Microbial Inoculants Research Articles

Synergistic Role of Streptomyces Composite Inoculants in Mitigating Wheat Drought Stress Under Field Conditions

Wheat (Triticum aestivum L.) is a globally important staple crop; however, its growth and yield are severely limited by drought stress. This study evaluated the effects of a combined microbial inoculant, Streptomyces pactum Act12 and Streptomyces rochei D74, on wheat photosynthesis, physiological traits, and yield under drought conditions. Key physiological and yield parameters were measured during the jointing, heading, and grain-filling stages. Drought stress significantly reduced chlorophyll content, maximum photochemical efficiency of photosystem II (PSII) (Fv/Fm), and antioxidant enzyme activities, while increasing malondialdehyde (MDA) levels, leading to a notable yield decline. In contrast, inoculation with Streptomyces strains alleviated these adverse effects, with the combined inoculant (Act12+D74) group demonstrating the most significant improvement. Chlorophyll content increased by up to 32.60%, Fv/Fm improved by 43.07%, and antioxidant enzyme activities were enhanced, with superoxide dismutase (SOD) activity increasing by 19.32% and peroxidase (POD) activity by 75.44%. Meanwhile, MDA levels were reduced by 61.61%. The proline content in the combined inoculant group increased by 90.44% at the jointing stage and the soluble protein content increased by 60.17% at the heading stage. Furthermore, it improved the yield by 26.19% by increasing both effective spikes and grains per spike. For the first time, this study revealed the synergistic effects of Act12 and D74 in enhancing photosynthesis, strengthening antioxidant defenses, and optimizing osmotic regulation under drought conditions. These findings provide a theoretical basis for developing environmentally friendly drought management strategies and highlight the potential applications of this inoculant in sustainable agriculture.

Isolation of Acetic Acid-Producing Bacterial Strains and Utilization as Microbial Inoculants in Sorghum Silages

Isolation of Acetic Acid-Producing Bacterial Strains and Utilization as Microbial Inoculants in Sorghum Silages

Changes in the nutritional composition of corn silage w ith the use of chemical additives and microbial inoculants

This work aimed to evaluate the use of different additives in the bromatological composition of corn silage. The experimental design was completely randomized with five additives evaluated: no additive; silage added with urea (3.0%); silage added with limestone (3.0%); silage added with granulated sugar (3.0%); and silage with bacterial inoculant. After 60 days of ensiling, the chemical-bromatological composition of the produced silages was evaluated. Lime-added silage had higher dry matter contents (not differing from silage with no additive), mineral matter, and lignin. Silage with urea had higher values of crude protein (19.55%) and lower levels of total carbohydrates (74.29%) and non-fiber carbohydrates (27.13%). Silage with no additive had lower levels of ADF (38.99%) and NDF (19.79%) and a higher content of total digestible nutrients (73.98%). Using additives in corn silage promotes higher concentrations of acid or neutral detergent fiber, cellulose, and hemicellulose and lower TDN levels in its bromatological composition. The addition of urea provides an increase in protein levels, while limestone increases mineral material levels.

Mitigating soil compaction and enhancing corn (Zea mays L.) growth through biological and non-biological amendments

Soil compaction is a pressing issue in agriculture that significantly hinders plant growth and soil health, necessitating effective strategies for mitigation. This study examined the effects of sugarcane bagasse, both in its raw form and as biochar, along with biological activators (Bacillus simplex UTT1 and Phanerochaete chrysosporium) on soil characteristics and corn (Zea mays L.) plant biomass in a compacted soil. Using a completely randomized factorial design, we assessed their impact on soil bulk density, porosity, nutrient concentrations, and plant growth parameters. Results indicated that combining organic amendments with microbial inoculation (B. simplex UTT1 + P. chrysosporium) significantly reduced soil bulk density (7–14%) (p < 0.05) and enhanced soil aggregate stability (38%), soil permeability coefficient (52%), and total porosity (40%) (p < 0.01) compared to controls. Notably, the application of biochar and microbial inoculants improved soil moisture retention (p < 0.05) and soil nutrient availability, particularly potassium and phosphorus, which increased by 18% and 23%, respectively. Plant biomass responses (10–35%) varied, with combined microbial treatments (B. simplex UTT1 + P. chrysosporium) yielding optimal outcomes (p < 0.01) compared to individual applications. The study emphasizes that integrating organic amendments with biological processes not only mitigates soil compaction but also fosters soil health and productivity. These findings support the need for sustainable agricultural practices, highlighting the role of effective resource management in enhancing soil structure and crop yields. Future research should focus on understanding the underlying mechanisms of these interactions and their long-term implications for soil health and agricultural sustainability.Clinical trial number Not applicable.

