LsGRP1, a class II glycine-rich protein of Lilium,confers plant resistance via mediating innate immune activation and inducing fungal programmed cell death

Defence-related LsGRP1 is a leaf-specific plant class II glycine-rich protein (GRP) involved in salicylic acid-induced systemic resistance against grey mould caused by necrotrophic Botrytis elliptica in lily (Lilium) cultivar Stargazer. The C-terminal region of LsGRP1 (LsGRP1^C) can inhibit fungal growth in vitro via a mechanism of inducing fungal apoptosis programmed cell death (PCD). In this study, the role of LsGRP1 in induced defence mechanism was investigated using LsGRP1-silenced Stargazer lily and LsGRP1-transgenic Arabidopsis thaliana. LsGRP1 silencing in lily was found to slightly inhibit plant growth and greatly increase the susceptibility to B. elliptica by suppressing callose deposition and early reactive oxygen species (ROS) accumulation. In contrast, LsGRP1-transgenic Arabidopsis showed higher resistance to Botrytis cinerea and also to Pseudomonas syringae pv. tomato DC3000 as compared to the wild type, accompanied with the enhancement of callose deposition and ROS accumulation. Additionally, LsGRP1 silencing increased plant cell death caused by B. elliptica secretion and reduced pathogen-associated molecular pattern (PAMP)-triggered defence activation in Stargazer lily. Consistently, LsGRP1 expression boosted PAMP-triggered defence responses and effector recognition-induced hypersensitive response in Arabidopsis. Moreover, fungal apoptosis PCD triggered by LsGRP1 in an LsGRP1^C-dependent manner was demonstrated by leaf infiltration with LsGRP1^C-containing recombinant proteins in Stargazer lily. Based on these results, we presume that LsGRP1 plays roles in plant defence via functioning as a pathogen-inducible switch for plant innate immune activation and acting as a fungal apoptosis PCD inducer to combat pathogen attack.     

两种葡萄孢属真菌对不同品种百合花瓣和叶片的侵染能力比较

为明确两种葡萄孢属真菌对不同百合品种叶片和花瓣的侵染能力,采用离体叶片接种法测定灰葡萄孢Botrytis cinerea和椭圆葡萄孢Botrytis elliptica对4个百合品种叶片和花瓣的侵染时间和病斑扩展速度。结果表明,供试百合花瓣接种灰葡萄孢病斑出现时间明显早于叶片,而不同品种花瓣接种椭圆葡萄孢病斑出现时间差异显著。此外,百合品种‘木门’叶片接种椭圆葡萄孢96 h后仍没有病斑出现,而花瓣接种后48 h病斑出现,说明‘木门’叶片对椭圆葡萄孢抗性较强,而花瓣较易感病。     

Formulation of Bacillus amyloliquefaciens B190 for Control of Lily Grey Mould (Botrytis elliptica)

Calcium hydroxide (0.1%) significantly increased the growth of Bacillus amyloliquefaciens B190, inhibited completely the germination of Botrytis elliptica, and decreased the disease severity caused by B. elliptica on lily. Spraying B. amyloliquefaciens B190 mixed with either 0.025% calcium hydroxide, 0.05% sodium carbonate or 0.025% ammonium nitrate decreased the grey mould disease on lily leaves. B. amyloliquefaciens B190 mixed with 0.025% calcium hydroxide and 0.05% sodium carbonate, or mixed with 0.025% calcium hydroxide and 0.025% ammonium nitrate controlled lily grey mould completely. When the concentration of tested adjuvants was below 0.1% (v/v), adhesive adjuvant, i.e. carboxymethyl cellulose and spreader, i.e. Tween 80 were equally effective to assist B. amyloliquefaciens B190 to control lily grey mould. Calcium hydroxide (0.025%) and 0.05% sodium carbonate mixed with 0.1% Tween 80 significantly controlled lily grey mould. B. amyloliquefaciens B190 mixed with 0.025% calcium hydroxide and 0.05% sodium carbonate, and these two chemicals plus or without 0.1% Tween 80 and 0.05% mineral oil (i.e. emulsion and wettable powder, respectively) was consistently able to control grey mould on lily as well as 100 p.p.m. flusilazole in greenhouse and field trials, respectively.     

