Progression of mycotoxin and nutrient concentrations in wheat after inoculation with Fusarium culmorum

The objective of this study was to follow the mycotoxin formation and changes in nutrient composition of wheat (cv. Ritmo) artificially inoculated with Fusarium culmorum. From anthesis until harvest, samples were taken once a week from the inoculated and control plots. The investigations were focused on monitoring the progression of the contamination of the wheat kernels with deoxynivalenol (DON) and zearalenone (ZON). Both the uncontaminated control kernels and the contaminated kernels were examined also for the presence of zearalenone-4-beta-D-glucopyranoside and several trichothecenes at harvest. Furthermore, the impact of the Fusarium inoculation on some nutrients as starch, crude protein, amino acid composition, crude ash, non starch polysaccharides (NSP) as well as viscosity and thousand seed weight (TSW) was examined. Also proteolytic and amylolytic activity as well as the NSP-degrading enzyme activities of inoculated and control samples were analysed at the time of harvest. DON was detected in higher concentrations and in earlier stages, while ZON was found later and in smaller amounts. On average 7.79?mg/kg DM of DON and 100?μg/kg DM of ZON were found in the inoculated kernels at the time of harvest. Neither in the contaminated nor in the control samples glucose conjugates of ZON (Zearalenone-4-beta-D-glucopyranoside) were detected. Moreover, the infection with Fusarium culmorum had pronounced effects on some quality parameters. The crude protein content of the inoculated kernels showed significantly higher values over the whole period compared to the control kernels. The protein content of the inoculated kernels amounted 13.9% DM at harvest, while only a concentration of 12.5% DM was detected in the control samples. Similarly, in almost all stages of development the crude ash content of inoculated samples was higher than in control samples. These distinct differences in kernel composition resulted possibly from the changes of the thousand seed weight. In the present work the grain harvested from the control plots showed a significantly higher TSW (24.2?g) as compared to their inoculated counterparts (15.5?g). Despite lower extract viscosity of inoculated samples at time of harvest, the content of soluble NSP of inoculated plots was higher than in control samples at the same time. Moreover, inoculation resulted in markedly increased activities of protease, amylase and several NSP-degrading enzyme activities. This would suggest that the cell wall penetrating properties of the fungus itself and/or that the fungus induced alterations of the metabolic activity of the embryo or other constituents of the wheat kernel could be responsible.     

Progression of mycotoxin and nutrient concentrations in wheat after inoculation with Fusarium culmorum

The objective of this study was to follow the mycotoxin formation and changes in nutrient composition of wheat (cv. Ritmo) artificially inoculated with Fusarium culmorum. From anthesis until harvest, samples were taken once a week from the inoculated and control plots. The investigations were focused on monitoring the progression of the contamination of the wheat kernels with deoxynivalenol (DON) and zearalenone (ZON). Both the uncontaminated control kernels and the contaminated kernels were examined also for the presence of zearalenone-4-beta-D-glucopyranoside and several trichothecenes at harvest. Furthermore, the impact of the Fusarium inoculation on some nutrients as starch, crude protein, amino acid composition, crude ash, non starch polysaccharides (NSP) as well as viscosity and thousand seed weight (TSW) was examined. Also proteolytic and amylolytic activity as well as the NSP-degrading enzyme activities of inoculated and control samples were analysed at the time of harvest. DON was detected in higher concentrations and in earlier stages, while ZON was found later and in smaller amounts. On average 7.79 mg/kg DM of DON and 100 microg/kg DM of ZON were found in the inoculated kernels at the time of harvest. Neither in the contaminated nor in the control samples glucose conjugates of ZON (Zearalenone-4-beta-D-glucopyranoside) were detected. Moreover, the infection with Fusarium culmorum had pronounced effects on some quality parameters. The crude protein content of the inoculated kernels showed significantly higher values over the whole period compared to the control kernels. The protein content of the inoculated kernels amounted 13.9% DM at harvest, while only a concentration of 12.5% DM was detected in the control samples. Similarly, in almost all stages of development the crude ash content of inoculated samples was higher than in control samples. These distinct differences in kernel composition resulted possibly from the changes of the thousand seed weight. In the present work the grain harvested from the control plots showed a significantly higher TSW (24.2 g) as compared to their inoculated counterparts (15.5 g). Despite lower extract viscosity of inoculated samples at time of harvest, the content of soluble NSP of inoculated plots was higher than in control samples at the same time. Moreover, inoculation resulted in markedly increased activities of protease, amylase and several NSP-degrading enzyme activities. This would suggest that the cell wall penetrating properties of the fungus itself and/or that the fungus induced alterations of the metabolic activity of the embryo or other constituents of the wheat kernel could be responsible.     

