Progress 04/15/18 to 12/31/21
Outputs
Target Audience:1. Students that worked on the project on a routine basis. 2. Volunteers and students that worked in various aspects of the project, including growing and inoculating seedlings in the greenhouse, transplanting seedlings to the field, and analyzing root colonization by arbuscular mycorrhizae dark septate fungi. 3. Scientists and managers from the Bureau of Land Management 4. Researchers, land managers and students that attended meetings where results of the project were presented or read the published articles. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the last year of the project, four undergraduates (Bailie Sirhall, Kateri Bilay, Christopher Bennett, and Jacob Venable) and one graduate student (Mathew Geisler) worked on the project. The undergraduates received training on experimental procedures related to plant water potential and photosynthesis measurements, microscopic analysis of root colonization, growing fungi in vitro, and preparation of samples for next-generation sequencing. They also used various computers programs for data analysis, including survival and image analysis. One of the undergraduates (Jacob Venable) became particularly interested in the work with DSE and will continue as a graduate student studying this topic. The PD provided training to the graduate student in statistical analyses using R and Python and guided him in writing his thesis, which is near completion. How have the results been disseminated to communities of interest?Two undergraduates (Kateri Bailey and Bailey Sirhall) presented results from the field experiment at a local meeting. The graduate student presented his last year's results from the greenhouse experiments at the 2021 annual meeting of the Botanical Society of America. 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: We completed all the work proposed under this objective. This work involved greenhouse experiments and resulted in two publications; another manuscript is in preparation. The experiments involved multiplying native arbuscular mycorrhizae (AMF) and the dark septate fungus (DSE) Darksidea sp., then inoculating A. tridentata seedlings with these fungi and analyzing seedlings' performance under drought. We observed differences in colonization between non-inoculated and inoculated seedlings in all cases. In addition, when seedlings were co-inoculated with AMF and Darksidea sp., colonization by AMF did not affect colonization by Darksidea and vice versa. The only exception was the density of vesicles, which increased in the presence of Darksidea. Under well-watered and water-stressed conditions, colonization by Darksidea sp. did not affect biomass, transpiration, and photosynthesis, indicating that the symbiosis was commensalistic. In contrast, colonization by AMF delayed the drought-induced decline in photosynthesis. We conducted two additional experiments to ascertain this result and quantified the decline by estimating the time when stomatal conductance and CO2 assimilation reached half their initial values (t1/2). Some variation was observed between experiments, but a meta-analysis of the three experiments indicated that AMF colonization increased the t1/2 for stomatal conductance and CO2 assimilation by 38 and 59%, respectively. Thus, as judged by the prolonged maintenance of photosynthesis, AMF reduced the sensitivity of the seedlings to drought. Efforts were also made to determine the mechanism by which AMF prolonged photosynthesis. For this purpose, we investigated in 3- and 18-month old seedlings the leaf water potential that caused a 50% reduction (Ψ50) in stomatal conductance or CO2 assimilation. We observed developmental differences in Ψ50 but no significant effect of AMF on this parameter. A more conclusive result was obtained when we analyzed stomatal conductance and CO2 assimilation in relationship to the decrease in soil water content. The soil water content at which these physiological parameters declined to half their initial values was lower in mycorrhizal than non-mycorrhizal plants. This effect may be due to extra-radical hyphae maintaining liquid continuity through soil air gaps. In the above experiments, we also analyzed the effect of water stress on fungal colonization of the roots. The extent of total AMF and DSE colonization was similar before and after the drought. However, water stress caused a reduction in arbuscular colonization, which might be advantageous by preventing the symbiosis from becoming parasitic as stomata close. In addition to colonization, we studied the fungal community composition of the roots in one of the experiments using next-generation sequencing approaches. Preliminary results from some of the samples suggest that drought did not alter the fungal community composition of the roots, but the analysis