Antagonistic activity and mechanism of Bacillus subtilis CG-6 suppression of root rot and growth promotion in Alfalfa.

Root rot is a common disease, that severely affects the yield and quality of alfalfa. Biocontrol is widely used to control plant diseases caused by pathogenic fungi, however, biocontrol strains for alfalfa root rot are very limited. In this study, a Bacillus subtilis CG-6 strain with a significant biocontrol effect on alfalfa root rot was isolated. CG-6 secretes antibacterial enzymes and siderophore, phosphate solubilization and indoleacetic acid (IAA). The inhibition rate of strain CG-6 against Fusarium oxysporum was 87.33%, and it showed broad-spectrum antifungal activity. Inoculation with CG-6 significantly reduced the incidence of alfalfa root rot, the control effect of greenhouse cultivation reached 58.12%, and CG-6 treatment significantly increased alfalfa plant height, root length, fresh weight, and dry weight. The treatment with CG-6 significantly increased the levels of antioxidant enzymes (catalase, peroxidase, superoxide dismutase, and lipoxygenase) in alfalfa leaves by 15.52%-34.03%. Defensive enzymes (chitinase and ß-1,3-glucanase) increased by 24.37% and 28.08%, respectively. The expression levels of regulatory enzyme genes (MsCAT, MsPOD, MsCu, Zn-SOD1, MsCu, Zn-SOD2, MsCu, Zn-SOD3, and MsLOX2) and systemic resistance genes (MsPR1, MsPDF1.2, and MsVSP2) increased by 0.50-2.85 fold, which were higher than those in the pathogen treatment group. Therefore, CG-6 could be used as a potential strain to develop biopesticides against alfalfa root rot.

Molybdenum-mediated nitrogen accumulation and assimilation in legumes stepwise boosted by the coexistence of arbuscular mycorrhizal fungi and earthworms.

Molybdenum (Mo) is a critical micronutrient for nitrogen (N) metabolism in legumes, yet the impact of Mo on legume N metabolism in the context of natural coexistence with soil microorganisms remains poorly understood. This study investigated the dose-dependent effect of Mo on soil N biogeochemical cycling, N accumulation, and assimilation in alfalfa under conditions simulating the coexistence of arbuscular mycorrhizal fungi (AMF) and earthworms. The findings indicated that Mo exerted a hormetic effect on alfalfa N accumulation, facilitating it at low concentrations (below 29.98 mg/kg) and inhibiting it at higher levels. This inhibition was attributed to Mo-induced constraints on C supply for nitrogen fixation. Concurrently, AMF colonization enhanced C assimilation in Mo-treated alfalfas by promoting nutrients uptake, particularly Mg, which is crucial for chlorophyll synthesis. This effect was further amplified by earthworms, which improved AMF colonization (p < 0.05). In the soil N cycle, these organisms exerted opposing effects: AMF enhanced soil nitrification and earthworms reduced soil nitrate (NO3--N) reduction to jointly increase soil phyto-available N content (p < 0.05). Their combined action improved alfalfa N assimilation by restoring the protein synthesis pathway that is compromised by high Mo concentrations, specifically the activity of glutamine synthetase. These findings underscored the potential for soil microorganisms to mitigate N metabolic stress in legumes exposed to elevated Mo levels.

Phosphatidylcholine-deficient suppressor mutant of Sinorhizobium meliloti, altered in fatty acid synthesis, partially recovers nodulation ability in symbiosis with alfalfa (Medicago sativa).

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.

Shifts of the soil microbiome composition induced by plant-plant interactions under increasing cover crop densities and diversities.

Interspecific and intraspecific competition and facilitation have been a focus of study in plant-plant interactions, but their influence on plant recruitment of soil microbes is unknown. In this greenhouse microcosm experiment, three cover crops (alfalfa, brassica, and fescue) were grown alone, in paired mixtures, and all together under different densities. For all monoculture trials, total pot biomass increased as density increased. Monoculture plantings of brassica were associated with the bacteria Azospirillum spp., fescue with Ensifer adhaerens, and alfalfa with both bacterial taxa. In the polycultures of cover crops, for all plant mixtures, total above-ground alfalfa biomass increased with density, and total above ground brassica biomass remained unchanged. For each plant mixture, differential abundances highlighted bacterial taxa which had not been previously identified in monocultures. For instance, mixtures of all three plants showed an increase in abundance of Planctomyces sp. SH-PL14 and Sandaracinus amylolyticus which were not represented in the monocultures. Facilitation was best supported for the alfalfa-fescue interaction as the total above ground biomass was the highest of any mixture. Additionally, the bulk soil microbiome that correlated with increasing plant densities showed increases in plant growth-promoting rhizobacteria such as Achromobacter xylosoxidans, Stentotrophomonas spp., and Azospirillum sp. In contrast, Agrobacterium tumefaciens, a previously known generalist phytopathogen, also increased with alfalfa-fescue plant densities. This could suggest a strategy by which, after facilitation, a plant neighbor could culture a pathogen that could be more detrimental to the other.

