Progress 01/01/10 to 06/30/13
Outputs
Target Audience: The results of the project were submitted for publication to a scientific journal and presented to researchers and land managers interested in mycorrhizal ecology and restoration of sagebrush habitats after fire. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Through this seed grant, the PD has been able to implement in his laboratory procedures for molecular identification of AMF, for multiplication of AMF in pot cultures and in vitro, and for the establishment of monospecific cultures. This expertise combined with the data collected on the structure of AMF communities and on the effects of AMF inoculation on seedlings performance will be basis for future funding proposals. The project has provided many research opportunities to graduate and undergraduate students A graduate student Keith Carter is near the completion of his M.S. thesis based on work funded by the project. He conducted much of the work described under objective 1 related to the molecular identification of AMF species in seven sagebrush habitats. Through his research he has received training on methods to extract DNA from roots and soil, DNA amplification and cloning, phylogenetic analyses, and statistical methods to compare the composition of microbial communities. Another graduate student Bill Davidson is also close to the completion of his M.S. work. Bill conducted most of the experiments described under objective 2. He received training in procedures to multiply mycorrhizae in pot cultures, analyze AMF root colonization, make measurements of photosynthetic characteristics, extract DNA from roots, and identify AMF using molecular techniques. The following undergraduates worked on the project and received training and gained experience on the procedures/techniques listed next to their names: Eric Roberts: extraction of DNA, PCR, cloning, and preparation of samples for sequencing Russell Holten: multiplication of mycorrhizae in pot cultures, analysis of mycorrhizal colonization, collection and separation of mycorrhizal spores, and chlorophyll fluorescence Ryan LaJoie: multiplication of mycorrhizae in pot cultures, PCR, and cloning Craig Carpenter: collection of mycorrhizal spores, surface sterilization of spores and root fragments, and in vitro culture of mycorrhizae Rachael Barron: DNA extraction, PCR, cloning, and generation of Ri T-DNA carrot roots How have the results been disseminated to communities of interest? The work has been disseminated through publications, presentations at professional meetings, and presentations to land managers and growers of native plants. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. This objective was completed and expanded over the work planned in the original proposal. Initially, the plan was to identify and multiply mycorrhizae from only one site. However, based on the suggestions of the reviewers we sampled Artemisia tridentata seedlings and soil from several sites. Specifically, we analyzed the arbuscular mycorrhizal (AMF) communities of seven sagebrush habitats in southwestern Idaho. Based on these analyses, we determined the overall AMF diversity within and among these communities and the extent to which preferential AMF-plant associations develop during seedling establishment. Sequences from the large subunit-D2 rDNA region and phylogenetic analyses revealed that the AMF diversity in sagebrush habitats of southwestern Idaho was similar to that reported for several mesic habitats. Overall, we identified seven phylotypes, two within the Claroideoglomeracea and five within the Glomeraceae. Some phylotypes showed high genetic variation. The most diverse was the phylotype that included R. irregularis. This phylotype included sequences showing pairwise differences up to 12% and could be subdivided into four groups with pairwise differences greater than 6%. Comparison of sequences obtained from the soil with those from A. tridentata roots revealed no significant differences between the AMF present in these samples. Most of the diversity in AMF sequences occurred within sites. However, some genetic differentiation was detected among sites and correlated with differences in soil texture. Another factor associated with differentiation of AMF communities was multiplication in pot cultures, which caused major shifts in the frequency of individual phylotypes. Objective 2. The goals under this objective were accomplished through two main experiments. The first experiment consisted of a factorial combination of two inoculation treatments (non-inoculated and inoculated seedlings) and two transplanting times (spring and fall). Initially, seedlings were grown in a greenhouse in sterile soil (non-inoculated seedlings) or soil from pot cultures of native mycorrhizae (inoculated seedlings). Three months old seedlings were transplanted to 24 L pots filled with soil collected from a sagebrush habitat and were grown under natural climatic conditions. Mycorrhizal colonization, seedling growth, photosynthetic characteristics, and seedling survival were monitored from the time of transplanting to up to eight months afterwards. During this period, seedlings that had been inoculated showed higher AMF colonization than non-inoculated seedlings, about 45% and 20% AMF colonization, respectively. Thus, inoculation increased AMF