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Improving the acclimatization and establishment of Arundo donax L. plantlets, a promising energy crop, using a mycorrhiza-based biofertilizer
Industrial Crops and Products 66 (2015) 299–304
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Improving the acclimatization and establishment of Arundo donax L. plantlets, a promising energy crop, using a mycorrhiza-based biofertilizer Maria Tauler, Elena Baraza ∗ University of Balearic Islands edf Guillem Colom Crta. de Valldemossa, km 7.5, Islas Baleares, ES-07122 Palma de Mallorca, Spain
a r t i c l e
i n f o
Article history: Received 20 October 2014 Received in revised form 16 December 2014 Accepted 19 December 2014 Keywords: Giant reed Mycorrhizal inoculation Micropropagated plants Acclimatization Open field transfer
a b s t r a c t Arundo donax has become one of the most promising species for cellulose paste, biomass, and secondgeneration biofuel production. Because this species is not able to produce viable seeds, micropropagation is the best method for large scale production. Several studies have demonstrated that arbuscular mycorrhizal fungi (AMF) can improve the acclimatization of micropropagated plantlets, enhancing their growth and survival. In this study, we evaluated the effect of AMF during the acclimatization period of A. donax plantlets and its posterior establishment to the field. Plantlets were inoculated with mycorrhizabased biofertilizer (AEGIS SYM® ) that contained Rhizophagus intraradices and Funneliformis mosseae. The inoculum was applied at the time of transplantation of plantlets to 28-cm3 cell trays filled with commercial nutrient-rich agricultural substrate. The results showed that during acclimatization, mycorrhized plantlets (AM) present higher number of leaves and height increments as compared to non-mycorrhized plantlets (control). Moreover, at the end of the hardening process, AM plantlets presented higher height, number of leaves, biomass, shoot:root ratio and chlorophyll a and b content than control plantlets. After open field establishment, AM plantlets presented greater size, number of stems and survival rate. These results demonstrate that inoculating micropropagated A. donax with AMF enhances its growth and development as well as produces better quality plantlets, which can be very useful for the large-scale production of this energetic crop. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Giant reed (Arundo donax) is a C3 rhizomatous perennial grass and is considered one of the most promising species for energy, cellulose paste and second generation biofuel production (Shatalov and Pereira, 2002; Lewandowski et al., 2003; Pilu et al., 2013). This fact is due to its high production of biomass for energy, which can be even higher than the yield of other C4 species grasses (Angelini et al., 2009; Mantineo et al., 2009; Kering et al., 2012), its ability to adapt to a wide variety of environments and its low input requirement after establishment (Cosentino et al., 2008; Pilu et al., 2013). Moreover, giant reed can be cultivated in marginal or sub-marginal lands due to its tolerance to environmental stresses, thus reducing
Abbreviations: AMF, arbuscular Mycorrhizal Fungi; AM, arbuscular mycorrhized. ∗ Corresponding author. Tel.: +34 971173177. E-mail addresses: [email protected] (M. Tauler), [email protected] (E. Baraza). http://dx.doi.org/10.1016/j.indcrop.2014.12.039 0926-6690/© 2014 Elsevier B.V. All rights reserved.
competition with food crops that generally require better-quality lands (Sims et al., 2010; Nassi o Di Nasso et al., 2013). Nevertheless, giant reed cannot produce viable seeds because its pollen is unfruitful (Boose and Holt, 1999). Therefore, propagation is only possible by vegetative means, using fragments of stems or rhizomes that present a high capacity to survive and to generate shoots and roots (Mann et al., 2013). Rhizomes are commonly the most used, but this method of propagation is time consuming and involves considerable cost and effort to dig-up, break-apart and replant the rhizomes. In addition, this method of propagation requires large land surface to produce enough plants for use in energy production programs, as each plant produces only a few rhizomes each year (Pilu et al., 2013). Ceotto and Di Candilo (2010) suggested that wherever irrigation water is abundantly available, planting shoot cuttings directly in the field is the most practical option; however, this situation is not common. Another propagation method is in vitro culture, which offers the potential required for the large-scale propagation of giant reed (Cavallaro et al., 2011). In vitro culture methods facilitate obtaining larger amounts of high-quality genetically homogeneous plants in less time with
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the guarantee of being free of pathogens (Cavallaro et al., 2011). However, micropropagation is a complex procedure that requires the development of appropriate techniques to ensure the success of the process to justify the investment. One of the phases of the in vitro plant production that has generated greater mortality is the acclimatization or hardening phase (Kapoor et al., 2008). Directly after transfer from in vitro to ex vitro conditions, micropropagated plants are susceptible to various stresses because of the sudden shock of environmental changes and the incompleteness of certain physiological activities and anatomical developments like photoheterotrophic nature, lack of cuticle or improper vascular connection (Alarcón and Ferrera-Cerrato, 2000). Some authors have proposed the use of Arbuscular Mycorrhizal Fungi (AMF) as a solution to improve the acclimatization process (Vestberg and Estaún, 1994; Estrada-Luna et al., 2000; Kapoor et al., 2008). AMF develop a symbiotic relationship with the plant that enhances the development of the root system; increases nutrient uptake, water conducting capacity and photosynthetic rates; and promotes defense against harmful pathogens and the synthesis of growth hormones (Rai, 2001; Kapoor et al., 2008; Parkash et al., 2011; Singh et al., 2012). The benefits associated with the use of AM inoculation for in vitro-raised plantlets have been reported in several horticultural, fruit, ornamental and forest species (Alarcón and Ferrera-Cerrato, 2000; Rai, 2001; Kapoor et al., 2008). However, little is known about the influence of AM fungi on the survival and growth of micropropagated bioenergy plant species. The present study evaluates the effect of AMF commercial inoculum on the survival, growth and chlorophyll content of micropropagated plantlets of A. donax during the acclimatization period and the survival and growth during land transfer. The objective is to determine the convenience of the use of mycorrhizal biotechnology to improve the plantlet production process of this promising energy crop.