Effects of nisin on bacterial community and fermentation profiles, in vitro rumen fermentation, microbiota, and methane emission of alfalfa silage.

BACKGROUND: Alfalfa (Medicago sativa L.) has been used widely in preparing silage. However, forage legumes are prone to contamination by spoilage bacteria during fermentation. Nisin has broad-spectrum antibacterial properties and has been applied as an inhibitor of rumen methane emissions. However, little research has been carried out on the application of nisin in silage. This study therefore aimed to investigate the impacts of different nisin concentrations on the bacterial community and fermentation dynamics, in vitro ruminal fermentation characteristics, microbiota, and methane emissions of alfalfa silage. RESULTS: The detection limits of organic acid in nisin-treated silages were not reached in 0.09 g kg-1 nisin (0.09 level) from days 1 to 7 of ensiling. With increasing nisin concentrations, the silage dry matter increased linearly (P < 0.05), and dry matter loss decreased linearly (P < 0.05). Moreover, both the 0.06 g kg-1 nisin (0.06 level) and 0.09 level treatments increased the relative abundance of Pediococcus acidilactici during ensiling. Concurrently, as the nisin concentrations increased, ruminal methane production decreased linearly (P < 0.05), while the relative abundances of ruminal Succinivibrio, Fibrobacter succinogenes and Ruminobacter amylophilus increased linearly (P < 0.05). The populations of ruminal total bacteria, methanogens, protozoa, and fungi decreased linearly with increasing nisin concentrations (P < 0.05). CONCLUSION: The addition of nisin delayed the fermentation process, preserved more nutrients in alfalfa silage, and promoted fermentation dominated by P. acidilactici in the late phase of ensiling. Moreover, nisin reduced in vitro rumen methane emissions without adverse effects on dry matter digestibility. © 2023 Society of Chemical Industry.

Arbuscular mycorrhizal fungus changes alfalfa (Medicago sativa) metabolites in response to leaf spot (Phoma medicaginis) infection, with subsequent effects on pea aphid (Acyrthosiphon pisum) behavior.

Plant disease occurs simultaneously with insect attack. Arbuscular mycorrhizal fungi (AMF) modify plant biotic stress response. Arbuscular mycorrhizal fungi and pathogens may modify plant volatile organic compound (VOC) production and insect behavior. Nevertheless, such effects are rarely studied, particularly for mesocosms where component organisms interact with each other. Plant-mediated effects of leaf pathogen (Phoma medicaginis) infection on aphid (Acyrthosiphon pisum) infestation, and role of AMF (Rhizophagus intraradices) in modifying these interactions were elucidated in a glasshouse experiment. We evaluated alfalfa disease occurrence, photosynthesis, phytohormones, trypsin inhibitor (TI) and total phenol response to pathogen and aphid attack, with or without AMF, and aphid behavior towards VOCs from AMF inoculated and non-mycorrhizal alfalfa, with or without pathogen infection. AM fungus enhanced alfalfa resistance to pathogen and aphid infestation. Plant biomass, root : shoot ratio, net photosynthetic rate, transpiration rate, stomatal conductance, salicylic acid, and TI were significantly increased in AM-inoculated alfalfa. Arbuscular mycorrhizal fungi and pathogen significantly changed alfalfa VOCs. Aphids preferred VOCs of AM-inoculated and nonpathogen-infected to nonmycorrhizal and pathogen-infected alfalfa. We propose that AMF alter plant response to multiple biotic stresses in ways both beneficial and harmful to the plant host, providing a basis for strategies to manage pathogens and herbivore pests.

