Accumulation of hydroxycinnamic acid amides in winter wheat under snow

It was found that the content of antifungal compounds p-coumaroylagmatine [1-(trans-4'-hydroxycinnamoylamino)-4-guanidinobutane] and p-coumaroyl-3-hydroxyagmatine [1-(trans-4'-hydroxycinnamoylamino)-3-hydroxy-4-guanidinobutane] in the crown of winter wheat (Triticum aestivum L. cv Chihokukomugi) significantly increased under snow cover. This finding suggests that the accumulation of these hydroxycinnamic acid amides was caused by winter stress and related to protecting the plant against snow mold under snow cover.     

An Antifungal Protein Purified from Pearl Millet Seeds Shows Sequence Homology to Lipid Transfer Proteins

In the course of a search for antifungal proteins from plant seeds, we observed inhibition of mycelial growth of Trichoderma viride with extracts of pearl millet. We have identified several proteins with antifungal properties in the seeds of pearl millet. One of these proteins has been purified to homogeneity and characterized. The purified protein has a molecular mass of 25 kDa. The N-terminal sequence of the protein (25 residues) shows homology to non-specific lipid transfer proteins (LTPs) of cotton, wheat and barley. The purified LTP inhibited mycelial growth of T. viride and the rice sheath blight fungus, Rhizoctonia solani in vitro.     

The Effect of Chitosan and Bion on Resistance to Pink Snow Mould in Perennial Ryegrass and Winter Wheat

The effects of chitosan on resistance to pink snow mould (Microdochium nivale) were studied in young winter wheat (Triticum aestivum L.) and perennial ryegrass (Lolium perenne L.) under controlled environmental conditions. In perennial ryegrass, the putative defence activator Bion was also tested. Resistance was measured as regrowth of plants after inoculation with M. nivale and incubation in darkness at 2°C. In winter wheat, pre‐treatment with chitosan at 1000 μg per plant increased resistance to subsequent infection by M. nivale, but this effect was less significant in a replicate experiment. Chitosan‐treated winter wheat plants expressed the gene for the pathogenesis‐related protein chitinase at higher levels than non‐treated plants. Chitinase gene expression was also stimulated by M. nivale infection in winter wheat. Perennial ryegrass pre‐treated with Bion or chitosan and inoculated with M. nivale did not display better regrowth after incubation than non‐treated, inoculated plants. Rather, regrowth was reduced in some of the Bion‐treated plants after incubation. We speculate that the cost or the mechanism of induced resistance makes Bion non‐effective in plants that are not actively growing. Bion at concentrations of 10, 100 and 1000 μg active ingredient per ml, and the highest concentration of chitosan used (2000 μg per ml) reduced in vitro growth of the pathogen, suggesting that both defence activators possess antifungal activity.     

Molecular characterization of the modular chitin binding protein Cbp50 from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> serovar <Emphasis Type="Italic">konkukian</Emphasis>

Bacillus thuringiensis is an insecticidal bacterium whose chitinolytic system may be exploited to improve the insecticidal system of Bt-crops. A nucleotide fragment of 1368 bp from B. thuringiensis serovar konkukian S4, containing the complete coding sequence of the chitin binding protein Cbp50, was cloned and sequenced. Analyses have shown the protein to contain a modular structure consisting of an N-terminal CBM33 domain, two copies of a fibronectin-like domain and a C-terminal chitin binding domain classified as CBM5. The Cbp50 protein was heterologously expressed in Escherichia coli, purified and assessed for chitin binding activity. A deletion mutant (CBD-N; containing only the N-terminal CBM33 domain) of Cbp50 was produced to determine the role of C-terminal domains in the binding activity of the protein. The full-length Cbp50 was shown to bind β-chitin most efficiently followed by α-chitin, colloidal chitin and cellulose. The polysaccharide binding activity of CBD-N was drastically decreased. The data demonstrate that both the N-terminal and C-terminal domains of Cbp50 are essential for the efficient binding of chitin. The purified Cbp50 showed antifungal activity against the phytopathogenic fungus Fusarium oxysporum and the opportunistic human pathogen Aspergillus niger. This is the first report of a modular chitin binding protein in bacteria.     