2. Material and methods 2.1. Experimental design This study was conducted between October 2013 and February 2014 in the experimental greenhouse of the Universitat de les Illes Balears, Majorca, Spain. The last phase of the study was conducted between March and May in marginal land, unused for the last 5 years, in the agricultural zone of Prat de Sant Jordi (Palma) located 14 km from Universitat de les Illes Balears. The plantlets that were used were Arundo-K12 clones provided by BIOTHEK ECOLOGIC FUEL S.L. Micropropagated plants were received bare-root, and 164 plantlets were immediately planted in a 28-cm3 cell planting tray and filled with agricultural substrate (KEKKILÄ DSM 1 W© ), which consisted of nutrient-rich black peat. At the moment of transplantation, 5 cm3 of AMF inoculum was added to 83 plantlets. The AMF commercial inoculum (AEGIS SYM© ) used contained 25 spores/g of Rhizophagus intraradices (before known as Glomus intraradices) and 25 spores/g of Funneliformis mosseae (before known as Glomus mosseae). To imitate the soil texture of inoculated cells, we added the same quantity of sterile zeolite to the others plantlets, which were used as a control. Plantlets were randomly distributed in the planting tray. Plantlets were maintained at a mean temperature of 18 ◦ C and 86% moisture in a greenhouse during the acclimatization period. The plantlets were watered according to their needs to always keep the substrate moist. In addition, we moved the tray periodically to avoid a position effect. After 46 days, 22 plants per treatment were used to measure fresh and dry biomass, chlorophyll content and mycorrhizal colo-
nization. The remaining 120 plants were transplanted to 300-cm3 cell planting trays. When climatic conditions were favorable (end of March), plantlets were transferred to open field. In the field, plants from the two treatments were randomly distributed, occupying the entire plot. Plants were watered every two-three days, but no fertilization treatment was made. During the first two months, mechanical weed control was implemented, but a great and rapid proliferation of weeds made the use of herbicides necessary. Sixty four days after transplantation to the field, systemic and selective post-emergent herbicide (CALLISTO® 480 SC) was applied for the control of weeds, primarily hardwoods. 2.2. Growth measurements During the acclimatization period, height (measured up to the highest leaf) and number of leaves was measured regularly every two weeks. After transplantation to 300-cm3 cell trays, we measured height and number of stems 4 times before transplantation to the field. Once in the field, 50 plants from each treatment were chosen randomly, and the length of the highest stem and the total number of stems were measured after 0, 8, 31, and 79 days. 2.3. Biomass measurements Twenty two plants per treatment were used to measure total, aerial and radicular fresh biomass at the time of destructive analysis. Plant material was maintained in the oven for 10 days at 40 ◦ C. After this period, we measured aerial and radicular dry biomass. We also calculated moisture percentage and shoot:root ratio. 2.4. Chlorophyll content The same plants were used to determinate chlorophyll content. A circle of 50.27 mm2 of fresh leave tissue was sampled, and pigments were extracted with 2 mL of 90% acetone. Absorbance values at 664, 647, 630, and 750 nm were read using a UV–visible spectrophotometer (CARY 50). Chlorophyll concentrations were calculated using the equations of Jeffrey and Humphrey (1975). 2.5. Mycorrhizal colonization A portion of root from 26 plants (13 per treatment) was used to determine the degree of mycorrhizal infection. Root samples were washed well with 10% KOH and 2% HCl and stained with Trypan blue (a modification of Phillips and Hayman (1970) method). AMF colonization was estimated using a modified line intersect method: 20 fragments of about 1 cm of root from each plant were placed on striped slide perpendicularly to the stripes and between 100 and 150 intersections between root and stripes were scored for the presence of any AM structures. These observations were made using light microscopy to rate the degree of root infection by AMF. The percentage of AM infection was calculated from the following equation: Percentage of AMF colonization = (
Root length infected ) × 100 Root length observed
2.6. Statistical analysis Each plantlet was considered as an experimental unit. The variation of height over time was analyzed by repeated measures ANOVA. When it was necessary, Greenhouse–Geisser epsilon values were used to correct for violations of the compound symmetry assumption of repeated measures ANOVA. Because of their distribution, variation in the number of leaves and stems was analyzed by a Generalized Linear Mixed Model, using the lmer function in the
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A 180
B AM
12 11
AM Control
Co ntro l
10
140
9
nº leaves
heig ht (mm)
160
301
120 100
8 7 6 5
80
4
60
3 d0
d8
d 22
d 36
d 48
d8
d 22
d 36
Fig. 1. Effect of mycorrhizal inoculation on the height (A) and number of leaves (B) of plants during the acclimatization period. Mean and standard error values for inoculated plants (AM) and control plants (Control) are shown.
