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Arbuscular mycorrhizal fungi improve plant growth of Ricinus communis by altering photosynthetic properties and increasing pigments under drought and salt stress
Industrial Crops & Products 117 (2018) 13–19
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Arbuscular mycorrhizal fungi improve plant growth of Ricinus communis by altering photosynthetic properties and increasing pigments under drought and salt stress Tao Zhanga,
T
⁎,1
, Yongjun Hub,1, Ke Zhangc, Changyan Tianc, Jixun Guoa
a
Institute of Grassland Science, Northeast Normal University, Key Laboratory of Vegetation Ecology, Ministry of Education, Changchun 130024, China School of Life Science, Changhun Normal University, Changchun 130032, China c State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Arbuscular mycorrhizal (AM) fungi Biofuel production Metabolites Physiological response Revegetation
Castor bean (Ricinus communis) is one of the most important candidate crops that can be used to increase the land cover and crop yield in the arid and saline region of China. To evaluate the effects of arbuscular mycorrhizal (AM) fungi on the growth of castor bean under drought and salt stresses, a pot experiment was carried out to examine how AM fungi improve the plant growth of castor bean by affecting the leaf gas exchange, pigments, and metabolites accumulation of castor bean seedlings. The results showed that AM fungi stimulated plant growth and increased the castor bean aboveground biomass. AM fungi significantly increased the net photosynthetic rate (A), stomatal conductance (gs) and transpiration rate (E) of castor bean and decreased the intercellular CO2 concentration (Ci) under drought and salt stress. The contents of chlorophyll a, chlorophyll b, chlorophyll a + b and carotenoids in leaves treated by AM fungi were higher than in those without the AM fungi treatment. AM fungi notably increased the soluble protein and proline contents and decreased the malondialdehyde (MDA) content of castor bean. The results indicated that AM fungi could protect castor bean against drought and salt stresses by improving its leaf gas exchanges and photosynthetic capacity and altering its concentrations of metabolites. Our results highlight that AM fungi might be a promising bio-approach that can be used to plant castor beans in northeast and northwest China.
1. Introduction Castor bean (Ricinus communis L. Euphorbiaceae) is one of the most important oilseed crops. The oil content in the castor bean seeds can reach 40–60%, and most of the oil is essentially the triglyceride of ricinoleic acid (Labalette et al., 1996). The castor bean is considered a promising candidate for biofuel production (Barnes et al., 2009a), and the oil obtained from castor beans is widely used to make many products, such as surfactants, coatings, greases, fungistats, pharmaceuticals, and cosmetics (Pinheiro et al., 2008). Moreover, the castor bean can also be used to reduce the soil degradation caused by heavy metals (Bauddh and Singh, 2012) and salt (Li et al., 2010; Wu et al., 2012). Because of its high economic and ecological value, castor bean is widely planted in many developing countries, such as India (Bauddh and Singh, 2012), South Africa (Barnes et al., 2009b), Argentina (Falasca et al., 2012) and Brazil (Pinheiro et al., 2008). Although castor bean has many important ecological and economic values, the castor bean
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1
cultivation area is not large, and the yield of castor bean is still not high because food security is prioritized over is cultivation, and most of good arable land is only used to grow crops (e.g, wheat, maize, and rice) in China. However, in northeast and northwest China there are approximately 35 million hectares of saline land that can be used to grow castor bean because of the high salt and drought tolerances of the plant. However, a previous study found that castor bean could not tolerate salt stress during its initial growth stages (Pinheiro et al., 2008). Therefore, it is important to increase the salt resistance at the early stage which is important to improve plant growth and yield. Arbuscular mycorrhizal (AM) fungi is one of most important soil microorganisms that can form mycorrhizal symbiosis with most of terrestrial plants. AM fungi can improve the growth of many plants by many pathways, known to increase nutrient uptake (van der Heijden et al., 2006; Zhang et al., 2011), improve drought tolerance (AlKaraki and AlRaddad, 1997; Auge et al., 2015), improve salt resistance (Feng et al., 2002; Evelin et al., 2009), and reduce plant vulnerability to
Corresponding authors at: No. 5268 Renmin Street, Changchun, 130024, China. E-mail address: [email protected] (T. Zhang). These authors contributed equally to this work.
https://doi.org/10.1016/j.indcrop.2018.02.087 Received 27 November 2017; Received in revised form 22 January 2018; Accepted 28 February 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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was maintained at 50% field capacity by weight in all treatment except the drought treatment. The experiment was carried out in phytotrons (LT/ACR-2002, E-Sheng Tech., Beijing, China) for 2 months with the temperature of 25 °C from 06:00–20:00, 18 °C from 20:00–06:00. The relative humidity was 40–60%, and the light intensity was 350 μ mol−2 S−1. The pots were placed the phytotrons randomly, and moved to a different place every week to eliminate the effect of light and temperature on plant growth. The plants were harvested after growing for three months.
pathogens (Wehner et al., 2011; Veresoglou and Rillig, 2012). There are few studies that report the association between AM fungi and castor bean (Machineski et al., 2011; Kurandawad et al., 2014). The previous studies found that AM fungi increased the growth of castor bean (AbdAllah and Khater, 2016), plant survival percentage, dry matter accumulation and phosphorus uptake and reduced the soil pH and salinity (Zhang et al., 2014a,b). However, the effects of AM fungi on the growth of castor bean are still not well understood, and the mechanism of how AM fungi improve plant growth under salt and drought stress remains unclear. The objectives of this study are: (a) to evaluate the effects of AM fungi on the leaf photosynthetic characteristics of castor bean under drought and salt stress condition; (b) to assess the influence of AM fungi on the leaf pigment contents under drought and salt stresses; and (c) to investigate the effects of AM fungi on protein content and proline and malondialdehyde (MDA) concentrations in castor bean leaves under drought and salt stresses. With these objectives, this study will elucidate the physiological mechanism by which AM fungi improves the growth of castor bean and evaluate the potential of AM fungi in castor bean cultivation in the arid or semiarid saline regions in northeast and northwest China.
2.3. Leaf photosynthesis
2. Material and methods
The leaf photosynthesis measurements were conducted on the 7th day between 08:00 to 12:00 am after the drought treatment by using a portable photosynthesis system (LI-6400, LI-COR Inc., USA). The leaf net photosynthetic rate (A), stomatal conductance (gs), transpiration rate (E), and intercellular CO2 concentration (Ci) were measured according to the methods described by Chen et al. (2005). Three fully opened leaves of were selected for each plant in each pot. We measured the values of these leaves and averaged them for each plant. The photosynthesis parameters were measured with photosynthetically active radiation (1200 μmol m−2 s−1), a CO2 concentration of 350 μmol mol−1, and a leaf temperature of 25 °C.
