Mulberry (Morus alba L.) is a woody perennial plant belonging to the Moraceae family that is highly adaptable and widely distributed throughout China. Parts of the mulberry plant, particularly the leaves, are frequently used in the practices of traditional Chinese medicine as they are rich in phenols, polysaccharides, alkaloids, and other phytochemicals such that they possess an array of anti-inflammatory, antioxidant, antidiabetic, antihelmintic, and antimicrobial properties (He et al., 2018; Zhang et al., 2018; Shan et al., 2022). Mulberry has also been regarded as a valuable source of forage for ruminants, as it exhibits high protein content, contains high levels of vitamins and minerals, and presents with a high biomass yield (Thaipitakwong et al., 2018). The high moisture content in fresh mulberry plants, however, makes them difficult to preserve, particularly during the rainy season in Southern China. While silage represents an effective approach to conserving fresh material, the fermentation process is complex and subject to numerous influencing factors, resulting in variability in silage quality that may require the incorporation of additives to properly manage the fermentation process (Yitbarek and Tamir, 2014). The generation of mulberry silage of superior quality remains difficult due to its low contents of water-soluble carbohydrates (WSC) and high buffering capacity (Wang et al., 2019a). There is thus a pressing need for the use of rational strategies to select appropriate additives that can be applied during ensiling in order to produce higher quality silage.

A variety of silage additives can be used to enhance fermentation, increase silage nutritional value, minimize losses, protect against aerobic deterioration, and thereby achieve improved aerobic stability (Yitbarek and Tamir, 2014). These additives can be broadly classified into stimulants and inhibitors of fermentation, inhibitors of aerobic deterioration, absorbents, and nutrients (McDonald et al., 1991). Glucose can serve as a fermentation stimulant by increasing the amount of fermentable sugar substrates accessible to lactic acid bacteria (LAB) during ensiling, supporting a rapid decrease in pH and enhanced lactic fermentation through the inhibition of proteolytic and butyric fermentation (Trabi et al., 2017). Li et al. (2014) found that glucose was able to improve king grass ensiling fermentation by facilitating the rapid accumulation of lactic acid during the early period of ensiling. LAB present in silage consist of homofermentative and heterofermentative LAB (Muck et al., 2018). Homofermentative LAB can rapidly and robustly reduce pH levels during ensiling through their conversion of WSC into lactic acid (Zhang et al., 2019). Lactobacillus buchneri is the dominant obligate heterofermentative LAB species used as a silage additive, functioning during the later stages of ensiling by the gradual conversion of lactic acid into acetic acid and 1,2-propanediol, thereby leading to enhanced aerobic stability (Muck et al., 2018). In addition, formic acid has been utilized as a fermentation inhibitor through its ability to achieve a rapid decline in pH through direct acidification, suppressing the growth of undesirable spoilage-associated microbes including enterobacteria and aerobic bacteria while allowing an optimal environment for more rapid LAB growth and improved silage preservation (Yitbarek and Tamir, 2014). Jiang et al. (2020) found that treating of whole-plant corn silage with formic acid, acetic acid, and potassium sorbate at a 7:1:2 ratio (6 L/t) was associated with increased lactic acid levels and a reduction in the levels of undesirable microbes including Klebsiella, Paenibacillus, and Enterobacter. Salts including sodium benzoate and potassium sorbate can effectively reduce the growth of yeast, molds, and spoilage-related bacteria in silage, thereby leading to increased aerobic stability. Knický and Spörndly (2009) found that when ensiling grasses with low dry matter (DM) content, sodium benzoate and potassium sorbate treatment were associated with higher lactic acid levels, together with lower levels of butyric acid and NH₃-N relative to untreated silage. This suggests that adding glucose may provide a means of overcoming the lack of WSC content in mulberry plants, while the application of LAB, organic acids, salts, or combinations thereof may help further improve fermentation quality for whole-plant mulberry silage.