of the sequences is still in progress. Objective 2 We completed the work described under this objective by conducting two field experiments; one started in the fall of 2018 and the other in the fall of 2019. A manuscript based on these experiments is near completion. In the first experiment, we could not increase AMF or DSE colonization levels over the background levels naturally occurring in the soil. We modified the inoculation method for the second experiment, which led to increased AMF colonization. Eight months after outplanting, AMF colonization in non-inoculated and inoculated seedlings was 24.1 and 48.5 %, respectively (p = 0.01). Significant differences in colonization were still present in the fall of 2020 and the spring of 2021. In contrast to AMF, we could not increase colonization by Darksidea. Independent of the inoculation treatment, colonization by DSE was relatively high in A. tridentata roots (~40%). However, this colonization was not due to Darksidea but rather a complex mixture of other DSE. Increased AMF colonization did not affect survival. In late July 2021, the percent survival of non-inoculated and inoculated plants was 92.9 and 95.8, respectively (p = 0.195). These survival rates were much higher than those typically observed in the region, which tend to be below 40%. Such high survival rates could have been due to mild winters facilitating root growth and our cultural practices that provided better protection against herbivory. Measurements of stomatal conductance and plant water potentials during the summers of 2020 and 2021 indicated that the plants experienced drought stress, but not to lethal levels. After an initial decrease from early April to June, average water potentials remained relatively constant at about -2.5 MPa through the summer despite the lack of precipitation and a continued reduction in soil moisture. A decrease in stomatal conductance was partially responsible for the seasonal homeostasis in water potential. Although AMF inoculation did not affect water potential or survival, the number of plants with inflorescences was higher in inoculated than non-inoculated seedlings. Albeit not significant, such a difference was observed in 2020, and it augmented and became significant during 2021. In July 2021, the percentage of plants developing inflorescences was 45.4 and 59.0% for non-inoculated and inoculated seedlings, respectively (p = 0.02). We plan to continue flowering measurements to determine whether dissimilarities persist in subsequent years. Such a result would indicate that early benefits of AMF colonization have a long-lasting competitive advantage. In summary, the results described under Objectives 1 and 2 indicate positive effects of AMF colonization on drought tolerance and reproductive development of A. tridentata seedlings. Based on the magnitude of the responses observed, these effects may not be sufficient to increase A. tridentata establishment markedly. However, they likely would contribute to creating a combination of conditions that facilitate recruitment in this species. Objective 3 In previous years, we have isolated three dark septate fungi from A. tridentata roots; Helminthosporium tilliae, Montagnula sp., and Cadophora sp. Furthermore, re-synthesis experiments in vitro indicated that they were root endophytes. During the past year, efforts concentrated on testing the effects of these fungi on plant growth and AMF colonization under greenhouse conditions. Attempts were made to inoculate seedlings in a 1:1 soil:sand mix using cultures or a fungal spawn of the isolated DSE fungi. Of these two methods, only the spawn led to the successful colonization of A. tridentata roots. However, significant colonization (between 20 and 30%) was only obtained with Montagula sp. Although the other two DSE readily colonized A. tridentata in vitro, colonization in soil did not occur or was within the margin of DSE colonization observed in the greenhouse without inoculation. Thus, we continued the experiment only with Montagnula. The results obtained with this fungus were similar to those with Darksidea. Colonization by Montagnula did not affect AMF colonization, and alone or co-inoculated with AMF, this DSE neither affected plant biomass. As indicated in the proposal, objective 3 is of a long-term nature. We will continue isolating and testing DSE but concentrating on those that, based on DNA analysis, appear common in A. tridentata roots. In sequences from roots collected during the field experiment, this was the case for Montagnula, but not for Helminthosporium or Cadophora.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Carpenter CL, White M, Serpe MD. 2021. Co-inoculation with a dark septate endophyte alters arbuscular mycorrhizal colonization of two widespread plants of the sagebrush steppe. Symbiosis 85: 343357.