Methicillin-resistant Staphylococcus aureus ST398 contamination and transmission in pigs after a low dose inoculation

Aims: Methicillin‐resistant Staphylococcus aureus (MRSA) ST398 has recently been described as a zoonotic agent. Its transmission between animals seems to be a pivotal factor in its emergence and dissemination. This experimental trial was performed to describe MRSA ST398 contamination and transmission in pigs after a low dose inoculation. Methods and results: Twelve specific pathogen‐free (SPF) pigs were randomly divided between two separate pens. Three pigs in each pen received a nasal inoculation of 2 × 10⁴ colony‐forming units per animal, and three naïve pigs were left in contact with them. Every 2 days and at necropsy, different samples were screened for MRSA. It was detected in nasal swabs from five inoculated and three naïve contact pigs, as early as 1 day after inoculation. MRSA was also found in environmental wipes but never in faecal samples. At necropsy, MRSA was detected in the lymph nodes of two contact pigs and in the tonsils and lymph nodes of three inoculated pigs. Twelve other SPF pigs were included as negative control in a separate room. Conclusion: This experiment showed that inoculation of a low dose of MRSA ST398 could lead to the horizontal transmission of the bacterium between pigs, the contamination of mandibular lymph nodes and the contamination of the environment without faecal carriage. Significance and Impact of the Study: The minimal inoculated dose via nasal route to observe transmission of MRSA ST398 between pigs is equal or lower to 2 × 10⁴ colony‐forming units per animal, and faecal excretion seems not to be a necessary condition for horizontal transmission.     

Incidence and levels of fumonisin contamination in Colombian corn and corn products

A survey was conducted to determine the levels of fumonisins B1 and B2 in corn and corn-based products available in Colombia for human and animal consumption. A total of 120 samples were analyzed by acetonitrile-water extraction, cleanup with a strong-anion-exchange column, and liquid chromatography with o-phthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. The samples of corn and corn-based products for animal intake were taken at different feed manufacturing plants, whereas the samples used for human foods where purchased from local retail stores. The number of positive samples for fumonisin B1 was 20.0% higher in corn and corn-based products for animal intake (75.0%) than in corn and corn-based products for human consumption (55.0%). The levels of fumonisin B1 were also higher in corn and corn-based products for animal intake (mean = 694 μg/kg; range = 32–2964 μg/kg), than in corn and corn-based products for human intake (mean = 218 μg/kg; range = 24–2170 μg/ kg). The incidence and levels of fumonisin B2 were lower than those for fumonisin B1. Corn and corn-based products for animal consumption had an incidence of fumonisin B2 of 58.3%, with a mean value of 283 μg/kg, and a range of 44–987 μg/kg. The incidence of fumonisin B2 in corn-based products for human intake was 35.0%, with a mean value of 118 μg/kg and a range of 21–833 μg/kg. The highest incidence and levels of fumonisins were found in samples of hominy feed, with concentrations ranging from 86 to 2964 μg/kg fumonisin B1 and 57 to 987 μg/kg fumonisin B2.     

Mould and mycotoxin contamination of stored corn in Turkey

A total of 167 corn samples, including imported and locally grown corn, were obtained from various regions and store houses in Turkey and surveyed for mould occurrence and mycotoxin content. The mould contamination level was 10⁵ — 10⁶ colonies/g.A. flavus, A. niger, F. oxyporum, P. variable, andRhizopus spp. However, the dominant flora showed significant differences between the imported and domestic corns. Afiatoxin B₁ was found in 16 % of the samples ranging from 2–74μg/kg. Ochratoxin A and sterigmatocystin were found at minimum detection levels. Mycotoxin production characteristics of mould isolates were also determined.     

Effects of wheat plant inoculation with cytokinin-producing microorganisms on plant growth at increasing level of mineral nutrition

Triticum durum Desf. plants were grown for 11 days in sand culture on nutrient solutions with optimum or lowered content of mineral nutrients. Thereafter, the level of mineral nutrition was increased in some portion of deficient plants. Two days before, plants were inoculated with cytokinin-producing microorganisms of the Bacillus genus. Nutrition deficiency resulted in a decrease in the rate of plant biomass accumulation, which was correlated with the level of active cytokinins in both roots and shoots. After improving the mineral nutrition of noninoculated plants, the rate of their biomass accumulation increased and, by the end of experiment, their shoot fresh weight was 1.5-fold higher than in deficient plants; however, it was still by 20% lower than in plants continuously growing at optimum mineral nutrition. Inoculation resulted in the considerable increase in the cytokinin content in shoots as compared with all other treatments. In this case, after the improvement of plant mineral nutrition, the rates of growth and relative biomass accumulation increased sharply; as a result, these plants had the highest dry and fresh weights. Thus, inoculation with cytokinin-producing bacteria was beneficial for plant growth after their transfer from deficient to sufficient mineral nutrition.     

