Chemical defenses of crucifers: elicitation and metabolism of phytoalexins and indole-3-acetonitrile in brown mustard and turnip

The metabolism of the cruciferous phytoalexins brassinin and cyclobrassinin, and the related compounds indole-3-carboxaldehyde, glucobrassicin, and indole-3-acetaldoxime was investigated in various plant tissues of Brassica juncea and B. rapa. Metabolic studies with brassinin showed that stems of B. juncea metabolized radiolabeled brassinin to indole-3-acetic acid, via indole-3-carboxaldehyde, a detoxification pathway similar to that followed by the "blackleg" fungus (Phoma lingam/Leptosphaeria maculans). In addition, it was established that tetradeuterated brassinin was incorporated into the phytoalexin brassilexin in B. juncea and B. rapa. On the other hand, the tetradeuterated indole glucosinolate glucobrassicin was not incorporated into brassinin, although the chemical structures of brassinins and indole glucosinolates suggest an interconnected biogenesis. Importantly, tetradeuterated indole-3-acetaldoxime was an efficient precursor of phytoalexins brassinin, brassilexin, and spirobrassinin. Elicitation experiments in tissues of Brassica juncea and B. rapa showed that indole-3-acetonitrile was an inducible metabolite produced in leaves and stems of B. juncea but not in B. rapa. Indole-3-acetonitrile displayed antifungal activity similar to that of brassilexin, was metabolized by the blackleg fungus at slower rates than brassinin, cyclobrassinin, or brassilexin, and appeared to be involved in defense responses of B. juncea.     

Phytoalexins from Thlaspi arvense, a wild crucifer resistant to virulent Leptosphaeria maculans: structures, syntheses and antifungal activity

Phytoalexins are inducible chemical defenses produced by plants in response to diverse forms of stress, including microbial attack. Our search for phytoalexins from cruciferous plants resistant to economically important fungal diseases led us to examine stinkweed or pennycress (Thlaspi arvense), a potential source of disease resistance to blackleg. We have investigated phytoalexin production in leaves of T. arvense under abiotic (copper chloride) and biotic elicitation by Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], and report here two phytoalexins, wasalexin A and arvelexin (4-methoxyindolyl-3-acetonitrile), their syntheses and antifungal activity against isolates of P. lingam/L. maculans, as well as the isolation of isovitexin, a constitutive glycosyl flavonoid of stinkweed, having antioxidant properties but devoid of antifungal activity.     

Resistance to Alternaria brassicicola in transgenic broccoli expressing a Trichoderma harzianum endochitinase gene

Transgenic broccoli plants expressing a Trichoderma harzianum endochitinase gene were obtained by Agrobacterium tumefaciens-mediated transformation. PCR and Southern blot analysis confirmed the presence of the gene in plants initially selected via resistance to kanamycin. Primary transformants (T₀) and selfed progeny (T₁) were examined for expression of the endochitinase gene using a fluorometric assay and for their resistance to the fungal pathogens Alternaria brassicicola and Sclerotinia sclerotiorum. All transgenic plants with elevated endochitinase activity had the expected 42 kDa endochitinase band in western blot analysis, whereas no such band was detected in the non-transgenic control. Leaves of most mature T₀ plants had 14–37 times higher endochitinase activity than controls; mature T₁ plants had higher endochitinase activity (100–200 times that in controls), in part because of lower control values. T₀ plantlets in vitro or young plants in soil had higher absolute and relative endochitinase activity. When detached leaves of T₀ plants were inoculated with A. brassicicola, lesion size showed a significant negative correlation with endochitinase levels. After inoculation of two-month old T₀ plants with A. brassicicola, all 15 transgenic lines tested showed significantly less severe disease symptoms than controls. In contrast, lesion size on petioles of T₀ and T₁ plants inoculated with S. sclerotiorum was not statistically different from controls.     

