Forestry and Forest Products

TECHNICAL REPORT No. 120 Carbon Isotope Discrimination Of Irrigated And Fertilised grandis Grown In Different Climates.

Ronni L. Korol, Miko U.F. Kirschbaum and Brian J. Myers

March 2000

CSIRO Forestry and Forest Products PO Box E4008 Kingston ACT 2604 Telephone: 02 6281 8211 (+61 2 6281 8211) Fax: 02 6281 8312 (+61 2 6281 8312)

CARBON ISOTOPE DISCRIMINATION OF IRRIGATED AND FERTILISED EUCALYPTUS GRANDIS GROWN IN DIFFERENT CLIMATES.

R.L. KOROL1,2, M.U.F. KIRSCHBAUM2, AND B.J. MYERS2

1USDA Forest Service Rocky Mountain Research Station 800 East Beckwith PO Box 8089 Missoula, Montana 59807 USA

2CSIRO Forestry and Forest Products PO Box E4008 Kingston ACT 2604 Australia

ABSTRACT Previous work suggests that growth of Eucalyptus grandis (Hill ex Maiden) grown in plantations in south-eastern Australia was negatively correlated with annual pan evaporation, with a 3-fold increase in growth across six sites corresponding to a 1000 mm decrease in annual evaporation. As both water and nutrients had been supplied in non-limiting quantities at these sites, these growth differences appeared to be caused by a tree response to the aerial environment. It was hypothesised that stomatal closure under the harsher atmospheric conditions might have been responsible for the observed growth suppression. This should have resulted in lower intercellular CO2 concentrations and lower carbon isotope discrimination at sites with higher evaporation rates. Wood samples from four locations were, therefore, analysed for their carbon isotope discrimination to identify whether there were differences in stomatal responses across the gradient in pan evaporation rates. The analysis showed that carbon isotope discrimination increased by only about 1‰ as annual pan evaporation increased from 1300 to 2040 mm. This -1 implies that mean effective intercellular CO2 concentration was about 16 µmol mol lower at the site with lowest pan evaporation than at the site with highest pan evaporation. This result was the opposite of what had been expected. Stomatal closure in response to higher vapour pressure deficits without direct effects on photosynthetic capacity, therefore, cannot explain the observed differences in growth across these sites. Instead, it appears that the inherent photosynthetic capacity of must have been affected similarly to stomatal conductance by increasing environmental stress.

Korol, Kirschbaum and Myers: Carbon isotope discrimination in Eucalyptus grandis 2

INTRODUCTION highest evaporation rates having the highest VPD. Consistent with that view, Leuning Eucalyptus grandis (Hill ex (1990) observed strong stomatal closure Maiden) is an important commercial with increasing VPD in E. grandis, which species that can achieve high growth rates if might have reduced potential nutrition and water are not limiting photosynthetic carbon gain at the sites with (Cromer et al. 1993). It can also transpire highest VPD. Substantial suppression of large quantities of water and accumulate a tree water use of E. grandis and Pinus large amount of nutrients, which in radiata has also been recorded at the effluent-irrigated plantations may be as intensively studied Wagga Wagga effluent- much a desired trait as a high wood-growth irrigated plantation during periods of high rate. However, we still have insufficient evaporative demand (Benyon et al. 1996a). knowledge of the conditions under which Myers et al. (1998) showed that daily rates of growth and water use can be transpiration of E. grandis was highly maximised, and to what environmental and/ correlated with mean daily VPD up to or management conditions these rates are about 1.5 kPa, but at higher VPDs, there most sensitive. was no further increase in transpiration. Myers et al. (1996) reported the Similarly, Sweeney and Stevens (1997) growth of E. grandis in six irrigated reported that for another irrigated eucalypt plantations established in different climates species, , there in eastern Australia. All these plantations was no significant correlation between daily were irrigated at or above their water-use water use and potential transpiration on rate and supplied with non-limiting levels days when potential transpiration was of nutrients either through application of greater than 3 mm d-1. municipal sewage effluent or fertiliser. Stomatal closure in response to high However, growth across the six sites VPD would have important implications for differed more than 3-fold, with highest the management of effluent-irrigated growth at Gympie in a subtropical climate plantations. One of the key aims in in southern , and lowest growth managing such plantations is to avoid at Robinvale in a semi-arid climate in oversupplying effluent. When it is western Victoria. oversupplied, excess nutrients might leach When growth was expressed as a below the root zone and eventually function of mean annual pan evaporation, a contaminate ground water reservoirs. If negative relationship was obtained. Gympie high VPD during summer months were to had the highest growth and lowest mean lead to stomatal closure, it would reduce annual pan evaporation rates, and trees’ water use at a time of the year when Robinvale had lowest growth and highest potentially the greatest water use is possible pan evaporation rates. Myers et al. (1996) and greatly reduce the total amount of therefore suggested that this might be water, and consequently nutrients, that trees caused by a tree response to vapour can utilise over a year. pressure deficit (VPD), with sites with

