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Evidences of inhibited arbuscular mycorrhizal fungal development and colonization in multiple lines of Bt cotton
Agriculture, Ecosystems and Environment 230 (2016) 169–176
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Evidences of inhibited arbuscular mycorrhizal fungal development and colonization in multiple lines of Bt cotton Xiu-Hua Chena,b,* , Feng-Ling Wangb , Rui Zhangb , Ling-Ling Jib , Zheng-Lun Yangb , Hui Lina , Bin Zhaoa a
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtse River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China b
A R T I C L E I N F O
Article history: Received 23 November 2015 Received in revised form 31 March 2016 Accepted 6 May 2016 Available online xxx Keywords: Bt cotton Arbuscular mycorrhizal fungi Pre-symbiotic hyphal differentiation Colonization
A B S T R A C T
A “sandwich system” was used to investigate the effect of transgenic Bacillus thuringiensis (Bt) cotton (Gossypium hirsutum L.) on early stages of arbuscular mycorrhizal (AM) fungal life cycle. Spore germination and pre-symbiotic hyphal differentiation of AM fungus Rhizophagus irregularis were decreased in the rhizosphere of transgenic Bt cotton isolines (Jin26, GK12 and Jin44) compared with in that of their corresponding parental non-transgenic isolines (Jin7, Si3 and Ji492); and the appressorium number, colonization intensity and arbuscule abundance were lower in Bt plant roots. More collapsed arbuscules were also observed in Bt roots. The results of statistical analysis demonstrate that Bt-trait significantly contributes to the inhibition of pre-symbiotic development and AM fungal colonization, which might be attributed to either Bt toxin toxicity or interference of signal perception between AM fungi and the hosts. The specific mechanism requires further study. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Cotton is one of the most important crops worldwide. In recent years, transgenic Bacillus thuringiensis (Bt) cotton varieties have been widely grown, which effectively controls cotton bollworm and reduces the application of insecticides (Lu et al., 2012). China is one of the largest producers of cotton in the world. In 2013, the cultivation area of Bt cotton reached 4.2 million hectares, accounting for 90% of the total area of cotton cultivation in China (James, 2014). During the entire growth period of Bt cotton, Bt toxin is introduced into the soil through root exudates, decomposing plant material and/or pollen deposition (Icoz and Stotzky, 2008). Bt toxin contains truncated genes that encode toxins rather than nontoxic crystalline protoxins produced by B. thuringiensis. It is not necessary to solubilize the protoxins at a high gut pH to gain the insecticidal activity. Since Bt toxin can be bound to clays and humic substances to resist microbial decomposition, its insecticidal activity can be maintained for several months in the soil
* Corresponding author at: College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China. E-mail address: [email protected] (X.-H. Chen). http://dx.doi.org/10.1016/j.agee.2016.05.008 0167-8809/ã 2016 Elsevier B.V. All rights reserved.
(Saxena and Stotzky, 2001). Besides, receptors for the toxins are present in both target and nontarget insects (Hoöfte and Whiteley, 1989). Bt toxin residue in soil may pose a potential risk to soil organisms such as arbuscular mycorrhizal (AM) fungi, a kind of non-target soil microorganisms fundamental for soil fertility and plant nutrition. AM fungi are obligate biotrophs that form one of the most widespread and ancient mutualistic symbioses with most land plants (Smith and Read, 2008). The symbionts have coexisted for 400 million years (Redeker et al., 2000). This evolutionary success can be traced to the role of AM fungi in providing multiple benefits for the plant, including uptake of nutrients (mainly phosphorus and nitrogen) and water (Smith and Read, 2008), and also tolerance to pathogens and abiotic stresses (Newsham et al., 1995; Smith et al., 2003; Chen and Zhao, 2007). In return, host plants transfer carbohydrates to the fungi to support their lifecycle (Ho and Trappe, 1973; Olsson et al., 2010). Because of their obligately biotrophic relationship with plant roots, AM fungi may be more sensitive to Bt crops than other freeliving organisms, even if they are not affected by Bt proteins directly. Recent studies have shown an altered relationship between some cultivars of Bt maize and AM fungi. Turrini et al.
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(2004) reported that transgenic Bt maize (event Bt 176) had a smaller number of viable AM fungal infection structures compared with its non-Bt parental isoline. Some studies showed reduced level of AM fungal colonization in Bt maize roots (Castaldini et al., 2005; Cheeke et al., 2012) and lower density of AM fungi spores in the plots with Bt maize cultivation history (Cheeke et al., 2014); while other studies demonstrated that there is no negative effect of Bt crop cultivation on AM fungi (de Vaufleury et al., 2007; Knox et al., 2008; Cheeke et al., 2013; Cheeke et al., 2015). There have been no evidences showing the direct effect of Bt crop on the early stages of AM fungal life cycle. Genetic changes within a plant (either through genetic engineering or traditional approaches) might alter plant metabolic activity (Schaarschmidt et al., 2007) and root exudate components (Bais et al., 2006; Broeckling et al., 2008), which might affect the chemical signals (Akiyama et al., 2005). Throughout the life cycle of AM fungi, from spore germination to fungal structure development within roots, AM fungi are exposed to root components from both extraradical and intraradical tissues. The inhibition of Bt crop cultivation on pre-symbiotic growth can lead to a decrease of AM fungal colonization, resulting in an alteration of abundance or diversity of AM fungal propagules in soil. These variations have potential impact on the growth and quality of subsequently cultivated plants (Arihara and Karasawa, 2000) and soil ecological function (Rillig et al., 2010). In this study, we explored the effect of three different transgenic Bt cotton (Gossypium hirsutum L.) cultivars on the events occurring prior to hyphal contact with the epidermal surface of the host roots. Spore germination and pre-symbiotic hyphal differentiation were examined. We also explored the impact of Bt cotton on AM fungal colonization and fungal structure development within roots. The major aim of the present study was to investigate the impairment of Bt cotton toward spore germination and fungal development, which could be attributed to Bt toxin effect or impediment of plant signaling recognition induced by Bt gene transformation. 2. Materials and methods 2.1. Plant cultivars Three different isolines of transgenic Bt cotton (Jin26, GK12 and Jin44 expressing Cry1Ac) and their corresponding parental nontransgenic isolines (Jin7, Si3 and Ji492) were obtained from Institute of Cotton, Chinese Academy of Sciences (Anyang, Henan, China). All transgenic isolines have been commercially planted in China. Similar-sized cotton seeds were selected and delinted in concentrated H2SO4 for 20 min, sterilized in 3% NaClO for 10 min, rinsed with deionized water, and were finally immersed in deionized water for 24 h. Seeds were germinated in sterile acidwashed quartz grit (chemical composition (%): SiO2 99, Al2O3 0.23, Fe2O3 0.024; diameter: 2–5 mm) at 25 C in dark. Similarsized seedlings were selected and transferred into pots containing 200 g sterile quartz grit, and were allowed to grow for 10 days before replanting, with one plant for each pot. 2.2. Fungal material The AM fungi species Rhizophagus irregularis (BGC AH01) was purchased from the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences (Beijing, China). The inoculums consisted of mycorrhizal roots, spores and extraradical mycelium, which were maintained in the pot culture collection.