2.1. Plant material, soil and AM fungi preparation
2.4. Leaf pigments
The seeds of castor bean (Ricinus communis) and the soil used in this experiment were collected from Xinjiang Institute of Ecology and Geography, China Academy of Sciences northwest China. The soil in the studied area is a saline desert soil, with a pH of 7.5–9.0. The average contents of soil total nitrogen and available soil phosphorus (0.5 mol L−1 NaHCO3-extractable) were 16.5 mg kg−1 and 3.0 mg kg−1, respectively. The collected soil was air dried and sieved (2 mm) to remove big stones, plant roots and other litters. The dry soil was sterilized at 121 °C for 2 h in order to remove AM fungi in the soil. The collected seeds were air dried and stored in a refrigerator at < 4 °C before use. The R. communis seeds were surface disinfested in 10% (v/v) hydrogen peroxide for 5 min, rinsed 5 times with deionized water, and then germinated at 20 °C for 48 h. Four germinated seeds were sown in pots. The seedlings were thinned to 2 per pot. Two dominant AM fungi species Funneliformis mosseae (Fm) and Rhizophagus intraradices (Ri) were used in this study. These two AM fungi species were isolated from the same sites where R. communis grew, and cultured individually using Medicago sativa for 4 months. The seeds of M. sativa were surface sterilized in 10% (v/v) hydrogen peroxide for 5 min and rinsed with deionized water. To maintain the purity of AM fungi, we cultured these two AM fungi species in different closed chambers. After that, the aboveground was removed and the two AM fungi species were mixed. We examined the purity of the two AM fungi species before inoculation and found that the cultured AM fungi were not contaminated by other fungi. The AM fungi inoculum consisted of a mixture of soil, hyphae, spores and infected root fragments (approximately 800 spores per 20 g soil).
After the leaf photosynthesis measurement, the leaf pigments were measured according to the methods of Zhang et al. (2016). Three fully expanded and undamaged leaves from each plant were sampled in each treatment, and wrapped immediately in aluminium foil to avoid pigments degradation. The whole leaves were cut into pieces and placed in a test tube containing 10 ml extractant (80% acetone and absolute alcohol at a ratio 1:1). The test tubes were incubated at 70 °C for 30 min (Hiscox and Israelstam, 1979), and then cooled in the dark. The cooled extract was analyzed using a spectrophotometer (UV-2201, Essentia, Japan) at 440, 649 and 665 nm. The chlorophyll a (Chl a), b (Chl b) and carotenoid concentrations were calculated according to the method of Wellburn (1994). 2.5. Leaf protein, proline and MDA The leaf soluble protein content was measured using the bovine serum albumin protein standard according to the previously reported method (Bradford, 1976). Fresh leaf samples (0.5 g) were homogenized with 4 ml Na-phosphate buffer (pH 7.2), and then centrifuged at 4 °C (Janmohammadi et al., 2012). The supernatants with dye were measured using a spectrophotometer (UV-2201, Essentia, Japan) at 595 nm. Proline was measured according to the methods described by Bates et al. (1973), and the malondialdehyde (MDA) content was measured according to Heath and Packer (1986). 2.6. Mycorrhizal colonization and biomass The roots were cut into 1-cm segments, and mixed thoroughly. A sub-sample of 0.5 g was cleared with 10% (w/v) KOH at 90 °C for 2 h and then stained with trypan blue. Thirty stained roots section were mounted on slides in polyvinyl alcohol–lactic acid glycerol and the arbuscule, vesicule and hyphae in the root were examined using a microscope at 100–400 magnification. The mycorrhizal colonization was calculated using the MYCOCALC program according to a previously described method (Zhang et al., 2011). Plant shoots and leaves were weighed after they were oven dried at 65 °C for 48 h.
2.2. Experimental design This experiment included control, drought stress and salt stress three main treatments, and each main treatment was applied to castor bean without AM fungi and those inoculated with AM fungi Fm and Ri treatments, and replicated four times. In the drought stress treatment, a seven days regime (seven days of withheld water) was applied to create drought stress before harvesting according to the methods described by Bauddh and Singh (2012). The control plants and the plants not subjected to the drought stress were watered routinely every 2–3 days. In the salt stress treatment, a sodium chloride (200 mM NaCl) solution was added to the soil. In order to keep the salt in the soil, the soil moisture
2.7. Statistical analysis All data were analyzed statistically by analysis of variance using the 14
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3.2. Leaf photosynthesis and gas exchange Drought and salt stresses significantly decreased A (P < 0.05, Fig. 2A), gs (P < 0.05, Fig. 2B) and E (P < 0.05, Fig. 2C), but they had no impact on Ci (P > 0.05, Fig. 2D). Under drought, AM fungi increased the mean values of A, gs and E by 37.1% (P < 0.05), 37.3% (P < 0.05) and 30.5% (P < 0.05), respectively, and decreased Ci by 11.1% (P < 0.05, Fig. 2D). Under salt stress conditions, AM fungi meanly increased A, gs and E by 34.7% (P < 0.05), 40.8% (P < 0.05) and 32.3% (P < 0.05), respectively, decreased Ci by 14.1% (P < 0.05, Fig. 2D). No significant differences in A were observed between plants inoculated by the two AM fungi species, except under drought. Under salt stress conditions, the increase in E was 12.5% (P < 0.05) greater in plants inoculated by Fm than in plants inoculated by Ri. No significant interactive effects of drought × AM fungi (P > 0.05, Table 1) and salt stress × AM fungi (P > 0.05, Table 2) on leaf photosynthesis were observed. 3.3. Leaf photosynthesis pigments Drought and salt stress both decreased Chl a (P < 0.05, Fig. 3A), Chl b (P < 0.05, Fig. 3B) and Chl a + b (P < 0.05, Fig. 3C) significantly, but had no impact on Car (P > 0.05, Fig. 3D). AM fungi significantly increased the leaf pigments. Under drought, the inoculations with Fm and Ri increased, on average, Chl a, Chl b, Chl a + b and Car by 30.9%, 31.7%, 32.3% and 39.9%, respectively, compared to the control without AM fungi (all P < 0.05). Under salt stress, AM fungi Fm and Ri increased on average, Chl a, Chl b, Chl a + b and Car by 33.9% (P < 0.05), 60.2% (P < 0.01), 25.9% (P < 0.05) and 134.2% (P < 0.01), respectively, compared to the control without AM fungi (Fig. 3). The mycorrhizal benefits of Fm and Ri differed under drought and salt stress conditions. Under drought, the contents of Chl a, Chl b, Chl a + b in the plants inoculated by Ri were higher than those in plants inoculated by Fm, but they were lower in the Ri inoculation treatment than the Fm inoculation treatment under salt stress. No significant differences in the mycorrhizal-induced increases in Car were detected between plants inoculated by Fm and Ri (P > 0.05, Fig. 3D). Significant interactive effects of drought × AM fungi on Chl a (P < 0.001) and Chl a + b (P < 0.001, Table 1) were detected. In addition, significant interactive effects of salt stress × AM on Chl a (P < 0.05) and Chl b (P < 0.01, Table 2) were detected.
Fig. 1. Myorrhizal colonization (A) in the root system of R. communis and aboveground biomass (B) under drought and salt stresses. Vertical bars indicate the standard error of the mean (n = 4). NM represents the control without arbuscular mycorrhizal (AM) fungi (white), Fm represents treatments inoculated with the AM fungi F. mosseae (gray), Ri represents treatments inoculated with the AM fungi R. intraradices (black). Different lowercase letters indicate significant differences between the two AM fungi treatments under control, drought and salt stress conditions at the 0.05 significance level.
software SPSS 16.0 (SPSS 16.0 for windows, USA) with the means separated using the Duncan test at P < 0.05 level.
