El gusano cabezudo, Capnodis tenebrionis (L.) (Coleoptera: Buprestidae), es una importante plaga de cultivos de frutales de hueso y de pepita en zonas mediterráneas. Este trabajo engloba los estudios llevados a cabo para probar la eficacia de los nematodos entomopatógenos en el control de Capnodis tenebrionis.Se realizó el aislamiento de nematodos entomopatógenos de cultivos de frutales de hueso en dos regiones mediterráneas de la Península Ibérica. Se recogieron un total de 630 muestras de suelo (de 210 campos) en Cataluña y 90 muestras de suelo (de 30 campos) en la región de Murcia, resultando un 5,2% y un 20% de suelos positivos para la presencia de nematodos entomopatógenos. Se recogieron diez aislados de steinernemátidos y tres heterorhabdítidos utilizando el método de Galleria mellonella. Las especies aisladas fueron identificadas como Steinernema feltiae y Heterorhabditis bacteriophora.Se evaluó la eficacia de los nematodos entomopatógenos contra la larva neonata de Capnodis tenebrionis. Las cepas de nematodos utilizadas en este ensayo fueron dos Steinernema carpocapsae, una S. feltiae, una S. arenarium y una Heterorhabditis bacteriophora. Las larvas neonatas fueron expuestas a 3 y 48 IJs/cm2 en tubos de ensayo con arena estéril, resultando una mortalidad de 60-91% y 96-100%, respectivamente. A la concentración de 150 IJs/larva, todas las cepas de nematodos fueron muy virulentas. Sin embargo, en el ensayo a baja concentración (10 IJs/larva), S. arenarium resulto ser la cepa más virulenta. Se evaluó también el rango de penetración como un indicador de la capacidad de infección de los nematodos. El porcentaje más elevado fue obtenido por S. arenarium (36%), seguido por H. bacteriophora (30.6%), S. feltiae (23.1%), y S. carpocapsae (20.7%).Se evaluó también la virulencia de cepas contra larvas, pupas y adultos de C. tenebrionis. La mortalidad larvaria varió de 50 a 100%, la de pupas de 0 a 70% y la de adultos de 66.7% a 100%. Los resultados de este estudio sugieren que algunos de los aislados de los nematodos testados pueden ser utilizados para cotrolar C. tenebrionis.Se evaluó la tolerancia al calor, desecación e hipoxia, el efecto de la temperatura en la capacidad de infección y reproducción y la capacidad de migración vertical en columnas de arena de diferentes cepas de nematodos entomopatógenos. Los resultados mostraron diferencias entre especies y dentro de cada una de las especies. Se analizaron todos los factores importantes para la presencia de C. tenebrionis, destacando tres cepas de S. feltiae.Se estudió la susceptibilidad de las larvas de C. tenebrionis en el interior de plántulas. Los nematodos fueron capaces de localizar y matar las larvas de C. tenebrionis en el interior de las raíces. S. feltiae obtuvieron una eficacia de entre 79.68% y 88.24%, las cepas de H. bacteriophora strains entre 71.66 y 76.47%. S. carpocapsae (B14) y S. affine (Gspe3) causaron el menor control de C. tenebrionis larvae (62.03% y 34.76%, respectivamente). Se realizó un ensayo de campo con una cepa de S. feltiae aislada de una larva muerta de C. tenebrionis. Se aplicaron los nematodos por riego superficial e inyección. En ambos tratamientos se utilizó una dosis de 1 millón de IJ por arbol durante 4 u 8 semanas. Se evaluó número, estadio y localización de cada insecto en el tronco del árbol. Steinernema feltiae Bpa redujo la población de C. tenebrionis en ambos tratamientos, con un cotrol entre 88,3% y 97%. No se observaron diferencias significativas entre ambos tratamientos. La persistencia de los nematodos se observó hasta 6 semanas después de haber sido aplicados. Los resultados indican que S. feltiae Bpa es adecuada para el control de C. tenebrionis en cultivos de cerezos. / The Mediterranean flat-headed rootborer, Capnodis tenebrionis (L.) (Coleoptera: Buprestidae), is an economically important pest of stone fruit and seed fruit in Mediterranean areas. This report summarizes the studies carried out to prove the efficacy of entomopathogenic nematodes to control C. tenebrionis:Entomopathogenic were isolated from stone-fruit orchards in two Mediterranean regions of Spain. A total of 630 soil samples from Catalonia and 90 soil samples from Murcia were evaluated resulting in 5.2% and 20% of the soils testing positive for nematodes, respectively. Ten steinernematid isolates and three heterorhabditid isolates were recovered using the Galleria mellonella baiting method. Based on morphometric data, molecular data and cross-breeding experiments the nematode species were identified as Steinernema feltiae and Heterorhabditis bacteriophora. The efficacy of five entomopathogenic nematode strains was tested against the neonate larvae of Capnodis tenebrionis. The nematode strains screened included two Steinernema carpocapsae, one S. feltiae, one S. arenarium and one Heterorhabditis bacteriophora. Exposure of neonate larvae to 3 and 48 IJs/cm2 resulted in mortality between 60-91% and 96-100%, respectively. At a concentration of 150 IJs/larva, all of the nematode strains were highly virulent. S. carpocapsae caused infection and mortality to larvae more quickly than the other strains. The penetration rate as an indicator of entomopathogenic nematode infection was also evaluated. The highest value was recorded for S. arenarium (36%), followed