colonization over the levels naturally occurring in the soil. This increase in colonization was associated with 25% increase in survival during the summer. Transplanting time also had an effect on survival. Independent of the mycorrhizal treatments, survival during the first eight months after transplanting was higher in seedlings transplanted during the fall than those transplanted during the spring. The second experiment was similar to the one described above, but with two exceptions. Instead of two, three inoculation treatments were used: non-inoculated seedlings, inoculated with native soil, and inoculated with pot cultures of native mycorrhizae. In addition, the seedlings were not transplanted to pots, but directly to the soil at a burnt site in southwestern Idaho. Plant survival was monitored for up to 18 months after transplanting and mycorrhizal colonization at the time of transplanting and three and seven months after the spring and fall transplanting, respectively. For the spring transplanting, inoculation did not increase the AMF colonization over the levels naturally occurring in the soil and no differences in survival were observed between inoculation treatments. This was in contrast with the results observed after fall transplanting. Eight months after transplanting inoculated seedlings maintained higher levels of colonization than non-inoculated ones. Similarly, the highest survival was observed in seedlings inoculated with pot cultures even after many losses of seedlings due to unusually dry conditions during winter, spring, and summer. The reasons for the differential responses between spring and fall transplanting are unclear, but may be attributed to differences in root system development. Following spring transplanting and in early summer, the roots predominantly grew in depth towards the deep and moist layer of the soil. Furthermore, AMF colonization was much higher in deep than shallow roots. In contrast, the roots of seedlings transplanted in early fall predominately grew and branched in the upper soil layers. Moist conditions during the fall may have favored the spread of the added inoculum and reinfection of developing roots. Overall, the results indicate that inoculation of seedlings with AMF can increase colonization after transplanting in both potted plants and in a natural setting. Moreover, the increase in colonization was correlated with an increase in survival. In addition to the measurements described above, we have extracted DNA from plant roots under the various treatments and used molecular methods to identify AMF taxa present in these samples. We are presently completing the analysis of these data with the goal of characterizing possible variations in AMF composition among seasons and between non-inoculated and inoculated seedlings after transplanting. Objective 3: Only part of the work described under this objective was completed because more efforts than anticipated were put towards objective 1 and 2. This was largely to address two valuable suggestions of the reviewers; which were to increase the number of sampled sites for AMF identification and to test the effect of AMF inoculation on seedling performance not only on potted plants, but also in a natural setting. Efforts under objective 3 concentrated on the implementation of procedures to establish monospecific cultures of AMF in pot cultures and in vitro. These efforts included using both spores and colonized root fragments as inoculum. Currently, we have established monospecific cultures of two AMF morphotypes in pot cultures and implemented procedures to multiply AMF in vitro using Ri T-DNA transformed carrot roots. Ongoing efforts are aimed at multiplying other morphotypes. Monospecific cultures will be used to test the effect of single species on A. tridentata seedling performance and to obtain a better taxonomic characterization of AMF.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Carter KA, Smith JF, White MM, Serpe MD, Assessing the diversity of arbuscular mycorrhizal fungi in semiarid shrublands dominated by Artemisia tridentata ssp. wyomingensis (submitted to Mycorrhiza)
- Type:
Other
Status:
Published
Year Published:
2013
Citation:
Serpe MD, Davidson BE Pre-inoculation of Wyoming big sagebrush seedlings with native arbuscular mycorrhizae: Effects on mycorrhizal colonization and community composition after transplanting. In Great Basin Native Plant Selection and Increase Project Annual Report 2012 (pp. 62-68)
- Type:
Other
Status:
Published
Year Published:
2012
Citation:
Serpe M Diversity of Mycorrhizal Fungi Associated with Artemisia tridentata ssp. wyomingensis. In Great Basin Native Plant Selection and Increase Project Annual Report 2011 (pp. 50-57)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Carter KA, Serpe MD, Smith JF (2013) Assessing the diversity of mycorrhizal communities in sagebrush steppes of southwestern Idaho. Annual meeting of the Botanical Society of America
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Davidson BE, Serpe MD (2013) Colonization of Artermisia tridentata ssp wyomingensis by native arbuscular mycorrhizae: persistence and consequence of inoculation. Great Basin Consortium Conference
