Tobacco Powdery Mildew Research Articles

Two New Isoindole Alkaloids from Aspergillus fumigatus as Potential Activators for Tobacco Powdery Mildew

Two New Isoindole Alkaloids from Aspergillus fumigatus as Potential Activators for Tobacco Powdery Mildew

Isoquinoline alkaloids from endophytic fungus Aspergillus puniceus and their potential as inhibitors for tobacco powdery mildew

The isoquinoline alkaloids have attracted widespread attention from chemists and biologists. With the aims of screening for biological compounds produced by fungi, three new (1–3), together with four known (4–7) isoquinolines were isolated from the cigar tobacco-derived endophytic fungus Aspergillus puniceus in this study. Their structures were determined by means of HRESIMS and NMR spectroscopic studies. Interestingly, the antifungal activity test revealed that compound 5 showed high antifungal activity against G. cichoracearum (DC) (the main pathogen of tobacco powdery mildew disease) with an inhibition rate of 76.3% ± 5.0. Compared to the negative control, compounds 1–4, 6, and 7 also showed notable anti-G. cichoracearum activities with inhibition rates in the range of 38.5–56.9%. The isolation of the above isoquinolines may provide materials for the study of more potent antifungal inhibitors and contribute to an improved utilisation of cigar tobacco-derived endophytic fungi.

Indole alkaloids isolated from the Nicotiana tabacum-derived Aspergillus fumigatus 0338 as potential inhibitors for tobacco powdery mildew and their mode of actions

To explore active natural products against tobacco powdery mildew caused by Golovinomyces cichoracearum, an extract from the fermentation of endophytic Aspergillus fumigatus 0338 was investigated. The mechanisms of action for active compounds were also studied in detail. As a result, 14 indole alkaloid derivatives were isolated, with seven being newly discovered (1–7) and the remaining seven previously described (8–14). Notably, compounds 1–3 are rare linearly fused 6/6/5 tricyclic prenylated indole alkaloids, with asperversiamide J being the only known natural product of this kind. The isopentenyl substitutions at the 5-position in compounds 4 and 5 are also rare, with only compounds 1-(5-prenyl-1H-indol-3-yl)-propan-2-one (8) and 1-(6-methoxy-5-prenyl-1H-indol3-yl)-propan-2-one currently available. In addition, compounds 6 and 7 are new framework indole alkaloid derivatives bearing a 6-methyl-1,7-dihydro-2H-azepin-2-one ring. The purified compounds were evaluated for their activity against G. cichoracearum, and the results revealed that compounds 7 and 9 demonstrated obvious anti-G. cichoracearum activities with an inhibition rate of 82.6% and 85.2%, respectively, at a concentration of 250 μg/mL, these rates were better than that of the positive control agent, carbendazim (78.6%). The protective and curative effects of compounds 7 and 9 were also better than that of positive control, at the same concentration. Moreover, the mechanistic study showed that treatment with compound 9 significantly increased the structural tightness of tobacco leaves and directly affect the conidiospores of G. cichoracearum, thereby enhancing resistance. Compounds 7 and 9 could also induce systemic acquired resistance (SAR), directly regulating the expression of defense enzymes, defense genes, and plant semaphorins, which may further contribute to increased plant resistance. Based on the activity experiments and molecular dockings, the indole core structure may be the foundation of these compounds' anti-G. cichoracearum activity. Among them, the indole derivative parent structures of compounds 6, 7, and 9 exhibit strong effects. Moreover, the methoxy substitution in compound 7 can enhance their activity. By isolating and structurally identifying the above indole alkaloids, new candidates for anti-powdery mildew chemical screening were discovered, which could enhance the utilization of N. tabacum-derived fungi in pesticide development.

