Fumonisin production in the maize pathogen Fusarium verticillioides: genetic basis of naturally occurring chemical variation

Fumonisins are polyketide-derived mycotoxins produced by the maize pathogen Fusarium verticillioides. Previous analyses identified naturally occurring variants of the fungus that are deficient in fumonisin C-10 hydroxylation or that do not produce any fumonisins. In the current study, gene deletion and genetic complementation analyses localized the C-10 hydroxylation deficiency to a cytochrome P450 monooxygenase gene in the fumonisin biosynthetic gene (FUM) cluster. Sequence analysis indicated that the hydroxylation deficiency resulted from a single nucleotide insertion that caused a frame shift in the coding region of the gene. Genetic complementation localized the fumonisin-nonproduction phenotype to the polyketide synthase gene in the FUM cluster, and sequence analysis indicated that the nonproduction phenotype resulted from a nucleotide substitution, which introduced a premature stop codon in the coding region. These results provide the first direct evidence that altered fumonisin production phenotypes of naturally occurring F. verticillioides variants can result from single point mutations in the FUM cluster.     

Fumonisin production by Fusarium verticillioides strains isolated from maize in Mexico and development of a polymerase chain reaction to detect potential toxigenic strains in grains

Fumonisins are mycotoxins produced by Fusarium verticillioides (Sacc. Nirenberg) in maize (Zea mays L.), a staple crop in Mexico. In this study, we report the isolation and identification of 67 Fusarium strains isolated from maize kernels collected in Northwest and Central Mexico. The strains were characterized regarding fumonisin B(1) production and the presence of the FUM1 gene. F. verticillioides was the predominant species isolated in both geographic regions, but the isolates from Northwest Mexico produced higher levels of fumonisin. A polymerase chain reaction (PCR)-based method, to detect a region of the FUM1 gene involved in fumonisin biosynthesis, was developed and employed to detect mycotoxigenic fungi in pure culture and in contaminated maize. The presence of the FUM1 gene was associated with fumonisin production in most isolates, except seven that did not synthesize fumonisin but contained the gene in their genome. The PCR method allowed the direct detection of fungal contamination in ground corn and could be employed to screen for the presence of potential mycotoxigenic fusaria.     

Fumonisin B(1)-nonproducing strains of Fusarium verticillioides cause maize (Zea mays) ear infection and ear rot

Fumonisins are polyketide mycotoxins produced by Fusarium verticillioides (synonym F. moniliforme), a major pathogen of maize (Zea mays) worldwide. Most field strains produce high levels of fumonisin B(1) (FB(1)) and low levels of the less-oxygenated homologues FB(2) and FB(3), but fumonisin B(1)-nonproducing field strains have been obtained by natural variation. To test the role of various fumonisins in pathogenesis on maize under field conditions, one strain producing FB(1), FB(2), and FB(3), one strain producing only FB(2), one strain producing only FB(3), and one fumonisin-nonproducing strain were applied to ears via the silk channel and on seeds at planting. Disease severity on the harvested ears was evaluated by visible symptoms and by weight percent symptomatic kernels. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of the applied FB(1)-nonproducing strains in kernels was determined by analysis of recovered strains for fumonisin production and other traits. All three FB(1)-nonproducing strains were able to infect ears following either silk-channel application or seed application at planting and were as effective as the FB(1)-producing strain in causing ear rot following silk-channel application. These results indicate that production of FB(1), FB(2), or FB(3) is not required for F. verticillioides to cause maize ear infection and ear rot.     

Incidence of Fusarium verticillioides and levels of fumonisins in corn from main production areas in Iran

A total of 52 corn samples collected in 2000 from four main corn production provinces of Iran (Fars, Kermanshah, Khuzestan, and Mazandaran) were analyzed for contamination with Fusarium verticillioides and fumonisins (FB(1), FB(2), FB(3), and 3-epi-FB(3)). The mean incidence of F. verticillioides (percent of kernels infected) for these four areas was 26.7, 21.4, 24.9, and 59.0%, respectively. The incidence in Mazandaran was significantly (p < 0.05) above that of the other areas. All samples from Mazandaran were contaminated with fumonisins with a mean level of total fumonisins of 10674 microg/kg. In contrast, the incidence of fumonisin contamination above 10 microg/kg was 53 (8/15), 42 (5/12), and 57% (8/14) in the samples from Fars, Kermanshah, and Khuzestan, respectively, and the corresponding mean total fumonisin levels were 215, 71, and 174 microg/kg, respectively. No statistical differences (p > 0.05) were observed in the fumonisin levels of the corn samples from these three provinces, which were significantly (p < 0.05) lower than the fumonisin contamination in samples from Mazandaran.     

