Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2017, 65, 9180-9185
Chemical Changes during Maize Tissue Aging and Its Relationship with Mediterranean Corn Borer Resistance Ana López-Malvar,† Bernardo Ordás,‡ Carlos Souto,§ Antonio Encina,∥ Rosa A. Malvar,‡ and Rogelio Santiago*,† †
Universidad de Vigo, Dpto. Biología Vegetal y Ciencias del Suelo, Unidad Asociada BVE1-UVIGO y Misión Biológica de Galicia (CSIC), Campus As Lagoas Marcosende, 36310 Vigo, Spain ‡ CSIC-Misión Biológica de Galicia, Grupo de Genética y Mejora de Maíz, Apartado 28, C.P., 36080 Pontevedra, Spain § E.E. Forestales, Dpto. Ingenieria Recursos Naturales y Medio Ambiente, Pontevedra 36005, Spain ∥ Universidad de León, Dpto. Ingeniería y Ciencias Agrarias, Á rea de Fisiología Vegetal, Campus de Vegazana s/n, 24071 León, Spain S Supporting Information *
ABSTRACT: The Mediterranean corn borer (MCB), Sesamia nonagrioides Lef, is an important pest of maize in temperate areas, causing significant stalk lodging and yield losses. The main goals of this study were to determine possible changes in chemical traits (phenols, flavonoids, anthocyanins, sugars, fibers, and lignin) during plant development after the flowering stage and to assess how those traits may differ in diverse genotypes of maize, such as MCB resistant and susceptible. Higher values for some particular traits in more mature tissues seemed to increase their effectiveness against the MCB attack. A decreased amount of borer damage in the field was recorded in the resistant inbred line and in older tissues (7.90 cm vs 31.70 cm as the mean for the stalk tunnel length). In accordance with these results, the resistant inbred line showed a higher degree of hemicellulose crosslinkage (due to ferulic and diferulic acids), higher soluble sugar content, and higher stalk strength. The use of resistant varieties and early sowings is highly recommended as an integrated approach to reduce the yield losses produced by this pest. KEYWORDS: Zea mays, Sesamia nonagrioides, resistance, plant development, cross-linkage, sowing
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INTRODUCTION In terms of production, maize (Zea mays) has the second highest worldwide agricultural yield, just after sugar cane and preceding rice, wheat, and potatoes. Maize plants are consumed by a large variety of insect herbivores1 that have diverse feeding habits and consume all plant parts. The Mediterranean corn borer (MCB), Sesamia nonagrioides Lef, is an important pest of maize in temperate areas.2 On the Iberian Peninsula, infestations commence at an early phenological stage, and after completing their first generation, stem borers of the second generation damage the plants during the reproductive stage.3 Maize yield is reduced by the second-generation larvae because tunneling in the stalks interferes with assimilate movement and increases the level of lodging.3 Several biochemical and physical characteristics (general plant traits, antibiotic compounds, and repellent or attractant metabolites) have been studied as indices of constitutive defense mechanisms against borers.3 Soluble compounds, such as hydroxamic acids, phenols, flavonoids, and sugars, have been consistently correlated with insect resistance. The most remarkable hydroxamic acid in terms of maize resistance to pests is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one.4 In MCB resistance, its effects include increased mortality rates of both larvae and pupae and a lengthening of the pupal stage.5 However, its concentration decreases during plant development, reducing its effectiveness against second-generation borer larvae.4 At the biochemical level, the roles of phenolic acids and flavonoids3,6,7 differ among species, acting in some cases as phagostimulants and in others as toxins. Similarly, the sugar © 2017 American Chemical Society
concentration in plants is correlated with the effects of feeding stimulation or deterrence against borers.8,9 At the physical level, resistance is translated into a reduction in the digestibility, together with an increase in tissue strength, creating an obstacle to the insect’s establishment and development.10 Physical opposition to corn borers can be measured using stem penetrometer resistance. The importance of this trait lies in avoiding or disturbing larval feeding in the stem by increasing the force required to penetrate the stems.11 Several cell wall components related to physical characteristics have been described. Cellulose, hemicelluloses, and lignin are the main fibrous components in cell walls, and the greater the amount of fiber, the tougher the tissue and the less convenient for the insects.3 In addition, cell wall hydroxycinnamates, which cross-link hemicellulose chains, have been consistently correlated with MCB resistance. The most common hydroxycinnamates consist of p-coumaric (p-CA) and ferulic acids (FAs), which, although structurally related, play different functional roles within the walls. While FA is esterified to arabinoxylan, the majority of p-CA is esterified to syringil units of lignin.12,13 Furthermore, FA can be coupled by peroxidaseor laccase-mediated oxidative coupling reactions to form a variety of dimers, which cross-link polysaccharide chains.14 The Received: Revised: Accepted: Published: 9180
