Potential use of lemongrass essential oil as fungicide against Aspergillus brasiliensis and as post-harvest protectant of wheat

Fungi are among the main responsible for damage and loss in stored grains, its control has been done through synthetic substances, which are harmful to man and the environment. Brazil, one of the leading countries in agriculture, has optimal environmental conditions for the development of mycotoxigenic fungi. Most of the synthetic chemicals used as preservatives have often been realized to be toxic to humans and also cause adverse environmental effects. Thus, it is necessary to search for alternative methods of controlling. In this study the aimed was to evaluate the efficacy of lemongrass essential oil in the control of the Aspergillus brasiliensis. In vitro and serial microdilution tests were carried out at different concentrations of essential oil and citral, which corresponds to 72% of the total oil composition. Inhibition of fungal growth on contaminated wheat grain was evaluated. The in vitro test results showed that the essential oil has fungicidal potential at concentrations from 0.6 956;L mL-1, the minimum inhibitory concentration was determined at 0.8 956;L mL-1. The tests with citral showed fungal control at concentrations from 0.6 956;L mL-1 onwards. For wheat grain, fungal growth inhibition was obtained at the concentration of 1.6 956;L mL-1. The essential oil of Cymbopogon flexuosus showed fungicidal activity against the fungus Aspergillus brasiliensis.

Impact of phosphate solubilizing bacteria on wheat (Triticum aestivum ) in the presence of pesticides / Impacto das bactérias solubilizantes de fosfato no trigo (Triticum aestivum ) na presença de pesticidas

Abstract Three phosphate solubilizing bacteria were isolated and identified by 16S rRNA sequencing as Pseudomonas putida, Pseudomonas sp and Pseudomonas fulva . The strains were subjected to plant biochemical testing and all the PGPR attributes were checked in the presence of pesticides (chlorpyrifos and pyriproxyfen). The phosphate solubilizing index of strain Ros2 was highest in NBRIP medium i.e 2.23 mm. All the strains showed acidic pH (ranges from 2.5-5) on both medium i.e PVK and NBRIP. Strain Ros2 was highly positive for ammonia production as well as siderophore production while strain Rad2 was positive for HCN production. The results obtained by the strains Rad1, Rad2 and Ros2 for auxin production were 33.1, 30.67 and 15.38 µg ml-1, respectively. Strain Rad1 showed 16% increase in percentage germination in comparison to control in the presence of pesticide stress. Most promising results for chlorophyll content estimation were obtained in the presence of carotenoids upto 6 mgg-1 without stress by both strains Rad1 and Rad2. Study suggests that especially strain Ros2 can enhance plant growth parameters in the pesticide stress.

Resumo Três bactérias solubilizantes de fosfato foram isoladas e identificadas por seqüenciamento de rRNA 16S como Pseudomonas putida, Pseudomonas sp e Pseudomonas fulva. As estirpes foram submetidas a testes bioquímicos de plantas e todos os atributos PGPR foram verificados na presença de pesticidas (clorpirifos e piriproxifeno). O índice de solubilização de fosfato da estirpe Ros2 foi mais elevado no meio NBRIP, isto é, 2,23 mm. Todas as estirpes apresentaram um pH ácido (varia de 2,5-5) em ambos os meios, isto é PVK e NBRIP. A estirpe Ros2 foi altamente positiva para a produção de amoníaco, bem como a produção de sideróforos enquanto a estirpe Rad2 foi positiva para a produção de HCN. Os resultados obtidos pelas estirpes Rad1, Rad2 e Ros2 para a produção de auxina foram 33,1, 30,67 e 15,38 μg ml-1 , respectivamente. A deformação Rad1 mostrou aumento de 16% na germinação percentual em comparação com o controlo na presença de stress de pesticida. Os resultados mais promissores para a estimativa do teor de clorofila foram obtidos na presença de carotenóides até 6 mgg-1 sem estresse por ambas as cepas Rad1 e Rad2. Estudo sugere que especialmente a estirpe Ros2 pode melhorar parâmetros de crescimento de plantas no estresse de pesticidas.