Volatiles from Fusarium verticillioides (Sacc.) nirenb. and their attractiveness to nitidulid beetles

It is known that sap beetles (Coleoptera: Nitidulidae) can vector the fungus Fusarium verticillioides (Sacc.) Nirenb. (= F. moniliforme Sheldon), which causes an important ear-rot disease in corn and also produces fumonisin mycotoxins. The volatiles produced by this fungus were studied to establish whether they could attract sap beetles. Such an association would suggest more than just an incidental role in transmission of the fungus by the beetles. F. verticillioides consistently produces a blend of five alcohols (ethanol, 1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, and 2-methyl-1-butanol), acetaldehyde, and ethyl acetate. Ethanol is the most abundant alcohol. The fungus also produces four phenolic compounds (the most abundant of which is ethylguaiacol), a series of presently unidentified sesquiterpene hydrocarbons, and an unidentified compound that is probably a 10-carbon ketone. Solid-phase microextraction was the key technique used in volatile analysis. The volatile profiles change over time and differ somewhat among fungal strains: The alcohols, aldehyde, and ester always appeared first and were present for each strain. Production of the phenolics lagged by several days, and in some strains these compounds were barely detectable. Volatile production eventually diminished in all strains. All strains were attractive to the sap beetle, Carpophilus humeralis (F.), in wind-tunnel bioassays. Attraction was correlated primarily to the presence of the alcohols, acetaldehyde, and ethyl acetate, rather than to the phenolics. To verify that the identified culture volatiles were responsible for beetle attraction, cultures were quantitatively simulated with synthetic chemicals, and the cultures and corresponding synthetic mixtures were then compared by bioassay. The comparisons were favorable. Volatile emission patterns from cultures were fairly robust with respect to inoculum level or incubation temperature, but some manipulation was possible. For example, after freeze-drying and rehydrating (a rapid simulation of winter/spring conditions), F. verticillioides produced ethyl acetate and other esters at unusually high levels. The fungus produced attractive volatiles following ear inoculation of milk-stage field corn as well as on sterile, mature kernels in the laboratory.     

Fumonisin disruption of ceramide biosynthesis in maize roots and the effects on plant development and Fusarium verticillioides-induced seedling disease

The fungus Fusarium verticillioides infects maize and produces fumonisins, inhibitors of ceramide synthase. Seeds of the cultivar Silver Queen were inoculated with fumonisin-producing or non-fumonisin-producing strains of F. verticillioides. Leaf lesion incidence and severity of effects on root and stalk growth were significantly correlated with fumonisin in roots and disruption of sphingolipid metabolism in roots. Uninoculated seeds grown in soil watered with solutions of fumonisin B1 exhibited above-ground symptoms indicative of F. verticillioides-induced seedling disease and dose-dependent reduction in root mass that was inversely correlated with fumonisin B1, sphingoid bases, and sphingoid base 1-phosphates in roots. There was also evidence of an adaptive response to disrupted sphingolipid metabolism in both the virulence and watering assays, suggesting induction of pathways responsible for metabolism of sphingoid base 1-phosphates after prolonged exposure. The results suggest that fumonisin, and its effects on sphingolipids, could contribute to all aspects of F. verticillioides maize seedling disease.     

