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Deep rooting and drought screening of cereal crops: A novel field-based method and its application
Field Crops Research 112 (2009) 165–171
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Deep rooting and drought screening of cereal crops: A novel field-based method and its application B.M. McKenzie a,*, A.G. Bengough a, P.D. Hallett a, W.T.B. Thomas a, B. Forster a,1, J.W. McNicol b a b
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK BioSS, Invergowrie, Dundee DD2 5DA, UK
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 September 2008 Received in revised form 11 February 2009 Accepted 22 February 2009
Lack of water is a major limitation to crop production, particularly where roots of cereal crops are not able to access water stored in the subsoil. One way that roots penetrate the subsoil to access water is by following natural biopores – paths created by roots from previous crops, or as burrows from soil fauna. Burying a mesh layer horizontally in the soil can prevent root penetration to the subsoil. We used this technique with the novel modification that the mesh was punctured to create a defined number of holes per unit area; controlling access to the subsoil and to the water therein. The holes were of similar size to biopores. Five barley genotypes were late sown and grown during a dry summer. Monitoring of crop performance included plant height, leaf area and Normalized Difference Vegetation Index (NDVI). Crops grown with unrestricted access to the subsoil outperformed crops with limited or no access to the subsoil. Crops grown with controlled, limited access to the subsoil performed better than those with no access and the performance was generally related to amount of access. Changes in soil water content were in line with the amount of root access to the subsoil; confirming the association between subsoil water and crop growth and development in drought conditions. While there were no significant interactions between the genotypes and treatments used here, the method offers promise for studying some aspects of cereal ecophysiology and could be used to identify promising germplasm that may be of interest in plant breeding. Further testing is required to adapt the method for a wider range of crop types and soil conditions and testing for crops grown to maturity. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Biopores Cereals Root growth Drought stress Subsoil water Plough pan
Even severely drought stressed crops may leave considerable plant available water in the subsoil (Passioura, 1985). High mechanical impedence of the soil, caused by compaction below the plough layer and the strengthening of soil as it dries, restricts root growth to shallow depths (Gregory, 2006). Therefore, of all phenotypic traits that can be altered to improve drought resistance of cereal crops, increased penetration and extension of root systems probably offers the greatest potential (Passioura, 2007). By penetrating deeper into soil, crop roots potentially access and exploit a greater volume of stored water. There is potential to learn from endemic plants that have an ecological niche in drought-prone climates and shallow soils. Poot and Lambers (2008) found rare endemic species with specialized root systems that are able to explore a large surface area at the soil-rock boundary for fissures so that roots can extend
* Corresponding author. Tel.: +44 1382 568 529; fax: +44 1382 568 502. E-mail address: [email protected] (B.M. McKenzie). 1 Current address: BioHybrids International Ltd., PO Box 2411, Earley, Reading RG6 5FY, UK. 0378-4290/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2009.02.012
to depth and access water. Arable soils can also contain fissures and other large pores that cross plough pans, compacted layers after machinery passage, or natural horizon boundaries (Vocanson et al., 2006). Biopores may be old root channels or burrows made by earthworms or other soil fauna. Cracks generated by shrinking and swelling may also leave planes of weakness that could be followed by roots (Passioura, 1991). There is considerable evidence that these pores provide preferential pathways for root growth and are extremely important to crop performance (Ehlers et al., 1983). The ability of roots of a specific crop cultivar to exploit these pores could thus be a major determinant of drought avoidance. Soil management can increase the abundance of fissures or macropores, with implications to crop performance in dry years. Lampurlanes et al. (2001) compared the root growth of barley and soil water status under deep and no-till systems. In years of low rainfall they found that the no till system performed better by creating an environment that encouraged roots deep into the soil profile. The deeper root proliferation with no-till was attributed to improved soil structure and particularly biopores providing pathways for roots.
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The nature and number of biopores per cross-sectional area have been considered by several authors. Smettem and CollisGeorge (1985) used a peel method to characterise numbers of biopores greater than 0.5 mm diameter and found at 10 cm depth there were approximately 470 biopores per square meter. Ehlers (1975) found biopore diameters of between 2 and 11 mm. The orientation of biopores in the soil profile may become nearer to vertical with depth (McKenzie and Dexter, 1993), thus providing suitable paths for roots to follow. Where hard soil layers impede root elongation Passioura (1991) has reported the clumping of several roots in a single biopore. Hirth et al. (2005) studied the behaviour of roots in artificial biopores and found that the orientation of the biopore and its surface properties influenced whether a root was able to move from the biopore into the bulk soil. Jakobsen and Dexter (1988) modelled the influence of biopores on root growth and cereal production for 2 contrasting environments. Their modelling suggested there may be an optimum density of biopores. In dry conditions too many biopores, providing unlimited access to the subsoil, could lead to early depletion of stored water and perhaps leave insufficient reserves for grain filling; while too few biopores prevent access by roots to subsoil water and limit yield. The modelling of Jakobsen and Dexter (1988) indicates that the optimum will be different for different plant densities per unit area. Thus the number of biopores can be described on per unit area or a per plant basis. To screen crops for deep rooting often requires soil samples be collected from a range of depths beneath a crop before roots are washed from the collected soil. The root length density at depth is an indication of the access the crop has to any subsoil water, however, the measurement of root material is very laborious (Gregory, 2006). Root pruning has been tried as a way to restrict lateral root spread and thus limit access to water and nutrients (Ma et al., 2008). Agronomic approaches to improve the drought tolerance of cereal crops include deep sowing so that the seedling emerges from depth and the root system is not exposed to hot dry conditions (Reynolds et al., 2005). Another possible mechanism for deep sown crops to be more drought tolerant is that they have more root axes at depth and thus have a greater chance of finding a biopore or other path into the subsoil where water may be available. In this paper we study the effect of restricting root growth in the subsoil using mesh (permeable to fluids) that contains varying densities of puncture holes (mimicking biopores). This confines root systems to the surface soil, except for roots that can elongate through the puncture holes in the mesh. The technique varies, in a controlled manner, the access of crop roots to water in the subsoil, providing a differential stress to the crop. This is proposed to allow study of the response of field-grown crops, to varying degrees of water stress by restriction of root growth in the subsoil. We tested the system for 5 barley (Hordeum vulgare) genotypes. 1. Materials and methods 1.1. Soil properties and meteorological data The experiment was conducted at the Scottish Crop Research Institute (SCRI); (56.27N 3.40W) during 2006. The soil type is Balrownie series, a Stagnic Cambisol in the FAO classification, with a sandy loam texture derived from red sandstone sediments (Bell and Hipkin, 1988). Plant available water of the surface soil was calculated from the water retention curve as the difference between field capacity (10 kPa matric suction) and wilting point (1500 kPa matric suction). For our site the surface soil stored 40 mm of plant available water in the surface 200 mm. Prior to establishing this experiment no crop had been grown in the soil for
several years. The area had been used for propagating horticultural plants in pots. Irrigation applied to the pots and the substantial winter and spring rainfall (data not shown) ensured that the subsoil stored considerable plant available water. Weather data were recorded at a meteorological station located within 50 m of the experiment site. Potential evapotranspiration, ETo, was taken from an automated weather station (Campbell Scientific) located less than 500 m from the site. 1.2. Experimental design and soil preparation A field experiment was established as a split-plot design with 5 main treatments repeated 3 times. The 5 main treatments were different barley (Hordeum vulgare) genotypes. Within each main treatment 6 levels of root restriction were applied as sub-plots; designed to provide different access to subsoil water. Each main treatment was a crop strip 6 m long and 1.2 m wide. The surface 0.2 m of soil was first removed and the exposed subsoil cultivated to a depth of 0.2 m (i.e. 0.4 m below the original surface) with a power harrow. Strips of nylon mesh (Clarcor, UK) were laid as 6 m long and 1.5 m wide. The mesh had holes of 40 mm to provide an open area of 26% that could be penetrated by water and gases, but not by barley roots. Preliminary experiments showed that the mesh provided a boundary to roots while permitting air and water transport. The 6 m lengths were divided into 6 m 1 m length sub-plots. For each genotype treatment and replicate, a different root restriction treatment was established. One of the 6 sections the mesh was removed completely (Unrestricted). The other 5 treatments had 0, 100, 200, 583 or 1167 holes m 2 inserted into the mesh to simulate biopores. Nail-boards were used to make puncture holes, which had a regular grid for all hole