Initial development and chemical components of sugarcane under water stress associated with arbuscular mycorrhizal fungi

Desenvolvimento inicial e componentes químicos da cana-de-açúcar sob estresse hídrico associado a fungos micorrízicos arbusculares R E S U M O Objetivou-se, neste estudo, avaliar os efeitos de níveis de estresse hídrico no solo e um mix (ou: um inóculo misto de quatro espécies: Claroideoglomus etunicatum, Gigasporas rosea, Acaulospora longula, Fuscutata heterogama) dos fungos micorrízicos arbusculares (FMA) no desenvolvimento vegetativo inicial, produção de biomassa fresca e seca, colonização micorrízica, teores de fósforo, proteínas, enzimas e aminoácido da cana-de-açúcar variedade RB 857515, em condições de casa de vegetação. O delineamento estatístico adotado foi blocos ao acaso em esquema fatorial 2 x 2 com 4 tratamentos (T150% CP – capacidade do pote com FMA; T2 -100% CP – com FMA; T3 50% CP sem FMA; T4 100% CP sem FMA) com 16 repetições. O nível de estresse hídrico de 50% CP diminuiu o diâmetro do colmo, a biomassa fresca da parte aérea e da raiz da cana-de-açúcar, além dos FMA no solo e raiz da planta; entretanto, os FMA e o nível de estresse hídrico de 50% CP, isoladamente ou em conjunto, não afetaram a altura da planta, número de folhas, matéria seca e teores de fósforo, proteínas solúveis totais, catalase, ascarbato peroxidase, polifenoloxidase, peroxidase nem a prolina da cana-de-açúcar variedade RB857515.


Introduction
The use of arbuscular mycorrhizal fungi (AMF) has major importance for the growth of various plant species, due to the great potential for a more efficient crop development and to the reduction in the use of fertilizers, especially in the beginning of plant development (Russomanno et al., 2008;Subramanian et al., 2011).AMF increase the absorption of soil nutrients, especially phosphorus, and plant resistance to biotic and abiotic stresses (Russomanno et al., 2008;Smith & Read, 2008;Kiriachek et al., 2009;Silva & Amorim, 2009;Ngwene et al., 2010;Subramanian et al., 2011).According to Abdel-Fattah & Asrar (2012), in addition to the nutritional effects, the association provides plants with tolerance to water stress, contributing to aggregate stability, increase in soil aeration and subsequent water availability (Borie et al., 2008), since plants with mycorrhizae maintain higher stomatal conductance during water deficit periods and, in return, higher diffusive dependence (Zhang et al., 2011).
Studies on the interaction between AMF and sugarcane, however, are scarce and, although the sugarcane response to AMF in field conditions is not expressive, there is a great potential to use this technology in the production of pathogenfree seedlings, with increases in plant survival indices and higher initial vigor, making them more adapted to the transplanting in the field (Silva et al., 2009).
On the other hand, it is important to develop studies on water use efficiency with the arbuscular mycorrhizal symbiosis aiming to increase the production with minimum use of the available water resources (Asrar et al., 2012).Given the above, this study aimed to evaluate the effect of water stress levels in the soil and inoculation with AMF on the initial vegetative development, fresh and dry biomass production, mycorrhizal colonization and contents of phosphorus, proteins, enzymes and amino acid of sugarcane.