3. Results 3.1. Plant growth and mycorrhizal colonization
3.4. Leaf protein, proline and MDA No mycorrhizal colonization was observed in the root of R. communis in all plants grown without AM fungi treatments (Fig. 1A). The mycorrhizal colonizations in the Fm inoculation control, drought and salt stress treatments treatment were 15.9% (P < 0.05), 21.9% (P < 0.05) and 14.6% (P < 0.05) lower, respectively, than in those inoculated by Ri. Drought and salt stress slightly decreased mycorrhizal colonization in the Fm inoculation treatments, but there was no significant difference between the treatments (P > 0.05). In the Ri inoculation treatments, salt stress decreased mycorrhizal colonization by 10.2% (P < 0.05), while drought had no impact on mycorrhizal colonization (P > 0.05). AM fungi stimulated the plant growth of R. communis in the control, drought and salt stress treatments (Fig. 1B). The aboveground biomass of plants inoculated by AM fungi treatment was 69.9% (P < 0.01) and 76.4% (P < 0.01) higher than plants without AM fungi under drought and salt stress, respectively. No significant difference in the mycorrhizal benefits to aboveground biomass induced by Fm and Ri was observed. No significant interactive effects of drought × AM fungi (P > 0.05, Table 1) and salt stress × AM fungi (P > 0.05, Table 2) on aboveground biomass were observed.
Drought and salt stress decreased protein contents (Fig. 4A), but increased proline (Fig. 4B) and MDA (Fig. 4C). Compared to plants without AM fungi, the AM fungi inoculation increased the mean protein and proline contents by 77.3% (P < 0.01) and 45.1% (P < 0.05), respectively, and reduced the MDA content by 45.1% (P < 0.05) under drought. Under salt stress, AM fungi meanly increased protein and proline contents by 80.7% (P < 0.01) and 23.9% (P < 0.05), respectively, and reduced the MDA content by 30.1% (P < 0.05). No significant difference in the mycorrhizal- induced changes in the protein, proline and MDA contents were observed between plants inoculated by Fm and Ri, except the mycorrhizal benefit of Ri was higher (P < 0.05) than Fm under salt stress. Significant interactive effects of drought × AM fungi (P < 0.05, Table 1) and salt stress × AM fungi (P < 0.01, Table 2) on MDA were observed. 4. Discussion It is well known that plant photosynthesis is very important to plant growth, especially in some extreme environments, such as regions affected by drought and saline soil. A large number of studies have reported that salt stress and drought can reduce the leaf net photosynthetic rate (A), stomatal conductance (gs) and transpiration rate (E) 15
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Table 1 Results of two-way ANOVA on the effects of drought, arbuscular mycorrhizal (AM) fungi, and their interactions on the contents of proline, protein and MDA,Pn, gs,Tr, Ci, Chl a, Chl b, Chl a + b, carotenoid (Car) and aboveground biomass. Source of variation
Proline
Protein
MDA
Pn
gs
Tr
Ci
Chl a
Chl b
Chl a + b
Car
Biomass
Drought AM fungi Salt × AM fungi
***
***
***
***
***
***
***
***
**
***
***
***
*
*
**
*
***
**
**
NS
**
NS
*
*
NS
NS
*
NS
NS
NS
NS
***
NS
***
NS
NS
“NS” indicates that the differences are not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.
result. The increases in chlorophyll and gas exchange indicate that AM fungi increase the carbon (C) fixed during photosynthesis, which would stimulate both the growth of the AM fungi and the plant photosynthetic rate due to the reciprocal C-P relationship between AM fungi and their host plants (Kiers et al., 2011; Jiang et al., 2017). Moreover, the present results suggest that AM fungi might reduce the destruction of the leaf cellular structure caused by drought and salt stress. In addition, the effects of different AM fungi species on leaf chlorophyll varied under drought and salt stress, suggesting that the ecological function of different AM fungi species differ the growth of castor bean. To optimize the positive effect of AM fungi on castor bean growth, future studies should determine whether a mixture of AM fungi species has a more positive effect on castor bean growth than single AM fungi species. Drought and salinity stress can cause oxidative damage to plants through the formation of reactive oxygen species, which damage proteins (Bauddh and Singh, 2012). Although several studies found that AM fungi did not affect proteins in plant leaves under drought (Subramanian and Charest, 1999; Porcel et al., 2003), there have been some studies that reported that AM fungi increased leaf proteins under water (Beltrano and Ronco, 2008) and salt stress (Hajiboland et al., 2010). Our results found that AM fungi increased the amount of protein in castor bean leaves, which is consistent with the latter results. The present results suggest that AM fungi could reduce the destruction of protein synthesis caused by drought and salt stress. Proline is one of one of the most important metabolites, and the high accumulation of proline can protect plant growth under drought and salt stress conditions. The present results found that AM fungi significantly leaf proline content, which suggests that AM fungi may play an important role in the protection of castor seedlings against drought and salt stress by increasing proline content and altering the osmo-regulation and osmotolerance of castor bean. MDA is a product of lipid peroxidation that can indicate oxidative damage (Meloni et al., 2003). In the current study, AM fungi decreased the MDA significantly under drought and salt stress. This result indicates that AM fungi might reduce serious plasma membrane lipid peroxidation of castor bean seedlings under drought and salt stresses, which would improve plant growth. AM fungi indeed improved the growth of castor bean, as the aboveground biomass of fungi-inoculated plants increased significantly. The current results suggest that AM fungi can improve the growth of castor bean seedling by altering metabolite contents, which play a key role in the
(Meloni et al., 2003; Hura et al., 2007; Yang et al., 2011; Yan et al., 2016). Some results found that leaf A, gs, and E increased under moderate salt stress condition (Maggio et al., 2000; Chen et al., 2009). The present study found that salt stress and drought decreased the leaf A, gs, and E of R. communis, which is consistent with a previous study that reported that salt reduced the A and gs of early castor bean seedlings (Pinheiro et al., 2008). The results suggest that drought and salt stress can suppress the growth of castor bean via the reduction of leaf gas exchange at the early seedling stage. Previous evidence has demonstrated that AM fungi can improve crops growth by increasing leaf A, E and gs under water and salt stress condition (Wu and Xia, 2006; Sheng et al., 2008; Zhu et al., 2014; He et al., 2017). However, whether AM fungi can improve the plant growth of R. communis by altering the leaf gas exchanges are still not clear. The current results found that AM fungi increased the leaf gas exchanges under drought and salt stresses, indicating AM fungi might improve leaf gas exchange in castor bean by increasing the stomatal conductance and transpiration in leaves. The improvement in leaf gas processes might be related to the increase in nutrient and water uptake caused by AM fungi (Zhu et al., 2010). Moreover, AM fungi significantly reduced the intercellular CO2 concentration (Ci) which is in agreement with previous studies on plants under drought (Sánchez-Díaz et al., 1990) and salt stresses (Sheng et al., 2008). Under drought and salt stress conditions, the increase in Ci indicates the reduction in leaf photosynthesis, and the present results suggest that AM fungi might alleviate the reduction in photosynthetic processes caused by drought and salt stress and improve plant growth. Chlorophyll is essential for plant photosynthesis, which enables plants to get energy from light (Zai et al., 2012). Previous studies have reported that the contents of chlorophyll significantly decreased under drought (Mafakheri et al., 2010) and salt stress (Li et al., 2010; Liu and Shi, 2010) conditions. Our study found that AM fungi enhanced the chlorophyll content in castor bean leaves under both drought and salt stress conditions, which is in agreement with previous studies under drought (Pinior et al., 2005; Fan and Liu, 2011) and salt stress (Zai et al., 2012; Talaat and Shawky, 2014). The increase of chlorophyll content might be related to the increase in phosphorus (P) and magnesium (Mg) uptakes caused by AM fungi (Zhu et al., 2014). A high chlorophyll content indicated that AM fungi increased the synthesis rate of chlorophyll and could increase leaf photosynthesis under drought and salt stress conditions, which is consistent with our above
Table 2 Results (F-value) of two-way ANOVA on the effects of salt, AM fungi, and their interactions on the contents of proline, protein and MDA, Pn, gs, Tr, Ci, Chl a, Chl b, Chl a + b, carotenoid (Car) and aboveground biomass. Source of variation
Proline
Protein
MDA
Pn
gs
Salt AM fungi Salt × AM fungi
***
***
***
***
***
**
*
**
**
**
*
NS
NS
**
NS NS
NS
NS
NS
Tr
“NS” indicates that the differences are not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.