Fumonisins: probable role as effectors in the complex interaction of susceptible and resistant maize hybrids and Fusarium verticillioides

Fusarium verticillioides is best known for its worldwide occurrence on maize resulting in highly variable disease symptoms, ranging from asymptomatic to severe rotting and wilting and fumonisin production. The aim of this study was to investigate the influence of hybrid genotypes in the early stages of F. verticillioides infection, and the role of fumonisins as effectors in the outcome of this complex interaction. Disease symptoms, growth parameters, root morphology, and fungal colonization were evaluated at 7, 14, and 21 days after planting in seedlings from maize seeds of resistant (RH) and susceptible (SH) hybrids inoculated with F. verticillioides or watered with solutions of fumonisins. F. verticillioides induced growth enhancement or retardation depending on the plant genetic background and the fungal colonization rate, while fumonisins caused severe reduction in biomass and fitness. Seedlings watered with high fumonisin concentrations displayed lesions similar to those seen in F. verticillioides maize seedling disease, and also elicited inhibitory effects on root growth and morphology and on functional properties. In summary, these data strongly suggest a dual role for fumonisins in the F. verticillioides-maize interaction, acting as pathogenic factors at high concentrations, or triggering the plant detoxification mechanisms at low levels.     

Carbon dynamics determined by natural C abundance in microcosm experiments with soils from long-term maize and rye monocultures

Understanding carbon dynamics in soil is the key to managing soil organic matter. Our objective was to quantify the carbon dynamics in microcosm experiments with soils from long-term rye and maize monocultures using natural ¹³C abundance. Microcosms with undisturbed soil columns from the surface soil (0-25 cm) and subsoil (25-50 cm) of plots cultivated with rye (C₃-plant) since 1878 and maize (C₄-plant) since 1961 with and without NPK fertilization from the long-term experiment ‘Ewiger Roggen’ in Halle, Germany, were incubated for 230 days at 8 °C and irrigated with 2 mm 10⁻² M CaCl₂ per day. Younger, C₄-derived and older, C₃-derived percentages of soil organic carbon (SOC), dissolved organic carbon (DOC), microbial biomass (C_mic) and CO₂ from heterothropic respiration were determined by natural ¹³C abundance. The percentage of maize-derived carbon was highest in CO₂ (42-79%), followed by C_mic (23-46%), DOC (5-30%) and SOC (5-14%) in the surface soils and subsoils of the maize plots. The percentage of maize-derived C was higher for the NPK plot than for the unfertilized plot and higher for the surface soils than for the subsoils. Specific production rates of DOC, CO₂-C and C_mic from the maize-derived SOC were 0.06-0.08% for DOC, 1.6-2.6% for CO₂-C and 1.9-2.7% for C_mic, respectively, and specific production rates from rye-derived SOC of the continuous maize plot were 0.03-0.05% for DOC, 0.1-0.2% for CO₂-C and 0.3-0.5% for C_mic. NPK fertilization did not affect the specific production rates. Strong correlations were found between C₄-derived C_mic and C₄-derived SOC, DOC and CO₂-C (r≥0.90), whereas the relationship between C₃-derived C_mic and C₃-derived SOC, DOC and CO₂-C was not as pronounced (r≤0.67). The results stress the different importance of former (older than 40 years) and recent (younger than 40 years) litter C inputs for the formation of different C pools in the soil.     

Reflectance and Transmittance Spectroscopy Applied to Detecting Fumonisin in Single Corn Kernels Infected with Fusarium verticillioides

Reflectance and transmittance visible and near‐infrared spectroscopy were used to detect fumonisin in single corn kernels infected with Fusarium verticillioides. Kernels with >100 ppm and <10 ppm could be classed accurately as fumonisin positive or negative, respectively. Classification results were generally better for oriented kernels than for kernels that were randomly placed in the spectrometer viewing area. Generally, models based on reflectance spectra have higher correct classification than models based on transmittance spectra. Statistical analyses indicated that including near‐infrared wavelengths in calibrations improved classifications, and some calibrations were improved by including visible wavelengths. Thus, the color and chemical constituents of the infected kernel contribute to classification models. These results show that this technology can be used to rapidly and nondestructively screen single corn kernels for the presence of fumonisin, and may be adaptable to on‐line detection and sorting.     

