Repellent activity of estrogenic compounds toward zoospores of the phytopathogenic fungus Aphanomyces cochlioides

Screening chemical compounds, we found that a xenoestrogen, bisphenol A, showed potent repellent activity against the zoospores of Aphanomyces cochlioides. Based on this finding, we tested a number of androgenic and estrogenic compounds (e.g. testosterone, progesterone, estradiols, diethylstilbestrol, estrone, estriol, pregnenolone, dienestrol etc.) on the motility behavior of A. cochlioides zoospores. Interestingly, most of the estrogenic compounds exhibited potent repellent activity (1 microg/ml or less by the "particle method") toward the motile zoospores of A. cochlioides. We derivatized some of the estrogens and discussed the relationship between the structure of active molecules and their repellent activity. Apparently, aromatization of the A ring with a free hydroxyl group at C-3 position of a steroidal structure is necessary for higher repellent activity. Interestingly, methylation of diethylstilbestrol (DES) yielded completely different activity i.e. both mono- and di-methyl ethers of DES showed attractant activity. Moreover, the attracted zoospores were encysted and then germinated in the presence of di-methyl ether of DES. The potential usefulness of this repellent test is discussed for the detection of estrogenic activity of naturally occurring compounds, and the possible role of phytoestrogens in host/parasite interactions. So far, this will be the first report of repellent activity of estrogenic compounds toward trivial fungal zoospores.     

Nicotinamide and structurally related compounds show halting activity against zoospores of the phytopathogenic fungus Aphanomyces cochlioides

In a survey of plant secondary metabolites regulating the behavior of phytopathogenic Aphanomyces cochlioides zoospores, we found that leaf extracts of Amaranthus gangeticus and cotyledon extracts of pea (Pisum sativum) remarkably halted the motility of zoospores. Bioassay-directed fractionation of A. gangeticus and pea constituents revealed that the halting activity was dependent on a single chemical factor (halting factor). The active principle was identified as nicotinamide (1) by comparing its biological activity and spectroscopic properties with those of the authentic compound. Nicotinamide (1) showed potent halting activity toward the zoospores of A. cochlioides and A. euteiches, but it exhibited very less activity against other Oomycetes, Pythium aphanidermatum and Phytophthora infestans zoospores. Interestingly, the zoospores halted by nicotinamide (1) encysted within 10-15 min and then the resulting cystospores regenerated zoospores instead of germination. Nicotinamide (1) and related compounds were subjected to the halting activity bioassay to elucidate the structure-activity relationships. These bioassays revealed that part structures of (A) the aromatic ring containing at least one nitrogen atom, (B) carbonyl-like group adjacent to the aromatic ring and (C) hydrogen atoms on the amide group are responsible for the strong activity. So far, this is the first report of halting activity of nicotinamide (1) against fungal zoospores.     

The third naturally occurring attractant toward zoospores of phytopathogenic Aphanomyces cochlioides from the Spinacia oleracea host plant

A bioassay-guided survey of spinach leaf constituents resulted in 5,4'-dihydroxy-3,3'-dimethoxy-6,7-methylenedioxyflavone being identified as the third naturally-occurring attractant in the host plant toward the zoospores of its pathogen, Aphanomyces cochlioides. The isolate showed attracting activity around Chromosorb W AW particles (60-80 mesh) coated with a 10(-5) M solution in a zoospore suspension. However, this activity was 1/100-1/1000 less than that of cochliophilin A, an attractant in the roots of spinach. Bioassays with the present isolate and related compounds revealed that 5,3',4'-trihydroxy-3-methoxy-6,7-methylenedioxyflavone did not possess attractant activity, but rather weak antagonistic activity toward the former two attractants from spinach.     