Evaluation of endophytic actinobacteria as antagonists of Gaeumannomyces graminis var. tritici in wheat

Endophytic actinobacteria isolated from healthy cereal plants were assessed for their ability to control fungal root pathogens of cereal crops both in vitro and in planta. Thirty eight strains belonging to the genera Streptomyces, Microbispora, Micromonospora, and Nocardioidies were assayed for their ability to produce antifungal compounds in vitro against Gaeumannomyces graminis var. tritici (Ggt), the causal agent of take-all disease in wheat, Rhizoctonia solani and Pythium spp. Spores of these strains were applied as coatings to wheat seed, with five replicates (25 plants), and assayed for the control of take-all disease in planta in steamed soil. The biocontrol activity of the 17 most active actinobacterial strains was tested further in a field soil naturally infested with take-all and Rhizoctonia. Sixty-four percent of this group of microorganisms exhibited antifungal activity in vitro, which is not unexpected as actinobacteria are recognized as prolific producers of bioactive secondary metabolites. Seventeen of the actinobacteria displayed statistically significant activity in planta against Ggt in the steamed soil bioassay. The active endophytes included a number of Streptomyces, as well as Microbispora and Nocardioides spp. and were also able to control the development of disease symptoms in treated plants exposed to Ggt and Rhizoctonia in the field soil. The results of this study indicate that endophytic actinobacteria may provide an advantage as biological control agents for use in the field, where others have failed, due to their ability to colonize the internal tissues of the host plant.     

Metabolism of the phytoalexins camalexins,their bioisosteres and analogues in the plant pathogenic fungus Alternaria brassicicola

The metabolism of the phytoalexins camalexin (1), 1-methylcamalexin (10) and 6-methoxycamalexin (11) by Alternaria brassicicola and their antifungal activity is reported. This work establishes that camalexins are slowly biotransformed (ca. six days) to the corresponding indole-3-thiocarboxamides, which are further transformed to the indole-3-carboxylic acids. These metabolites are substantially less inhibitory to A. brassicicola than the parent camalexins, indicating that these enzyme-mediated transformations are detoxifications. In addition, analyses of the metabolism of synthetic isomers and bioisosteres of camalexin (1) indicate that isomers of camalexin in the thiazole ring are not metabolized. Based on these results, the potential intermediates that lead to formation of indole-3-thiocarboxamides are proposed.     

<Emphasis Type="Bold">Characterization of nitrile hydratation catalysed by </Emphasis><Emphasis Type="BoldItalic">Nocardia</Emphasis><Emphasis Type="Bold"> sp. 108</Emphasis>

Abstract Nocardia sp. 108 exhibited strong acrylonitrile-hydrating activity and its nitrile hydratase was Co²⁺-dependent. Nocardia sp. 108 was active within a broad pH range from 6.0 to 10.0 at 30°C and thermostable at temperatures below 35°C, but became unstable at temperatures above 45°C. Furthermore, it was found that Nocardia sp. 108 can hydrate indole-3-acetonitrile, p-chlorobenzonitrile, p-hydroxybenzylcyanide, 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, 3-cyanopyridine, o-chlorobenzonitrile to the corresponding amides and hence displayed a broad substrate specificity. The temperature and pH optima for these hydrations were 28°C and pH 7.0–7.5, respectively. At the observed concentrations, acrylonitrile was completely converted within 5 min, while 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, indole-3-acetonitrile, p-chlorobenzonitrile were approximately 21.71, 8.98, 34.44, 93.10% hydrated. p-Chlorobenzonitrile appeared to be the preferred aromatic nitrile for Nocardia sp. 108.     

