Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Fungistatic Activity of Multiorigin Humic Acids in Relation to Their Chemical Structure Shiping Wei,†,‡ Meng Wu,†,‡ Guilong Li,† Ming Liu,†,‡ Chunyu Jiang,†,‡ and Zhongpei Li*,†,‡ †
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, No. 71, East Beijing Road, P.O. Box 821, Nanjing 210008, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China
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S Supporting Information *
ABSTRACT: Humic acid (HA) has an inhibitory effect on phytopathogenic fungi, but the structure−activity relationship remains unclear. HAs were extracted from 14 different materials, and their fungistatic activities and elemental C, N, S, and O contents were measured. Cross-polarization magic-angle spinning 13C nuclear magnetic resonance (CPMAS 13C NMR) was used to measure the organic carbon composition. The results showed that all HAs suppressed phytopathogenic fungi growth, with Yunnan lignite HAs showing the highest inhibition (85.3%) against Physalospora piricola. The soil and compost HA aromaticity (ARO) was 60%. The ARO of meadow and moss peat HAs was 60%), while compost and soil HAs were generally aliphatic (ARO < 50%), except black soil (ARO = 59%). The woody peat HA (M1) was partially aromatic with ARO = 50.6%, but the meadow peat HA (M2) and the moss peat HA (M3) were aliphatic. The lignite HA from Inner Mongolia (M4) had the largest ARO and the lowest O-alkyl C/alkyl C, which indicated that it had the most stable aromatic compounds of the HAs investigated. Paddy soil HA (M10) had the lowest aromatic content and aromatic C−O. Wheat straw compost HA was the most degradable of the HAs tested and had the largest O-alkyl C/alkyl C. The 13C NMR results indicated that there were substantial differences in the chemical structure of HAs from selected materials. CPMAS 13C NMR spectroscopy was used to monitor the carbon composition of HAs. The aliphaticity of soil HAs was reported in a previous study, which indicated that large quantities of soil HAs had aliphatic moieties.33 The authors also considered that the existence of aromatic chemical structures most probably originated from lignin degradation D
DOI: 10.1021/acs.jafc.8b01931 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
which have led to sulfur being widely used in fungicides.39−41 A negative correlation between the O content and the inhibitory proportion has been reported in previous studies in which the authors determined the percentage inhibition of HAs against soil-borne phytopathogenic fungi such as F. oxysporum, Trichoderma viride, Trichoderma harzianum, and Pythium ultimum, and hypothesized this ability might be related to the hydrophobic properties of humic substances.18,42,43 Hence, the sulfur content and extent of oxidation of HAs plays an important role during the inhibition of fungal growth. Organic compounds consisting of carbonyl, such as carboxylic acid/aldehyde, are associated with the fungistatic activities of humic substances.44 Furthermore, quinones as electron-accepting groups play an important role in forming reactive oxygen species (ROS), which are responsible for the fungistatic/bactericidal features of HAs because they cause oxidative stress and induce apoptosis in fungi/bacteria.31 Siddiqui et al.30 used Fourier-transform infrared spectroscopy (FTIR) to semiquantify the HA content oxygen-containing groups, and concluded that COOH groups were positively correlated with the inhibitory action. Loffredo et al.18 found that HAs with fewer COOH groups had greater inhibitory effects on two strains of F. oxysporum. Combined with Figure 1a, we further speculated that when the C content was constant, the lower O content of the carbonyl structure in the same HA could offer a stronger suppression against phytopathogenic fungi. Carbohydrates can provide nutrients for phytopathogenic fungi growth, and therefore the inhibitory action can be negated. Aromatic components with neutral hydrophobic organic matter have been speculated to exert a hormone-like activity on plant growth.45 However, Nardi et al.46 considered the hydrophilic and available components in humic matter to enable a more efficient diffusion of bioactive molecules at the cellular membrane level. This was probably the reason that phytopathogenic fungal growth was inhibited because the presence of more aromatic components would cause lower hydrophilicity and diffusion.
Table 4. Mantel Statistics between the Inhibition Rates for the Elemental Content and Relative Intensity of the 13C Nuclear Magnetic Resonance (13C NMR) Carbon Fractions element
r
P
carbon type
r
P
C N S O C/N C/S N/S C/O N/O O/S
−0.014 0.256 0.390 0.312 −0.010 0.041 −0.086 0.089 0.331 0.065
0.584 0.001b 0.001b 0.001b 0.504 0.260 0.899 0.108 0.001b 0.155
carbonyl C aromatic C−O aromatic C anomeric C O-alkyl C O−CH3/NCH alkyl C ARO O-alkyl C/alkyl C
0.123 0.117 −0.086 0.087 0.047 0.039 0.011 −0.034 0.017
0.006b 0.010a 0.877 0.045a 0.131 0.194 0.387 0.678 0.353
a
Indicates a significant correlation at the 0.05 level. bIndicates a significant correlation at the 0.01 level.
