The Effect of Chitosan and Bion on Resistance to Pink Snow Mould in Perennial Ryegrass and Winter Wheat

The effects of chitosan on resistance to pink snow mould (Microdochium nivale) were studied in young winter wheat (Triticum aestivum L.) and perennial ryegrass (Lolium perenne L.) under controlled environmental conditions. In perennial ryegrass, the putative defence activator Bion was also tested. Resistance was measured as regrowth of plants after inoculation with M. nivale and incubation in darkness at 2°C. In winter wheat, pre‐treatment with chitosan at 1000 μg per plant increased resistance to subsequent infection by M. nivale, but this effect was less significant in a replicate experiment. Chitosan‐treated winter wheat plants expressed the gene for the pathogenesis‐related protein chitinase at higher levels than non‐treated plants. Chitinase gene expression was also stimulated by M. nivale infection in winter wheat. Perennial ryegrass pre‐treated with Bion or chitosan and inoculated with M. nivale did not display better regrowth after incubation than non‐treated, inoculated plants. Rather, regrowth was reduced in some of the Bion‐treated plants after incubation. We speculate that the cost or the mechanism of induced resistance makes Bion non‐effective in plants that are not actively growing. Bion at concentrations of 10, 100 and 1000 μg active ingredient per ml, and the highest concentration of chitosan used (2000 μg per ml) reduced in vitro growth of the pathogen, suggesting that both defence activators possess antifungal activity.     

SABP2, a methyl salicylate esterase is required for the systemic acquired resistance induced by acibenzolar-S-methyl in plants

Tobacco SABP2, a 29 kDa protein catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) to induce SAR. Pretreatment of plants with acibenzolar-S-methyl (ASM), a functional analog of salicylic acid induces systemic acquired resistance (SAR). Data presented in this paper suggest that SABP2 catalyzes the conversion of ASM into acibenzolar to induce SAR. Transgenic SABP2-silenced tobacco plants when treated with ASM, fail to express PR-1 proteins and do not induce robust SAR expression. When treated with acibenzolar, full SAR is induced in SABP2-silenced plants. These results show that functional SABP2 is required for ASM-mediated induction of resistance.     

Overexpression of salicylic acid carboxyl methyltransferase reduces salicylic acid-mediated pathogen resistance in Arabidopsis thaliana

We cloned a salicylic acid/benzoic acid carboxyl methyltransferase gene, OsBSMT1, from Oryza sativa. A recombinant OsBSMT1 protein obtained by expressing the gene in Escherichia coli exhibited carboxyl methyltransferase activity in reactions with salicylic acid (SA), benzoic acid (BA), and de-S-methyl benzo(1,2,3)thiadiazole-7-carbothioic acid (dSM-BTH), producing methyl salicylate (MeSA), methyl benzoate (MeBA), and methyl dSM-BTH (MeBTH), respectively. Compared to wild-type plants, transgenic Arabidopsis overexpressing OsBSMT1 accumulated considerably higher levels of MeSA and MeBA, some of which were vaporized into the environment. Upon infection with the bacterial pathogen Pseudomonas syringae or the fungal pathogen Golovinomyces orontii, transgenic plants failed to accumulate SA and its glucoside (SAG), becoming more susceptible to disease than wild-type plants. OsBSMT1-overexpressing Arabidopsis showed little induction of PR-1 when treated with SA or G. orontii. Notably, incubation with the transgenic plant was sufficient to trigger PR-1 induction in neighboring wild-type plants. Together, our results indicate that in the absence of SA, MeSA alone cannot induce a defense response, yet it serves as an airborne signal for plant-to-plant communication. We also found that jasmonic acid (JA) induced AtBSMT1, which may contribute to an antagonistic effect on SA signaling pathways by depleting the SA pool in plants. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Isolation and characterization of isonicotinic acid hydrazide-resistant mutants of Nicotiana tabacum

Summary Twenty stable variant lines resistant to isonicotinic acid hydrazide (INH), an inhibitor of the conversion of glycine to serine in the glycolate pathway, were isolated in cell cultures initiated from allodihaploid Nicotiana tabacum. Plants were regenerated from 13 of these lines and explants were tested for resistance. For some lines virtually all of the regenerated plants scored as resistant; for others a mixed population of sensitive and resistant plants were obtained. One or more plants from 5 lines were fertile, presumably as a result of spontaneous diploidization of cells in the plant or culture. Callus initiated from the seed progeny of these plants was resistant to INH confirming the characteristic as a stable mutation. Seedlings from all INH-resistant plants were small and slow-growing, but the slow-growth trait could be separated from resistance in backcrosses of hybrids. In one case (line I21) crosses with sensitive lines show the resistant trait in that line to be dominant.     

