Yunhao Sun,Dianxian Cai,Di Qin,Jialiang Chen,Yutong Su,Xiaoying Zheng,Zhen Meng,Jie Zhang,Lina Xiong,Zhangyong Dong,Ping Cheng,Xiaoming Peng,Guohui Yu
The plant protection preparation GZM improves crop immunity, yield, and quality
Plant pathology,Plant development,Omics
Summary Lauryl alcohol, a natural compound found in plants and other organisms, is widely used to make surfactants, food, and pharmaceuticals. GZM, a plant protection preparation with lauryl alcohol as its major component is thought to establish a physical barrier on the plant surface, but its physiological functions are unknown. Here, we show that GZM improves the performance of peanut (Arachis hypogaea) plants in both the laboratory and the field. We demonstrate that the treatment with GZM or lauryl alcohol raises the contents of several specific lysophospholipids and induces the biosynthesis of phenylpropanoids, flavonoids, and wax in various plant species. In the field, GZM improves crop immunity, yield, and quality. In addition, GZM and lauryl alcohol can inhibit the growth of some pathogenic fungi. Our findings provide insights into the physiological and biological effects of GZM treatment on plants and show that GZM and lauryl alcohol are promising preparations in agricultural production.
The plant protection preparation GZM improves crop immunity, yield, and quality

Lauryl alcohol, a natural compound found in plants and other organisms, is widely used to make surfactants, food, and pharmaceuticals. GZM, a plant protection preparation with lauryl alcohol as its major component is thought to establish a physical barrier on the plant surface, but its physiological functions are unknown. Here, we show that GZM improves the performance of peanut (Arachis hypogaea) plants in both the laboratory and the field. We demonstrate that the treatment with GZM or lauryl alcohol raises the contents of several specific lysophospholipids and induces the biosynthesis of phenylpropanoids, flavonoids, and wax in various plant species. In the field, GZM improves crop immunity, yield, and quality. In addition, GZM and lauryl alcohol can inhibit the growth of some pathogenic fungi. Our findings provide insights into the physiological and biological effects of GZM treatment on plants and show that GZM and lauryl alcohol are promising preparations in agricultural production.

During their entire life cycle, plants are exposed to many stresses, including biotic and abiotic factors, which have a negative effect on their germination, growth, development, productivity, and postharvest preservation.,, Indeed, these stresses contribute significantly to the loss of crop production worldwide with an average yield decline of 65%–85%.,, Therefore, plants have acquired a series of effective mechanisms that can provide protection or become activated under stress conditions. In addition to the cell wall, synthetic physical barriers that coat the plant surface are critical for crops to resist biotic and abiotic stresses, such as drought, low temperature, ultraviolet (UV) damage, diseases, and pests., Dozens of such physical protective agents have been developed and form a film on the plant surface, with good effects on yield and performance following their applications.,,, In the 1980s, a reagent under the tradename gao-zhi-mo in Chinese (GZM, meaning “high-quality lipid membrane”) was developed and achieved good results for plant protection in the field, with a lower cost and less environmental damage than those in traditional plant protection preparations. The main component of GZM is lauryl alcohol; 1-hexadecanol and Tween 80 are auxiliary components for emulsification and film formation. Based on tests conducted by the Chemical Testing Institute of the Ministry of Agriculture of China, GZM is considered to be relatively innocuous, with no known toxic effects on the environment at the concentrations used in the field. GZM is generally thought to act as a physical barrier to protect plants from pathogen invasion, as it forms a continuous liquid film or membrane on the plant surface when applied at a very low concentration. However, action as a physical barrier cannot solely explain the efficacy of GZM. Despite the low cost of production and application of GZM, the lack of understanding of the specific mechanism behind the effectiveness of this chemical has hindered its popularization. Lauryl alcohol (1-dodecanol), the main component of GZM, is a natural product found in female ginseng (Angelica sinensis), sacred lotus (Nelumbo nucifera), and other organisms. It is widely used to make surfactants and pharmaceuticals, as well as to enhance flavor in food.,, However, the physiological effects of lauryl alcohol on plants are unknown. High-throughput “omics” technologies, such as transcriptomics and metabolomics have become powerful approaches to analyze genes, metabolites, and pathways related to the responsiveness of a plant to potential physiological intervention agents.,, In plants, the general phenylpropanoid biosynthetic pathway is one of the most important metabolic pathways related to biotic and abiotic stimuli. Phenylpropanoid-based secondary products, like lignin and flavonoids, derived from the general phenylpropanoid biosynthetic pathway contribute substantially to the mechanical stability and robustness of plants against environmental damage, like drought or wounding, and they have multiple roles in plant defense against pathogens and herbivores,,, antioxidant activity,, protection against UV damage,,, legume nodulation,, and control of auxin transport. Plants that exhibit increased biosynthesis of phenylpropanoid-based secondary products under biotic and abiotic stresses usually acclimate better to adverse environments., Therefore, the transcript levels of the key genes involved in the general phenylpropanoid biosynthetic pathway and concentrations of phenylpropanoid-based secondary products in plant tissue are ideal indicators of the extent of stress tolerance of various plants. The enzyme phenylalanine ammonia-lyase (PAL) catalyzes the formation of cinnamic acid from phenylalanine, which acts as the critical regulatory step between primary and secondary metabolism., Numerous studies have shown the corresponding response of PAL activity to various biotic and abiotic stresses,,, Lysophosphatidylethanolamine (LPE) is a biologically active lipid that can enhance PAL activity, prime the plant immune system, promote basal resistance against hemibiotrophic pathogens, regulate plant senescence and aging,,, and induce plant growth. Lysophosphatidylcholine (LPC), another biologically active lysophospholipid, like LPE can also mediate a variety of cellular responses, including plant growth, development, systemic responses after wounding, and responses to stress and defense.,,, Thus, activation of the general phenylpropanoid biosynthetic pathway and stimulation of specific plant metabolites may offer environmentally friendly and natural solutions, to reduce losses from crop production and improve crop performance through various approaches, including application of plant bio-stimulant products. The main component of GZM is lauryl alcohol, which is a biosurfactant. Biosurfactants are considered to be less toxic and more eco-friendly than traditional chemical pesticides and use of green compounds to achieve sustainability in agriculture is a priority., We show here, from work conducted on multiple crops grown in the laboratory and in field experiments that treating plants with GZM improves crop immunity, yield, and quality. Moreover, we demonstrate that the biosynthetic pathways for phenylpropanoids, flavonoids, and wax are activated upon GZM application and that several specific lysophospholipids accumulate. These results offer new clues as to the physiological effects of GZM and lauryl alcohol on plants and hold important theoretical significance for the promotion and application of GZM as a promising product in agriculture.