A photoaffinity probe designed for host-specific signal flavonoid receptors in phytopathogenic Peronosporomycete zoospores of Aphanomyces cochlioides

Aphanomyces cochlioides zoospores show chemotaxis to cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone, 1), a host derived attractant, and also respond to 5,7-dihydroxyflavone (2) known as an equivalent chemoattractant. To investigate the chemotactic receptors in the zoospores, we designed photoaffinity probes 4'-azido-5,7-dihydroxyflavone (3) and 4'-azido-7-O-biotinyl-5-hydroxyflavone (4) considering chemical structure of 2. Both 3 and 4 had zoospore attractant activity which was competitive with that of 1. When zoospores were treated with the biotinylated photoaffinity probe followed by UV irradiation and streptavidin-gold or peroxidase-conjugated streptavidin, probe-labeled proteins were detected on the cell membrane. This result indicated that the 1-specific-binding proteins, a candidate for hypothetical cochliophilin A receptor, were localized on the cell membrane of the zoospores. This is the first experimental evidence of flavonoid-binding proteins being present in zoospores, using chemically synthesized azidoflavone as photoaffinity-labeling reagent.     

Host-specific plant signal and G-protein activator, mastoparan, trigger differentiation of zoospores of the phytopathogenic oomycete Aphanomyces cochlioides

We found that the gradient of a host-specific attractant, cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone) isolated from the roots of spinach triggered encystment followed by germination of zoospores of Aphanomyces cochlioidesat a concentration less than micromolar order. This compound did not affect the growth and reproduction of this phytopathogen up to 10^–6 M concentration in the culture medium. We also observed that mastoparan, an activator of heterotrimeric G-protein could inhibit the motility of zoospores and then strikingly effect encystment followed by 60–80% germination of cysts. Concomitant application of cochliophilin A and mastoparan showed stronger encystment followed by 100% germination of cysts. In addition, we have observed that chemicals interfering with phospholipase C activity (neomycin) and Ca²⁺ influx/release (EGTA and loperamide) suppress cochliophilin A or mastoparan induced encystment and germination. These results suggest that G-protein mediated signal transduction mechanism may be involved in the differentiation of the A. cochlioides zoospores. This is the first report on the differentiation of oomycete zoospores initiated by a host-specific plant signal or a G-protein activator.     

Chemotaxis of fungal zoospores, with special reference to Aphanomyces cochlioides.

Zoospores of phytopathogenic fungi accumulate at the potential infection sites of host roots by chemotaxis. The aggregated spores then adhere, encyst, germinate, and finally penetrate into the root tissues to initiate infection. Some of the host-specific attractants have already been identified. The host-specific attractants also induce cell differentiation of certain zoospores under laboratory conditions. This indicates that a signal released from the roots of the host plant guides the pest propagules for orientation and prepares them for establishing a host-pathogen relationship by necessary physiological changes. Some non-host plant secondary metabolites were found to markedly regulate behavior and viability of zoospores, suggesting that non-host compounds may also play a role in protecting the non-host plants from the attack of zoosporic fungi. We hypothesized that zoospores perceive the host signal(s) by specific G-protein-coupled receptors and translate it into responses by way of the phosphoinositide-Ca2+ signaling cascade. The details of the signal transduction mechanism in fungal zoospores are yet to be discovered. In this report, we review the signaling and communications between phytopathogenic fungal zoospores and host and non-host plants with special reference to Aphanomyces cochlioides.     

Dynamic rearrangement of F-actin organization triggered by host-specific plant signal is linked to morphogenesis of Aphanomyces cochlioides zoospores

Cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone), a root releasing host-specific plant signal triggers chemotaxis and subsequent morphological changes in pathogenic Aphanomyces cochlioides zoospores before host penetration. The present study illustrates time-course changing patterns of cytoskeletal filamentous actin (F-actin) organization in the zoospores of A. cochlioides during rapid morphological changes (encystment and germination) after exposure to cochliophilin A. Confocal laser scanning microscopic analysis revealed that F-actin microfilaments remained concentrated at ventral groove and diffusely distributed in peripheral cytoplasm of the zoospore. These microfilaments dramatically rearranged and changed into granular F-actin plaques interconnected with fine arrays during encystment. A large patch of actin arrays accumulated at one pole of the cystospores just before germination. Then the actin plaques moved to the emerging germ tube where a distinct cap of microfilaments was seen at the tip of the emerging hypha. Zoospores treated with an inhibitor of F-actin polymerization, latrunculin B or motility halting and regeneration inducing compound nicotinamide, displayed different patterns of F-actin in both zoospores and cystospores than those obtained by the induction of cochliophilin A. Collectively, these results indicate that the host-specific plant signal cochliophilin A triggers a dynamic polymerization/depolymerization of F-actin in pathogenic A. cochlioides zoospores during early events of plant-peronosporomycete interactions.     

