Carbon Inputs by Wheat and Vetch Roots to an Irrigated Vertosol

1 CARBON INPUTS BY ROOTS OF WHEAT AND VETCH SOWN IN ROTATION WITH IRRIGATED COTTON ON PERMANENT BEDS IN A VERTISOL

N. R. Hulugalle, T.B. Weaver and L.A. Finlay Australian Cotton Research Institute, NSW Department of Primary Industries and Cotton Catchment Communities Co-operative Research Centre, Locked bag 1000, Narrabri, NSW 2390, Australia

Abstract Research on the amounts of carbon that can be added to Vertosols of NSW and Queensland by crop roots in irrigated cotton farming systems is sparse. The objective of this study was to determine the amounts of carbon added to soil by roots of wheat (Triticum aestivum L.) and purple vetch (Vicia benghalensis) sown in rotation with irrigated cotton (Gossypium hirsutum L.). Measurements were made from 2008 to 2010 in an on-going experiment near Narrabri, northern NSW using a combination of soil cores and minirhizotron observations. The experimental treatments were: cotton monoculture, cotton-vetch, cotton-wheat where wheat stubble was incorporated into the beds with a disc-hiller, and cotton-wheat-vetch where wheat stubble was retained as in-situ mulch. Vetch in cotton-vetch and cotton-wheat- vetch rotations was killed by a combination of mowing and contact herbicides and the residues retained as in situ mulch. Root length per unit area of vetch in cotton-wheat-vetch and both wheat treatments was comparable, although wheat had a higher concentration of roots in surface 0.10 m. Root growth of the vetch in cotton-vetch was sparse. Root carbon available for addition to soil was greater with vetch than with wheat and was in the order of vetch in cotton-wheat-vetch (5.1 t C/ha/year) > vetch in cotton-vetch (1.9 t C/ha/year) > wheat in cotton-wheat (1.6 t C/ha/year) = wheat in cotton-wheat-vetch (1.7 t C/ha/year). Intra-seasonal root mortality accounted for 12% of total root carbon in vetch and 36% in wheat. The remaining fraction consisted of carbon in the root mass at the end of the growing season. Carbon sequestered by root inputs of the rotation crops was estimated to be of the order of 0.34 t C/ha/year for the vetch and wheat crops in the cotton-wheat-vetch rotation, 0.10 t C/ha for the vetch in the cotton-vetch rotation and 0.08 t C/ha for wheat in the cotton- wheat rotation. The cotton-whet-vetch sequence, was therefore, potentially the most effective in sequestering carbon from roots. Key words Haplustert; Sequestration; Rotation; Cotton; Farming system Introduction Sowing cereal crops such as wheat (Triticum aestivum L.)or leguminous crops such as vetch (Vicia villosa Roth.) or faba bean (V. faba L.) in rotation with cotton (Gossypium hirsutum L.) in irrigated Vertosols can be beneficial to soil quality in terms of improving soil structure (i.e. better soil water storage, aeration and drainage) and soil N, although expected increases in soil carbon have not occurred (1,2). To the contrary, in most locations soil carbon stocks in cotton-based farming systems have declined or at best, have stabilised (2). These results are similar to those from many other row cropped semi-arid annual cropping systems, irrigated and dryland (3,4,5,6,7). In contrast, increases in soil carbon stocks with time have been reported for land that has been under pasture or where a pasture is sown in rotation with an annual crop (3,4,5,6,7). Chan et al (4) have suggested that this may be related to the carbon added to soil through the roots of the pasture. Furthermore, some authors (8,9) have reported that carbon derived from crop roots contribute more to soil carbon and its stability than that from above-ground residues. Few studies have, however, examined the amounts of carbon that can be added to soil by the roots of various crops in cotton farming systems to furrow- irrigated Vertosols. These suggest that depending on seasonal conditions, genotype, cropping system and crop health, the amounts of carbon added per season by cotton and rotation crops 2 can differ: 2.3 t C/ha (range of 0.5 to 4 t C/ha) by cotton, 7.7 t C/ha by corn (Zea mays L.) (range of 5.5 to 9.3 t C/ha) and 10.9 t C/ha by grain sorghum (Sorghum bicolour (L.) Moench) (range of 9 to 12.5 t C/ha) (2,10,11,12). Assuming that sequestration rates were of the order of 5% of total carbon inputs, carbon from root materials that could be sequestered in soil