Article pubs.acs.org/JAFC
Pod Mildew on Soybeans Can Mitigate the Damage to the Seed Arising from Field Mold at Harvest Time Jiang Liu,*,†,‡,§ Juncai Deng,†,‡ Ke Zhang,‡,∥ Haijun Wu,‡ Caiqiong Yang,†,‡ Xiaowen Zhang,‡ Junbo Du,‡ Kai Shu,‡,§ and Wenyu Yang*,‡ ‡
Key Laboratory of Crop Ecophysiology and Farming System in Southwest, Ministry of Agriculture, Chengdu, Sichuan 611130, People’s Republic of China § Institute of Ecological Agriculture, Sichuan Agricultural University, Chengdu, Sichuan 611130, People’s Republic of China ∥ College of Pharmacy, Central South University, Changsha, Hunan 410013, People’s Republic of China S Supporting Information *
ABSTRACT: Seedpods are the outermost barrier of legume plants encountered by pests and pathogens, but research on this tissue, especially regarding their chemical constituents, is limited. In the present study, a mildew-index-model-based cluster analysis was used to evaluate and identify groups of soybean genotypes with different organ-specific resistance against field mold. The constituents of soybean pods, including proteins, carbohydrates, fatty acids, and isoflavones, were analyzed. Linear regression and correlation analyses were also conducted between these main pod constituents and the organ-specific mildew indexes of seed (MIS) and pod (MIP). With increases in the contents of infection constituents, such as proteins, carbohydrates, and fatty acids, the MIP increased and the MIS decreased. The MIS decreased with increases in the contents of glycitein (GLE)-type isoflavonoids, which act as antibiotic constituents. Although the infection constituents in the soybean pods caused pod mildew, they also helped mitigate the corresponding seed mildew to a certain extent. KEYWORDS: soybean pod, field mold, protein, fatty acid, isoflavone
■
INTRODUCTION Soybean (Glycine max L. Merr.) is an important oilseed and protein crop.1 As a result of their high protein and oil contents, soybean seeds are very susceptible to mildew deterioration. Soybean mildew can be divided into three main categories based on the location of the mildew outbreak: field, storage, and processing mold.2 In southwest China, largely a karst landscape, the rainfall in autumn is approximately one-quarter of the annual precipitation.3 Although the number of rainy days have decreased significantly over almost the entire southwestern region,4 the number of extremely heavy rains have increased in most of the region.5 In these areas, soybean is sown at the beginning of June and harvested at the end of October to be exposed to the autumn rainfall over western China.6 During the wet season, soybeans await harvest in the field, and the prolonged and continuous rainfall causes abnormally cold (13−21 °C) and humid (85− 100% humidity) weather that is detrimental to crop production.2 Soybeans that are fully or nearly mature during this time become heavily infected with fungus in the field. This preharvest deterioration, known as field mold, has appeared in not only southwest China but also other soybean production areas around the world. For example, the first systematic report of field mold was related to the abnormally warm, humid weather that lasted from Sept 18 to Oct 7, 1986 and resulted in a delayed soybean harvest in five U.S. states.7 Our previous studies revealed that the mildew resistance of soybean seeds was correlated with their chemical constituents, including fatty acids, proteins, and polysaccharides.2 On the one hand, the nutrients, especially fatty acids, which consist of five © 2016 American Chemical Society
main compounds, palmitic acid (PA), stearic acid (SA), linoleic acid (LA), oleic acid (OA), and α-linolenic acid (ALA), act as the energy supply for fungal growth.8 On the other hand, the phenolic acids in soybean seeds, especially isoflavones, which consist of 12 isoflavone compounds, including daidzin (DG), glycitin (GLG), genistin (GEG), malonyldaidzin (MD), malonylglycitin (MGL), acetyldaidzin (AD), acetylglycitin (AGL), malonylgenistin (MG), daidzein (DE), acetylgenistin (AG), glycitein (GLE), and genistein (GE), act as antibacterial agents.9 Previous studies focused more attention on the seed coat, which was exclusively considered as the main physical and chemical barrier.10 However, information on the soybean pod and the involvement of its chemical constituents in plant defense remains relatively scarce. As the first seed barrier encountered by pests and pathogens, the seedpod also plays an important role in the resistance physiology of soybeans. The present study tested the hypothesis that the potential resistance functions are closely related to the chemical constituents of the seedpod. The objective of this study was to evaluate the chemical profiles of soybean pods in different genotypes, especially related to proteins, carbohydrates, fatty acids, and isoflavones. The relationships between the chemical constituents and the Received: Revised: Accepted: Published: 9135
August 11, 2016 November 3, 2016 November 14, 2016 November 14, 2016 DOI: 10.1021/acs.jafc.6b03561 J. Agric. Food Chem. 2016, 64, 9135−9142
Article
Journal of Agricultural and Food Chemistry
