Source: MICHIGAN STATE UNIV submitted to NRP
BENEFICIAL PLANT-MICROBE INTERACTIONS OF AGRICULTURAL IMPORTANCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0075609
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Sep 1, 2009
Project End Date
Aug 31, 2014
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Microbiology & Molecular Genetics
Non Technical Summary
Agriculture uses chemical fertilizers to achieve the highest crop yields possible. However, their adverse effects show the clear need to change production methods to achieve sustainability, economic success, conservation of natural resources and avoidance of adverse disturbances to the agroecosystem and neighboring centers of human population. Plant-associated microbes can help to increase grain yields world wide without compromising food production to accommodate population growth by improving the success of sustainable agricultural practices that use biological processes to enhance the crop's output without irreparably damaging the natural resource base where the crop can be grown. Some soil microbes are admirably suited for use in "biofertilization" programs that boost crop yields while reducing chemical fertilizers in biosafe and sustainable ways. Cereals are the major source of food for human nutrition. Rice (Oryza sativa L.) is most prominent staple diet for the world's population. Wheat (Triticum aestivum) is 2nd in importance as a major carbohydrate source. Their high grain production has only been possible with high N fertilizer inputs. The environmental problems associated with inorganic fertilizer usage could be mitigated if the cereal crops established a more direct and intimate association with beneficial bacteria that promote their growth and increase their grain yield while simultaneously reducing their dependence on chemical N-fertilizer inputs. Although some agricultural biofertilizers (e.g., Rhizobium for legumes, Azospirillum for wheat, and cyanobacteria for rice) are already used in crop production, none till now have satisfactorily replaced the full fertilizer N demand to maximize yield potentials under field conditions. Therefore, discovery and development of more effective biofertilizers that can reduce the dependence on chemical N-fertilizer inputs to maximize cereal crop yields is a high priority area to help achieve the goals of sustainable agriculture worldwide. Our development of new, reliable agricultural biofertilizers addresses the important real-world problem of limited biologically available N in cereal crop production and shows that exploitation of natural beneficial plant-microbe associations is environmentally sound and has high intrinsic probability for success. We will continue basic research to increase knowledge of beneficial plant-microbe interactions of agricultural importance, especially those involving Rhizobium and cereal crops, and utilize this knowledge in real-world, sustainable agroecosystems. The expected outcomes will provide crucial information needed to succeed in implementing environmentally sound, biofertilizer inoculants into sustainable agroecosystems. Major beneficiaries of the applied information are Ag extension agents, low-income farmers in developing and 3rd-world countries who commonly produce cereal crops on marginally fertile soils but cannot afford to utilize chemical fertilizers, and worldwide manufacturers of biofertilizer inoculants in both the general and private sectors.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1024010110025%
1330120106010%
2051530106015%
2051549106015%
2061530102015%
2061549102010%
9034010106010%
Knowledge Area
206 - Basic Plant Biology; 133 - Pollution Prevention and Mitigation; 102 - Soil, Plant, Water, Nutrient Relationships; 205 - Plant Management Systems; 903 - Communication, Education, and Information Delivery;

Subject Of Investigation
0120 - Land; 1549 - Wheat, general/other; 4010 - Bacteria; 1530 - Rice;

Field Of Science
1060 - Biology (whole systems); 1100 - Bacteriology; 1020 - Physiology;
Goals / Objectives
The goal of this project is to increase our knowledge of beneficial plant-microbe interactions of agricultural importance, especially those involving Rhizobium and cereal crops, and to utilize this knowledge in real-world, sustainable agroecosystems. The specific objectives of this project cycle are (1) to complete the field inoculation trials that establish how well do our most promising candidate strains of endophytic rhizobia perform as superior biofertilizer inoculants for rice and wheat grain production, and (2)to continue development and release of CMEIAS software tools for computer-assisted microscopy of plant-microbe interactions and apply them to a quantitative assessment of endophytic plant growth-promotive rhizobacteria that colonize crop plants. The expected outputs will be research publications that advance basic knowledge on beneficial plant-microbe associations and provide crucial information to succeed in implementing environmentally sound, biofertilizer inoculants into sustainable agroecosystems involving legume-cereal production, and public release of CMEIAS software that strengthens microscopy-based approaches for understanding microbial ecology.
Project Methods
Objective 1: we collaborate with Dr. Youssef Yanni (Sakha Agricultural Research Station, Kafr El-Sheikh, Egypt [Nile delta]) to conduct field inoculation trials with rice and wheat using selected indigenous strains of endophytic rhizobia isolated from these crop plants. Agronomic parameters (grain yield, straw yield, harvest index and agronomic fertilizer-N use efficiency) measured at maturity reveal the influence of rhizobial inoculation on vegetative and reproductive growth of the cereal crop, the strain/variety specificity in field inoculation responses, the efficiency in which the bacterial inoculum modifies the crop plant's ability to exploit nutrient reserves in the root zone for grain production, and the amount of chemical fertilizer-N that can be replaced by the bacterial inoculum to achieve the same increase in crop yield. We will also explore the benefits and tradeoffs of consortia inoculants including selected endophytic rhizobia with other plant growth-promoting rhizobacteria (e.g., cyanobacteria, Pseudomonas sp.)in promoting crop production. We use a variety of microscopy methods (fluorescence, electron) to examine the colonization patterns of our best performing inoculant strains on rice and wheat roots, and then quantify their spatial ecology using CMEIAS image analysis software that we develop. Results are published in refereed journals, book chapters, CRIS reports and international scientific conferences. Extension delivery of the biofertilization technology resulting from the translational research is targeted to low-income farmers in developing and 3rd-world countries who commonly produce cereal crops on marginally fertile soils but cannot afford to utilize chemical fertilizers.

Progress 09/01/09 to 08/31/14

Outputs
Target Audience: Scientists interested in beneficial plant-bacterial associations of agricultural importance, sustainable agriculture, microbial ecology, soil microbiology, plant microbiology, agricultural microbiology, plant growth-promotive rhizobacteria, studies on the soil and plant microbiomes. Agronomists working together with Ag Extension agents to recommend the use of biofertilizer inoculants to optimize sustainable agricultural practices that result in high-yielding cereal crops (especially rice and wheat). Microbial ecologists interested in learning about new technological developments and applications of CMEIAS computer-assisted microscopy software in their quantitative biology research, especially as it applies to plant microbiology and microbial biofilm ecology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This research has provided opportunities for training and professional development of 2 international visiting scholars (Dr. Abu-Bakr Gomaa, Egypt; Dr. Prabhat Jha, India) and 18 undergraduate microbiology majors whose miniproject research activities fulfilled the capstone research requirement for their bachelor of science degree in microbiology at Michigan State University. This project represented an international research collaboration between Prof. Frank Dazzo at Michigan State University and Dr. Youssef Yanni at the Sakha Agricultural Research Station, Egypt. Dr. Yanni spent 1-2 months per year of this project working with Dr. Dazzo at MSU. How have the results been disseminated to communities of interest? Publications in refereed scientific journals, invited book chapters in books with a focus on agricultural microbiology and microbial ecology, news of pertinent information at our CMEIAS project website, presentation of scientific posters at local, national and international conferences, and discussion of research findings using CMEIAS software in microbiology coursework at Michigan State University (senior level undergraduate Prokaryotic Microbial Physiology course MMG 421; 1st year graduate level Integrated Microbial Biology course MMG 801). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. We performed numerous large-scale field inoculation trials to assess the performance of our superior biofertilizer inoculant strains of rhizobia isolated from roots of rice and wheat grown in fields within the Nile delta of Egypt. These rhizobial isolates are natural root endophytes of the crop plants that have been rotated successfully with berseem clover for centuries. The results of our extensive field inoculation trials conclusively show, with high statistical significance, the improvement in crop vegetative and grain yields of rice and wheat when inoculated with our biofertilizer strains and cultivated in a wide variety of soils differing in fertility and salinity. The results are very strain-variety specific, identify which biofertilizer inoculant strains can significantly reduce the demand for chemical N fertilizer to achieve high grain yield in ways that are fully consistent with sustainable agriculture, and demonstrate the cultivation safety used in the assessments. These accomplishments are unique in comparison to most other studies on plant growth-promotive rhizobacteria in that they include EXTENSIVE field inoculation studies covering farmers' large fields in many locations under real-world agronomic growing conditions. This accomplishment is significant since it fills the major gap in providing extensive translational assessment of the benefits of microbial biofertilization in sustainable agriculture. We have published our findings on the beneficial Rhizobium-cereal associations of agricultural importance in both refereed scientific journals and in invited book chapters, and have a few more manuscripts currently in preparation to complete our dissemination of that scientifically successful story. The pertinent publications are indicated in this final report. 2. Microscopy and digital image analysis are important investigative tools in microbial ecology. However, they are underutilized because of the lack of freely-available, well-documented and reliable investigative tools of computer-assisted microscopy that can process and analyze complex digital images of microbial populations and communities at single cell resolution, and directly compute quantitative information about the in situ ecology of the microbes’ world from their own perspective and spatial scale, without the need for their laboratory cultivation. Commonly, images in the microbial ecology literature are only accompanied with visual, descriptive interpretation. To address this challenge, we have been developing image analysis software designed to extract the full information content in digital images of actively growing microbial populations and communities. Our suite of CMEIAS software applications represents a computing technology of new and improved tools for image bioinformatics, including improvements in image acquisition, processing and segmentation, object analysis and classification, data processing, statistical analysis and exploratory data-mining. When finalized, CMEIAS software applications and their various documentations (refereed journal publications, illustrated user manuals, help topic search files, audio-visual tutorials and training macros/scripts with training images) are released as free downloads for research and educational purposes at our MSU CMEIAS website . During this period of the project, we made considerable progress in CMEIAS software development, documented in our project website with links to pertinent scientific literature in its "Publications using CMEIAS" webpage. Reference citation # 88 provides a thorough technical summary of our accomplishments up to April 2013 and is available online at . Three major milestones in CMEIAS development included our completion and release of the software executable and user support files for the (1) CMEIAS Color Segmentation application designed to facilitate the complex task of color image segmentation, which opens new opportunities of imaging applications where discriminating color recognition is important; (2) CMEIAS Quadrat Maker designed to alleviate the nontrivial problem of optimizing the grid-lattice dimensions and automating the production of size-optimized quadrat images for plot-based spatial pattern analysis in landscape ecology; and (3) we are near completion of our CMEIAS JFrad software application designed to provide the computing tools to analyze the fractal dimension of landscape architectures and spatial patterns of individual cells in microbial biofilms. Other noteworthy accomplishments are (4) major upgrades of the core CMEIAS image analysis program, including improvements in the GUI layout, (5) user-friendly improvements in selections/settings, (6) addition of several new discriminating measurement features in landscape and spatial ecology, (7) multiple additions to the CMEIAS data analysis toolpack that compiles and analyzes CMEIAS data within Microsoft Excel, and (8) a comprehensive analysis and optimization of the image analysis protocol to quantify the colonization of crop roots by superior biofertilizer inoculant strains of plant growth-promoting rhizobacteria. We are currently using our newly developed technology of CMEIAS computer-assisted microscopy and digital image analysis to examine if the spatial intensity of root colonization by biofertilizer inoculants can predict their performance in promoting crop yield under field conditions.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Folland, I., D. Trione and Frank B. Dazzo. 2014. Accuracy of biovolume formulas for CMEIAS computer-assisted microscopy and body size analysis of morphologically diverse microbial populations and communities. Microbial Ecology 68:596-610
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Trevisan, R., M. Picarella, F. B. Dazzo, S. Bona, G. Morabito, and A. Squartini. 2014. Using a morpho-functional approach to assess phytoplankton dynamics in two adjacent high-mountain lakes: a 10-year survey. Journal of Limnology 73(3): 409-420
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Ji, Z., K, Card and F. B. Dazzo. 2014. CMEIAS JFrad: a new computing toolkit to discriminate the fractal geometry of landscape architecture and spatial patterns of individual cells in microbial biofilms. Microbial Ecology, DOI: 10.1007/s00248-014-0495-1
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Dazzo, FB, I. Ganesan and Y. Yanni 2014. Spatial ecology of rhizobacterial colonization on roots analysed by CMEIAS computer-assisted microscopy at single-cell resolution. In: D. P. Singh and H. Singh (eds.) Trends in Soil Ecology, Chpt. 4, pp. 67-92, Studium Press, Houston
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: Y. G. Yanni and F. B. Dazzo 201-. Occurrence and ecophysiology of the natural endophytic Rhizobium-rice association, and translational assessment of its biofertilizer performance within the Egypt Nile delta. In: F. DeBruijn (Ed.), Biological Nitrogen Fixation, Wiley/Blackwell, in press.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: M. Robledo, L. Rivera, E.Men�ndez, P. Mart�nez-Hidalgo, R.Rivas, E. Vel�zquez, F.B. Dazzo, E. Mart�nez-Molina and P. F. Mateos. 201-. Role of Rhizobium cellulase CelC2 in host root colonization and infection In: Frans J. DeBruijn (Ed). Biological Nitrogen Fixation, Wiley/Blackwell. In press.
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: Y. G. Yanni, F. B. Dazzo, A. Squartini, M. Zidan, A. E. Alsadany. 201-. Assessment of a natural endophytic association between wheat and Rhizobium and its ability to promote wheat production. In preparation for Plant & Soil.
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: Y. G. Yanni, F. B. Dazzo, M. Zidan, A. E. Alsadany. 201-. Enhancement of rice crop production using consortia of inoculants containing species of Rhizobium, Pseudomonas and Nostoc. In preparation for Plant & Soil.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2009 Citation: Y.G.Yanni & F.B.Dazzo 2009 Prospects of contributed biofertilization technology for agricultural sustainability and environmental bio-safety.(poster) 19th International Symposium on Environmental Biogeochemistry - Environmental Changes and Sustainability of Biogeochemical Cycling, Sept.14 - 18, 2009, University of Hamburg, Germany.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2010 Citation: Lina P. Rivera, Marta Robledo, Encarna Vel�zquez, Youssef G. Yanni, Frank Dazzo, Eustoquio Mart�nez-Molina, Pedro F. Mateos. 2010, Overexpresion of endoglucanase (CelC) increase infectivity of the rice endophyte Rhizobium leguminosarum bv. trifolii strain E11. 9th European Nitrogen Fixation Conference. September, 2010. Geneva, Switzerland
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Card, K. and F. Dazzo. 2013. Discrimination of natural microbial biofilm architectures using CMEIAS landscape ecology metrics. (Scientific poster presented at the 15th Annual MSU Undergraduate Student Research & Arts Forum), April 12, 2013
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Ganesan, I. and F. Dazzo. 2013. Spatial analysis of microbial community images at single-cell resolution using CMEIAS software. (Scientific poster presented at the 15th Annual MSU Undergraduate Student Research & Arts Forum), April 12, 2013.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2010 Citation: Lina P. Rivera, Marta Robledo, Encarna Vel�zquez, Youssef G. Yanni, Frank Dazzo, Eustoquio Mart�nez-Molina, Pedro F. Mateos. 2010 Rhizobium leguminosarum bv trifolii E11 as multifunctional bioinoculant in legumes and non-legumes. XIII National Meeting of the Spanish Society of Nitrogen Fixation and II Portuguese-Spanish Congress on Nitrogen Fixation. June, 2010. Zaragoza, Spain.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Adia-Nimuwa, U., D. Colbry, and F. Dazzo. 2013. Optimizing CMEIAS Iterations, a novel computing tool for microbial ecology research. (Scientific poster presented at the 15th Annual MSU Undergraduate Student Research & Arts Forum), April 12, 2013
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Jha, Prabhat, Y. Yanni, T. Stedtfeld, B. Stedtfeld, A.B. Gomaa, S. Gantner, B. Chai, S. Hashsham and F. B. Dazzo. 2014. Inoculation with plant growth-promoting Rhizobium biofertilizer and urea N fertilizer treatments restructure the endophytic microbiome of rice roots grown in soil. (poster) American Society for Microbiology 114th Ann. Mtg, Boston.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Scientists interested in bendficial plant-bacteria associations of agricultural importance. Microbial ecologists interested in learning about new developments and applications of CMEIAS computer-assisted microscopy software developments that we have made on this project. Changes/Problems: The major problems encountered were the complex and lengthy delays in processing the paperwork for the visa for the International reseach collaborator to come from Egypt to Michigan State University with research samples for analysis, and the USDA APHIS permits to bring the research samples. Even after spending months doing all the redtape paperwork, the customs inspector said the permit documents were not fully satisfactory. Also the funding of this project on the Egypt side started significantly later than funding on the USA side, so we will need to obtain the no-cost extensions to put the 2 portions of this international collaboration project in phase so they can be completed at the same time for both investigators. All the detail is provided in the annual report documents submitted to the funding program (US-Egypt Science & Technology Joint Program) during this reporting period. What opportunities for training and professional development has the project provided? The project during the past reporting period has trained 18 students and one International Visiting Professor. How have the results been disseminated to communities of interest? Various scientific publications cited in this report, our project website http://cme.msu.edu/cmeias/, and where appropriate, lectures in undergraduate and graduate microbiology courses. What do you plan to do during the next reporting period to accomplish the goals? Continue pursuing the same major goals; continue to develop and apply the software to the project goals, publish the scientific results and release the software when fully developed and documented.

