Mycorrhiza DOI 10.1007/s00572-017-0795-7 ORIGINAL PAPER Ectomycorrhizal fungi reduce the light compensation point and promote carbon fixation of Pinus thunbergii seedlings to adapt to shade environments Liang Shi1 & Jie Wang1 & Binhao Liu1 & Kazuhide Nara2 & Chunlan Lian3 & Zhenguo Shen1 & Yan Xia 1 & Yahua Chen 1 Received: 10 April 2017 /Accepted: 3 August 2017 # The Author(s) 2017. This article is an open access publication Abstract We examined the effects of three ectomycorrhizal significantly increased chlorophyll content of needles and (ECM) symbionts on the growth and photosynthesis capacity higher Pi concentrations compared to NM seedlings. of Japanese black pine (Pinus thunbergii) seedlings and esti- Overall, ECM symbionts promoted growth and photosynthe- mated physiological and photosynthetic parameters such as sis while reducing the LCP of P. thunbergii seedlings. These the light compensation point (LCP), biomass, and phosphorus findings indicate that ECM fungi can enhance the survival and (Pi) concentration of P. thunbergii seedlings. Through this competitiveness of host seedlings under low light. investigation, we documented a new role of ectomycorrhizal (ECM) fungi: enhancement of the survival and competitive- Keywords Ectomycorrhiza . Photosynthetic rate . Light ness of P. thunbergii seedlings under low-light condition by compensation point . Japanese black pine . Light limitation reducing the LCP of seedlings. At a CO2 concentration of 400 ppm, the LCP of seedlings with ECM inoculations was − − 40–70 μmol photons m 2 s 1, significantly lower than that of Abbreviation μ −2 −1 non-mycorrhizal (NM) seedlings (200 mol photons m s ). ACO2 assimilation In addition, photosynthetic carbon fixation (Pn) increased AQY Apparent quantum yield with light intensity and CO2 level, and the Pn of ECM seed- CCP CO2 compensation point lings was significantly higher than that of NM seedlings; Chl Chlorophyll Pisolithus sp. (Pt)- and Laccaria amethystea (La)-mycorrhizal CSP CO2 saturation point seedlings had significantly lower Pn than Cenococcum ECM Ectomycorrhizal geophilum (Cg)-mycorrhizal seedlings. However, La- Fv/Fm Maximal photochemical efficiency mycorrhizal seedlings exhibited the highest fresh weight, rel- LCP Light compensation point ative water content (RWC), and the lowest LCP in the mycor- LSP Light saturation point rhizal group. Concomitantly, ECM seedlings showed NM Non-mycorrhizal Pi Phosphorus Pn Photosynthetic carbon fixation * Yan Xia RWC Relative water content [email protected] ΦPS II Actual PS II efficiency * Yahua Chen [email protected] 1 College of Life Sciences, Nanjing Agricultural University, Introduction Nanjing 210095, China 2 Department of Natural Environmental Studies, Graduate School of Mycorrhizal fungi play crucial roles in shaping the develop- Frontier Science, The University of Tokyo, 5-1-5 Kashiwanoha, ment of forest ecosystems (Clark and St Clair 2011). In for- Kashiwa, Chiba 277-8563, Japan ests, light is the greatest limiting factor for seedling survival 3 Asian Natural Environmental Science Center, The University of and growth (Kolb et al. 1990). Today, conifer trees within Tokyo, 1-1-8 Midoricho, Nishitokyo, Tokyo 188-0002, Japan native ranges often account for less than 15% of standing Mycorrhiza stock, primarily due to shade intolerance and slow initial Kubota et al. 2017). However, the limited study to date has growth rates (Kabrick et al. 2015). In old-growth forests of investigated the shade tolerance of ECM P. thunbergii seed- Eastern Asia, Taiwania cryptomerioides is a shade-intolerant lings. Meanwhile, the response of soil to increased carbon and long-lived conifer that experiences intense light competi- availability is largely driven by root-associated ECM fungi tion, yet eventually emerges from the canopy (40–70 m), in forest ecosystems, as they partition host-derived carbon which comprises more shade-tolerant evergreen broad- belowground (Fransson 2012). Aspen trees with mycorrhizal leaved trees (He et al. 2015). Paper birch also competes well associations exhibit higher net photosynthetic rates and an for light and inhibits the growth of shade-intolerant conifers ability to maintain higher sucrose levels in their leaf tissue (Callaway and Walker 1997). These previous studies indicate compared to those without mycorrhizal associations (Einig that some conifer species do not possess a competitive advan- et al. 1997; Loewe et al. 2000). Furthermore, shading can lead tage in forest ecosystems under natural conditions. Therefore, to carbon limitation in aspen through decreased photosynthet- the protection and restoration of shade-intolerant conifers re- ic rates (Calder et al. 2011). For example, light reductions and/ main challenging but essential endeavors. or shifts