Korol, Kirschbaum and Myers: Carbon isotope discrimination in Eucalyptus grandis 3

If stomatal closure occurs in direct measure of the intercellular CO2 response to high VPD, it is also of interest concentration at the time when the carbon to know whether this is a response to was fixed. Moreover, by analysing the lowered photosynthetic capacity, or is itself wood laid down over a whole season, or a the cause of reduced photosynthetic rates. number of seasons, an integrated measure There are essentially two possibilities: of the gas exchange over the whole period either, stomata respond directly to VPD and and the whole canopy can be obtained. reduce photosynthetic rates through These measures would be weighted by the lowered intercellular CO2 concentration, or amount of carbon fixed at different times of both stomatal conductance and inherent the year. photosynthetic capacity may be similarly The present study used carbon affected by VPD so that photosynthesis is isotope ratios to infer the average reduced with little change in intercellular intercellular CO2 concentration (ci) from CO2 concentration. If stomatal conductance four of the plantations reported by Myers et is more sensitive to VPD than the al. (1996). This was done in order to photosynthetic capacity of leaves then distinguish between the possibilities of stomatal closure in response to an external reductions in photosynthesis being caused condition can force the intercellular CO2 by reduced intercellular CO2 concentration, concentration to low values which restricts or occurring without changes in photosynthetic carbon gain, but increases intercellular CO2 concentration. transpiration efficiency. This has been observed in P. radiata in response to water METHODS stress (Korol et al. 1999). On the other Carbon isotope ratios were hand, if both stomatal conductance and the measured on wood samples as described in leaves’ inherent photosynthetic capacity detail by Korol et al. (1999). According to a were similarly sensitive to external theoretical relationship, the carbon isotope conditions, then any reduced photosynthetic 13 composition of a C3 (δ C) is related capacity would not be accompanied by to the intercellular CO2 concentration by altered intercellular CO2 concentration and (Francey and Farquhar 1982; Farquhar et transpiration efficiency. al., 1982, 1989): It is possible to distinguish between 13 13 . δ C (‰) = δ Ca - a - (b-a) (ci/ca) (1) these possibilities by measuring the carbon 13 isotope discrimination in wood of trees where δ Ca is the isotopic composition of grown under these different conditions. CO2 in the atmosphere, approximately - Carbon isotope discrimination of carbon 8.0‰ for current atmospheric conditions, fixed in photosynthesis is directly related to ‘a’ is the discrimination associated with the intercellular CO2 concentration diffusion (4.4‰), ‘b’ is a fitted parameter (Farquhar et al. 1982, 1989). If there are no the value of which is determined primarily further discriminations during subsequent by the discrimination of the enzyme biological reactions in the plant, the isotope Ribulose bisphosphate carboxylase/ ratio of carbon laid down in wood is a oxygenase (27‰), and ci/ca is the ratio of CO2 concentrations inside the and that

