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Article

Effect of Harvest Maturity and Lactiplantibacillus plantarum Inoculant on Dynamics of Fermentation Characteristics and Bacterial and Fungal Community of Triticale Silage

by
Run Gao
1,2,
Yi Liu
3,
Bo Wu
1,2,
Chunlin Jia
1,2,
Zhu Yu
3 and
Guoliang Wang
1,2,*
1
Institute of Leisure Agriculture, Shandong Academy of Agricultural Sciences, Jinan 250100, China
2
Key Laboratory of Urban Agriculture in East China, Ministry of Agriculture and Rural Affairs, Jinan 250100, China
3
College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(10), 1707; https://doi.org/10.3390/agriculture14101707 (registering DOI)
Submission received: 26 August 2024 / Revised: 16 September 2024 / Accepted: 24 September 2024 / Published: 29 September 2024
(This article belongs to the Section Farm Animal Production)
Browse Figures
Versions Notes

Abstract

:
(1) Background: Suitable harvest maturity stage selection and microbial inoculation during anaerobic fermentation are effective strategies for improving the quality of triticale (×Triticosecale) silage for ruminant nutrition. (2) Methods: In the present study, the fermentation characteristics, microbial communities, and their correlations were evaluated for triticale silages, as affected by Lactiplantibacillus plantarum (LP) inoculation at the heading, flowering, filling, milk-ripening, and wax-ripening stages. (3) Results: The results indicate that the filling and milk-ripening stages without LP inoculation resulted in lower pH and higher lactic acid than other harvest maturity stages (p < 0.05). Inoculating with LP decreased the pH at each harvest maturity stage, except for the filling stage, and increased the lactic acid concentration at the heading and filling stages (p < 0.05). The bacterial dynamics indicated that the abundances of Lactiplantebacilli and Monascus of the triticale silages without the LP inoculation were different between the harvest maturity stages (p < 0.05), and the abundance of Enterobacters was different between the harvest maturity stages in the triticale silage (p < 0.05). Remarkably, negative correlations were found between the Lactiplantebacillus, Monascus, and pH and positive correlations were found between the Lactiplantebacillus, Monascus, and lactic acid content (p < 0.05). (4) Conclusions: The filling and milk-ripening stages were the most suitable harvest maturity stages for the triticale silage. Inoculation with LP could enhance the fermentation quality, increase the abundances of beneficial microorganisms, and inhibit harmful microorganisms in triticale silage.

1. Introduction

Triticale (×Triticosecale) is a new species that is artificially synthesized from Triticum and Secale [1] and is favored by an increasing number of breeders for ruminant nutrition [2,3] due to its high biomass and rich nutritional value [4]. It can be planted during the winter and spring fallow seasons after corn harvesting and is grown in many countries as a forage crop [5]. Therefore, the preservation of triticale is urgently needed to effectively improve this source of high-quality forage. Silage is an effective method for preserving forage [6]. However, deteriorated silage seriously affects the growth and development of ruminant animals and poses certain risks to their physical health [7,8]. Therefore, reasonable silage harvest maturity stage and effective additives are needed for high-quality fermented forage.
The appropriate harvest maturity stage is an important factor in obtaining high-quality anaerobic fermented forage, as the crude protein (CP) content gradually decreases and the neutral detergent fiber (αNDF) and acid detergent fiber (ADF) contents increase as the harvest maturity stage progresses [9], which also affect the silage fermentation quality and the digestibility and intake of the forage. Furthermore, there are differences in the microbial community attached to the surface of the forage during different harvest maturity stages, and the ability to produce organic acids under anaerobic conditions varies [10]. Exogenous lactic acid bacteria (LAB) can be added to forage surfaces at certain harvest maturity stages to improve organic acids production in anaerobic environments, thereby rapidly reducing the pH and preserving high-quality forage [6,11].
A thorough understanding of the bacterial and fungal composition and its correlation with fermentation indicators can reveal the main functions of the dominant microorganisms in the anaerobic fermentation process of ensiled forage [6,12]. Consequently, an adequate understanding of the microbial composition of anaerobic fermented forage can help farmers to choose the appropriate harvest maturity stage for ensiling [10]. Beneficial microorganisms can accelerate organic acid production and further improve the fermentation performance of fermented forage [13]. Undesirable microorganisms not only deplete the nutrients of fermented forage but may also generate substances that are harmful to the health of ruminants [7,14]. Lactiplantibacillus plantarum (LP) is a widely used lactic acid bacterium that can improve the production rates of organic acids, rapidly reduce the pH in the initial stages of forage fermentation, and improve the fermentation quality of forage [15]. Nevertheless, Escherichia coli, Clostridium, and yeast may generate harmful substances and compete for carbohydrates with LAB, thus preventing the formation of organic acids and resulting in the inability of the pH to expeditiously decrease in the initial stages of ensiling, and the quality of forage will deteriorate [7,16]. A lower pH can prevent the reproduction of undesirable microorganisms and enhance the aerobic stability [12].
There are types of LAB used in forage ensiling, and LP is one of the effective LAB. It has been reported that Lactobacillus rhamnosus, which is one of the LAB, improved anaerobic fermentation profiles, enriched Lactiplantibacillus spp., and decreased microbial richness with diversity, which led to an increased efficiency of lactic acid fermentation of triticale silage at early heading and heading stages [10]. The effect of LP on the fermentation characteristics and dynamics of microbial communities during triticale anaerobic fermentation at different harvest maturity stages has not been reported. Additionally, fungal community dynamics in triticale silage at different harvest maturity stages have also not been studied. In our study, we investigated the impacts of LP on the fermentation performance and microbial community (both bacterial and fungal) dynamics of triticale silage at five harvest maturity stages. The findings of our study were beneficial for screening the appropriate harvest maturity stages and improving the fermentation profiles by inoculating with LP for high-quality triticale silage. The bacterial and fungal community dynamics and their correlation with fermentation characteristics after anaerobic fermentation were studied in an attempt to elucidate the influence of bacteria and fungi on the fermentation characteristics of triticale silage.

2. Materials and Methods

2.1. Triticale Sowing and Overview

The experimental site was located at the Jiyang Experimental Demonstration Base of the Shandong Academy of Agricultural Sciences (36°59′ N, 116°59′ E), in Jinan, Shandong, China. This region belongs to the warm temperate semihumid and semiarid monsoon zone, with four distinct seasons and simultaneous rainfall and heat. Triticale (Shida No. 1) was planted on 27 October 2021 with no fertilizers or herbicides throughout the cultivation, with a sowing rate of 150 kg/ha and a sowing line spacing of 30 cm.

2.2. Silage Material and Ensiling

The triticale was harvested at the heading, flowering, filling, milk-ripening, and wax-ripening stages. At each harvest maturity stage, approximately 10 kg of the triticale was harvested with a sickle by hand and 5 cm of stubble was left. Subsequently, a chopper (9Z-0.4, Jinniu Machinery Factory Rongyang, Zhengzhou, China) was instantly used to chop the triticale into 1 to 2 cm pieces. Three replicates of the prepared material were kept to evaluate the chemical composition of the triticale before silage. The LP additive was screened from sheepskin-soaking liquid by the team that were responsible for the forage production, processing, and utilization at the China Agricultural University [17]. The LP (1 × 106 cfu/g FW) was dissolved in distilled water (1% of the fresh weight) and sprayed on the prepared triticale. Simultaneously, the same volume of distilled water was sprayed onto the prepared triticale as a control (CK). A total of 750 g of the mixed triticale from the heading, flowering, and filling stages; 650 g of the mixed triticale from the milk-ripening stage; and 530 g of the mixed triticale from the wax-ripening stage were weighed and packed into a 1 L polyethylene bottle. Meanwhile, 7.5 mL of distilled water and diluted liquid from the heading, flowering, and filling stages; 6.5 mL of distilled water and diluted liquid from the milk-ripening stage; and 5.3 mL of distilled water and diluted liquid from the wax-ripening stage were sprayed into a 1 L polyethylene bottle. We used a hammer to strike a wooden stick and place the weighed samples of the prepared triticale into polyethylene bottles layer by layer. The bottles were covered and sealed with plastic wrap, and then the outer cover was placed on the bottles and sealed with transparent tape. A total of 75 bottles (5 harvest maturity stages × 2 additives × 3 duplicates) were prepared in our study. All the filled and sealed silage bottles were stored at room temperature (25 °C) for 45 d until opening.