Seed coating is a promising technique for the large-scale inoculation of crop seeds with adhesive agents, including GZM, to improve seed performance, alleviate biotic and abiotic stresses, and reduce production costs.,,,,, We investigated whether GZM deployed as a seed coating agent might improve the germination rate of seeds and early performance during the seedling stage. We coated peanut (Arachis hypogaea) seeds (cultivar Yuhua 9805) with different concentrations of GZM 18 months after seed harvest, a storage interval normally associated with poor germination, and scored germination rates and seedling growth under normal conditions in soil and under drought (simulated with 15% [w/v] polyethylene glycol [PEG] solution) or high-salt (provided by 1.5% [w/v] NaCl solution) conditions. Seeds treated with GZM showed greater germination potential and the resulting seedlings fared better than did controls, as evidenced by seedling height after 7 days of growth (Figures 1A–1C and Table S1). Furthermore, under drought and high-salt conditions, seedlings whose seeds were coated with GZM grew taller than control seedlings 3 days after germination (Figures 1D and 1E and Table S1). According to the dose effects observed at 7 days after germination, the seeds coated with higher concentrations of GZM showed greater germination potential and the seedlings fared better than those obtained from the seeds treated with lower concentrations of GZM (Figures 1D and 1E and Table S1). Lauryl alcohol, but not Tween 80, also improved seedling growth (Figure S1 and Table S1). These results demonstrate that GZM in general and lauryl alcohol in particular, improves peanut seed germination and seedling growth, even under stress conditions.

To assess the potential usefulness of GZM in practical applications, we measured multiple parameters for peanut and soybean (Glycine max, cultivar Huachun No.1) plants grown in the field, where they suffered a severe drought during the growing period. Plants treated with GZM (four concentrations at different growth stages) accumulated more plant biomass and produced more seeds than did control plants (Figure 2). In addition, in-field GZM treatment diminished the occurrence of disease symptoms on the leaves of peanut plants and increased the number of root nodules on soybean plants (Figures 2A and 2D). Using peanut as a representative crop, we specifically measured a series of physiological and biochemical indicators. Peanut plants treated with GZM exhibited a marked increase in physiological indexes related to plant growth, such as plant height, number of tillers, diameter of straws, total chlorophyll contents, blade size, and percentage of fresh active root nodules (Figures 3A–3H and Table S2). GZM treatment also increased seed yield, fresh weight of pods, ratio of pods, and oil content of pods relative to control plants (Figures 3I–3K and Table S2). The numbers of mildewed pods (Figure S2 and Table S2) and rotten pods decreased after GZM treatment compared to controls (Figures 3L and 3M and Table S2). GZM treatment also decreased the incidence of disease spots on leaves (Figure 3N and Table S2) caused by Cercospora leaf spot disease. To explore the effects of GZM treatment on pest and disease prevention, we grew GZM-treated and control peanut plants under laboratory conditions and fed their leaves to third-stage tobacco cutworm (Spodoptera litura) (Fabricus) larvae, which constitute a serious threat to peanut plants, in the same insect breeding environment. We detected fewer instances of leaf consumption on the leaves of plants treated with GZM than on those of control plants (Figure 3O and Table S2), suggesting that GZM treatment increased plant resistance against herbivores. As greater tolerance against pests is often associated with reactive oxygen species (ROS) contents, we measured the activity of several redox-related enzymes, as well as proline and malondialdehyde (MDA) contents. Proline (PRO) is associated with increased tolerance against pathogens and abiotic stress, while MDA is a marker of lipid peroxidation due to the accumulation of ROS. Compared to the control group, GZM-treated fresh leaves showed greater accumulation of PRO and higher activity levels for superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) but lower MDA content (Figures 3P–3T and Table S2). Taken together, these results indicate that treatment with GZM improves multiple related morphological and physio-biochemical traits of field crops in agricultural field settings. We also assessed whether GZM treatment affected phytohormone levels in the leaves of peanut plants grown in the field. Many plant hormones involved in biotic and abiotic stress responses, such as salicylic acid, gibberellin A19, jasmonic acid, and abscisic acid, were more abundant in GZM-treated plants than in control plants, whereas cytokinin and auxin, which are related to growth regulation, were less abundant in GZM-treated plants than in control plants (Figure 4A and Table S3). As the soil microbiome can influence plant fitness, we purified DNA from the rhizosphere soil and analyzed the composition of the rhizosphere microbiome associated with field-grown peanut plants. Gammaproteobacteria, Alphaproteobacteria, and Bacteroidia, all known to play important roles in disease resistance and growth promotion,, were enriched in the rhizosphere of GZM-treated plants relative to controls (Figures 4B and S3). These results demonstrate that GZM treatment affects phytohormone contents and the composition of rhizosphere microorganisms in plants grown under field production conditions.