Korol, Kirschbaum and Myers: Carbon isotope discrimination in Eucalyptus grandis 4 in the air surrounding the leaf. Carbon sandy loam or sandy clay-loam overlying isotope discrimination, ∆, was calculated sand to medium clays in the B horizon. as: Trees were planted at a spacing of 3x2 m -1 13 13 for a total of 1667 trees ha . δ Ca - δ Cp ∆ = ------(2) 13 Adelaide, South Australia 1 + δ Cp This plantation was established in STUDY SITES early 1990 at the Bolivar sewage treatment works north of Adelaide in eastern South Wood samples were collected from Australia (Hanna et al. 1992). This is the E. grandis trees in four of the irrigated most arid of the four sites with the lowest plantations reported by Myers et al. (1996) annual rainfall of 440 mm and the highest representing a range of climates and growth annual pan evaporation during the rates. Three of the plantations were experiment of 2040 mm. Evaporation was irrigated with nutrient-rich municipal strongly seasonal, ranging from 320 mm in sewage effluent at, or above, their rate of January to 55 mm in June. Mean annual water use while one plantation (Gympie) daily maximum temperature was 22°C, was heavily fertilised and irrigated with ranging from 29°C in January to 15°C in bore water. In all four plantations, water July. The site is located 2 km from the coast and nutrients were considered to have been and is exposed to strong on-shore salt-laden non-limiting to growth over the first three winds. The duplex soils comprise 15-25 cm years, which was the period over which of loamy sand overlying a calcareous clay growth was observed. sub-soil. The sub-soil is saline and sodic and overlies a shallow watertable less than Wagga Wagga, NSW 2 m deep with salinity in excess of 1500 mS m-1. Trees were planted at a spacing of This plantation was established in -1 3x1.5 m or 2222 trees ha . mid-1991 at Wagga Wagga in central New South Wales (Myers et al. 1994). The site Gympie, South Queensland is moderately arid with a mean annual rainfall of about 550 mm which is slightly This plantation was established at winter dominant, ranging from 63 mm in Toolara State Forest near Gympie in south- June to 37 mm in December. Annual pan eastern Queensland in early 1987 (Cromer evaporation during the experiment was et al. 1993). This sub-tropical site is the 1640 mm and was strongly seasonal, most mesic of the four sites with the ranging from 280 mm in January to 23 mm highest mean annual rainfall of 1150 mm in June. Mean annual daily maximum and the lowest mean annual pan temperature was 22°C, ranging from 31°C evaporation of 1300 mm. Mean annual in January to 12°C in July. The site was daily maximum temperature was 27°C, formerly an improved pasture. The duplex ranging from 31°C in January and February soils are a mixture of well-drained red to 21°C in July. The site had grown pine earths and red podsolics. The A horizon is a

Korol, Kirschbaum and Myers: Carbon isotope discrimination in Eucalyptus grandis 5 plantations for more than 35 years before Australia does not form recognisable the current trees were established. The soil annual growth rings so that an analysis is a well-drained, sandy yellow earth with based on annual rings was not possible. no impeded drainage above 1.5 m. Trees Carbon analyses were therefore done were planted at a spacing of 3.4 x 2.6 m. separately on each of the four sections, and Total stand density was 1131 trees ha-1. the mean diameter for each section was recorded. Wodonga, Victoria Between-plot differences were This plantation was established in analysed using standard t-tests or an mid 1980 on the Murray River flood plain ANOVA. The statistical tests were done at α near Wodonga in north-eastern Victoria the 95% confidence level ( = 0.05), and ∆ (Stewart et al. 1988). The site has a the probabilities (p) that the average plot moderately mesic climate with a mean are from the same population are presented. annual rainfall of 713 mm which is winter The stable carbon isotope dominant, ranging from 82 mm in June to composition was determined on finely 38 mm in January. Average annual pan ground wood samples using a VG Isomass evaporation during the experiment was isotope ratio mass spectrometer, using an 1572 mm. Mean annual daily maximum internal standard that was related in turn to temperature was 22°C, ranging from 32°C PDB as the usual reference (Farquhar et al. in January to 13°C in July. The site was 1989). Whole-wood samples were used for formerly an improved pasture. The soil is a analyses because research has shown that pale loam. Trees were planted at a spacing the same trends in δ13C, and subsequently of 3x1.5 m; stand density was 2222 trees ∆, occur in whole wood as in the cellulose ha-1. component, or in leaf material (Leavitt and Long 1982, 1986; Francey 1986). DATA COLLECTION AND ANALYSIS RESULTS AND DISCUSSION