densities. The nails were 2 mm in diameter, similar to that of an earthworm burrow (Ehlers, 1975; Smettem and Collis-George, 1985). To drive the nails through the mesh, a pressure of approximately 13 kPa was applied. This was simply generated by a person standing on the board. Blank nailboards were used to apply the same stress to the sub-plots with 0 holes m 2 and no mesh. This light compaction ensured good contact between the mesh and the sub-soil and the surface soil was then returned to cover the mesh (with no compaction by vehicular traffic). The replaced soil was raked before sowing the barley seed. 1.3. Genotypes and crop management Five modern barley genotypes (Golden Promise, Optic, Derkado, B83-12/21/5 and Morex) were allocated randomly to each replicate and sown into the 6 m long strips. Golden Promise is a semi-dwarf barley carrying a dwarfing allele at the ari-e locus and is relatively shallow rooting (AlMenaie, 2003). Derkado and B83-12/21/5 are parents of a mapping population that segregates for 2 different dwarfing genes. Derkado carries the sdw1 dwarfing gene whereas B83-12/21/5 carries a similar ari-e dwarfing gene to Golden Promise. The sdw1 dwarfing gene is associated with increased root mass during the vegetative growth period (Chloupek et al., 2006). Optic carries the sdw1 dwarfing gene. It was the most popular spring barley malting cultivar in the UK during the late 1990s and is still widely grown. A large number of ESTs from a variety of stress treatments have been derived from Optic and structured mutant populations have also been developed from it (Caldwell et al., 2004). Morex is a spring malting barley that was widely grown in the Pacific North West region of the USA. Unlike the other genotypes, it has a 6-row head and is relatively early heading. A number of barley genomics tools have also been based on Morex, notably a Bacterial Artificial Chromosome library that is estimated to provide 6.3 coverage of
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the genome (Yu et al., 2000), a resource that is of great value in gene isolation. The plots were sown in 8 rows, with an effective width of 1.2 m and to a target density of 365 established plants m 2. The number of holes through the mesh per plant was approximately 0, 0.27, 0.55, 1.60, and 3.20. These holes per plant values are used to describe the treatments. The treatment without a mesh is referred to as unrestricted. The barley was sown late (23 June 2006), at least 10 weeks after the usual sowing date, but at a time when drought conditions were most likely. The result was that early crop growth occurred in summer when water stress from lack of rain and high evaporative demand was most severe, and the advantage for plants gaining access to subsoil water was maximal. However, this late sowing also meant that the crop started maturing in late autumn when rainfall was considerable and the short day length limited grain filling. Thus the crop never reached maturity. Fertiliser was applied at sowing as a single application of 100 kg ha 1 N, 20 kg ha 1 P and 70 kg ha 1 K.
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Fig. 1. Weekly rainfall and potential evapotranspiration, ETo, for days after sowing (DAS) until harvest.
1.4. Measurements 1.6. Soil water Plant heights were measured on 4 tagged plants in each subplot; 1 plant from each of the 4 inner rows of the crop. Height was initially from the ground to the tip of the tallest leaf and then, if a seed head appeared, to the collar. Heading date was measured as the day after sowing on which 50% of the plot had reached growth stage 53 (HGCA, 2006). Tiller number was measured by counting the number of tillers for each of the tagged plants on 4 dates. Weekly measurements of plant height commenced 26 days after sowing (DAS). If any of these plants died, from stress or other cause, a near neighbour of similar stature in the same row was tagged and used for subsequent measurements. To test whether plant-to-plant differences might have been caused by the location of individual plants relative to the holes, the heights of all Morex plants from rows 3 and 6 were determined along with location in the row on 48–49 DAS. With more than 100 plants from each treatment statistical analysis for height variability was conducted. Leaf area index (LAI) was measured using a model PAR-80 AccuPARTM Ceptometer (Decagon Devices Inc., Pullman, WA, USA) (Murphy et al., 2008) on each sub-plot from 55 to 99 DAS. After 99 DAS leaf area measurements were not possible due to extensive cloud cover, persistent rain and shortening day length. Crop health was estimated by crop colour, or ‘‘greenness’’, on each subplot usually on a weekly basis using a GreenseekerTM hand held optical sensor unit model 505 (NTech Industries Inc., USA) to estimate Normalised Difference Vegetative Index (NDVI). The unit lights the crop at 2 specific wavelengths and the colour is determined from the light reflected. Similar methodology has been used to study the effect of nitrogen deficiency and senescence in wheat (Adamsen et al., 1999) and the relationship between rainfall and NDVI for a range of vegetation types (Nicholson and Farrar, 1994). 1.5. Harvest measurements The individual plants studied during the experiment were harvested 110 DAS. Due to the late sowing the crop did not reach maturity, and compensation through late harvest would have risked severe lodging and disease because of large rainfall (Fig. 1) and decreasing temperatures. Individual tagged plants were harvested, their development parameters recorded and oven dry mass (60 8C) taken. Leaves from one tagged plant per sub-plot were ground, digested in acid and analysed by ICP-MS for elemental concentration. Total aboveground dry mass was estimated from quadrat samples of the inner 4 rows of the crop along a 0.5 m section of each sub-plot, harvested by hand 110–112 DAS.
Soil water sensors (ML2x Theta-Probe, Delta-T Ltd., Cambridge, UK) were used to record volumetric water content at 2 depths for the Optic cultivar only. In all 3 replicates, they were inserted at 30 cm depth (10 cm below the mesh) when the mesh was installed and 0–6 cm depth after the crop was sown for the Unrestricted and 0 holes per plant treatments. In one replicate, probes were inserted at 30 cm depth for all six root restriction treatments. The probes were connected to dataloggers (DL6, Delta-T Ltd., Cambridge, UK) with readings taken every 6 h. 1.7. Calculations and statistical analysis Statistical analysis was performed in Genstat Tenth Edition (VSN International, Herts, UK). Treatments were compared by analysis of variance assuming a split-plot design. Plant height, leaf area and crop health were each statistically analysed separately at each measurement date. Throughout this paper, where differences are mentioned they are significant at p < 0.05 or better. 2. Results 2.1. Weather during crop growth Fig. 1 presents the weekly rainfall and potential evapotranspiration data from sowing until harvest. Rainfall was limited during the early growth of the crop (average approximately 8 mm/ week) but after about 56 DAS weekly rainfall averaged around 20 mm until harvest (110–112 DAS); or around half of the plant available water storage possible for the top 20 cm of soil. For most of the first 8 weeks of crop growth potential evapotranspiration was much greater than rainfall. Sunshine was only 4.2 h per day during September (70–99 DAS) and daily mean maximum temperatures in the same period were 19 8C. 2.2. Growing season measurements on individual plants As early as 26 DAS differences existed between plant height (p < 0.01; Fig. 2) in the different sub-plots. Plants grown without restriction were tallest, but there were no differences between the other treatments. Totally confining roots to a limited soil depth has elsewhere been reported to decrease shoot growth (Beyrouty and Oosterhuis, 1989). At 26 DAS there were height differences between genotypes (p < 0.001) with, in order: Morex, 26.5 cm: Derkado, 22.4 cm: B83-12/21/5, 19.2 cm: Optic, 17.5 cm: Golden
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Fig. 2. Mean plant height (4 plants from each of 5 genotypes per treatment) as a function of days after sowing (DAS). The average number of holes per plant in the restricting mesh is indicated in the legend. NS, not significant; **p < 0.01; *** p < 0.001. Bars are 5% LSD for stress on each DAS.
Promise 17.4 cm: LSD 5% 2.2 cm. At no measurement date was there an interaction between genotype and root restriction, indicating that there were no early differences in the ability to overcome the root restriction and exploit subsoil water. At no measurement date was there an effect of crop row on plant height. By 41 DAS the differences between plant height, in response to the root restrictions, were more significant (p < 0.001; Fig. 2) than at earlier dates with differences between the other treatments. The order for the holes per plant treatments and heights (using the 5 genotypes) was: Unrestricted, 37.0 cm: 3.20, 29.8 cm: 1.60, 29.9 cm: 0.55, 29.1 cm: 0.27, 28.0 cm: 0, 27.7 cm; LSD 5% = 2.0 cm. This is the first demonstration that by controlling the extent of root restriction and thus access to the subsoil, a water-stress gradient can be applied to a crop. At 55 DAS strong differences persisted (p < 0.001), with enhanced separation between the root restriction treatments. The order for the holes per plant treatments and heights (using the 5 genotypes) was: Unrestricted, 40.8 cm: 3.20, 35.4 cm: 1.60, 35.4 cm: 0.55, 33.9 cm: 0.27, 33.1 cm: 0, 31.9 cm; LSD 5% = 2.5 cm. At 68 DAS with the substantial rain (Fig. 1), height differences between root restriction treatments were still strong (p = 0.002) but were diminishing. Differences persisted through 75 DAS (p = 0.003) but had disappeared with the continued rainfall by 91 DAS. At 55, 68, 96 and 110 DAS the measured total number of tillers for each of the individual tagged plants was determined. At no measurement date was there a significant effect of root restriction. Differences between genotypes existed at all dates (p < 0.01) with the Optic and B83-12/21/5 consistently having most tillers and Morex consistently fewest tillers (data not shown). Optic overtook Morex as the tallest genotype from 62 DAS. Differences in height between the genotypes persisted to harvest, indicating that it was the removal of drought by rainfall that allowed all genotypes to recover. Ear emergence for all genotypes was complete by 72 DAS and while there were differences between genotypes (p < 0.05) there were no differences with root restriction.