lme4 package of R. We considered treatment and time as fixed factors and the identity of the plant as a random factor with a Poisson distribution of residuals. When analyzing isolated measurements of the number of stems, we used a generalized linear model with Poisson or quasi-Poisson distribution, depending on the overdispersion of residuals (Zuur et al., 2009), with treatment as a factor. The significance of fixed factors was calculated using likelihood ratio tests of nested models (Zuur et al., 2009). Means of final height, total and aerial dry biomass, moisture percentage, shoot-root ratio and chlorophyll contents were compared by T-test. Because of nonhomogeneous variances (AMF colonization percentage), we used Kruskal–Wallis tests to compare means of AMF colonization percentage and Spearman’s correlations to relate AMF colonization percentage with final height, total dry biomass and shoot:root ratio. Experimental data were analyzed with the R and JMP® statistical programs. 3. Results Plantlet survival was 100% for both treatments during the acclimatization period and maintenance in trays. In the field, after herbicide treatment, 5 control plants died, and 2 plants presented white spots on their leaves, whereas no effect was observed in the AM plants. The following results are divided into the acclimatization period, the posterior maintenance in greenhouse and establishment in the field. 3.1. Acclimatization period 3.1.1. Growth There were no initial differences of height between treatments (Fig. 1a). However, there was a significant treatment effect on height variation (F1,162 = 55.65, P < 0.0001 rmANOVA), with higher increments of height for the inoculated plants (treatment × time F1.62/261 = 73.12, P < 0.0001 rmANOVA). Twenty two days after the beginning of the experiment, the height of the AM plants was higher
than the control plants, and this trend was maintained during all periods (Fig. 1a). There was a significant effect of AMF inoculation on the variation of the number of leaves (2 = 6.45, P = 0.01 GLMM likelihood ratio tests), which changed over time (treatment × time 2 = 21.31, P < 0.0001 GLMM likelihood ratio tests). The number of leaves was similar for both treatments between day 0 and day 8, but there was a decrease in the number of leaves on the control plants after day 22 (Fig. 1b). 3.1.2. Morphology Mycorrhizal inoculation positively affected plant total, aerial and root dry weight (Table 1), which increased by 44%, 58% and 22% respectively. Aerial biomass comprised 70% of the total biomass in inoculated A. donax, while it constituted 64% in the control plants. As a result, the shoot:root ratio was significantly greater in the AM plants than in the control plants (Table 1). However, the moisture content of the plants was not affected by inoculation (Table 1). 3.1.3. Chlorophylls content The AM plants had significantly higher chlorophyll a (AM 0.076 g/mm2 ± 0.0025; control 0.049 g/mm2 ± 0.0020; P < 0.0001 T-test) and chlorophyll b (AM 0.018 g/mm2 ± 0.0007; control 0.011 g/mm2 ± 0.0005; P < 0.0001, T-test) contents than those of the control plants. 3.1.4. AMF infection Arbuscular mycorrhizal was found in 100% of the inoculated plants. A total of 46% of the control plants showed AMF infection, most likely from contamination by a few presence of AMF on substrate used or by the proximity of plantlets from both treatments. Because the mean percentage of AMF infection in the control plants was very low (Table 1), we can consider that no effects from mycorrhization occurred in this group of plants. However, the percentage of AMF infection was highly variable among inoculated plants with minimum value of 4.85% and maxim of 77.67%.
Table 1 Effect of mycorrhizal inoculation on the final height, total, aerial and root dry biomass, moisture content, shoot:root ratio and percentage of AMF colonization. AM Final height (cm) Total dry biomass (g) Aerial dry biomass (g) Root dry biomass (g) Moisture percentage Shoot:root ratio AMF colonization percentage
16.89 0.23 0.16 0.07 86.63 2.35 35.91
Control ± ± ± ± ± ± ±
0.67 a 0.01 a 0.01 a 0.00 a 0.11 a 0.71 a 7.43 a
12.94 0.16 0.10 0.05 86.80 1.80 1.32
± ± ± ± ± ± ±
0.52 b 0.01b 0.01 b 0.00 b 0.09 a 0.43 b 0.52 b
Mean and standard error values followed by different lower-case letters within a row are significantly different (P < 0.05, T-test or Kruskal–Wallis) for AMF colonization between AM plants and control plants.
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Fig. 2. Effect of mycorrhizal inoculation on the height (A) and number of stems (B) of the plants after transplantation to 300 cm2 cells. Mean and standard error values for inoculated plants (AM) and control plants (Control) are shown.
In addition to AMF, an obligate biotrophic root-associated fungus Olpidium sp. colonized the AM roots. The Olpidium infection appeared in some of the AM roots (both inoculated and noninoculated) because it was likely present in the environment. The effects of Olpidium on the plants are reported to be relatively neutral (Powell, 1993; Weber and Webster, 2000). There was a significant correlation between AMF colonization percentage and final height ( = 0.46, P = 0.018 Spearman correlation) and shoot:root ratio ( = 0.39, P = 0.046 Spearman correlation) but not between AMF colonization percentage and total dry biomass ( = 0.29, P = 0.142 Spearman correlation).
3.2. Maintenance in greenhouse After transplantation to 300-cm3 cells, the height increment of the control plants was higher than that of the AM plants (treatment × time F2.27/266.6 = 20.37, P < 0.0001 rmANOVA). However, the AM plants presented significantly larger heights over all subsequent time points (F1,162 = 55.65, P < 0.0001 rmANOVA) (Fig. 2a). The difference in the number of leaves between treatments was still significant after the transplantation of the plants, being higher for the AM plants (AM 20.38 leaves ± 0.56; control 17.23 ± 0.61; P = 0.0007 GLM quasi-Poisson). The variation of the number of stems was significantly affected by mycorrhization (2 = 4.71, P = 0.03 GLMM likelihood ratio tests). The number of stems was similar 18 days after transplantation for both treatments (Fig. 2b). Nevertheless, the AM plants presented a greater number of stems in subsequent measurements (Fig. 2b).
3.3. Establishment in the open field Although no differences in the length of the highest stem between the AM and control plants were found at the end of the experiment (AM 32.26 ± 0.84 cm; control 31.36 ± 0.95 cm), there was a significant effect of AM treatment over height variation (F1,93 = 7 .06, P = 0.009 rmANOVA); control plants were smaller during the first month (AM 21.3 cm ± 0.60; control 17.99 ± 0.58 cm 31 days post-transplantation). The number of stems was similar 8 days after transplantation for both treatments (AM 6.31 stems ± 0.21; control 5.88 stems ± 0.30). Nevertheless, the AM plants presented a higher number of stems 79 days post-transplantation (AM 20.4 stems ± 0.70; control 17.96 stems ± 0.79). As a result the increment of the number of stems was significantly affected by mycorrhization (2 = 21.77, P < 0.0001 GLMM likelihood ratio tests).
