Trypsin-like proteinase produced by Fusarium culmorum grown on grain proteins

The fungal disease Fusarium head blight occurs on wheat (Triticum spp.) and barley (Hordeum vulgare L.) and is one of the worldwide problems of agriculture. It can be caused by various Fusarium species. We are characterizing the proteinases of F. culmorum to investigate how they may help the fungus to attack the grain. A trypsin-like proteinase has been purified from a gluten-containing culture medium of F. culmorum. The enzyme was maximally active at about pH 9 and 45 degrees C, but was not stable under those conditions. It was stabilized by calcium ions and by the presence of other proteins. The proteinase was most stable at pH 6-7 at ambient temperatures, but was quickly inactivated at 50 degrees C. It was strongly inhibited by p-amidino phenylmethylsulfonyl fluoride (p-APMSF), and soybean trypsin and Bowman-Birk inhibitors, and it preferentially hydrolyzed the peptide bonds of the protein substrate beta-purothionin on the C-terminal side of Arg (mainly) and Lys residues. These characteristics show that it is a trypsin-like proteinase. In addition, its N-terminal amino acid sequence was 88% identical to that of the F. oxysporum trypsin-like enzyme. The proteinase hydrolyzed the D hordein and some of the C hordeins (the barley storage proteins). This enzyme, and a subtilisin-like proteinase that we recently purified from the same organism, possibly play roles in helping the fungus to colonize grains.     

Metabolites inhibiting germination of Orobanche ramosa seeds produced by Myrothecium verrucaria and Fusarium compactum

Myrothecium verrucaria and Fusarium compactum were isolated from diseased Orobanche ramosa plants collected in southern Italy to find potential biocontrol agents of this parasitic weed. Both fungi grown in liquid culture produced metabolites that inhibited the germination of O. ramosa seeds at 1-10 muM. Eight metabolites were isolated from M. verrucaria culture extracts. The main metabolite was identified as verrucarin E, a disubstituted pyrrole not belonging to the trichothecene group. Seven compounds were identified by spectroscopic methods as macrocyclic trichothecenes, namely, verrucarins A, B, M, and L acetate, roridin A, isotrichoverrin B, and trichoverrol B. The main metabolite produced by F. compactum was neosoloaniol monoacetate, a trichothecene. All the trichothecenes proved to be potent inhibitors of O. ramosa seed germination and possess strong zootoxic activity when assayed on Artemia salina brine shrimps. Verrucarin E is inactive on both seed germination and zootoxic assay.     

Effects of novel bioorganic fertilizer produced by Bacillus amyloliquefaciens W19 on antagonism of Fusarium wilt of banana

Banana production has been severely hindered by the long-term practice of monoculture agriculture. Fusarium wilt, caused by the Fusarium oxysporum f. sp. cubense (FOC), is one of the most destructive diseases that can afflict banana plants. It is both necessary and urgent to find an efficient method for protecting banana production worldwide. In this study, 57 antagonistic bacterial strains were isolated from the rhizospheres of healthy banana plants grown in a heavily wilt-diseased field; of the 57 strains, six strains with the best survival abilities were chosen for further study. Compared with the control and the other strains in the greenhouse experiment, W19 strain was found to observably decrease the incidence of Fusarium wilt and promote the growth of banana plants when combined with the organic fertilizer (OF). This strain was identified as Bacillus amyloliquefaciens based on its morphological, physiological, and biochemical properties, as well as 16S rRNA analysis. Two kinds of antifungal lipopeptides (iturin and bacillomycin D) produced by W19 strain were detected and identified using HPLC–ESI-MS. Another lipopeptide, called surfactin, was also produced by the thick biological film forming W19 strain. In addition to lipopeptides, 18 volatile antifungal compounds with significant antagonistic effect against F. oxysporum were detected and identified using gas chromatography–mass spectrometer (GC–MS). The work described herein not only highlights how the bioorganic fertilizer with B. amyloliquefaciens can be used to control Fusarium wilt of banana but also examines some of the potential mechanisms involved in the biocontrol of Fusarium wilt.     

