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Genetically modified cotton has no effect on arbuscular mycorrhizal colonisation of roots
Field Crops Research 109 (2008) 57–60
Contents lists available at ScienceDirect
Field Crops Research journal homepage: www.elsevier.com/locate/fcr
Short communication
Genetically modified cotton has no effect on arbuscular mycorrhizal colonisation of roots O.G.G. Knox a,c,*, D.B. Nehl b,c, T. Mor b, G.N. Roberts d, V.V.S.R. Gupta c,e a
CSIRO Entomology, Australian Cotton Research Institute, Locked Bag 59, Narrabri, NSW 2390, Australia NSW Department of Primary Industries, Australian Cotton Research Institute, Locked Bag 1000, Narrabri, NSW 2390, Australia c Cotton Catchment Communities CRC, Locked Bag 1001, Narrabri, NSW 2390, Australia d CSIRO Plant Industries, Australian Cotton Research Institute, Locked bag 59, Narrabri, NSW 2390, Australia e CSIRO Entomology, Gate 5, Waite Road, Narrabri, SA 5460, Australia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 May 2008 Received in revised form 10 June 2008 Accepted 11 June 2008
There is conjecture that genetically modified (GM) plants, expressing insecticidal or herbicide tolerance traits, do not form mycorrhizal symbioses. For cotton, Gossypium hirsutum, which is grown worldwide as a high and low input crop, such an issue would be of concern because it depends upon symbiosis with arbuscular mycorrhizal (AM) fungi for uptake of immobile elements, such as phosphorus and zinc, and GM cotton varieties are widely grown. We compared mycorrhizal development in commercial cultivars of cotton expressing genes for insect resistance (Cry1Ac and Cry2Ab), glyphosate tolerance (5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS)), or both, and their conventional parent line. AM development in cotton roots increased rapidly in the first three weeks after sowing, reaching a plateau level of around 70–80% root length. The observed pattern of colonisation was virtually identical among both conventional and GM cultivars of cotton at each assessment, clearly indicating that colonisation by AM fungi were not affected by the expressed transgenic traits. Crown Copyright ß 2008 Published by Elsevier B.V. All rights reserved.
Keywords: Gossypium hirsutum GM Transgenic AM
1. Introduction Since the introduction of genetically modified (GM) crops, there has been a steady increase in the global agricultural uptake and use of this technology (Brookes and Barfoot, 2005). However, whilst the benefits of the adoption of the various GM traits within the specific cropping systems often seem apparent from an economic and environmental perspective (Brookes and Barfoot, 2005; Knox et al., 2006), there remains a degree of concern over the safety of these GM crops (Azevedo and Araujo, 2003; Hilbeck and Schmidt, 2006). The ramifications of such concerns can be profound, with some nations refusing to utilise GM crops or accept food aid because of the European Union’s non-acceptance of the technology (Bodulovic, 2005). In Australia, the cotton industry quickly adopted genetically modified (GM) cotton, Gossypium hirsutum, in the late 1990s. In recent years an average of 70% of the Australian crop was sown with GM cotton cultivars having either insecticidal, herbicide tolerance or both traits (Monsanto Australia Limited, 2004).
* Corresponding author. Present address: SAC, Kings Buildings, West Mains Road, Edinburgh, EH 3JG, United Kingdom. Tel.: +44 131 5354170; fax: +44 131 5354144. E-mail address: [email protected] (O.G.G. Knox).
According to GMO Compass (http://www.gmo-compass.org/eng/ home/), GM cotton accounts for more than 40% of the current global cotton crop. GM cotton, as with all transgenic crops, has the potential to impact upon the soil microbiota through (i) the exudation of transgenic proteins from the root system, (ii) the release of the transgenic proteins as roots senesce, die and are broken down or (iii) when above-ground plant material becomes incorporated into soil, and (iv) through differences in exudation chemistry that might have resulted through either the gene insertion or backcrossing events used in producing the GM cultivar (Gupta and Watson, 2004; Knox et al., 2007; Saxena and Stotzky, 2001). Effects of GM technology on plant and associated microbe interactions have been reported (Baumgarte and Tebbe, 2005; Hilbeck and Schmidt, 2006; Powell et al., 2007; Wei et al., 2006; Zwahlen et al., 2003) although most have occurred in response to environmental or varietal differences rather than the inserted GM event and their significance to crop productivity has not been demonstrated. Glandorf et al. (1997) raised the possibility that the GM traits could influence symbiosis with arbuscular mycorrhizal (AM) fungi and this non-target effect has been a consideration by the European Union (Anderson et al., 2005) and by nongovernment organisations (Vogel, 2005) in deliberations about the acceptance of GM technology. Since then, there have been a
0378-4290/$ – see front matter . Crown Copyright ß 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2008.06.005
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few reports that mycorrhizal fungi had a reduced colonisation potential on some GM cultivars of maize (Castaldini et al., 2005; Turrini et al., 2004). In contrast to this, Powell et al. (2007) found that conventional and GM soybeans had different levels of rhizobium and mycorrhizal colonisation and that differences were attributed to varieties and not the GM status of the plant. Our study was conducted in light of a lack of available scientific evidence and in response to concerns about negative effects of GM plants on mycorrhizal symbiosis, both in the scientific community and within the Australian Cotton industry. No scientific publications exist for mycorrhizal development in GM cotton and conjecture in the existing literature suggests the need for case by case evaluation, both in terms of the plant species and inserted gene events. Given that cotton is an AM dependant plant (Smith and Roncadori, 1986; Rich and Bird, 1974), is grown in both high and low input systems, and that poor establishment of the symbiosis may have a significant impact on the crop from a nutritional perspective. AM fungi assist cotton with the acquisition of phosphorus (P), zinc (Zn) and several other trace nutrients (Nehl et al., 1996; Rochester et al., 2001). Slow mycorrhizal development and a deficiency in P and Zn uptake have been observed in association with a growth disorder of cotton in which early growth in the crop is stunted, crop maturity delayed and yield decreased (Nehl et al., 1996). However, the lack of mycorrhizal development was not due to a lack of AM fungi in the soil, suggesting that edaphic factors can have substantial impacts on mycorrhizal development in cotton (Nehl et al., 1996). In response to the lack of scientific data (Walter, 2005) and concerns that GM crops may have negative impacts on mycorrhizal symbiosis (Davidson, 2005; Shaw, 2005; Velkov et al., 2005) we conducted a study of AM colonisation of roots of several cultivars of cotton to establish if colonisation was affected by any of the transgenic modifications. 