4. Discussion Our results provide evidence of the benefits of early inoculation with mycorrhizal fungi during the acclimatization period on the development and growth of micropropagated A. donax plantlets. Height, number of leaves, number of stems and weight of the plantlets has been improved by mycorrhizal inoculation, which is most likely due to higher photosynthetic rates (EstradaLuna et al., 2000; Singh et al., 2012) as a result of greater uptake and mobilization of nutrients and water (Panwar, 1991). Because micropropagated plants live by means of the absorption of carbohydrates from the culture medium during the culture phase, they must change from heterotrophic to autotrophic conditions during the acclimatization period. By enhancing CO2 fixation capacity through mycorrhizal inoculation, we can facilitate plant conversion to an autotrophic metabolism and therefore enhance plantlet success during the acclimatization phase (Alarcón and Ferrera-Cerrato, 2000). The possibility of higher photosynthetic rates in AM plants is reinforced by the fact that mycorrhizal inoculation also increases the chlorophyll content of A. donax plantlets, as has been observed in other studies with diverse species (Sharma et al., 2008; Yadav et al., 2013; Yang et al., 2014). The increase in the chlorophyll content of leaves could be attributed to enhanced uptake of Mg, Cu and Fe, which are essential for chlorophyll synthesis (Krishna et al., 2005; Sharma et al., 2008). The time required for AMF to colonize and establish symbiosis with a plant varies between 2 and 6 weeks depending on the inoculums (Corkidi et al., 2004). In our case, within 22 days, the AM plants showed greater height and number of leaves; therefore, 3 weeks was necessary for our AMF inoculum to colonize and benefit the A. donax plantlets. Other authors reported higher AMF infection levels than those observed in this study, such as 94% for micropropagated Psidium guajava plantlets (Estrada-Luna et al., 2000). The degree of infection by AMF is dependent on both the elapsed time post-inoculation and the species of fungus used. For instance Singh et al. (2012) found that the rate of infection doubled between 60 and 90 days after infection when infection levels rose above 80%. In our case, infection was evaluated 46 days after inoculation; therefore, we can expect an increase of infection with time, which may be accompanied by an increase of the effect of fungi on plants, as we found that the percentage of AMF infection was positively correlated with heights and shoot:root ratios. Despite the fact that the AMF are not species specific, different species of fungi generate different infection rates and plant responses in the same plant species (Krishna et al., 2005; Singh et al., 2012; Yang et al., 2014). Several commercial mycorrhizal inoculants of the same species could generate significantly different
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infection rates in the same species (Corkidi et al., 2004). Moreover, the percentage of AMF infection was low and highly variable among inoculated plants, which indicated a low homogeneity on commercial inoculum used. Probably will be necessary doses higher than those recommended by the manufacturer to ensure a larger and more homogeneous percentage of infection. Therefore, the selection of the inoculants could be critical for determining the final result. After transplantation to 300 cm3 cells, differences in the height of the AM and control plantlets were reduced because the control plantlets experienced faster growth. However, the final height during the maintenance period of the AM plantlets was still greater than the control plantlets, and it was accompanied by a greater number of stems after land transfer. This result suggests that mycorrhization influences biomass distribution; the AM plants showed slower height growth but an increased number of stems, while the control plants gave priority to increasing their height. In fact, the shoot:root ratio was higher in the AM plantlets, as would be expected in response to greater increments of shoot mass relative to root mass (Azcón-Aguilar and Barea, 1997). This increased investment in the production of new stems could have important implications for the production of biomass in adult plants and would benefit from additional long-term studies. Micropropagated plantlets of A. donax do not require AMF inoculation to reach high survival rates during the acclimatization period, as evidenced by the survival of all control plants in our study. This result is consistent with Cavallaro et al. (2011), who reported survival rates exceeding 95% during the acclimatization period of micropropagated giant reed. However, inoculation with AMF improves the growth and physiological status of the plantlets, ensuring greater field survival. Another advantage is that the acclimatization period can be shortened by inoculating plantlets with AMF, as AM plantlets reach larger sizes and greater status in less time than control plantlets. This advantage has also been reported by Salamanca et al. (1992), who succeeded in shortening the acclimatization process for micropropagated woody legumes from 18 to 10 weeks by introducing mycorrhizas. Moreover, since low input cultivation is adopted for this perennial energy crop, in the following years, this initial inoculum may guarantee a better use efficiency of the low quantities of water and fertilizers supplied. Our results reflect the potential gain in time and cost that is made possible through the use of mycorrhizal technology. The price of commercial quantities of AMF inoculum is low (for instance, the commercial inoculum used in this assay would cost 0.007D per plant). Moreover, the inoculation of plantlets is a singlestep process, only involving the addition of AMF inoculum to the substrate at the time of transplantation of the micropropagated plantlets. Therefore, inoculation with AMF commercial inoculum would have little impact on production costs.
5. Conclusions We conclude that early inoculation of an AMF biofertilizer of micropropagated plants of A. donax can be an effective method to improve the acclimatization process and plantlet quality. Moreover, this study opens new lines of research to investigate the long-term effect of inoculation on A. donax aerial biomass production, the role of AMF on plantlet herbicide resistance and the search for more effective AMF inoculants. Mycorrhizal technology can be a good strategy for improving the plantlet production process of this promising energy crop and merits further exploration.
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Acknowledgments We thank Antonia Romero, Catina Alomar and Miquel Nolla for their help on laboratory work. We thank EMAYA for providing farmland and irrigation facilities. This study was supported by the “European Project Optimization of Perennial Grasses for Biomass Production” (FP7 ; 289642).