2.3. Experimental design 2.3.1. Spore germination and pre-symbiotic fungal growth Spores were manually extracted with forceps under a dissecting microscope by wet-sieving (Gerdemann and Nicolson, 1963) and surface-sterilized according to Bécard and Fortin (1988). A “sandwich system” with minor modifications was used for investigating the early stages of AM fungal life cycle (Giovannetti and Mosse, 1980). Briefly, the surface-sterilized spores were placed on 47-mm diameter cellulose ester Millipore TM membranes (0.45 mm diameter pores). Each membrane containing 30 spores was covered with an empty membrane to make sandwiched spore membranes. The cotton seedlings, which had been grown for 10 days in sterile quartz grit, were extracted and placed onto the sandwiched spore membranes. The sandwiched membranes were folded around the seedling roots and the seedlings were transplanted into sterile quartz grit in 10 cm diameter pots with one plant for each pot. In this way, the spores were exposed to root exudates in rhizosphere, but had no contact with the roots. The experiment was replicated 9 times for each plant line. Plants were grown in a growth chamber under a 16-h-light (25 C)/8-h-dark (22 C) regime. Sterilized water was added as required daily and 1/10 phosphate strength Hoagland solution (Hoagland and Arnon, 1950) was supplied weekly. At harvest, the sandwiched membranes were extracted from three pots for each cotton line at 10, 20 and 30 days postinoculation (dpi), respectively. To visualize the spore germination and hyphal growth and branching of the AM fungus, the folded sandwich membranes were gently flushed with tap water to remove quartz grit. To prevent the spores from floating off, the membranes were not opened until the finish of flushing, and then the membranes were stained with 0.05% Trypan blue in a culture dish. After 5 min, the membranes were destained with tap water. Germination percentage and hyphal growth were observed with a dissecting microscope. The spores displaying the growth of new hyphae were considered to be germinated (Bartolome-Esteban and Schenck, 1994). Images were captured using a Zeiss Axiocam MRc5 digital camera (Carl Zeiss Inc. Germany). To obtained high contrast images for quantification, images were imported into Image-Pro Plus, Version 4.03 (Media Cybernetics, America), and the hyphal networks were traced using magnetic pencil tool for analysis of hyphal branching and growth (Twanabasu et al., 2013). 2.3.2. Mycorrhizal colonization and plant growth The 10-day cotton seedlings were transplanted into 10-cm diameter pots containing 600 g sterile quartz grit and 50 g mycorrhizal inoculums (consisting of mycorrhizal roots, spores and extraradical mycelium) with one plant for each pot. The experiment was replicated 15 times for each plant line for mycorrhizal colonization assessment, and another 3 times for plant growth examination. Plants were grown in the same chamber as above. Sterilized water was added as required daily and 1/10 phosphate strength Hoagland solution (Hoagland and Arnon, 1950) was supplied weekly. Plants shoots and roots were harvested separately at 18, 21, 24, 27, 30 dpi, with three replicates for each harvest. Sub-samples of root were prepared for staining according to Phillips and Hayman (1970), and were respectively stained in 0.05% Trypan blue and 0.2 mg ml1 WGAAlexafluor 488 (Molecular Probes, Eugene, OR) to visualize the appressorium formation and fungal development within the roots. Intensity of mycorrhizal colonization and arbuscule abundance in the root system were monitored according to Trouvelot et al. (1986).
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To evaluate the effect of Bt-trait on plant growth, shoots and roots were harvested at 30 dpi. Root samples were carefully washed with tap water followed by rinsing with deionized water to remove adhering particles. The shoot and root samples were weighed after oven drying to a constant weight at 70 C for 48 h. 2.4. Data analysis Data were analyzed with SPSS software package (version 19.0, IBM SPSS Statistics 19, SAS Institute, Cary, North Carolina, USA). Prior to test of significant differences, the data were checked for normality and homogeneity of variance and, if necessary, they were arcsine-transformed (the percentage data). Although some analyses were conducted based on transformed data, the tables and graphs presented in the results are based on the means of raw data. Within each pair of Bt and the corresponding non-Bt isoline, multiple comparisons were conducted using Duncan's multiple range test at P 0.05 after a one-way ANOVA analysis. The effects of cotton varieties, harvest time and their interactions on presymbiosis and colonization were tested by a two-way ANOVA and comparisons among means were performed by Duncan’s multiple range test at P 0.05.
