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.     

New sesquiterpenic phytotoxins establish unprecedented relationship between different groups of blackleg fungal isolates

A comprehensive search for sesquiterpenic metabolites produced by isolates of the blackleg fungus [Leptosphaeria maculans (Desm.)] Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.] revealed that an isolate pathogenic on both canola and brown mustard (IBCN 18) and two isolates pathogenic on brown mustard (Laird 2 and Mayfair 2) produced similar sesquiterpenes. The isolation, chemical structure elucidation, and phytotoxicity of these new sesquiterpenes with silphinene and selinene type skeletons is reported. This is the first time that an isolate virulent on canola and brown mustard is found to produce metabolites characteristic of both virulent (sirodesmins) and avirulent (phomalairdenones) L. maculans/P. lingam. In the context of grouping the various isolates of L. maculans/P. lingam, this work suggests an additional pathogenicity group comprising isolates that produce both sirodesmins and phomalairdenones and are virulent on both canola and brown mustard.     

Phomapyrones from blackleg causing phytopathogenic fungi: isolation, structure determination, biosyntheses and biological activity

The isolation and structure determination of phomapyrones D-G, three 2-pyrones and a coumarin, from a group of isolates of the fungal pathogen Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm, is reported. As well, phomenin B, infectopyrone, and polanrazines B and C were also obtained for the first time from these isolates. In addition, based on results of incorporations of 13C-labeled acetate and malonate, and deuterated methionine, a polyketide pathway is proposed for the biosyntheses of phomapyrones.     

Isolation, structure determination, and phytotoxicity of unusual dioxopiperazines from the phytopathogenic fungus Phoma lingam.

The isolation, chemical structure elucidation, and bioactivity of polanrazines B-F, five new dioxopiperazines produced by isolates of the blackleg fungus [Phoma lingam, perfect stage Leptosphaeria maculans (Desm.) Ces. et de Not.] originating from Poland, are reported. Polanrazines C and E showed moderate but selective toxicity, causing necrotic and chlorotic lesions (1-3 mm diameter) on brown mustard leaves.     

Detoxification of the phytoalexin brassinin by isolates of Leptosphaeria maculans pathogenic on brown mustard involves an inducible hydrolase

Brassinin is a phytoalexin produced by plants from the family Brassicaceae that displays antifungal activity against a number of pathogens of Brassica species, including Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.] and L. biglobosa. The interaction of a group of isolates of L. maculans virulent on brown mustard (Brassica juncea) with brassinin was investigated. The metabolic pathway for degradation of brassinin, the substrate selectivity of the putative detoxifying hydrolase, as well as the antifungal activity of metabolites and analogs of brassinin are reported. Brassinin hydrolase activity was detectable only in cell-free homogenates resulting from cultures induced with brassinin, N'-methylbrassinin, or camalexin. The phytoalexin camalexin was a substantially stronger inhibitor of these isolates than brassinin, causing complete growth inhibition at 0.5mM.     

HPLC analyses of cultures of Phoma spp.: differentiation among groups and species through secondary metabolite profiles

The metabolite profiles of 26 isolates of the blackleg fungus (Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm.), obtained from diverse parts of the world (part of the International Blackleg Crucifer Network collection), were studied utilizing specific culture conditions, HPLC analysis, and a set of chemical markers. This fungus is the causative agent of blackleg disease of brassica oilseeds; a virulent strain of the pathogen has caused significant rapeseed (Brassica napus L., and B. rapa L.) and canola (B. napus L., and B. rapa L.) losses in Canada, and is also considered a serious agricultural problem worldwide. Effective surveys of blackleg epidemics require simple and reliable analytical methodology to differentiate among the diverse groups of isolates. The chemical analysis of phytotoxins and related secondary metabolites is perhaps one of the most discriminating and the least ambiguous methods for differentiation of Phoma blackleg isolates. Following HPLC analyses, the 26 isolates could be placed in three main groups, irrespective of country of origin: isolates producing phomamide and sirodesmins, isolates producing indolyl dioxopiperazines, and isolates producing polyketides. Discussion of the implications of our findings and suggestions for species reclassification are provided.     