Improvements in silage quality through the use of any additive types are ultimately attributable to effects on the activity of beneficial or undesirable microbes (Kung et al., 2018; Liu et al., 2019). The development of high-throughput sequencing has fueled a growing number of studies exploring how various additives impact microbial communities in stylo (He et al., 2021), amaranth (Zhao et al., 2022), paper mulberry (Cheng et al., 2022), and native grass (Li et al., 2022). As far as we know, previous studies mainly used mulberry leaves as raw materials to prepare silage, focusing on the effects of additives on chemical composition, fermentation quality and part of microorganisms, rather than the whole plant (He et al., 2019; Wang et al., 2019a; Dong et al., 2020; Wang C et al., 2020; Cui et al., 2022). Little remains known, however, on the influence of different types of additive on fermentation and variations in microbial communities associated with whole-plant mulberry silages. As such, the present study was conceived with the goal of assessing how glucose, LAB, organic acids, salts and mixtures thereof impact fermentation quality and bacterial communities during mulberry ensiling through a high-throughput sequencing approach. The findings provided by this study can serve as a reference for addressing feed shortages, enhancing the quality of forage feed, and improving the economic management of ruminant husbandry.

Greater CP and lower cell wall content (NDF, ADF, and ADL) can often serve as indicators associated with high-quality forage (Huang et al., 2019). Fresh whole-plant mulberry exhibited a CP content of 14.48% in this study, in line with values reported for traditional forage such as silage derived from alfalfa and soybean (Ni et al., 2018; Özüretmen et al., 2022). The higher CP contents, together with the low ADF and NDF content observed in whole-plant mulberry, suggests that it may be a valuable protein-rich forage option for ruminants. However, whole-plant mulberry exhibited lower CP content than that for mulberry leaves (Wang et al., 2019a; He et al., 2020), together with higher NDF and ADF content, potentially suggesting that the nutritional composition of mulberry plants varies across various parts of these plants. Relative to the fresh material, mulberry silages exhibited expected reductions in DM and WSC content. This is attributable to oxygen consumption by plant cells and aerobic microorganisms during the early stages of ensiling together with the fermentation of WSC into lactic acid by LAB over the course of ensiling (Dunière et al., 2013). The observed decreases in silage NDF, ADF, and ADL content were likely primarily attributable to the hydrolytic breakdown of cell walls by organic acids and enzymes produced during ensiling (Larsen et al., 2017).

Ensiling entails inevitable changes in chemical composition resulting from soluble carbohydrate conversion into organic acids and the breakdown of proteins and fibers (Wei et al., 2021). The higher levels of DM content in the A, S, GA, and GS groups suggest lower DM loss primarily stemming from the ability of formic acid and salts (sodium benzoate + potassium sorbate) to more efficiently inhibit the growth of undesirable microbes, thereby minimizing nutrient loss (Muck et al., 2018). As reported previously (Li et al., 2022; Wu C et al., 2022; Zhao et al., 2022), additives failed to impact NDF content in this study. Cheng et al. (2022), however, found that the L. plantarum inoculation of paper mulberry silage led to an increase in NDF content after ensiling for 60 days, while Jiang et al. (2020) reported no change in NDF content in corn silage after treatment with LAB and organic acid after 45 days but a decrease following 90 days of ensiling. Adding formic acid, molasses, and fibrolytic enzymes also reportedly reduced NDF content in Napier grass samples (Desta et al., 2016). These discrepant results may be attributable to differences in the particular grass species, additives, and ensilage times in individual studies. WSC content was increased in groups treated with glucose relative to other groups in the present study, as expected. However, WSC content changes dynamically over the course of ensiling, with the acid hydrolysis of the fiber fraction leading to the release of WSC that can be used by LAB for the production of organic acids (Jiang et al., 2020).

Analyzing pH, organic acid content, and NH₃-N content can provide valuable insights into silage fermentation quality (Li et al., 2019; Gao et al., 2022). Relative to the C group, the groups treated with additives in the present study exhibited lower pH values and NH₃-N content, together with higher lactic acid content, suggesting that utilizing these additives led to significant improvements in silage fermentation quality. While the lactic acid content in silage found in the current investigation was consistent with that reported for corn silage in past reports (Liu et al., 2021; Wang et al., 2022), the pH (4.28) was substantially higher than in these prior reports (3.50–3.88). This further emphasizes the challenges of directly ensiling whole mulberry plants, potentially owing to their high buffering capacity, inhibiting drops in the pH of the resultant silage (Hao et al., 2021). Epiphytic LAB counts >10⁵ CFU/g of FM and a WSC content >6% DM are essential for successful fermentation (Oliveira et al., 2017). Adding glucose and LAB can thus overcome the limitations in WSC and epiphytic LAB found in the fresh mulberry material, leading to superior fermentation. Here, the A and GA groups exhibited the best fermentation quality, potentially because the formic acid was able to rapidly lower the pH, thereby inhibiting undesirable microbe growth while providing an environment suitable for the growth of LAB (Lv et al., 2020).