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Improving the acclimatization and establishment of Arundo donax L. plantlets, a promising energy crop, using a mycorrhiza-based biofertilizer Maria Tauler, Elena Baraza ∗ University of Balearic Islands edf Guillem Colom Crta. de Valldemossa, km 7.5, Islas Baleares, ES-07122 Palma de Mallorca, Spain
a r t i c l e
i n f o
Article history: Received 20 October 2014 Received in revised form 16 December 2014 Accepted 19 December 2014 Keywords: Giant reed Mycorrhizal inoculation Micropropagated plants Acclimatization Open field transfer
a b s t r a c t Arundo donax has become one of the most promising species for cellulose paste, biomass, and secondgeneration biofuel production. Because this species is not able to produce viable seeds, micropropagation is the best method for large scale production. Several studies have demonstrated that arbuscular mycorrhizal fungi (AMF) can improve the acclimatization of micropropagated plantlets, enhancing their growth and survival. In this study, we evaluated the effect of AMF during the acclimatization period of A. donax plantlets and its posterior establishment to the field. Plantlets were inoculated with mycorrhizabased biofertilizer (AEGIS SYM® ) that contained Rhizophagus intraradices and Funneliformis mosseae. The inoculum was applied at the time of transplantation of plantlets to 28-cm3 cell trays filled with commercial nutrient-rich agricultural substrate. The results showed that during acclimatization, mycorrhized plantlets (AM) present higher number of leaves and height increments as compared to non-mycorrhized plantlets (control). Moreover, at the end of the hardening process, AM plantlets presented higher height, number of leaves, biomass, shoot:root ratio and chlorophyll a and b content than control plantlets. After open field establishment, AM plantlets presented greater size, number of stems and survival rate. These results demonstrate that inoculating micropropagated A. donax with AMF enhances its growth and development as well as produces better quality plantlets, which can be very useful for the large-scale production of this energetic crop. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Giant reed (Arundo donax) is a C3 rhizomatous perennial grass and is considered one of the most promising species for energy, cellulose paste and second generation biofuel production (Shatalov and Pereira, 2002; Lewandowski et al., 2003; Pilu et al., 2013). This fact is due to its high production of biomass for energy, which can be even higher than the yield of other C4 species grasses (Angelini et al., 2009; Mantineo et al., 2009; Kering et al., 2012), its ability to adapt to a wide variety of environments and its low input requirement after establishment (Cosentino et al., 2008; Pilu et al., 2013). Moreover, giant reed can be cultivated in marginal or sub-marginal lands due to its tolerance to environmental stresses, thus reducing
Abbreviations: AMF, arbuscular Mycorrhizal Fungi; AM, arbuscular mycorrhized. ∗ Corresponding author. Tel.: +34 971173177. E-mail addresses: [email protected] (M. Tauler), [email protected] (E. Baraza). http://dx.doi.org/10.1016/j.indcrop.2014.12.039 0926-6690/© 2014 Elsevier B.V. All rights reserved.
competition with food crops that generally require better-quality lands (Sims et al., 2010; Nassi o Di Nasso et al., 2013). Nevertheless, giant reed cannot produce viable seeds because its pollen is unfruitful (Boose and Holt, 1999). Therefore, propagation is only possible by vegetative means, using fragments of stems or rhizomes that present a high capacity to survive and to generate shoots and roots (Mann et al., 2013). Rhizomes are commonly the most used, but this method of propagation is time consuming and involves considerable cost and effort to dig-up, break-apart and replant the rhizomes. In addition, this method of propagation requires large land surface to produce enough plants for use in energy production programs, as each plant produces only a few rhizomes each year (Pilu et al., 2013). Ceotto and Di Candilo (2010) suggested that wherever irrigation water is abundantly available, planting shoot cuttings directly in the field is the most practical option; however, this situation is not common. Another propagation method is in vitro culture, which offers the potential required for the large-scale propagation of giant reed (Cavallaro et al., 2011). In vitro culture methods facilitate obtaining larger amounts of high-quality genetically homogeneous plants in less time with
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the guarantee of being free of pathogens (Cavallaro et al., 2011). However, micropropagation is a complex procedure that requires the development of appropriate techniques to ensure the success of the process to justify the investment. One of the phases of the in vitro plant production that has generated greater mortality is the acclimatization or hardening phase (Kapoor et al., 2008). Directly after transfer from in vitro to ex vitro conditions, micropropagated plants are susceptible to various stresses because of the sudden shock of environmental changes and the incompleteness of certain physiological activities and anatomical developments like photoheterotrophic nature, lack of cuticle or improper vascular connection (Alarcón and Ferrera-Cerrato, 2000). Some authors have proposed the use of Arbuscular Mycorrhizal Fungi (AMF) as a solution to improve the acclimatization process (Vestberg and Estaún, 1994; Estrada-Luna et al., 2000; Kapoor et al., 2008). AMF develop a symbiotic relationship with the plant that enhances the development of the root system; increases nutrient uptake, water conducting capacity and photosynthetic rates; and promotes defense against harmful pathogens and the synthesis of growth hormones (Rai, 2001; Kapoor et al., 2008; Parkash et al., 2011; Singh et al., 2012). The benefits associated with the use of AM inoculation for in vitro-raised plantlets have been reported in several horticultural, fruit, ornamental and forest species (Alarcón and Ferrera-Cerrato, 2000; Rai, 2001; Kapoor et al., 2008). However, little is known about the influence of AM fungi on the survival and growth of micropropagated bioenergy plant species. The present study evaluates the effect of AMF commercial inoculum on the survival, growth and chlorophyll content of micropropagated plantlets of A. donax during the acclimatization period and the survival and growth during land transfer. The objective is to determine the convenience of the use of mycorrhizal biotechnology to improve the plantlet production process of this promising energy crop.