would range from 0.1 to 0.6 t C/ha. Published information on the amounts of carbon added to Vertosols by root systems of the more common rotation crops such as wheat and vetch have not been reported in the literature. The objective of this study, therefore, was to determine the amounts of carbon added to soil by winter rotation crops such as wheat and purple or Popany vetch (Vicia benghalensis L., syn. Vicia atropurpurea Desf.) sown in rotation with irrigated cotton through root turnover during the growing season and decay of root systems thereafter. Measurements were made from 2008 to 2010 in a long-term experiment using a combination of soil cores and minirhizotron observations. Materials and methods Site and experimental layout Wheat and vetch root growth were measured during the growing seasons (autumn-springr) of 2008, 2009 and 2010 in an experiment that had commenced in 2002 at the Australian Cotton Research Institute (ACRI), near Narrabri (149o47’E, 30o13’S) in New South Wales (NSW), Australia. Narrabri has a sub-tropical semi-arid climate, BSh (13) and experiences four distinct seasons with a mild winter and a hot summer. The hottest month is January (mean daily maximum of 35 oC and minimum of 19 oC) and July the coldest (mean daily maximum of 18 oC and minimum of 3 oC). Mean annual rainfall is 593 mm. The soil at the experimental site is an alkaline, self-mulching, grey clay, classified as a self-mulching, grey Vertosol; very-fine (14) or a fine, thermic, smectitic, Typic Haplustert (15). Mean particle size distribution in the 0-1 m depth was: 64 g/100g clay, 11 g/100g silt and 25 g/100g sand. Average ESP in the 0.6-1.2 m depth was 15 but did not exceed 6 in the shallower depths. The experimental treatments consisted of four cotton-based rotation systems sown on permanent beds: cotton monoculture (summer cotton-winter fallow-summer cotton), cotton- vetch (summer cotton-winter vetch-summer cotton), cotton-wheat (summer cotton-winter wheat-summer and winter fallow-summer cotton ) where wheat stubble was incorporated into the beds after harvest with a disc-hiller, and cotton-wheat-vetch (summer cotton-winter wheat-summer fallow-autumn and winter vetch-summer cotton) where wheat stubble was retained as an in-situ mulch into which the following vetch crop was sown. Vetch, which is prostrate, leguminous crop, in cotton-vetch and cotton-wheat-vetch rotations was killed during or just prior to flowering through a combination of mowing and contact herbicides (16), and the residues retained as in situ mulch into which the following cotton was sown. The experiment was laid out as a randomized complete block with three replications and designed such that both cotton and rotation crop phases in the last two rotation treatments were sown every year. Individual plots were 165 m long and 20 rows wide. The rows (beds) were spaced at 1-m intervals with vehicular traffic being restricted to the furrows. All treatments were furrow irrigated with about 100 mm of water when rainfall was insufficient to meet evaporative demand. Crop management Cotton: In NSW cotton is sown in October. Roundup-Ready® cotton was sown in the experiment from 2002 to 2005, and Bollgard® II –Roundup-Ready®-Flex® cotton thereafter. Cotton was picked during late April or early May with a 2-row picker after defoliation in early April. After cotton-picking, the cotton was slashed and incorporated into the beds with a disc-hiller. Details of cotton management in this experiment have been reported by Hulugalle et al. (10). 3 Wheat and vetch: Namoi woolly pod vetch (Vicia villosa Roth.) was sown in the experiment from 2002 to 2006 and purple or Popany vetch thereafter. Wheat was sown at a rate of 60 kg/ha and vetch at 20 kg/ha. Wheat received 20 kg N/ha as urea by broadcasting at sowing during late May or early June, and 60 kg N/ha during late July or early August. Vetch in the cotton-wheat-vetch rotation was sown into wheat stubble during late February or early March following summer rains, and that in cotton-vetch rotation, after cotton picking and pupae- busting, in late May or early June. N fertiliser was not applied to vetch. Phosphorus was applied only during September 2010 to all plots at a rate of 25 kg P/ha as single superphosphate. Depending on in-crop rainfall and stored soil water, wheat and vetch received up to two irrigations of 