Effects of salicylic and jasmonic acids on the components of pro/antioxidant system in potato plants infected with late blight

The effects of salicylic acid (SA) and jasmonic acid (JA) on plant defense responses were studied with aseptic potato (Solanum tuberosum L.) plantlets infected with Phytophthora infestans (Mont.) de Bary. Plant treatment with 10⁻⁶ M SA or 10⁻⁷ M JA induced plant resistance; the mixture of these acids was most efficient. After treatment with these compounds, phenolic compounds were accumulated and peroxidase was activated in the sites of pathogen localization, and this might be the reason of resistance enhancement. In addition, more H₂O₂ was accumulated in infected plants treated with JA or its mixture with SA but not in plants treated with SA alone. It might occur because of observed inhibition of catalase and activation of isoperoxidase with the isoelectric point (pI) of ∼9.3, which manifests an affinity for the pathogen cell wall. The data obtained allow us to recommend the application of these compounds for potato plant protection against late blight.     

In vitro development and transfer of resistance to chlortetracycline in Bacillus subtilis

The present criteria and rules controlling the approval of the use of probiotics are limited to antibiotic resistance patterns and the presence of antibiotic resistance genes in bacteria. There is little information available in the literature regarding the risk of the usage of probiotics in the presence of antibiotic pressure. In this study we investigated the development and transfer of antibiotic resistance in Bacillus subtilis selected in vitro by chlortetracycline in a stepwise manner. Bacillus subtilis was exposed to increasing concentrations of chlortetracyclineto induce in vitro resistance to chlortetracycline, and the minimal inhibitory concentrations were determinedfor the mutants. Resistant B. subtilis were conjugated with Escherichia coli NK5449 and Enterococcus faecalis JH2-2 using the filter mating. Three B. subtilis tetracycline resistant mutants (namely, BS-1, BS-2, and BS-3) were derived in vitro. A tetracycline resistant gene, tet (K), was found in the plasmids of BS-1 and BS-2. Three conjugates (BS-1N, BS-2N, and BS-3N) were obtained when the resistant B. subtilis was conjugated with E. coli NK5449. The conjugation frequencies for the BS-1N, BS-2N, and BS-3N conjugates were 4.57×10^?7, 1.4×10^?7, and 1.3×10^?8, respectively. The tet(K) gene was found only in the plasmids of BS-1N. These results indicate that long-term use of probiotics under antibiotic selection pressure could cause antibiotic resistance, and the resistance gene could be transferred to other bacteria. The risk arising from the use of probiotics under antibiotic pressure should be considered in the criteria and rules for the safety assessment of probiotics.     

Defense Responses and Changes in Symbiotic Gut Microflora in the Colorado Potato Beetle <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis> under the Effect of Endophytic Bacteria from the Genus Bacillus

Phytophagous insects and host plants have a complex of microsymbionts and make up a united co-evolving system with them. Microsymbiotic complexes are actively involved in stress responses of macrosymbionts. We established that a treatment of potato plants with endophytic bacterial strains Bacillus thuringiensis var. thuringiensis-5689, B. th. var. kurstaki-5351, and Bacillus subtilis 26D decreased the survival rate of the plant feeder, Colorado potato beetle Leptinotarsa decemlineata Say. The B. th. strains suppressed phenoloxidase and acetylcholinesterase activities in the beetle hemolymph. An antagonistic relationship was found between endophytic bacteria B. subtilis 26D and beetle symbiotic bacteria from the genera Acinetobacter and Enterobacter, with the former being able to suppress the growth of endophytic colonies. The recombinant B. subtilis strain 26D Cry, containing the B. th. var. kurstaki δ-endotoxin cry1Ia gene, combined the ability of the original B. subtilis 26D strain to suppress the development of beetle symbionts and immune responses with a production of the Cry toxin, thus leading to a high mortality of the phytophage.     

Integrated control of tobacco black shank by combined use of riboflavin and <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain Tpb55

We investigated the effect of riboflavin on the biocontrol activity of Bacillus subtilis Tpb55 against Phytophthora nicotianae (Pn), which causes tobacco black shank. Riboflavin (0.2 mg ml^?1) significantly improved the biocontrol activity of Tpb55 (2.0 × 10⁸ cfu ml^?1). Riboflavin (0.02–0.5 mg ml^?1) alone could not significantly inhibit Pn growth. However, it enhanced the B. subtilis population, both in vitro and in tobacco roots and significantly increased the activity of defense enzymes, peroxidase, catalase, superoxide dismutase, and β-1,3-glucanase, in the roots of B. subtilis-treated tobacco seedlings. Our results indicate that riboflavin can stimulate the growth of B. subtilis Tpb55 and induce resistance to Pn in tobacco plants. These findings should boost the prospects for practical application of B. subtilis Tpb55 as a biocontrol agent against black shank of tobacco.     