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Progress 04/15/20 to 04/14/21
Outputs
Target Audience:1. Students that worked on the project on a routine basis 2. Student volunteers who worked in various aspects of the project, including planting and harvesting seedlings and analyzing roots' fungal colonization. 3. Land managers from the Bureau of Land Management 4. Researchers and student attending a presentation and one meeting where results of the project were presented Changes/Problems:The experiments were conducted with no significant changes. The major problems were limited access to the lab and restrictions on vehicle occupancy. These problems slowed down field work and the analysis of samples and prevented us from conducting some experiments, particularly those under objective 3. What opportunities for training and professional development has the project provided?Two graduate students (Craig Carpenter and Mathew Geisler) and four undergraduates (Nichole Hoffman, Bailie Sirhall, Kateri Bilay, and Christopher Bennett) received supervision related to various aspects of this project. This supervision involved periodic observation of the students' work, such as assessing their accuracy in analyzing fungal colonization and measuring plant photosynthesis and water potential. In addition, the PI met weekly with the students to ascertain they implemented the different steps of the experiments correctly. The PI also assisted the students with data analysis and writing a manuscript and a thesis. How have the results been disseminated to communities of interest?Based on the project's results, a graduate student, Craig Carpenter, presented his work to a local audience, which included BLM land managers. In addition, he prepared and submitted a manuscript for publication. The PI presented a summary of the project's results at the 2020 NIFA PD meeting. What do you plan to do during the next reporting period to accomplish the goals?For Objective 1, we have two primary goals. One is to complete a greenhouse experiment to determine the effect of AMF on drought tolerance of very young (2 to 3 months old) A. tridentata seedlings. One of the graduate students, Mathew Geissler, is conducting this work. The results of this experiment combined with similar experiments he completed with older seedlings are the basis of his MS thesis. Thus, the second goal under objective 1 is to complete the thesis and submit a manuscript based on this work. He aims to graduate and submit a manuscript during the fall of 2021. Concerning Objective 2, we will continue collecting data for the field experiment. For the roots that we collected last year in late spring and early fall, we are presently analyzing the fungal taxa present in the roots. These data will be valuable in evaluating how inoculation and summer drought affected the endophyte community and may help identify fungal symbioses more tolerant of drought. In addition, we plan to continue analyzing plant survival and water potential during the summer because forecasts predict that 2021 will be drier and hotter than average. These conditions may provide an opportunity to assess the extent to which relative young plants can cope with drought. The final goal under this objective will be to prepare a manuscript that combines the results we obtained at the field site since the initial planting in October 2019. The central goal under Objective 3 will be to assess the effect of the isolated dark septate fungi on plant growth and AMF colonization.
Impacts
What was accomplished under these goals?
Objective 1: In previous years, we investigated the effect of root endophytes on drought tolerance of A. tridentata seedlings under conditions where water deficits developed 10 to 15 days after withholding watering. At these times, stomatal conductance and CO2 assimilation were minimal, and there was a marked decline in the operation efficiency of photosystem II (ΦPSII). A significant result of these experiments was that AMF inoculation delayed the drought-induced decrease in photosynthesis. During this reporting period, we conducted an additional experiment to study the impact of AMF on photosynthesis for plants exposed to more gradual drought stress. For this purpose, non-inoculated and AMF-inoculated seedlings were grown in larger pots, and a collar foam covered the pots to minimize evaporative water loss from the soil. The method used to inoculate seedlings with AMF was effective in increasing colonization by these fungi. Median values of colonization in non-inoculated and inoculated seedlings were 1.0 and 30.9 %, respectively (p = 0.0006). Also, the development of drought was more gradual. Reductions in CO2 assimilation and ΦPSII to half of their values under well-watered conditions took an average of 32 and 37 days, respectively. However, contrary to previous experiments, AMF colonization did not delay the decrease in photosynthesis. Similarly, no differences were observed in the leaf water potential that caused a 50% reduction (Ψ50) in CO2 assimilation, which was -2.23 (± 0.21) and -2.47 (± 0.43) MPa for non-inoculated and inoculated seedlings, respectively. However, AMF inoculation had an effect on the Ψ50 for ΦPSII. A 50% reduction in ΦPSII occurred at -3.79 (± 0.73) MPa in non-inoculated seedlings and -5.37 (± 0.53) MPa in inoculated ones. Also, AMF colonization increased water use efficiency, which was 2.07 and 2.73 µmol CO2 per mmol H2O for non-inoculated and inoculated seedlings, respectively (p = 0.02). Thus, even though AMF did not delay the decrease in photosynthesis