Impacts
What was accomplished under these goals? Abstract for the Dazzo/Yanni published chapter entitled "CMEIAS:An Improved Computing Technolgy for Quantitative Image Analysis of Root colonization by Rhizbacteria In Situ at Single-Cell Resolution": This chapter describes how computer-assisted microscopy can enhance studies on bacterial colonization of roots by defining ecologically significant events at single-cell resolution and the spatial scale at which they occur in situ. The acquired data are supported by rigorous statistical analyses, have high signal-to-noise outputs and provide deeper insights into rhizobacterial colonization behavior. Point pattern, plot-based quadrat-lattice, and geostatistical analyses are used to distinguish bacterial distributions that are completely random (Null hypothesis) versus nonrandom patterns that cannot be explained by chance. The latter involve positive, aggregated interactions or negative interactions resulting in self-avoiding colonization behavior. In the first of two experimental examples, image analyses of a scanning electron micrograph illustrate how the colonization pattern of a superior inoculant strain of Rhizobium leguminosarum bv. trifolii forms discontinuous biofilms on the rice root surface, represented by spatially discrete bacterial aggregates that influence local densities of neighboring cells over a radial separation distance of ~12 um, which encapsulates all 494 bacteria in that image. The second example investigates the spatial heterogeneity in intensity of bacterial gene expression activated by N-acylhomoserine lactone-mediated cell-to-cell communication during colonization of wheat roots by reporter strains of Pseudomonas putida. Both examples show how computer-assisted microscopy can provide ecologically important information when included in studies of rhizobacterial colonization of plant root surfaces. Abstract for the Dazzo/Klemmer/Chandler/Yanni published article in the Diversity journal is as follows: This paper describes the utility of CMEIAS (Center for Microbial Ecology Image Analysis System) computer-assisted microscopy to extract data from accurately segmented images that provide 63 different insights into the ecophysiology of microbial populations and communities within biofilms and other habitats. Topics include quantitative assessments of: (i) morphological diversity as an indicator of impacts that substratum physicochemistries have on biofilm community structure and dominance-rarity relationships among populations; (ii) morphotype-specific distributions of biovolume body size that relate microbial allometric scaling, metabolic activity and growth physiology; (iii)fractal geometry of optimal cellular positioning for efficient utilization of allocated nutrient resources; (iv) morphotype-specific stress responses to starvation, environmental disturbance and bacteriovory predation; (v) patterns of spatial distribution indicating positive and negative cell–cell interactions affecting their colonization behavior; and (vi)significant methodological improvements to increase the accuracy of color-discriminated ecophysiology, e.g., differentiation of cell viability based on cell membrane integrity, cellular respiratory activity, phylogenetically differentiated substrate utilization, and N-acyl homoserine lactone-mediated cell–cell communication by bacteria while colonizing plant roots. The intensity of these ecophysiological attributes commonly varies at the individual cell level, emphasizing the importance of analyzing them at single-cell resolution and the proper spatial scale at which they occur in situ. Abstract of the Dazzo/Gross publication in the Journal of Ecosystems & Ecography: This paper describes CMEIAS Quadrat Maker, a new digital computing tool designed to alleviate the nontrivial problem of optimizing the grid-lattice dimensions and automating the production of size-optimized quadrat images for plot-based spatial pattern analysis in landscape ecology. The program is written for 32-bit and 64-bit Window’s operating systems and handles both 8-bit grayscale and 24-bit color input images. Following a brief user interaction, the software application transforms a copy of the input landscape image into an annotated, color index image with optimized grid overlay and column-row labeling of individual quadrats, cuts a copy of the landscape image into quadrats defined by the optimized grid raster, and then saves each individual quadrat image with a file name indicating its unique location within the landscape domain, now ready for stack building and automated image analysis. Version 1.0 of this computing technology is implemented into a software package containing the executable file, user manual and tutorial images that will be freely available at http://cme.msu.edu/cmeias/. This new computing technology will facilitate quadrat-based analyses of how spatial patterns vary with the scale at which they are measured, and will also strengthen microscopy-based approaches for understanding the spatial ecology of microbial biofilm communities. In addition, several field inoculation trials were conducted in the Nile delta to assess the performance of our Rhizobium biofertilizers on rice under real-world agronomic conditions. Analysis of the output data are in progress.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Dazzo, F.B. and C. Gross. 2013. CMEIAS Quadrat Maker: a digital software tool to optimize grid dimensions and produce quadrat images for landscape ecology spatial analysis. J. Ecosystems & Ecography 3(4)(1000136): 1-4. DOI: 10.4172/2157-7625.1000136
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2013 Citation: Dazzo, FB, I. Ganesan and Y. Yanni 2013. Spatial ecology of rhizobacterial colonization on roots analysed by CMEIAS computer-assisted microscopy at single-cell resolution. In: D. P. Singh and H. Singh (eds.), Trends in Soil Ecology, Studium Press, India, in press.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Dazzo, F.B., J. Liu, C. Gross, C. Reddy, C. Monosmith, N. Philips, C. Radek, K. Klemmer, Zhou Ji, P. Smith, I. Ganesan, D. McGarrell, M. Thanyakarn, I. Folland, S. Xia, M. Cavanaugh, K Ogbenna, A Turmo, J Ho, N Duque-Feghali, K Card, A Baruti, N. Haque, U. Adia-Nimuwa, D. Colbry, T. McCann, C. Hagen, I. Leader, S. Zamani, R. Verhelst, S. Gantner and Y. Yanni.. 2013. CMEIAS v3.10: advanced computational tools of image analysis software designed to strengthen microscopy-based approaches for understanding microbial ecology. 2013 All Scientist Meeting, KBS-Long Term Ecological Research, MSU Kellogg Conference Center, April 4, 2013. Online at:
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: F.B . Dazzo and S. Gantner. 2013. In situ calling distances and high population independent N-acylhomoserine lactone-mediated communication on plant root surfaces. In: F. DeBruijn (editor), Molecular Microbial Ecology of the Rhizosphere, Vol. 2, Chapter 74, pp. 785-788. J Wiley & Sons, NY.
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: F. B. Dazzo and Y .G. Yanni. 2013. CMEIAS: an improved computing technology for quantitative image analysis of root colonization by Rhizobacteria in situ at single-cell resolution. In: F. DeBruijn (editor), Molecular Microbial Ecology of the Rhizosphere, Vol. 2, Chapter 69, pp. 733-742. J Wiley & Sons, NY.
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: N. Uphoff, F. Chi, F.B. Dazzo, and R. J. Rodriguez. 2013. Soil fertility as a contingent rather than inherent characteristic: considering the contributions of crop-symbiotic soil biota. In: Principles of Sustainable Soil Management in Agroecosystems, Advances in Soil Sciences Series, edited by R. Lal and B. Stewart. Chapter 6, pp. 141-166 CRC Press, Taylor & Francis, Boca Raton FL
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Dazzo, F.B., K. Klemmer, R. Chandler and Y. G. Yanni. 2013. In situ ecophysiology of microbial biofilm communities analysed by CMEIAS computer-assisted microscopy at single-cell resolution. Diversity 5: 426-460. DOI:10.3390/d5030426


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: During year 2012, we continued development of CMEIAS image analysis software and application of this computing tool to analyze the ecophysiology of rhizobacteria colonizing roots of agriculturally important crop plants at single-cell resolution and the spatial scales of their interactions in situ. Documents describing the development and ecological applications of this software for computer-assisted microscopy during year 2012 include 3 papers published in refereed journals, 1 published book chapter, 2 manuscripts submitted for publication and 5 invited book chapters that are currently in press (scheduled for publication in 2013). PARTICIPANTS: Frank Dazzo and Youssef Yanni TARGET AUDIENCES: Researchers investigating the microbial ecology of beneficial plant-bacterial interactions, ecologists interested in spatial ecology research and support software for that field. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Thirty-nine different spatial pattern analyses were used to evaluate the in situ distribution of a superior biofertilizer inoculant strain of Rhizobium leguminosarum bv. trifolii colonized on a rice rhizoplane landscape. Individual cells were located by immunofluorescence microscopy using a strain-specific immunofluorescent antibody probe. Methods of spatial statistics applied to the biogeography data were designed to distinguish bacterial spatial patterns that are completely random from nonrandom patterns that cannot be explained by chance, using plot-less point pattern, gridded quadrat-lattice, and geostatistical tests. The data had high signal-to-noise outputs, were supported by rigorous statistical analyses, and provided quantitative insights into rhizobacterial colonization behavior on the root habitat. The results indicated a discontinuous biofilm pattern of bacterial aggregates with significant spatially autocorrelated clusters exhibiting discrete hot-spots of anisotropy, fractal geometry, and clustered point alignments of colonization behavior that influence local densities of neighboring cells over a radial separation distance of up to 13 micrometers, a dimension that encapsulates 96.7% of the bacteria on the root landscape examined. Other experiments using engineered reporter sensor strains expressing green fluorescent protein detected bacterial cell-to-cell communication during their colonization of roots, with variations in intensity ranging from "soft whisper" to "intense shouting". The results provide (in the broadest sense) both indirect and direct evidence of bacterial cell-to-cell communications mediated by environmental sensing phenomena, the geospatial scales and intensities at which they occur, and the positive impacts they have on rhizobacterial colonization behavior, all measured in situ at individual, single-cell resolution. This is cutting-edge research in development of software for computer-assisted microscopy to support studies in rhizosphere microbial ecology.