Mycotoxins and other secondary metabolites produced in vitro by Penicillium paneum Frisvad and Penicillium roqueforti Thom isolated from baled grass silage in Ireland

Secondary metabolites produced by Penicillium paneum and Penicillium roqueforti from baled grass silage were analyzed. A total of 157 isolates were investigated, comprising 78 P. paneum and 79 P. roqueforti isolates randomly selected from more than 900 colonies cultured from bales. The findings mostly agreed with the literature, although some metabolites were not consistently produced by either fungus. Roquefortine C, marcfortine A, and andrastin A were consistently produced, whereas PR toxin and patulin were not. Five silage samples were screened for fungal metabolites, with two visually moldy samples containing up to 20 mg/kg of roquefortine C, mycophenolic acid, and andrastin A along with minor quantities (0.1-5 mg/kg) of roquefortines A, B, and D, festuclavine, marcfortine A, and agroclavine. Three visually nonmoldy samples contained low amounts of mycophenolic acid and andrastin A. The ability of both molds to produce a diverse range of secondary metabolites in vitro and in silage should be a concern to livestock producers.     

Cyanide production by rhizobacteria as a possible mechanism of plant growth inhibition

Summary Volatile metabolites from a number of rhizosphere pseudomonads prevented lettuce root growth in a seedling bioassay. One of these metabolites was identified as cyanide. Direct contact between rhizobacteria and plant roots produced, with one exception, similar responses. However, not all cyanogenic isolates were plant-growth-inhibitory rhizobacteria. When grown in liquid culture, cyanogenic strains produced an average of 37 nmol HCN ml^–1 over a 36-h period and inhibition of root growth occurred at concentrations as low as 20 nmol ml^–1. Cyanogenic strains introduced into sand or soil also produced HCN. Two cyanogenic strains ofPseudomonas fluorescens, one (5241) a plant-growth inhibitory rhizobacterium and the other (S97) a plant-growth-promotory rhizobacterium, were used to treat bean and lettuce seedlings prior to planting in soil. Lettuce dry weight was reduced by 49.2% (day 28) and 37.4% (day 49) when inoculated with S241 whereas S97 increased growth initially (+64.5% at day 28, no difference from control at day 49). Equivalent figures for inoculated bean plants were: –52.9% and –65.1% (5241); +40.7% and +23.3% (S97). A more detailed experiment using only bean plants confirmed these contrasting affects. Inhibition by S241 was related to consistently higher levels of rhizosphere cyanide in comparison with S97-treated plants and control soils. S241 also survived in the rhizosphere at higher densities and for a longer period of time than S97. The possible contribution of rhizobacterial cyanogenesis to plant growth inhibition is discussed.     

Structural analysis of a new cytotoxic demethylated analogue of neo-N-methylsansalvamide with a different peptide sequence produced by Fusarium solani isolated from potato

A novel cytotoxic cyclic pentadepsipeptide, neosansalvamide, was produced by Fusarium solani KCCM90040 isolated from Fusarium -contaminated potato in Korea. The molecular formula of neosansalvamide was analyzed as C??H??N?O? by electrospray ionization tandem mass spectrometry and combined structural analysis. The one- and two-dimensional nuclear magnetic resonance and absolute configuration of amino acid spectral data allowed for the resolution of cyclic five subunits linked in the following order: (S)-leucic acid, two L-leucine, L-valine, and L-phenylalanine, and this sequence shows a molecular structure as a new demethylated analogue of neo-N-methylsansalvamide but having a different peptide sequence. The cytotoxic effects of neosansalvamide were investigated by sulforhodamine B bioassay on four human cancer cell lines. The IC?? value of neosansalvamide required to inhibit cell growth in vitro by 50% for A549 (lung cancer), SK-OV-3 (ovarian cancer), SK-MEL-2 (skin melanoma), and MES-SA (uterine sarcoma) cell lines were 11.70 ± 0.55, 10.38 ± 0.64, 13.99 ± 1.32, and 11.75 ± 0.13 μM, respectively (mean ± standard error).     