Figure 1. Two-dimensional coordinates of germplasm clustering. The blue and red markers represent the different soybean germplasms. The red triangles, blue boxes, blue circle, and blue triangles represent samples with high MIS and MIP, high MIS and low MIP, low MIS and MIP, and low MIS and high MIP, respectively. corresponding mold grades, n is the survey numbers, and smas is the top mold grade). These mildew indexes were subjected to cluster analysis, which categorized the germplasms into several groups. Protein and Carbohydrate Measurements. Soybeans were harvested at approximately the R8 stage (full maturity stage) of seed development. The fruits from the middle plant portion of the control samples grown under normal conditions were collected; the pod and seed were peeled manually and frozen in liquid nitrogen immediately; and all of the freeze-dried samples were then stored in airtight tubes at −80 °C until analysis. The total protein content (TPC) of the soybean pods was calculated by converting the nitrogen content, which was quantified by Kjeldahl’s method using an automatic Kjeldahl system (Kjeltec 8400, FOSS, Sweden). The nitrogen percentage was converted to crude protein by multiplying the percentage by 6.25.13 The total carbohydrate content (TCC) of the soybean pods was determined by the phenol−sulfuric acid method. The TCCs were expressed as glucose equivalents.14 Chromatographic Analyses of Isoflavones. The extraction of isoflavones and fatty acids was based on our previously published methods, with certain modifications.15 Isoflavone identification and quantification were performed using an Agilent 1260 series highperformance liquid chromatography (HPLC) system equipped with a mass spectrometric detector (Agilent Quadrupole LC/MS 6120). The main chromatographic conditions were as follows: mobile phase, A (0.1% acetate solution, v/v) and B (acetonitrile); gradient elution, 15− 20% B (0−30 min), 20−40% B (30−60 min), and 40% B (60−70 min); injection volume, 5 μL; chromatographic column, 4.6 × 250 mm, 5 μm, 12 nm (YMC-pack ODS-AQ); and column temperature, 30 °C. Gradient elution was performed at flow rates of 1.0−0.8 mL min−1 (0−30 min), 0.8 mL min−1 (30−60 min), and 0.8−1.0 mL min−1 (60−70 min). Selected ion monitoring (SIM) mode was used in the mass spectral acquisition, and the scan range was 100−700 amu. The mass spectra were measured under the following conditions: electrospray ionization (ESI), positive ion mode; desolvation temperature, 350 °C; desolvation pressure, 35 psig; and capillary voltage, 3.8
mildew indexes of seed (MIS) and pod (MIP) are also discussed.
■
EXPERIMENTAL SECTION
Plant Materials and Experimental Design. A total of 14 soybean genotypes grown in Sichuan, China, numbered A3, 2162, C103, E60, 2080, N256-1, E70, G378-1, GX1, ND12, D49, D15, A13, and D1141 (Table S1 of the Supporting Information) were used in this study, 2 of which were conventional cultivars in southwestern China, Gongxuan 1 (GX1) and Nandou 12 (ND12). All of the above genotypes were grown in pots in the experimental field of the Sichuan Agricultural University (SICAU) in Ya’an, China. Six seeds were sown in the pots and thinned to three plants per pot 2 weeks after seeding, and typical agronomic management practices were used. Half of the potted soybean plants were transferred from the field to a solar greenhouse approximately 5 days before the growth stage R7 (beginning maturity stage), and other plants at the same developmental progression were used as a control under normal conditions (20−30 °C and 60−70% humidity). The plants in the greenhouse were exposed to a day/night temperature of 21/13 °C and 85−100% humidity for 7 days during the remainder of their seed development and maturation period according to the method of Keigley et al., with slight modifications.11 The above experiments represented three biological replicates for all 14 soybean genotypes. Mildew Survey and Germplasm Categorization. After treatment, five randomly selected plants from each of the 14 soybean genotypes were removed from their pots and the mold levels in their seeds and pods were determined according to the method of Li et al., with slight modifications.12 The seed and pod injuries caused by field mold were visually evaluated by estimating the mildew percentage of the seed/pod area, considering the entire plot for grade judgment, when the infestation had reached levels that allowed for germplasm discrimination. These operations were described in our previous study.2 Mildew rate = f/n × 100 (where f is the mold seed/pod numbers and n is the survey numbers). Mildew index = ∑f isi/nsmas × 100 (where f i is the mold seed/pod numbers of various grades, si is the 9136
DOI: 10.1021/acs.jafc.6b03561 J. Agric. Food Chem. 2016, 64, 9135−9142
Article