Publications

  • Dazzo, F. and Gantner, S. 2012. Rhizosphere. In T. Schmidt and M. Schaechter (eds.), Topics in Ecological and Environmental Microbiology. Elsevier Press, Waltham, MA, p. 467-479.
  • Dazzo, F.B. 2012. CMEIAS-aided microscopy of the spatial ecology of individual bacterial interactions involving cell-to-cell communication within biofilms. Sensors, 12: 7047-7062.
  • Robeldo, M., Rivera, L., Jimenez-Zurdo, J., Rivas, R., Dazzo, F. B., Velazquez, E., Martinez-Molina, E., Hirsch, A., and Mateos, P. F. 2012. Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces. Microbial Cell Factories, 11: 125-132.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: This project seeks to advance knowledge of beneficial plant-microbe interactions of agricultural importance, especially those involving Rhizobium, and where appropriate, transform that new knowledge into useful technology applied to sustainable agroecosystems. Three research directions of this project were pursued during this reporting period. First, we explored the involvement of a Rhizobium cell-bound cellulase isozyme during primary and secondary infection events required to establish a dinitrogen-fixing symbiosis with its host legume, white clover. Second, we continued field inoculation trials to identify superior consortia of plant growth-promoting biofertilizer inoculants (R. leguminosarum bv. trifolii, Pseudomonas stutzeri, Nostoc sp.) that enhance rice and wheat grain productivity in real-world agroecosystems while reducing their dependence on chemical fertilizers. Third, we continued the development and testing of innovative CMEIAS image analysis software designed to strengthen microscopy-based approaches for understanding the ecology of plant growth-promoting rhizobacteria during their colonization of crop plant roots. PARTICIPANTS: Frank Dazzo at Michigan State University collaborates with Pedro Mateos and colleagues at the University of Salamanca, Spain on studies of rhizobial infection events in the Rhizobium-white clover symbiosis. Frank Dazzo collaborates with Youssef Yanni at the Sakha Agricultural Research Station, Kafr El-Sheikh, Egypt on basic and applied studies of the beneficial Rhizobium-cereal association, including field inoculation trials in the Egyptian Nile Delta where these crops have been cultivated for millennia. Frank Dazzo collaborates with a large international team of microbiologists, mathematicians and computer scientists in development of innovative CMEIAS image analysis software for microbial ecology research. The lengthy list of collaborators is indicated in the online, extended summary cited above. TARGET AUDIENCES: The target audiences of this project include agricultural microbiologists, agronomists, industrial producers of biofertilizers, scientists in multidisciplinary fields studying basic aspects of beneficial plant-bacteria interactions of agricultural importance, and scientist who develop and utilize image analysis software in microbial ecology research. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We found that R. leguminosarum bv. trifolii uses its CelC2 cellulase to erode the non-crystalline wall at the apex of root hairs thereby creating the primary portal of its entry into roots of white clover during primary host infection. In subsequent studies, we found that this same enzyme is also important for secondary infections during dissemination of the bacterial symbiont within root nodules of the clover host. Multiple field inoculation studies strengthen previous results indicating that inoculation treatments increase grain yield, straw production, harvest index and agronomic fertilizer N-use efficiency of rice and wheat, showing statistical evidence that certain consortial inoculants containing rhizobia, pseudomonads and cyanobacteria produced the highest yield responses on specific cultivars. This has high potential impact on our biofertilizer technological development program. Developments of CMEIAS image analysis software during this reporting period include refinements of analytical systems to measure root architecture by their fractal dimension, and measure the colonization behavior of plant growth-promoting rhizobacteria on rice roots in gnotobiotic culture and field-grown plants using plotless, plot-based and geostatistical methods of spatial pattern analysis.

Publications

  • Robeldo, M., Jiminez-Zurdo, J., Soto, M.J., Velazquez, E., Dazzo, F. B., Martinez-Molina, E., and Mateos, P. F. (2011). Development of functional symbiotic white clover root hairs and nodules requires tightly regulated production of rhizobial cellulase CelC2. Molecular Plant-Microbe Interactions. 24: 798-807 (DOI: 10.1094/MPMI-10-10-0249).
  • Yanni, Y.G., and Dazzo, F.B. (2011). Beneficial endophytic Rhizobia as biofertilizer inoculants for rice and the spatial ecology of this bacteria-plant association. In D.K. Maheshwari (ed.), Bacteria in Agrobiology, Chapter 10, pp. 265-294.(DOI 10.1007-978-3-642-18357-7).
  • Dazzo, F.B., Liu, J., Tang, G., Gross, C., Reddy, C., Monosmith, C., Zhu, G., Wang, J., Li, M., Philips, N., Baruti, A., Leader, I., Zamani, S., Verhelst, R., Radek, C., Klemmer, K., Farrell, K., McCully, J., Krasnov, B., Ji, Z., Smith, P., Kneeshaw, S., Ganesan, I., McGarrell, D., Leader-Wineland, M., Hollingsworth, R., Smucker, A., Nakano, S., Squartini, A., Mateos, P., Gantner, S., and Yanni, Y. G. (2011). CMEIAS v3.1: Advanced computational tools of image analysis software designed to strengthen microscopy-based approaches for understanding microbial ecology at multiple spatial scales. All Investigator Meeting, Long-Term Ecological Research program, April 15, 2011, Kellogg Biological Station, Hickory Corners, MI. (http://lter.kbs.msu.edu/abstracts/414).


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: This project seeks to advance knowledge of beneficial plant-microbe interactions of agricultural importance, especially those involving Rhizobium and cereal crops, and transform that new knowledge into useful technology applied to sustainable agroecosystems. Our previous studies have shown that the clover root-nodule occupant Rhizobium leguminosarum bv. trifolii participates in a natural, beneficial association with rice roots (Oryza sativa L.) that can significantly improve rice growth, grain productivity and the agronomic fertilizer-use efficiency with less chemical fertilizer inputs and independent of nodule formation and biological N2-fixation. During year 2010, we published studies on performance of selected endophytic strains in 24 large-scale field inoculation trials in 11 counties at the central and northern regions of the Nile delta (one of the highest rice producing regions in the world), using 7 inoculant strains tested on 5 rice varieties during 5 annual growing seasons (plant May/June, harvest September/October). PARTICIPANTS: Frank Dazzo and Youssef Yanni are collaborator / PIs on this project. We work together to isolate, screen and test candidate biofertilizer inoculant strains of endophytic rhizobia, collect data and analyze them statistically, prepare posters to communicate results at international symposia, and prepare / submit / publish research findings in refereed journals and invited book chapters. TARGET AUDIENCES: Agricultural microbiologists, agronomists, extension agents, industrial producers of biofertilizers, and peasant farmers and policy makers who deal with rice, the world's most important food crop. Scientists in multidisciplinary fields studying basic aspects of beneficial plant-bacteria interactions of agricultural importance should see the details provided above on our studies accomplished and their impact on sustainable agriculture. PROJECT MODIFICATIONS: No-cost extensions have been made on both the USA and Egypt sides to eliminate the problem of an out-of-phase project start date to obtain and analyze biological materials for the project.

Impacts
Inoculation with single strains or multi-strain consortia significantly increased grain yield in 19 of the 24 field trials under real-world agricultural conditions, including in salt-affected soils and in certain locations where chemical N-fertilization was less profitable. By combining superior rhizobial inoculants with agricultural extension training, grain yield increased up to 47% in farmers' fields, with an average increase of 19.5%. In 7 experiments, inoculation increased the paddy grain yield to levels that exceeded the world's highest national mean recorded in 2008. Data on rice straw production, harvest index and the agronomic fertilizer N-use efficiency also indicated positive agronomic benefits of rhizobial inoculation in these studies. This extensive testing program of large-scale field inoculation trials establishes the merit of deploying our biofertilizer strategy using selected rhizobial strains to promote rice production capacity while reducing the need for chemical N-fertilizer inputs under actual farming conditions to maintain agricultural sustainability and acceptable production economy. Technology transfer of this important translational research can significantly help to alleviate hunger and meet the nutritional needs of many people in developing countries.

Publications

  • Yanni, Y. G., and Dazzo, F. B. (2010). Enhancement of Rice Production using Endophytic Strains of Rhizobium leguminosarum bv. trifolii in Extensive Field Inoculation Trials within the Egypt Nile Ddelta. Plant and Soil, 336: 129-142. (DOI: 1007-s1104-010-7)
  • Gross, C. A., Reddy, C., and Dazzo, F. B. (2010). CMEIAS Color Segmentation: an Improved Computing Technology to Process Color Images for Quantitative Microbial Ecology Studies at Single-Cell Resolution. Microbial Ecology, 59(2): 400-414. (DOI:10.1007/s00248-009-9616-7).
  • Bano, A., Batool, R., and Dazzo, F. B. (2010). Adaptation of Chickpea to Desiccation Stress is Enhanced by Symbiotic Rhizobia. Symbiosis, 50:129-133.(DOI: 10.1007/s13199-010-0051-9).


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: This project seeks to advance knowledge of beneficial plant-microbe interactions of agricultural importance, especially those involving Rhizobium and cereal crops, and transform that new knowledge into useful technology applied to sustainable agroecosystems. Specific objectives are to identify endophytic strains of rhizobia that perform as superior biofertilizer inoculants for rice and wheat grain production, and to continue development of CMEIAS software tools for computer-assisted microscopy of plant-microbe interactions and their application to quantitative assessments of plant growth-promotive rhizobacteria that colonize crop plants. For objective 1, we have completed large-scale field inoculation experiments during 5 growing seasons in the Nile delta, including sites that produced the world's record in rice grain yield per unit area. Inoculation with certain endophytic rhizobial strains significantly increased rice grain yield while reducing the need for N-fertilizer inputs in 19 out of the 24 trials performed. We reported this work at the 8th Int. Plant-Growth Promotive Rhizobacteria Workshop, and have described it in detail in a manuscript submitted for publication. For objective 2, we developed an improved computing technology to alleviate the difficulty of color segmentation for digital image analysis of microorganisms in environmental samples. The system's uniqueness is its ability to edit digital images accurately when presented with the common challenge of removing background pixels whose 3-dimensional color space overlaps the range that defines foreground objects. Image segmentation is accomplished by utilizing algorithms that address color and spatial relationships of user-selected foreground object pixels. Performance of the color segmentation algorithm evaluated on 26 complex micrographs at single pixel resolution had an overall pixel classification accuracy of 99+%. Several applications illustrate how this improved computing technology can successfully resolve numerous challenges of complex color segmentation in order to produce images from which quantitative information can be accurately extracted, thereby gain new perspectives on the in situ ecology of microorganisms. Examples include improvements in the quantitative analysis of (i) microbial abundance and phylotype diversity of single cells classified by their discriminating color within heterogeneous communities, (ii) cell viability, (iii) spatial relationships and intensity of bacterial gene expression involved in cellular communication between individual cells within rhizoplane biofilms, and (iv) biofilm ecophysiology based on ribotype-differentiated radioactive substrate utilization. We plan to release this freely available, stand-alone software plus user support files for this color segmentation computing application at our CMEIAS software project website located at http://cme.msu.edu/cmeias/. PARTICIPANTS: Frank Dazzo and Youssef Yanni are collaborator / PIs on this project. We work together to isolate, screen and test candidate biofertilizer inoculant strains of endophytic rhizobia, collect data and analyze them statistically, prepare posters to communicate results at international symposia, and prepare / submit / publish research findings in refereed journals and invited book chapters. Frank Dazzo directs a team of investigators to design, develop, test and release CMEIAS software designed to strengthen microscopy-based approaches for understanding microbial ecology. TARGET AUDIENCES: (1) agricultural microbiologists, agronomists, extension agents, industrial producers of biofertilizers, and peasant farmers who grow rice, the world's most important food crop. Scientists in multidisciplinary fields studying basic aspects of beneficial plant-bacteria interactions of agricultural importance. (2) microbial ecologists, environmental microbiologists, plus investigators in all scientific disciplines that use digital color classification in their work, e.g., forensic science, turfgrass science, biomedical imaging, clinical pathology, eukaryotic cell biology, astronomy, astrobiology, etc. PROJECT MODIFICATIONS: This project is externally funded by a competitive grant award from the US-Egypt Science and Technology Joint Program (funds from US State Department, administered through the USDA). The budget is split approximately equally to both PI's programs. The funding was significantly delayed on the Egypt side, causing an out-of-phase project start date to obtain and analyze biological materials for the project. Adjustments have been made by a 1-year no-cost extension on the USA side, but another 1-year no-cost extension will likely be necessary to fully complete the collaborative project since it requires a close partnership of synergistic research activities at both locations.

