Comparative proteomic analysis of resistant and susceptible cotton genotypes in response to leaf hopper infestation.

The cotton leaf hopper is a major pest in cotton, causing a hopper burn in leaves. In this study, a comparative proteomic analysis of NDLH2010 (Resistant) and LRA5166 (Susceptible), infected with leaf hopper, was employed using a nano LC-MS/MS approach. A total of 1402 proteins varied significantly between leaf hopper-infected and control plants. The resistant and susceptible genotypes had differentially expressed proteins (DEPs) of 743 and 659, respectively. Functional annotation of DEPs revealed that the DEPs were primarily associated with stress response, hormone synthesis, photosynthesis, cell wall, and secondary metabolites. Notably, DEPs such as polyphenol oxidase, carboxypeptidase, heat shock proteins, protein BTR1-like isoform X2, chaperone protein ClpB1, and ß glucosidase factors associated with environmental stress response were also detected. Quantitative real-time PCR (qRT-PCR) analysis confirmed a positive correlation between protein abundances and transcripts for all genes. Collectively, this study provides the molecular mechanisms associated with cotton defense responses against leaf hopper. SIGNIFICANCE STATEMENT: Cotton, a natural fiber, assumes a pivotal role as a raw material for textile industries, thereby bearing significant importance in the global economy. The cotton production sector is considerably affected by both biotic and abiotic stresses. The cotton leaf hopper (Amrasca biguttula biguttula (Ishida)) stands as a polyphagous insect, emerging as a dominant sap-feeding pest of the cotton crop. The continuous onslaught of sap-feeding insects on cotton plants has a detrimental impact, with leaf hoppers potentially causing yield reductions of up to 50%. Therefore, comprehending the molecular interplay between cotton and leaf hopper, elucidated at the proteome level, holds promise for more effective pest management strategies. This approach holds the potential to offer insights that contribute to the development of leaf hopper-resistant cotton varieties.

Ecotoxicity of Cadmium along the Soil-Cotton Plant-Cotton Bollworm System: Biotransfer, Trophic Accumulation, Plant Growth, Induction of Insect Detoxification Enzymes, and Immunocompetence.

Cadmium (Cd) is a hazardous element that may jeopardize environmental safety and human health through biotransfer and trophic accumulation. Here, we tested Cd toxicity on cotton plants, cotton bollworms, and their responses. Results demonstrated that Cd accumulated in plant roots, aerial parts, insect larvae, pupae, and frass in a dose-dependent pattern. The ∼9.35 mg kg-1 of Cd in plant aerial parts, ∼3.68 in larvae, ∼6.43 in pupae, and high transfer coefficient (∼5.59) indicate significant mobility. The ∼19.61 mg kg-1 of Cd in larvae frass suggests an effective detoxification strategy, while BAFcotton (∼1.14) and BAFworm (∼0.54) indicated low bioaccumulation. Cadmium exposure resulted in compromised plant growth and yield as well as alterations in photosynthetic pigment contents, antioxidant enzyme activities, and certain life history traits of cotton bollworms. Furthermore, carboxylesterase activity and encapsulation rates of insect larvae decreased with increasing Cd concentrations, whereas acetylcholinesterase, phenol oxidase, glutathione S-transferase, and multifunctional oxidase exhibited hormesis responses.

Genome-wide analyses of member identification, expression pattern, and protein-protein interaction of EPF/EPFL gene family in Gossypium.