Micronutrient availability in soils of Northwest Bosnia and Herzegovina in relation to silage maize production

Maize (Zea mays L.) is the most widely grown crop in Bosnia and Herzegovina especially in Northwest part of the country. Considering that, the maize is extremely sensitive to micronutrient deficiency the main aim of this study was to asses: (1) micronutrient availability in soil, (2) micronutrient status in silage maize; and (3) the relationship between micronutrient soil availability and maize plant concentration. Soil samples for micronutrient availability (n?=?112) were collected from 28 farms in 7 municipalities. Plant available micro- and macro- nutrients in soil were extracted using Mehlich-3, except plant available Se was extracted using 0.1M KH₂PO₄. Result showed that on average there was no significant difference between different soil types regarding their potential in plant available nutrients. P deficiency was present both, in soil and plants in whole region. Soil extractable P was ranging from 0.003–0.13?g?kg^?1 and total plant P was ranging from 0.79–4.95?g?kg^?1. Zinc deficiency was observed in two locations both in soil (0.71?mg?kg^?1; 0.79?mg?kg^?1) and plant (11.5?mg?kg^?1; 15.8?mg?kg^?1). Potential Se soil deficiency was observed on some locations, while Se plant status is not high enough to meet daily requirements of farm animals. Extractable soil nutrients could be used as relatively good predictor of potential soil and plant deficiencies, but soil nutrient interactions and climate conditions are highly effecting the plant uptake potential.     

Mobilization of phosphorus contributes to positive rotational effects of leguminous cover crops on maize grown on soils from northern Nigeria

Previous research has demonstrated a positive rotational effect of tropical leguminous cover crops on maize growth on a luvisol from Nigeria. This effect could not be explained by a better N supply. The objective of the present work was to further clarify whether improved P nutrition has been a contributing factor. Nine cover crops and maize were studied in nutrient solution‐culture with 1 and 20 μM P and with NO₃‐N as N source for root physiological parameters that may affect P mobilization. Zea mays, Lablab purpureus, and Centrosema pubescens responded to P deficiency by higher rates of proton excretion. Clitoria ternatea excreted OH^— with only small differences due to P nutrition. At low P supply, Chamaecrista rotundifolia, Clitoria, and Centrosema had the highest exudation rates of organic acid anions, especially citrate and malate. A major difference between plant species was found in root‐surface acid phosphatase activity. Cajanus cajan expressed the highest phosphatase activity. V_max of P uptake increased markedly under P deficiency, particularly for maize. Compared to the other plant species Cajanus, Chamaecrista, and Clitoria were characterized by a greater capability to absorb P at low external P concentrations. The nine cover crops and maize were also grown in pot experiments using two soils from northern Nigeria low in available P. The ultisol from Jos had a high P fixation capacity and was more acidic than the alfisol from Zaria. All plant species were precultured in the first season at 100 (Zaria) and 250 (Jos) mg P per pot. In the subsequent season, maize was uniformly seeded into all pots containing the remaining roots and the incorporated shoot dry mass (according to 15 mg P per pot) of the pre‐crop. No P and 250 (Zaria) or 500 (Jos) mg P per pot were freshly applied. Maize growth and P uptake were enhanced after legumes in Zaria soil. Cajanus showed the highest residual effect, and also Clitoria, Chamaecrista, and Lablab showed effects superior to the mean. In Jos soil, Clitoria, Cajanus, and Lablab enhanced maize growth above average. Also, a highly significant positive correlation between P uptake and biomass production was obtained. These results indicate that enhancement of maize growth after leguminous cover crops, at the low P supply of the soil used, mainly depends on P mobilization capacity of the cover crop.     

Role of maize hybrids and their chemical composition in Fusarium infection and fumonisin production

This study was designed to investigate the role of hybrids in maize Fusarium section Liseola interaction and fumonisin production, with particular emphasis on the occurrence and accumulation of hidden fumonisins in maize (masking phenomenon). In this 2 year study, naturally infected field crops were chosen with 10 maize hybrids, six of them grown in both years. Maize samples collected in 2010 showed a higher incidence of fungal infection as well as higher fumonisin contamination than those obtained in 2009 but a very similar incidence of F. section Liseola. Fumonisin masking was confirmed in raw maize, with a lower amount of hidden forms as compared to free fumonisins detected in the year with higher contamination. The chemical composition of the different hybrids was determined and correlated with the contamination data: the results obtained highlight the main role of fatty acids, with a higher fumonisin contamination in hybrids showing a higher linoleic acid content and a higher masking action in hybrids with higher oleic to linoleic ratio. These results represent a good basis to explain maize hybrid susceptibility to F. section Liseola infection, fumonisin contamination, and masking not related to a specific commercial hybrid but extendable to all hybrids.     