In previous studies, GZM application formed a protective film on the plant surface. We noticed that spraying peanut, soybean, and Arabidopsis (Arabidopsis thaliana) leaves with a mixture of lauryl alcohol and Tween 80 (1.3 mM lauryl alcohol, 23.3 μM Tween 80) was effective in covering the leaf surface, even that of hairy leaves (Figure S4). However, during test experiments, we discovered that spraying the leaves of Arabidopsis and soybean plants with high concentrations of GZM (five times higher than the typically used concentration) results in a cell death–like phenotype within 10 h of treatment (Figure S5). This observation suggested that GZM might not only form a physical barrier on leaves but also quickly change the physiological properties of treated plants. We performed transcriptome and metabolome profiling of leaves from 4-week-old peanut plants treated with GZM diluted 1,000 times (corresponding to concentrations of 1.3 mM lauryl alcohol, 123.7 μM 1-hexadecanol, and 23.3 μM Tween 80). We identified 529 differentially expressed genes (DEGs) between plants treated with GZM and control plants (Figure 5A and Table S4). Among them, 428 genes were upregulated in GZM-treated plants, while the remaining 101 genes were downregulated (Table S4). A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that these DEGs were significantly enriched in the pathways of plant–pathogen interaction, mitogen-activated protein kinase (MAPK) signaling, isoflavonoid biosynthesis, and alpha-linolenic acid metabolism (Figure 5B and Table S5). We also identified 62 metabolites with greater abundance and 53 metabolites with lower abundance in GZM-treated peanut leaves than in controls (Figure 5C and Table S6). KEGG analysis of these differentially abundant metabolites also revealed an enrichment for the isoflavonoid biosynthesis pathway in GZM-treated leaves compared to controls (Figure 5D and Table S7). Most of the 62 more abundant metabolites were flavonoids and phenolic acids (Table S6). Of these, the top 10 more abundant metabolites belonged to lignans and coumarins, amino acids and derivatives, flavonoids, phenolic acids, nucleotides and derivatives, and lipids (Figure 5E and Table S6). Together, the transcriptome and metabolome results indicate the enrichment of the isoflavonoid biosynthesis pathway in leaves treated with GZM compared to controls (Figure 5F). In addition, we observed the transcriptional upregulation of genes encoding various enzymes involved in isoflavonoid biosynthesis (Figure S6, Tables S4 and S6). These results suggest that GZM treatment induces isoflavonoid biosynthesis in peanut plants. To assess whether this effect is conserved across other members of the Leguminosae family, we measured metabolite contents in kudzu (Pueraria lobata [Willd.] Ohwi), with high flavonoid contents accumulating in its dried roots (named Radix Puerariae). Among 141 metabolites with significant differences in abundance, 135 were more abundant in GZM-treated samples than in controls (Figure 6A and Table S8). Consistent with the results in peanuts, isoflavonoid biosynthesis was the most highly enriched pathway in P. lobata (Figure 6B and Table S9), with isoflavonoids, anthocyanins, flavones, and flavonols accumulating in GZM-treated plants compared to controls (Figures S7–S9 and Table S8). The effects of GZM treatment on the isoflavonoid biosynthesis pathway also extended outside the Leguminosae, as leaves of Nicotiana benthamiana, from the Solanaceae family, displayed greater contents of flavonoids when treated with GZM than when treated with water (Figure S10, Tables S10 and S11). We expanded our transcriptome analysis to soybean (Leguminosae family), Arabidopsis (Cruciferae family), wheat (Triticum aestivum L.; Poaceae family), and tomato (Solanum lycopersicum; Solanaceae family), as well as the epidermis of navel orange (Citrus sinensis Osb. Var. brasiliensis Tanaka) fruits (Rutaceae family), which accumulate variable levels of flavonoids without GZM treatment. Genes participating in the phenylpropanoid and flavonoid biosynthesis pathways in these plants were significantly upregulated in samples treated with GZM compared to controls (Figures S12–S28 and Table S12. DEGs between GZM-treated and control soybean leaves, related to Figure 6, Table S13. Enriched KEGG pathways using the differentially abundant metabolites between GZM-treated and control soybean leaves, related to Figure 6, Table S14. DEGs between Arabidopsis thaliana leaves treated with GZM or with sterile water as control, related to Figure 6, Table S15. Enriched KEGG pathways based on the differentially abundant metabolites between Arabidopsis thaliana leaves treated with GZM or with sterile water as control, related to Figure 6, Table S16. DEGs between Triticum aestivum L. leaves treated with GZM or with sterile water as control, related to Figure 6, Table S17. Enriched KEGG pathways based on the differentially abundant metabolites in Triticum aestivum L. leaves treated with GZM or with sterile water as control, related to Figure 6, Table S18. DEGs in Solanum lycopersicum leaves treated with GZM or with sterile water as control, related to Figure 6, Table S19. Enriched KEGG pathways based on the differentially abundant metabolites in Solanum lycopersicum leaves treated with GZM or with sterile water as control, related to Figure 6, Table S20. DEGs in the fruit epidermis of Citrus sinensis Osb. var. brasiliensis Tanaka treated with GZM or with sterile water as control, related to Figure 6, Table S21. Enriched KEGG pathways based on the differentially abundant metabolites in the fruit epidermis of Citrus sinensis Osb. var. brasiliensis Tanaka treated with GZM or with sterile water as control, related to Figure 6), suggesting that GZM has a widespread and shared effect on plants. GZM is used as a spray preparation and comes into direct contact with the plant surface. Considering that the wax layer in plants plays an important protective role against biotic and abiotic stresses,, we used scanning electron microscopy to observe the effect of GZM treatment on the wax crystals on the surface of peanut leaves. We noticed more wax crystals on the surface of leaves treated with GZM three times during leaf development than on the surface of control leaves (Figure 7A). We also observed the significant upregulation of key genes in the cutin and wax biosynthesis pathways in GZM-treated tissues compared to controls (Figure 7B and Table S4). To assess whether this effect is conserved across different crops, we analyzed the transcriptome data of crops treated with GZM and detected the same molecular signature in all treated plants, indicating that the physiological role of GZM is conserved in different crops (Figure 7B, Tables S4, S12, S16, S18, and S20). Taken together, these results demonstrate that GZM treatment can regulate phenylpropanoid, flavonoid, and wax biosynthesis in a conserved manner across different plant species.

