Susceptibility of Anthonomus grandis (cotton boll weevil) and Spodoptera frugiperda (fall armyworm) to a cry1ia-type toxin from a Brazilian Bacillus thuringiensis strain.

Different isolates of the soil bacterium Bacillus thuringiensis produce multiple crystal (Cry) proteins toxic to a variety of insects, nematodes and protozoans. These insecticidal Cry toxins are known to be active against specific insect orders, being harmless to mammals, birds, amphibians, and reptiles. Due to these characteristics, genes encoding several Cry toxins have been engineered in order to be expressed by a variety of crop plants to control insectpests. The cotton boll weevil, Anthonomus grandis, and the fall armyworm, Spodoptera frugiperda, are the major economically devastating pests of cotton crop in Brazil, causing severe losses, mainly due to their endophytic habit, which results in damages to the cotton boll and floral bud structures. A cry1Ia-type gene, designated cry1Ia12, was isolated and cloned from the Bt S811 strain. Nucleotide sequencing of the cry1Ia12 gene revealed an open reading frame of 2160 bp, encoding a protein of 719 amino acid residues in length, with a predicted molecular mass of 81 kDa. The amino acid sequence of Cry1Ia12 is 99% identical to the known Cry1Ia proteins and differs from them only in one or two amino acid residues positioned along the three domains involved in the insecticidal activity of the toxin. The recombinant Cry1Ia12 protein, corresponding to the cry1Ia12 gene expressed in Escherichia coli cells, showed moderate toxicity towards first instar larvae of both cotton boll weevil and fall armyworm. The highest concentration of the recombinant Cry1Ia12 tested to achieve the maximum toxicities against cotton boll weevil larvae and fall armyworm larvae were 230 microg/mL and 5 microg/mL, respectively. The herein demonstrated insecticidal activity of the recombinant Cry1Ia12 toxin against cotton boll weevil and fall armyworm larvae opens promising perspectives for the genetic engineering of cotton crop resistant to both these devastating pests in Brazil.

Molecular cloning and expression of an alpha-amylase inhibitor from rye with potential for controlling insect pests.

Alpha-amylase inhibitors have important roles in plant defense mechanisms, particularly against insects, and several of these inhibitors have been expressed in different crops to increase their resistance to particular insects. In this work, we report the cloning and expression of a gene encoding for a new alpha-amylase inhibitor (BIII) from rye (Secale cereale) seeds. The BIII gene contains 354 nucleotides that encode for 118 amino acids sequence. A 313 bp fragment of the gene was expressed in Escherichia coli and resulted in a functional inhibitor that reduced the activity of alpha-amylases of larvae of the coleopteran pests Acanthoscelides obtectus, Zabrotess subfasciatus and Anthonomus grandis. In contrast, the inhibitor did not inhibit the activity of porcine pancreatic alpha-amylase. Although the amino acid sequence of BIII showed high identity with those of bifunctional inhibitors, the recombinant protein was unable to inhibit trypsin-like serine proteinases. The effects of recombinant BIII were evaluated in vivo against A. grandis. When first instar larvae were reared on an artificial diet containing four different concentrations of BIII, a reduction in larval weight and a mortality of 83% were observed at the highest concentration.

Inibidores de amilase em híbridos de milho como fator de resistência a Sitophilus zeamais ( Coleoptera: Curculionidae ) / Amylase inhibitors in corn hybrids as a resistance factor to Sitophilus zeamais ( Coleoptera: Curculionidae )

Sitophilus zeamais Motschulsky é uma das principais pragas do milho armazenado, sendo controlado, principalmente, com inseticidas. Porém, devido às conseqüências indesejáveis desses químicos sobre o meio ambiente e a saúde humana, outras táticas de controle têm sido investigadas, especialmente o desenvolvimento de plantas resistentes. A presença de inibidores de amilase nos grãos de milho pode interferir na digestão do inseto. Esta pesquisa foi desenvolvida com o objetivo de avaliar a resistência de híbridos de milho ao ataque de S. zeamais, correlacionada com a presença de inibidores de amilase e com a composição nutricional dos grãos. Adultos do gorgulho-do-milho foram expostos a amostras de grãos de diferentes híbridos a 27 ± 1°C e 75 ± 5 por cento de UR. Avaliou-se o ciclo biológico, peso dos adultos, índice de suscetibilidade e consumo dos grãos. Os resultados foram correlacionados com a composição dos grãos (teores de umidade, cinzas, lipídios, proteínas e carboidratos) e com a atividade de inibidores de amilase. A atividade inibidora de amilase foi determinada incubando-se o inibidor com 0,2 unidades de amilase durante 30 min. a 25°C, medindo-se posteriormente a atividade amilásica residual pelo método iodométrico. Dos parâmetros nutricionais avaliados, somente o conteúdo de lipídios correlacionou-se significativamente com o índice de suscetibilidade (r = 0,46). Os inibidores de amilase obtidos pela extração com etanol ou tampão correlacionaram-se negativa e significativamente com o índice de suscetibilidade (r = -0,58), sugerindo que esses inibidores contribuem para a resistência de híbridos de milho ao ataque de S. zeamais.

