Bicycloalternarenes produced by the phytopathogenic fungus Alternaria alternata

Eleven compounds, ACTG toxins A and B and nine new substances named bicycloalternarenes (BCAs), were isolated and characterized from the culture filtrate of the phytopathogenic fungus Alternaria alternata. Under acidic conditions, these mixed terpenoids convert to pairs of tricycloaltenarenes that can be used for identification of the native compounds. We could not confirm the phytotoxic effects reported with ACTG-toxins in the past. However, BCA 2 at concentrations of 10(-4) M strongly inhibited germination and growth of Spirodela polyrhiza turions.     

The role of pigmentation in survival of the leaf spot fungus Cercospora beticola

Both germination and survival time of spores of a pigmented strain of Cercospora beticola were directly correlated with relative humidity. Pigmented cultures, under conditions of low water availability (15%) survived 242 days' continuous exposure to light (110 lx). The ability of various strains of the fungus to survive in soil was found to be influenced by pigmentation, rate of growth, inoculum ratio in the soil and the physiological state of the organism before exposure to the soil environment.     

Isolation and characterization of a clay-dispersing polysaccharide produced by the phytopathogenic fungus, Colletotrichum gloeosporioides

The fungus, Colletotrichum gloeosporioides, which is pathogenic to peppers produced an extracellular polysaccharide in liquid culture which possessed clay-dispersing activity. The polysaccharide could bind cationic dyes, Ruthenium Red and Alcian Blue, indicating it to be polyanionic. The polysaccharide dispersed kaolin in water and the dispersion was maintained for more than 7 days at 25 °C. Kaolin dispersion by the polysaccharide was stable from pH 3 to 10 but the addition of divalent metals at 1 mM inhibited half of the dispersion activity comparing to the control. The polysaccharide could disperse bentonite, calcium carbonate and other fine particles but did not possess emulsifying activity.     

Inhibition of the phytopathogenic fungus Fusarium proliferatum by volatile compounds produced by Pseudomonas

The Fusarium head blight of grain cereals is a significant disease worldwide. In Argentina, high levels of contamination with Fusarium proliferatum have been found in crops. Many strains of the Pseudomonas genus antagonize the growth of fungi by different mechanisms, such as the production of antibiotics, siderophores, volatiles, and extracellular enzymes. In this work, we have designed a new system for studying the growth inhibition of F. proliferatum—namely by volatile compounds produced by Pseudomonas fluorescens MGR12. In both rich and minimal media, the bacterium released volatiles that negatively affected the mycelial growth of that phytopathogenic fungus. These bacterial compounds were analyzed by gas chromatography–mass spectrometry, but only a few could be identified by comparing their mass spectra with the libraries of the National Institutes of Standards and Technology MS search.     

Grifolisin, a member of the sedolisin family produced by the fungus Grifola frondosa

The pepstatin-insensitive carboxyl proteinase grifolisin was purified from fruiting bodies of the fungus Grifola frondosa, a maitake mushroom. The enzyme had an optimum pH of 3.0 for the digestion of hemoglobin and 2.8 for milk casein digestion. Its molecular mass was determined to be 43kDa by SDS-PAGE and 40kDa by gel chromatography on Superose 12, and its isoelectric point was found to be 4.6 by isoelectric focusing. The enzyme hydrolyzed four major bonds in the oxidized insulin B-chain: Phe1-Val2, Ala14-Leu15, Gly20-Glu21 and Phe24-Phe25 at pH 3.0. The first 15 amino acid residues in the N-terminal region were AVPSSCASTITPACL, and the coding region of the grifolisin gene (gfrF) has a 1960-base pair cDNA. The predicted mature grifolisin protein consisted of 365 residues and was 26% identical to that of sedolisin from Pseudomonas sp. 101 and 34% identical to that of aorsin from Aspergillus oryzae. Grifolisin is a member of the sedolisin S53 family and is not inhibited by pepstatin.     

Metabolites produced by the phytopathogenic fungus Rhizoctonia solani: isolation, chemical structure determination, syntheses and bioactivity

The isolation, structure determination, syntheses and biological activity of Nb-acetyltryptamine and three proline containing dioxopiperazines, cyclo(S-Pro-S-Leu) (2), cyclo(S-Pro-S-Ile) (3), and cyclo(S-Pro-S-Val) (4), from cultures of Rhizoctonia solani Kuhn are reported here for the first time. Despite the small amounts isolated, the absolute stereochemistry of these naturally occurring dioxopiperazines was established by 1H NMR using for the first time the chiral solvating agent (R)-(-)-2,2,2-trifluoro-1-(9-anthryl)ethanol.     

Growth inhibition of a phytopathogenic fungus, Colletotrichum species by acetic acid

Acetic, oxalic, malic, and citric acids significantly inhibited the growth of Colletotrichum, gloeosporioides, a phytopathogenic fungus, and acetic acid showed the strongest inhibition with no growth at 50 mM. The growth inhibition by these organic acids was closely related with the inhibition of respiration, as tested using three species, C. gloeosporioides, C. coccodes, and C. dematium. Optimum growth of C. gloeosporioides was observed around pH 6.0. The inhibition of growth by acetic acid accelerated along with a decrease in pH from 6.0 to 4.0, suggesting that the inhibition might be more enhanced by undissociated form of acetic acid. Despite of growth inhibition by acetic acid, the fungus was able to grow in a normal medium when acetic acid was eliminated, implying that the growth inhibition may be resulted from an acetic acid-mediated inhibition of respiration than a structural damage of cell. Catalase activity of the fungus increased in response to 0.1% hydrogen peroxide, but addition of this together with 30 mM acetic acid brought about a decrease in the activity. The fungus which showed no grow at 30 mM acetic acid or 0.5% hydrogen peroxide began to grow after the elimination of these. But the fungus added simultaneously by these two compounds did not grow at all despite the elimination of these. Thus, controlling of Colletotrichum might be developed using acetic acid which is generally less dangerous than chemical reagents.     