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3. Results 3.1. Spore germination and pre-symbiotic fungal growth 3.1.1. Spore germination Spore germination percentage increased with the increase of culture time. It was significantly lower at 10 dpi compared with at 20 and 30 dpi (Fig. 1). And the spore germination percentage was lower in the rhizosphere of transgenic Bt cotton varieties Jin26, GK12 and Jin44 at 20 and 30 dpi compared with in that of their corresponding non-transgenic varieties (Fig. 1). 3.1.2. Hyphal branching and growth The number of hyphal branches and total hyphal length increased significantly over time for all the cotton lines (Figs. 2 and 3). Compared with their corresponding non-Bt lines, the Bt cotton varieties Jin26, GK12 and Jin44 presented a smaller number of hyphal branches and a shorter total hyphal length at 20 and 30 dpi. 3.2. Mycorrhizal colonization 3.2.1. Appressorium development Appressorium formation was delayed on Bt roots compared with on the roots of their non-Bt lines. The appressorium number
Fig. 1. Spore germination percentage on the sandwiched spore membrane in the rhizosphere of Bt cotton (Bt) and non-Bt parental cotton (P) at different harvest times. Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
Fig. 2. Number of hyphal branches on the sandwiched spore membrane in the rhizosphere of Bt cotton (Bt) and non-Bt parental cotton (P). Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
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Fig. 3. Hyphal length on the sandwiched spore membrane in the rhizosphere of Bt cotton (Bt) and non-Bt parental cotton (P). Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
on Bt roots was significantly lower than that on the roots of non-Bt parental lines at most time points, with a 33.3%, 8.0% and 3.3% reduction at 30 dpi for Jin26, GK12 and Jin44, respectively (Fig. 4). 3.2.2. AM fungal colonization The root systems of the plants were analyzed to determine the symbiosis development. Colonization intensity in Bt lines was significantly lower compared with in their non-Bt parental lines,
with a 44.4%, 25.0% and 51.3% reduction for Jin26, GK12 and Jin44 at 30 dpi, respectively (Fig. 5). 3.2.3. Arbuscule frequency and fungal structure Arbuscule frequency increased significantly with the increase of culture time (Fig. 6). Lower arbuscule frequency was observed in Bt lines (Fig. 6), with a 45.7%, 21.6%, 68.2% reduction at 30 dpi for Jin26, GK12, Jin44, respectively. Furthermore, there were more
Fig. 4. Density of AM fungal appressoria on the root cortex of Bt cotton (Bt) and non-Bt parental cotton (P) inoculated with R. irregularis. Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
Fig. 5. Intensity of mycorrhizal colonization in the root system of Bt cotton (Bt) and non-Bt parental cotton (P) inoculated with R. irregularis. Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
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Fig. 6. Arbuscule abundance within roots of Bt cotton (Bt) and non-Bt parental cotton (P) inoculated with R. irregularis. Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
degenerated arbuscules within Bt roots relative to within the nonBt roots (Fig. 7). 3.2.4. Overall effect of Bt-trait on AM fungal development and colonization A two-way ANOVA analysis and comparisons were performed to evaluate the effect of cotton varieties and harvest time on AM fungus. The results showed that both cotton varieties and harvest time had significant impact on the hyphal branch number, hyphal
length, appressorium density, colonization intensity and arbuscule abundance (Table 1). To identify whether it is the Bt-trait or cultivar difference that plays the dominant role in affecting hyphal differencition and colonization, comparisons of the means of different variables over all sampling periods were conducted. The consistently lower germination percentage, branch number, hyphal length, appressorium density and colonization intensity of Bt cotton cultivars are indicative of a Bt-related impairment on the AM fungal develop-
Fig. 7. Laser scanning confocal microscope images of representative arbuscules in mycorrhizal roots of non-Bt cotton (Gossypium hirsutum L.) (A, Jin7; B, Si3; C, Ji492) and the corresponding transgenic Bt cotton (D, Jin26; E, GK12; F, Jin44) grown in sterile quartz grit harvested at 30 dpi. Roots were stained with WGA-Alexafluor 488 to visualize fungal structures within the roots. Arrowheads indicate large arbuscules in non-Bt cotton, a large majority of fully developed, highly branched arbuscules with distinct hyphal branches. Arrows indicate collapsed arbuscules in Bt roots, with many arbuscules degenerated and only trunk left in cortical cell. ih, intercellular hyphae (Scale bars: 25 mm).
Table 1 Two factor ANOVA (cotton varieties (V) and time (T) for spore germination percentage, number of hyphal branches, hyphal length, appressorium density, colonization intensity and arbuscule abundance (F values (P values)).
Germination percentage Number of hyphal branches Hyphal length Density of appressoria Colonization intensity Arbuscule abundance
Varieties (V)
Time (T)
2.4 (0.058) 21.6 (<0.001) 28.6 (<0.001) 46.9 (<0.001) 84.4 (<0.001) 77.5 (<0.001)
137.8 (<0.001) 339.0 (<0.001) 487.1 (<0.001) 77.0 (<0.001) 200.7 (<0.001) 180.8 (<0.001)
Interaction V T 0.8 (0.662) 5.1 (<0.001) 7.1 (<0.001) 3.8 (<0.001) 4.7 (<0.001) 5.3 (<0.001)
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ment and colonization (Table 2). Besides, the significant difference in arbuscule abundance between Bt and non-Bt cotton cultivars also suggests the impediment of plant signal reception and response in Bt isolines (Table 2). 3.2.5. Effect of Bt-trait on plant growth Bt-trait did not significantly affect plant root biomass for both inoculated and non-inoculated plants, except that Jin7 had a significantly higher root yield than others (Table 3). However, as for shoot biomass, the non-Bt varieties had significantly higher yield than the Bt varieties for the inoculated plants. The non-inoculated plants followed the similar trend as the inoculated ones with the exception of Ji492, which showed an equal biomass with the Bt lines Jin26 and GK12 (Table 3). 4. Discussion The life cycle of AM fungi begins with the germination of fungal spores and growth of hyphae toward the host root. In the presence of potential host, signal exchange between the fungus and plant occurs. A molecular dialogue is initiated by the host plant via strigolactones, which can promote the branching of fungal hyphae and activate fungal metabolism (Akiyama et al., 2005; Besserer et al., 2006). Subsequent recognition of AM fungi by the host involves the perception of a series of diffusible signals (Kosuta et al., 2003). Zhang et al. (2010) speculated that strigolactones not only promote the branching of fungal hyphae, but also induce the strong ramification of fungal hyphae that leads to arbuscule formation. Transferring Bt gene into cotton may result in alteration of the components of root exudates, such as organic acids, amino acids and soluble sugars (Yan et al., 2007). In this study, the reduced spore germination percentage and hyphal differentiation in the rhizosphere of Bt cotton might indicate a direct effect of plant signal compounds on pre-symbiotic development, although there has been no evidence about the alteration of signaling components activated by the introduction of Bt gene. Our results are consistent with the findings of Turrini et al. (2004), who reported that root exudates of Bt maize (event Bt 176) significantly reduced the pre-