Prenylated flavonoids isolated from the twigs of Artocarpus champeden as potential activators for tobacco powdery mildew and their mode of actions

BackgroundGolovinomyces cichoracearum (DC.) is the main pathogen for tobacco powdery mildew fungus disease. Its outbreaks often result in severe harvest losses for the yield and quality of tobacco. Artocarpus champeden is rich in prenylated flavonoids, which are important for the plant’s defensive strategies. With the aim of continuously exploring bioactive natural metabolites for agricultural chemicals, the chemical investigations on the twigs of A. champeden were carried out.ResultsSix new (1–6) and five known (7–11) prenylated flavonoids were isolated. Compound 1 is the first example of flavone whose prenylated side-chain is converted into an unusual 1H-pyrrol-2-yl functional group. Compounds 2 and 3 are rare flavones bearing a 4-methylfuran-2-yl moiety. The frameworks of the above three flavones are reported in natural products for the first time. Interestingly, compound 1 showed high anti-G. cichoracearum activity with an inhibition rate of 88.3% ± 6.2. This rate is higher than that of the positive control (with an inhibition rate of 81.5% ± 6.3) compared to the negative control, compounds 2–11 also showed potential activities with inhibition rates in the range of 50.9%–72.0%. In addition, the mechanistic studies on 1 revealed that it has a potent direct effect on conidiospores of G. cichoracearum and induces systemic acquired resistance for tobacco plants, which may be the reasons for its significant effects against G. cichoracearum.ConclusionsPowdery mildew is a fungal disease harmful to tobacco. Flavonoids have been identified as the sources of promising antifungal agents. For prenylated flavonoids, the combination of a flavonoid skeleton with prenylated side-chain can give the resultant more potential for biological activities. The successful isolation and structure identification of the above prenylated flavonoids provide new materials for the screening of powdery mildew inhibitors, and also contribute to the improved utilization of A. champeden.Graphical

Biocontrol of Lysobacter enzymogenes CQ18 against the tobacco powdery mildew fugus, Erysiphe cichoracearum

BackgroundPowdery mildew is a common leaf disease of crops worldwide. A large quantity of chemical fungicides is used to control this disease in horticulture and agriculture, producing serious safety and environmental problems. To suppress this disease in safe and environment-friendly ways, the biocontrol of a self-isolated new strain of Lysobacter enzymogenes (CQ18) was studied against flue-cured tobacco powdery mildews.ResultsL. enzymogenes CQ18 produced chitinase, protease, β-1,3-glucanase, phosphatase, and siderophore, which may enable this biocontrol bacterium to degrade pathogen cell membranes and walls and deprive pathogens of iron. HPLC/MS analysis identified 14 antifungal metabolites present in L. enzymogenes CQ18 fermentation liquid (LEFL), which were grouped into organic acids, azoles, and pyrimidines. The variable targets in or on pathogen cells and combinative effects of these multiple metabolites may potently suppress the powdery mildew and be less likely to make Erysiphe cichoracearum develop resistance. LEFL was rich in L-pyroglutamate. Both LEFL and L-pyroglutamate inhibited the germination of E. cichoracearum conidia in vitro and reduced the powdery mildew index in the greenhouse and field. L-Pyroglutamate at a concentration of 0.50% achieved the same control efficacy as the chemical fungicide triadimefon (91–94%).ConclusionsL. enzymogenes CQ18 and the metabolite L-pyroglutamate effectively controlled flue-cured tobacco powdery mildew. L. enzymogenes CQ18 grows rapidly and is resilient to adversity. L-Pyroglutamate has no toxicity to humans and is easy to synthesize at a low cost. Both show potential use in controlling plant powdery mildews.Graphical

Bacillus amyloliquefaciens YN201732 Produces Lipopeptides With Promising Biocontrol Activity Against Fungal Pathogen Erysiphe cichoracearum.

Bacillus amyloliquefaciens YN201732 is an endophytic bacteria with high biocontrol efficiency and broad-spectrum antimicrobial activities. In order to clarify the main active ingredients and their antifungal mechanisms against powdery mildew of tobacco, this study is focused on lipopeptide obtained through acid precipitation and organic solvent extraction. HPLC and LCMS-IT-TOF were used to separate and identify antimicrobial lipopeptides. Findings revealed that bacillomycin D plays an important role against surrogate fungal pathogen Fusarium solani. Synthetic pathways of sfp, bacillomycin D, and fengycin were separately disrupted. The sfp gene knockout mutant B. amyloliquefaciens YN201732M1 only showed minor antagonistic activity against F. solani. While Erysiphe cichoracearum spore germination was inhibited and pot experiments displayed a significant decrease in tobacco powdery mildew. The spore inhibition rate of YN201732M1 was only 30.29%, and the pot experiment control effect was less than 37.39%, which was significantly lower than that of the wild type. The inhibitory effect of mutant YN201732M2 (deficient in the production of bacillomycin D) and mutant YN201732M3 (deficient in the production of fengycin) on the spore germination of E. cichoracearum were 50.22% and 53.06%, respectively, suggesting that both fengycin and bacillomycin D had potential effects on spore germination of powdery mildew. Interestingly, in a greenhouse assay, both B. amyloliquefaciens YN201732M2 and YN201732M3 mutants displayed less of a control effect on tobacco powdery mildew than wild type. The results from in vitro, spore germination, and greenhouse-pot studies demonstrated that antimicrobial lipopeptides especially bacillomycin D and fengycin may contribute to the prevention and control of tobacco powdery mildew. In addition, gene mutation related to lipopeptide synthesis can also affect the biofilm formation of strains.

