Proceeding of The National Conference On Undergraduate Research (NCUR) 2002 University of Wisconsin-Whitewater Whitewater, Wisconsin April 25-27, 2002

Enhanced Tree Fern Prothalli Formation and Development Using Soil Mycorrhiza
1

Rhonda Riddle, 1 Dr. Nancy Brooker, and 2 Dr. David Pruitt 1 Department of Biology Pittsburg State University 1701 S. Broadway Pittsburg, Kansas, 66762 2 Hot Springs, Arkansas Faculty Advisor: Dr. Nancy Brooker Abstract

Dicksonia antartica is a tree fern native to Australia. Previous studies on ferns have shown that some have unique relationships with fungi. It has been hypothesized that these relationships include mycorrhizal interactions. To test this hypothesis, a mycorrhizal fungal culture was isolated from soil samples taken from around the roots of a vigorously growing Christmas tree fern (Polystichum acrostichoides). This fungal culture (Trichoderma sp.) was isolated and used as inoculum to examine fungal dosage effects on the formation and development of tree fern prothalli. Fungal spore dosages ranging from 0 to 1 x 106 spores/ml were used in combination with 1 x 103 fern spores per treatment. Prothalli number and size was monitored bi-monthly, and the experiment was repeated in duplicate. Results of these studies indicate that fungal co-culturing increases prothalli development by as much as 91% as compared with the fungus-free control treatment. In addition, the fungal co-cultured treatments exhibited denser growth with larger size and development. This data suggest that there is a synergistic relationship between D. antartica and the mycorrhizal isolate that reaches an optimum rate at 1 x 103 fungal sp/ml. Keywords: 1.Prothalli, 2. Mycorrhiza, 3. Dicksonia.

1. Introduction
Dicksonia antartica is a slow growing tree fern native to Australia and New Zealand. D. antartica can have a life span of over 400 years, growing 1 to 3 cm a year, and it is capable of reaching heights of 20 to 50 feet with fronds commonly about 8 to 10 feet in length [1]. Many of these tree ferns are now being imported into the United States and Europe as ornamental plants. D. antartica is known for its ease of cultivation and great beauty, making it one of the best known and most sought after tree ferns around the world [2]. Previous studies have reported that some ferns have displayed unique relationships with fungi, including endophytic and parasitic [3]. One relationship seldom mentioned is that of mycorrhizal fungi in the rhizosphere of the plant. In order to examine the possibility of tree fern mycorrhizal relationships, this study was undertaken to explore the rhizosphere flora and diversity, and to study its impact on tree fern growth and development. One group of fungi that has been found to be active in the rhizosphere of plants is the genus Trichoderma [4]. Trichoderma fungi have been used to protect the rhizosphere of plants and previous studies have shown that these fungi provide competition against pathogenic fungi also found in the rhizosphere of the plant [4]. As a result of these biological control studies, commercialization of Trichoderma seed treatments now offer a novel and natural approach to controlling soil borne diseases [5].

This study used a Trichoderma fungal isolate to explore the impact of this soil borne mycorrhizae on D. antarticas early growth and development.

2. Materials & Methods 2.1. tree fern spores and quantification

Dicksonia antartica spores were obtained from Dr. David Pruitts private collection. A preliminary trial was conducted to determine the optimum amount of tree fern spores to use in subsequent experiments. This trial used varying orders of magnitude concentrations of tree fern spores ranging from 0 to1 x 105 spores/ml quantified by hemacytometer counts. These fern spores were applied to fern culturing media and monitored for growth over a 4 month period. Results indicated that a spore concentration of 1 x 103 sp/ml produced the most uniform, uncrowded conditions for cultivating D. antartica in vitro, and this concentration would be best suited for the remainder of the experiments.

2.2. fungal isolation and culturing

The fungal isolate used to inoculate each treatment was taken from soil samples surrounding the roots of vigorously growing crowns of Christmas ferns (Polystichum acrostichoides). This fungal culture was purified as a single spore isolate and grown on half strength Potato Dextrose Agar (PDA) plates until a dense surface layer of spore growth was achieved. Using sterile water, the fungal spores were carefully scraped off of the plate, placed in a sterile water solution, and quantified using a hemacytometer. Once the quantity of spores was established, a series of dilutions were made in order to make a range of fungal dosage concentrations ranging from 1 x 101 to 1 x 106 fungal spores per milliliter. The fungal isolate was also grown on Cellulose Asparagine (CA) medium to aid in taxonomic identification. The fungal isolate was able to grow using cellulose as its sole carbon source. This evidence, along with a wet-mount slide of the isolate distinguishing the structure of the fungal spore, classified the fungus as a Trichoderma species. In order to verify that this Trichoderma did colonize the developing tree ferns, soil samples were taken from the treatments with optimum growth and the fungus was re-isolated using serial dilutions. These re-isolates were grown on the CA medium, and these fungi were also able to use cellulose as their sole carbon source. These fungal cultures were identified as the same Trichoderma species originally used as inoculum in this study.

