Journal of Applied Botany and Food Quality 88, 22 - 28 (2015), DOI:10.5073/JABFQ.2015.088.005 1 CEBra - Centre for Energy Technology Brandenburg e.V., Cottbus, Germany 2 Brandenburg University of Technology Cottbus-Senftenberg, Chair of Soil Protection and Recultivation, Cottbus, Germany Effects of nitrogen and phosphate fertilization on leaf nutrient content, photosynthesis, and growth of the novel bioenergy crop Fallopia sachalinensis cv. ‘Igniscum Candy’ Laurie Anne Koning1, 2, Maik Veste1, 2, *, Dirk Freese2, Stefan Lebzien3 (Received October 12, 2014) * Corresponding author Summary The aim of the study was to determine the effects of nitrogen and phos- phate fertilization on the growth performance of the novel bioenergy crop Fallopia sachalinensis cv. ‘Igniscum Candy’ (Polygonaceae). In a controlled pot experiment various nitrogen (0, 50, 150, 300 kg N ha-1) and phosphate (20, 40, 80 kg P ha-1) fertilizer amounts were applied to measure the effect on the biomass, plant height, leaf area, and leaf nutrient (N and P) content. Furthermore, the ecophysiologi- cal processes of chlorophyll content, chlorophyll fluorescence, and gas exchange were measured. The application of nitrogen correlated positively with biomass production, while phosphate fertilization did not show a significant effect on plant growth or ecophysiological parameters. The leaf nitrogen contents were significantly correlated with the nitrogen applications, while the leaf phosphate contents did not show a correlation with the P fertilizations, but increased with the leaf nitrogen contents. A significant linear correlation between N-Tester chlorophyll meter values and chlorophyll contents as well as with leaf nitrogen contents could be determined. Under the influ- ence of the nitrogen fertilization, net photosynthesis increased from 3.7 to 6.6 μmol m-2 s-1. The results of this experiment demonstrated that nitrogen fertilization has an overall positive correlation with leaf nitrogen content, photosynthesis, and growth of the bioenergy crop Fallopia sachalinensis var. Igniscum Candy. Introduction Renewable energy production from biomass is of growing impor- tance worldwide and will increase in the next decades (Kaltschmitt et al., 2009). Biomass can be utilized in diverse manners, such as for fuel, heating, and power. In Germany, maize is currently dominating agricultural biomass production for use in biogas plants because of its high methane production (schittenhelm et al., 2008; Vetter et al., 2009; FrancK, 2012). High biomass production, biomass quality, optimal energy yield of the harvested biomass and existing harvesting technology are key factors for the selection of new plant species for bioenergy production. To avoid monospecific biomass production based on maize, other species need to also be considered. The major second generation bioenergy crops include e.g., Miscan- thus giganteus (lewandowsKi and schmidt, 2006), Panicum vir- gatum (GuretzKy et al., 2011), Sida hermaphrodita (BorKowsKy and molas, 2012; FranzarinG et al., 2014) and Silphium perfo- liatum (Vetter et al., 2009; GansBerGer et al., 2014; Franzar- inG et al., 2014). In this context, knotweed species (Fallopia spp., syn. Reynoutria spp.) can play important roles in the production of biomass for bioenergy in the near future (seppälä, 2013). The new cultivars Igniscum Basic and Igniscum Candy have been cultivated from wild forms of Fallopia sachalinensis hybrids for use as bio- energy crops (leBzien et al., 2012). Igniscum Basic is intended for use in combined heat and power plants, while Igniscum Candy can be harvested up to two times during the growing season for biogas production. These cultivars can be alternatives to maize, adding to the portfolio of bioenergy crops. As members of a pioneer species, the cultivars are able to grow in a wide range of habitats (adachi et al., 1996) and are characterized by high biomass production (pude and FranKen, 2001; Veste et al., 2011). Knotweed species have proven themselves to achieve comparable biomass outputs to Mis- canthus. Already in the third year of an experiment in which Fal- lopia x bohemica and Miscanthus x giganteus were grown under field conditions, yields were 24.2 Mg DM -1 and 21.2 Mg DM ha-1, respectively (pude and FranKen, 2001). Other reported biomass production has ranged from 13.2 to 25 Mg DM ha-1 depending on the particular knotweed species, soil, climate and planting density (Vetter et al., 2009; strašil and Kára, 2010). It is crucial to understand plant responses to combinations of wa- ter and nutrient