Cryopreservation at -75 °C of Agaricus subrufescens on wheat grains with sucrose

Abstract Agaricus subrufescens is a basidiomycete which is studied because of its medicinal and gastronomic importance; however, less attention has been paid to its preservation. This study aimed to evaluate the effect of sucrose addition to substrate and cryotube on the viability of Agaricus subrufescens cryopreserved at -20 °C and at -75 °C for one and two years. Zero, 10% or 20% sucrose was added to potato dextrose agar or wheat grain. The mycelia were cryopreserved in the absence of cryoprotectant or with sucrose solutions at 15%, 30% or 45%. After one or two years at -75 °C or at -20 °C, mycelia were thawed and evaluated about viability, initial time of growth, colony diameter and genomic stability. Cryopreservation at -20 °C is not effective to keep mycelial viability of this fungus. Cryopreservation at -75 °C is effective when sucrose is used in substrates and/or cryotubes. Without sucrose, cryopreservation at -75 °C is effective only when wheat grains are used. Physiological characteristic as mycelial colony diameter is negatively affected when potato dextrose agar is used and unaffected when wheat grain is used after two-year cryopreservation at -75 °C. The fungus genome does not show alteration after two-year cryopreservation at -75 °C.

Plant growth promoting rhizobacteria reduce aphid population and enhance the productivity of bread wheat

Abstract Plant growth promoting rhizobacteria increase plant growth and give protection against insect pests and pathogens. Due to the negative impact of chemical pesticides on environment, alternatives to these chemicals are needed. In this scenario, the biological methods of pest control offer an eco-friendly and an attractive option. In this study, the effect of two plant growth promoting rhizobacterial strains (Bacillus sp. strain 6 and Pseudomonas sp. strain 6K) on aphid population and wheat productivity was evaluated in an aphid susceptible (Pasban-90) and resistant (Inqlab-91) wheat cultivar. The seeds were inoculated with each PGPR strain, separately or the combination of both. The lowest aphid population (2.1 tiller−1), and highest plant height (85.8 cm), number of spikelets per spike (18), grains per spike (44), productive tillers (320 m−2), straw yield (8.6 Mg ha−1), and grain yield (4.8 Mg ha−1) were achieved when seeds were inoculated with Bacillus sp. strain 6 + Pseudomonas sp. strain 6K. The grain yield of both varieties was enhanced by 35.5-38.9% with seed inoculation with both bacterial strains. Thus, the combine use of both PGPR strains viz. Bacillus sp. strain 6 + Pseudomonas sp. strain 6K offers an attractive option to reduce aphid population tied with better wheat productivity.

Combined application of bio-organic phosphate and phosphorus solubilizing bacteria (Bacillus strain MWT 14) improve the performance of bread wheat with low fertilizer input under an arid climate

Abstract This study was aimed to investigate the effect of bio-organic phosphate either alone or in combination with phosphorus solubilizing bacteria strain (Bacillus MWT-14) on the growth and productivity of two wheat cultivars (Galaxy-2013 and Punjab-2011) along with recommended (150-100 NP kg ha−1) and half dose (75-50 NP kg ha−1) of fertilizers. The combined application of bio-organic phosphate and the phosphorous solubilizing bacteria strain at either fertilizer level significantly improved the growth, yield parameters and productivity of both wheat cultivars compared to non-inoculated control treatments. The cultivar Punjab-2011 produced the higher chlorophyll contents, crop growth rate, and the straw yield at half dose of NP fertilizer; while Galaxy-2013, with the combined application of bio-organic phosphate and phosphorous solubilizing bacteria under recommended NP fertilizer dose. Combined over both NP fertilizer levels, the combined use of bio-organic phosphate and phosphorous solubilizing bacteria enhanced the grain yield of cultivar Galaxy-2013 by 54.3% and that of cultivar Punjab-2011 by 83.3%. The combined application of bio-organic phosphate and phosphorous solubilizing bacteria also increased the population of phosphorous solubilizing bacteria, the soil organic matter and phosphorous contents in the soil. In conclusion, the combined application of bio-organic phosphate and phosphorous solubilizing bacteria offers an eco-friendly option to harvest the better wheat yield with low fertilizer input under arid climate.