2.3. Fermentation Profile and Chemical Composition

On the opening day, 6 bottles (3 replicates × 2 additives) were opened, and 20 g of each sample was weighed into 180 mL of distilled water, homogenized with a blender for 60 s, and consecutively filtered with 4 layers of nylon gauze and qualitative filter paper. Immediately after, a pH meter (Five Easy Plus FE28, Mettler Toledo Co., Ltd., Shanghai, China) was used to measure the silage pH. The content of ammonia nitrogen (NH3-N) was measured with the method of Broderick and Kang [18]. High-performance liquid chromatography (HPLC) was used to test the contents of lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) [19].
The dry matter (DM) of the triticale was produced with an oven (GZX-9140MBE, Shanghai Boxun Co., Ltd., Shanghai, China) at 65 °C for 48 h. The dried triticale samples were then passed through a 1 mm sieve for chemical composition analysis. According to AOAC [20] standards, the crude protein (CP) content was detected with an automatic Kjeldahl nitrogen apparatus (Kjeltec 2300 AutoAnalyzer, FOSS Analytical AB, Hoganas, Sweden). A fiber analyzer (A2000I, Ankom Technology, Macedon, NY, USA) was used to detect the contents of αNDF and ADF [21]. The αNDF analysis used sodium sulfite and thermostable α-amylase. The anthrone method was used to detect the water-soluble carbohydrate (WSC) content [22].

2.4. Microbial Community Composition

All the silage samples were analyzed by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The microbial genomic DNA extraction of the triticale silage samples was carried out according to the manufacturer’s instructions. Then, 1.0% agarose gel electrophoresis and a NanoDrop® ND-2000 spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA) were used to test the quality and concentration of the DNA, and the DNA was saved at −80 °C until the next procedure. The primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [23] were amplified at the hypervariable region V3-V4 of the bacterial 16S rRNA gene by an ABI GeneAmp® 9700 PCR thermocycler (ABI, Foster City, CA, USA). The fungal ITS gene primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) were amplified in the in vivo transcriptional spacer 1 (ITS1) region of ribose. The PCR mixture was amplified and extracted according to the previous method [23]. On an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego, CA, USA), the purified amplicons were combined in equimolar amounts and pair-end sequenced according to standard protocols by the biological company. The data processing was undertaken according to the previous method [10].

2.5. Statistical Analyses

All statistical analyses were implemented using SAS 9.4 software (SAS Institute Inc., Cary, NC, USA), and the results are given as mean values. The ANOVA procedure was used to test the effects of the additive for each harvest maturity stage or harvest maturity stage for each additive. The model used was
Y = μ + Si + eij or Y = μ + Ai + eij,
where Y is the response variable, μ is the overall mean, Si is the effect of harvest maturity stage, Ai is the effect of additive, and εij is the residual error. Duncan’s multiple range test was used for the post hoc mean comparisons.
The effects of the additive treatments, harvest maturity stages, and their interactions on each parameter were tested with the GLM procedure in SAS. The model used was
Yijk = μ + Si + Aj + (S × A)ij + eijk,
where Yijk is the dependent variable representing the response for the harvest maturity stage i observed in additive j, μ is the mean, Si is the harvest maturity stage effect, Aj is the additive effect, (S × A)ij is the interaction effect, and eijk is the residual error. Significance was considered at p ≤ 0.05. The microbial data analysis was conducted on the Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) Cloud platform (https://cloud.majorbio.com) and can be accessed on 11 September 2024.

3. Results

3.1. Chemical Compositions of Triticale before Ensiling

The chemical compositions of the triticale before ensiling at different harvest maturity stages are shown in Table 1. As the harvest maturity stage progressed, the DM content before ensiling the triticale tended to gradually increase and the CP content tended to gradually lower (p < 0.001). The αNDF and ADF contents first became lower and then became greater but not significantly, while the WSC content first tended to increase and then tended to decrease (p < 0.001).

3.2. Fermentation Characteristics of Triticale after Ensiling

The impacts of the different harvest maturity stages and LP on the fermentation characteristics of the triticale silage are shown in Table 2. The harvest maturity stage and additive exhibited effects on the pH and LA, AA, PA, BA, and NH3-N contents, while the harvest maturity stage and additive showed interactive effects on the pH and AA content (p < 0.05). As the harvest maturity stage progressed, the pH of the CK group gradually lowered, and the pH of the wax-ripening stage was undesirable, the pH of which was significantly greater than that of the other harvest maturity stages. However, the pH of the LP group decreased to less than 4.2 during the wax-ripening stage. The pH of the LP group was lower than that of the CK group (p < 0.01), except for the filling stage. The LA contents in the filling and milk-ripening stages were greater than those in the other harvest maturity stages (p < 0.01). The AA content in the CK group during the filling stage was greater than those in the flowering, milk-ripening, and wax-ripening stages, while the AA contents in the LP group during the heading and filling stages were greater than those in the other harvest maturity stages (p < 0.01). The PA contents in the CK and LP groups during the milk-ripening stage was greater than those in the other harvest maturity stages (p < 0.01). Notably, in the CK group, only a small amount of BA was detected during the flowering stage, while in the LP group, a small amount of BA was detected during all the harvest maturity stages. The possible reason for this was that the inoculation with LP increased the richness of the LAB, which produced more LA than did the CK group and provided more substrates for the Clostridium, which resulted in the production of a small amount of BA. The NH3-N content gradually increased with the delay in the harvest maturity stage, regardless of whether the silages were inoculated with the LP or not, and the NH3-N content in the wax-ripening stage was greater than those in the other harvest maturity stages (p < 0.01).