One of the top 10 more abundant metabolites in peanut leaves treated with GZM did not belong to either flavonoids or phenolic acids: the lipid lysophosphatidylcholine (LysoPC; 12:0) (Figures 5E and 8A). LysoPC and lysophosphatidylethanolamine (LysoPE) are lysophospholipids, important signaling molecules in response to biotic and abiotic stresses,, that delay plant senescence and aging,, and promote plant growth. To determine whether LysoPC or other lipids accumulate in other plants, we looked for this compound in metabolome data obtained for GZM-treated peanut leaves, the fruit epidermis of navel oranges grown in the field, and tomato leaves grown in the laboratory. GZM treatment led to increased abundance of LysoPC (20:4) in peanut leaves, LysoPE (18:2) in navel orange fruit epidermis, and LysoPC (12:0) in tomato leaves (Figures 8B–8D and Tables S22, S23, and S24). To determine the individual contributions of the main components of GZM to the accumulation of these metabolites, we treated leaves from various plant species with either lauryl alcohol or Tween 80 and carried out metabolome analysis of treated and control samples. Several lipids accumulated upon treatment with lauryl alcohol compared to treatment with Tween 80: LysoPC (12:0) and LysoPE (16:3) in peanut, LysoPC (12:0) in Arabidopsis, LysoPE (16:1, 2n isomer) and LysoPC (12:0) in rice (Oryza sativa), and LysoPC (12:0) and LysoPC (20:5) in wheat and N. benthamiana leaves (Figures 8E–8I and Table S25. Differentially abundant metabolites between the leaves of peanut plants treated with lauryl alcohol or Tween 80, related to Figure 8, Table S26. Differentially abundant metabolites between the leaves of Arabidopsis thaliana plants treated with lauryl alcohol or Tween 80, related to Figure 8, Table S27. Differentially abundant metabolites between the leaves of Oryza sativa L. plants treated with lauryl alcohol or Tween 80, related to Figure 8, Table S28. Differentially abundant metabolites between the leaves of Triticum aestivum L. plants treated with lauryl alcohol or Tween 80, related to Figure 8, Table S29. Differentially abundant metabolites between the leaves of Nicotiana benthamiana plants treated with lauryl alcohol or Tween 80, related to Figure 8). Similarly, LysoPC (12:0), LysoPE (14:0, 2n isomer), and LysoPE (14:0) accumulated in tomato leaves treated with lauryl alcohol relative to control leaves (Figure 8J and Table S30). These results indicate that GZM and its main component lauryl alcohol can induce the accumulation of specific lysophospholipids in various plants.

We next assessed whether GZM treatment improves plant growth and postharvest preservation performance of crops and seeds. The diameter of rice straws from plants grown in the field and treated with GZM was 22.9% greater than that of controls; we observed no significant difference in plant height between the groups (Figure S29 and Table S31). Likewise, GZM treatment of tobacco (Nicotiana tabacum) increased growth rate, growth potential, leaf size, straw thickness, and root biomass (Figures S10A–S10D). In three independent field tests, GZM treatment improved tobacco yield by 20.3% relative to controls (Figure S10E). GZM treatment also affected fruit production. The fruit epidermis of Tribute Citru (Citrus sinensis) treated with GZM was smoother than that of controls (Figure 9A). Furthermore, the storage period of GZM-treated fruits at room temperature was prolonged compared to that of control fruits (Figures 9B and 9C). The occurrence of physiological premature drop of fruits and sunburned fruits markedly decreased following GZM treatment (Figures 9D–9F and Table S32), as did the extent of damage caused by the citrus leafminer Phyllocnistis citrella (Figure 9F and Table S32). We obtained similar results for Japanese plum (Prunus salicina) production: GZM treatment decreased the occurrence of physiological premature drop of fruits and the incidence of fruit cracking (Figures 9G and 9H and Table S32). Taken together, these results demonstrate that GZM has a good application value in production practices (Table S33).

Does GZM directly block the growth of pathogenic microbes rather than (or in addition to) providing a physical barrier against pathogens? We established that GZM and lauryl alcohol does inhibit growth of the pathogenic fungi Fusarium oxysporum f. sp. cubense and Sclerotium rolfsii in a dose-dependent manner (Table S34). Other components of GZM, such as 1-hexadecanol and Tween 80, had no effect (Figure 10). These results suggest that GZM and its main component lauryl alcohol can inhibit the growth of some pathogenic fungi.