June 23, 2017 September 27, 2017 October 2, 2017 October 2, 2017 DOI: 10.1021/acs.jafc.7b02911 J. Agric. Food Chem. 2017, 65, 9180−9185
Article
Journal of Agricultural and Food Chemistry
colorimetric method.20 Briefly, 50 mg of the ground sample was extracted with 1.5 mL of ethanol (80%) at 4 °C over 24 h. Samples were then centrifuged at 4200g for 10 min. Three aliquots of 100 μL were diluted to 1 mL each with distilled water. Next, 0.125 mL of Folin−Ciocalteu reagent (1:1 dilution with water) and 0.5 mL of Na2CO3 (20%) were added to each tube, and the mixture was incubated at 45 °C for 15 min. The sample’s absorbance was measured at 650 nm in a spectrophotometer. Data were expressed in units of milligrams per gram of dry weight (DW) using gallic acid as the standard. Determination of Flavonoid Contents. The total flavonol determination method was the same as the phenolic procedure until the aliquots were collected. In that step, three 100 μL aliquots were diluted to 2 mL with absolute methanol and sequentially mixed with 0.1 mL of AlCl3 (10% water solution), 0.1 mL of 1 M C3H3KO2, and 2.8 mL of distilled water. After a 30 min incubation at room temperature, the absorbance was recorded at 415 nm.21 The flavonol content was expressed in units of milligrams per gram of DW on the basis of a calibration curve using rutin as the standard. Determination of Anthocyanin Contents. Briefly, 50 mg of the sample was extracted with 1.5 mL of extraction solvent (7:2:1 methanol/water/hydrochloric acid) at 4 °C for 24 h.22 Samples were then centrifuged at 4200g for 20 min at 4 °C. The absorbance of the supernatants was measured at 530 nm, and the anthocyanin content was expressed in units of micrograms per milligram of DW on the basis of a calibration curve using cyanidin chloride as the standard. Determination of Soluble Sugar Contents. To determine the soluble sugar content, 50 mg of the sample was weighed and subsequently extracted twice with 1 mL of 70% EtOH at room temperature for 24 h in a rotator. The supernatants were mixed and concentrated in a SpeedVac system (Savant Instruments, Holbrook, NY) until they were dry. Samples were dissolved into 2 mL of distilled water before being partitioned against ethyl acetate (1:1 by volume). The total sugar content was assayed on the aqueous fraction using the phenol−sulfuric acid method and expressed in units of milligrams per gram of DW on the basis of a calibration curve using glucose as the standard.23 Determination of Soluble and Cell Wall Hydroxycinnamate Contents. Here, 500 mg of the sample was extracted in 30 mL of 80% methanol and mixed using a Polytron mixer (Brinkman Instruments, Westbury, NY). Samples were extracted for 1 h and then centrifuged for 10 min at 1000g. After centrifugation, the supernatant was collected and the remaining precipitate was washed with 20 mL of distilled water and centrifuged as described above. The pellets contained the cell wall-bound hydroxycinnamates, and the supernatants contained the soluble hydroxycinnamates. The supernatants containing soluble phenolic acids were combined and concentrated in a SpeedVac to 20 mL. These aqueous solutions were acidified using 6 N HCl to a pH of 2.0 before being extracted with 20 mL of ethyl acetate. The ethyl acetate extract was dried in a SpeedVac at a medium setting without a radiant cover, and the resulting precipitate was dissolved in 1.5 mL of high-performance liquid chromatography (HPLC) grade methanol. This solution was used to determine free phenolic compounds by HPLC. The remaining pellet containing the cell wall-bound material was then shaken in 20 mL of 2 N NaOH under a nitrogen flow for 4 h. Digested samples were neutralized with 6 N HCl, and the pH was decreased to 2.0. After centrifugation, the supernatant was collected and the pellet washed twice with distilled water (10 mL each). Supernatants were pooled and then extracted twice with ethyl acetate (40 mL each). Collected organic fractions were combined and reduced to dryness using a SpeedVac for 6 h at a medium setting without a radiant cover. The final extract was dissolved in 1.5 mL of HPLC grade methanol and stored at −20 °C prior to HPLC analysis. Standards and samples were filtered through a 22 μm tetrafluoroethylene filter (Chromatographic Specialties, Brockville, ON) before being analyzed. Analyses were performed using a 2690 Waters Separations Module (Waters, Milford, MA) equipped with a model 996 photodiode array detector (Waters) with a Waters YMC ODS-AM narrow bore column (100 × 2 mm inside diameter; 3 μm particle size). The solvent system consisted of acetotrinile (solvent A) and trifluoroacetic acid (0.05%) in