3.3. Bacterial Changes in Triticale after Ensiling

A total of 1,308,955–560,926,484 optimized sequences were obtained, with an average sequence length of 428 bp, and the observed richness of the OTUs was a 97% sequence similarity. The alpha diversities of the bacteria in the triticale silages at different harvest maturity stages are shown in Table 3. The coverage values of all the samples were greater than 0.99, which suggests that the sequencing depth was sufficient to reveal the complete bacterial diversity of the samples. The harvest maturity stage had an impact on the Sobs, Simpson, and Chao indices; the additive had an impact on the Shannon and Simpson indices; and the harvest maturity stage and additive had an interactive effect on the Simpson index (p < 0.05). The bacteria Shannon indices and Simpson indices of the triticale inoculated with LP or not differed during the different harvest maturity stages (p < 0.01). The Shannon indices during the milk and wax-ripening stages were greater than those during the heading and filling stages in the CK group, and those during the heading and flowering stages were greater than those during the filling stage in the LP group (p < 0.05). The Shannon index of the CK group was greater than that of the LP group during the flowering stage. The Simpson indices at the heading and filling stages were greater than those at the other harvest maturity stages in the CK group, and those at the heading and flowering stages were lower than those at the other harvest maturity stages in the LP group (p < 0.05). The Simpson indices of the CK group were lower than those of the LP group during the flowering, milk-ripening, and wax-ripening stages. The Ace index of the LP group during the flowering stage was lower than those during the other harvest maturity stages (p < 0.05).
The PCoA of the bacterial diversity of the triticale silages at different harvest maturity stages is shown in Figure 1A. The PCoA proclaimed that component 1 and component 2 explained 62.44% and 19.35% of the total data variance, respectively. The separation distances of the CK group during the flowering, milk-ripening, and wax-ripening stages were greater than those of the other harvest maturity stages, which indicates that the species composition differed significantly between these three harvest maturity stages.
At the OTU level, the numbers of common and unique bacterial species after the ensiling of triticale at different harvest maturity stages are shown in Figure 2A. There were 28 common bacterial communities in all the silages. During the heading stage, the CK and LP groups were unique to 7 and 9 species, the flowering stage was unique to 4 and 5 species, the filling stage was unique to 65 and 38 species, the milk-ripening stage was unique to 59 and 28 species, and the wax-ripening stage was unique to 42 and 13 species.
In total, 21 bacterial phyla, 41 classes, 105 orders, 186 families, 398 genera, and 540 species were found in all the triticale silage samples. Among these phyla, Firmicutes and Proteobacteria had relatively high abundances, and among the genera, Lactiplantibacillus and Enterobacter had relatively high abundances.
At the phylum level, the main species of the triticale silage at different harvest maturity stages were Firmicutes and Proteobacteria (Figure 3A). The abundances of Firmicutes, Proteobacteria, and Cyanobacteria were significantly different in the CK group during the different harvest maturity stages (Figure 4A–C). The abundance of Firmicutes in the CK group tended to first increase and then decrease with the postponement of the harvest maturity stage (p < 0.05). The richnesses of Firmicutes at the heading, flowering, and filling stages were greater than 83%, and the richness at the flowering stage reached 92% (Supplementary Table S1). The richnesses of Firmicutes at the milk-ripening and wax-ripening stages decreased to 56% and 43%, respectively (Figure 4A, Supplementary Table S1). However, the abundance of Firmicutes after the addition of LP was greater than 90% and there was no significant difference (Supplementary Table S1). In addition, the richnesses of Proteobacteria increased to 42% and 54% during the milk-ripening and wax-ripening stages, respectively (Figure 4B, Supplementary Table S1).
At the genus level, the most abundant bacterial flora were Lactiplantiscillus and Enterobacter (Figure 3B). Except for the wax-ripening stage (accounting for 31%), the abundance of Lactiplantebacilli in the CK treatment exceeded 50% at all other harvest maturity stages (Figure 4D, Supplementary Table S2). However, the abundance of Lactipllantibacillus in the LP treatment group was greater than 82% (Supplementary Table S3). The abundances of Enterobacters and unclassified_f__Enterobacteriaceae were greater in the CK treatment than in the LP treatment and decreased after the LP treatment, but with no significant difference (Supplementary Table S4). During the flowering stage, the richness of Leuconostoc was relatively high and decreased after the addition of LP, but with no significant difference (Supplementary Table S4). During the heading and flowering stages, the richness of Levilactobacillus accounted for approximately 3.6% (Figure 4F). However, after the addition of LP during the heading stage, its richness decreased, while it increased during the flowering stage (Figure 5C). The abundance of Levilactobacillus in the LP group was higher during the flowering stage, and the abundances of the Leuconostoc and Latilactobacillus decreased (Supplementary Table S4). There was no difference in the bacteria genera between the CK and LP groups at each harvest maturity stage (Supplementary Table S4).
The Spearman correlation between the bacteria at the genus level and fermentation characteristics is shown in Figure 6A. Lactiplantibacillus was positively correlated with the LA and AA and negatively correlated with the pH. This indicates that Lactiplantibacillus was beneficial for producing more organic acids, which reduced the pH of the silage. Enterobacter and unclassified_f_Enterobacteriaceae were positively correlated with the pH; the Enterobacter was negatively correlated with the LA, PA, and BA; and unclassified_f_Enterobacteriaceae was negatively correlated with both the LA and PA. The abundance of the Enterococcus was positively correlated with the pH and negatively correlated with the BA. Leuconostoc was negatively correlated with the AA content, which was different from the findings that Leuconostoc was positively correlated with the LA content and negatively correlated with the pH. Levilactobacillus was negatively correlated with the LA content. Furthermore, Levilactobacillus, Lentilactobacillus, and Latilactobacillus were all negatively correlated with the AN content. Weissella was positively correlated with the pH, and negatively correlated with the LA, AA, PA, and BA contents.

3.4. Fungal Changes in Triticale after Ensiling

A total of 1,813,750–41,365,1791 optimized sequences were obtained, with an average sequence length of 228 bp and OTUs with a 97% sequence similarity. The alpha diversity of the fungal diversity of the triticale silages at different harvest maturity stages is shown in Table 4. The coverage values of all the samples were greater than 0.99, which suggests that the sequencing depth was sufficient to reveal the complete fungal diversity of the samples. The harvest maturity stage had an impact on the Shannon and Simpson indices, while the additives had an impact on the Sobs, Shannon, Simpson, and Chao indices. The harvest maturity stage and additives had an interactive effect on the Shannon indices (p < 0.05). The Shannon indices of the CK group at the heading and flowering stages were greater than those at the milk-ripening stage (p < 0.05), and the Shannon indices at the filling and milk-ripening stages were lower than those at the other harvest maturity stages in the LP group (p < 0.01). And the Shannon index of the LP group was lower than that of the CK group during the milk-ripening stage (p < 0.01). The Simpson indices of the LP group during the filling and milk-ripening stages were significantly greater than those of the other harvest maturity stages. And the Simpson index of the LP group was higher than that of the CK group during the filling stage (p < 0.05).
The PCoA results of the fungal diversity of the triticale silages at different harvest maturity stages is shown in Figure 1B. The PCoA revealed that component 1 and component 2 explained 45.61% and 15.16% of the total data variance, respectively. There was perfect separation and differences in the distribution and structure of the fungal population between each harvest maturity stage and treatment. This indicated that the fungal communities of the different harvest maturity stages and treatments were distinct. The CK and LP sample points during the heading and flowering stages were relatively close, mostly in the third quadrant, which indicates a similar fungal composition. The wax-ripening stage samples were all in the second quadrant, and the distance was relatively close. The filling and milk-ripening stage sample points were separated in the first and second quadrants, which indicates a relatively rich fungal community.
At the OTU level, the number of common and unique fungal species after the ensiling of triticale at different harvest maturity stages are shown in Figure 2B. There were 28 common fungal communities in all the silages. During the heading stage, the CK and LP groups were unique to 93 and 52 species, respectively; the flowering stage was unique to 53 and 24 species; the filling stage was unique to 37 and 8 species; the milk-ripening stage was unique to 20 and 11 species; and the wax-ripening stage was unique to 245 and 28 species.
In total, 11 fungal phyla, 38 classes, 90 orders, 204 families, 393 genera, and 641 species were found in all the triticale silage samples. Among these phyla, Ascomycota and Basidiomycota had relatively high abundances, and among the genera, Monascus, Cladosporium, and Filobasidium had relatively high abundances.
At the phylum level, the main species in the triticale silage at different harvest maturity stages were Ascomycota and Basidiomycota (Figure 7A). The abundances of Ascomycota and Basidiomycota in the CK group showed no significant difference during the different harvest maturity stages (Figure 8, Supplementary Table S5). After the addition of LP, the abundances of Ascomycota were significant difference during different harvest maturity stages, where the wax-ripening stage was significantly lower than the heading, filling, and milk-ripening stages (Figure 9A, Supplementary Table S5). The abundance of Basidiomycota in the LP group showed a significant difference during the different harvest maturity stages, where the wax-ripening stage was significantly higher than the heading, filling, and milk-ripening stages (Figure 9B; Supplementary Table S5).
At the genus level, the most abundant fungal flora were Monascus, Cladosporium, Filobasidium, Vishniacozyma, and Tetrapisispora (Figure 7B). The most common fungal communities during the heading and wax-ripening stage, as well as those in the CK group during the flowering stage, were Cladosporium and Alternaria, which represented more than 14% and 12%, respectively (Supplementary Tables S6 and S7). During the filling and milking stages, as well as in the LP group during the flowering stage, the fungal genus with the greatest abundance was Monascus, which exceeded 27% (Supplementary Tables S6 and S7). The abundance of Monascus in the milk-ripening stage was significantly higher than in the heading, flowering, and wax-ripening stages in the CK group (Figure 8A). In addition, the abundance of Monascus in the milk-ripening stage was significantly higher than in the heading and wax-ripening stages in the LP group and the abundance of Monascus in the filling stage was significantly higher than in the heading and wax-ripening stages in the LP group (Figure 9C). Additionally, after inoculating with the LP, the abundance of Monascus increased, but no significant difference was found between the CK and LP groups during all the harvest maturity stages (Supplementary Table S8). During the wax-ripening stage, Cladosporium and Filobasidium were the fungal genera with the greatest abundances and they displayed significant differences during the harvest maturity stages, no matter whether they were with or without LP (Figure 8B,C and Figure 9D,F). There was no difference in the fungi genera between the CK and LP groups at each harvest maturity stage (Supplementary Table S8).
The Spearman correlation between the fungi at the genus level and the fermentation characteristics is shown in Figure 6B. It was found that the pH was negatively correlated with Monascus and Tetrapisispora and positively correlated with Cladosporium, Alternaria, Penicillium, and Sporidiobolus. In addition, the LA and PA contents were negatively correlated with Cladosporium, Alternaria, Cystofilobasidium, Penicillium, Sporidiobolus, and Epicoccum and positively correlated with Monascus and Tetrapisispora. This suggests that Monascus and Tetrapisispora may play a positive role in the acid production of silage fermentation, while Cladosporium, Alternaria, Penicillium, and Sporidiobolus were not conducive to producing organic acid during the fermentation process of silage.

