Antivirulence effects of cell-free culture supernatant of endophytic bacteria against grapevine crown gall agent, Agrobacterium tumefaciens, and induction of defense responses in plantlets via intact bacterial cells.

BACKGROUND: Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control plant diseases via the use of several mechanisms. This research examined the potential for controlling crown gall by three endophytic bacteria that were previously isolated from healthy cultivated and wild grapevines including Pseudomonas kilonensis Ba35, Pseudomonas chlororaphis Ba47, and Serratia liquefaciens Ou55. RESULT: At various degrees, three endophytic bacteria suppressed the populations of A. tumefaciens Gh1 and greatly decreased the symptoms of crown gall. Furthermore, biofilm production and motility behaviors of A. tumefaciens Gh1were greatly inhibited by the Cell-free Culture Supernatant (CFCS) of endophytic bacteria. According to our findings, CFCS may reduce the adhesion of A. tumefaciens Gh1 cells to grapevine cv. Rashe root tissues as well as their chemotaxis motility toward the extract of the roots. When compared to the untreated control, statistical analysis showed that CFCS significantly reduced the swimming, twitching, and swarming motility of A. tumefaciens Gh1. The findings demonstrated that the endophytic bacteria effectively stimulated the production of plant defensive enzymes including superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and total soluble phenols at different time intervals in grapevine inoculated with A. tumefaciens Gh1. The Ba47 strain markedly increased the expression levels of defense genes associated with plant resistance. The up-regulation of PR1, PR2, VvACO1, and GAD1 genes in grapevine leaves indicates the activation of SA and JA pathways, which play a role in enhancing resistance to pathogen invasion. The results showed that treating grapevine with Ba47 increased antioxidant defense activities and defense-related gene expression, which reduced oxidative damage caused by A. tumefaciens and decreased the incidence of crown gall disease. CONCLUSION: This is the first study on how A. tumefaciens, the grapevine crown gall agent, is affected by CFCS generated by endophytic bacteria in terms of growth and virulence features. To create safer plant disease management techniques, knowledge of the biocontrol processes mediated by CFCS during microbial interactions is crucial.

Strawberry crinkle (SCV) and Strawberry mottle (SMoV) viruses affect yield and physio-biochemical responses of three strawberry cultivars under different seasonal conditions.

BACKGROUND: The current study was performed to evaluate the impact of Strawberry crinkle virus (SCV) and Strawberry mottle virus (SMoV) viruses, in single and mixed infections, on the yield and physio-biochemical traits of strawberry plants with different genetic backgrounds. Accordingly, the virus-free plantlets were produced via meristem culture and were then transplanted in a research field in different seasons. METHODS AND RESULTS: The results indicated that genetic background clearly affects the responses of the strawberry plants to the viral infection. In addition, considering the effects of changing environmental factors, different seasons had significant influences on the strawberry responses to the virus infection. The results indicated that the components indicating destructive effects were significantly higher during the summer season. Reverse transcription-polymerase chain reaction using viral specific primers revealed 345 and 461 bp fragments for SCV and SMoV viruses, respectively, where all plants were positive for studied viruses. The comparison between the influence of SCV and SMoV viruses on the host plants indicated that SCV caused more damaging effects on the strawberry fruit yield and physio-biochemical traits. Simultaneous infection with SCV and SMoV severely impacted the strawberry plants compared with the effect of each virus individually. CONCLUSION: It is recommended to keep the temperature of the greenhouses producing strawberry at the cooler levels to prevent the spreading of the aphid-transmitted viruses.

Effect of volatile compounds produced by endophytic bacteria on virulence traits of grapevine crown gall pathogen, Agrobacterium tumefaciens.

The volatile organic compounds (VOCs) produced by endophytic bacteria have a significant role in the control of phytopathogens. In this research, the VOCs produced by endophytic bacteria including Serratia sp. Ba10, Pantoea sp. Sa14, Enterobacter sp. Ou80, Pseudomonas sp. Ou22, Pseudomonas sp. Sn48 and Pseudomonas sp. Ba35, which were previously isolated from healthy domesticated and wild-growing grapevine were evaluated in terms of their effects on the virulence traits of Agrobacterium tumefaciens Gh1, the causal agent of crown gall disease. Based on the gas chromatography-mass spectrometry analysis, 16, 15, 14, 7, 16, and 15 VOCs have been identified with high quality in strains of Ba10, Sa14, Ou80, Ou22, Sn48, and Ba35, respectively. All endophytic bacteria produced VOCs that significantly reduced crown gall symptoms and inhibited the populations of A. tumefaciens Gh1 at different levels. Moreover, scanning electron microscopy analysis revealed various morphological abnormalities in the A. tumefaciens cells exposed to the VOCs produced by Ba35, Ou80, and Sn48 strains. The VOCs significantly reduced swarming-, swimming-, twitching motility and biofilm formation by A. tumefaciens Gh1. Our results revealed that VOCs could reduce the attachment of A. tumefaciens Gh1 cells to root tissues of grapevine cultivars Rashe and Bidane sefid, as well as chemotaxis motility towards root extract of both cultivars. Based on our results, it was shown that the antibacterial VOCs produced by endophytic bacteria investigated in the current study can manage crown gall disease and increase our knowledge on the role of VOCs in microbial interactions.

