Absence of non-target effects of two Bacillus thuringiensis coleopteran active δ-endotoxins on the bulb mite, Rhizoglypus robini (Claparède) (Acari, Acaridae)

Transgenic crops with plant‐incorporated protectants are often more specific than synthetic insecticides and have the potential to reduce impacts on non‐target organisms. In this study we assessed the impact of Cry3Aa and Cry3Bb1 coleopteran‐active δ‐endotoxins on the bulb mite, Rhizoglypus robini. The effect of Cry3Aa prototoxin in solutions of the biopesticide Novodor^® on mite survival was assessed in laboratory studies. Survival of R. robini exposed to Cry3Aa in a short‐term contact and ingestion experiment was not affected, although R. robini was significantly affected by the insecticide Fipronil^® used as a positive control. Similarly, R. robini exposed in a longer duration feeding trial to the Cry3Aa toxin in artificial diet were also not significantly affected. When Cry3Aa was tested on the positive control insect, Leptinotarsa decemlineata, reduced weight of larvae and increased mortality was recorded. The effect of Cry3Bb1 toxin in transgenic corn tissues on R. robini food choice was assessed in a laboratory study. In no‐choice tests a greater proportion of R. robini were found on garlic roots than on Cry3Bb1 transgenic corn and a near‐isogenic non‐transgenic corn. In a choice test, more R. robini was recovered on garlic roots than on either corn variety, and on Cry3Bb1 corn than on non‐transgenic corn. In large field plots using specific mite traps across the growing season, R. robini mite populations were not significantly different between Cry3Bb1 corn and non‐transgenic corn alone or non‐transgenic corn treated with different combinations of two insecticides. Our results, combined with results from other studies, suggest that transgenic plants expressing the Cry3Aa or Cry3Bb1 Bacillus thuringiensis toxins are likely to have negligible impact on R. robini mite populations.     

Foraging activity of bumblebees (Bombus terrestris L.) on Bt-expressing eggplants

A greenhouse experiment was setup to study foraging behavior of the bumblebee Bombus terrestris L. on Cry3Bb-expressing genetically modified (GM) eggplants and their near-isogenic control. Commonly, more bumblebees visited GM eggplants compared to near-isogenic control, but this difference was only marginally significant. The mean length of feeding bouts was similar on the two treatments. Neither the number of flowers produced nor their size could explain bumblebees?? tendency to prefer GM eggplants. Volatile compounds were extracted from five plants per genotype and separated using gas chromatography. Thirteen compounds were identified and five of them appeared significantly more abundant in GM eggplants. Six of the identified compounds [(+)-limonene, Z-jasmone, p-cymene, ??-pinene, methyl-salicilate, and (?)-limonene] were tested in electrophysiological bioassays with antennas detached from young bumblebees, and a response was recorded in all six cases. Experimental results indicate that pollination activity of bumblebees is compatible with this GM eggplant event as a food source and that chemical cues may have an important role in plant identification. The implications for environmental risk assessment of GM plants are discussed.     

Evaluation of adult emergence and larval root injury for Cry3Bb1‐resistant populations of the western corn rootworm

The transgenic maize (Zea mays L.) event MON 88017 produces the Bacillus thuringiensis Berliner (Bt) toxin Cry3Bb1 to provide protection from western corn rootworm (Diabrotica virgifera virgifera LeConte) larval feeding. In response to reports of reduced performance of Cry3Bb1‐expressing maize at two locations in Illinois, we conducted a two‐year experiment at these sites to characterize suspected resistance, as well as to evaluate root injury and adult emergence. Single‐plant bioassays were performed on larvae from each population that was suspected to be resistant. Results indicate that these populations had reduced mortality on Cry3Bb1‐expressing maize relative to susceptible control populations. No evidence of cross‐resistance between Cry3Bb1 and Cry34/35Ab1 was documented for the Cry3Bb1‐resistant populations. Field studies were conducted that included treatments with commercially available rootworm Bt hybrids and their corresponding non‐Bt near‐isolines. When compared with their near‐isolines, larval root injury and adult emergence were typically reduced for hybrids expressing Cry34/35Ab1 either alone or in a pyramid. In many instances, larval root injury and adult emergence were not significantly different for hybrids expressing mCry3A or Cry3Bb1 alone when compared with their non‐Bt near‐isolines. These findings suggest that Cry34/35Ab1‐expressing Bt maize may represent a valuable option for maize growers where Cry3Bb1 resistance is either confirmed or suspected. Consistent trends in adult size (head capsule width and dry mass) for individuals recovered from emergence cages were not detected during either year of this experiment. Because of the global importance of transgenic crops for managing insect pests, these results suggest that improved decision‐making for insect resistance management is needed to ensure the durability of Bt maize.     