2. Materials and methods The cotton cultivar Sicot 189 was used as the conventional representative for this study because several transgenic varieties, with virtually identical agronomic traits, had been derived from it. The derived GM varieties were: Sicot 189 Roundup Ready1 (189RR), Sicot 289 Bollgard II1 (289B), and Sicot 289 Bollgard II1 Roundup Ready1 (289BR). The specific traits carried by the various varieties were the Bacillus thuringiensis (Bt) Cry1Ac and Cry2Ab insecticidal Bt genes in Bollgard1 varieties, and the 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS), from Agrobacterium sp. Strain CP4, for tolerance to glyphosate in the Roundup Ready1 varieties. Seed for all varieties was obtained from Cotton Seed Distributors Ltd. (Wee Waa, NSW). All seed was coated with a coloured seed dressing (Peridiam, Bayer CropScience Limited), containing the fungicides metalaxyl-m (Apron1, Syngenta Crop Protection), at 150 mg/kg seed, and PCNB (Quintozene, Bayer Crop Science), at 1.075 g/kg seed, according to standard commercial practice at the time. Cotton was sown on 8 October 2004, at 15 seeds/m of row, using a Norseman planter (Edgeroi, Australia) fitted with Almaco cones (Nevada, USA) in pre-irrigated fields that had 21% gravimetric water content in the top 15 cm of soil, at the Australian Cotton Research Institute (ACRI), Narrabri, NSW. The field history at the site included cotton in the summer of 2002– 2003, followed by wheat in the winter of 2003 and then fallow until the experiment was sown in October 2004. The soil was a grey self-mulching Vertosol (clay 55%, organic C 0.7%, pH 8, P 12–42 mg/ kg). The planted plots were 7 m long 4 m wide (i.e. four rows of cotton) with 1 m buffers between plots. The cultivars were sown in a completely randomised block design with four replicates.
Assessments started 14 days after sowing (DAS) and were continued weekly until 56 DAS, with one further assessment made in January 2005. Assessments involved recovering the roots from composite samples of four soil cores (90 mm diameter 150 mm deep) from each plot. Each core included the roots of two or more plants, depending on plant proximity to each other. The shoots of plants, above the four cored sites in each plot, were cut at soil level and combined as a composite sample. The number of shoots in each sample was recorded before oven drying 3 days at 70 8C in a Hurricane Force air drier (Wessberg and Tulander Pty. Ltd.) for dry mass determination. The large plants recovered in January 2005, had roots ramified through much of the soil bed so soil cores were taken adjacent to the large tap root of these plants. In all cases, shoots and soil cores were taken in the first and fourth rows of each plot, with subsequent samples taken towards the centre and not immediately adjacent to previous soil cores. Recovered soil cores were placed in polythene bags and stored in a portable cooler for no more than 2 h, prior to return to the laboratory for root recovery and staining. To recover the roots, 0.2% (w/v) sodium hexametaphosphate (Calgon1) was added to the bagged soil cores in sufficient quantity to submerse the soil. After soaking for 2 h, the soil was dispersed by vigorously shaking the bag by hand, allowing to settle for approximately 30 s and then the supernatant, containing suspended roots, was poured into a 130 mm sieve and washed with tap water. The trapped roots were recovered from the sieve, cut into 1 cm long sections and a subsample of at least 0.3 g (fresh mass) was stored in 70% ethanol. Roots were cleared and stained with 0.5% trypan blue by the method of Koske and Gemma (1989), modified by the use of 10% KOH solution to clear the roots and 2% HCl solution to acidify the roots prior to staining for 20 min. The stained roots were stored in acidified 50% glycerol solution prior to assessment of the percent of root length colonised by arsbucules, by scoring 100 root-gridline intersects (Giovannetti and Mosse, 1980) under 20 magnification for the presence of arbuscules. Data were analysed using the statistical program Genstat (Version 8, VSN International Ltd.) to compare cultivars by oneway analysis of variance (ANOVA). Graphical representations of mycorrhizal colonisation were plotted using the statistical program SYSTAT (Version 10, SPSS Inc.) and Gompertz curves fitted using least squares regression with quasi-Newton minimisation. At the end of the growing season, machine-picked cotton mass from the middle two rows of each plot was measured on a suspended load cell mounted within a modified cotton picker (Case International 1822). These weights were adjusted for an average content of 40% lint and used to estimate kg/ha harvested lint weights. Estimated yield weights were compared using ANOVA in Genstat 8. 3. Results Shoot dry weight analysis showed no difference between cotton cultivars at each of the sampling times (P = 0.27) and comparisons of mean shoot weights over time indicated that plant growth trends were similar (data not presented). Cotton plant growth for all cultivars, as assessed by above ground biomass dry weight, grew in an exponential fashion over the period of assessments, described by the equation y = 0.0142e0.0892x with R2 = 0.95, n = 128. In all four cultivars, AM colonisation developed in a pattern of logistic growth, with a rapid increase during the first three weeks after sowing being followed by a plateau level of around 70–80%
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Fig. 1. Similarity of arbuscular mycorrhizal development in roots of cotton cultivars; Sicot 189 (A), Sicot 189RR (B), Sicot 289B (C) and Sicot 289BR (D), sown at the Australian Cotton Research Institute in the 2004–2005 season.