Alternate gene expression profiling of monoterpenes in Hymenocrater longiflorus as a novel pharmaceutical plant under water deficit.

Hymenocrater longiflorus (surahalala) is a wild plant species with potential pharmaceutical and ornamental interest. To date, the genomics of this plant is unknown and the gene expression profiling of the genes related to its metabolite has never been studied before. In order to study the responses of in vitro-grown surahalala plants to abiotic stresses and the differential expression of the genes related to its essential oils under exogenous proline application; three levels of PEG600 (0, 10, and 20%) and five levels of proline (0, 5, 10, 15, and 20 µm) were combined in the culture media. Thus, water deficit increased oxidants levels and decreased fresh weight of surahalala tissues, whereas addition of proline up to 15 µm was able to relatively compensate the negative effect of water deficit. Contrarily, high proline level (20 µm) had a negative effect on surahalala plants probably due to the stress simulation (nutrition) under high proline concentration. In addition, the best combination for achieving highest essential oils content was 10 µm proline plus 10% PEG. The expressional profiling of the genes TPS27, L3H, TPS2, TPS1, OMT and GDH3 were successfully carried out and their involvement in 1,8-cineole, carvone, α-pinene, thymol, estragole and ß-Citronellol biosynthesis, respectively, was verified. In addition, our results indicated that these genes could also be involved in the synthesis of other metabolites under water deficit condition.

Uncommon Terpenoids from Salvia Species: Chemistry, Biosynthesis and Biological Activities.

The search for new bioactive compounds from plant sources has been and continues to be one of the most important fields of research in drug discovery. However, Natural Products research has continuously evolved, and more and more has gained a multidisciplinary character. Despite new developments of methodologies and concepts, one intriguing aspect still persists, i.e., different species belonging to the same genus can produce different secondary metabolites, whereas taxonomically different genera can produce the same compounds. The genus Salvia L. (Family Lamiaceae) comprises myriad distinct medicinal herbs used in traditional medicine worldwide that show different pharmacological activities due to the presence of a variety of interesting specialized metabolites, including mono-, sesqui-, di-, sester-, tri-, tetra-, and higher terpenoids as well as phenylpropanoids, phenolic acid derivatives, lignans, flavonoids, and alkaloids. We herein summarize the research progress on some uncommon terpenoids, isolated from members of the genus Salvia, which are well recognized for their potential pharmacological activities. This review also provides a current knowledge on the biosynthesis and occurrence of some interesting phytochemicals from Salvia species, viz. C23-terpenoids, sesterterpenoids (C25), dammarane triterpenoids (C30), and uncommon triterpenoids (C20+C10). The study was carried out by searching various scientific databases, including Elsevier, ACS publications, Taylor and Francis, Wiley Online Library, MDPI, Springer, Thieme, and ProQuest. Therefore, 106 uncommon terpenoids were identified and summarized. Some of these compounds possessed a variety of pharmacological properties, such as antibacterial, antiviral, antiparasitic, cytotoxic and tubulin tyrosine ligase inhibitory activities. Due to the lack of pharmacological information for the presented compounds gathered from previous studies, biological investigation of these compounds should be reinvestigated.

Superoxide dismutase (SOD) as a selection criterion for triticale grain yield under drought stress: a comprehensive study on genomics and expression profiling, bioinformatics, heritability, and phenotypic variability.