Genome composition of asymmetric hybrids in relation to the phylogenetic distance between the parents. Nucleus-chloroplast interaction

Summary A series of fusion experiments were performed between protoplasts of a cytoplasmic albino mutant of tomato, Lycopersicon esculentum (ALRC), and gamma-irradiated protoplasts of L. hirsutum and the Solanum species S. commersonii, S. etuberosum and S. nigrum. These species were chosen for their different phylogenetic relationships to tomato. In all fusion combinations except from those between ALRC and S. nigrum, green calli were selected as putative fusion products and shoots regenerated from them. They were subsequently analyzed for their morphology, nuclear DNA composition and chloroplast DNA origin. The hybrids obtained between ALRC and L. hirsutum contained the chloroplasts of L. hirsutum and had the flower and leaf morphology of L. esculentum. After Southern blot analysis, using 13 restriction fragment length polymorphisms (RFLPs) randomly distributed over all chromosomes, all hybrids showed L. esculentum hybridization patterns. No chromosomes of L. hirsutum were found. These results indicate that these hybrids were true cybrids.The putative asymmetric hybrids, obtained with S. commersonii and S. etuberosum, showed phenotypic traits of both parents. After hybridization with species-specific repetitive nuclear DNA probes it was found that nuclear material of both parents was present in all plants. In the case of S. nigrum, which combination has the greatest phylogenetic distance between the fusion parents, no hybrid plants could be obtained. The chloroplast DNA of all hybrid plants was of the donor type suggesting that chloroplast transfer by asymmetric protoplast fusion can overcome problems associated with large phylogenetic distances between parental plants.     

A biotechnology‐based male‐sterility system for hybrid seed production in tomato

Incorporating male sterility into hybrid seed production reduces its cost and ensures high varietal purity. Despite these advantages, male‐sterile lines have not been widely used to produce tomato (Solanum lycopersicum) hybrid seeds. We describe the development of a biotechnology‐based breeding platform that utilized genic male sterility to produce hybrid seeds. In this platform, we generated a novel male‐sterile tomato line by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐mediated mutagenesis of a stamen‐specific gene SlSTR1 and devised a transgenic maintainer by transforming male‐sterile plants with a fertility‐restoration gene linked to a seedling‐colour gene. Offspring of crosses between a hemizygous maintainer and the homozygous male‐sterile plant segregated into 50% non‐transgenic male‐sterile plants and 50% male‐fertile maintainer plants, which could be easily distinguished by seedling colour. This system has great practical potential for hybrid seed breeding and production as it overcomes the problems intrinsic to other male‐sterility systems and can be easily adapted for a range of tomato cultivars and diverse vegetable crops.     

Methyl salicylate production in tomato affects biotic interactions

The role of methyl salicylate (MeSA) production was studied in indirect and direct defence responses of tomato (Solanum lycopersicum) to the spider mite Tetranychus urticae and the root‐invading fungus Fusarium oxysporum f. sp. lycopersici, respectively. To this end, we silenced the tomato gene encoding salicylic acid methyl transferase (SAMT). Silencing of SAMT led to a major reduction in SAMT expression and MeSA emission upon herbivory by spider mites, without affecting the induced emission of other volatiles (terpenoids). The predatory mite Phytoseiulus persimilis, which preys on T. urticae, could not discriminate between infested and non‐infested SAMT‐silenced lines, as it could for wild‐type tomato plants. Moreover, when given the choice between infested SAMT‐silenced and infested wild‐type plants, they preferred the latter. These findings are supportive of a major role for MeSA in this indirect defence response of tomato. SAMT‐silenced tomato plants were less susceptible to a virulent strain of F. oxysporum f. sp. lycopersici, indicating that the direct defense responses in the roots are also affected in these plants. Our studies show that the conversion of SA to MeSA can affect both direct and indirect plant defence responses.     