Impacts
(1) Our extensive testing program of large-scale field inoculation trials establishes the merit of deploying our biofertilizer strategy using selected rhizobial strains to promote rice production capacity while reducing the need for chemical N-fertilizer inputs under real-world, actual farming conditions to maintain agricultural sustainability and acceptable production economy. Technology transfer of this important translational research can significantly help to meet the nutritional needs of many people in developing countries.(2) Our improved software computing technology opens new opportunities of imaging applications where discriminating colors really matter most, thereby strengthening quantitative microscopy-based approaches to advance microbial ecology in situ at individual single-cell resolution.

Publications

  • Gross, C., Reddy, C., and Dazzo, F.B. 2009. CMEIAS color segmentation: an improved software computing technology to process color images for quantitative microbial ecology studies. Microbial Ecology, Online First DOI 10.1007/s00248-009-96167, (in press).
  • Yanni, Y.G., and Dazzo, F. B. 2009. Enhanced rice production by Rhizobium leguminosarum bv. trifolii in extensive field inoculation trials in the Egypt Nile delta. Abstract Book 8th International Plant Growth-Promotive Rhizobacterial Meeting, Portland Oregon (P62).


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Rice is one of the world's most important food crops, providing staple diet for almost half the world's human population. However, high rice grain production is only possible with high inputs of chemical fertilizers, creating environmental, economical, and health risk problems. Our research project seeks to reduce this nutrient availability problem by exploiting the natural, beneficial associations between rice and selected soil bacteria. The Egyptian Nile delta has been ideal to test this model, where rice production has benefited since antiquity by rotation with the legume, berseem clover in ways extending beyond the benefits of symbiotic nitrogen fixation by Rhizobium. Our studies conducted at MSU and in the Nile delta of Egypt show that clover rhizobia (R. leguminosarum bv. trifolii) naturally colonize rice plants in field rotations with legumes, and these plant growth-promoting rhizobacteria can be exploited to significantly improve cereal crop growth in a strain/variety-specific manner, resulting in increased grain productivity and agronomic fertilizer N-use efficiency with less dependence on nitrogen fertilizer inputs to maximize crop yield. To date, we have conducted 24 field inoculation trials on rice, using selected strains of endophytic rhizobia in small replicated plots (20 square meters each) and in large farmers' fields. Of these, 20 separate field tests (83% of total) have revealed statistically significant increases in rice paddy grain yield production at a confidence level of 95%, as compared to the corresponding non-inoculated counterparts. Autecological biogeography studies in the Nile delta indicate distinct patchiness in distribution of one rhizobial strain that repeatedly enhances rice grain yield in field inoculation trials, providing further evidence indicating the importance of field inoculation. In some cases, defined consortia inoculants of multiple test strains evoked the highest inoculation response to grain yield. More studies using consortia inoculants are currently being conducted to define the optimal Rhizobium-rice biofertilizer inoculants for public release. PARTICIPANTS: Frank Dazzo and Youssef Yanni are collaborative principal investigators who designed and performed the laboratory and field microbial ecology studies. R. Hollingsworth listed as a participant in the original project description did not participate in the work reported as progress for this project during year 2008. TARGET AUDIENCES: microbial ecologists, especially those studying beneficial plant-bacterial interactions and rhizosphere associations PROJECT MODIFICATIONS: none noted

Impacts
These studies increase our understanding and knowledge of the intricate interactions between bacteria and their beneficial association with cereal crop plants, especially relevant to exploitation of the natural Rhizobium-cereal association represented by some of the world's most important food crops in sustainable agriculture.

Publications

  • Mishra, R., Singh, R.K., Jaiswal, H.K., Singh, M.K.,Yanni, Y.G., and Dazzo, F.B. 2008. Rice-Rhizobia association: evolution of an alternate niche of beneficial plant-bacteria association. In I. Ahmad, J. Pichtel, and S. Hayat (eds.), Plant-Bacteria Interactions: Strategies and Techniques to Promote Plant Growth. Wiley-VCH Verlag GmbH, Weinheim, Germany, p. 165-194.
  • Yanni, Y.G., Dazzo, F.B., Rizk, R., Zidan, M., and Fattah, F.A. 2008. Enhancement of nitrogen acquisition, plant growth and crop performance of rice by a natural, beneficial plant-microbe association. Proc. 1st Int. Conference on Biological and Environmental Sciences, Hurghada, Egypt.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Our earlier studies have shown that the soil microorganism, Rhizobium leguminosarum bv. trifolii, develops a natural, intimate, endophytic association with rice plants (within both below-ground and above-ground plant tissues), and can promote their grain production under field conditions in a strain-cultivar specific manner. Current evidence suggests that the main mechanism of that beneficial association involves the rhizobial modulation of phytohormone levels affecting whole plant growth physiology, thereby increasing its efficiency in nutrient acquisition and biomass production. Here we report that this same rhizobial biovar also forms a natural, endophytic association with wheat, another very important cereal crop. Lab and field microbial ecology studies were conducted in Michigan and the Nile delta of Egypt, where legume-cereal crops have been rotated since antiquity. A pure culture collection of forty-seven isolates of endophytic rhizobia were isolated from surface-sterilized, field-grown wheat roots using the legume trap method tested with the legume hosts traditionally rotated with that cereal crop in the Nile delta (fababean, lentil, lupine, fenugreek, pea, berseem clover, soybean and chickpea). Interestingly, this inoculant of endophytes induced true root nodules infected with rhizobia only on the legume host, berseem clover. Plasmid profiling, BOX-PCR and 16S rDNA sequencing of the clover nodule isolates indicated 3 groups of R. leguminosarum bv. trifolii genotypes. Group-2 isolates were Fix+ on berseem clover whereas groups -1 and -3 were ineffective (Fix-) on this host. Some isolates promoted wheat growth in bioassays conducted under lab gnotobiotic conditions. Four consecutive years of inoculation studies in large farmers' fields indicated that some of the effective group-2 isolates significantly increased wheat grain yield from 8.1% to 39.0%, depending on the wheat variety, rhizobial endophyte strain and geographical location. Certain consortial inoculants containing selected wheat-adapted rhizobial endophytes performed better in promoting wheat grain production than did other consortia, or than did some inoculants containing single isolates alone. Also, several inoculant formulations significantly increased the agronomic fertilizer-N use efficiency of wheat, indicating that those specific bacterial inoculants enhanced the wheat plant's ability to produce grain while reducing its dependence on chemical fertilizer-N inputs to achieve high yields. Considered collectively, these studies indicate that Rhizobium leguminosarum bv. trifolii is particularly adapted to develop natural endophytic associations with wheat and rice roots, and some isolates adapted to occupy this ecological niche have high potential for development as biofertilizer inoculants that can improve grain production of these important cereal crops. These results were disseminated in a scientific poster presented at the 2nd Internation Rhizosphere Conference held in Montpellier, France during year 2007. PARTICIPANTS: Frank Dazzo and Youssef Yanni (principal investigators). Designed and performed the laboratory and field microbial ecology studies. Andrea Squartini (collaborator). Performed the molecular analysis of strain diversity in the culture collection. TARGET AUDIENCES: Microbial ecologists, especially those studying beneficial plant-bacterial interactions and rhizosphere associations. PROJECT MODIFICATIONS: none noted.

Impacts
These studies increase our understanding of the intricate interactions between bacteria during their beneficial association with crop plants, especially relevant to exploitation of the natural Rhizobium-cereal association represented by some of the world's most important food crops in sustainable agriculture.

Publications

  • Yanni, Y.G., Squartini, A. and Dazzo, F.B. 2007. Natural endophytic association between Rhizobium leguminosarum bv. trifolii and wheat and its potential to promote wheat plant growth and crop performance. Rhizosphere-2 International Conference Program and Abstract Book, Montpellier, France (P-769).
  • Dazzo, F. B., Schmid, M. and Hartmann A. 2007. Immunofluorescence microscopy and fluorescence in situ hybridization combined with CMEIAS and other image analysis tools for soil- and plant-associated microbial autecology, Chapter 59 In J. Garland, C. Hurst, D. Lipson, A. Mills, L. Stetzenbach, and R. Crawford (eds.), Manual of Environmental Microbiology, 3rd ed. American Society for Microbiology Press, Washington, DC, p. 712-733.


Progress 01/01/06 to 12/31/06

Outputs
We are studying the autecological biogeography of a candidate biofertilizer inoculant strain of Rhizobium leguminosarum bv. trifolii during colonization of rice roots, conducted at micrometer scale and single cell resolution using CMEIAS computer-assisted microscopy and digital image analysis. For this study, we have built new computing tools to facilitate these spatial distribution analyses. A major component developed for this purpose is the CMEIAS Cluster Index, designed to measure the local density of clustered neighbors near each individual bacterial cell over the spatial domain of the substratum surface upon which they have colonized. This parameter has been evaluated using scanning electron microscopy and confocal laser scanning microscopy of bacteria colonized on rice and white clover root surfaces. We found that values of this CMEIAS measurement attribute of the bacteria colonized on roots extracted from digital images is autocorrelated with their spatial distribution over a separation distance of up to ca. 9 um from bacterial neighbors in gnotobiotic tube cultures, and of up to ca. 52 um when grown in soil. The semivariograms produced by the geostatistical analysis of this Z-variate are best described by exponential and spherical isotropic models, providing predictive information on their colonization behavior and in situ spatial scale of microbe-microbe interaction. We conclude from this study that bacteria influence their bacterial neighbors' ability to develop into clusters (e.g., microcolonies) at these stated in situ spatial scales while colonizing plant roots. This powerful spatial modeling technique is particularly useful for studies of microbial colonization of roots, especially because it produces statistically defendable interpolation results even for areas of the root epidermis that cannot be physically sampled (in this case, areas obscured beneath overlying root hairs). In other studies, we measured the in situ calling distances between Rfp- and Gfp- reporter strains of bacteria conducting cell-to-cell communication while colonizing plant roots. CMEIAS image analysis indicated that this bacterial interaction occurs between as few as 2 individual cells, over a separation range of up to 78 um, equivalent to two adults talking to each other while located in opposite end-zones of a football field in a stadium containing no other people. Geostatistical analysis of these results predict that this type of bacterial communication is governed primarily by positioning of the bacteria within gradients of the signal molecules produced by neighboring bacteria, rather than by high population densities per se, and that it occurs much more commonly than predicted by conventional thinking.

Impacts
These studies increase our understanding of the intricate interactions between bacteria during their beneficial association with crop plants, especially relevant to exploitation of the natural Rhizobium-cereal association represented by some of the world's most important food crops in sustainable agriculture.

Publications

  • Dazzo, F.B., Yanni, Y.G. 2006. The natural Rhizobium-cereal crop association as an example of plant-bacteria interaction. In: N. Uphoff, A. Ball, E. Fernandes, H. Herren, O. Husson, M. Laing, C. Palm, J. Pretty, P. Sanchez, N. Sanginga, and J. Thies (eds.). Biological Approaches to Sustainable Soil Systems, pp. 109-127, CRC Taylor & Francis, Boca Raton, FL.
  • Gantner, S., Schmid, M., Durr, C., Schuhegger, R., Steidle, A., Hutzler, P., Langebartels, C., Eberl, L., Hartmann, A., Dazzo, F.B. 2006. In situ spatial scale of calling distances and population density-independent N-acylhomoserine lactone mediated communication by rhizobacteria colonized on plant roots. Federation of European Microbiological Societies Microbiology and Ecology 56: 188-194.