Identification of an antifungal chitinase from a potential biocontrol agent, Bacillus cereus 28-9

Bacillus cereus 28-9 is a chitinolytic bacterium isolated from lily plant in Taiwan. This bacterium exhibited biocontrol potential on Botrytis leaf blight of lily as demonstrated by a detached leaf assay and dual culture assay. At least two chitinases (ChiCW and ChiCH) were excreted by B. cereus 28-9. The ChiCW-encoding gene was cloned and moderately expressed in Escherichia coli DH5alpha. Near homogenous ChiCW was obtained from the periplasmic fraction of E. coli cells harboring chiCW by a purification procedure. An in vitro assay showed that the purified ChiCW had inhibitory activity on conidial germination of Botrytis elliptica, a major fungal pathogen of lily leaf blight.     

Antifungal Activity of Thymol against Aspergillus awamori and Botrytis aclada Isolated from Stored Onion Bulbs

The antifungal activity of thymol against Aspergillus awamori F23 and Botrytis aclada F15 in onions was examined through direct treatment with amended media and gaseous treatment with I-plates (plastic plates containing central partitions). The protective and curative control efficacy of thymol was examined 24 h before and after the inoculation of onion bulbs with the fungal isolates. Mycelial growth, sporulation, and spore germination of the isolates were inhibited on potato dextrose agar amended with various concentrations of thymol or acetic acid (positive control). Overall, thymol produced a stronger inhibitory effect on the mycelial growth and development of the isolates than acetic acid. Following gaseous treatment in I-plates, mycelial growth, sporulation, and spore germination of the isolates were inhibited at higher concentrations of thymol or acetic acid; however, acetic acid showed a little effect on the sporulation and spore germination of the isolates. Following the treatment of onion bulbs with 1000 mg L⁻¹ of thymol 24 h before and after fungal inoculation, lesion diameter was greatly reduced compared with that following treatment with 0.5% ethanol (solvent control). Onion bulbs sprayed with thymol 24 h before fungal inoculation generally showed reduced lesion diameters by isolate F23 but not in isolate F15 compared with those sprayed 24 h after fungal inoculation. Collectively, thymol effectively inhibited the growth and development of A. awamori and B. aclada on amended media and in I-plates. In addition, spraying or fumigation of thymol is more desirable for effectively controlling these postharvest fungal pathogens during long-term storage conditions.     

Microbial Oxidation of Cephalosporins to Cephalosporin Sulfoxides

The highest inhibition rate of conidial germination of Pyricularia oryzae was shown by extracts of rice plant leaves inoculated by a pathogen after treatment with probenazole, a rice blast controlling agent. Four anti-conidial germination substances were isolated from these extracts. Substances A, C and D inhibited the germination of the conidia at concentrations between 100 and 200 mcg/ml, and substance B caused morphological changes in the germination tubes of the conidia with a little inhibition of germination. These substances were differentiated from momilactone A, B and the degraded or metabolized products of probenazole. Besides anti-conidial germination activity, they showed antimicrobial activities against several kinds of phytopathogenic bacteria of fungi on agar plates by diffusion method.     

Identification and transcriptional analysis of genes involved in <Emphasis Type="Italic">Bacillus cereus</Emphasis>-induced systemic resistance in <Emphasis Type="Italic">Lilium</Emphasis>