4. Discussion

In our study, the DM content of the triticale increased and the CP content gradually decreased before ensiling as the harvest maturity stage progressed; this finding is similar to a previous result [10]. Evidence shows that as the harvest maturity stage is delayed, the αNDF and ADF contents of forage gradually increase [24]. Nevertheless, the αNDF and ADF contents of triticale before ensiling decreased first and then increased as the harvest maturity stage progressed in our study, while the WSC content first increased and then decreased. This difference was possibly because as the harvest maturity stage progressed, the increase in the WSCs and starch contents in the triticale resulted in a gradual decrease in the αNDF and ADF contents. Then, as the maturity of the triticale progressed and the degree of lignification increased, the contents of the αNDF and ADF tended to increase again. Therefore, the triticale showed lower αNDF and ADF contents and higher WSC content during the filling and milking stages, which indicates that the triticale exhibited greater nutritional value between these two maturity stages and was conducive to successful silage fermentation.
The fermentation characteristics showed a similar trend with the chemical composition content of the triticale before ensiling. It is widely believed that a pH higher than 4.2 is undesirable for silage [25]. In our study, the pH of the triticale silage was lower than 4.2 at all harvest maturity stages, except for the CK group during the wax-ripening stage (4.81). In addition, the silage LA content was lower and the NH3-N content was higher in the CK group during the wax-ripening stage. It is considered that a silage pH higher than 4.2 is not a good silage for ruminants. However, after the addition of LP, the pH of the triticale silage was lowered to 4.2 at the wax-ripening stage. It is considered that the addition of LP resulted in a rapid growth of the LAB, which led to a well-conserved silage [26]. The interactive analysis showed that the pH of the LP group was significantly lower than that of the CK group. This indicates that the addition of LP was necessary during the wax-ripening stage to improve the fermentation characteristics of the triticale. It was found in the interactive analysis that the LA content in the LP group was significantly greater than that in the CK group in our study, which indicates that inoculating with LP was beneficial for lactic acid fermentation to produce more LA, which was more conducive to producing successful triticale silage. This was similar to a previous finding where L. rhamnosus could increase the LA content [10,27]. Notably, we found that a small amount of BA was detected only during the flowering stage in the CK group, while a small amount of BA was detected at all harvest maturity stages in the LP group. This was different from a finding where the LAB addition could reduce the BA content [10]. The possible reason was that compared with the CK group, the LP inoculation increased the abundance of Lactiplantebacilli, which produced more LA; meanwhile, this provided more substrates for the Clostridium, which resulted in the production of a small amount of BA. The NH3-N content gradually increased as the harvest maturity stage progressed, possibly because as the maturity of the triticale progressed, the moisture content decreased, which was not conducive to the rapid reproduction of the LAB, reduction in the pH, and production of organic acids to inhibit protein hydrolysis, which resulted in the amount of protein hydrolysis being increased. Meanwhile, the CP content gradually decreased as the maturity progressed, which therefore resulted in a gradual decrease in the content of NH3-N. The NH3-N content of the CK group was greater than that of the LP group during the heading and flowering stages, which was possibly because LP inoculation during the two harvest maturity stages resulted in rapid LA accumulation and formed an acidic environment that could effectively inhibit the proteolysis caused by proteinases and/or proteolytic microorganisms [27,28].
In our study, the interactive analysis of the bacterial alpha diversity showed that the Shannon index of the LP group was significantly lower than that of the CK group. The decrease in the Shannon and Simpson indices indicates that the diversity of the bacteria decreased, which may have been due to the addition of the LP, which caused rapidly forming anaerobic and acidic environments and a rapid decrease in the pH of the triticale silage, which resulted in a large increase in the quantity of Lactiplantebacilli and the diversity of aerobic epiphytic bacteria decreased [29]. It has been reported that the addition of L. rhamnosus was shown to reduce the Simpson index; this finding was opposite to our study [10].
It has been reported that most of the dominant bacterial populations involved in silage belong to Firmicutes [16]. In our study, the abundance of Firmicutes in the CK group first increased and then decreased with the postponement of the harvest maturity stages. This indicates that as the harvest maturity stage progressed, the beneficial bacteria on the surface of the triticale for silage tended to decrease. Moreover, the abundance of Firmicutes after the addition of LP was greater than 90%, which indicates that inoculating with LP significantly increased the abundance of beneficial bacteria and decreased the abundance of undesirable bacteria. This is similar to our earlier reported result that Lactiplantibacillus is the dominant bacteria in silage [6]. We found that except for the CK group during the wax-ripening stage, Lactiplantebacilli was the dominant bacteria genera, where the abundance of Lactiplantebacilli exceeded 50%, and in the LP group, the abundance was greater than 82%. It has been reported that grass and maize silage inoculated with LAB showed reductions in E. coli and Enterobacteria with increased LAB levels [30]. The abundances of Enterobacters and unclassified_f__Enterobacteriaceae were significantly lower in the LP treatment than in the CK treatment in our study. This indicates that successful silage includes the dominant Lactipllantibacillus and the disappearance of Enterobacters [31]. It has been reported that Leuconostoc is one of the most dominant bacteria in silages [32]. In our study, we found that the abundance of Leuconostoc was higher during the flowering stage, which decreased significantly after the addition of LP. It has been reported that Levilactobacillus was a minor taxon in high- and low-moisture whole-corn silage, where it accounted for 0.31% and 0.14%, respectively [33]. We found that the abundance of Levilactobacillus in the CK group was approximately 3.6% during the heading and flowering stages, and its abundance decreased after the addition of LP during the heading stage, while the abundance increased during the flowering stage. The possible reason for this was that after the addition of the LP, the abundance of Lactipllantibacillus was dominant during the heading stage and inhibited the proliferation of Levilactobacillus and Enterobacters [34]. Nevertheless, the addition of LP resulted in an increased abundance of Levilactobacillus during the flowering stage, which inhibited the reproduction of Leuconostoc and Latilactobacillus. It was found that the control group showed a lower abundance of Leuconostoc than the fructose group in alfalfa silage [35]. Interestingly, we found that the abundance of Leuconostoc was greater in the CK group during the flowering stage of the triticale silage, and lower after the addition of the LP. This indicates that adding fructose may increase the abundance of Leuconostoc in silage, while adding LP may reduce its abundance.
It has been reported that Lactiplantibacillus is beneficial for producing more organic acids, thereby reducing the pH of silage. In our study, we found that Lactiplantibacillus was positively correlated with the LA and AA and negatively correlated with the pH. This finding was similar to a previous study that showed that the LA concentration was positively correlated with Lactiplantibacillus [36]. We found that the abundance of Enterobacter, unclassified_f_Enterobacteriaceae, and Enterococcus were positively correlated with the pH, and the abundance of Enterobacter and unclassified_f_Enterobacteriaceae were negatively correlated with the LA and PA contents. This indicates that Enterobacter, unclassified_f_Enterobacteriaceae, and Enterococcus were undesirable in the silage fermentation [37]. It was found that the abundance of Leuconostoc was positively correlated with the LA content and negatively correlated with the pH [6]. However, we found that the abundance of Leuconostoc was negatively correlated with the AA content, which was different from a previous finding. Furthermore, we found that the abundances of Levilactobacillus, Lentilactobacillus, and Latilactobacillus were negatively correlated with the NH3-N content. This suggests that these bacterial genera may inhibit protein degradation in silage, thereby improving the fermentation performance, but this needs further research. In our study, the abundance of Weissella was positively correlated with pH and negatively correlated with the LA, AA, PA, and BA contents, which indicates that Weissella may have been unfavorable for the triticale silage. This finding was inconsistent with the finding that the abundance of Weissella was negatively correlated with pH [6]. This may have been due to the different roles played by the same bacteria in the fermentation process of different forage silages.
A PCoA demonstrates the similarity or difference in the composition of a population [32]. In our study, the PCoA results showed better separation and differences in the distribution and structure of the fungal population between each harvest maturity stage and treatment. This indicates that the fungal communities of different harvest maturity stages and treatments were distinct. Furthermore, we found that the abundance of Monascus exceeded 27% during the filling and milking stages, as well as in the LP group during the flowering stage. It has been reported that Monascus fuliginosus was the dominant fungus in the oat silage treated with the PA, which accounted for 25%; this indicates that Monascus was the dominant genera after aerobic exposure and its aerobic stability deteriorated [12]. Another study showed that Monascus ruber was the most common fungus in grass and maize silage, where it accounted for 37% and 29%, respectively [7]. In addition, it has also been reported that Monascus purpureus was one of the predominant fungal genera in high- and low-moisture whole-corn silage, where it accounted for 26.38% and 19.02%, respectively [33]. This indicates that Monascus is a commonly occurring fungus in forage silage, but there is no unified research on its specific function and needs further research. Simultaneously, we found that the abundance of Filobasidium was greater in the LP group than in the CK group during the wax-ripening stage. This indicates that inoculating with the LP was beneficial for the reproduction of Filobassidium, though it may have been detrimental to the aerobic stability of the silages. It has been reported that the abundance of Filobasidium in oat silage supplemented with sodium benzoate was 25%, but no function was found [12]; this needs further research.
We found that the abundances of Monascus and Tetrapisispora were negatively correlated with the pH and positively correlated with the LA and PA contents. This suggests that Monascus and Tetrapisispora may play a positive role in the production during the fermentation process of triticale silage. This was similar to a report that there was a positive correlation with the LA content of Monascus fuliginosus [12]. It has been reported that during fermentation, Monascus can produce various secondary metabolites, such as pigments, lovastatin/monacolin K, polysaccharides, γ-aminobutyric acid (GABA), ergosterol, and citrinin [38]. Monacolin K is a natural statin that acts as an inhibitor of the enzyme 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), which prevents the formation of mevalonate from HMG-CoA during cholesterol biosynthesis [39]. However, citrinin is a mycotoxin of food safety concern that can be produced by some Monascus spp., such as Monascus purpureus, during fermentation [40]. The studies revealed aerobic deterioration by the combined activities of Monascus and Candida species in whole-plant corn silages [29]. However, the function of Monascus in the fermentation process of silage is unclear. Therefore, further research on the function of Monascus in silage fermentation is needed. Additionally, Tetrapisispora is an ascomycetous yeast species, the function of which in the fermentation process of silage is not yet clear and further research is needed.

