Nitrogen fixation in Pinus (pines)

Nitrogen Fixation in Pinus (pines)

The understanding, role and application of nitrogen fixing plants has not changed much in the last 30 years in the context of forest gardening and permaculture communities – advice written in the 1990s is largely the same as now. Here begins a series to re-introduce both new and orphaned knowledge of nitrogen cycling to update the view of the forest gardener – there are many articles to be written so stay tuned.

There are usually two main groups of nitrogen fixers that are discussed – leguminous plants that symbiose with Rhizobium and actinorhizal plants that symbiose with Frankia. Sometimes mentioned are ‘outliers’ such as Gunnera that symbiose with Nostoc cyanobacteria. Unlike the previous two large categories, there are no nodules that form on the roots of Gunnera, just a specialized mucus-producing gland on the stems – Nostoc cyanobacteria live inside and then largely forgo their previous photosynthetic lifestyle in order to feed directly from the rich carbohydrate mucus and in return provide their nitrogen fixation sercices. It took approximately 70 years from the date of the hypothesis that Gunnera was involved in a strong nitrogen fixing symbiosis to be conclusively proven – so it can take a while for knowledge in nitrogen cycling to change. [1]

The next group of nitrogen fixing plants identified were those without obvious physical characteristics, and also formed less-coupled symbioses – thus much more difficult to identify. The two main zones of the plant where these nitrogen-fixing symbionts live are in the rhizosphere, or within the plant leaves and stems (an endophyte). The study of nitrogen fixation is discovering new examples of these symbioses regularly, and the evidence is mounting that nitrogen fixation is much more common than previously thought (despite there being large amounts of observations and experiments starting from 70 years ago that there were unexplainable inputs of large amounts of nitrogen into many agricultural and ecological systems).

Pines have long been known as a pioneer species, colonizing difficult environments all over the Northern hemisphere – from growing on acidic, nutrient-poor bog soils, to fire ecology and newly exposed glacial till. The speed of growth has also been noted with many species being grown as commodity timbers.

Research with pines

The first strong hints that pines formed strong nitrogen fixing relationships come from the 1950s, when nitrogen accounting found unexplainable increases in nitrogen in certain forests and ecosystems all over the world. In the introduction of Fixation of Nitrogen by Non-nodulated plants is mentioned In New Zealand the rapid growth of exotic pines on depleted mountain country and even on sand dunes is striking. The writer measured the nitrogen in a 25-year-old Pinus radiata forest on depleted hill country near Nelson, and found the gain in nitrogen per acre to be approximately 300 lb. in the standing trees and 500 lb. in the litter, and average increment of 32 lb. N/ac./yr. This amount (35.8 kg N/ha/y) is a significant amount for exposed hill terrain. This same paper also references a site of predominantly Pinus ponderosa with a gain of 56 lb/ac/year (62.7 kg N/ha/y) [I was unable to find this referenced study to read additional details] [2]

A series of semi-controlled pot experiments by Greta Stevenson in New Zealand, were published in 1959. These were aimed at determining where this nitrogen fixation took place, Pinus radiata seedlings were sterilized and inoculated with various fungi, or a sterile soil. These seedlings were then grown in perlite and fed with a nutrient mixture lacking only N (so that nitrogen was guaranteed to be the limiting growth factor). The sterile-soil inoculated plants died quickly, and the Amanita-inoculated plants took longer but also died – but all the others were thriving after 6 months – providing evidence evidence that the mycorrhiza were responsible for fixing nitrogen for these trees. Additional experiments with 15N suggested that Pinus roots could fix nitrogen, but not leaves. Combined these results suggested that the mycorrhizal fungi were responsible for the addition of nitrogen to the soil. [2]

A 1964 study by Richards and Voigt using mycorrhiza inoculated Pinus radiata seedlings growing in quartz sand also found strong evidence of nitrogen fixation, despite the experiment running for only half a year. [3]

In 1967 a study by Richards and Bevege in Australia found that nitrogen fixing legumes did not benefit the growth rate of Pinus elliottii, P. taeda, and P. caribaea – but they did benefit those of the native conifers Araucaria cunninghamii and Agathis robusta. They go on to state: There is evidence of substantial accretion of nitrogen, amounting to 20-45 lb/acre/year, to ecosystems dominated by Pinus spp., even in the absence of legumes – this is up to 50.4 kg N / ha/y, but they also remarked this was almost all present in the trees and not in the litter (this is expected given that pine foliage can live for years, and the experiment running for only 5 years). [4]