BACKGROUND: The main objectives of this study were to find the possible structural association between the activity of enzymatic antioxidants and the grain yield of triticale plants as well as identifying the genotypic variability which might be effective on this association. Accordingly, expression levels of superoxide dismutase (SOD) isozymes (Mn-SOD, Cu/Zn-SOD, and Fe-SOD) were appraised to distinguish any possible relationship between SOD expression and drought resistance of triticale. A novel analytical method for distinguishing elite genotypes based on measured features was proposed. Additionally, a new programing based on SAS-language (IML) was introduced to estimate the genetic parameters rooted from combined ANOVA model (linear mixed model), which is capable of being used in any field study other than the current one. METHODS: Thirty genotypes of triticale were studied under normal and drought stress conditions during 6 years (three different locations). Accordingly, based on the results of genetic variability, heatmap analysis, biplot graph, and clustering technique, two genotypes with the highest genetic distance were selected to appraise the differential expression profiling of three SOD isozyme in shoot and root organs. RESULTS: Field experiments and bioinformatics results showed that superoxide dismutase (SOD) was the most influential antioxidant in resistance of triticale to drought stress; therefore, it could be used as an indirect selection index in early stages to distinguish resistant genotypes to drought stress. Additionally, Mn-SOD and Fe-SOD showed roughly similar expression levels for both genotypes under drought stress. However, Cu/Zn-SOD expression level was higher in root and shoot of the tolerant genotype than the susceptible genotype. CONCLUSION: Heatmap analysis that is applied for the first time to screen suitable genotypes, showed to be highly capable of distinguishing elite genotypes and pointing out the proper features for selection criteria. Bioinformatics results indicated that SOD is more important than other enzymatic antioxidant for being considered as selection criteria or candidate gene for transgenic purposes. Based on expressional results, Mn-SOD announced as a general isozyme that is probably highly expressed in most of the species, while, Cu/Zn-SOD was introduced as a genotype specific isozyme that is likely more expressed in tolerant genotypes.

Production of synthetic wheat lines to exploit the genetic diversity of emmer wheat and D genome containing Aegilops species in wheat breeding.

Due to the accumulation of various useful traits over evolutionary time, emmer wheat (Triticum turgidum subsp. dicoccum and dicoccoides, 2n = 4x = 28; AABB), durum wheat (T. turgidum subsp. durum, 2n = 4x = 28; AABB), T. timopheevii (2n = 4x = 28; AAGG) and D genome containing Aegilops species offer excellent sources of novel variation for the improvement of bread wheat (T. aestivum L., AABBDD). Here, we made 192 different cross combinations between diverse genotypes of wheat and Aegilops species including emmer wheat × Ae. tauschii (2n = DD or DDDD), durum wheat × Ae. tauschii, T. timopheevii × Ae. tauschii, Ae. crassa × durum wheat, Ae. cylindrica × durum wheat and Ae. ventricosa × durum wheat in the field over three successive years. We successfully recovered 56 different synthetic hexaploid and octaploid F2 lines with AABBDD, AABBDDDD, AAGGDD, D1D1XcrXcrAABB, DcDcCcCcAABB and DvDvNvNvAABB genomes via in vitro rescue of F1 embryos and spontaneous production of F2 seeds on the Fl plants. Cytogenetic analysis of F2 lines showed that the produced synthetic wheat lines were generally promising stable amphiploids. Contribution of D genome bearing Aegilops and the less-investigated emmer wheat genotypes as parents in the crosses resulted in synthetic amphiploids which are a valuable resource for bread wheat breeding.

Hairy root induction and Farnesiferol B production of endemic medicinal plant Ferula pseudalliacea.

The effects of medium, gibberellic acid (GA3) and stratification treatments on the seed germination of Ferula pseudalliacea were evaluated. Filter paper medium, 500 micro molar GA3 and 8 week chilling treatment were resulted in significantly more seed germination than others. F. pseudalliacea was also transformed by Agrobacterium rhizogenes. Explants from young leaves, stems, cotyledon, and embryo were inoculated with A. rhizogenes strains ATCC 15834, 1724, A4, LB9402 and Ar318. Hairy roots were induced only from 10 to 12-days embryo explants using strains ATCC 15824 and 1724. Although, the transformation efficiency of ATCC 15834 (4%) strain was higher than 1724 (2%). Maximum hairy root transformation frequency (25%) was obtained in infection time of 10 min compared to that of 20 (20%) and 30 (5%) min. In addition, the transformation rate was significantly higher at the inoculation time of 72 h (29%) compared to that of 48 h (22%) and 24 h (6%). Transgenic hairy root lines were confirmed by PCR amplification of rolB gene. Hairy root lines were produced higher biomass in half B5 medium compared to that of half MS medium. Hairy roots lines from the strain ATCC 15834 produced more hairy root numbers and fresh and dried biomass compared to that of the strain 1724. Analyses of transgenic hairy root and natural roots extracts using HPLC showed that all the hairy root lines produced farnesiferol B.