Genetic engineering of wheat for increased resistance to powdery mildew disease

 Fungal wheat (Triticum aestivum) diseases greatly affect crop productivity and require the economically and ecologically undesirable application of fungicides in wheat agriculture. We have generated transgenic wheat plants constitutively expressing an antifungal barley-seed class II chitinase. The transgene was stably expressed and the chitinase properly localized in the apoplast of the transgenic lines. The engineered wheat plants showed increased resistance to infection with the powdery mildew-causing fungus Erysiphe graminis. Received: 20 October 1998 / Accepted: 26 October 1998     

Mycelial growth of the snow mold fungus, Sclerotinia borealis, improved at low water potentials: an adaption to frozen environment

The snow mold fungus, Sclerotinia borealis, shows optimal growth at 4°C on potato dextrose agar (PDA) and can grow even at subzero temperature. Its mycelial growth was improved on frozen PDA at −1°C and on PDA containing potassium chloride (KCl) (water potential, −4.27 to −0.85 MPa) or d(−) sorbitol (−3.48 to −0.92 MPa). Its optimal growth temperature shifted from 4 to 10°C on PDA amended with KCl or sorbitol, indicating that inherent optimal growth occurs at high temperatures. These results suggest that S. borealis uses concentrated nutrients in the frozen environment and that such physiologic characteristics are critical for the fungus to prevail at subzero temperatures.     

Molecular Characterization of the Rice Pathogenesis-related Protein, OsPR-4b, and Its Antifungal Activity Against Rhizoctonia solani

We cloned and identified a new rice pathogenesis‐related (PR)‐4 gene, OsPR‐4b. OsPR‐4b encodes a 151 amino acid protein with a predicted molecular mass of 16.47 kDa and pI of 4.42. The putative OsPR‐4b shows high similarity to PR‐4 type proteins from various plant species and belongs to the Barwin family. Like other PR‐4s from monocot plants, OsPR‐4b contains a conserved Barwin domain and has a signal peptide at its N‐terminus. Recombinant OsPR‐4b protein expressed in Escherichia coli showed antifungal activity in vitro against the sheath blight fungus, Rhizoctonia solani. The results suggest that the OsPR‐4b may play a role in the disease resistance responses of rice against pathogen attacks through its antifungal activity.     

Identification of LY83583 as a specific inhibitor of Candida albicans MPS1 protein kinase

Candida albicans is the most common and virulent fungus causing candidiasis in various parts of the body and can be lethal to immunocompromised patients. All currently known antifungal therapies are drugs which cause serious side effects in the host. An inhibitor specific for fungus survival is an ideal therapeutic. C. albicans MPS1 (monopolar spindle 1) has been reported as a kinase essential to its survival. Because CaMps1p shares limited sequence homology with the human ortholog (hMps1p), we screened for a chemical inhibitor in anticipation of finding one with Candida specific cytotoxicity. In vitro screening using a recombinant catalytic domain of CaMps1p identified LY83583 (6-anilino-5,8-quinolinedione), known as a guanylate cyclase inhibitor, to be blocking CaMps1p kinase activity. In addition to its in vitro kinase inhibition, LY83583 reduced the growth rate of C. albicans. Finally, we compared the inhibitory activity on CaMps1p and hMps1p among inhibitors against those kinases. LY83583 showed specific inhibition for CaMps1p with no effect on hMps1p activity. Conversely, the CaMps1p activity was not affected by known hMps1p inhibitors. These findings suggest that CaMps1p may well be an ideal target molecule for antifungal therapy.     

Variation within isolates ofTyphula incarnata from localities differing in winter climate

Isolates ofTyphula incarnata, a snow mold fungus, were collected from four localities with different winter climates. Their ecological traits such as mycelial growth rate, sclerotium size, carpogenic germination of sclerotia, and aggressiveness were compared between populations in order to reveal infraspecific differentiation associated with climatic differences. Population variability was evident only in sclerotium germination: isolates from more snowy localities germinated faster than those from less snowy localities.T. incarnata is regarded as a versatile pathogen with no specialized forms in contrast withT. ishikariensis. The germination rate of sclerotia is considered very critical in the life history ofT. incarnata.     

Recombinant Wheat Antifungal PR4 Proteins Expressed in Escherichia coli

PR proteins are soluble and host-coded molecules with antifungal activity induced by a variety of agents. Wheat contains several PR proteins and among them are those of the class 4 coded wheatwin1 and wheatwin2; the two native proteins have been isolated from wheat kernel and the coding cDNA clones have been recently characterized. Herein, we report the expression of recombinant wheatwin1 and wheatwin2 in Escherichia coli-insoluble fractions; a new protocol for the purification in high yields and correct processing of the two proteins was developed. The recombinant proteins have molecular weights identical to that of the native proteins, indicating that the removal of the N-terminal methionine and cyclization of glutamine to pyroglutamate was complete. Both recombinant proteins inhibited in vitro the growth of Fusarium culmorum exhibiting antifungal properties similar to those of the native proteins.     