Pro domain peptide of HGCP-Iv cysteine proteinase inhibits nematode cysteine proteinases.

Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides. A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form. The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region. HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs. Recombinant propeptides of HGCP-Iv, expressed in E. coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems. Cysteine proteinases from other species produced no noticeable activity.

Trends of fumonisin contamination and animal intoxication through monitoring 1991 to 1997 corn crop in the State of Paraná, Brazil.

Eleven feed samples associated with six animal (horse and poultry) intoxication outbreaks (1991) in the state of Paraná, Brazil, were evaluated for fungal and fumonisin contamination. In order to estimate the trend of livestock intoxication, fumonisin contamination was monitored in corn produced both at the commercial level (1991, 1995 crop), and in an experimental field at a local Agronomy Institute (1997 crop). The total mould count in the feed samples ranged from 2.9 x 10(3) to 1.9 x 10(7) CFU/g, with Fusarium verticillioides as the predominant species, at a high count of 2.4 x 10(4)-6.5 x 10(5) CFU/g. Fumonisins (FB1 + FB2) were detected in all corn-based feed samples at levels ranging from 2.89 to 14.54 microg/g. All 27 Northern corn samples (1991 crop) were contaminated with fumonisins at levels ranging from 2.32 to 16.64 microg/g. Twenty-six (96.3%) out of 27 corn samples from the Central-Southern region (1995 crop) were positive for fumonisins (FB1+FB2), with the range of 0.07-3.66 microg/g, while all 37 Northern samples (1995 crop) were contaminated with fumonisins ranging from 0.57 to 9.97 microg/g. Twenty-one out of 37 corn samples from the Northern region (1997 crop) were positive for fumonisins, but at low level (range of 0.05-2.67 microg/g). The results showed a decreasing trend in fumonisin contamination over the years. Nowadays animal intoxication outbreaks rarely occur in this State, as both animal producers and feed industries have become conscious about monitoring of corn and other raw materials at the quality control level.

New Amylase Inhibitor Present in Corn Seeds Active In Vitro Against Amylase from Fusarium verticillioides.

A screening for specific amylase inhibitor levels against amylase from Fusarium verticillioides (Fusarium moniliforme), the most relevant mycotoxigenic fungus in corn, was conducted on 37 corn hybrids. The amylase inhibitor levels in these hybrids ranged from 5.5 to 16.0 amylase inhibitor units per gram of corn (AIU/g) in the MASTER and AG5011 hybrids, respectively. The hybrid with the maximum content of inhibitor was used as the source of this new protein. The inhibitor was partially purified using fractional precipitation, gel filtration on Sephadex G-75 column, high performance liquid chromatography (HPLC) Superose HR 10/30 column, and HPLC anion exchange chromatography, obtaining a 20.7-fold purification. Electrophoresis after denaturing and heating under reductive conditions showed an apparent 23.8 kDa molecular mass and an acidic isoelectric point of 5.4, which differs from previous molecular masses reported for other inhibitors present in corn seeds (14 and 22 kDa). This inhibitor showed activity against amylases from human saliva and pancreas, from the fungi F. verticillioides and Aspergillus flavus, and from the insects Acanthoscelides obtectus, Zabrotes subfasciatus, Tribolium castaneum, and Sitotroga cerealella. The mycoflora found in the corn grain indicated Fusarium sp. as the most prevalent fungi (81.1% of the samples), with a count ranging from 1.5 × 102 to 2.4 × 106 CFU/g of corn. The presence of fumonisins was detected in 21 out of the 37 hybrids studied, ranging from 0.05 to 2.67 µg of FB per gram of corn. No correlation could be established between this amylase inhibitor level in the corn seeds and the presence of Fusarium sp. or with the fumonisin content under the experimental conditions of the test.

Characterization of a Hydrophobic Amylase Inhibitor from Corn (Zea mays) Seeds with Activity Against Amylase from Fusarium verticillioides.

ABSTRACT A hydrophobic 19.7-kDa amylase inhibitor (AI) was purified from corn kernels by 95% ethanol extraction and anionic exchange chromatography. The AI has an isoelectric point of 3.6 and was very stable at different pH values and high temperatures, maintaining 47.6% activity after heating to 94 degrees C for 60 min. Amino acid analysis indicated high valine, leucine, glycine, alanine, and glutamic acid/glutamine content, and especially high valine content (41.2 mol%). This inhibitor is not a glycoprotein. It required 30-min preincubation to maximize complex enzyme-inhibitor formation when the amylase from Fusarium verticillioides was tested. The optimal pH of interaction was 6.5. It showed broad-spectrum activity including the following amylases: human saliva, porcine pancreas, F. verticillioides, as well as those from some insects of agricultural importance (Acanthoscelides obtectus, Zabrotes subfasciatus, Sitophilus zeamais, and Prostephanus truncatus). This novel hydrophobic protein not only inhibited the amylase from F. verticillioides but also decreased the conidia germination. Thus, this protein represents an approach to decrease the production of fumonisin in corn, either by using it as a molecular marker to detect fungal resistance or through genetic engineering.