greater the level of dimerization in the wall, the more effective it is as a physical barrier against pathogens and pests.15 While diverse mechanisms exist in plants to resist pests, it remains unknown if these defense mechanisms have the same levels of effectiveness during different plant developmental stages, mainly because most of these compounds vary during plant development and/or environmental fluctuations. For example, the cell walls of many tissues undergo dramatic shifts in chemical characteristics during maturation. Initially, plants have small amounts of hemicellulose and pectin present in primary walls and lignin is absent, while mature tissues have greater levels of cross-linkage and lignification when the secondary walls are added.16 Differences in borer damage correlate with differences in the date of sowing in maize, and early sowing has been proposed as a method for controlling corn borer attacks.17,18 Different dates of planting (early and late) result in plants being at different developmental stages during the MCB life cycle. An early sowing results in plants being at advanced developmental stages when the second generation of MCB larvae attack. Our working hypothesis is that less plant development results in less mature and more tender tissues on which the larvae can feed. In a more mature state, the plants are more capable of minimizing the damage. Thus, the main objectives of this study were (1) to determine changes in diverse resistant traits during the two stages of plant development after silking (corresponding to young and more mature tissues) and (2) to assess the occurrences and degrees of trait changes in MCB resistant and susceptible inbred lines of maize.
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MATERIALS AND METHODS
Plant Materials and Experimental Design. Two inbred lines of maize, EP39 and EP42, resistant and susceptible to MCB, respectively,19 were evaluated at two planting dates, early and late. These genotypes were evaluated in 2010 and 2011 using a split plot design with two replications. The dates of planting were assigned as the main plots, and the genotypes were assigned as the subplots. All of the experiments were conducted at Pontevedra in northwestern Spain (42°25′ N, 8°38′ W and 20 m above sea level). Each plot had two rows spaced 0.80 m apart, and each row consisted of 21 two-kernel hills spaced 0.21 m apart. After the plots had been thinned to one plant per hill, the plant density was ∼60000 plants ha−1. The soil type is acid sandy loam. Trials were irrigated once, and cultural operations, fertilization, and weed control were performed according to local practices. Infestation with MCB. MCB eggs were placed between the main ear and the stem when the late planting maize were about to reach the protruding female inflorescence state.19 The early planting maize were in the milky/lacteous reproductive state. The difference in ages between late and early planting maize was 2 weeks. Twenty-one days after infestation, 10 plants were collected from each plot. The stalks were split lengthwise, and the lengths of the galleries produced by the larvae were measured. In addition, the numbers of larvae were recorded. The measurements were repeated 42 and 72 days after MCB infestation. Sample Collection and Biochemical Determinations. Samples for analytical determinations were collected when the infestationrelated parameters were recorded. The second internodes under the main ears were collected from five plants in each plot. To evaluate the main stem tissues subjected to larval feeding, the pith tissues were assessed. For each harvested internode, the pith was manually detached and frozen at −20 °C. Then, samples were lyophilized and ground in a Wiley (Arthur H. Thomas, Philadelphia, PA) mill with a 0.75 mm screen before being analyzed. Determination of Soluble Phenolic Contents. Total phenolic quantification was performed using a modified Folin−Ciocalteu 9181
DOI: 10.1021/acs.jafc.7b02911 J. Agric. Food Chem. 2017, 65, 9180−9185
Article
Journal of Agricultural and Food Chemistry
Table 1. Mean Values for the Biochemical and Physical Traits Evaluated in Two Inbred Lines (EP39 and EP42) for Two Tissues of Different Ages (young and old)a genotype traitb
EP39
phenolics (mg/g of DW) flavonoids (mg/g of DW) anthocyanins (μg/mg of DW) sugars (mg/g of DW)
38.0 a 9.4 a 9.5 a 252.4 a
soluble p-CA soluble FA cell wall p-CA cell wall FA cell wall DFAs
26.0 a 10.0 a 6334 a 4786 a 51 a
ADF NDF ADL hemicellulose
14.8 a 29.3 b 2.7 a 14.5 b
pith puncture resistance rind puncture resistance
1.16 a 2.49 a
tissue aging
EP42
LSD
Soluble Compounds 40.0 a 5.0 20.1 a 11.3 12.8 a 4.8 187.4 b 32.9 Hydroxycinnamates (μg/g of DW) 46.0 b 16.0 1.0 b 7.0 5932 a 107 3700 b 306 324 b 67 Fibers and Lignin (%)c 22.7 a 13.5 46.3 a 9.6 5.9 a 4.2 23.5 a 3.8 Stalk Toughness (load−kg/section) 0.62 b 0.06 2.10 b 0.17
young
old
LSD
37.0 a 12.3 a 11.7 a 203.2 b
38.0 a 15.7 a 10.6 a 236.6 a
5.0 11.2 4.8 32.9
39.3 a 2.0 b 6558 a 4182 a 453 a
31.9 a 9.0 a 5709 a 4305 a 382 b
16.0 7.0 1073 307 67
− − − −
− − − −
− − − −
0.94 a 2.22 a
0.82 b 2.33 a
0.06 0.17
a Means within a column followed by the same letter are not significantly different (P ≤ 0.05). bLegend: p-CA, p-coumaric acid; FA, trans-ferulic acid; DFAs, sum of all the different DFA regioisomers quantified (5−5-DFA, 8−O−4-DFA, 8−5-cyclic-DFA, and 8−5-noncyclic); NDF, neutral detergent fiber; ADF, acid detergent fiber; ADL, acid detergent lignin. cAnalyses between genotypes for fibers and lignin correspond to young tissue samples (see the Supporting Information for details).