availability for the development of sustainable plant production systems. Nitrogen is the most common limiting factor in plant production systems. As a consequence, it is standard to add nitrogen fertilizer to a crop. But, the uptake and biomass produc- tion is crop specific e.g., switchgrass (Panicum virgatum) has con- sistently high biomass yields and relatively low nutrient removal rates, while giant reed (Arundo donax) with its high biomass pro- duction needs increased fertilization rates over time (KerinG et al., 2012) and Silphium perfoliatum requires 130-160 kg N ha-1 (aur- Bacher et al., 2012; GansBerGer et al., 2014). Furthermore, there are environmental concerns about the losses of nitrogen due to leach- ing and gaseous emissions (denitrification, ammonia volatilization and NOx emission), which are influenced by fertilization, soil type, crop species and management practices (paustian et al., 1990; helleBrand et al., 2008; cameron et al., 2013). The benefit of growing second generation bioenergy crops, like Miscanthus, Sida, Silphium and Igniscum, is their ability to store nutrients in their rhi- zomes over the winter months, which enables them to grow back strong in the spring (pude and FranKen, 2001; heaton et al., 2009). This feature lowers the required nitrogen application in the early growing season. Furthermore, there can be a reduction of greenhouse gas emissions depending on the crop management (hudiBurG et al., 2014). The other major limiting factor in plant production systems is phosphorus. Only around 15 % of the phosphate fertilizer applied to a soil is taken up by a plant from the soil solution immediately after fertilizing and the remainder relatively quickly converts to an in- soluble form in the non-labile fraction (menGel and KirKBy, 2001). The development of second generation bioenergy crops and optimi- zation of their nitrogen use efficiencies will help fulfill the sustain- ability criteria for biomass in the production of biofuels (erisman et al., 2010). Proper development of crop production systems based on specific crop and environmental quality information can lead to maximum economic returns and reductions of negative environmen- tal consequences. The objective of our study was to investigate the relationship between nutrient supply, the ecophysiological processes and biomass production. Materials and methods Experimental conditions for plant cultivation One-year-old Igniscum Candy saplings grown in plastic trays were obtained from Conpower Rohstoffe GmbH and cultivated in indi- Influence of Nitrogen and phosphate on Igniscum 23 vidual 7 L plastic pots in the greenhouse of the Brandenburg Uni- versity of Technology Cottbus-Senftenberg under semi-controlled conditions. Each pot was filled with 7 kg of silty sand with a texture of 67.7 % sand, 27.0 % silt, and 5.3 % clay. Soil nitrogen content was 0.6 - 0.9 g N kg-1dry soil and phosphate content was 0.3 g P kg-1dry soil. The average soil pH was 7.8. Every pot received 200 mL of a modified Hoagland’s solution (with- out the basic N and P content; hoaGland and arnon, 1950) as a basic fertilizer. After sixty days of the estabishment of the young plant in the greenhouse, the plants were cut down to allow a re- sprouting from the rhizomes; this event marked the start of the ex- periment. Nitrogen and phosphate fertilizers were applied to the soil surfaces. Twelve treatments with ten plants each involving every possible combination of commonly obtained nitrogen (calcium am- monium nitrate) and phosphate (superphosphate) pellet fertilizers at rates of 0, 50, 150 and 300 kg N ha-1 and 20, 40 and 80 kg P ha-1, respectively, were evaluated in the experiment. Volumetric soil moisture was measured with a frequency domain reflectometry probe (�M300, �oil Moisture �ensor Delta�T De�(�M300, �oil Moisture �ensor Delta�T De- vices, Cambridge, UK) and was kept just below the field capacity for sandy soils (14 % soil moisture). Temperature in the greenhouse ranged between 20 °C and 25 °C. Natural light was supplemented by high-pressure mercury lamps to provide photosynthetic photon flux density (PPFD) of 350�450 μmol m-2 s-1 at plant level for 10 hours per day. The pots were spatially sorted by treatment, while the treatments themselves were randomized in the greenhouse. Twice a week the pots were shifted in order to avoid possible spatial effects on the plants. Measurements of chlorophyll contents Leaf chlorophyll contents were assessed with a hand-held chloro- phyll meter and through chemical analysis. Relative indices for the chlorophyll