Alan Chambers and Dr. Brad Geary, Plant and Animal Sciences
Introduction
Endophyte infected, drought resistant crops are gaining in popularity with growers because irrigation can be expensive and rainfall unpredictably sparse. For this reason there has been a recent explosion of research relating to the benefits of plant microbe interactions. Our investigations were focused on fungal endophytes that produce alkaloid compounds which confer drought resistance in forage grasses.
Progress until Problems
Early this year we were able to positively identify the presence of Erogvaline in seven of our grass varieties. We had isolated endophytes from all 12 different grasses and were working on creating endophyte free lines using Tebuconizole. The drought project had some unexpected difficulties after a few months of growing and checking for endophyte infection. At the end of May we lost nearly all of our germplasm on short notice, except for a salt tolerant bluegrass and MaxQ, for proprietary reasons. With the loss of germplasm we also lost the data that would have been collected relating to generating endophyte free lines – and we had to start again.
As of the beginning of fall semester we have built up our germplasm based on diversity. We now have 15 varieties of different grasses from GRIN and other sources. Festuca arizonica (United States), Festuca rubra subsp. Fallax (Netherlands and New Zealand), Festuca rubra (Afghanistan), Brachypodium distachyon (Iraq), Festuca arundinacea (Morocco, 2 varieties), Agrostis clavata (Mongolia), and Lolium perenne (Russian Federation) were all obtained from GRIM germplasm resources. Our goal was to obtain a broad array of grasses and hopefully endophytes. MaxQ and a drought tolerant tall fescue were purchased, and salt tolerant bluegrass was collected as an environmental sample. Kentucky 31 E+ and E-, Hi Loline, and Matrix were donated for incorporation into our research.
Current Progress
We currently have healthy supplies of growing grasses. Fungal endophytes have been positively identified by staining in at least eight varieties, and the absence of fungal endophyte has been confirmed in Kentucky 31 E-. We are still investigating the remaining grasses where no endophytes were found by preliminary staining. Leaf sheath isolations have produced pure lines of fungal endophytes from nine different grasses and this work is continuing as well. We have learned that staining alone is insufficient to provide information about the presence of fungal endophytes so both methods are useful.
We are using either heat or various fungicides to create endophyte free lines. We are currently testing these methods for potential to eradicate fungal endophytes. Bayleton and Propiconizole were provided by Bayer and BASF and were applied at 42 g ai per 93m2 (Siegel, 1984) and 11 kg ai per ha (Hill, 1990) respectively. Applications will be made a second time two weeks after the initial application and new tillers will be examined for infection after 5 weeks.
We have also obtained the use of a pressure bomb, chlorophyll fluorescence meter, and a CO2 gas/transpiration analyzer. We will spend more time becoming proficient at using our machines over the next few weeks while our plants are growing.
Future Work and Goals
We will run alkaloid extractions on all 13 varieties before the end of November this year. Protocols for Loline extraction and troubleshooting are being provided by the University of Kentucky from Chris Schardl’s lab. It is anticipated that this round of Loline alkaloid extractions will be cleaner than the first and simplified by our past experience. The Ergots will also be extracted and it is anticipated that we will have few problems based on the successes we have also already had with this class of alkaloid. Peramine will be confirmed by TLC, but not quantified due to the costs of buying a new, special column for the GC.
Future Timeline
The endophyte free lines should be ready for use as a control group against all 13 varieties by the end of this year. The drought program will then continue as outlined in the project proposal, and most likely without recurrent problems. We expect data collection during drought conditions to start in January of 2007 and this initial trial will wind up from late February until the end of March. The data should be finished and analyzed in time to submit for presentation at the annual APS meetings in the summer of 2007.
Conclusion
We have had many successes even though we have not completed many of our initial research goals by the original timeline. There has been great value, though, in learning what it is like to have a project reset, yet continued. Once again, this fulfills the purpose of the ORCA grants to provide an experience beyond what can be done in the classroom alone. I have never attended a class where all the scores were reset after a few months and we had to begin again.
I would also like to report that I was able to present research funded by a previous ORCA grant at the annual APS meeting this year in Quebec City, Canada. I was fortunate to be able to give an oral presentation on the diversity of our endophytes. It was extremely rewarding to attend the meetings, and also to have the opportunity of sharing the information that we have generated. Many other attendees were impressed that BYU had such a strong mentoring program for undergraduates. Of course, this is nothing new to us.