Postharvest Infiltration of BTH Reduces Infection of Mango Fruits (Mangifera indica L. cv. Tainong) by Colletotrichum gloeosporioides and Enhances Resistance Inducing Compounds

Mango fruits (Mangifera indica L.) were treated by vacuum infiltration of 1.0 mm benzo‐(1,2,3)‐thiadiazole‐7‐carbothioic acid s ‐methyl ester (BTH) after harvest. Seventy‐two hours after the treatment with BTH, the fruit were inoculated with 15 μl of conidial suspension of Colletotrichum gloeosporioides (1 × 10⁵ conidia/ml) and incubated at 13°C, 85–90% RH for disease development. Disease incidence and lesion diameter in mango fruit after the inoculation were significantly (P < 0.05) reduced by the BTH treatment during the incubation. Peroxidase, polyphenoloxidase, phenylalanine ammonia‐lyase, chitinase and β‐1,3‐glucanase activities and total phenolic compounds content in the fruits were all enhanced by the BTH treatment during the incubation. The catalase activity in the fruit was inhibited, whereas the level of hydrogen peroxide was increased by the BTH treatment during the infection. These responses may be involved in the induced resistance against the pathogen infection in mango fruit by BTH treatment. Application of BTH in fruit possesses promising results in the control of postharvest diseases as an alternative to traditional methods.     

Naphthylphthalamic acid-binding sites in cultured cells from Nicotiana tabacum

Naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, binds with high affinity to membrane preparations from callus and cell suspension cultures derived from Nicotiana tabacum (K _d approx. 2·10^–9 M). The concentration of membrane-bound binding sites is higher in cell suspension than in callus cultures. The binding of NPA to these sites seems to be a simple process, in contrast to the binding of the synthetic auxin naphthylacetic acid (1-NAA) to membrane preparations from callus cultures, which is more complex (A.C. Maan et al., 1983, Planta 158, 10–15). Naphthylacetic acid, a number of structurally related compounds and the auxin-transport inhibitor triiodobenzoic acid were all able to compete with NPA for the same binding site with K _d values ranging from 10^–6 to 10^–4 M. On the other hand, NPA was not able to displace detectable amounts of NAA from the NAA-binding site. A possible explantation is the existence of two different membrane-bound binding sites, one exclusively for auxins and one for NPA as well as auxins, that differ in concentration. The NPA-binding site is probably an auxin carrier.Abbreviations 1-NAA 1-Naphthylacetic acid - 2-NAA 2-Naphthylacetic acid - NPA N-1-Naphthylphthalamic acid     

Ultraviolet light and ozone stimulate accumulation of salicylic acid,pathogenesis-related proteins and virus resistance in tobacco

In tobacco (Nicotiana tabacum L. cv. Xanthinc), salicylic acid (SA) levels increase in leaves inoculated by necrotizing pathogens and in healthy leaves located above the inoculated site. Systemic SA increase may trigger disease resistance and synthesis of pathogenesis-related proteins (PR proteins). Here we report that ultraviolet (UV)-C light or ozone induced biochemical responses similar to those induced by necrotizing pathogens. Exposure of leaves to UV-C light or ozone resulted in a transient ninefold increase in SA compared to controls. In addition, in UV-light-irradiated plants, SA increased nearly fourfold to 0.77 g·g^–1 fresh weight in leaves that were shielded from UV light. Increased SA levels were accompanied by accumulation of an SA conjugate and by an increase in the activity of benzoic acid 2-hydroxylase which catalyzes SA biosynthesis. In irradiated and in unirradiated leaves of plants treated with UV light, as well as in plants fumigated with ozone, PR proteins 1a and 1b accumulated. This was paralleled by the appearance of induced resistance to a subsequent challenge with tobacco mosaic virus. The results suggest that UV light, ozone fumigation and tobacco mosaic virus can activate a common signal-transduction pathway that leads to SA and PR-protein accumulation and increased disease resistance.Abbreviations PR protein pathogenesis-related protein - SA salicylic acid - TMV tobacco mosaic virus - UV ultraviolet This work was financed by grants from the U.S. Department of Agriculture (Competitive Research Grants Office), Division of Energy Biosciences of U.S. Department of Energy, the Rockefeller Foundation, the New Jersey Commission for Science and Technology, and the New Jersey Agricultural Experiment Station.     