5. Conclusions

The filling to milk-ripening stages were the most suitable harvest maturity stages for the triticale silage, which was characterized by greater lactic acid contents and high abundances of Lactiplantebacillus and Monascus. Adding the LP significantly decreased the pH and produced more lactic acid, which increased the abundance of Lactiplantebacillus and inhibited Enterobacter proliferation during the triticale anaerobic fermentation. Lactiplantebacilli and Monascus were negatively correlated with the pH and positively correlated with the lactic acid content. The silages from the filling to milk-ripening stages were good enough even without the use of a microbial additive containing Lactiplantebacillus. This should be considered by forage processors and breeders to reduce the production cost of silages.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14101707/s1, Table S1: The relative abundances of the top 10 bacterial phyla among different harvest maturity stages; Table S2: The relative abundances of the top 20 bacterial genera among different harvest maturity stages of CK group; Table S3: The relative abundances of the top 20 bacterial genera among different harvest maturity stages of LP group; Table S4: The relative abundances of the top 10 bacterial genera between CK and LP during different harvest maturity stages; Table S5: The relative abundances of the top 10 fungal phyla among different harvest maturity stages; Table S6: The relative abundances of the top 10 fungal genera among different harvest maturity stages of CK group; Table S7: The relative abundances of the top 10 fungal genera among different harvest maturity stages of LP group; Table S8: The relative abundances of the top 10 fungal genera between CK and LP during different harvest maturity stages.