Mycorrhizal fungi and earthworms reduce antioxidant enzyme activities in maize and sunflower plants grown in Cd-polluted soils

Changes in plant antioxidant enzymes (AOEs) in response to cadmium (Cd) pollution are an important mechanism for plant growth and tolerance to Cd-induced stress. The main objective of this greenhouse study was to determine the combined influence of earthworm and arbuscular mycorrhiza (AM) fungal inoculation and their interactions with Cd on AOEs and proline accumulation in leaves of two major crops under Cd stress. Maize (Zea mays L.) and sunflower (Helianthus annuus L.) plants were exposed to Cd stress (10 and 20 mg kg⁻¹ soil), inoculated with either earthworm (Lumbricus rubellus L.) or AM fungi (Glomus intraradices and Glomus mosseae species) in a pot experiment for three months. Exposure to Cd decreased shoot dry weights, increased shoot Cd and P concentrations, leaf proline accumulation and the activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and polyphenol oxidase (PPO) in both mycorrhizal and non-mycorrhizal plants and both in the presence and absence of earthworms. Inoculation of both model plants with earthworms and AM fungi decreased shoot Cd concentrations and the activity of all AOEs, except PPO. Although earthworm activity enhanced the proline content of sunflower in Cd-polluted soils, the proline level of both plants remained unaffected by AM fungi. AM fungi and earthworms may decrease the activity of AOEs through a decline in shoot Cd toxicity and concentration, confirming that plant inoculation with these soil organisms improves maize and sunflower tolerance and protection against Cd toxicity. Generally, the effect of AM fungal inoculation on plant responses to Cd addition was greater than that of earthworm activity. Nonetheless, the interactive effect of AM fungus and earthworm is of minor importance for most of the plant AOEs in Cd-polluted soils.     

Organic compounds in soils and sediments from unlined waste disposal pits for natural gas production and processing

Samples of soil in dry waste pits and of sediment from waste pits currently in-use for natural gas production in the San Juan Basin of northwest New Mexico were collected for characterization of the presence and concentration of organic compounds. Samples were dried at 25 °C and about 50 g were extracted successively in a Soxhlet apparatus using hexane, benzene, and methanol. Extracts were filtered and condensed to 10 mL. Analyses of hexane extracts of samples using high-resolution gas chromatography showed presence of a complex mixture of 40 to 50 organic compounds in dry soil and in sediment from in-use pits. The range of size for compounds was C₁₀ to C₂₅. Although no compounds were detected in GC analyses of subsequent benzene and methanol extracts of the same soil samples, these extracts were highly colored and contained 5 to 10% of total absorptivity at 254 nm. Total extractable residues in the hexane extracts ranged from 49 to 110 mg g^?1 of soil. Polycyclic aromatic hydrocarbons were detected in hexane extracts at concentrations of 270 to 870μg g^?1 of dry sample.     

Variations in nitrogen,zinc, and sugar concentrations in Chinese fir seedlings grown on shrubland and plowed soils in response to arbuscular mycorrhizae-mediated process

One-year-old seedlings of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) are not colonized with ectomycorrhizal (EM) fungi, but often colonized with arbuscular mycorrhizal (AM) fungi. The AM fungi could be important for nutrient acquisition in Chinese fir seedlings. Previous studies show that feedbacks between aboveground and belowground plant tissues play a fundamental role in controlling the interaction between plants and AM fungi. Our results indicate significant feedback in seedlings grown on shrubland soils, but not on plowed soils. The amounts of sugar in fir leaves in the shrubland soils were significantly lower than those in plowed soils. Leaf zinc (Zn) and nitrogen (N) concentrations were significantly higher in seedlings in shrubland soils than in plowed soils. In mycorrhizal seedlings growing in shrubland soils, leaf N:P ratios were significantly higher than those in plowed soils, likely because of enhanced N absorption through AM-mediated process. Leaf N:P ratios in seedlings grown on plowed soils were below the threshold levels, because of low metabolic activity of feedback in AM-mediated process. The results suggested that the presence of feedback between Chinese fir seedlings and AM fungi should be benefit in transplanting Chinese fir seedlings.     