contents of leaves were obtained with the N-Tester chlo- rophyll meter from Yara (Yara International ASA, Oslo, Norway) that is based on leaf transmittance of the wavelengths 650 nm (red) and 940 nm (IR) (netto et al., 2005). In the standard mode the measure- ments of 30 leaves is averaged. Another mode offered by the device is to sample individual leaves. This individual leaf mode produces a relative index result comparable to results obtained with �PAD chlorophyll meters from Konica Minolta. Readings were taken in the middle portion of fully expanded leaves on the day of harvest. Five plants from each treatment were assessed in the standard mode while every single plant in the experiment was assessed in the indi- vidual mode. For comparison between both modes, we calculated the chlorophyll index values of the Yara-N-Tester (YNT) follow the formula: YNT = 15*CHL – 90, where YNT is the relative N-Tester index value and CHL is the chlorophyll meter value of a single leaf (Yara GmbH, Dülmen, Germany, F. Brentrup, personal communica- tion). The same leaves that were assessed in the N-Tester individual mode were subjected to chlorophyll a and b extraction with 80 % acetone and analyzed spectrophotometerically with a Lambda 2 UV/ VIS Spectrophotometer to determine total chlorophyll contents (Per- kin Elmer, Norwalk, USA) after lichtenthaler (1987). Chlorophyll fluorescence For the measurements of in vivo photosynthesis on harvest day, a PAM (pulse amplitude modulation) fluorescence system (MINI� PAM Heinz Walz GmbH, Effeltrich, Germany; Veste et al. (2000) with a 6 mm diameter standard fibre optic was used. The fibre optic was fixed at a distance of 10 mm from the leaf surface with a leaf clip holder at an angle of 60°. �tandard routine programs within the PAM were used to determine effective quantum yield of photosystem II (ΦP�II; (F´m – F´t) * F´m-1) in the presence of light on a photosyn- thesizing sample (for nomenclature of fluorescence signals used see schreiBer et al., 1994; Von willert et al., 1995). All chlorophyll fluorescence parameters were instantly calculated, displayed on a LCD screen and stored on an internal data logger. Photosynthetic photon flux density (PPFD) was determined with a calibrated light sensor connected with the leaf clip holder. An external halogen lamp was used to illuminate the leaf sample with PPFD ≥1100 μmol m-2 s-1 for 2 minutes prior to PAM measurements. The linear electron transport rate (ETR) for photosystem II can be calculated by multi- plying the effective quantum yield of P�II (ΦP�II) and the incident PPFD (ETR = ΦP�II * PPFD * α * 0.5; see schreiBer et al., 1994; Von willert et al., 1995). The leaf absorption for light (α) was set to 0.84 (Veste et al., 2000). Leaf CO2 gas exchange The gas exchange of fully expanded leaves was measured with a compact minicuvette system (CM� 400, Heinz Walz GmbH, Effel- trich, Germany; midGley et al., 1997) on the harvest day. The gas exchange measurements were conducted at the same leaves as the chlorophyll fluorescence measurements (10 leaves, 5 plants). The gas exchange measurements were carried out under ambient CO2 concentrations (Veste and herppich, 1995). Illumination was set to a constant PPFD ≥1100 μmol m-2 s-1 with an external halogen lamp to ensure a light-saturated photosynthesis. Air temperature in the cuvette was 25 °C and water vapor pressure deficit (VPD) of 17.4 mPa Pa-1 was set with a cooling trap to correspond to the cli- matic conditions in the greenhouse. Changes of CO2 concentration were determined with an infra-red gas analyzer (BINOS 100-4P, Rosemount, Hanau, Germany). Net CO2 exchanges were calculated using the standard software DIAGA� 2.0 (Heinz Walz GmbH, Ef- feltrich, Germany) and CO2 flux was expressed on the projected leaf area (Von willert et al., 1995). Biomass The plants were harvested 173 days after the application of nitrogen and phosphate fertilizers. The fresh matter (directly after harvest) and dry weight (oven drying at 60 °C) of every plant was obtained for the biomass assessment. The harvesting procedure was carried out as follows in order to accommodate the various leaf assessments made pre- and post-harvest: I) The leaves on which individual N- Tester measurements (one leaf per plant) had been made were cut from each plant at the base of the leaves (petioles considered stem components and not leaf components) and stored in a freezer in in- dividual plastic freezer bags. These leaves would later be subjected to a chemical analysis of chlorophyll content in the laboratory. II) The five plants