GZM is believed to form a protective coat over leaves, providing a physical barrier against pathogens. We confirmed here that spraying plant leaves with a mixture of lauryl alcohol and Tween 80 covered the leaf surface, even that of hairy leaves (Figure S4). This physical effect makes GZM a possible mixing agent with other pesticides to increase their plant surface adsorption and action. We determined that GZM directly induced strong physiological and physio-biochemical responses in plants. These results not only reveal the physiological mechanism of action of GZM but also suggest that other reagents, used in field production or during development to protect plants by forming a physical film, might also have practical applications in inducing plant physiological and physio-biochemical responses. Another practical reason for GZM use is its affinity toward the leaf surface, which increases the absorption and diffusion of traditional pesticides. We also established in this study that leaves treated with GZM showed a lower incidence of disease spots (Figure 3N and Table S2) caused by Cercospora leaf spot disease, as well as diminished damage caused by the citrus leafminer P. citrella (Figure 9F and Table S32). We cannot currently distinguish between a direct effect of GZM as an insect repellent or its effect on plant immunity, underscoring the need for further research. However, at the application level, these results showed that GZM is a promising preparation for boosting the affinity of agents for the plant surface during mixed application and might also serve as a carrier in the development of mixed green agents in the future. In general, plants must balance their needs for growth with the demand for resources dedicated toward their immunity., However, biological agents or plant endophytes can promote plant growth and even improve yield and product quality while also inducing immunity.,,, These reports suggest that the trade-off between defensive phenotypes and growth may be constrained by the allocation of limiting resources, while under stress conditions, the same trade-offs may become mutually supportive. The phenylpropanoid and flavonoid biosynthesis pathways play critical roles in plant stress responses., For instance, the induction of phenylpropanoid biosynthesis leads to an increase in lignin contents and raises plant resistance against diseases and pests., Flavonoids are important plant metabolites that impart greater tolerance to UV damage and drought stress,,,,, which may explain why straw diameter, chlorophyll contents, plant growth, and yield increased under stress conditions, while the incidence of plant diseases decreased (Figure 2). In addition, we showed that LysoPC and LysoPE were among the metabolites displaying the largest increase in contents in GZM-treated plants (Figure 8, Table S22. Differentially abundant metabolites between the leaves of peanut plants grown in the field and treated with GZM or with sterile water as control, related to Figure 8, Table S23. Differentially abundant metabolites between the fruit epidermis of Citrus sinensis Osb. var. brasiliensis Tanaka treated with GZM or with sterile water as control, related to Figure 8, Table S24. Differentially abundant metabolites between the leaves of Solanum lycopersicum treated with GZM or with sterile water as control, related to Figure 8, Table S25. Differentially abundant metabolites between the leaves of peanut plants treated with lauryl alcohol or Tween 80, related to Figure 8, Table S26. Differentially abundant metabolites between the leaves of Arabidopsis thaliana plants treated with lauryl alcohol or Tween 80, related to Figure 8, Table S27. Differentially abundant metabolites between the leaves of Oryza sativa L. plants treated with lauryl alcohol or Tween 80, related to Figure 8, Table S28. Differentially abundant metabolites between the leaves of Triticum aestivum L. plants treated with lauryl alcohol or Tween 80, related to Figure 8, Table S29. Differentially abundant metabolites between the leaves of Nicotiana benthamiana plants treated with lauryl alcohol or Tween 80, related to Figure 8, and S33). The transcriptome data showed that in some crops, such as peanuts, wheat, and navel orange, the transcript levels of phospholipase genes involved in the biosynthesis of LPE (Lysophosphatidylethanolamine) and LPC (Lysophosphatidylcholine), were significantly upregulated in tissues treated with lauryl alcohol (Table S35). These results may provide an explanation for the increase in LPC and LPE contents in these species, but determining why the related genes are not all induced in all crops will require further research. LPE and LPC exist only in relatively small amounts in natural organisms., They have a fatty acid group, and the results of metabolomics indicated that the composition of this group is similar to that of lauryl alcohol or n-hexadecanol, suggesting that the main components of GZM may act as precursors of LPE and LPC and participate in the physiological biosynthesis pathway in plants after external application. Further research, for example isotope labeling and tracing, is required to analyze differences in the types of accumulated lysophosphatides across different crops and to understand how lauryl alcohol increases the contents of specific lysophosphatides. LysoPC and LysoPE are lysophospholipids that promote plant growth, delay plant senescence and aging,, and induce stress and defense responses.,, The consistent accumulation of lysophospholipids upon GZM treatment suggests they are key factors in the effects of GZM treatment on plant physiology. Lysophospholipids can raise activity of phenylalanine ammonia-lyase (PAL), the rate-limiting enzyme in phenylpropanoid, and flavonoid biosynthesis. This observation may explain the increased abundance of both lysophospholipid and flavonoid in various plants upon treatment with GZM. In this study, we showed that PAL1 transcript levels were induced in peanuts, Arabidopsis, wheat, tomato, and navel orange upon GZM treatment, whereas we detected no changes in PAL1 expression in soybean, kudzu, rice, tobacco, or N. benthamiana. However, the aim of the transcriptome sequencing was to specifically examine PAL1 transcript levels; in the future, targeted exploration of changes in the transcript levels of other PAL paralogs in each crop will potentially indicate whether the level of PAL in cells is altered by GZM treatment. In studying the rhizosphere microbiome of peanut plants, we determined that the abundance of Gammaproteobacteria, Alphaproteobacteria, and Bacteroidia increased significantly in the GZM-treated group relative to controls (Figure 4). Gammaproteobacteria are important in disease suppressiveness, while Bacteroidia population diversity can increase with the addition of sawdust in the soil. It is possible that GZM altered the rhizosphere microbiome after leaching into the soil during spraying. Alternatively, GZM may induce physiological changes in plants that in turn alter the contents and/or types of root exudates, which would also affect the complement of rhizosphere microorganisms. The fact that we counted more effective nodules on the roots of Leguminous plants treated with GZM relative to control plants (Figures 2B and 3H) might support the second hypothesis. We showed here that GZM and lauryl alcohol had almost the same inhibitory effect on the growth of F. oxysporum f. sp. cubense, and GZM was more effective than lauryl alcohol at preventing the growth of S. rolfsii (Figure 10 and Table S34). These results suggest that the small amount of 1-hexadecanol in GZM might contribute to the inhibitory effect of GZM on some specific microorganisms. However, 1-hexadecanol alone did not inhibit the growth of S. rolfsii, suggesting that it might have an auxiliary role during physical film-forming or that lauryl alcohol and 1-hexadecanol have synergistic effects. In this study, we showed that GZM and lauryl alcohol deployed as a seed coating agent on peanut seeds improved seed germination rate and early performance, even under stress conditions, during the seedling stage. A recent study revealed that mitochondrial GPAT (glycerol-3-phosphate acyltransferase)-derived LPA controls auxin-dependent embryonic and postembryonic development. Considering that GZM and its main component lauryl alcohol induce the accumulation of lysophospholipids in various plant species, and in light of the phytohormone contents of GZM-treated plants, we observed the accumulation of some plant hormones, including 3-indolebutyric acid, which can promote seed germination, stress tolerance, and root growth.,, Therefore, we speculate that the effect of GZM and lauryl alcohol on seed germination and early plant performance may be due to the accumulation of specific lysophospholipids that lead to changes in the content of related phytohormones. In practical applications, the recommended dilution of the GZM stock solution is 1,000 times (corresponding to 1.3 mM lauryl alcohol, 123.7 μM 1-hexadecanol, and 23.3 μM Tween 80). In our test experiments, we sprayed Arabidopsis and soybean leaves with a concentration of GZM solution that was five times higher than this recommended concentration for practical implementations (corresponding to 6.5 mM lauryl alcohol, 618.5 μM 1-hexadecanol, and 116.5 μM Tween 80), but observed a phenotype similar to cell death within 10 h (Figure S5). This phenomenon may be due to severe physiological and biochemical reactions upon treatment with GZM. Biosurfactants can damage plant cells at high doses,, and so this result might also suggest that GZM can act as a surfactant. In addition to the effects of GZM on plant pathogens, leaves treated with GZM appeared to be less palatable to herbivores than control leaves (Figure 3O and Table S2), which was reflected in the contents of metabolites and phytohormones in treated leaves. Lauryl alcohol is one of the metabolites in the entomopathogenic fungus Conidiobolus coronatus that can affect the fatty acid composition and cellular immunity of the greater wax moth (Galleria mellonella) and blue blow flies (Calliphora vicina), suggesting that lauryl alcohol may play an important role during C. coronatus infection. These observations raise the possibility that GZM might directly affect pest physiology. However, this hypothesis is not consistent with our observations in the field, where we failed to detect any effect for GZM on predatory insects or insect pollination, suggesting that lauryl alcohol may influence insect food intake instead. In this study, we reported that GZM treatment induces the accumulation of several phytohormones, such as gibberellin (Figure 4A), and antioxidant enzymes (Figures 3Q, 3R and 3T), which are important for plant defenses against biotic and abiotic stresses.,,,,