Phytochemical composition and in vitro antioxidant potential of Cynodon dactylon leaf and rhizome extracts as affected by drying methods and temperatures.

The effect of five drying methods including shade drying (SHD), solar drying (SOD), and oven drying at 30 (OD30), 40 (OD40) and 50 °C (OD50) on the phytochemical composition and antioxidant potential of C. dactylon leaf and rhizome was assessed. Among drying methods, OD50 resulted in the shortest drying time (18.3 and 12 h for rhizome and leaf, respectively), when compared with SHD and SOD. Based on GC-MS analyses, 15 and 17 constituents were identified in leaf and rhizome extracts, respectively, accounting for ~ 99% of all components. Fatty acids (palmitic acid and linoleic acid) along with their methyl esters (ethyl palmitate, ethyl linoleate and ethyl oleate) and other derivatives (dihomo-γ-linoleic acid) were the main identified constituents shortly after drying procedures; however, other components such as 5-hydroxymethylfurfural, maltol, retinol and phytol were also traced. Some of C. dactylon phytochemicals including 5-hydroxymethylfurfural and ethyl linoleate were sensitive to high drying temperatures. Besides, higher drying temperatures lead to the production or increasing the level of substances such as 2,3-dihydrobenzofuran, tricyclopentadeca-3,7-dien and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one and diacetin. Based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, the IC50 values were generally higher (significance level of 0.05) for oven-dried rhizome compared with shade-dried leaves and rhizomes that quenched more than 84% of the DPPH at the concentration of 400 mg/ml (IC50 59.12). Our findings suggest that OD30 is a versatile drying method not only to reduce drying time but also to preserve the main phytochemicals and antioxidant activity of C. dactylon during dehydration.

Grape response to salinity stress and role of iron nanoparticle and potassium silicate to mitigate salt induced damage under in vitro conditions.

Grape softwood cuttings of Khoshnaw cultivar were cultured using tissue-culture methods to study the effect of iron nanoparticles and potassium silicate under salinity conditions during the 2015-2016 growing season. The treatments consisted of salinity stress (0, 50, and 100 mM NaCl), nanoparticles of iron (0, 0.08, and 0.8 ppm), and potassium silicate (0, 1, 2 mM). The results also showed that the application of iron nanoparticles and potassium silicate significantly increased the total protein content and reduced proline, enzymatic antioxidant activity and hydrogen peroxide. Salinity stress reduced membrane stability index while increased malondialdehyde content. Increase of membrane stability index and reduction of malondialdehyde content were obtained for 2 mM potassium silicate and 0.8 ppm iron nanoparticle. Iron and potassium silicate were shown to lower the sodium content and increase the potassium content under salinity-stress conditions. The highest ratio of sodium to potassium was observed in plants under salinity conditions (100 mM) treated with neither iron nanoparticles nor potassium silicate; conversely, the lowest ratio was achieved in plants treated with both 0.8 ppm iron nanoparticles with 1 mM and 2 mM potassium silicate under non-stress conditions. These results indicate that the application of micronutrients in stressful conditions is a suitable method to compensate for the negative effects of salinity stress. Tissue culture in this study was shown to be an economically efficient and applicable technique for producing grape softwood cuttings to be used in experiments.

Mitigation by sodium nitroprusside of the effects of salinity on the morpho-physiological and biochemical characteristics of Rubus idaeus under in vitro conditions.

This study examined the changes brought about by sodium nitroprusside (SNP) in the effects of salinity on the morpho-physiological and biochemical characteristics of Rubus idaeus var. Danehdrosht. Raspberry shoot-tip explants were cultured on Murashige and Skoog medium supplemented with a growth regulator that combined benzyleadenine (1 mg/l), indol-3-butyric acetic acid (0.2 mg/l), SNP (0, 50 and 100 µM) and sodium chloride (0, 50 and 100 mM). The results showed that salinity stress significantly decreased morpho-physiological and biochemical characteristics such as RWC, MSI and total protein content in regenerated explants and significantly increased the total soluble sugar, proline contents, peroxidase and superoxide dismutase activity in compared to the control. However, SNP treatments mitigated the impacts of salinity on morphological and physiological characteristics in raspberry shoot-tip explants by increasing the accumulation of proline content, total protein content and total soluble sugar in line with increasing antioxidant enzyme activity under salinity conditions.

Antioxidant Bioactive Compounds Changes in Fruit of Quince Genotypes Over Cold Storage.