Resistance to Wheat streak mosaic virus generated by expression of an artificial polycistronic microRNA in wheat

Wheat streak mosaic virus (WSMV) is a persistent threat to wheat production, necessitating novel approaches for protection. We developed an artificial miRNA strategy against WSMV, incorporating five amiRNAs within one polycistronic amiRNA precursor. Using miRNA sequence and folding rules, we chose five amiRNAs targeting conserved regions of WSMV but avoiding off-targets in wheat. These replaced the natural miRNA in each of five arms of the polycistronic rice miR395, producing amiRNA precursor, FanGuard (FGmiR395), which was transformed into wheat behind a constitutive promoter. Splinted ligation detected all five amiRNAs being processed in transgenic leaves. Resistance was assessed over two generations. Three types of response were observed in T(1) plants of different transgenic families: completely immune; initially resistant with resistance breaking down over time; and initially susceptible followed by plant recovery. Deep sequencing of small RNAs from inoculated leaves allowed the virus sequence to be assembled from an immune transgenic, susceptible transgenic, and susceptible non-transgenic plant; the amiRNA targets were fully conserved in all three isolates, indicating virus replication on some transgenics was not a result of mutational escape by the virus. For resistant families, the resistance segregated with the transgene. Analysis in the T(2) generation confirmed the inheritance of immunity and gave further insights into the other phenotypes. Stable resistant lines developed no symptoms and no virus by ELISA; this resistance was classified as immunity when extracts failed to transmit from inoculated leaves to test plants. This study demonstrates the utility of a polycistronic amiRNA strategy in wheat against WSMV.     

Resistance of wheat genotypes to boron toxicity is expressed at the cellular level

The effects of toxic boron (B) concentrations on the growth of wheat genotypes at organ level and cellular level were investigated using excised root culture techniques. At the organ level, wheat genotypes differed for root elongation and lateral root development in response to toxic B concentrations. Genotypes classified as resistant from field studies had longer root axes and more lateral roots than sensitive or moderately sensitive genotypes, but there was no difference in axis elongation between sensitive and moderately sensitive genotypes. At the cellular level, callus production of root explants among genotypes still different at toxic B concentrations: resistant genotypes could produce more callus than sensitive or moderately sensitive genotypes. These results suggest that differences among genotypes in resistance to toxic B concentrations may be related to cell membrane permeability to B since they were also expressed in undifferentiated callus cells. The distinct and consistent differences among genotypes in response to B toxicity both at the organ level and at the cellular level could serve as a basis for selection in a breeding program. The methods were precise and efficient.     

Subcellular localization of proteases in wheat and corn mesophyll protoplasts

Mesophyll protoplasts were isolated from the leaves of wheat and corn seedlings. After purification the protoplasts were judged to be free of contaminating proteases in the isolation enzymes based on specific activity of the proteases in comparison to leaf tissue and their response to inhibitors that “differentiated” between leaf and isolation enzyme proteases. Wheat protoplasts showed rates of photosynthesis of 95 to 100 micromoles O₂ per milligram chlorophyll per hour, while corn exhibited rates of 35 to 85 micromoles O₂ per milligram chlorophyll per hour, indicating the intactness of the chloroplasts within the protoplasts. These chloroplasts were isolated from the protoplasts using the procedure of Robinson and Walker (1979 Arch Biochem Biophys 196: 319-323). Yields of 91 and 82% intact chloroplasts were obtained from wheat and corn, respectively, based on the distribution of ribulose bisphosphate carboxylase in wheat and NADP-malate dehydrogenase in corn. Vacuoles were obtained from the protoplasts using a modification of the techniques of Wagner and Siegelman (1975 Science 190: 1298-1299) and Saunders (1979 Plant Physiol 64: 74-78). The vacuoles were at least 98% free of protoplast contamination as determined by assaying for “marker” enzymes of chloroplasts, mitochondria, and endoplasmic reticulum. Assuming one vacuole per protoplast, the vacuoles contained 4% of the soluble protein of the protoplasts in wheat and 8% in corn. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuoles. Although the isolated chloroplasts always retained about 13% of the proteolytic activity of the protoplasts, this could be accounted for by that which became associated with the chloroplasts during their isolation.     