Phellinstatin, a new inhibitor of enoyl-ACP reductase produced by the medicinal fungus Phellinus linteus

A new trimeric hispidin derivative, phellinstatin, was isolated from a culture broth of the medicinal fungus Phellinus linteus and its structure was established by various spectral analysis. Phellinstatin strongly inhibited Staphylococcus aureus enoyl-ACP reductase with an IC₅₀ of 6 μM and also showed antibacterial activity against S. aureus and MRSA.     

Biosynthesis of abscisic acid by the direct pathway via ionylideneethane in a fungus, Cercospora cruenta

We examined the biosynthetic pathway of abscisic acid (ABA) after isopentenyl diphosphate in a fungus, Cercospora cruenta. All oxygen atoms at C-1, -1, -1', and -4' of ABA produced by this fungus were labeled with (18)O from (18)O(2). The fungus did not produce the 9Z-carotenoid possessing gamma-ring that is likely a precursor for the carotenoid pathway, but produced new sesquiterpenoids, 2E,4E-gamma-ionylideneethane and 2Z,4E-gamma-ionylideneethane, along with 2E,4E,6E-allofarnesene. The fungus converted these sesquiterpenoids labeled with (13)C to ABA, and the incorporation ratio of 2Z,4E-gamma-ionylideneethane was higher than that of 2E,4E-gamma-ionylideneethane. From these results, we concluded that C. cruenta biosynthesized ABA by the direct pathway via oxidation of ionylideneethane with molecular oxygen following cyclization of allofarnesene. This direct pathway via ionylideneethane in the fungus is consistent with that in Botrytis cinerea, except for the positions of double bonds in the rings of biosynthetic intermediates, suggesting that the pathway is common among ABA-producing fungi.     

大豆灰斑病菌毒素的分离与提取*

用不同极性的有机溶剂均未萃取到具有致病组分的化合物。但用甲醇、乙醇、丙醇和硫酸铵等两种沉淀大分子物质的方法,却提取到了使大豆叶片产生典型病斑和致萎的化合物。该粗提物能引起大豆感病品种产生典型的蛙眼形病斑,由此可以肯定从培养滤液中提取的粗提物含有大豆灰斑病菌所产生的毒素。以二倍体积的甲醇来提取大豆灰斑病菌毒素是最为简单、快速的方法。     

REPAT, a new family of proteins induced by bacterial toxins and baculovirus infection in Spodoptera exigua

Insect larvae spend most of their time eating and the digestive tract is the most crucial barrier for the entrance of many pathogens. In our study, suppression subtractive hybridization (SSH) was used to compare Spodoptera exigua midgut gene expression between larvae exposed to the Bacillus thuringiensis Cry1Ca toxin and non-exposed insects. Based on the SSH results, full cDNA sequences coding for four homologous proteins were obtained. Quantitative and semi-quantitative RT-PCR showed the increased expression of the genes coding for these proteins after exposure to different B. thuringiensis toxins as well as after infection with baculovirus. The proteins were named REPAT after their increased expression in Response to Pathogen. REPAT1, a member of this family, was recombinantly expressed using the baculovirus expression system, revealing the glycosylated nature of the protein. Recombinant baculoviruses expressing REPAT1 were used to infect larvae from S. exigua, showing that expression of REPAT1 was reducing the virulence of baculovirus to the infected larvae. Together, these results suggest a role for REPAT1 in mitigating pathological effects.     

Acrolein produced from polyamines as one of the uraemic toxins

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.     

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea.

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing.     

Ceriporic acid C, a hexadecenylitaconate produced by a lignin-degrading fungus, Ceriporiopsis subvermispora

A lignin-degrading basidiomycete, Ceriporiopsis subvermispora produces a series of alkyl- and alkenylitaconates (ceriporic acids). Previously, two alkylitaconic acids with tetradecyl and hexadecyl side chains were isolated and identified as 1-heptadecene-2,3-dicarboxylic acid (ceriporic acid A) and 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B). In the present study, one hexadecenylitaconate (ceriporic acid C) was isolated and its chemical structure was analyzed by glycolation and subsequent (1) trimethylsilation, or (2) acetalation with acetone and acetone-d6. Analyses of the isolated metabolite demonstrated that the hexadecenylitaconic acid was (Z)-1,10-nonadecadiene-2,3-dicarboxylic acid. The structure of the side chain in ceriporic acid C was the same as that of hexadecenylcitraconate, chaetomellic acid B. Thus, it was found that ceriporic acids share close structural similarity with alk(en)yl citraconate derivatives, chaetomellic acids and other lichen lactones, protolichesterinic, lichesterinic, and murolic acids.