Stimulation of Cellular Mechanisms of Potato Antivirus Resistance by the Action of a Preparation Based on <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Bacteria

A comparative study of the antiviral activity of a new biopreparation based on bacteria Bacillus subtilis (strain 47) and commercial biopesticides (beta-protectin, phyto-protectin, frutin) in potato plants of Belarusian selection (Lileya and Scarb) was carried out in vivo and ex vitro. Pretreatment of plants with B. subtilis biopreparation and biopesticides prevents infection by potato X-viruses. The antiviral efficacy of B. subtilis does not depend on the conditions of plant growth and is more effective than biopesticides. Increased potato plant resistance to viral infection was accompanied by an effect leading to an increase in the mass of minitubers and the dry matter content in them. Treatment of potato plants with B. subtilis did not affect the molecular heterogeneity of peroxidase and superoxide dismutase, but it changed the relative activity of their isoforms. Treatment with a bacterial preparation increased the activity of superoxide dismutase when it was applied both to intact plants and those preinfected with virus. The results indicate that pretreatment of potato plants with the B. subtilis drug prevents virus infection, inducing the antiviral resistance of the potato, and is accompanied by a change in the activity of redox enzymes.     

Strains of <Emphasis Type="Italic">Bacillus</Emphasis> ssp. regulate wheat resistance to <Emphasis Type="Italic">Septoria nodorum</Emphasis> Berk.

The ability of Bacillus subtilis Cohn and Bacillus thuringiensis Berliner to induce systemic resistance in wheat plants to the casual agent of Septoria nodorum Berk., blotch has been studied. It has been shown that strains of Bacillus ssp. that possess the capacity for endophytic survival have antagonistic activity against this pathogen in vitro. A reduction of the degree of Septoria nodorum blotch development on wheat leaves under the influence of Bacillus spp. was accompanied by the suppression of catalase activity, an increase in peroxidase activity and H₂O₂ content, and expression of defence related genes such us PR-1, PR-6, and PR-9. It has been shown that B. subtilis 26 D induces expression levels of wheat pathogenesis-related (PR) genes which marks a SA-dependent pathway of sustainable development and that B. thuringiensis V-5689 and V-6066 induces a JA/ET-dependent pathway. These results suggest that these strain Bacillus spp. promotes the formation of wheat plant resistance to S. nodorum through systemic activation of the plant defense system. The designed bacterial consortium formed a complex biological response in wheat plants infected phytopathogen.     

Efficient induction of microspore embryogenesis using abscisic acid,jasmonic acid and salicylic acid in Brassica napus L

The stress hormones abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) play an important role in the regulation of physiological processes and are often used in tissue culture to promote somatic embryogenesis and to enhance the quality of somatic embryos. Despite many studies on Brassica napus microspore culture, the effects of stress hormones (ABA, JA and SA) on microspore embryogenesis are not well explored. In this study, the effects of three incubation periods (6, 12 and 24 h) at different levels of ABA, JA and SA (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1) on microspore embryogenesis of rapeseed (B. napus L.) cv. ‘Regent’ were investigated. ABA (0.5 mg l^?1 for 12 h) enhanced microspore embryogenesis by about threefold compared with untreated cultures and increased normal plantlet regeneration by 68 %. ABA treatment also effectively reduced secondary embryo formation at all concentrations tested but enhanced callusing at high levels, for example 67 % at 1.0 mg l^?1 for 24 h. Highest embryo yield (286.0 embryos Petri dish^?1) was achieved using 1.0 mg l^?1 JA for 24 h and highest normal plantlet regeneration (54 %) was observed in cultures exposed to 0.5 mg l^?1 JA for 12 h. JA (5.0 mg l^?1 for 24 h) also reduced the germination of microspore-derived embryos on regeneration medium by 21 %. SA at 0.2 and 0.5 mg l^?1 for 6 h increased microspore embryogenesis (184.0 and 193.4 embryos Petri dish^?1) relative to the control (136.2 embryos Petri dish^?1). However, SA did not improve normal regeneration, secondary embryo formation or callusing. Microspore embryogenesis and plant regeneration could be improved by ABA, JA as well as SA when the appropriate level and duration of incubation were selected.     