100 mm per season. Wheat was harvested with a grain harvester during late November or early December. Crop root measurements Root growth in the surface 0.10 m was measured with the core-break method using 0.10 m diameter cores (17). Soil cores were used for the surface 0.10 m because minirhizotron measurements underestimated root growth in this depth, presumably due to light leakage and temperature effects (18). A sub-sample of the cores taken from the surface 0.10 m in each plot at each time of sampling was transported to the laboratory in labelled and sealed plastic bags and stored in a cold room (4o C) for root washing and separation. The root samples were soaked in a warm water solution containing a 2:1 10% sodium hexametaphosphate: 0.1 M sodium hydroxide for a period of ~4 hours. Once dispersed, root and other organic material were separated from soil by flotation and decantation and by washing through a 0.212 mm sieve. The organic material obtained (including roots) was then stained with a 0.1% congo red solution for a period of 4-8 hours (depending on age of crop), followed by washing in absolute alcohol (19, 20). The congo red stains the live roots a bright red colour, whereas the dead organic material remains black. The live roots were separated from the dead material under a bright light using a forceps after spreading the sample in a shallow white, plastic tray filled with ~5 mm of water. The live roots were then stored in a 25% alcohol solution until their length was measured using a modified Newman’s line interception method during 2008 with a 100 mm x 100 mm grid (18,20) and scanned and measured with WINRHIZO® software during 2009 and 2010. The root samples were then oven-dried and weighed. Relationships were derived between root number, root length and root weight, and the root length and weight in each core estimated. Relative root length (root weight/root length) was also calculated. Carbon concentration in the oven-dried root material was measured by combustion with a LECO CHN 2000® analyser. Root growth in the 0.10 to 1.0 m depth was measured at 0.10 m depth intervals with a “Bartz” BTC-2 minirhizotron video camera® and I- CAP image capture system® . The video camera was inserted into clear, plastic acrylic minirhizotron tubes (50 mm diameter) installed within each plot, 30o from the vertical. The operating and measurement procedures used were those described by Johnson et al. (21). Depending on crop growth stage and environmental conditions, measurements were taken at intervals of approximately 3 to 5 weeks. Root images were captured in two orientations, left and right side of each tube, at each time of measurement and analysed with RooTracker 2.03® (22) to estimate selected root growth indices. The results for each orientation at each depth and over the entire measured profile were summed to provide an assessment of root growth over a 360o plane of vision. The indices evaluated were the length and number of live roots at each time of measurement, number of roots which changed length, number and length of roots which died (i.e. disappeared between times of measurement), new roots initiated between times of measurement and net change in root numbers and length. The above, together with relative root lengths and root C concentrations of samples taken from the previously described soil cores, were then used to calculate several other indices of root growth; viz. (1) Root length density, LV, for individual depth intervals and root length per unit area to a depth of 1 m, LA; (2) Root carbon at end of season, Croot = Sum of net changes 4 in root carbon between times of measurement in all depths where, for individual depths and between times of measurement, the net change in root carbon was calculated as: Net change in root length x Relative root length x Root carbon concentration) (3) Root carbon added to the soil during season, Clost = Sum of root carbon added to soil due to root death between times of measurement in all depths where, for individual depths and between times of measurement, root carbon added to soil was calculated as: Length of roots which died x Relative root length x Root carbon concentration; (4) Root carbon which could be potentially added to soil, Ctotal = Croot (2) + Clost (3). Details of the previously mentioned calculations are reported in Hulugalle et al. (10).