from each treatment which had been used in the eco� physiological assessments, which included: N-Tester in the stan- dard and individual modes, chlorophyll fluorescence, and CO2 gas exchange were harvested in three steps. The first step was to acquire a representative mixed leaf sample for the chemical analysis of ni- trogen and phosphate contents in the laboratory by cutting off ten mature leaves from halfway up the plants and packaging them in paper bags. The second step was to cut off the rest of the leaves and package them in a paper bag for the biomass assessment. The third step was to cut down the stem to just above the soil surface and pack- age it in a paper bag for the biomass assessment. III) The other five plants in each treatment were individually harvested for the biomass assessment by cutting off the leaves and cutting down the stems, then packaging the two components separately in paper bags. Analysis of nitrogen and phosphorus contents The representative mixed leaf samples (n=10) obtained in step II of the harvesting procedure were oven-dried at 60 °C and ground up for nutrient analysis. The carbon and nitrogen contents were analyzed 24 L.A. Koning, M. Veste, D. Freese, �. Lebzien with a CNS element analyzer (Vario EL III, Elementar Analysen- systeme GmbH, Hanau, Germany). The phosphorus contents of the samples were determined using a Lambda 2 UV/VIS spectropho- tometer (Perkin Elmer, Norwalk, U�A) following the DIN EN I�O 6878 method. Statistics The relationships between the treatments and the independent vari- ables were statistically assessed in the R software suite (r deVelop- ment core team, 2008) and Grapher 2.0 (Golden Software Inc, Golden, USA) with linear regression analysis by applying the Pear- son correlation and accepting significance at p<0.05. Results and discussion Leaf nitrogen and phosphorus contents In a linear regression analysis, the nitrogen contents of the leaves were significantly correlated with the nitrogen treatments in a Pear- son correlation (R2= 0.55) (Fig. 1). In contrast to this strong relation- ship, no correlation existed between the leaf phosphorus contents and the phosphate treatments (R2= 0.01) (Fig. 2). A linear regression analysis of the leaf phosphorus and nitrogen contents demonstrated a weak positive trend (R2= 0.16) (Fig. 3). The nitrogen contents of the Igniscum Candy leaves in the green- house experiment compared well with the results of wild Fallopia x bohemica in Belgium, both having mean values ranging from mid 1 % to mid 2 % nitrogen (herpiGny et al., 2011). The nitrogen con- tents of Igniscum Candy leaves from field trial sites in Germany with various management regimes (maturely rooted crops planted in 2008 and sampled in 2010 and 2011) were higher than the greenhouse leaves, having mean values ranging from 2.8 % to 3.1 % (Veste, unpublished data). Base nutrient levels in the respective soils may be a factor in the variance of nitrogen content (menGel and KirKBy, 2001). The starting soil condition in the greenhouse experiment be- fore fertilizer was applied averaged 0.06 - 0.09 % N. Soil analysis of the sites where Fallopia was found in Belgium also showed the soil to be poor in nitrogen (mean of 0.11 - 0.19 % N) and no ad- ditional fertilizer or management was applied to these wild plants before sampling took place. The amount of influence of the P fertilizer in this experiment is in- conclusive. All three P treatments (20 P, 40 P, and 80 P) delivered similarly uneventful results for nutrient contents and biomass. A plot of the P content in the leaves against the N treatments showed no significant trends (R2=0.01 and R2=0.07, respectively), suggesting that nutrient uptake was not influenced by the P fertilization rates. A plot of dry matter against the P treatments also produced a lack of trend (R2=0.00) (data not shown). Phosphorus uptake is not well understood (schachtman et al., 1998). An investigation into the availability of phosphorus in the soil or a biological analysis of the phosphorus distribution throughout the entirety of the plants (e.g. VeneKlaas et al., 2012) may have shed more light on the phospho- rus uptake by Igniscum. Chlorophyll content Previous studies have shown that the chlorophyll contents of leaves correlates strongly with the nitrogen contents of leaves (Gianquinto Fig. 1: Relationship between applied nitrogen fertilizer and leaf nitrogen content at various phosphate levels (20, 40, 80 kg P ha-1). Linear regression y = 0.001477 x + 0.692778, R2 = 0.3429) Fig. 2: Relationship between applied phosphate fertilizer