Cost-effective production of bacterial cellulose using acidic food industry by-products

Abstract To reduce the cost of obtaining bacterial cellulose, acidic by-products of the alcohol and dairy industries were used without any pretreatment or addition of other nitrogen sources. Studies have shown that the greatest accumulation of bacterial cellulose (6.19 g/L) occurs on wheat thin stillage for 3 days of cultivation under dynamic conditions, which is almost 3 times higher than on standard Hestrin and Schramm medium (2.14 g/L). The use of whey as a nutrient medium makes it possible to obtain 5.45 g/L bacterial cellulose under similar conditions of cultivation. It is established that the pH of the medium during the growth of Gluconacetobacter sucrofermentans B-11267 depends on the feedstock used and its initial value. By culturing the bacterium on thin stillage and whey, there is a decrease in the acidity of the waste. It is shown that the infrared spectra of bacterial cellulose obtained in a variety of environments have a similar character, but we found differences in the micromorphology and crystallinity of the resulting biopolymer.

Fusarium and Aspergillus mycotoxins contaminating wheat silage for dairy cattle feeding in Uruguay

Abstract Wheat is one of the most important cultivated cereals in Uruguay for human consumption; however, when harvest yields are low, wheat is usually used in ensiling for animal feeding. Ensiling is a forage preservation method that allows for storage during extended periods of time while maintaining nutritional values comparable to fresh pastures. Silage is vulnerable to contamination by spoilage molds and mycotoxins because ensilage materials are excellent substrates for fungal growth. The aim of the study was to identify the mycobiota composition and occurrence of aflatoxins and DON from wheat silage. A total of 220 samples of wheat were collected from four farms in the southwest region of Uruguay were silage practices are developed. The main fungi isolated were Fusarium (43%) and Aspergillus (36%), with Fusarium graminearum sensu lato and Aspergillus section Flavi being the most prevalent species. Aflatoxin concentrations in silo bags ranged from 6.1 to 23.3 µg/kg, whereas DON levels ranged between 3000 µg/kg and 12,400 µg/kg. When evaluating aflatoxigenic capacity, 27.5% of Aspergillus section Flavi strains produced AFB1, 5% AFB2, 10% AFG1 and 17.5% AFG2. All isolates of F. graminearum sensu lato produced DON and 15-AcDON. The results from this study contribute to the knowledge of mycobiota and mycotoxins present in wheat silage.

Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the rhizosphere of wheat

ABSTRACT The main objective of the present study was to isolate phytohormone-producing, phosphate-solubilizing strains of Azospirillum from wheat to be used as inoculants for plant growth promotion. Five Azospirillum strains were isolated from the rhizosphere of field-grown wheat (Triticum aestivum L.), and it was confirmed by BOX-polymerase chain reaction (PCR) that the isolates were different and not re-isolates of the same strain. Sequence analysis of the PCR-amplified 16S rRNA gene indicated that four isolates showed maximum similarity to Azospirillum brasilense and one isolate showed maximum similarity to Azospirillum zeae. This is the first report indicating the presence of an A. zeae like isolate in the wheat rhizosphere in Pakistan. The bacterial isolates were characterized for their plant growth-promoting traits, phosphate solubilization, and indole-3-acetic acid (IAA) production. None of the isolates showed phosphate solubilization activity in the commonly used Pikovskaya medium. However, all strains (except AzoK4) exhibited ability to solubilize tricalcium phosphate (TCP) in modified Pikovskaya medium in which sucrose was replaced by Na-malate, as well as in TCP-supplemented Luria-Bertani (LB) medium. Organic acids, such as acetic, citric, lactic, malic, and succinic acids, were detected in culture supernatants of the tested Azospirillum strains. All strains exhibited ability to produce IAA in the growth medium, except Azospirillum sp. AzoK1. Among the strains tested, the maximum IAA production (30.49 ± 1.04 mg L-1) and phosphate solubilization (105.50 ± 4.93 mg L-1) were shown by a pure culture of Azospirillum sp. AzoK2. In pot experiments, single-strain inocula of Azospirillum sp. AzoK1 and AzoK2 improved wheat plant growth.

Alleviation of salt stress by halotolerant and halophilic plant growth-promoting bacteria in wheat (Triticum aestivum)

ABSTRACT In the current study, 18 halotolerant and halophilic bacteria have been investigated for their plant growth promoting abilities in vitro and in a hydroponic culture. The bacterial strains have been investigated for ammonia, indole-3-acetic acid and 1-aminocyclopropane-1-carboxylate-deaminase production, phosphate solubilisation and nitrogen fixation activities. Of the tested bacteria, eight were inoculated with Triticum aestivum in a hydroponic culture. The investigated bacterial strains were found to have different plant-growth promoting activities in vitro. Under salt stress (200 mM NaCl), the investigated bacterial strains significantly increased the root and shoot length and total fresh weight of the plants. The growth rates of the plants inoculated with bacterial strains ranged from 62.2% to 78.1%.Identifying of novel halophilic and halotolerant bacteria that promote plant growth can be used as alternatives for salt sensitive plants. Extensive research has been conducted on several halophilic and halotolerant bacterial strains to investigate their plant growth promoting activities. However, to the best of my knowledge, this is the first study to inoculate these bacterial strains with wheat.