Journal of Agricultural and Food Chemistry kV. Nitrogen was used in the ion source and as the collision gas, and the desolvation gas flow rate was 10 L min−1. Identification of the isoflavones was achieved by comparing the sample retention times and mass spectra to those of standard compounds. In this study, five main isoflavones (DE, GE, GLE, GLG, and MGL) were quantified in absolute terms via linear regression of their corresponding standards. Chromatographic Analyses of Fatty Acids. Gas chromatography−mass spectrometry (GC−MS) analyses of fatty acids was also performed following previously described methods, with certain modifications,15 using a QP2010 GC−MS system (Shimadzu, Japan) in SIM mode. The main chromatographic conditions on a 30 m × 0.25 mm × 0.25 μm Rtx-5Ms capillary column were as follows. The linear oven temperature program increased from 130 to 170 °C (6.5 °C min−1), followed by a hold for 6 min, increased from 170 to 215 °C (3 °C min−1), followed by a hold for 13 min, and increased to 230 °C (3 °C min−1), followed by a hold for 10 min. The injection port temperature was 270 °C; the helium flow was 1.0 mL min−1; the injection volume was 1.0 μL; the ionization potential was 70 eV; and the scan range was 30−450 amu. The fatty acids were identified by comparing the sample retention times and mass spectra to those of standard compounds in a fatty acid methyl ester (FAME) mixture. All samples were run in triplicate. In this study, five main fatty acids (PA, SA, LA, OA, and ALA) were quantified in absolute terms via linear regression using the corresponding FAME standard. Statistical Analyses. All tests were conducted in nonuplicate, and the results are reported as the mean ± standard deviation. Variance analyses were performed using the general linear model procedure in SPSS (version 20.0, SPSS, Chicago, IL, U.S.A.). Duncan’s multi-range test was used when the samples exhibited significantly different metabolite concentrations at the p < 0.05 level of significance. The results of the different constituents in the soybean pods were subjected to regression and correlation analyses to construct mathematical models using all of the experimental data in Microsoft Excel 2013 and SPSS software.
high content of phenolic constituents, especially anthocyanin in the dark seed coats, and seed hardness may be responsible for the mold resistance.16−18 Pod Protein and Carbohydrate Contents and Their Relationship to the Organ-Specific Mildew Index. In the current experiment, protein and carbohydrates were detected in the pods, and the contents in different soybeans with varying field mildew resistance are shown in Table 1. There were large Table 1. Total Protein and Soluble Polysaccharide Contents (mg g−1) in Soybean Pods with Different Field Mildew Resistance Gradesa resistance grade
code
HR
C103 2162 D49 A3 ND12 GX1 G378-1 N256-1 2080 E60 E70 D15 A13 D1141
R S
HS
TPC 31.839 48.357 32.034 28.630 21.661 24.897 26.367 18.366 24.248 26.308 28.570 16.316 20.805 21.595
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.404 0.451 0.841 1.839 1.739 1.784 0.404 0.832 0.381 0.067 0.402 0.224 1.003 0.277
TCC b a b c ef d cd gh de cd c h fg ef
23.395 25.651 29.998 36.195 25.430 22.338 21.534 25.453 30.037 22.354 35.273 20.489 22.457 21.484
± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.455 1.920 0.712 1.532 6.382 2.173 0.748 1.445 2.182 0.060 1.210 1.705 0.851 2.167
bc bc ab a bc c bc ab abc bc a c bc c
a
All samples were measured in triplicate. Data are expressed as the mean ± standard deviation (n = 9) based on dry weight. Values marked by the same letter within the same line are not significantly different (p > 0.05). HR, high resistance; R, resistance; S, susceptibility; and HS, high susceptibility.
■
RESULTS Mildew Survey and Germplasm Categorization. Zeromean normalization was applied to the original MIS and MIP (Table S1 of the Supporting Information), which conform a mean of 0 and standard deviation of 1. All of the normalized data were projected onto a two-dimensional coordinate space. Figure 1 illustrates that all of the soybean genotypes were categorized into four major types/groups based on their MIS and MIP (Figure 1 and Table S1 of the Supporting Information). Most of the soybean germplasms clustered in the same first quadrant of the coordinate plot (quadrant I of Figure 1). Their MIS and MIP values were both high (e.g., 32.90−54.60 and 57.52−84.57, respectively). Three germplasms with high MIS (e.g., 51.13−58.79) and low MIP (e.g., 31.98−47.10) values, D15, A13, and D1141, were clustered in the second quadrant (quadrant II of Figure 1). A special germplasm D49 was classified in the third quadrant (quadrant III of Figure 1), with low MIS and MIP values (e.g., 12.04 and 5.85, respectively). Other germplasms, including three black soybeans 2162, C103, and A3, were clustered in the fourth quadrant (quadrant IV of Figure 1) with low MIS values (e.g., 1.61−12.27) and high MIP values (e.g., 70.19−72.52). Interestingly, as shown in Figure 1, all samples were discriminated by their different seed coat colors based on the x and y axes. The samples with dark (black and brown) seed coats clustered into the bottom half of the plot and had lower MIS values than the soybeans with light (green and yellow) seed coats in the top half of the plot. This clustering agrees with our previous study, which found that mildew resistance was significantly correlated with the seed coat color and soybean seeds with dark coats showed higher field mold resistance.8 The
