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.     

Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to pathogens,salinity and drought,and increases photosynthesis and tiller number

Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF‐Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding β‐glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H₂O₂), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H₂O_2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up‐regulation of defence‐related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers.     

Controlling and Defence‐related Mechanisms of Bacillus Strains Against Bacterial Leaf Blight of Rice

Bacillus strains are broadly studied for their beneficial role in plant growth and biological control of plant disease and pest; however, little is known about their underlying mechanisms. In this study, we assessed the controlling and defence‐related mechanisms of three Bacillus strains including rice seed‐associated strain B. subtilis A15, rhizobacterial strains B. amyloliquefaciens D29 and B. methylotrophicus H8, all of which are against bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae. Results indicated that all three strains showed strong biofilm formation ability. The culture filtrates of each strain significantly suppressed the growth and biofilm formation of X. oryzae, while changes in bacterial cell morphology such as cell swell and severe cell wall alterations were observed through the transmission electron microscopy images. PCR analysis revealed that all three strains harbour the antimicrobial‐associated genes that are responsible for biosynthesis of bacillomycin, fengycin, iturin and surfactin. Subsequent real‐time qPCR analysis revealed the upregulated expression of fenD and srfAA genes in D29 and H8, and fenD and ituC genes in A15 during their in vitro interaction with X. oryzae. It suggests that the antibacterial mechanisms of the three strains may be at least partially associated with their ability to secrete corresponding lipopeptides. Interestingly, the applications of the three strains in greenhouse conditions were found to be effective in controlling the BLB disease, which was achieved through the activation of inducing systemic resistance resulted from the enhanced activities of defence‐related enzymes. This is the first report of demonstration of the mode of antibacterial effect of Bacillus strains against X. oryzae. Overall, data from the current study provide valuable information for biological control of BLB disease in rice.     

Mycorrhizal colonization alleviates drought-induced oxidative damage and lignification in the leaves of drought-stressed perennial ryegrass (Lolium perenne)

To investigate the effects of arbuscular mycorrhizal (AM) fungus Glomus intraradices on antioxidative activity and lignification under drought‐stressed (DS) conditions, the enzyme activities, growth, lignin contents and some stress symptomatic parameters as affected by drought treatment were compared in AM colonized or non‐colonized (non‐AM) perennial ryegrass plants for 28 days. Drought significantly decreased leaf water potential (Ψ_w), photosynthesis rate and biomass. The negative impact of drought on these parameters was much highly relived in AM plants compared to non‐AM ones. Drought increased H₂O₂, lipid peroxidation, phenol and lignin levels, with significantly higher in non‐AM relative to AM plants at day 28 after drought treatment. The enhanced activation of guaiacol peroxidase (GPOX), coniferyl alcohol peroxidase (CPOX), syringaldazine peroxidase (SPOX) and polyphenol oxidase (PPO) was closely related with the decrease in Ψ_w in both AM and non‐AM plants. GPOX, CPOX, SPOX and PPO highly activated with a concomitant increase in lipid peroxidation and lignin as the Ψ_w decreased below ?2.11 MPa in non‐AM plants, while much less activated by maintaining Ψ_w≥?1.15 MPa in AM ones. These results indicate that AM symbiosis plays an integrative role in drought stress tolerance by alleviating oxidative damage and lignification, which in turn mitigate the reduction of forage growth and digestibility under DS conditions.     

The Role of Toxin(s) Produced by Germinating Spores of Pyricularia oryzae in Pathogenesis

Pyricularia oryzae produced toxin(s) during spore germination which induced susceptibility to infection by non-pathogenic Alternaria alternata of rice leaves. The induced susceptibility was independent of the compatibility between the races of blast fungi used for obtaining the toxin(s) and the rice cultivars used for bioassay. Susceptibility was also induced in other susceptible species (e.g. barley, Italian ryegrass, perennial ryegrass and wheat), results suggesting that the toxin(s), are host-selective and determine the host specificity at plant species level.     