Screening and characterization of endophytic Bacillus and Paenibacillus strains from medicinal plant Lonicera japonica for use as potential plant growth promoters

Abstract A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid production). The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inoculation. Results showed that the number of endophytic bacteria in L. japonica varied among different tissues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L) and aminocyclopropane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h). Strain 170 had the highest indoleacetic acid (IAA) production (49.2 mg/L) and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase) activity and inhibition rate on Fusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase). The six endophytic bacterial strains were identified to be Paenibacillus and Bacillus strains based on the results of 16S rRNA gene sequencing analysis and their physiological and biochemical characteristics. Results indicate the promising application of endophytic bacteria to the biological control of pathogenic fungi and the improvement of wheat crop growth.

Diversidad genética de aislamientos de Fusarium graminearum sensu lato asociados con la fusariosis de la espiga en trigo de diversas localidades argentinas / Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina

La fusariosis de la espiga de trigo es una importante enfermedad para la región pampeana Argentina; Fusarium graminearum es el principal patógeno asociado. Se estudió el polimorfismo del ADN de un conjunto de aislamientos utilizando las técnicas de IGS-RFLP e ISSR. La técnica de IGS-RFLP produjo 41 bandas, 30 de ellas fueron polimórficas. El análisis de los ISSR mostró 87 bandas con 47 bandas polimórficas. La primera de estas metodologías fue más eficiente, ya que detectó mayor promedio polimórfico (59,91%) que la segunda (44,11%). Los valores promedio del contenido de información polimórfica (PIC) fueron 0,211 y 0,129, respectivamente. Se identificaron 20 haplotipos por IGS-RFLP, mientras que el análisis de los ISSR reveló 15 haplotipos. La agrupación de genotipos obtenida en ambos dendrogramas fue diferente. Los grupos genéticos obtenidos por la técnica de IGS-RFLP mostraron una asociación parcial con el origen geográfico. Este es el primer reporte que analiza la variabilidad genética en poblaciones de F. graminearum de trigo empleando marcadores IGS-RFLP e ISSR en Argentina

Fusarium Head Blight is an important wheat disease in the Argentine Pampas region, being Fusarium graminearum the predominant pathogen. DNA polymorphism of the isolates was analyzed by IGS-RFLP and ISSR. IGS-RFLP and ISSR profiling were carried out using six endonucleases and eight primers, respectively. IGS-RFLP yielded 41 bands, 30 of which were polymorphic while ISSR produced 87 bands with 47 polymorphic bands. Both markers showed genetic variability among the analyzed isolates; however, IGS-RFLP was more efficient than ISSR, showing a higher polymorphic average (59.91%) than the latter (44.11%). The averages of polymorphic information content (PIC) were 0.211 and 0.129, respectively. Twenty haplotypes were identified by IGS-RFLP and 15 haplotypes by ISSR. Genotype clustering within dendrograms was different for both types of markers. The genetic groups obtained by IGS-RFLP showed a partial association to geographic origin. This is the first report on genetic variability of F. graminearum isolates from wheat in Argentina using IGS-RFLP and ISSR markers

Fermentação em estado sólido de Aspergillus parasiticus e produção de aflatoxinas / Solid state fermentation of Aspergillus parasiticus and aflatoxin production