Biocontrol potential of the endophytic Bacillus amyloliquefaciens YN201732 against tobacco powdery mildew and its growth promotion

The effectiveness of Bacillus amyloliquefaciens YN201732, a beneficial endophyte isolated from tobacco seeds was explored for control of tobacco powdery mildew and the systemic resistance mechanisms involved. The results of the study showed that endophyte YN201732 has antagonistic effects on 12pathogenic fungi. The fermentation broth can inhibit the spore germination and has good control effect on tobacco powdery mildew in the greenhouse. The control effect of the protective test and the therapeutic test at 14 days reached up to 71.09% and 86.11%, respectively. Moreover, endophyte YN201732 can also promote seed germination and seedling plant growth, and improve plant photosynthesis. Compare with water treatment, the root length, plant height, maximum leaf length, leaf width, fresh weight, dry weight, chlorophyll and net photosynthetic rate of tobacco seedlings increased by 32.27%, 86.67%, 154.92%, 107.25%, 664.71%, 207.69%, 150.33%, and 240%, respectively. Additionally, the expression of NPR1 in tobacco leaves was stable when inoculated with YN201732 at different time points, while the PDF1.2 gene was up-regulated rapidly, suggesting that jasmonic acid/ethylene signaling pathway of plant might be induced, which enhance plant resistance to powdery mildew. The chit and PPO genes were highly expressed while the PAL and POD defense enzyme genes were down-regulated, the expression of chit was up-regulated 30–70 times after induction treatment. In addition, we revealed that polyphenol oxidase and chitinases are closely related to tobacco induced resistance to powdery mildew. Thus, our study showed that B. amyloliquefaciens YN201732 has high potential to be used as biocontrol agent and plant-growth promoting bacterium in tobacco agriculture.

Transcriptome analysis reveals pathways facilitating the growth of tobacco powdery mildew in Arabidopsis

Powdery mildew (PM) fungi are biotrophic pathogens that rely on living hosts to survive and thrive. However, their colonization is restricted by host defenses at both the penetration and post-penetration stages. The tobacco PM strain Golovinomyces cichoracearum (Gc) SICAU1 has overcome penetration resistance of Arabidopsis but its growth is arrested by post-penetration resistance. While Gc SICAU1 only poorly grows in Arabidopsis Col-0 wild-type plants, it can sustainably grow for more than 20 days on the same infected leaves of the double mutant pad4–1 sid2–1 that is defective in both the synthesis and signaling of salicylic acid (SA). To understand the underlying molecular mechanisms, we conducted a comparative transcriptome analysis between Col-0 and pad4–1 sid2–1 in response to Gc SICAU1. We found that 4811 genes were differentially expressed more than four-fold between any two of the measured seven time points (0, 1, 3, 6, 8, 10 and 12 days post-inoculation). Gene expression pattern analysis suggests that differential expression of 348 genes and 190 genes may explain resistance in Col-0 and susceptibility in pad4–1 sid2–1, respectively. Gene Ontology (GO) analysis suggests that Gc SICAU1 might be arrested in Col-0 by both pattern-triggered immunity and SA-dependent defense. By contrast, its sustained growth in pad4–1 sid2–1 may be attributable to the activation of a detoxification pathway that is normally repressed by the SA-signaling pathway. Taken together, our results suggest that multiple distinct, yet interconnected pathways control the growth of tobacco powdery mildew in Arabidopsis.

