41st Annual Meeting of the Society for Invertebrate Pathology
and 9th International Conference on Bacillus thuringiensis

The use of transgenic Bt-maize is increasing yearly (last year accounting for about 19% of the total maize planted area in the world) because of the efficient control of the corn borers, in especial Ostrinia nubilalis . Resistance to Bacillus thuringiensis (Bt) insecticidal toxins has been linked to the 12-domain cadherin locus in 3 lepidopteran species. The O. nubilalis cadherin gene has been revealed as a complex gene of about 20 kbp in length, with 34 introns. In the present work, we have studied the size polymorphism of the gene in a Spanish population, by amplifying the genomic sequence of the gene in 16 overlapping regions. The variability observed was not uniformly distributed, with a maximum in region 14 and a minimum (no polymorphism) in region 4. All this size variability must be due to changes in the intronic regions because we found no detectable size differences in mRNA. This variability can be useful to select appropriate polymorphic regions to be used as markers of this gene in experiments such us to determine the genetic linkage of the cadherin to Bt resistance traits.

An Ostrinia nubilalis colony was selected for resistance to Bacillus thuringiensis Cry1Ab protoxin. Previous work evidenced the implication of more than one genetic locus and the reduction of the cadherin receptor. We have now determined the contribution of the cadherin gene to the overall Cry1Ab resistance in this strain. Individual larval midguts from susceptible (Europe-S) and resistant (Europe-R) insects were used to prepare cDNAs from the cadherin gene. We found major mutations that suggested highly structural deficient proteins because they introduced premature termination codons (PTC) and/or large deletions (1383-1701 bp). In the resistant strain, these mutations were found in 13 out of 20 insects analyzed. In the susceptible strain, only one PTC was detected among the major mutations, but always in heterozygotes. To check for the contribution of the major mutations to the resistance, Europe-R insects were subjected to a high dose of Cry1Ab protoxin. The analysis of the survivors showed that major mutations were absent. These results support a polygenic inheritance of resistance in the Europe-R strain, in which mutations in the cadherin gene would contribute to resistance by means of an additive effect.

The effective control of European corn borer (ECB) is a very important aspect in food safety programmes in maize growing systems. Making holes in the stalks and in ears the pest larvae are a primary cause of maize infections by toxinogenic micromycetes dangerous both for human consumers and animals. The efficacy of selective insecticides (metoxyfenozide, indoxacarb), Bt maize and Trichogramma wasp (Tw) as preparation Trichocap ® against ECB was evaluated on several different locations during period 2002-2007. Before the harvest the plant injuries (tunnels and stalk breakage) caused by ECB were evaluated. The highest biological efficacy was achieved in Bt- maize (100%) in all experimental years. The effect of insecticide treatments was very good ranging from 80% to 95%. Tw applications resulted in satisfactory effect (cca 50%) in the most of years. The exception was previous season (2007) when the efficacy nearly 80% was observed. In addition, the concept of an antiresistant strategy in ECB control is proposed. In this concept Tw and selective insecticides can be applied in refugees used in insect resistant management in the protection against European corn borer. The work was funded by the project No 1B53043 of the Ministry of Agriculture of the Czech Republic.

The control of European corn borer (ECB) plays the most important in the prevention of mycotoxin accumulation in maize. Except of this, the occurrence of insecticide residues must be studied if chemical control used. Bt-maize, insecticides, and Trichogramma wasp were tested against ECB during period 2002-2007. To this purpose Bt-maize (‘MON 810’) and non-Bt hybrid (‘Monumental’) were used. The content of mycotoxins (NIV, DON, ADONs, T-2, HT-2, FUS-X and ZEA) in maize grain was evaluated. In addition, the incidence of insecticide residues (metoxyfenozide, indoxacarb) used in ECB chemical control of sweet maize was analysed. In Bt maize the lower occurence of toxinogenic micromycetes was observed. From the fungi linked to injuries by ECB to the most frequent species belonged: Fusarium subglutinans, F. verticillioides, F. proliferatum, F. sporotrichioides. Generally, the most frequent mycotoxin found in product was DON. NIV and ZEA appeared in some seasons only and the content of other mycotoxins was mostly bellow LOQ. A slight contamination by residues was observed in ear coats sampled during vegetation season. Before the harvest the metoxyfenozide residues was detected whereas indoxacarb declined better. Negligible contents (<MRL) of pesticide residues left by the tested insecticides were found in harvested sweet maize grain.

Aminopeptidases N (APNs) have been identified as Bacillus thuringiensis endotoxins receptor candidates in several Lepidopteran species. Employing the RACE PCR technique we obtained two complete cDNAs corresponding to two APNs expressed in the midgut of Ostrinia nubilalis larvae. One of the sequences was 3624 bp long, and the predicted protein was composed by 940 aminoacids, whereas the other cDNA was 3226 nucleotides long, leading a putative protein composed by 994 aminoacids. The in silico study of the sequences, showed in both proteins a signal peptide, a GPI-anchor domain, a zinc-binding region HEXXHX18E and a GAMEN motif, characteristic of the gluzincin aminopeptidases. Moreover, several glicosilation sites were identified. The phylogram tree derived from ClustalW alignment grouped Lepidopteran APNs in five classes. The first sequence would belong to class 1 while the second one would belong to class 2. The expression of the two O. nubilalis APNs was studied during the larval development. Total RNA was purified from neonate larvae and from larvae 5, 10, 15 and 25 days old. RT-PCR reactions showed that both APNs where expressed during the whole larval growth.

Samplings of natural populations of European corn borer (ECB) Ostrinia spp. collected in France were examined for microsporidian infection. Parallel light microscopic and PCR-based detection carried out for 30 ECB larvae resulted in 30 and 57% parasite’s prevalence rate, respectively. PCR-based detection using SSU rRNA primers could yield specific product even at 1000-fold dilution of initial infected host DNA sample. Further PCR-detection of microsporidia was performed using O. nubilalis larvae collected from maize (N=602) and O. scapulalis larvae collected from mugwort (N=286) in 2000-2003. Microsporidia infection levels of larvae from both maize and mugwort were low, although some were present in both species. Among six samples of 40-50 larvae from mugwort, infection was found only in one population with 8% prevalence rate. PCR products (1123 b.p.) from two samples were sequenced and showed 99.9% similarity to Nosema pyrausta SSU rRNA. Among 12 samples of 47-50 ECB larvae from maize, microsporidia were found in 7 populations with prevalence rate ranging from 2 to 10%. Of 10 PCR products sequenced, six showed highest (99.9%) similarity to Nosema pyrausta, three – (96%) to N. bombi, and one – (82%) to Encephalitozoon hellem. Supported by RFBR n°07-04-92170, n°07-04-00269; CNRS, PICS n°3864; and RF President’s grant n°MK-653.2007.4.