Bacillus cereus C1L was demonstrated to induce systemic resistance in Lilium formosanum against leaf blight caused by Botrytis elliptica. Suppression subtractive hybridization library of L. formosanum triggered by B. cereus C1L were screened and 3 differentially expressed genes were identified. Based on sequence analysis, these genes encoding putative glycine-rich protein, metallothionein-like protein, and PsbR protein of photosystem 2, were designated LfGRP1, LfMT1, and LfPsbR, respectively. The results of Northern blot analysis showed that expressions of LfGRP1, LfMT1 and LfPsbR increased in response to B. elliptica infection. On the other hand, expression of LfMT1 increased but expressions of LfGRP1 and LfPsbR decreased when the rhizosphere of L. formosanum was drenched with suspension of B. cereus C1L with or without subsequent challenge with B. elliptica on lily leaves. Similar expression profiles of homologues of LfGRP1, LfMT1, and LfPsbR (named LsGRP1, LsMT1, and LsPsbR, respectively) were presented in Lilium oriental hybrid Star Gazer. In addition, application of the photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, on the leaves reduced disease severity and expressions of LsGRP1 and LsPsbR just as that in response to B. cereus C1L treatment.     

Syringomycin, a bacterial phytotoxin, closes stomata

The effects of the bacterial phytotoxin, syringomycin, on stomata were investigated using detached leaves of Xanthium strumarium and isolated epidermes of Vicia faba. Syringomycin is known to cause K⁺ efflux in fungal and higher plant cells. Doses of syringomycin as low as 0.3 unit per square centimeter (about 0.88 pmole per square centimeter) resulted in measurable stomatal closure when applied through the transpiration stream of detached leaves; higher doses produced larger reductions in stomatal conductance. Stomatal apertures of isolated epidermes were also reduced by low concentrations (3.2 units per milliliter; 10⁻⁸ molar) of syringomycin. The effects of syringomycin were similar to those of ABA. Both compounds closed stomata at a similar rate and at similar concentrations. In addition, neither compound significantly affected the relationship between photosynthesis and intercellular CO₂ based on data taken after stomatal conductance had stabilized following the treatment. It is possible that syringomycin and ABA activate the same K⁺ export system in guard cells, and syringomycin may be a valuable tool for studying the molecular basis of ABA effects on guard cells.     

Endophytic blastospores of Beauveria bassiana provide high resistance against plant disease caused by Botrytis cinerea

The entomopathogenic fungus Beauveria bassiana is widely used for insect pest control and can produce three distinct infective unit types under different nutritional and environmental conditions: aerial conidia, blastospores, and submerged conidia. Here, we identified endophytic colonization ability and existing forms of the three types of B. bassiana infective units after inoculating Arabidopsis plants via soil drenching, and tested their effects on their presence mold disease caused by Botrytis cinerea. We found that all B. bassiana infective unit types colonized Arabidopsis leaves, with germinating and producing hyphae by hydrophilic blastopores and submerged conidia; further, we showed that blastospores were more effective in defending against B. cinerea, compared with aerial conidia. These findings suggest that in addition to aerial conidia, the colonization of other two types of entomopathogenic fungal infective units could also have important impacts on plant resistance. This study contributes to better understanding on the function of B. bassiana as fungal endophytes, which could lead to a new paradigm for how to successfully use these organisms in biological control against plant diseases.     

Signal cross talk in Arabidopsis exposed to cadmium, silicon, and Botrytis cinerea

The role of defence gene expression triggered by Cd toxicity in the plant’s response to Botrytis cinerea was investigated in Arabidopsis thaliana Columbia 0. Silicon (0 or 1.5 mM) and Cd (0, 1 or 10 μM) were supplied to 3-month-old solution-cultured plants. After 3 days, half of the plants of each treatment were inoculated with Botrytis. Supplied Cd concentrations were below the toxicity threshold and did not cause shoot growth inhibition or evidence of oxidative stress, while Botrytis infection severely decreased plant growth in all treatments. The expression of marker genes PR1 and BGL2 for the salicylic acid (SA) and the PDF1.2 for the jasmonic acid–ethylene (JA–ET) signalling pathways was enhanced in 10 μM Cd-treated non-infected plants. Twenty hours after inoculation, PDF1.2 expression showed a strong increase in all treatments, while enhanced PR1, BGL2, and CHIB expression was only found 7 days after infection. A great synergistic effect of Cd and Botrytis on PDF1.2 expression was found in 10 μM Cd-treated plants. Silicon decreased PR1, BGL2, and CHIB, while increasing PDF1.2 expression, which indicates its role as a modulator of the signalling pathways involved in the plant’s response to fungal infection. Botrytis growth decreased in 10 μM Cd-treated plants, which could be due to the combined effects of Cd and Botrytis activating the SA and JA–ET-mediated signalling pathways. Taken together, our results provide support for the view that Cd concentrations close to the toxicity threshold induce defence signalling pathways which potentiate the plant’s response against fungal infection.     

Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1

Bacillus atrophaeus CAB-1 displays a high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. We extracted and identified lipopeptides and secreted proteins and volatile compounds produced by strain CAB-1 to investigate the mechanisms involved in its biocontrol performance. In vitro assays indicated all three types of products contributed to the antagonistic activity against the fungal pathogen Botrytis cinerea. Each of these components also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the major bioactive lipopeptide was fengycin A (C15–C17). We isolated the crude-secreted proteins of CAB-1 and purified a fraction with antifungal activity. This protein sequence shared a high identity with a putative phage-related pre-neck appendage protein, which has not been reported as an antifungal factor. The volatile compounds produced by CAB-1 were complex, including a range of alcohols, phenols, amines, and alkane amides. O-anisaldehyde represented one of the most abundant volatiles with the highest inhibition on the mycelial growth of B. cinerea. To our knowledge, this is the first report on profiling three types of antifungal substances in Bacilli and demonstrating their contributions to plant disease control.     

Biological control of strawberry crown rot,root rot and grey mould by the beneficial fungus Aureobasidium pullulans

Utilization of biocontrol agents is a sustainable approach to reduce plant diseases caused by fungal pathogens. In the present study, we tested the effect of the candidate biocontrol fungus Aureobasidium pullulans (De Bary) G. Armaud on strawberry under in vitro and in vivo conditions to control crown rot, root rot and grey mould caused by Phytophthora cactorum (Lebert and Cohn) and Botrytis cinerea Pers, respectively. A dual plate confrontation assay showed that mycelial growth of P. cactorum and B. cinerea was reduced by 33–48% when challenged by A. pullulans as compared with control treatments. Likewise, detached leaf and fruit assays showed that A. pullulans significantly reduced necrotic lesion size on leaves and disease severity on fruits caused by P. cactorum and B. cinerea. In addition, greenhouse experiments with whole plants revealed enhanced biocontrol efficacy against root rot and grey mould when treated with A. pullulans either in combination with the pathogen or pre-treated with A. pullulans followed by inoculation of the pathogens. Our results demonstrate that A. pullulans is an effective biocontrol agent to control strawberry diseases caused by fungal pathogens and can be an effective alternative to chemical-based fungicides.

     

Development of novel 2-substituted acylaminoethylsulfonamide derivatives as fungicides against Botrytis cinerea

Botrytis cinerea is an economically important fungal pathogen with a host range of over 200 plant species. Unfortunately, gray mold disease caused by B. cinerea has not been effectively controlled because of its high risk for fungicide resistance development. As a part of our ongoing efforts to develop novel sulfonamides as agricultural fungicides against Botrytis cinerea, we introduced 2-aminoethanesulfonic acid (taurine) substructure, designed and synthesized a series of novel 2-substituted acylaminoethylsulfonamides. The newly synthesized sulfonamides were evaluated in vitro and in vivo for their fungicidal activity against Botrytis cinerea, of which the 2-ethoxyacetylamide derivative (V-A-12, EC₅₀ = 0.66 mg·L⁻¹) exhibited the highest potency in vitro and superior fungicidal activity compared with procymidone (EC₅₀ = 1.06 mg·L⁻¹). In vivo bioassay indicated that compound V-A-12 could be effective for the control of tomato gray mold. Moreover, the structure-activity relationship of these sulfonamides was analyzed by establishing a three-dimensional quantitative structure-activity relationship (3D-QSAR) model, which can provide guidance for the development of sulfonamides as fungicides. Finally, the effeicacy of sulfonamide derivatives was again verified in the activity evaluation against resistant Botrytis cinerea strains. These results further enhance the development value of 2-substituted acylaminoethylsulfonamides to control the tomato gray mold.     