and leaf phos- phate content at various nitrogen levels (0, 50, 150, 300 kg N ha-1). Linear regression y = -6.071* 10-5 x + 0.0787, R2 = 2.752 * 10-5) Fig. 3: Relationship between leaf nitrogen content and leaf phosphate con- tent at various nitrogen levels (0, 50, 150, 300 kg N ha-1). Linear regression y = 0.019396 x + 1.06893, R2 = 0.474 Influence of Nitrogen and phosphate on Igniscum 25 et al., 2004; samBorsKi, 2009) and based on this relationship, hand- held devices measuring the chlorophyll in living plants can reflect the nitrogen status and physiological activity of plants (BullocK et al., 1998; netto et al., 2005). The relative indices measured by the N�Tester were in significant linear correlation to the chlorophyll contents of the leaves (R2=0.65) (Fig. 4) and the nitrogen contents of the leaves (R2=0.52) (Fig. 5). As expected, a significant linear cor- relation also existed between the chlorophyll contents of the leaves and the nitrogen contents of the leaves (R2=0.42) (not shown). The theory behind the N-Tester was supported by the current research through strong positive trends in the relationships between the chlo- rophyll and nitrogen contents as well as the N-Tester results and ni- trogen contents. between the different nitrogen treatments (Fig. 6b). Even though photosynthesis depends on phosphate containing compounds and phosphate efficiency should influence photosynthetic activity (Vene- Klaas et al., 2012), we did not find any relation between the photo- synthesis parameters and the phosphate fertilization. However, CO2 exchange rates and electron transport rates showed a high variance within each treatment. The measured net photosynthesis of Fallopia is lower compared to other perennial bioenergy crops or highly pro- ductive annual crops. mantoVani et al. (2014) measured mean CO2 exchange rates of 7.5 to 9.7 μmol m-2 s-1 for Igniscum in a lysimeter experiment. For the perennial Sida hermaphrodita, which is con- sidered a highly productive bioenergy crop, mean net photosynthesis rates from 6 �17 μmol m-2 s-1 were measured (FranzarinG et al., 2014; Veste et al., 2014), while annual crops like sunflowers or to- matoes reach net photosynthesis rates of 12 � 24 μmol m-2 s-1 and 18 � 26 μmol m-2 s-1, respectively (BrecKle et al., 2003). In general, the photosynthesis and related physiological processes are directly linked to the leaf nitrogen content (eVans, 1983; saGe et al., 1987; lawlor et al., 2001). In our experiment a linear relationship be- tween the leaf N content and the net CO2 exchange could be shown (Fig. 7a), while the electron transport rate showed no correlation with the leaf N content (Fig. 7b). However, a clear linear influence of nitrogen leaf content on photosynthesis could be observed under various field conditions in Germany (Veste et al., 2011). Further- more, nitrogen also alters leaf morphology (lawlor, 2001). Since CO2 exchange rates are area related, leaf morphological changes in response to nitrogen needs to be taken into account to understand the plant CO2 uptake. Hereby, the leaf area demonstrated a strong posi- tive trend over the increasing N treatments (Fig. 8c) and increased total carbon uptake. It needs to be considered that nutrient deficiency Fig. 4: Relationship between Yara N-Tester values and chlorophyll content (linear regression y = 0.4924 x + 11.871, R2 = 0.645) at nitrogen levels (0, 50, 150, 300 kg N ha-1). Fig. 5: Relationship between leaf nitrogen content and Yara N-Tester value (linear regression y = 384.918 x + 14.2766, R2 = 0.518) at various nitrogen levels (0, 50, 150, 300 kg N ha-1). Leaf photosynthesis The mean net photosynthesis rates varied between 3.7 and 6.6 μmol m-2 s-1 and increased (R2=0.251) with increasing nitrogen applica- tion (Fig. 6a), while the electron transport rates showed no changes Fig. 6: Influence of the applied nitrogen fertilizer on net CO2 exchange (A) and electron transport rate (B). (A) Linear regression y = 0.00216963 x + 1.133749, R2 = 0.2013), (B) Linear regression y = 0.022849 x + 56.83, R2 = 0.004). 