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Davidson BE, Serpe MD (2012) Improvement in colonization and seedling survival of Wyoming big sagebrush following inoculation with native arbuscular mycorrhizal fungi. Annual meeting of the Mycological Society of America. Inoculum 63: 15
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Davidson B, Serpe MD (2012) Improvement in colonization and seedling survival of Wyoming big sagebrush following inoculation with native arbuscular mycorrhizal fungi. Annual Meeting of Northwest Science. Program and abstracts p. 29
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Holten R, Roberts E, Serpe MD (2012) Survival of native arbuscular mycorrhizal inoculum following transplanting of Wyoming big sagebrush. National Conference of Undergraduate Research. Program p. 125
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Carter K, White M, Serpe M (2011) Identification of mycorrhizal species associated with Artemisia tridentata ssp. wyomingensis in southwestern Idaho. Annual meeting of the Mycological Society of America. Inoculum 62 (3): 12-13
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2011
Citation:
Davidson D, Cragin J, Serpe M (2011) Seasonal changes in gas exchange and chlorophyll fluorescence parameters in Artemisia tridentata ssp. wyomingensis. Annual meeting of the Great Basin native plant selection and increase project
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2010
Citation:
Carter K, Serpe M (2010) Identification and multiplication of native mycorrhizal species that colonize Artemisia tridentata ssp. Wyomingensis seedlings. Annual Meeting Intermountain Seedling Growers Association
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2010
Citation:
Carter K, Davidson B, White M, Shaw N, Serpe M (2010) Identification of mycorrhizae associated with Artermisia tridentata ssp. Wyomingensis in Southwestern Idaho. National Native Seed Conference
|
Progress 01/01/13 to 06/30/13
Outputs
Target Audience: The results of the project were presented to researchers and land managers interested in restoration of sagebrush habitats after fire. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students, Keith Carther and Bill Davidson, learned how to conduct phylogenetic analyses of DNA sequences and received training on various approaches used for the comparison of mycorrhizal communities. Two undergraduates, Eric Roberts and Rachael Barron, learned various molecular methods related to extraction of DNA from soils and roots, PCR amplification, and cloning. In addition, these undergradutes helped with the analysis of mycorrhizal colonization. A third undergraduate, Craig Carpenter, received training in tissue cultures procedures used to multiply mycorrhizae in organ cultures. How have the results been disseminated to communities of interest? Yes, we presented our results at the following meetings: Davidson B, Serpe M. (2013) Colonization of Artemisia tridentata ssp wyomingensis bynative arbuscular-mycorrhizae: persistence and consequence of inoculation. The Great Basin Consortium 2nd Annual Conference Roberts E, Barron R, Serpe M (2013). Mycorrhizal colonization and community composition in shallow and deep roots of Wyoming big sagebrush seedlings. Boise State Undergraduate Research Conference What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal 1. We have completed the characterization of arbuscular mycorrhizal (AMF) communities from seven sagebrush habitats in southwestern Idaho. This characterization was based on the analysis of AMF sequences obtained from soil samples, Artemisia tridentata seedlings, and pot cultures. The sequences were used to identify AMF phylotypes present in sagebrush habitats and to determine the overall mycorrhizal diversity within and among the studied sites. In addition, comparison of the AMF phylotypes present in the soil with those present in the roots of seedlings was used to test the possibility that some phylotypes preferentially associate with A. tridentata. Similarly, comparison of the AMF communities of field samples with those obtained after multiplication in pot cultures was used to characterize the extent to which pot culture cultivation alters the AMF community. Possible differences in phylotype composition among sites, between soil and roots, or between field samples and pot cultures were evaluated by analysis of molecular variance (AMOVA) and using non-metric multidimensional scaling (NMDS). Goal 2. In 2013, the work under Goal 2involved continuing an experiment initiated in 2012. The experiment consisted of a factorial combination of 3 inoculation treatments (non-inoculated control and two levels of inoculation) and 2 transplanting times (spring and fall). Initially, we grew seedlings in a greenhouse for three months and have them under one of three inoculation treatments: sterile soil from the field site (control treatment), untreated soil from the field site (native treatment), or soil from pot cultures of native mycorrhizae (inoculated treatment). Subsequently, the seedlings were transplanted to a burnt site in southwestern Idaho. The goals were to determine the effect of inoculation and transplanting time on seedling survival, AMF colonization, and AMF community composition. During 2013, we continued the measurements of these response variables. Goal 3: We have continued our efforts to multiply monospecific cultures of AMF in pot cultures of leeks and in Ri T-DNA transformed carrot roots.