The Role of Toxin(s) Produced by Germinating Spores of Pyricularia oryzae in Pathogenesis

Pyricularia oryzae produced toxin(s) during spore germination which induced susceptibility to infection by non-pathogenic Alternaria alternata of rice leaves. The induced susceptibility was independent of the compatibility between the races of blast fungi used for obtaining the toxin(s) and the rice cultivars used for bioassay. Susceptibility was also induced in other susceptible species (e.g. barley, Italian ryegrass, perennial ryegrass and wheat), results suggesting that the toxin(s) are host-selective and determine the host specificity at plant species level.     

氧化石墨烯对多年生黑麦草逆境生理及光合特征的影响

为探讨不同浓度氧化石墨烯（GO）对多年生黑麦草生长、生理及光合特征的影响,该文采用盆栽试验,在土壤中添加0、10、20、30、40、50 mg·g^-1 GO进行多年生黑麦草培养,并测定植物生长指标、光合色素含量、保护酶活性、丙二醛（MDA）含量、叶片质膜透性、可溶性蛋白含量和光合参数。结果表明：（1）10、20 mg·g^-1 GO处理对多年生黑麦草生长无显著影响;30～50 mg·g^-1 GO处理对多年生黑麦草生长具有抑制作用,在50 mg·g^-1 GO浓度下多年生黑麦草株高和生物量均最小,较对照分别降低了16.8%和27.1%。（2）当GO浓度达到30 mg·g^-1时,总叶绿素和类胡萝卜素的含量显著降低,在50 mg·g^-1 GO处理时达到最低。（3）高浓度的GO处理(40、50 mg·g^-1)虽降低了多年生黑麦草的叶片净光合速率（P_n）、气孔导度（G_s）和蒸腾速率（T_r     

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.     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

Two thiadiazole compounds promote rice defence against Xanthomonas oryzae pv. oryzae by suppressing the bacterium's production of extracellular polysaccharides

Thiazole, isothiazole, thiadiazole, and their derivatives are used to control various human, animal and plant diseases. In addition to having direct anti‐microbial and anti‐fungal properties, these compounds are thought to induce host defences, but the mechanism of defence induction remains poorly understood. This article reports that the thiadiazoles of zinc thiazole and bismerthiazol induce H₂O₂ accumulation, up‐regulation of defence‐related genes, callose deposition and hypersensitive response‐like cell death in rice leaves infected with Xanthomonas oryaze pv. oryzae (Xoo) strain ZJ173, but not in non‐infected leaves. These defence responses in Xoo‐infected leaves were suppressed by the exogenous application of catalase, which reduces H₂O₂ accumulation. The application of extracellular polysaccharides (EPSs) extracted from strain ZJ173 significantly compromised rice defence against ZJ173 with or without thiadiazole treatment. The EPS‐deficient Xoo mutant ?gumH triggered a stronger defence than its parent strain ZJ173. The thiadiazole treatments reduced EPS production by strain ZJ173, but not by the thiadiazole‐resistant strain 2‐1‐1, which is thiadiazole resistant in vivo, but not in vitro; moreover, enhanced defence was not detected in thiadiazole‐treated rice inoculated with 2‐1‐1. Based on these data, we infer that zinc thiazole and bismerthiazol promote rice defence against Xoo by inhibiting the production of bacterial EPS.     

Sugar homeostasis mediated by cell wall invertase GRAIN INCOMPLETE FILLING 1 (GIF1) plays a role in pre‐existing and induced defence in rice