26 L.A. Koning, M. Veste, D. Freese, �. Lebzien might also affect the carbon source-sink interrelation. Understanding the carbon-sink interrelation as well as carbon allocation is impor- tant for understanding the growth performance of plants in relation to their photosynthesis (Körner, 2013; Fatichi et al., 2014). In the case of the studied Fallopia, the formation of the underground rhi- zome to store carbon and nutrients during the establishment of young plants is such an example of carbon and nutrient flows into different sink organs. However, in mature established plants the interrelation between aboveground and belowground organs is important to un- derstand biomass production under different nutrient supply und har- vest regimes (adachi et al., 1996; li et al., 1998; araVindhaKshan et al., 2011) and needs further investigations in herbaceous perennial bioenergy plants. Biomass production The dry weight, plant height, and leaf area (Fig. 8) all had quadratic relationships with the nitrogen treatments (R2=0.32, 0.34, 0.32, re- spectively). The peaks of the parabolic curves, or the point of opti- mum yield for dry weight, were reached around 170 kg N ha-1. A com- plete lack of correlation (R2=0.00) existed between the dry weight and phosphate treatments (not shown). Our experiment showed that increasing nitrogen fertilization to young knotweed plants leads to higher biomass yields, as shown previously by schmitt (1994). The strongest effect of the nitrogen supply was from 100 to 150 kg kg N ha-1, which could also be shown in a lysimeter experiment (manto- Vani et al., 2014) and is in the same range as observed for Silphium perfoliatum (GansBerGer et al., 2014). The positive effects of the nitrogen fertilization on biomass, plant height and leaf area showed a general decline between the 150 and 300 kg N ha-1 treatments, possibly due to nitrogen uptake entering the zone of luxury nutrient consumption (westerman, 1990). pude and FranKen (2001) ob- served an increase in biomass production with up to 200 kg N ha-1, while on agricultural fields of the �oester Börde (Northrhine West� falia, Germany) the growth response to nitrogen fertilization was indifferent (Veste et al., 2011). As shown in Fig. 8c, the leaf area of the Igniscum plants in this experiment ranged between 50 and 100 cm2. Consequently, plants in the current experiment did not reach their full leaf area potentials between 150 and 350 cm2 (herpiGny et al., 2011) or 300 cm2 (Bailey and BimoVa, 2009) as described for F. x bohemica. Just as the plants in the greenhouse did not reach as high of nitrogen contents nor produce as high of relative indices with the N�tester as the plants in field trials, the small size of the leaves could likely be contributed to the more limited greenhouse condi- tions as compared to the outdoor sites (weih and nordh, 2005). Conclusions The results of this experiment demonstrated that nitrogen fertiliza- tion has an overall positive correlation with leaf nitrogen content, photosynthesis, and growth of the bioenergy crop Fallopia sacha- linensis var. Igniscum Candy. The existence of significant trends in the influence of phosphate fertilizer on Igniscum Candy could not be demonstrated and requires further investigation. Fig. 8: Influence of the applied nitrogen fertilizer on aboveground biomass (A), plant height (B) and total leaf area (C) at various phosphate lev- els (20, 40, 80 kg P ha-1). (A) Biomass (n = 10, R2 = 0.32), (B) plant height (n = 10, R2 = 0.34), and (C) leaf area (n = 5, R2 = 0.32). Fig. 7: Relationship between leaf nitrogen content and net CO2 exchange (A) and electron transport rate (B) at various phosphate levels (20, 40, 80 kg P ha-1). (A) Linear regression y= 2.518 x + 2.378, R2 = 0.251), (B) Linear regression y= 13.173 x + 48.117, R2 = 0.134). Influence of Nitrogen and phosphate on Igniscum 27 Acknowledgement Thanks to the students Christian Halke and Mirko Milke for their help with the harvest as well as Mandy Turski, Philipp Lange and Thomas Fischer (BTU Cottbus-Senftenberg) for their support with the chemical analyses. Thanks to Yara Research Centre Hanning- hof, Dülmen, Germany for providing the N�Tester. The research was funded by Conpower Rohstoffe GmbH, Planegg, Germany. Thanks to the two reviewers for their helpful recommendations for improve- ments to the manuscript. References adachi, n., terashima, i., taKahashi, m., 1996: Nitrogen translocation via rhizome systems in monoclonal stands of Reynoutria japonica in an oligotrophic desert on Mt Fuji: Field experiments. Ecological Research 11 (2), 175-186. araVindhaKshan, s.c., epplin, F.m., taliaFerro, c.m., 2011: Switch- grass, Bermudagrass, Flaccidgrass, and Lovegrass biomass yield re- sponse to nitrogen for single and double harvest. Biomass Bioenerg. 35, 308-319. aurBacher, J., BenKe, m., Formowitz, B., Glauert, t., heiermann, m., herrmann, c., idler, c., Kornatz, p., nehrinG, a., riecKmann, c., riecKmann, G., reus, d., Vetter, a., Vollrath, B., wilKen, F., willms, m., 2012: Energiepflanzen für Biogasanlagen. Regional- broschüre Niedersachsen. Fachagentur Nachwachsende Rohstoffe e.V. (FNR), Gülzow�Prüzen. BorKowsKy, h., molas, r., 2012: Two extremely different crops, Salix and Sida, as source for renewable bioenergy. Biomass Bioenerg. 