Author Contributions

Conceptualization, R.G., Z.Y., and G.W.; methodology, R.G.; software, R.G.; validation, C.J., Z.Y., and G.W.; formal analysis, R.G.; investigation, R.G., Y.L., and B.W.; resources, G.W.; data curation, R.G. and Y.L.; writing—original draft preparation, R.G.; writing—review and editing, R.G.; visualization, R.G.; supervision, R.G.; project administration, R.G.; funding acquisition, G.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Shandong Academy of Agricultural Sciences Agricultural Science and Technology Innovation Engineering, funding number CXGC2023F12; China Forage and Grass Research System, funding number CARS-34; Integration and Demonstration of Smart Ecological Agriculture Technology Models in Binhai Saline Alkali Land, funding number 2023YFD2001405.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this paper are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Zillinsky, F.J. The development of Triticale. Adv. Agron. 1974, 26, 315–348. [Google Scholar] [CrossRef]
  2. Harper, M.T.; Oh, J.; Giallongo, F.; Roth, G.W.; Hristov, A.N. Inclusion of wheat and triticale silage in the diet of lactating dairy cows. J. Dairy. Sci. 2017, 100, 6151–6163. [Google Scholar] [CrossRef] [PubMed]
  3. Jr, V.; Guimares, V.; Ribeiro, E.; Silva, L.; Zanin, E. Productive performance of lambs fed with high-moisture triticale grain ensiled with different additives. Can. J. Anim. Sci. 2019, 100, 323. [Google Scholar] [CrossRef]
  4. Soundharrajan, I.; Kim, D.; Kuppusamy, P.; Muthusamy, K.; Lee, H.J.; Choi, K.C. Probiotic and triticale silage fermentation potential of Pediococcus pentosaceus and Lactobacillus brevis and their impacts on pathogenic bacteria. Microorganisms 2019, 7, 318. [Google Scholar] [CrossRef]
  5. Yang, Y.Q.; Liu, H.C.; Tian, X.H.; Du, W.H. Lodging resistance and feeding quality of triticale and cereal rye lines in an alpine pastoral area of P. R. China. Agron. J. 2022, 114, 1284–1297. [Google Scholar] [CrossRef]
  6. Wu, B.; Ai, J.; Li, T.; Qin, W.; Hu, Z.; Siqin, T.; Wu, T.; Wang, C.; Niu, H. Fermentation quality, aerobic stability, and microbiome structure and function of Caragana korshinskii silage inoculated with/without Lactobacillus rhamnosus or Lactobacillus buchneri. Front. Sustain. Food Syst. 2023, 7, 1255936. [Google Scholar] [CrossRef]
  7. Penagos-Tabares, F.; Khiaosa-Ard, R.; Schmidt, M.; Pacífico, C.; Faas, J.; Jenkins, T.; Nagl, V.; Sulyok, M.; Labuda, R.; Zebeli, Q. Fungal species and mycotoxins in mouldy spots of grass and maize silages in Austria. Mycotoxin Res. 2022, 38, 117–136. [Google Scholar] [CrossRef]
  8. Smith, D.; Lynch, G. Aspergillus fumigatus in samples of moldy silage. J. Dairy. Sci. 1973, 56, 828–829. [Google Scholar] [CrossRef]
  9. Maxin, G.; Andueza, D.; Le Morvan, A.; Baumont, R. Effect of intercropping vetch (Vicia sativa L.), field pea (Pisum sativum L.) and triticale (X Triticosecale) on dry-matter yield, nutritive and ensiling characteristics when harvested at two growth stages. Grass Forage Sci. 2017, 72, 777–784. [Google Scholar] [CrossRef]
  10. Jung, J.S.; Ravindran, B.; Soundharrajan, I.; Awasthi, M.K.; Choi, K.C. Improved performance and microbial community dynamics in anaerobic fermentation of triticale silages at different stages. Bioresour. Technol. 2022, 345, 126485. [Google Scholar] [CrossRef]
  11. Wan, J.C.; Xie, K.Y.; Wang, Y.X.; Liu, L.; Yu, Z.; Wang, B. Effects of wilting and additives on the ensiling quality and in vitro rumen fermentation characteristics of sudangrass silage. Asian-Australas. J. Anim. Sci. 2020, 34, 56–65. [Google Scholar] [CrossRef] [PubMed]
  12. Jia, T.; Yun, Y.; Yu, Z. Propionic acid and sodium benzoate affected biogenic amine formation, microbial community, and quality of oat silage. Front. Microbiol. 2021, 12, 750920. [Google Scholar] [CrossRef] [PubMed]
  13. Pieper, R.; Hackl, W.; Korn, U.; Zeyner, A.; Souffrant, W.B.; Pieper, B. Effect of ensiling triticale, barley and wheat grains at different moisture content and addition of Lactobacillus plantarum (DSMZ 8866 and 8862) on fermentation characteristics and nutrient digestibility in pigs. Anim. Feed Sci. Technol. 2011, 164, 96–105. [Google Scholar] [CrossRef]
  14. Vissers, M.M.M.; Driehuis, F.; Giffel, M.C.T.; De Jong, P.; Lankveld, J.M.G. Concentrations of butyric acid bacteria spores in silage and relationships with aerobic deterioration. J. Dairy. Sci. 2007, 90, 928–936. [Google Scholar] [CrossRef]
  15. Mu, L.; Xie, Z.; Hu, L.; Chen, G.; Zhang, Z. Cellulase interacts with Lactobacillus plantarum to affect chemical composition, bacterial communities, and aerobic stability in mixed silage of high-moisture amaranth and rice straw. Bioresour. Technol. 2020, 315, 123772. [Google Scholar] [CrossRef]
  16. Bai, J.; Xu, D.; Xie, D.; Wang, M.; Li, Z.; Guo, X. Effects of antibacterial peptide-producing Bacillus subtilis and Lactobacillus buchneri on fermentation, aerobic stability, and microbial community of alfalfa silage. Bioresour. Technol. 2020, 315, 123881. [Google Scholar] [CrossRef] [PubMed]
  17. Gao, R.; Luo, Y.; Xu, S.; Wang, M.; Sun, Z.; Wang, L.; Yu, Z. Effects of replacing ensiled-alfalfa with fresh-alfalfa on dynamic fermentation characteristics, chemical compositions, and protein fractions in fermented total mixed ration with different additives. Animals 2021, 11, 572. [Google Scholar] [CrossRef]
  18. Broderick, G.A.; Kang, J.H. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy. Sci. 1980, 63, 64–75. [Google Scholar] [CrossRef]
  19. He, L.; Zhou, W.; Wang, Y.; Wang, C.; Chen, X.; Zhang, Q. Effect of applying lactic acid bacteria and cellulase on the fermentation quality, nutritive value, tannins profile and in vitro digestibility of Neolamarckia cadamba leaves silage. J. Anim. Physiol. Anim. Nutr. 2018, 102, 1429–1436. [Google Scholar] [CrossRef]
  20. AOAC. Official Methods of Analysis, 15th ed.; Association of Official Analytical Chemists: Arlington, VA, USA, 1990. [Google Scholar]
  21. Van Soest, P.J.; Robertson, J.B.; Lewis, B.A. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy. Sci. 1991, 74, 3583–3597. [Google Scholar] [CrossRef]
  22. Murphy, R.P. A method for the extraction of plant samples and the determination of total soluble carbohydrates. J. Food Agric. 2010, 9, 714–717. [Google Scholar] [CrossRef]
  23. Liu, C.; Zhao, D.; Ma, W.; Guo, Y.; Wang, A.; Wang, Q.; Lee, D.-J. Denitrifying sulfide removal process on high-salinity wastewaters in the presence of Halomonas sp. Appl. Microbiol. Biotechnol. 2016, 100, 1421–1426. [Google Scholar] [CrossRef] [PubMed]
  24. Deng, X.; Jia, Y.; Ge, G.; Wang, Z.; Liu, M.; Bao, J.; Zhao, M.; Si, Q.; Liu, Y.; Zhao, W. Microbiomics and volatile metabolomics-based investigation of changes in quality and flavor of oat (Avena sativa L.) silage at different stages. Front. Plant Sci. 2023, 14, 1278715. [Google Scholar] [CrossRef] [PubMed]
  25. Ahmadi, F.; Lee, Y.H.; Lee, W.H.; Oh, Y.-K.; Park, K.; Kwak, W.S. Long-term anaerobic conservation of fruit and vegetable discards without or with moisture adjustment after aerobic preservation with sodium metabisulfite. Waste Manag. 2019, 87, 258–267. [Google Scholar] [CrossRef]
  26. He, L.; Wang, C.; Xing, Y.; Zhou, W.; Pian, R.; Chen, X.; Zhang, Q. Ensiling characteristics, proteolysis and bacterial community of high-moisture corn stalk and stylo silage prepared with Bauhinia variegate flower. Bioresour. Technol. 2020, 296, 122336. [Google Scholar] [CrossRef]
  27. Ren, H.; Feng, Y.; Pei, J.; Li, J.; Wang, Z.; Fu, S.; Zheng, Y.; Li, Z.; Peng, Z. Effects of Lactobacillus plantarum additive and temperature on the ensiling quality and microbial community dynamics of cauliflower leaf silages. Bioresour. Technol. 2020, 307, 123238. [Google Scholar] [CrossRef]
  28. Zhao, J.; Dong, Z.; Li, J.; Chen, L.; Bai, Y.; Jia, Y.; Shao, T. Evaluation of Lactobacillus plantarum MTD1 and waste molasses as fermentation modifier to increase silage quality and reduce ruminal greenhouse gas emissions of rice straw. Sci. Total Environ. 2019, 688, 143–152. [Google Scholar] [CrossRef]
  29. Keshri, J.; Chen, Y.; Pinto, R.; Kroupitski, Y.; Weinberg, Z.G.; Sela, S. Microbiome dynamics during ensiling of corn with and without Lactobacillus plantarum inoculant. Appl. Microbiol. Biotechnol. 2018, 102, 4025–4037. [Google Scholar] [CrossRef]
  30. Queiroz, O.C.M.; Ogunade, I.M.; Weinberg, Z.; Adesogan, A.T. Silage review: Foodborne pathogens in silage and their mitigation by silage additives. J. Dairy. Sci. 2018, 101, 4132–4142. [Google Scholar] [CrossRef]
  31. Xiao, Y.; Sun, L.; Xin, X.; Xu, L.; Du, S. Physicochemical characteristics and microbial community succession during oat silage prepared without or with Lactiplantibacillus plantarum or Lentilactobacillus buchneri. Microbiol. Spectr. 2023, 11, e0222823. [Google Scholar] [CrossRef]