colonised root length (Fig. 1). Analysis of variance at each assessment time showed that there were no significant differences in colonisation among cultivars over the entire duration of the experiment (P = 0.145, P = 0.622, P = 0.439, P = 0.151, P = 0.788, P = 0.131, P = 0.828 and P = 0.896 for each assessment, respectively). The yield of lint in the experiment ranged from 2565 to 3022 kg/ ha and there was no significant difference in yield among the four varieties. 4. Discussion The lack of differences in growth and yield among the cultivars reflects the agronomic similarity of the GM cultivars to their parent line and does not indicate that the gene insertions for insect resistance were not effective, as the pressure from lepidopteran pests during the summer of the experiment was very low. Deficiency of either P or Zn can cause stunting of the cotton plants (Rochester et al., 2001), but these symptoms were not observed during any of the crop assessments. The absence of symptoms of nutritional deficiency in conjunction with previous soil tests (data not shown) indicates that, although not tested directly, all cultivars had adequate P and Zn nutrition. The yields were well within expected cultivar performance boundaries, indicating that agronomic practices during the experiment were consistent with commercial practice. The status of arbuscular mycorrhizas as a symbiont rather than pathogen can vary according to the compatibility of the host plant and the species or strain of arbuscular fungus (Kiers et al., 2002). For example, the capacity for AM fungi to transport P to the plant
varies with the species of fungus (Pearson and Jakobsen, 1993) and host (Ravnskov and Jakobsen, 1995). Accordingly, the abundance and spectrum of species of arbuscular fungi in soil will vary with agricultural practices, such as crop rotation (Vestberg et al., 2005). In our experiment, it was assumed that the level of AM fungal inoculum in the trial site was relatively uniform, given the trial site’s previous cropping history. Upon sowing cotton into this soil, AM fungi colonised both the GM and the conventional cotton cultivars equally, providing firm evidence that both the GM and non-GM cotton cultivars were equally capable of establishing mycorrhizal symbiosis. The possibility that the gene insertions may have influenced the function of the AM symbiosis (Glandorf et al., 1997) cannot be ruled out. However, if any variation in symbiotic function did occur among the cultivars, such variation was not expressed in either the level of colonisation of roots by the fungi or the growth and yield of the host. AM colonisation of cotton normally progresses quickly during the first four weeks of growth, reaching a plateau level approximately in the range of 50–70% root length colonised by arsbucules (Nehl et al., 1996, 1999; Pattinson and McGee, 1997). In this regard, the pattern of development of mycorrhizal colonisation in our assessment was typical and grew in a sigmoid pattern of logistic growth over the 14 weeks of the assessment to levels of approximately 80% colonisation per unit root length for all cultivars (Fig. 1). The lack of differences in colonisation between GM and conventional cotton that we observed corroborates that reported for GM and conventional soybean (Powell et al., 2007), but is in contrast to reports of differential mycorrhizal colonisation of GM corn (Castaldini et al., 2005; Turrini et al., 2004). Whilst there were
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major differences between the cropping system in our experiment (C3 plants in the field) and these other studies (C3 or C4 plants in the glasshouse), the mechanisms by which GM crops might be influencing mycorrhizal colonisation remains speculative (Powell et al., 2007) and requires further investigation. Additionally, the fact that conventional and GM cotton, soybean and some corns are equally as mycorrhizal, whilst a few GM corns have reduced formation of this symbiosis, serves as a reminder that evaluation of GM technology should be conducted on a case by case scenario. 5. Conclusion In conclusion, the findings of the experiment reported in this paper corroborate the study of Walter (2005) and clearly indicate that field grown cotton, regardless as to whether it is conventional or GM for either insecticidal or herbicide tolerance or both traits, is mycorrhizal. Acknowledgements The reported work was funded by the Cotton Research and Development Corporation, NSW Department of Primary Industries and the CSIRO divisions of Land and Water, Entomology and Plant Industry, with support from the Cotton CRC. Technical assistance by Ms Vicki Bourke is gratefully acknowledged. References Anderson, H.C., Bartsch, D., Buhk, H., Davies, H., De Loose, M., Gasson, M., Hendriksen, N., Heritage, J., Karenlampi, S., Krypsin-Sorensen, I., Kuiper, H., Nuti, M., O’gara, F., Puigdomenech, P., Sakellaris, G., Schiemann, J., Seinen, W., Sessitsch, A., Sweet, J., van