Microbes are vital in the ensiling process such that efforts to monitor the bacterial community during ensiling can inform efforts to better understand and enhance the process of fermentation (Xu et al., 2017). Here, ensiling led to increases in Shannon index values together with decreased Simpson index values, consistent with reduced diversity in bacterial communities and in line with earlier findings (Guan et al., 2018; Wu B et al., 2022). Méndez-García et al. (2015) posited that this may reduction may be linked to the reduction in the pH of the resultant silage, limiting the ability of microbes to grow under acidic conditions. Reductions in unique bacterial OTUs following ensiling were also evident in the present study, consistent with past reports (Ren et al., 2019; Su et al., 2021). The magnitude of this reduction in community diversity was greatest in the A and GA groups, supporting the ability of formic acid to improve the quality of the resultant fermentation more effectively than other additives.

The epiphytic bacterial communities found in the fresh material are closely associated with the type of plant (Jiang et al., 2020). Fresh native grasses, for example, are frequently dominated by Pantoea, Pseudomonas, and Erwinia (Li et al., 2022), whereas Moringa oleifera leaves are dominated by Exiguobacterium, Acinetobacter, and Pseudomonas (Wang et al., 2018). In the present study, the dominant genera associated with fresh material were Bacillus, Sphingomonas, and Hymenobacter. Bacillus species are capable of producing antifungal compounds and facilitating the degradation of anti-nutritional factors and macromolecular nutrients through exoenzyme secretion (Chi and Cho, 2016). Phyllosphere microbiomes including those associated with fruits and flowers are often rich in Sphingomonas and Hymenobacter (Olimi et al., 2022). Epiphytic bacteria also exhibit a high degree of sensitivity to climatic, regional, and environmental factors. Guan et al. (2018), for instance, found that epiphytic bacterial communities were significantly correlated with temperature, humidity, and rainfall. Interestingly, despite the variations in epiphytic bacteria reported in the above studies, all of these species decreased sharply in abundance during the ensiling process together with prominent Lactobacillus outgrowth.

Additives can have a profound impact on silage quality through their effects on microbial communities present therein. LAB are the primary microorganisms that shape the process of fermentation, and these species can be morphologically classified into bacilli including Lactobacillus species, as well as cocci such as Enterococcus, Lactococcus, Leuconostoc, Pediococcus, and Weissella species (Yang et al., 2022). As expected, greater Lactobacillus abundance was evident in the G, A, and GA groups in the present study, likely owing to the addition of glucose as a source of WSC and the ability of formic acid to suppress harmful microbe growth, thereby generating conditions favorable to LAB growth. Higher Lactobacillus abundance has also been reported in native grass silage after treatment with molasses (Li et al., 2022) or in corn silage to which formic, acetic, and propionic acids were added at a 7:1:2 ratio (Jiang et al., 2020). Strikingly, the addition of LAB in the L and GL groups led to a decrease in Lactobacillus levels relative to the C group while increasing the proportion of Weissella in the resultant silage. This coincided with enhanced fermentation quality in these L and GL groups, as determined by the observed reduction in pH and elevation of lactic acid levels. In line with these results, Mu et al. (2021) also determined that adding Lactobacillus plantarum and molasses to mixed rice straw and amaranth silage led to a reduction in relative Lactobacillus abundance together with an increase in Weissella abundance. The mechanisms underlying this observation remain uncertain and warrant further study. The proportions of Lactobacillus and Weissella in the S and GS groups tended to exhibit similar trends in their variability to those observed in the L and GL groups. This is likely attributable to the ability of potassium sorbate and sodium benzoate to inhibit undesirable microbe growth while also limiting Lactobacillus growth, ultimately establishing conditions conducive to Weissella growth. Strikingly, all of the additives employed in this study resulted in decreased Enterobacter abundance, which is important given that these undesirable bacteria can induce fermentation of lactic acid to produce succinic acid, acetic acid, and certain endotoxins, thereby compromising the nutritional quality of the silage and leading to its contamination (Wang et al., 2022). Relative to other tested additives, the formic acid (A and GA) and salt (S and GS) groups exhibited more effective inhibition of harmful bacterial growth. Adding formic acid also increases the relative abundance of Pediococcus, which is frequently utilized to enhance silage quality due to its ability to produce lactic acid and to tolerate acidic conditions (Wang et al., 2019b). Relative to other additives, formic acid thus provides the most effective approach to enhancing Lactobacillus and Pediococcus abundance while limiting the growth of potentially harmful microbial species.