Publications
- Type:
Other
Status:
Published
Year Published:
2013
Citation:
Serpe MD, Davidson BE. Pre-inoculation of Wyoming big sagebrush seedlings with native arbuscular mycorrhizae: effects on mycorrhizal colonization and community composition after transplanting. Great Basin Native Plant Selection and Increase Project Annual Report 2012 (pp. 62-68)
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Objective 1, Multiplication and identification of native mycorrhizal species: We have continued extracting DNA from seedlings of Artemisia tridentata ssp. wyomingensis (sagebrush) and amplifying mycorrhizal DNA present in these samples with Glomeromycota specific primers. Approximately, an additional 500 mycorrhizal sequences were obtained and used to characterize the diversity of mycorrhizae colonizing this species. Characterization of mycorrhizal diversity involved phylogenetic analysis and determination of pairwise distances among the sequences. In addition, we have analyzed soil characteristics of the various sites from where we collected samples. Objective 2, Determine whether addition of a native AMF mixture to soils improves mycorrhizal colonization and seedling survival: In 2012, the work under Objective 2 involved two main experiments. One of the experiments was initiated in 2011 and finished in 2012. This experiment initially involved growing seedlings in a greenhouse for three months and exposing them to either of two treatments: no-inoculation or inoculation with a mixture of native mycorrhizae that has been multiplied in pot cultures. In early fall 2011, the seedlings were transplanted to 24 L pots filled with soil collected from a sagebrush habitat and were grown under natural climatic conditions until the spring of 2012. Seedling survival was measured monthly and mycorrhizal colonization eight month after transplanting. In addition, DNA was extracted from the roots at this time to analyze the mycorrhizal community composition under the two treatments. The goal was to determine whether inoculation altered the mycorrhizal composition of the roots that developed after transplanting. A second experiment was initiated in 2012. This experiment was similar to the one described above, but with seedlings transplanted to soil at a burnt site in southwestern Idaho. The experiment consisted of a factorial combination of three inoculation treatments (no-inoculation, inoculation with native soil, and inoculation with pot cultures of native mycorrhizae) and two transplanting times (spring and fall). The goals were to determine the effect of inoculation and transplanting time on seedling survival under conditions where root growth is not limited by soil volume. Objective 3, Effect of single mycorrhizal species on seedling establishment: We have continued our efforts to develop monospecific cultures of AMF in root organ cultures. Initially, we attempted to initiate these cultures on Ri T-DNA transformed roots of Medicago truncatula. With our samples, growth of mycorrhizae on these roots proved to be fairly limited. To improve this situation, we have continued the development of root organ culture using a different host, Ri T-DNA transformed roots of carrot, which is a widely used species for growing mycorrhizae in roots cultures. During 2012, the results of the project were presented at four meetings: Annual meeting of the Mycological Society of America, Annual meeting of the Great Basin native plant selection and increase project, Annual Meeting of Northwest Science, and the National Conference of Undergraduate Research. PARTICIPANTS: Marcelo Serpe (PD): training and supervision of students and conduction of various aspects of the experimental work including DNA extraction, planting and inoculation of seedlings, development of root organ cultures, and analysis of data. Merlin White (Co-PI): analysis of DNA sequences using phylogenetic approaches. Keith Carter: graduate student; Keith conducted much of the work described under objective 1 related to the molecular identification of mycorrhizal species. Bill Davidson: graduate student; Bill conducted most of the experiments described under objective 2. Eric Roberts; undergraduate; Eric helped graduate students with the amplification of mycorrhizal genes, cloning, and preparation of samples for sequencing. Russell Holten: undergraduate; Russell multiplied mycorrhizae