symbiotic hyphal growth of Glomus mosseae when compared with another Bt maize hybrid (event Bt 11) and non-Bt maize. Cheeke et al. (2012) demonstrated a reduction of AM fungal colonization in multiple Bt maize lines. In this study, compared with the roots of the corresponding non-Bt cotton, Bt cotton roots showed significantly inhibited fungal development, with a 44.4%, 25.0% and 51.3% reduction in colonization intensity, and a 45.7%, 21.6% and 68.2% reduction in arbuscule frequency at 30 dpi for Jin26, GK12 and Jin44, respectively. Taking all varieties into consideration, the Bt cultivars showed inhibitory effect on the spore germination, hyphal differentiation and colonization of AM fungus, suggesting that Bt-trait has significant contribution to the differences among cotton cultivars. Furthermore, more degenerated arbuscules observed in Bt lines also indicate that Bt cultivars have inhibitory effect on the development of AM fungus. Arbuscule is known as a hallmark of the symbiosis, in which phosphate, NH4+ and other nutrients are transported to root cortical cells (Gianinazzi-Pearson, 1996; Harrison et al., 2002). Besides, it was predicted that carbon transfer occurs at the arbuscule/cortical cell interface (Smith and Smith, 1990; Helber et al., 2011; Fiorilli et al., 2013). Arbuscule development and degeneration are partially controlled by the host (Parniske, 2004). In this study, the low arbuscule frequency and more collapsed arbuscules in the Bt lines might suggest that genetically modified Bt cotton affects the development of R. irregularis within the root and arbuscular lifespan, which could have an impact on plant nutrient uptake and transportation. This speculation is supported by the result that Bt lines had significantly lower shoot yield for the inoculated plants. Indeed, high levels of Bt toxins in Bt maize have been reported. For example, 1.7–2.9 mg kg1 and 2.1–3.8 mg kg1 fresh weight of Cry1Ab protein were detected in Bt11 maize and MON810 (Tan et al., 2011), and 80.63 mg Cry1Ab/g protein was detected in Bt176 (Turrini et al., 2004). High levels of Bt proteins in Bt plants may pose potential risk to the development and growth of the intraradical part of AM fungi (Liu, 2010). Several mechanisms by which Bt toxins induce cytotoxicity to invertebrates have been reported. There are two acknowledged models: the first is that Bt toxin causes an osmotic imbalance in
Table 2 Means of spore germination percentage, number of hyphal branches, hyphal length, appressorium density, colonization intensity and arbuscule abundance over all sampling periods. Cotton varieties Jin7 (P) Si3 (P) Ji492 (P) Jin26 (Bt) GK12 (Bt) Jin44 (Bt)
Germination percentage (%)
Number of branches (unit spore1)
Hyphal length (mm)
Density of appressoria (unit cm1)
Colonization intensity (%)
41.45a 41.29a 38.61ab 37.54ab 35.36b 35.95b
14.80a 13.00b 10.62c 9.67c 9.56c 9.70c
336.17ab 309.30 b 351.69a 262.77c 241.37cd 225.92d
10.30a 8.53c 8.80b 8.30c 7.74d 8.49c
32.42a 25.47b 23.90c 23.73c 15.74d 15.43d
Arbuscule abundance (%) 30.25a 23.26b 22.21b 19.29c 14.45d 13.67d
Note: Values are means of three replicates of each plant line. Different letters within a column indicate significant difference (P < 0.05).
Table 3 Shoot and root dry matter yield of Bt cotton and non-Bt cotton harvested at 30 days postinoculation. Cotton varieties Jin7 (P) Si3 (P) Ji492 (P) Jin26 (Bt) GK12 (Bt) Jin44 (Bt)
Shoot biomass (g) R. irregularis
Non-mycorrhizal
R. irregularis
Root biomass (g) Non-mycorrhizal
0.34a 0.33a 0.30b 0.27c 0.27c 0.25c
0.30a 0.31a 0.26b 0.26b 0.26b 0.20c
0.29a 0.19b 0.21b 0.20b 0.17b 0.19b
0.25a 0.16b 0.18b 0.16b 0.16b 0.18b
Note: Values are means of three replicates of each plant line. Different letters within a column indicate significant difference (P < 0.05).
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response to the formation of pores in the cell membrane, and the second is that Bt toxin causes the opening of ion channels, which activates the process of cell death (Melo et al., 2016). The action mode of Bt cotton on AM fungi is still unclear; however, the induced reduction of pre-symbiotic hyphal differentiation and AM fungal colonization suggests that it might be either the direct mycotoxic effect of Bt toxin or the lack of signal compounds in the root exudates that causes the alteration of signal perception between the host and AM fungi. Future investigation of the mechanism by which Bt plants affect AM fungus development will help to elucidate the interplays between these plant-fungus partners. 5. Conclusion This study presents the evidence of the influences of Bt cotton on the events occurring prior to hyphal contact with the epidermal surface of the host root. The inhibition of spore germination and hyphal branching and growth in the rhizosphere of Bt isolines indicates that it is either interference of signal perception or Bt toxin toxicity that affects the development of AM fungi. The reduced colonization level and more collapsed arbuscules in Bt roots could affect plant nutrient cycling and the ecosystem function of AM fungi. Our findings will be valuable for future studies aimed at evaluating the ecological risk of Bt crop cultivation. Acknowledgements The present work was supported by grants from National Natural Science Foundation of China (Grant Nos: 41271474,41330852), the Fundamental Research Funds for the Central Universities (Program No.: 2010QC036), National Basic Scientific and Technological Project of China (Program No.: 2015FY110700), and the open funds of the State Key Laboratory of Agricultural Microbiology (Program No.: AMLKF201301). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version at http://dx.doi.org/10.1016/j.agee.2016.05.008. References Akiyama, K., Matsuzaki, K., Hayashi, H., 2005. Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature 435, 824–827. Arihara, J., Karasawa, T., 2000. Effect of previous crops on arbuscular mycorrhizal formation and growth of succeeding maize. Soil Sci. Plant Nutr. 46, 43–51. Bécard, G., Fortin, J.A., 1988. Early events of vesicular-arbuscular mycorrhizal formation on Ri T-DNA transformed roots. New Phytol. 108, 211–218. Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S., Vivanco, J.M., 2006. The role of root exudates in rhizosphere interations with plants and other organisms. Ann. Rev. Plant Biol. 57, 233–266. Bartolome-Esteban, H., Schenck, N.C., 1994. Spore germination and hyphal growth of arbuscular mycorrhizal fungi in relation to soil aluminumsaturation. Mycologia 86, 217–226. Besserer, A., Puech-Pagès, V., Kiefer, P., Gomez-Roldan, V., Jauneau, A., Roy, S., Portais, J.C., Roux, C., Bécard, G., Séjalon-Delmas, N., 2006. Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria. PLoS Biol. 4, 226. Broeckling, C.D., Broz, A.K., Bergelson, J., Manter, D.K., Vivanco, J.M., 2008. Root exudates regulate soil fungal community composition and diversity. Appl. Environ. Microbiol. 74, 738–744. 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. Cheeke, T.E., Rosenstiel, T.N., Cruzan, M.B., 2012. Evidence of reduced arbuscular mycorrhizal fungal colonization in multiple lines of Bt maize. Am. J. Bot. 99, 700–707.
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Bt corn has higher lignin content than non-Bt corn. Am. J. Bot. 88, 1704–1706. Schaarschmidt, S., Kopka, J., Ludwig-Muller, J., Hause, B., 2007. Regulation of arbuscular mycorrhization by apoplastic invertases: enhanced invertase activity in the leaf apoplast affects the symbiotic interaction. Plant J. 51, 390– 405. Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis. Academic Press, San Diego, CA. Smith, S.E., Smith, F.A., 1990. Structure and function of the interfaces in biotrophic symbioses as they relate on nutrient transport. New Phytol. 114, 1–38. Smith, S.E., Smith, F.A., Jakobsen, I., 2003. Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses. Plant Physiol. 133, 16–20.