Components of Pink Snow Mould Resistance in Winter Wheat are Expressed Prior to Cold Hardening and in Detached Leaves

Resistance to pink snow mould, caused by Microdochium nivale, was investigated in four resistant winter wheat lines from the USDA World Cereal Collection (CI9342, CI14106, PI173440 and PI181268) and three Nordic wheat lines (Bjørke, Rida and V1004). Pink snow mould resistance was tested in non‐hardened and cold‐hardened plants incubated under artificial snow cover and in detached leaf segments mounted on water agar and incubated at either 3°C in darkness or at room temperature with light during the day. The wheat lines CI9342, CI14106 and PI181268 were more resistant than the Nordic lines, both before and after cold hardening. Thus, although cold hardening strongly increases the level of snow mould resistance in all the wheat lines, some resistance mechanisms are also present prior to cold hardening in some of the resistant lines. CI9342, CI14106 and PI181268 also had a higher level of resistance than the other lines in the detached leaf assay, indicating that these lines have some resistance mechanisms acting in the leaves. The resistance of PI173440 was expressed only in intact hardened plants and not in non‐hardened plants or in detached leaves. This indicates that this line relies on cold hardening‐related changes in the crown for its resistance. In the detached leaf assay the rate of lesion development varied greatly between leaves of different order. The highest correlation with the whole plant test was obtained when using secondary leaves and incubation at 3°C in the dark.     

Metabolites from <Emphasis Type="Italic">Pseudomonas brassicacearum</Emphasis> with activity against the pink snow mould causing pathogen <Emphasis Type="Italic">Microdochium nivale</Emphasis>

Bioassay-guided fractionation of cell-free culture supernatants of the bacterium Pseudomonas brassicacearum MA250 yielded three bioactive compounds (1–3). Compound 1 was identified as the unsaturated fatty acid γ-lactone piliferolide A, compound 2 as the not previously described open acid form of 1, and 3 as the compound SB-253514, which is an imide of a 3-O-rhamnosyl fatty acid and a bicyclic carbamate. All three compounds displayed moderate activity towards the pink snow mould causing pathogen Microdochium nivale, and may thus contribute to the previously observed biological control of this strain on M. nivale on wheat. Compound 1 further exhibited activity towards the human pathogen Aspergillus fumigatus, while compound 3 showed antifungal as well as antibacterial activity.     

Endophytic fungus <Emphasis Type="Italic">Trichothecium roseum</Emphasis> LZ93 antagonizing pathogenic fungi <Emphasis Type="Italic">in vitro</Emphasis> and its secondary metabolites

The endophytic fungus Trichothecium roseum LZ93 from Maytenus hookeri was found to antagonize other pathogenic fungi in vitro. To identify which compound contributed substantially to the antagonism, we fermented the strain and purified its fermentation products. Eleven compounds were obtained, including two trichothecenes, five rosenonolactones, two cardiotonic cyclodepsipeptides, and two sterols. Compound 11β-hydroxyrosenonolactone (1) was assigned according to 1D and 2D-NMR data for the first time. At the same time, the ¹H and ¹³C-NMR assignments for 6β-hydroxyrosenonolactone (2) were revised. Of all of them, only trichothecin (6) showed strong antifungal activity. Based on our observations of the antagonistic activity and the other experimental results, we suggest that the antifungal compound trichothecin was the main contributor to the antagonistic action of T. roseum LZ93.     

小麦锌指蛋白基因TaLOL2的克隆及特征分析

通过电子克隆与RT-PCR相结合的方法,在条锈菌诱导的小麦叶片中克隆获得1个新的LSD1型锌指蛋白基因TaLOL2,并用qRT-PCR技术分析了其转录表达特征。结果显示:(1)小麦锌指蛋白基因TaLOL2的cDNA全长1 095bp,编码179个氨基酸。(2)TaLOL2含有3个典型的zf-LSD1型(CxxCxRxxLMYxxGASxVxCxxC)保守结构域,与水稻、拟南芥、大麦等植物LSD1型锌指蛋白序列具有高度相似性,其中与水稻OsLOL2相似度达86.0%。(3)进化树分析表明,TaLOL2与水稻、拟南芥和大麦中部分含有3个保守zf-LSD1锌指结构的基因亲缘关系较近,而与其它包含不同数目的zf-LSD1锌指结构的基因亲缘关系较远。(4)qRT-PCR定量分析表明,TaLOL2在条锈菌侵染前期呈上调表达,在亲和及非亲和反应中差异表达。研究表明,TaLOL2参与了条锈菌诱导的小麦抗病防卫反应,很可能作为正调控因子参与了小麦-条锈菌非亲和互作中对条锈菌的抗性信号途径。     