In 1982 the Hubbard Brook sandbox study was started, an attempt to deduce and quantify more accurately the existence of unexplained nitrogen sources in model ecosystems (mesocosms) – by careful accounting of the initial nitrogen and the outputs and inputs. Box-shaped pits were dug, lined with landfill-liner plastic, and filled with glacier sand. Planted in these were 16 pines in a grid pattern (as well as alder and black locust in other identical boxes). These were left to grow for 6 years. The amount of water that drained was carefully measured (including nitrogen leaching losses), and upon the end of the experiment – trees were removed and sampled for nitrogen content. The initial experiment conclusion was These results provide strong evidence for N2 fixation in pine systems of almost-equal-to 50 kg . ha-1 . yr-1 N – but due to the conventional understanding, such a large amount of non-nodulated nitrogen fixation was largely dismissed as an error.
The original paper was later re-examined for these sources of errors, the initial soil checked and the original conclusion was strengthened: New estimates for accumulation of N in the entire sandbox soil (0–135 cm), based on the fixed-mass method, were also large with small confidence intervals: 70 +- 21 kg ha/y for red pine and 63 +- 16 kg ha/y for pitch pine. This was the lower-end estimate – the maximum was 149 +- 23 kg N/ha/y and 130 +- 43 kg N/ha/y for red pine and pitch pine respectively. The same experimental conditions resulted in 175 kg N/ha/y for Alnus glutinosa (below its maximal rate in other experiments – perhaps suggesting conservative measurements all around).
The conclusion that nitrogen fixation rates of above 25 kg N / ha/y in unknown sources of nitrogen was not at all accepted as wisdom in the 2000 review by Binkley et al, despite all other studies to the contrary being less controlled observation studies (not mesocosm studies) [5][6]

A 2012 study from British Columbia found very strong indirect evidence of nitrogen fixation in wild stands of Pinus contorta. Trees observed growing in gravel pits had almost identical rates of growth to those growing in more fertile soil – however foliar 15N ratio was much lower in the gravel pit trees than the fertile soil trees. The simplest explanation for this discrepancy is if the gravel pit trees were receiving substantial amounts of biologically fixed nitrogen. The total nitrogen content of tissues was virtually identical between the two sites. The gravel also had higher nitrate content than the fertile soil, while the fertile soil was much higher in ammonium. This is perhaps due to accumulated BNI effects – a topic I will write about in future on the second pine discussion.
The study authors concluded: Based on our previous work which demonstrates high levels of acetylene reduction in tuberculate mycorrhizae in P. contorta, we conclude that the simplest and most likely explanation for comparable growth and nitrogen levels between trees growing on intact soil and those growing on very low nitrogen gravel is that this pine species, in conjunction with certain symbionts, is capable of fixing biologically significant quantities of nitrogen. [7]

Finally some direct evidence for nitrogen fixation was presented in a 2013 study also performed in British Columbia [8]. Pinus contorta var. latifolia seed was first sterilized, then inoculated with Paenibacillus polymyxa (a bacteria isolated from within the pine tissues, and that has been determined to fix nitrogen) and grown on a sand-clay mixture (fertilized once with some 15N-tagged fertilizer) for 13 months. The innoculated pines grew approximately twice as massive as the non-inoculated ones and had a much higher survival rate. What was interesting about this study – is that there was no mycorrhizal development in the roots, but there were large amounts of P. polymyxa in the root tissue. This study suggests that bacterial endophytes are a strong nitrogen-fixing partner of pines.

Further direct evidence of nitrogen fixation, along with some quantization was presented in a 2016 study [9]- using the acetylene reduction assay of subalpine wild-collected Pinus flexilis needles (one site in forest, the other at tree-line).

This amount was low however, perhaps partially due to the extreme habitat of the limber pine: Based on the ARA rates we measured and the calculations reported above…, we estimate an annual N input of 0.2–20.4 g N ha⁻¹ yr⁻¹ to a P. flexilis stand (assuming a 150‐d growing season). This is vastly less than that estimated for lower elevation pines, which were estimated on the order of 50kg N/ha/y. [8]

A 2019 study on post-fire recovery rates of Pinus contortavar. latifolia in Yellowstone National Park found that nitrogen had increased in all measured areas (foliage and soil horizons) – counter to the expectation that tree recovery would remove soil nitrogen. For live biomass they calculated an increase of 7.2 kg N/ha/y, in the soil (0-15 cm) N accumulation of [34.3 kg N/ha/y]. The O-horizon accumulated approximately 1.1 kg N/ha/y – for a total of 42.6 kg N/ha/y accumulated. – from which the study authors concluded: The large increases in N pools cannot be explained by atmospheric N deposition or presence of known N fixers. These results suggest an unmeasured N source andare consistent with recent reports of N fixation in young lodgepole pine.