Association Analysis of the Genomic and Functional Characteristics of Halotolerant Glutamicibacter endophyticus J2-5-19 from the Rhizosphere of Suaeda salsa.

Halotolerant plant growth-promoting bacteria (HT-PGPB) have attracted considerable attention for their significant potential in mitigating salt stress in crops. However, the current exploration and development of HT-PGPB remain insufficient to meet the increasing demands of agriculture. In this study, an HT-PGPB isolated from coastal saline-alkali soil in the Yellow River Delta was identified as Glutamicibacter endophyticus J2-5-19. The strain was capable of growing in media with up to 13% NaCl and producing proteases, siderophores, and the plant hormone IAA. Under 4‱ salt stress, inoculation with strain J2-5-19 significantly increased the wheat seed germination rate from 37.5% to 95%, enhanced the dry weight of maize seedlings by 41.92%, and notably improved the development of maize root systems. Moreover, this work presented the first whole-genome of Glutamicibacter endophyticus, revealing that G. endophyticus J2-5-19 resisted salt stress by expelling sodium ions and taking up potassium ions through Na+/H+ antiporters and potassium uptake proteins, while also accumulating compatible solutes such as betaine, proline, and trehalose. Additionally, the genome contained multiple key plant growth-promoting genes, including those involved in IAA biosynthesis, siderophore production, and GABA synthesis. The findings provide a theoretical foundation and microbial resources for the development of specialized microbial inoculants for saline-alkali soils.

Insights into nitrogen metabolism and humification process in aerobic composting facilitated by microbial inoculation.

To enhance the retention of compost nutrients, specifically in nitrogen metabolism and humification, compound microbial agents were added during the aerobic composting of bagasse pith and buffalo manure. The introduction of the microbial agent successfully colonized the mixture, boosted the degradation capacity of organic matter, and facilitated the formation of nitrogenous substances and humic substances (HSs). The incorporation of a composite microbial inoculum led to a substantial rise in total Kjeldahl nitrogen (TKN) by 62.04%, nitrate nitrogen (NO- 3-N) by 291.65%, and amino acid (AA) by 78.77%. Furthermore, this intervention resulted in achieving a humic acid (HA) to fulvic acid (FA) ratio of 1.64. Metagenomic sequencing revealed enhanced synergistic interactions among microorganisms through inoculation, increasing the abundance of functional genes related to nitrification and nitrogen fixation compared to the uninoculated control. Spearman correlation analysis identified unclassified_c__Deltaproteobacteria, unclassified_f__Planctomycetaceae, Chryseosolibacter, unclassified_f__Hyphomicrobiaceae as the principal producers of HA. This research provides insights into the interactions between nitrogen metabolism and humification in composting, aiming to effectively retain compost nutrients and support the long-term sustainability of agriculture.

Phenotypic Profiling of Selected Cellulolytic Strains to Develop a Crop Residue-Decomposing Bacterial Consortium.

Slow decomposition rates of cereal crop residues can lead to agronomic challenges, such as nutrient immobilization, delayed soil warming, and increased pest pressures. In this regard, microbial inoculation with efficient strains offers a viable and eco-friendly solution to accelerating the decomposition process of crop residues. However, this solution often focuses mostly on selecting microorganisms based on the appropriate enzymic capabilities and neglects the metabolic versatility required to utilize both structural and non-structural components of residues. Therefore, this study aimed to address these limitations by assessing the metabolic profiles of five previously identified cellulolytic bacterial strains, including Bacillus pumilus 1G17, Micromonospora chalcea 1G49, Bacillus mobilis 5G17, Streptomyces canus 1TG5, and Streptomyces achromogenes 3TG21 using Biolog Phenotype Microarray analysis. Moreover, this study evaluated the impact of wheat straw inoculation with single strains and a bacterial consortium on soil organic carbon and nitrogen content in a pot experiment. Results revealed that, beyond the core subset of 12 carbon sources, the strains exhibited diverse metabolic capacities in utilizing 106 carbon sources. All strains demonstrated effective straw biomass degradation compared to the negative control, with significant differences detected only in oil seed rape straw biodegradation estimations. Furthermore, wheat straw inoculated with a bacterial consortium showed a significant increase in soil organic carbon content after 180 days in the pot experiment. Overall, these findings underscore the critical role of metabolic profiling in gaining a deeper understanding of microbial capabilities and addressing the complexities of residue composition and environmental variability.