water (solvent B) as follows: initial conditions of 10:90 A:B, changing to 30:70 over 3.5 min, 32:68 over 6.5 min, 100:0 over 4 min, isocratic elution at 100:0 for 4.5 min, and finally returning to the initial conditions (10:90) over 3 min. The mobile phase flow rate was 0.3 mL/min, and the total analysis time was 17.5 min. The sample injection volume was 4 μL. Standards of the most common phenolics (ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, and vanillic acid) were purchased from Sigma (St. Louis, MO). Identities of ferulic acid dimers were confirmed by comparison with the authentic 5−5 standard or retention time and UV spectra previously published.24 The DFAT concentration was calculated as the sum of three regioisomers of diferulates identified and quantified by this analytical procedure: 8− O−4-DFA, 5−5-DFA, and 8−5-DFA. The 8−5-DFA concentration was calculated as the sum of 8−5-cyclic (or benzofuran)-DFA and 8− 5-noncyclic (or open)-DFA. Determinations of Fiber and Lignin Contents. Fiber is composed largely of cellulose, hemicellulose, and lignin, which are the primary components of plant cell walls. Neutral detergent fiber (NDF) is composed of cellulose, hemicellulose, and lignin. Acid detergent fiber (ADF) is composed of mostly cellulose and lignin, while acid detergent lignin (ADL) is composed primarily of lignin.16 Hemicellulose was estimated as NDF minus ADF. Determinations of NDF, ADF, and ADL were performed using AOAC Official Method 973.18.25 Determinations of Stalk Toughness. Rind and pith puncture resistance (RPR and PPR, respectively; load−kg/section) are the measured maximum forces required to puncture the rind and pith, respectively, on one side of the stalk. The pith was obtained by the manual elimination of the rind. The second internode under the main ear was punctured at the time of collection using an Accuforce Cadet Force Gauge (Ametek, Mansfield and Green Division, Largo, FL).26 The needle was inserted perpendicular to the stalk to measure RPR and perpendicular to the pith to measure PPR. Statistical Analyses. A combined analysis of variance following the generalized linear model of SAS designed for fixed models was performed. Every source of variation was considered as a fixed factor except for replications. The comparisons of the average values by genotypes or tissue ages for MCB resistance, and stem biochemical or
physical traits, were performed using the least significant difference tests. All statistical analyses were performed using the SAS software package.27
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RESULTS
Differences in the average values of biochemical and physical traits under study for genotypes and tissues are listed in Table 1. Among soluble compounds, significant differences were found in only the sugar concentration, with it being greater in resistant inbred line EP39 (252.4 mg/g of DW) than in susceptible EP42 (187.4 mg/g of DW). Older tissues showed a concentration of sugars (236.6 mg/g of DW) significantly higher than that of younger tissues (203.2 mg/g of DW). The concentrations of cell wall-bound FA and DFA, as well as soluble FA, were significantly higher in the EP39 inbred line than in the EP42 line. In contrast, the soluble p-CA content was significantly higher in the EP42 inbred line (26 mg/g of DW) than in the EP39 line (46 mg/g of DW). No difference in the level of p-CA bound to the cell wall was detected between the inbred lines. Significant differences between the concentrations of hydroxycinnamates and tissue aging were observed for only free FA, with it being greater in older (9 μg/g of DW) than in younger (2 μg/g of DW) tissues, and for DFA, with it being higher in younger (453 μg/g of DW) than in older (382 μg/g of DW) tissues. Because of the tissue amounts, it was not possible to estimate the percentages of ADF, NDF, and ADL or the concentrations of hemicellulose in older tissues. Therefore, differences between genotypes correspond to young tissues. The percentages of NDF and hemicellulose were significantly higher in EP42 (22.7% NDF and 23.5% hemicellulose) than in EP39 (14.8% NDF and 14.5% hemicellulose). Finally, for stalk toughness, significant differences in the PPR were found, being highest in the EP39 inbred line (1.16 load−kg/section compared with 0.62 load−kg/section in the EP42 inbred 9182