Dry matter content and inoculant alter the metabolome and bacterial community of alfalfa ensiled at high temperature.

Alfalfa silage fermentation quality, metabolome, bacterial interactions, and successions as well as their predicted metabolic pathways were explored under different dry matter contents (DM) and lactic acid bacteria (LAB) inoculations. Silages were prepared from alfalfa with DM contents of 304 (LDM) and 433 (HDM) g/kg fresh weight and inoculated with Lactiplantibacillus plantarum (L. plantarum, LP), Pediococcus pentosaceus (P. pentosaceus, PP), or sterile water (control). The silages were stored at a simulated hot climate condition (35°C) and sampled at 0, 7, 14, 30, and 60 days of fermentation. The results revealed that HDM significantly improved the alfalfa silage quality and altered microbial community composition. The GC-TOF-MS analysis discovered 200 metabolites in both LDM and HDM alfalfa silage, mainly consisting of amino acids, carbohydrates, fatty acids, and alcohols. Compared with LP and control, PP-inoculated silages had increased concentrations of lactic acid (P < 0.05) and essential amino acids (threonine and tryptophan) as well as decreased pH, putrescine content, and amino acid metabolism. However, alfalfa silage inoculated with LP had higher proteolytic activities than control and PP-inoculated silage, as revealed by a higher concentration of ammonia nitrogen (NH3-N), and also upregulated amino acid and energy metabolism. HDM content and P. pentosaceus inoculation significantly altered the composition of alfalfa silage microbiota from 7 to 60 days of ensiling. Conclusively, these results indicated that inoculation with PP exhibited great potential in enhancing the fermentation of silage with LDM and HDM via altering the microbiome and metabolome of the ensiled alfalfa, which could help in understanding and improving the ensiling practices under hot climate conditions. KEY POINTS: • HDM improved fermentation quality and declined putrescine content of alfalfa silage • P. pentosaceus inoculation enhanced the fermentation quality of alfalfa silage • P. pentosaceus is an ideal inoculant for alfalfa silage under high temperature.

Coaggregative interactions between rhizobacteria are promoted by exopolysaccharides from Sinorhizobium meliloti.

Bacterial surface components and extracellular compounds such as exopolysaccharides (EPSs) are crucial for interactions between cells, tolerance to different types of stress, and host colonization. Sinorhizobium meliloti produces two EPSs: Succinoglycan (EPS I), which is involved in the establishment of symbiosis with Medicago sativa, and galactoglucan (EPS II), associated with biofilm formation and the promotion of aggregation. Here, we aimed to assess their role in aggregative interactions between cells of the same strain of a given species (auto-aggregation), and between genetically different strains of the same or different species (intra- or intergeneric coaggregation). To do this, we used S. meliloti mutants which are defective in the production of EPS I, EPS II, or both. Macroscopic and microscopic coaggregation tests were performed with combinations or pairs of different bacterial strains. The EPS II-producing strains were more capable of coaggregation than those that cannot produce EPS II. This was true both for coaggregations between different S. meliloti strains, and between S. meliloti and other common rhizobacteria of agricultural relevance, such as Pseudomonas fluorescens and Azospirillum brasilense. The exogenous addition of EPS II strongly promoted coaggregation, thus confirming the polymer's importance for this phenotype. EPS II may therefore be a key factor in events of physiological significance for environmental survival, such as aggregative interactions and biofilm development. Furthermore, it might be a connecting molecule with relevant properties at an ecological, biotechnological, and agricultural level.

Effects of Pyroligneous Acid on Diversity and Dynamics of Antibiotic Resistance Genes in Alfalfa Silage.

