Phytotoxicity of pathogenic fungi and their mycotoxins to cereal seedling viability

Aspergillus flavus, Alternaria alternata and Fusarium oxysporum were the pathogenic fungi most reduced cereal (barley, sorghum and wheat) seedlings. The pathogens have the ability to produce aflatoxin B₁ and G₁, diacetoxyscirpenol, kojic acid and tenuazonic acid that reduced seedling viability. The inhibition dose for 50% reduction (LD₅₀) was recorded by aflatoxins at 0.83 mg L^-1 for barley, 1.74 mg L^-1 for wheat and 2.75 mg L^-1 for sorghum. Diacetoxyscirpenol produced its inhibition at 1.26 mg L^-1 for barley, 3.98 mg L^-1 for wheat and 10 mg L^-1 for sorghum. Kojic acid induced 50% inhibition at 63 mg L^-1 for barley, 105 mg L^-1 for wheat and 251 mg L^-1 for sorghum. However, tenuazonic acid was less toxic where the toxicity ranged between 79–550 mg L^-1. The germination inhibition was more pronounced in barley followed by wheat and was negligible in sorghum for all tested mycotoxins. This inhibition was attributed to the reduction in the seedling amylase activity, where amylase was also reduced in the same trend: barley > wheat > sorghum. Grain treated with carboxin-captan and thiophanatemethyl-thiram at 1 g kg^-1 grain increased the seedlings vigour of wheat in sterilized soil by 45 and 22%, barley by 24 and 33% and sorghum by 15 and 30%, respectively. These fungicides also had a positive effect on cereal when the soil was inoculated with A. flavus, A. alternata and F. oxysporum, but the improvement was still below normal. This revised version was published online in June 2006 with corrections to the Cover Date.     

Solid Substrate Production of Alternaria alternata f. sp. sphenocleae Conidia

Sphenoclea zeylanica (gooseweed), a major weed of paddy rice in Southeast Asia, is one of the targets in a biological weed control research program in the Philippines. A fungal pathogen, Alternaria alternata f. sp. sphenocleae , is being evaluated as a biological control agent for this weed. The feasibility of solid substrate fermentation for the mass production of A. alternata f. sp. sphenocleae has been examined. Conidia production and virulence of A. alternata f. sp. sphenocleae were affected by temperature, light, and incubation period. Abundant conidia were produced under continuous light on seeds of sorghum, hard red spring wheat, and barley at 28 o C. The greatest number of conidia was produced on sorghum seed followed by barley and oats seeds at 28 o C exposed to near-ultraviolet (NUV). More conidia were produced at 28 o C under NUV light on sorghum, barley, oats, and hard red spring wheat seeds, cornmeal, and polished rice grains than on the other substrates. Less conidia were produced on these substrates under light. At 28 o C, large numbers of virulent conidia were produced on sorghum seeds after 4 weeks of incubation under either constant light or dark. A mix of equal quantities of sorghum seeds and water (w/v) maximized conidial production. Conidia produced on sorghum seeds had a shelf life of at least 12 months when stored in production flasks under room conditions (24 ±2 o C). The use of sorghum seeds as a solid substrate for production of A. alternata f. sp. sphenocleae could be a feasible method to produce conidia in a village co-operative or cottage industry type scenario in Southeast Asia.     

Shoot apical meristem: A sustainable explant for genetic transformation of cereal crops

Summary Immature zygotic embryo has been the widely used explant source to develop embryogenic callus lines, cell suspensions and protoplasts for transformation of cereal crops including maize, wheat, rice, oat, barley, sorghum, and millet. However, the lack of competence of immature embryos in certain elite lines is still a barrier to rontine production of transgenic cereal crops in certain commercial cultivars. In addition, a great deal of effort is required to produce immature embryos, manipulate cultures, of immature embryos or their cell suspensions, and cryoperserve cultures for further use. In addition, undifferentiated cells may have reduced regenerability after a few months, of in vitro culture. Alternative explants and regeneration systems for efficient transformation of cereal crops are needed to avoid or reduce the above limitations. During the past decade, scientists have successfully manipulated the shoot apical meristerms from seedlings of maize oat, sorghum, millet, wheat, and barley in an effort to develop a less genetype-dependent and efficient cereal regneration system that can be maintained in vitro for long pertiods of time without the need for cryopreservation. Furthermore, apical mesistem regeneration systems were used to stably transform maize, wheat, rice, oat, barley, sorghum, and millet.     

