Efficacy of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Berliner) in controlling the tomato borer, <Emphasis Type="Italic">Tuta absoluta</Emphasis> (Meyrick) (Lepidoptera: Gelechiidae)

The tomato borer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is considered to be one of the most devastating pests affecting tomato crops in South America, where crop losses range from 60 to 100%. After its detection in the Spanish tomato-growing area at the end of 2006, it spread quickly to other European and northern African countries. Currently, T. absoluta management in these countries is mainly based on chemical treatments. Nonetheless, special emphasis is being placed on implementing environmentally safe strategies. Commercial formulates based on Bacillus thuringiensis may be a good alternative, as they have been used to control other insect pests successfully. The laboratory, greenhouse, and open-field experiments presented in this work are evidence that B. thuringiensis is highly efficient in controlling T. absoluta. First instar larvae were the most susceptible, while susceptibility was lower in second and third instar larvae. Our results have shown that the impact of T. absoluta can be greatly reduced by spraying only B. thuringiensis-based formulates, with no need for chemical insecticides. Furthermore, the integration of this technology with other biological control methods focused on T. absoluta eggs, such as the use of mirid predators or parasitoids, could reduce the number of B. thuringiensis treatments and the use of chemicals, with the consequent reduction of residues on fruits.     

Plant Resistance to the Moth <Emphasis Type="Italic">Tuta absoluta</Emphasis> (Meyrick) (Lepidoptera:Gelechiidae) in Tomato Cultivars

The resistance of 11 tomato cultivars (Ps-6515, Berlina, Poolad, Petoprid-5, Zaman, Matin, Golsar, Sandokan-F1, Golshan-616, Sadeen-95 and Sadeen-21) to the tomato moth, Tuta absoluta (Meyrick) (Lepidoptera:Gelechiidae) was investigated under field conditions. A randomized complete block design was used with three replications. Data analysis indicated that there were significant differences (P?<?0.05) among cultivars regarding leaflet damage, leaf damage, overall plant damage, number of mines per leaf, number of holes on the stem, and fruit. Our findings revealed that the cultivars Berlina, Golsar, Poolad, and Zaman were less suitable cultivars, suggesting that they are more resistant to the tomato moth than the other cultivars. The high density of leaf trichomes present in the cultivars Berlina, Zaman, and Golsar can be one of the possible causes of resistance to T. absoluta. Knowledge of the extent of susceptibility or resistance of cultivars to a pest on a crop is one of the fundamental components of integrated pest management (IPM) programs for any crop.     

Molecular and biological characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains for controlling tomato leafminer (<Emphasis Type="Italic">Tuta absoluta</Emphasis> Meyrick) (Lepidoptera: Gelechiidae) in Colombia

Twenty-eight soil samples were obtained from open fields and greenhouses used for tomato cultivation in various regions of Colombia. For functional characterization, 99 Bacillus thuringiensis (Bt) strains were isolated and characterized by abundance and morphology of microscopic crystals, SDS–PAGE of protein extracts and M-PCR analyses of genes of the cry1 family, as well as for their insecticidal activity against Tuta absoluta second instar larvae. Native Bt strains had amorphous (5%), bi-pyramidal (27%), square (8%), spherical (38%) and triangular (22%) crystal forms. Based on the presence of 1–4 different crystal forms, 18 different profiles were established. The SDS–PAGE analyses of protein extracts established ten different strain groups based on their protein band weight and potential biological activity. The M-PCR technique identified 35 native Bt strains based on the presence of the 6 genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D, whose frequency of occurrence was 76, 26, 21, 35, 32 and 8.8%, respectively. Thirteen different PCR profiles were found in native Bt strains. Several gene combinations tended to co-occur with elevated frequency, such as the pairs cry1Ac/cry1C, cry1Ab/cry1Ac and cry1Ab/cry1B, for which Pearson correlation coefficients were 0.69, 0.52 and 0.54, respectively. Native strains ZBUJTL39 and ZCUJTL11 had up to three times higher biological activity against T. absoluta second instar larvae than the reference strain Bt var. kurstaki HD1, with an LD₅₀ of 2.4 μg/ml (P < 0.05) for native Bt strain ZCUJTL11. This study suggests a high biodiversity of native Bt strains from tomato growing regions in Colombia, which has important implications for designing biological control strategies for T. absoluta.     