BACKGROUND: Epidermal patterning factor / -like (EPF/EPFL) gene family encodes a class of cysteine-rich secretory peptides, which are widelyfound in terrestrial plants.Multiple studies has indicated that EPF/EPFLs might play significant roles in coordinating plant development and growth, especially as the morphogenesis processes of stoma, awn, stamen, and fruit skin. However, few research on EPF/EPFL gene family was reported in Gossypium. RESULTS: We separately identified 20 G. raimondii, 24 G. arboreum, 44 G. hirsutum, and 44 G. barbadense EPF/EPFL genes in the 4 representative cotton species, which were divided into four clades together with 11 Arabidopsis thaliana, 13 Oryza sativa, and 17 Selaginella moellendorffii ones based on their evolutionary relationships. The similar gene structure and common motifs indicated the high conservation among the EPF/EPFL members, while the uneven distribution in chromosomes implied the variability during the long-term evolutionary process. Hundreds of collinearity relationships were identified from the pairwise comparisons of intraspecifc and interspecific genomes, which illustrated gene duplication might contribute to the expansion of cotton EPF/EPFL gene family. A total of 15 kinds of cis-regulatory elements were predicted in the promoter regions, and divided into three major categories relevant to the biological processes of development and growth, plant hormone response, and abiotic stress response. Having performing the expression pattern analyses with the basic of the published RNA-seq data, we found most of GhEPF/EPFL and GbEPF/EPFL genes presented the relatively low expression levels among the 9 tissues or organs, while showed more dramatically different responses to high/low temperature and salt or drought stresses. Combined with transcriptome data of developing ovules and fibers and quantitative Real-time PCR results (qRT-PCR) of 15 highly expressed GhEPF/EPFL genes, it could be deduced that the cotton EPF/EPFL genes were closely related with fiber development. Additionally, the networks of protein-protein interacting among EPF/EPFLs concentrated on the cores of GhEPF1 and GhEPF7, and thosefunctional enrichment analyses indicated that most of EPF/EPFLs participate in the GO (Gene Ontology) terms of stomatal development and plant epidermis development, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of DNA or base excision repair. CONCLUSION: Totally, 132 EPF/EPFL genes were identified for the first time in cotton, whose bioinformatic analyses of cis-regulatory elements and expression patterns combined with qRT-PCR experiments to prove the potential functions in the biological processes of plant growth and responding to abiotic stresses, specifically in the fiber development. These results not only provide comprehensive and valuable information for cotton EPF/EPFL gene family, but also lay solid foundation for screening candidate EPF/EPFL genes in further cotton breeding.

Promotion of apoplastic oxidative burst by artificially selected GhCBSX3A enhances Verticillium dahliae resistance in upland cotton.

Verticillium wilt (VW) is a devasting disease affecting various plants, including upland cotton, a crucial fiber crop. Despite its impact, the genetic basis underlying cotton's susceptibility or defense against VW remains unclear. Here, we conducted a genome-wide association study on VW phenotyping in upland cotton and identified a locus on A13 that is significantly associated with VW resistance. We then identified a cystathionine ß-synthase domain gene at A13 locus, GhCBSX3A, which was induced by Verticillium dahliae. Functional analysis, including expression silencing in cotton and overexpression in Arabidopsis thaliana, confirmed that GhCBSX3A is a causal gene at the A13 locus, enhancing SAR-RBOHs-mediated apoplastic oxidative burst. We found allelic variation on the TATA-box of GhCBSX3A promoter attenuated its expression in upland cotton, thereby weakening VW resistance. Interestingly, we discovered that altered artificial selection of GhCBSX3A_R (an elite allele for VW) under different VW pressures during domestication and other improved processes allows specific human needs to be met. Our findings underscore the importance of GhCBSX3A in response to VW, and we propose a model for defense-associated genes being selected depending on the pathogen's pressure. The identified locus and gene serve as promising targets for VW resistance enhancement in cotton through genetic engineering.

GHCU, a Molecular Chaperone, Regulates Leaf Curling by Modulating the Distribution of KNGH1 in Cotton.

Leaf shape is considered to be one of the most significant agronomic traits in crop breeding. However, the molecular basis underlying leaf morphogenesis in cotton is still largely unknown. In this study, through genetic mapping and molecular investigation using a natural cotton mutant cu with leaves curling upward, the causal gene GHCU is successfully identified as the key regulator of leaf flattening. Knockout of GHCU or its homolog in cotton and tobacco using CRISPR results in abnormal leaf shape. It is further discovered that GHCU facilitates the transport of the HD protein KNOTTED1-like (KNGH1) from the adaxial to the abaxial domain. Loss of GHCU function restricts KNGH1 to the adaxial epidermal region, leading to lower auxin response levels in the adaxial boundary compared to the abaxial. This spatial asymmetry in auxin distribution produces the upward-curled leaf phenotype of the cu mutant. By analysis of single-cell RNA sequencing and spatiotemporal transcriptomic data, auxin biosynthesis genes are confirmed to be expressed asymmetrically in the adaxial-abaxial epidermal cells. Overall, these findings suggest that GHCU plays a crucial role in the regulation of leaf flattening through facilitating cell-to-cell trafficking of KNGH1 and hence influencing the auxin response level.