Contribution to the fungal flora of cereal grains in Egypt

30 genera and 77 species, in addition to 4 varieties were isolated from 25 samples of each of barley, wheat, maize and sorghum grains collected from different places in Egypt. The broadest spectrum of genera and species was recorded in wheat (25 genera and 59 species + 4 varieties) followed by barley (21 genera and 52 species + 2 varieties), sorghum (14 genera and 33 species + 2 varieties) and maize grains (11 genera and 29 species + 2 varieties). Aspergillus, Fusarium, Penicillium and Rhizopus were the most common genera in the four grains, except maize where Penicillium emerged in low frequency. Aspergillus was the main component of the fungal flora of the four grains and contributed 79–94.4% of the gross total count of fungi. From the preceding genera A. niger, A. flavus, A. fumigatus, F. oxysporum, P. chrysogenum, P. corylophilum, P. notatum and R. stolonifer were the most frequent.     

Bright Greenish-Yellow Fluorescence and Aflatoxin in Agricultural Commodities

The corn milling industry has widely accepted the presence of bright greenish-yellow fluorescence under a black light as a presumptive indicator of aflatoxin (a poison produced by the mold Aspergillus flavus). This test was applied to wheat, oats, barley, rice, coconut, white corn, yellow corn, peanuts, sorghum, and soybeans, and evaluated in the laboratory. Our study supported the use of bright greenish-yellow fluorescence as a presumptive test for aflatoxin in wheat, oats, barley, corn, and sorghum.     

Tenuazonic acid production by Alternaria alternata and Alternaria tenuissima isolated from cotton.

Cultures of Alternaria alternata (three isolates) and Alternaria tenuissima (three isolates) obtained from cottonseeds and bolls were toxigenic when cultured on various laboratory media. A mycotoxin was isolated and identified as tenuazonic acid by using solvent partition, thin-layer chromatography, and instrument analyses. Toxicity was monitored with brine shrimp and chicken embryo bioassays. All cultures except A. alternata 938 produced tenuazonic acid when grown on cottonseed and on yeast extract-sucrose broth. The most toxin (266 mg/kg) was produced by A. tenuissima 843 on cottonseed.     

Tenuazonic acid production by Alternaria alternata and Alternaria tenuissima isolated from cotton

Cultures of Alternaria alternata (three isolates) and Alternaria tenuissima (three isolates) obtained from cottonseeds and bolls were toxigenic when cultured on various laboratory media. A mycotoxin was isolated and identified as tenuazonic acid by using solvent partition, thin-layer chromatography, and instrument analyses. Toxicity was monitored with brine shrimp and chicken embryo bioassays. All cultures except A. alternata 938 produced tenuazonic acid when grown on cottonseed and on yeast extract-sucrose broth. The most toxin (266 mg/kg) was produced by A. tenuissima 843 on cottonseed.     

日本曲霉产生的黑麦酮酸F对玉米的化感作用

日本曲霉（Aspergillus japonicus)是土壤和谷物种子表面的一种常见真菌。研究结果表明。日本曲霉所产生的大量黑麦酮酸F(SAF)对玉米(Zea mays)有很强的化感作用,低浓度显著促进玉米幼苗生长,高浓度则抑制．在0．0375mmol·L^-1SAF下,玉米幼苗根长增长31．7％,根数量增加13．2％,根活力提高4．73倍,并促进玉米对P、K、Ca、Mg、S等5种营养元素的吸收．高浓度SAF(0．3mmol·L^-1)下玉米根活力受抑制(72．1％),根对N、P、K、Ca、Mg、Fe等营养元素的吸收也受抑制.0．6mmol·L^-1的SAF下根完全失去活力,电镜观察表明,有SAF的情况下玉米叶绿体片层结构模糊、混乱,双层膜不完整。     

Tolerance of antibiotic and susceptible cereal seedlings to the aphids Metopolophium dirhodum and Rhopalosiphum padi

