Zhen-Yu Liu

Introduction: Adverse abiotic environmental conditions including excess salt in the soil, constantly challenge plants and disrupt the function of plants, even inflict damage on plants. Salt stress is one of the major limiting factors for agricultural productivity and severe restrictions on plant growth. One of the critical ways to improve plant salt tolerance is halotolerant bacteria application. However, few such halotolerant bacteria were known and should be explored furtherly.Methods: Halophilic bacterium strain was isolated from saline soil with serial dilution and identified with classical bacteriological tests and 16S rRNA analysis. Perennial ryegrass (Lolium perenne L) was used in this study to evaluate the potential effect of the bacteria.Results and discussion: A halophilic bacterium strain GDHT17, was isolated from saline soil, which grows in the salinities media with 1.0%, 5.0%, and 10.0% (w/v) NaCl, and identified as Gracilibacillus dipsosauri. Inoculating GDHT17 can significantly promote ryegrass’s seedling height and stem diameter and increase the root length, diameter, and surface area at different salt concentrations, indicating the significant salt stress alleviating effect of GDHT17 on the growth of ryegrass. The alleviating effect on roots growth showed more effective, especially on the root length, which increased significantly by 26.39%, 42.59%, and 98.73% at salt stress of 100 mM, 200 mM, and 300 mM NaCl when the seedlings were inoculated with GDHT17. Inoculating GDHT17 also increases perennial ryegrass biomass, water content, chlorophyll and carotenoid content under salt stress. The contents of proline and malonaldehyde in the seedlings inoculated with GDHT17 increased by 83.50% and 6.87%, when treated with 300 mM NaCl; however, the contents of MDA and Pro did not show an apparent effect under salt stress of 100 mM or 200 mM NaCl. GDHT17-inoculating maintained the Na⁺/K⁺ ratio in the salt-stressed ryegrass. The Na⁺/K⁺ ratio decreased by 26.52%, 6.89%, and 29.92% in the GDHT17-inoculated seedling roots treated with 100 mM, 200 mM, and 300 mM NaCl, respectively. The GDHT17-inoculating increased the POD and SOD activity of ryegrass seedlings by 25.83% and 250.79%, respectively, at a salt stress of 300 mM NaCl, indicating the properties of GDHT17, improving the activity of antioxidant enzymes of ryegrass at the salt-stress condition. Our results suggest that G. dipsosauri GDHT17 may alleviate salt stress on ryegrass in multiple ways; hence it can be processed into microbial inoculants to increase salt tolerance of ryegrass, as well as other plants in saline soil.

Paenibacillus polymyxa is a plant growth–promoting rhizobacteria (PGPR) that has significant biocontrol properties. Wheat sheath blight caused by Rhizoctonia cerealis is a significant soil–borne disease of wheat that causes significant losses in wheat production, and the biological control against the disease has received extensive attention. P. polymyxa ZYPP18 was identified using morphological and molecular characterization. An antagonistic activity experiment verified that ZYPP18 inhibits the growth of R. cerealis on artificial growth media. A detached leaf assay verified that ZYPP18 inhibits the expansion of wheat sheath blight on the detached leaf. ZYPP18 has been found to possess plant growth–promoting properties, as well as the ability to solubilize phosphate and generate indole–3–acetic acid. Results from hydroponic experiments showed that wheat seedlings treated with ZYPP18 grew faster. Additionally, pot experiments and field experiments demonstrated that ZYPP18 effectively controls the occurrence of wheat sheath blight. ZYPP18 reduced the incidence of wheat sheath blight in wheat seedlings by 37.37% and 37.90%, respectively. The control effect of ZYPP18 on wheat sheath blight was 56.30% and 65.57%, respectively. These findings provide evidence that P. polymyxa ZYPP18 is an effective biological factor that can control disease and promote plant growth.

During their growth, bacteria respond to complex environmental changes through equally com-plex and orderly regulatory mechanisms. sRNA has an important role as a post-transcriptional regulator in the environmental adaptation of Bacillus. In this study, we identified an sRNA Bvs091 located in the intergenic region of the Bacillus velezensis, which is widely present in the genus Bacillus and whose expression is upregulated by salt treatment. The deletion of bvs091 in B. ve-lezensis PEBA20 led to a reduced growth rate and a change in the phenotypes, with simple colony structures and low and easily degradable biofilm. Salt tolerance tests indicated that the deletion of bvs091 results in a reduced salt tolerance. galT2 mRNA is supposed to be a direct target of Bvs091 according to results obtained by using the bioinformatic method and performing qRT-PCR analysis. The microscale thermophoresis (MST) analysis showed that Bvs091 could directly bind 28‒51 bases in front of the promoter of galT2. Subsequently, the deletion of galT2 in B. velezensis resulted in increased salt tolerance. Based on the techniques of double plasmids and a half-life period, sRNA Bvs091 is confirmed as a trans-encoded sRNA that negatively regulates the mRNA expression of galT2. In brief, the trans-encoded sRNA Bvs091 acts by pairing the incomplete complementary bases of galT2 mRNA, reducing galT2 mRNA’s stability and affecting the trans-lation efficiency.

