Faba bean root exudates alter pea root colonization by the oomycete Aphanomyces euteiches at early stages of infection.

Aphanomyces euteiches is an oomycete pathogen that causes the pea root rot. We investigated the potential role of early belowground defense in pea (susceptible plant) and faba bean (tolerant plant) at three days after inoculation. Pea and faba bean were inoculated with A. euteiches zoospores. Root colonization was examined. Root exudates from pea and faba bean were harvested and their impact on A. euteiches development were assessed by using in vitro assays. A. euteiches root colonization and the influence of the oomycete inoculation on specialized metabolites patterns and arabinogalactan protein (AGP) concentration of root exudates were also determined. In faba bean root, A. euteiches colonization was very low as compared with that of pea. Whereas infected pea root exudates have a positive chemotaxis index (CI) on zoospores, faba bean exudate CI was negative suggesting a repellent effect. While furanoacetylenic compounds were only detected in faba bean exudates, AGP concentration was specifically increased in pea.This work showed that early in the course of infection, host susceptibility to A. euteiches is involved via a plant-species specific root exudation opening new perspectives in pea root rot disease management.

Italian acacia honey exhibits lytic effects against the crayfish pathogens Aphanomyces astaci and Fusarium avenaceum.

This study purpose was to evaluate the in vitro inhibitory properties of Italian acacia honey extracts against pathogenic aquatic oomycete/fungal isolates that cause different diseases in crayfish, resulting in an elevated mortality rate. The antimycotic activity of acacia honey aqueous extracts was evaluated against the strain UEF88662 of Aphanomyces astaci (oomycete) and the strain SMM2 of Fusarium avenaceum (fungus). The extracts preparation was carried out with water by a cheap, not complex and organic solvent-free procedure, with low environmental impact and the higher possibility of large-scale reproducibility. The anti-oomycete and antifungal activities were quantitatively evaluated by growth, survival and sporulation microbiological assays. The extracts displayed a dose-dependent inhibitory efficacy on oomycete and fungal growth and survival, as well as on the production of oomycete and fungal spores. Supported by future in vivo studies, our results encourage the use of natural extracts like honey as innovative tools to counteract mycotic infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The continuous spread of aquatic fungal disease as the 'crayfish plague' and the 'burn spot disease' has severe ecological and commercial repercussions. Critical factor to prevent further spread is the availability of effective antifungals possibility derived from local natural resources to use in innovative strategies of control and eradication of these diseases. This study provides relevant information about the in vitro anti-oomycete and antifungal activity of Italian acacia honey aqueous extracts against two highly infectious and dangerous pathogenic species, Aphanomyces astaci and Fusarium avenaceum, that are responsible for important crayfish diseases.

An anti-apoptotic B-cell lymphoma-2 (BCL-2) from Channa striatus: Sequence analysis and delayed and advanced gene expression in response to fungal, bacterial and poly I:C induction.

B-cell lymphoma-2 (BCL-2) is a suppressor of apoptosis and inhibits the caspase dependent apoptosis pathway. In this study, we report molecular characterization of a cDNA sequence encoded of BCL-2 from striped murrel, Channa striatus. A partial cDNA sequence of CsBCL-2 was identified from the striped murrel cDNA library during annotation. Subsequently, the full length CsBCL-2 cDNA sequence was obtained by an internal sequencing method using a forward primer. The sequence contains 699 nucleotide base pairs which encode 232 amino acid residues. The domain and motif analysis revealed that the CsBCL-2 polypeptide consists of BCL-2 homologous domain BH4 at the N-terminal region between 4 and 21 and the BCL-2 homologous domains BH1, BH2 and BH3 between 87 and 187. The CsBCL-2 polypeptide sequence does not have a signal peptide region, but it consists of two novel transmembrane regions at 134-152 and 209-226. The sequence analysis showed that the CsBCL-2 has highest sequence identity (70%) with BCL-2 like protein 1 (BCL-2 L1) from pufferfish Takifugu rubripes. The phylogenetic analysis showed that the CsBCL-2 was situated in the BCL-2 L1 fish clade. The secondary analysis showed that the CsBCL-2 protein consists of 132 amino acid residues in the α-helical region and 100 amino acid residues in the random coil region. The validated 3D structure of CsBCL-2 showed the active residues Gly(135) and Arg(136) in the 7th α-helical position, whereas Trp(178) is in the 9th α-helical region. CsBCL-2 mRNA transcription is predominately present in spleen and is upregulated upon being induced with fungus Aphanomyces invadans, bacteria Aeromonas hydrophila, Escherichia coli LPS, Laminaria digitata beta-1,3-glucan and poly I:C. Overall, the CsBCL-2 mRNA transcription results indicate the potential involvement of CsBCL-2 in immune system of C. striatus. However, further research at proteomic level is necessary to examine these predictions.