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Agriculture, Ecosystems and Environment journal homepage: www.elsevier.com/locate/agee
Evidences of inhibited arbuscular mycorrhizal fungal development and colonization in multiple lines of Bt cotton Xiu-Hua Chena,b,* , Feng-Ling Wangb , Rui Zhangb , Ling-Ling Jib , Zheng-Lun Yangb , Hui Lina , Bin Zhaoa a
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtse River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China b
A R T I C L E I N F O
Article history: Received 23 November 2015 Received in revised form 31 March 2016 Accepted 6 May 2016 Available online xxx Keywords: Bt cotton Arbuscular mycorrhizal fungi Pre-symbiotic hyphal differentiation Colonization
A B S T R A C T
A “sandwich system” was used to investigate the effect of transgenic Bacillus thuringiensis (Bt) cotton (Gossypium hirsutum L.) on early stages of arbuscular mycorrhizal (AM) fungal life cycle. Spore germination and pre-symbiotic hyphal differentiation of AM fungus Rhizophagus irregularis were decreased in the rhizosphere of transgenic Bt cotton isolines (Jin26, GK12 and Jin44) compared with in that of their corresponding parental non-transgenic isolines (Jin7, Si3 and Ji492); and the appressorium number, colonization intensity and arbuscule abundance were lower in Bt plant roots. More collapsed arbuscules were also observed in Bt roots. The results of statistical analysis demonstrate that Bt-trait significantly contributes to the inhibition of pre-symbiotic development and AM fungal colonization, which might be attributed to either Bt toxin toxicity or interference of signal perception between AM fungi and the hosts. The specific mechanism requires further study. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Cotton is one of the most important crops worldwide. In recent years, transgenic Bacillus thuringiensis (Bt) cotton varieties have been widely grown, which effectively controls cotton bollworm and reduces the application of insecticides (Lu et al., 2012). China is one of the largest producers of cotton in the world. In 2013, the cultivation area of Bt cotton reached 4.2 million hectares, accounting for 90% of the total area of cotton cultivation in China (James, 2014). During the entire growth period of Bt cotton, Bt toxin is introduced into the soil through root exudates, decomposing plant material and/or pollen deposition (Icoz and Stotzky, 2008). Bt toxin contains truncated genes that encode toxins rather than nontoxic crystalline protoxins produced by B. thuringiensis. It is not necessary to solubilize the protoxins at a high gut pH to gain the insecticidal activity. Since Bt toxin can be bound to clays and humic substances to resist microbial decomposition, its insecticidal activity can be maintained for several months in the soil
* Corresponding author at: College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China. E-mail address: [email protected] (X.-H. Chen). http://dx.doi.org/10.1016/j.agee.2016.05.008 0167-8809/ã 2016 Elsevier B.V. All rights reserved.
(Saxena and Stotzky, 2001). Besides, receptors for the toxins are present in both target and nontarget insects (Hoöfte and Whiteley, 1989). Bt toxin residue in soil may pose a potential risk to soil organisms such as arbuscular mycorrhizal (AM) fungi, a kind of non-target soil microorganisms fundamental for soil fertility and plant nutrition. AM fungi are obligate biotrophs that form one of the most widespread and ancient mutualistic symbioses with most land plants (Smith and Read, 2008). The symbionts have coexisted for 400 million years (Redeker et al., 2000). This evolutionary success can be traced to the role of AM fungi in providing multiple benefits for the plant, including uptake of nutrients (mainly phosphorus and nitrogen) and water (Smith and Read, 2008), and also tolerance to pathogens and abiotic stresses (Newsham et al., 1995; Smith et al., 2003; Chen and Zhao, 2007). In return, host plants transfer carbohydrates to the fungi to support their lifecycle (Ho and Trappe, 1973; Olsson et al., 2010). Because of their obligately biotrophic relationship with plant roots, AM fungi may be more sensitive to Bt crops than other freeliving organisms, even if they are not affected by Bt proteins directly. Recent studies have shown an altered relationship between some cultivars of Bt maize and AM fungi. Turrini et al.
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(2004) reported that transgenic Bt maize (event Bt 176) had a smaller number of viable AM fungal infection structures compared with its non-Bt parental isoline. Some studies showed reduced level of AM fungal colonization in Bt maize roots (Castaldini et al., 2005; Cheeke et al., 2012) and lower density of AM fungi spores in the plots with Bt maize cultivation history (Cheeke et al., 2014); while other studies demonstrated that there is no negative effect of Bt crop cultivation on AM fungi (de Vaufleury et al., 2007; Knox et al., 2008; Cheeke et al., 2013; Cheeke et al., 2015). There have been no evidences showing the direct effect of Bt crop on the early stages of AM fungal life cycle. Genetic changes within a plant (either through genetic engineering or traditional approaches) might alter plant metabolic activity (Schaarschmidt et al., 2007) and root exudate components (Bais et al., 2006; Broeckling et al., 2008), which might affect the chemical signals (Akiyama et al., 2005). Throughout the life cycle of AM fungi, from spore germination to fungal structure development within roots, AM fungi are exposed to root components from both extraradical and intraradical tissues. The inhibition of Bt crop cultivation on pre-symbiotic growth can lead to a decrease of AM fungal colonization, resulting in an alteration of abundance or diversity of AM fungal propagules in soil. These variations have potential impact on the growth and quality of subsequently cultivated plants (Arihara and Karasawa, 2000) and soil ecological function (Rillig et al., 2010). In this study, we explored the effect of three different transgenic Bt cotton (Gossypium hirsutum L.) cultivars on the events occurring prior to hyphal contact with the epidermal surface of the host roots. Spore germination and pre-symbiotic hyphal differentiation were examined. We also explored the impact of Bt cotton on AM fungal colonization and fungal structure development within roots. The major aim of the present study was to investigate the impairment of Bt cotton toward spore germination and fungal development, which could be attributed to Bt toxin effect or impediment of plant signaling recognition induced by Bt gene transformation. 2. Materials and methods 2.1. Plant cultivars Three different isolines of transgenic Bt cotton (Jin26, GK12 and Jin44 expressing Cry1Ac) and their corresponding parental nontransgenic isolines (Jin7, Si3 and Ji492) were obtained from Institute of Cotton, Chinese Academy of Sciences (Anyang, Henan, China). All transgenic isolines have been commercially planted in China. Similar-sized cotton seeds were selected and delinted in concentrated H2SO4 for 20 min, sterilized in 3% NaClO for 10 min, rinsed with deionized water, and were finally immersed in deionized water for 24 h. Seeds were germinated in sterile acidwashed quartz grit (chemical composition (%): SiO2 99, Al2O3 0.23, Fe2O3 0.024; diameter: 2–5 mm) at 25 C in dark. Similarsized seedlings were selected and transferred into pots containing 200 g sterile quartz grit, and were allowed to grow for 10 days before replanting, with one plant for each pot. 2.2. Fungal material The AM fungi species Rhizophagus irregularis (BGC AH01) was purchased from the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences (Beijing, China). The inoculums consisted of mycorrhizal roots, spores and extraradical mycelium, which were maintained in the pot culture collection.