between the relative intensity of the 13C NMR carbon fractions and fungistatic activity was also conducted (Figure 1b). The RDA1 and RDA2 axis explained 40.0% and 30.4% of the variation, respectively. Figure 1b shows that the relative intensity of the aliphatic C (alkyl C, O-alkyl C, and O−CH3/ NCH) and carbonyl C in HAs expressed a positive effect on the RDA1 and RDA2 axes, respectively. In contrast, the aromatic fractions (ARO, aromatic C, and aromatic C−O) and anomeric C expressed a negative effect in the two axes. The effects of carbon composition on the RDA2 axes were similar to those of the Mantel test. Therefore, the main positive effect was from carbonyl C and the main negative effect was from anomeric C and aromatic C−O. The results of the Mantel test and RDA analysis revealed significant positive correlation factors (S and carbonyl C) and negative correlation factors (O, anomeric C, and aromatic C− O). In previous studies, sulfur and sulfide, especially organic sulfur compounds, have been confirmed to display fungistatic activity in connection with bioactivity and molecular structure, such as the active groups −CS2− in the dithiocarbamates,
Figure 1. Redundancy analysis between the fungistatic activities of humic acids (HAs) from different materials and their elemental content (a) or the relative intensity of the 13C nuclear magnetic resonance (13C NMR) carbon fraction (b). An ANOVA test was conducted and the significance was obtained for the two RDA models (P < 0.001). The P value of the first models RDA1 and RDA2 were both less than 0.001, while the second models RDA1 and RDA2 were both less than 0.01. E
DOI: 10.1021/acs.jafc.8b01931 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Goldsworthy et al.47 reported that the toxicity to fungal activity was dependent on alkyl radicals. Because this part of alkyl C might be derived from the selective preservation of recalcitrant alkyl C associated with plant-derived biopolymers and microbially originated products, it could not be used in the growth and metabolism of pathogens.32 In this study, the effect of alkyl C on the fungistatic activity of HAs was not significant because it was related to the different materials and fungi species tested. In summary, the S content and the structure of oxygenated organic compounds had an important effect on fungistatic activity. The HAs with higher fungistatic activity had a higher C content in terms of carboxyl organic compounds, fewer carbohydrates, and fewer aromatic compounds. The proportional content of carbohydrates, aromatic compounds, aliphatic compounds, and organic compounds consisting of carbonyl could directly determine the fungistatic activity of HAs. Phytotoxicity Test for HAs. Figure 2 shows the average GI value for every treatment. The GI values were all more than
suppression against phytopathogenic fungi, more information is needed about their effectiveness in the plant rhizosphere and the response of beneficial micro-organisms. This information will be critical in the design of HA-based products. The results of this study should encourage the use of HAs from a wider range of source materials.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b01931. CPMAS 13C NMR spectra of humic acids extracted from 14 materials (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +86 25 86881323. E-mail:
[email protected] (Z.P.L.). ORCID
Shiping Wei: 0000-0003-4445-8702 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the grants from the National Natural Science Foundation of China (Nos. 41430859 and 41771298) and National Key Research and Development Program of China (2016YFD0200309).
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ABBREVIATIONS USED HA, humic acid; CPMAS 13C NMR, cross-polarization magicangle spinning 13C nuclear magnetic resonance; ARO, aromaticity; RDA, redundancy analysis; FA, fulvic acid; HM, humin; DMSO, dimethyl sulfoxide; IR, inhibition rate; GI, germination index; ANOVA, one-way analysis of variance; ROS, reactive oxygen species; FTIR, Fourier-transform infrared spectroscopy
Figure 2. Germination indexes of pak choi with the addition of humic acids (HAs) analyzed by a one-way ANOVA. The test concentration was 50 mg·L−1. HAs powders were dissolved in dimethyl sulfoxide (DMSO) solution.
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80% except the danty HA (M6), which was just 74.94%. Some HAs, such as M4, M7, and M11, even had a GI of more than 100%. There were no differences (P > 0.05) among the different HAs. There was no significant promotion or inhibition of seed germination and seedling root elongation, which indicated that all HAs from the selected materials were safe to use. Phytotoxicity test is the base index to assess the applicability of HAs. No significant promoting or suppressing effect was identified in the study, and therefore the HAs from the selected materials were found to be nontoxic to plants. HAs play the role of plant regulators, and therefore the toxicity of HAs to plant seed germination and root elongation as a basic measurement trait was evaluated.48 The role of HAs on plant root growth regulation might be correlated with the polarity alterations induced by the interactions between soluble HAs and root exudates, the molecular size of the HAs, H+-ATPase activity in membrane vesicles, mineral use, and nutrients and water reserved by HAs.7,8,49,50 Vlckova et al.51 indicated that lignite HAs as natural products were efficient, were less expensive compared to commercial fertilizers, and provided environmental protection when used in soil maintenance. It is therefore feasible to further develop and use the fungistatic activity of HAs. Before the application of HAs to enhance soil
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DOI: 10.1021/acs.jafc.8b01931 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