Aflatoxinas são metabólitos secundários de fungos com grande potencial carcinogênico, produzidos principalmente por Apergillus flavus e Aspergillus parasiticus. Em vista da ampla variedade de alimentos em que se encontram essas micotoxinas, o presente estudo teve como objetivo determinar condições de cultivo para o Aspergillus parasiticus e produção das quatro principais aflatoxinas (AFB1, AFB2, AFG1 e AFG2) considerando diferentes substratos conhecidos pela contaminação por estas micotoxinas, entre eles arroz branco, arroz cateto, amendoim, milho e farinha de trigo integral, e diferentes valores de umidade e pH. Ao analisar por cromatrografia em camada delgada os extratos dos diferentes substratos, verificou-se a produção de aflatoxinas em todos os alimentos, porém na cromatografia líquida de alta eficiência foi possível perceber a maior produção de aflatoxinas no arroz cateto e na farinha de trigo integral. Para a continuidade do trabalho, utilizou-se o arroz cateto e então preparou- se diferentes meios sólidos com valores de pH entre 3,5 e 7,5 e umidade entre 42 % e 62 %. Ao analisar por CLAE, todas as amostras apresentaram produção de AF, porém as amostras com o maior valor de água agregada (62%) apresentaram maior produção enquanto a variação de pH não apresentou influência nesta produção.

Aflatoxins are secondary metabolites of fungi with great carcinogenic potencial, mainly produced by Aspergillus flavus and Aspergillus parasiticus. In order of the wide variety of foods where mycotoxins are found in, this study aimed to determine growth conditions for Aspergillus parasiticus and production of four major aflatoxins (AFB1, AFB2, AFG1 and AFG2) considering different substrates known for contamination by these aflatoxins, including white rice, cathetus rice, peanuts, maize and whole wheat flour, and different pH and humidity values. When extracts of different substrates were analyzed by thin layer chromatography, it has been verified the production of aflatoxins in all foods; however, when high performance liquid chromatography (HPLC) analysis was performed, the greater production of aflatoxins was observed in cathetus rice and whole-wheat flour. Cathetus rice was then selected to continue the study and different solid medium with pH values between 3.5 and 7.5 and humidity percentages between 42 % and 62 % were prepared. When analyzed by HPLC, all samples showed production of aflatoxins, but the samples with higher humidity value (62%) showed greatest production while the pH changes had no effect on this production.

Efficiency of treatments for controlling Trichoderma spp during spawning in cultivation of lignicolous mushrooms

Trichoderma spp is the cause of the green mold disease in mushroom cultivation production. Many disinfection treatments are commonly applied to lignocellulose substrates to prevent contamination. Mushroom growers are usually worried about the contaminations that may occur after these treatments during handling or spawning. The aim of this paper is to estimate the growth of the green mold Trichoderma sp on lignocellulose substrates after different disinfection treatments to know which of them is more effective to avoid contamination during spawning phase. Three different treatments were assayed: sterilization (121 ºC), immersion in hot water (60 and 80 ºC), and immersion in alkalinized water. Wheat straw, wheat seeds and Eucalyptus or Populus sawdust were used separately as substrates. After the disinfection treatments, bagged substrates were sprayed with 3 mL of suspension of conidia of Trichoderma sp (10(5) conidia/mL) and then separately spawned with Pleurotus ostreatus or Gymnopilus pampeanus. The growth of Trichoderma sp was evaluated based on a qualitative scale. Trichoderma sp could not grow on non-sterilized substrates. Immersions in hot water treatments and immersion in alkalinized water were also unfavorable treatments for its growth. Co- cultivation with mushrooms favored Trichoderma sp growth. Mushroom cultivation disinfection treatments of lignocellulose substrates influence on the growth of Trichoderma sp when contaminations occur during spawning phase. The immersion in hot water at 60 ºC for 30 min or in alkalinized water for 36 h, are treatments which better reduced the contaminations with Trichoderma sp during spawning phase for the cultivation of lignicolous species.

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies

Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

Improvement of yield of the edible and medicinal mushroom Lentinula edodes on wheat straw by use of supplemented spawn