by H. bacteriophora (30.6%), S. feltiae (23.1%), and S. carpocapsae (20.7%).The virulence of 14 Steinernema feltiae isolates, one S. carpocapsae and three Heterorhabditis bacteriophora isolates was compared in the laboratory against larvae, pupae and adults of Capnodis tenebrionis. Larval mortality ranged from 50 to 100% and pupae mortality from 0 to 70% and adults from 66.7% to 100%. Significant differences in mortality between C. tenebrionis males and females were observed. The results of this study suggest that some of the isolate strains have potential for improved biological control of C. tenebrionis.Environmental tolerance to heat, desiccation and hypoxia, the effect of temperature on infectivity and reproduction and nematode migration in sand columns were compared among isolates and one S. carpocapsae strain. Results showed differences among species and a great variability within species. Beneficial traits for each strain were added up to identify a superior candidate to control Mediterranean flat-headed rootborer, Capnodis tenebrionis. When all analyzed factors were considered, three S. feltiae isolates (Bpa, Sor and M116) obtained the best scores, and when hypoxia was removed, two of the strains (Bpa and Sor) continued ranking superior to other strains.The susceptibility of larvae of Capnodis tenebrionis to entomopathogenic nematodes was examined using GF-677@ potted trees (peach x almond hybrid) as the host plant. The nematode strains tested included nine Steinernema feltiae, one S. affine, one S. carpocapsae and two Heterorhabditis bacteriophora. Nematodes showed the ability to locate and kill larvae of C. tenebrionis just after they enter into the roots of the tree. S. feltiae strains provided an efficacy ranging from 79.68% to 88.24%. H. bacteriophora strains resulted in control of 71.66-76.47%. S. carpocapsae (B14) and S. affine (Gspe3) caused lower control of C. tenebrionis larvae (62.03% and 34.76%, respectively). The influence of foraging strategy and the use of native nematodes to control C. tenebrionis larvae inside the roots is discussed.The potential control of the entomopathogenic nematode Steinernema feltiae (Filipjev) (strain Bpa), isolated from a dead C. tenebrionis larva, was tested in a cherry tree orchard in Ullastrell, Barcelona (Spain). Nematode infective juveniles (IJs) were applied by drench and injection. In both the treatments, a rate of 1 million IJs was applied per tree every week during 4 or 8 weeks, with a total dose of 4 x 106 IJs/tree and 8 x 106 IJs/tree. Number, stage and localization of insects in each tree trunk were recorded. In both the experiments, S. feltiae significantly reduced the population of C. tenebrionis providing control ranging from 88.3% to 97%. No significant differences were recorded between the different treatments. Persistence of nematodes was recorded until 6 weeks after application. Results indicate that the application of S. feltiae (Bpa) provides adequate control of C. tenebrionis in cherry trees.

Cry3Aa is a 73 kDa protoxin toxic to insect larvae of Coleoptera order. It is processed to form a stable 65 kDa &amp / #61540 / -endotoxin by endogenous proteases. The first part of this study involved the determination of the patterns of biosynthesis of Coleoptera-specific &amp / #61540 / -endotoxin by a local isolate of Bacillus thuringiensis subsp. tenebrionis (Btt) in relation to its growth and sporulation. Among four different media compared (DSM, GYS, HCT and C2) Cry3Aa production was the highest in DSM, especially at 72nd h and 120th h of incubation. For improvement of Cry3Aa production, the effects of different carbon and nitrogen sources, inorganic phosphate and other mineral elements were tested. Increasing concentrations (5-10 g.L-1) of glucose or sucrose decreased the toxin yield probably by suppressing sporulation. Inorganic phosphate was found to have the most striking effect on toxin biosynthesis. 200 mM inorganic phosphate concentration resulted in 5 fold increase in Cry3Aa yield. Cry3Aa production was greatly reduced when various combinations of organic and inorganic nitrogen sources, especially ammonium sulphate and Casamino acids were replaced with Nutrient broth in DSM. The highest Cry3Aa production was obtained in the media containing 10-5-10-7 M MnCl2, 10-5 M FeSO4 and 5.10-4 M MgSO4, corresponding to their original concentrations in DSM. Decrease of iron concentration or its omission from the medium decreased the toxin yield. Toxin production capacity of our local isolate was compared with those of 30 different anti-Coleopteran Bt strains. Most of the strains producing this protein gave general protein banding patterns quite similar to that of our local isolate. Lastly, the cry3Aa gene of the Btt local isolate was PCR-amplified and cloned into the E. coli/Bacillus shuttle vector pNW33N. The recombinant plasmid was amplified in E. coli and the sequence of the cry3Aa was determined. Amino acid sequence deduced was found to be 97.4 %-99.2 % identical to the cry3Aa sequences (GenBank) of 10 different quaternary ranks. In this respect, the gene has to represent the 11th quaternary rank of the cry3Aa ones. The recombinant plasmid carrying cry3Aa gene was next used to transform Bs 168 as an intermediate host and low level of expression was seen.