A Lignan-type Stress Compound in Potato Infected with Nematode (Globodera rostochiensis)

Rustmicin, a new antibiotic active against the wheat stem rust fungus, was isolated from a cultured broth of Micromonospora chalcea 980-MC₁. Rustmicin showed strong inhibitory activity against the wheat stem rust fungus both in vitro and in pot tests in a greenhouse with MIC being 1 and 0.8/ig/ml, respectively. Its structure was determined by NMR spectroscopy to be a new 14-membered macrolide antibiotic lacking sugar substituents.     

Effect of Cottony Snow Mold on Mortality and Biomass of Calamagrostis canadensis under Controlled-Environment Conditions

A low-temperature basidiomycete (LTB), the causal agent of cottony snow mold, was evaluated as a potential biological control agent for marsh reed grass (Calamagrostis canadensis [Michx.] P. Beauv.), a native grass of the North American boreal forest. Marsh reed grass and 2-year-old white spruce trees (Picea glauca [Moench] Voss) were inoculated with LTB and incubated for 12 weeks in a cold chamber to determine whether the fungus was pathogenic to these species. The fungus was not pathogenic to white spruce but was to marsh reed grass, causing 15% mortality and 64% reduction in dry weight of aboveground plant matter. In a second experiment, two clones of marsh reed grass were inoculated with an isolate of LTB and incubated for 8, 14, and 20 weeks in a cold chamber. Maximum response in the grass to the LTB treatment occurred after 20 weeks of incubation, with decreases of 97, 97, and 63%, respectively, in tiller number, dry weight of aboveground plant matter, and plant height. The LTB is a potential biological control agent of marsh reed grass.     

Antifungal activity of methanol extracts from spikes of Triticum spelta and Triticum aestivum genotypes differing in their response to Fusarium culmorum inoculation

The total concentrations of free phenolic compounds and peroxidase were determined in spikes (collected at the flowering stage) of some spelt and common wheat cultivars differing in their response to F. culmorum infection. The antifungal activity of methanol extracts obtained from spikes was also evaluated. The tested genotypes differed significantly in their response to inoculation. The most resistant were Torka and Zebra among common wheat cultivars, and Weisser Grannenspelz among spelt cultivars. The average content of free phenolic compounds in spikes of spelt and common wheat was 1246.56 μg g⁻¹ and 1236.58 μg g⁻¹, respectively. The cultivars whose spikes contained the largest amounts of phenols showed the weakest response to F. culmorum infection. No significant differences were observed with regard to peroxidase content, which was 5.22 U g⁻¹ in common wheat spikes and 5.14 U g⁻¹ in spelt spikes. Methanol extracts from spikes of all wheat cultivars contained antifungal substances. The extracts from spelt spikes inhibited the growth of F. culmorum on PDA to a lesser degree than the extracts from common wheat spikes. This corresponds to the results of field trials, in which T. spelta generally exhibited a stronger response to F. culmorum infection than common wheat. The high correlation (r = 0.816) between mycelium growth inhibition on the medium and F. culmorum infection indicates that an evaluation of the antifungal activity of extracts from spikes may be used for the selection of breeding materials directed towards increased resistance to Fusarium head blight.     

A unique β-agarase,AgaA, from a marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. strain PO-303

The agaA gene encoding β-agarase-a (AgaA) was cloned from the chromosomal DNA of a marine bacterium, Vibrio sp. strain PO-303. The nucleotide sequence of the agaA gene consists of 2,958 bp and encodes a protein of 985 amino acids with a molecular mass of 106,062 Da. The deduced enzyme protein contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 266 amino acid sequence that is homologous to catalytic module of family 16 glycoside hydrolases, a bacterial immunoglobulin group 2 (Big-2)-like domain of 52 amino acid residues, two carbohydrate-binding modules of family 6 separated from Big-2-like domain by nine times repeated GDDTDP amino acid sequence. AgaA is the first agarase that was identified to possess a Big-2-like domain. The recombinant AgaA (rAgaA) expressed in Escherichia coli exhibited maximal activity around 40°C and pH 7.5, with a specific activity of 16.4 units mg⁻¹, a K _m of 1.10 mg ml⁻¹, and a V _max of 22.5 μmol min⁻¹ mg⁻¹ for agarose. The rAgaA hydrolyzed neoagarohexaose, but did not act on neoagarotetraose and neoagarobiose.