2.3. Experimental design 2.3.1. Spore germination and pre-symbiotic fungal growth Spores were manually extracted with forceps under a dissecting microscope by wet-sieving (Gerdemann and Nicolson, 1963) and surface-sterilized according to Bécard and Fortin (1988). A “sandwich system” with minor modifications was used for investigating the early stages of AM fungal life cycle (Giovannetti and Mosse, 1980). Briefly, the surface-sterilized spores were placed on 47-mm diameter cellulose ester Millipore TM membranes (0.45 mm diameter pores). Each membrane containing 30 spores was covered with an empty membrane to make sandwiched spore membranes. The cotton seedlings, which had been grown for 10 days in sterile quartz grit, were extracted and placed onto the sandwiched spore membranes. The sandwiched membranes were folded around the seedling roots and the seedlings were transplanted into sterile quartz grit in 10 cm diameter pots with one plant for each pot. In this way, the spores were exposed to root exudates in rhizosphere, but had no contact with the roots. The experiment was replicated 9 times for each plant line. Plants were grown in a growth chamber under a 16-h-light (25 C)/8-h-dark (22 C) regime. Sterilized water was added as required daily and 1/10 phosphate strength Hoagland solution (Hoagland and Arnon, 1950) was supplied weekly. At harvest, the sandwiched membranes were extracted from three pots for each cotton line at 10, 20 and 30 days postinoculation (dpi), respectively. To visualize the spore germination and hyphal growth and branching of the AM fungus, the folded sandwich membranes were gently flushed with tap water to remove quartz grit. To prevent the spores from floating off, the membranes were not opened until the finish of flushing, and then the membranes were stained with 0.05% Trypan blue in a culture dish. After 5 min, the membranes were destained with tap water. Germination percentage and hyphal growth were observed with a dissecting microscope. The spores displaying the growth of new hyphae were considered to be germinated (Bartolome-Esteban and Schenck, 1994). Images were captured using a Zeiss Axiocam MRc5 digital camera (Carl Zeiss Inc. Germany). To obtained high contrast images for quantification, images were imported into Image-Pro Plus, Version 4.03 (Media Cybernetics, America), and the hyphal networks were traced using magnetic pencil tool for analysis of hyphal branching and growth (Twanabasu et al., 2013). 2.3.2. Mycorrhizal colonization and plant growth The 10-day cotton seedlings were transplanted into 10-cm diameter pots containing 600 g sterile quartz grit and 50 g mycorrhizal inoculums (consisting of mycorrhizal roots, spores and extraradical mycelium) with one plant for each pot. The experiment was replicated 15 times for each plant line for mycorrhizal colonization assessment, and another 3 times for plant growth examination. Plants were grown in the same chamber as above. Sterilized water was added as required daily and 1/10 phosphate strength Hoagland solution (Hoagland and Arnon, 1950) was supplied weekly. Plants shoots and roots were harvested separately at 18, 21, 24, 27, 30 dpi, with three replicates for each harvest. Sub-samples of root were prepared for staining according to Phillips and Hayman (1970), and were respectively stained in 0.05% Trypan blue and 0.2 mg ml1 WGAAlexafluor 488 (Molecular Probes, Eugene, OR) to visualize the appressorium formation and fungal development within the roots. Intensity of mycorrhizal colonization and arbuscule abundance in the root system were monitored according to Trouvelot et al. (1986).
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To evaluate the effect of Bt-trait on plant growth, shoots and roots were harvested at 30 dpi. Root samples were carefully washed with tap water followed by rinsing with deionized water to remove adhering particles. The shoot and root samples were weighed after oven drying to a constant weight at 70 C for 48 h. 2.4. Data analysis Data were analyzed with SPSS software package (version 19.0, IBM SPSS Statistics 19, SAS Institute, Cary, North Carolina, USA). Prior to test of significant differences, the data were checked for normality and homogeneity of variance and, if necessary, they were arcsine-transformed (the percentage data). Although some analyses were conducted based on transformed data, the tables and graphs presented in the results are based on the means of raw data. Within each pair of Bt and the corresponding non-Bt isoline, multiple comparisons were conducted using Duncan's multiple range test at P 0.05 after a one-way ANOVA analysis. The effects of cotton varieties, harvest time and their interactions on presymbiosis and colonization were tested by a two-way ANOVA and comparisons among means were performed by Duncan’s multiple range test at P 0.05.