2. Material and methods 2.1. Experimental design This study was conducted between October 2013 and February 2014 in the experimental greenhouse of the Universitat de les Illes Balears, Majorca, Spain. The last phase of the study was conducted between March and May in marginal land, unused for the last 5 years, in the agricultural zone of Prat de Sant Jordi (Palma) located 14 km from Universitat de les Illes Balears. The plantlets that were used were Arundo-K12 clones provided by BIOTHEK ECOLOGIC FUEL S.L. Micropropagated plants were received bare-root, and 164 plantlets were immediately planted in a 28-cm3 cell planting tray and filled with agricultural substrate (KEKKILÄ DSM 1 W© ), which consisted of nutrient-rich black peat. At the moment of transplantation, 5 cm3 of AMF inoculum was added to 83 plantlets. The AMF commercial inoculum (AEGIS SYM© ) used contained 25 spores/g of Rhizophagus intraradices (before known as Glomus intraradices) and 25 spores/g of Funneliformis mosseae (before known as Glomus mosseae). To imitate the soil texture of inoculated cells, we added the same quantity of sterile zeolite to the others plantlets, which were used as a control. Plantlets were randomly distributed in the planting tray. Plantlets were maintained at a mean temperature of 18 ◦ C and 86% moisture in a greenhouse during the acclimatization period. The plantlets were watered according to their needs to always keep the substrate moist. In addition, we moved the tray periodically to avoid a position effect. After 46 days, 22 plants per treatment were used to measure fresh and dry biomass, chlorophyll content and mycorrhizal colo-
nization. The remaining 120 plants were transplanted to 300-cm3 cell planting trays. When climatic conditions were favorable (end of March), plantlets were transferred to open field. In the field, plants from the two treatments were randomly distributed, occupying the entire plot. Plants were watered every two-three days, but no fertilization treatment was made. During the first two months, mechanical weed control was implemented, but a great and rapid proliferation of weeds made the use of herbicides necessary. Sixty four days after transplantation to the field, systemic and selective post-emergent herbicide (CALLISTO® 480 SC) was applied for the control of weeds, primarily hardwoods. 2.2. Growth measurements During the acclimatization period, height (measured up to the highest leaf) and number of leaves was measured regularly every two weeks. After transplantation to 300-cm3 cell trays, we measured height and number of stems 4 times before transplantation to the field. Once in the field, 50 plants from each treatment were chosen randomly, and the length of the highest stem and the total number of stems were measured after 0, 8, 31, and 79 days. 2.3. Biomass measurements Twenty two plants per treatment were used to measure total, aerial and radicular fresh biomass at the time of destructive analysis. Plant material was maintained in the oven for 10 days at 40 ◦ C. After this period, we measured aerial and radicular dry biomass. We also calculated moisture percentage and shoot:root ratio. 2.4. Chlorophyll content The same plants were used to determinate chlorophyll content. A circle of 50.27 mm2 of fresh leave tissue was sampled, and pigments were extracted with 2 mL of 90% acetone. Absorbance values at 664, 647, 630, and 750 nm were read using a UV–visible spectrophotometer (CARY 50). Chlorophyll concentrations were calculated using the equations of Jeffrey and Humphrey (1975). 2.5. Mycorrhizal colonization A portion of root from 26 plants (13 per treatment) was used to determine the degree of mycorrhizal infection. Root samples were washed well with 10% KOH and 2% HCl and stained with Trypan blue (a modification of Phillips and Hayman (1970) method). AMF colonization was estimated using a modified line intersect method: 20 fragments of about 1 cm of root from each plant were placed on striped slide perpendicularly to the stripes and between 100 and 150 intersections between root and stripes were scored for the presence of any AM structures. These observations were made using light microscopy to rate the degree of root infection by AMF. The percentage of AM infection was calculated from the following equation: Percentage of AMF colonization = (
Root length infected ) × 100 Root length observed
2.6. Statistical analysis Each plantlet was considered as an experimental unit. The variation of height over time was analyzed by repeated measures ANOVA. When it was necessary, Greenhouse–Geisser epsilon values were used to correct for violations of the compound symmetry assumption of repeated measures ANOVA. Because of their distribution, variation in the number of leaves and stems was analyzed by a Generalized Linear Mixed Model, using the lmer function in the
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A 180
B AM
12 11
AM Control
Co ntro l
10
140
9
nº leaves
heig ht (mm)
160
301
120 100
8 7 6 5
80
4
60
3 d0
d8
d 22
d 36
d 48
d8
d 22
d 36
Fig. 1. Effect of mycorrhizal inoculation on the height (A) and number of leaves (B) of plants during the acclimatization period. Mean and standard error values for inoculated plants (AM) and control plants (Control) are shown.