The most direct mechanism of the effect of GZM on plant physiology has not been clarified. Whether GZM is recognized by plant receptors or is directly involved in specific physiological metabolic pathways in treated plants will need further exploration.



Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Guohui Yu (ygh76411@zhku.edu.cn).

This study did not generate new unique reagents.

Plants of A. hypogaea L. (Yuhua 9805), O. sativa L. (Huanghua Zhan), T. aestivum L. (Yunong 211), N. benthamiana, S. lycopersicum (Money Maker), and G. max L. (Huachun No.1) were grown (26 ± 2°C with a 16-h light/8-h dark photoperiod and 70–80% relative humidity) in the greenhouse. A. thaliana plants (accession Columbia-0 [Col-0]) were grown under different conditions (21 ± 2°C with a 14-h light/10-h dark photoperiod and 70% relative humidity) in an artificial climate box. F. oxysporum f. sp. cubense (FOC009) and S. rolfsii (ZHKSR) were cultured on PDA medium at 30°C.

For F. oxysporum f. sp. cubense and S. rolfsii, one piece (5 mm in diameter) of agar plate covered with a fresh fungal mass was cut with a hole puncher and placed in the center of a PDA (Potato Dextrose Agar) plate (with different concentrations of reagents or an equivalent volume of sterile water). Plates were incubated at 30°C until the mycelium of the control group filled the Petri plate; the diameter of the mycelial cover was then measured in each treatment group and representative plates were photographed.