Stemwood samples were collected Figure 1 shows that there was a from nine trees at breast height at each site positive relationship between ∆ and using an increment borer. Tree diameters evaporative demand, with Gympie having had been recorded annually since plantation the lowest discrimination (18.69 ± 0.16‰; establishment. The rings representing the mean ± standard error), and Adelaide first three years of growth were separated having the highest (19.71 ± 0.22‰). While and bulked for isotopic analysis. For the difference between sites of analysis of the effect of tree size on carbon approximately 1‰ is not large, the value of isotope discrimination, samples from trees ∆ at Gympie was significantly different grown at Gympie were used. Nine trees from that at Wagga Wagga (19.64 ± were selected, and for each tree, the stem 0.23‰; p = 0.003) and Adelaide (p = was divided into four concentric sections 0.002), but not significantly different from with equal diameters. E. grandis grown in the value at Wodonga (19.20 ± 0.29‰; p = irrigated plantations in south-eastern 0.15). The Adelaide, Wagga Wagga and

Korol, Kirschbaum and Myers: Carbon isotope discrimination in Eucalyptus grandis 6

reduction in photosynthetic capacity. Had Figure 1: Stand volume (upper panel) and carbon the high VPD led to stomatal closure ∆ isotope discrimination, , (lower panel) at age 3 for without affecting the leaves’ inherent four sites in south-eastern Australia, expressed as a function of annual pan evaporation rate. For photosynthetic capacity, then the sites with Gympie, this is the mean annual pan evaporation high VPD should have had lowest rate, and for the other three sites, it is the amount intercellular CO2 concentrations and lower recorded in the relevant years. Error bars given for carbon isotope discrimination refer to the standard carbon isotope discrimination, which is the error of the data. Lines drawn in the Figure are opposite of what was observed. regression lines fitted to the data. One possible explanation for this surprising result relates to the influence of

100 tree size on stomatal responses. Panek and 80 Waring (1995), for example, found that ) -1 60 carbon isotope discrimination can decrease ha 3 40 with tree size, suggesting lower (m 20 intercellular CO2 concentrations as trees

Stand volume age 3 at volume Stand 0 age. As trees in the present study were all 20.0 sampled over 3 years of growth, and as ∆ ∆ 19.5 ∆ ∆ there were significant differences in ( o /

oo growth, any size induced differences in 19.0 ) stomatal responses might possibly have 18.5 contributed to the observed pattern. 1200 1400 1600 1800 2000 An analysis of the Gympie trees by Annual pan evaporation quarter sections showed that for very young (mm yr-1) trees, when diameters were below 3 cm, ∆

averaged 16.97 ± 0.16‰, while for samples taken when trees were older with diameters Wodonga sites had mean ∆ values that were between 3-7 cm, average discrimination not significantly different from each other had increased to 18.66 ± 0.25‰ but (p = 0.31). changed little with further growth within More important than the between- the observed range (Fig. 2). The 1.7‰ ∆ plot differences observed was the direction difference in between the two smallest of the change; it was opposite of what had size classes was highly significant (p < been expected. Gympie, with the most 0.001). Once diameters were greater than 3 mesic climate and lowest salinity, had the cm, there was no further significant ∆ lowest carbon isotope discrimination and difference in (p = 0.85). therefore the lowest inferred intercellular The effect of tree size on carbon CO2 concentration. Consequently, the data isotope discrimination was not investigated suggest that the high VPDs (from a at the other sites. However, trees were correlation with high pan evaporation rate) largest at Gympie, and if the same size at Wagga Wagga and Adelaide did not lead dependence of carbon isotope to stomatal closure independent of any discrimination occurred at all four sites,

Korol, Kirschbaum and Myers: Carbon isotope discrimination in Eucalyptus grandis 7 then it would have marginally increased the rates observed at Gympie. The data imply that trees had higher transpiration efficiency Figure 2: Carbon isotope discrimination (∆) as a at Gympie, but that would have been no function of stem diameter for trees from Gympie. advantage as water was available in non- Four samples were taken from each of nine trees at limiting quantities. different positions within the stem. Data were pooled for the ranges <3 cm, 3-7 cm, 7-10 cm and >10 cm. In addition to the gradient in VPD Data show means and standard errors. The curve is a across the four sites, there was a gradient in relationship fitted to all individual data and given salinity. This gradient, however, is more by: difficult to quantify. The Gympie plantation 2 ∆ = 18.70 - 3.96 exp(-0.535 d); r = 0.489. would have experienced the lowest salinity because it was irrigated with low salinity bore water and benefited from soil leaching 19 from the high annual rainfall of 1150 mm. The Wodonga plantation also would have