Temperature-related effects of treatments with jasmonic and salicylic acids on Arabidopsis infected with cucumber mosaic virus

Plant-virus interactions are affected by environmental factors, including temperature. Plant defenses are often inhibited by high or low temperature. In this study, oxidative damage and gene expression were detected in Arabidopsis thaliana infected with cucumber mosaic virus (CMV) at different temperatures. Before virus inoculation, plants were treated with jasmonic acid (JA) and salicylic acid (SA), both of which are important signaling molecules in plant defense responses. The levels of MDA and hydrogen peroxide (H₂O₂), and electrolyte leakage were significantly higher in CMV-infected leaves at 15 and 37°C. The accumulation of H₂O₂ and superoxide radical (O ₂ ^·? ) was obviously suppressed by spraying with JA followed by SA (JA → SA) at different temperatures. The CMV-CP expression analysis showed that virus replication was inhibited efficiently in the (JA → SA) treatment. Therefore, many JA- and SA-responsible resistance genes were quantified; MPK4 was expressed highly and steadily in the (JA → SA) treatment. To further confirm the role of MPK4, the CMV-CP gene expression was evaluated in wild-type Arabidopsis and its mpk4 mutant infected with CMV. The results suggested that MPK4 might play an important role in the antagonism between JA and SA at temperature fluctuation.     

Impact of Chemical,Organic and Bio-Fertilizers Application on Bell Pepper, <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. and Biological Parameters of <Emphasis Type="Italic">Myzus persicae</Emphasis> (Sulzer) (Hem.: Aphididae)

Myzus persicae (Sulzer) is a polyphagous aphid that causes chlorosis, necrosis, stunting, and reduce growth rate of the host plants. In this research, the effects of Zinc sulfate and vermicompost (30%), Bacillus subtilis, Pseudomonas fluorescens, Glomus intraradices, G. intraradices × B. subtilis, and G. intraradices × P. fluorescens compared to control was investigated on the growth characters of Capsicum annuum L. and biological parameters of M. persicae. Different fertilizers caused a significant effect on growth characters of C. annuum and biological parameters of M. persicae. The highest plant growth was observed on Zinc sulfate and B. subtilis treated plants, and the lowest was on control. Increase in the amount of specific leaf area (SLA) (0.502 mm² mg^?1) was significantly higher in the B. subtilis than other fertilizer treatments. The longest (10.3 days) and the shortest (5.3 days) developmental times of M. persicae nymphs were observed on 30% vermicompost and Zinc sulfate treatments, respectively. The lowest adult longevity periods of M. persicae (11.2 and 11.3 days) were observed on G. intraradices × B. subtilis and 30% vermicompost treatments, respectively, and the longest ones (16.4 days) on Zinc sulfate. The highest rate of nymphal mortality and the lowest amount of nymphal growth index (NGI) were recorded on 30% vermicompost. The nymphs reared on Zinc sulfate treatment had the lowest rate of nymphal mortality and the highest amount of NGI. Thus, amending the soil with 30% vermicompost had a significantly negative effect on the biological parameters of M. persicae that can be used as an ecological control tactic for this pest.     

In vitro genetic labeling of Bacillus subtilis cryptic plasmid pHV400.

A DNA segment which encodes resistance to tetracycline, and cannot replicate autonomously, was excised by HindIII endonuclease from plasmid pT127 and joined to the cryptic Bacillus subtilis plasmid pHV400. The analysis of resulting chimerae has allowed us to identify a 1.8 × 10⁶ segment of pHV400 which carried the replication functions of the cryptic plasmid. Another DNA segment, designated pHV32, which can replicate in Escherichia coli but not in B. subtilis has also been used for genetic labeling of the replication region of pHV400. pHV32 is convenient for use in isolating cryptic replicons active in B. subtilis because (1) it can be prepared in large quantities, free from any interferring B. subtilis replicons, from an appropriate E. coli strain; (2) it carries unique sites for various restriction endonucleases; (3) the chloramphenicol resistance gene which it specifies can transform B. subtilis at a high efficiency (10⁶–10⁷ transformants/μg of DNA).     

Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine

Non‐self‐recognition of microorganisms partly relies on the perception of microbe‐associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA‐regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long‐lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP‐non‐producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.     

Degradation of nicosulfuron by Bacillus subtilis YB1 and Aspergillus niger YF1

The optimal degrading conditions for the nicosulfuron degradation by Bacillus subtilis YB1 and Aspergillus niger YF1, and site of their action on nicosulfuron were studied. The results showed that the degradation efficiency of free cells of B. subtilis YB1 and A. niger YF1 was respectively 87.9 and 98.8% in basic medium III containing 2 mg/l of nicosulfuron after inoculation with 1 ml of culture containing 2.3 × 10⁷ CFU ml^?1 and incubation for 5 days at 35°C. Moreover, the degradation rate of nicosulfuron by the mixture of microorganisms was much higher than for every of them taken separately in the same conditions. The mass spectrometric analysis of the products degraded by B. subtilis YB1 revealed that the sulfonylurea bridge in nicosulfuron molecule had been broken. Extracellular (EXF) and endocellular (ENF) fractions obtained from bacterium and fungus were tested for the ability to degrade nicosulfuron. The degradation efficiency of fractions extracted from B. subtilis YB1 was 66.8% by EXF and 15.8% by ENF, but neither EXF nor ENF extracted from A. niger YF1 had the activity of degrading nicosulfuron.     