A Genome-Wide Analysis of the CEP Gene Family in Cotton and a Functional Study of GhCEP46-D05 in Plant Development.

C-TERMINALLY ENCODED PEPTIDEs (CEPs) are a class of peptide hormones that have been shown in previous studies to play an important role in regulating the development and response to abiotic stress in model plants. However, their role in cotton is not well understood. In this study, we identified 54, 59, 34, and 35 CEP genes from Gossypium hirsutum (2n = 4x = 52, AD1), G. barbadense (AD2), G. arboreum (2n = 2X = 26, A2), and G. raimondii (2n = 2X = 26, D5), respectively. Sequence alignment and phylogenetic analyses indicate that cotton CEP proteins can be categorized into two subgroups based on the differentiation of their CEP domain. Chromosomal distribution and collinearity analyses show that most of the cotton CEP genes are situated in gene clusters, suggesting that segmental duplication may be a critical factor in CEP gene expansion. Expression pattern analyses showed that cotton CEP genes are widely expressed throughout the plant, with some genes exhibiting specific expression patterns. Ectopic expression of GhCEP46-D05 in Arabidopsis led to a significant reduction in both root length and seed size, resulting in a dwarf phenotype. Similarly, overexpression of GhCEP46-D05 in cotton resulted in reduced internode length and plant height. These findings provide a foundation for further investigation into the function of cotton CEP genes and their potential role in cotton breeding.

The proteomic landscape of fall armyworm oral secretion reveals its role in plant adaptation.

BACKGROUND: The fall armyworm (FAW, Spodoptera frugiperda (J.E. Smith)) is a polyphagous agricultural pest with rapidly evolving adaptations to host plants. We found the oral secretion (OS) of FAW from different plants influences plant defense response differentially, suggesting its role in adapting to host plants. However, the protein expression profile of FAW OS respond to different plants is largely unknown. RESULTS: Here, from the mass spectrometry assay, we identified a total of 256 proteins in the OS of FAW fed on cotton (Gossypium hirsutum L.), tobacco (Nicotiana benthamiana Domin), maize (Zea mays L.) and artificial diet. The FAW OS primarily comprise of 60 proteases, 32 esterases and 92 non-enzymatic proteins. It displays high plasticity across different diets. We found that more than half of the esterases are lipases which have been reported as insect elicitors to enhance plant defense response. The lipase accumulation in cotton-fed larvae was the highest, followed by maize-fed larvae. In the presence of lipase inhibitors, the enhanced induction on defense genes in wounded leaves by OS was attenuated. However, the putative effectors were most highly accumulated in the OS from FAW larvae fed on maize compared to those fed on other diets. We identified that one of them (VRLP4) reduces the OS-mediated induction on defense genes in wounded leaves. CONCLUSION: Together, our investigation presents the proteomic landscape of the OS of FAW influenced by different diets and reveals diet-mediated plasticity of OS is involved in FAW adaptation to host plants. © 2024 Society of Chemical Industry.

Genome-Wide and Expression Pattern Analysis of the HIT4 Gene Family Uncovers the Involvement of GHHIT4_4 in Response to Verticillium Wilt in Gossypium hirsutum.