Elsas, J.D., Wal, J., 2005. Opinion of the scientific panel on genetically modified organisms on a request from the commission related to the notification (reference C/F?96/05.10) for the placing on the market of insect resistant genetically modified maize Bt11, for cultivation, feed and industrial processing, under Part C of directive 2001/18/EC from Syngenta seeds. EFSA J. 213, 1–33. Azevedo, J.L., Araujo, W.L., 2003. Genetically modified crops: environmental and human health concerns. Mutat. Res. 544, 223–233. Baumgarte, S., Tebbe, C.C., 2005. Field studies on the environmental fate of the Cry1ab Bt-toxin produced by transgenic maize (Mon810) and its effect on bacterial communities in the maize rhizosphere. Mol. Ecol. 14, 2539–2551. Bodulovic, G., 2005. Is the European attitude to GM products suffocating African development? Funct. Plant Biol. 32, 1069–1075. Brookes, G., Barfoot, P., 2005. GM crops: the global economic and environmental impact–the first nine years 1996–2004. Agric. Biol. Forum 8, 187–196. Castaldini, M., Turrini, A., Sbrana, C., Benedetti, A., Marchionni, M., Mocali, S., Fabiani, A., Landi, S., Santomassimo, F., Pietrangeli, B., Nuti, M.P., Miclaus, N., Giovannetti, M., 2005. Impact of Bt corn on rhizospheric and soil eubacterial communities and on beneficial mycorrhizal symbiosis in experimental microcosms. Appl. Environ. Microbiol. 71, 6719–6729. Davidson, B., 2005. Bio Nutrient Solutions. Cotton Consultants Australia Inc. Newsletter (Winter). CCA Inc.. Giovannetti, M., Mosse, B., 1980. An evaluation of techniques for measuring vesicular–arbuscular mycorrhizal infection in roots. New Phytologist 84, 489–500. Glandorf, D.C.M., Bakker, P.A.H.M., VanLoon, L.C., 1997. Influence of the production of antibacterial and antifungal proteins by transgenic plants on the saprophytic soil microflora. Acta Bot. Neerland. 46, 85–104. Gupta, V., Watson, S., 2004. Ecological impacts of GM cotton on soil biodiversity. Final report for a project funded by Australian Government Department of the Environment and Heritage, CSIRO Land and Water.
Hilbeck, A., Schmidt, J.E.U., 2006. Another view of Bt proteins—how specific are they and what else might they do? Biopesticides Int. 2, 1–50. Kiers, E.T., West, S.A., Denison, R.F., 2002. Mediating mutualisms: farm management practices and evolutionary changes in symbiont co-operation. J. Appl. Ecol. 39, 745–754. Knox, O.G.G., Constable, G.A., Pyke, B., Gupta, V.V.S.R., 2006. Environmental impact of conventional and Bt insecticidal cotton expressing one and two cry genes in Australia. Aust. J. Agric. Res. 57, 501–509. Knox, O.G.G., Gupta, V.V.S.R., Nehl, D.B., Stiller, W.N., 2007. Constitutive expression of cry proteins in roots and border cells of transgenic cotton. Euphytica 154, 83– 90. Koske, R.E., Gemma, J.N., 1989. A modified procedure for staining roots to detect V-A mycorrhizas. Mycol. Res.V 92 486–488. Monsanto Australia Limited, 2004. Area of biotech cotton doubles: Australian growers embrace cleaner and greener biotechnology [online]. Available from http://www.monsanto.com.au/content/links_resources/media/04/ 04_20_12.pdf. Nehl, D.B., Allen, S.J., Brown, J.F., 1996. Mycorrhizal colonisation, root browning and soil properties associated with a growth disorder of cotton in Australia. Plant Soil 179, 171–182. Nehl, D.B., Mcgee, P.A., Torrisi, V., Pattinson, G.S., Allen, S.J., 1999. Patterns of arbuscular mycorrhiza down the profile of a heavy textured soil do not reflect associated colonization potential. New Phytologist 142, 495–503. Pattinson, G.S., McGee, P.A., 1997. High densities of arbuscular mycorrhizal fungi maintained during long fallows in soils used to grow cotton except when soil is wetted periodically. New Phytologist 136, 571–580. Pearson, J.N., Jakobsen, I., 1993. The relative contribution of hyphae and roots to phosphorous uptake by arbuscular mycorrhizal plants, measured by dual labeling with P-32 and P-33. New Phytologist 124, 489–494. Powell, J.R., Gulden, R.H., Hart, M.M., Campbell, R.G., Levy-Booth, D.J., Dunfield, K.E., Pauls, K.P., Swanton, C.J., Trevors, J.T., Klironomos, J.N., 2007. Mycorrhizal and rhizobial colonization of genetically modified and conventional soybeans. Appl. Environ. Microbiol. 73, 4365–4367. Ravnskov, S., Jakobsen, I., 1995. Functional compatibility in arbuscular mycorrhizas measured as hyphal P transport to the plant. New Phytologist 129, 611–618. Rich, J.R., Bird, G.W., 1974. Association of early-seasons vesicular–arbuscular mycorrhizae with increased growth and development of cotton. Phytopathology 64. Rochester, I.J., Rea, M., Dorahy, C., Constable, G.A., Wright, P., Deutscher, S., Thongbai, P., Larsen, D., 2001. NUTRIpak–A Practical Guide to Cotton Nutrition. Australian Cotton CRC, CSIRO Publishing, Melbourne. Saxena, D., Stotzky, G., 2001. Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. Soil Biol. Biochem. 33, 1225–1230. Shaw, G., 2005. Soil Health Issues for Australian Cotton Production—Growers Perspective. Cotton Research and Development Corporation. Smith, G.S., Roncadori, R.W., 1986. Response of three vesicular–arbuscular mycorrhizal fungi at four soil temperatures and their effects on cotton growth. New Phytologist 104, 89–95. Turrini, A., Sbrana, C., Nuti, M.P., Pietrangeli, B.M., Giovannetti, M., 2004. Development of a model system to assess the impact of genetically modified corn and aubergine plants on arbuscular mycorrhizal fungi. Plant Soil 266, 69–75. Velkov, V.V., Medvinsky, A.B., Sokolov, M.S., Marchenko, A.I., 2005. Will transgenic plants adversely affect the Environment? J. Biosci. 30, 515–548. Vestberg, M., Saari, K., Kukkonen, S., Hurme, T., 2005. Mycotrophy of crops in rotation and soil amendment with peat influence the abundance and effectiveness of indigenous arbuscular mycorrhizal fungi in field soil. Mycorrhiza 15, 447–458. Vogel, B., 2005. Auswirkungen des Anbaus von gentechnisch vera¨nderten Pflanzen auf die biologische Vielfalt. NABU, Deutschland. Agro-Gentechnik & Naturschutz. Deutschland, NABU. Walter, D., 2005. The environmental impact of genetically modified crop plants on the microbiology of the rhizosphere. Ph.D. thesis. Flinders University, South Australia. Wei, X.D., Zou, H.L., Chu, L.M., Liao, B., Ye, C.M., Lan, C.Y., 2006. Field released transgenic papaya affects microbial communities and enzyme activities in soil. Plant Soil 285, 347–358. Zwahlen, C., Hilbeck, A., Howald, R., Nentwig, W., 2003. Effects of transgenic Bt corn litter on the earthworm lumbricus terrestris. Mol. Ecol. 12, 1077–1086.
Contents lists available at ScienceDirect
Field Crops Research journal homepage: www.elsevier.com/locate/fcr
Short communication
Genetically modified cotton has no effect on arbuscular mycorrhizal colonisation of roots O.G.G. Knox a,c,*, D.B. Nehl b,c, T. Mor b, G.N. Roberts d, V.V.S.R. Gupta c,e a
CSIRO Entomology, Australian Cotton Research Institute, Locked Bag 59, Narrabri, NSW 2390, Australia NSW Department of Primary Industries, Australian Cotton Research Institute, Locked Bag 1000, Narrabri, NSW 2390, Australia c Cotton Catchment Communities CRC, Locked Bag 1001, Narrabri, NSW 2390, Australia d CSIRO Plant Industries, Australian Cotton Research Institute, Locked bag 59, Narrabri, NSW 2390, Australia e CSIRO Entomology, Gate 5, Waite Road, Narrabri, SA 5460, Australia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 May 2008 Received in revised form 10 June 2008 Accepted 11 June 2008
There is conjecture that genetically modified (GM) plants, expressing insecticidal or herbicide tolerance traits, do not form mycorrhizal symbioses. For cotton, Gossypium hirsutum, which is grown worldwide as a high and low input crop, such an issue would be of concern because it depends upon symbiosis with arbuscular mycorrhizal (AM) fungi for uptake of immobile elements, such as phosphorus and zinc, and GM cotton varieties are widely grown. We compared mycorrhizal development in commercial cultivars of cotton expressing genes for insect resistance (Cry1Ac and Cry2Ab), glyphosate tolerance (5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS)), or both, and their conventional parent line. AM development in cotton roots increased rapidly in the first three weeks after sowing, reaching a plateau level of around 70–80% root length. The observed pattern of colonisation was virtually identical among both conventional and GM cultivars of cotton at each assessment, clearly indicating that colonisation by AM fungi were not affected by the expressed transgenic traits. Crown Copyright ß 2008 Published by Elsevier B.V. All rights reserved.
Keywords: Gossypium hirsutum GM Transgenic AM
1. Introduction Since the introduction of genetically modified (GM) crops, there has been a steady increase in the global agricultural uptake and use of this technology (Brookes and Barfoot, 2005). However, whilst the benefits of the adoption of the various GM traits within the specific cropping systems often seem apparent from an economic and environmental perspective (Brookes and Barfoot, 2005; Knox et al., 2006), there remains a degree of concern over the safety of these GM crops (Azevedo and Araujo, 2003; Hilbeck and Schmidt, 2006). The ramifications of such concerns can be profound, with some nations refusing to utilise GM crops or accept food aid because of the European Union’s non-acceptance of the technology (Bodulovic, 2005). In Australia, the cotton industry quickly adopted genetically modified (GM) cotton, Gossypium hirsutum, in the late 1990s. In recent years an average of 70% of the Australian crop was sown with GM cotton cultivars having either insecticidal, herbicide tolerance or both traits (Monsanto Australia Limited, 2004).