16
Ci
Chl a
Chl b
Chl a + b
Car
Biomass
**
***
NS
***
***
***
*
**
*
***
*
*
**
NS
NS NS
NS
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Fig. 2. The effects of AM fungi on the plant net photosynthetic rate (A), stomatal conductance (B), transpiration rate (C), and intercellular CO2 concentration (D) under drought and salt stress. Vertical bars indicate the standard error of the mean (n = 4). NM represents the control without AM fungi (white), Fm represents treatments inoculated with the AM fungi F. mosseae (gray), Ri represents treatments inoculated with the AM fungi R. intraradices (black). Different lowercase letters indicate significant differences between the two AM fungi treatments under control, drought and salt stress conditions at the 0.05 significance level.
Fig. 3. The effects of AM fungi on the leaf photosynthesis pigments: Chl a (A), Chl b (B), Chl a + b (C) and carotenoid (D) under drought and salt stress. Vertical bars indicate the standard error of the mean (n = 4). NM represents the control without AM fungi (white), Fm represents treatments inoculated with the AM fungi F. mosseae (gray), Ri represents treatments inoculated with the AM fungi R. intraradices (black). Different lowercase letters indicate significant differences between the two AM fungi treatments under control, drought and salt stress conditions at the 0.05 significance level.
inoculated by Ri, for both drought and salt stress treatments, was higher than that in plants inoculated by Fm. However, the mycorrhizal benefits on the physiological responses of castor bean to stress were not higher for the Ri inoculation than the Fm inoculation; in fact, for some responses, the benefits of Ri were even lower than those of Fm. The results suggest that the contributions of different AM fungi species to castor bean physiological response to drought and salt stresses are different.
growth of castor bean in regions affected by drought and salinization, however, drought and salinization are often simultaneously present. The effects of AM fungi on castor bean growth under drought and salt stresses are still not clear; in addition, castor bean is an important oilseed crop, and whether AM fungi can increase the oil content in castor bean seeds is still not understood and should be studied in the future. Our results also found that the mycorrhizal colonization of plants 17
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Acknowledgements This work was supported by the National Natural Science Foundation of China (31770359 and 31470405), State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences and the Program of Introducing Talents of Discipline to Universities (B16011). We thank the anonymous reviewers for helpful comments on revision of this manuscript. The authors have declared that no competing interests exist. References Abd-Allah, W.H., Khater, R.M., 2016. Effect of rock phosphate and mycorrhiza on vegetative growth and productivity of Ricinus communis var Red Arish under North Sinai Conditions. Middle East J. 5, 412–421. AlKaraki, G.N., AlRaddad, A., 1997. Effects of arbuscular mycorrhizal fungi and drought stress on growth and nutrient uptake of two wheat genotypes differing in drought resistance. Mycorrhiza 7, 83–88. Auge, R.M., Toler, H.D., Saxton, A.M., 2015. 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Fig. 4. The effects of AM fungi on contents of leaf protein (A), proline (B) and MDA (C) under drought and salt. Vertical bars indicate the standard error of the mean (n = 4). NM represents the control without AM fungi (white), Fm represents treatments inoculated with the AM fungi F. mosseae (gray), Ri represents treatments inoculated with the AM fungi R. intraradices (black). Different lowercase letters indicate significant differences between the two AM fungi treatments under control, drought and salt stress conditions at the 0.05 significance level.
Therefore, it is necessary to screen AM fungi species before their practical use. Moreover, the interactions between AM fungi and other soil microbes could improve soil properties and the growth of castor bean (Zhang et al., 2014a,b). However, the effect of interactions between AM fungi and other soil microbiomes on the physiological response of castor bean to drought and salt stress are still not clear, so this subject requires further studies. In conclusion, our results found that AM fungi could increase the resistance of castor bean seedlings to drought and salt stress by improving leaf gas exchange properties (A, gs, E), increasing the contents of chlorophyll, increasing the protein and proline contents, and reducing the MDA contents. The results suggest that AM fungi play a vital role in the survival and growth of castor bean seedlings, which might increase the yield of castor been seeds and increase C sequestration in regions affected by drought and salinization.