variations in the contents of pod protein (e.g., 16.3−48.4 mg g−1) and carbohydrates (e.g., 20.5−35.3 mg g−1) among the different soybean samples. The TPC in the pod of the highly resistant germplasm “2162” was the highest (e.g., 48.357 mg g−1) and was significantly different from the other soybeans. The carbohydrate contents in the pods of susceptible germplasms “E70” and “D15” were the highest (e.g., 35.273 mg g−1) and lowest (e.g., 20.489 mg g−1), respectively, and the pod carbohydrate contents did not significantly differ among the various soybean germplasms. As shown in Table 1 and Table S1 of the Supporting Information, in comparison to the variation of the MIS, the carbohydrate content in the pods did not change significantly. Moreover, the pod protein contents in soybeans with resistant seeds and low MIS values were significantly higher than those in germplasms with susceptible seeds and high MIS values. On the basis of in-depth linear regression analysis, the MIP value increased and the MIS value visibly decreased with the increase in the pod protein content, with determination coefficients of 0.0567 and 0.5518, respectively (Figure 2a). Correlation analysis also showed that the protein content in the pod was most significantly negatively correlated with the MIS value (r = −0.652; Table S2 of the Supporting Information). A similar tendency was detected in the correlation between the pod carbohydrate content and the mildew indexes, although their determination coefficients were low (Figure 2b; 0.0367 and 0.0935, respectively). The MIS and MIP values obviously decreased and increased, respectively, as the TCC increased (Figure 2b). This “scissors difference” phenomenon between 9137
DOI: 10.1021/acs.jafc.6b03561 J. Agric. Food Chem. 2016, 64, 9135−9142
Article
Journal of Agricultural and Food Chemistry
Figure 2. Linear regression analysis between the chemical constituents of soybean pods and their organ-specific mildew indexes: (a) TPC, (b) TCC, (c) TFA, (d) UFAs, (e) total content of isoflavones (TIS), and (f) To-GL (GLE + GLG + MGL).
(panels c and d of Figure 2). Correlation analysis also showed that the TFA and UFAs in the pods were significantly positively correlated with the MIP values (r = 0.554 and 0.519, respectively; Table S2 of the Supporting Information). As the main nutrient contained in soybean seeds, fatty acids play a key role in seed mildew production, providing the energy source for mold development on the soybean pod. Therefore, fatty acids are the most important inducer of field mold outbreaks on soybean seeds.2 The above experiments imply that the fatty acids in soybean pods lead to pod mildew and, to some extent, ease the damage to soybean seeds. Isoflavone Profiles in Soybean Pods in Different Germplasms. In this proposed experiment, 12 typical isoflavones and several isomers were detected (Figure 3a), and the contents of these soybean isoflavones in the pods are shown in Table 3. A total of 46.685−881.892 μg g−1 isoflavones were contained in the soybean pods, which amounts to an approximate 2−20% isoflavone content in the soybean seeds.15 β-Glucosides and malonylglucosides are the main constituents of isoflavones in soybean seeds.15 Differently, aglycones dominated in the soybean pods in the present study, including DE (e.g., 4.753−465.849 μg g−1), GE (e.g., 4.532−411.301 μg g−1), and GLE (e.g., 7.810−291.916 μg g−1). Additionally, GLG (e.g., 7.176−33.238 μg g−1) and MGL (e.g., 11.436−79.961 μg g−1) were also detected as main compounds in the soybean pods (Table 3 and Figure 3). In general, the main isoflavones in the soybean pods can be summarized as two groups: aglycones (DE + GE + GLE) and GL-type isoflavonoids (GLE + GLG +
the MIS and MIP demonstrates direct or indirect links between the pod protein and carbohydrate contents and their organspecific MIS and MIP values. Fatty Acid Profiles in Soybean Pods and Their Relationship to the Organ-Specific Mildew Index. On the basis of the fatty acid analysis of the soybean pods, approximately 2.8−9.5 mg g−1 fatty acids was contained in the soybean pods (Table 2), corresponding to an approximate 4− 10% fatty acid content in the soybean seeds.15 Similar to the fatty acid profile in soybean seeds, the fatty acid constituents in the pod consisted of five major fatty acids, including two saturated fatty acids (SFAs), palmitic acid (PA) (e.g., 1.345− 1.827 mg g−1) and stearic acid (SA) (e.g., 0.973−1.224 mg g−1), and three unsaturated fatty acids (UFAs), linoleic acid (LA) (e.g., 0.000−1.929 mg g−1), oleic acid (OA) (e.g., 0.000− 1.267 mg g−1), and α-linolenic acid (ALA) (e.g., 0.000−0.590 mg g−1). The fatty acid contents in the soybean pods are shown in Table 2. Different from soybean seeds with a high ratio of UFAs, the SFAs were the major fatty acids in the soybean pods (e.g., 2.801−4.197 mg g−1). UFAs were even not detected in the pods of some soybean germplasms. Similar to the pod protein and carbohydrate analyses, the “scissors difference” phenomenon was apparent in the linear regression analysis between the fatty acid content and organspecific MIS and MIP values. With the increase in the total content of fatty acids (TFA) and UFAs in the soybean pods, the MIP values increased sharply with a good fit (R2 = 0.2974 and 0.2578, respectively), and the MIS values visibly decreased 9138
DOI: 10.1021/acs.jafc.6b03561 J. Agric. Food Chem. 2016, 64, 9135−9142
Article
c cd
a
de
de
c
a
f
f
d
a
d
d
HS
S
R
All samples were measured in triplicate. Data are expressed as the mean ± standard deviation (n = 9) based on dry weight. Values marked by the same letter within the same line are not significantly different (p > 0.05).
