Soil Biology and Biochemistry 144 (2020) 107779

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Soil Biology and Biochemistry

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Short Communication Differences in soil ammonia oxidizing bacterial communities under unpalatable (Stellera chamaejasme L.) and palatable (Elymus nutans Griseb.) growing on the Qinghai Tibetan Plateau

Jianguo Ma a, Saman Bowatte a,*, Yufang Wang a, Paul Newton b, Fujiang Hou a a State Key Laboratory of Grassland Agro-ecosystems, Key Laboratory of Grassland Industry Innovation, Ministry of Agriculture and Rural Affairs, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, 730020, b AgResearch Limited, Grasslands Research Centre, Tennent Drive, Private Bag 11008, Palmerston North, 4442, New Zealand

ARTICLE INFO ABSTRACT

Keywords: A comparison of ammonia oxidizing bacterial (AOB) communities in the root zone soil of the unpalatable weedy Poisonous plants species Stellera chamaejasme L. and the palatable species Elymus nutans Griseb. showed Stellera had different AOB Ammonia oxidizing bacteria present and that these AOB were characterized by having low ammonia oxidation rates. This difference is N transformations consistent with a lower nitrification rate in Stellera soils when urine was added to simulate the dynamics in animal urine patches. Lower nitrificationis expected to reduce losses of nitrogen (N) to the environment leading to greater N availability for growth. This may provide a competitive advantage for Stellera and help explain its success as an invasive plant in the Qinghai Tibetan Plateau. Interestingly, it also implies a reduced envi­ ronmental footprint of this apparently undesirable species through reduced emissions of nitrous oxide and leaching of nitrate.

À À The spread of poisonous, unpalatable plants represents a serious NO3 . The NO3 -N produced by nitrification can be taken up by plants, threat to the ecological stability and productivity of grazed alpine move downward through the soil profile where it becomes subject to meadows of the Qinghai Tibetan plateau (QTP) in China (Wang et al., being leached or may provide a substrate for the production of nitrous 2015; Gao et al., 2019). Stellera chamaejasme L. is one of the major un­ oxide (N2O), an important gas. To establish differences be­ palatable weed species (Xing et al., 2002; Liu et al., 2004). Stellera is a tween Stellera and palatable species we compared its AOB community to perennial herbaceous plant belonging to the family that that of the palatable grass Elymus nutans Griseb. During the peak plant produces toxins in both above ground herbage and roots (Shi, 1997) and growth period in August of 2018, Stellera and Elymus patches were releases an odour that deters livestock (Sun et al., 2009). A feature of identified in 6 areas (each about 20 m apart) of an alpine meadow � 0 � 0 Stellera is that it is associated with different soil processes to grassland at the Azi Research station in Maqu county (33 59 N, 102 00 co-occurring palatable species. The differences result in greater avail­ E, 3500 m.a.s.l.) on the eastern QTP. This unfertilized grassland area is ability of nutrients (particularly nitrogen (N)) in Stellera patches (Sun grazed year-round by yaks. After removing above ground herbage, a soil et al., 2009) which likely confers increased competitiveness against sample from the root zone was taken in adjacent Stellera and Elymus other plant species in the meadow community. patches (10 cm diam, 10 cm depth). AOB abundance and composition Management of these threatened grasslands is enhanced by an un­ were measured. The methods are given in detail in the supplementary derstanding of the biological processes at play. In this paper we examine file. potential soil microbial processes that may drive the differences in soil N The abundance of AOB was significantlylower (P < 0.05) in Stellera dynamics in Stellera patches. soil compared to Elymus soil (6.25 � 0.23 cf. 8.35 � 0.50 Log amoA gene À We carried out a meta-barcoding study using Illumina MiSeq copies g 1 dry soil). Soil AOB community diversity and composition of sequencing analysis focusing on the ammonia oxidizing bacteria (AOB). Stellera and Elymus soils were assessed by a total of 87,214 high quality þ AOB control the rate limiting step of soil nitrificationby oxidizing NH4 sequencing reads with an average read of 14,535 per replicate sample. A À to NO2 , which is subsequently oxidized by bacterial nitrite oxidizers to total of 8979 AOB OTUs were detected in this study. Of these, 490 were