Fig. 3. Leaf area index relative to the mean of the unrestricted treatment at 99 days after sowing (DAS). The average number of holes per plant in the restricting mesh is indicated in the legend. Values are means of 5 genotypes. ***p < 0.001; Bars are 5% LSD for stress on each DAS.
different with different root restriction, and whether there was any pattern to the heights of the plants that could be related to the spacing of the holes in the mesh. Given that the holes in the mesh were on a regular grid we might expect 2 types of plant response; those plants with a root that finds a hole and gains access to subsoil water and those plants from which no root found a hole and thus gained no access to subsoil water. This might lead to 2 distributions of plant height, formally a mixture of plant heights with an unknown proportion in each category. The histograms of plant heights (data not shown) showed no evidence of 2 distributions. 2.4. Leaf area index (LAI) Leaf area is expressed relative to the mean of the unrestricted treatment at 99 DAS (Fig. 3). There were differences in leaf area between root restriction treatments and between genotypes but there were no interactions between root restriction and genotype at any date. The rank order for the holes per plant treatments at 55 DAS from greatest to least leaf area was: Unrestricted: 3.2:1.6:0.55:0.27:0 (Table 1). This trend is consistent with the order for plant height differences at the same time. The rank order for the genotypes was Optic: Derkado: B83-12/21/5: Morex: Golden Promise (Table 1). As stated above there were no root restriction by genotype interactions (Table 1) however for some genotypes there appears a non-significant critical or threshold level of root restriction. For Optic there is a noticeable step in LAI between 1.60 and 3.20 holes per plant; while for B83-12/21/5 a similar step is between 0.55 and 1.60 holes per plant. From 69 DAS (Fig. 3) differences resulting from the stress of root restriction persisted with unrestricted plants consistently performing best, the totally restricted having lowest LAI, but with no significant differences between the intermediate root restriction treatments. Differences in leaf area with root restriction treatments persisted (p < 0.001) until 99 DAS, which is after differences in height had disappeared due to stem elongation in response to rainfall. 2.5. Crop greenness as Normalised Difference Vegetative Index (NDVI)
2.3. Variability of individual plant height The heights measured on the Morex plants at 48 and 49 DAS were analysed to determine whether the variability in height was
Differences in crop colour with root restriction were observed between 33 DAS and 54 DAS (NDVI in Fig. 4), during an extended period with little rain. Larger values for NDVI indicate a greener
B.M. McKenzie et al. / Field Crops Research 112 (2009) 165–171 Table 1 Crop and plant characters at 55 DAS. Genotype
Holes per plant
Plant height (cm) mean of 4 plants
Leaf area index
NDVI
Golden Golden Golden Golden Golden Golden
0.00 0.27 0.55 1.60 3.20 Unrestricted
26.9 27.2 29.9 32.3 31.9 34.8
1.28 1.21 1.34 1.41 1.54 1.90
0.49 0.21 0.59 0.58 0.53 0.57
Optic Optic Optic Optic Optic Optic
0.00 0.27 0.55 1.60 3.20 Unrestricted
40.2 41.8 35.8 44.2 39.3 43.9
1.91 2.14 2.12 2.14 2.43 2.79
0.57 0.57 0.54 0.80 0.69 0.87
Derkado Derkado Derkado Derkado Derkado Derkado
0.00 0.27 0.55 1.60 3.20 Unrestricted
32.8 30.2 36.2 35.0 35.2 42.2
1.65 1.50 1.77 1.76 1.64 2.13
0.47 0.58 0.49 0.68 0.55 0.70
B83-12/21/5 B83-12/21/5 B83-12/21/5 B83-12/21/5 B83-12/21/5 B83-12/21/5
0.00 0.27 0.55 1.60 3.20 Unrestricted
24.9 27.8 29.2 29.2 29.4 33.1
1.29 1.70 1.62 1.91 2.04 2.14
0.54 0.84 0.88 0.72 0.70 0.80
Morex Morex Morex Morex Morex Morex
0.00 0.27 0.55 1.60 3.20 Unrestricted
34.6 38.1 38.2 36.3 41.2 50.0
1.46 1.25 1.62 1.50 1.53 2.30
0.42 0.33 0.51 0.33 0.40 0.73
2.5 3.4
0.20 0.54
0.11 0.16
Promise Promise Promise Promise Promise Promise
LSD 5% stress LSD 5% genotype
crop. The smaller NDVI may be due to a combination of slower crop development leaving some bare soil exposed in the root restricted treatments and to the crop being less green. In both these scenarios it is a stress response resulting from the treatments that is being determined. From 56 DAS rain fell in most weeks (Fig. 1) and differences in the NDVI disappeared. No differences (p > 0.05) in
Fig. 4. Normalized Difference Vegetation Index (NDVI) as a function of days after sowing (DAS). The average number of holes per plant in the restricting mesh is indicated in the legend. Values are means of 5 genotypes. NS, not significant; ** p < 0.01; ***p < 0.001. Bars are 5% LSD for stress on each DAS.
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NDVI with root restriction occurred after 61 DAS. The NDVI showed a more rapid response of the crop to rain than did plant height where differences persisted until 75 DAS, which was a more rapid response than leaf area where differences persisted until 98 DAS. Differences in NDVI existed between genotypes on all but 2 recording dates. The genotypes split into groups with Morex and Golden Promise having lower NDVI values than the other 3 genotypes, Optic, B83 and Derkado, which were all similar. A diagnostic biplot of leaf area and NDVI for each measurement date (not shown) showed similar separation between genotypes. Table 1 shows the height, leaf area and NDVI results by stress and genotype at 55 DAS, when major differences between genotypes existed. All genotypes appeared to be similarly stressed relative to their unrestricted treatment. There was thus no evidence to suggest that genotypes differed in their ability to exploit holes in the mesh and obtain subsoil water. Morex, the only genotype without a dwarfing gene, was tallest in the unrestricted treatment but the relative heights with restriction were consistent with trends for the other genotypes. 2.6. Harvest At 110 DAS the plants had not reached maturity and different genotypes were at different stages of ear formation and filling. The dry weight of the individual plants grown with unrestricted roots was greater than for all levels of root restriction which did not differ significantly from each other. The total dry weight of plant material based on the quadrat samples was greatest in the unrestricted plots but no other differences occurred. Elemental analysis by ICP-MS showed no differences with either stress or genotype for Fe, Cl, Cs, K, Mg, Mn, Ni, P, or S. Minor differences between genotypes were found for Cu and Zn. The only element to show an effect of stress treatment was Ca, but this was not in an order consistent with treatments or other results (data not shown). 2.7. Soil water content Fig. 5 shows the water contents in the surface soil (above the mesh) and in the subsoil (below the mesh) relative to the mean water contents of each depth for the unrestricted treatment at 55 DAS for Optic. There was a clear trend that the soil was drier under
Fig. 5. Relative volumetric water content in the topsoil (0–6 cm) and subsoil (30 cm) of the Optic genotype at 55 days after sowing (DAS). Relative water content is water content in the treatment/water content in the unrestricted treatment.
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3. Discussion
plant height were still present and leaf area remained for several more weeks. This clearly demonstrates that to understand crop physiological responses to stress requires more than a single character to be quantified. All crops in this experiment had the same applied fertiliser.