Progress 01/01/05 to 12/31/05

Outputs
This project focuses on basic and applied studies of the Rhizobium-cereal association that is now known to benefit cereal production worldwide. Collaborative studies published in 2005 were done to gain a better understanding of the route(s) used by rhizobia to enter, disseminate and colonize the interior of rice plants. For these studies, constituitively-expressed Gfp derivatives of various species of wild-type rhizobia were inoculated on rice plants and their colonization and dissemination within the plant tissues were studied by a combination of laser scanning confocal microscopy, CMEIAS in situ image analysis, and viable plate counts on media containing appropriate antibiotics. The results indicated a dynamic infection process in rice, beginning with preferential colonization and entry of the bacteria at lateral root emergence, followed by their endophytic ascending migration into and transient growth to high local populations within aerial plant tissues that include the stem base, leaf sheath and leaves of rice. Thus, the endophytic rhizobia-cereal association is far more dynamic and invasive than previously thought. Other collaborative studies indicated that the strain-variety variation in ability of rhizobial endophytes to promote rice growth correlated with the ability of the plan's root exudates to stimulate production of indoleacetic acid and gibberellin growth phytohormones by the rhizobia in vitro, and also correlated with the elevation in phytohormone levels in vivo within the plants inoculated with the rhizobia. Finally, recent collaborative field inoculation trials using our best biofertilizer candidate strains of cereal-adapted rhizobia and scaled up to the size of farmers fields have indicated overall increases in crop grain yield that ranged between 12.8 - 47.1 % for rice and 16.2% for wheat.

Impacts
The results of these studies heighten the intrinsic interest of the natural Rhizobium-cereal association and its potential value for exploitation in sustainable agriculture to produce some of the world's most important cereal crops.

Publications

  • Dazzo, F.B., Yanni, Y.G., Rizk, R., Zidan, M., Gomaa, A.M., Squartini, A., Jing, Y.X., Chi, F., Shen, S.H. 2005. Recent studies on the Rhizobium-cereal association. In Y-P. Wang, M. Lin, Z-X Tian, C. Elmerich and WE Newton (eds). Biological Nitrogen Fixation, Sustainable Agriculture and the Environment, Springer, Dordrecht, The Netherland. 379-380pp.
  • Chi, F., Shen, S.H., Cheng, H.P., Jing, Y.X., Dazzo, F.B. 2005. Ascending migration of endophytic rhizobia from roots to leaves inside rice plants. In Y-P. Wang, M. Lin, Z-X Tian, C. Elmerich and WE Newton (eds). Biological Nitrogen Fixation, Sustainable Agriculture and the Environment, Springer, Dordrecht, The Netherlands. 381-382pp.
  • Chi, F., Shen, S.H., Cheng, H.P., Jing, Y.X., Yanni, Y.G., Dazzo, F.B. 2005. Ascending migration of endophytic rhizobia from roots to leaves inside rice plants and assessment of their benefits to the growth physiology of rice. Applied and Environmental Microbiology 71: 7271-7278.


Progress 01/01/04 to 12/31/04

Outputs
This project focuses on basic and applied studies of the Rhizobium-cereal association that is now known to benefit cereal production worldwide. The recent collaborative studies (with Y.X. Jing and colleagues) reported here have been done to gain a better understanding of the route(s) used by rhizobia to enter, disseminate and colonize the interior of rice plants. For these studies, constituitively-expressed gfp and antibiotic resistance markers were introduced into various wildtype rhizobia (Sinorhizobium meliloti, R. leguminosarum bv. viciae, Azorhizobium caulinodans, Mesorhizobium haukuii). Populations of these marked rhizobia were inoculated into the rhizosphere of rice seedlings and later their association with various rice tissues were analyzed by a combination of fluorescence confocal microscopy, CMEIAS in situ image processing and analysis, and viable plate counts on media containing appropriate antibiotics. The new technology of color image segmentation recently introduced into CMEIAS image analysis software was used for these studies to discriminate between positive signals derived from foreground pixels of gfp-labeled bacterial cells and background pixels representing autofluorescence from host plant tissues. The results indicated a dynamic infection process in rice, beginning with preferential colonization and entry of the bacteria at lateral root emergence, followed by their endophytic ascending migration into and growth within aerial plant tissues that include the stem base, leaf sheath and leaves of rice. These bacteria remained metabolically active during rice development from the vegetative to the reproductive stages, even though their colony forming ability eventually declined with time. This intimate interaction results in elevated levels of growth-regulating phytohormones in the aerial tissues of rice. Thus, the endophytic rhizobia-cereal association is far more dynamic and invasive than previously thought.

Impacts
These studies heighten the interest of the Rhizobium-cereal association and its potential value for exploitation in sustainable agriculture to produce some of the most important cereal crops for the future.

Publications

  • Dazzo, F.B., Yanni, Y.G., Rizk, R., Zidan, M., Gomaa, A.M., Squartini, A., Jing, Y.X., Chi, F., and Shen, S.H. 2004. Recent studies on the Rhizobium-cereal association. Published Abstract L39 (p. 40). 14th International Congress on Nitrogen Fixation, Program and Abstract Book, Beijing China.
  • Chi, F., Shen, S.H., Cheng, H.P., Jing, Y.X., and Dazzo, F.B. 2004. Ascending migration of endophytic rhizobia from roots to leaves inside rice plants. Published abstract L40 (p. 40). 14th International Congress on Nitrogen Fixation, Program and Abstract Book, Beijing China.
  • Dazzo, F.B. 2004. New CMEIAS image analysis software for computer-assisted microscopy of microorganisms and their ecology. Microscopy Today 12:18-23.
  • Reddy, C.K. and Dazzo, F.B. 2004 Computer-assisted segmentation of bacteria in color micrographs. Microscopy & Analysis 18: 5-7.
  • Dazzo, FB. 2004. Applications of quantitative microscopy in studies of plant surface microbiology. In: Varma, A., Abbott, L., Werner, D., and Hampp, R. (eds.), Plant Surface Microbiology. Springer-Verlag, Germany, 503-550pp.
  • Dazzo, F.B. 2004. Production of anti-microbial antibodies and their utilization in studies of microbial autecology by immunofluorescence microscopy and in situ CMEIAS image analysis. In: Kowalchuk, G., deBruijn, F., Head, I., Akkermans, A., and Elsas, J. (eds.), Molecular Microbial Ecology Manual, 2nd Ed., Chapter 4.04, 911-932pp. Kluwer Publishers, Dordrecht, Netherlands.


Progress 01/01/03 to 12/31/03

Outputs
We have developed two new indices to examine the autecological biogeography of selected strains of rhizobia that occupy beneficial endophytic niches with roots of cereals rotated with legume crops. The strain under investigation is considered a strong candidate plant growth-promotive biofertilizer inoculant. The first is an Autecological Biogeography Index developed to operate at a macro scale in order to map the distribution of the rhizobia in cereal-legume agroecosystems. This index is based on quantitative immunofluorescence microscopy of nodule occupants obtained from the field using uninoculated legume trap hosts and a strain-specific fluorescent antibody. The second is a Cluster Index designed to operate at a micro scale relevant to colonization of the cereal roots by the bacterium. This latter index is introduced to analyze the in situ spatial pattern of root colonization by the bacteria at single-cell resolution using our custom CMEIAS (Center for Microbial Ecology Image Analysis System) software for computer-assisted microscopy. When sampled at multiple georeferenced sites (i.e., at known Cartesian x,y coordinates relative to a landmark origin), these two indices provide quantitative values that are suitable for general use as the Z variate in spatial geostatistical analyses to model bacterial dispersion and colonization behavior, and to produce interpolated maps of the continuous distribution of the microbial symbiont within the defined spatial domain of the geographical region or root substratum, including areas that cannot be physically sampled.

Impacts
Information gained by use of these indices will help to model the outcome of biofertilizer inoculant trials under field conditions. Both indices also have the potential for broad applications in spatial distribution analyses and microbial ecology.

Publications

  • Dazzo, F.B., Joseph, A.R., Gomaa, A.B., Yanni, Y.G., and Robertson, G.P. 2003. Quantitative indices for the autecological biogeography of a Rhizobium endophyte of rice at macro and micro spatial scales. Symbiosis 35: 147-158.
  • Rivas, R., Willems, A., Subba-Rao, N.S., Mateos, P.F., Dazzo, F.B., Kroppenstedt, R., Martinez-Molina, E., Gillis, M. and Velazquez, E. 2003. Description of Devosia neptuniae sp. nov. that nodulates and fixes nitrogen in symbiosis with Neptunia natans, an aquatic legume from India. Systematic and Applied Microbiology 26: 47-53.
  • Dazzo, F., Liu, J., Prabhu, A., Reddy, C., Wadekar, M., Peretz, R., Bollempalli, R., Trione, D., Marshall, E., Zurdo, J., Hammoud, H., Wang, J., Li, M., McGarrell, D., Gore, A., Maya-Flores, J., Gantner, S., and Hollingsworth, N. 2003. CMEIAS v. 3.0: Integrative software package to strengthen microscopy-based approaches for understanding microbial ecology (abstract). 2003 Long-Term Ecological Research in Row-Crop Agriculture. Michigan State Univ. East Lansing, MI. .


Progress 01/01/02 to 12/31/02

Outputs
Earlier studies established that rhizobia can occupy 3 ecological niches in fields where the corresponding legume host is rotated successfully with a cereal crop. These include the nitrogen-fixing endosymbiont within the legume root nodule, the free-living saprophyte in the soil, and the plant growth-promoting endophyte within the cereal roots. We first described this latter niche in the Nile Delta of Egypt, where rice has been rotated with berseem clover since antiquity. Recently, we extended these findings by showing that rhizobia also naturally associate endophytically with wheat roots, and have begun to accumulate evidence indicating that some strain/variety combinations of this rhizobia-wheat association can benefit wheat growth. We conducted two cycles of isolation from inside surface-sterilized field-grown wheat roots and have produced a pure culture collection of 36 isolates of wheat-adapted rhizobia. Interestingly, although wheat is rotated with various legumes (berseem clover, favabean, alfalfa, lentils, and soybean) in the Nile delta, the clover microsymbiont Rhizobium leguminosarum bv. trifolii was the only type of rhizobia isolated endophytically in field-grown wheat roots by the legume trap technique. Eight different isolates were evaluated in bioassays to assess whether they can establish intimate associations with wheat roots in gnotobiotic culture and beneficially promote the growth of this important cereal crop. The results indicated statistically significant positive enhancement of wheat growth due to inoculation with some of these wheat-adapted rhizobial isolates. Two of these rhizobial isolates were then tested in replicated field trials, which indicated that they can enhance growth and performance on certain wheat varieties under field conditions. These results add further evidence of the ability of rhizobia to serve as a dual biofertilizer agent that can benefit both legumes and cereals in rotation, fully consistent with sustainable agriculture.

Impacts
This work carries both basic and applied impacts: on the basic side it add new information that changes our understanding of the microbial ecology of soil rhizobia - this new concept has already been introduced into two year-2002 microbiology textbooks. On the applied side, it contributes to the scientific documentation supporting the benefits of using selected rhizobial isolates as plant growth-promoting biofertilizers that can reduce the need for applying chemical fertilizer to achieve high yield for both legume and cereal crops, fully consistent with sustainable agriculture.

Publications

  • McDermott, T. R. and Dazzo, F. B. 2002. Use of fluorescent antibodies for studying the ecology of soil- and plant-associated microbes. In: Hurst, C, Crawford, R. C., Knudsen, G. R., McInerney, M. J., Stetzenbach, L. D. (eds.), Manual of Environmental Microbiology, Chapter 28, p. 615-626, American Society for Microbiology Press, Washington, DC.


Progress 01/01/01 to 12/31/01

Outputs
This project is to search for natural, intimate associations between rhizobia and rice (Oryza sativa L.), assess their impact on plant growth, and exploit those combinations that can enhance grain yield with less dependence on inputs of nitrogen fertilizer. Diverse, indigenous populations of R. leguminosarum bv. trifolii (the clover root-nodule endosymbiont) intimately colonize rice roots in the Egyptian Nile delta where this cereal has been rotated successfully with berseem clover (Trifolium alexandrinum) since antiquity. The association of certain rhizobial strain / rice variety combinations promotes root and shoot growth thereby significantly improving seedling vigor that carries over to significant increases in grain yield at maturity. Three field inoculation trials in the Nile delta indicated that a few strain / variety combinations significantly increased rice grain yield, agronomic fertilizer N-use efficiency, and harvest index. The benefits of this association more likely involve rhizobial modulation of the plant's root architecture for more efficient acquisition of certain soil nutrients (e.g., N, P, K, Mg, Ca, Zn, Na, and Mo) rather than biological nitrogen fixation. Inoculation increased total protein quantity per ha in field-grown grain, thereby increasing its nutritional value without altering the ratios of nutritionally important proteins. Rice endophyte rhizobial strain E11 produced indoleacetic acid auxin and gibberellin (tentatively identified as GA7) phytohormones. Rice root exudate significantly enhanced E11's production of this auxin. E11 extensively colonized the rice root surface with a spatial distribution that favored its erosion of the epidermal surface, colonization of small crevices at epidermal junctions as a possible portal of entry into the root, and quorum sensing of diffusable signal molecules indicating that their nearest bacterial neighbors are in close proximity in situ. Selected rhizobial endophytes of rice produced cell-bound cellulase and polygalacturonase enzymes and non-trifolitoxin bacteriocin(s) that can inhibit other strains of clover rhizobia. E11 also endophytically colonized rice roots of varieties commonly used by Filipino peasant farmers, and stimulated genotype-specific growth-promotion of corn (Zea mays, maize) under field conditions. These results indicate that some rhizobia have evolved an additional ecological niche enabling them to form a 3-component life cycle including a free-living heterotrophic phase in soil, a nitrogen-fixing endosymbiont phase within legume root nodules, and a beneficial growth-promoting endocolonizer phase within cereal roots in the same crop rotation. The results also indicate the potential opportunity to exploit this newly described, plant-rhizobia association by developing biofertilizer inoculants that may assist low-income farmers in increasing cereal production [especially rice] with less fertilizer-N inputs, fully consistent with both sustainable agriculture and environmental safety.