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3. Results 3.1. Spore germination and pre-symbiotic fungal growth 3.1.1. Spore germination Spore germination percentage increased with the increase of culture time. It was significantly lower at 10 dpi compared with at 20 and 30 dpi (Fig. 1). And the spore germination percentage was lower in the rhizosphere of transgenic Bt cotton varieties Jin26, GK12 and Jin44 at 20 and 30 dpi compared with in that of their corresponding non-transgenic varieties (Fig. 1). 3.1.2. Hyphal branching and growth The number of hyphal branches and total hyphal length increased significantly over time for all the cotton lines (Figs. 2 and 3). Compared with their corresponding non-Bt lines, the Bt cotton varieties Jin26, GK12 and Jin44 presented a smaller number of hyphal branches and a shorter total hyphal length at 20 and 30 dpi. 3.2. Mycorrhizal colonization 3.2.1. Appressorium development Appressorium formation was delayed on Bt roots compared with on the roots of their non-Bt lines. The appressorium number
Fig. 1. Spore germination percentage on the sandwiched spore membrane in the rhizosphere of Bt cotton (Bt) and non-Bt parental cotton (P) at different harvest times. Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
Fig. 2. Number of hyphal branches on the sandwiched spore membrane in the rhizosphere of Bt cotton (Bt) and non-Bt parental cotton (P). Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
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Fig. 3. Hyphal length on the sandwiched spore membrane in the rhizosphere of Bt cotton (Bt) and non-Bt parental cotton (P). Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
on Bt roots was significantly lower than that on the roots of non-Bt parental lines at most time points, with a 33.3%, 8.0% and 3.3% reduction at 30 dpi for Jin26, GK12 and Jin44, respectively (Fig. 4). 3.2.2. AM fungal colonization The root systems of the plants were analyzed to determine the symbiosis development. Colonization intensity in Bt lines was significantly lower compared with in their non-Bt parental lines,
with a 44.4%, 25.0% and 51.3% reduction for Jin26, GK12 and Jin44 at 30 dpi, respectively (Fig. 5). 3.2.3. Arbuscule frequency and fungal structure Arbuscule frequency increased significantly with the increase of culture time (Fig. 6). Lower arbuscule frequency was observed in Bt lines (Fig. 6), with a 45.7%, 21.6%, 68.2% reduction at 30 dpi for Jin26, GK12, Jin44, respectively. Furthermore, there were more
Fig. 4. Density of AM fungal appressoria on the root cortex of Bt cotton (Bt) and non-Bt parental cotton (P) inoculated with R. irregularis. Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
Fig. 5. Intensity of mycorrhizal colonization in the root system of Bt cotton (Bt) and non-Bt parental cotton (P) inoculated with R. irregularis. Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
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Fig. 6. Arbuscule abundance within roots of Bt cotton (Bt) and non-Bt parental cotton (P) inoculated with R. irregularis. Values are means of three replicates of each plant line. Bars represent standard errors of the means. Different letters above the bars indicate significant difference (P < 0.05).
degenerated arbuscules within Bt roots relative to within the nonBt roots (Fig. 7). 3.2.4. Overall effect of Bt-trait on AM fungal development and colonization A two-way ANOVA analysis and comparisons were performed to evaluate the effect of cotton varieties and harvest time on AM fungus. The results showed that both cotton varieties and harvest time had significant impact on the hyphal branch number, hyphal
length, appressorium density, colonization intensity and arbuscule abundance (Table 1). To identify whether it is the Bt-trait or cultivar difference that plays the dominant role in affecting hyphal differencition and colonization, comparisons of the means of different variables over all sampling periods were conducted. The consistently lower germination percentage, branch number, hyphal length, appressorium density and colonization intensity of Bt cotton cultivars are indicative of a Bt-related impairment on the AM fungal develop-
Fig. 7. Laser scanning confocal microscope images of representative arbuscules in mycorrhizal roots of non-Bt cotton (Gossypium hirsutum L.) (A, Jin7; B, Si3; C, Ji492) and the corresponding transgenic Bt cotton (D, Jin26; E, GK12; F, Jin44) grown in sterile quartz grit harvested at 30 dpi. Roots were stained with WGA-Alexafluor 488 to visualize fungal structures within the roots. Arrowheads indicate large arbuscules in non-Bt cotton, a large majority of fully developed, highly branched arbuscules with distinct hyphal branches. Arrows indicate collapsed arbuscules in Bt roots, with many arbuscules degenerated and only trunk left in cortical cell. ih, intercellular hyphae (Scale bars: 25 mm).
Table 1 Two factor ANOVA (cotton varieties (V) and time (T) for spore germination percentage, number of hyphal branches, hyphal length, appressorium density, colonization intensity and arbuscule abundance (F values (P values)).
Germination percentage Number of hyphal branches Hyphal length Density of appressoria Colonization intensity Arbuscule abundance
Varieties (V)
Time (T)
2.4 (0.058) 21.6 (<0.001) 28.6 (<0.001) 46.9 (<0.001) 84.4 (<0.001) 77.5 (<0.001)
137.8 (<0.001) 339.0 (<0.001) 487.1 (<0.001) 77.0 (<0.001) 200.7 (<0.001) 180.8 (<0.001)
Interaction V T 0.8 (0.662) 5.1 (<0.001) 7.1 (<0.001) 3.8 (<0.001) 4.7 (<0.001) 5.3 (<0.001)
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ment and colonization (Table 2). Besides, the significant difference in arbuscule abundance between Bt and non-Bt cotton cultivars also suggests the impediment of plant signal reception and response in Bt isolines (Table 2). 3.2.5. Effect of Bt-trait on plant growth Bt-trait did not significantly affect plant root biomass for both inoculated and non-inoculated plants, except that Jin7 had a significantly higher root yield than others (Table 3). However, as for shoot biomass, the non-Bt varieties had significantly higher yield than the Bt varieties for the inoculated plants. The non-inoculated plants followed the similar trend as the inoculated ones with the exception of Ji492, which showed an equal biomass with the Bt lines Jin26 and GK12 (Table 3). 4. Discussion The life cycle of AM fungi begins with the germination of fungal spores and growth of hyphae toward the host root. In the presence of potential host, signal exchange between the fungus and plant occurs. A molecular dialogue is initiated by the host plant via strigolactones, which can promote the branching of fungal hyphae and activate fungal metabolism (Akiyama et al., 2005; Besserer et al., 2006). Subsequent recognition of AM fungi by the host involves the perception of a series of diffusible signals (Kosuta et al., 2003). Zhang et al. (2010) speculated that strigolactones not only promote the branching of fungal hyphae, but also induce the strong ramification of fungal hyphae that leads to arbuscule formation. Transferring Bt gene into cotton may result in alteration of the components of root exudates, such as organic acids, amino acids and soluble sugars (Yan et al., 2007). In this study, the reduced spore germination percentage and hyphal differentiation in the rhizosphere of Bt cotton might indicate a direct effect of plant signal compounds on pre-symbiotic development, although there has been no evidence about the alteration of signaling components activated by the introduction of Bt gene. Our results are consistent with the findings of Turrini et al. (2004), who reported that root exudates of Bt maize (event Bt 176) significantly reduced the pre-