3.1. Responses to restricted access to subsoil water
3.2. Do differences in subsoil exploitation exist?
In dry conditions plants may rely on extracting water from the subsoil (Passioura, 2006). Subsoil water will not be available in all environments, notably semi-arid regions, but practices such as fallowing are designed to store water in the soil profile (Koohafkan and Stewart, 2008). Periods of drought sufficient to affect crop growth and yield can occur in nearly all environments where cereals are produced, but it is especially important that genotypes grown in dryland environments are able to exploit stored water. Fig. 2 shows the importance of subsoil access across 5 common barley genotypes. As early as 26 DAS height differences occurred. Plants with unrestricted access to the subsoil were taller with a greater leaf area. Crops with plants having a tall dense canopy are better able to intercept light and thus out-compete weed populations or survive attack by pathogens. The advantage of access to subsoil water persisted through periods without rain and the plant heights and leaf area responded to the increased subsoil access. Our results are consistent with those of Beyrouty and Oosterhuis (1989) who used a mesh layer to totally restrict access and found that crops with no access to subsoil water performed less well that those with unrestricted access. After about 55 DAS, rainfall and decreased evaporative (Fig. 1) demand created an environment where the water needs of the crop could be supplied by rainfall without the crop needing to access subsoil water. This provided an environment whereby the crop was no longer water stressed. For the differences in height resulting from stress to be overcome the plants had to elongate the single main tiller and flag leaf. This requires relatively little resource and so plant height responded quickly to improved water availability. That there was no evidence of separation into 2 distributions of plant heights for plants that found a hole and those plants from which a root did not find the hole might be explained by the diameter of the holes relative to the diameter of a barley root at 20 cm depth in the soil. Typically barley roots are less than 1 mm diameter leaving sufficient space for more than one root to access any individual hole (2 mm diameter): indeed, it is common for roots to cluster together in naturally occurring biopores (Passioura, 1991). Leaf area index showed a response consistent with plant height. The crops with best access to subsoil water had a fuller canopy and those with restricted access had decreased leaf area proportional to the degree of restriction. Even though significant rain fell after 55 DAS the differences in leaf area index persisted until harvest. For the crop to recover from the imposed stress and to achieve the same canopy development as the unstressed crop requires increased tillering and expansion of total leaf area. This would require more photosynthate and mineral resources than required to simply elongate a single stem and flag leaf. Hence the differences in leaf area index persisted even after differences in plant height had disappeared. NDVI, a measure of crop colour, is influenced by multiple factors including exposed soil visible through the crop canopy, water status, nitrogen status, and leaf senescence. Despite the range of factors involved for any given date the NDVI’s were broadly consistent with the imposed stress treatment, i.e. increased crop stress resulting in lower NDVI’s. Because the NDVI is controlled by multiple factors the loss of differences between stress treatments after rain from 55 DAS could be a response of several factors. Differences in NDVI were not significant by 61 DAS while those for
All 5 modern genotypes of barley responded similarly to water stress applied by controlling access to subsoil water. Had the water stress differences persisted it is possible that interactions between the genotypes and stress may have occurred in important features such as final grain yield. Controlled application of water and protection from rainfall could improve future studies to allow more control of plant available water in the topsoil and subsoil. Differences in the interaction of roots with soil and biopores could provide improved exploitation of subsoil water. Firstly, plants that have more roots reaching an impeding soil or mesh layer will have greater chance of at least one root finding a biopore and thus gaining access to subsoil water. The modern genotypes used here all produce similar numbers of seminal roots. Secondly, there may be differences in the likelihood that a root reaching a barrier finds and enters a biopore. Dexter (1986) using model experiments and computer simulations found that the numbers of roots encountering holes in an artificial barrier was not different than on the basis of random chance. However, he found limited but sufficient evidence that in poorly aerated systems the possibility of roots growing preferentially to biopores could not be ruled out. In a similar model system Hirth et al. (1997) found no evidence that roots of ryegrass were likely to elongate to earthworm cast material. So there remains a possibility for differences between genotypes in sensing oxygen or other gradients around roots. Thirdly, roots elongating through a biopore and reaching subsoil water may have different plasticities or ability to branch. While root plasticity to nutrient localised supply has been well studied since Drew (1975), examples of root plasticity to localised differences in water supply, such as occur with water in subsoils, are relatively scarce. So there are at least 3 features of cereal root systems (number of roots, ability to find biopores and plasticity to subsoil water) that could differ with genotype and which provide hypotheses for future comparisons.
crops grown in treatments with more access through the mesh to the subsoil, while the topsoil was drier under crops that had less or no access to the subsoil.
3.3. Opportunities to modify soil to improve subsoil access Soil is a heterogeneous medium and situations where root access to the subsoil is totally constrained are likely to be rare. However, there may be scope to improve crop production in areas where root-impeding layers exist in the soil. This could either be done by improving cultivation systems, including the use of subsoil slotting and deep-ripping (Hartmann et al., 2008), or using crop rotation to increase the numbers of stable biopores (McCallum et al., 2004). 3.4. Implementation of the method and potential applications This first report of a novel method clearly raises questions about how to improve its effectiveness and what adaptation is needed to make it widely applicable. The layout of the experiment was done to be consistent with common cereal trials units of 6 m length, divided into 1 m2 subplots. While this offers utility it also requires sufficient seed to be available. Heights were measured on plants in the inner 4 (of 8) rows. As there were no effect of row on height it is probable that none of the measured plants had roots that grew beyond the mesh. Smaller units requiring less seed could be tested, offering the ability to test more genotypes per mesh area.
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Similarly questions of the number, size and arrangement of the holes in the mesh could be investigated based on understanding of root architecture and biopore distributions within soil. There may be scope to develop the method by placing the mesh at the same depth as local impeding layers occur. The methodology to establish this screening method in the field was relatively straightforward. The removal of the soil to position the mesh was done by machine, but in areas where labour is cheaper it could also be done by hand. Making the holes in the mesh and replacing the soil required no more care than required for careful establishment of any field experiment. The maximum number of holes in the mesh, expressed on a per plant basis was 3.2. Figs. 2–4 all show that at most times plants in this treatment grew less well than those in the unrestricted treatment. This suggests that to optimise cereal production soils should be managed to have no impeding layer, or if an impeding layer exists biopore numbers should be greater than 3.2 per cereal plant. We need to better understand the manner in which plant roots exploit the soil resource and importantly how soil can be managed to provide an environment conducive to root proliferation. Characterizing and quantifying the soil conditions at multiple depths, using modern soil quality descriptors, e.g. the Least Limiting Water Range (Da Silva and Kay, 2004), allow for quantification of suitable or non-suitable, restricting environments. In environments where management options are limited the need to understand the role of biopores and consider how they might be best used to provide options for roots to access subsoil water will be increasingly important. 4. Conclusions The results from this experiment support that by puncturing a mesh buried horizontally in the soil we are able to mimic biopores through hard soil layers and thus control access to subsoil water. As crops often rely on the subsoil for water to alleviate drought stress by varying the number of holes through the mesh we are able to differentially stress field-grown cereal crops for drought. The ability to regulate drought stress in a field environment provides a new method for plant breeders to screen germplasm. By identifying promising germplasm with root characteristics able to explore impeded layers for biopores, an important mechanism in drought tolerance could be better exploited. Further refinement of the method for adaptation to a range of crop types and soil conditions is now needed. Acknowledgments We thank Rowan Forster, Lawrie Brown, Richard Keith and Dennis Gordon for technical support. We thank Euan Caldwell and the estate staff at SCRI for crop management. The Scottish Crop Research Institute receives grant-in-aid support from the Scottish Government Rural and Environment Research and Analysis Directorate. References Adamsen, F.J., Pinter, P.J., Barnes, E.M., LaMorte, R.L., Wall, G.W., Leavitt, S.W., Kimball, B.A., 1999. Measuring wheat senescence with a digital camera. Crop Sci. 39, 719–724.