in soil chemistry limited height growth, biomass gain, Pinus thunbergii Parlat. is a major shade-intolerant ever- photosynthesis, and the production of defense compounds green conifer species in Japan and has been introduced to (phenolic glycosides and condensed tannins). China and America (Choi 1986;Masakaetal.2010). Photosynthetic parameters such as CO2 assimilation (A), Taniguchi et al. (2008) reported that when P. thunbergii seed- actual PS-II efficiency (ΦPS II), gas exchange, and needle ne- lings were planted in soil obtained from a black locust- crosis have typically been the foci of previous studies dominated (Robinia pseudoacacia) area, all seedlings died (Bucking and Heyser 2003;Nguyenetal.2006;Heinonsalo under low-light intensity conditions. Furthermore, inhibition et al. 2015). Among these parameters, determining in vitro Pn of the regeneration of P. thunbergii seedlings was strongly in conifers often involves cutting branches (Zeibig et al. 2005; mediated by shading (Taniguchi et al. 2007). To adequately Renninger et al. 2013), isolating needle chloroplasts (Huang protect and restore this species, it is critical to improve the and Tao 2004), or collecting needles from seedlings in a leaf ability of P. thunbergii seedlings to survive under light- chamber cuvette (Thompson and Wheeler 1992; Ibell et al. limiting conditions by enhancing their shade tolerance. A 2013). However, these photosynthetic parameters, including key trait affecting the survival of seedlings grown under in vitro LCP, may not be accurate. Some studies have shown light-limiting conditions is the light compensation point that leaves collected in vitro make their water supply be (LCP) (Kitao et al. 2016), i.e., the light intensity at which interrupted, leading to the limitation of stomatal or non- the photosynthetic rate of plant leaves is equivalent to the stomatal factors (Saliendra et al. 1995; Flexas and Medrano respiration rate (Taiz and Zeiger 2010). Although the benefits 2002), stomatal closure, reducing adenosine triphosphate of mycorrhizal symbionts are well established, whether (ATP), and ribulose-1,5-diphosphate (RuBP) levels, decreas- ectomycorrhizal (ECM) fungal inoculation improves the ca- ing content and activity of ribulose-1,5-biphosphate pacity of P. thunbergii seedlings to utilize low light has not carboxylase/oxygenase (Rubisco) and reducing the photosyn- been examined. thetic rate ultimately (Gimenez et al. 1992; Flexas and Photosynthesis is the process by which plants convert car- Medrano 2002). bon dioxide (CO2) and water into sugars and oxygen using In this study, our objectives were to determine (1) whether solar energy; this reaction is highly sensitive to environmental inoculation with ECM fungi improves the capacity of changes (Taiz and Zeiger 2010). In some broad-leaved plants, P. thunbergii seedlings to utilize low light; (2) how ECM such as poplars and Mediterranean orchids, mycorrhizal fungi fungal inoculation enhances the shade tolerance of effectively improve Pn and leaf chlorophylls a and b,aswell P. thunbergii seedlings; and (3) the effects of ECM fungi on as the maximal photochemical efficiency (Fv/Fm)ofhost thecarbonfixation(Pn)ofP. thunbergii seedlings. plants (Gambini and Vellini 2007; Smith and Read 2008). Similarly, the ECM plants of Helianthemum sessiliflorum ex- hibited higher rates of photosynthesis (35%), transpiration Materials and methods (18%), and dark respiration (49%) than non-mycorrhizal plants (Turgeman et al. 2011). Most previous studies have Preparation of ectomycorrhizal P. thunbergii seedlings focused on the effects of ECM inoculation on the salt toler- ance of P. thunbergii seedlings (Kim et al. 2016) or their re- Source of strain Three isolates of ECM fungi, Cenococcum sistance to damage caused by pathogenic microorganisms geophilum (KY075873), Pisolithus sp. (KY075875), and (Ichihara et al. 2001) because of its high salt tolerance (Kim Laccaria amethystea (KY075878), were obtained from et al. 2016) and high capacity to intercept salt spray (Kim Sanqing Mountain in Jiangxi Province, China (Table 1). 2010), and a few studies have examined the photosynthetic These fungi were chosen because they are easily cultivated, responses of ECM P. thunbergii (Nazir and Khan 2012; grow rapidly, and readily form ECM associations. Mycorrhiza Table 1 Sources of and information regarding ECM Strains Sites Isolation source Lat and Lon Sequence ID fungal strains used in this study Cenococcum geophilum Mountain Sanqing Mycorrhizal root tips 29.03 N 118.26 E KY075873 Pisolithus sp. Mountain Sanqing Sporocarps 28.54 N 118.03 E KY075875 Laccaria amethystina Mountain Sanqing Sporocarps 28.55 N 118.09 E KY075878 Furthermore, they are globally distributed, and they maintain thinned to 30 per pot,