Microalgae and microbial inoculant as partial substitutes for chemical fertilizer enhance Polygala tenuifolia yield and quality by improving soil microorganisms

Microalgae and microbial inoculant as partial substitutes for chemical fertilizer enhance Polygala tenuifolia yield and quality by improving soil microorganisms

The Promotion of Dark Septate Endophytes on the Performance and Active Ingredients Accumulation of Dendranthema morifolium Under Cd Stress

Dark septate endophytes (DSE) may facilitate plant growth and stress tolerance in stressful ecosystems. However, little is known about the response of medicinal plants to DSE, especially under heavy metal stress. This study aimed to investigate how DSE affects the growth of Dendranthema morifolium in medicinal plants under cadmium (Cd) stress. In this investigation, the sterile and non-sterile inoculations were carried out to evaluate the effect of three DSE strains on D. morifolium stressed with Cd. For the root, DSE15 sterile or non-sterile inoculation resulted in enhanced root biomass, root volume, the Cd content of roots, and the indoleacetic acid (IAA) levels in D. morifolium under Cd stress. DSE7 non-sterile inoculation significantly enhanced the Cd content of roots at 1 and 5 mg Cd/kg soil. Regarding impact stems and leaves, under sterile conditions, DSE7 and DSE15 effectively regulated the shoot biomass, plant height, chlorophyll level, and superoxide dismutase (SOD) content. Under sterile conditions, DSE15 positively influenced shoot biomass and plant height, while DSE7 had no significant effect on them when subjected to Cd stress. For effects on flowers under non-sterile conditions, DSE7 and DSE15 significantly increased the flower biomass under Cd stress, while DSE7 reduced the Cd transfer coefficient of flowers at 1 and 5 mg Cd/kg soil. Importantly, at 1 mg Cd/kg soil, DSE7 and DSE15 non-sterile inoculations promoted the 1, 5-dicaffeoylquinic acid content by 18.29% and 21.70%. The interaction between DSE and soil factors revealed that DSE species had significant effects on soil organic carbon and available nitrogen in D. morifolium non-sterile soil. The DSE15 inoculation enhanced soil organic carbon content, while the inoculation of DSE7 and DSE15 reduced soil available nitrogen content under Cd stress. These results contribute to a better understanding of DSE-plant interactions in habitats contaminated by heavy metals and demonstrate the potential utility of DSE strains for cultivating medicinal plants.

Isolation and Characterization of Lactic Acid Bacteria: A Strategy to Produce Inoculum for Forage Ensiling

Purpose: Sri Lankan dairy sector faces challenges due to seasonal variability and suboptimal quality of forage for dairy cattle feeding. While silage offers year-round availability of forage, concerns arise regarding potential quality degradation during forage ensiling. Microbial inoculants are utilized to expedite the ensiling and minimize quality deterioration. This study involves the isolation and characterization of lactic acid bacteria (LAB) from silage, with the aim of identifying potential candidates to be used as silage inoculants.Research Method: Lactobacillus were isolated from 10 silage samples of each forage type: sorghum, maize, and guinea grass. They were characterized using the 16S rRNA gene sequencing procedure. The anaerobic sugar fermentation ability of the isolated LAB was evaluated by inoculating them into sterilized sugar solutions, and measuring the titratable acidity (TA) and pH value over time.Findings: The isolates were identified to be non-motile, non-spore-forming and Gram-positive rods. Further, they tested negative for catalase, triple sugar iron, citrate, urease, indole, gelatine hydrolysis, motility, and oxidase tests. The LAB isolated from guinea grass, sorghum, and maize corresponded to Lactobacillus oris, Lactobacillus rhamnosus, and Lactobacillus plantarum, respectively. The TA in all inoculated solutions was higher compared to the control (0.55-0.66% vs. 0.48%), with L. plantarum showing the highest TA (0.66%) at 30 hours incubation. Additionally, all sugar solutions treated with LAB isolates exhibited a lower pH value at 30 hours compared to the control (4.70-5.07 vs. 5.75). Notably, L. plantarum demonstrated the most significant pH value reduction at 18 hours (5.85 vs. 6.29-6.64).Value: Lactobacillus oris, L. rhamnosus, and L. plantarum, isolated from guinea grass, sorghum, and maize silage, respectively, have the potential to be developed as inoculants for ensiling forage.