Quince fruit has many benefits to human health and is excellent source of bioactive compounds. The fruit of 15 quince genotypes stored at 2 °C for 5 mo to study fruit quality changes during cold storage. Fruit were sampled monthly and stored at 20 °C for 24 h. Fruit ascorbic acid (AA), total phenol (TP), and total flavonoid (TF) concentrations, total antioxidant activity (TAA), flesh browning (FB) incidence, polyphenol oxidase (PPO), peroxidase (POX), and superoxide dismutase (SOD) activities were measured during storage. A high variation in bioactive compounds was observed across genotypes. The range of 26.8 to 44.4 mg/100 g FW for AA, 86.7% to 98.2% for TAA, 157.7 to 380.7 mg GAE 100(-1) g FW for TP, and 5.3 to 10.7 mg/100 g FW for TF were observed across genotypes at harvest time. The overall AA, TAA, TP, TF, and SOD decreased while PPO and POX increased during storage. FB was first observed after 4 mo and increased thereafter while the FB index was different across genotypes. Higher bioactive content may prevent or reduce FB index so that a negative correlation was found between FB and AA, TAA, TP, TF, and SOD.

Protective effect of ethyl pyruvate on mice sperm parameters in phenylhydrazine induced hemolytic anemia.

The aim of the present study was to assess the protective effect of ethyl pyruvate (EP) on sperm quality parameters, testosterone level and malondialdehyde (MDA) in phenylhydrazine (PHZ) treated mice. For this purpose, 32 NMRI mice with the age range of 8 to 10 weeks, weight average 26.0 ± 2.0 g, were randomly divided into four equal groups. The control group (1) received normal saline (0. 1 mL per day) by intraperitoneal injection (IP). Group 2 (PHZ group) was treated with initial dose of PHZ (8 mg 100 g(-1), IP) followed by 6 mg 100 g(-1) , IP every 48 hr. Group 3, (Group PHZ+EP) received PHZ (according to the previous prescription) with EP (40 mg kg(-1), daily, IP). Ethyl pyruvate group (4) received only EP (40 mg kg(-1), daily, IP). Treatment period was 35 days. After euthanasia, sperms from caudal region of epididymis were collected and the total mean sperm count, sperm viability, motility and morphology were determined. Testis tissue MDA and serum testosterone levels of all experimental groups were also evaluated. A considerable reduction in mean percentage of number, natural morphology of sperm, sperm motility and viability and serum testosterone concentration besides DNA injury increment among mice treating with PHZ in comparison with control group were observed. However, in PHZ+EP group the above mentioned parameters were improved. This study showed that PHZ caused induction of toxicity on sperm parameters and reduction of testosterone as well as the increment of MDA level and EP as an antioxidant could reduce destructive effects of PHZ on sperm parameters, testosterone level and lipid peroxidation.

In vitro propagation and conservation of Satureja avromanica Maroofi-an indigenous threatened medicinal plant of Iran.

An efficient and rapid in vitro propagation system for Satureja avromanica, a rare and endangered folk medicinal plant of Iran was developed through the shoot tip and leaf disc explants. Nodal and leaf explants from wild plants were established on MS and WPM media supplemented with BA, BAP and TDZ (0, 0.1, 0.5, 1, 1.5, 2, 5 and 10 mgl(-1)) alone or by application of BA and TDZ (0, 2, 5 and 10 mgl(-1)) in combination with IBA and 2,4-D (0, 0.1, 0.5 and 1 mgl(-1)), respectively. Based on results, the highest mean shoot number (6.21) was obtained on MS medium supplemented with 2 mgl(-1) BA. Regarding the shoot elongation, MS supplemented with 2 mgl(-1) TDZ and MS containing 5 mgl(-1) BA showed the longest shoots (4.82 and 4.39 cm, respectively) after 6 weeks of culture. As a matter of fact, increasing all three tested cytokinins levels led to enhancement of explant response frequency and regenerated shoot number. On the other side, WPM medium supplemented with 0.1 mgl(-1) IBA was found suitable for rooting of regenerated shoots. RAPD molecular analysis revealed genetic stability of in vitro raised plants. In conclusion, individual application of BA, BAP and TDZ were in favor of S. avromanica direct shoot regeneration while treatment media with a combination of IBA and BA as well as 2,4-D and TDZ resulted in callogenesis in most explants. Finally, the in vitro raised plantlets were acclimatized and successfully established in the greenhouse conditions. Our developed protocol can be employed for the large-scale micropropagation and conservation of S. avromanica as a threatened medicinal plant.