Alan Chambers and Dr. Brad Geary, Plant and Animal Sciences

Plant microbe interactions have been shown to enhance plant growth (Nassar, 2005) and it is well established that fungal endophytes affect beneficial phenotypes like resistance to drought or insects (Bacon 1997). Plants have been selected for beneficial phenotypic traits for thousands of years, but selection for beneficial microbe interactions is under studied. We desire to investigate improving crop yield and quality, while minimizing the expenditure of natural resources, by growing maize artificially infected with fungal endophytes from plants adapted to stressful environments. We report our early success here.

Endophytes from Great Basin Wild Rye (Elymus cinereus) (WR) and Fourwing saltbush (Atriplex canescens) are of interest because of their host’s general stress tolerance. Callus was initiated on Murishige and Skoog media with 2 mg/L of 2,4-D. Callus induction was slow; taking months to establish stock cultures large enough for experimental trials. Inoculation events were conducted by placing a ~5 mm piece of callus tissue directly on the radical of recently germinated maize seed growing aseptically on MS hormone-free media. Genetic analysis was performed on DNA extracted from two month old plants growing in a greenhouse. SEM data was collected 5-7 days after initial inoculation.

Our initial attempt to use AFLP’s to show the transfer of endophytes into maize failed. We therefore decided to use simple PCR methods. Amplifying fungal specific ITS regions will allow future research to quickly identify transferred endophytes. To date, we have found that something fungal in nature from WR does transfer to maize roots (fig 1); confirming visual assessment (fig 2).

The DNA for lane 2 was from control plant roots. There was no fungal endophyte picked up by our primer. The DNA for lane 3 was from our maize inoculated with WR callus. This band agrees with previous data for the expected fragment coming from WR fungal endophytes. This asymptomatic association is maintained for at least 2 months in greenhouse conditions.

Figure 2 shows the phenotypic difference that initially intrigued our lab. DNA evidence suggests that this result could be caused by an endophyte transfer.

The SEM data shows an incredibly close and extensive relationship between maize and an endophyte from WR (data not shown). The association seems symptomless as there are no visible stress exhibited by experimental groups. Figure 3 shows a fungal infection structure and the difference in size between root hairs, secondary roots, and the fungal mycelium.

This project was started de novo this year and we are confident that much more progress will be made before the end of this year. We have shown a fungal association with maize roots originating from WR. Future work will include sequence results identifying our fungal endophytes, and hopefully evidence about the nature of internal colonization.

References

• Bacon C, Richardson M, White J (1997) Modification and uses of endophyte-enhanced turfgrasses: A role for molecular technology. Crop Science 37:1415-1425
• Nassar A, El-Tarabily K, Sivasithamparam (2005) Promotion of plant growth by an auxin-producing isolate of the yeast Williopsis saturnus endophytic in maize (Zea mays L.) roots. Biology and Fertility of Soils 42:97-108