Secondary metabolites of plants from the genus cipadessa: chemistry and biological activity

The plants of genus Cipadessa, which are distributed across India and southwest of China, have been used as natural insecticides, as well as folk medicines for the treatment of a range of maladies such as diabetes, dysentery, malaria, piles, snake poison, rheumatism, etc. This article reviews the chemical constituents that have been isolated from Cipadessa species to date, including their biological activities. The compounds listed are tetranortriterpenoids (limonoids), diterpenoids, sesquiterpenoids, flavonoids, steroids, and some others.     

Factors in seminal plasma of bulls that affect the viability and motility of spermatozoa

Bound sialic acids on rat spermatozoa were assayed by oxidation with 1 mM-NaIO4 at 0 degree C, liberating C-9 as formaldehyde which was further quantitated using 3-methyl-2-benzothiazolinone. The mean +/- s.d. (n = 20) content of bound sialic acids of spermatozoa from the caput and cauda epididymidis was 50.9 +/- 8.0 and 25.2 +/- 3.8 nmol/10(8) spermatozoa respectively. About 85% of the former and 75% of the latter could be extracted by 1% Triton X-100 and 2 mM-dithiothreitol. About 70% of the former and 20% of the latter were released by neuraminidase from Vibrio cholerae. About 40% of the former and 30% of the latter were sensitive to trypsin. During sperm maturation, the decrease in the total bound sialic acids was due to the decrease in the neuraminidase-sensitive but not the neuraminidase-resistant sialic acids.     

Secondary metabolites in in vitro cultured plants of the genus Drosera

Extracts from plantlets of different species of the genus Drosera, grown as in vitro cultures, were evaluated for the level of phenolic secondary metabolites from the group of naphthoquinones and flavonols. The profiles of natural products in the extracts obtained from different species were monitored by HPLC with UV detection at 260 and 330 nm. On the basis of the data obtained, Drosera binata, the species with the highest amount of plumbagin, was selected for further studies. The most effective method of extraction of quinones was established and the composition of phenolic secondary metabolites in the tissues was determined. For the identification of phenolic compounds, HPLC-UV and HPLC-ESI/MS were applied.     

Identification of effector genes from the phytopathogenic Oomycete Plasmopara viticola through the analysis of gene expression in germinated zoospores

Grapevine downy mildew caused by the Oomycete Plasmopara viticola is one of the most important diseases affecting Vitis spp. The current strategy of control relies on chemical fungicides. An alternative to the use of fungicides is using downy mildew resistant varieties, which is cost-effective and environmentally friendly. Knowledge about the genetic basis of the resistance to P. viticola has progressed in the recent years, but little data are available about P. viticola genetics, in particular concerning the nature of its avirulence genes. Identifying pathogen effectors as putative avirulence genes is a necessary step in order to understand the biology of the interaction. It is also important in order to select the most efficient combination of resistance genes in a strategy of pyramiding. On the basis of knowledge from other Oomycetes, P. viticola effectors can be identified by using a candidate gene strategy based on data mining of genomic resources. In this paper we describe the development of Expressed Sequence Tags (ESTs) from P. viticola by creating a cDNA library from in vitro germinated zoospores and the sequencing of 1543 clones. We present 563 putative nuclear P. viticola unigenes. Sequence analysis reveals 54 ESTs from putative secreted hydrolytic enzymes and effectors, showing the suitability of this material for the analysis of the P. viticola secretome and identification of effector genes. Next generation sequencing of cDNA from in vitro germinated zoospores should result in the identification of numerous candidate avirulence genes in the grapevine/downy mildew interaction.     