Using an RDA approach, the relationships between bacterial community composition and fermentation parameters were explored in greater detail. Across all groups, longer arrows were observed for lactic acid and acetic acid, indicating that these two organic acids had the greatest impact on the bacterial communities present in silage, in line with what has been reported in paper mulberry silage (Wu C et al., 2022) and alfalfa silage (Bai et al., 2020). Lactic acid and acetic acid were also found to be positively associated with the samples in groups A and GA, suggesting that adding formic acid led to more effective increases in the content of these organic acids. In line with expectations, lactic acid concentrations were negatively correlated with pH, butyric acid levels, and NH₃-N concentrations, as lactic acid can reduce pH values and suppress the growth of spoilage-related bacterial species including Clostridia, thereby preventing protein degradation and the generation of butyric acid (Wang et al., 2019). Lactate-producing bacteria tended to be positively linked to the concentrations of lactic acid in these samples. However, Lactobacillus abundance was not significantly correlated with lactic acid concentrations in the present study, while a negative relationship was reported in alfalfa silage by Wang B et al. (2020). The contributions of Lactobacillus to increases in lactic acid levels may be less significant than the contributions of other silage bacteria. Leuconostoc, Enterococcus, and Lactococcus were also found to be negatively associated with concentrations of lactic acid while they were positively associated with pH, butyric acid, and NH₃-N contents, in contrast to the overall findings. This may suggest that the growth of these beneficial species of bacteria coincided with the increased growth of certain undesirable species. Li et al. (2020) determined that when initial lactic acid fermentation-induced acidification failed to prevent Clostridia growth in silage, Clostridia fermentation can occur, leading to the production of butyric acid or to ammonia accumulation. Formic acid, in contrast, exhibits a greater capacity for acidification such that it can more effectively suppress the outgrowth of Clostridia and certain other undesirable species of bacteria (Lv et al., 2020). Consistent with this possibility, the pH and community diversity indices in the A and GA groups were lower. Additionally, Pediococcus, which was more abundant in samples from the A and GA groups, was positively associated with lactic acid concentrations but negatively associated with pH, butyric acid, and NH₃-N levels, in line with what has previously been described in corn silage by Guan et al. (2018).

In summary, the present results revealed an increase in DM content in the A, S, GA, and GS treatment groups. Decreased pH and NH₃-N levels were observed in all additive treatment groups. The A and GA groups presented with the lowest pH values and the greatest lactic acid contents. The various tested additives modified bacterial community structures through increases in the abundance of beneficial bacteria including Weissella and Pediococcus. A and GA addition was also sufficient to profoundly inhibit Enterobacter growth. As such, the additives utilized in this study offer promise as tools to improve whole-plant mulberry silage to varying degrees. A and GA treatments were found to be the most efficient approach to reducing pH levels, increasing lactic acid content, and preventing the growth of undesirable bacteria in ensiled mulberry samples.

The 16S rRNA sequence data were submitted to the NCBI Sequence Read Archive (SRA; https://submit.ncbi.nlm.nih.gov/subs/sra/) database with the accession number of PRJNA1024408 for open access.

LH: Writing – review & editing, Writing – original draft, Conceptualization. FJ: Writing – review & editing, Writing – original draft, Conceptualization. YW: Writing – review & editing, Project administration. HW: Writing – review & editing, Resources, Data curation. HH: Writing – review & editing, Project administration, Data curation. WY: Writing – review & editing, Formal analysis, Data curation. XH: Writing – review & editing, Formal analysis, Data curation. HC: Writing – review & editing, Formal analysis, Data curation. CW: Writing – review & editing, Writing – original draft, Investigation, Funding acquisition. ES: Writing – review & editing, Writing – original draft, Investigation, Funding acquisition.