The combined use of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Nesidiocoris tenuis</Emphasis> against the tomato borer <Emphasis Type="Italic">Tuta absoluta</Emphasis>

Since Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) was first detected at the end of 2006 in the Mediterranean Basin, several endemic natural enemies have been reported to prey on this exotic pest. The predator Nesidiocoris tenuis Reuter (Hemiptera: Miridae) can regulate T. absoluta populations, because it is able to prey efficiently on T. absoluta eggs. Furthermore, previous studies have demonstrated that first-instar larvae of T. absoluta are highly susceptible to Bacillus thuringiensis (Bt) treatments. In this work, we tested the combination of both approaches under greenhouse conditions. B. thuringiensis formulations were sprayed weekly for two months, three months or throughout the growing cycle, and in all cases, one N. tenuis per plant was also released. Control plants were completely destroyed by the infestation levels reached by T. absoluta. In contrast, all treatments based on B. thuringiensis treatments and releases of N. tenuis reduced leaf damage by more than 97% when compared to the untreated control, with no significant differences among them. Furthermore, yield in the control plants was significantly reduced when compared with all Bt–N. tenuis treatments. Our results demonstrate that when B. thuringiensis treatments are applied immediately after the initial detection of T. absoluta on plants, they do not interfere with N. tenuis establishment in the crop because T. absoluta eggs are available. According to our data, treatments with B. thuringiensis later in the growing season would no longer be necessary because mirids alone would control the pest.     

Flight behavior and oviposition of <Emphasis Type="Italic">Tuta absoluta</Emphasis> on susceptible and resistant genotypes of <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Plants produce volatile chemical compounds that can negatively affect the preference (antixenosis) or the performance (antibiosis) of herbivorous insects. It is thought that these volatile compounds are used as cues by herbivorous insects to determine the suitability of the plant for egg deposition and, hence, offspring performance. Here, we investigated whether volatiles produced by tomato plants play a role in modulating the flight and oviposition behavior of mated Tuta absoluta females. We found that the behavioral steps displayed by mated females did not differ when they flew toward resistant or susceptible tomato genotypes, but they reached the susceptible genotypes faster than the resistant ones. Moreover, females landed more often and laid more eggs on the most susceptible genotype, the Santa Clara variety. Because this variety is known to be of high quality for the development of T. absoluta larvae, the female’s decision to land and lay more eggs on this genotype seems to be mainly to maximize offspring performance. However, this is not so straightforward because the proportion of landings and eggs laid by T. absoluta on another susceptible genotype tested in this study was not significantly higher than on the resistant genotypes. Finally, although future studies are still needed, considering the antixenotic and antibiotic traits of the resistant genotypes studied here, they are likely to succeed if used in integrated pest management.     

<Emphasis Type="Italic">Hsp70</Emphasis> genes of the <Emphasis Type="Italic">Megaphragma amalphitanum</Emphasis> (Hymenoptera: Trichogrammatidae) parasitic wasp

Miniaturization is an evolutionary process that is widely represented in both invertebrates and vertebrates. Miniaturization frequently affects not only the size of the organism and its constituent cells, but also changes the genome structure and functioning. The structure of the main heat shock genes (hsp70 and hsp83) was studied in one of the smallest insects, the Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) parasitic wasp, which is comparable in size with unicellular organisms. An analysis of the sequenced genome has detected six genes that relate to the hsp70 family, some of which are apparently induced upon heat shock. Both induced and constitutively expressed hsp70 genes contain a large number of introns, which is not typical for the genes of this family. Moreover, none of the found genes form clusters, and they are all very heterogeneous (individual copies are only 75–85% identical), which indicates the absence of gene conversion, which provides the identity of genes of this family in Drosophila and other organisms. Two hsp83 genes, one of which contains an intron, have also been found in the M. amalphitanum genome.     

Dispersion and Persistence of <Emphasis Type="Italic">Trichogrammatoidea bactrae</Emphasis> (Nagaraja) over <Emphasis Type="Italic">Tuta absoluta</Emphasis> (Meyrick), in Tomato Greenhouses

Inundative biological control depends on the ability of natural enemies to disperse and persist in the environment. The objective was to evaluate the dispersion and persistence of Trichogrammatoidea bactrae (Nagaraja) on Tuta absoluta (Meyrick) eggs. Inundative releases of this parasitoid were performed in experimental tomato greenhouses. For vertical dispersion, leaves of the upper and middle third of plants were artificially infested with T. absoluta eggs; for horizontal dispersion, plants at increasing distances from a release point were infested. These eggs were renewed at days 2 and 4 to evaluate persistence. The amount of parasitized patches was registered. Logistic regression analysis was used. The position of the eggs in the plant did not affect the DE (discovery efficiency: proportion of parasitized patches). Time since release negatively affected the DE, while distance affected it only when plants were higher. These results could be used to adjust the release methodology of T. bactrae.     

<Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> in its Environment

Plasmodiophora brassicae Wor. is viewed in this article from the standpoint of a highly evolved and successful organism, well fitted for the ecological niche that it occupies. Physical, chemical, and biological components of the soil environment are discussed in relation to their effects on the survival, growth, and reproduction of this microbe. It is evident that P. brassicae is well equipped by virtue of its robust resting spores for survival through many seasonal cycles. Germination is probably triggered as a result of signals initiated by root exudates. The resultant motile zoospore moves rapidly to the root hair surface and penetration and colonization follow. The short period between germination and penetration is one of greatest vulnerability for P. brassicae. In this phase survival is affected at the very least by soil texture and structure; its moisture; pH; calcium, boron, and nitrogen content; and the presence of active microbial antagonists. These factors influence the inoculum potential (sensu Garrett, 1956) and its viability and invasive capacity. There is evidence that these effects may also influence differentially the survival of some physiologic races of P. brassicae. Considering the interaction of P. brassicae with the soil environment from the perspective of its biological fitness is an unusual approach; most authors consider only the opportunities to destroy this organism. The approach adopted here is borne of several decades spent studying P. brassicae and the respect that has been engendered for it as a biological entity. This review stops at the point of penetration, although some of the implications of the environment for successful colonization are included because they form a continuum. Interactions with the molecular and biochemical cellular environment are considered in other sections in this special edition.

Molecular Biology of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Initially, molecular techniques were used to detect and distinguish Plasmodiophora pathotypes in soil. Meanwhile, chromosomes from 2.2 Mb to 680 kb are characterized and the total genome size is estimated to be approximately 20 Mb. Furthermore, the genomic gene structure and the cDNA structure of several genes have been revealed, and the expression of those genes has been linked to development of clubroot to some extent. In addition, the sequence data have reinforced the inclusion of the plasmodiophorids within the Cercozoa. The recent successes in molecular biology have produced new approaches for clubroot research.     

Efficiency of spinosad, <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Trichogramma brassicae</Emphasis> against the tomato leafminer in greenhouse

The objective of this study was to evaluate the efficacy of three eco-friendly control agents, either singly or in a pairwise combination, for the control of the tomato leafminer, Tuta absoluta (Meyrick) (Lep: Gelechiidae). They include the naturally derived pesticide spinosad, a commercially available formulation of Bacillus thuringiensis var. Kurstaki (Bt), and a native population of Trichogramma brassicae Bezdenko (Hym: Trichogrammatidae). Tomato plants containing the T. absoluta were treated with one of the seven following treatments in a greenhouse: (1) a single release of T. brassicae against the eggs; (2) two applications of Bt (2 kg ha^?1); (3) and (4) one application of spinosad at two rates (60 and 120 g a.i. ha^?1); (5) T. brassicae release?+?Bt spray; (6) T. brassicae release?+?spinosad spray; and (7) spinosad spray?+?Bt spray. The highest mortality rate was recorded for the spinosad?+?Bt (88.33?±?1.43%) and T. brassicae?+?spinosad (78.33?±?3.74%) combinations, respectively; while the lowest mortality rate was obtained through the single application of T. brassicae (31.67?±?4.84%). Based on our results, the Bt and spinosad seem to be suitable candidates for combination with other biological and cultural techniques towards an integrated management of the tomato leafminer.     

Development,Reproduction, Survival,and Demographic Patterns of <Emphasis Type="Italic">Tuta absoluta</Emphasis> (Meyrick) (Lepidoptera: Gelechiidae) on Different Commercial Tomato Cultivars

The increase in the production of tomato, Solanum lycopersicon Mill. (Solanaceae), has favored the proliferation of pests, especially Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). In this study, the development, reproduction, survival, and demographic parameters of T. absoluta reared on six commercial tomato cultivars (Cherry, Cordilheira, Giuliana, Nemoneta, Paron, and Santa Clara) were evaluated. Tuta absoluta completed its development in all tomato cultivars. Development from newly hatched caterpillar to newly emerged adult varied between 24.8 and 28.2 days. Female fecundity ranged from 126.3 to 166.9 eggs, with fertility from 54.2 to 84.1%. Mortality during egg-adult development varied between 21.4 and 46.4% for insects reared on cultivars Cherry and Giuliana, respectively. The cultivars Cordilheira, Giuliana, and Santa Clara are promising options to tomato producers in order to decrease the attack and proliferation of T. absoluta. However, the development and population growth of T. absoluta is faster on the tomato cultivar Cherry.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Life Cycle of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Plasmodiphora brassicae is a soil-borne obligate parasite. The pathogen has three stages in its life cycle: survival in soil, root hair infection, and cortical infection. Resting spores of P. brassicae have a great ability to survive in soil. These resting spores release primary zoospores. When a zoospore reaches the surface of a root hair, it penetrates through the cell wall. This stage is termed the root hair infection stage. Inside root hairs the pathogen forms primary plasmodia. A number of nuclear divisions occur synchronously in the plasmodia, followed by cleavage into zoosporangia. Later, 4–16 secondary zoospores are formed in each zoosporangium and released into the soil. Secondary zoospores penetrate the cortical tissues of the main roots, a process called cortical infection. Inside invaded roots cells, the pathogen develops into secondary plasmodia which are associated with cellular hypertrophy, followed by gall formation in the tissues. The plasmodia finally develop into a new generation of resting spores, followed by their release back into soil as survival structures. In vitro dual cultures of P. brassicae with hairy root culture and suspension cultures have been developed to provide a way to nondestructively observe the growth of this pathogen within host cells. The development of P. brassicae in the hairy roots was similar to that found in intact plants. The observations of the cortical infection stage suggest that swelling of P. brassicae-infected cells and abnormal cell division of P. brassicae-infected and adjacent cells will induce hypertrophy and that movement of plasmodia by cytoplasmic streaming increases the number of P. brassicae-infected cells during cell division.     

