Isolation and Characterization of Nitrogen Fixing Bishop Pine Bacteria

C. Kitchell1, R. Walker2, and R. Chakraborty2

Contact Information celiakitchell@gmail.com(415) 516-2892

Methods

Support Information This work was funded by National Science Foundation Award ECCS-1461157 &ECCS-0939514

Abstract Endophytes form a mutualistic association with conifers, providing them with labile nitrogen through biological nitrogen fixation (BNF) [1] [2]. This has important implications for agriculture as BNF is viewed as a sustainable alternative to anthropogenic fertilizers and for understanding forest nitrogen budgets. To investigate what species of endophytes are responsible for nitrogen fixation in lodgepole pine, we used pine needle samples collected from the Ecological Staircase at Mendocino that Moyes et al. tested positive for nitrogen fixation using acetylene reduction assay. Several bacteria were isolated from surface sterilized needles and grown with media we designed based on the carbon utilization of Gluconacetobacter sp., a potential candidate for N-fixation. Strains that formed nitrogen-fixing pellicles in anaerobic gradient vials were restreaked according to several methods and identified using 16S rRNA gene analyses. We also sequenced for the nifH gene to identify the ability to encode nitrogenase. Utilization of nitrogen and carbon compounds was analyzed by monitoring the reduction of a tetrazolium dye by NADH using the Omnilog system, which allowed us to further differentiate the isolates. We isolated ten strains of Burkholderia sordidicola with unique nitrogen and carbon utilization patterns. Many of these strains showed nifH amplification in the gels but not in the sequencing results.

Results

2016 Transfer-to-Excellence Research Experiences for Undergraduates Program (TTE REU Program)

Table 1. NFb & LGI ingredients. We adapted 2 media recipes from Baldani et al. (2014) [4]. We substituted mannitol, a sugar alcohol widely distributed in plants, as the carbon source. The LGI was particularly successful in cultivating plate isolates

Figure 2. Design for isolation protocol to eliminate all epiphytic bacteria and fungi and select only for the endophytes, those organisms living inside the pine needles.

Figure 1. The ecological staircase at Mendocino where the samples were collected is a succession of marine terraces derived from the same mineralogical parent material [3].

Conclusions

References

1. Carrell, A.A. and A.C. Frank, Pinus flexilis and Picea engelmannii share a simple and consistent needle endophyte microbiota with a potential role in nitrogen fixation. Frontiers in Microbiology, 2014. 5: p. 333.

2. Moyes, A.B., et al., Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer. New Phytologist, 2016. 210(2): p. 657-668.

3. Uroz, S., et al., Structure and function of bacterial communities in ageing soils: Insights from the Mendocino ecological staircase. Soil Biology and Biochemistry, 2014. 69: p. 265-274

4. Baldani et al., The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semi-solid media: a practical guide for microbiologists. Plant and Soil, 2014.

Isolation Protocol

Sampling & Growth Media

• We succesfully isolated 10 endophytes from pine needles.• The 16S rRNA gene sequence placed these isolates in the Paraburkholderia

genus and were most closley related to P. sordidicola (Fig. 4).• Evidence of nitrogen fixation as observed as a band of growth in N-deplete

media. This media allows nitrogen fixing bacteria to fix atmospheric N2 in a zone where O2 is not limiting the reaction.

• Evidence of nifH PCR amplification, but sequence could not be obtained due to non-specific binding of primers amplifying multiple fragments.

• The Paraburkholderia appears to be highly abundant and dominant, isolating the N-fixers will require more selective methods of cultivation if in fact they are culturable.

Acknowledgements

The authors would like to thank the National Science Foundation and the DOE for financial support. Thanks also to Lara Kueppers and the Moyes lab for their initial work on this project collecting and testing the samples.

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MilliQ waterRemove water + add 100mL NFb

liquid media

Blend to break up needles

Four of the nitrogen-deplete anaerobic gradient vials saw the growth of a pellicle (a thin membrane of microbial growth), indicating the presence of N-fixing organisms.

However, we were unable to isolate the N-fixer(s) in this pellicle. We tried direct streaking onto LGI and NFb plates and serial dilutions. Both methods yielded only Burkholderiasordidicola. We used 16S rRNA sequencing to identify the isolates, and referenced them against NCBI Blast.

Although all isolates were identified as the same species, phenotypic differences were detected in the different strains. At least 5 distinct morphologies were detected, and differences in nitrogen utilization of 4 of the strains were measured by the Omnilog.

Figure 4. A 16S rRNA gene phylogenetic reconstruction by maximum likehood following a general time reversible model of selected isolates. Branch support is show as a percentage using Approximate Liklihood Ratio test.

Figure 3. Pellicle of nitrogen fixing zone of bacterial endophyes

Figure 5. Fragments obtained by gel electrophoresis of the PCR products of endophyte isolates obtained through direct streaking of the pellicle (top row) and through serial dilutions (bottom row) showed the presence of PCR fragments of similar size to nifH of Azospirillum lipoferum positive control.

The only sample used in this study came a Bishop pine (Pinus muricata) found in Terrace 5.

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N25 2 0 2 2 2 1 3 0 1 3 2 3 2 2 3 0 1 2 3 2 0 2 1 2 1 0 0 0 1 0 3 1 2 3Ne5 2 1 1 3 3 3 2 3 3 1 1 3 3 3 1 1 2 3 3 3 2 0 1 1 1 1 1 1 1 0 2 0 3 0Nc6.2 3 1 0 3 3 2 0 0 0 2 2 3 2 2 1 2 2 1 0 1 2 1 2 2 1 2 1 1 0 0 2 0 2 0Nd5#3 2 2 0 3 3 3 3 2 2 2 2 3 3 3 1 2 2 3 2 3 2 1 1 1 1 2 1 1 2 1 2 0 3 0

Table 2. Omnilog data showing nitrogen utilization of 4 of the isolates. Sources of nitrogen are listed above. Zero represents growth above the negative control; positive growth is quantified on a scale of 1 to 3.

We also tested isolates for nitrogen fixation by sequencing for nifH, the gene responsible for the ability to encode nitrogenase (the enzyme complex that catalyzes the conversion of N2 gas to ammonia).

1City College of San Francisco, 2Lawrence Berkeley National Laboratory