Interactive effects of methyl jasmonate and salicylic acid on floret opening in spikelets of Sorghum

Floret opening of excised spikelets in Sorghum bicolor (L.) Moench and Sorghum Sudanesis (Piper) Stapf was significantly stimulated by immersing into 2 mM methyl jasmonate (MeJA) solution. In male sterile (MS) lines of Sorghum bicolor (L.) Moench, floret opening was more sensitive to MeJA than that in maintainer (MT) lines. Salicylic acid (SA) could abolish the effect of MeJA on the opening of spikelets. These data indicate that MeJA plays a role in floret opening in sorghum, and that SA interacts with MeJA in regulating of this process.     

OsBISAMT1, a gene encoding S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, is differentially expressed in rice defense responses

We isolated and identified a full-length cDNA, OsBISAMT1 [Oryza sativa L. benzothiadiazole (BTH)-induced SAMT 1], which encodes a putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase (SAMT) from rice. OsBISAMT1 contains an ORE of 1128 bp, which predicts to encode a 375 aa protein. The OsBISAMT1 protein sequence shows a high level of identity to known plant SAMTs and contains a conserved characteristic methyltransferase domain. OsBISAMT1 is a member of a small gene family in the rice genome. Expression of OsBISAMT1 in rice leaves was induced by treatments with benzothiadiazole and salicylic acid, which are capable of inducing rice disease resistance. OsBISAMT1 was also up-regulated in both incompatible and compatible interactions between rice and the blast fungus, Magnaporthe grsiea, but the induced expression of OsBISAMT1 was greater and more rapid in the incompatible interaction than that in the compatible one. Moreover, mechanical wounding also activated OsBISAMT1 expression. The results suggest that OsBISAMT1 may be involved in disease resistance responses as well as in wound response in rice.     

Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance to N. tabacum plastids

Summary Protoplasts of Nicotiana tabacum SRI (streptomycin resistant) and of Nicotiana knightiana (streptomycin sensitive) were fused using polyethylene glycol treatment. From three heterokaryons 500 clones were obtained. From the 43 which were further investigated, 6 resistant, 3 sensitive, and 34 chimeric (consisting of resistant and sensitive sectors) calli were found. From eight clones, a total of 39 plants were regenerated and identified as somatic hybrids. Chloroplast type (N. tabacum = NT or N. knightiana = NK) in the plants was determined on the basis of the species specific EcoRI restriction pattern of the chloroplast DNA. Regenerates contained NT (13 plants) or NK (15 plants) plastids but only the plants with the NT chloroplasts were resistant to streptomycin. This finding and our earlier data on uniparental inheritance points to the chloroplasts as the carriers of the streptomycin resistance factor.     

Anthranilate synthase forms in plants and cultured cells of Nicotiana tabacum L.

Tobacco (N. tabacum cv. Xanthi) cell lines contained two forms of anthranilate synthase (AS; EC 4.1.3.27) which could be partially separated by gel-filtration chromatography. One form was resistant to feedback inihibition by 10 M tryptophan (trp) while the other form was almost completely inhibited by trp at the same concentration. Cell lines selected as resistant to 5-methyltryptophan (5MT) had more of the trp-resistant AS form. Only the trp-sensitive form was detected in plants regenerated from both normal and 5MT-resistant cell lines. Overexpression of the trp-resistant form in 5MT-resistant tobacco cells disappeared during plant regeneration but reappeared when callus was initiated from the leaves of these plants. The trp-sensitive form was localized in the particulate fraction and the trp-resistant form in the cytosol of tobacco cultured cell protoplasts. The trp-resistant form of AS from tobacco had an estimated MW of 200 000, determined by Sephacryl S-200 chromatography, compared to an estimated MW of 150 000 for the trp-sensitive form. The estimated molecular weights of AS from carrot and corn were 160 000 and 150 000, respectively. Analysis of AS activity from the diploid Nicotiana species Nicotiana otophora (chromosome number 2n=24) by high-performance liquid chromatography showed two activity peaks identical in elution time and trp inhibition characteristics to the activity from N. tabacum (chromosome No. 48). Thus the two enzyme forms found in tobacco did not appear to have originated individually from the progenitor species genomes which combined to make up the tobacco genome.Abbreviations AS anthranilate synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - HPLC high-performance liquid chromatography - 5MT D1-5-methyltryptophan - trp L-tryptophan     