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Influence of soil type on the relationships between NDVI, rainfall and soil moisture in semi-arid Botswana 1 NDVI response to rainfall. Remote Sens. Environ. 50, 107–120. Passioura, J.B., 1985. Roots and water economy of wheat. In: Day, W., Atkin, R.K. (Eds.), Wheat Growth and Modelling. Plenum Press, New York, pp. 185–199. Passioura, J.B., 1991. Soil structure and plant growth. Aust. J Soil Res. 29, 717–728. Passioura, J.B., 2006. Increasing crop productivity when water is scarce—from breeding to field management. Agric. Water Manage. 80, 176–196. Passioura, J.B., 2007. The drought environment: physical, biological and agricultural perspectives. J. Exp. Bot. 58, 113–117. Poot, P., Lambers, H., 2008. Shallow-soil endemics: adaptive advantages and constraints of a specialized root-system morphology. New Phytol. 178, 371–381. Reynolds, M.J., Mujeeb-Kazi, A., Sawkins, M., 2005. Prospects for utilising plantadaptive mechanisms to improve wheat and other crops in drought- and salinity-prone environments. Ann. Appl. Biol. 146, 239–259. Smettem, K.R.J., Collis-George, N., 1985. Statistical characterization of soil biopores using a soil peel method. Geoderma 36, 27–36. Vocanson, A., Roger-Estrade, J., Boizard, H., Jeuffray, M-H., 2006. Effects of soil structure on pea (Pisum sativum L.) root development according to sowing data and cultivar. Plant Soil 281, 121–135. Yu, Y., Tompkins, J.P., Waugh, R., Frisch, D.A., Kudrna, D., Kleinhofs, A., Brueggeman, R.S., Muehlbauer, G.J., Wise, R.P., Wing, R.A., 2000. A bacterial artificial chromosome library for barley (Hordeum vulgare L.). Plant J. 101, 1093–1099.
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Field Crops Research journal homepage: www.elsevier.com/locate/fcr
Deep rooting and drought screening of cereal crops: A novel field-based method and its application B.M. McKenzie a,*, A.G. Bengough a, P.D. Hallett a, W.T.B. Thomas a, B. Forster a,1, J.W. McNicol b a b
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK BioSS, Invergowrie, Dundee DD2 5DA, UK
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 September 2008 Received in revised form 11 February 2009 Accepted 22 February 2009
Lack of water is a major limitation to crop production, particularly where roots of cereal crops are not able to access water stored in the subsoil. One way that roots penetrate the subsoil to access water is by following natural biopores – paths created by roots from previous crops, or as burrows from soil fauna. Burying a mesh layer horizontally in the soil can prevent root penetration to the subsoil. We used this technique with the novel modification that the mesh was punctured to create a defined number of holes per unit area; controlling access to the subsoil and to the water therein. The holes were of similar size to biopores. Five barley genotypes were late sown and grown during a dry summer. Monitoring of crop performance included plant height, leaf area and Normalized Difference Vegetation Index (NDVI). Crops grown with unrestricted access to the subsoil outperformed crops with limited or no access to the subsoil. Crops grown with controlled, limited access to the subsoil performed better than those with no access and the performance was generally related to amount of access. Changes in soil water content were in line with the amount of root access to the subsoil; confirming the association between subsoil water and crop growth and development in drought conditions. While there were no significant interactions between the genotypes and treatments used here, the method offers promise for studying some aspects of cereal ecophysiology and could be used to identify promising germplasm that may be of interest in plant breeding. Further testing is required to adapt the method for a wider range of crop types and soil conditions and testing for crops grown to maturity. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Biopores Cereals Root growth Drought stress Subsoil water Plough pan
Even severely drought stressed crops may leave considerable plant available water in the subsoil (Passioura, 1985). High mechanical impedence of the soil, caused by compaction below the plough layer and the strengthening of soil as it dries, restricts root growth to shallow depths (Gregory, 2006). Therefore, of all phenotypic traits that can be altered to improve drought resistance of cereal crops, increased penetration and extension of root systems probably offers the greatest potential (Passioura, 2007). By penetrating deeper into soil, crop roots potentially access and exploit a greater volume of stored water. There is potential to learn from endemic plants that have an ecological niche in drought-prone climates and shallow soils. Poot and Lambers (2008) found rare endemic species with specialized root systems that are able to explore a large surface area at the soil-rock boundary for fissures so that roots can extend
* Corresponding author. Tel.: +44 1382 568 529; fax: +44 1382 568 502. E-mail address: [email protected] (B.M. McKenzie). 1 Current address: BioHybrids International Ltd., PO Box 2411, Earley, Reading RG6 5FY, UK. 0378-4290/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2009.02.012
to depth and access water. Arable soils can also contain fissures and other large pores that cross plough pans, compacted layers after machinery passage, or natural horizon boundaries (Vocanson et al., 2006). Biopores may be old root channels or burrows made by earthworms or other soil fauna. Cracks generated by shrinking and swelling may also leave planes of weakness that could be followed by roots (Passioura, 1991). There is considerable evidence that these pores provide preferential pathways for root growth and are extremely important to crop performance (Ehlers et al., 1983). The ability of roots of a specific crop cultivar to exploit these pores could thus be a major determinant of drought avoidance. Soil management can increase the abundance of fissures or macropores, with implications to crop performance in dry years. Lampurlanes et al. (2001) compared the root growth of barley and soil water status under deep and no-till systems. In years of low rainfall they found that the no till system performed better by creating an environment that encouraged roots deep into the soil profile. The deeper root proliferation with no-till was attributed to improved soil structure and particularly biopores providing pathways for roots.
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The nature and number of biopores per cross-sectional area have been considered by several authors. Smettem and CollisGeorge (1985) used a peel method to characterise numbers of biopores greater than 0.5 mm diameter and found at 10 cm depth there were approximately 470 biopores per square meter. Ehlers (1975) found biopore diameters of between 2 and 11 mm. The orientation of biopores in the soil profile may become nearer to vertical with depth (McKenzie and Dexter, 1993), thus providing suitable paths for roots to follow. Where hard soil layers impede root elongation Passioura (1991) has reported the clumping of several roots in a single biopore. Hirth et al. (2005) studied the behaviour of roots in artificial biopores and found that the orientation of the biopore and its surface properties influenced whether a root was able to move from the biopore into the bulk soil. Jakobsen and Dexter (1988) modelled the influence of biopores on root growth and cereal production for 2 contrasting environments. Their modelling suggested there may be an optimum density of biopores. In dry conditions too many biopores, providing unlimited access to the subsoil, could lead to early depletion of stored water and perhaps leave insufficient reserves for grain filling; while too few biopores prevent access by roots to subsoil water and limit yield. The modelling of Jakobsen and Dexter (1988) indicates that the optimum will be different for different plant densities per unit area. Thus the number of biopores can be described on per unit area or a per plant basis. To screen crops for deep rooting often requires soil samples be collected from a range of depths beneath a crop before roots are washed from the collected soil. The root length density at depth is an indication of the access the crop has to any subsoil water, however, the measurement of root material is very laborious (Gregory, 2006). Root pruning has been tried as a way to restrict lateral root spread and thus limit access to water and nutrients (Ma et al., 2008). Agronomic approaches to improve the drought tolerance of cereal crops include deep sowing so that the seedling emerges from depth and the root system is not exposed to hot dry conditions (Reynolds et al., 2005). Another possible mechanism for deep sown crops to be more drought tolerant is that they have more root axes at depth and thus have a greater chance of finding a biopore or other path into the subsoil where water may be available. In this paper we study the effect of restricting root growth in the subsoil using mesh (permeable to fluids) that contains varying densities of puncture holes (mimicking biopores). This confines root systems to the surface soil, except for roots that can elongate through the puncture holes in the mesh. The technique varies, in a controlled manner, the access of crop roots to water in the subsoil, providing a differential stress to the crop. This is proposed to allow study of the response of field-grown crops, to varying degrees of water stress by restriction of root growth in the subsoil. We tested the system for 5 barley (Hordeum vulgare) genotypes. 1. Materials and methods 1.1. Soil properties and meteorological data The experiment was conducted at the Scottish Crop Research Institute (SCRI); (56.27N 3.40W) during 2006. The soil type is Balrownie series, a Stagnic Cambisol in the FAO classification, with a sandy loam texture derived from red sandstone sediments (Bell and Hipkin, 1988). Plant available water of the surface soil was calculated from the water retention curve as the difference between field capacity (10 kPa matric suction) and wilting point (1500 kPa matric suction). For our site the surface soil stored 40 mm of plant available water in the surface 200 mm. Prior to establishing this experiment no crop had been grown in the soil for