Impact of insecticidal proteins expressed in transgenic plants on bumblebee microcolonies

In order to assess the risk that insecticidal transgenic plants may pose for bumblebees, we tested whether Bombus terrestris (L.) (Hymenoptera: Apidae) workers are able to detect insecticidal proteins dissolved in sucrose solution and whether consumption of these proteins will affect survival and offspring production. Feeders containing either Bacillus thuringiensis toxin (Cry1Ab), Kunitz soybean trypsin inhibitor (SBTI), or Galanthus nivalis agglutinin (GNA) were offered to bumblebee colonies at low (0.01% wt/vol for SBTI and GNA, 0.001% for Cry1Ab) and high concentrations (0.1% for SBTI and GNA, 0.01% for Cry1Ab) together with a control (pure sucrose solution) in a glasshouse chamber. No difference was found in the number of visits and the duration of visits among the different concentrations for each of the insecticidal proteins, indicating that bumblebees do not discriminate the compounds. To investigate the impact of the different insecticidal proteins on B. terrestris, microcolonies were established by placing three newly emerged bumblebee workers in wooden boxes. Within a few days, a hierarchy in each microcolony was established and the dominant worker developed its ovaries and laid haploid eggs. Bumblebees were fed with Cry1Ab (0.01%), SBTI, or GNA (both at 0.01 and 0.1%) dissolved in sucrose solution and also fed mixed floral pollen for a maximum period of 80 days. Additionally, microcolonies with three drones each were established to measure individual bee longevity. While the Cry1Ab did not affect microcolony performance, the consumption of SBTI and especially GNA affected survival of B. terrestris workers and drones and caused a significant reduction in the number of offspring. The use of microcolonies appears to be well‐suited to measure lethal and sublethal effects of insecticidal proteins expressed in transgenic plants on bumblebees.     

Compact tomato seedlings and plants upon overexpression of a tomato chromatin remodelling ATPase gene

Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the AtCHR12/23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this approach to the horticultural crop tomato (Solanum lycopersicum). We identified and cloned the single tomato ortholog of the two Arabidopsis Snf2 genes, designated SlCHR1. Transgenic tomato plants (cv. Micro‐Tom) that constitutively overexpress the coding sequence of SlCHR1 show reduced growth in all developmental stages of tomato. This confirms that SlCHR1 combines the functions of both Arabidopsis genes in tomato. Compared to the wild type, the transgenic seedlings of tomato have significantly shorter roots, hypocotyls and reduced cotyledon size. Transgenic plants have a much more compact growth habit with markedly reduced plant height, severely compacted reproductive structures with smaller flowers and smaller fruits. The results indicate that either GMO‐based or non‐GMO‐based approaches to modulate the expression of chromatin remodelling ATPase genes could develop into methods to control plant growth, for example to replace the use of chemical growth retardants. This approach is likely to be applicable and attractive for any crop for which growth habit reduction has added value.     

Competence of the pollen of wild species and the cultivar of tomato to affect fertilization at low temperature

Summary The competitive ability of the pollen of wild tomato species (Solanum pennellii, Lycopersicum hirsutum and L. minutum) and a cultivar of tomato to affect fertilization at low temperature was investigated. Pollen grains of wild species were mixed with those of the cultivar and pollinated onto the stigma of the cultivar. The number of seedlings resembling the wild species, excluding L. minutum, were significantly higher when the plants were maintained at a low temperature (8°–12° C) than when they were maintained at a normal temperature (22°–28° C). This indicates that the pollen of S. pennellii and L. hirsutum wild species can compete better with pollen of the cultivar at a low temperature, while there was no change in the competitive ability of L. minutum pollen under these conditions.     

Effects of adult emergence timing on susceptibility and fitness of Cry3Bb1‐resistant western corn rootworms

Field‐evolved resistance by the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte to the Cry3Bb1 trait expressed in maize, has been documented in areas of Nebraska USA. Currently, only limited information is available on life‐history traits of Cry3Bb1‐resistant field populations. Therefore, the Gassmann on‐plant bioassay was used to investigate the potential variability among four Cry3Bb1‐resistant WCR field collections made in 2011–2012 by focusing on the key parameters: larval survival, developmental stage and weight with specific emphasis on the impact of adult emergence timing on these parameters in subsequent progeny. Key results: In three of four collections, the susceptibility of larval progeny from adults that emerged early or late within a generation from Cry3Bb1 plants was similar. Each of the three collections exhibited complete resistance; that is, survival on Cry3Bb1 plants was greater or equal to survival on non‐Bt isoline plants. Bioassays from an additional field collection from one site 2 years (2013) after the original collection (2011) (both from Cry3Bb1 maize) indicated that resistance to Cry3Bb1 was maintained over time at the site despite Bt trait rotation in 2012. In general, comparative WCR life‐history parameter data from Cry3Bb1 and isoline maize indicate that fitness of field collections exhibiting complete resistance was similar on each hybrid. The mean proportion of larvae in third instar and mean weight of larvae recovered in bioassays from progeny of early‐ and late‐emerged adults was not significantly affected by emergence period. This suggests that delays in development and associated mean adult emergence commonly observed in populations that are susceptible to Cry3Bb1 may become smaller as populations become resistant to Cry3Bb1. Results from this article will inform Cry3Bb1 resistance mitigation efforts and contribute to the development of sustainable WCR management programmes.     