The promoter of an antifungal protein gene from Gastrodia elata confers tissue -specific and fungus-inducible expression patterns and responds to both salicylic acid and jasmonic acid

Gastrodia antifungal proteins (GAFPs) are a group of mannose-binding lectins purified from Gastrodia elata that show strong resistance against a wide spectrum of fungi. The GAFP-2 promoter was analyzed for its ability to control the expression of the reporter gene, -glucuronidase (GUS) in transgenic tobacco plants. The GUS assays revealed that the GAFP-2 promoter is expressed in a tissue-specific manner, which mainly expressed in the vascular cells. The highest GUS activity was observed in roots, followed by stems. GAFP-2-GUS expression was strongly induced by the fungus Trichoderma viride and by the plant stress regulators, salicylic acid and jasmonic acid in the stably transformed tobacco plants. The –537 region of the GAFP-2 promoter was sufficient for its tissue-specific and inducible expression of the promoter.Communicated by H.S. Judelson     

Capillary electrophoresis-based profiling and quantitation of total salicylic acid and related phenolics for analysis of early signaling in Arabidopsis disease resistance

A capillary electrophoresis-based method for quantitation of total salicylic acid levels in Arabidopsis leaves was developed. Direct comparison to previous high-performance liquid chromatography (HPLC)-based measurements showed similar levels of salicylic acid. Simultaneous quantitation of trans-cinnamic acid, benzoic acid, sinapic acid, and an internal recovery standard was achieved. A rapid, streamlined protocol with requirements for plant tissue reduced relative to those of HPLC-based protocols is presented. Complicated, multiparameter experiments were thus possible despite the labor-intensive nature of inoculating plants with bacterial pathogens. As an example of this sort of experiment, detailed time course studies of total salicylic acid accumulation by wild-type Arabidopsis and two lines with mutations affecting salicylic acid accumulation in response to either of two avirulent bacterial strains were performed. Accumulation in the first 12h was biphasic. The first phase was partially SID2 and NDR1 dependent with both bacterial strains. The second phase was largely independent of both genes with bacteria carrying avrB, but dependent upon both genes with bacteria carrying avrRpt2. Virulent bacteria did not elicit salicylic acid accumulation at these time points. Application of this method to various Arabidopsis pathosystems and the wealth of available disease resistance signaling mutants will refine knowledge of disease resistance and associated signal transduction.     

The frequency of embryogenic pollen grains is not increased by in vitro anther culture in Nicotiana tabacum L.

The numbers of embryogenic (S) grains present in in-situ mature anthers of Nicotiana tabacum L. were compared to the numbers of embryos and plantlets produced in cultured anthers excised at the optimal mitotic stage of development for anther culture. The Feulgen technique of staining embryos caused a considerable loss of grains from cultured anthers but this did not seriously affect the determination of the percentage of embryos present. In no instance did the numbers of embryos produced exceed the maximum number of S grains found, and the distributions of S grain and embryo frequencies in anthers were similar. In rare instances S grains which had undergone the first embryogenic division were observed in situ. The results indicate that all grains capable of embryogenesis are determined during early flower formation and that their number is not increased by in vitro culture.     

Altered feedback sensitivity of acetohydroxyacid synthase from valine-resistant mutants of tobacco (Nicotiana tabacum L.)

Acetohydroxyacid synthase (EC 4.1.3.18) has been extracted from leaves of three valine-resistant (Val^r) tobacco (Nicotiana tabacum) mutants, and compared with the enzyme from the wild-type. The enzyme from all three mutants is appreciably less sensitive to inhibition by leucine and valine than the wild-type. Two of the mutants, Val^r-1 and Val^r-6, have very similar enzymes, which under all conditions are inhibited by less than half that found for the wild-type. The other mutant, Val^r-7, has an enzyme that only displays appreciably different characteristics from the wild-type at high pyruvate or inhibitor concentrations. Enzyme from Val^r-7 also has a higher apparent K_m for pyruvate, threefold greater than the value determined for the wild-type and the other mutants. The sulphonylurea herbicides strongly inhibit the enzyme from all the lines, though the concentrations required for half-maximal inhibition of enzyme from Val^r-1 and Val^r-6 are higher than for Val^r-7 or the wildtype. No evidence has been found for multiple isoforms of acetohydroxyacid synthase, and it is suggested that the valine-resistance of these mutant lines is the result of two different mutations affecting a single enzyme, possibly involving different subunits.     