Increasing Productivity and Recovering Nutritional, Organoleptic, and Nutraceutical Qualities of Major Vegetable Crops for Better Dietetics.

The intensive use of chemical fertilizers for vegetable cultivation to achieve higher productivity causes soil degradation, resulting in an alarming decline (25-50%) in nutritional quality and a reduction in a wide variety of nutritionally essential minerals and nutraceutical compounds in high-yielding vegetable crops over the last few decades. To restore the physio-chemical and biological qualities of soil as well as the nutritional and nutraceutical qualities of fresh produce, there is a growing desire to investigate the remedial impacts of organic sources of nutrition. This study specifically focused on the impact of six different ratios of chemical fertilizers and organic sources with microbial inoculation on vegetable productivity, nutrition quality, and soil health parameters. Results show that replacing chemical fertilizers with organic sources in the presence of a microbial consortium supports the proliferation of the microbial population in the soil rhizosphere and improves the nutritional status and physico-chemical quality of soil, which is the area around the roots of plants where maximum nutrient uptake occurs. This combination of factors significantly recovers overall soil quality, increasing crop productivity by 13.58 to 18.32 percent in tomato, brinjal, and okra. Experimental findings likewise indicate that an assortment of organic sources with a microbial consortium significantly recovers the abundance of beneficial microbes and earthworms in the rhizosphere, which leads to an improvement in nutritional, organoleptic, and nutraceutical quality, with higher antioxidant contents in all three vegetables grown in arid climate conditions.

Exploring Optimal Combinations of Green Manures, Composts, and Microbial Inoculums to Boost Soil Biological Properties, Nutrient Release, and Basmati Rice Yield

Exploring Optimal Combinations of Green Manures, Composts, and Microbial Inoculums to Boost Soil Biological Properties, Nutrient Release, and Basmati Rice Yield

Microbial Inoculants in Sustainable Agriculture: Advancements, Challenges, and Future Directions.

The rapid growth of the human population has significantly increased the demand for food, leading to the intensification of agricultural practices that negatively impact the environment. Climate change poses a significant threat to global food production, as it can disrupt crop yields and modify the lifecycle stages of phytopathogens and pests. To address these challenges, the use of microbial inoculants, which are bioproducts containing beneficial microorganisms known as plant growth promotion microorganisms (PGPMs), has emerged as an innovative approach in sustainable agriculture. This review covers the isolation and identification of beneficial strains, the screening and selection process, the optimization of production techniques, and the importance of quality control and field testing. It also discusses the key points for the development and formulation of high-quality microbial inoculants, as well as highlights their advancements, current challenges, and future directions for research and application.

Closed genomes of commercial inoculant rhizobia provide a blueprint for management of legume inoculation.

Inoculation of cultivated legumes with exotic rhizobia is integral to Australian agriculture in soils lacking compatible rhizobia. The Australian inoculant program supplies phenotypically characterized high-performing strains for farmers but in most cases, little is known about the genomes of these rhizobia. Horizontal gene transfer (HGT) of symbiosis genes from inoculant strains to native non-symbiotic rhizobia frequently occurs in Australian soils and can impact the long-term stability and efficacy of legume inoculation. Here, we present the analysis of reference-quality genomes for 42 Australian commercial rhizobial inoculants. We verify and classify the genetics, genome architecture, and taxonomy of these organisms. Importantly, these genome sequences will facilitate the accurate strain identification and monitoring of inoculants in soils and plant nodules, as well as enable detection of horizontal gene transfer to native rhizobia, thus ensuring the efficacy and integrity of Australia's legume inoculation program.

Physicochemical Properties, Flavor and Microbial Community of Clean Low-Temperature Daqu Originated from Synthetic Autochthonous Microbiota