* Corresponding author. College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, 730020, China. E-mail address: [email protected] (S. Bowatte). https://doi.org/10.1016/j.soilbio.2020.107779 Received 21 November 2019; Received in revised form 8 March 2020; Accepted 9 March 2020 Available online 10 March 2020 0038-0717/© 2020 Elsevier Ltd. All rights reserved. J. Ma et al. Soil Biology and Biochemistry 144 (2020) 107779 shared by Stellera and Elymus soils; 5842 were unique to Elymus soil and 2647 were unique to Stellera soil. Only 5% of AOB OTUs were shared between Stellera and Elymus soils. At the species level, 30 out of 35 AOB species identifiedin this study were significantly different between the two soils. Eleven species were significantly more and 19 species significantly less abundant in Stellera soil (Fig. 1). Specific differences included greater abundance of Nitro­ somonas and Nitrosovibrio in Stellera soil and the appearance of Nitro­ sococcus only in the Stellera soil (Fig. 1). The rarefaction analysis of richness index; Chao1 (Fig. 2A), was consistently lower for Stellera soil indicating a lower diversity compared to Elymus soil. Beta diversity, explored using NMDS analysis, suggested dissimilar AOB communities for Stellera and Elymus soils although high variation between samples meant that the difference was non-significant (Fig. 2B). The phylogenetic analysis by the neighbour joining tree (Fig. 1) indicated that the AOB species which were significantlymore abundant in Elymus soil were mostly clustered to AOB cluster 3b. This cluster has been characterized as a group of AOB with fast ammonia oxidation ki­ netics as opposed to the AOB cluster 3a which shows significant delays in initiating ammonia oxidation after the supply of substrate (Webster et al., 2005). Nitrosospira TCH11 and Nitrosovibrio sp. RY3C clustered within the AOB cluster 3a were among the most dominant AOB species found in Stellera soil. As a preliminary test of any functional differences arising from the difference in AOB communities we conducted an experiment where animal urine was added to the soil and the subsequent dynamics of soil N were measured. The relevance of this test is that in grazed grasslands animals ingest N from across the landscape but then return it in discrete patches through urination. The N is concentrated in these urine patches often to a point that exceeds the amount that can be taken up by plants (Ball et al., 1979; Selbie et al., 2015). On the positive side, the high N availability in urine patches results in enhanced plant growth compared to non-urine patches but on the negative side, the excess of N can result in losses of N to the environment through volatilization, leaching of nitrate or emissions of N2O. Consequently, N transformations in urine patches are of high importance in terms of productivity and the envi­ ronmental footprint of grasslands. Of particular importance is the rate of change in soil nitrate and ammonium as this is an indication of the rate of nitrification which is determined largely by the activity of AOB. Soil nitrate increased with time in both soils indicating that soil nitrification was occurring (Fig. 3A). There was significantly lower nitrate concen­ tration in the Stellera soil at all sampling times with the difference get­ ting larger over time due to a lower rate of change in Stellera (Fig. 3A). Ammonium in the Stellera soil started at a lower amount than in the Elymus soil suggesting rapid immobilization and ammonia volatilization after urine application (Carran et al., 1982; Selbie et al., 2015). There­ after, the rate of change in ammonium was slower in the Stellera soil (Fig. 3B). Lower nitrificationunder the unpalatable Stellera, is important as it implies reduced losses of N through nitrate leaching and N2O emissions (Coskun et al., 2017) resulting in more N available for plant growth leading to higher plant and litter production (Sun et al., 2009) thus providing a ready source of re-cycled nutrients and a conserved nutrient cycle. The outcome of these changes is likely to increase competitiveness of Stellera (Sun et al., 2009). Similar impacts of exotic plants on soil communities have been observed in other ecosystems by Fig. 1. Neighbour joining tree of the partial amoA sequences retrieved from Wolfe and Klironomos (2005). this study and reference sequences for AOB clusters from Fan et al. (2011). Although we cannot identify why Stellera and Elymus have con­ Filled black circles represent AOB species significantlymore abundant in Elymus trasting AOB communities the fact that Stellara can exude toxins from its soils, filledblack triangles represent AOB species significantlymore abundant in roots (Shi, 1997) may be relevant. Plant root exudates that specifically Stellera soils and filled black diamonds represent AOB species not significantly inhibit soil nitrification, now termed as Biological Nitrification Inhibi­ different in abundance between soils. tion (BNI), has been identified in Brachiaria grass (Subbarao et al., 2009), (Sun et al., 2016) and sorghum (Subbarao et al., 2013). In our study, the marked difference in AOB population size and identity and the concomitant effect on soil nitrification suggest a potential role for BNI in Stellera plants.