Conclusions

Based on these results, there seems to be strong evidence for nitrogen fixation in pines – likely through a combination of both endophytes and mycorrhiza in the roots (which may in turn be utilizing the same endophytes), depending on the species, inoculation conditions and climate. The amount fixed seems to be approximately 50 kg N/ha/y as a conservative estimate, for faster growing pioneer pines for lower elevation sites.

There is limited evidence on whether pines are facultative (they fix nitrogen only to meet their needs) versus obligate (they always fix nitrogen at the same rate) nitrogen fixers, but 2012 British Columbia study with 15N amounts indicates that pines with sufficient soil nitrogen still obtained a significant amount of their nitrogen needs from fixed nitrogen. The 2019 study also indicated some obligate tendencies.

How does this knowledge changes the usage of pines in agroforestry systems?

I will refrain from making sweeping statements until we have revisited pines in the second (or third?) part of this series. We can however state with high confidence with just this knowledge that:

– Pines may be expected to fix at least 50 kg N/ha/y (for known pioneer species) and perhaps up to twice that amount in a lower elevation site (although it’s difficult to estimate what the amount fixed would be if there is sufficient soil nitrogen).
– However unlike other N-fixing species, the C:N ratio is much higher in pines (because the total biomass of pines is higher – they are also ‘carbon fixers’), and the foliage is much longer lived too – to liberate this nitrogen to the system will require the tree to be felled and perhaps used for hugelkultur (this allows the temporary nitrogen deficit to decompose the lignin to be spread out over time, and for the nitrogen from the pine to enter the soil slowly).

References

[1] BERGMAN, B. , JOHANSSON, C. and SÖDERBÄCK, E. (1992), The Nostoc–Gunnera symbiosis. New Phytologist, 122: 379-400. doi:10.1111/j.1469-8137.1992.tb00067.x
Retrieve from: https://nph.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1469-8137.1992.tb00067.x

[2] STEVENSON, G. (1959). Fixation of Nitrogen by Non-nodulated Seed Plants. Annals of Botany, 23(4), 622–635. doi:10.1093/oxfordjournals.aob.a083680

[3] RICHARDS, B. N., & VOIGT, G. K. (1964). Role of Mycorrhiza in Nitrogen Fixation. Nature, 201(4916), 310–311. doi:10.1038/201310a0

[4] Richards BN Bevege DI (1967) The productivity and nitrogen economy of artificial ecosystems comprising various comninations of perennial legumes and coniferous tree species. Australian Journal of Botany 15, 467-480.
Retrieved from: https://www.publish.csiro.au/bt/bt9670467   

[5] Bormann, Tabata & Bormann, F. & Bowden, William & Pierce, R. & Hamburg, Steve & Wang, Deane & Snyder, Michael & Li, C. & Ingersoll, Rick. (1993). Rapid N^2 Fixation in Pines, Alder, and Locust: Evidence From the Sandbox Ecosystems Study. Ecology. 74. 583-598. 10.2307/1939318.

[6] Bormann, B., Keller, C., Wang, D., & Bormann, F. (2002). Lessons from the Sandbox: Is Unexplained Nitrogen Real? Ecosystems, 5(8), 727-733. Retrieved from http://www.jstor.org/stable/3658876

[7] Chapman, W. K., & Paul, L. (2012). Evidence that northern pioneering pines with tuberculate mycorrhizae are unaffected by varying soil nitrogen levels. Microbial ecology, 64(4), 964–972. doi:10.1007/s00248-012-0076-0
Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3474912/

[8] Anand, R., Grayston, S., & Chanway, C. (2013). N2-Fixation and Seedling Growth Promotion of Lodgepole Pine by Endophytic Paenibacillus polymyxa. Microbial Ecology, 66(2), 369–374. doi:10.1007/s00248-013-0196-1

[9] Moyes, A. B., Kueppers, L. M., Pett‐Ridge, J. , Carper, D. L., Vandehey, N. , O’Neil, J. and Frank, A. C. (2016), Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer. New Phytol, 210: 657-668. doi:10.1111/nph.13850

[10] Turner, M. G., Whitby, T. G., & Romme, W. H. (2019). Feast not famine: Nitrogen pools recover rapidly in 25-yr old postfire lodgepole pine. Ecology, e02626. doi:10.1002/ecy.2626