caused by drought, it improved photosynthetic electron transport at low water potentials and the trade-off between carbon gain and water loss. In the experiment described above, we also investigated the effect of drought on AMF colonization. For AMF-inoculated seedlings, there were no differences in total AMF colonization between well-watered and water-stressed plants. In contrast, the percent of arbuscules was lower under drought than under well-watered conditions, 5.7 and 19.7 %, respectively (p = 0.009). Given that arbuscules are the primary site of nutrient exchange, the decline in arbuscules suggests that possible benefits of the symbiosis would tend to diminish as the drought develops. Overall, the results of the greenhouse experiments revealed positive effects of AMF colonization on the performance of A. tridentata plants under drought. However, these effects varied depending on the speed at which water stress developed. Objective 2. In October of 2019, we started a second field experiment at Kuna butte, ID (43° 26' 47.32" N, 116° 26' 48.61" W). At this time, 150 seedlings were transplanted for each of four inoculation treatments. In 2020, we analyzed the effects of these treatments on root colonization by three groups of fungi: AMF, septate, and thin-non-septate. Samples for these analyses were collected in June and October 2020, eight and twelve months after transplanting, respectively. Also, we assessed the impact of the treatments on the plants by monitoring leaf water potential, the percent of plants with flowers, and survival. Eight months after transplanting, seedlings inoculated with a mixture of native AMF have higher AMF colonization levels than non-inoculated seedlings, 48.5 and 24.1 %, respectively (p = 0.01). Based on the measurements made in early fall, differences in AMF colonization were maintained throughout the summer. For the samples harvested in October, AMF colonization was 11.6% in non-inoculated seedlings and 37% in inoculated ones (p = 0.013). Between seedlings inoculated with Darksidea sp. and those that were not, differences in colonization by septate fungi were only observed for samples harvested in the fall, which had 28.8 and 51.9 % colonization for non-inoculated and inoculated seedlings, respectively. However, molecular analyses of the septate fungi present in these samples indicated that the differences were due to septate fungi other than Darksidea; the latter was not detected in these analyses. The third type of fungal endophyte, thin-non-septate, showed somewhat lower colonization levels than AMF or septate fungi, about 22%, and no differences were noted between treatments. Since transplanting, we have measured survival monthly and pre-dawn and midday leaf water potential during the summer. Precipitation was minimal from the beginning of July to the beginning of October 2020. During this period, seedlings had median midday water potential that ranged between -2.0 and -2.8 MPa, and no differences in leaf water potential were apparent between non-inoculated and AMF-inoculated seedlings. Even though leaf water potentials were relatively low, with some seedlings reaching values as low as -5.0 MPa, mortality was minimal. As of April 2021, survival was about 95 % in all treatments. Also, during the spring and early summer of 2020, many plants developed inflorescences, 37.7 and 44.3% of non-inoculated and AMF-inoculated seedlings, respectively (p = 0.2). In summary, the field experiment indicated that inoculation with AMF increased colonization by these fungi over the natural background levels. In contrast, the attempt to increase colonization by Darksidea sp. was unsuccessful, perhaps reflecting the high abundance at which septate fungi are naturally present in A. tridentata roots. In addition, the survival levels obtained were much higher than those usually observed in the region, which tend to be below 40%. Given that there were no differences in survival between inoculation treatments, the reasons for high seedling survival are unclear. A possibility is that the AMF inoculum spread to non-inoculated plants, and although colonization was lower in them than in the inoculated ones, it somewhat contributed to drought tolerance. In addition, precipitation in the spring of 2020 was higher than average, and our methods of outplanting and seedling protection from herbivory were different from those used by federal and state agencies. Current work is aimed at identifying factors that contributed to high seedling survival. However, independent of the reasons, the results suggest that for the conditions the plants experienced in the field, AMF inoculation was not critical for survival. Objective 3. In previous years, we have isolated three dark septate fungi from A. tridentata roots; Helminthosporium tilliae, Montagnula sp., and Cadophora sp. Furthermore, re-synthesis experiments in vitro indicated that they are root endophytes. We have maintained these fungi during the past year and isolated two more that are currently identifying and testing. In addition, we implemented a faster method using transformed carrot roots to initially screen for endophytes. However, the main plan under objective 3 was to analyze whether inoculation with the isolated dark septate endophytes affects plant growth and AMF colonization. Unfortunately, the laborious nature of this work and limited access to the lab due to COVID restrictions prevented us from conducting the experiments related to this aspect of the project. An extension was requested and granted to complete this work.
