Progress 10/01/15 to 09/30/19
Outputs
Target Audience:This project benefits truffle farmers across North Carolina and beyond. Company servicing the truffle industry will also benefit from the results of this project. The students participated in this project acquired skills for conducting research on truffles, as well as for working in truffle farming/industry. Changes/Problems:The major challenges this project faced were: 1. Power failure that ruined all samples collected from the first experiment. 2. The unavailability of greenhouse space due to the renovation and repairs which lasted over 12 months. 3. Next generation sequencing data analysis which is slow because it is outsourced. 4. Facilities and equipment: Most of our major equipment (autoclave, freeze dryer, Real-time PCR machine) were broken. Most of them are over ten years old and relatively not serviceable. What opportunities for training and professional development has the project provided? Co-Pi and PhD. Student attended a six weeks training on metagenomics at Duke University. The PI visited a lab of researchers doing similar research in Italy The PhD. student is now accepted into one-year training in the area of metalgenomics at Duke University. A master's degree student has been trained in field sampling to collect soil and root samples from field plots; extract DNA and run PCR analysis of samples collected. Three Research Assistants have been trained in sample collection and processing from field plots, and trees aeration, which is necessary for mycorhization of new roots in the field plots. How have the results been disseminated to communities of interest? Sharing and discussion on the data collected so far had been done at the Genomics group meeting with our peers across the University and the Joint School of Nanoscience and Nanotechnology. A poster presentation authored by the Master's student, co-PI and PI was presented at the mycology conference in Chicago. Got coverage in several news and media- A newspaper publication on Times news on the actual fruiting of Tuber borchii in reduced times was published; NC A &T magazine publication ; Fox 8 TV News report: https://myfox8.com/2019/10/25/north-carolina-at-mushroom-researcher-hopes-to-revolutionize-livestock-feed-industry/ What do you plan to do during the next reporting period to accomplish the goals?This project has ended, however the seedlings that were planted and inoculated just at the end of this project has been rolled over to a new project. Essentially the objective one of this project is expanded in a new project that will see the seedling study go beyond the initial scope of objective one to include longer time monitoring of the effect of fertilizer on seedlings growth, mycorrhizae development and persistence on loblolly pine seedlings.
Impacts
What was accomplished under these goals?
Specific objectives met: There are two major objectives of this project: 1. To determine the level of nutrient supplementation for enhanced seedling growth and mycorrhization by T. borchii Seedling stratification and germination: Seedling inoculation and fertilizer application were completed and sampling was also completed. 2. To monitor truffle establishment in field plots consisting of T. borchii inoculated pine seedlings Established protocols and optimizations were applied in screening selected soil samples from field plots. Random pine trees were selected. Soil sample were collected from each selected trees at 3 different distances, 1ft, 2ft, and 3ft. DNA was isolated from soil samples of pines from pine plots via MOBIO PowerSoil DNA Isolation Kit. DNA was quantified with Promega Quantus Fluormeter and diluted for Next Generation Sequencing (NGS) at the University of North Carolina, Chapel Hill Mocrobiome Core Facility. NGS specs include 250 base pair, paired end reads with 16s rRNA primers on the Illumina MiSeq. Similarly, samples collected were sent to UC Irvine for NGS sequencing that included the fungi diversity in the field plots. Diagnostic PCR From 15 randomly selected trees, soil cores were taken from one foot, two feet, and three feet away from the base of each tree and at 2 centimeters depth from coordinates 0° North, 90° East, 180° South, and 270° West. The soil cores were used for DNA extraction as above. DNA from the samples were also used to run regular PCR with Tuber borchii specific primers. PCR products were ran on agarose gel to detect amplification. Key outcomes Objective 1. Experimental set up and sample collection for objective 1 was completed and samples stored for analysis. The samples included soil, roots, and shoots. Plant growth parameters we also collected. However, all samples were lost due to power failure in our minus 20 C refrigerator which lasted for over four days. The samples all grew mold and were not possible to recover. Attempt to repeat this experiment ran into another hitch when the greenhouse to be used was under reconstruction which lasted beyond expectation. Objective 2. Results show various bacterial phyla, classes, orders, families. However, bacteria from the genera of Agrobacterium, Novosphingobium, Rhodoplanes, Pseudonocardia and Streptomyces were most abundant in the soils tested. There were 5 trees that showed a decrease in diversity with the increase in distance from the tree, 4 trees that showed an increase in diversity with the increase in distance from the tree and 1 tree where the diversity remained the same. This data took into account the removal of bacteria where it was less than 0.40% across all soil samples Tuber borchii specific primers turned out to be very efficient at detecting the presence and colonization of out-planted inoculated loblolly pines in the orchard. After 5 years in the field, the presence of the T. borchii could be detected in all trees except those which are 1, 2 and 3 ft distances from the tree. This outcome represents an inexpensive fast test to detect and monitor progress of colonization and persistence of T. borchii fungi in the roots of inoculated trees in truffle orchards.