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France. In: Janick, J. (Ed.), Progress in new crops, Alexandria. ASHS Press. Li, G., Wan, S., Zhou, J., Yang, Z., Qin, P., 2010. Leaf chlorophyll fluorescence hyperspectral reflectance, pigments content, malondialdehyde and proline accumulation responses of castor bean (Ricinus communis L.) seedlings to salt stress levels. Ind. Crops Prod. 31, 13–19. Liu, J., Shi, D.C., 2010. Photosynthesis, chlorophyll fluorescence, inorganic ion and organic acid accumulations of sunflower in responses to salt and salt-alkaline mixed stress. Photosynthetica 48, 127–134. Machineski, O., Balota, E.L., de Souza, J.R.P., 2011. Response of castor bean to arbuscular mycorrhizal fungi and levels of phosphorus. Semin-Cienc Agrar. 32, 1855–1862. Mafakheri, A., Siosemardeh, A., Bahramnejad, B., Struik, P., Sohrabi, Y., 2010. Effect of drought stress on yield, proline and chlorophyll contents in three chickpea cultivars. Aust. J. Crops Sci. 4, 580. Maggio, A., Reddy, M.P., Joly, R.J., 2000. Leaf gas exchange and solute accumulation in the halophyte Salvadora persica grown at moderate salinity. Environ. Exp. Bot. 44, 31–38. Meloni, D.A., Oliva, M.A., Martinez, C.A., Cambraia, J., 2003. Photosynthesis and activity of superoxide dismutase: peroxidase and glutathione reductase in cotton under salt stress. Environ. Exp. Bot. 49, 69–76. Pinheiro, H.A., Silva, J.V., Endres, L., Ferreira, V.M., de Albuquerque Câmara, C., Cabral, F.F., Oliveira, J.F., de Carvalho, L.W.T., dos Santos, J.M., dos Santos Filho, B.G., 2008. Leaf gas exchange, chloroplastic pigments and dry matter accumulation in castor bean (Ricinus communis L.) seedlings subjected to salt stress conditions. Ind. Crops Prod. 27, 385–392. Pinior, A., Grunewaldt-Stöcker, G., von Alten, H., Strasser, R.J., 2005. Mycorrhizal impact on drought stress tolerance of rose plants probed by chlorophyll a fluorescence, proline content and visual scoring. Mycorrhiza 15, 596. Porcel, R., Barea, J.M., Ruiz-Lozano, J.M., 2003. Antioxidant activities in mycorrhizal soybean plants under drought stress and their possible relationship to the process of nodule senescence. New Phytol. 157, 135–143. Sánchez-Díaz, M., Pardo, M., Antolin, M., Pena, J., Aguirreolea, J., 1990. Effect of water stress on photosynthetic activity in the Medicago-Rhizobium-Glomus symbiosis. Plant Sci. 71, 215–221. Sheng, M., Tang, M., Chen, H., Yang, B.W., Zhang, F.F., Huang, Y.H., 2008. Influence of arbuscular mycorrhizae on photosynthesis and water status of maize plants under salt stress. Mycorrhiza 18, 287–296. Subramanian, K.S., Charest, C., 1999. Acquisition of N by external hyphae of an arbuscular mycorrhizal fungus and its impact on physiological responses in maize under drought-stressed and well-watered conditions. Mycorrhiza 9, 69–75. Talaat, N.B., Shawky, B.T., 2014. Protective effects of arbuscular mycorrhizal fungi on wheat (Triticum aestivum L.) plants exposed to salinity. Environ. Exp. Bot. 98, 20–31. van der Heijden, M.G.A., Streitwolf-Engel, R., Riedl, R., Siegrist, S., Neudecker, A., Ineichen, K., Boller, T., Wiemken, A., Sanders, I.R., 2006. The mycorrhizal
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Arbuscular mycorrhizal fungi improve plant growth of Ricinus communis by altering photosynthetic properties and increasing pigments under drought and salt stress Tao Zhanga,
T
⁎,1
, Yongjun Hub,1, Ke Zhangc, Changyan Tianc, Jixun Guoa
a
Institute of Grassland Science, Northeast Normal University, Key Laboratory of Vegetation Ecology, Ministry of Education, Changchun 130024, China School of Life Science, Changhun Normal University, Changchun 130032, China c State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Arbuscular mycorrhizal (AM) fungi Biofuel production Metabolites Physiological response Revegetation
Castor bean (Ricinus communis) is one of the most important candidate crops that can be used to increase the land cover and crop yield in the arid and saline region of China. To evaluate the effects of arbuscular mycorrhizal (AM) fungi on the growth of castor bean under drought and salt stresses, a pot experiment was carried out to examine how AM fungi improve the plant growth of castor bean by affecting the leaf gas exchange, pigments, and metabolites accumulation of castor bean seedlings. The results showed that AM fungi stimulated plant growth and increased the castor bean aboveground biomass. AM fungi significantly increased the net photosynthetic rate (A), stomatal conductance (gs) and transpiration rate (E) of castor bean and decreased the intercellular CO2 concentration (Ci) under drought and salt stress. The contents of chlorophyll a, chlorophyll b, chlorophyll a + b and carotenoids in leaves treated by AM fungi were higher than in those without the AM fungi treatment. AM fungi notably increased the soluble protein and proline contents and decreased the malondialdehyde (MDA) content of castor bean. The results indicated that AM fungi could protect castor bean against drought and salt stresses by improving its leaf gas exchanges and photosynthetic capacity and altering its concentrations of metabolites. Our results highlight that AM fungi might be a promising bio-approach that can be used to plant castor beans in northeast and northwest China.
1. Introduction Castor bean (Ricinus communis L. Euphorbiaceae) is one of the most important oilseed crops. The oil content in the castor bean seeds can reach 40–60%, and most of the oil is essentially the triglyceride of ricinoleic acid (Labalette et al., 1996). The castor bean is considered a promising candidate for biofuel production (Barnes et al., 2009a), and the oil obtained from castor beans is widely used to make many products, such as surfactants, coatings, greases, fungistats, pharmaceuticals, and cosmetics (Pinheiro et al., 2008). Moreover, the castor bean can also be used to reduce the soil degradation caused by heavy metals (Bauddh and Singh, 2012) and salt (Li et al., 2010; Wu et al., 2012). Because of its high economic and ecological value, castor bean is widely planted in many developing countries, such as India (Bauddh and Singh, 2012), South Africa (Barnes et al., 2009b), Argentina (Falasca et al., 2012) and Brazil (Pinheiro et al., 2008). Although castor bean has many important ecological and economic values, the castor bean
⁎
1
cultivation area is not large, and the yield of castor bean is still not high because food security is prioritized over is cultivation, and most of good arable land is only used to grow crops (e.g, wheat, maize, and rice) in China. However, in northeast and northwest China there are approximately 35 million hectares of saline land that can be used to grow castor bean because of the high salt and drought tolerances of the plant. However, a previous study found that castor bean could not tolerate salt stress during its initial growth stages (Pinheiro et al., 2008). Therefore, it is important to increase the salt resistance at the early stage which is important to improve plant growth and yield. Arbuscular mycorrhizal (AM) fungi is one of most important soil microorganisms that can form mycorrhizal symbiosis with most of terrestrial plants. AM fungi can improve the growth of many plants by many pathways, known to increase nutrient uptake (van der Heijden et al., 2006; Zhang et al., 2011), improve drought tolerance (AlKaraki and AlRaddad, 1997; Auge et al., 2015), improve salt resistance (Feng et al., 2002; Evelin et al., 2009), and reduce plant vulnerability to
Corresponding authors at: No. 5268 Renmin Street, Changchun, 130024, China. E-mail address: [email protected] (T. Zhang). These authors contributed equally to this work.
https://doi.org/10.1016/j.indcrop.2018.02.087 Received 27 November 2017; Received in revised form 22 January 2018; Accepted 28 February 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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was maintained at 50% field capacity by weight in all treatment except the drought treatment. The experiment was carried out in phytotrons (LT/ACR-2002, E-Sheng Tech., Beijing, China) for 2 months with the temperature of 25 °C from 06:00–20:00, 18 °C from 20:00–06:00. The relative humidity was 40–60%, and the light intensity was 350 μ mol−2 S−1. The pots were placed the phytotrons randomly, and moved to a different place every week to eliminate the effect of light and temperature on plant growth. The plants were harvested after growing for three months.
pathogens (Wehner et al., 2011; Veresoglou and Rillig, 2012). There are few studies that report the association between AM fungi and castor bean (Machineski et al., 2011; Kurandawad et al., 2014). The previous studies found that AM fungi increased the growth of castor bean (AbdAllah and Khater, 2016), plant survival percentage, dry matter accumulation and phosphorus uptake and reduced the soil pH and salinity (Zhang et al., 2014a,b). However, the effects of AM fungi on the growth of castor bean are still not well understood, and the mechanism of how AM fungi improve plant growth under salt and drought stress remains unclear. The objectives of this study are: (a) to evaluate the effects of AM fungi on the leaf photosynthetic characteristics of castor bean under drought and salt stress condition; (b) to assess the influence of AM fungi on the leaf pigment contents under drought and salt stresses; and (c) to investigate the effects of AM fungi on protein content and proline and malondialdehyde (MDA) concentrations in castor bean leaves under drought and salt stresses. With these objectives, this study will elucidate the physiological mechanism by which AM fungi improves the growth of castor bean and evaluate the potential of AM fungi in castor bean cultivation in the arid or semiarid saline regions in northeast and northwest China.