Chromatin remodelers are essential for regulating plant growth, development, and responses to environmental stresses. HIT4 (HEAT-INTOLERANT 4) is a novel stress-induced chromatin remodeling factor that has been less studied in abiotic stress and stress resistance, particularly in cotton. In this study, we conducted a comprehensive analysis of the members of the HIT4 gene family in Gossypium hirsutum using bioinformatics methods, including phylogenetic relationships, gene organization, transcription profiles, phylogenetic connections, selection pressure, and stress response. A total of 18 HIT4 genes were identified in four cotton species, with six HIT4 gene members in upland cotton. Based on the evolutionary relationships shown in the phylogenetic tree, the 18 HIT4 protein sequences were classified into four distinct subgroups. Furthermore, we conducted chromosome mapping to determine the genomic locations of these genes and visually represented the structural characteristics of HIT4 in G. hirsutum. In addition, we predicted the regulatory elements in HIT4 in G. hirsutum and conducted an analysis of repetitive sequences and gene collinearity among HIT4 in four cotton species. Moreover, we calculated the Ka/Ks ratio for homologous genes to assess the selection pressure acting on HIT4. Using RNA-seq, we explored the expression patterns of HIT4 genes in G. hirsutum and Gossypium barbadense. Through weighted gene co-expression network analysis (WGCNA), we found that GHHIT4_4 belonged to the MEblue module, which was mainly enriched in pathways such as DNA replication, phagosome, pentose and glucuronate interconversions, steroid biosynthesis, and starch and sucrose metabolism. This module may regulate the mechanism of upland cotton resistance to Verticillium wilt through DNA replication, phagosome, and various metabolic pathways. In addition, we performed heterologous overexpression of GH_D11G0591 (GHHIT4_4) in tobacco, and the results showed a significant reduction in disease index compared to the wild type, with higher expression levels of disease resistance genes in the transgenic tobacco. After conducting a VIGS (virus-induced gene silencing) experiment in cotton, the results indicated that silencing GHHIT4_4 had a significant impact, the resistance to Verticillium wilt weakened, and the internode length of the plants significantly decreased by 30.7% while the number of true leaves increased by 41.5%. qRT-PCR analysis indicated that GHHIT4_4 mainly enhanced cotton resistance to Verticillium wilt by indirectly regulating the PAL, 4CL, and CHI genes. The subcellular localization results revealed that GHHIT4_4 was predominantly distributed in the mitochondria and nucleus. This study offers preliminary evidence for the involvement of the GHHIT4_4 in cotton resistance to Verticillium wilt and lays the foundation for further research on the disease resistance mechanism of this gene in cotton.

StoMYB41 positively regulates the Solanum torvum response to Verticillium dahliae in an ABA dependent manner.

MYB transcription factor despite their solid involvement in growth are potent regulator of plant stress response. Herein, we identified a MYB gene named as StoMYB41 in a wild eggplant species Solanum torvum. The expression level of StoMYB41 was higher in root than the tissues including stem, leaf, and seed. It induced significantly by Verticillium dahliae inoculation. StoMYB41 was localized in the nucleus and exhibited transcriptional activation activity. Silencing of StoMYB41 enhanced susceptibility of Solanum torvum against Verticillium dahliae, accompanied by higher disease index. The significant down-regulation of resistance marker gene StoABR1 comparing to the control plants was recorded in the silenced plants. Moreover, transient expression of StoMYB41 could trigger intense hypersensitive reaction mimic cell death, darker DAB and trypan blue staining, higher ion leakage, and induced the expression levels of StoABR1 and NbDEF1 in the leaves of Solanum torvum and Nicotiana benthamiana. Taken together, our data indicate that StoMYB41 acts as a positive regulator in Solanum torvum against Verticillium wilt.

GhRCD1 promotes cotton tolerance to cadmium by regulating the GhbHLH12-GhMYB44-GhHMA1 transcriptional cascade.

Heavy metal pollution poses a significant risk to human health and wreaks havoc on agricultural productivity. Phytoremediation, a plant-based, environmentally benign, and cost-effective method, is employed to remove heavy metals from contaminated soil, particularly in agricultural or heavy metal-sensitive lands. However, the phytoremediation capacity of various plant species and germplasm resources display significant genetic diversity, and the mechanisms underlying these differences remain hitherto obscure. Given its potential benefits, genetic improvement of plants is essential for enhancing their uptake of heavy metals, tolerance to harmful levels, as well as overall growth and development in contaminated soil. In this study, we uncover a molecular cascade that regulates cadmium (Cd2+) tolerance in cotton, involving GhRCD1, GhbHLH12, GhMYB44, and GhHMA1. We identified a Cd2+-sensitive cotton T-DNA insertion mutant with disrupted GhRCD1 expression. Genetic knockout of GhRCD1 by CRISPR/Cas9 technology resulted in reduced Cd2+ tolerance in cotton seedlings, while GhRCD1 overexpression enhanced Cd2+ tolerance. Through molecular interaction studies, we demonstrated that, in response to Cd2+ presence, GhRCD1 directly interacts with GhbHLH12. This interaction activates GhMYB44, which subsequently activates a heavy metal transporter, GhHMA1, by directly binding to a G-box cis-element in its promoter. These findings provide critical insights into a novel GhRCD1-GhbHLH12-GhMYB44-GhHMA1 regulatory module responsible for Cd2+ tolerance in cotton. Furthermore, our study paves the way for the development of elite Cd2+-tolerant cultivars by elucidating the molecular mechanisms governing the genetic control of Cd2+ tolerance in cotton.