The phytopathogenic fungi Leptosphaeria maculans and Leptosphaeria biglobosa: chemotaxonomical characterization of isolates and metabolite production in different culture media

Previous molecular chemotaxonomic analyses of isolates of the plant pathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. (asexual stage Phoma lingam (Tode ex Fr.) Desm.) in a chemically defined medium suggested that this species complex was composed of at least three distinct groups. Subsequently, a group within L. maculans was classified as Leptosphaeria biglobosa, on the basis of morphologic characteristics and the lack of sexual crossing. To obtain clarification regarding the metabolite profiles of the various groups or species of blackleg fungi, the objectives of this work were (i) to determine the chemical structures of metabolites produced by Canadian V isolates and Polish-type isolates in potato dextrose broth (PDB) and (ii) to determine the chemotaxonomic relationship among French isolates of L. biglobosa and among Canadian W isolates and Thlaspi isolates of L. maculans. Here, we report for the first time that Canadian V isolates grown in PDB produced 2,4-dihydroxy-3,6-dimethylbenzaldehyde, a metabolite never reported from L. maculans, but none of the usual phytotoxins (sirodesmins). In addition, we report a new metabolite, 2-[2-(5-hydroxybenzofuranyl)]-3-(4-hydroxyphenyl)propanenitrile, from Polish-type isolates of L. maculans grown in PDB and the metabolite profiles of 16 Thlaspi isolates. The metabolite profiles of Thlaspi isolates indicate that these are part of two distinct groups, the Polish W group and the Canadian W group, i.e., L. biglobosa. Finally, we demonstrate that the metabolite profiles of the French isolates classified as L. biglobosa are similar to those of Canadian W isolates.     

Phomalairdenone: a new host-selective phytotoxin from a virulent type of the blackleg fungus Phoma lingam

The chemical structure and bioactivity of phomalairdenone (7), a new sesquiterpenic host-selective phytotoxin produced by an unusual virulent type isolate of the blackleg fungus [Phoma lingam, perfect stage Leptosphaeria maculans (Desm.) Ces. et de Not.] are reported.     

山茶灰斑病病原菌斑污拟盘多毛孢的研究

山茶灰斑病病原菌的正名为斑污拟盘多毛孢Pestalotiopsis maculans(Cda.)Nag Raj,山茶盘多毛孢Pestalotia guepini Desm.和山茶拟盘多毛孢Pestalotiopsis guepini(Desm.)Stey.均为其异名。本文根据作者对自1978年以来采集的大量标本和分离菌株的研究,记述了斑污拟盘多毛孢的形态学、培养性状、致病性及寄主范围、电泳图谱等特征,并附有讨论。     

Camalexin induces detoxification of the phytoalexin brassinin in the plant pathogen Leptosphaeria maculans

The impact of the phytoalexins camalexin and spirobrassinin on brassinin detoxification by Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], a pathogenic fungus prevalent on crucifers, was investigated. Brassinin is a plant metabolite of great significance due to its dual role both as an effective phytoalexin and as an early biosynthetic precursor of the majority of the phytoalexins produced by plants of the family Brassicaceae (Cruciferae). The rate of detoxification of brassinin in cultures of L. maculans increased substantially in the presence of camalexin, whereas spirobrassinin did not appear to have a detectable effect. In addition, the brassinin detoxifying activity of cell-free extracts obtained from cultures incubated with camalexin was substantially higher than that of control cell-free extracts or cultures incubated with spirobrassinin, and correlated positively with brassinin oxidase activity. The discovery of a potent synthetic modulator of brassinin oxidase activity, 3-phenylindole, and comparison with the commercial fungicide thiabendazole is also reported. The overall results indicate that brassinin oxidase production is induced by camalexin and 3-phenylindole but not by spirobrassinin or thiabendazole. Importantly, our work suggests that introduction of the camalexin pathway into plants that produce brassinin might make these plants more susceptible to L. maculans.     