一株高效拮抗向日葵菌核菌的细菌菌株YC16的筛选及其作用效果研究

[目的]筛选高效拮抗向日葵菌核菌的细菌菌株,为开发防治菌核菌病害、提高向日葵产量的生物菌剂提供菌种资源。[方法]以羧甲基纤维素钠(CMC)、小麦秸秆纤维素为唯一碳源的无机盐培养基,分离高效降解纤维素的细菌菌株;采用纤维素降解菌与菌核菌的平板对峙方法,进一步筛选拮抗菌核菌的菌株;利用16S rDNA序列鉴定菌株、PDYA平板对峙实验检验上述所选拮抗菌株的抑菌谱;采用离体向日葵新鲜叶片、草炭土基质盆栽实验,观察拮抗菌菌株抑制菌核菌生长的能力;温室盆栽和田间试验条件下,研究其防治向日葵菌核菌病害、促进生长和提高产量的效果。[结果]筛选了一株高效抑制菌核菌的细菌YC16,经过16S rDNA序列分析,鉴定为解淀粉芽孢杆菌。YC16菌株能够抑制8种病原真菌生长,包括齐整小核菌、腐皮镰孢菌、尖孢镰刀菌、稻梨孢、辣椒疫霉、镰刀菌、尖镰孢黄瓜专化型和向日葵菌核菌;抑制菌核菌感染叶片,抑制率达到了80.42%;抑制盆栽基质中菌核菌的菌丝生长,基质表面菌丝密度比对照减少了50%以上。盆栽接种YC16的向日葵生物量比对照提高54.9%,田间向日葵接种YC16菌剂对菌核菌引发的盘腐病防治效果达39%-100%,产量提高24.4%-30.2%。[结论]YC16生物菌剂施用于土壤,能够有效防治向日葵的茎腐病和盘腐病,展现了防治向日葵菌核病和提高产量的双重效果,是一株具有良好应用前景的高效菌种资源。     

Microbial conversion and in vitro and in vivo antifungal assessment of bioconverted docosahexaenoic acid (bDHA) used against agricultural plant pathogenic fungi

Microbial modification of polyunsaturated fatty acids can often lead to special changes in their structure and in biological potential. Therefore, the aim of this study was to develop potential antifungal agents through the microbial conversion of docosahexaenoic acid (DHA). Bioconverted oil extract of docosahexaenoic acid (bDHA), obtained from the microbial conversion of docosahexaenoic acid (DHA) by Pseudomonas aeruginosa PR3, was assessed for its in vitro and in vivo antifungal potential. Mycelial growth inhibition of test plant pathogens, such as Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum, was measured in vitro. bDHA (5 μl disc⁻¹) inhibited 55.30–65.90% fungal mycelium radial growth of all the tested plant pathogens. Minimum inhibitory concentrations (MICs) of bDHA against the tested plant pathogens were found in the range of 125–500 μg ml⁻¹. Also, bDHA had a strong detrimental effect on spore germination for all the tested plant pathogens. Further, three plant pathogenic fungi, namely C. capsici, F. oxysporum and P. capsici, were subjected to an in vivo antifungal screening. bDHA at higher concentrations revealed a promising antifungal effect in vivo as compared to the positive control oligochitosan. Furthermore, elaborative study of GC-MS analysis was conducted on bioconverted oil extract of DHA to identify the transformation products present in bDHA. The results of this study indicate that the oil extract of bDHA has potential value of industrial significance to control plant pathogenic fungi.     

Phytoalexins and polar metabolites from the oilseeds canola and rapeseed: differential metabolic responses to the biotroph Albugo candida and to abiotic stress

