Background: Symbiotic Methylobacterium strains comprise a significant part of plant microbiomes. Their presence enhances plant productivity and stress resistance, prompting classification of these strains as plant growth-promoting bacteria (PGPB). Methylobacteria can synthesize unusually high levels of plant hormones, called cytokinins (CKs), including the most active form, trans-Zeatin (tZ). Results: This study provides a comprehensive inventory of 46 representatives of Methylobacterium genus with respect to phytohormone production in vitro, including 16 CK forms, abscisic acid (ABA) and indole-3-acetic acid (IAA). High performance-liquid chromatography—tandem mass spectrometry (HPLC–MS/MS) analyses revealed varying abilities of Methylobacterium strains to secrete phytohormones that ranged from 5.09 to 191.47 pmol mL⁻¹ for total CKs, and 0.46 to 82.16 pmol mL⁻¹ for tZ. Results indicate that reduced methanol availability, the sole carbon source for bacteria in the medium, stimulates CK secretion by Methylobacterium. Additionally, select strains were able to transform L-tryptophan into IAA while no ABA production was detected. Conclusions: To better understand features of CKs in plants, this study uncovers CK profiles of Methylobacterium that are instrumental in microbe selection for effective biofertilizer formulations.

While a lot is known about cytokinins (CKs) and their actions at the molecular and cellular levels in plants, much less is known about the function of CKs in other kingdoms such as fungi. CKs have been detected in a wide range of fungal species where they play roles ranging from enhancing the virulence of phytopathogens to fortifying plant growth when secreted from fungal symbionts. However, the role of CKs where they concern fungal physiology, apart from plant associations, remains largely uncharacterized. Profiling by UHPLC-HRMS/MS (ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry) revealed that Pleurotus ostreatus (oyster mushroom) produces CKs in vitro in both liquid and solid cultures. During fungal growth, CK profiling patterns were consistent with previous suggestions that tRNA degradation products might play a role in the physiological development of fungi. It confirms that those products are CKs that act as fungal growth regulators. Moreover, P. ostreatus was shown to respond to exogenous applications of aromatic and isoprenoid CKs, and their effects were dependent on the dose and CK type in a biphasic manner consistent with hormone action. N⁶-benzyladenine (BAP), kinetin (KIN), N⁶-isopentenyladenine (iP), and trans-zeatin (tZ) bioassays all revealed hormesis-type responses. Accordingly, at low doses, mycelium colony diameter, biomass accumulation, and changes in morphology were stimulated, whereas at high doses only inhibitory effects were observed. Thus, CKs may act as "mycohormones" and consequently have potential for applications in fungal agriculture and medicinal compound production.

Magneto-priming (MP) has been reported as a sustainable method to enhance crop yield and resistance to adverse environmental conditions; however, any physiochemical basis for these findings remains unavailable. In the present study, soybean (Glycine max) seeds (n = 232) were exposed to a static magnetic field (SMF) of 150–205 mT for 60 min. The effect of SMF exposure on seedling mass, hypocotyl length, radicleemergence rate, total seedling height, leaf area, chlorophyl content, and physiological attributes was evaluated. Differential analysis of the cytokinin (CK) profile of seedling tissues was achieved using ultra-high-performance liquid chromatography coupled with electrospray-ionization high-resolution tandem mass spectrometry (UHPLC-(ESI)-HRMS/MS). Results indicate that MP seedlings achieved radicle emergence earlier; however, no other statistically significant differences could be established. The absence in significant improvement in the agronomic and physiological attributes measured is in stark contrast to the results reported in other studies. Nonetheless, significant differences were found in the concentrations of trans-Zeatin (tZ) and cis-Zeatin (cZ) between control and MP plants. These results are incongruent as it would be assumed that insignificant differences in agronomic and physiological properties would align with phytohormone profiles. Future work should be performed to determine if there are any meaningful bioeffects that can be assigned to MP-generated hormone changes.

Symbiotic Methylobacterium comprise a significant part of the plant microbiome and are known to benefit host plant growth, development, tolerance to abiotic stress, and enhanced disease resistance. The wide application of commercial broad-spectrum fungicide formulations in contemporary agriculture practices has necessitated the investigation of compatibility between popular pesticide products and bacterial endophytes, especially as the Methylobacterium are increasingly considered for agronomic use, including biocontrol of phytopathogens. This study provides an evaluation of compatibility between a extensive inventory of 40 Methylobacterium strains in response to commercial pesticide formulations, each containing different agtive ingredients: DYNASTY® and QUADRIS® (azoxystrobin), MAXIM®480 (fludioxonil), and APRON XL® LS (metalaxyl-M). Using a diffusion disk assay, no sensitivity of tested Methylobacterium strains could be detected against any fungicide product, at doses within and above the recommended therapeutic window (1–100 µg). Potency of formulations across the same range were confirmed using the sensitive phytopathogen Fusarium graminearum. As Methylobacterium spp. continue to emerge as suitable candidates for various roles in biotechnology, including agriculture, a better understanding on the compatibility between this important genus and commercial fungicide products has become a relevant consideration for integrated pest management practices.

The biocontrol potential of Methylobacterium spp. against key phytopathogenic fungi was evaluated using in vitro antagonism assays and confined germination bioassays. Across 40 Methylobacterium isolates, inhibitory activity was highly variable and strain-dependent. Fusarium graminearum (UAMH 3329) was the most consistently suppressed pathogen; however, only M. thiocyanatum (NBRC 103124) and M. aminovorans (LMG 21752) produced significant inhibition (>30%, p < 0.05) across all three Fusarium species tested. Growth suppression was enhanced on nutrient-minimum agar amended with cell-free bacterial broth, supporting the involvement of diffusible inhibitory factors in fungal growth suppression. However, marked isolate- and substrate-dependent effects suggest that antagonism is contingent on specific bacterium–pathogen combinations. In germination assays, M. organophilum (LMG 6083) conferred protection to soybean ( Glycine max) seeds challenged with F. graminearum, improving germination and seedling development in both artificial and soil substrates. Co-inoculated seeds exhibited lower disease severity scores (DSS), fewer necrotic lesions, and reduced fungal colonization during germination and emergence. Collectively, these findings identify candidate Methylobacterium strains with antagonistic activity against Fusarium spp. and provide a foundation for future characterization of their effectors relevant to integrated crop protection.