Antibiotic resistance genes (ARGs) are recognized as contaminants due to their potential risk for human and environment. The aim of the present study is to investigate the effects of pyroligneous acid (PA), a waste of biochar production, on fermentation characteristics, diversity, and dynamics of ARGs during ensiling of alfalfa using metagenomic analysis. The results indicated that PA decreased (P < 0.05) dry matter loss, pH value, gas production, coliform bacteria count, protease activity, and nonprotein-N, ammonia-N, and butyric acid contents and increased (P < 0.05) lactic acid content during ensiling. During fermentation, Bacteria, Firmicutes, and Lactobacillus were the most abundant at kingdom, phylum, and genus levels, respectively. Pyroligneous acid reduced the relative abundance of Bacteria and Firmicutes and increased that of Lactobacillus. The detected ARGs belonged to 36 drug classes, including mainly macrolides, tetracycline, lincosamides, and phenicol. These types of ARGs decreased during fermentation and were further reduced by PA. These types of ARGs were positively correlated (P < 0.05) with fermentation parameters like pH value and ammonia-N content and with bacterial communities. At the genus level, the top several drug classes, including macrolide, tetracycline, lincosamide, phenicol, oxazolidinone, streptogramin, pleuromutilin, and glycopeptide, were positively correlated with Staphylococcus, Streptococcus, Listeria, Bacillus, Klebsiella, Clostridium, and Enterobacter, the potential hosts of ARGs. Overall, ARGs in alfalfa silage were abundant and were influenced by the fermentation parameters and microbial community composition. Ensiling could be a feasible way to mitigate ARGs in forages. The addition of PA could not only improve fermentation quality but also reduce ARG pollution of alfalfa silage. IMPORTANCE Antibiotic resistance genes (ARGs) are considered environmental pollutants posing a potential human health risk. Silage is an important and traditional feed, mainly for ruminants. ARGs in silages might influence the diversity and distribution of ARGs in animal intestinal and feces and then the manure and the manured soil. However, the diversity and dynamics of ARGs in silage during fermentation are still unknown. We ensiled alfalfa, one of the most widely used forages, with or without pyroligneous acid (PA), which was proved to have the ability to reduce ARGs in soils. The results showed that ARGs in alfalfa silage were abundant and were influenced by the fermentation parameters and microbial community. The majority of ARGs in alfalfa silage reduced during fermentation. The addition of PA could improve silage quality and reduce ARG pollution in alfalfa silage. This study can provide useful information for understanding and controlling ARG pollution in animal production.

The "Molecular Biodiversity of Bacteria Isolated from Medicago sativa Rhizosphere in Hail District, Saudi Arabia.

Global biodiversity is affected remarkably by global climate change, which in turn its effect reflected on all life aspects. Identifying microorganisms in environmental samples, particularly soil could be a valuable interest to study their effect on soil quality and plant growth. Through this study, we conducted a molecular characterization of bacteria found in the rhizosphere of Medico sativa plants grown in Hail soil and we highlighted their main properties. The sequences analyses revealed that the main bacterial isolates Pseudarthrobacter, Metabacillus, Priestia, and Massilia species. According to the sequences analysis and the phylogeny tree results, some of the identified bacteria were classified at the species level: Pseudarthrobacter was identified clearly as Pseudarthrobacter phenanthrenivorans; Metabacillus isolates grouped with Metabacillus sediminilitoris and the two Priestia isolates closely related to Priestia aryabhattai. We concluded that Hail soil is a niche of diverse bacteria with a high interest in soil environment and ecosystems. Further studies are required for further classification of all identified bacteria and to define their specific role in the environment.

Changes to the microbiome of alfalfa during the growing season and after ensiling with Lentilactobacillus buchneri and Lentilactobacillus hilgardii inoculant.

AIMS: This study evaluated changes in epiphytic microbial population of alfalfa (Medicago sativa) during the growing season. First cut forage was harvested to study the effects of an inoculant combining two obligate heterofermentative lactic acid bacteria strains on the bacterial and fungal communities and the fermentation of alfalfa silage. METHODS AND RESULTS: The epiphytic microbiome of alfalfa was evaluated 10-times during the growing season. Alfalfa wilted to 395.0 g/kg was treated with water (Control) or with a combination of L. buchneri NCIMB 40788 and L. hilgardii CNCM-I-4785 (LBLH). Mini-silos were opened after 1, 4, 8, 16, 32, and 64 days of ensiling. The relative abundance (RA) of the epiphytic bacterial and fungal families varied during the growing season. After 1 day, Weissella was the most abundant genus and present at similar RA in the two treatments (average 80.4%). Compared with Control, LBLH had a higher RA of Lactobacillus at day 1, 16, 32, and 64, and a lower RA of Weissella from day 8 to 64. Control contained more bacteria belonging to the Enterobacteriales than LBLH up to day 16. Inoculated silage had more acetate than Control at day 32 and 64. The fungal population were similar between treatments. The enhanced development and dominance of Lactobacillus in inoculated silage led to greater accumulation of acetate and propionate, which reduced the numbers of culturable yeasts but did not markedly affect the fungal community structure. CONCLUSIONS: The bacterial community composition of alfalfa stands in the filed changed over time and was affected by cutting. For the ensiling trial, inoculation modified the composition of the bacterial community of alfalfa, increasing the RA of Lactobacillus while reducing the RA of Weissella and of Enterobacteriaceae. SIGNIFICANCE AND IMPACT OF STUDY: Inoculation increased the RA of Lactobacillus, hampering the dominance of Weissella in the early stages of ensiling, improving antifungal compounds production and reducing the numbers of culturable yeasts.