Material and Methods
The experiment was carried out in a greenhouse (8° 01' 05" S; 34° 56' 48" W; 4 m) of the Federal Rural University of Pernambuco (UFRPE) in Recife-PE, Brazil, with minimum and maximum temperatures of 23.8 and 36.7 ºC, respectively, and average relative air humidity of 77.38%.
The sugarcane variety used in the experiment was RB 857515.The micropropagated seedlings were provided by the Biofactory of the Northeast Center for Strategic Technologies (CETENE).The seedlings remained 20 days in acclimation in pots with humus, in a greenhouse, and then were transplanted to pots with autoclaved soil.
The pot capacity (PC) was considered as being the content of water retained by the soil after saturation and the action of gravity, until drainage stopped (Souza et al., 2000).The irrigation of the pots was controlled by daily weighing, using a precision scale with sensitivity of 1 g, between 7 and 9 a.m., for later replenishment of the water evapotranspired in the period, maintaining the soil in the pots close to the field capacity, which began 50 days after transplanting the seedlings into the pots.
The The AMF were separately multiplied, for three months, in pots planted with millet (Panicum miliaceum L.), maize (Zea mays L.), sunflower (Helianthus annus L.) and sorghum (Sorghum bicolor L. Moench).The pots contained 3 kg of soil sterilized in an autoclave at 120 ºC, in two periods of 1 h and 30 min, on consecutive days.The AMF (200 spores plant -1 ) were deposited around the roots of the sugarcane seedlings, 20 days after transplantation into a greenhouse.The harvest was performed 90 days after AMF inoculation.
The evaluated variables were based on the morphological growth (stem length, stem diameter and number of leaves), on shoot and root fresh matter, shoot dry matter, number of glomerospores in the soil, mycorrhizal colonization rate, shoot phosphorus content, total soluble proteins, the enzymes catalase, ascorbate peroxidase, polyphenoloxidase and peroxidase, and on proline.
The morphological growth was evaluated by measurements using a tape measure and a caliper rule, on the 90 th day after mycorrhizal inoculation.For dry matter determinations, the collected samples were stored in previously identified paper bags, and then weighed and dried in a forced-air oven at 65 ºC, until constant mass.After determining shoot dry mass, the samples were ground and placed in polyethylene bags, while aliquots were used for phosphorus (P) analysis using the method of Miyazawa et al. (1984), at the Plant Chemistry Laboratory of the UFRPE.
The biochemical analysis was performed using the third leaf of each plant, which, after harvested, was submersed in liquid nitrogen and stored in a freezer.Sample extract was prepared for the analysis through the homogenization of 0.1 g of fresh matter in 4 mL of the buffer sodium phosphate 0.1 M (pH 6.5), mixed with 0.05 g of polyvinylpyrrolidone (PVP).The homogenate was centrifuged at 10.000 × g at 4 ºC for 10 min (Zeraik et al., 2008), and then analysed for total soluble proteins (Bradford, 1976) and the enzymes catalase (Berrs & Sizer 1952), ascorbate peroxidase (Nakano & Asada, 1981), polyphenoloxidase (Kar & Mishra, 1976) and peroxidase (Fatibelho Filho & Vieira, 2002).For the analysis of the amino acid proline, an extract obtained from 1 g of dry matter in 5 mL of sulfosalicylic acid at 3% was used.The homogenate was centrifuged at 2.000 x g for 10 min, filtered using No. 2 filter paper and determined according to Bates et al. (1973).
The data were subjected to analysis of variance and, when significant, subjected to Tukey test (p ≤ 0.05) using the statistical software SAS.

