Development and evaluation of temperature-sensitive Mycoplasma anserisalpingitidis clones as vaccine candidates.

Mycoplasma anserisalpingitidis is economically the most important pathogenic Mycoplasma species of waterfowl in Europe and Asia. The lack of commercially available vaccines against M. anserisalpingitidis had prompted this study with the aim to produce temperature-sensitive (ts+) clones as candidates for an attenuated live vaccine. The production of ts+ clones was performed by N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis of Hungarian M. anserisalpingitidis field isolates. The clones were administered via eye-drop and intracloacally to 33-day-old geese. Colonization ability was examined by PCR and isolation from the trachea and cloaca, while the serological response of the birds was tested by ELISA. Pathological and histopathological examinations were performed in the eighth week after inoculation. Whole-genome sequence (WGS) analysis of the selected clone and its parent strain was also performed. NTG-treatment provided three ts+ mutants (MA177/1/11, MA177/1/12, MA271). MA271 was detected at the highest rate from cloacal (86.25%) and tracheal (30%) samples, while MA177/1/12 and MA271 elicited remarkable serological responses with 90% of the birds showing seroconversion. Re-isolates of MA271 remained ts+ throughout the experiment. Based on these properties, clone MA271 was found to be the most promising vaccine candidate. WGS analysis revealed 59 mutations in the genome of MA271 when compared to its parent strain, affecting both polypeptides involved in different cellular processes and proteins previously linked to bacterial fitness and virulence. Although further studies are needed to prove that MA271 is in all aspects a suitable vaccine strain, it is expected that this ts+ clone will contribute to the control of M. anserisalpingitidis infection.RESEARCH HIGHLIGHTS Three M. anserisalpingitidis ts+ vaccine candidates were produced by NTG-mutagenesis.Clone MA271 was able to colonize geese and induce a serological response.MA271 re-isolates remained ts+ during the 8-week-long experiment.WGS analysis revealed 59 mutations in the genome of MA271.

High red/far-red ratio promotes root colonization of Serratia plymuthica A21-4 in tomato by root exudates-stimulated chemotaxis and biofilm formation.

Effective colonization on plant roots is a prerequisite for plant growth promoting rhizobacterias (PGPR) to exert beneficial activities. Light is essential for plant growth, development and stress response. However, how light modulates root colonization of PGPR remains unclear. Here, we found that high red/far red (R/FR) light promoted and low R/FR light inhibited the colonization and growth enhancement of Serratia plymuthica A21-4 (S. plymuthica A21-4) on tomato, respectively. Non-targeted metabolomic analysis of root exudates collected from different R/FR ratio treated tomato seedlings with or without S. plymuthica A21-4 inoculation by UPLC-MS/MS showed that 64 primary metabolites in high R/FR light-grown plants significantly increased compared with those determined for low R/FR light-grown plants. Among them, 7 amino acids, 1 organic acid and 1 sugar obviously induced the chemotaxis and biofilm formation of S. plymuthica A21-4 compared to the control. Furthermore, exogenous addition of five artificial root exudate compontents (leucine, methionine, glutamine, 6-aminocaproic acid and melezitose) regained and further increased the colonization ability and growth promoting ability of S. plymuthica A21-4 on tomato under low R/FR light and high R/FR light, respectively, indicating their involvement in high R/FR light-regulated the interaction of tomato root and S. plymuthica A21-4. Taken together, our results, for the first time, clearly demonstrate that high R/FR light-induced root exudates play a key role in chemotaxis, biofilm formation and root colonization of S. plymuthica A21-4. This study can help promote the combined application of light supplementation and PGPR to facilitate crop growth and health in green agricultural production.

Development of a Real-Time Quantitative PCR Assay for the Specific Detection of Bacillus velezensis and Its Application in the Study of Colonization Ability.