  32. Zeng, T.; Li, X.; Guan, H.; Yang, W.; Liu, W.; Liu, J.; Du, Z.; Li, X.; Xiao, Q.; Wang, X.; et al. Dynamic microbial diversity and fermentation quality of the mixed silage of corn and soybean grown in strip intercropping system. Bioresour. Technol. 2020, 313, 123655. [Google Scholar] [CrossRef] [PubMed]
  33. Bai, C.; Wang, C.; Sun, L.; Xu, H.; Jiang, Y.; Na, N.; Yin, G.; Liu, S.; Xue, Y. Dynamics of bacterial and fungal communities and metabolites during aerobic exposure in whole-plant corn silages with two different moisture levels. Front. Microbiol. 2021, 12, 663895. [Google Scholar] [CrossRef] [PubMed]
  34. Wang, T.; Teng, K.; Cao, Y.; Shi, W.; Xuan, Z.; Zhou, J.; Zhang, J.; Zhong, J. Effects of Lactobacillus hilgardii 60TS-2, with or without homofermentative Lactobacillus plantarum B90, on the aerobic stability, fermentation quality and microbial community dynamics in sugarcane top silage. Bioresour. Technol. 2020, 312, 123600. [Google Scholar] [CrossRef] [PubMed]
  35. Wang, B.; Gao, R.; Wu, Z.; Yu, Z. Functional analysis of sugars in modulating bacterial communities and metabolomics profiles of Medicago sativa silage. Front. Microbiol. 2020, 11, 641. [Google Scholar] [CrossRef] [PubMed]
  36. Lian, T.; Zhang, W.; Cao, Q.; Wang, S.; Dong, H.; Yin, F. Improving production of lactic acid and volatile fatty acids from dairy cattle manure and corn straw silage: Effects of mixing ratios and temperature. Bioresour. Technol. 2020, 359, 127449. [Google Scholar] [CrossRef]
  37. Wang, M.; Gao, R.; Franco, M.; Hannaway, D.B.; Ke, W.; Ding, Z.; Yu, Z.; Guo, X. Effect of mixing alfalfa with whole-plant corn in different proportions on fermentation characteristics and bacterial community of silage. Agriculture 2021, 11, 174. [Google Scholar] [CrossRef]
  38. Srianta, I.; Ristiarini, S.; Nugerahani, I.; Sen, S.K.; Zhang, B.B.; Xu, G.R.; Blanc, P. Recent research and development of Monascus fermentation products. Int. Food Res. J. 2014, 21, 1–12. [Google Scholar]
  39. Suraiya, S.; Kim, J.-H.; Tak, J.Y.; Siddique, M.P.; Young, C.J.; Kim, J.K.; Kong, I.-S. Influences of fermentation parameters on lovastatin production by Monascus purpureus using Saccharina japonica as solid fermented substrate. Lwt-Food Sci. Technol. 2018, 92, 1–9. [Google Scholar] [CrossRef]
  40. Silva, L.J.G.; Pereira, A.M.P.T.; Pena, A.; Lino, C.M. Citrinin in foods and supplements: A review of occurrence and analytical methodologies. Foods 2021, 10, 14. [Google Scholar] [CrossRef]
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Figure 1. PCoA (principal co-ordinates) results based on OTU levels of the bacteria (A) and fungi (B) in the triticale silage (n = 3). PC1, principal co-ordinate 1; PC2, principal co-ordinate 2; CK, group with same volume of distilled water added; LP, group with Lactiplantibacillus plantarum added; H, heading stage; F, flowering stage; F1, filling stage; M, milk-ripening stage; W, wax-ripening stage.
Figure 1. PCoA (principal co-ordinates) results based on OTU levels of the bacteria (A) and fungi (B) in the triticale silage (n = 3). PC1, principal co-ordinate 1; PC2, principal co-ordinate 2; CK, group with same volume of distilled water added; LP, group with Lactiplantibacillus plantarum added; H, heading stage; F, flowering stage; F1, filling stage; M, milk-ripening stage; W, wax-ripening stage.
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Figure 2. Petal chart of common and unique bacteria (A) and fungi (B) populations in the triticale silage at different harvest maturity stages. Different colors represent different treatments, with petals representing the number of species unique to the corresponding treatment and the center representing the number of species common to all treatments. CK, group with the same volume of distilled water added; LP, group with Lactiplantibacillus plantarum added; H, heading stage; F, flowering stage; F1, filling stage; M, milk-ripening stage; W, wax-ripening stage.
Figure 2. Petal chart of common and unique bacteria (A) and fungi (B) populations in the triticale silage at different harvest maturity stages. Different colors represent different treatments, with petals representing the number of species unique to the corresponding treatment and the center representing the number of species common to all treatments. CK, group with the same volume of distilled water added; LP, group with Lactiplantibacillus plantarum added; H, heading stage; F, flowering stage; F1, filling stage; M, milk-ripening stage; W, wax-ripening stage.
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Figure 3. Relative abundances of the bacterial community proportions at the phylum (A) and genus (B) levels and in the triticale silage at different harvesting stages. HCK, control (CK) in the heading period; HLP, Lactiplantibacillus plantarum (LP) added in the heading period; FCK, CK in the flowering period; FLP, LP added in the flowering period; F1CK, CK in the filling period; F1LP, LP added in the filling period; MCK, CK in the milk-ripening period; MLP, LP added in the milk-ripening period; WCK, CK in the wax-ripening period; WLP, LP added in the wax-ripening period.
Figure 3. Relative abundances of the bacterial community proportions at the phylum (A) and genus (B) levels and in the triticale silage at different harvesting stages. HCK, control (CK) in the heading period; HLP, Lactiplantibacillus plantarum (LP) added in the heading period; FCK, CK in the flowering period; FLP, LP added in the flowering period; F1CK, CK in the filling period; F1LP, LP added in the filling period; MCK, CK in the milk-ripening period; MLP, LP added in the milk-ripening period; WCK, CK in the wax-ripening period; WLP, LP added in the wax-ripening period.
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Figure 4. The differential bacterial species of the triticale silage at the phylum and genus (among the top 10 bacterial genera) levels during the different harvest maturity stages of the CK group. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.001.
Figure 4. The differential bacterial species of the triticale silage at the phylum and genus (among the top 10 bacterial genera) levels during the different harvest maturity stages of the CK group. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.001.
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Figure 5. The different bacterial species of the triticale silage at the genus (among the top 10 bacterial genera) level during the different harvest maturity stages of the LP group. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.001.
Figure 5. The different bacterial species of the triticale silage at the genus (among the top 10 bacterial genera) level during the different harvest maturity stages of the LP group. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.001.
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Figure 6. Spearman correlation heatmaps for the fermentation characteristics with the top 10 enriched bacteria (A) and fungi (B) at the genus level. Positive correlations are shown in purple and negative correlations are shown in orange. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.001.
Figure 6. Spearman correlation heatmaps for the fermentation characteristics with the top 10 enriched bacteria (A) and fungi (B) at the genus level. Positive correlations are shown in purple and negative correlations are shown in orange. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.001.
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Figure 7. Relative abundances of fungal community proportions at the phylum (A) and genus (B) levels and in triticale silage at different harvesting stages. HCK, control (CK) in the heading period; HLP, Lactiplantibacillus plantarum (LP) added in the heading period; FCK, CK in the flowering period; FLP, LP added in the flowering period; F1CK, CK in the filling period; F1LP, LP added in the filling period; MCK, CK in the milk-ripening period; MLP, LP added in the milk-ripening period; WCK, CK in the wax-ripening period; WLP, LP added in the wax-ripening period.
Figure 7. Relative abundances of fungal community proportions at the phylum (A) and genus (B) levels and in triticale silage at different harvesting stages. HCK, control (CK) in the heading period; HLP, Lactiplantibacillus plantarum (LP) added in the heading period; FCK, CK in the flowering period; FLP, LP added in the flowering period; F1CK, CK in the filling period; F1LP, LP added in the filling period; MCK, CK in the milk-ripening period; MLP, LP added in the milk-ripening period; WCK, CK in the wax-ripening period; WLP, LP added in the wax-ripening period.