Many putative sRNAs have been characterized using bioinformatic analysis and high-throughput sequencing in Gram-positive Bacillus strains, but there are only a few functional studies on the sRNAs involved in the spore formation developmental process. In particular, there is no sRNA confirmed experimentally to regulate the late stages of sporulation. Bvs196 is an sRNA with a length of 294 nucleotides that is abundantly expressed in the stationary phase of several media and independently transcribed in Bacillus velezensis strain PEBA20, as validated by RNA-seq and Northern blot,. It is also confirmed, by qRT-PCR, that Bvs196 is transcribed abundantly throughout the intermediate and late stages of sporulation. Using the gfpmut3a gene transcriptional reporter demonstrates that Bvs196 is expressed specifically in the forespore during sporulation and controlled by σ^F and σ^G (mainly by σ^G). This was observed by fluorescence microscopy and multi-function microplate reader. Further evolutionary conservation analysis found that Bvs196 is widely present in Bacillus with a strongly conserved and stable secondary structure. Resistance phenotypic assays of spores formed from the Bvs196 deletion mutant, the overexpressed Bvs196 mutant, and the wild-type strain revealed that the absence of Bvs196 led to reduced heat and UV resistance and enhanced formaldehyde resistance. We determined, by MST analysis, that Bvs196 can directly interact with spo0A and sspN-tlp mRNAs in vitro, and that short incomplete complementary paired bases affect the binding affinity of Bvs196 to target mRNAs. Our results suggest that Bvs196 is a novel sporulation-specific sRNA of B. velezensis, 294 nt in length, independently transcribed under the control of σ^F and σ^G in the forespore during sporulation, and that it affects spore resistance, and is able to directly interact with spo0A and sspN-tlp mRNAs. The remarkable conservation and impressive expression level of Bvs196 imply that it acts as an important conservative regulator, presumably by interacting with many other unknown targets in the forespore, and therefore contributing to spore properties. This work provides new clues for further understanding of the spore formation regulatory network.

Ophiostomatoid fungi are known for their associations with bark beetles, and some species are important sources of tree diseases. Ceratocystiopsis is a genus of the ophiostomatoid fungi in order Ophiostomatales. The shortage of DNA barcodes for many species in this genus has resulted in the presence of many unnamed cryptic species. In this study, Ceratocystiopsis subelongati sp. nov. associated with Ips subelongatus infesting Pinus sylvestris var. mongolica in Inner Mongolia, China, was identified and described based on phylogenetic inference of multi-gene DNA sequences and morphological characteristics. The species is characterized by a hyalorhinocladiella- to sporothrix-like asexual state and an optimal growth temperature of 30 °C. Artificial inoculation tests in the field showed that it is mildly pathogenic to five-year-old larch trees, the main host of I. subelongatus. It is also the first described Ceratocystiopsis species associated with I. subelongatus in China. This discovery should provide new avenues for studying the symbiosis between bark beetles and ophiostomatoid fungi.

Bacillus velezensis PEBA20 is a poplar endophyte with biocontrol activities and plant growth-promoting effects. The genome of B. velezensis PEBA20 was sequenced and the draft genome assembled, with a length of 4,249,176 bp and 4,487 genes.

Prunus serrulata (Japanese Cherry) belongs to the Rosaceae, subgenus Cerasus, and is a well-known ornamental plant worldwide. In May 2010, a stem canker disease on this plant was found in parks in Ji’nan city, Shandong province, China. The disease infected the trees severely, causing branches to wither and defoliate, flowers to fade and trees die. To isolate fungi, about forty diseased stem tissues were dipped in 70% ethanol for 30 s, sterilized with 0.1% (w/v) HgCl2 for 2 min, washed three times in sterilized water and cultured on potato dextrose agar (PDA). Fungal isolates were identified based on morphological characteristics and molecular features. Cultures grown on PDA were initially white with abundant aerial mycelia, and gradually became grey to dark grey after four days before finally becoming black, with a few scattered pycnidia formed occasionally in the colony 30 days later. The reverse side of the colony appeared light yellow, then became dark green in the center, and finally became black green. Conidia were hyaline, aseptate, thin walled, fusiform, and measured 18.2 ~ 28.84 μm× 4.14 ~ 7.42 μm. All of the morphological characteristics were consistent with Botryosphaeria sp. (Slippers et al. 2004). Genomic DNA was extracted and used for PCR to amplify the internal transcribed spacer (ITS) region, beta-tubulin (β-tub) gene, and partial translation elongation factor 1-alpha (EF1-α) gene, using primers ITS1/ITS4, βt2a/βt2b and EF1-728F/EF1-986R (De Wet et al. 2008), respectively. The obtained ITS sequence (GenBank Accession No. KT026967), β-tub sequence (KT026968) and EF1-α sequence (KT026969) showed 100%, 100%, 100% identity with those of the reference sequences of Botryosphaeria dothidea submitted in GenBank (KF293950, KR260833, HQ660485), respectively. Morphological and molecular results confirmed the fungus as B. dothidea. Pathogenicity tests were conducted on excised branches maintained fresh by placing one end in water. Healthy fresh branches without leaves of P. serrulata approximately 10 mm in diameter and 30-40 cm in length were washed in deionized water and surface sterilized in 10% sodium hypochlorite for 1 min. The branches were inoculated with a quarter of a 5-mm-diameter mycelial-agar disc, using three inoculation methods, burn inoculation, needle inoculation, and injury-free inoculation, respectively. There were 10 branches used for each method. Similarly, branches inoculated with a quarter of sterile a 5-mm-diameter PDA-disc using the three methods served as the negative control. After inoculation, the branches were kept moist by enclosing them in plastic wrap at 25°C. Twelve days after inoculation, the disease incidence reached 100%, 58.33%, and 0%, respectively, for the burn inoculation, needle inoculation and injury-free inoculations, while no symptoms appeared on the control branches, indicating the pathogenicity of the fungal isolate on P. serrulata. To our knowledge, this is the first report of P. serrulata stem canker caused by B. dothidea in China. This finding not only alerts local gardeners to have a serious concern of this disease, but may also lay the foundation for future disease diagnosis and control efforts in China. Reference: (1) Slippers, B., et al. 2004. Mycologia. 96:83. (2) De Wet, J., et al. 2008. Molecular Phylogenetics and Evolution. 46:116.