2.3. fern culturing

Soil medium used for each treatment consisted of one part perlite and two parts peat moss mixed together and damp ed with a minimal volume of water and then separated into 400 ml beakers. No unbound water collected in the bottom of the beakers. The amount of soil in each beaker was enough to make each sample roughly 1 1/2 to 2 inches deep when loosely compacted. Compacting each soil sample allowed for a flat, level surface and even distribution of spores that also aided in quantifying the fern growth. The beakers were then sealed with foil, autoclaved, and kept sterile until inoculated. One ml of D. antartica was added to 4 mls of sterile water and was used to inoculate each beaker of soil. The inoculum was sprayed onto the surface of the culture media using a DeVilbiss spray unit and pressured air at a spray rate of 1 ml/second. Each beaker also received one of the fungal spore concentration treatments sprayed in 5 ml aliquots on the surface of the growing media. The beakers were then covered with clear plastic wrap and placed under a 12 hour a day fluorescent grow light and monitored bi-monthly for growth. This experiment was conducted in January of 2001 and was repeated again in May of 2001. In each experiment, fresh soil mix and new solutions of the tree fern and fungal isolate were prepared, however the same batch of D. antartica spores were used in each trial.

3. Results
In Experiment 1, the first growth of prothalli was noted 10 weeks post inoculation. Experiment 2 displayed first growth of prothalli after 10 weeks. Differences in coloration of prothalli between the fungus-free control group and the co-cultured treatment groups were immediately noticeable. Prothalli in the fungus-free control groups were a light green-yellow, almost translucent, whereas, in treatments co-cultured with fungi, most prothalli appeared a

rich, dark green. In addition, each co-cultured treatment showed an increased life span when compared to the fungus-free control group. After 7 months of growth, the fungus-free control group began to senesce and die. Over a year later, the tree ferns in the co-cultured groups still continue to grow and thrive. Co-cultured treatments also yielded prothalli about 2 times the size of those in the control group. The quantity of prothalli in the co-cultured groups with higher fungal dosages (1 x 104 - 1 x 106 sp/ml) averaged 75% greater in number and size than the amount of prothalli in the fungus-free control group. Co-cultured treatments yielded prothalli about 2 times the size of those in the control group. In addition, the quantity of prothalli in the co-cultured groups with lower dosage (1 x 101 to 1 x 103 sp/ml) treatments are on the average 86% greater in number than the amount of prothalli in the fungus-free control group, and 56% greater in number than the higher concentration co-cultured treatments (1 x 104 1 x 106 sp/ml). As seen in Figure 1, prothalli growing in the fungal spore concentrations 1 x 101 to 1 x103 are 3 to 4 times larger than prothalli in the fungus free control group. The lower spore concentrations yielded prothalli about twice the size as compared to prothalli in fungal spore concentrations of 1 x 104 to 1 x 106 sp/ml. Table 1 summarizes the quantity and size of prothalli from each cocultured treatment.

Figure 1. Prothalli growth and development in fungus-free cultures (0 sp/ml) and in Trichoderma fungal co-culture (1x103 sp/ml).

Table 1. Density and size of fungal dosage treatments on prothalli growth and development. Fungal Dosage (sp/ml) Experiment #1: Experiment #2: Average Quantity of D. antartica prothalli D. antartica prothalli D. antartica prothalli Quantity Avg size (mm) Quantity Avg size (mm) Control: 0 24 2-4 0 0 12 1 x 101 57 5-7 15 5-7 36 1 x 102 177 7-12 0 0 88.5 1 x 103 119 10-15 155 7-12 137 1 x 104 34 4-7 25 4-7 29.5 1 x 105 93 4-7 24 4-7 58.5 1 x 106 86 4-7 30 4-7 58 Table 2 is a histogram showing the optimal dosages of fungal spores for prothalli germination. The lower concentration set, specifically at the 1 x 103 sp/ml treatment, appears to be the best treatment for prothalli germination. Overall, the optimal fungal concentrations are the lower ones, ranging from 1 x 101 to 1 x 103 sp/ml. This group of lower fungal spore concentrations showed increased germination rates, and overall larger prothalli than the fungus-free control group. The higher fungal spore concentration treatments, ranging from 1 x 104 to 1 x 106 sp/ml, showed improved germination rates and improved development when compared to the fungus-free control set. However, the least effective fungal spore concentration was the 1 x 104 sp/ml treatment with only a 59% increase in the prothalli germination as compared with the fungus-free control.