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Agriculture 14 01707 g008
Figure 8. The different fungal species of the triticale silage at the phylum (no different fungal species were found) and genus (among the top 10 bacterial genera) level during the different harvest maturity stages of the CK group. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.001.
Figure 8. The different fungal species of the triticale silage at the phylum (no different fungal species were found) and genus (among the top 10 bacterial genera) level during the different harvest maturity stages of the CK group. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.001.
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Agriculture 14 01707 g009
Figure 9. The different fungal species of the triticale silage at the phylum and genus (among the top 10 bacterial genera) levels during the different harvest maturity stages of the LP group. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.001.
Figure 9. The different fungal species of the triticale silage at the phylum and genus (among the top 10 bacterial genera) levels during the different harvest maturity stages of the LP group. * means 0.01 < p ≤ 0.05, ** means 0.001 < p ≤ 0.01, *** means p ≤ 0.001.
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Table 1. Chemical compositions of triticale before ensiling at different harvest maturity stages.
Table 1. Chemical compositions of triticale before ensiling at different harvest maturity stages.
Harvest Maturity StagesDMCPαNDFADFWSC
%%DM
Heading20.59 d11.88 a63.4536.801.70 c
Flowering20.93 d12.40 a59.4534.422.33 bc
Filling33.64 c7.91 b55.4032.856.52 a
Milk ripening39.15 b6.74 bc56.4133.813.46 b
Wax ripening59.96 a5.13 c60.6137.062.05 bc
SEM3.8690.7931.0390.6550.501
p-value<0.001<0.0010.0790.163<0.001
Note: DM, dry matter; CP, crude protein; αNDF, neutral detergent fiber; ADF, acid detergent fiber; WSC, water-soluble carbohydrate. SEM, standard error of the means. a–d in the same column indicate significant differences between the different harvest maturity stages on the same chemical composition (p < 0.05).
Table 2. Fermentation characteristics of triticale silage treated with or without LP after 45 d of ensiling at different harvest maturity stages.
Table 2. Fermentation characteristics of triticale silage treated with or without LP after 45 d of ensiling at different harvest maturity stages.
ItemsAdditivesHarvest Maturity StagesSEMp-ValueInteraction
HeadingFloweringFillingMilk RipeningWax RipeningSA1S*A1
pHCK4.17 bA3.99 cA3.89 cd3.79 dA4.81 aA0.098<0.001<0.001<0.001<0.001
LP3.88 bB3.75 cB3.84 b3.71 cB4.04 aB0.031<0.001
SEM0.0690.0570.0130.0180.175
p-value0.0120.0110.1380.0340.013
LACK1.62 bB1.50 b2.99 a2.61 a0.47 c0.247<0.001<0.001<0.0010.134
%DMLP2.16 bA1.52 b3.74 a3.16 a1.69 b0.2550.002
SEM0.1410.0850.2540.2380.317
p-value0.0260.8250.0340.0970.111
AACK0.73 abB0.50 cd0.77 aB0.57 bc0.36 d0.0440.001<0.001<0.0010.002
%DMLP1.38 aA0.44 b1.30 aA0.79 b0.52 b0.1130.001
SEM0.1520.0410.1340.0820.076
p-value0.0080.6040.0140.1310.482
PACK0.48 cB0.52 b0.51 bB1.22 a0.26 c0.091<0.001<0.0010.0060.357
%DMLP0.82 bA0.44 b0.83 bA1.45 a0.64 b0.1090.015
SEM0.0880.0250.0810.1460.126
p-value0.0480.2570.0360.4810.246
BACK0.000.020.000.000.000.0020.0530.0030.0320.971
%DMLP0.010.020.010.010.010.0020.156
SEM0.0010.0040.0010.0010.001
p-value0.1840.4230.1840.4230.184
NH3-NCK2.69 eA3.01 dA3.93 c4.17 b5.52 a0.267<0.001<0.0010.0050.449
%TNLP2.43 cB2.66 cB3.66 b4.17 b4.97 a0.262<0.001
SEM0.0600.0880.0930.0360.218
p-value0.0090.0150.0911.0000.231
Note: LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; NH3-N, ammonia nitrogen; DM, dry matter; TN, total nitrogen. CK, group with the same volume of distilled water added; LP, group with Lactiplantibacillus plantarum added. SEM, standard error of the means. a–e in the same row indicate significant differences between the different harvest maturities (p < 0.05). A–B in the same column indicate significant differences between two additive treatments (p < 0.05). S, harvest maturity stages; A1, additives; S*A1 denotes the interaction between the harvest maturity stages and additives.
Table 3. Bacterial alpha diversity of triticale silages at different harvest maturity stages.
Table 3. Bacterial alpha diversity of triticale silages at different harvest maturity stages.
ItemsAdditivesHarvesting StagesSEMp-ValueInteraction
HeadingFloweringFillingMilk RipeningWax RipeningSA1S*A1
SobsCK70.0066.33167.67121.67120.6712.9410.0570.0040.3260.500
LP80.6773.33118.33110.67105.676.3940.098
SEM3.8962.19716.87810.24818.265
p-value0.1620.2770.2420.4710.809
ShannonCK0.78 b1.38 abA0.82 b1.45 a1.54 a0.1190.0490.073<0.0010.086
LP0.73 a0.74 aB0.36 b0.49 ab0.53 ab0.0590.036
SEM0.1730.1480.1160.2340.259
p-value0.9100.0260.0700.0690.120
SimpsonCK0.60 a0.36 bB0.69 a0.38 bB0.33 bB0.0450.0060.002<0.0010.017
LP0.72 b0.70 bA0.90 a0.85 aA0.82 aA0.0270.003
SEM0.0550.0790.0540.1070.116
p-value0.3470.0280.0900.0300.044
AceCK136.39132.00202.37148.52191.0312.6010.3570.0770.0910.557
LP145.01 a108.30 b142.79 a139.38 a158.45 a5.4840.030
SEM11.4827.53020.92313.34115.037
p-value0.7940.2460.2330.5460.490
ChaoCK110.7999.23195.34147.46161.9213.1190.1700.0240.3520.649
LP120.84102.35145.85139.42143.326.8310.281
SEM8.1505.91218.09513.72317.863
p-value0.5460.8440.2810.5710.761
CoverageCK0.99930.99920.99870.99920.99890.0000.2260.0780.6940.396
LP0.99910.99920.99910.99910.99900.0000.441
SEM0.0000.0000.0000.0000.000
p-value0.1831.0000.2140.8600.774
Note: CK, group with the same volume distilled water added; LP, group with Lactiplantibacillus plantarum added. SEM, standard error of the means. a–b in the same row indicate significant differences between the different harvest maturities (p < 0.05). A–B in the same column indicate significant differences between two additive treatments (p < 0.05). S, harvest maturity stages; A1, additives; S*A1 denotes the interaction between the harvest maturity stages and additives.
Table 4. Fungal alpha diversity of triticale silages at different harvest maturity stages.
Table 4. Fungal alpha diversity of triticale silages at different harvest maturity stages.
ItemsAdditivesHarvesting StagesSEMp-ValueInteraction
HeadingFloweringFillingMilk RipeningWax RipeningSA1S*A1
SobsCK172.33126.33131.0070.67212.0021.5860.3690.1330.0260.711
LP118.6778.0065.0068.33105.009.3640.383
SEM25.59915.43521.3238.53146.417
p-value0.3750.2430.1610.9260.336
ShannonCK3.54 a3.53 a2.41 ab1.88 bA2.49 ab0.2150.039<0.0010.0010.009
LP3.32 a2.59 a0.70 b1.03 bB2.38 a0.2820.001
SEM0.0990.2550.4520.3290.144
p-value0.4090.0900.0530.0050.706
SimpsonCK0.070.060.24 B0.380.230.0430.157<0.0010.0120.089
LP0.09 b0.17 b0.70 aA0.49 a0.16 b0.0770.007
SEM0.0110.0400.1210.1100.025
p-value0.3830.2170.0290.1000.591
AceCK179.30145.28139.3477.99217.7321.5140.4080.1880.0510.704
LP124.43111.1283.8373.43108.098.8750.476
SEM25.16610.83720.5638.23147.151
p-value0.3650.3570.2690.8560.326
ChaoCK184.00133.05140.4874.95219.1722.0680.3780.1420.0260.705
LP121.5086.8376.2972.72109.588.8640.458
SEM27.23713.49221.3788.68947.274
p-value0.3690.1760.1720.9320.318
CoverageCK0.99980.99980.99970.99980.99980.0000.5060.0540.3510.271
LP0.9999 a0.9999 a0.9996 b0.9999 a0.9999 a0.0000.028
SEM0.0000.0000.0000.0000.000
p-value0.1940.7740.4360.8600.173
Note: CK, group with the same volume of distilled water added; LP, group with Lactiplantibacillus plantarum added. a–b in the same volume indicate significant differences (p < 0.05). A–B in the same column indicate significant differences between two additive treatments (p < 0.05). SEM, standard error of the means. S, harvest maturity stages; A1, additives; S*A1 denotes the interaction between the harvest maturity stages and additives.
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MDPI and ACS Style

Gao, R.; Liu, Y.; Wu, B.; Jia, C.; Yu, Z.; Wang, G. Effect of Harvest Maturity and Lactiplantibacillus plantarum Inoculant on Dynamics of Fermentation Characteristics and Bacterial and Fungal Community of Triticale Silage. Agriculture 2024, 14, 1707. https://doi.org/10.3390/agriculture14101707

AMA Style

Gao R, Liu Y, Wu B, Jia C, Yu Z, Wang G. Effect of Harvest Maturity and Lactiplantibacillus plantarum Inoculant on Dynamics of Fermentation Characteristics and Bacterial and Fungal Community of Triticale Silage. Agriculture. 2024; 14(10):1707. https://doi.org/10.3390/agriculture14101707

Chicago/Turabian Style

Gao, Run, Yi Liu, Bo Wu, Chunlin Jia, Zhu Yu, and Guoliang Wang. 2024. "Effect of Harvest Maturity and Lactiplantibacillus plantarum Inoculant on Dynamics of Fermentation Characteristics and Bacterial and Fungal Community of Triticale Silage" Agriculture 14, no. 10: 1707. https://doi.org/10.3390/agriculture14101707

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