lme4 package of R. We considered treatment and time as fixed factors and the identity of the plant as a random factor with a Poisson distribution of residuals. When analyzing isolated measurements of the number of stems, we used a generalized linear model with Poisson or quasi-Poisson distribution, depending on the overdispersion of residuals (Zuur et al., 2009), with treatment as a factor. The significance of fixed factors was calculated using likelihood ratio tests of nested models (Zuur et al., 2009). Means of final height, total and aerial dry biomass, moisture percentage, shoot-root ratio and chlorophyll contents were compared by T-test. Because of nonhomogeneous variances (AMF colonization percentage), we used Kruskal–Wallis tests to compare means of AMF colonization percentage and Spearman’s correlations to relate AMF colonization percentage with final height, total dry biomass and shoot:root ratio. Experimental data were analyzed with the R and JMP® statistical programs. 3. Results Plantlet survival was 100% for both treatments during the acclimatization period and maintenance in trays. In the field, after herbicide treatment, 5 control plants died, and 2 plants presented white spots on their leaves, whereas no effect was observed in the AM plants. The following results are divided into the acclimatization period, the posterior maintenance in greenhouse and establishment in the field. 3.1. Acclimatization period 3.1.1. Growth There were no initial differences of height between treatments (Fig. 1a). However, there was a significant treatment effect on height variation (F1,162 = 55.65, P < 0.0001 rmANOVA), with higher increments of height for the inoculated plants (treatment × time F1.62/261 = 73.12, P < 0.0001 rmANOVA). Twenty two days after the beginning of the experiment, the height of the AM plants was higher
than the control plants, and this trend was maintained during all periods (Fig. 1a). There was a significant effect of AMF inoculation on the variation of the number of leaves (2 = 6.45, P = 0.01 GLMM likelihood ratio tests), which changed over time (treatment × time 2 = 21.31, P < 0.0001 GLMM likelihood ratio tests). The number of leaves was similar for both treatments between day 0 and day 8, but there was a decrease in the number of leaves on the control plants after day 22 (Fig. 1b). 3.1.2. Morphology Mycorrhizal inoculation positively affected plant total, aerial and root dry weight (Table 1), which increased by 44%, 58% and 22% respectively. Aerial biomass comprised 70% of the total biomass in inoculated A. donax, while it constituted 64% in the control plants. As a result, the shoot:root ratio was significantly greater in the AM plants than in the control plants (Table 1). However, the moisture content of the plants was not affected by inoculation (Table 1). 3.1.3. Chlorophylls content The AM plants had significantly higher chlorophyll a (AM 0.076 g/mm2 ± 0.0025; control 0.049 g/mm2 ± 0.0020; P < 0.0001 T-test) and chlorophyll b (AM 0.018 g/mm2 ± 0.0007; control 0.011 g/mm2 ± 0.0005; P < 0.0001, T-test) contents than those of the control plants. 3.1.4. AMF infection Arbuscular mycorrhizal was found in 100% of the inoculated plants. A total of 46% of the control plants showed AMF infection, most likely from contamination by a few presence of AMF on substrate used or by the proximity of plantlets from both treatments. Because the mean percentage of AMF infection in the control plants was very low (Table 1), we can consider that no effects from mycorrhization occurred in this group of plants. However, the percentage of AMF infection was highly variable among inoculated plants with minimum value of 4.85% and maxim of 77.67%.
Table 1 Effect of mycorrhizal inoculation on the final height, total, aerial and root dry biomass, moisture content, shoot:root ratio and percentage of AMF colonization. AM Final height (cm) Total dry biomass (g) Aerial dry biomass (g) Root dry biomass (g) Moisture percentage Shoot:root ratio AMF colonization percentage
16.89 0.23 0.16 0.07 86.63 2.35 35.91
Control ± ± ± ± ± ± ±
0.67 a 0.01 a 0.01 a 0.00 a 0.11 a 0.71 a 7.43 a
12.94 0.16 0.10 0.05 86.80 1.80 1.32
± ± ± ± ± ± ±
0.52 b 0.01b 0.01 b 0.00 b 0.09 a 0.43 b 0.52 b
Mean and standard error values followed by different lower-case letters within a row are significantly different (P < 0.05, T-test or Kruskal–Wallis) for AMF colonization between AM plants and control plants.
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Fig. 2. Effect of mycorrhizal inoculation on the height (A) and number of stems (B) of the plants after transplantation to 300 cm2 cells. Mean and standard error values for inoculated plants (AM) and control plants (Control) are shown.
In addition to AMF, an obligate biotrophic root-associated fungus Olpidium sp. colonized the AM roots. The Olpidium infection appeared in some of the AM roots (both inoculated and noninoculated) because it was likely present in the environment. The effects of Olpidium on the plants are reported to be relatively neutral (Powell, 1993; Weber and Webster, 2000). There was a significant correlation between AMF colonization percentage and final height ( = 0.46, P = 0.018 Spearman correlation) and shoot:root ratio ( = 0.39, P = 0.046 Spearman correlation) but not between AMF colonization percentage and total dry biomass ( = 0.29, P = 0.142 Spearman correlation).
3.2. Maintenance in greenhouse After transplantation to 300-cm3 cells, the height increment of the control plants was higher than that of the AM plants (treatment × time F2.27/266.6 = 20.37, P < 0.0001 rmANOVA). However, the AM plants presented significantly larger heights over all subsequent time points (F1,162 = 55.65, P < 0.0001 rmANOVA) (Fig. 2a). The difference in the number of leaves between treatments was still significant after the transplantation of the plants, being higher for the AM plants (AM 20.38 leaves ± 0.56; control 17.23 ± 0.61; P = 0.0007 GLM quasi-Poisson). The variation of the number of stems was significantly affected by mycorrhization (2 = 4.71, P = 0.03 GLMM likelihood ratio tests). The number of stems was similar 18 days after transplantation for both treatments (Fig. 2b). Nevertheless, the AM plants presented a greater number of stems in subsequent measurements (Fig. 2b).
3.3. Establishment in the open field Although no differences in the length of the highest stem between the AM and control plants were found at the end of the experiment (AM 32.26 ± 0.84 cm; control 31.36 ± 0.95 cm), there was a significant effect of AM treatment over height variation (F1,93 = 7 .06, P = 0.009 rmANOVA); control plants were smaller during the first month (AM 21.3 cm ± 0.60; control 17.99 ± 0.58 cm 31 days post-transplantation). The number of stems was similar 8 days after transplantation for both treatments (AM 6.31 stems ± 0.21; control 5.88 stems ± 0.30). Nevertheless, the AM plants presented a higher number of stems 79 days post-transplantation (AM 20.4 stems ± 0.70; control 17.96 stems ± 0.79). As a result the increment of the number of stems was significantly affected by mycorrhization (2 = 21.77, P < 0.0001 GLMM likelihood ratio tests).