* Corresponding author. Present address: SAC, Kings Buildings, West Mains Road, Edinburgh, EH 3JG, United Kingdom. Tel.: +44 131 5354170; fax: +44 131 5354144. E-mail address: [email protected] (O.G.G. Knox).
According to GMO Compass (http://www.gmo-compass.org/eng/ home/), GM cotton accounts for more than 40% of the current global cotton crop. GM cotton, as with all transgenic crops, has the potential to impact upon the soil microbiota through (i) the exudation of transgenic proteins from the root system, (ii) the release of the transgenic proteins as roots senesce, die and are broken down or (iii) when above-ground plant material becomes incorporated into soil, and (iv) through differences in exudation chemistry that might have resulted through either the gene insertion or backcrossing events used in producing the GM cultivar (Gupta and Watson, 2004; Knox et al., 2007; Saxena and Stotzky, 2001). Effects of GM technology on plant and associated microbe interactions have been reported (Baumgarte and Tebbe, 2005; Hilbeck and Schmidt, 2006; Powell et al., 2007; Wei et al., 2006; Zwahlen et al., 2003) although most have occurred in response to environmental or varietal differences rather than the inserted GM event and their significance to crop productivity has not been demonstrated. Glandorf et al. (1997) raised the possibility that the GM traits could influence symbiosis with arbuscular mycorrhizal (AM) fungi and this non-target effect has been a consideration by the European Union (Anderson et al., 2005) and by nongovernment organisations (Vogel, 2005) in deliberations about the acceptance of GM technology. Since then, there have been a
0378-4290/$ – see front matter . Crown Copyright ß 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2008.06.005
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few reports that mycorrhizal fungi had a reduced colonisation potential on some GM cultivars of maize (Castaldini et al., 2005; Turrini et al., 2004). In contrast to this, Powell et al. (2007) found that conventional and GM soybeans had different levels of rhizobium and mycorrhizal colonisation and that differences were attributed to varieties and not the GM status of the plant. Our study was conducted in light of a lack of available scientific evidence and in response to concerns about negative effects of GM plants on mycorrhizal symbiosis, both in the scientific community and within the Australian Cotton industry. No scientific publications exist for mycorrhizal development in GM cotton and conjecture in the existing literature suggests the need for case by case evaluation, both in terms of the plant species and inserted gene events. Given that cotton is an AM dependant plant (Smith and Roncadori, 1986; Rich and Bird, 1974), is grown in both high and low input systems, and that poor establishment of the symbiosis may have a significant impact on the crop from a nutritional perspective. AM fungi assist cotton with the acquisition of phosphorus (P), zinc (Zn) and several other trace nutrients (Nehl et al., 1996; Rochester et al., 2001). Slow mycorrhizal development and a deficiency in P and Zn uptake have been observed in association with a growth disorder of cotton in which early growth in the crop is stunted, crop maturity delayed and yield decreased (Nehl et al., 1996). However, the lack of mycorrhizal development was not due to a lack of AM fungi in the soil, suggesting that edaphic factors can have substantial impacts on mycorrhizal development in cotton (Nehl et al., 1996). In response to the lack of scientific data (Walter, 2005) and concerns that GM crops may have negative impacts on mycorrhizal symbiosis (Davidson, 2005; Shaw, 2005; Velkov et al., 2005) we conducted a study of AM colonisation of roots of several cultivars of cotton to establish if colonisation was affected by any of the transgenic modifications. 2. Materials and methods The cotton cultivar Sicot 189 was used as the conventional representative for this study because several transgenic varieties, with virtually identical agronomic traits, had been derived from it. The derived GM varieties were: Sicot 189 Roundup Ready1 (189RR), Sicot 289 Bollgard II1 (289B), and Sicot 289 Bollgard II1 Roundup Ready1 (289BR). The specific traits carried by the various varieties were the Bacillus thuringiensis (Bt) Cry1Ac and Cry2Ab insecticidal Bt genes in Bollgard1 varieties, and the 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS), from Agrobacterium sp. Strain CP4, for tolerance to glyphosate in the Roundup Ready1 varieties. Seed for all varieties was obtained from Cotton Seed Distributors Ltd. (Wee Waa, NSW). All seed was coated with a coloured seed dressing (Peridiam, Bayer CropScience Limited), containing the fungicides metalaxyl-m (Apron1, Syngenta Crop Protection), at 150 mg/kg seed, and PCNB (Quintozene, Bayer Crop Science), at 1.075 g/kg seed, according to standard commercial practice at the time. Cotton was sown on 8 October 2004, at 15 seeds/m of row, using a Norseman planter (Edgeroi, Australia) fitted with Almaco cones (Nevada, USA) in pre-irrigated fields that had 21% gravimetric water content in the top 15 cm of soil, at the Australian Cotton Research Institute (ACRI), Narrabri, NSW. The field history at the site included cotton in the summer of 2002– 2003, followed by wheat in the winter of 2003 and then fallow until the experiment was sown in October 2004. The soil was a grey self-mulching Vertosol (clay 55%, organic C 0.7%, pH 8, P 12–42 mg/ kg). The planted plots were 7 m long 4 m wide (i.e. four rows of cotton) with 1 m buffers between plots. The cultivars were sown in a completely randomised block design with four replicates.