several years. The area had been used for propagating horticultural plants in pots. Irrigation applied to the pots and the substantial winter and spring rainfall (data not shown) ensured that the subsoil stored considerable plant available water. Weather data were recorded at a meteorological station located within 50 m of the experiment site. Potential evapotranspiration, ETo, was taken from an automated weather station (Campbell Scientific) located less than 500 m from the site. 1.2. Experimental design and soil preparation A field experiment was established as a split-plot design with 5 main treatments repeated 3 times. The 5 main treatments were different barley (Hordeum vulgare) genotypes. Within each main treatment 6 levels of root restriction were applied as sub-plots; designed to provide different access to subsoil water. Each main treatment was a crop strip 6 m long and 1.2 m wide. The surface 0.2 m of soil was first removed and the exposed subsoil cultivated to a depth of 0.2 m (i.e. 0.4 m below the original surface) with a power harrow. Strips of nylon mesh (Clarcor, UK) were laid as 6 m long and 1.5 m wide. The mesh had holes of 40 mm to provide an open area of 26% that could be penetrated by water and gases, but not by barley roots. Preliminary experiments showed that the mesh provided a boundary to roots while permitting air and water transport. The 6 m lengths were divided into 6 m 1 m length sub-plots. For each genotype treatment and replicate, a different root restriction treatment was established. One of the 6 sections the mesh was removed completely (Unrestricted). The other 5 treatments had 0, 100, 200, 583 or 1167 holes m 2 inserted into the mesh to simulate biopores. Nail-boards were used to make puncture holes, which had a regular grid for all hole densities. The nails were 2 mm in diameter, similar to that of an earthworm burrow (Ehlers, 1975; Smettem and Collis-George, 1985). To drive the nails through the mesh, a pressure of approximately 13 kPa was applied. This was simply generated by a person standing on the board. Blank nailboards were used to apply the same stress to the sub-plots with 0 holes m 2 and no mesh. This light compaction ensured good contact between the mesh and the sub-soil and the surface soil was then returned to cover the mesh (with no compaction by vehicular traffic). The replaced soil was raked before sowing the barley seed. 1.3. Genotypes and crop management Five modern barley genotypes (Golden Promise, Optic, Derkado, B83-12/21/5 and Morex) were allocated randomly to each replicate and sown into the 6 m long strips. Golden Promise is a semi-dwarf barley carrying a dwarfing allele at the ari-e locus and is relatively shallow rooting (AlMenaie, 2003). Derkado and B83-12/21/5 are parents of a mapping population that segregates for 2 different dwarfing genes. Derkado carries the sdw1 dwarfing gene whereas B83-12/21/5 carries a similar ari-e dwarfing gene to Golden Promise. The sdw1 dwarfing gene is associated with increased root mass during the vegetative growth period (Chloupek et al., 2006). Optic carries the sdw1 dwarfing gene. It was the most popular spring barley malting cultivar in the UK during the late 1990s and is still widely grown. A large number of ESTs from a variety of stress treatments have been derived from Optic and structured mutant populations have also been developed from it (Caldwell et al., 2004). Morex is a spring malting barley that was widely grown in the Pacific North West region of the USA. Unlike the other genotypes, it has a 6-row head and is relatively early heading. A number of barley genomics tools have also been based on Morex, notably a Bacterial Artificial Chromosome library that is estimated to provide 6.3 coverage of
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the genome (Yu et al., 2000), a resource that is of great value in gene isolation. The plots were sown in 8 rows, with an effective width of 1.2 m and to a target density of 365 established plants m 2. The number of holes through the mesh per plant was approximately 0, 0.27, 0.55, 1.60, and 3.20. These holes per plant values are used to describe the treatments. The treatment without a mesh is referred to as unrestricted. The barley was sown late (23 June 2006), at least 10 weeks after the usual sowing date, but at a time when drought conditions were most likely. The result was that early crop growth occurred in summer when water stress from lack of rain and high evaporative demand was most severe, and the advantage for plants gaining access to subsoil water was maximal. However, this late sowing also meant that the crop started maturing in late autumn when rainfall was considerable and the short day length limited grain filling. Thus the crop never reached maturity. Fertiliser was applied at sowing as a single application of 100 kg ha 1 N, 20 kg ha 1 P and 70 kg ha 1 K.
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Fig. 1. Weekly rainfall and potential evapotranspiration, ETo, for days after sowing (DAS) until harvest.
1.4. Measurements 1.6. Soil water Plant heights were measured on 4 tagged plants in each subplot; 1 plant from each of the 4 inner rows of the crop. Height was initially from the ground to the tip of the tallest leaf and then, if a seed head appeared, to the collar. Heading date was measured as the day after sowing on which 50% of the plot had reached growth stage 53 (HGCA, 2006). Tiller number was measured by counting the number of tillers for each of the tagged plants on 4 dates. Weekly measurements of plant height commenced 26 days after sowing (DAS). If any of these plants died, from stress or other cause, a near neighbour of similar stature in the same row was tagged and used for subsequent measurements. To test whether plant-to-plant differences might have been caused by the location of individual plants relative to the holes, the heights of all Morex plants from rows 3 and 6 were determined along with location in the row on 48–49 DAS. With more than 100 plants from each treatment statistical analysis for height variability was conducted. Leaf area index (LAI) was measured using a model PAR-80 AccuPARTM Ceptometer (Decagon Devices Inc., Pullman, WA, USA) (Murphy et al., 2008) on each sub-plot from 55 to 99 DAS. After 99 DAS leaf area measurements were not possible due to extensive cloud cover, persistent rain and shortening day length. Crop health was estimated by crop colour, or ‘‘greenness’’, on each subplot usually on a weekly basis using a GreenseekerTM hand held optical sensor unit model 505 (NTech Industries Inc., USA) to estimate Normalised Difference Vegetative Index (NDVI). The unit lights the crop at 2 specific wavelengths and the colour is determined from the light reflected. Similar methodology has been used to study the effect of nitrogen deficiency and senescence in wheat (Adamsen et al., 1999) and the relationship between rainfall and NDVI for a range of vegetation types (Nicholson and Farrar, 1994). 1.5. Harvest measurements The individual plants studied during the experiment were harvested 110 DAS. Due to the late sowing the crop did not reach maturity, and compensation through late harvest would have risked severe lodging and disease because of large rainfall (Fig. 1) and decreasing temperatures. Individual tagged plants were harvested, their development parameters recorded and oven dry mass (60 8C) taken. Leaves from one tagged plant per sub-plot were ground, digested in acid and analysed by ICP-MS for elemental concentration. Total aboveground dry mass was estimated from quadrat samples of the inner 4 rows of the crop along a 0.5 m section of each sub-plot, harvested by hand 110–112 DAS.
Soil water sensors (ML2x Theta-Probe, Delta-T Ltd., Cambridge, UK) were used to record volumetric water content at 2 depths for the Optic cultivar only. In all 3 replicates, they were inserted at 30 cm depth (10 cm below the mesh) when the mesh was installed and 0–6 cm depth after the crop was sown for the Unrestricted and 0 holes per plant treatments. In one replicate, probes were inserted at 30 cm depth for all six root restriction treatments. The probes were connected to dataloggers (DL6, Delta-T Ltd., Cambridge, UK) with readings taken every 6 h. 1.7. Calculations and statistical analysis Statistical analysis was performed in Genstat Tenth Edition (VSN International, Herts, UK). Treatments were compared by analysis of variance assuming a split-plot design. Plant height, leaf area and crop health were each statistically analysed separately at each measurement date. Throughout this paper, where differences are mentioned they are significant at p < 0.05 or better. 2. Results 2.1. Weather during crop growth Fig. 1 presents the weekly rainfall and potential evapotranspiration data from sowing until harvest. Rainfall was limited during the early growth of the crop (average approximately 8 mm/ week) but after about 56 DAS weekly rainfall averaged around 20 mm until harvest (110–112 DAS); or around half of the plant available water storage possible for the top 20 cm of soil. For most of the first 8 weeks of crop growth potential evapotranspiration was much greater than rainfall. Sunshine was only 4.2 h per day during September (70–99 DAS) and daily mean maximum temperatures in the same period were 19 8C. 2.2. Growing season measurements on individual plants As early as 26 DAS differences existed between plant height (p < 0.01; Fig. 2) in the different sub-plots. Plants grown without restriction were tallest, but there were no differences between the other treatments. Totally confining roots to a limited soil depth has elsewhere been reported to decrease shoot growth (Beyrouty and Oosterhuis, 1989). At 26 DAS there were height differences between genotypes (p < 0.001) with, in order: Morex, 26.5 cm: Derkado, 22.4 cm: B83-12/21/5, 19.2 cm: Optic, 17.5 cm: Golden
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Fig. 2. Mean plant height (4 plants from each of 5 genotypes per treatment) as a function of days after sowing (DAS). The average number of holes per plant in the restricting mesh is indicated in the legend. NS, not significant; **p < 0.01; *** p < 0.001. Bars are 5% LSD for stress on each DAS.