a
d
d
c c d c
abc a de cd e e bcd bcd bcd de ab e de e 0.027 0.029 0.022 0.003 0.031 0.026 0.045 0.053 0.037 0.016 0.035 0.010 0.006 0.010 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.168 1.224 1.026 1.082 0.979 0.973 1.112 1.100 1.105 1.024 1.184 0.988 1.057 0.975 b a def c f ef c cd c def a ef de def 0.023 0.027 0.027 0.007 0.031 0.033 0.051 0.048 0.031 0.013 0.026 0.013 0.012 0.019 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.641 1.827 1.402 1.546 1.345 1.359 1.548 1.484 1.537 1.403 1.786 1.371 1.440 1.414 C103 2162 D49 A3 ND12 GX1 G378-1 N256-1 2080 E60 E70 D15 A13 D1141 HR
MGL). As shown in Figure 3, susceptible germplasms, such as E60 (Figure 3b) and A13 (Figure 3c), with high MIS values contained high ratios of aglycones and low ratios of GL-type isoflavonoids. Resistant germplasms, such as D49 (Figure 3d) and C103 (Figure 3e), with low MIS values contained high ratios of GL-type isoflavonoids and low ratios of aglycones. Several other isoflavone glucosides, such as DG, GEG, and MD, were also detected in the resistant germplasms. Linear regression analysis between the pod isoflavone content and the organ-specific mildew indexes (Figure 2e) revealed that the MIS and MIP values slightly increased as the pod isoflavone content increased with low determination coefficients (e.g., 0.0941 and 0.0598, respectively). Correlation analysis also showed that the total content of GL-type isoflavonoids (To-GL) was significantly negatively correlated with the MIS value (r = −0.510; Table S2 of the Supporting Information). On the basis of further linear regression analysis between the GL-type isoflavonoid content in the pods and the organ-specific mildew indexes, the MIP value did not obviously change as the pod GL-type isoflavonoid content increased (R2 = 0.0078). Inversely, the MIS value sharply decreased (R2 = 0.2903) as the pod GL-type isoflavonoid content increased (Figure 2f). Numerous studies have revealed that isoflavonoids are associated with a wide range of biological activities, especially antioxidant19 and antifungal20 activities. The biological activities of isoflavonoids are determined by their chemical structures with varied functional groups.21 Our recent study indicated that GL-type isoflavones with methoxylation at the C-6 position of the A ring are enriched in the seeds of resistant soybean germplasms, which have greater potential resistance against field mildew.9 In summary, the above analysis and available literature imply that the GL-type isoflavonoids in soybean pods might play an important role in the field mold tolerance of soybean fruit. Although the isoflavonoids in soybean pods cannot reverse the pod mildew damage caused by high pod contents of nutrients, such as proteins, carbohydrates, and fatty acids, the antimycotic isoflavonoids can mitigate the mildew damage to soybean seeds to some extent.