Secondary metabolites from the liverwort Heteroscyphus coalitus

A new dihydroisocoumarin derivative, R-(?)-heteroscyphide (1), together with three known terpenoid derivatives (2–4), were isolated from the liverwort Heteroscyphus coalitus. Their structures were elucidated by spectroscopic methods and the biogenetic pathway of compound 1 was proposed. Cytotoxic test revealed that they exhibited moderate inhibitory activity to three human tumor cell lines.     

Secondary metabolites from the liverwort Tylimanthus renifolius

Two new herbertane sesquiterpenoids, (-)-herbertene-1,12-diol and (-)-gamma-herbertenol, a new aromadendrane, 9-acetoxy-10-hydroxyaromadendrane, a new rosane diterpenoid, 5,15-rosadiene-3,11-dione and a new phytane, 2Z-phytene-1,15,20-triol, along with the known (-)-alpha-herbertenol have been isolated from the liverwort Tylimanthus renifolius. Several flavones and flavanones with an unsubstituted ring B were also identified. This is the first report of this class of flavonoids from the Hepaticae. All structures were elucidated by NMR spectroscopy.     

Secondary metabolites from the liverwort Jamesoniella colorata

Six new labdane type diterpenoids, three seco-clerodane diterpenoids, jamesoniellide I, along with the two new jamesoniellides K and L, the sesquiterpene waitziacuminone and a new chlorinated bisbibenzyl, 6,6',10,10',12,12'-hexachloroisoperrottetin A, have been isolated from the liverwort Jamesoniella colorata. Their structures were elucidated by NMR spectroscopy. The absolute configuration of 3-oxo-labda-8(17),13(16),14-triene (1) was established by CD spectroscopy.     

2,4-Diacetylphloroglucinol suppresses zoosporogenesis and impairs motility of Peronosporomycete zoospores

2,4-Diacetylphloroglucinol (DAPG) produced by Pseudomonas fluorescens, shows toxicity to many microorganisms including fungi, bacteria, and peronosporomycetes. Zoosporogenesis and motility of zoospores are critical for a complete disease cycle and pathogenicity of the peronosporomycete phytopathogens. The aim of this study was to test the effects of DAPG and its derivatives on zoosporogenesis and motility of zoospores of a downy mildew pathogen, Plasmopara viticola, and a damping-off pathogen, Aphanomyces cochlioides. In both cases, DAPG inhibited zoosporogenesis (5 μg/ml) and the motility of zoospores (10 μg/ml) in a dose-dependent manner. Generally, zoospores became immotile shortly after exposure to DAPG followed by lysis. However, a fraction of DAPG treated A. cochlioides zoospores formed round cystospores instead of lysis and then germinated with excessively-branched germ tubes. All derivatives of DAPG had similar inhibitory activities but at varying doses. Among them, 2,4-dipropylphloroglucinol exerted the highest inhibitory activity against both zoosporogenesis and motility of zoospores. This revealed that the degree of hydrogen atoms substitution in the benzene ring by acyl groups and the length of substituted acyl groups were related to the level of bioactivity. This is the first report of inhibitory activities of DAPG and its derivatives against zoosporogenesis and motility of zoospores of two important peronosporomycete phytopathogens.     

Cyanobacterial chemotaxis to extracts of host and nonhost plants

Chemotaxis may be important when forming cyanobacterial symbioses. However, knowledge of cyanobacterial attraction towards plants and factors affecting chemotaxis is limited. Chemo-attraction was observed in Nostoc strains 8964:3 and PCC 73102 towards exudate or crushed extract of the natural hosts Gunnera manicata, Cycas revoluta and Blasia pusilla, and the nonhost plants Trifolium repens, Arabidopsis thaliana and Oryza sativa. As all tested plant extracts generated chemotaxis, the possibility to attract cyanobacteria may be widespread in plants. Chemotaxis was reduced by increased temperature and darkness and was stimulated by phosphorous and iron starvation and elevated salt concentration. Sugars (arabinose, galactose, and glucose) had a positive effect on chemotaxis, whereas flavonoids (chrysin and naringenin) and amino acids (methionine, glycine, serine, phenylalanine, glutamine, and lysine) had no effect.     