) 18 experienced low-salinity conditions. oo / o Despite being irrigated with effluent ( ∆ ∆ ∆ ∆ 17 averaging 70 mS m-1 EC, the rate of application generally exceeds the rate of 16 tree water use (Stewart et al. 1988). 02468101214 Therefore, salt would have regularly been Stem diameter (cm) leached below the root zone and trees

would have generally been exposed to salinities not much higher than that of the trend apparent in Figure 1 even further. effluent. The Wagga Wagga plantation was irrigated with slightly lower salinity -1 INFERRED INTERCELLULAR CO2 effluent (average 60 mS m ), but because it was irrigated at or below the water use rate CONCENTRATION AND of the plantation, salt accumulated in the root zone to the extent that soil salinity rose TRANSPIRATION EFFICIENCY to between 250 and 300 mS m-1 in mid The observed carbon isotope summer of the fourth year (Benyon et al. discrimination implies that the difference of 1996b). The Adelaide plantation µ -1 experienced the highest salinity because of ca - ci was about 16 mol mol greater at -1 Gympie than at Adelaide, which is the salty effluent (230 mS m ), salt-laden air opposite of what might have been expected from the coast and a very saline water table on the basis of observed leaf-level at 1.5 m depth. responses to VPD. The fact that trees at It might be expected that trees Gympie operated at a lower ci should have experiencing higher salinity may have reduced photosynthesis and consequently reduced stomatal conductance. This could growth. Therefore, the inferred low ci can, then lead to reduced ci and increased therefore not explain the higher growth transpiration efficiency. Hence, the

Korol, Kirschbaum and Myers: Carbon isotope discrimination in Eucalyptus grandis 8 differences in salinity between sites, just highest annual evaporation rates having like the differences in climatic conditions, highest intercellular CO2 concentration, but would lead to the expectation that trees at poorest growth. These results were Gympie should have operated at the highest surprising and inconsistent with other lines ci and trees at Adelaide at the lowest. This of evidence that suggest strong effects of trend is again the opposite of what was the aerial environment on stomatal observed (Fig. 1), and leaves open the behaviour. question why trees in these environments ACKNOWLEDGMENTS showed such marked differences in growth response. Our analysis would exclude The work of Ronni Korol was supported by reduced stomatal conductance leading to the USDA Forest Service RWU-INT-4154, reduced photosynthesis as a possibility. while the work of Miko Kirschbaum was This has important implications for supported by the NGAC Dedicated the management of water application on Greenhouse Research Grants Scheme. We effluent-irrigated plantations. Myers et al. would like to thank Sue Shaw (South (1996) showed that plantation water use Australia Dept Primary Industry) for the could not be calculated as a simple fraction Adelaide samples and Carl Garland for the of pan evaporation rates, and that growth of Gympie samples. We would like to thank plantation trees was severely reduced in Partap Khanna for help in the collection of climates with higher VPD. The present the Wodonga and Wagga Wagga samples, study shows that these reductions in growth John Smith, and Vijay Koul for preparing were accompanied by only small wood samples, Sue Wood for conducting the carbon isotope analyses and Chris differences in intercellular CO2 concentration. The reduced growth under Beadle, Nico Marcar and Melinda Moeur conditions of high VPD presumably was for useful comments on the manuscript. accompanied by reduced water use, but REFERENCES water use was not further reduced by trees Benyon, R.G., Myers, B.J. and Theiveyanathan, operating at lower intercellular CO 2 S. (1996a). Transpiration rates of effluent- concentration, which would have increased irrigated flooded gum and radiata pine the efficiency with which water was used. plantations. Proc. Land Application of CONCLUSIONS Wastes in Australia and New Zealand: Research and Practice, NZ Land Treatment Our results imply that there were Collective Annual Conference, CSIRO only small changes in carbon isotope Canberra, 29 September-5 October. pp discrimination across four sites with widely 228-233. differing climates, and that those changes Benyon, R.G., Myers, B.J., Theiveyanathan, S., indicated trees to operate at highest Smith, C.J., Falkiner, R.A., Polglase, P.J. intercellular CO2 concentration on sites and Bond, W.J. (1996b). Effects of salinity where their growth was poorest. They can on water use, water stress and growth of therefore not help to explain the observed effluent-irrigated Eucalyptus grandis. 3-fold differences in growth, with sites with Proc. Land Application of Wastes in Australia and New Zealand: Research and