■
DISCUSSION Seedpods are the outermost barrier of legume plants, but previous studies on this tissue were limited, especially regarding their chemical constituents. In previous studies, more attention was paid to the physical and chemical features of the seed coat. The present study systematically analyzed the chemical constituents in seedpods from different soybean germplasms that had varied organ-specific mold tolerance. Similar to soybean seeds, constituents, such as proteins, carbohydrates, fatty acids, and isoflavonoids, were detected in soybean pods. Differently, the contents of these pod constituents were significantly lower than the corresponding contents in soybean seeds, and the pod and seed compositions were different. These main constituents can be divided into two types: infection constituents, including proteins, carbohydrates, and fatty acids, and antibiotic constituents, such as isoflavonoids, of which five aglycones and GL-type isoflavonoids were detected in the soybean pods. In-depth linear regression and correlation analyses between the contents of the infection constituents and the organ-specific MIS and MIP values presented similar “scissors difference” phenomena. This “scissors difference” indicates a trade-off
a
f b gh c i h d g d gh a h gh e 0.083 0.023 0.045 0.037 0.062 0.058 0.135 0.103 0.063 0.029 0.091 0.022 0.019 0.083 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.006 8.536 3.563 7.187 2.801 3.465 6.700 3.721 6.658 3.557 9.519 3.494 3.631 6.265 2.057 ± 0.037 4.339 ± 0.077 0g 3.422 ± 0.035 0g 0g 2.905 ± 0.039 0g 2.884 ± 0.032 0g 5.413 ± 0.111 0g 0g 3.392 ± 0.054 0.051 0.054 0.045 0.010 0.062 0.058 0.096 0.103 0.070 0.029 0.060 0.022 0.019 0.030 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0f 0.477 ± 0.022 0f 0.423 ± 0.003 0f 0f 0.397 ± 0.004 0f 0.394 ± 0.002 0f 0.590 ± 0.006 0f 0f 0.411 ± 0.004 e b
0.925 ± 0.010 1.215 ± 0.007 0h 1.015 ± 0.010 0h 0h 0.869 ± 0.007 0h 0.871 ± 0.007 0h 1.267 ± 0.019 0h 0h 1.064 ± 0.016 0.519 ± 0.013 1.192 ± 0.019 0g 0.838 ± 0.011 0g 0g 0.704 ± 0.017 0g 0.680 ± 0.012 0g 1.929 ± 0.057 0g 0g 0.721 ± 0.013
f b
OA LA SA PA code resistance grade
Table 2. Fatty Acid Contents (mg g−1) in Soybean Pods with Different Field Mildew Resistance Gradesa
ALA
b
3.948 4.197 3.563 3.765 2.801 3.465 3.794 3.721 3.775 3.557 4.106 3.494 3.631 2.873
SFAs
bc a efg d h g cd de d efg ab fg defg h
UFAs
f b
total content
Journal of Agricultural and Food Chemistry
9139
DOI: 10.1021/acs.jafc.6b03561 J. Agric. Food Chem. 2016, 64, 9135−9142
Article
Journal of Agricultural and Food Chemistry
Figure 3. Total ion chromatograms (TICs) from HPLC−MS/SIM analysis of soy isoflavone standards and soybean pod samples: (a) standard chromatogram, (b) soybean germplasm “E60” (high MIS and high MIP), (c) soybean germplasm “A13” (high MIS and low MIP), (d) soybean germplasm “D49” (low MIS and low MIP), and (e) soybean germplasm “C103” (low MIS and high MIP). Peak assignments: (1) daidzin (DG), (2) glycitin (GLG), (3) genistin (GEG), (4) malonyldaidzin (MD), (5) malonylglycitin (MGL), (6) acetyldaidzin (AD), (7) acetylglycitin (AGL), (8) malonylgenistin (MG), (9) daidzein (DE), (10) acetylgenistin (AG), (11) glycitein (GLE), and (12) genistein (GE).
between the mildew contents of the seeds and pods. High contents of the infection constituents in the soybean pods caused pod mildew, which mitigated the seed mildew to a certain extent. Additionally, the antibiotic constituents, especially GL-type isoflavonoids, played an important role in the field mold tolerance. Although GL-type isoflavonoids cannot completely reverse serious mold damage on soybean
pods, they were able to mitigate the seed mildew to a certain extent. In conclusion, soybean seeds and pods were easily affected by mildew in the field because of their high contents of infection constituents, such as proteins, carbohydrates, and fatty acids, and the soybean pods were more susceptible. However, soy isoflavonoids, especially GL-type derivatives, which have been demonstrated to be a phytoalexin in previous studies, can 9140
DOI: 10.1021/acs.jafc.6b03561 J. Agric. Food Chem. 2016, 64, 9135−9142
Article