Stock solutions of GZM or its constituent reagents were used at the same concentrations; lauryl alcohol (1.3 M), 1-hexadecanol (123.7 mM), and Tween 80 (23.3 mM). Sterile water was used as the solvent for the stock solutions and for dilution. A series of diluted GZM stock solutions were used in this study. For 400× dilution of GZM, the final concentrations of the constituent reagents were 3.3 mM lauryl alcohol, 309.3 μM 1-hexadecanol, and 58.3 μM Tween 80. For 800× dilution of GZM, the final concentrations of the constituent reagents were 1.6 mM lauryl alcohol, 154.7 μM 1-hexadecanol, and 29.1 μM Tween 80. For 1,000× dilution of GZM, the final concentrations of the constituent reagents were 1.3 mM lauryl alcohol, 123.7 μM 1-hexadecanol, and 23.3 μM Tween 80. For 1,600× dilution of GZM, the final concentrations of the constituent reagents were 815.0 μM lauryl alcohol, 77.3 μM 1-hexadecanol, and 14.5 μM Tween 80. For peanut seeds treated under laboratory conditions, seeds (n = 390) (cultivar Yuhua 9805, 18 months after collection) were soaked in a series of diluted GZM stock solutions (400x, 800x or 1,600x dilutions of stock solutions with sterile water) or 800x dilutions of lauryl alcohol stock solution for 1 min, surface-dried, and then sown on soil under normal growth conditions, or seeds were half soaked in 15% (w/v) PEG2000 solution or 1.5% (w/v) NaCl solution for drought and high-salt pressure growth conditions. Seeds soaked in sterile water or Tween 80 (29.1 μM) served as the control (CK) groups. Seedling growth was analyzed on days 3, 5 and 7 after seed germination. Root lengths of seedlings or plants were measured. In actual production practice, peanut and soybean seeds were coated with GZM solution (400x dilution) as treatment group or soaked in sterile water as the control (CK) group before being sown in farmland. Three biological replicates were conducted each time. For the treatment of leaves under laboratory conditions, the leaves of 4- to 5-week-old plants (n = 18) were fully sprayed with 1,000x diluted GZM (corresponding to 1.3 mM lauryl alcohol, 123.7 μM 1-hexadecanol, and 23.3 μM Tween 80) or lauryl alcohol (corresponding to 1.3 mM lauryl alcohol and 23.3 μM Tween 80) by manual atomizing spray. Leaves sprayed with sterile water or Tween 80 (23.3 μM) served as the control (CK) groups. Leaves were sprayed again 48 h later. Six leaves with similar status per plant were cut 72 h after the first spraying and quickly frozen in liquid nitrogen for 5 min and then transferred to a dry ice environment for storage until use. For the growth inhibition tests of microorganisms (F. oxysporum f. sp. cubense and S. rolfsii), different concentrations of reagents or an equivalent volume of sterile water were mixed with the agar medium before pouring the plates. During the test using the Oxford cup method, 0.1 mL diluted solution or an equivalent volume of sterile water was added to the corresponding cup. For the treatment of GZM on annual crops during field production, seeds were coated with GZM solution (400× dilution, corresponding to 3.3 mM lauryl alcohol, 309.3 μM 1-hexadecanol, and 58.3 μM Tween 80) as treatment group or sterile water as the control (CK) group before being sown; the leaves of established plants were then sprayed with 1,000x diluted GZM (corresponding to 1.3 mM lauryl alcohol, 123.7 μM 1-hexadecanol, and 23.3 μM Tween 80) four times at the seedling stage, flowering stage, seed setting stage, and seed mature phase, with a vapor-pressure type knapsack sprayer. For fruit production in the field, the same concentration of GZM solution was sprayed four times on leaves and fruits during the new bud formation stage, flowering stage, fruit setting stage, and fruit mature phase. Plants were sprayed with sterile water as the control (CK) group. Plants (n = 18) in different groups were randomly selected, and plant tissues and soil samples were collected 1 week before harvest (1 week after the fourth spraying) in field production, quickly frozen in liquid nitrogen for 5 min, and then transferred to a dry ice environment for storage until use. Field experiments of peanut (A. hypogaea L.), soybean (G. max L.), and navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) were performed in Zhangshi Town, Shaoguan City, Guangdong Province, China. Field production experiments for Citrus fruits were conducted in Tangtang Town, Guangzhou City, Guangdong Province, China. Experiments with Japanese plum (P. salicina) were performed in Dungang Town, Shaoguan City, Guangdong Province, China. Field experiments with tobacco (K326) and rice were performed in Mashi Town, Shaoguan City, Guangdong Province, China.

Samples were collected and immediately frozen in liquid nitrogen for 5 min and then transferred to a dry ice environment for storage until the determination of all omics data and phytohormone contents. The processing and testing of the samples were completed by MetWare (Wuhan, China) (http://www.metware.cn/). For RNA extraction, library construction, and sequencing, Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for total RNA extraction from all samples. RNA integrity was assessed using an RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). mRNA was isolated with oligo(dT) beads, then fragmented and reverse-transcribed into first-strand cDNA with random primers. The second strand of cDNAs was produced using DNA polymerase I, then purified with a QiaQuick PCR extraction kit (Qiagen, Hilden, Germany). The products were selected by electrophoresis and PCR-amplified with Phusion High-Fidelity DNA polymerase, Index (X) Primer, and Universal PCR primers. The PCR products were purified using AMPure XP beads and clustered with a cBot Cluster Generation System, according to the manufacturer’s instructions. Libraries were sequenced on an Illumina Hiseq platform, as paired-end reads of 125–150 bp. The raw data were filtered using fastp v 0.19.3. HISAT v2.1.0 was used to construct the demultiplex index and map the clean reads to the reference genome. The alignment and FPKM of each gene were calculated using featureCounts v1.6.2. For sample preparation and extraction of widely targeted metabolomics, vacuum freeze-drying was used; the samples were placed in a lyophilizer (Scientz-100F) and then ground (30 Hz, 1.5 min) to powder form with a grinder (MM 400, Retsch). Then, 50 mg of each sample was added to 1,200 μL 70% (v/v) methanol pre-chilled to −20°C. Extracts were vortexed once for 30 s every 30 min, for a total of 6 times. After centrifugation at 10,000 g for 3 min (4°C), the supernatant was transferred to a new tube, filtered using a microporous membrane (0.22-μm pore size) and stored in an injection vial for UPLC-MS/MS analysis. The extracts were analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLC AD, https://sciex.com.cn/; MS, Applied Biosystems 4500 Q TRAP, https://sciex.com.cn/). The analytical conditions were as follows: UPLC column, Agilent SB-C18 (1.8 μm, 2.1 mm ∗ 100 mm). l-2-chlorophenylalanine was used as the internal standard (Burlingway, Cas No: 103616-89-3, 1 ppm). The mobile phase consisted of solvent A, pure water with 0.1% (v/v) formic acid, and solvent B, acetonitrile with 0.1% (v/v) formic acid. Sample measurements were performed with a gradient program with starting conditions of 95% A, 5% B. Within 9 min, a linear gradient to 5% A, 95% B was programmed, and a composition of 5% A, 95% B was kept for 1 min. Subsequently, a composition of 95% A, 5.0% B was adjusted within 1.1 min and kept for 2.9 min. The flow velocity was set as 0.35 mL per min; The column oven was set to 40°C; The injection volume was 4 μL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS. For ESI-Q TRAP-MS/MS analysis, the ESI source operation parameters were as follows: source temperature 550°C; ion spray voltage (IS) 5500 V (positive ion mode)/–4500 V (negative ion mode); ion source gas I (GSI) 50 psi; gas II (GSII) 60 psi; curtain gas (CUR) 25 psi; collision-activated dissociation (CAD) high. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to medium. DP (declustering potential) and CE (collision energy) for individual MRM transitions was done with further DP and CE optimization. A specific set of MRM transitions was monitored for each period according to the metabolites eluted within this period. Both hierarchical cluster analysis (HCA) and Pearson’s correlation coefficient (PCC) analysis of the samples and metabolites were carried out using the ComplexHeatmap package in R. For HCA, a color spectrum was used for normalizing the signal intensities of the metabolites. Variable importance in projection (VIP) scores >1 and absolute Log2FC (|Log2FC|) ≥ 1.0 were used to determine differentially abundant metabolites. The VIP values were extracted from the result of Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA). Genes with transcript levels or metabolites with contents that showed an absolute FC ≥ 2 with a false discovery rate (FDR) of 0.05 were regarded as differentially expressed genes (DEGs) or differentially abundant metabolites, respectively. KOBAS software was used to determine the statistical enrichment of terms from Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Significantly enriched KEGG pathways were defined with an FDR of 0.05 as threshold. The analysis of the rhizosphere microbiome was conducted using the Uparse algorithm (Uparse v7.0.1001, http://www.drive5.com/uparse/) to cluster all effective tags across all samples. By default, sequences were clustered into operational taxonomic units (OTUs) with at least 97% identity. Representative OTU sequences were selected. The sequence with the highest frequency in OTUs was selected as the representative sequence of OTUs and then annotated with the species name by Mothur method and SILVA138 (http://www.arb-silva.de/) (with a threshold set to 0.8–1)., For determination of phytohormone contents, the samples were placed in a lyophilizer (Scientz-100F) and then ground (30 Hz, 1.5 min) to a powder with a grinder (MM 400, Retsch). Then, 50 mg (fresh weight) of each sample was extracted using methanol:water:formic acid (15:4:1, v/v/v). Under a nitrogen gas stream, the extracts were evaporated to dryness, followed by reconstitution in 80% (v/v) methanol and filtration with polytetrafluoroethylene with a pore size of 0.22 μm. The QTRAP 6500 LC-MS/MS platform (SCIEX, Macclesfield, UK) was used to analyze the extracts of each sample. The qualitative analysis of sample data was based on a standard sample database (Metware Biotechnology, MWDB). Quantification was performed through the multiple reaction monitoring mode (MRM) of triple quadrupole mass spectrometry.