The metabolites produced in leaves of the oilseeds canola and rapeseed (Brassica rapa L.) inoculated with either different races of the biotroph Albugo candida or sprayed with CuCl(2) were determined. This investigation established consistent phytoalexin (spirobrassinin, cyclobrassinin, and rutalexin) and phytoanticipin (indolyl-3-acetonitrile, arvelexin, caulilexin C, and 4-methoxyglucobrassicin) production in canola and rapeseed in response to both biotic and abiotic elicitation. In addition, a wide number of polar metabolites were isolated from infected leaves, including six new phenylpropanoids and two new flavonoids. The extractable chemical components of zoosporangia of A. candida and the anti-oomycete activity of phytoalexins were determined as well. Overall, the results suggest that during the initial stage of the interaction, leaves of B. rapa have a similar response to virulent and avirulent races of A. candida, with respect to the accumulation of chemical defenses. After this stage, despite the higher phytoalexin concentration, the "compatible" races could overcome the plant defense system for further infection, but growth of the "incompatible" races was inhibited. Since results of bioassays showed that cyclobrassinin and brassilexin were more inhibitory to A. candida than rutalexin, the apparent redirection of the phytoalexin pathway towards rutalexin, avoiding cyclobrassinin and brassilexin accumulation might be caused by the pathogen. Alternatively, A. candida might be able to detoxify both cyclobrassinin and brassilexin, similar to necrotrophic plant pathogens. Overall, the correlation between phytoalexin production in infected or stressed leaves and the outcome of the plant-pathogen interaction suggested that A. candida was able to elude the plant defense mechanisms by, for example, redirecting the phytoalexin biosynthetic pathway.     

Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties

Myrosinases (EC 3.2.1.147) are β-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1–TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS–PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation–inhibition responses towards ascorbic acid with maximal activity around 0.7–1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-β-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.     

解淀粉芽胞杆菌HZ-12中ysnE参与吲哚-3-乙酸合成的代谢途径研究

【目的】吲哚-3-乙酸是调控植物生长发育和生理活动的重要激素,吲哚-3-乙酸N-乙酰转移酶YsnE在吲哚-3-乙酸合成中发挥重要作用,本研究拟解析解淀粉芽胞杆菌中YsnE参与吲哚-3-乙酸合成的代谢途径。【方法】通过基因ysnE缺失和强化表达,分析ysnE对吲哚-3-乙酸合成影响,结合吲哚-3-乙酸合成中间物(吲哚丙酮酸、吲哚乙酰胺、色胺和吲哚乙腈)添加和体外酶转化实验,解析ysnE参与吲哚-3-乙酸合成的代谢途径。【结果】明确了YsnE在解淀粉芽胞杆菌HZ-12吲哚-3-乙酸合成中发挥重要作用。发现ysnE缺失菌株中的吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈利用显著降低,揭示了YsnE主要发挥吲哚丙酮酸脱羧酶YclB和吲哚乙酰胺水解酶/腈水解酶/腈水合酶YhcX的功能,并通过参与吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径来影响吲哚-3-乙酸合成。【结论】初步揭示了YsnE通过影响吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径参与吲哚-3-乙酸合成的代谢机理,为吲哚-3-乙酸合成途径解析和代谢工程育种构建吲哚-3-乙酸高产菌株奠定了基础。     

Control of blast and sheath blight diseases of rice using antifungal metabolites produced by Streptomyces sp. PM5

Two antifungal aliphatic compounds, SPM5C-1 and SPM5C-2 with a lactone and ketone carbonyl unit, respectively obtained from Streptomyces sp. PM5 were evaluated under in vitro and in vivo conditions against major rice pathogens, Pyricularia oryzae and Rhizoctonia solani. These compounds were dissolved in distilled water/medium to get the required concentrations. The well diffusion bioassay indicated that the of SPM5C-1 remarkably inhibited the mycelial growth of P. oryzae and R. solani in comparison to SPM5C-2. Though SPM5C-2 showed low antifungal activity against P. oryzae, it was not active against R. solani. Further, SPM5C-1 completely inhibited the growth of P. oryzae and R. solani at concentrations of 25, 50, 75 and 100 μg/ml. Greenhouse experiments revealed that spraying of SPM5C-1 at 500 μg/ml on rice significantly decreased blast and sheath blight development by 76.1% and 82.3%, respectively, as compared to the control with a corresponding increase in rice grain yield.     