2.3. Leaf photosynthesis
2. Material and methods
The leaf photosynthesis measurements were conducted on the 7th day between 08:00 to 12:00 am after the drought treatment by using a portable photosynthesis system (LI-6400, LI-COR Inc., USA). The leaf net photosynthetic rate (A), stomatal conductance (gs), transpiration rate (E), and intercellular CO2 concentration (Ci) were measured according to the methods described by Chen et al. (2005). Three fully opened leaves of were selected for each plant in each pot. We measured the values of these leaves and averaged them for each plant. The photosynthesis parameters were measured with photosynthetically active radiation (1200 μmol m−2 s−1), a CO2 concentration of 350 μmol mol−1, and a leaf temperature of 25 °C.
2.1. Plant material, soil and AM fungi preparation
2.4. Leaf pigments
The seeds of castor bean (Ricinus communis) and the soil used in this experiment were collected from Xinjiang Institute of Ecology and Geography, China Academy of Sciences northwest China. The soil in the studied area is a saline desert soil, with a pH of 7.5–9.0. The average contents of soil total nitrogen and available soil phosphorus (0.5 mol L−1 NaHCO3-extractable) were 16.5 mg kg−1 and 3.0 mg kg−1, respectively. The collected soil was air dried and sieved (2 mm) to remove big stones, plant roots and other litters. The dry soil was sterilized at 121 °C for 2 h in order to remove AM fungi in the soil. The collected seeds were air dried and stored in a refrigerator at < 4 °C before use. The R. communis seeds were surface disinfested in 10% (v/v) hydrogen peroxide for 5 min, rinsed 5 times with deionized water, and then germinated at 20 °C for 48 h. Four germinated seeds were sown in pots. The seedlings were thinned to 2 per pot. Two dominant AM fungi species Funneliformis mosseae (Fm) and Rhizophagus intraradices (Ri) were used in this study. These two AM fungi species were isolated from the same sites where R. communis grew, and cultured individually using Medicago sativa for 4 months. The seeds of M. sativa were surface sterilized in 10% (v/v) hydrogen peroxide for 5 min and rinsed with deionized water. To maintain the purity of AM fungi, we cultured these two AM fungi species in different closed chambers. After that, the aboveground was removed and the two AM fungi species were mixed. We examined the purity of the two AM fungi species before inoculation and found that the cultured AM fungi were not contaminated by other fungi. The AM fungi inoculum consisted of a mixture of soil, hyphae, spores and infected root fragments (approximately 800 spores per 20 g soil).
After the leaf photosynthesis measurement, the leaf pigments were measured according to the methods of Zhang et al. (2016). Three fully expanded and undamaged leaves from each plant were sampled in each treatment, and wrapped immediately in aluminium foil to avoid pigments degradation. The whole leaves were cut into pieces and placed in a test tube containing 10 ml extractant (80% acetone and absolute alcohol at a ratio 1:1). The test tubes were incubated at 70 °C for 30 min (Hiscox and Israelstam, 1979), and then cooled in the dark. The cooled extract was analyzed using a spectrophotometer (UV-2201, Essentia, Japan) at 440, 649 and 665 nm. The chlorophyll a (Chl a), b (Chl b) and carotenoid concentrations were calculated according to the method of Wellburn (1994). 2.5. Leaf protein, proline and MDA The leaf soluble protein content was measured using the bovine serum albumin protein standard according to the previously reported method (Bradford, 1976). Fresh leaf samples (0.5 g) were homogenized with 4 ml Na-phosphate buffer (pH 7.2), and then centrifuged at 4 °C (Janmohammadi et al., 2012). The supernatants with dye were measured using a spectrophotometer (UV-2201, Essentia, Japan) at 595 nm. Proline was measured according to the methods described by Bates et al. (1973), and the malondialdehyde (MDA) content was measured according to Heath and Packer (1986). 2.6. Mycorrhizal colonization and biomass The roots were cut into 1-cm segments, and mixed thoroughly. A sub-sample of 0.5 g was cleared with 10% (w/v) KOH at 90 °C for 2 h and then stained with trypan blue. Thirty stained roots section were mounted on slides in polyvinyl alcohol–lactic acid glycerol and the arbuscule, vesicule and hyphae in the root were examined using a microscope at 100–400 magnification. The mycorrhizal colonization was calculated using the MYCOCALC program according to a previously described method (Zhang et al., 2011). Plant shoots and leaves were weighed after they were oven dried at 65 °C for 48 h.
2.2. Experimental design This experiment included control, drought stress and salt stress three main treatments, and each main treatment was applied to castor bean without AM fungi and those inoculated with AM fungi Fm and Ri treatments, and replicated four times. In the drought stress treatment, a seven days regime (seven days of withheld water) was applied to create drought stress before harvesting according to the methods described by Bauddh and Singh (2012). The control plants and the plants not subjected to the drought stress were watered routinely every 2–3 days. In the salt stress treatment, a sodium chloride (200 mM NaCl) solution was added to the soil. In order to keep the salt in the soil, the soil moisture
2.7. Statistical analysis All data were analyzed statistically by analysis of variance using the 14
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3.2. Leaf photosynthesis and gas exchange Drought and salt stresses significantly decreased A (P < 0.05, Fig. 2A), gs (P < 0.05, Fig. 2B) and E (P < 0.05, Fig. 2C), but they had no impact on Ci (P > 0.05, Fig. 2D). Under drought, AM fungi increased the mean values of A, gs and E by 37.1% (P < 0.05), 37.3% (P < 0.05) and 30.5% (P < 0.05), respectively, and decreased Ci by 11.1% (P < 0.05, Fig. 2D). Under salt stress conditions, AM fungi meanly increased A, gs and E by 34.7% (P < 0.05), 40.8% (P < 0.05) and 32.3% (P < 0.05), respectively, decreased Ci by 14.1% (P < 0.05, Fig. 2D). No significant differences in A were observed between plants inoculated by the two AM fungi species, except under drought. Under salt stress conditions, the increase in E was 12.5% (P < 0.05) greater in plants inoculated by Fm than in plants inoculated by Ri. No significant interactive effects of drought × AM fungi (P > 0.05, Table 1) and salt stress × AM fungi (P > 0.05, Table 2) on leaf photosynthesis were observed. 3.3. Leaf photosynthesis pigments Drought and salt stress both decreased Chl a (P < 0.05, Fig. 3A), Chl b (P < 0.05, Fig. 3B) and Chl a + b (P < 0.05, Fig. 3C) significantly, but had no impact on Car (P > 0.05, Fig. 3D). AM fungi significantly increased the leaf pigments. Under drought, the inoculations with Fm and Ri increased, on average, Chl a, Chl b, Chl a + b and Car by 30.9%, 31.7%, 32.3% and 39.9%, respectively, compared to the control without AM fungi (all P < 0.05). Under salt stress, AM fungi Fm and Ri increased on average, Chl a, Chl b, Chl a + b and Car by 33.9% (P < 0.05), 60.2% (P < 0.01), 25.9% (P < 0.05) and 134.2% (P < 0.01), respectively, compared to the control without AM fungi (Fig. 3). The mycorrhizal benefits of Fm and Ri differed under drought and salt stress conditions. Under drought, the contents of Chl a, Chl b, Chl a + b in the plants inoculated by Ri were higher than those in plants inoculated by Fm, but they were lower in the Ri inoculation treatment than the Fm inoculation treatment under salt stress. No significant differences in the mycorrhizal-induced increases in Car were detected between plants inoculated by Fm and Ri (P > 0.05, Fig. 3D). Significant interactive effects of drought × AM fungi on Chl a (P < 0.001) and Chl a + b (P < 0.001, Table 1) were detected. In addition, significant interactive effects of salt stress × AM on Chl a (P < 0.05) and Chl b (P < 0.01, Table 2) were detected.