Assessments started 14 days after sowing (DAS) and were continued weekly until 56 DAS, with one further assessment made in January 2005. Assessments involved recovering the roots from composite samples of four soil cores (90 mm diameter 150 mm deep) from each plot. Each core included the roots of two or more plants, depending on plant proximity to each other. The shoots of plants, above the four cored sites in each plot, were cut at soil level and combined as a composite sample. The number of shoots in each sample was recorded before oven drying 3 days at 70 8C in a Hurricane Force air drier (Wessberg and Tulander Pty. Ltd.) for dry mass determination. The large plants recovered in January 2005, had roots ramified through much of the soil bed so soil cores were taken adjacent to the large tap root of these plants. In all cases, shoots and soil cores were taken in the first and fourth rows of each plot, with subsequent samples taken towards the centre and not immediately adjacent to previous soil cores. Recovered soil cores were placed in polythene bags and stored in a portable cooler for no more than 2 h, prior to return to the laboratory for root recovery and staining. To recover the roots, 0.2% (w/v) sodium hexametaphosphate (Calgon1) was added to the bagged soil cores in sufficient quantity to submerse the soil. After soaking for 2 h, the soil was dispersed by vigorously shaking the bag by hand, allowing to settle for approximately 30 s and then the supernatant, containing suspended roots, was poured into a 130 mm sieve and washed with tap water. The trapped roots were recovered from the sieve, cut into 1 cm long sections and a subsample of at least 0.3 g (fresh mass) was stored in 70% ethanol. Roots were cleared and stained with 0.5% trypan blue by the method of Koske and Gemma (1989), modified by the use of 10% KOH solution to clear the roots and 2% HCl solution to acidify the roots prior to staining for 20 min. The stained roots were stored in acidified 50% glycerol solution prior to assessment of the percent of root length colonised by arsbucules, by scoring 100 root-gridline intersects (Giovannetti and Mosse, 1980) under 20 magnification for the presence of arbuscules. Data were analysed using the statistical program Genstat (Version 8, VSN International Ltd.) to compare cultivars by oneway analysis of variance (ANOVA). Graphical representations of mycorrhizal colonisation were plotted using the statistical program SYSTAT (Version 10, SPSS Inc.) and Gompertz curves fitted using least squares regression with quasi-Newton minimisation. At the end of the growing season, machine-picked cotton mass from the middle two rows of each plot was measured on a suspended load cell mounted within a modified cotton picker (Case International 1822). These weights were adjusted for an average content of 40% lint and used to estimate kg/ha harvested lint weights. Estimated yield weights were compared using ANOVA in Genstat 8. 3. Results Shoot dry weight analysis showed no difference between cotton cultivars at each of the sampling times (P = 0.27) and comparisons of mean shoot weights over time indicated that plant growth trends were similar (data not presented). Cotton plant growth for all cultivars, as assessed by above ground biomass dry weight, grew in an exponential fashion over the period of assessments, described by the equation y = 0.0142e0.0892x with R2 = 0.95, n = 128. In all four cultivars, AM colonisation developed in a pattern of logistic growth, with a rapid increase during the first three weeks after sowing being followed by a plateau level of around 70–80%
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Fig. 1. Similarity of arbuscular mycorrhizal development in roots of cotton cultivars; Sicot 189 (A), Sicot 189RR (B), Sicot 289B (C) and Sicot 289BR (D), sown at the Australian Cotton Research Institute in the 2004–2005 season.