Long chain alkanes in silk extracts of maize genotypes with varying resistance to Fusarium graminearum

The alkane content of the silks of nine maize genotypes was analyzed to investigate the role of silk wax in resistance to Fusarium graminearum. Silk samples were collected 2, 4, 6, and 8 days after silk emergence and divided into three sections: exposed silk, silk channel silk, and silk that is under the husk and overlying the kernels. Four major unbranched alkanes (C(25), C(27), C(29), and C(31)) and three isoalkanes (C(27i), C(29i), and C(31i)) were identified. Total alkane contents were highest in the exposed silk followed by the silk channel silk, with the lowest in the youngest silk closest to the kernels. In the silk channel and overlying kernel silks, the moderately resistant inbred CO272 consistently had the highest alkane content. None of the other inbreds with improved resistance had as high a level of alkanes as CO272, indicating that alkane content is not a major mechanism of resistance.     

Differences in the growth and ectomycorrhizal community of Dryobalanops lanceolata (Dipterocarpaceae) seedlings grown in ultramafic and non-ultramafic soils

Ultramafic soils have naturally high concentrations of metals and are often low in major plant nutrients. Plant species of non-ultramafic origin, such as Dryobalanops lanceolata (Dipterocarpaceae), generally grow less well on these soils. I found minimal changes in growth, but a 17% reduction in foliar potassium, when seedlings of D. lanceolata were grown in a non-native ultramafic soil when compared with a ‘normal’ tropical ultisol. There were, however, marked changes in the ectomycorrhizal community structure on the roots of D. lanceolata. Cenococcum geophilum was at least 10 times more common and Inocybe sp. was one and a half times more common in non-ultramafic soils, whereas Boletales sp. was over 30 times more common in the non-ultramafic soil. These changes may have been brought about by a number of edaphic differences between the two soil types, including high metal concentrations and differences in organic matter content.     

Elemental content relationships in greenhouse grown apple seedlings supplemented with copper and peat

Abstract

Apple seedlings from a greenhouse pot experiment, investigating the effects of copper (Cu) and peat amendments on crop performance growing on a Cu‐and potassium (K)‐deficient soil, were characterized for a range of major and trace elements. Concentrations of barium (Ba), calcium (Ca), Cu, iron (Fe), K, magnesium (Mg), manganese (Mn), molybdenum (Mo), sodium (Na), rubidium (Rb), strontium (Sr), and zinc (Zn) in leaf and stem tissues were correlated with treatment and tissue. Addition of Cu, peat and the nature of the tissue had significant impacts on many element concentrations. Generally, increasing Cu resulted in elevated Ba, Fe, Mo, and Sr as well as Cu levels. The presence of peat resulted in reduced levels, generally in both leaf and stem, of Ba, Mg, Mn, Rb, and Zn and increased levels of Fe, K, and Mo. Finally, the vast majority of elemental concentrations were higher in leaf tissue rather than stem, with the exceptions of Na and Zn. Elemental concentration ranges, over all tissues and conditions of added Cu and peat were (mg kg^‐1) Ba 9–49, Ca 6380–16340, Cu 2–11, Fe 10–57, K 4070–16950, Mg 900–4260, Mn 22–197, Mo 0.02–0.19, Na 28–124, Rb 0.7–12, Sr 41–58, Zn 18–48.     

Predicting bioavailability of PAHs in soils to wheat roots with triolein-embedded cellulose acetate membranes and comparison with chemical extraction

Triolein-embedded cellulose acetate membrane (TECAM) was buried in 15 field-contaminated soils in parallel with the cultivation of wheat to predict bioavailability of naphthalene, phenanthrene, pyrene, and benzo[a]pyrene to wheat roots, and the method was compared with chemical extraction methods. Although a good linear relationship was found between PAH concentrations in chemical extractants and wheat roots, the percentage of PAH in soil removed by chemical extraction was much higher than the corresponding percentage removed by wheat roots. In contrast to chemical extraction, a nearly 1:1 relationship was found between the amount of each PAH taken up by TECAMs and wheat roots (r(2) = 0.798-0.925, P < 0.01). Furthermore, the uptake of PAHs by TECAMs and wheat roots had the same pathway of passive transport via the soil solution. Moreover, TECAM caused minimal disturbance to the soil and was easy to deploy. Therefore, TECAM is believed to be a useful tool to predict bioavailability of PAHs to wheat roots grown in contaminated soils.