Fig. 1. Myorrhizal colonization (A) in the root system of R. communis and aboveground biomass (B) under drought and salt stresses. Vertical bars indicate the standard error of the mean (n = 4). NM represents the control without arbuscular mycorrhizal (AM) fungi (white), Fm represents treatments inoculated with the AM fungi F. mosseae (gray), Ri represents treatments inoculated with the AM fungi R. intraradices (black). Different lowercase letters indicate significant differences between the two AM fungi treatments under control, drought and salt stress conditions at the 0.05 significance level.
software SPSS 16.0 (SPSS 16.0 for windows, USA) with the means separated using the Duncan test at P < 0.05 level.
3. Results 3.1. Plant growth and mycorrhizal colonization
3.4. Leaf protein, proline and MDA No mycorrhizal colonization was observed in the root of R. communis in all plants grown without AM fungi treatments (Fig. 1A). The mycorrhizal colonizations in the Fm inoculation control, drought and salt stress treatments treatment were 15.9% (P < 0.05), 21.9% (P < 0.05) and 14.6% (P < 0.05) lower, respectively, than in those inoculated by Ri. Drought and salt stress slightly decreased mycorrhizal colonization in the Fm inoculation treatments, but there was no significant difference between the treatments (P > 0.05). In the Ri inoculation treatments, salt stress decreased mycorrhizal colonization by 10.2% (P < 0.05), while drought had no impact on mycorrhizal colonization (P > 0.05). AM fungi stimulated the plant growth of R. communis in the control, drought and salt stress treatments (Fig. 1B). The aboveground biomass of plants inoculated by AM fungi treatment was 69.9% (P < 0.01) and 76.4% (P < 0.01) higher than plants without AM fungi under drought and salt stress, respectively. No significant difference in the mycorrhizal benefits to aboveground biomass induced by Fm and Ri was observed. No significant interactive effects of drought × AM fungi (P > 0.05, Table 1) and salt stress × AM fungi (P > 0.05, Table 2) on aboveground biomass were observed.
Drought and salt stress decreased protein contents (Fig. 4A), but increased proline (Fig. 4B) and MDA (Fig. 4C). Compared to plants without AM fungi, the AM fungi inoculation increased the mean protein and proline contents by 77.3% (P < 0.01) and 45.1% (P < 0.05), respectively, and reduced the MDA content by 45.1% (P < 0.05) under drought. Under salt stress, AM fungi meanly increased protein and proline contents by 80.7% (P < 0.01) and 23.9% (P < 0.05), respectively, and reduced the MDA content by 30.1% (P < 0.05). No significant difference in the mycorrhizal- induced changes in the protein, proline and MDA contents were observed between plants inoculated by Fm and Ri, except the mycorrhizal benefit of Ri was higher (P < 0.05) than Fm under salt stress. Significant interactive effects of drought × AM fungi (P < 0.05, Table 1) and salt stress × AM fungi (P < 0.01, Table 2) on MDA were observed. 4. Discussion It is well known that plant photosynthesis is very important to plant growth, especially in some extreme environments, such as regions affected by drought and saline soil. A large number of studies have reported that salt stress and drought can reduce the leaf net photosynthetic rate (A), stomatal conductance (gs) and transpiration rate (E) 15
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Table 1 Results of two-way ANOVA on the effects of drought, arbuscular mycorrhizal (AM) fungi, and their interactions on the contents of proline, protein and MDA,Pn, gs,Tr, Ci, Chl a, Chl b, Chl a + b, carotenoid (Car) and aboveground biomass. Source of variation
Proline
Protein
MDA
Pn
gs
Tr
Ci
Chl a
Chl b
Chl a + b
Car
Biomass
Drought AM fungi Salt × AM fungi
***
***
***
***
***
***
***
***
**
***
***
***
*
*
**
*
***
**
**
NS
**
NS
*
*
NS
NS
*
NS
NS
NS
NS
***
NS
***
NS
NS
“NS” indicates that the differences are not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.