An insight into the gene expression evolution in Gossypium species based on the leaf transcriptomes.

BACKGROUND: Gene expression pattern is associated with biological phenotype and is widely used in exploring gene functions. Its evolution is also crucial in understanding species speciation and divergence. The genus Gossypium is a bona fide model for studying plant evolution and polyploidization. However, the evolution of gene expression during cotton species divergence has yet to be extensively discussed. RESULTS: Based on the seedling leaf transcriptomes, this work analyzed the transcriptomic content and expression patterns across eight cotton species, including six diploids and two natural tetraploids. Our findings indicate that, while the biological function of these cotton transcriptomes remains largely conserved, there has been significant variation in transcriptomic content during species divergence. Furthermore, we conducted a comprehensive analysis of expression distances across cotton species. This analysis lends further support to the use of G. arboreum as a substitute for the A-genome donor of natural cotton polyploids. Moreover, our research highlights the evolution of stress-responsive pathways, including hormone signaling, fatty acid degradation, and flavonoid biosynthesis. These processes appear to have evolved under lower selection pressures, presumably reflecting their critical role in the adaptations of the studied cotton species to diverse environments. CONCLUSIONS: In summary, this study provided insights into the gene expression variation within the genus Gossypium and identified essential genes/pathways whose expression evolution was closely associated with the evolution of cotton species. Furthermore, the method of characterizing genes and pathways under unexpected high or slow selection pressure can also serve as a new strategy for gene function exploration.

The importance of species-specific and temperature-sensitive parameterisation of A/Ci models: A case study using cotton (Gossypium hirsutum L.) and the automated 'OptiFitACi' R-package.

Leaf gas exchange measurements are an important tool for inferring a plant's photosynthetic biochemistry. In most cases, the responses of photosynthetic CO2 assimilation to variable intercellular CO2 concentrations (A/Ci response curves) are used to model the maximum (potential) rate of carboxylation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, Vcmax) and the rate of photosynthetic electron transport at a given incident photosynthetically active radiation flux density (PAR; JPAR). The standard Farquhar-von Caemmerer-Berry model is often used with default parameters of Rubisco kinetic values and mesophyll conductance to CO2 (gm) derived from tobacco that may be inapplicable across species. To study the significance of using such parameters for other species, here we measured the temperature responses of key in vitro Rubisco catalytic properties and gm in cotton (Gossypium hirsutum cv. Sicot 71) and derived Vcmax and J2000 (JPAR at 2000 µmol m-2 s-1 PAR) from cotton A/Ci curves incrementally measured at 15°C-40°C using cotton and other species-specific sets of input parameters with our new automated fitting R package 'OptiFitACi'. Notably, parameterisation by a set of tobacco parameters produced unrealistic J2000:Vcmax ratio of <1 at 25°C, two- to three-fold higher estimates of Vcmax above 15°C, up to 2.3-fold higher estimates of J2000 and more variable estimates of Vcmax and J2000, for our cotton data compared to model parameterisation with cotton-derived values. We determined that errors arise when using a gm,25 of 2.3 mol m-2 s-1 MPa-1 or less and Rubisco CO2-affinities in 21% O2 (KC 21%O2) at 25°C outside the range of 46-63 Pa to model A/Ci responses in cotton. We show how the A/Ci modelling capabilities of 'OptiFitACi' serves as a robust, user-friendly, and flexible extension of 'plantecophys' by providing simplified temperature-sensitivity and species-specificity parameterisation capabilities to reduce variability when modelling Vcmax and J2000.

Genome-Wide and Expression Pattern Analysis of the DVL Gene Family Reveals GhM_A05G1032 Is Involved in Fuzz Development in G. hirsutum.