Aphid induction of phytohormones in <Emphasis Type="Italic">Medicago truncatula</Emphasis> is dependent upon time post-infestation,aphid density and the genotypes of both plant and insect

This study examined the induction of the defence-related hormones jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA) and the phytoalexin medicarpin in Medicago truncatula when challenged by the pea aphid Acyrthosiphon pisum. There was some induction of hormones in the compatible interaction between A. pisum clone N116 and M. truncatula cultivar DZA315, whereas JA, SA and medicarpin exhibited more significant increases in foliage concentration during the incompatible interaction between A. pisum clone PS01 and M. truncatula cultivar Jemalong A17. Foliar concentration of JA, SA and medicarpin exhibited a positive relationship with aphid density after 3-day feeding, whereas ABA was not affected by the presence of aphids. When aphids were restricted to a single leaf using plastic tubes, JA, SA and medicarpin displayed strong local induction, whereas there were no significant systemic increases in uninfested leaves. Medicarpin and SA appeared to increase with duration of aphid feeding, whereas JA showed a more transient increase in concentration 24 h after challenge commenced. Results suggest that increases in JA, SA and medicarpin are associated with M. truncatula resistance to particular clones of A. pisum. The variation in concentration of the defence-related compounds recorded with regard to aphid density, duration of challenge, genotypes of plant and aphids, and between locally challenged and distant leaves reinforces the need for consideration of these experimental factors when generalizing about the plant defence processes that occur during aphid–plant interactions.     

Evaluation of Bacillus subtilis (JN032305) biofungicide to control chilli anthracnose in pot controlled conditions

Colletotrichum gloeosporioides (Penz.) causing anthracnose is a potent pathogen of chilli resulting in significant yield loss. The in vitro root colonisation study showed an increase in root bacterial count by 10 × 10⁵ colony forming units/cm root for Bacillus subtilis after 15 days of germination. Population level of the antagonist was stable in talc till the 180th day (30°C – 1.6 × 10⁸; 4°C – 1.9 × 10⁸) and in lignite till the 150th day (30°C – 1.5 × 10⁸; 4°C – 1.3 × 10⁸). Combined application of B. subtilis and carbendazim enhanced all biometric parameters with reduction in disease incidence. Soil, seed, root dip and foliar spray treatment significantly enhanced the growth parameters of chilli in B. subtilis inoculated treatments in comparison to the untreated control. Seed application resulted in highest plant fresh weight (76.84 g) and dry weight (34.17 g) compared to the untreated control (50 g and 21 g fresh and dry weight, respectively). Highest plant height ranging from 70 cm (soil application) to 77 cm (dip treatment) with Bacillus inoculation was comparable with carbendazim treatment (61 cm with soil application) and 78 cm (dip treatment) and significantly higher than the untreated control (58.2 cm with soil and 61 cm with application, respectively). Dip treatment resulted in significant increase in root length with B. subtilis (33 cm) and carbendazim (32.5 cm) in comparison to untreated control (15 cm). Co-inoculation of pathogen with B. subtilis (singly and with carbendazim) not only reduced the disease incidence but also improved all the biometric parameters in comparison to challenge inoculation. Root dip application was effective in promoting growth while seed application was effective in disease control.     

Muscle Contraction : (Outline Studies in Biology) by C.R. Bagshaw Chapman & Hall; London,New York, 1982 79 pages. £2.75

On incubation of B. subtilis RM125(arg15 leuA8 r_M^? m_M^?) with DNA from alkalophilic Bacillus, the transformants (Arg⁺Leu^? or Leu^?Arg⁺) appeared at pH 10. The transformants were able to grow even at pH 7. Alkalophilic Bacillus was resistant to bacteriophages π105D1C2·1012 grown on B. subtilis 1012(r^-m_M⁺) and π105D1C2·ISMR4 grown on B. subtilis ISMR4r_M⁺r_R⁺m_M⁺m_R⁺), but the recipient B. subtilis and the transformant(Arg⁺Leu^?) were susceptible to both the of the bacteriophages. The results indicate that the transformant is a B. subtilis derivative and that alkalophilicity of alkalophilic Bacillus was transferred to B. subtilis.     