Bacillus velezensis is a widely used biocontrol agent closely related to B. amyloliquefaciens, and the two species cannot be distinguished by universal primers that are currently available. The study aimed to establish a rapid, specific detection approach for B. velezensis. Many unique gene sequences of B. velezensis were selected through whole genome sequence alignment of B. velezensis strains and were used to design a series of forward and reverse primers, which were then screened by PCR and qPCR using different Bacillus samples as templates. The colonization ability of B. velezensis ZF2 in different soils and different soil environmental conditions was measured by qPCR and a 10-fold dilution plating assay. A specific primer pair targeting the sequence of the D3N19_RS13500 gene of B. velezensis ZF2 was screened and could successfully distinguish B. velezensis from B. amyloliquefaciens. A rapid specific real-time qPCR detection system for B. velezensis was established. B. velezensis ZF2 had a very strong colonization ability in desert soil, and the optimal soil pH was 7-8. Moreover, the colonization ability of strain ZF2 was significantly enhanced when organic matter from different nitrogen sources was added to the substrate. This study will provide assistance for rapid specificity detection and biocontrol application of B. velezensis strains.

Decoding the potential of a new Pseudomonas putida strain for inducing drought tolerance of tomato (Solanum lycopersicum) plants through seed biopriming.

A total of 11 potential plant growth promoting rhizobacteria previously isolated from naturally stressed environments were evaluated for various traits of interest for a beneficial symbiosis with plants, including colonization ability, biofilm formation, motility, exopolysaccharide production and salt tolerance. The vast majority of the strains were found to possess multiple plant growth promoting traits. Nevertheless, the intensity varied among isolates, with those originated from tomato plants being more efficient colonizers. The strain SAESo11, genetically characterized as a Pseudomonas putida member was selected for further investigation of its potential to alleviate drought stress in tomato seedlings. Inoculation with SAESo11 mitigated the negative effects of drought stress as indicated by growth and photosynthetic indices. Furthermore, bacterial inoculation enhanced H2O2 content and malondialdehyde levels in colonized plants. Drought treatment did not further alter the oxidative status of these plants. Similarly, total phenolic content and antioxidant enzyme activity were induced in plant tissues in response to drought stress only at the absence of inoculum. These results indicated that inoculation with the selected strain imposed plants at a priming state, that enabled them to respond more robustly at the exposure to drought stress and efficiently attenuated the drought-induced injury. This state of plant alertness mediated by SAESo11 occurred at no cost to growth, highlighting its role as a potential plant priming agent.

Genomic Insights Into the Antifungal Activity and Plant Growth-Promoting Ability in Bacillus velezensis CMRP 4490.

The main objective of this study was to evaluate Bacillus velezensis strain CMRP 4490 regarding its ability to inhibit soil-borne plant pathogens and to increase plant growth. The study included evaluation of in vitro antifungal control, sequencing the bacterial genome, mining genes responsible for the synthesis of secondary metabolites, root colonization ability, and greenhouse studies for the assessment of plant growth-promoting ability. The strain was obtained from soil samples in the north of Paraná in Brazil and was classified as a B. velezensis, which is considered a promising biological control agent. In vitro assay showed that B. velezensis CMRP 4490 presented antagonistic activity against Sclerotinia sclerotiorum, Macrophomina phaseolina, Botrytis cinerea, and Rhizoctonia solani with a mycelial growth inhibition of approximately 60%, without any significant difference among them. To well understand this strain and to validate its effect on growth-promoting rhizobacteria, it was decided to explore its genetic content through genome sequencing, in vitro, and greenhouse studies. The genome of CMRP 4490 was estimated at 3,996,396 bp with a GC content of 46.4% and presents 4,042 coding DNA sequences. Biosynthetic gene clusters related to the synthesis of molecules with antifungal activity were found in the genome. Genes linked to the regulation/formation of biofilms, motility, and important properties for rhizospheric colonization were also found in the genome. Application of CMRP 4490 as a coating film on soybean increased from 55.5 to 64% on germination rates when compared to the control; no differences were observed among treatments for the maize germination. The results indicated that B. velezensis CMRP 4490 could be a potential biocontrol agent with plant growth-promoting ability.