Soil structures produced by tillage as affected by soil water content and the physical quality of soil

Tillage experiments were carried out in order to study the effect of water content on the aggregate size distribution produced by tillage, and to investigate the relationship between the soil structures produced by tillage and Dexter's index of soil physical quality, S. Tillage with a mouldboard plough was done on four different soils over a range of naturally occurring water contents. The aggregate size distribution and the specific surface area produced by tillage were obtained by sieving. We define the optimum water content for tillage, θ_OPT, as the water content at which the specific surface area of the aggregates produced is maximum. This is consistent with the water content at which the amount of small aggregates produced is greatest and the proportion of clods produced is smallest. For the four investigated soils, θ_OPT was found to be close to the water content at the inflection point of the water retention curve, and in the vicinity of 0.8θ_PL (where θ_PL is the lower plastic limit). At water contents either lower or higher than θ_OPT, the specific surface area produced was smaller. The specific surface area produced at θ_OPT was found to be strongly correlated with the index of soil physical quality, S. The specific surface area produced is larger the greater S, i.e. the better the soil physical quality. Consistently, the proportion of small aggregates produced at θ_OPT is larger and the proportion of clods produced at θ_OPT smaller, the greater S. No clods (>50 mm) are produced on soils with good physical quality.     

长期施用牛粪对松嫩平原盐渍化土壤质量的影响

在盐渍化土壤中,普遍存在土壤结构性和养分状况差以及土壤酶活性低等现象.这种现象主要是由于土壤中过量的交换性钠离子和较高的土壤pH所引起.长期施用有机肥(牛粪)是一项提升盐渍化土壤质量的重要措施.本研究依托东北农业大学盐碱土改良长期定位试验站,以腐熟的牛粪为改良材料,依据牛粪施用年限共设置5个处理,采用完全随机区组设计,每处理3次重复,供试作为玉米,各处理分别为:施用牛粪2年、6年、13年和18年,以不施用牛粪的盐渍化土壤作为对照.分别测定各处理的土壤理化指标和酶活性.采用因子分析法与土壤质量指数法评价长期施用有机肥对盐渍化土壤质量的影响.研究结果表明:长期施用有机肥能够改善盐渍化土壤的物理性状、提高土壤养分状况、降低土壤pH和盐分以及增加土壤酶活性.根据特征根>1原则,经因子分析后可提取2个公因子,分别表征"土壤结构性"(因子1)和"土壤盐碱性质"(因子2).与未施用有机肥的土壤相比,施用有机肥13年和18年的土壤具有较好的土壤结构性和较低的盐碱性质,而施用有机肥2年和6年的土壤仅具有较低的盐碱性质.比较土壤质量指数(SOI)可知:盐渍化土壤质量随有机肥施用年限而增加,有机肥施用18年处理的土壤质量最高.总之,土壤结构性差和盐碱性质高是影响松嫩平原盐渍化土壤质量的关键限制因子,其中以土壤结构性差最为主导;土壤容重、pH和盐分的降低是长期有机培肥措施下盐渍化土壤质量得以提升的重要特征,尤其是以土壤容重的降低最为重要.     

Use of cholinesterase activity in monitoring organophosphate pesticide exposure of cattle produced in tropical areas.

The use of cholinesterase activity as a biochemical method for monitoring organophosphate pesticide exposure in cattle is described herein. Determination of cholinesterase activity of whole blood, erythrocyte, and plasma was carried out according to the Ellman modified kinetic method. The mean baseline acetylcholinesterase activities of 9.549 +/- 3.619 IU/mL in whole blood, 9.444 +/- 3.006 IU/mL in erythrocytes, and 0.149 +/- 0.063 IU/mL in plasma were estimated for steers from the control group. Results of multivariate analysis showed that the general responses between the control and experimental groups (in vivo, monitoring and case studies) treated with Coumaphos and Fenthion were statistically different, and the general responses of these experimental groups were statistically different over time as well. Among the fractions that were analyzed, the erythrocyte acetylcholinesterase activity could be adequate for the diagnosis of exposure or acute poisoning in cattle as it showed a good within-run and between-run precision with CVs <10% better than those in plasma.     