in pot cultures and conducted measurements of mycorrhizal colonization. Ryan LaJoie: undergraduate; Ryan grew and inoculated sagebrush seedlings in the greenhouse prior to transplanting and conducted measurements of mycorrhizal colonization. Craig Carpenter: undergraduate: Craig initiated and maintained root organ cultures of mycorrhizae. Collaborators: Dr. Nancy Shaw, USDA Forest Service, Rocky Mountain Research Station, Boise, Idaho. Dr. Shaw and the USDA Forest Service provided transportation to collect soil and the space to conduct the field experiments in large pots. Anne S. Halford, Restoration Ecologist, BLM Office: Four Rivers Field Office, Boise, ID 83705. Ms. Harlford organized volunteers and provided tools to conduct the transplanting of more than 600 sagebrush seedlings to the burnt site where we conducted the experiment described under Objective 2. This site is located at BLM-Morley Nelson Snake River Birds of Prey National Conservation Area. TARGET AUDIENCES: The results of the project were presented to researchers interested in restoration ecology and land managers involved in revegetation of degraded sagebrush habitats. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objective 1: Phylogenetic analyses of the sequences obtained from the various sites resulted in the identification of seven phylotypes with Bayesan posterior probabilities larger than 90%. The phylotypes belong to two families: Glomeraceae and Claroideoglomeraceae. Four of the phylotypes clustered with known species and the other three did not, which suggests that they may be new species. When grouped based on pairwise distance analysis, the sequences clustered into 17 OTUs, each OTU defined as having similarities ≥ 94%. Sequences within a particular OTU were always present within one phylotype, but some phylotypes included several OTUs. For example, a phylotype that clustered with Rhizophagus irregularis included 4 OTUs, indicating large genetic diversity within this clade. As estimated by AMOVA, there were differences in mycorrhizal community composition among sites. The sites also differed in soil characteristics including texture and nutrient levels. Currently, we are determining whether differences in mycorrhizal communities among sites are related to differences in soil characteristics. Objective 2: For the experiment in pots, mycorrhizal colonization at the time of transplanting was 0.16 and 69% for non-inoculated and inoculated seedlings, respectively. Differences in colonization remained significant eight months after transplanting; 23 and 44 % for non-inoculated and inoculated seedlings, respectively. On the other hand, inoculation did not affect the mycorrhizal composition of the roots. Similarly, no differences were observed on seedling survival. Both treatments had 100% survival by June 2012, when the experiment was ended because plant growth and survival became limited by the size of the pots. The experiment involving transplanting to soil at a burnt site showed a significant interaction between inoculation treatments and transplanting times. For the seedlings transplanted in spring, no difference in colonization was detected three months after transplanting and survival was similar among treatments. In early fall 2012, all inoculation treatments had survival rates of about 40% and no seedling mortality occurred during the subsequent fall and winter. In contrast, seedling transplanted in early fall showed significant differences in seedling survival (p < 0.01). In March 2013, percent survival was 44, 62, and 81% for non-inoculated seedlings, inoculated with native soil, and inoculated with pot cultures, respectively. Analysis of colonization in these seedlings is in progress. Overall, the results indicate that transplanting during the fall combined with inoculation with native mycorrhizae can significantly improve seedling survival with respect to spring transplanting or transplanting of non-inoculated seedlings. Objective 3: We have generated Ri T-DNA roots of carrots and initiated mycorrhizal cultures using spores collected from pot cultures. Material from the in vitro cultures is being used to complement taxonomic studies of mycorrhizae and to inoculate sagebrush seedlings with single phylotypes. This last approach will allow us to investigate possible functional differences among species/phylotypes of mycorrhizae.