symbiotic hyphal growth of Glomus mosseae when compared with another Bt maize hybrid (event Bt 11) and non-Bt maize. Cheeke et al. (2012) demonstrated a reduction of AM fungal colonization in multiple Bt maize lines. In this study, compared with the roots of the corresponding non-Bt cotton, Bt cotton roots showed significantly inhibited fungal development, with a 44.4%, 25.0% and 51.3% reduction in colonization intensity, and a 45.7%, 21.6% and 68.2% reduction in arbuscule frequency at 30 dpi for Jin26, GK12 and Jin44, respectively. Taking all varieties into consideration, the Bt cultivars showed inhibitory effect on the spore germination, hyphal differentiation and colonization of AM fungus, suggesting that Bt-trait has significant contribution to the differences among cotton cultivars. Furthermore, more degenerated arbuscules observed in Bt lines also indicate that Bt cultivars have inhibitory effect on the development of AM fungus. Arbuscule is known as a hallmark of the symbiosis, in which phosphate, NH4+ and other nutrients are transported to root cortical cells (Gianinazzi-Pearson, 1996; Harrison et al., 2002). Besides, it was predicted that carbon transfer occurs at the arbuscule/cortical cell interface (Smith and Smith, 1990; Helber et al., 2011; Fiorilli et al., 2013). Arbuscule development and degeneration are partially controlled by the host (Parniske, 2004). In this study, the low arbuscule frequency and more collapsed arbuscules in the Bt lines might suggest that genetically modified Bt cotton affects the development of R. irregularis within the root and arbuscular lifespan, which could have an impact on plant nutrient uptake and transportation. This speculation is supported by the result that Bt lines had significantly lower shoot yield for the inoculated plants. Indeed, high levels of Bt toxins in Bt maize have been reported. For example, 1.7–2.9 mg kg1 and 2.1–3.8 mg kg1 fresh weight of Cry1Ab protein were detected in Bt11 maize and MON810 (Tan et al., 2011), and 80.63 mg Cry1Ab/g protein was detected in Bt176 (Turrini et al., 2004). High levels of Bt proteins in Bt plants may pose potential risk to the development and growth of the intraradical part of AM fungi (Liu, 2010). Several mechanisms by which Bt toxins induce cytotoxicity to invertebrates have been reported. There are two acknowledged models: the first is that Bt toxin causes an osmotic imbalance in
Table 2 Means of spore germination percentage, number of hyphal branches, hyphal length, appressorium density, colonization intensity and arbuscule abundance over all sampling periods. Cotton varieties Jin7 (P) Si3 (P) Ji492 (P) Jin26 (Bt) GK12 (Bt) Jin44 (Bt)
Germination percentage (%)
Number of branches (unit spore1)
Hyphal length (mm)
Density of appressoria (unit cm1)
Colonization intensity (%)
41.45a 41.29a 38.61ab 37.54ab 35.36b 35.95b
14.80a 13.00b 10.62c 9.67c 9.56c 9.70c
336.17ab 309.30 b 351.69a 262.77c 241.37cd 225.92d
10.30a 8.53c 8.80b 8.30c 7.74d 8.49c
32.42a 25.47b 23.90c 23.73c 15.74d 15.43d
Arbuscule abundance (%) 30.25a 23.26b 22.21b 19.29c 14.45d 13.67d
Note: Values are means of three replicates of each plant line. Different letters within a column indicate significant difference (P < 0.05).
Table 3 Shoot and root dry matter yield of Bt cotton and non-Bt cotton harvested at 30 days postinoculation. Cotton varieties Jin7 (P) Si3 (P) Ji492 (P) Jin26 (Bt) GK12 (Bt) Jin44 (Bt)
Shoot biomass (g) R. irregularis
Non-mycorrhizal
R. irregularis
Root biomass (g) Non-mycorrhizal
0.34a 0.33a 0.30b 0.27c 0.27c 0.25c
0.30a 0.31a 0.26b 0.26b 0.26b 0.20c
0.29a 0.19b 0.21b 0.20b 0.17b 0.19b
0.25a 0.16b 0.18b 0.16b 0.16b 0.18b
Note: Values are means of three replicates of each plant line. Different letters within a column indicate significant difference (P < 0.05).
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response to the formation of pores in the cell membrane, and the second is that Bt toxin causes the opening of ion channels, which activates the process of cell death (Melo et al., 2016). The action mode of Bt cotton on AM fungi is still unclear; however, the induced reduction of pre-symbiotic hyphal differentiation and AM fungal colonization suggests that it might be either the direct mycotoxic effect of Bt toxin or the lack of signal compounds in the root exudates that causes the alteration of signal perception between the host and AM fungi. Future investigation of the mechanism by which Bt plants affect AM fungus development will help to elucidate the interplays between these plant-fungus partners. 5. Conclusion This study presents the evidence of the influences of Bt cotton on the events occurring prior to hyphal contact with the epidermal surface of the host root. The inhibition of spore germination and hyphal branching and growth in the rhizosphere of Bt isolines indicates that it is either interference of signal perception or Bt toxin toxicity that affects the development of AM fungi. The reduced colonization level and more collapsed arbuscules in Bt roots could affect plant nutrient cycling and the ecosystem function of AM fungi. Our findings will be valuable for future studies aimed at evaluating the ecological risk of Bt crop cultivation. Acknowledgements The present work was supported by grants from National Natural Science Foundation of China (Grant Nos: 41271474,41330852), the Fundamental Research Funds for the Central Universities (Program No.: 2010QC036), National Basic Scientific and Technological Project of China (Program No.: 2015FY110700), and the open funds of the State Key Laboratory of Agricultural Microbiology (Program No.: AMLKF201301). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version at http://dx.doi.org/10.1016/j.agee.2016.05.008. References Akiyama, K., Matsuzaki, K., Hayashi, H., 2005. Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature 435, 824–827. Arihara, J., Karasawa, T., 2000. Effect of previous crops on arbuscular mycorrhizal formation and growth of succeeding maize. Soil Sci. Plant Nutr. 46, 43–51. Bécard, G., Fortin, J.A., 1988. Early events of vesicular-arbuscular mycorrhizal formation on Ri T-DNA transformed roots. New Phytol. 108, 211–218. Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S., Vivanco, J.M., 2006. The role of root exudates in rhizosphere interations with plants and other organisms. Ann. Rev. Plant Biol. 57, 233–266. Bartolome-Esteban, H., Schenck, N.C., 1994. Spore germination and hyphal growth of arbuscular mycorrhizal fungi in relation to soil aluminumsaturation. Mycologia 86, 217–226. Besserer, A., Puech-Pagès, V., Kiefer, P., Gomez-Roldan, V., Jauneau, A., Roy, S., Portais, J.C., Roux, C., Bécard, G., Séjalon-Delmas, N., 2006. Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria. PLoS Biol. 4, 226. Broeckling, C.D., Broz, A.K., Bergelson, J., Manter, D.K., Vivanco, J.M., 2008. Root exudates regulate soil fungal community composition and diversity. Appl. Environ. Microbiol. 74, 738–744. 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. Cheeke, T.E., Rosenstiel, T.N., Cruzan, M.B., 2012. Evidence of reduced arbuscular mycorrhizal fungal colonization in multiple lines of Bt maize. Am. J. Bot. 99, 700–707.
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