Evaluating the potential of lignosulfonates and chitosans as alfalfa hay preservatives using in vitro techniques.

Our objectives were to compare the antifungal activity of 5 lignosulfonates, and 2 chitosans against fungi isolated from spoiled hay, and assess the effects of an optimized lignosulfonate, chitosan, and propionic acid (PRP) on high-moisture alfalfa hay. In experiment 1, we determined the minimum inhibitory concentration and minimum fungicidal concentration of 4 sodium lignosulfonates, 1 magnesium lignosulfonate, 2 chitosans, and PRP (positive control) against Aspergillus amoenus, Mucor circinelloides, Penicillium solitum, and Debaromyces hansenii at pH 4 and 6. Among sodium lignosulfonates, the one from Sappi Ltd. (NaSP) was the most antifungal at pH 4. However, chitosans had the strongest fungicidal activity with the exception of M. circinelloides at both pH 4 and 6. PRP had more antifungal effects than NaSP and was only better than chitosans for M. circinelloides. In experiment 2, we evaluated the effects of 3 additives (ADV): optimized NaSP (NaSP-O, UMaine), naïve chitosan (ChNv, Sigma-Aldrich), and PRP on high-moisture alfalfa hay. The experimental design was a randomized complete block design replicated 5 times. Treatment design was the factorial combination of 3 ADV× 5 doses (0, 0.25, 0.5, 1, and 2% w/w fresh basis). Additives were added to 35 g of sterile alfalfa hay (71.5 ± 0.23% DM), inoculated with a mixture of previously isolated spoilage fungi (5.8 log cfu/fresh g), and aerobically incubated in vitro for 23 d (25°C). After incubation, DM losses were reduced by doses as low as 0.25% for both NaSP-O and PRP (x¯=1.61) vs. untreated hay (24.0%), partially due to the decrease of mold and yeast counts as their doses increased. Also, hay NH3-N was lower in NaSP-O and PRP, with doses as low as 0.25%, relative to untreated hay (x¯=1.13 vs. 7.80% of N, respectively). Both NaSP-O and PRP increased digestible DM recovery (x¯=69.7) and total volatile fatty acids (x¯=94.3), with doses as low as 0.25%, compared with untreated hay (52.7% and 83.8 mM, respectively). However, ChNv did not decrease mold nor yeast counts (x¯=6.59 and x¯=6.16 log cfu/fresh g, respectively) and did not prevent DM losses relative to untreated hay. Overall, when using an alfalfa hay substrate in vitro, NaSP-O was able to prevent fungal spoilage to a similar extent to PRP. Thus, further studies are warranted to develop NaSP-O as a hay preservative under field conditions.

In our first experiment, we assessed the antifungal activity of two major types of byproducts, one known as lignosulfonates (5 types), which are generated by paper mills, and another known as chitosans (2 types), which are generated from shellfish. These were tested against four fungi isolated from spoiled hay. We observed that acidic conditions are not necessary for chitosans but are crucial to activate the antifungal properties of lignosulfonates. Also, we found that sodium lignosulfonate from Sappi Ltd. was the most antifungal relative to other sodium lignosulfonates from other manufacturers. Chitosans had stronger fungicidal activity than propionic acid or lignosulfonates against all but one mold tested. In our second experiment, we compared the best treatments from experiment 1 against propionic acid using alfalfa hay as a substrate to grow the same fungi tested in experiment 1. None of the doses of chitosan prevented spoilage on high moisture hay, showing results similar to untreated hay. In contrast, an optimized sodium lignosulfonate and propionic acid prevented fungal spoilage of alfalfa hay with doses as low as 0.25%.

Combined effects of intermittent radio frequency heating with cinnamon oil vapor on microbial control and quality changes of alfalfa seeds.