Based on the 23 strains of low-temperature Daqu, microbial inoculants were prepared and inoculated into crushed barley and pea to prepare clean low-temperature Daqu (XQ). The physicochemical properties, flavor profile, and microbial community of XQ were investigated, with traditional low-temperature Daqu (CQ) and production requirements as controls. The results indicated that there was no significant difference in moisture and acidity between the two types of Daqu: CQ exhibited a moisture of 10.6% and an acidity of 1.1 mmol/10 g, while XQ demonstrated a moisture of 10.9% and an acidity of 1.2 mmol/10 g. The fermenting activity (1.36 g/0.5 g·72 h) and liquefying activity (1.13 g/g·h) of XQ surpassed those of CQ; however, its saccharifying activity (780 mg/g·h) and esterifying activity (808 mg/50 g·7 d) were lower. In general, the physicochemical properties of XQ align with the production requirements. HS-SPME-GC-MS analysis indicated that the flavor profiles of the two types of Daqu were largely similar, with 84.85% of the flavor components of CQ being reproducible in XQ. Microbiota community analysis revealed there were some differences in relative abundance of microbes and COG (Clusters of Orthologous Groups) function between the two Daqu. The dominant microorganisms in XQ identified were Bacillus, Pichia, Transversalis, and Monascus, meanwhile the dominant microorganisms in CQ identified were Pediococcus, Rhizomucor, Wickerhamomyces. This study establishes a robust experimental basis for producing clean Daqu and provides valuable insights for the development of safer microbial fermented foods.

The dissimilarity between multiple management practices drives the impact on soil properties and functions

Higher dissimilarity between restoration factors boosted soil microbial activities when multiple factors jointly applied.More diverse management practices enhanced soil aggregate stability and improved soil pH.Increasing restoration factors up to 8 factors only influenced soil properties (water stable aggregates and soil pH) but not soil microbial activities.A range of land management practices are available to achieve better soil quality, but their combined effects remain understudied. We hypothesize that more diverse management practices, meaning higher dissimilarity, lead to stronger effects on soil functions and properties. Eight practices (biochar, compost, clay, amorphous silica, basalt, microbial inoculum, reduced physical disturbance and organic matter diversity) were selected with 20 replicates for treatments involving 2, 4, or 6 factors and 10 replicates for 8 factor treatments. We investigated the impact of individual factors, factor number, factor dissimilarity and factor composition on soil respiration, soil enzymatic activities (β-glucosidase, β-D-cellobiosidase, β-N-acetylglucosaminidase and phosphatase), soil pH, water stable aggregates and permanganate oxidizable carbon fraction. By including dissimilarity in addition to factor number, variance explained for soil respiration and enzymatic activities increased up to 54.21%. For soil pH and water-stable aggregates, explained variability increased to 65.57% and 57.38%, respectively. More diverse management practices boosted soil microbial activities, enhanced soil aggregate stability, improved soil pH while reducing labile carbon, whereas factor number only influenced water stable aggregates and soil pH. Our study highlights the importance of management practices diversity in soil functions and properties and calls for further research on synergistic combinations of diverse interventions.

Inoculation with effective microorganisms agent enhanced fungal diversity in the secondary fermentation process.

Microbial inoculations have emerged as a key approach to address the low natural microbial activity of traditional composting technologies. It is crucial for successfully promoting manure composting to understand the influences of microbial inoculations on fungal communities and its mechanisms. To investigate the effects of microbial inoculation on diversity characteristics, tropic mode, and co-occurrence network of fungal communities during composting, an aerobic composting experiment of chicken manure inoculated with microbial agents was performed. The results showed that microbial inoculations enhanced fungal richness and diversity during the secondary fermentation, promoted beneficial fungi, and restrained pathogenic microbes. Microbial inoculation facilitated saprophytic fungi and symbiotic fungi, augmented fungal network complexity and cooperation during the first fermentation, concurrently impeding fungal network complexity and cooperation during the secondary fermentation. These results provide technical guidance for composting process optimization and compost product quality improving, which was beneficial to promote soil quality and mitigating agricultural non-point source pollution.

Integrating plant growth microorganisms in potato farming reduce soil CO2 emissions and improve crop quality

AbstractCurrent horticultural practices typically require high demands of fertilizers, leading to significant environmental impacts and increased production costs. Alternatives based on microbial inoculants have garnered considerable interest owing to their potential to enhance soil quality whilst reducing external inputs and costs, all without compromising productivity. This study aimed to compare the impact of four fertilizer application strategies, including mineral fertilizers and microbial inoculants, on crop yield, soil fertility and functionality and soil greenhouse gas emissions in potato production in Southern Spain. Four treatments were tested: (i) mineral fertilizers added to meet the crop's nutritional needs (F100); (ii) a 50% reduction of the F100 rate (F50); (iii) a 50% reduction of the F100 rate combined with the application of a formulation containing nutrient‐solubilizing bacteria, nitrogen‐fixing bacteria and non‐mycorrhizal fungi (BAI+FU) and (iv) a 50% reduction of the F100 rate combined with a formulation of N, P and K solubilizing bacteria (BAII). Results showed that crop yield was unaffected by the different fertilizer application treatments. However, the mean tuber weight and firmness were significantly higher under the BAI+FU treatment, indicating improved product quality. CO2 release rates decreased by 25%, 34% and 42% with F50, BAI+FU and BAII treatments, respectively, compared with F100. The N2O and CH4 emissions or soil nutrient contents were not affected by treatments, except for ammonium content, which was highest under the BAI+FU treatment. Additionally, whilst overall soil bacterial and fungal abundance was not significantly affected, the BAI+FU treatment resulted in a higher number of nirK (denitrification) and cbbL (carbon fixation) gene copies. Therefore, combining biofertilizers with reduced chemical fertilizer rates could represent a sustainable strategy for mitigating climate change whilst enhancing crop quality in potato production.