Impacts
This research impacts both basic and applied research on beneficial plant-microbe interactions of major significance to agriculture. It has revealed a new type of natural biological association that is now known to be widespread worldwide where cereal and legume crops are rotated successfully; the bacterial symbiont not only benefits the legume but also the cereal crop. Our studies suggest that this association can be exploited by using biofertilizer inoculants to assist farmers in increasing rice production with less chemical fertilizer-N inputs, fully consistent with sustainable agriculture.

Publications

  • Yanni,Y.G., Rizk, R.Y., Abd El-Fattah, F.K., Squartini, A., Corich, V., Giacomini, A., deBruijn, F., Rademaker, J., Maya-Flores, J., Ostrom, P., Vega-Hernandez, M., Hollingsworth, R.I., Martinez-Molina, E., Mateos, P., Velazquez, E., Wopereis, J., Triplett, E.,Umali-Garcia, M., Anarna, J.A., Rolfe, B.G., Ladha, J.K., Hill, J., Mujoo, R., Ng, P.G., and Dazzo, F.B. 2001. The beneficial plant growth-promoting association of Rhizobium leguminosarum bv. trifolii with rice roots. Austr. J. Plant Physiol. 28: 845-870.
  • Perrine, F.M, Prayitno, J., Weinman, J., Dazzo, F.B., and Rolfe B.C. 2001. Rhizobium plasmids are involved in the inhibition or stimulation of rice growth and development. Austr. J. Plant Physiol. 28:923-937.


Progress 01/01/00 to 12/31/00

Outputs
A diverse, natural population of R. leguminosarum bv. trifolii intimately associates with roots of rice grown in the Egyptian Nile Delta where this cereal has been rotated with berseem clover since antiquity. Growth chamber and greenhouse studies of several rice-adapted rhizobia and rice genotypes have indicated that certain strain / variety combinations significantly improve seedling vigor expressed as increased root and shoot biomass, expanded root architecture, uptake of several plant nutrients, and N-content which carry-over to significant increases in grain yield at maturity. Acetylene reduction assays and 15N-based studies do not support a major role of biological nitrogen fixation in this plant growth-promotion response. Some of these rhizobial strains secrete indoleacetic acid and gibberellin(s) when grown in vitro, extracellularly solubilize precipitated inorganic and organic phosphates, extensively colonize rice root surfaces, and also grow in planta. Most importantly, three cycles of field inoculation trials in the Egyptian Nile Delta have indicated that some of these rice-adapted rhizobia can significantly increase rice grain yield and agronomic N-fertilizer use efficiency for certain rice varieties. Studies in progress are designed to identify the superior inoculant strains of rhizobia that promote rice growth and production reliably and repeatedly under field conditions, and to better understand the ecological and physiological interactions occurring in this newly discovered, beneficial plant-microbe association. This ability of selected rhizobial inoculants to increase rice grain productivity while reducing chemical N-fertilizer inputs demonstrates their potential importance as biofertilizer inoculants for sustainable production of rice, the most vital food source for half the world's population.

Impacts
This research impacts both basic and applied research on beneficial plant-microbe interactions of major significance to agriculture. It has revealed a new type of natural biological association that is now known to be widespread worldwide where cereal and legume crops are rotated successfully; the bacterial symbiont not only benefits the legume but also the cereal crop. Our studies suggest that this association can be exploited by using biofertilizer inoculants to assist farmers in increasing rice production with less chemical fertilizer-N inputs, fully consistent with sustainable agriculture.

Publications

  • Biswas, J., Ladha J.K., and Dazzo F.B. 2000. Rhizobia inoculation improves nutrient uptake and growth in lowland rice. Soil Sci. Soc. Amer. J. 64: 1644-1650.
  • Biswas J.C., Ladha J.K., Dazzo F.B., Yanni Y.G., and Rolfe B.G. 2000. Rhizobial inoculation influences seedling vigor and yield of rice. Agronomy Journal 92: 880-886.
  • Dazzo, F., and Wopereis J. 2000. Unraveling the infection process in the Rhizobium-legume symbiosis by microscopy. In E. Triplett (ed.), Prokaryotic nitrogen fixation: a model system for the analysis of a biological process; pp. 295-347. Horizon Scientific Press, UK.
  • Dazzo, F.B., Yanni Y.G., and 28 others. 2000. Progress in multi-national collaborative studies on the beneficial association between Rhizobium leguminosarum bv. trifolii and rice. In J.K. Ladha and P. M. Reddy (eds.), The Quest for Nitrogen Fixation in Rice. International Rice Research Institute, Los Banos, The Philippines, pp. 167-189.
  • Rolfe B.G., Mathesius U., Prayitno J., Perrine F., Weinman J., Stefaniak J., Djordjevic M., Guerreiro N., and Dazzo F.B. 2000. Rhizobium nodulation and interaction with legumes and nonlegumes. In J.K. Ladha and P. M. Reddy (eds.), The Quest for Nitrogen Fixation in Rice. International Rice Research Institute, Los Banos, The Philippines, pp. 291-309.
  • Dazzo, F.B., Yanni Y.G., Rizk R., de Bruijn F., Squartini A., Mateos P., Martinez-Molina E., Velazquez E., Biswas J., Ladha J.K., Weinman J., Rolfe B., Vega-Hernandez M., Hollingsworth R.I., Marshall E., Jain T., Leon-Barrios M., and Perez-Galdona R. 2000. The natural beneficial association between Rhizobium leguminosarum bv. trifolii and rice and its exploitation in sustainable agriculture. In A. Abdel-Hafez, M. El-Leithy, and A. El-Nawawy (eds), Proc. International Symposium on Biological Nitrogen Fixation and Crop Production, pp. 135-140. FAO/RNE, Egypt.


Progress 01/01/99 to 12/31/99

Outputs
The interactions between several rice cultivars and various rice endophytic bacterial strains of Rhizobium leguminosarum bv. trifolii originally isolated from rice plants grown in Egypt were investigated. Inoculation experiments with rice seedlings showed that specific isolates of these rice-associated bacteria could either promote, inhibit, or have no influence on rice plant growth, confirming previously reported results. Furthermore, these growth effects were greatly influenced by the environmental growth conditions used. Rhizobium strains were constructed with a plasmid expressing the green fluorescent protein to examine root colonization patterns. Fluorescence microscopy showed that the bacteria preferentially colonize rice seedling surfaces mainly in microcolonies, along grooves on the root surface, or at emerging lateral roots and root tips. Some rhizobia could also colonize intercellularly in lateral roots formed on the main roots near the culm region of the seedling. A bioassay to measure bacterial multiplication in rice leaves showed that the rice-associated strains could multiply and survive within these tissues. The results suggest that some of these rice-associated rhizobia possess important genes that enhance their ability to intimately colonize niches on and within rice tissues, and promote rice plant growth.

Impacts
Rice is the most important food crop of the developing world. This research may lead to development of biofertilizer inoculants that improve rice production with less dependence on chemical fertilizer inputs, fully consistent with sustainable agriculture. This translates to new ways for the poor farmer to produce more rice for the dinner table, especially in developing countries where rice is the staple diet (ca. 40% of the world's population).

Publications

  • Dazzo, F.B., Yanni, Y.G., Rizk, R., de Bruijn, F., Corich, V., Squartini, A., Mateos, P., Martinez-Molina, E., Biswas, J., Ladha, J.K., Weinman, J., Rolfe, B. G., Hartmann, A., Glagoleva, O., Vega-Hernandez, M., Hollingsworth R.I., Leon-Barrios, M., and Perez-Galdona, R. 1999. Ecology and plant growth-promoting activities of the natural association between Rhizobium leguminosarum bv. trifolii and rice roots. In E. Martinez and G. Hernandez (eds.), Highlights in Nitrogen Fixation Research, pp. 101-104, Kluwer Academic Publishers, New York.
  • Prayitno, J., Stefaniak, J., McIver, J., Weinman, J., Dazzo, F. B., Ladha, J.K., Barraquio, W., Yanni, Y.G., and Rolfe, B. G. 1999. Interactions of rice seedlings with bacteria isolated from rice roots. Australian J. Plant Physiol. 26: 521-535.


Progress 01/01/98 to 12/31/98

Outputs
A detailed microscopical analysis of primary host infection, nodule organogenesis, infection thread dissemination, bacterial release, and development of nitrogen-fixing endosymbiotic bacteroids in wild-type Lotus japonicus ecotype Gifu B-129-S9 plants inoculated with wild-type (Meso)rhizobium loti NZP2235 was performed to provide the necessary framework for evaluation of altered phenotypes of L. japonicus symbiotic mutants. Eleven morphologically distinct stages of symbiotic development, including several that were peculiar to the M. loti-L. japonicus symbiosis, were illustrated by a combination of brightfield and phase contrast light microscopy, stereomicroscopy, and transmission electron microscopy. The bacteria entered L. japonicus roots through infection threads initiated within deformed root hairs, thus triggering foci of cell divisions in the outer cortex that developed into spherical or lobed-shaped nodules exhibiting typical determinant histology. Unlike soybean, infection threads in L. japonicus typically developed in elongated, markedly curled root hairs, and their outer surface developed papillae-like structures as the normal case. Infection threads within root nodules had an unusually wide lumen that could enclose several vegetative bacteria side by side, and which branched to thinner threads of more typical diameter. Bacteria released into symbiosomes from localized sites of wall dissolution along the sides of the thicker threads and the ends of the thinner threads only occasionally underwent cell division and only slightly enlarged into bacteroids. Unlike in soybean, no evidence for a matrix of fibrillar polymeric material surrounding bacteroids of M. loti was found in lotus symbiosomes. An undesirable characteristic of L. japonicus roots was its resistance to clearing with even concentrated hypochlorite solution, making the evaluation of Fccd and Noi events difficult.

Impacts
(N/A)

Publications

  • Szczyglowski, K., Shaw, R.S., Wopereis, J., Copeland, S., Hamburger, D., Kasiborski, B., Dazzo, F. B., and de Bruijn, F. J. 1998. Nodule organogenesis and symbiotic mutants of the model legume Lotus japonicus. Molec. Plant-Microbe Interact. 11: 684-697.


Progress 01/01/97 to 12/31/97

Outputs
Rhizobium chitolipooligosaccharides (CLOSs) are heterogeneous fatty acylated N-acetyl glucosamine oligomers with variations in both the polar (hydrophilic) oligosaccharide head group and the non-polar (hydrophobic) fatty acyl chain. They trigger root hair deformation and cortical cell divisions in legume roots during development of the nitrogen-fixing root-nodule symbiosis. It has been proposed that only certain unique molecular species of CLOSs made by a particular rhizobia can elicit these responses on the corresponding legume host, suggesting that receptor-mediated perception of CLOSs serves as a basis of symbiotic specificity. We evaluated the relative symbiotic importance of the hydrophilic and hydrophobic structural domains of CLOSs by comparing the biological activities of CLOSs from wild type R. leguminosarum bv. trifolii ANU843 with that of various synthetic analogs. These tests were performed in axenic bioassays on the compatible symbiotic host, white clover (Trifolium repens) and the incompatible non-host legume, alfalfa (Medicago sativa). Fluorochrome-tagged derivatives of the native CLOSs and the analogs were also prepared in order to evaluate the uptake and localization patterns of these molecules within host root cells. The results indicate a direct link between uptake and biological activities of Rhizobium CLOSs on legume roots. The smallest CLOS analog taken up and biologically active on white clover and alfalfa was a N-fattyacylglucosamine, without an essential requirement of oligomerization, fatty N-acyl unsaturation, or acetate / sulfate functionalization. This suggests that N-fattyacylglucosamine is the common minimum structure required and sufficient for uptake and biological activity of CLOS glycolipids in these legumes, and that the various specific modifications of its polar head group and hydrophobic tail modulate its inherent ability to further express these activities, thus influencing which legumes are capable of responding to CLOSs rather than dictating their biological activities per se.