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Fig. 2. (A) rarefaction analysis of richness index; Chao1 of AOB community (B) NMDS analysis of AOB in soils originating under Stellera or Elymus.

Fig. 3. Changes in A) soil nitrate and B) soil ammonium after the application of sheep urine. Values are means � sem.

The lower nitrification rate under Stellara also raises the interesting Carran, R.A., Ball, P.R., Theobald, P.W., Collins, M.E.G., 1982. Soil nitrogen balances in proposition that the presence of the unpalatable, poisonous Stellera urine-affected areas under two moisture regimes in Southland. New Zealand Journal of Experimental Agriculture 10, 377–381. could potentially have a positive environmental benefitthrough reduced Coskun, D., Britto, D.T., Shi, W.M., Kronzucker, H.J., 2017. Nitrogen transformations in nitrate leaching and lower N2O emissions. Further direct measurement modern agriculture and the role of biological nitrification inhibition. Nature Plants of these losses would be necessary to confirm this. 3, 17074. Fan, F.L., Zhang, F.S., Lu, Y.H., 2011. Linking plant identity and interspecificcompetition to soil nitrogen cycling through ammonia oxidizer communities. Soil Biology and – Declaration of competing interest Biochemistry 43, 46 54. Gao, F.Y., Li, K., Zhao, C.Z., Ren, H., Nie, X.Y., Jia, D.Y., Li, L.F., Li, Q.F., 2019. The expansion process of a Stellera chamaejasme population in a degraded alpine meadow The authors declare that they have no known competing financial of Northwest China. Environmental Science and Pollution Research 26, interests or personal relationships that could have appeared to influence 20469–20474. Liu, Y., Long, R.J., Yao, T., 2004. Research progress on Stellera chamajasme L. in the work reported in this paper. grassland. Pratacultural Science 6, 55–61 (in Chinese with English abstract). Selbie, D.R., Buckthought, L.E., Shepherd, M.A., 2015. The challenge of the urine patch Acknowledgements for managing nitrogen in grazed pasture systems. Advances in Agronomy 129, 229–292. Shi, Z.C., 1997. Important Toxic Plants of China Grassland. China Agriculture Ture Press, This study was supported by the Project of the Second Tibetan Beijing. Plateau Scientific Expedition (2019QZKK0302), the National Natural Subbarao, G.V., Nakahara, K., Hurtado, M.P., Ono, H., Moreta, D.E., Salcedo, A.F., Yoshihashi, A.T., Ishikawa, T., Ishitani, M., Ohnishi-Kameyama, M., Yoshida, M., Science Foundation of China (31672472), Lanzhou University grant; Rondon, M., Rao, I.M., Lascano, C.E., Berry, W.L., Ito, O., 2009. Evidence for Fundamental Research Funds for Central Universities (lzujbky-2017- biological nitrificationinhibition in Brachiaria pastures. Proceedings of the National ot21), and the program for Changjiang Scholars and Innovative Academy of Sciences 106, 17302–17307. Subbarao, G.V., Nakahara, K., Ishikawa, T., Ono, H., Yoshida, M., Yoshihashi, T., Zhu, Y. Research Team in University (IRT_17R50). Y., Zakir, H.A.K.M., Deshpande, S.P., Hash, C.T., Sahrawat, K.L., 2013. Biological nitrification inhibition (BNI) activity in sorghum and its characterization. Plant and Appendix A. Supplementary data Soil 366, 243–259. Sun, G., Luo, P., Wu, N., Qiu, P.F., Gao, Y.H., Chen, H., Shi, F.S., 2009. Stellera chamaejasme L. increases soil N availability, turnover rates and microbial biomass in Supplementary data to this article can be found online at https://doi. an alpine meadow ecosystem on the eastern Tibetan Plateau of China. Soil Biology org/10.1016/j.soilbio.2020.107779. and Biochemistry 41, 86–91. Sun, L., Lu, Y.F., Yu, F.W., Kronzucker, H.J., Shi, W.M., 2016. Biological nitrification inhibition by rice root exudates and its relationship with N-use efficiency. New References Phytologist 212, 646–656. Wang, P., Lassoie, J.P., Morreale, S.J., Dong, S.K., 2015. A critical review of Ball, R., Keeney, D.R., Thoebald, P.W., Nes, P., 1979. Nitrogen balance in urine-affected socioeconomic and natural factors in ecological degradation on the Qinghai-Tibetan – areas of a New Zealand pasture 1. Agronomy Journal 71, 309–314. Plateau, China. The Rangeland Journal 37, 1 9.

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