Publications
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Progress 04/15/19 to 04/14/20
Outputs
Target Audience:1. Students that worked on the project on a routine basis 2. Student volunteers that helped with planting activities in the greenhouse and in the field 3. Community volunteers that assisted with field transplanting 4. Land managers from the Bureau of Land Management and US Forest Service 5. Researchers and studentsattending two meetings were results of the project were presented Changes/Problems:No significant changes were made to the project. The greenhouse experiment was changed from a completely randomized design to a completely randomized design with replications within blocks. This change allowed us to make the experiment more manageable and ultimately have better measurements of plant responses to drought and more replications. Still, the treatments and objectives are the same as those described in the proposal. What opportunities for training and professional development has the project provided?Two graduate students (Mathew Geisler and Craig Carpenter) and four undergraduates (Erika Petzinger, Sierra Moiza, Nichole Hoffman, and Bailie Sirhall) received training related to various aspects of this project. This training involved guiding the students through multiple experimental procedures. Such procedures included measuring plant water potential and photosynthesis, inoculating seedlings with AMF and DSE, assessing AMF and DSE colonization, cultivating A. tridentata seedlings in vitro, and isolating and culturing DSE fungi. The PI and Co-PI guided the students until they could work independently. The PI also assisted the students with data analysis, the preparation of poster and oral presentations, and the editing of a thesis. In addition, in consultation with colleagues and through individual study and experimentation, we have established improved and non-destructive methods to detect mild water deficits in big sagebrush seedlings. For our experiments, this is particularly important because fungal symbioses appear to be most beneficial to the seedlings during these periods. How have the results been disseminated to communities of interest?Based on the results of the project, thegraduate students made two presentations at the 2019 Mycological Society of America Meeting, while two undergraduates presented their work at a local meeting. In addition, volunteers from the Bureau of Land Management (BLM), Fish and Game, and Boise State students helped us with the field transplanting. We explained the project to these volunteers and to BLM land managers in various informal meetings. What do you plan to do during the next reporting period to accomplish the goals?For the first objective of the project, we have two primary goals. One is to submit a publication based on the thesis that one of my students already completed. This work describes interactions between Darksidea sp. and AMF in big sagebrush roots. The second goal is to complete the greenhouse experiment, which at this point, involves conducting two more replications of the three trials already completed. We hope these replications will help to ascertain the findings thus far obtained on the effects of AMF and Darksidea sp. on seedling responses to drought. Concerning the second objective, we will continue with the field experiment. This involves analyzing fungal colonization at the beginning of summer and fall and measuring survival until the end of the project period. During the summer, we will also measure stomatal conductance and plant water potential to determine whether the responses to fungal inoculation observed in the greenhouse occur under field conditions. A graduate student is currently conducting the greenhouse and field experiments and writing his thesis based on this work. The plan is to submit at least one manuscript from his research. To complete the third objective of the proposal, we will focus our efforts on determining the effect of the isolated DSE on AMF colonization.
Impacts
What was accomplished under these goals?
The shrubArtemisia tridentata(big sagebrush) plays a critical role in semiarid habitats of western North America by providing habitat and forage for local animals, which depend on big sagebrush for most of their winter diet. Over the past century, invasion by annual grasses and an increase in the frequency of wildfires have resulted in widespread loss of big sagebrush. Attempts to reestablish this shrub have usually failed due to high seedling mortality, particularly during summer drought. The ability of plant roots to extract water and nutrients from the soil can improve via associations with soil fungi such as arbuscular mycorrhizae (AMF) and dark septate endophytes (DSE). We have isolated and grown some of these fungi and subsequently used them to inoculate big sagebrush seedlings. Under greenhouse conditions, we have also tested the effect of AMF and DSE on seedling growth and drought tolerance. Growth of the seedlings was not affected by these fungi, but colonization by AMF increased drought tolerance. As the drought developed, AMF-inoculated seedlings maintained photosynthesis and survived longer than control plants. We are currently conducting additional experiments to determine whether similar effects occur in the field. Also, we are testing other dark septate endophytes for their impact on sagebrush growth and drought tolerance. Identification of sagebrush-fungal associations that improve plant survival would lead to more effective practices to reestablish this shrub. These practices would, in turn, help to maintain biodiversity and enhance the ecological services that the sagebrush steppe provides. Objective 1: We completed greenhouse experiments aimed at analyzing the effect of native AMF and the DSE fungusDarksideasp. on drought tolerance ofsagebrushseedlings. The four inoculation treatments were seedlings without inoculum, or seedlings inoculated with AMF,Darksidea, or both of these fungi. We evaluated the effects of these treatments on seedling responses to drought through daily measurements of stomatal