Structure and biological activity of maculansin A, a phytotoxin from the phytopathogenic fungus Leptosphaeria maculans

During a search for elicitors and phytotoxins produced by virulent isolates of the phytopathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phomalingam (Tode ex Fr.) Desm.], the selective phytotoxin maculansin A was isolated and its structure determined by analysis of spectroscopic data and chemical degradation. Maculansin A, a unique derivative of mannitol containing the unusual chromophore 2-isocyano-3-methyl-2-butenoyl, was isolated from potato dextrose cultures of L. maculans virulent on canola (Brassica napus L. cv. Westar). Surprisingly, maculansin A was more toxic to resistant plants (B. juncea L. cv. Cutlass, brown mustard) than to susceptible plants (canola). Maculansin A, however, did not elicit the production of phytoalexins either in resistant or susceptible plants. In addition, other maculansin type structures and the metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde were isolated and the latter was found to be a strong inhibitor of root growth of both brown mustard and canola. Considering that L. maculans seems to be expanding its host range to infect brown mustard as well, maculansins could assist in chemotaxonomic studies to group the diverse isolates.     

Phomenoic acid and phomenolactone, antifungal substances from Phoma lingam (Tode) Desm.: kinetics of their biosynthesis, with an optimization of the isolation procedures.

Phomenoic acid and phomenolactone, previously isolated from the fungus Phoma lingam (Tode) Desm., have shown moderate antifungal and antibacterial properties in vitro. To rationalize the production of phomenoic acid, a kinetic study of its biosynthesis in the mycelium was performed. Phomenoic acid and phomenolactone appear in the mycelium after a prolonged incubation, a phenomenon which may be of particular interest for the production of these substances or in the study of the mechanism of their biosynthesis. The isolation procedure was optimized for phomenoic acid. Through a series of SiO2 column chromatographies, high-pressure liquid chromatography, and transformation of the mixture of phomenoic acid and phomenolactone into methyl phomenoate (BF3), the final yield of phomenoic acid reached 160 mg/liter of culture medium. An alternative method for the isolation of both phomenoic acid and phomenolactone is also reported in detail.     

Phomenoic acid and phomenolactone, antifungal substances from Phoma lingam (Tode) Desm.: kinetics of their biosynthesis, with an optimization of the isolation procedures

Phomenoic acid and phomenolactone, previously isolated from the fungus Phoma lingam (Tode) Desm., have shown moderate antifungal and antibacterial properties in vitro. To rationalize the production of phomenoic acid, a kinetic study of its biosynthesis in the mycelium was performed. Phomenoic acid and phomenolactone appear in the mycelium after a prolonged incubation, a phenomenon which may be of particular interest for the production of these substances or in the study of the mechanism of their biosynthesis. The isolation procedure was optimized for phomenoic acid. Through a series of SiO2 column chromatographies, high-pressure liquid chromatography, and transformation of the mixture of phomenoic acid and phomenolactone into methyl phomenoate (BF3), the final yield of phomenoic acid reached 160 mg/liter of culture medium. An alternative method for the isolation of both phomenoic acid and phomenolactone is also reported in detail.     

6-BA,NAA和Vc浓度配比对山葵试管苗增殖体系的影响

山葵为十字花科山嵛菜属多年生草本植物,具有强烈的香辛味,是一种高级蔬菜和药用植物。在山葵生产中存在的一个最大问题即健壮种苗的供给,通过山葵的茎尖培养建立组培快繁体系是解决该问题的有效手段。本文通过311-A最优回归设计研究6-BA,NAA和Vc浓度配比对山葵试管苗增殖体系的影响,对11种不同配方培养基中山葵苗的增殖系数进行调查,通过DPS3.01数据统计软件建立回归方程,并对回归方程进行主效因子及其互作效应分析。结果发现:当6-BA(X1)、NAA(X2)、Vc(X3)的浓度分别为1.2504mg/L、0mg/L、1.9668mg/L时,增殖系数可以达到6.8380,进一步优化了山葵试管苗增殖的体系。     

Phoma nigrificans (P. Karst.) Boerema et al., a New Species for Poland

Phoma nigrificans was isoiated from leaves of Thlaspi arvense. The cultural characteristics are described, and results of comparative pathogenicity tests including Phoma lingam isolates on B. napus var. oleifera and T. arvense are given. This is the first report of P. nigrificans from Poland.     