DOI: 10.1021/acs.jafc.7b02911 J. Agric. Food Chem. 2017, 65, 9180−9185
Article
Journal of Agricultural and Food Chemistry
Table 2. Mean Values of Borer Resistance Traits after Infestation of Two Inbred Lines (EP39 and EP42) and at Two Tissue Aging Stages (young and old)a genotype damage trait
time
tunnel length (cm)
no. of larvae per plant
a
21 42 70 21 42 70
EP39
daib dai dai dai dai dai
0.70 11.10 12.90 0.20 0.44 0.34
EP42
a b b a b b
0.80 32.80 41.10 0.21 2.19 2.06
a a a a a a
tissue aging LSD
young
0.90 5.87 7.89 0.32 0.44 0.76
1.20 26.70 34.30 0.29 1.79 1.65
a a a a a a
old 0.40 17.20 19.80 0.13 0.84 0.75
LSD a b b a b b
0.90 5.86 7.88 0.32 0.44 0.76
Means within a column followed by the same letter are not significantly different (LSD, P ≤ 0.05). bdai means days after infestation.
plant.17 We confirmed that younger tissues are more favorable for larval development. The tissue’s maturation interferes negatively with larval progress, while on the other hand, more tender and more succulent tissues are more palatable and easily pierced by the larvae.28 The toughness of the plant tissues increases as they develop, interfering with European corn borer (ECB) feeding.29 Those results corroborate that the developmental stage of the plant plays an important role in the evaluation of borer resistance. Significant interactions emphasized that the beneficial effect of an early sowing time against borer damage increases when combined with the use of a resistant inbred line. At the biochemical level, free soluble phenolics have low molecular weights and are partially soluble in many solvents, which are necessary properties for the transfer of plant toxins through cell membranes. The antibiotic roles of phenolic compounds are variable among borers. Some studies have shown that p-CA and FA have no effects on ECB, while others noted a phagostimulant effect on the spotted stem borer, Chillo partellus (Swinhoe).30−32 In the MCB, four phenols (p-CA, FA, p-hydroxybenzaldehyde, and vanillin) in their free forms and under laboratory conditions did not have negative effects on the growth of MCB larvae.33 However, a possible role as precursors for cell wall-linked phenolics was proposed. In agreement with that, in the study presented here the highest concentrations of soluble FA were observed in resistant inbred line EP39 and older tissues. However, no differences between genotypes or tissue ages were found for total phenolics, flavonoids, or anthocyanins, suggesting that those compounds, or at least their total contents, have no roles in MCB resistance. Likewise, the role that soluble sugars play in plant defenses against borers remains uncertain. In rice, coincident lower borer incidence and larval survival rates, as well as sugar contents, were observed.34 Some maize inbred lines with low soluble sugar contents were moderately susceptible, and those with a higher sugar content were either resistant or moderately resistant.35 Here, the highest concentrations of soluble sugars were found in resistant inbred line EP39 and older tissues. Thus, we hypothesize that soluble sugars act as deterrents, improving plant defenses. However, another plausible hypothesis is that the amount of sugar in the tissue reduces the level of damage because the larvae need to feed on a smaller proportion of tissue to obtain a reasonable amount of nutrients. In this sense, previous studies noted that MCB larvae maintained with the pith of the maize inbred line F473 reached weights that were heavier than those of larvae fed with another inbred line, but the F473 plants showed smaller MCB galleries in the field.36 For cell wall-bound hydroxycinnamates, monomers and dimers play significant roles in MCB resistance, and we have consistently identified higher concentrations of these com-
line) and in younger tissues (0.94 load−kg/section) compared with older tissues (0.82 load−kg/section). Moreover, EP39 had a RPR value (2.29 load−kg/section) higher than that of EP42 (2.10 load−kg/section) (Table 1). The damage produced by MCB larvae accumulated from the infestation date, covering