Promise 17.4 cm: LSD 5% 2.2 cm. At no measurement date was there an interaction between genotype and root restriction, indicating that there were no early differences in the ability to overcome the root restriction and exploit subsoil water. At no measurement date was there an effect of crop row on plant height. By 41 DAS the differences between plant height, in response to the root restrictions, were more significant (p < 0.001; Fig. 2) than at earlier dates with differences between the other treatments. The order for the holes per plant treatments and heights (using the 5 genotypes) was: Unrestricted, 37.0 cm: 3.20, 29.8 cm: 1.60, 29.9 cm: 0.55, 29.1 cm: 0.27, 28.0 cm: 0, 27.7 cm; LSD 5% = 2.0 cm. This is the first demonstration that by controlling the extent of root restriction and thus access to the subsoil, a water-stress gradient can be applied to a crop. At 55 DAS strong differences persisted (p < 0.001), with enhanced separation between the root restriction treatments. The order for the holes per plant treatments and heights (using the 5 genotypes) was: Unrestricted, 40.8 cm: 3.20, 35.4 cm: 1.60, 35.4 cm: 0.55, 33.9 cm: 0.27, 33.1 cm: 0, 31.9 cm; LSD 5% = 2.5 cm. At 68 DAS with the substantial rain (Fig. 1), height differences between root restriction treatments were still strong (p = 0.002) but were diminishing. Differences persisted through 75 DAS (p = 0.003) but had disappeared with the continued rainfall by 91 DAS. At 55, 68, 96 and 110 DAS the measured total number of tillers for each of the individual tagged plants was determined. At no measurement date was there a significant effect of root restriction. Differences between genotypes existed at all dates (p < 0.01) with the Optic and B83-12/21/5 consistently having most tillers and Morex consistently fewest tillers (data not shown). Optic overtook Morex as the tallest genotype from 62 DAS. Differences in height between the genotypes persisted to harvest, indicating that it was the removal of drought by rainfall that allowed all genotypes to recover. Ear emergence for all genotypes was complete by 72 DAS and while there were differences between genotypes (p < 0.05) there were no differences with root restriction.
Fig. 3. Leaf area index relative to the mean of the unrestricted treatment at 99 days after sowing (DAS). The average number of holes per plant in the restricting mesh is indicated in the legend. Values are means of 5 genotypes. ***p < 0.001; Bars are 5% LSD for stress on each DAS.
different with different root restriction, and whether there was any pattern to the heights of the plants that could be related to the spacing of the holes in the mesh. Given that the holes in the mesh were on a regular grid we might expect 2 types of plant response; those plants with a root that finds a hole and gains access to subsoil water and those plants from which no root found a hole and thus gained no access to subsoil water. This might lead to 2 distributions of plant height, formally a mixture of plant heights with an unknown proportion in each category. The histograms of plant heights (data not shown) showed no evidence of 2 distributions. 2.4. Leaf area index (LAI) Leaf area is expressed relative to the mean of the unrestricted treatment at 99 DAS (Fig. 3). There were differences in leaf area between root restriction treatments and between genotypes but there were no interactions between root restriction and genotype at any date. The rank order for the holes per plant treatments at 55 DAS from greatest to least leaf area was: Unrestricted: 3.2:1.6:0.55:0.27:0 (Table 1). This trend is consistent with the order for plant height differences at the same time. The rank order for the genotypes was Optic: Derkado: B83-12/21/5: Morex: Golden Promise (Table 1). As stated above there were no root restriction by genotype interactions (Table 1) however for some genotypes there appears a non-significant critical or threshold level of root restriction. For Optic there is a noticeable step in LAI between 1.60 and 3.20 holes per plant; while for B83-12/21/5 a similar step is between 0.55 and 1.60 holes per plant. From 69 DAS (Fig. 3) differences resulting from the stress of root restriction persisted with unrestricted plants consistently performing best, the totally restricted having lowest LAI, but with no significant differences between the intermediate root restriction treatments. Differences in leaf area with root restriction treatments persisted (p < 0.001) until 99 DAS, which is after differences in height had disappeared due to stem elongation in response to rainfall. 2.5. Crop greenness as Normalised Difference Vegetative Index (NDVI)
2.3. Variability of individual plant height The heights measured on the Morex plants at 48 and 49 DAS were analysed to determine whether the variability in height was
Differences in crop colour with root restriction were observed between 33 DAS and 54 DAS (NDVI in Fig. 4), during an extended period with little rain. Larger values for NDVI indicate a greener
B.M. McKenzie et al. / Field Crops Research 112 (2009) 165–171 Table 1 Crop and plant characters at 55 DAS. Genotype
Holes per plant
Plant height (cm) mean of 4 plants
Leaf area index
NDVI
Golden Golden Golden Golden Golden Golden
0.00 0.27 0.55 1.60 3.20 Unrestricted
26.9 27.2 29.9 32.3 31.9 34.8
1.28 1.21 1.34 1.41 1.54 1.90
0.49 0.21 0.59 0.58 0.53 0.57
Optic Optic Optic Optic Optic Optic
0.00 0.27 0.55 1.60 3.20 Unrestricted
40.2 41.8 35.8 44.2 39.3 43.9
1.91 2.14 2.12 2.14 2.43 2.79
0.57 0.57 0.54 0.80 0.69 0.87
Derkado Derkado Derkado Derkado Derkado Derkado
0.00 0.27 0.55 1.60 3.20 Unrestricted
32.8 30.2 36.2 35.0 35.2 42.2
1.65 1.50 1.77 1.76 1.64 2.13
0.47 0.58 0.49 0.68 0.55 0.70
B83-12/21/5 B83-12/21/5 B83-12/21/5 B83-12/21/5 B83-12/21/5 B83-12/21/5
0.00 0.27 0.55 1.60 3.20 Unrestricted
24.9 27.8 29.2 29.2 29.4 33.1
1.29 1.70 1.62 1.91 2.04 2.14
0.54 0.84 0.88 0.72 0.70 0.80
Morex Morex Morex Morex Morex Morex
0.00 0.27 0.55 1.60 3.20 Unrestricted
34.6 38.1 38.2 36.3 41.2 50.0
1.46 1.25 1.62 1.50 1.53 2.30
0.42 0.33 0.51 0.33 0.40 0.73
2.5 3.4
0.20 0.54
0.11 0.16
Promise Promise Promise Promise Promise Promise
LSD 5% stress LSD 5% genotype
crop. The smaller NDVI may be due to a combination of slower crop development leaving some bare soil exposed in the root restricted treatments and to the crop being less green. In both these scenarios it is a stress response resulting from the treatments that is being determined. From 56 DAS rain fell in most weeks (Fig. 1) and differences in the NDVI disappeared. No differences (p > 0.05) in
Fig. 4. Normalized Difference Vegetation Index (NDVI) as a function of days after sowing (DAS). The average number of holes per plant in the restricting mesh is indicated in the legend. Values are means of 5 genotypes. NS, not significant; ** p < 0.01; ***p < 0.001. Bars are 5% LSD for stress on each DAS.
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NDVI with root restriction occurred after 61 DAS. The NDVI showed a more rapid response of the crop to rain than did plant height where differences persisted until 75 DAS, which was a more rapid response than leaf area where differences persisted until 98 DAS. Differences in NDVI existed between genotypes on all but 2 recording dates. The genotypes split into groups with Morex and Golden Promise having lower NDVI values than the other 3 genotypes, Optic, B83 and Derkado, which were all similar. A diagnostic biplot of leaf area and NDVI for each measurement date (not shown) showed similar separation between genotypes. Table 1 shows the height, leaf area and NDVI results by stress and genotype at 55 DAS, when major differences between genotypes existed. All genotypes appeared to be similarly stressed relative to their unrestricted treatment. There was thus no evidence to suggest that genotypes differed in their ability to exploit holes in the mesh and obtain subsoil water. Morex, the only genotype without a dwarfing gene, was tallest in the unrestricted treatment but the relative heights with restriction were consistent with trends for the other genotypes. 2.6. Harvest At 110 DAS the plants had not reached maturity and different genotypes were at different stages of ear formation and filling. The dry weight of the individual plants grown with unrestricted roots was greater than for all levels of root restriction which did not differ significantly from each other. The total dry weight of plant material based on the quadrat samples was greatest in the unrestricted plots but no other differences occurred. Elemental analysis by ICP-MS showed no differences with either stress or genotype for Fe, Cl, Cs, K, Mg, Mn, Ni, P, or S. Minor differences between genotypes were found for Cu and Zn. The only element to show an effect of stress treatment was Ca, but this was not in an order consistent with treatments or other results (data not shown). 2.7. Soil water content Fig. 5 shows the water contents in the surface soil (above the mesh) and in the subsoil (below the mesh) relative to the mean water contents of each depth for the unrestricted treatment at 55 DAS for Optic. There was a clear trend that the soil was drier under
Fig. 5. Relative volumetric water content in the topsoil (0–6 cm) and subsoil (30 cm) of the Optic genotype at 55 days after sowing (DAS). Relative water content is water content in the treatment/water content in the unrestricted treatment.