Journal of Agricultural and Food Chemistry Table 3. Isoflavone Profile and Contents (μg g−1) in Soybean Pods with Different Field Mildew Resistance Gradesa resistance grade
code
HR
C103 2162 D49 A3 ND12 GX1 G378-1 N256-1 2080 E60 E70 D15 A13 D1141
R S
HS
DE 4.753 98.755 24.256 31.343 28.659 21.271 243.806 20.271 351.169 435.346 22.061 10.914 465.849 13.293
± ± ± ± ± ± ± ± ± ± ± ± ± ±
GE 0.000 0.005 0.001 0.001 0.001 0.001 0.004 0.000 0.010 0.020 0.001 0.001 0.021 0.000
f e f f f f d f c b f f a f
4.532 38.107 14.779 25.461 19.513 11.342 259.923 18.813 326.439 411.301 23.985 10.814 326.834 15.366
± ± ± ± ± ± ± ± ± ± ± ± ± ±
GLE 0.000 0.001 0.001 0.002 0.001 0.001 0.010 0.001 0.018 0.017 0.001 0.000 0.013 0.001
f e ef ef ef ef d ef c a ef ef c ef
16.938 291.916 33.575 48.730 20.936 18.555 7.810 19.729 10.868 13.153 59.456 33.083 13.767 17.429
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.000 0.180 0.002 0.003 0.001 0.001 0.000 0.000 0.007 0.001 0.039 0.002 0.000 0.001
GLG b a b b b b b b b b b b b b
9.026 33.238 13.766 31.867 13.590 9.544 7.176 18.894 11.699 6.266 4.032 20.150 6.612 16.883
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.000 0.001 0.005 0.002 0.000 0.000 0.001 0.001 0.002 0.000 0.000 0.001 0.000 0.001
MGL efg a cde a cde efg fg bc def fg g b fg bcd
11.436 14.076 59.804 79.961 28.770 18.563 11.951 44.682 31.647 15.825 42.680 49.909 14.048 44.954
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.002 0.003 0.003 0.004 0.006 0.005 0.000 0.002 0.001 0.001 0.004 0.008 0.000 0.003
total content f f b a de ef f c d f c bc f c
46.685 476.092 146.179 217.362 111.468 79.274 530.665 122.389 731.821 881.892 152.213 124.870 827.109 107.926
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.002 0.173 0.012 0.007 0.009 0.006 0.009 0.002 0.021 0.039 0.045 0.012 0.026 0.005
d b cd c cd c b cd a a cd cd a cd
a All samples were measured in triplicate. Data are expressed as the mean ± standard deviation (n = 9) based on dry weight. Values marked by the same letter within the same line are not significantly different (p > 0.05).
(2) Deng, J.; Liu, J.; Lei, T.; Yang, F.; Su, B.; Cui, L.; Wan, Y.; Huang, S.; Yang, W. Effect of seed mildew in field on yield and quality of soybean during harvest season. Chin. J. Oil Crop Sci. 2015, 37, 077−82. (3) Liu, B.; Chen, C.; Lian, Y.; Chen, J.; Chen, X. Long-term change of wet and dry climatic conditions in the southwest karst area of China. Glob. Planet. Change 2015, 127, 1−11. (4) Gemmer, M.; Fischer, T.; Jiang, T.; Su, B.; Liu, L. L. Trends in precipitation extremes in the Zhujiang River Basin, South China. J. Clim. 2011, 24, 750−761. (5) Zhai, P.; Zhang, X.; Wan, H.; Pan, X. Trends in total precipitation and frequency of daily precipitation extremes over China. J. Clim. 2005, 18, 1096−1108. (6) Yang, F.; Huang, S.; Gao, R.; Liu, W.; Yong, T.; Wang, X.; Wu, X.; Yang, W. Growth of soybean seedlings in relay strip intercropping systems in relation to light quantity and red:far-red ratio. Field Crops. Res. 2014, 155, 245−253. (7) Jacobsen, B. J.; Harlin, K. S.; Swanson, S. P.; Lambert, R. J.; Beasley, V. R.; Sinclair, J. B.; Wei, L. S. Occurrence of fungi and mycotoxins associated with field mold damaged soybeans in the Midwest. Plant Dis. 1995, 79, 86−89. (8) Deng, J. C.; Lei, T.; Zhong, L.; Wu, H. J.; Yang, F.; Liu, W. G.; Huang, S.; Liu, J.; Yang, W. Y. The analysis of correlation and path coefficient between agronomic and quality traits with the resistance of soybean to seed mildew in field during harvest time. Soybean Sci. 2015, 34, 837−842. (9) Zhang, X. W.; Luo, H.; Wu, H. J.; Yang, C. Q.; Liu, W. G.; Liu, J.; Yang, W. Y. Correlation analysis between chemical profiles and field mold resistance in different soybean genotypes. Nat. Prod. Res. Dev. 2016, 28, 1001−1007. (10) Moïse, J. A.; Han, S.; Gudynaitę-Savitch, L.; Johnson, D. A.; Miki, B. L. A. Seed coats: Structure, development, composition, and biotechnology. In Vitro Cell. Dev. Biol.: Plant 2005, 41, 620−644. (11) Keigley, P. J.; Mullen, R. E. Changes in soybean seed quality from high temperature during seed fill and maturation. Crop Sci. 1986, 26, 1212−1216. (12) Li, H.; Liu, X.; Zhen, H. Studies on quality and yield loss caused by Cercospora sojina Hara in soybean. Chin. J. Oil Crop Sci. 2005, 27, 66−69. (13) Comai, S.; Bertazzo, A.; Bailoni, L.; Zancato, M.; Costa, C. V. L; Allegri, G. Protein and non-protein (free and protein-bound) tryptophan in legume seeds. Food Chem. 2007, 103 (103), 657−661. (14) Zhu, Z. Y.; Zhang, J. Y.; Chen, L. J.; Liu, X. C.; Liu, Y.; Wang, W. X.; Zhang, Y. M. Comparative evaluation of polysaccharides isolated from astragalus, oyster mushroom, and yacon as inhibitors of α-glucosidase. Chin. J. Nat. Med. 2014, 12, 290−293. (15) Liu, J.; Yang, C. Q.; Zhang, Q.; Lou, Y.; Wu, H. J.; Deng, J. C.; Yang, F.; Yang, W. Y. Partial improvements in the flavor quality of