Results and Discussion
There was no interaction between AMF and water stress, and no effect of AMF on stem length, stem diameter, number of leaves, shoot and root fresh matter, shoot dry matter and P content in the plant (Tables 1 and 2).Similar results were obtained by Russomanno et al. (2008), in rosemary (Rosmarinus officinalis L.) and basil plants (Ocimum basilicum L.), and by Silva et al. (2009) in sweet passion fruit plants (Passiflora alata).
Water stress significantly affected stem diameter and shoot and root fresh matter.On the other hand, there was interaction between water stress and AMF; water stress affected the number of glomerospores in the soil and the AMF root colonization rate of sugarcane seedlings.However, the concentration of assimilable P in the studied soil was equal to 8 mg kg -1 , which is considered as intermediate for sugarcane, since high P availabilities would inhibit AMF root colonization (Zhang et al., 2011), considering that plants would not need to spend photoassimilates with AMF.In this case, the fungus would behave as a parasite and could interfere with plant development.For this reason, most studies on AMF are performed in soils either poor or dependent on phosphorus (Smith & Read, 2008;Kiriacher et al., 2009).Beltrano & Ronco (2008) reported significant increases in maize (Zea mays L.) and wheat (Triticum spp.), respectively under moderate and extreme water stress, inoculated with Glomus claroideum.According to Sánchez-Blanco et al. ( 2004) and Goicoechea et al. (2005), this symbiosis (AMF) has proved to increase the tolerance to water stress, although the mechanisms involved are controversial, because under drought conditions the leaf water potential may or may not be lower in plants with AMF.This decrease, however, is lower in plants with AMF and under stress, possibly due to the fact that in low soil water potentials, plants with AMF can absorb more water compared with AMF-free plants, as mentioned by Porcel & Ruiz-Lozano (2004).
The stress level of 50% PC was enough to change the development and the vegetative growth of sugarcane seedlings, even in the presence of AMF, with decrease of 75, 83.55 and 80.54% in stem diameter, shoot fresh matter and root fresh matter, respectively (Table 3).Russomanno et al. (2008) obtained the opposite result for root fresh biomass, in rosemary plants, with no differences between the AMF-inoculated species (C.etunicatum and G. clarum), while in basil plants, root fresh biomass increased in 93.10% when compared with the control, non-inoculated, which also occurred in other crops associated with AMF (Silva et al., 2009).
The obtained results are contrary to the ones found by Silva et al. (2009), who observed that seedlings of yellow passion fruit did not show differences in stem diameter when inoculated with G. albida and S. heterogama.
The highest results for AMF population occurred when sugarcane was not subjected to water stress, indicating that AMF develop better (both in roots and in soil) when sugarcane is supplied with water, i.e., when the soil is at field capacity (Table 4).
With the water stress, both colonization and sporulation were reduced in 25 and 35%, respectively (Table 4).Inoculation was performed with 200 glomerospores per pot, which corresponds to approximately 6.66 glomerospores per 100 g soil.The percentage of root colonization was low for the plants in the two water systems, which suggests that a few * Significant at 0.05 probability level; ns -not-significant by F test Table 2. Summary of analysis of variance of shoot dry matter (SDM), number of glomerospores (NGS), AMF root colonization rate (RCR) and plant phosphorus content (PP) of RB 857515 sugarcane seedlings subjected to water stress levels and inoculated with AMF, in greenhouse.*Significant at 0.05 probability level; ns -not-significant by F test Table 1.Analysis of variance for stem length (SL), stem diameter (SD), number of leaves (NL), shoot fresh matter (SFM), root fresh matter (RFM) of RB85751 sugarcane seedlings subjected to water stress levels and inoculated with AMF, in greenhouse.
Different letters in the columns have significantly different means by Tukey test at 0.05 probability level; With -50% of the pot capacity; Without -100% of the pot capacity; LSD -Least significant difference Table 3. Mean values of stem diameter (SD), shoot fresh biomass (SFB) and root fresh biomass (RFB) of sugarcane variety RB857515 under water stress (50% PC) 90 days after inoculation in greenhouse glomerospores managed to germinate, penetrate and colonize the roots.Usually, mycorrhizal efficiency is related to the amount of external mycelium formed in soil, varying with AMF and plant species (Rossumanno et al., 2008).
Other factors that reflect on mycorrhizal efficiency are related to edaphoclimatic conditions and aspects of the fungusplant relationship, i.e., they depend on fungus species, plant species and environmental conditions (Zhang et al., 2011;Asrar et al., 2012).Ngwene et al. (2010), studying cowpea (Vigna unguiculata) inoculated with AMF, found that the colonization was severely reduced when plants received phosphate and nitrogen fertilization.Bonfim et al. (2010) observed higher number of AMF spores in the system with arboreal component (larger root area) and in coffee crop (Coffea arabica L.) in dry period, indicating that the restriction in water availability in this season has induced microorganism association with plants and the manifestation of adaptation mechanisms, like increase in sporulation.However, for both evaluated seasons (dry and rainy), there was no correlation between number of spores and leaf water potential, indicating that, regardless of water regime, AMF act normally in the crop and in the soil.Similar results were observed by Amorim et al. (2004), in Brazilian pepper (Schinus terebinthifolius Raddi) and 'umbu' trees (Spondias tuberosa L.) under water stress conditions.
The summary of analysis of variance of the enzymes present in tissues of sugarcane plants subjected to different levels of water stress and inoculated with AMF is shown in Table 5.
There was no interaction between AMF and water stress, and no isolated effect of AMF and water stress for the enzymes catalase, peroxidase, ascorbate peroxidase, polyphenoloxidase, soluble protein and the amino acid proline.Similar results for total protein activity in wheat leaves, with and without water stress, were observed by Beltrano & Ronco (2008), although plants with AMF had higher protein concentration than plants without AMF under severe stress.

Conclusions
1.The water stress levels of 50 and 100% pot capacity were not enough to interfere with the arbuscular mycorrhizal colonization, and with growth and initial development of the sugarcane variety RB857515.
2. The water stress level of 50% pot capacity (PC) affected the initial development of sugarcane seedlings and the AMF colonization in the soil and in plant roots.The AMF species Claroideoglomus etunicatum, Gigaspora rosea, Acaulospora longula and Fuscutata heterogama, and the water stress level of 50% PC, separately or combined, did not affect the initial development, dry matter production and the contents of P, total soluble proteins, catalase, ascorbate peroxidase, polyphenoloxidase, peroxidase and proline of the sugarcane variety RB857515.

Table 4 .
Mean values of the number of glomerospores of arbuscular mycorrhizal fungi (AMF) in the soil (NGS) and AMF root colonization rate (RCR) in the sugarcane variety RB 857515 under water stress (50% PC) 90 days after inoculation in greenhouse Different letters have significantly different means for each variable by Tukey test at 0.05 probability level, and lowercase letters refer to rows and uppercase letters refer to columns; LSD -least significant difference.PC = pot capacity, according to Souza et al. (2000) *Significant at 0.05 probability level; ns -not-significant by F test Table 5. Analysis of variance for the enzymes catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), polyphenoloxidase (PPO), soluble protein (SP) and the amino acid proline (PRO) in RB 857515 sugarcane plants subjected to water stress levels and inoculated with AMF in greenhouse