aid for the identification and phylogenetic study of Colletotrichum 1999. National Chung-Hsing University. (Master thesis)