Biological and molecular characterization of two tomato strains of potato virus Y (PVY)

Two PVY tomato strains (LYE 84 and LYE 84.2), arising from the same natural isolate, and a strain originating from a wild Solanaceous host, Solanum nigrum (SON 41.2), were compared for host range and symptomatology. All strains induced mosaic without necrosis on tobacco as PVY^O strains. The two tomato strains behaved similarly on pepper, infecting only susceptible pepper cultivars (pathotype 0), whereas SON 41.2 was able to overcome the two alleles of the recessive resistance gene pvr2 (pathotype 1.2). On the other hand, only LYE 84.2 was virulent on tomato and broke the resistance of the wild genitor Lycopersicon hirsutum PI 247087. Sequence determination of the capsid gene and the 3′ non-coding region of LYE 84 and LYE 84.2 showed a total homology at both nucleic acid and amino acid levels. This suggests that LYE 84.2 has probably derived from LYE 84, that both strains have very similar sequences and that the capsid protein does not play a direct role in the resistance-breaking capacity of LYE 84.2.     

Pepino mosaic virus: a successful pathogen that rapidly evolved from emerging to endemic in tomato crops

Taxonomy: Pepino mosaic virus (PepMV) belongs to the Potexvirus genus of the Flexiviridae family. Physical properties: PepMV virions are nonenveloped flexuous rods that contain a monopartite, positive‐sense, single‐stranded RNA genome of 6.4 kb with a 3′ poly‐A tail. The genome contains five major open reading frames (ORFs) encoding a 164‐kDa RNA‐dependent RNA polymerase (RdRp), three triple gene block proteins of 26, 14 and 9 kDa, and a 25‐kDa coat protein. Genome diversity: Four PepMV genotypes, with an intergenotype RNA sequence identity ranging from 78% to 95%, can be distinguished: the original Peruvian genotype (LP); the European (tomato) genotype (EU); the American genotype US1; and the Chilean genotype CH2. Transmission: PepMV is very efficiently transmitted mechanically, and a low seed transmission rate has been demonstrated. In addition, bumblebees have been associated with viral transmission. Host range: Similar to other Potexviruses, PepMV has a rather narrow host range that is thought to be largely restricted to species of the Solanaceae family. After originally being isolated from pepino (Solanum muricatum), PepMV has been identified in natural infections of the wild tomato species S. chilense, S. chmielewskii, S. parviflorum and S. peruvianum. PepMV is causing significant problems in the cultivation of the glasshouse tomato Solanum lycopersicum, and has been identified in weeds belonging to various plant families in the vicinity of tomato glasshouses. Symptomatology: PepMV symptoms can be very diverse. Fruit marbling is the most typical and economically devastating symptom. In addition, fruit discoloration, open fruit, nettle‐heads, leaf blistering or bubbling, leaf chlorosis and yellow angular leaf spots, leaf mosaic and leaf or stem necrosis have been associated with PepMV. The severity of PepMV symptoms is thought to be dependent on environmental conditions, as well as on the properties of the viral isolate. Minor nucleotide sequence differences between isolates from the same genotype have been shown to lead to enhanced aggressiveness and symptomatology. Control: Prevention of infection through strict hygiene measures is currently the major strategy for the control of PepMV in tomato production. Cross‐protection can be effective, but only under well‐defined and well‐controlled conditions, and the effectiveness depends strongly on the PepMV genotype.     

Meiotic recombination profiling of interspecific hybrid F1 tomato pollen by linked read sequencing

Genome wide screening of pooled pollen samples from a single interspecific F1 hybrid obtained from a cross between tomato, Solanum lycopersicum and its wild relative, Solanum pimpinellifolium using linked read sequencing of the haploid nuclei, allowed profiling of the crossover (CO) and gene conversion (GC) landscape. We observed a striking overlap between cold regions of CO in the male gametes and our previously established F6 recombinant inbred lines (RILs) population. COs were overrepresented in non‐coding regions in the gene promoter and 5′UTR regions of genes. Poly‐A/T and AT rich motifs were found enriched in 1 kb promoter regions flanking the CO sites. Non‐crossover associated allelic and ectopic GCs were detected in most chromosomes, confirming that besides CO, GC represents also a source for genetic diversity and genome plasticity in tomato. Furthermore, we identified processed break junctions pointing at the involvement of both homology directed and non‐homology directed repair pathways, suggesting a recombination machinery in tomato that is more complex than currently anticipated.     