4. Discussion Our results provide evidence of the benefits of early inoculation with mycorrhizal fungi during the acclimatization period on the development and growth of micropropagated A. donax plantlets. Height, number of leaves, number of stems and weight of the plantlets has been improved by mycorrhizal inoculation, which is most likely due to higher photosynthetic rates (EstradaLuna et al., 2000; Singh et al., 2012) as a result of greater uptake and mobilization of nutrients and water (Panwar, 1991). Because micropropagated plants live by means of the absorption of carbohydrates from the culture medium during the culture phase, they must change from heterotrophic to autotrophic conditions during the acclimatization period. By enhancing CO2 fixation capacity through mycorrhizal inoculation, we can facilitate plant conversion to an autotrophic metabolism and therefore enhance plantlet success during the acclimatization phase (Alarcón and Ferrera-Cerrato, 2000). The possibility of higher photosynthetic rates in AM plants is reinforced by the fact that mycorrhizal inoculation also increases the chlorophyll content of A. donax plantlets, as has been observed in other studies with diverse species (Sharma et al., 2008; Yadav et al., 2013; Yang et al., 2014). The increase in the chlorophyll content of leaves could be attributed to enhanced uptake of Mg, Cu and Fe, which are essential for chlorophyll synthesis (Krishna et al., 2005; Sharma et al., 2008). The time required for AMF to colonize and establish symbiosis with a plant varies between 2 and 6 weeks depending on the inoculums (Corkidi et al., 2004). In our case, within 22 days, the AM plants showed greater height and number of leaves; therefore, 3 weeks was necessary for our AMF inoculum to colonize and benefit the A. donax plantlets. Other authors reported higher AMF infection levels than those observed in this study, such as 94% for micropropagated Psidium guajava plantlets (Estrada-Luna et al., 2000). The degree of infection by AMF is dependent on both the elapsed time post-inoculation and the species of fungus used. For instance Singh et al. (2012) found that the rate of infection doubled between 60 and 90 days after infection when infection levels rose above 80%. In our case, infection was evaluated 46 days after inoculation; therefore, we can expect an increase of infection with time, which may be accompanied by an increase of the effect of fungi on plants, as we found that the percentage of AMF infection was positively correlated with heights and shoot:root ratios. Despite the fact that the AMF are not species specific, different species of fungi generate different infection rates and plant responses in the same plant species (Krishna et al., 2005; Singh et al., 2012; Yang et al., 2014). Several commercial mycorrhizal inoculants of the same species could generate significantly different
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infection rates in the same species (Corkidi et al., 2004). Moreover, the percentage of AMF infection was low and highly variable among inoculated plants, which indicated a low homogeneity on commercial inoculum used. Probably will be necessary doses higher than those recommended by the manufacturer to ensure a larger and more homogeneous percentage of infection. Therefore, the selection of the inoculants could be critical for determining the final result. After transplantation to 300 cm3 cells, differences in the height of the AM and control plantlets were reduced because the control plantlets experienced faster growth. However, the final height during the maintenance period of the AM plantlets was still greater than the control plantlets, and it was accompanied by a greater number of stems after land transfer. This result suggests that mycorrhization influences biomass distribution; the AM plants showed slower height growth but an increased number of stems, while the control plants gave priority to increasing their height. In fact, the shoot:root ratio was higher in the AM plantlets, as would be expected in response to greater increments of shoot mass relative to root mass (Azcón-Aguilar and Barea, 1997). This increased investment in the production of new stems could have important implications for the production of biomass in adult plants and would benefit from additional long-term studies. Micropropagated plantlets of A. donax do not require AMF inoculation to reach high survival rates during the acclimatization period, as evidenced by the survival of all control plants in our study. This result is consistent with Cavallaro et al. (2011), who reported survival rates exceeding 95% during the acclimatization period of micropropagated giant reed. However, inoculation with AMF improves the growth and physiological status of the plantlets, ensuring greater field survival. Another advantage is that the acclimatization period can be shortened by inoculating plantlets with AMF, as AM plantlets reach larger sizes and greater status in less time than control plantlets. This advantage has also been reported by Salamanca et al. (1992), who succeeded in shortening the acclimatization process for micropropagated woody legumes from 18 to 10 weeks by introducing mycorrhizas. Moreover, since low input cultivation is adopted for this perennial energy crop, in the following years, this initial inoculum may guarantee a better use efficiency of the low quantities of water and fertilizers supplied. Our results reflect the potential gain in time and cost that is made possible through the use of mycorrhizal technology. The price of commercial quantities of AMF inoculum is low (for instance, the commercial inoculum used in this assay would cost 0.007D per plant). Moreover, the inoculation of plantlets is a singlestep process, only involving the addition of AMF inoculum to the substrate at the time of transplantation of the micropropagated plantlets. Therefore, inoculation with AMF commercial inoculum would have little impact on production costs.
5. Conclusions We conclude that early inoculation of an AMF biofertilizer of micropropagated plants of A. donax can be an effective method to improve the acclimatization process and plantlet quality. Moreover, this study opens new lines of research to investigate the long-term effect of inoculation on A. donax aerial biomass production, the role of AMF on plantlet herbicide resistance and the search for more effective AMF inoculants. Mycorrhizal technology can be a good strategy for improving the plantlet production process of this promising energy crop and merits further exploration.
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Acknowledgments We thank Antonia Romero, Catina Alomar and Miquel Nolla for their help on laboratory work. We thank EMAYA for providing farmland and irrigation facilities. This study was supported by the “European Project Optimization of Perennial Grasses for Biomass Production” (FP7 ; 289642).
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