The research evaluated the interactions of two main factors (strain / types of spawn) on various parameters with the purpose to assess its effect on yield and biochemical composition of Lentinula edodes fruiting bodies cultivated on pasteurized wheat straw. The evaluation was made with four strains (IE-40, IE-105, IE-124 and IE-256). Different types of spawns were prepared: Control (C) (millet seed, 100%), F1 (millet seed, 88.5%; wheat bran, 8.8%; peat moss, 1.3%; and CaS0(4), 1.3%) and F2 (the same formula as F1, but substituting the wheat bran with powdered wheat straw). Wheat straw was pasteurized by soaking it for 1 h in water heated to 65 °C. After this the substrate (2 kg wet weight) was placed in polypropylene bags. The bags were inoculated with each spawn (5% w/w) and incubated in a dark room at 25 °C. A proximate analysis of mature fruiting bodies was conducted. The mean Biological Efficiency (BE) varied between 66.0% (C-IE-256) and 320.1% (F1-IE-124), with an average per strain of 125.6%. The highest mean BE was observed on spawn F1 (188.3%), significantly different from C and F2. The protein content of fruiting bodies was high, particularly in strain IE-40-F1 (17.7%). The amount of fat varied from 1.1 (F1-IE-40) to 2.1% (F2-IE-105) on dry matter. Carbohydrates ranged from 58.8% (F1-IE-40) to 66.1% (F1-IE-256). The energy value determined ranged from 302.9 kcal (F1-IE-40) to 332.0 kcal (F1-IE-256). The variability on BE observed in this study was significantly influenced by the spawn's formulation and genetic factors of the different strains.

Toxigenic profile and AFLP variability of Alternaria alternata and Alternaria infectoria occurring on wheat

The objectives of this study were to evaluate the ability to produce alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) by A. alternata and A. infectoria strains recovered from wheat kernels obtained from one of the main production area in Argentina; to confirm using AFLPs molecular markers the identify of the isolates up to species level, and to evaluate the intra and inter-specific genetic diversity of these two Alternaria species. Among all the Alternaria strains tested (254), 84% of them were able to produce mycotoxins. The most frequent profile of toxin production found was the co-production of AOH and AME in both species tested. TA was only produced by strains of A. alternata. Amplified fragment polymorphism (AFLPs) analysis was applied to a set of 89 isolates of Alternaria spp (40 were A. infectoria and 49 were A. alternata) in order to confirm the morphological identification. The results showed that AFLPs are powerful diagnostic tool for differentiating between A. alternata and A. infectoria. Indeed, in the current study the outgroup strains, A. tenuissima was consistently classified. Characteristic polymorphic bands separated these two species regardless of the primer combination used. Related to intraspecific variability, A. alternata and A. infectoria isolates evaluated seemed to form and homogeneous group with a high degree of similarity among the isolates within each species. However, there was more scoreable polymorphism within A. alternata than within A. infectoria isolates. There was a concordance between morphological identification and separation up to species level using molecular markers. Clear polymorphism both within and between species showed that AFLP can be used to asses genetic variation in A. alternata and A. infectoria. The most important finding of the present study was the report on AOH and AME production by A. infectoria strains isolated from wheat kernels in Argentina on a semisynthetic media for the first time. Also, specific bands for A. alternata and A. infectoria have been identified; these may be useful for the design of specific PCR primers in order to differentiate these species and to detect them in cereals.

Aflatoxin contamination in wheat flour samples from Golestan province, Northeast of Iran

Due to the high toxicity of aflatoxin and its effects on public health, determination of aflatoxin level in Wheat flour samples in the Golestan province, north of Iran was investigated. To examine the effect of seasonal changes, summer and winter sampling was performed with standard sampling methods. A total of 200 flour samples were collected from 25 factories. HPLC method with immunoaffinity chromatography was used to measure aflatoxin types [G2, G1, B2 and B1]. Statistical analysis was performed by the Pearson correlation test, One-way ANOVA and multivariate regression analysis. Mean total aflatoxin levels of samples were 0.82 and 1.99 ng/g in summer and winter, respectively. Aflatoxin B1 levels were detected in 3.1%, 7.4% over permissible limits by worldwide regulations in samples collected in summer and winter, respectively. Aflatoxins in winter were higher than summer. The highest frequency of aflatoxin contamination in winter was B2 [98%] and in summer G1 [51%]. The relationship between humidity and rate of aflatoxin B1 and total aflatoxin was significant in winter. Results of multivariate regression were showed the strongest relationship with humidity and aflatoxin level. Despite the contamination of flour samples, there was no contamination higher than the standard limit of Iran Standard Institute. But it was significantly higher than similar studies from other regions. Therefore, with regard to negative impacts of aflatoxin on health, aflatoxin contamination should be considered in future programs. Decrease of aflatoxin contamination may be made practical through reducing wheat storage duration and controlling humidity

Identification of leaf rust resistance genes in selected Argentinean bread wheat cultivars by gene postulation and molecular markers