Daily rhythms in parasitization of the Angoumois grain moth <Emphasis Type="Italic">Sitotroga cerealella</Emphasis> Oliv. (Lepidoptera,Gelechiidae) eggs by the egg parasitoid <Emphasis Type="Italic">Trichogramma principium</Emphasis> Sug. et Sor. (Hymenoptera,Trichogrammatidae) females

Laboratory experiments conducted at L: D = 16: 8 have shown that the observed temporal pattern of parasitization of the Angoumois grain moth, Sitotroga cerealella eggs by Trichogramma principium females represents a resultant of arrhythmic age-related trends and circadian rhythms. Most of females delayed parasitization. The daily number of females starting to parasitize was maximal on the first day of contact with the host and then gradually declined. Practically all of the females started parasitization during the photophase. Moreover, when the first contact with the host fell on the scotophase, the total cumulative percentage of females that started parasitization during four days of the experiment significantly decreased. Oviposition activities of parasitizing females also occurred mainly during photophase. However, under constant light, these circadian rhythms were damped out after one cycle. In addition, anticipatory period of darkness during photophase directly inhibited parasitization. This suggests that the observed rhythms can be easily modified by the direct environmental influence. Under natural conditions, such a flexible oviposition rhythm may be of advantage for these parasitoids enabling them to use any opportunity for reproduction. In biocontrol practice, the lability of parasitization rhythms may enable Trichogramma females to adapt immediately to any new light-dark regimes, although darkness may have negative effects on their efficiency.     

Parasitism of <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Lepidoptera: Noctuidae) by <Emphasis Type="Italic">Microplitis prodeniae</Emphasis> (Hymenoptera: Braconidae)

Spodoptera litura Fabricius is a major vegetable pest that is widely distributed throughout tropical, subtropical and temperate regions. Microplitis prodeniae Rao and Chandry is a solitary endoparasitoid of S. litura. To assess the potential use of this parasitoid as a biological control agent, the reproductive schedule, fecundity and functional response of M. prodeniae were investigated under conditions of 28 ± 1°C and 70 ± 10% relative humidity with a 14:10-h L:D photoperiod. The parasitoid’s average lifetime fecundity was 171.0 ± 10.4 eggs, of which approximately 50% were laid within the first 3 days. Additionally, M. prodeniae exhibited a Holling type II functional response, and the estimated maximum numbers of the 1st, 2nd and 3rd instar larvae that were parasitized by a single M. prodeniae female were 71.6, 78.4 and 41.5 larvae over a 24-h period, respectively. The results of this study suggest that M. prodeniae has great potential as a candidate for controlling S. litura and can guide efforts in its mass production.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Detection and Measurement of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Clubroot, caused by Plasmodiophora brassicae, is one of the most important diseases of brassicas. Management of clubroot is difficult, and the best means of avoiding the disease include planting in areas where P. brassicae is not present and using plants and growing media free from pathogen inoculum. As P. brassicae is not culturable, its detection has traditionally relied on plant bioassays, which are time-consuming and require large amounts of glasshouse space. More recently, fluorescence microscopy, serology, and DNA-based methods have all been used to test soil, water, or plant samples for clubroot. The use of fluorescence microscopy to detect and count pathogen spores in the soil requires significant operator skill and is unlikely to serve as the basis for a routine diagnostic test. By contrast, serologic assays are inexpensive and amenable to high-throughput screening but need to be based on monoclonal antibodies because polyclonal antisera cannot be reproduced and are therefore of limited quantity. Several polymerase chain reaction (PCR)-based assays have also been developed; these are highly specific for P. brassicae and have been well-correlated with disease severity. As such, PCR-based diagnostic tests have been adopted to varying extents in Canada and Australia, but wide implementation has been restricted by sample processing costs. Efforts are underway to develop inexpensive serologic on-farm diagnostic kits and to improve quantification of pathogen inoculum levels through real-time PCR. Proper detection and quantification of P. brassicae will likely play an increasingly important role in the development of effective clubroot management strategies.