Some cereal seedlings exhibit antibiotic and antixenotic resistance to the aphids Metopolophium dirhodum (Walker) and Rhopalosiphum padi (L.), because the seedlings contain hydroxamic acids or gramine. The association between tolerance to aphids and aphid antibiosis was investigated for three cereals, Dollarbird wheat Vulcan wheat and Yagan barley. The dry biomass gained by the aphids and the simultaneous reduction in the biomass of the plants (biomass conversion ratio) quantified tolerance. Biomass production and the density dependence of biomass production by the aphids quantified antibiosis more effectively than fecundity. Vulcan wheat, which has more hydroxamic acid than Dollarbird wheat showed the highest level of antibiosis, and the barley was not antibiotic for either aphid. The biomass conversion ratio was a constant; the biomass of an infested plant was reduced by 3 mg for each mg of aphid biomass gained, regardless of aphid species, plant cultivar, or aphid density. The three plants showed no differential tolerance to the aphids, and therefore tolerance is not associated with antibiosis in this case.     

Composition of the fungal flora of four cereal grains in Saudi Arabia

Using the grain-plate method and on glucose-Czapek's agar at 28°C, fifty-eight species belonging to 26 genera were collected from barley (42 species and 19 genera), maize (29 species and 16 genera), sorghum (32 species and 17 genera) and wheat grains (42 species and 18 genera).The most frequent genera were Aspergillus, Penicillium, Rhizopus, Fusarium, and Mucor followed by Alternaria, Drechslera, and Curvularia. From the preceding genera Aspergillus flavus, A. niger, Penicillium notatum, Rhizopus stolonifer, Fusarium moniliforme, Mucor racemosus, Alternaria alternata, Drechslera spicifera, and Curvularia lunata were the most prevalent species in the four types of grains tested.     

Toxicity of mycotoxins produced by four Alternaria species toArtemia salina larvae

22 isolates ofAlternaria alternata, A raphani, A consortiale, andA chartarum were examined for the production of alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin I (ATX I), and tenuazonic acid (TA) on wheat grain and for toxicity of culture extracts toArtemia salina larvae. The total amount of 5 toxins produced under laboratory conditions ranged from 5 mg/kg to 11.112mg/kg. The toxic extracts showed EC50 values in the range of 3.3 to 144.5 mg/mL. There was no correlation between toxicity of extracts toArtemia salina and the amount of mentioned mycotoxins in culture.     

Gas chromatographic determination of 2(3)-benzoxazolinones from cereal plants

2(3)-Benzoxazolinone (BOA), 6-methoxy-2(3)-benzoxazolinone (MBOA) and 6,7-dimethoxy-2(3)-benzoxazolinone (dimethoxy-BOA) could be separated by GC. The identity of these components was verified by combined GC-MS. BOA and MBOA were determined quantitatively in 0·1 g samples of corn seedling. The presence of analogs not previously reported was demonstrated in seedlings of wheat, rye and in leaves of Job's tears. Seedlings of rice, barley, oat and sorghum did not contain any detectable amount of benzoxazolinones.     

Enhancement of NaCl tolerance in Arabidopsis thaliana by exogenous L-asparagine and D-asparagine

The tolerances of Columbia Arabidopsis thaliana (L.) Heynh. to NaCl, L-asparagine (L-Asn) and D-asparagine (D-Asn) during seedling establishment on sterile agar medium were determined. Germination and the establishment of upright seedlings with expanded green cotyledons were increasingly inhibited by NaCl concentrations from 20 to 180 m M and radicle growth was prevented at 225 m M NaCl. Tolerance of established seedlings to NaCl was similar at these concentrations. Seedling establishment was prevented at 20 m M L-Asn and 60 m M D-Asn, but L-Asn was not toxic to established seedlings. At lower concentrations, exogenous L- and D-Asn enhanced NaCl tolerance during germination and seedling establishment. Inhibition of seedling establishment by NaCl concentrations below 225 m M was reduced by the addition of L- and D-Asn to the medium. Maximal reduction of NaCl inhibition occurred between 2 and 4 m M for both L- and D-Asn. Higher concentrations of NaCl prevented establishment whether exogenous Asn was present or not. Reduction of NaCl inhibition occurred to the same extent whether L-Asn was presented simultaneously with the NaCl or preloaded for up to 24 h. The total seedling content of Na⁺ increased about 4-fold to 55 μg (mg dry weight)⁻¹ as the medium concentration of NaCl was increased from 9 μ M to 150 m M NaCl. Total K⁺ content declined about 80% from about 34 μg (mg dry weight)⁻¹ over the same range of NaCl concentrations. The Na⁺ uptake and K⁺ efflux by whole seedlings were similar whether or not NaCl tolerance was increased by exogenous Asn.     