Concentration- and time-dependent effects of isothiocyanates produced from Brassicaceae shoot tissues on the pea root rot pathogen Aphanomyces euteiches.

Isothiocyanates (ITCs) hydrolyzed from glucosinolates (GSLs) in Brassicaceae tissue are toxic to soil organisms. In this study, the effect of aliphatic and aromatic ITCs from hydrated dry Brassicaceae shoot tissues on the mycelium and oospores of the pea root rot pathogen Aphanomyces euteiches was investigated. The profile and concentrations of GSLs in two test Brassicaceae species, Sinapis alba and Brassica juncea, and the ITCs from the dominant hydrolyzed parent GSLs were monitored. The concentrations of dominant ITCs and pathogen exposure time were evaluated in in vitro experiments. The greatest effect on the pathogen was observed from aliphatic ITCs hydrolyzed from B. juncea tissue, and the effect depended on the ITC concentration and exposure time. ITCs were more effectively hydrolyzed from B. juncea GSLs than from S. alba GSLs; i.e., the ITC/GSL ratio was higher in B. juncea than in S. alba tissue, giving a different release pattern. The release of phenylethyl isothiocyanate, which was common to both species, followed a pattern similar to that of the dominant ITC in each crop species. This suggests that traits other than GSL content, e.g., plant cell structure, may affect the release of ITCs and should therefore influence the choice of species used for biofumigation purposes.

Production of antioomycete compounds active against the phytopathogens Phytophthora sojae and Aphanomyces cochlioides by clavicipitoid entomopathogenic fungi.

A total of 412 strains belonging to 14 genera of clavicipitoid entomopathogenic fungi (EPF) were screened for activities against two economically important plant pathogenic oomycetes, Phytophthora sojae and Aphanomyces cochlioides. To identify the antioomycete compounds produced by EPF, the extracts of 13 highly active EPF strains were characterized in detail by high performance liquid chromatography with diode array detection and high-resolution mass spectrometric detection and antioomycete assay. The antioomycete activity of several Metarhizium extracts was associated with previously isolated aurovertins, fungerin, N-(methyl-3-oxodec-6-enoyl)-2-pyrroline, and N-(methyl-3-oxodecanoyl)-2-pyrroline. The depsipeptide beauvericin was confirmed to be one of the active principles of three strains of Isaria tenuipes, which strongly inhibited mycelial growth of both P. sojae and A. cochlioides. Two known bioactive metabolites, paecilosetin and aranorosinol A, together with a novel and potent antioomycete compound, farinomalein, were isolated from the extracts of Isaria farinosa and all compounds were confirmed to have antioomycete activity. Identification of 8 antioomycete compounds from 13 clavicipitioid EPF demonstrated a new potential use of EPF as a source of compounds for the control of soil-borne plant pathogenic oomycetes.

Zoosporicidal metabolites from an endophytic fungus Cryptosporiopsis sp. of Zanthoxylum leprieurii.

Two polyketides, cryptosporiopsin A (1) and hydroxypropan-2',3'-diol orsellinate (3), and a natural cyclic pentapeptide (4), together with two known compounds were isolated from the culture of Cryptosporiopsis sp., an endophytic fungus from leaves and branches of Zanthoxylum leprieurii (Rutaceae). The structures of these metabolites were elucidated on the basis of their spectroscopic and spectrometric data. Cryptosporiopsin A and the other metabolites exhibited motility inhibitory and lytic activities against zoospores of the grapevine downy mildew pathogen Plasmopara viticola at 10-25µg/mL. In addition, the isolated compounds displayed potent inhibitory activity against mycelial growth of two other peronosporomycete phytopathogens, Pythium ultimum, Aphanomyces cochlioides and a basidiomycetous fungus Rhizoctonia solani. Weak cytotoxic activity on brine shrimp larvae was observed.

Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination.

Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions.

Ultrastructure of Aphanomyces cochlioides zoospores and changes during their developmental transitions triggered by the host-specific flavone cochliophilin A.

Aphanomyces cochlioides is a serious damping-off causing pathogen of sugar beet, spinach and some other members of Chenopodiaceae and Amaranthaceae. The biflagellated motile zoospores of the pathogen locate their host roots by perceiving the host-specific flavone cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone), transiently modify into cystospores that germinate prior to penetration. This study for the first time illustrated ultrastructure of the zoospores and morphological modification during their developmental transitions triggered by cochliophilin A using transmission electron microscopy (TEM). TEM revealed that zoospores had two heterokont flagella inserted laterally into a ventral groove of their body where each is attached to a kinetosome. In the cross sections of flagellar axonemes, two single and nine peripheral microtubules in doublets were clearly observed. Mitochondria, the Golgi complexes, finger print vesicles, and vesicles with striated electron opaque inclusion and vesicles containing a granular cortex and center were also detected. The latter vesicles disappeared and two flagella were shed when zoospores converted to spherical cystsopores. The shape, size and number of mitochondria were dynamically changed during the encystment of zoospores presumably through fission and fusion processes. The dynamics of mitochondria observed in this study indicated its distinct role in the signal transduction pathway of the zoospore encystment. This study also revealed the transformation of shape of nuclei from pyriform in zoospores to spherical in cystospores and lanceolate in the hyphae.

Variation in chemotactic preferences of Aphanomyces cochlioides zoospores to flavonoids.

The zoospores of the phytopathogenic Aphanomyces cochlioides are chemotactically attracted by a host-specific flavone, cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone), and repelled from the mammalian estrogens or estrogenic compounds. This study further examined the responses of A. cochlioides zoospores to some flavonoids structurally related to cochliophilin A or compounds known as phytoestrogens. The bioassay revealed that some synthetic flavones (such as 6-methyl-4'-methoxyflavone, 3-hydroxy-4'-methoxyflavone, 7-hydroxy-5-methylflavone, 3-hydroxy-4'-methoxy-6-methylflavone) elicited attractant activity at concentrations as low as picomolar (10 pM), which was an 100-fold lower concentration than that of the threshold concentration of the host-specific attractant cochliophilin A. Apparently, a hydrophobic B-ring with an alkylated (methylated) A-ring or a methoxylated B-ring with an unsubstituted A-ring in the flavone skeleton played a significant role in higher attractant activity. On the other hand, all known estrogenic flavonoids (such as equol, 3'- or 8-prenylated naringenins) displayed potent repellent activity toward zoospores. Surprisingly, zoospores were attracted by non-estrogenic 6-prenylated naringenin indicating that repellent activity is linked to the estrogenic activity of the phytoestrogens.

Actin filaments predominate in morphogenic cell stages, whereas plaques predominate in non-morphogenic cell stages in Peronosporomycetes.

We investigated the structural distribution of both types of actin arrays, filaments and plaques, in a soil-borne phytopathogenic peronosporomycete (oomycete), Aphanomyces cochlioides, under standardized host-free bioassays. The phenomenon was monitored during progression through all the asexual developmental processes of the organism. It was noted that the filamentous-form of actin was predominant during the morphogenic (morphologically active) stages of development. Conversely, during non-morphogenic (morphologically quiescent) stages, plaques dominated. From these analyses, we proposed a criterion that predominance of an actin form relates to, and precedes the morphological behaviour of a cellular stage in Peronosporomycetes. A decrease in the quantity of plaques in the encysted zoospore (non-morphogenic stage) during its developmental progression into morphogenic stages, both in germination and regeneration processes, asserted the notion that plaques function as the organization centres and are related to the reorganization of cell structure and the transition of the cell into a new stage. Furthermore, polymerization of filamentous-form during emergence stages in zoospore regeneration process revealed that filaments render motility to a developing zoospore. This unprecedented function of filaments in the developing zoospores was demonstrated using nicotinamide (0.8 x 10(-6)m), which did not cause actin disruption, but could induce zoospore encystment, and its further replacement with water triggered the zoospore emergence process. Additionally, by using latrunculin B, an actin polymerization inhibitor, we also demonstrated the functional necessity of actin during various developmental processes in Aphanomyces.

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.