Alfalfa sprouts have high nutritional values when consumed raw, but are easily contaminated by food pathogens. This study aimed to evaluate effects of essential oil (EO) vapors, RF heating and their combinations on microbial inactivation, germination rate and generated sprouts' quality of alfalfa seeds at moisture contents of 7.52%, 9.53% and 11.45% wet basis (w.b.). Results showed that cinnamon oil and oregano oil vapors both had antimicrobial effects against Salmonella on alfalfa seeds and cinnamon oil vapor had a little better activity. Weibull model well fitted the Salmonella inactivation curves under RF heating. The rate of Salmonella inactivation increased but germination rate decreased with increasing temperature and moisture content of seeds. The intermittent RF treatments improved the heating uniformity and germination rate of the whole batch of seeds as compared to the continuous RF heating. The combination of intermittent RF heating and cinnamon oil vapor exhibited additive antimicrobial effects, up to 3.99-4.12 log CFU/g Salmonella reductions, and maintained the germination rate above 90%. However, for natural microbial decontamination, combined treatments only caused 0.56-0.82 log CFU/g reductions of total bacterial counts. The fresh weight, length, flavor, the content of phenolics and ascorbic acid, and antioxidant capacity of generated sprouts were not significantly impacted. The study provided an effective method for microbial control on sprouting seeds.

Effect of Alfalfa Cultivars and Ages on the Occurrence of Verticillium Wilt Caused by Verticillium alfalfae.

In August 2019, Verticillium wilt was observed in commercial alfalfa fields in Jinta County, Jiuquan, located to the west of Gansu, China, where Verticillium wilt of alfalfa was first observed in this region. This study was conducted to evaluate the effect of alfalfa cultivars (Galaxie Max, Liangmu No. 2, and Danon VNS, planted in 2017) and ages (cultivar Adrenalin, planted in 2014, 2015, and 2016) on the occurrence of Verticillium wilt caused by Verticillium alfalfae. The results showed that V. alfalfae was successfully isolated from both symptomatic and asymptomatic plants. The percentage of V. alfalfae colonization ranged from 22 to 83% in symptomatic plants and 19 to 31% in asymptomatic plants. Among the three cultivars tested, the lowest incidence of disease symptoms was observed in the plants of cultivar Galaxie Max, In addition, the plants of Galaxie Max had a lower rate of infection with V. alfalfae in the field than the cultivar Danon VNS. Moreover, the diseased plants of Galaxie Max had a higher shoot dry weight and levels of nitrogen (N), phosphorus (P), starch, sucrose, and chlorophyll than the diseased plants of Liangmu No. 2 and Danon VNS. This demonstrated that Galaxie Max has higher resistance/tolerance to Verticillium wilt than Danon VNS. An examination of the different ages of Adrenalin indicated that the plants in 2014 had a higher incidence of disease and rate of infection in the field than the plants in 2016. In addition, the diseased plants in 2016 had a higher shoot dry weight and contents of N, P, sucrose, and starch than the diseased plants in 2014 and 2015. This result indicated that an increase in the age of alfalfa plants contributes to the occurrence and development of Verticillium wilt. The infection of V. alfalfae significantly decreased the shoot dry weights and the contents of chlorophyll, N, P, and starch of alfalfa plants. These results provide a better understanding of the physiological mechanisms of the response of alfalfa plants to Verticillium wilt caused by V. alfalfae.

Metagenomic analysis reveals the linkages between bacteria and the functional enzymes responsible for potential ammonia and biogenic amine production in alfalfa silage.

AIMS: To clarify the molecular mechanisms underlying ammonia (NH3 ) and biogenic amines (BAEs) formation in alfalfa silage, whole metagenomic sequencing analysis was performed to identify the linkages between functional bacteria and their responsible enzymes in alfalfa silage prepared with and without sucrose addition. METHODS AND RESULTS: Genes encoding nitrite reductase (nirB) resulting in NH3 formation were the most abundant and were mostly assigned to Enterobacter cloacae and Klebsiella oxytoca. Putrescine-related genes, classified mainly to encode ornithine decarboxylase (odcA), were predominantly carried by Escherichia coli, Ent. cloacae and Citrobacter sp. Escherichia coli and Kl. oxytoca were the important species responsible for cadaverine and tyramine formation. Ent. cloacae, E. coli, and Kl. oxytoca dominated the bacterial community in naturally fermented alfalfa silage, whilst sucrose-treated silages greatly inhibited the growth of these species by promoting the dominance of Lactobacillus plantarum, thus decreasing the concentrations of NH3 , cadaverine, putrescine and tyramine. CONCLUSIONS: Enterobacteriaceae bacteria are mainly responsible for the NH3 , putrescine, cadaverine and tyramine formations in alfalfa silage. SIGNIFICANCE AND IMPACT OF THE STUDY: Whole metagenomic sequencing analysis served as a useful tool to identify the linkages between functional bacteria and associated enzymes responsible for NH3 and BAEs formation.