EDS1-mediated basal defense and SA-signaling contribute to post-invasion resistance against tobacco powdery mildew in Arabidopsis

Arabidopsis-powdery mildew is a unique pathosystem in dissecting the molecular mechanism of plant-biotrophic fungus interactions. To increase the diversity of powdery mildew pathogens, we identified a tobacco powdery mildew, designated Gc SICAU1, which belonged to Golovinomyces cichoracearum (Gc) based on the internal transcribed spacer (ITS) rDNA fragment sequences. Inoculation test in laboratory conditions revealed that Gc SICAU1 could colonize on wild type Arabidopsis Col-0 leaves, but failed to grow as abundantly on tobacco leaves, indicating that Gc SICAU1 has completely overcome the pre-invasion resistance and its colonization is hindered by the post-invasion resistance in Arabidopsis. The growth of Gc SICAU1 in the fls2, efr-1 and pen1-1 mutants displayed no obvious difference from that of wild type plants. In contrast, salicylic acid (SA)-signaling deficient mutants such as pad4-1 and eds5-1, but not ethylene-signaling mutants, exhibited increased susceptibility to Gc SICAU1. Moreover, eds1-2 and pad4-1/sid2-1 were fully susceptible to Gc SICAU1 and the disease symptom was comparable to Gc SICAU1 on tobacco leaves. These data indicate that EDS1-mediated basal defense and SA-signaling play critical roles for post-invasion resistance against this pathogen. Taken together, our data demonstrated that Gc SICAU1 is a pathogen weakly virulent to Arabidopsis and can be used as a tool to finely dissect post-invasion resistance mechanisms.

Role of Reactive Oxygen Species (ROS) in Non-host Resistance Against Phytopathogens

The majority of the plants in the natural conditions are resistant to most of the incompatible pathogens (viral, fungal and bacterial infections). This phenomenon is called ״non-host resistance״. This type of resistance is very important however, not enough research was conducted to explain the mechanism of this type of resistance. When the authors inoculated several non-host plants with incompatible pathogens, levels of hydrogen peroxide (H2O2) and superoxide (O2.-) were increased and elevated early after inoculation. This phenomenon was found in tomato, datura, tobacco, cucumber, squash and Chenopodium which were inoculated with tobacco powdery mildew, Papaya ringspot virus (PRSV), cucumber powdery mildew, tobacco mosaic virus (TMV), tomato powdery mildew and PRSV, respectively. Interestingly that, when some hosts from those mentioned above were inoculated, each with its compatible pathogen, no accumulation of H2O2 and O2.- was occurred. The authors concluded that reactive oxygen species (ROS) mainly H2O2 and O2.- could have a key role in inhibiting or killing the pathogens early in the non-host plants. The authors recommend giving more attention to the application of H2O2 and O2.- either with direct application or with applying compounds which induce or produce ROS against phytopathogens.

Resistance to Powdery Mildew (Erysiphe cichoracearum DC.) in Oriental and Semi-Oriental Tobacco Germplasm under Field Conditions

Powdery mildew (Erysiphe cichoracearum DC.) is an important disease of tobacco (Nicotiana tabacum L.) in many countries. Losses in yield and quality have been reported to be around 30% to 80% in severe cases. Northwestern Iran is a hotspot for tobacco powdery mildew. To identify resistant genotypes, tobacco germplasm, including recombinant inbred lines (RILs) derived from the cross Basma S.31 × Dubec566, some Iranian landraces, and cultivars from different countries were evaluated under field conditions using a simple lattice design (10×10) with two replications. Each plot comprised 3 rows, 5 meters long. Spacing between rows was 65 cm and between plants within rows 20 cm. Susceptible line (Basma S.31) was planted throughout the experiment to guarantee homogeneous inoculation. The most resistant genotypes were 12 RILs (PD205, PD209, PD379, PD324, PD328, PD329, PD336, PD345, PD364, PD365, PD371, and PD381), three Iranian landraces (SPT413, Jahrom15, and Jahrom12), and cultivars (Basma104-1 and Pobeda1). Three cultivars (Kuklen6, Nevrokop261, and L17), two mutants (Mutant 2 and Mutant 3), and Matianus were included in the resistant group. The more resistant RILs (PD205 and PD209) were evaluated across three years and showed consistent behavior across years. Most genotypes studied were susceptible or highly susceptible to the disease. This information should be helpful in tobacco breeding programs.