Publications
- Davidson B, Serpe M. (2012) Improvement in colonization and seedling survival of Wyoming big sagebrush following inoculation with native arbuscular mycorrhizal fungi. Inoculum 63: 15
- Holten R, Roberts E, Serpe MD (2012) Survival of native arbuscular mycorrhizal inoculum following transplanting of Wyoming big sagebrush. National Conference of Undergraduate Research Program p. 14
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Objective 1, Multiplication and identification of native mycorrhizal species: Seedlings of Artemisia tridentata ssp. wyomingensis (sagebrush) and soil samples were collected from seven sites in southwestern Idaho. From these samples, we extracted DNA and identified arbuscular mycorrhizal fungi (AMF) using molecular methods. The presence of mycorrhizal DNA in the extracts was determined via PCR with Glomeromycota specific primers that amplify a region of the large subunit ribosomal RNA gene (LSU rDNA). The PCR products were cloned and the inserts sent for sequencing to a commercial facility. The sequences obtained were analyzed using two approaches. First, we grouped the AMF sequences into operational taxonomic units with sequence similarities larger than 97% (OTUs 0.03). These OTUs were then used to estimate differences in AMF communities between roots and soil samples and between sites. In addition, we conducted a phylogenetic analysis of the DNA sequences to identify species based on comparison with a reference data set for phylotaxonomy of AMF (http://www.lrz.de/~schuessler/amphylo/). Maximum parsimony analyses were performed using PRAP2. Objective 2, Determine whether addition of a native AMF mixture to soils improves mycorrhizal colonization and seedling survival: Native AMF were multiplied from soil collected at Kuna butte-Idaho using Sudan grass pot cultures. To generate differences in colonization, sagebrush seedlings were first grown in a greenhouse in 50 ml containers containing pot cultures (inoculated seedlings) or sterilized pot cultures (control seedlings). In early spring, three-month old seedlings were transplanted to 24 L pots filled with soil collected from a sagebrush habitat and were grown under natural climatic conditions until the fall. Mycorrhizal colonization was measured 2.5 and 5 months after transplanting, while seedling survival was measured biweekly. An experiment similar to the one just described was also started five month later. This resulted in transplanting during the early fall, which allowed us to compare the effect of differences in transplanting time on AMF colonization and seedling survival. Objective 3, Effect of single mycorrhizal species on seedling establishment: We have worked on the development of monospecific cultures of AMF in root organ cultures. For this purpose, we have genetically modified and established root cultures of Medicago truncatula following published protocols. Furthermore, fungal spores or root fragments colonized with mycorrhizae were surface sterilized and transferred to Petri dishes containing the transformed roots to initiate the root cultures. The results of the project were presented at the following meetings:Carter K, Serpe M, White M (2011) Identification of mycorrhizal species associated with Artemisia tridentata ssp. wyomingensis in southwestern Idaho. Annual meeting of the Mycological Society of America. Davidson D, Cragin J, Serpe M (2011) Seasonal changes in gas exchange and chlorophyll fluorescence parameters in Artemisia tridentata ssp. wyomingensis. Annual meeting of the Great Basin native plant selection and increase project PARTICIPANTS: Marcelo Serpe (PD): training and supervision of students and conduction of various aspects of the experimental work including DNA extraction, planting and inoculation of seedlings, development of root organ cultures, and analysis of data. Merlin White (Co-PI): analysis of DNA sequences using phylogenetic approaches. Keith Carter: graduate student; Keith conducted most of the experiments described under objective 1 related to the molecular identification of mycorrhizal species. Bill Davidson: graduate student; Bill conducted most of the experiments described under objective 2. Russell Holten: undergraduate; Russell multiplied mycorrhizae in pot cultures and conducted measurements of mycorrhizal colonization and seedling survival. Eric Roberts: undergraduate; Eric collected spores, surface sterilized spores and root fragments, and inoculated root organ cultures with spores. Collaborators: Dr. Nancy Shaw Nancy L. Shaw, USDA Forest Service, Rocky Mountain Research Station, Boise, Idaho. Dr. Shaw and the USDA Forest Service provided transportation to collect soil and the space to conduct the field experiments. TARGET AUDIENCES: The results of the project were presented to land managers involved in the restoration of sagebrush habitats. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objective 1: From the DNA extracted from the various sites, we obtained 345 AMF sequences that grouped into 34 OTUs0.03. Of these, 25 were from samples directly collected from the field, while the rest were detected after AMF multiplication in pot cultures. As estimated by the Jaccard index, the similarity in AMF OTUs varied between the sites, ranging from 66 to 16 %. Of the 25 OTUs identified in field samples, ten were found in soil and roots, eleven only in the soil, and four only in the roots, which suggests some preference in the OTUs that colonize sagebrush. The phylogenetic analysis revealed that all the sequences obtained belonged to the family Glomeraceae. In the phylogenetic tree, the sequences grouped into twelve terminal clades with posterior probability values larger than 80%. Two of these clades showed high sequence similarity with Claroideoglomus claroideum, three with Glomus intraradices, and one with Rhizophagus irregularis. The other clades exhibited less than 94% similarity with published LSU rDNA sequences. These clades may represent new species or alternatively known species whose sequences have not been reported for the LSU rDNA gene. Overall, the results revealed genetic diversity in the AMF composition of sagebrush habitats and differences between sites. Objective 2: For the spring experiment, the percent colonization at the time of transplanting was 0.3 and 57% for control and inoculated seedlings, respectively. Differences in colonization remained significant 2.5 and 5 months after transplanting. In July, the percent colonization was 20 and 44 % for control and inoculated seedlings, respectively. Similarly, in October the percent colonization was 19 and 50% for control and inoculated seedlings, respectively. These results indicate that the inoculum contributed to the colonization of the roots that developed after transplanting resulting in higher levels of colonization than those naturally occurring in the soil. The differences in colonization did not affect plant biomass or the shoot over root ratio. In contrast, differences in colonization were associated with differences in seedling survival, which was 24% higher in inoculated than control seedlings (p<0.01). Differences in survival were observed towards the end of the summer, when water deficits are more severe. This suggests that colonization by AMF increases sagebrush tolerance to drought conditions. For the fall transplanting experiment, the analysis of colonization is in progress, while survival has been monitored biweekly. As of December 31, 2011, no seedling mortality has been observed. This indicates that sagebrush seedlings can cope with the cold temperatures of winter much better than with the dry summer conditions. Objective 3: Various attempts were made to initiate organ cultures of AMF using spores and root fragments colonized with mycorrhizae. Thus far, we have only been successful in initiating root cultures using spores. The AMFs present in these cultures will be characterized using molecular methods and used to inoculate sagebrush seedlings with single AMF species.
Publications
- Carter K, Serpe M, White M (2011) Identification of mycorrhizal species associated with Artemisia tridentata ssp. wyomingensis in southwestern Idaho. Inoculum 62 (3): 12-13
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Objective 1: Multiplication and identification of native mycorrhizal species During the first year of work, we have identified native mycorrhizal species from several sites in sagebrush steppe habitats of southwestern Idaho. The species were identified using molecular approaches starting with DNA extracted from the roots of 3 to 6 month old seedlings of Artemisia tridentata ssp. wyomingensis and soil samples collected at the different sites. Samples were collected in seven sites and the presence of mycorrhizal DNA in the root and soil extracts was determined via nested PCR. General fungal primers LR1 and FLR2 were used in the first amplification and their products were amplified in a second PCR reaction with the Glomeromycota specific primers FLR3 and FLR4. These primers amplify regions of the large subunit ribosomal RNA gene (LSU rDNA). Some of the products amplified by PCR were cloned and sent for sequencing to a commercial facility. The resulting nucleotide sequences were used for identification of AMF species based on comparison with published sequences via phylogenic analysis. To increase the chances of detecting species that may be present at very low density, the soil was also used to multiply mycorrhizae in pot cultures using Sudan grass as a host. DNA was then extracted from Sudan grass roots and soil from the pot cultures. Objective 2: Determine whether addition of a native AMF mixture to soils improves mycorrhizal colonization and seedling survival With soil collected at one site (Kuna butte), various experiments were conducted to compare mycorrhizal colonization without and with addition of pot culture inoculum. Seeds were planted in pots containing either sterile soil, native soil (untreated), or native soil supplemented with pot culture. On the seedlings, time courses of colonization were analyzed over a three month period. Seedlings were also grown under outdoor conditions to determine their survival and photosynthetic performance from mid spring to winter. Objective 3: Effect of single mycorrhizal species on seedling establishment No progress was made on this aspect of the project. The results of the study were presented at meetings of land managers and growers of native seeds: K. Carter, B. Davidson, M. White, M. Serpe Identification of mycorrhizae associated with Artemisia tridentata ssp. wyomingensis in Southwestern Idaho. National native seed conference 2010 K. Carter, M. Serpe Identification and multiplication of native mycorrhizal species that colonize Artemisia tridentata ssp. wyomingensis seedlings. 