Dynamics of the bacterial communities and predicted functional profiles in wilted alfalfa silage.

AIMS: To investigate the fermentation characteristics, bacterial community and predicted functional profiles during the ensiling of wilted alfalfa (Medicago sativa L.). METHODS AND RESULTS: First-cutting alfalfa was harvested at the early bloom stage, wilted for 6 h, and ensiled in laboratory-scale silos (1 L capacity). Triplicate silos were sampled after 1, 3, 7, 15, 30 and 60 days of ensiling, respectively. The bacterial communities of wilted alfalfa and silages on day 3 and 60 were assessed through high throughput sequencing technology, and their functional characteristics were evaluated according to the Kyoto Encyclopedia of Genes and Genomes using Tax4Fun. After 60 days of ensiling, alfalfa silage showed a moderate fermentation quality, indicated by high lactic acid (56.7 g kg-1 dry matter [DM]) and acetic acid (39.4 g kg-1 DM) contents, and low concentrations of butyric acid (2.12 g kg-1 DM) and ammonia nitrogen (128 g kg-1 total nitrogen). Lactobacillus rapidly became predominant on day 3 and increased to 60.4% on day 60. Results of functional prediction analyses showed that the metabolism of amino acid, energy, cofactors and vitamins were reduced, while metabolism of nucleotide and carbohydrate were increased during ensiling. Fructokinase, 1-phosphofructokinase and pyruvate kinase played important roles in producing lactic acid. The production of acetic acid may be correlated with the enhancement of 6-phosphogluconate dehydrogenase and acetyl-CoA synthetase. CONCLUSIONS: Knowledge regarding bacterial dynamics and their metabolic pathways during alfalfa ensiling is important for understanding the fermentation process and may contribute to the production of nutritious and stable alfalfa silage. SIGNIFICANCE AND IMPACT OF THE STUDY: High throughput sequencing technology combined with 16S rRNA gene-predicted functional analyses could provide a new and comprehensive insight into bacterial community dynamics and functional profiles to further improve the silage quality.

Silage fermentation characteristics and microbial diversity of alfalfa (Medicago sativa L.) in response to exogenous microbiota from temperate grasses.

The objective of this study was to explore the microbiological factors that cause the difference in silage fermentation characteristics between grass and legume. Specifically, the effects of epiphytic microbiota from alfalfa, oat and Italian ryegrass on ensiling characteristics and microbial community of alfalfa were assessed. By γ-ray irradiation sterilization and microbiota transplantation technology, the sterile alfalfa was inoculated as follows: (i) aseptic water (STAL); (ii) epiphytic bacteria from alfalfa (ALAL); (iii) epiphytic bacteria from oat (ALOT); (iv) epiphytic bacteria from Italian ryegrass (ALIR). Alfalfa at the initial flowering stage was ensiled in laboratory-scale silos for 1, 3, 7, 14, 30 and 60 days. Compared with ALAL and ALIR, higher lactic acid contents and ratio of lactic acid to acetic acid, and lower acetic acid, propionic acid, ethanol and ammonia nitrogen contents were observed in ALOT after 60 days of fermentation. In each treated group, Lactobacillus was the most dominant genus after 60 days of ensiling. Relatively higher abundance of Weissella, Hafnia-Obesumbacterium, Enterobacteriaceae or hetero-fermentative Lactobacillus was found in ALAL and ALIR after 60 days. Co-occurrence network analysis proved Pediococcus and Lactococcus were pivotal in deciding the fermentation pattern of alfalfa silage. According to the 16S rRNA gene-predicted functional profiles, the metabolism of amino acids was inhibited by the epiphytic microbiota from oat. Overall, ALOT showed a homo-fermentative process, whereas ALAL and ALIR exhibited a hetero-fermentative pattern. Furthermore, the exogenous microorganisms inhibiting the metabolism of amino acids can be a good potential source to improve the silage quality of legume forage.