Novel mode of resistance to Fusarium infection by a mild dose pre-exposure of cadmium in wheat.

Exposure of healthy wheat seeds (Triticum aestivum var Sonalika) to mild dose of cadmium (Cd(2+)) given as 50 microM CdCl(2) for 48 h and then washed off Cd(2+) offered resistance to the subsequent infection by Fusarium oxysporum inoculum. Seven days old seedlings having two primary leaves were aseptically inoculated with fungus, F. oxysporum (1 x 10(6)) spores. The seedlings pre-exposed to low level of Cd(2+) survived the Fusarium infection, while plantlets without Cd(2+) stress wilted and then perished due to Fusarium infection. The stress associated proteins induced by Cd(2+) (50 microM), F. oxysporum and by the co-stress (50 microM Cd(2+) and then with F. oxysporum) treatments were observed to be of same molecular weight (51 kDa). Antibody was raised against the purified Cd(2+)-stress associated protein (CSAP). Immuno-gold labeling of wheat seedling root tissue showed the presence of this CSAP in Cd(2+) pre-exposed and in co-stressed tissues and to be located predominantly on the inner linings of the cell membranes. We also observed that the anti-CSAP-antibody also labeled the root tissue of only Fusarium inoculated seedlings and the gold labeling was intensely located on the membrane. This cross-reaction of anti-CSAP suggests that Fusarium-induced stress protein (FISP) possibly has close homology to CSAP. We thus show for the first time the over expression of a high molecular mass protein by mild dose of Cd(2+) pre-exposure to wheat seeds which subsequently provided protection against Fusarium infection. This mode of resistance developed by an abiotic stress-causing agent against pathogen infection is novel.     

RNAi‐suppression of barley caffeic acid O‐methyltransferase modifies lignin despite redundancy in the gene family

Caffeic acid O‐methyltransferase (COMT), the lignin biosynthesis gene modified in many brown‐midrib high‐digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl‐to‐guaiacyl ratio was reduced by ~50%, the 5‐hydroxyguaiacyl (5‐OH‐G) unit incorporated into lignin at 10‐–15‐fold higher levels than normal, and the amount of p‐coumaric acid ester‐linked to cell walls was reduced by ~50%. No brown‐midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown‐midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.     

72份青稞氨基酸组成与营养价值评价

为了解青稞氨基酸的组成及营养品质,本研究测定了72份青稞材料籽粒的17种氨基酸(色氨酸除外)含量,通过对供试青稞材料氨基酸含量进行聚类分析,并比较育成品种与地方品种的氨基酸组成差异,对青稞的氨基酸进行了营养价值评价。结果表明,青稞氨基酸总含量平均为87.454 mg/g DW,变幅为47.8~178.7 mg/g DW,其中必需氨基酸含量占37.15%,谷氨酸的含量最高且变异大,蛋氨酸含量最低,赖氨酸含量变异小,91.67%的青稞材料的第一限制性氨基酸为赖氨酸。青稞氨基酸的贴近度为0.903,与世界粮农组织(FAO)、世界卫生组织(WHO)、联合国大学(UNU)提出的模式蛋白质的必需氨基酸组成较接近,氨基酸比值系数分SRC(73.14)较高。非必需氨基酸占青稞总氨基酸含量的62.85%,其中鲜味和甜味氨基酸含量分别为26.58 mg/g DW和21.85 mg/g DW,分别占总氨基酸含量的30.04%和24.43%。不同青稞材料的氨基酸含量和营养价值有很大的差异,地方品种各氨基酸含量均高于育成品种。供试材料中有4份青稞的氨基酸营养价值及风味氨基酸含量均较高。研究结果可为优质氨基酸组成的青稞选育及青稞加工提供指导。     

Root exudates of plants

Summary Comparisons have been made of the amount and composition of seed and seedling exudates in barley, wheat, cucumber, and bean. Except in the case of wheat a greater proportion of the total nitrogen content in the seed exudates was formed by protein and peptide nitrogen than by nitrogen of free amino acids. In contrast, the greater part of the total nitrogen in the exudates of seedlings was formed by free amino acid nitrogen, except in the case of barley. Peptides represented 8 to 26 per cent of the protein and peptide fraction in seed exudates, the highest amount being found in bean. On an equal weight basis, the spectrum of amino acids released from seeds and seedlings differed little between barley, wheat and cucumber; greater differences were observed in bean exudates. Seedlings exuded reducing substances to a considerably greater extent than seeds. The spectrum of reducing sugars in seed and root exudates differed greatly, especially in the case of keto sugars. Differences in the organic acid spectra were small, except for bean plants, the seedling exudate of which contained more organic acids with a richer spectrum than seed exudate. Both seeds and seedlings of cucumber exuded a small quantity and a poor spectrum of organic acids.     