31st Intermountain Seedling Grower's Association Meeting 2010 PARTICIPANTS: Marcelo Serpe (PD) training and supervision of students and conduction of various aspects of the experimental work including collection of seedling and soil samples, DNA extraction, inoculation of seedlings, and measurements of chlorophyll fluorescence parameters and CO2 assimilation. Merlin White (Co-PI) Analysis of DNA sequences using phylogenetic approaches Keith Carter: graduate student; Keith conducted most of the experiments described under objective I related to the molecular identification of mycorrhizal species. Jacob Cragin: undergraduate; he conducted analyses of mycorrhizal colonization and made measurements of survival and chlorophyll fluorescence parameters in sagebrush seedlings Bill Davidson: undergraduate; cleaning and planting of seeds, multiplication of mycorrhizae in pot cultures, inoculation of seedlings, and quantification of mycorrhizal colonization Nick Waters: undergraduate; conducted measurements of mycorrhizal colonization and chlorophyll fluorescence parameters Collaborators: Dr. Nancy Shaw Nancy L. Shaw, USDA Forest Service, Rocky Mountain Research Station, Boise, Idaho Dr. Shaw and the USDA Forest Service provided seeds of Artemisia tridentata var. wyomingensis and indicated the location of sites with plants of this species. They also provided transportation to collect soil from various sites and space in their field station for us to conduct the field experiments. TARGET AUDIENCES: The results of this project have been presented to people involved in restoration projects including land managers, researchers involve in collecting and characterizing native seeds, and private growers of native plants. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objective 1: From the DNA extracted from the roots and soil, we obtained PCR products of the expected length, which ranges from 300 to 380 bp. Thus far, we have obtained sequences from five sites. A bootstrap analysis conducted with these sequences showed that Glomus intraradices is the most common mycorrhizal species in both the sagebrush roots and in the soil. Other prevalent species include G. microaggregatum and G. claroideum. In addition, there are several clusters in the analysis that denote previously unsequenced strains or species, potentially representing novel mycorrhizae. Between the various sites, we have detected two genera and at least five mycorrhizal species, this number is common for sites in semiarid regions. Objective 2: The inoculated soil resulted in higher levels of colonization than native and sterile soil. After two and half months from seeding, the percent colonization was 1.25 (sd 0.1), 5.3 (sd 0.4), and 45 (sd 8.3) for the sterile, native, and inoculated soil, respectively. Thus, for the soil collected at Kuna butte, a significant increase in colonization can be achieved by the addition of inoculum. An additional goal under objective 2 was to compare seasonal changes in survival and chlorophyll fluorescence parameters. For the spring of 2010, we were not able to obtain seedlings with high percent colonization, which limited the value of comparing mycorrhizal treatments. Nevertheless, we considered worthwhile to investigate survival rates and chlorophyll fluorescence parameters in non-inoculated seedlings. The seedlings transplanted in spring showed high values of Fv/Fm (>0.7) and survival rates above 80% until early summer. During late June and July, seedlings that did not receive irrigation showed a decrease in Fv/Fm values and the survival rate was less than 5% at the end of July. To increase seedling survival a set of seedlings was watered from the middle of June to the end of July. On these seedlings, we continued measurements of survival and photosynthesis through the fall and winter. Survival through this period was 98% and the seedlings maintained their photosynthetic capacity. The decrease in leaf water potential after the cessation of watering had little effect on Fv/Fm, but decreased the effective quantum yield (ΔF/Fm'), indicating a decrease in the proportion of incident light used for electron transport. During late fall and winter, Fv/Fm decreased. However, the Fv/Fm values markedly changed on a daily basis. At temperatures below OC, the Fv/Fm values were low (<0.3) and the leaves did not photosynthesize. However, as temperatures increased above 5C, within hours Fv/Fm increased to about 0.6 as well as the rate of CO2 assimilation. With temperatures between 5 and 10C, the rate of CO2 assimilation was about half of that observed under warm temperatures and well watered conditions. The results of survival and photosynthetic characteristics suggest that transplanting during the fall may be a good alternative to spring transplanting. This hypothesis will be tested in 2011 with inoculated and non-inoculated seedlings.
Publications
- No publications reported this period
|
|