The proportion of disease spots on peanut leaves was scored in ImageJ. To score the refusal of S. litura to eat leaves, the leaves of 4- to 5-week-old peanut plants (n = 18) were fully sprayed with a 1,000x diluted GZM solution (corresponding to 1.3 mM lauryl alcohol, 123.7 μM 1-hexadecanol, and 23.3 μM Tween 80) by manual atomizing spray. Leaves sprayed with sterile water served as the control (CK) groups. Leaves were sprayed again 48 h later. After 72 h from the first spraying, six leaves with similar status per plant were collected. Five S. litura third-stage larvae were placed into a feeding box without food for 2 h to induce starvation. Six leaf blades for the treatment group and six blades for the CK (water) group were placed in far corners of the feeding box at the same time. After 12 h of feeding under dark conditions, chewing marks on the leaves were recorded and converted to a five-level scale (level 1, leaves were not eaten; level 2, slight feeding traces and 1–2 feeding plaques; level 3, slight feeding traces and 3–4 feeding plaques; level 4; 4–5 feeding traces, with obvious feeding traces; level 5, clear outline and large area of feeding plaques, and more than 5 feeding traces). Six biological replicates were conducted, and the experiments were carried out as three independent biological repeats. The contents for proline and malondialdehyde (MDA) in fresh leaves were determined with kits (proline, A107-1-1; MDA, A003-1; Nanjing Jiancheng Bioengineering Institute). For the determination of SOD and POD enzyme activities in fresh leaves, kits A001-3 (SOD) and A084-3 (POD) were used (Nanjing Jiancheng Bioengineering Institute). A catalase (CAT) activity assay kit (D799598, Sangon Biotech) was used.

The leaves of 3-week-old peanut seedlings (n = 12) were fully sprayed with a 1,000x diluted GZM solution (corresponding to 1.3 mM lauryl alcohol, 123.7 μM 1-hexadecanol, and 23.3 μM Tween 80) by manual atomizing spray. Leaves sprayed with sterile water served as the control (CK) groups. Leaves were sprayed again at 5 and 7 weeks after seed germination. Then, leaves with similar status per plant were collected 1 week after the third spraying, immersed immediately in fixative solution (G1102-100 ML, Servicebio) for 2 h at room temperature, then transferred to 4°C for preservation. Tissues were washed with 0.1 M phosphate buffer (PB, pH 7.4) 3 times for 15 min each. Then tissues were transferred into 1% (w/v) OsO4 in 0.1 MPB (pH 7.4) for 1–2 h at room temperature. Tissues were then washed in 0.1 MPB (pH 7.4) 3 times for 15 min each. The samples were gradually dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, 100%, 100%, all v/v) for 15 min each time and isoamyl acetate for 15 min. Samples were dried with a critical point dryer, followed by sputter-coating with gold for 30 s. Photographs were taken on an SEM Hitachi SU8100.

Unless stated otherwise, at least three replicates were conducted per experiment, and representative results are presented. Statistical analysis was performed in Microsoft Excel (version Office 365) and GraphPad Prism (version 8.02). Levene’s and Shapiro-Wilk tests were used to assess group homoscedasticity and normality, respectively. Statistical significance was set to p ≤ 0.05. Duncan’s test were used to quantify statistically significant differences between datasets. All p-values are presented on the compared groups, with results considered statistically significant when p < 0.05.