Toxins of molds from decaying tomato fruit.

Among 27 mold isolates from decaying tomatoes, culture filtrates or ethyl acetate extracts of 8 isolates grown in yeast extract-sucrose medium were markedly toxic (mortality, greater than 50%) to brine shrimp larvae. The toxicity of six of these isolates could be attributed to the presence of citrinin, tenuazonic acid, or T-2 toxin. Ethyl acetate extracts of five Alternaria isolates and one Fusarium isolate were mutagenic for Salmonella typhimurium strains. In ripe tomatoes inoculated with toxin-producing isolates and incubated at 25 degrees C, one Alternaria alternata isolate produced tenuazonic acid in seven of seven tomatoes at levels of up to 106 micrograms/g and alternariol methyl ether in one of the seven tomatoes at 0.8 microgram/g. Another A. alternata isolate produced tenuazonic acid or alternariol methyl ether at much lower levels in only three of seven tomatoes. Patulin and citrinin were produced by a Penicillium expansum isolate at levels of up to 8.4 and 0.76 microgram/g, respectively. In tomatoes incubated at 15 degrees C, a Fusarium sulphureum isolate produced T-2 toxin, HT-2 toxin, and neosolaniol at levels of up to 37.5, 37.8 and 5.6 micrograms/g, respectively. If these mycotoxins are thermostable, they may occur at detectable levels in tomato products whenever partially moldy tomatoes are used as raw material.     

Preliminary Study of Fat Oxidation in Sorghum and Maize Brewing

Superoxide dismutase (SOD) activity in the white sorghum farafara and ICRISAT sorghum variety – ICSV 400 was at a high activity in the embryo at about 8 SOD units/tissue. This activity was almost completely destroyed at 80 °C. Totox index of the brewing grains were 366 for ICSV 400, 312 for farafara, 112 for maize grits and 90 for barley malt. Worts from sorghum/maize and sorghum/barley malt brews all had hydroperoxy linoleic acid (15–19 μM) which remained undetected after wort boiling. Sorghum/maize brews formed very little trub (wort proteinous sediments) in the whirlpool and trub increased in sorghum/barley brews with increased usage of barley malt. Sorghum/maize brews had free fatty acids (FFA) at 22 mg/l in pitching wort but in sorghum/barley brew (50/50) only 9 mg/l. This revised version was published online in July 2006 with corrections to the Cover Date.     

Toxins of molds from decaying tomato fruit.

Among 27 mold isolates from decaying tomatoes, culture filtrates or ethyl acetate extracts of 8 isolates grown in yeast extract-sucrose medium were markedly toxic (mortality, greater than 50%) to brine shrimp larvae. The toxicity of six of these isolates could be attributed to the presence of citrinin, tenuazonic acid, or T-2 toxin. Ethyl acetate extracts of five Alternaria isolates and one Fusarium isolate were mutagenic for Salmonella typhimurium strains. In ripe tomatoes inoculated with toxin-producing isolates and incubated at 25 degrees C, one Alternaria alternata isolate produced tenuazonic acid in seven of seven tomatoes at levels of up to 106 micrograms/g and alternariol methyl ether in one of the seven tomatoes at 0.8 microgram/g. Another A. alternata isolate produced tenuazonic acid or alternariol methyl ether at much lower levels in only three of seven tomatoes. Patulin and citrinin were produced by a Penicillium expansum isolate at levels of up to 8.4 and 0.76 microgram/g, respectively. In tomatoes incubated at 15 degrees C, a Fusarium sulphureum isolate produced T-2 toxin, HT-2 toxin, and neosolaniol at levels of up to 37.5, 37.8 and 5.6 micrograms/g, respectively. If these mycotoxins are thermostable, they may occur at detectable levels in tomato products whenever partially moldy tomatoes are used as raw material.