Data were analysed after loge transformation with analysis of variance using a randomised complete block design. During the 2010 winter, vetch in replicate one of the cotton-wheat- vetch rotation was severely damaged by cotton aphids (Aphis gossypii). The aphid damaged plot was analysed as a separate treatment using a mixed models approach to quantify the effect of the aphids on root growth. Results and discussion Vetch root length

Fig. 1. Seasonal variation in root length per unit area, LA of rotation crop. ●, cotton-vetch; (vetch); ○, cotton- wheat-vetch (vetch); ▼, cotton-wheat (wheat); ∆, cotton-wheat-vetch (wheat); ■, cotton-wheat-vetch (aphid- damaged vetch).

Peak LA of vetch (early flowering) in the cotton-wheat-vetch rotation (“early-sown vetch”) exceeded that of vetch in the cotton-vetch rotation (“Late-sown vetch”) during 2009 and 2010 by an average of 2½ times whereas during 2008 values in both treatments were generally similar with that in early-sown vetch being marginally higher (by 12%) (Fig. 1, Table 1). These inter-seasonal differences may be due to differences in the length of growing season (5-6 months for the early-sown vetch and 3-4 months for the later-sown vetch), in- crop rainfall and soil water storage, and increasing winter weed numbers in the late-sown vetch with increasing duration of the experiment. Due to high populations of winter weeds such as dead nettle (Lamium amplexicaule L.) and to a lesser extent, milk thistle (Sonchus oleraceus L.) in the late-sown vetch, control measures included application of herbicides (Roundup®, Diuron®). Although relatively minor damage occurred to above-ground parts of vetch, vegetative growth recovered. It appears, however, that inhibition to root growth may have been greater and more long-lasting. The effect of winter weeds on the early-sown vetch was limited because it was able to establish itself during later autumn before the abovementioned winter weeds germinated, and was thus able to outcompete them. 5 Aphid-damage during 2010 to the early-sown Table 1. Analyses of variance for ln LA, root vetch resulted in a significant reduction in LA length per unit area. Back-transformed values (Fig. 1, Table 1). Average LA during late are shown in Figs. 1. *, ** and *** indicate vegetative growth and early flowering in June that means differed significantly at P < 0.05, P 2 < 0.01 and P < 0.001, respectively. 2010 was of the order of 54 km/m in undamaged vetch and 21 km/m2 in aphid- Year and date F value damaged vetch, i.e. a reduction of 61%. 2008 Reduction of root biomass due above-ground 27 June (df = 3,30) 18.75*** herbivory has been reported for many plant 5 August (df = 3.30) 4.21* 11 September (df = 2,22) 0.44 species, ranging from aphid damage in annual 26 September (df = 2,22) 0.63 grasses such as Poa annua (23) to damage 30 September (df =1,14) 9.09** caused by pine sawflies (Neodiprion spp.) to 2009 woody perennials such as Pinus ponderosa (24). 6 July (df = 3,30) 72.66*** Van Dam and Bezemer (25) have suggested that 4 August (df = 3.30) 51.95*** 31 August (df = 2,22) 27.91*** this response is mediated through an alteration 7 October (df =1,14) 0.60 in the internal plant hormone balance. Insect 2010 damage to above-ground plant organs may, 14 April (df = 1,7) 0.38 therefore, inhibit root functions such as water 5 May (df = 1,7) 0.43 and nutrient extraction and ultimately, amounts 10 June (df = 1,7) 5.79* 23 June (df = 2,29) 53.66*** of carbon in crop roots with the intensity of the 16 August (df = 2,22) 28.12*** changes varying between tap and feeder roots 21 September (df = 2,22) 13.61*** (26). 27 October (df = 1,14 ) 0.58 25 November (df = 1,14 ) 0.03 Wheat root length

LA of wheat was comparable to that of the early- sown vetch but was generally higher than the late-sown vetch (Fig. 1, Table 1). Consistent differences in LA were absent between wheat treatments. Significant differences were present only on three occasions: during grain filling/ripening in 2008 and flowering (anthesis)/milking during 2010 when LA of wheat in the cotton-wheat rotation was greater than that in the cotton-wheat-vetch rotation by 2.6 and 1.7 times, respectively (Fig. 1, Table 1). This was mainly due to a high concentration of roots in the beds (0-0.1 m) of the former treatment. During stem elongation in 2009, LA of wheat in the cotton-wheat-vetch rotation was greater than that in the cotton-wheat by 3.4 times. Again, this was due to a higher concentration of roots in the surface 0.2 m in the latte rotation. Overall, wheat tended to have higher concentrations of roots in the surface 0.1 m than the either of the vetch crops. This may be related to the application of N fertiliser to wheat. Root proliferation in soil patches enriched with water and nutrients is a strategy that annual crops employ to improve their survivability and competitiveness (27). Root carbon indices of vetch and wheat

Over the three years, average total root C potentially available for addition to soil (Ctotal) was in the order of vetch in cotton-wheat-vetch (510 g/m2) > vetch in cotton-vetch (192 g/m2) > wheat in cotton-wheat (160 g/m2) = wheat in cotton-wheat-vetch (173 g/m2) (Fig. 2, Table 3). Ctotal was, thus, more with vetch than with wheat: vetch in cotton-wheat vetch was 3⅓ times greater and that in cotton-vetch 1.2 times greater. Consequently, wheat was less effective in returning carbon to the soil. As LA of both wheat crops was comparable to that of early-sown vetch and greater than that of the later-sown vetch, this was probably related to the finer root system of the wheat. Averaged over the three years, relative root weight of vetch was 29.8 mg/m and that of wheat, 9.7 mg/m (t = 3.15, df = 35, P < 0.01). Assuming an average soil carbon sequestration rate of 5% (the literature proposes values ranging between 3 and 15% of plant inputs, albeit for above-ground residues), carbon sequestered by root inputs of the 6