Phenotypic and biological properties of two antagonist <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains

Bacillus subtilis KB-1111 and KB-1122 were studied to illustrate their phenotypic and biological properties. Comparison of KB-1111 with KB-1122 in morphology was carried out by microscopy and agar plate assays. Biological assay of the test strains showed that they may possess different physiological pathways from those of reference strain ATCC6501. The assessment of antagonism against the indicator fungi showed that both test strains had broad antifungal characteristics against eight phytopathogenic fungi. Of those fungal species, Magnaporthe grisea P131, Sclerotinia sclerotiorum, and F. oxysporium exhibited high sensitivity to the test strains.     

Auxin Activity of Glucobrassicin

The indole-glucosinolate, glucobrassicin, which is found in large amounts in members of the genus Brassica, was subjected to tests in the standard Avena curvature bioassay. Both the tetramethylammonium salt of glucobrassicin and the non-crystallizted material induced curvature at a concentration of 5 × l0^?4M. The curvature from the salt was, however, only half of that induced by the acid form. Bioassays of chromatograms of fresh methanolic extracts of savoy cabbage indicated the presence of indole-3-acetic acid as well as indole-3-acetonitrile in small amounts along with large amounts of glucobrassicin. Segments of green hypocotyl tissue of savoy cabbage respond with limited growth to high concentrations of glucobrassicin. Segments of etiolated hypocotyls or green epicotyls give no growth response to glucobrassicin. Pea stem segments which do not respond to indoleacetonitrile responded with similar growth to glucobrassicin and indoleacetic acid. Nitrilase activity was found in savoy hypocotyl and epicotyl tissue by chromatography of incubation media. The growth regulation in savoy cabbage in conjunction with data on the distribution of glucobrassicin in the plant, leads to the conclusion that glucobrassicin is a special, inactive storage and transport form of the active auxin in cabbage.     

AtWRKY63基因过表达拟南芥对核盘菌抗性的研究

为研究草酸在核盘菌致病过程中可能的作用,以模式植物拟南芥为材料,采用30mmol/L草酸喷施3周龄拟南芥,发现草酸显著诱导拟南芥AtWRKY63的表达。通过构建AtWRKY63过表达载体转化拟南芥,获得过表达AtWRKY63的纯系转基因植株,再用核盘菌活体接种拟南芥,结果表明过表达AtWRKY63植株对核盘菌的抗性显著增强。组织化学染色结果表明,AtWRKY63是通过诱导植物的氧爆发,抑制核盘菌菌丝的生长来抵御核盘菌的侵染;qRT-PCR对拟南芥转录水平分析表明,AtWRKY63可能激活了过表达植株的水杨酸与茉莉酸依赖的抗病信号途径,从而增强对核盘菌的抗性。     

Physiology and costs of resistance to herbivory and disease in Brassica

We used artificial selection experiments to study genetic allocation costs and physiological mechanisms of resistance to herbivory and fungal disease. Genetic costs to resistance were present in some instances and absent in others. Genetic resistance to the fungal pathogen, Leptosphaeria maculans was cost-free, while resistance to Peronospora parasitica showed a negative genetic correlation between disease resistance and growth rate. Leptosphaeria resistant genotypes had 13% higher chitinase activity. Genetic increases in myrosinase activity were correlated with increased resistance to flea beetles (Phyllotreta cruciferae), but resulted in lower plant fecundity, presumably due to production costs of myrosinase. Genetic costs of resistance may maintain genetic variation in natural plant populations. These studies demonstrate the predictive and explanatory power of a functional approach to plant-herbivore and plant-pathogen interactions.     