Sugar metabolism and sugar signalling are not only critical for plant growth and development, but are also important for stress responses. However, how sugar homeostasis is involved in plant defence against pathogen attack in the model crop rice remains largely unknown. In this study, we observed that the grains of gif1, a loss‐of‐function mutant of the cell wall invertase gene GRAIN INCOMPLETE FILLING 1 (GIF1), were hypersusceptible to postharvest fungal pathogens, with decreased levels of sugars and a thinner glume cell wall in comparison with the wild‐type. Interestingly, constitutive expression of GIF1 enhanced resistance to both the rice bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. The GIF1‐overexpressing (GIF1‐OE) plants accumulated higher levels of glucose, fructose and sucrose compared with the wild‐type plants. More importantly, higher levels of callose were deposited in GIF1‐OE plants during pathogen infection. Moreover, the cell wall was much thicker in the infection sites of the GIF1‐OE plants when compared with the wild‐type plants. We also found that defence‐related genes were constitutively activated in the GIF1‐OE plants. Taken together, our study reveals that sugar homeostasis mediated by GIF1 plays an important role in constitutive and induced physical and chemical defence.     

Early Detection of Bacterium-induced Basal Resistance in Tobacco Leaves with Diaminobenzidine and Dichlorofluorescein Diacetate

The present study demonstrate that in tobacco leaves the diaminobenzidine (DAB) and 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) staining is a useful indicator of the basal (also known as general or innate) defence‐associated reactions, especially of the early developing form of basal resistance (EBR). DAB and DCFH‐DA, in the presence of H₂O₂ and peroxidase converts to a brown polymer and fluorescent DCF respectively. In the present study, the hypersensitive response (HR)‐inducing avirulent Pseudomonas syringae pv. syringae 61, its HR‐negative hrp/hrc mutants and even non‐pathogenic bacteria such as P. fluorescens and Escherichia coli caused DAB and DCFH‐DA staining, if the dyes were injected 3–4 h after bacterial inoculation into tobacco leaves. The conditions that enable the staining of plant leaves infiltrated with HR‐negative bacteria were persisted for 1 to several days depending on the physiological state of the plant, and plant activity was required to the development of the staining. The live virulent P. syringae pv. tabaci was able to suppress the development of the staining reaction. Bacteria that induced more intensive staining reaction triggered stronger local resistance response, which was verified by its ability to inhibit the HR by challenging avirulent bacteria and by expression analysis of genes that are activated during the basal defence response. The peroxidase enzyme activity increased in bacterially treated tobacco tissue, and inhibition of peroxidase activity blocked the development of the staining. The results showed that in tobacco leaves the staining reactions were associated with the general recognition and basal defence reaction of tobacco plant and can be used as markers in tobacco leaves for testing the occurrence of this type of defence.     

Improvement of fitness and biocontrol properties of Pseudomonas putida via an extracellular heme peroxidase

The extracellular 373-kDa PehA heme peroxidase of Pseudomonas putida KT2440 has two enzymatic domains which depend on heme cofactor for their peroxidase activity. A null pehA mutant was generated to examine the impact of PehA in rhizosphere colonization competence and the induction of plant systemic resistance (ISR). This mutant was not markedly hampered in colonization efficiency. However, increase in pehA dosage enhanced colonization fitness about 30 fold in the root and 900 fold in the root apex. In vitro assays with purified His-tagged enzymatic domains of PehA indicated that heme-dependent peroxidase activity was required for the enhancement of root tip colonization. Evaluation of live/dead cells confirmed that overexpression of pehA had a positive effect on bacterial cell viability. Following root colonization of rice plants by KT2440 strain, the incidence of rice blast caused by Magnaporthe oryzae was reduced by 65% and the severity of this disease was also diminished in comparison to non-treated plants. An increase in the pehA dosage was also beneficial for the control of rice blast as compared with gene inactivation. The results suggest that PehA helps P. putida to cope with the plant-imposed oxidative stress leading to enhanced colonization ability and concomitant ISR-elicitation.     