Induction of an anionic peroxidase in cowpea leaves by exogenous salicylic acid

Two isoperoxidases were detected in cowpea (Vigna unguiculata) leaves. Treatment of the primary leaves with 10mM salicylic acid increased the total peroxidase activity contributed by the anionic isoform. To isolate both the anionic and cationic peroxidases the leaf crude extract was loaded on a Superose 12 HR 10/30 column followed by chromatography on Mono-Q HR 5/5. Both enzymes were stable in a pH range from 5 to 7. The optimum-temperatures for the cationic and anionic peroxidase isoforms were, respectively, 20-30 degrees C and 30 degrees C. The dependence of guaiacol oxidation rate varying its concentration at constant H(2)O(2) concentration showed, for both enzymes, Michaelis-Menten-type kinetic. Apparent K(m)(s) were 0.8 and 4.8 microM for the cationic and anionic isoperoxidases, respectively.     

Response of barley grains to the interactive e.ect of salinity and salicylic acid

Effect of grain soaking presowing in 1 mM salicylic acid (SA) and NaCl (0, 50, 100, 150 and 200 mM) on barley (Hordeum vulgare cv Gerbel) was studied. Increasing of NaCl level reduced the germination percentage, the growth parameters (fresh and dry weight), potassium, calcium, phosphorus and insoluble sugars content in both shoots and roots of 15-day old seedlings. Leaf relative water content (RWC) and the photosynthetic pigments (Chl a, b and carotenoids) contents also decreased with increasing NaCl concentration. On the other hand, Na, soluble sugars, soluble proteins, free amino acids including proline content and lipid peroxidation level and peroxidase activity were increased in the two plant organs with increasing of NaCl level. Electrolyte leakage from plant leaves was found to increase with salinity level. SA-pretreatment increased the RWC, fresh and dry weights, water, photosynthetic pigments, insolube saccharides, phosphorus content and peroxidase activity in the stressed seedlings. On the contrary, Na⁺, soluble proteins content, lipid peroxidation level, electrolyte leakage were markedly reduced under salt stress with SA than without. Under stress conditions, SA-pretreated plants exhibited less Ca²⁺ and more accumulation of K⁺, and soluble sugars in roots at the expense of these contents in the plant shoots. Exogenous application (Grain soaking presowing) of SA appeared to induce preadaptive response to salt stress leading to promoting protective reactions to the photosynthetic pigments and maintain the membranes integrity in barley plants, which reflected in improving the plant growth.     

Differential expression of HSP70 and SAPK/JNK genes in the cabbage aphid upon salicylic and dilute sulfuric acid treatments in canola

Investigating the interaction between insect pests and host plants at molecular level, provides an insight for effective implementation of plant resistance-based strategies in insect pest control programs. Heat shock proteins (HSPs) are found in all living organisms which protect them from biotic and abiotic stresses. Stress-activated protein kinases (SAPKs) are members of MPK kinases which also are activated upon cellular stresses. We investigated the effect of seed-primed canola plants with salicylic acid (SA) and the plants treated with dilute sulfuric acid on the regulation of two stress-related genes, HSP70 and SAPK/JNK genes in the cabbage aphid. The results of qRT-PCR revealed a statistically significant up-regulation of HSP70 gene expression in aphids feeding on sulfuric acid-treated plants in comparison with controls (P < 0.01). However, this gene was not regulated in aphids feeding on salicylic acid-treated plants. In addition, quantification of SAPK/JNK gene showed a significant effect in aphids feeding on SA-primed plants (P < 0.05) and a marginally significant effect in sulfuric acid treatment. Further, the expression of both genes were significantly changed in interaction effect between SA and sulfuric acid treatments. Finally, the induction of HSP70 and SAPK/JNK genes in this research might be related to activation of systemic acquired resistance in canola.