conductance, photosynthesis, transpiration, and chlorophyll fluorescence. Fungal colonization was negligible in non-inoculated seedlings, (20 ±5)% in Darksidea inoculated seedlings, and 50 (±7)% in AMF-inoculated seedlings. Either before or during the drought, Darksidea colonization did not affect the photosynthetic parameters measured. This response differed from that observed with AMF. Under well-watered conditions, photosynthesis and transpiration were similar in -AMF and +AMF seedlings. However, as the drought developed, the rates of photosynthesis and stomatal conductance declined sooner in -AMF than +AMF seedlings. The combined results from three reiterations of the experiment indicate that in non-inoculated seedlings, the decrease in stomatal conductance and photosynthesis occurred two days earlier than in AMF-inoculated seedlings, this represents about 15% of the drought duration. After withholding water, we also measured chlorophyll fluorescence parameters. These parameters showed less sensitivity to drought than stomatal conductance. Evident changes in chlorophyll fluorescence occurred approximately six days after the decrease in stomatal conductance. This decline was associated with plant death and happened sooner in control than AMF-inoculated seedlings.One factor that affected the response to AMF was the initial value of photosynthesis.When the analysis was limited to seedlings with initial photosynthetic rates above 10 µmol CO2m-2s-1, the effect of AMF on postponing drought symptoms doubled. Also, the response to AMF colonization was affected by the rate at which the drought developed. The AMF-induced delay in drought symptoms was most marked in the trial when drought developed more gradually due to milder greenhouse conditions. It is likely that in thefield, the development of drought is more gradual than in potted plants due to the larger volume of soil in the former. Under this scenario, AMF would tend to maintain photosynthesis and survival for a longer period than that observed in the greenhouse experiments. Objective 2: To accomplish this objective, we started a field experiment in October 2018. However, at this time, the seedlings have not reached adequate levels of colonization, and we implemented an alternative inoculation protocol. Unfortunately, this protocol resulted in only small differences in colonization between control and inoculated seedlings. Furthermore, the spring of 2019 was unusually wet, and mortality due to summer drought was very low. As of April 2020, control and inoculated seedlings had similar levels of survival of 77.5 (±5) and 85 (±6.5) %, respectively. Thus, neither the treatments nor weather conditions allowed us to test the effect of fungal inoculation on seedling drought tolerance adequately. In an attempt to gain more knowledge about the effect of fungal inoculation on seedling drought tolerance and survival, we started two new field experiments. For these experiments, we used the inoculation protocols described in the proposal, but also experimented with a new approach to increase colonization. This work resulted in a publication in the Journal of Rangeland Ecology and Management. For the first field experiment, the seedlings were transplanted to the field in April 2019. We carried out this experiment with a small number of seedlings, about 25 per treatment; the aim was ascertaining whether seedlings inoculated in the greenhouse maintained a higher level of fungal colonization than control seedlings after three months in the field. Overall this was the case, particularly for AMF. After three months in the field, AMF colonization in control and AMF inoculated seedlings was 9 (±5) and 44 (±18)%, respectively (p= 0.001). The second experiment is aimed at analyzing the effect of fungal colonization on seedlings survival. After achieving adequate levels of colonization in the greenhouse, seedlings were transplanted to the field at Kuna butte, ID (43° 26' 47.32" N, 116° 26' 48.61" W) in October of 2019, fall transplanting is the typical transplanting time for big sagebrush seedlings. One hundred and fifty seedlings were planted for each of the four treatments. As of April 2020, only five plants have died with no difference in survival between treatments. Measurements of survival will continue until April 2021, and colonization will be measured in early summer and early fall to determine whether the initial differences in fungal colonization between control and inoculated seedlings persist in the field. Objective 3: We continued isolating and culturing DSE fungi fromArtemisia tridentataroots. However, the main effort was to conduct re-synthesis experiments with the fungi isolated and identified during the first year of the project. This work involved growingA. tridentantaseedlings in vitro and inoculating them with each of the three DSE previously identified. The three fungi colonized sagebrush roots without apparent adverse effects on the seedlings; the growth of inoculated seedlingsin vitrowas similar to that of non-inoculated controls. We also conducted anatomical analyses to ascertain that the fungi penetrated the roots, The three fungi tested,Helminthosporium tilliae, Montagnulasp., andCadophorasp. were mainly present in epidermal and cortical cells. Their localization in the cortex indicates that they are root endophytes. After inoculation in vitro, we transplanted seedlings to an autoclaved soil: sand mix to determine the extent the symbiosis continues in a more natural substrate. Two of the fungi tested,Montagnulasp., andCadophorasp., maintained adequate colonization rates of about 20%. Experiments are currently in progress to determine whether colonization by these two fungi affects AMF colonization.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Serpe MD, Thompson A, Petzinger E (2020) Effects of a companion plant on the formation of mycorrhizal propagules in Artemisia tridentata seedlings. Rangeland Ecology & Management 73: 138-146
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2020
Citation:
A symbiosis between a dark septate fungus, an arbuscular mycorrhiza, and two plants native to the sagebrush steppe.