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:This project benefits truffle farmers across North Carolina and beyond. Company servicing the truffle industryare also benefit from the results of this project. The students participating in this project acquire skills for conducting research on truffles, as well as for working in truffle farming/industry. Changes/Problems:Facilities and equipment failures are still a major problem: Most of our major equipment (autoclave, freeze dryer, Real-time PCR machine) are broken. Most of them are over ten years old and relatively not serviceable. The greenhouse was out due to renovation which took over six months. These have great limitations on the rate at which we can do our work. What opportunities for training and professional development has the project provided?1. Co-PI and PhD student Janelle Robinson attended one week course on real-time PCR at University of North Carolina. 2. A master's degree student, Ese Ekhator has been trained in field sampling to collect soil and root samples from field plots; extract DNA and run PCR analysis of samples collected. 3. Research Assistants (Melvin Bonsu) have been trained in sample collection from field plots, and tree root aeration. How have the results been disseminated to communities of interest? Sharing and discussion of the data collected so far has been done at the Genomics group meeting with our peers across the University and the Joint School of Nanoscience and Nanotechnology. A poster presentation authored by the Co-PI, PI and students was presented at the BEACON Conference, Michigan State University. The breakthrough and harvest of an 6 oz Tuber borchii truffle by one of our truffle farmers was reported on NC A&T facebook page (#AggiesInnovate#AggiesDo) and it got 176 views, 60 comments and 65 shares. Published the research in N C A & T research magazine. What do you plan to do during the next reporting period to accomplish the goals?1. Complete objective one, which will determine the effect of fertilizer on mycorrhization of inoculated seedlings in the greenhouse. 2. Nest Generation Sequencing (NGS) analysis of fungi and bacteria in soil samples from field plots. 3. Write manuscript for peer review publications.
Impacts
What was accomplished under these goals?
Experiment for objective 1. Inoculation failure due to inadequate autoclave and non-availability of greenhouse has made it necessary to repeat this experiment. Seedling germination and inoculation is ongoing. Field plots were sampled to detect and monitor truffle establishment in field plots consisting of T. borchii inoculated pine seedlings as per objective 2. Specific objectives met: There are two major objectives of this project: 1. To determine the level of nutrient supplementation for enhanced seedling growth and mycorrhization by T. borchii- Seedling stratification and germination followed by first round of inoculation is complete. Next round of inoculation failed and a repeat of that experiment is ongoing. 2. To monitor truffle establishment in field plots consisting of T. borchii inoculated pine seedlings- Established protocols were applied in screening selected soil samples from field plots for the second time in 2018. Seedling randomly selected from previous year were sampled again. Soil sample were collected from each selected trees at 3 different distances, 1ft, 2ft, and 3ft. DNA was isolated from soil samples and processed as per previous year and PCR conducted to test the presence of truffle DNA. Next generation sequence analysis of 24 soil samples was completed. Key outcomes: Seedling response to stratification and germination successful. The first round of inoculation is ongoing. The first set of results on NGS based diversity studies on samples indicated significant changes in soil microbial community due to introduction of truffle-inoculated tress in the field plots. The Tuber borchii specific primer based PCR for tracking growth into truffle orchard field indicated that substantial development into the soil has taken place. Most samples tested showed PCR amplification bands in the electrophoreses. Truffle DNA were detected even at 3 ft distances from the tress, compared to previous year's results where implication were mainly restricted 1 and rarely 2 ft from the base of inoculated trees.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Anike FN, Panchagavi R, Pollard S, Robinson JR, Harrison SH, Isikhuemhen OS. Investigating Soil Microbiome Diversity in Truffle Producing plot, BEACON Conference, August 8-11, 2018 at Michigan State University, East Lansing, MI. (Poster Presentation).