Table 2 Dosage effects of Trichoderma on D. antartica

mean germination value

150 100 50 0 0 2 log (dose) 4 6 mean

4. Discussion
In comparison to the control groups of each experiment, fungal dosages resulted in improved formation and development of the tree fern prothalli. The higher concentration set of fungal dosages of 1 x 104 to 1 x 106 sp/ml showed increase prothalli quantity ranging from 59 - 79% increases and these treatments also increased the size of prothalli compared to the fungus-free control group. The lower fungal concentration set ranging from 1 x 101 to 1 x 103 sp/ml increased prothalli germination by 67 - 91%. Lower fungal concentrations also resulted in the most improved formation and development of D. antartica prothalli. The optimal fungal dosage for prothalli germination and development occurred at 1 x 103 sp/ml, resulting in over 11 times the amount of growth seen in the fungus-free

control. The next optimal dose was 1 x 102 sp/ml, which resulted in about 7 times the amount of growth as in the fungus-free control dose. This would suggest that there is something provided from the Trichoderma culture that at lower dosages improves the tree fern prothalli growth and development. With all treatment data combined, the co-cultured treatments averaged prothalli roughly 3 times larger than the prothalli in the fungus-free control group. In general, the higher fungal spore concentrations did not promote prothalli growth and development as well as lower fungal spore concentrations. From these findings, they suggest that whatever substance(s) is promoting growth by co-culturing the fern spores with fungi, may have dosage dependent characteristics. The data also suggests that at high concentrations the fungal derived substance(s) may become inhibitory for prothalli growth and development. Results show that the least effective fungal dose occurred at 1 x 104 sp/ml. However, even though the higher spore concentrations (>104 sp/ml) did not promote prothalli growth and development as well as lower spore concentrations (<103 sp/ml), these fungal co-cultures still improved prothalli germination by twice the quantity and size as the fungus-free control. In order to determine the repeatability of these findings, this study was repeated 5 months after the initial study using the same D. antartica spawn as the first experiment. The 5 month age difference in the spawn inoculum may be the cause of the slightly lower germination results in the second experiment. This may also explain why there was not any germination in some of the treatments in this experiment. Despite these differences, the same coculturing enhancement phenomenon was duplicated in this second study. Results showed the same fungal dosage dependent responses in tree fern prothalli growth and development, with the optimal growth at lower fungal spore concentrations. Therefore, this study does indicate the importance of Trichoderma fungi for enhanced tree fern growth and development and the need for further research in this area. In order to further study the mechanism(s) of how this Trichoderma fungus enhances prothalli germination and development, several experiments, using the same fern and fungus inoculum will need to be conducted. This would give more precise results with a better basis of comparison between each fungal dosage group. It might also be possible to extract and purify the active agent(s) from Trichoderma cultures that enhances tree fern growth and development. This fungal derived compound could then be used to elucidate how the fungus promotes plant growth and development at the physiological level. In addition, this purified fungal compound would be of considerable interest to commercial tree fern growers. The results of this research impact several botanical and commercial areas. D. antartica is a highly valued tree fern and is desired in botanical gardens around the world. Mycorrhizal relationships improving the development and growth of the tree fern have the potential of increasing its profitability by enhancing growth and development of this rare and beautiful ornamental plant. In addition, this research has provided greater insight into the ecological, physiological, and developmental roles that fungi play in nature. As a result, one cannot underestimate the importance of these fungal plant relationships in nature and their impact on fern fitness and survival. Results of this study certainly merit further research of this unique fungal plant relationship.

5. Acknowledgements
The authors wish to express their appreciation to Dr. Ananda Jayawardhana, Assistant Professor of Mathematics, Dr. James Dawson, Professor of Biology, and Malcom Turner, University Communications, all of Pittsburg State University, and to Mr. Jim Croft of Australia who shared initial information about the tree fern, Dicksonia antartica.

6. References
[1] Tropical Gardner Encyclopedia: Dicksonia antartica. http://www.tropicalgardner.co.uk/encyc/ferns/dickant/dick-ant.htm. [2] Dicksonia antartica. http://www.angelfire.com/wa/margate/antartica.html. [3] Swatzell, Lucinda J., Powell, Martha J., and Kiss, John Z. 1996. The Relationship of Endophytic Fungi to the Gametophyte of the Fern Schizaea Pusilla. International Journal of Plant Science 157(1):53-62. [4] Wolffhechel, H. and Jensen D. 1992. Use of Trichoderma harzianum and Gliocladium virens for the Biological Control of Post-emergence Damping-off and Root Rot of Cucumbers Caused by Pythium ultimum. Journal of Phytopathology. 136 221-230. [5] Lorito, M. et al. 1993. Chitinolytic Enzymes Produced by Trichoderma harzianum: Antifungal Activity of Purified Endochitinase and Chitobiosidase. Molecular Plant Pathology 83(3):302-307.