36, 234-240. BullocK, d.G., anderson, d.s., 1998: Evaluation of the Minolta �PAD� 502 chlorophyll meter for nitrogen management in corn. J. Plant Nutr. 21 (4), 741-755. cameron, K.c., di, h.J., moir, J.l., 2013: Nitrogen losses from the soil/ plant system: a review. Ann. Appl. Biol. 162, 145-173. doi: 10.1111/ aab.12014 erisman, J., GrinsVen, h., leip, a., mosier, a., BleeKer, a., 2010: Nitro- gen and biofuels: an overview of the current state of knowledge. Nutr. Cycl. Agroecosys. 86, 211-223. Fatichi, s., leuzinGer, s., Körner, c., 2014: Moving beyond photosynthe- sis: from carbon source to sink-driven vegetation modeling. New Phytol. 201, 1086-1095. FrancK, e., 2012: Maisanbau in Niedersachsen. �tandort 36, 194�198. FranzarinG, J., schmid, i., Bäuerle, l., Gensheimer, G., FanGmeier, a., 2014: Investigations on plant functional traits, epidermal structures and the ecophysiology of the novel bioenergy species Sida hermaphrodita Rusby and Silphium perfoliatum L. J. Appl. Bot. Food Qual. 87, 36-45. GansBerGer, m., montGomery, l.F.r., lieBhard, p., 2014: Botanical characteristics, crop management and potential of Silphium perfoliatum L. as a renewable resource for biogas production: A review. Industrial Crops and Products, http://dx.doi.org/10.1016/j.indcrop.2014.09.047. Gianquinto, G., GoFFart, J.p., oliVier, m., Vos, J., macKerron, d.K.l., 2004: The use of hand-held chlorophyll meters as a tool to assess the nitrogen status and to guide nitrogen fertilization of potato crop. Potato Res. 47, 35-80. GuretzKy, J., Biermacher, J. cooK, B., KierinG, m., mosali, J., 2011: Switchgrass for forage and bioenergy: harvest and nitrogen rate effects on biomass yields and nutrient composition. Plant Soil 339, 69-81. heaton, e.a., dohleman, F.G., lonG, s.p., 2008: Meeting U� biofuel goals with less land: the potential of Miscanthus. Glob. Change Biol. 14, 2000� 2014. heaton, e.a., dohleman, F.G., lonG, s.p., 2009: Seasonal nitrogen dy- namics of Miscanthus x giganteus and Panicum virgatum. GCB Bioen- ergy 1, 297-307. helleBrand, h.J., scholz, V., Kern, J., 2008: Nitrogen conversion and nitrous oxide hot spots in energy crop cultivation. Res. Agri. Eng. 54, 58-67. hoaGland, d.r., arnon, d.i., 1950: The water-culture method for growing plants without soil. California Agricultural Experiment Station, Circular 347, University of California, 1-32. hudiBurG, t.w., daVis, s.c., parton, w., delucia, e.h., 2014: Bioenergy crop greenhouse gas mitigation potential under a range of management practices. GCB Bioenergy. doi: 10.1111/gcbb.12152 Kaltschmitt, m., hartmann, h., hoFBauer h., 2009: Energie aus Bio- masse. Grundlagen, Techniken und Verfahren. Springer. Berlin, Heidel- berg, New York. Körner, c., 2013: Growth controls photosynthesis – mostly. Nov. Acta Leo-Nov. Acta Leo- poldina NF 114, Nr. 391, 273-283. lawlor, d.w., 2001: Carbon and nitrogen assimilation in relation to yield: mechanisms are the key to understanding production systems. J. Exp. Bot. 53, 773-787. lawlor, d.w., lemaire, G., Gastal, F., 2001: Nitrogen, plant growth and crop yield. In: Lea, P.J., Morot�Gaudry, J.�F. (eds.), Plant nitrogen, 343� 367. Springer-Verlag, Berlin. leBzien, s., Veste, m., Fechner, h., KoninG, l., mantoVani, d., Freese, d., 2012: The Giant Knotweed (Fallopia sachalinensis var. Igniscum) as a new plant resource for biomass production for bioenergy. Geophys. Res. Abstr. 14, EGU2012-6060. lewandowsKi, i., Böhmel, c., Vetter, a., hartmann, h., 2009: Land- wirtschaftlich produzierte Lignocellulosepflanzen. In: Kaltschmidt, M., Hartmann, H., Hofbauer, H. (eds.), Energie aus Biomasse. Grundlagen, Techniken und Verfahren, 88-108. Springer, Heidelberg. lewandowsKi, i., cliFton-Brown, J.c., scurlocK, J.m.o., huisman, w., 2000: Miscanthus: European experience with a novel energy crop. Bio- mass Bioenerg. 19, 209-227. li, r., werGer, m.J.a., durinG, h.J., zhonG, z.c., 1998: Carbon and nu- trient dynamics in relation to growth rhythm in the giant bamboo Phyl- lostachys pubescens. Plant Soils 201, 113-123. lichtenthaler, h.K., 1987: Chlorophyll and carotenoids: pigments of pho- tosynthetic biomembranes. Methods Enzymol. 