Root microbes can improve plant tolerance to insect damage: A systematic review and meta-analysis.

To limit damage from insect herbivores, plants rely on a blend of defensive mechanisms that includes partnerships with beneficial microbes, particularly those inhabiting roots. While ample evidence exists for microbially mediated resistance responses that directly target insects through changing phytotoxin and volatile profiles, we know surprisingly little about the microbial underpinnings of plant tolerance. Tolerance defenses counteract insect damage via shifts in plant physiology that reallocate resources to fuel compensatory growth, improve photosynthetic efficiency, and reduce oxidative stress. Despite being a powerful mitigator of insect damage, tolerance remains an understudied realm of plant defenses. Here, we propose a novel conceptual framework that can be broadly applied across study systems to characterize microbial impacts on expression of tolerance defenses. We conducted a systematic review of studies quantifying the impact of rhizosphere microbial inoculants on plant tolerance to herbivory based on several measures-biomass, oxidative stress mitigation, or photosynthesis. We identified 40 studies, most of which focused on chewing herbivores (n = 31) and plant growth parameters (e.g., biomass). Next, we performed a meta-analysis investigating the impact of microbial inoculants on plant tolerance to herbivory, which was measured via differences in plant biomass, and compared across key microbe, insect, and plant traits. Thirty-five papers comprising 113 observations were included in this meta-analysis, with effect sizes (Hedges' d) ranging from -4.67 (susceptible) to 18.38 (overcompensation). Overall, microbial inoculants significantly reduce the cost of herbivory via plant growth promotion, with overcompensation and compensation comprising 25% of observations of microbial-mediated tolerance. The grand mean effect size 0.99 [0.49; 1.49] indicates that the addition of a microbial inoculant increased plant biomass by ~1 SD under herbivore stress, thus improving tolerance. This effect was influenced most by microbial attributes, including functional guild and total soil community diversity. Overall, results highlight the need for additional investigation of microbially mediated plant tolerance, particularly in sap-feeding insects and across a more comprehensive range of tolerance mechanisms. Such attention would round out our current understanding of anti-herbivore plant defenses, offer insight into the underlying mechanisms that promote resilience to insect stress, and inform the application of microbial biotechnology to support sustainable agricultural practices.

Quitosana diminui as perdas fermentativas e melhora a estabilidade aeróbica da silagem de milho reidratado

ABSTRACT: Ensilage of rehydrated corn kernels (RC) has been used to improve nutritional value and facilitate on-farm storage. This study evaluated the effects of chitosan and lactic acid microbial inoculants on rehydrated corn silage microbiology, fermentation profile and losses, chemical composition, in vitro degradation, and aerobic stability. Forty experimental silos were used in a completely random design to evaluate the following treatments: 1) Control (CON): RC silage without additives; 2) Chitosan (CHI): RC silage with 6 g/kg dry matter (DM) of chitosan; 3) Lactobacillus buchneri (LB): RC ensiled with 5 × 105 colony forming units (CFU) of L. buchneri per gram fresh weight; and 4) Lactobacillus plantarum and Pediococcus acidilactici (LPPA): RC ensiled 1.6 × 105 of L. plantarum and 1.6 × 105 P. acidilactici per gram fresh weight. Additives increased lactic acid bacteria and concentration of lactic and propionic acid, decreased mold and yeast count and gas and fermentative losses, and improved DM recovery. The CHI-silos had lower silage pH, Ammonia-N concentration, fermentative losses, and higher acetic acid concentration compared to microbial inoculated-silos. In addition, CHI and LB decreased silage pH and temperature after aerobic exposure. Although, treatments showed slight effects on the nutritional value of RC, CHI improved aerobic stability and decreaseds fermentation losses.