Leaf rust, caused by Puccinia triticina Eriks. is a common and widespread disease of bread wheat (Triticum aestivum L.), in Argentina. Host resistance is the most economical, effective and ecologically sustainable method of controlling the disease. Gene postulation helps to determine leaf rust resistance genes (Lr genes) that may be present in a large group of wheat germplasm. Additionally presence of Lr genes can be determined using associated molecular markers. The objective of this study was to identify Lr genes that condition leaf rust resistance in 66 wheat cultivars from Argentina. Twenty four differential lines with individual known leaf rust resistance genes were tested with 17 different pathotypes of leaf rust collected from Argentina. Leaf rust infection types produced on seedling plants of the 66 local cultivars were compared with the infection types produced by the same pathotypes on Lr differentials to postulate which seedling leaf rust genes were present. Presence of Lr9, Lr10, Lr19, Lr20, Lr21, Lr24, Lr25, Lr26, Lr29, Lr34, Lr35, Lr37, Lr47 and Lr51 was also determined using molecular markers. Eleven different Lr genes were postulated in the material: Lr1, Lr3a, Lr3ka, Lr9, Lr10, Lr16, Lr17, Lr19, Lr24, Lr26, Lr41. Presence of Lr21, Lr25, Lr29, and Lr47 could not be determined with the seventeen pathotypes used in the study because all were avirulent to these genes. Eleven cultivars (16.7 percent) were resistant to all pathotypes used in the study and the remaining 55 (83.3 percent) showed virulent reaction against one or more local pathotypes. Cultivars with seedling resistance gene combinations including Lr16 or single genes Lr47 (detected with molecular marker), Lr19 and Lr41, showed high levels of resistance against all pathotypes or most of them. On the opposite side, cultivars with seedling resistance genes Lr1, Lr3a, Lr3a + Lr24, Lr10, Lr3a + Lr10, Lr3a + Lr10 + Lr24 showed the highest number of virulent reactions against local...

Validity of method for determining deoxynivalenol through high performance liquid chromatography with detection in the ultraviolet in wheat samples / Validación de un método para la determinación de deoxinivalenol por cromatografía líquida de alta resolución con deteccíon ultravioleta en muestras de trigo

With the purpose of evaluating the wheat grain and wheat flour contamination by Deoxynivalenol (DON) in the municipality of Chapeco-SC, and standardize an useful method to detect this mycotoxin by High Performance Liquid Chromatography (HPLC), six samples of wheat grains from different storage location, and one sample of wheat grain from the flour milling industry were obtained during in the month of august 2008. Samples belong to a corporation from Chapeco-SC that stores and processes wheat grains, flour and wheat middlings, among other products. The extraction has been carried out with methanol: water (100: 100 v / v), filtered paper filter and applied in immunoaffinity column specific DON. After the wash with water column, the toxin was eluted with methanol. The detection and quantification Deoxynivalenol in samples was carrid out through the method of HPLC in the UV-visible with detection 244 nm. The 6 analyzed samples of wheat grain showed DON levels within 7.0 and 10.1 ppb, while the wheat flour contained 90.2 ppb. DON contents in wheat grains and wheat flour are lower than the limits claimed by the studied corporated importers and the international legislation.

Con el objetivo de evaluar la contaminación por Deoxinivalenol (DON) en granos y harina del trigo en la municipalidad de Chapeco-SC y estandarizar un método de deteccíon para este micotoxina por cromatografía líquida de alta resolución (CLAR), se procesaron durante el mes de agosto de 2008, seis muestras de granos del trigo en diferentes situaciones de almacenamiento y una muestra de harina de trigo de un molino . Las muestras pertenecen a una cooperativa de Chapeco-SC que procesa y almacena granos y harinas de trigo entre otros productos. La extracción de la micotoxina se obtuvo con metanol: agua (100: 100 v / v), filtrado en papel filtro y aplicado a una columna de inmunoafinidad específica (DON). Después del lavado de la columna con agua, la toxina fue elucidada con metanol. La detección y cuantificación de Deoxinivalenol en las muestras se determinó por el método CLAR en el UV- visible con una longitud de onda de 244 nm. Las 6 muestras analizadas del grano, mostraron que el DON nivela entre 7,0 y 10, 1 ppb, mientras la harina del trigo alcanzó las 90,2 ppb. Los niveles de DON en los granos y harina de trigo tienen límites menores que los exigidos por las cooperativas importadoras estudiadas y la legislación internacional.