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Progress 04/15/18 to 04/14/19
Outputs
Target Audience:The target audience included the following: Students that worked on the project on a routine basis Student volunteers that helped with planting activities in the greenhouse and in the field Community volunteers that assisted with field transplanting Land managers from the Bureau of Land Management Students and researchers attending two meetings were results of the project were presented Changes/Problems:No major changes were made to the project other than the method used to inoculate the fileld-transplated seedlings. As indicated earlier, if this method is not adequate to increase colonization over the background levels naturally occurring in the soil, the experiment will be repeated with greenhouse inoculated seedlings. What opportunities for training and professional development has the project provided?Training activities: Two graduate students (Mathew Geisler and Craig Carpenter) and four undergraduates (Adam Thompson, Erika Petzinger, Jenna Bishop, and Noah Poulin) received training related to various aspects of this project. The PI and Co-PI guided thestudents through various procedures until they could work independently. These procedures included methods forassessing AMF and DSE colonization, isolation of AMF spores, inoculation of seedlings with AMF and DSE, cultivation ofA. tridentataseedlings in vitro and in soil, isolation and culture of DSE fungi, DNA extraction, PCR, and taxonomic identification of fungi based on DNA sequences.In addition, graduate students supervised the work of undergraduates. The students were also assisted in the analysis of data and the preparation of posters that they presented at a conference for undergraduate research. How have the results been disseminated to communities of interest?Some of the work was presented at a research conference for undergraduates. Most of the attendees at this conference were not aware of the ecological value of A. tridentata or of the variety of symbioses that this plant forms with fungal endophytes. Similarly, volunteers from the Bureau of Land Management, Fish and Game, and Boise State students helped us with the field transplanting. An overview of the project was presented to these volunteers. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to finish the greenhouse experiment aimed at assessing the effects of AMF and Darksidea on plant drought tolerance.In addition, we will continue with the field experiment. We hope that the method used for inoculation in the field proves adequate to increase colonization over the background levels naturally occurring in the soil. However, if this is not the case, a new transplanting experiment is planned. Presently, we are growing and inoculating A. tridentata seedlings in the greenhouse that could be used for such experiment. With respect to the third goal of the proposal, we will attempt to isolate other DSE from sagebrush roots. However, the main work will be on characterizing the nature of the DSE-sagebrush symbiosis for the fungi that we have already isolated and the extent that colonization by these fungi affect AMF colonization.
Impacts
What was accomplished under these goals?
The shrub Artemisia tridentata (big sagebrush) plays a critical role in semiarid habitats of western North America by providing habitat and forage for local animals including greater sage grouse and pronghorn, which depend on big sagebrush for most of their winter diet. Over the past century, invasion by annual grasses and an increase in the frequency of wildfires have resulted in widespread loss of big sagebrush. Attempts to reestablish this shrub in burned habitats have usually failed due to high seedling mortality, particularly during summer drought. The ability of plant roots to extract water and nutrients from the soil can be enhanced by the formation of beneficial association with certain soil fungi such as arbuscular mycorrhizae and dark septate endophytes. In this project, we have first grown some of these fungi and subsequently used them to inoculate sagebrush seedlings. Currently, we are conducting experiments to determine the extent to which plant growth and drought tolerance is affected by the inoculation treatments. Identification of sagebrush-fungal associations that improve plant survival under drought would readily lead to more effective practices to reestablish this shrub. This, in turn, would help to maintain biodiversity and enhance the ecological services that the sagebrush steppe provides. Objective 1:To compare drought tolerance among seedlings under the following root fungal endophyte treatments: control seedlings (without root fungal endophytes), seedlings colonized by a DSE within theDarksideagenus, seedlings colonized by a mixture of native AMF, and seedlings colonized by both theDarksideasp and the native AMF. Major activities completed toward this goal included growing Darksidea sp. and AMF, growing sagebrush seedlings, and inoculating seedlings with these fungi. After inoculation, the first part of the experiment involved cultivating plants under well-watered conditions for about four months and then analyzing AMF and Darksidea colonization of roots as well as plant biomass. The results of these analyses are summarized in Table 1. Table 1. Percent colonization of Artermisia tridentata roots by AMF and Darksidea sp and total dry biomass under the different inoculation treatments. Mean (± se) of six replications. Treatment AMF colonization DSE colonization Biomass (g) Control 0. 