Soil compaction in cropping systems: A review of the nature, causes and possible solutions

Soil compaction is one of the major problems facing modern agriculture. Overuse of machinery, intensive cropping, short crop rotations, intensive grazing and inappropriate soil management leads to compaction. Soil compaction occurs in a wide range of soils and climates. It is exacerbated by low soil organic matter content and use of tillage or grazing at high soil moisture content. Soil compaction increases soil strength and decreases soil physical fertility through decreasing storage and supply of water and nutrients, which leads to additional fertiliser requirement and increasing production cost. A detrimental sequence then occurs of reduced plant growth leading to lower inputs of fresh organic matter to the soil, reduced nutrient recycling and mineralisation, reduced activities of micro-organisms, and increased wear and tear on cultivation machinery. This paper reviews the work related to soil compaction, concentrating on research that has been published in the last 15 years. We discuss the nature and causes of soil compaction and the possible solutions suggested in the literature. Several approaches have been suggested to address the soil compaction problem, which should be applied according to the soil, environment and farming system.

The following practical techniques have emerged on how to avoid, delay or prevent soil compaction: (a) reducing pressure on soil either by decreasing axle load and/or increasing the contact area of wheels with the soil; (b) working soil and allowing grazing at optimal soil moisture; (c) reducing the number of passes by farm machinery and the intensity and frequency of grazing; (d) confining traffic to certain areas of the field (controlled traffic); (e) increasing soil organic matter through retention of crop and pasture residues; (f) removing soil compaction by deep ripping in the presence of an aggregating agent; (g) crop rotations that include plants with deep, strong taproots; (h) maintenance of an appropriate base saturation ratio and complete nutrition to meet crop requirements to help the soil/crop system to resist harmful external stresses.     

Acyl-homoserine lactones produced by Pantoea sp. isolated from the "maize white spot" foliar disease

The "maize white spot" foliar disease is a problem of increasing importance to Brazilian maize crops. A bacterium isolated from water-soaked lesions from infected maize leaves was pathogenic in biological assays in vivo. It was identified as a Gram-negative, nonsporulating, facultative anaerobic bacterium, belonging to the genus Pantoea. Chemical study of the extracts from bacterial cultivation media allowed the identification of (S)-(-)-N-butanoyl-homoserine lactone and trace amounts of N-hexanoyl-homoserine lactone, widely recognized quorum-sensing signaling substances employed in cell-to-cell communication systems. The absolute configuration of natural (S)-(-)-N-butanoyl-homoserine lactone was determined by gas chromatography-flame ionization detection with a chiral stationary phase and by comparison of circular dichroism spectroscopic data with enantiopure synthetic substances. Biological evaluations with reporter Agrobacterium tumefaciens NTL4(pZLR4) were carried out with synthetic and natural products and also with extracts from maize leaves contaminated with the isolated bacterium, as well as from healthy leaves.     

Control of Fusarium wilt disease of cucumber plants with the application of a bioorganic fertilizer

Two field experiments were conducted to evaluate the effect of organic fertilizer application either with or without antagonistic bacteria (Bacillus subtilis SQR-5 and Paenibacillus polymyxa SQR-21) on the control of Fusarium oxysporum f. sp. Cucumerinum J. H. Owen wilt disease in cucumber. The incidence of Fusarium wilt disease was 5.3–13.5% for cucumber plants treated with bioorganic fertilizer, while it was 30.3–51% in controls (only with organic fertilizer). Higher yields and lower disease incidences were observed in the dry season when compared with the wet season for both types of organic fertilizer treatments. Biolog analysis showed a significant change in soil bacterial composition and activity after bioorganic fertilizer application. The numbers of colony-forming units of F. oxysporum f. sp. Cucumerinum J. H. Owen for bioorganic-fertilizer-treated soils were significantly decreased compared with control. Scanning electron micrographs of cucumber basal stems showed a presence of mycelia-like mini strands accompanied by an amorphous substance within the xylem vessels. This amorphous substance and mini strands were richer in calcium and phosphorus but had low carbon and oxygen than the living mycelia. Reverse-phase high-pressure liquid chromatography and mass spectroscopic analysis showed that the antagonistic bacteria produced the antifungal compounds fusaricidin A, B, C, and D with molecular weights of 883.5, 897.5, 947.5, and 961.5 Da, respectively. The application of bioorganic fertilizer has a great potential for the control of F. oxysporum wilt disease in cucumber plants.     

