A Study of the Moss Parasite Eocronartium muscicola By: Alicia Knudson Advisor: Dr. Elizabeth Frieders Abstract The genus Eocronartium contains a single described species of parasitic fungus on moss plants but character observations suggest that there may be more than one. Various specimens of moss from numerous geographic regions were examined for comparable features. Light microscopy was performed to identify shape and form differences of the fungus in different portions of the moss plant. Molecular sequencing was carried out on two genes in the fungal DNA to reveal genetic similarities. The fungus was found to inhabit two distinct sections of moss plants: the reproductive structure (sporophyte) and the vegetative body of the plant (gametophyte). These regions are dissimilar enough that the fungus would need to behave drastically different in each section. The two regions of DNA examined were used to build relationship trees to determine if there was enough of a genetic difference to consider the fungus two species. The fungi in the two regions were not found to have enough of a significant genetic difference to separate them into two species. Introduction Eocronartium muscicola, a fungal parasite of mosses, is the only described species in the genus, but it can be found in two distinctly different portions of the plant. A related genus of moss parasites (Jola) may also be found in different areas of the plant but this genus contains several different species. The different species exhibit morphological and genetic distinction. The objective was to examine the single Eocronartium species to determine if it was actually two distinct species. Dr. Frieders has done previous work with on the life cycles of both genera of moss parasites. Eocronartium is parasitic in over thirty different species of moss. The perennial photosynthetic portion of the moss plant is called the gametophyte. This leafy structure nutritionally supports a reproductive portion called the sporophyte with special transfer cells. The sporophyte is temporary and will produce spores. 1
Methods and Materials Fifteen specimens of Eocronartium (Eo) were examined; the specimens came from locations such as England, Finland, and the United States (Michigan, Wisconsin, Minnesota, Vermont, and New York). The specimens that were collected were entire moss plant with the fungal infection. Pieces of the moss plant were excised and placed within plastic micropipette tubes to undergo chemical staining that would dye any present fungal tissue. Chemical Staining: first included fixation in a 50% ethanol, 2% glutaraldehyde, and 4% formaldehyde solution. After being rinsed three times with a K 2 HPO 4 buffer (or sterilized water) the specimens were cleared using a 2.5% to 10% KOH solution and heated to 90 Celsius for 10 to 30 minutes. The samples were rinsed with sterilized water three times and a 3 ml mixture of 20% ammonium hydroxide in 3% H 2 O 2 was added. After rinsing with water three more times, 1% HCl was added to the tubes for 1-24 hours. A lactic acid fuchsia stain is lastly added to the solution. Light Microscopy: When removed from the stain solution, the pieces were placed, with a forceps, upon glass slides for initial light microscope observations. A % glycerol solution and a coverslip were added to each slide. Sketches and notes were made at three objectives of the microscope (4x, 10x, 40x). After notes were taken on intact pieces, the specimen was slightly smashed under the coverslip to separate fungal and 2
moss tissue. The specimens are observed to determine the area of moss plant infection (sporophyte or gametophyte) and how the fungal tissue behaved in the infected areas. DNA extraction and sequencing: Fruit bodies (fungal reproductive structures) were removed from the moss specimens and placed into plastic micropipette tubes. DNA was removed using the E.Z.N.A. Forensic DNA Kit #D-3591-02 (Omega Biotek). Nuclear-encoded ribosomal genes LSU (large ribosomal subunit) and ITS (internally transcribed spacer region) were chosen for analysis. ITS is a non-coding region of the fungal DNA and is highly variable between species. That region was most important for distinguishing genetic difference between species. Restriction enzymes For 25 µl JM's Cocktail Components reactions (per tube) dntps (datp, dctp,dgtp,dttps) 4 H 2 O 3.25 10X PCR buffer (15 mm MgCl 2 ) 2.5 BSA 2.5 Primer 1 1.25 Primer 2 1.25 Amplitaq (Taq Polymerase) 0.25 Total cocktail volume 15 DNA template (.1-1 ng/µl) 10 *PCR buffers/primers and amounts used for excision of desired genes varied depending on the lab that performed the sequencing. Polymerase Chain Reaction (PCR) was performed to increase the quantity of DNA from the specimens. The recipe (JM s cocktail) for the mixture used in the PCR amplification is in the table on the left. A 1% agarose gel was used to check if PCR operated correctly. For each specimen, 3 µl PCR DNA and 1 µl of loading dye were combined and placed into the agar wells. A ladder, control and negative control were also placed in separate wells of the gel. 3