Korol, Kirschbaum and Myers: Carbon isotope discrimination in Eucalyptus grandis 9

Practice, NZ Land Treatment Collective Leavitt, S.W., and Long, A. (1982). Evidence of Annual Conference, CSIRO Canberra, 29 13C/12C fractionation between tree leaves September-5 October. pp. 169-174. and wood. Nature 298: 742-744. Cromer, R.N., Cameron, D.M, Rance, S.J., Leuning, R. (1990) Modelling stomatal Ryan, P.A. and Brown, M. (1993). behaviour and photosynthesis of Response to nutrients in Eucalyptus Eucalyptus grandis. Australian Journal of grandis. 1. Biomass accumulation. Forest Plant Physiology 17: 159-175. Ecology and Management 62: 211-230. Myers, B.J., Benyon, R.G., Theiveyanathan, S., Farquhar, G.D., Ehleringer, J.R., and Hubick, Criddle, R.S., Smith, C.J. and Falkiner, K.T. (1989). Carbon isotope discrimination R.A. (1998). Response of effluent-irrigated and photosynthesis. Annual Review of Plant Eucalyptus grandis and Pinus radiata to Physiology and Plant Molecular Biology salinity and vapour pressure deficits. Tree 40: 503-537. Physiology 18: 565-573. Farquhar, G.D., O’Leary, M.H. and Berry, A. Myers, B.J., Bond, W.J., Falkiner, R.A., (1982). On the relationship between carbon O'Brien, N.D., Polglase, P.J., Smith, C.J. isotope discrimination and the intercellular and Theiveyanathan, S. (1994). Wagga carbon dioxide concentration in leaves. Wagga Effluent Plantation Project - Australian Journal of Plant Physiology 9: Technical Report, September 1994. CSIRO 121-137. Division of Forestry Canberra. User Francey, R.J. (1986). Carbon isotope Series No. 17, 123pp. measurements in baseline air, forest Myers, B.J., Theiveyanathan, S., O’Brien, N.D. canopies and . In The Changing and Bond, W.J. (1996). Growth and water Carbon Cycle: A Global Analysis. (Eds. use of effluent-irrigated Eucalyptus grandis J.R. Trabalka, and D.E. Reichle Springer- and Pinus radiata plantations. Tree Verlag, New York, NY. USA.). Physiology 16: 211-219. Francey, R.J. and Farquhar, G.D. (1982). An Panek, J.A. and Waring, R.H. (1995). Carbon explanation of 13C/12C variations in tree isotope variation in Douglas-fir foliage: rings. Nature 297: 28-31. improving the delta13C-climate Hanna, D., Shaw, S. and Boardman, R. (1992). relationship. Tree Physiology. 15: 657-663. Early performance of effluent irrigated Stewart, H.T.L., Hopmans, P., Flinn, D.W., hardwood plantations at Bolivar, South Hillman, T.J. and Collopy, J. (1988). Australia. Proc. Catchments of Green Evaluation of irrigated tree crops for land Conference, Adelaide, 23-26 March.. disposal of municipal effluent at Wodonga. Greening Australia, Canberra. pp. 207-214. Albury-Wodonga Develop-ment Corp. Korol, R.L., Kirschbaum, M.U.F., Farquhar, Tech. Rep No. 7, 28 pp. G.D. and Jeffreys, M. (1999). The effect of Sweeney, S.M. and Stevens, R.M. (1997). water status and soil fertility on the C- Growth and water-use of eucalypt trees isotope signature in Pinus radiata. Tree irrigated with saline drainage water. Physiology 19: 551-562. Irrigation Science 17: 173-181. Leavitt, S.W., and Long, A. (1986). Stable- carbon isotope variability in tree foliage and wood. Ecology 67(4): 1002-1010.