result. The increases in chlorophyll and gas exchange indicate that AM fungi increase the carbon (C) fixed during photosynthesis, which would stimulate both the growth of the AM fungi and the plant photosynthetic rate due to the reciprocal C-P relationship between AM fungi and their host plants (Kiers et al., 2011; Jiang et al., 2017). Moreover, the present results suggest that AM fungi might reduce the destruction of the leaf cellular structure caused by drought and salt stress. In addition, the effects of different AM fungi species on leaf chlorophyll varied under drought and salt stress, suggesting that the ecological function of different AM fungi species differ the growth of castor bean. To optimize the positive effect of AM fungi on castor bean growth, future studies should determine whether a mixture of AM fungi species has a more positive effect on castor bean growth than single AM fungi species. Drought and salinity stress can cause oxidative damage to plants through the formation of reactive oxygen species, which damage proteins (Bauddh and Singh, 2012). Although several studies found that AM fungi did not affect proteins in plant leaves under drought (Subramanian and Charest, 1999; Porcel et al., 2003), there have been some studies that reported that AM fungi increased leaf proteins under water (Beltrano and Ronco, 2008) and salt stress (Hajiboland et al., 2010). Our results found that AM fungi increased the amount of protein in castor bean leaves, which is consistent with the latter results. The present results suggest that AM fungi could reduce the destruction of protein synthesis caused by drought and salt stress. Proline is one of one of the most important metabolites, and the high accumulation of proline can protect plant growth under drought and salt stress conditions. The present results found that AM fungi significantly leaf proline content, which suggests that AM fungi may play an important role in the protection of castor seedlings against drought and salt stress by increasing proline content and altering the osmo-regulation and osmotolerance of castor bean. MDA is a product of lipid peroxidation that can indicate oxidative damage (Meloni et al., 2003). In the current study, AM fungi decreased the MDA significantly under drought and salt stress. This result indicates that AM fungi might reduce serious plasma membrane lipid peroxidation of castor bean seedlings under drought and salt stresses, which would improve plant growth. AM fungi indeed improved the growth of castor bean, as the aboveground biomass of fungi-inoculated plants increased significantly. The current results suggest that AM fungi can improve the growth of castor bean seedling by altering metabolite contents, which play a key role in the
(Meloni et al., 2003; Hura et al., 2007; Yang et al., 2011; Yan et al., 2016). Some results found that leaf A, gs, and E increased under moderate salt stress condition (Maggio et al., 2000; Chen et al., 2009). The present study found that salt stress and drought decreased the leaf A, gs, and E of R. communis, which is consistent with a previous study that reported that salt reduced the A and gs of early castor bean seedlings (Pinheiro et al., 2008). The results suggest that drought and salt stress can suppress the growth of castor bean via the reduction of leaf gas exchange at the early seedling stage. Previous evidence has demonstrated that AM fungi can improve crops growth by increasing leaf A, E and gs under water and salt stress condition (Wu and Xia, 2006; Sheng et al., 2008; Zhu et al., 2014; He et al., 2017). However, whether AM fungi can improve the plant growth of R. communis by altering the leaf gas exchanges are still not clear. The current results found that AM fungi increased the leaf gas exchanges under drought and salt stresses, indicating AM fungi might improve leaf gas exchange in castor bean by increasing the stomatal conductance and transpiration in leaves. The improvement in leaf gas processes might be related to the increase in nutrient and water uptake caused by AM fungi (Zhu et al., 2010). Moreover, AM fungi significantly reduced the intercellular CO2 concentration (Ci) which is in agreement with previous studies on plants under drought (Sánchez-Díaz et al., 1990) and salt stresses (Sheng et al., 2008). Under drought and salt stress conditions, the increase in Ci indicates the reduction in leaf photosynthesis, and the present results suggest that AM fungi might alleviate the reduction in photosynthetic processes caused by drought and salt stress and improve plant growth. Chlorophyll is essential for plant photosynthesis, which enables plants to get energy from light (Zai et al., 2012). Previous studies have reported that the contents of chlorophyll significantly decreased under drought (Mafakheri et al., 2010) and salt stress (Li et al., 2010; Liu and Shi, 2010) conditions. Our study found that AM fungi enhanced the chlorophyll content in castor bean leaves under both drought and salt stress conditions, which is in agreement with previous studies under drought (Pinior et al., 2005; Fan and Liu, 2011) and salt stress (Zai et al., 2012; Talaat and Shawky, 2014). The increase of chlorophyll content might be related to the increase in phosphorus (P) and magnesium (Mg) uptakes caused by AM fungi (Zhu et al., 2014). A high chlorophyll content indicated that AM fungi increased the synthesis rate of chlorophyll and could increase leaf photosynthesis under drought and salt stress conditions, which is consistent with our above
Table 2 Results (F-value) of two-way ANOVA on the effects of salt, AM fungi, and their interactions on the contents of proline, protein and MDA, Pn, gs, Tr, Ci, Chl a, Chl b, Chl a + b, carotenoid (Car) and aboveground biomass. Source of variation
Proline
Protein
MDA
Pn
gs
Salt AM fungi Salt × AM fungi
***
***
***
***
***
**
*
**
**
**
*
NS
NS
**
NS NS
NS
NS
NS
Tr
“NS” indicates that the differences are not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.
16
Ci
Chl a
Chl b
Chl a + b
Car
Biomass
**
***
NS
***
***
***
*
**
*
***
*
*
**
NS
NS NS
NS
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Fig. 2. The effects of AM fungi on the plant net photosynthetic rate (A), stomatal conductance (B), transpiration rate (C), and intercellular CO2 concentration (D) under drought and salt stress. Vertical bars indicate the standard error of the mean (n = 4). NM represents the control without AM fungi (white), Fm represents treatments inoculated with the AM fungi F. mosseae (gray), Ri represents treatments inoculated with the AM fungi R. intraradices (black). Different lowercase letters indicate significant differences between the two AM fungi treatments under control, drought and salt stress conditions at the 0.05 significance level.
Fig. 3. The effects of AM fungi on the leaf photosynthesis pigments: Chl a (A), Chl b (B), Chl a + b (C) and carotenoid (D) under drought and salt stress. Vertical bars indicate the standard error of the mean (n = 4). NM represents the control without AM fungi (white), Fm represents treatments inoculated with the AM fungi F. mosseae (gray), Ri represents treatments inoculated with the AM fungi R. intraradices (black). Different lowercase letters indicate significant differences between the two AM fungi treatments under control, drought and salt stress conditions at the 0.05 significance level.
inoculated by Ri, for both drought and salt stress treatments, was higher than that in plants inoculated by Fm. However, the mycorrhizal benefits on the physiological responses of castor bean to stress were not higher for the Ri inoculation than the Fm inoculation; in fact, for some responses, the benefits of Ri were even lower than those of Fm. The results suggest that the contributions of different AM fungi species to castor bean physiological response to drought and salt stresses are different.
growth of castor bean in regions affected by drought and salinization, however, drought and salinization are often simultaneously present. The effects of AM fungi on castor bean growth under drought and salt stresses are still not clear; in addition, castor bean is an important oilseed crop, and whether AM fungi can increase the oil content in castor bean seeds is still not understood and should be studied in the future. Our results also found that the mycorrhizal colonization of plants 17
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Acknowledgements This work was supported by the National Natural Science Foundation of China (31770359 and 31470405), State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences and the Program of Introducing Talents of Discipline to Universities (B16011). We thank the anonymous reviewers for helpful comments on revision of this manuscript. The authors have declared that no competing interests exist. References Abd-Allah, W.H., Khater, R.M., 2016. Effect of rock phosphate and mycorrhiza on vegetative growth and productivity of Ricinus communis var Red Arish under North Sinai Conditions. Middle East J. 5, 412–421. AlKaraki, G.N., AlRaddad, A., 1997. Effects of arbuscular mycorrhizal fungi and drought stress on growth and nutrient uptake of two wheat genotypes differing in drought resistance. Mycorrhiza 7, 83–88. Auge, R.M., Toler, H.D., Saxton, A.M., 2015. 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Fig. 4. The effects of AM fungi on contents of leaf protein (A), proline (B) and MDA (C) under drought and salt. Vertical bars indicate the standard error of the mean (n = 4). NM represents the control without AM fungi (white), Fm represents treatments inoculated with the AM fungi F. mosseae (gray), Ri represents treatments inoculated with the AM fungi R. intraradices (black). Different lowercase letters indicate significant differences between the two AM fungi treatments under control, drought and salt stress conditions at the 0.05 significance level.
Therefore, it is necessary to screen AM fungi species before their practical use. Moreover, the interactions between AM fungi and other soil microbes could improve soil properties and the growth of castor bean (Zhang et al., 2014a,b). However, the effect of interactions between AM fungi and other soil microbiomes on the physiological response of castor bean to drought and salt stress are still not clear, so this subject requires further studies. In conclusion, our results found that AM fungi could increase the resistance of castor bean seedlings to drought and salt stress by improving leaf gas exchange properties (A, gs, E), increasing the contents of chlorophyll, increasing the protein and proline contents, and reducing the MDA contents. The results suggest that AM fungi play a vital role in the survival and growth of castor bean seedlings, which might increase the yield of castor been seeds and increase C sequestration in regions affected by drought and salinization.
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