In the tripartite combination Botrytis cinerea–Arabidopsis–Eurydema oleracea,the fungal pathogen alters the plant–insect interaction via jasmonic acid signalling activation and inducible plant-emitted volatiles

In ecosystems, plants are continuously challenged by combined stress conditions more than by a single biotic or abiotic factor. Consequently, in recent years studies on plant relationships with multiple stresses have aroused increasing interest. Here, the impact of inoculation with fungal pathogens with different lifestyles on Arabidopsis plants response to the following infestation with the invasive crop pest Eurydema oleracea was investigated. In particular, as fungal pathogens the necrotroph Botrytis cinerea and the biotroph Golovinomyces orontii were used. Plants exposed to B. cinerea, but not to G. orontii, showed reduced herbivore feeding damage. This difference was associated to different hormonal pathways triggered by the pathogens: G. orontii only induced the salicylate-mediated pathway, while B. cinerea stimulated also the jasmonate-dependent signalling, which persisted for a long time providing a long-term defence to further herbivore attack. In particular, the lower susceptibility of B. cinerea-infected Arabidopsis plants to E. oleracea was related to the stimulation of the JA-induced pathway on the production of plant volatile compounds, since treatment with VOCs emitted by B. cinerea inoculated plants inhibited both insect plant choice and feeding damage. These results indicate that necrotrophic plant pathogenic fungi modulate host volatile emission, thus affecting plant response to subsequent insect, thereby increasing the knowledge on tripartite plant–microbe–insect interactions in nature.

     

Vapours from plant essential oils to manage tomato grey mould caused by Botrytis cinerea

Tomato grey mould has been a great concern during tomato production. The in vitro antifungal activity of vapours emitted from four plant essential oils (EOs) (cinnamon oil, fennel oil, origanum oil, and thyme oil) were evaluated during in vitro conidial germination and mycelial growth of Botrytis cinerea, the causal agent of grey mould. Cinnamon oil vapour was the most effective in suppressing conidial germination, whereas the four EOs showed similar activities regarding inhibiting mycelial growth in dose-dependent manners. The in planta protection effect of the four EO vapours was also investigated by measuring necrotic lesions on tomato leaves inoculated by B. cinerea. Grey mould lesions on the inoculated leaves were reduced by the vapours from cinnamon oil, origanum oil and thyme oil at different levels, but fennel oil did not limit the spread of the necrotic lesions. Decreases in cuticle defect, lipid peroxidation, and hydrogen peroxide production in the B. cinerea-inoculated leaves were correlated with reduced lesions by the cinnamon oil vapours. The reduced lesions by the cinnamon oil vapour were well matched with arrested fungal proliferation on the inoculated leaves. The cinnamon oil vapour regulated tomato defence-related gene expression in the leaves with or without fungal inoculation. These results suggest that the plant essential oil vapours, notably cinnamon oil vapour, can provide eco-friendly alternatives to manage grey mould during tomato production.     

Heat-induced oxidative injury contributes to inhibition of Botrytis cinerea spore germination and growth

The inhibitory effect of heat treatment (HT) on Botrytis cinerea, a major postharvest fungal pathogen, and the possible mode of action were investigated. Spore germination and germ tube elongation of B. cinerea were both increasingly and significantly inhibited by HT (43 °C) for 10, 20 or 30 min. HT-induced gene expression of NADPH oxidase A, resulted in the intracellular accumulation of reactive oxygen species. HT-treated B. cinerea spores exhibited higher levels of oxidative damage to proteins and lipids, compared to the non-HT control. These findings indicate that HT resulted in oxidative damage which then played an important role in the inhibitory effect on B. cinerea. In the current study, HT was effective in controlling gray mold, caused by B. cinerea, in pear fruits. Understanding the mode of action by which HT inhibits fungal pathogens will help in the application of HT for management of postharvest fungal diseases of fruits and vegetables.