Analysis of DNA product: Sequences were completed at the University of Wisconsin-Madison lab and imported into the software program DNA Sequencher. LSU and ITS sequences were aligned using this program. The arranged sequences from each specimen were compared using Phylogenetic Analysis Using Parsimony (PAUP). The program arranges the specimens by how closely related they are to one another in phylogenetic (relationship) trees. Specimens from one species will form a distinct cluster (clade); two species should yield two distinct clades, suggesting significant genetic difference. Results and Discussion Microscopy: Eo was found to infect both the sporophyte and gametophyte portion of the moss plant. In the sporophyte, the fungus will infect along the border of the transfer cells and travels up through the sporophyte to emerge out of the top and create a sheath around it. The fungus will stop the sporophyte development and will use the sporophyte as a support structure for its own fruit body. Infecting the sporophyte would seem the best option for the fungus. A constant nutrient flow is being shuttled up by the gametophyte transfer cells to support the sporophyte. The fungus is capable of halting sporophyte growth and using the nutrients for its own development. In the gametophyte, the fungus was found throughout the stem tissue but never crosses into sporophyte tissue. Fruit bodies were made in a mass of hyphae (thin strands of fungal tissue) at the apex of the stem. The gametophyte is a less desirable option for 4
the fungus because it does not contain a concentrated food source. But species of moss that have gametophyte infections (Climacium) have very large gametophytes (several times greater than most other species). This much larger gametophyte might be capable of supplying the fungus with the same amount of nutrients that it would receive in the sporophyte. The following table (below) displays the specimen number and portion infected by Eo. The picture illustrates the size difference between the moss species Climacium and another sample moss species. Specimen ID # of Fruit Bodies Examined Site of Infection CUP 167 3 gametophyte CUP 21609 1 sporophyte CUP 30976 3 gametophyte CUP 30980 1 sporophyte CUP 33828 1 sporophyte CUP 9056 1 gametophyte EMF 008 1 sporophyte EMF 018 2 sporophyte EMF 019 1 sporophyte KEW 78686 3 sporophyte KEW 84242 2 sporophyte MIN 795107 3 gametophyte MIN 795110 3 sporophyte MIN 795111 4 Sporophyte MIN 796433 5 Sporophyte *Specimen ID number and the type of infection identified. *Moss species Climacium (left) compared to other moss species. DNA Sequencing and Phylogenetics: The sixteen specimens yielded useable DNA and the DNA was aligned. Several phylogenetic trees were made using different methods of comparison. 5
For the different trees that were constructed using PAUP, they all failed to place the gametophyte-infecting specimens in a distinct cluster (clade). Several sporophyteinfecting specimens appeared between the gametophyte specimens. The phylogenetic trees below show the closeness of relation (lack of branching) and the gametophyte specimens (at the arrows). Majority rule 74 74 74 55 77 63 86 85 99 94 57 58 CUP 30976 KEW 84242 CUP 30980 Eo MIN796433 ITS5 Eo MIN795110 ITS Eo MIN795111 ITS MIN 796447 Eocronartium L20280 CUP 9056_ITS KEW 84240_ITS CUP 21609_ITS CUP 167_ITS CUP 33828 Eo_EMF008-4_ITS Eo Brazil Eo MIN795107 ITS KEW 78686 Eo-EMF 018 EMF 019 ITS Eocronartium 1 Eocronartium muscicola - Bryopsida ga Jola - DJM 739 Jola_EMF013-6A_its Jola EMF014 ITS Jola EMF015 ITS Jola_EMF013-1_its JD1990 ITS NYBG398 ITS Jola EMF012 ITS Jola_EMF012B-1_its Jola EMF006 ITS Jola hook EMF 017-1 Platygloea pustulata Platygloea disciformis Insolibasidium_183-2_ITS Vienna 3179 Herpobasidium filicinum Bootstrap 73 61 83 59 99 87 76 67 86 55 68 CUP 30976 KEW 84242 CUP 30980 Eo MIN796433 ITS5 Eo MIN795110 ITS Eo MIN795111 ITS Eocronartium 1 Eocronartium muscicola - Bryopsida gamet MIN 796447 Eocronartium L20280 CUP 9056_ITS KEW 84240_ITS CUP 21609_ITS Vienna 3179 KEW 78686 CUP 167_ITS CUP 33828 Eo-EMF 018 EMF 019 ITS Eo_EMF008-4_ITS Eo Brazil Eo MIN795107 ITS Jola - DJM 739 Jola_EMF013-6A_its Jola EMF014 ITS Jola EMF015 ITS Jola_EMF013-1_its JD1990 ITS NYBG398 ITS Jola EMF012 ITS Jola_EMF012B-1_its Jola EMF006 ITS Jola hook EMF 017-1 Platygloea pustulata Platygloea disciformis Insolibasidium_183-2_ITS Herpobasidium filicinum 6
*Above are two types of analysis of the specimen DNA. The gametophyte-infecting specimens are not found to be significantly genetically different from the other specimens. The specimens of Eocronartium that are found in the sporophyte do not form a separate clade from those found in the gametophyte. There is not a significant genetic difference in either gene (ITS, LSU) between the two morphologically distinct specimens. The only species is Eocronartium muscicola. The generated phylogenetic trees were the result of only two analyses. Additional analyses may yield a different arrangement of the specimens but would still not place the gametophyte specimens into a separate clade. Eocronartium muscicola has the ability to infect both portions of the moss plant. Infecting the sporophyte will give access to a continuous flow of nutrients from the gametophyte (intended to support sporophyte development). Species of moss with gametophyte infection (Climacium) are considerably larger than other species and the fungus may be able to derive a similar amount of nutrients from the much large gametophyte. 7
References: Boehm, E. W. A., and McLaughlin, D. J. (1988). Eocronartium muscicola: a basidiomycetous moss parasite exploiting gametophytic transfer cells. Can. J. Bot. 66 762-770. Fitzpatrick, H. M. (1918). The life history and parasitism of Eocronartium muscicola. Phytopathology. Vol. 8. 5. 197-219 Frieders, E.M. (1997). An integrated approach to understanding the moss parasites and their role in basidiomycete evolution. (thesis, University of Minnesota, 1997). 8