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:This project will benefit truffle farmers across North Carolina and beyond. Companies servicing the truffle industry will also benefit from the results of this project. The students participating in this project will acquire skills to conduct research on truffles, and for working in the truffle-farming industry. Changes/Problems:Facilities and equipment: Most of our major equipment (autoclave, freeze dryer, Real-time PCR machine) is broken. Most of these technologies are more than 10 years old and not serviceable. Equipment restrictions greatly limit the rate at which we can accomplish our work. What opportunities for training and professional development has the project provided? PI, Co-PI and Ph. D. student (Janelle Robinson) attended a one-week course on Next Generation Sequencing, sample preparation and processing for bacteria and Fungi at University of California, Irvine. PI and Co-PI attended a one-week course on Next Generation Sequencing, sample preparation and processing for bacteriaat the Michigan State University Sequencing Core facility. Master's degree student Leisha Wainwright has been trained in field sampling to collect soil and root samples from field plots; extract DNA and run PCR analysis of samples collected. Research assistant Dominic Beckoe has been trained in sample collection from field plots, and tree aeration, which is necessary for mycorrhization of new roots in the field plots. How have the results been disseminated to communities of interest? Sharing and discussion on the data collected so far has been conducted at the Genomics group meeting with our peers across the University, including the Joint School of Nanoscience and Nanoengineering. A poster presentation authored by the master's student (Leisher), the co-PI and PI was presented at the mycology conference in Chicago. A newspaper publication on Times News on the actual fruiting of Tuber borchii in reduced times was published Re:search (College of Agriculture and Environmental Sciences) magazine publication What do you plan to do during the next reporting period to accomplish the goals? Complete objective No. 1, to determine the effect of fertilizer on the mycorrhization of inoculated seedlings in the greenhouse Metagenomics analysis of fungi and bacteria in soil samples from field plots Master technologies for doing metagenomics analysis of fungi in field plots Confirm the accuracy of specific primer PCR tracking of truffle growth in soil through the sequencing of PCR products already acquired
Impacts
What was accomplished under these goals?
Experiment for objective 1 is set up and we are currently conducting the inoculation cycle and data collection. This process was necessitated after the accidental loss of previous experiments due to power failure in the laboratory. Field plots were sampled to detect and monitor establishing truffle in field plots consisting of T. borchii inoculated pine seedlings, as per objective No. 2. Specific objectives met: There are two major objectives of this project: To determine the level of nutrient supplementation for enhanced seedling growth and mycorrhization by T. borchii Seedling stratification and germination: Seedling stratification, germination completed. First round of inoculation is complete. Next round of inoculation is ongoing. 2. To monitor truffle establishment in field plots consisting of T. borchii inoculated pine seedlings Established protocols were applied in screening selected soil samples from field plots. Random pine trees were selected. Soil samples were collected from each of the selected trees at three different distances: 1 foot, 2 feet and 3feet. DNA was isolated from soil samples of pines from pine plots via Qiagen PowerSoil DNA Isolation Kit. DNA was quantified with Promega Quantus Fluorometer. Purified DNA is now being processed for Next Generation Sequencing (NGS) according to the training we just received from University of California, Irvine. We are waiting for results from the first set of DNA samples sent for sequencing on their platform. The change to this platform was necessary because we will be able to also determine fungal diversity, instead of only bacteria; and fungal diversity is most critical for determining competing organisms at play in the rhizosphere of inoculated trees Isolated DNA from field samples were PCR amplified with specific primers to determine the extent of growth from the inoculated plants into field soils. Key outcomes Seedling response to the first round of inoculation is satisfactory since mycorrhizae can already be seen on of the roots of the inoculated seedlings. We are awaiting the first set of results on NGS-based diversity studies on samples submitted through UC Irvine to the sequencing facility at the University of California-Davis. The Tuber borchii specific primer-based PCR for tracking growth into truffle-orchard field indicated that substantial development into the soil has taken place. Most samples tested showed PCR amplification bands in the electrophoreses. We are waiting confirmation from DNA sequencing. This will become a powerful tool to monitor the establishment of truffle fungus in field plots.