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3. Discussion
plant height were still present and leaf area remained for several more weeks. This clearly demonstrates that to understand crop physiological responses to stress requires more than a single character to be quantified. All crops in this experiment had the same applied fertiliser.
3.1. Responses to restricted access to subsoil water
3.2. Do differences in subsoil exploitation exist?
In dry conditions plants may rely on extracting water from the subsoil (Passioura, 2006). Subsoil water will not be available in all environments, notably semi-arid regions, but practices such as fallowing are designed to store water in the soil profile (Koohafkan and Stewart, 2008). Periods of drought sufficient to affect crop growth and yield can occur in nearly all environments where cereals are produced, but it is especially important that genotypes grown in dryland environments are able to exploit stored water. Fig. 2 shows the importance of subsoil access across 5 common barley genotypes. As early as 26 DAS height differences occurred. Plants with unrestricted access to the subsoil were taller with a greater leaf area. Crops with plants having a tall dense canopy are better able to intercept light and thus out-compete weed populations or survive attack by pathogens. The advantage of access to subsoil water persisted through periods without rain and the plant heights and leaf area responded to the increased subsoil access. Our results are consistent with those of Beyrouty and Oosterhuis (1989) who used a mesh layer to totally restrict access and found that crops with no access to subsoil water performed less well that those with unrestricted access. After about 55 DAS, rainfall and decreased evaporative (Fig. 1) demand created an environment where the water needs of the crop could be supplied by rainfall without the crop needing to access subsoil water. This provided an environment whereby the crop was no longer water stressed. For the differences in height resulting from stress to be overcome the plants had to elongate the single main tiller and flag leaf. This requires relatively little resource and so plant height responded quickly to improved water availability. That there was no evidence of separation into 2 distributions of plant heights for plants that found a hole and those plants from which a root did not find the hole might be explained by the diameter of the holes relative to the diameter of a barley root at 20 cm depth in the soil. Typically barley roots are less than 1 mm diameter leaving sufficient space for more than one root to access any individual hole (2 mm diameter): indeed, it is common for roots to cluster together in naturally occurring biopores (Passioura, 1991). Leaf area index showed a response consistent with plant height. The crops with best access to subsoil water had a fuller canopy and those with restricted access had decreased leaf area proportional to the degree of restriction. Even though significant rain fell after 55 DAS the differences in leaf area index persisted until harvest. For the crop to recover from the imposed stress and to achieve the same canopy development as the unstressed crop requires increased tillering and expansion of total leaf area. This would require more photosynthate and mineral resources than required to simply elongate a single stem and flag leaf. Hence the differences in leaf area index persisted even after differences in plant height had disappeared. NDVI, a measure of crop colour, is influenced by multiple factors including exposed soil visible through the crop canopy, water status, nitrogen status, and leaf senescence. Despite the range of factors involved for any given date the NDVI’s were broadly consistent with the imposed stress treatment, i.e. increased crop stress resulting in lower NDVI’s. Because the NDVI is controlled by multiple factors the loss of differences between stress treatments after rain from 55 DAS could be a response of several factors. Differences in NDVI were not significant by 61 DAS while those for
All 5 modern genotypes of barley responded similarly to water stress applied by controlling access to subsoil water. Had the water stress differences persisted it is possible that interactions between the genotypes and stress may have occurred in important features such as final grain yield. Controlled application of water and protection from rainfall could improve future studies to allow more control of plant available water in the topsoil and subsoil. Differences in the interaction of roots with soil and biopores could provide improved exploitation of subsoil water. Firstly, plants that have more roots reaching an impeding soil or mesh layer will have greater chance of at least one root finding a biopore and thus gaining access to subsoil water. The modern genotypes used here all produce similar numbers of seminal roots. Secondly, there may be differences in the likelihood that a root reaching a barrier finds and enters a biopore. Dexter (1986) using model experiments and computer simulations found that the numbers of roots encountering holes in an artificial barrier was not different than on the basis of random chance. However, he found limited but sufficient evidence that in poorly aerated systems the possibility of roots growing preferentially to biopores could not be ruled out. In a similar model system Hirth et al. (1997) found no evidence that roots of ryegrass were likely to elongate to earthworm cast material. So there remains a possibility for differences between genotypes in sensing oxygen or other gradients around roots. Thirdly, roots elongating through a biopore and reaching subsoil water may have different plasticities or ability to branch. While root plasticity to nutrient localised supply has been well studied since Drew (1975), examples of root plasticity to localised differences in water supply, such as occur with water in subsoils, are relatively scarce. So there are at least 3 features of cereal root systems (number of roots, ability to find biopores and plasticity to subsoil water) that could differ with genotype and which provide hypotheses for future comparisons.
crops grown in treatments with more access through the mesh to the subsoil, while the topsoil was drier under crops that had less or no access to the subsoil.
3.3. Opportunities to modify soil to improve subsoil access Soil is a heterogeneous medium and situations where root access to the subsoil is totally constrained are likely to be rare. However, there may be scope to improve crop production in areas where root-impeding layers exist in the soil. This could either be done by improving cultivation systems, including the use of subsoil slotting and deep-ripping (Hartmann et al., 2008), or using crop rotation to increase the numbers of stable biopores (McCallum et al., 2004). 3.4. Implementation of the method and potential applications This first report of a novel method clearly raises questions about how to improve its effectiveness and what adaptation is needed to make it widely applicable. The layout of the experiment was done to be consistent with common cereal trials units of 6 m length, divided into 1 m2 subplots. While this offers utility it also requires sufficient seed to be available. Heights were measured on plants in the inner 4 (of 8) rows. As there were no effect of row on height it is probable that none of the measured plants had roots that grew beyond the mesh. Smaller units requiring less seed could be tested, offering the ability to test more genotypes per mesh area.
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Similarly questions of the number, size and arrangement of the holes in the mesh could be investigated based on understanding of root architecture and biopore distributions within soil. There may be scope to develop the method by placing the mesh at the same depth as local impeding layers occur. The methodology to establish this screening method in the field was relatively straightforward. The removal of the soil to position the mesh was done by machine, but in areas where labour is cheaper it could also be done by hand. Making the holes in the mesh and replacing the soil required no more care than required for careful establishment of any field experiment. The maximum number of holes in the mesh, expressed on a per plant basis was 3.2. Figs. 2–4 all show that at most times plants in this treatment grew less well than those in the unrestricted treatment. This suggests that to optimise cereal production soils should be managed to have no impeding layer, or if an impeding layer exists biopore numbers should be greater than 3.2 per cereal plant. We need to better understand the manner in which plant roots exploit the soil resource and importantly how soil can be managed to provide an environment conducive to root proliferation. Characterizing and quantifying the soil conditions at multiple depths, using modern soil quality descriptors, e.g. the Least Limiting Water Range (Da Silva and Kay, 2004), allow for quantification of suitable or non-suitable, restricting environments. In environments where management options are limited the need to understand the role of biopores and consider how they might be best used to provide options for roots to access subsoil water will be increasingly important. 4. Conclusions The results from this experiment support that by puncturing a mesh buried horizontally in the soil we are able to mimic biopores through hard soil layers and thus control access to subsoil water. As crops often rely on the subsoil for water to alleviate drought stress by varying the number of holes through the mesh we are able to differentially stress field-grown cereal crops for drought. The ability to regulate drought stress in a field environment provides a new method for plant breeders to screen germplasm. By identifying promising germplasm with root characteristics able to explore impeded layers for biopores, an important mechanism in drought tolerance could be better exploited. Further refinement of the method for adaptation to a range of crop types and soil conditions is now needed. Acknowledgments We thank Rowan Forster, Lawrie Brown, Richard Keith and Dennis Gordon for technical support. We thank Euan Caldwell and the estate staff at SCRI for crop management. The Scottish Crop Research Institute receives grant-in-aid support from the Scottish Government Rural and Environment Research and Analysis Directorate. References Adamsen, F.J., Pinter, P.J., Barnes, E.M., LaMorte, R.L., Wall, G.W., Leavitt, S.W., Kimball, B.A., 1999. Measuring wheat senescence with a digital camera. Crop Sci. 39, 719–724.
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