Influence of aFusarium culmorum inoculation of wheat onin sacco dry matter degradation of wheat straw and wheat chaff

The aim of this study was to investigate the effect of aFusarium culmorum inoculation of wheat on thein sacco dry matter degradation (DG) of wheat straw and wheat chaff in dairy cows. The ruminal disappearance of dry matter was measured with thein situ nylon bag technique. Samples of wheat straw and wheat chaff from non-inoculated andFusarium-inoculated wheat were used to examine the ruminal dry matter degradability. Samples were subjected to ruminal incubation in two dairy cous fitted with a permanent rumen fistula and incubated for 4, 8, 16, 24, 48, 72, 96 and 120 h. To describe the degradation kinetics, the equation by Ørskov and McDonald (1979) was used. DG rates obtained for contaminates straw and chaff were higher compared to the corresponding rates of the non-contaminated samples, which is assumed to be due to the activity of fungal enzymes. It can be concluded that an infection of wheat withF. culmorum may have an influence on the dry matter degradation of straw and chaff.     

Biocontrol of aflatoxin in corn by inoculation with non-aflatoxigenic Aspergillus flavus isolates

The ability of two non-aflatoxigenic Aspergillus flavus Link isolates (CT3 and K49) to reduce aflatoxin contamination of corn was assessed in a 4-year field study (2001–2004). Soil was treated with six wheat inoculant treatments: aflatoxigenic isolate F3W4; two non-aflatoxigenic isolates (CT3 and K49); two mixtures of CT3 or K49 with F3W4; and an autoclaved wheat control, applied at 20 kg ha^?1. In 2001, inoculation with the aflatoxigenic isolate increased corn grain aflatoxin levels by 188% compared to the non-inoculated control, while CT3 and K49 inoculation reduced aflatoxin levels in corn grain by 86 and 60%, respectively. In 2002, the non-toxigenic CT3 and K49 reduced aflatoxin levels by 61 and 76% compared to non-inoculated controls, respectively. In 2001, mixtures of aflatoxigenic and non-aflatoxigenic isolates had little effect on aflatoxin levels, but in 2002, inoculation with mixtures of K49 and CT3 reduced aflatoxin levels 68 and 37% compared to non-inoculated controls, respectively. In 2003 and 2004, a low level of natural aflatoxin contamination was observed (8 ng g^?1). However, inoculation with mixtures of K49?+?F3W4 and CT3?+?F3W4, reduced levels of aflatoxin 65–94% compared to the aflatoxigenic strain alone. Compared to the non-sclerotia producing CT3, strain K49 produces large sclerotia, has more rapid in vitro radial growth, and a greater ability to colonize corn when artificially inoculated, perhaps indicating greater ecological competence. Results indicate that non-aflatoxigenic, indigenous A. flavus isolates, such as strain K49, have potential use for biocontrol of aflatoxin contamination in southern US corn.     

Depolarization of transmembrane potential of corn and wheat coleoptiles under reduced water potential and after IAA application

The influence of IAA and temporarily reduced water potentialon the transmembrane potential (PD) of corn and wheat coleoptilesections was investigated. The studies were performed by twodifferent methods: discontinuous PD measurements with microelectrodesin corn coleoptile sections and continuous measurements withmicroelectrodes in wheat coleoptile sections. Both methods showeda very rapid depolarization of PD within a period of 2 to 5min. In corn coleoptile sections, a PD oscillation was observedwithin an average period of 14.5 min after IAA application andof 14.0 min after reduction of water potential. The PD amplitudewas about 20 to 25 mV. In wheat coleoptile sections, an oscillationof PD could not be definitely established. After an IAA-induceddepolarization period of 4 to 7 min, the initial potential sometimeswas restored, while in some other cases, a subsequent hyperpolarizationwas found although in most cases the depolarization continued.The discussion covers the possibility that a decrease in PDcaused by IAA and water stress may induce activation of H⁺ secretionby a plasmalemma H₊ pump. (Received November 24, 1978; )     

Cefotaxime prevents microbial contamination and improves microspore embryogenesis in wheat and triticale

Key message

Cefotaxime (100 mg/l) mitigate occasional gram negative bacterial contamination in wheat and triticale microspore culture and most importantly it increases cell growth and green plant production.