Effect of allopurinol treatment on biotrophic growth of some powdery mildew fungi in their specific hosts

When supplied via roots to tobacco host plants, aqueous solutions of allopurinol, a specific inhibitor of xanthine oxidase and dehydrogenase, markedly reduced or completely prevented the visible development of tobacco powdery mildew fungus Erysiphe cichoracearum(E. orontii) at concentrations as low as 25–50μM administered for periods as short as 2 days. The duration of this allopurinol-dependent protection from the pathogen was of at least 20 days after the last administration. If supplied as foliar sprays at concentrations up to 1600μM, allopurinol was not absorbed by the leaf tissues and did not affect the fungal infection. Histological observations revealed that on tobacco plants treated with allopurinol via roots, a sharp reduction inE. cichoracearum mycelial development started only 48h after inoculation (after formation of the first haustoria) and was accompanied by a strong delay and reduction of sporulation. The proportion of haustoria partially or entirely embedded in thick translucent sheaths (possibly callosic in nature) increased significantly in allopurinol irrigated plants reaching about 41% of the total haustoria (compared to 9% in controls) 7 days post-inoculation. No effects of allopurinol supply was observed on Blumeria graminis and Erysiphe trifolii development on wheat and clover plants, respectively. The present results are consistent with the view that allopurinol affects pathogen growth and sporulation by inhibition of xanthine dehydrogenase in the plant, the pathogen or both organisms.

Expression of a defence-related intercellular barley peroxidase in transgenic tobacco

Tobacco plants ( Nicotiana benthamiana L.) have been transformed with a T-DNA vector construct carrying the cDNA pBH6-301, encoding the major pathogen induced leaf peroxidase (Prx8) of barley, under control of an enhanced CaMV 35S promoter. Progeny from three independent transformants were analyzed genetically, phenotypically and biochemically. The T-DNA was steadily inherited through three generations. The barley peroxidase is expressed and sorted to the intercellular space in the transgenic tobacco plants. The peroxidase can be extracted from the intercellular space in two molecular forms from both barley and transgenic tobacco. The tobacco expressed forms are indistinguishable from the barley expressed forms as determined by analytical isoelectric focusing (pI 8.5) and Western-blotting. Staining for N-glycosylation showed that one form only was glycosylated. The N-terminus of purified Prx8 from transgenic tobacco was blocked by pyroglutamate, after the removal of which, N-terminal sequencing verified the transit signal-peptide cleavage site deduced from the cDNA sequence. Phenotype comparisons show that the constitutive expression of Prx8 lead to growth retardation. However, an infection assay with the tobacco powdery mildew pathogen Erysiphe cichoracearum did not indicate that the transgenic plants had achieved enhanced resistance.

Induction of Systemic Resistance to Tobacco Powdery Mildew by Tobacco Mosaic Virus, Tobacco Necrosis Virus or Ethephon

AbstractLocal infections of either TMV or TNV in tobacco plants cv. Havana 425 (hypersensitive to TMV) proved effective in inducing systemic resistance to subsequent inoculation with the powdery mildew fungus Erysiphe cichoracearum DC. The proportion of leaf surface invaded by this pathogen and the amount of conidia it produced were both significantly lower in virus inoculated plants than in non‐inoculated controls. However, the decrease in sporulation rate was less regularly observed than the reduction in leaf area infected. TMV was more effective than TNV in protecting tobacco plants from powdery mildew. E. cichoracearum is thus added to the list of challenge pathogens to which TMV or TNV are known to induce resistance in the host plants.Necrotic lesions caused to the leaves by local treatment with Ethephon (an ethylene‐releasing compound) also conferred to tobacco some degree of systemic resistance to the same fungal pathogen, more frequently visible as a reduction of leaf area invaded. The protection due to the Ethephon lesions was in present experiments less marked than that of TMV. No effects against subsequent powdery mildew infection were obtained when point freeze necrotic lesions were provoked on the plants.

A preformed chemical inhibitor of tobacco powdery mildew on leaves of Nicotiana glutinosa

A diterpene identified as 13(s)-hydroxylabda-8(20),14-dien-2-one (2-ketoepimanool) (C20H32O2) was isolated from epicuticular leaf “waxes” of Nicotiana glutinosa, a species immune to powdery mildew of tobacco. When externally applied to leaf surfaces of susceptible tobacco plants this diterpene strongly suppressed the emergence of conidial germ tubes 2 and 3 (but not of germ tube 1) and hence inhibited mildew development (ED90 = 1 μg cm−2). The compound was not detected in N. debneyii or N. tomentosiformis, two other species immune to mildew, nor in three immune cultivars of N. tabacum.