Secondary metabolites from Ganoderma lucidum and Spongiporus leucomallellus

The hydrodistillates and solvent extracts of the fruit bodies of Ganoderma lucidum (Fr.) P. Karst. and Spongiporus leucomallellus (Murril) A. David were investigated. The constituents in both oils comprised hydrocarbons, monoterpenes, sesquiterpenes, and fatty acids. Major volatiles of G. lucidum were trans-anethol, R-(-)-linalool, S-(+)-carvone and alpha-bisabolol, while the essential oil of S. leucomallellus contained relatively large amounts of R-(-)-1-octene-3-ol, R-(-)-linalool, 1-hepten-3-one and (Z)-nerolidol. From the n-hexane extract of G. lucidum, the steroid ester ergosta-7,22-diene-3beta-yl pentadecanoate could be identified. From S. leucomallellus two constituents showing structures of 3,4-seco-lanostane type triterpene acids were identified as (+)-23-oxo-3,4-seco-lanosta-4(28),7(8),9(11),24(31)-tetraene-3,26-dicarboxylic acid and (+)-20-hydroxy-23-oxo-3,4-seco-lanosta-4(28),7(8),9(11),24(31)-tetraene3,26-dicarboxylic acid, respectively. Cytotoxicity and antimicrobial activity of selected compounds were investigated using standard tests.     

Secondary metabolites from species of the biocontrol agent Trichoderma

Trichoderma species are free-living fungi that are highly interactive in root, soil and foliar environments and have been used successfully in field trials to control many crop pathogens. Structural and biological studies of the metabolites isolated from Trichoderma species are reviewed. This review, encompassing all the literature in this field up to the present and in which 269 references are cited, also includes a detailed study of the biological activity of the metabolites, especially the role of these metabolites in biological control mechanisms. Some aspects of the biosynthesis of these metabolites and related compounds are likewise discussed.     

Secondary metabolites from the leaves of Feijoa sellowiana Berg

The investigation of the lipid extract of leaves of Feijoa sellowiana cultivated along the east coast of Sicily has yielded in addition to the widespread secondary metabolites: alpha-tocopherol, flavone, stigmasterol and beta-carotene, an inseparable mixture of tyrosol esters of lignoceric (1a), cerotic (1b) and montanic (1c) acids, and a novel galactolipid identified as (2S)-1,2,6'-tri-O-[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl]-3-O-beta-D-galactopyranosyl glycerol (2).     

Effect of some phytopathogenic fungi and their metabolites on growth of Heliothis virescens (F.) and its host plants

Eleven fungal isolates and their secondary metabolites incorporated into artificial diet were tested for oral toxicity to the tobacco budworm (TBW) by examining larval weight, efficiency of conversion of ingested food to body tissue (ECI), pupal weight, days to pupation, and mortality. Two isolates of Altemaria alternata, two isolates of Fusarium moniliforme, three isolates of F. oxysporum and an isolated of F. solani reduced larval weight 90–99% after 7 days and inhibited pupation. ECI was reduced 34–96% in control groups. One isolate of A. alternata reduced pupal weight by 67% and increased the time to pupation three‐fold. One isolate of Cladosporium cladosporioides reduced larval weights by 56% and pupal weights by 7%. In a preference test of these isolates incorporated at a 1:4 ratio into artificial diet, 48% of the larvae were found on diet cubes containing autoclaved rice, 19% on standard diet, 10% on C. cladosporioides, 6–9% on F. solani, 8% on A. alternata and 3% on F. moniliforme. The fusarial toxins, T‐2 and diacetoxyscirpenol (DAS), were the most active compounds against TBW larvae among the 10 microbial toxins tested. T‐2 toxin reduced larval weight by 87%, reduced ECI by 62%, reduced pupal weight by 33% and delayed pupation by 1 week DAS caused similar but less severe effects than T‐2 toxin. AAL‐toxin inhibited larval growth and reduced pupal weights by 20% and 13%, respectively. A. alternata, F. moniliforme and F. solani were also phytotoxic to alfalfa (Medicago sativa,), crimson clover (Trifolium incarnatum) and wild geranium (Geranium dissectum,), which are early season plant hosts of TBW.