Characterizing potential growth promoting bacterial endophytes associated with Pinus contorta, Pinus ponderosa and Pinus nigra

Background/Question/Methods

Aboveground bacterial endophytes—symbionts inside healthy plant leaf and stem tissue—have been shown to fix nitrogen, promote plant growth and development, and protect against stress. To date, most studies on bacterial endophytes have focused on root endophytes and their benefits in agricultural plants. In contrast, the role and diversity of aboveground endophytes, in particular those in ecologically and economically important natural ecosystems such as forests, remain unknown. Initial studies suggest that Pinus sylvestris consistently hosts growth-promoting intracellular bacteria in their shoot meristem. Conifers tolerate harsh and nutrient poor sites, and often microbial symbionts accelerate adaptation for ecological success in limiting environments. A better understanding of these unexplored symbioses is imperative as they may influence whole ecosystem processes such as carbon and nitrogen cycling. Questions directing our work include: 1) Do additional Pinus species also consistently host certain bacterial species in their shoot tissues?  2) Can we culture bacteria from Pinus shoot tissues? To answer these questions we developed culture methods to maximize recovery of conifer shoot endophyte isolates. We identified isolates by sequencing full-length 16S rRNA genes. Shoot tissues were sampled from Pinus contorta—Tuolumne Meadows, CA, Pinus ponderosa —Yosemite Valley, CA, and Pinus nigra—Merced, CA.

Results/Conclusions

Bacterial endophytes were isolated most successfully with minimal R2A agar media supplemented with dextrose, sucrose and glucose. More isolates were recovered when shoot tissue was cryoprotected before liquid nitrogen tissue preparation. Overlapping bacterial endophytes were found across sampling locations and tree species, suggesting that these bacterial endophytes could be important to the host trees. Several Kocuria sp. were found in P. contorta and P. nigra. Two Bacillus sp. were both found in P. ponderosa as well as P. nigra and other endophytes previously found in agricultural plants were isolated from conifer shoot tissue. With improvements to culture methods in the second phase of the project we were able to culture Methylobacterium spp. from P. contorta. Methylobacterium extorquens isolated from P. sylvestris shoots in Finland have positive impacts on growth and development of P. sylvestris seedlings. Our results, that culturable bacteria from the genera Kocuria, Bacillus and Methylobacterium are associated with pine shoot tissues across species and geographic areas suggests long-term associations and mutualistic interactions. Future work will involve testing the effects of these isolates on host growth as well as mining their genomes for known and novel genes and pathways with roles in plant growth.