Revealing nutrient limitation status of microorganisms in the soil of Robinia pseudoacacia plantation through soil stoichiometry and enzyme metrology.

Exploring nutrient limitation in forest soil holds significant implications for forest tending and management. However, current research on nutrient limitation status of microorganisms in Robinia pseudoacacia plantations within the Loess Plateau remains insufficient. To investigate soil microbial nutrient limitation of R. pseu-doacacia plantations on the Loess Plateau, we selected R. pseudoacacia plantations with different afforestation time series (15, 25, 35, and 45 years) and a pile of barren slope cropland (control) in Yongshou County, Shaanxi Province as the research objects. We analyzed the contents of soil organic matter, total nitrogen, and total phosphorus, and the activities of ß-1,4-glucosidase (BG), cellobiose hydrolase (CBH), leucine aminopeptidase (LAP), ß-1,4-N-acetylglucoside (NAG) and phosphatase (AP). We analyzed the soil nutrient limitation by stoichiometry and enzyme metrology. The results showed a shift in soil pH from alkaline to acidic during vegetation restoration process, and that total phosphorus exhibited a gradual decrease over the course of 0 to 25 years. Soil orga-nic matter, total nitrogen and enzyme activities exhibited an increasing trend during the same time frame. However, between 25 and 45 years of age, soil total phosphorus, soil organic matter, total nitrogen, AP and LAP gradually declined while NAG, BG, and CBH initially increased and then decreased. Notably, the values of (BG+CBH)/(LAP+NAG), (BG+CBH)/AP and (LAP+NAG)/AP in R. pseudoacacia plantations were higher than the global average throughout the process of vegetation restoration. In the study area, the vector length was less than 1 and gradually increased, indicating that a progressive increase in microbial carbon limitation during the process of vegetation restoration. The vector angle exceeded 45° and exhibited an overall decreasing trend, suggesting that soil microorganisms were constrained by phosphorus (P) with a gradual deceleration of P limitation, without any nitrogen (N) limitation. The restoration of R. pseudoacacia plantation resulted in significant change in soil physical and chemical properties, while the time series of afforestation also influenced nutrient limitation of soil microorganisms.

Herbaspirillum robiniae sp. nov., isolated from root nodules of Robinia pseudoacacia in a lead-zinc mine.

A novel endophytic bacterium, designated strain HZ10T, was isolated from root nodules of Robinia pseudoacacia growing in a lead-zinc mine in Mianxian County, Shaanxi Province, China. The bacterium was Gram-stain-negative, aerobic, motile, slightly curved- and rod-shaped, methyl red-negative, catalase-positive, and did not produce H2S. Strain HZ10T grew at 4-45 °C (optimum, 25-30 °C), pH 5-9 (optimum, pH 7-8) and 0-1 % (w/v) NaCl. The major fatty acids were identified as C16 : 0, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), and the quinone type was Q-8. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of the genomic DNA was 64.9 mol% based on the whole genome sequence. According to the 16S rRNA gene sequence analysis, the closest phylogenetic relative to strain HZ10T is Herbaspirillum chlorophenolicum CPW301T (98.72 % sequence identity). Genome relatedness of the type strains H. chlorophenolicum CPW301T, Herbaspirillum seropedicae Z67T and Herbaspirillum aquaticum IEH 4430T, was quantified by using the average nucleotide identity (86.9-88.0 %) and a genome-to-genome distance analysis (26.6 %-29.3 %), with both strongly supporting the notion that strain HZ10T belongs to the genus Herbaspirillum as a novel species. Based on the results from phylogenetic, chemotaxonomic and physiological analyses, strain HZ10T represents a novel Herbaspirillum species, for which the name Herbaspirillum robiniae sp. nov. is proposed. The type strain is HZ10T (=JCM 31754T=CCTCC AB 2014352T).

Mitsuaria noduli sp. nov., isolated from the root nodules of Robinia pseudoacacia in a lead-zinc mine.

A novel endophytic bacterium, designated strain HZ7T, was isolated from the root nodules of Robinia pseudoacacia growing in a lead-zinc mine in Mianxian County, Shaanxi Province, China. Cells were Gram-reaction-negative, aerobic, motile, rod-shaped, methyl-red-negative, catalase-positive, positive for chitosan-degrading activity and did not produce H2S. Strain HZ7T grew at 4-45 °C (optimum 25-30 °C), at pH 5-9 (optimum pH 7-8) and with 0-1 % (w/v) NaCl. The quinone type was ubiquinone 8 (UQ-8). The major fatty acids were identified as C16 : 0, C17 : 0 cyclo and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The G+C content of the genomic DNA was 68.5 mol% by whole genome sequencing. According to 16S rRNA gene sequence analysis, the closest phylogenetic relative was Mitsuaria chitosanitabida 3001T (99.05 % similarity). Genome relatedness was computed using average nucleotide identity and genome-to-genome distance analysis, both of which strongly supported strain HZ7Tas belonging to the genus Mitsuaria as a representative of a novel species. On the basis of phylogenetic analysis, chemotaxonomic data and physiological characteristics, strain HZ7T represents a novel species of the genus Mitsuaria, for which the name Mitsuaria noduli sp. nov. is proposed. The type strain is HZ7T (=JCM 31671T=CCTCC AB 2014353T).

Micromonospora nickelidurans sp. nov., isolated from soil from a nickel-mining site.

An actinomycete, strain K55T, was isolated from a composite soil sample from a nickel mine,collected from Yueyang, Shaanxi Province, PR China. Strain K55T showed 16S rRNA gene sequence similarities of 98.73 %­98.51 % to species of the genus Micromonospora, including Micromonospora haikouensis 232617T, Micromonospora coxensis 2-30-b(28)T, Micromonospora wenchangensis 2602GPT1-05T, Micromonospora matsumotoense IMSNU22003T, Micromonospora maoerensis NEAU-MES19T, and Micromonospora humi P0402T. This strain harboured meso-diaminopimelic acid, alanine and glycine as the major cell-wall amino acids, xylose and glucose as the characteristic whole-cell sugars, and iso-C15 : 0(20.53 %),iso-C17 : 0 (12.74 %), iso-C16 : 0 (12.15 %), anteiso-C17 : 0 (7.97 %), C17 : 1ω8c(7.49 %) and C17 : 0 (6.63 %) as the dominant fatty acids. The major menaquinones were MK-10(H4) and MK-10(H6). The phospholipid profile comprised phosphatidylethanolamine,diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol and unknown phosphoglycolipids. The DNA G+C content was 71.4 mol%. A comprehensive analysis ofseveral physiological and biochemical traits and DNA­DNA relatedness indicated that strainK55T was different from closely related species. These phenotypic, genotypic and chemotaxonomic data suggest that strain K55T represents a novel species of the genus Micromonospora, for which the name Micromonospora nickelidurans sp. nov., is proposed. The type strain is K55T (5JCM 30559T5ACCC19713T).