Diverse mechanisms adopted by fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> PGC2 during the inhibition of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Rhizoctonia solani and Phytophthora capsici are two of the most destructive phytopathogens occurring worldwide and are only partly being managed by traditional control strategies. Fluorescent Pseudomonas isolates PGC1 and PGC2 were checked for the antifungal potential against R. solani and P. capsici. Both the isolates were screened for the ability to produce a range of antifungal compounds. The results of this study indicated the role of chitinase and β-1,3-glucanase in the inhibition of R. solani, however, antifungal metabolites of a non-enzymatic nature were responsible for inhibition of P. capsici. The study confirmed that multiple and diverse mechanisms are adopted by the same antagonist to suppress different phytopathogens, as evidenced in case of R. solani and P. capsici.     

A pathogenesis related protein, AhPR10 from peanut: an insight of its mode of antifungal activity

A pathogenesis related protein (AhPR10) is identified from a clone of 6-day old Arachis hypogaea L. (peanut) cDNA library. The clone expressed as a ∼20 kDa protein in E. coli. Nucleotide sequence derived amino acid sequence of the coding region shows its homology with PR10 proteins having Betv1 domain and P loop motif. Recombinant AhPR10 has ribonuclease activity, and antifungal activity against the peanut pathogens Fusarium oxysporum and Rhizoctonia solani. Mutant protein AhPR10-K54N where lys54 is mutated to asn54 loses its ribonuclease and antifungal activities. FITC labeled AhPR10 and AhPR10-K54N are internalized by hyphae of F. oxysporum and R. solani but the later protein does not inhibit the fungal growth. This suggests that the ribonuclease function of AhPR10 is essential for its antifungal activity. Energy and temperature dependent internalization of AhPR10 into sensitive fungal hyphae indicate that internalization of the protein occurs through active uptake.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .The nucleotide sequence of AhPR10 reported in this paper is submitted to NCBI Nucleotide Sequence Database under the Accession number AY726607.     

Metabolic processes and carbon nutrient exchanges between host and pathogen sustain the disease development during sunflower infection by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Interactions between the necrotrophic fungus Sclerotinia sclerotiorum and one of its hosts, Helianthus annuus L., were analyzed during fungal colonization of plant tissues. Metabolomic analysis, based on ¹³C- and ³¹P-NMR spectroscopy, was used to draw up the profiles of soluble metabolites of the two partners before interaction, and to trace the fate of metabolites specific of each partner during colonization. In sunflower cotyledons, the main soluble carbohydrates were glucose, fructose, sucrose and glutamate. In S. sclerotiorum extracts, glucose, trehalose and mannitol were the predominant soluble carbon stores. During infection, a decline in sugars and amino acids was observed in the plant and fungus total content. Sucrose and fructose, initially present almost exclusively in plant, were reduced by 85%. We used a biochemical approach to correlate the disappearance of sucrose with the expression and the activity of fungal invertase. The expression of two hexose transporters, Sshxt1 and Sshxt2, was enhanced during infection. A database search for hexose transporters homologues in the S. sclerotiorum genome revealed a multigenic sugar transport system. Furthermore, the composition of the pool of reserve sugars and polyols during infection was investigated. Whereas mannitol was produced in vitro and accumulated in planta, glycerol was exclusively produced in infected tissues and increased during colonization. The hypothesis that the induction of glycerol synthesis in S. sclerotiorum exerts a positive effect on osmotic protection of fungal cells and favors fungal growth in plant tissues is discussed. Taken together, our data revealed the importance of carbon–nutrient exchanges during the necrotrophic pathogenesis of S. sclerotiorum.     

Zerumbone: a potential fungitoxic agent isolated from Zingiber cassumunar Roxb.

The rhizomes of Zingiber cassumunar exhibited strong fungitoxic action against Rhizoctonia solani, the damping-off pathogen. On chemical and spectral investigations, the antifungal compound was found to be zerumbone — a sesquiterpene. Its minimum effective dose against R. solani was 1000 ppm, much lower than some commercial fungicides. Zerumbone had fungistatic activity, a narrow fungitoxic spectrum and was not phytotoxic. Moreover, when used as a seed treatment, zerumbone could control damping-off disease of Phaseolus aureus caused by Rhizoctonia solani by 85.7%.