Evaluation of Pochonia chlamydosporia and Purpureocillium lilacinum for Suppression of Meloidogyne enterolobii on Tomato and Banana.

Meloidogyne enterolobii is one of the most important root-knot nematode in tropical regions, due to its ability to overcome resistance mechanisms of a number of host plants. The lack of new and safe active ingredients against this nematode has restricted control alternatives for growers. Egg-parasitic fungi have been considered as potential candidates for the development of bionematicides. In tissue culture plates, Pochonia chlamydosporia (var. catenulata and chlamydosporia) and Purpureocillium lilacinum strains were screened for their ability to infect eggs of the root-knot nematode M. enterolobii on water-agar surfaces. Reduction in the hatching of J2 varied from 13% to 84%, depending on strain. The more efficacious strains reduced hatchability of J2 by 57% to 84% when compared to untreated eggs, but average reductions were only 37% to 55% when the same strains were applied to egg masses. Combinations of fungal isolates (one of each species) did not increase the control efficacy in vitro. In experiments in which 10,000 nematode eggs were inoculated per plant, reductions in the number of eggs after 12 months were seen in three of four treatments in banana plants, reaching 34% for P. chlamydosporia var. catenulata. No significant reductions were seen in tomato plants after 3 mon. In another experiment with tomato plants using either P. chlamydosporia var. catenulata or P. lilacinum, the number of eggs was reduced by 34% and 44%, respectively, when initial infestation level was low (500 nematode eggs per plant), but tested strains were not effective under a moderate infestation level (5,000 eggs per plant). Under all infestation levels tested in this work, gall and egg mass indexes (MI) did not differ from the untreated controls, bringing concerns related to the practical adoption of this control strategy by farmers. In our opinion, if the fungi P. chlamydosporia and P. lilacinum are to be used as biocontrol tools toward M. entorolobii, they should focus on agricultural settings with low soil infestation levels and within an IPM approach.

Evaluating the elimination of Brazilian entomopathogenic Bacillus by non-target aquatic species: an experimental study.

Ecotoxicity tests are key to predict environmental hazards resulting from chemical and biological pesticides in non-target species. In order to assess the effects of microbial pesticides it is important to determine if they cause infection in test organisms. At present the microbial elimination rate or clearance is not included in ecotoxicological regulatory protocols. This study evaluated the elimination of Bacillus thuringiensis and Bacillus sphaericus from fish and snails, after 30 days' exposure to commercial formulations of such entomopathogens. Data obtained showed that in clean water the tendency to eliminate microbial agents from the body of the exposed organisms is gradual over time but after 7 days the fish and snails were free of the two tested Bacillus spp.

Toxicity assessment and clearance of Brazilian microbial pest control agents in mice.

The environmental toxicology of chemical pesticides have increased interest in the development and use of microbial pest control agents. In the present study four new Brazilian strains of Bacillus and one fungus were tested to evaluate the acute oral toxicity and clearance of these microbials in C57BL6 mice. No mortality was observed after exposure for any of the microorganisms tested. Clearance was significant after 30 days but for one strain of B. thuringiensis and one of B. sphaericus this time was not enough to completely eliminate the spores.

The Cry48Aa-Cry49Aa binary toxin from Bacillus sphaericus exhibits highly restricted target specificity.

The Cry48Aa/Cry49Aa binary toxin of Bacillus sphaericus was recently discovered by its ability to kill Culex quinquefasciatus mosquito larvae through a novel interaction between its two components. We have investigated the target specificity of this toxin and show it to be non-toxic to coleopteran, lepidopteran and other dipteran insects, including closely related Aedes and Anopheles mosquitoes. This represents an unusually restricted target range for crystal toxins from either B. sphaericus or Bacillus thuringiensis. Gut extracts from Culex and Aedes larvae show differential processing of the Cry48Aa protein, with the location of cleavage sites in Culex reflecting those previously shown for the activation of Cry4 toxins in mosquitoes. Pre-activation of Cry48Aa/Cry49Aa with Culex extracts, however, fails to induce toxicity to Aedes larvae. Co-administration of Cry49Aa with Cry4Aa gives higher than predicted toxicity, perhaps suggesting weak synergism against Culex larvae between Cry49Aa and other three-domain Cry toxins.

A recombinant truncated Cry1Ca protein is toxic to lepidopteran insects and forms large cuboidal crystals in insect cells.

A truncated version of the cry1Ca gene from Bacillus thuringiensis was introduced into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) under the control of two promoters. A recombinant virus (vSyncry1c) was isolated and used to infect insect cells in culture and insect larvae. Structural and ultrastructural analysis of insects infected with vSyncry1C showed the formation of large cuboidal crystals inside the cytoplasm of insect cells in culture and in insect cadavers late in infection. Infected insect cell extracts were analyzed by SDS-PAGE and Western blot and showed the presence of a 65-kDa polypeptide probably corresponding to the protease processed form of the toxin. Bioassays using purified recombinant toxin crystals showed a CL(50) of 19.49 ng/ml for 2(nd) instar A. gemmatalis larvae and 114.1 ng/ml for S. frugiperda.

Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds.

Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to alpha-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian alpha-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil.

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated.

Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis).

The cotton boll weevil, Anthonomus grandis, is an economically important pest of cotton in tropical and subtropical areas of several countries in the Americas, causing severe losses due to their damage in cotton floral buds. Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities. Furthermore, in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Previously, in vivo effects of black-eyed pea trypsin chymotrypsin inhibitor (BTCI) have been demonstrated towards the boll weevil pest. Here, when neonate larvae were reared on an artificial diet containing SKTI at three different concentrations, a reduction of larval weight of up to 64% was observed for highest SKTI concentration 500 microM. The presence of SKTI caused an increase in mortality and severe deformities of larvae, pupae and adult insects. This work therefore represents the first observation of a Kunitz trypsin inhibitor active in vivo and in vitro against A. grandis. Bioassays suggested that SKTI could be used as a tool in engineering crop plants, which might exhibit increased resistance against cotton boll weevil.

Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis.

The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.