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:This project will benefit truffle farmers across North Carolina and beyond. Companies servicing the truffle industry will also benefit from the results of this project. The students participating in this project will acquire skills for conducting research on truffles as well as knowledge for working in truffle farming/industry. Changes/Problems: Facilities and equipment are dated and relatively not serviceable. This has great limitations on the rate at which we can do our work. There was power failure in our lab with the -20ºC freezer where samples are stored and all DNA extracted from soils for over four days. Therefore, all samples were loss due to molds that grew on them and the DNA stored were degraded. Three years of work and materials collected were loss. This is a major setback that will not allow for timely completion of this project in the scheduled period. The power situation in the affected lab is well known for the past five years, but no attention has been given to fix it. What opportunities for training and professional development has the project provided? Co-project director and a PhD student attended a six weeks course on metagenomics training at Duke University. The project director visited a lab of researchers in the same field in Italy The PhD student is now accepted into one-year training in the area of metal genomics at Duke University. AMS degree student has been trained in field sampling to collect soil and root samples from field plots; extract DNA and run PCR analysis of samples collected. Research assistants have been trained in sample collection from field plots, and trees aeration, which is necessary for mycorhization of new roots in the field plots. How have the results been disseminated to communities of interest?Sharing and discussion on the data collected so far has been done at the genomics group meeting with peers across the University and the Joint School of Nanoscience and Nanotechnology. No publication of data in per reviewed journal yet. What do you plan to do during the next reporting period to accomplish the goals?Plans for the next reporting period are to: Study root sample morphology and metagenomics analysis of samples from field plots. Do more metagenomics analysis of same soil samples, but on bacteria communities? Master technologies for doing metagenomics analysis of fungi in field plots. Start all over again the experiments associated with Objective 1.
Impacts
What was accomplished under these goals?
Major activities completed experimental set up and sample collection for Objective 1 was completed and samples stored for analysis. The samples included soil, roots, and shots. Plant growth parameters were also collected. However, all samples were loss due to power failure in a minus 20º C refrigerator which lasted for over four days. The samples all grew mold and were not possible to recover. Field plots were sampled to detect and monitor truffle establishment in field plots consisting of T. borchii inoculated pine seedlings was done. Specific objectives met: There are two major objectives of this project: To determine the level of nutrient supplementation for enhanced seedling growth and mycorrhization by T. borchii. Seedling stratification and germination: Seedling inoculation and fertilizer application were completed and sampling was also completed. To monitor truffle establishment in field plots consisting of T. borchii inoculated pine seedlings Established protocols and optimizations from last year were applied in screening selected soil samples from field plots. Random pine trees were selected. Soil sample were collected from each selected tree at three different distances, 1 foot, 2 feet, and 3feet. DNA was isolated from soil samples of pines from pine plots via MOBIO PowerSoil DNA Isolation Kit. DNA was quantified with Promega Quantus Fluormeter and diluted for Next Generation Sequencing (NGS) at the University of North Carolina, Chapel Hill Mocrobiome Core Facility. NGS specs include 250 base pair, paired end reads with 16s rRNA primers on the Illumina MiSeq. The NGS was done and analyzed for microbial diversity (Bacteria at UNC Chapel Hill. Key outcomes Results show various bacterial phyla, classes, orders, and families. However, bacteria from the genera of Agrobacterium, Novosphingobium, Rhodoplanes, Pseudonocardia and Streptomyces were most abundant in the soils tested. There were five trees that showed a decrease in diversity with the increase in distance from the tree; four trees that showed an increase in diversity with the increase in distance from the tree; and one tree where the diversity remained the same. This data took into account the removal of bacteria where there was abundance less than 0.40% across all soil samples.Researchers plan to do more sampling and sequencing to reach better conclusions. Also, researchers plan to study changes in fungi in the same samples next.
Publications
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