Mycolicibacterium sp. strain PAM1, an alfalfa rhizosphere dweller, catabolizes PAHs and promotes partner-plant growth.

This research was focused on the isolation and characterization of a PAH-catabolizing mycobacterial strain from the petroleum hydrocarbon-contaminated rhizosphere of alfalfa, as well as on revealing some points of interaction between the microorganism and the plant. Mycolicibacterium sp. PAM1, a pyrene degrader isolated from the niche of interest to us, can catabolize fluoranthene, anthracene, fluorene, and phenanthrene. On the basis of curves of PAM1 growth with different PAHs as the sole carbon sources and on the basis of PAH-degradation rates, we found that pollutant availability to the strain decreased in the sequence phenanthrene > fluorene > fluoranthene ∼ pyrene > anthracene. For each PAH, the catabolic products were identified. PAM1 was found to have the functional genes nidA and nidB. New data modeling the 2D and 3D structures, intrinsic structural disorder, and molecular dynamics of the nidA and nidB gene products were obtained. The identified genes and intermediates of pyrene degradation indicate that PAM1 has a PAH catabolic pathway that is peculiar to known mycobacterial pyrene degraders. PAM1 utilized some components of alfalfa root exudates as nutrients and promoted plant growth. The use of mycobacterial partners of alfalfa is attractive for enhancing the phytoremediation of PAH-contaminated soils.

The influence of alfalfa-switchgrass intercropping on microbial community structure and function.

The use of nitrogen fertilizer on bioenergy crops such as switchgrass results in increased costs, nitrogen leaching and emissions of N2 O, a potent greenhouse gas. Intercropping with nitrogen-fixing alfalfa has been proposed as an environmentally sustainable alternative, but the effects of synthetic fertilizer versus intercropping on soil microbial community functionality remain uncharacterized. We analysed 24 metagenomes from the upper soil layer of agricultural fields from Prosser, WA over two growing seasons and representing three agricultural practices: unfertilized switchgrass (control), fertilized switchgrass and switchgrass intercropped with alfalfa. The synthetic fertilization and intercropping did not result in major shifts of microbial community taxonomic and functional composition compared with the control plots, but a few significant changes were noted. Most notably, mycorrhizal fungi, ammonia-oxidizing archaea and bacteria increased in abundance with intercropping and fertilization. However, only betaproteobacterial ammonia-oxidizing bacteria abundance in fertilized plots significantly correlated to N2 O emission and companion qPCR data. Collectively, a short period of intercropping elicits minor but significant changes in the soil microbial community toward nitrogen preservation and that intercropping may be a viable alternative to synthetic fertilization.

alfaNET: A Database of Alfalfa-Bacterial Stem Blight Protein-Protein Interactions Revealing the Molecular Features of the Disease-causing Bacteria.

Alfalfa has emerged as one of the most important forage crops, owing to its wide adaptation and high biomass production worldwide. In the last decade, the emergence of bacterial stem blight (caused by Pseudomonas syringae pv. syringae ALF3) in alfalfa has caused around 50% yield losses in the United States. Studies are being conducted to decipher the roles of the key genes and pathways regulating the disease, but due to the sparse knowledge about the infection mechanisms of Pseudomonas, the development of resistant cultivars is hampered. The database alfaNET is an attempt to assist researchers by providing comprehensive Pseudomonas proteome annotations, as well as a host-pathogen interactome tool, which predicts the interactions between host and pathogen based on orthology. alfaNET is a user-friendly and efficient tool and includes other features such as subcellular localization annotations of pathogen proteins, gene ontology (GO) annotations, network visualization, and effector protein prediction. Users can also browse and search the database using particular keywords or proteins with a specific length. Additionally, the BLAST search tool enables the user to perform a homology sequence search against the alfalfa and Pseudomonas proteomes. With the successful implementation of these attributes, alfaNET will be a beneficial resource to the research community engaged in implementing molecular strategies to mitigate the disease. alfaNET is freely available for public use at http://bioinfo.usu.edu/alfanet/.