Abstract

Isolated microspore culture is a promising option to rapidly fix the product of meiotic recombination of F₁ hybrids, in the process of varietal development. Clean culture and high embryogenesis rate are essential to commercial triticale and wheat microspore cultures. So, this study investigated (1) contaminants from isolated microspores cultures, (2) two antibiotics to control bacterial growth, and (3) the contribution of antibiotics to increased microspore-derived embryo-like structures (ELS), green and albino plants. Five species of bacteria were identified in contaminated cultures (Erwinia aphidicola, Pantoea agglomerans, Pseudomonas sp., Staphylococcus epidermis and Staphylococcus warneri) using fatty acid analysis and 16S ribosomal RNA sequences analysis, and yeast. Antibacterial susceptibility test using Cefotaxime and Vancomycin resulted in strong inhibition of 24 bacterial isolates, using Cefotaxime at 100 mg/l, but not Pseudomonas sp. Other antibiotic treatments inhibited bacterial growth at least partially. Microspore induction medium supplemented with the same antibiotics treatments resulted in successful microspore embryogenesis and green plant production. Antibiotic treatments were first tested in triticale and then validated in wheat cultivars AC Carberry and AC Andrew. Induction medium supplemented with Cefotaxime at 50 and 100 mg/l substantially increased the formation of ELS and green plants in triticale and wheat, respectively. Incidentally, it also affected the occurrence of albinism in all genotypes. Our results demonstrated dual purpose of Cefotaxime for isolated microspore culture, most importantly it increases cell growth and success of microspore cultures in triticale and wheat genotypes, but would also prevent accidental loss of cultures with most common bacterial contaminants.     

Fate of Escherichia coli during ensiling of wheat and corn

A recombinant Escherichia coli strain carrying a plasmid with an antibiotic resistance marker and expressing the green fluorescent protein was inoculated at a concentration of 3.8 x 10(8) CFU/g into direct-cut wheat (348 g of dry matter kg(-1)), wilted wheat (450 g of dry matter kg(-1)), and corn (375 g of dry matter kg(-1)). The forages were ensiled in mini-silos. The treatments included control (no E. coli added), application of tagged E. coli, and delayed sealing of the inoculated wheat. Three silos per treatment were sampled on predetermined dates, and the numbers of E. coli were determined on Chromocult TBX medium with or without kanamycin. Colonies presumptively identified as E. coli were also tested for fluorescence activity. Addition of E. coli at the time of ensiling resulted in a more rapid decrease in the pH but had almost no effect on the chemical composition of the final silages or their aerobic stability. E. coli disappeared from the silages when the pH decreased below 5.0. It persisted longer in silages of wilted wheat, in which the pH declined more slowly. Control silages of all crops also contained bacteria, presumptively identified as E. coli, that were resistant to the antibiotic, which suggests that some epiphytic strains are naturally resistant to antibiotics.     

Drought stress amelioration in wheat through inoculation with Burkholderia phytofirmans strain PsJN

Plant growth promoting endophytic bacteria Burkholderia phytofirmans PsJN was used to investigate the potential to ameliorate the effects of drought stress on growth, physiology and yield of wheat (Triticum aestivum L.) under natural field conditions. Inoculated and uninoculated (control) seeds of wheat cultivar Sahar 2006 was sown in the field. The plants were exposed to drought stress at different stages of growth (tillering stage and flowering stage) by skipping the respective irrigation. The results showed that drought stress adversely affected the physiological, biochemical and growth parameters of wheat seedlings. It decreased the CO₂ assimilation, stomatal conductance, relative water content, transpiration rate and chlorophyll contents in wheat. Inoculation of wheat with PsJN significantly diluted the adverse effects of drought on relative water contents and CO₂ assimilation rate thus improving the photosynthetic rate, water use efficiency and chlorophyll content over the uninoculated control. Grain yield was also decreased when plants were exposed to drought stress at the tillering and flowering stage, but inoculation resulted in better grain yield (up to 21 and 18 % higher, respectively) than the respective uninoculated control. Similarly, inoculation improved the ionic balance, antioxidant levels, and also increased the nitrogen, phosphorus, potassium and protein concentration in the grains of wheat. The results suggested that B. phytofirmans strain PsJN could be effectively used to improve the growth, physiology and quality of wheat under drought conditions.     

小麦抗白粉病基因

到目前为止,小麦中已经鉴定出31个主效抗白杨病基因位点(Pm1-Pm31),对这些小麦抗白粉病基因位点的来源、染色体定位、遗传特点以及载体品种等方面进行了概括性综述。