48, 350�382. mantoVani, d., Veste, m., Gypser, s., halKe, c., KoninG, l., Freese, d., leBzien, s., 2014: Transpiration and biomass production of the bio- energy crop Giant Knotweed Igniscum under various supplies of water and nutrients. Journal of Hydrology and Hydromechanics 62, 316-323. menGel, K., KirKBy, e., 2001: Principle’s of plant nutrition. Dordrecht: Kluwer Academic Publishers. midGley, G., Veste, m., Von willert, d.J., daVis, G.w., steinBerG, m., powrie, l.w., 1997: Comparative field performance of three different gas exchange systems. Bothalia 27 (1), 83-89. netto, a.l., campostrini, e., GoncalVes de oliVerira, J., Bressan- smith, r.e., 2005: Photosynthetic pigments, nitrogen, chlrophyll a fluo- rescence and �PAD�502 readings in coffee leaves. �ci. Horti. 104 (1), 199-209. paustian, K., andrén, o., clarholm, m., hansson, a.-c., Johansson, G., laGerloF, J. , lindBerG, t., pettersson, r., sohlenius, B., 1990: Carbon and nitrogen budgets of four agro-ecosystems with annual and perennial crops,with and without N fertilization. J. Appl. Ecol. 27 (1), 60-84. pude, r., FranKen, h., 2001: Reynoutria bohemica − eine Alternative zu Miscanthus x giganteus? Bodenkultur 52 (1), 19-27. r deVelopment core team, 2008: R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org. saGe, r.F., pearcy, r.w., seemann J.r, 1987: The nitrogen use efficiency of C3 and C4 plants. I. Leaf nitrogen, growth, and biomass partitioning in Chenopodium album (L.) and Amaranthus retroflexus (L.). Plant Phy- siol. 84, 954-958. seppälä, m., antti, l., JuKKa, r., 2013: Screening of novel plants for bio- gas production in northern conditions. Bioresource Technol. 139, 355- 362. schachtman, d.p. reid, r.J., aylinG, s.m., 1998: Phosphorus uptake by 28 L.A. Koning, M. Veste, D. Freese, �. Lebzien plants: from soil to cell. Plant Physiol. 116, 447-453. schittenhelm, s., 2008: Chemical composition and methane yield of maize hybrids with contrasting maturity. Eur. J. Agron. 29, 72-79. schmitt, a., 1994: Qualitätssicherung von feldmäßig angebautem Pflanzen- material aus Reynoutria sachaliensis unter Berücksichtigung der resis� tenzinduzierenden Eigenschaften. Abschlussbericht zum Forschungsauf- trag 91-NR 002. schreiBer, u., BilGer, w., neuBauer, c., 1994: Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthe- sis. In: �chulze, E.D., Caldwell, M.M. (eds.), Ecophysiology of Photo�In: �chulze, E.D., Caldwell, M.M. (eds.), Ecophysiology of Photo- synthesis, Ecological Studies 100, 49-70. Springer, New York. strašil, z., Kára, J., 2010: Study of knotweed (Reynoutria) as pos- sible phytomass resource for energy and industrial utilization. Res. Agric. Eng. 56 (3), 85-91. VeneKlaas, e.J., lamBers, h., BraGG, J., FinneGan, p.m., loVelocK, c.e., plaxton, w.c., price, c.a., scheiBle, w.-r., shane, m.w., white, p.J., raVen, J.a., 2012: Opportunities for improving phospho- rus�use efficiency in crop plants. New Phytologist 195, 306�320. Veste, m., herppich, w., 1995: Influence of diurnal and seasonal fluctations in the atmospheric CO2 concentration on the photosynthesis of Populus tremula. Photosynthetica 31 (3), 371-378. Veste, m., mantoVani, d., KoninG, l., leBzien, s., Freese, d., 2011: Im- proving nutrient and water use efficiency of Igniscum – a new bioenergy crop. Berichte der Deutschen Bodenkundlichen Gesellschaft. DBG, 4p. http://eprints.dbges.de/739/ Veste, m., Ben-Gal, a., shani, u., 2000: Impact of thermal stress and high vpd on gas exchange and chlorophyll fluorescence of Citrus grandis un- der desert conditions. Acta Hortic. 531, 143-149. Veste, m., quinKenstein, a., Freese, d., 2014: BioSida – Anbau von Sida als neue Kultur für Bioenergie und zur Inwertsetzung degradierter �tand- orte. Arbeitsgemeinschaft industrielle Forschung, Report, Cottbus, 1-35. Vetter, a., heiermann, m., toews, t., 2009: Anbausysteme für Energie- pflanzen; DLG Verlag Frankfurt/Main. Von willert, d.J., herppich, w.B., matyyseK, r., 1995: Experimentelle Pflanzenökologie. Grundlagen und Anwendungen. Thieme Verlag, �tutt- gart. weiland, p., 2010: Biogas production: current state and perspectives. Appl. Microbiol. Biot. 85, 849�860. westerman, r., 1990: �oil testing and plant analysis. Madison, Wisconsin, USA: Soil Science Society of America, Inc. Addresses of the authors: Laurie A. Koning, Maik Veste, CEBra − Centre for Energy Technology Bran- denburg e.V., Friedlieb-Runge-Straße 3, 03046 Cottbus, Germany; Branden- burg University of Technology Cottbus-Senftenberg, Chair of Soil Protection and Recultivation, Konrad�Wachsmann�Allee 6, 03046 Cottbus, Germany Dirk Freese, Brandenburg University of Technology Cottbus��enftenberg, Chair of �oil Protection and Recultivation, Konrad�Wachsmann�Allee 6, 03046 Cottbus, Germany �tefan Lebzien, ENAGRA Biomasse GmbH, Auf der Grub 1, 54472 Mon- zelfeld, Germany