DVL is one of the small polypeptides which plays an important role in regulating plant growth and development, tissue differentiation, and organ formation in the process of coping with stress conditions. So far, there has been no comprehensive analysis of the expression profile and function of the cotton DVL gene. According to previous studies, a candidate gene related to the development of fuzz was screened, belonging to the DVL family, and was related to the development of trichomes in Arabidopsis thaliana. However, the comprehensive identification and systematic analysis of DVL in cotton have not been conducted. In this study, we employed bioinformatics approaches to conduct a novel analysis of the structural characteristics, phylogenetic tree, gene structure, expression pattern, evolutionary relationship, and selective pressure of the DVL gene family members in four cotton species. A total of 117 DVL genes were identified, including 39 members in G. hirsutum. Based on the phylogenetic analysis, the DVL protein sequences were categorized into five distinct subfamilies. Additionally, we successfully mapped these genes onto chromosomes and visually represented their gene structure information. Furthermore, we predicted the presence of cis-acting elements in DVL genes in G. hirsutum and characterized the repeat types of DVL genes in the four cotton species. Moreover, we computed the Ka/Ks ratio of homologous genes across the four cotton species and elucidated the selective pressure acting on these homologous genes. In addition, we described the expression patterns of the DVL gene family using RNA-seq data, verified the correlation between GhMDVL3 and fuzz development through VIGS technology, and found that some DVL genes may be involved in resistance to biotic and abiotic stress conditions through qRT-PCR technology. Furthermore, a potential interaction network was constructed by WGCNA, and our findings demonstrated the potential of GhM_A05G1032 to interact with numerous genes, thereby playing a crucial role in regulating fuzz development. This research significantly contributed to the comprehension of DVL genes in upland cotton, thereby establishing a solid basis for future investigations into the functional aspects of DVL genes in cotton.

Identification, evolution, and expression of GDSL-type Esterase/Lipase (GELP) gene family in three cotton species: a bioinformatic analysis.

BACKGROUND: GDSL esterase/lipases (GELPs) play important roles in plant growth, development, and response to biotic and abiotic stresses. Presently, an extensive and in-depth analysis of GELP family genes in cotton is still not clear enough, which greatly limits the further understanding of cotton GELP function and regulatory mechanism. RESULTS: A total of 389 GELP family genes were identified in three cotton species of Gossypium hirsutum (193), G. arboreum (97), and G. raimondii (99). These GELPs could be classified into three groups and eight subgroups, with the GELPs in same group to have similar gene structures and conserved motifs. Evolutionary event analysis showed that the GELP family genes tend to be diversified at the spatial dimension and certain conservative at the time dimension, with a trend of potential continuous expansion in the future. The orthologous or paralogous GELPs among different genomes/subgenomes indicated the inheritance from genome-wide duplication during polyploidization, and the paralogous GELPs were derived from chromosomal segment duplication or tandem replication. GELP genes in the A/D subgenome underwent at least three large-scale replication events in the evolutionary process during the period of 0.6-3.2 MYA, with two large-scale evolutionary events between 0.6-1.8 MYA that were associated with tetraploidization, and the large-scale duplication between 2.6-9.1 MYA that occurred during diploidization. The cotton GELPs indicated diverse expression patterns in tissue development, ovule and fiber growth, and in response to biotic and abiotic stresses, combining the existing cis-elements in the promoter regions, suggesting the GELPs involvements of functions to be diversification and of the mechanisms to be a hormone-mediated manner. CONCLUSIONS: Our results provide a systematic and comprehensive understanding the function and regulatory mechanism of cotton GELP family, and offer an effective reference for in-depth genetic improvement utilization of cotton GELPs.

Evaluation of sample extracting methods of FCSM by Lactobacillus acidophilus based on a UPLC-Q-TOF-MS global metabolomics analysis

Abstract The study of metabolomics requires extracting as many metabolites as possible from a biological sample. This study aimed to determine the optimal method for the extraction of metabolites from solid-state fermented cottonseed meal (FCSM). The UPLC-Q-TOF-MS global metabolomics technology was used to detect the metabolites in FCSM, and the extraction quantity and extraction efficiency of seven different extraction methods, specifically the WA, 50MeOH, 50MeOHB, 50MeCNB, 80MeOHB, 80MeOH and AMF methods were evaluated. The results showed that the number of VIP metabolites extracted by AMF method are 196 and 184 in ESI+ and ESI- mode respectively, it is the largest number of all exacted methods; and the AMF methods also provided a higher extraction efficiency compared with the other methods, especially in indoleacrylic acid, dl-tryptophan and epicatechin (p < 0.01). As a result, AMF/-4 °C method was identified as the best method for the extraction of metabolites from FCSM by Lactobacillus acidophilus. Our study establishes a technical basis for future metabolomics research of fermented feed.