Effects of non-thermal plasma technology on Diaporthe longicolla cultures and mechanisms involved.

BACKGROUND: The Diaporthe/Phomopsis complex (D/P) is a group of soybean seed-borne fungi. The use of chemical fungicides, either for seed treatment or during the crop cycle, is the most adopted practice for treating fungal diseases caused by this complex. Worldwide, there is a search for alternative seed treatments that are less harmful to the environment than chemicals. Non-thermal plasma (NTP) is a novel seed treatment technology for pathogen removal. This research aimed to evaluate the effects of NTP on the in vitro performance of pure cultures of Diaporthe longicolla and elucidate the mechanisms underlying these effects. RESULTS: Active D. longicolla mycelium, growing in vitro, was exposed to different NTP treatments, employing a dielectric barrier discharge arrangement with different carrier gases (N2 or O2 ). Fungal growth, fresh biomass and colony appearance were negatively affected by plasma treatments (TN3 and TO3). Lipid peroxidation and antioxidant activities were higher in plasma-treated colonies comparison with non-exposed colonies (control). Fungal asexual spores (conidia) were also exposed to NTP, showing high susceptibility. CONCLUSION: Exposure of D. longicolla colonies to NTP severely compromised fungal biology. Ozone production during treatment and lipid peroxidation of fungal cell membranes appeared to be involved in the observed effects. © 2020 Society of Chemical Industry.

Enhancement of soybean nodulation by seed treatment with non-thermal plasmas.

Soybean (Glycine max (L.) Merrill) is one of the most important crops worldwide providing dietary protein and vegetable oil. Most of the nitrogen required by the crop is supplied through biological N2 fixation. Non-thermal plasma is a fast, economical, and environmental-friendly technology that can improve seed quality, plant growth, and crop yield. Soybean seeds were exposed to a dielectric barrier discharge plasma operating at atmospheric pressure air with superimposed flows of O2 or N2 as carrying gases. An arrangement of a thin phenolic sheet covered by polyester films was employed as an insulating barrier. We focused on the ability of plasma to improve soybean nodulation and biological nitrogen fixation. The total number of nodules and their weight were significantly higher in plants grown from treated seeds than in control. Plasma treatments incremented 1.6 fold the nitrogenase activity in nodules, while leghaemoglobin content was increased two times, indicating that nodules were fixing nitrogen more actively than control. Accordingly, the nitrogen content in nodules and the aerial part of plants increased by 64% and 23%, respectively. Our results were supported by biometrical parameters. The results suggested that different mechanisms are involved in soybean nodulation improvement. Therefore, the root contents of isoflavonoids, glutathione, auxin and cytokinin, and expansin (GmEXP1) gene expression were determined. We consider this emerging technology is a suitable pre-sowing seed treatment.

Erratum to "Improvement of growth and yield of soybean plants through the application of non-thermal plasmas to seeds with different health status" [Heliyon Volume 5, Issue 4, April 2019, e01495].

[This corrects the article DOI: 10.1016/j.heliyon.2019.e01495.].

Improvement of growth and yield of soybean plants through the application of non-thermal plasmas to seeds with different health status.

Soybean (Glycine max (L.) Merrill) is a globally important crop, providing oil and protein. Diaporthe/Phomopsis complex includes seed-borne pathogens that affect this legume. Non-thermal plasma treatment is a fast, cost-effective and environmental-friendly technology. Soybean seeds were exposed to a quasi-stationary (50 Hz) dielectric barrier discharge plasma operating at atmospheric pressure air. Different carrying gases (O2 and N2) and barrier insulating materials were used. This work was performed to test if the effects of non-thermal plasma treatment applied to healthy and infected seeds persist throughout the entire cycle of plants. To this aim, lipid peroxidation, activity of catalase, superoxide dismutase and guaiacol peroxidase, vegetative growth and agronomic traits were analysed. The results here reported showed that plants grown from infected seeds did not trigger oxidative stress due to the reduction of pathogen incidence in seeds treated with cold plasma. Vegetative growth revealed a similar pattern for plants grown from treated seeds than that found for the healthy control. Infected control, by contrast, showed clear signs of damage. Moreover, plasma treatment itself increased plant growth, promoted a normal and healthy physiological performance and incremented the yield of plants. The implementation of this technology for seeds treatment before sowing could help reducing the use of agrochemicals during the crop cycle.

Heme oxygenase up-regulation under ultraviolet-B radiation is not epigenetically restricted and involves specific stress-related transcriptions factors.

Heme oxygenase-1 (HO-1) plays a protective role against oxidative stress in plants. The mechanisms regulating its expression, however, remain unclear. Here we studied the methylation state of a GC rich HO-1 promoter region and the expression of several stress-related transcription factors (TFs) in soybean plants subjected to ultraviolet-B (UV-B) radiation. Genomic DNA and total RNA were isolated from leaves of plants irradiated with 7.5 and 15kJm-2 UV-B. A 304bp HO-1 promoter region was amplified by PCR from sodium bisulfite-treated DNA, cloned into pGEMT plasmid vector and evaluated by DNA sequencing. Bisulfite sequencing analysis showed similar HO-1 promoter methylation levels in control and UV-B-treated plants (C: 3.4±1.3%; 7.5: 2.6±0.5%; 15: 3.1±1.1%). Interestingly, HO-1 promoter was strongly unmethylated in control plants. Quantitative RT-PCR analysis of TFs showed that GmMYB177, GmMYBJ6, GmWRKY21, GmNAC11, GmNAC20 and GmGT2A but not GmWRK13 and GmDREB were induced by UV-B radiation. The expression of several TFs was also enhanced by hemin, a potent and specific HO inducer, inferring that they may mediate HO-1 up-regulation. These results suggest that soybean HO-1 gene expression is not epigenetically regulated. Moreover, the low level of HO-1 promoter methylation suggests that this antioxidant enzyme can rapidly respond to environmental stress. Finally, this study has identified some stress-related TFs involved in HO-1 up-regulation under UV-B radiation.