Impacts
(N/A)

Publications

  • PHILIP-HOLLINGSWORTH, S., ET AL. 1997. Structural requirements of Rhizobium chitolipooligosaccharides for uptake and bioactivity in legume roots as revealed by synthetic analogs and fluorescent probes. J. Lipid Res. 38: 1229-1241.
  • REDDY P.M., ET AL. 1997. Rhizobial communication with rice roots: induction of phenotypic changes, mode of invasion, and extent of colonization. Plant and Soil 194: 81-98.
  • SANCHEZ-ANDUJAR, C., ET AL. 1997. Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv. trifolii. Mol. Gen. Genet. 255: 131-140.
  • YANNI, Y.G., ET AL. 1997. Natural endophytic association between Rhizobium leguminosarum bv. trifolii and rice roots and assessment of its potential to promote rice growth. Plant and Soil 194: 99-114.


Progress 01/01/96 to 12/30/96

Outputs
We used brightfield, time-lapse video, cross-polarized, phase contrast, & fluorescence microscopies to test isolated chitolipooligosaccharides (CLOSs) from wild type R. leguminosarum bv. trifolii on development of white clover root hairs, & the role of bioactive glycolipids in primary host infection. CLOS action caused a 3-fold increase in differentiation of root epidermal cells into root hairs. At maturity, root hairs were longer due to an extended period of active elongation without a change in the rate itself. Time-series image analysis showed the basis of CLOS-induced root hair deformation is a redirection of tip growth displaced from the medial axis. Further studies showed newly described infection-related root hair responses to CLOSs, including localized disruption of normal crystallinity in cell wall architecture & induction of new infection sites. The application of CLOS enabled a NodC- mutant of R. leguminosarum bv. trifolii to progress in the infection process by inducing bright refractile spot modifications of deformed root hair walls. CLOSs didn't rescue the ability of the NodC- mutant to induce marked curlings or infection threads in root hairs. These results indicate chitolipooligosaccharide Nod factors elicit several host responses that modulate growth dynamics & symbiont infectibility of white clover root hairs but CLOSs alone are not sufficient to permit successful entry of bacteria into root hairs during primary host infection in Rhizobium-clover symbiosis.

Impacts
(N/A)

Publications

  • DAZZO, F. B., et al. 1996. Modulation of dev. growth dynamics, wall crystallinity, & inf. sites in white clover root hairs by membrane chitolipooligosaccharides from R. leguminosarum bv. trifolii. J. Bact. 178:3621-3627.


Progress 01/01/95 to 12/30/95

Outputs
Fractionation of membrane extracts from of R. leguminosarum bv. trifolii wild type strain ANU843 led to the isolation of a diverse family of chitolipooligosaccharides (CLOSs) in high yield (> 15 mg/L of culture), whereas all attempts to isolate CLOSs from the corresponding culture supernatant failed. Structural analyses revealed that these membrane CLOSs consist of O-acetylated or non O-acetylated chito- tri-,-tetra- and pentasaccharides bearing an N-acyl moiety (predominantly cis-vaccenic acid, and lesser proportions of other saturated, unsaturated, and 3-hydroxy fatty acids) at the non-reducing glucosamine residue. No nodE -dependent CLOSs were found. ANU843 membrane CLOSs induced foci of cortical cell divisions in axenic roots white clover, but not hairy vetch or alfalfa. These results indicate that wild type R. leguminosarum bv. trifolii accumulates a very diverse family of CLOS Nod factors primarily in its cell membranes rather than secretes them, and our protocol which isolates these biologically active glycolipids in high yield from this source eliminates the need to use recombinant "overproducing" strains to obtain sufficient quantities for structural analyses. We predict that CLOS glycolipids perform important membrane functions for wild type rhizobia in the host root environment, and that these bacterial factors are likely to operate primarily at short range rather than as freely diffusible extracellular molecules in the Rhizobium-legume symbiosis.

Impacts
(N/A)

Publications


    Progress 01/01/94 to 12/30/94

    Outputs
    We investigated the accumulation of a diglycosyl diacylglycerol (BF-7) in wild type R. leguminosarum bv. trifolii ANU843 and its involvement in the root nodule symbiosis with white clover. BF-7 accumulates in cells grown with nod-activating 4',7-dihydroxyflavone, and this effect of flavone is suppressed in nodA::Tn5 and nodD::Tn5 mutants. BF-7 at 10-9 to 10-11 M elicits thick-short roots, root hair deformation, and collaroid foci of cortical cell divisions on white clover roots. Alfalfa and vetch were much less responsive to BF-7 at these low concentrations. A different diglycosyl diacylglycerol did not induce these responses on white clover. BF-7 elicited a meristematic rather than collaroid mitogenic response in the root cortex when ethylene production by clover was suppressed by aminoethoxyvinylglycine. These results indicate an involvement of flavone-activated nod expression in membrane accumulation of BF-7 in bv. trifolii and a potent ability of this glycolipid to activate segments of its host's symbiotic program during early development of the root nodule symbiosis.

    Impacts
    (N/A)

    Publications


      Progress 01/01/93 to 12/30/93

      Outputs
      The phospholipid and associated fatty acid compositions of the bacterial symbiont of clover, Rhizobium leguminosarum biovar trifolii wild type ANU843 was analyzed by two-dimensional silica thin-layer chromatography, fast atom bombardment-mass spectrometry, flame-ionization detection gas-liquid chromatography and combined gas-liquid chromatography mass spectrometry. The phospholipid composition included phosphatidylethanolamine (15%), N-methylphosphatidyl-ethanolamine (47%), N,N-dimethylphosphatidylethanolamine (9%), phosphatidyl-glycerol (19%), cardiolipin (5%), and phosphatidylcholine (2%). Fatty acid composition included predominantly cis-11-octadecenoic acid, lower levels of cis-9-hexadecenoic acid, hexadecenoic acid, 11-methyl-11-octadecenoic acid, octadecanoic acid, 11,12-methylene-octadecanoic acid, eicosanoic acid and traces of branched, and di- and triunsat-urated fatty acids. The influence of expression of the "nodulation" genes encoding symbiotic functions on the composition of these membrane lipids was examined in wildtype cells grown with or without the flavone inducer, 4-7-dihydroxyflavone and in mutated cells lacking the entire symbiotic plasmid where these genes reside, or containing single transposon insertions in selected nodulation genes. No significant changes in phospholipid or associated fatty acid compositions were detected by the above methods of analysis.

      Impacts
      (N/A)

      Publications


        Progress 01/01/92 to 12/30/92

        Outputs
        The involvement of Rhizobium enzymes that degrade plant cell wall polymers has long been an unresolved question about the infection process in root nodule symbiosis. Here we report the production of enzymes from Rhizobium leguminosarum bv. trifolii that degrade carboxymethyl cellulose and polypectate model substrates with sensitive methods that reliably detect the enzyme activities: a double-layer plate assay, quantitation of reducing sugars with a bicinchoninate reagent, and activity gel electrophoresis-isoelectric focusing. Both enzyme activities were (i) produced commonly by diverse wild-type strains, (ii) cell bound with at least some of the activity associated with the cell envelope, and (iii) not changed appreciably by growth in the presence of the model substrates or a flavone that activates expression of nodulation (nod) genes on the resident symbiotic plasmid (pSym). Equivalent levels of carboxymethyl cellulase activity were found in wild-type strain ANU843 and its pSym-cured derivative, ANU845. However, polygalacturonase activity was lower in ANU845 and was not restored to wild-type levels in the recombinant derivative of pSym(superscript -) ANU845 containing the common and host-specific nod genes within a 14-kb HindIII DNA fragment of pSym from ANU843 cloned on plasmid pRt032. Activity gel electrophoresis resolved three carboxymethyl cellulase isozymes of approximately 102, 56, and 33 kDa in cell extracts from ANU843.

        Impacts
        (N/A)

        Publications


          Progress 01/01/91 to 12/30/91

          Outputs
          The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. Rhizobium trifolii 0403 released LPS into the external root environment, which bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 (mu)g per plant) but inhibited at a higher dose. This biological activity of LPS was higher with LPS from cells in the early stationary phase than in the mid-exponential phase, and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. R. trifolii LPS induced acceleration of cytoplasmic streaming and production of novel proteins in root hairs. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.

          Impacts
          (N/A)

          Publications


            Progress 01/01/90 to 12/30/90

            Outputs
            We used a biochemical and dual genetic approach (gain or loss of function) to evaluate the contribution of certain pSym nod gene products in modifying the acidic capsular heteropolysaccharide (CPS) of Rhizobium trifolii. The CPS of wild type R. trifolii ANU843 was compared to various recombinant strains and Tn5 insertion mutants altered in certain pSym nod genes (obtained from B. Rolfe, Australian National University, Canberra, Australia). Cells were grown with, and in some cases, without 4'-7 dihydroxyflavone to control the level of pSym nod gene expression in defined BIII medium. Quantitative NMR studies of enzyme-cleaved octasaccharide units of CPS showed that they were substituted with less 3-hydroxybutyrate (3-HB) in the wild type and recombinant strains when grown to express pSym nodI. In contrast, the CPS oligomers contained more 3-HB substitution when produced by recombinant strains lacking nodI or the nodI::Tn5 mutant derivative of wild type ANU843. All strains produced CPS octasaccharide units with the same glycosyl sequence. The data suggest that the products of nodIJ, and especially nodI, modulate the level of 3-HB substitution in the acidic CPS synthesized by R. trifolii ANU843.

            Impacts
            (N/A)

            Publications


              Progress 01/01/89 to 12/30/89

              Outputs
              Host specificity in the Rhizobium-legume symbiosis is controlled in the bacterium by host specific nodulation (Hsn) genes residing on its symbiotic plasmid. We have examined the structure of the major acidic heteropolysaccharide (CPS) produced by recombinant hybrid strains of Rhizobium leguminosarum carrying cloned R. trifolii hsn genes with those produced by the parent donor and recipient strains. Alteration of the gene composition of R. leguminosarum strain 300 by introduction of an 8-kilobase set of hsn genes (nodFERL and nodMN) from R. trifolii strain ANU843, resulted in a hybrid strain which conferred efficient white clover infection and nodulation, production of the R. trifolii-types CPS, and an increased proportion of bacterial cells which bound to the white clover lectin, trifoliin A, in external root environment. H NMR studies indicated that the structure of the CPS from the hybrid recombinant differed from that of theR. leguminosarum strain 300 recipient in acetate and 3-hydroxybutyrate substituents. In contrast, the polysaccharide from a different hybrid recombinant strain containing only R. trifolli nodFERL genes had the acetylation pattern of the R. leguminosarum recipient but was substituted with 3-hydroxybutyrate at a level between that made by R. trifolii andR. leguminosarum. This latter recombinant strain displays sparse infection and nodulation of white clover roots. The R. leguminosarum recombinant strain containing the R.

              Impacts
              (N/A)

              Publications


                Progress 01/01/88 to 12/30/88

                Outputs
                Proton NMR and fast atom bombardment/mass spectrometry analyses of enzymaticallyderived oligosaccharides from acidic EPS of three strains of R. leguminosarus (300, 128C63) showed that they have an identical structure which is similar but not identical to acidic EPS from seven strains of R. trifolii. Three types of EPS structures could be distinguished among the R. trifolii strains. The type 1 R. trifolii EPS, made by strains LPR5035 and 0403, most closely resembled R. leguminosarum EPS. For both Type 1 and Type 2 EPS of R. trifolii, at most only half of the terminal branching galactose residues are substituted with 3-hydroxybutyrate whereas almost all of these galactose residues bear this substitution in R. leguminosarum EPS. The type 2 R. trifolii EPS, made by strains ANU843, TA1, and NA30, also differs from R. leguminosarum EPS in glycosyl site of acetylation. The Type 3 R. trifolii EPS, made by strains 4S and USDA 20.102 have unique patterns of acylations compared to each other and to all the other strains. Interestingly, this group has very different kinetics of nodulation on certain rare species of clovers such as Trifolium semipilosum. A major conclusion of this work is that the acidic EPS of pea rhizobia is similar (sometimes very) but not identical to the EPS of clover rhizobia. Therefore, the bacterial acidic EPS could play a role in determining host specificity.