Interannual variation in nitrous oxide emissions from perennial ryegrass/white clover grassland used for dairy production

Nitrous oxide (N₂O) emissions are subject to intra‐ and interannual variation due to changes in weather and management. This creates significant uncertainties when quantifying estimates of annual N₂O emissions from grazed grasslands. Despite these uncertainties, the majority of studies are short‐term in nature (<1 year) and as a consequence, there is a lack of data on interannual variation in N₂O emissions. The objectives of this study were to (i) quantify annual N₂O emissions and (ii) assess the causes of interannual variation in emissions from grazed perennial ryegrass/white clover grassland. Nitrous oxide emissions were measured from fertilized and grazed perennial ryegrass/white clover grassland (WC) and from perennial ryegrass plots that were not grazed and did not receive N input (GB), over 4 years from 2008 to 2012 in Ireland (52°51′N, 08°21′W). The annual N₂O‐N emissions (kg ha^?1; mean ± SE) ranged from 4.4 ± 0.2 to 34.4 ± 5.5 from WC and from 1.7 ± 0.8 to 6.3 ± 1.2 from GB. Interannual variation in N₂O emissions was attributed to differences in annual rainfall, monthly (December) soil temperatures and variation in N input. Such substantial interannual variation in N₂O emissions highlights the need for long‐term studies of emissions from managed pastoral systems.     

Elevated CO2 changes the interactions between nematode and tomato genotypes differing in the JA pathway

Interactions between the root‐knot nematode Meloidogyne incognita and three isogenic tomato (Lycopersicon esculentum) genotypes were examined when plants were grown under ambient (370 ppm) and elevated (750 ppm) CO₂. We tested the hypothesis that, defence‐recessive genotypes tend to allocate ‘extra’ carbon (relative to nitrogen) to growth under elevated CO₂, whereas defence‐dominated genotypes allocate extra carbon to defence, and thereby increases the defence against nematodes. For all three genotypes, elevated CO₂ increased height, biomass, and root and leaf total non‐structural carbohydrates (TNC):N ratio, and decreased amino acids and proteins in leaves. The activity of anti‐oxidant enzymes (superoxide dismutase and catalase) was enhanced by nematode infection in defence‐recessive genotypes. Furthermore, elevated CO₂ and nematode infection did not qualitatively change the volatile organic compounds (VOC) emitted from plants. Elevated CO₂ increased the VOC emission rate only for defence‐dominated genotypes that were not infected with nematodes. Elevated CO₂ increased the number of nematode‐induced galls on defence‐dominated genotypes but not on wild‐types or defence‐recessive genotypes roots. Our results suggest that CO₂ enrichment may not only increase plant C : N ratio but can disrupt the allocation of plant resources between growth and defence in some genetically modified plants and thereby reduce their resistance to nematodes.     

Role of ethylene signalling in growth and systemic resistance induction by the plant growth‐promoting fungus Penicillium viridicatum in Arabidopsis

The plant growth‐promoting fungi (PGPF) have long been known to improve plant growth and suppress plant diseases. The PGPF Penicillium viridicatum GP15‐1 elicited plant growth and induced systemic resistance (ISR) in Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000 (Pst), leading to a restriction of pathogen growth and disease development. Examination of local and systemic genes indicated that GP15‐1 did not modulate the expression of any of the tested defence‐related marker genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene signalling pathways. Subsequent challenge of GP15‐1‐colonized plants with Pst bacterium primed Arabidopsis plants for enhanced activation of the JA‐inducible Atvsp (vegetative storage protein) gene at a later stage of infection. To assess the contribution of different signalling pathways in GP15‐1‐elicited plant growth and ISR, Arabidopsis genotypes implicated in SA signalling expressing the nahG transgene (NahG) or carrying disruption in NPR1 (npr1), JA signalling (jar1) and ethylene signalling (ein2) were tested. The GP15‐1‐induced plant growth and ISR were fully compromised in an ein2 mutation. Root colonization assay revealed that the inability of the ein2 mutant to express GP15‐1‐induced plant growth and ISR was not associated with reduced root colonization by GP15‐1. In conclusion, our results demonstrate the ethylene signalling pathway is involved in plant growth promotion and ISR elicitation by the PGPF P. viridicatum GP15‐1 in Arabidopsis. These results provide evidence that ethylene signalling has a substantial role in plant growth and disease resistance.