Activation of elicitor defensive properties by systemic signal molecules during the interaction between potato and the late blight agent

The elicitor arachidonic acid in combination with jasmonic acid (JA) induced a higher level of defense against the late blight agent in potato (Solanum tuberosum L.) tissues than in combination with salicylic acid (SA). On the contrary, the elicitor chitosan displayed a higher inductive effect in combination with SA as compared with JA. The optimal concentrations of tested compounds were selected for designing the compositions activating wound repair, induction of proteinase inhibitors, and resistance to the biotrophic pathogen Phytophthora infestans (Mont.) de Bary. It was demonstrated that the compositions of elicitor and systemic signal molecules provided a faster spreading of an inducing effect in the potato tissues.     

Rhizosphere mediated growth enhancement using phosphate solubilizing rhizobacteria and their tri-calcium phosphate solubilization activity under pot culture assays in Rice (Oryza sativa.)

Phosphate solubilizing rhizobacteria are considered as an important alternative to increase the availability of accumulated phosphates through solubilization. These increase the growth of plant by enhancing the efficiency of fixing biological nitrogen. This was studied through a pot experiment involving two Phosphate Solubilizing Rhizobacteria (PSRB) isolates, Pseudomonas aeruginosa and Bacillus subtilis along with Tri-calcium phosphate (TCP) on availibity of nutrients, biological composition of soil and yield attributes of rice crop at its growth stages. Experiment was laid in factorial completely randomized design (CRD) comprising of eight treatments replicated thrice with two factors viz. factor 1 with or without TCP (1 g^?1soil) and factor 2 with single or combined inoculation of PSRB isolates. Considerable enhancement in available content of potassium (K), phosphorous (P), nitrogen (N) in soil was found with TCP 1 g^?1soil (P₁) and consortium of Pseudomonas aeruginosa and Bacillus subtilis broth culture at crop growth stages. Highest increase in available N (17.13% and 19.1%), available P (232% and 265%), available K (19.6% and 29.2%) over control were recorded in B₃ (consortium of Pseudomonas aeruginosa and Bacillus subtilis broth culture). Similarly, maximum nutrient uptake N (6.4%), P (15.8%) and K (8.9%) were recorded with same treatment. A considerable growth in soil microbial biomass carbon and dehydrogenase activity at crop growth stages was recorded on application of TCP 1 g^?1soil (P₁) and consortium of PSRB isolates' Pseudomonas aeruginosa and Bacillus subtilis (B₃). Highest increase in microbial biomass carbon (16.4% and 16.5%) and dehydrogenase activity 34.7% and 43.8% over control were recorded in B₃ (consortium of PSRB isolates Pseudomonas aeruginosa and Bacillus subtilis) and was found best among all treatments in terms of yield (63.2%) and yield attributes; number of panicles^?1plant (54.8%), number of grains^?1panicle (156%) and average panicle length (63.9%).     

Overexpressing the N-terminus of CATALASE2 enhances plant jasmonic acid biosynthesis and resistance to necrotrophic pathogen Botrytis cinerea B05.10

Salicylic acid (SA) acts antagonistically to jasmonic acid (JA) in plant immunity. We previously reported that CATALASE2 (CAT2) promotes JA-biosynthetic acyl-CoA oxidase (ACX) activity to enhance plant resistance to necrotrophic Botrytis cinerea, and SA represses JA biosynthesis through inhibiting CAT2 activity, while the underlying mechanism remains to be further elucidated. Here, we report that the truncated CAT2 N-terminus (CAT2-N) interacts with and promotes ACX2/3, and CAT2-N-overexpressing plants have increased JA accumulation and enhanced resistance to B. cinerea B05.10, but compromised antagonism of SA on JA. Catalase inhibitor treatment or mutating CAT2 active amino acids abolished CAT2 H₂O₂-decomposing activity but did not affect its promotion of ACX2/3 activity via interaction. CAT2-N, a truncated protein with no catalase activity, interacted with and promoted ACX2/3. Overexpressing CAT2-N in Arabidopsis plants resulted in increased ACX activity, higher JA accumulation, and stronger resistance to B. cinerea B05.10 infection. Additionally, SA dramatically repressed JA biosynthesis and resistance to B. cinerea in the wild type but not in the CAT2-N-overexpressing plants. Together, our study reveals that CAT2-N can be utilized as an accelerator for JA biosynthesis during plant resistance to B. cinerea B05.10, and this truncated protein partly relieves SA repression of JA biosynthesis in plant defence responses.