protect soybeans against Colletotrichum gloeosporioides attack at concentrations as low as 0.0025% (25 mg L−1)22 and also present significant inhibitory activity against Aspergillus ochraceus at 5 × 10 −4 mol L −1 .23 Although GL-type isoflavonoids were detected in the soybean pods, their contents may never reach the antimycotic threshold. Undoubtedly, the serious mold on soybean pods mitigated the mildew damage to seeds to a certain extent.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b03561. Clustering analysis of soybeans based on MIS and MIP (Table S1) and correlation analysis between the pod constituents and mildew indexes (Table S2) (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*Telephone: +28-86290960. Fax: +28-86290870. E-mail:
[email protected]. *Telephone: +28-86290960. Fax: +28-86290870. E-mail:
[email protected]. ORCID
Jiang Liu: 0000-0001-8791-3053 Author Contributions †
Jiang Liu, Juncai Deng, and Caiqiong Yang contributed equally to this work. Funding
This study was financially supported by the National Natural Science Foundation of China (Grant 31401329) and the China Postdoctoral Science Foundation (Grant 2014M560724). Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Vollmann, J.; Fritz, C. N.; Wagentristl, H.; Ruckenbauer, P. Environmental and genetic variation of soybean seed protein content under Central European growing conditions. J. Sci. Food Agric. 2000, 80, 1300−1306. 9141
DOI: 10.1021/acs.jafc.6b03561 J. Agric. Food Chem. 2016, 64, 9135−9142
Article
Journal of Agricultural and Food Chemistry soybean seeds using intercropping systems with appropriate shading. Food Chem. 2016, 207, 107−114. (16) Mulero, J.; Martínez, G.; Oliva, J.; Cermeño, S.; Cayuela, J. M.; Zafrilla, P.; Martínez-Cachá, A.; Barba, A. Phenolic compounds and antioxidant activity of red wine made from grapes treated with different fungicides. Food Chem. 2015, 180, 25−31. (17) Zhang, R. F.; Zhang, F. X.; Zhang, M. W.; Wei, Z. C.; Yang, C. Y.; Zhang, Y.; Tang, X. J.; Deng, Y. Y.; Chi, J. W. Phenolic composition and antioxidant activity in seed coats of 60 Chinese Black soybean (Glycine max L. Merr.) varieties. J. Agric. Food Chem. 2011, 59, 5935− 5944. (18) Zhou, S.; Sekizaki, H.; Yang, Z.; Sawa, S.; Pan, J. Phenolics in the seed coat of wild soybean (Glycine soja) and their significance for seed hardness and seed germination. J. Agric. Food Chem. 2010, 58, 10972−10978. (19) Wolfe, K. L.; Liu, R. H. Structure-activity relationships of flavonoids in the cellular antioxidant activity assay. J. Agric. Food Chem. 2008, 56, 8404−8411. (20) Friedman, M. Antibacterial, antiviral, and antifungal properties of wines and winery byproducts in relation to their flavonoid content. J. Agric. Food Chem. 2014, 62, 6025−6042. (21) Chen, Y. H.; Yang, Z. S.; Wen, C. C.; Chang, Y. S.; Wang, B. C.; Hsiao, C. A.; Shih, T. L. Evaluation of the structure-activity relationship of flavonoids as antioxidants and toxicants of zebrafish larvae. Food Chem. 2012, 134, 717−724. (22) Lee, J. H.; Jeong, S. W.; Cho, Y. A.; Park, S.; Kim, Y. H.; Bae, D. W.; Chung, J. I.; Kwak, Y. S.; Jeong, M. J.; Park, S. C.; Shim, J. H.; Jin, J. S.; Shin, S. C. Determination of the variations in levels of phenolic compounds in soybean (Glycine max Merr.) sprouts infected by anthracnose (Colletotrichum gloeosporioides). J. Sci. Food Agric. 2013, 93, 3081−3086. (23) Krämer, R. P.; Hindorf, H.; Jha, H. C.; Kallage, J.; Zilliken, F. Antifungal activity of soybean and chickpea isoflavones and their reduced derivatives. Phytochemistry 1984, 23, 2203−2205.
9142
DOI: 10.1021/acs.jafc.6b03561 J. Agric. Food Chem. 2016, 64, 9135−9142