Enhancement of trichothecene production in Fusarium graminearum by cobalt chloride

The effects of cobalt chloride on the production of trichothecene and ergosterol in Fusarium graminearum were examined. Incorporation experiments with (13)C-labeled acetate and leucine confirmed that both 3-acetyldeoxynivalenol and ergosterol were biosynthesized via a mevalonate pathway by the fungus, although hydroxymethyl-glutaryl CoA (HMG-CoA) from intact leucine was able to be partially used for ergosterol production. Addition of cobalt chloride at concentrations of 3-30 μM into liquid culture strongly enhanced 3-acetyldeoxynivalenol production by the fungus, whereas the amount of ergosterol and the mycelial weight of the fungus did not change. The mRNA levels of genes encoding trichothecene biosynthetic proteins (TRI4 and TRI6), ergosterol biosynthetic enzymes (ERG3 and ERG25), and enzymes involved in the mevalonate pathway (HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR)) were all strongly up-regulated in the presence of cobalt chloride. Precocene II, a specific trichothecene production inhibitor, suppressed the effects of cobalt chloride on Tri4, Tri6, HMGS, and HMGR, but did not affect erg3 and erg25. These results indicate that cobalt chloride is useful for investigating regulatory mechanisms of trichothecene and ergosterol production in F. graminearum.     

Impact of competitive fungi on Trichothecene production by Fusarium graminearum

Bioassays were used to determine the production of the trichothecene mycotoxin, deoxynivalenol (DON), by two isolates of Fusarium graminearum when grown in association with potentially competitive fungi and an antifungal chemical, 6-pentyl-alpha-pyrone (6PAP). The presence of 6PAP in the culture medium reduced DON production by as much as 80%, but this effect was reduced for the F. graminearum isolate that most efficiently metabolized the added 6PAP. A 6PAP-producing Trichoderma isolate grown in a competition assay system with the F. graminearum isolates was also able to substantially reduce DON production. When Fusarium isolates (F. crookwellense, F. culmorum, F. subglutinans, F. poae, F. equiseti, F. avenaceum, and F. sambucinum), which co-occur with F. graminearum in New Zealand maize plants (Zea mays), were grown in competition assays, the effect on DON production was variable. However, all isolates of F. subglutinans tested were shown to cause reductions in DON production (by 13-76%, mean = 62%). F. subglutinans frequently co-occurs with F. graminearum, but its presence can vary with location and time of the season. When the competitive fungus tested was also a trichothecene producer (e.g., of nivalenol), both toxins were produced in the assay medium. The results indicate that mycotoxin production by F. graminearum can be affected by the presence of particular competitive fungi. These results have implications for an ecological understanding of pathogenicity and of mycotoxin accumulation in plants. Early establishment of F. subglutinans, for example, may act as a biological control mechanism providing a temporary protection against invasion by more commonly toxigenic fusaria such as F. graminearum.     

Behavior of nitrogen in the media supplied with the aqueous phase produced by methane fermentation as liquid fertilizer

pp. 859–864

Behavior of nitrogen in the media was investigated when the aqueous phase produced by methane fermentation was supplied as liquid fertilizer.

When the aqueous phase was supplied to masa-soil,  included in masa-soil was nitrified to . Additionally, in masa-soil,  absorption was observed. On the other hand, in the case of coconut fiber and rockwool, has been hardly nitrified. From above results, it was determined that  toxic effect from the aqueous phase on tomato plants growth was inhibited by means of the progress of nitrification and  absorption by masa-soil.

However, nitrification was restrained so that  was fixed in masa-soil. On the other hand, in the sampled field soil,  was not fixed, and was nitrified immediately. In the field soil, there was a great deal of generated . In comparison with masa-soil.     

Behavior of nitrogen in the media supplied with the aqueous phase produced by methane fermentation as liquid fertilizer

pp. 859–864
Behavior of nitrogen in the media was investigated when the aqueous phase produced by methane fermentation was supplied as liquid fertilizer.
When the aqueous phase was supplied to masa-soil,      included in masa-soil was nitrified to      . Additionally, in masa-soil,       absorption was observed. On the other hand, in the case of coconut fiber and rockwool,     has been hardly nitrified. From above results, it was determined that       toxic effect from the aqueous phase on tomato plants growth was inhibited by means of the progress of nitrification and       absorption by masa-soil.
  However, nitrification was restrained so that       was fixed in masa-soil. On the other hand, in the sampled field soil,       was not fixed, and was nitrified immediately. In the field soil, there was a great deal of generated      . In comparison with masa-soil.