Evaluation of native bacteria and manganese phosphite for alternative control of charcoal root rot of soybean.

Plant growth promoting rhizobacteria (PGPR) are potential agents to control plant pathogens and their combined use with biopesticides such as phosphites may constitute a novel strategy to incorporate in disease management programs. In the present study, 11 bacterial isolates were selected on the basis of their antagonistic activity against Macrophomina phaseolina in dual-culture tests, and their plant growth promoting traits. Selected isolates were characterised on the basis of auxin and siderophore production, phosphate solubilisation and rep-PCR genomic fingerprinting. Two of these isolates, identified as Pseudomonas fluorescens 9 and Bacillus subtilis 54, were further evaluated for their inhibitory capacity against M. phaseolina using in vitro (on soybean seeds) and in vivo (greenhouse assay) tests. Both bacteria were applied individually as well as in combined treatment with manganese phosphite as seed treatments. Damage severity on soybean seeds was significantly reduced, compared with the untreated control, by both bacterial strains; however, the individual application of phosphite showed to be least effective in controlling M. phaseolina. Interestingly, the phosphite treatment improved its performance under greenhouse conditions compared to the results from the in vitro assays. In the greenhouse trials, the greatest reductions in disease severity were achieved when strain P. fluorescens 9 was applied singly or when strain B. subtilis 54 was combined with manganese phosphite, achieving 82% of control in both cases. This work is the first to report the control of M. phaseolina using combined treatment with PGPR and phosphite under greenhouse conditions.

Nitric oxide induces specific isoforms of antioxidant enzymes in soybean leaves subjected to enhanced ultraviolet-B radiation.

Antioxidant enzymes play a key role in plant tolerance to different types of stress, including ultraviolet-B (UV-B) radiation. Here we report that nitric oxide (NO) enhances antioxidant enzymes gene expression and increases the activity of specific isoforms protecting against UV-B radiation. Pre-treatments with sodium nitroprussiate (SNP), a NO-donor, prevented lipid peroxidation, ion leakage and H2O2 and superoxide anion accumulation in leaves of UV-B-treated soybean plants. Transcripts levels of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were significantly induced by SNP. These data correlated with the enhancement of particular antioxidant enzyme isoforms, such as one CAT isoform and two APX isoforms. Moreover, SNP induced the expression of three new isoforms of SOD, identified as Mn-SOD subclass. Further results showed that total activities of SOD, CAT and APX significantly increased by 2.2-, 1.8- and 2.1-fold in SNP-treated plants compared to controls, respectively. The protective effect of SNP against UV-B radiation was negated by addition of the specific NO scavenger cPTIO, indicating that NO released by SNP mediates the enhancement of antioxidant enzymes activities. In conclusion, NO is involved in the signaling pathway that up-regulates specific isoforms of antioxidant enzymes protecting against UV-B-induced oxidative stress.

Symbiotic association between soybean plants and Bradyrhizobium japonicum develops oxidative stress and heme oxygenase-1 induction at early stages.

We have previously demonstrated that the induction of heme oxygenase-1 (HO-1) (EC 1.14.99.3) plays a protective role against oxidative stress in leaves and nodules of soybean plants subjected to cadmium, UV-B radiation, and salt stress. Here, we investigated HO-1, localization and their relationship with oxidative stress in different growth stages of soybean plants roots inoculated with Bradyrhizobium japonicum (3, 5, 7, 10, and 20 days post-inoculation) and nodules. After 7 days of inoculation, we observed a 70% increase in thiobarbituric acid-reactive substances that correlates with an enhancement in the gene expression of HO-1, catalase, and superoxide dismutase. Furthermore, the inhibition of HO-1 activity by Zn-protoporphyrin IX produced an increase in lipid peroxidation and a decrease in glutathione content suggesting that, in this symbiotic process, HO-1 may act as a signal molecule that protects the root against oxidative stress. We determined, for the first time, the tissular localization of HO-1 in nodules by electron-microscope examination. These results undoubtedly demonstrated that this enzyme is localized only in the plant tissue and its overexpression may play an important role as antioxidant defense in the plant. Moreover, we demonstrate that, in roots, HO-1 is induced by oxidative stress produced by inoculation of B. japonicum and exerts an antioxidant response against it.

Heme Oxygenase Contributes to Alleviate Salinity Damage in Glycine max L. Leaves.

Plants are frequently subjected to different kinds of stress, such as salinity and, like other organisms, they have evolved strategies for preventing and repairing cellular damage caused by salt stress. Glycine max L. plants were subjected to different NaCl concentrations (0-200 mM) for 10 days. Treatments with 100 and 200 mM NaCl induced ion leakage and lipid peroxidation augmentation, loss in chlorophyll content, and accumulation of O(2) (*-) and H(2)O(2). However, 50 mM NaCl did not modify these parameters, which remains similar to control values. Catalase, superoxide dismutase, and heme oxygenase (HO-1) activities and gene expressions were increased under 100 mM NaCl, while no differences were observed with respect to controls under 50 mM salt. Treatment with 200 mM NaCl caused a diminution in the enzyme activities and gene expressions. Results here reported let us conclude that HO also plays a leading role in the defense mechanisms against salinity.