3 ± (0.3)b* 3.0 ± (2.1)b 1.13 ± (0.03)a AMF inoculated 51.0 ± (6.8)a 0.3 ± (0.3)b 1.15 ± (0.03)a Darksidea inoculated 0.0 ± (0.0)b 29.0 ± (4.5)a 1.16 ± (0.03)a AMF + Darksidea inoculated 59.8 ± (4.7)a 22.7 ± (4.4)a 1.15 ± (0.02)a *Within a column values followed by different letters are significantly different (p < 0.01) The results indicate that inoculation with DSE did not affect AMF colonization. Similarly, inoculation with AMF did not affect DSE colonization. In addition, the values of biomass were virtually identical among the treatments. An important outcome of these analyses was the finding that AMF and Darksidea sp. do not have a negative effect on each other. The results also indicate that the association between Darksidea sp. and A. tridentata is of a commensalistic nature. However, the small size of the cone-tainers (150 ml) may have limited the ability of both Darksidea sp. and AMF to have a positive effect on growth. Plants growing initially on cone-tainers have been transplanted to 1 m long PVC pots and are still growing under well-watered conditions. The plan is to start the drought treatment within the next month. Consequently, we do not know yet the effect of the inoculation treatments on seedling drought tolerance. Obective 2: To increase the background levels of DSE and AMF colonization naturally occurring in sagebrush seedlings and assess the effect of these increases on seedling survival under field conditions. To accomplish this goal, the plan was to transplant seedlings to the field in early fall, which is the most common time for outplanting of A. tridentata seedlings. However, at this time, the A. tridentata seedlings grown in the greenhouse were too small for field transplanting and had not reached adequate levels of either AMF or Darksidea colonization. Nevertheless, the Bureau of Land Management provided us with seedlings that have been grown in a local nursery. With these seedling, we initiated an experiment but with changes in the inoculation procedure. Rather than inoculating seedlings in the greenhouse, AMF and/or Darksidea inoculum was placed beneath the root ball of some of the transplanted seedlings, those that had been randomly assigned to either AMF, Darksidea, or the AMF plus Darksidea treatment. Transplanting occurred during Oct 2018 at Kuna butte, ID (43° 26' 47.32" N, 116° 26' 48.61" W) and two hundred seedlings were transplanted per treatment. From October to mid-February, when survival was last measured, seedling mortality has been minimal . We will continue the monthly measurements of survival and in early spring we will also add more AMF and Darksidea inoculum. This inoculum will be placed at about 20 cm from the soil surface via PVC pipes that had been installed next to each seedling. Colonization will be measured in early summer and early fall to determine whether the added inoculum increased colonization over the background levels naturally occurring in the soil. Objective 3: To isolate and identify other DSE from sagebrush seedlings and test their effects on AMF colonization and seedling growth with the ultimate aim of identifying DSEs that form mutualistic symbioses with the seedlings. Major activities completed toward this goal included isolating dark septate fungi (DSE) from field collected A. tridentata roots, culturing these fungi, and subsequently identifying them using molecular methods. Initially, root fragments were surface-sterilized and incubated in water agar. A somewhat surprising observation was the high proportion of root fragments (~70%) showing hyphae growing from their cut ends. This suggests that fungal root endophytes are ubiquitous in field grown A. tridentata seedlings. The hyphae growing from the cut ends were then transferred to PDA medium, about 100 plates were prepared at this stage. Following growth, the fungi were examined for the presence of melanized and septate hyphae, which are typical morphological characteristics of DSE fungi. Many of the isolates did not have septate hyphae and were discarded. From those showing typical DSE characteristics, DNA was extracted, and the fungi identified to the genus level based on sequences from the ITS and LSU regions of ribosomal RNA genes. Several of the plates have the same fungus and none of them was Darksidea. The three DSE that we isolated and identified are listed in Table 1. Currently, we are conducting re-synthesis experiments withA. tridentata and each of the isolated DSE to ascertain that these fungi are root endophytes and do not have pathogenic effects. Species Family Order Helminthosporium tilliae Massarinaceae Pleosporales Montagnula sp. Montagnulaceae Pleosporales Cadophora sp. Incertae sedis Helotiales
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