Table 2. Analyses of variance for root carbon indices. Back-transformed values are shown in Fig. 6. *, ** and *** indicate that means differed significantly at P < 0.05, P < 0.01 and P < 0.001, respectively Parameter F-value 2008 (df = 3,30) 2009 (df = 3,30) 2010 (df = 4,29) Ln (Carbon in crop roots at end of 10.07*** 21.10*** 3.52* season) Ln (Carbon in roots that died) 4.18* 19.58*** 19.93*** Ln (Total carbon available for addition to 12.71*** 26.52*** 3.87* soil) rotation crops can estimated to be of the order of 0.34 t C/ha/year for the vetch-wheat component of the cotton-wheat-vetch rotation, 0.10 t C/ha for the vetch in the cotton-vetch rotation and 0.08 t C/ha for wheat in the cotton-wheat rotation. As the C/N ratio of vetch roots were of the order of 18 (2), and thus, readily decomposable, the values for rotations that contained vetch may well be lower. Nonetheless, the abovementioned estimates may understate the potential amounts sequestered by root systems in all the crop rotations as Katterer et al. (9) in a cold temperate, subhumid environment reported that C sequestration by root inputs were 2.3 times more than that by above-ground inputs. Although a similar study has not been conducted for the irrigated cotton-farming environment of northern New South Wales (warm to hot temperatures, high N inputs and frequent wet/dry cycles), it is unlikely that the same magnitude of difference between C sequestration rates for above- and below ground residues reported by Katterer et al. (9) would prevail due to enhanced microbial activity and thus, higher soil respiration rates (3,5).

Fig. 2. Effect of crop rotation on root C indices

C in root mass remaining at the end of the season (Croot) followed a similar trend to Ctotal such that vetch in cotton-wheat-vetch > vetch in cotton-vetch > wheat in cotton-wheat = wheat in cotton-wheat-vetch. C added to soil through intra-seasonal root death (Clost), however, differed, and was in the order of wheat in cotton-wheat = wheat in cotton-wheat-vetch > vetch in cotton-wheat-vetch > vetch in cotton-vetch . Although there was much variation across seasons, average Croot in vetch crops accounted for 88% of Ctotal and Clost, 12%, whereas in wheat crops average Croot and Clost accounted for 64% and 36%, respectively of Ctotal. The difference between crop species with respect to these indices is probably largely related to their management; viz. wheat was allowed to mature until grain ripening whereas vetch was terminated at flowering, although a species effect cannot be excluded. Values of Clost for wheat are comparable to those reported for temperate zone wheat (~40-60%) and unstressed cotton (25-49%) bit are lower than that of corn (11%) (11,12,28). 7 It was previously noted that the weed control measures implemented in the cotton-vetch rotation appeared to have inhibited vetch root growth. This was also reflected in the vetch’s root carbon indices. In comparison with values for 2008, average Croot during 2009 was 66% lower and during 2010, 72% lower. At the same time, in comparison with vetch in cotton- wheat-vetch, average Croot of vetch in cotton-vetch was 68% lower, Clost 78% and Ctotal 62%, with greatest differences occurring during 2009 and 2010. In comparison with undamaged vetch, damage by aphids during 2010 to vetch resulted in Ctotal decreasing by 73% (Fig. 6). The proportion of root mass at termination during 2010 (relative to Ctotal) decreased from 94% in undamaged plants to 80% in aphid-damaged plants. Proportional root mortality, in contrast, increased from 6% when undamaged to 20% in aphid-damaged vetch. Aphid-damage to vetch resulted, therefore, in a root system that contributed less carbon to soil due to an overall reduction in biomass (see discussion of LA results) and suffered from greater level of intra-season root mortality. Conclusions Root length per unit area of early sown vetch and wheat were comparable, although the latter tended to have a higher concentration of roots in surface 0.10 m. Root growth of the later- sown vetch was sparse. Root growth was inhibited by aphid damage to above-ground parts of vetch. Root carbon available for addition to soil was greater with vetch than with wheat and was in the order of vetch in cotton-wheat-vetch (5.1 t C/ha/year) > vetch in cotton-vetch (1.9 t C/ha/year) > wheat in cotton-wheat (1.6 t C/ha/year) = wheat in cotton-wheat-vetch (1.7 t C/ha/year). Intra-seasonal root mortality accounted for 12% of total root carbon in vetch and 36% in wheat. The remaining fraction consisted of carbon in the root mass at the end of the growing season. Carbon sequestered by root inputs of the rotation crops was estimated to be of the order of 0.34 t C/ha/year for the vetch and wheat crops in the cotton-wheat-vetch rotation, 0.10 t C/ha for the vetch in the cotton-vetch rotation and 0.08 t C/ha for wheat in the cotton-wheat rotation. The cotton-whet-vetch sequence, was therefore, potentially the most effective in sequestering carbon from roots. Acknowledgements Funding for this research was provided by the Cotton Catchment Communities Co-operative Research Centre and Cotton Research and Development Corporation of Australia. References 1. Hulugalle, N.R., and Scott, F. 2008. A review of the changes in soil quality and profitability accomplished by sowing rotation crops after cotton in Australian Vertosols from 1970 to 2006. 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