                Impacts
                (N/A)

                Publications


                  Progress 01/01/87 to 12/30/87

                  Outputs
                  A symbiotically defective mutant strain of Rhizobium trifolii, UR251, was obtained by transposon Tn5 mutagenesis of R. trifolii 0403 rif and recognized by its partially ineffective (Fix) phenotype on white clover plants. UR251 had a single Tn5 insertion in plasmid DNA, a wild-type plasmid pattern, and no detectable Mu DNA sequences originally present in the vector used for Tn5 mutagenesis. Agglutination by the clover lectin trifoliin A and attachment to clover root hairs was higher with UR251 than with the wild-type strain. The capsular polysaccharide (CPS) of UR251 was altered as shown by (i)a slower rate of CPS depolymerization with a CPS beta-lyase, PD-I; (ii) more pyruvate and less acetate and 3-hydroxybutanoate noncarbohydrate substitutions as quantitated by H nuclear magnetic resonance; and (iii) a higher pyruvyl transferase activity (enzymatic pyruvylation of UR251 was on the terminal galactose of the branch of the repeating oligosaccharide unit. These results show that the level of noncarbohydrate substitutions of the CPS as well as pyruvyl transferase activity are altered in R. trifolii UR251 and that trifoliin A-binding ability and clover root hair attachment are improved in this mutant strain of R. trifolii 0403 rif.

                  Impacts
                  (N/A)

                  Publications


                    Progress 01/01/86 to 12/30/86

                    Outputs
                    A particulate form of the lectin, trifoliin A., has been isolated from the root exudate of axenically grown seedlings of white clover (Trifolium repens L. cv. Louisiana Nolin). The majority of trifoliin A active in binding to R. trifolii 0403 was sedimented from root exudate by centrifugation at 15,000 g for 30 min, indicating that it was particulate. Immunofluorescence, electron and immunoelectron microscopy using antibody prepared against trifoliin A from seeds, suggested that trifoliin A was associated with the particles in root exudate that bound specifically to the acidic capsular polysaccharides of Rhizobium trifolii 0403. Electron microscopic examination also showed that trifoliin A-colloidal gold conjugates bound to these same particles, indicating that they also have affinity for the lectin. The particles could be dislodged from intact seedling by vigorous shaking in isotonic plant growth medium. Isolated particles fractionated by ultracentrifugation through a metrizamide gradient had a mean density of 1.12g cm. These isolated particles retained the ability to bind to R. trifolii 0403 as shown by immunofluorescence using anti-trifoliin A antibody. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the isolated particles are a mixture of proteins including one with an approximate molecular weight of trifoliin A.

                    Impacts
                    (N/A)

                    Publications


                      Progress 01/01/85 to 12/30/85

                      Outputs
                      A polysaccharide depolymerase isolated from the phage lysate of Rhizobium trifolii 4S was used to fragment capsular polysaccharides (CPS) and extracellular polysaccharides (EPS) of R. trifolii 0403 into oligosaccharides. These products were analyzed for clover lectin (trifoliin A)-binding ability, effect on infection of white clover root hairs, and changes in glycosyl and noncarbohydrate composition with culture age. The oligosaccharides from CPS of cultures grown on agar plates for 3, 5, and 7 days exhibited lectin-binding ability at levels similar to those of the corresponding intact CPS. The intact EPS did not bind to clover lectin, although the oligosaccharide fragments from EPS did. In contrast, oligosaccharides from deacetylated CPS had less than half the lectin-binding ability of the native polysaccharide substrate. The CPS from 5-day-old cultures, its corresponding oligosaccharide fragments, and the oligosaccharide fragments of EPS from 5-day-old cultures, all at a concentration of 2.5 mu g per seedling, stimulated infection thread formation in root hairs of clover seedlings inoculated with R. trifolii 0403. Thus, this bacteriophage-induced polysaccharide depolymerase converted the acidic CPS and EPS of R. trifolii 0403 into biologically active oligosaccharides capable of binding trifoliin A and stimulating root hair infection.

                      Impacts
                      (N/A)

                      Publications


                        Progress 01/01/84 to 12/30/84

                        Outputs
                        A polysaccharide depolymerase isolated from the phage lysate of Rhizobium trifolii 4S was used to fragment capsular polysaccharides (CPS) and extracellular polysaccharides (EPS) of R. trifolii 0403 into oligosaccharides. These products were analyzed for clover lectin (trifoliin A)-binding ability, effect on infection of white clover root hairs, and changes in glycosyl and noncarbohydrate composition with culture age. The oligosaccharides from CPS of cultures grown on agar plates for 3, 5, and 7 days exhibited lectin binding ability at levels similar to those of the corresponding intact CPS. The intact EPS did not bind to clover lectin although the oligosaccharide fragments from EPS did. In contrast, oligosaccharides from deacetylated CPS had less than half the lectin-binding ability of the native polysaccharide substrate. The CPS from 5-day-old cultures, its corresponding oligosaccharide fragments, and the oligosaccharide fragments of EPS from 5-day-old cultures, all at a concentration of 2.6 mu g per seedling, stimulated infection thread formation in root hairs of clover seedlings inoculated with R. trifolii 0403. Thus, this bacteriophage-induced polysaccharide depolymerase converted the acidic CPS and EPS of R. trifolii 0403 into biologically active oligosaccharides capable of binding trifoliin A and stimulating root hair infection.

                        Impacts
                        (N/A)

                        Publications


                          Progress 01/01/83 to 12/30/83

                          Outputs
                          Lipopolysaccharide (LPS) isolated from Rhizobium trifolii in early stationary phase binds specifically to the clover lectin, trifoliin A. Kamberger (1979) proposed that the specific interaction of lectin with LPS of fast-growing rhizobia triggers successful infection of root hairs after attachment via cross-bridging of lectin and capsular polysaccharides. To test this hypothesis, sterile white clover seedlings were preincubated with purified LPS, rinsed, and used to prepare Fahraeus slide cultures with standardized inocula of R. trifolii 0403. Purified LPS isolated from R. trifolii 0403 in early stationary phase bound specifically to clover root hairs within 2 hr as shown by immunofluorescence microscopy. Pretreatment with this LPS significantly increased the frequency of root hair infections (infection threat formation). LPS-mediated enhancement of root hair infection was dose-dependent (in the ng-mug range), time-dependent, host- and R. trifolii-specific. LPS-mediated enhancement was significantly less with LPS which did not bind trifoliin A, incapable of reversing the inhibition of root hair infection by 15 mM nitrate, and restricted to the region of the seedling root present at the time of exposure to LPS. These results support the hypothesis that LPS plays a specific role in root hair infection by fast-growing rhizobia.

                          Impacts
                          (N/A)

                          Publications


                            Progress 01/01/82 to 12/30/82

                            Outputs
                            The effect of white clover root exudate on capsules of Rhizobium trifolii 0403 was examined. Trifoliin A bound uniformly to encapsulated, heat-fixed cells during 1 h. of incubation with root exudate from 2 clover varieties. After 4 to 8 h. trifoliin A was only bound to one pole of the cells, and by 12 h. it could no longer be detected on the bacteria. Transmission E.M. showed that the capsule itself was altered, beginning in the equatorial center of the rod-shaped cell and then progressed toward the poles at unequal rates. Trifoliin A was detected in situ on one pole of cells grown for 4 days in the clover root environment of Fahraeus slide cultures. Inhibition studies with the hapten 2-deoxy-D-glucose showed that trifoliin A in root exudate had a higher affinity for one of the cell poles. In the presence of concentrated root exudate, extracellular polysaccharides from R. trifolii 0403 were converted into products which eventually lost their ability to immunoprecipitate with homologous antibody. This alteration proceeded more rapidly with root exudate from seedlings grown under nitrogen-free conditions than with root exudate from plants grown with 15 mM KNO(3). The root exudate, depleted of trifoliin A by immunoaffinity chromatography, was still able to alter the capsule of R. trifolii 0403. Reconstitution experiments showed that the proteins in root exudate which induced this alteration of the capsule were antigenically unrelated to trifoliin A.

                            Impacts
                            (N/A)

                            Publications


                              Progress 01/01/81 to 12/30/81

                              Outputs
                              The lipopolysaccharide (LPS) from Rhizobium trifolii 0403 was isolated at different stages of growth and was examined for its (i) ability to bind a white clover lectin (trifoliin A), (ii) immunochemical properties, and (iii) composition. There was significantly more binding of trifoliin A to purified LPS and cells in the early stationary phase than to cells in the exponential phase. New antigenic determinants of the LPS appeared for brief periods on cells at the end of the lag phase and the beginning of the stationary phase. These new antigens were not detected on cells in mid-exponential or late stationary phase. Fab fragments of IgG raised against the unique antigenic determinants in the LPS competively blocked the binding of trifoliin A to cells in the early stationary phase. GLC analysis showed that several glycosyl components in the LPS increased as the culture advanced from the mid-exponential to the early stationary phase. In addition, cells in the early stationary phase had a larger LPS. Quinovosamine (2-amino-2,6-dideoxyglucose) was identified by combined gas chromatography-mass spectrometry as a sugar component of the LPS which had not been previously reported. D-Quinovosamine, N-acetyl-D-quinovosamine, and its n-propyl-beta-glycoside were effective hapten sugars which inhibited the binding of trifoliin A, anti-clover root antibody, and homologous antibody to these new determinants in the LPS.

                              Impacts
                              (N/A)

                              Publications


                                Progress 01/01/80 to 12/30/80

                                Outputs
                                This portion of the project has focused on the cell walls of white clover roots grown in hydroponic solution containing 15 mM NO -(3), at which concentration root hairs are not infected by Rhizobium trifolii 0403. Purified cell walls bind 3-4 fold less trifoliin A (a clover lectin) when the plants are grown with 15 mM NO -(3) rather than N-free solution. In addition, wall-bound hydroxyproline, an indicator of the wall glycoprotein, extensin, increases significantly (70-100%) in walls grown with 15 mM NO -(3). Fractionation of the Hyp-arabinosides on Bio-Gel P-2 suggests that more Hyp-Ara(4) accounts for this increase, but more studies are necessary to establish this finding. These results focus on significant NO -(3)-modulated alterations in the root cell walls which may be correlated with the inability of cell walls to accumulate lectins that bind the symbiont rhizobia. Trifoliin A levels (per mg total protein) in clover root exudate are 30-fold lower when roots are grown with 15 mM NO -(3) rather than in N-free medium. Thus, both free and wall-bound trifoliin A are affected by the presence of NO -(3) in the rooting medium. Immunofluorescence studies show that trifoliin A binds to one cell pole of R. trifolii 0403 in the clover rhizosphere, and this significant result may relate to the distinctively polar attachment of these bacteria to the root epidermis.

                                Impacts
                                (N/A)

                                Publications


                                  Progress 01/01/79 to 12/30/79

                                  Outputs
                                  (2nd in series). The long-term goal of this project is to determine how NO(3) -controls the level of trifoliin, a clover lectin on the root surface which binds specifically to Rhizobium trifolii. The first part of this project was to determine if there is a direct interaction between NO(3) - and trifoliin or its homologous antibody. The results of 4 experimental approaches indicate that there is no such direct interaction. First, the specific agglutinating activity of trifoliin for R. trifolii 0403 is unaffected by 15 mM NO(3) -. Second, NO(3) - does not bind directly to trifoliin as shown by ligand-binding studies using radioactive 1 3NO(3) -. Third, the quantitative immunoprecipitin curves of trifoliin and its homologous antibody are identical in the presence or absence of NO(3) - (15mM). And fourth, 1 hr assays of Phase I adherence (docking) indicate that what NO(03) - does not directly interact with trifoliin on root hairs in a way which could prevent selective adhesion of the rhizobial symbiont. Thus, there is some intervening process modulated by NO(3) which regulates the levels of trifoliin on clover roots. Part 2 of this project is to determine if the trifoliin receptor on the root cell walls undergoes alteration when seedlings are fed NO(3); thereby preventing the accumulation of trifoliin.

                                  Impacts
                                  (N/A)

                                  Publications


                                    Progress 06/01/78 to 12/30/78

                                    Outputs
                                    Fixed nitrogen, e.g., NO(3) -, is a key nutrient in the soil which limits the development of the nitrogen fixing Rhizobium-legume symbiosis. One objective of this project is to determine how NO(3) - controls the level of trifolin, a Rhizobium recognition protein, on the root surface of the legume clover. Ultrafiltration ligand-binding studies, using 1 3NO(3) - and trifoliin on Amicon PM-10 ultramembranes, have shown that NO(3) - does not bind directly with trifolin, thus eliminating one possible mechanism of regulation. Project 1314H also includes studies on the mechanism of cellular adhesion in the Rhizobium-clover symbiosis. Saccharide determinants on R. trifolii responsible for adhesion to clover root hairs were found for only transient periods of time when cells were grown on Bergersen's agar or broth. These transiently appearing receptors were associated with a fibrillar polyanionic capsule surrounding the rhizobial cell. The architecture of the rhizobial cell surface is not constant in composition, but changes with the phase of growth.

                                    Impacts
                                    (N/A)

                                    Publications