Inheritance of leaf rust and stem rust resistance in 'Roblin' wheat.

The Canadian common wheat (Triticum aestivum L.) cultivar 'Roblin' is resistant to both leaf rust (Puccinia recondita Rob. ex. Desm.) and stem rust (Puccinia graminis Pers. f. sp. tritici Eriks. and E. Henn.). To study the genetics of this resistance, 'Roblin' was crossed with 'Thatcher', a leaf rust susceptible cultivar, and RL6071, a stem rust susceptible line. A set of F6 random lines was developed from each cross. The random lines and the parents were grown in a field rust nursery artificially inoculated with a mixture of P. recondita and P. graminis isolates and scored for rust reaction. The same material was tested with specific races of leaf rust and stem rust. These data indicated that 'Roblin' has Lr1, Lr10, Lr13, and Lr34 for resistance to P. recondita and Sr5, Sr9b, Sr11, and possibly Sr7a and Sr12 for resistance to P. graminis. In a 'Thatcher' background, the presence of Lr34 contributes to improve stem rust resistance, which appears also to occur in 'Roblin'.     

Sporenkeimung von Phoma lingam (Tode ex. Fr.) Desm. und Resistenzprüfung bei Raps im Gewächshaus

Spore germination of Phoma lingam (Tode ex. Fr.) Desm. and methods to determine resistance of oil seed rape in the greenhouse It was the aim of this investigation to obtain more insight into the epidemiology of Phoma lingam (Tode ex. Fr.) Desm. (stat. gen. Leptosphaeria maculans (Desm.) Ces. et de Not.) and to improve the existing methods for resistance testing. In laboratory experiments the differing demands on temperature of both, the sexual as well as the non sexual phase were observed. Many ascospores developed germ tubes after eight hours at 4-8°C whilst pycnidiospores needed 24 hours at 16°C to have similar development. In greenhouse experiments young plants were infected by spraying or by placing a droplet of spore suspension onto cotyledons or leaves. Generally, ascospores were more virulent than pycnidiospores. The ascospores were obtained from old rape stalks which could be stored at -18°C without losing virulence. The most severe attack was observed after incorporating infested oat kernels (3 % w/ w) into soil, but the difference between cultivars vanished which was already low with the other methods, and which did not always correspond with results obtained in the field at stage 85, so that all these methods are not as suitable as those in the field. The distribution of pycnidiospores is also possible by adhering to the seed after threshing. The infection of the seedlings from this source was more pronounced in steamed than in unsteamed soil. The re-isolation of P. lingam increased as well from plants grown in steamed soil. Furthermore, pycnidiospores are distributed by wind during combining to neighbouring fields, already prepared at that time for rape sowing.     

Remarkable microfungi from Oaxaca (Mexico) of Acacia species

In the state of Oaxaca (Mexico, 10 km north-west of Puerto Escondido 15 degrees 55' N, 97 degrees 09' W) we were able to collect some microfungi living as parasites or saprophytes on Acacia species, some of them are causing attention for Oaxaca. Many belong to the Deuteromycotina (Hyphomycetes, Coelomycetes) and Ascomycotina. On A. hindsii: Calonectria pseudopeziza (Desm.) Sacc., Hypoxylon truncatum (Schwein. Fr.) J.H. Miller, Epicoccum nigrum Link., Zygosporium gibbum (Sacc., M. Roussau & E. Bommer) S.J. Hughes and on A. cornigera: Phyllosticta acaciicola P. Henn., Taeniolella alta (Ehrenb. ex Pers.) S.J. Hughes, Cephaliophora tropica Thaxt., Diplodia mutila (Fr. Fr.) Mont., Pleospora herbarum (Pers. Fr.) Rabenh., Gliocladium roseum Bainier, Ulocladium atrum Preuss., and different others. All species collected are listed in text.