colonised root length (Fig. 1). Analysis of variance at each assessment time showed that there were no significant differences in colonisation among cultivars over the entire duration of the experiment (P = 0.145, P = 0.622, P = 0.439, P = 0.151, P = 0.788, P = 0.131, P = 0.828 and P = 0.896 for each assessment, respectively). The yield of lint in the experiment ranged from 2565 to 3022 kg/ ha and there was no significant difference in yield among the four varieties. 4. Discussion The lack of differences in growth and yield among the cultivars reflects the agronomic similarity of the GM cultivars to their parent line and does not indicate that the gene insertions for insect resistance were not effective, as the pressure from lepidopteran pests during the summer of the experiment was very low. Deficiency of either P or Zn can cause stunting of the cotton plants (Rochester et al., 2001), but these symptoms were not observed during any of the crop assessments. The absence of symptoms of nutritional deficiency in conjunction with previous soil tests (data not shown) indicates that, although not tested directly, all cultivars had adequate P and Zn nutrition. The yields were well within expected cultivar performance boundaries, indicating that agronomic practices during the experiment were consistent with commercial practice. The status of arbuscular mycorrhizas as a symbiont rather than pathogen can vary according to the compatibility of the host plant and the species or strain of arbuscular fungus (Kiers et al., 2002). For example, the capacity for AM fungi to transport P to the plant
varies with the species of fungus (Pearson and Jakobsen, 1993) and host (Ravnskov and Jakobsen, 1995). Accordingly, the abundance and spectrum of species of arbuscular fungi in soil will vary with agricultural practices, such as crop rotation (Vestberg et al., 2005). In our experiment, it was assumed that the level of AM fungal inoculum in the trial site was relatively uniform, given the trial site’s previous cropping history. Upon sowing cotton into this soil, AM fungi colonised both the GM and the conventional cotton cultivars equally, providing firm evidence that both the GM and non-GM cotton cultivars were equally capable of establishing mycorrhizal symbiosis. The possibility that the gene insertions may have influenced the function of the AM symbiosis (Glandorf et al., 1997) cannot be ruled out. However, if any variation in symbiotic function did occur among the cultivars, such variation was not expressed in either the level of colonisation of roots by the fungi or the growth and yield of the host. AM colonisation of cotton normally progresses quickly during the first four weeks of growth, reaching a plateau level approximately in the range of 50–70% root length colonised by arsbucules (Nehl et al., 1996, 1999; Pattinson and McGee, 1997). In this regard, the pattern of development of mycorrhizal colonisation in our assessment was typical and grew in a sigmoid pattern of logistic growth over the 14 weeks of the assessment to levels of approximately 80% colonisation per unit root length for all cultivars (Fig. 1). The lack of differences in colonisation between GM and conventional cotton that we observed corroborates that reported for GM and conventional soybean (Powell et al., 2007), but is in contrast to reports of differential mycorrhizal colonisation of GM corn (Castaldini et al., 2005; Turrini et al., 2004). Whilst there were
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major differences between the cropping system in our experiment (C3 plants in the field) and these other studies (C3 or C4 plants in the glasshouse), the mechanisms by which GM crops might be influencing mycorrhizal colonisation remains speculative (Powell et al., 2007) and requires further investigation. Additionally, the fact that conventional and GM cotton, soybean and some corns are equally as mycorrhizal, whilst a few GM corns have reduced formation of this symbiosis, serves as a reminder that evaluation of GM technology should be conducted on a case by case scenario. 5. Conclusion In conclusion, the findings of the experiment reported in this paper corroborate the study of Walter (2005) and clearly indicate that field grown cotton, regardless as to whether it is conventional or GM for either insecticidal or herbicide tolerance or both traits, is mycorrhizal. Acknowledgements The reported work was funded by the Cotton Research and Development Corporation, NSW Department of Primary Industries and the CSIRO divisions of Land and Water, Entomology and Plant Industry, with support from the Cotton CRC. Technical assistance by Ms Vicki Bourke is gratefully acknowledged. References Anderson, H.C., Bartsch, D., Buhk, H., Davies, H., De Loose, M., Gasson, M., Hendriksen, N., Heritage, J., Karenlampi, S., Krypsin-Sorensen, I., Kuiper, H., Nuti, M., O’gara, F., Puigdomenech, P., Sakellaris, G., Schiemann, J., Seinen, W., Sessitsch, A., Sweet, J., van Elsas, J.D., Wal, J., 2005. Opinion of the scientific panel on genetically modified organisms on a request from the commission related to the notification (reference C/F?96/05.10) for the placing on the market of insect resistant genetically modified maize Bt11, for cultivation, feed and industrial processing, under Part C of directive 2001/18/EC from Syngenta seeds. EFSA J. 213, 1–33. Azevedo, J.L., Araujo, W.L., 2003. Genetically modified crops: environmental and human health concerns. Mutat. Res. 544, 223–233. Baumgarte, S., Tebbe, C.C., 2005. Field studies on the environmental fate of the Cry1ab Bt-toxin produced by transgenic maize (Mon810) and its effect on bacterial communities in the maize rhizosphere. Mol. Ecol. 14, 2539–2551. Bodulovic, G., 2005. Is the European attitude to GM products suffocating African development? Funct. Plant Biol. 32, 1069–1075. Brookes, G., Barfoot, P., 2005. GM crops: the global economic and environmental impact–the first nine years 1996–2004. Agric. Biol. Forum 8, 187–196. Castaldini, M., Turrini, A., Sbrana, C., Benedetti, A., Marchionni, M., Mocali, S., Fabiani, A., Landi, S., Santomassimo, F., Pietrangeli, B., Nuti, M.P., Miclaus, N., Giovannetti, M., 2005. Impact of Bt corn on rhizospheric and soil eubacterial communities and on beneficial mycorrhizal symbiosis in experimental microcosms. Appl. Environ. Microbiol. 71, 6719–6729. Davidson, B., 2005. Bio Nutrient Solutions. Cotton Consultants Australia Inc. Newsletter (Winter). CCA Inc.. Giovannetti, M., Mosse, B., 1980. An evaluation of techniques for measuring vesicular–arbuscular mycorrhizal infection in roots. New Phytologist 84, 489–500. Glandorf, D.C.M., Bakker, P.A.H.M., VanLoon, L.C., 1997. Influence of the production of antibacterial and antifungal proteins by transgenic plants on the saprophytic soil microflora. Acta Bot. Neerland. 46, 85–104. Gupta, V., Watson, S., 2004. Ecological impacts of GM cotton on soil biodiversity. Final report for a project funded by Australian Government Department of the Environment and Heritage, CSIRO Land and Water.
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