Behavior and biology of the tomato psyllid, Bactericerca cockerelli, in response to the Mi-1.2 gene

The Mi‐1.2 gene, identified from wild varieties of tomato, Solanum peruvianum (Mill) (Solanaceae), has been incorporated into near‐isogenic commercial varieties of tomato and has been shown to confer resistance to three different species of phloem feeders: aphids, whiteflies, and nematodes. The results presented here show that plants bearing Mi‐1.2 were also resistant to the tomato psyllid, Bactericerca [Paratrioza] cockerelli (Sulc) (Homoptera: Psyllidae), a serious pest of tomato, Solanum lycopersicon (Mill), in the western half of North America. In choice studies, tomato psyllids preferred to settle on plants that did not contain the gene [Moneymaker (mi‐1.2)] compared to near‐isogenic plants with the gene [Motelle (Mi‐1.2)]. As a result, total oviposition was higher on the susceptible variety, although no‐choice studies indicated that there were no differences in numbers of eggs laid by individual females on either variety. Survival from egg to adult was higher on plants lacking the gene compared to plants containing the gene. However, there were no differences in total development time of individuals reared from either variety. The results suggest that mechanisms of resistance to the tomato psyllid observed in plants bearing the Mi‐1.2 gene are distinct from the mechanisms of resistance to the three phloem feeders examined in other studies.     

Screening of non-tuber bearing Solanaceae for resistance to and induction of juvenile hatch of potato cyst nematodes and their potential for trap cropping

Ninety accessions of non‐tuber bearing Solanaceae were screened for (i) resistance to and (ii) stimulatory effect on juvenile hatch of potato cyst nematodes, and (iii) their growth under temperate climatic conditions. All plant species belonging to the genus Solanum tested induced hatching but this effect was most pronounced for plant species of the Solanum nigrum complex. Hatching of juveniles was hardly or not stimulated by other plant genera of the Solanaceae. Solanum sisymbriifolium combined a high hatching effect with complete resistance to both Globodera rosiochiensis and G pallida. Two S. nigrum varieties showed full resistance to G rostochiensis and a high level of resistance to G pallida. Moreover, S. sisymbriifolium and the two varieties of S. nigrum performed very well under Dutch field conditions and, therefore, they are suggested as candidate trap crops for the control of potato cyst nematodes.     

Interrelated responses of tomato plants and the leaf miner Tuta absoluta to nitrogen supply

Plant–insect interactions are strongly modified by environmental factors. This study evaluates the influence of nitrogen fertilisation on the tomato (Solanum lycopersicum L.) cv. Santa clara and the leafminer (Tuta absoluta (Meyrick), Lepidoptera: Gelechiidae). Greenhouse‐grown tomato plants were fed hydroponically on a complete nutrient solution containing either a high nitrogen concentration (HN) sustaining maximum growth or a low nitrogen concentration (LN) limiting plant growth. Insect‐free plants were compared with plants attacked by T. absoluta. Seven and 14 days after artificial oviposition leading to efficacious hatching and larvae development, we measured total carbon, nitrogen and soluble protein as well as defence compounds (phenolics, glycoalkaloids, polyphenol oxidase activity) in the HN versus LN plants. Only in the HN treatment did T. absoluta infestation slightly impair leaf growth and induce polyphenol oxidase (PPO) activity in the foliage. Neither the concentration of phenolic compounds and proteins nor the distribution of nitrogen within the plant was affected by T. absoluta infestation. In contrast, LN nutrition impaired T. absoluta‐induced PPO activity. It decreased protein and total nitrogen concentration of plant organs and enhanced the accumulation of constitutive phenolics and tomatine. Moreover, LN nutrition impaired T. absoluta development by notably decreasing pupal weight and lengthening the development period from egg to adult. Adjusting the level of nitrogen nutrition may thus be a means of altering the life cycle of T. absoluta. This study provides a comprehensive dataset concerning interrelated responses of tomato plants and T. absoluta to nitrogen nutrition.     

Identification of a short rDNA spacer sequence highly specific of a tomato line containing Tm-1 gene introgressed from Lycopersicon hirsutum

Summary We studied rDNA restriction fragment length polymorphism between two tomato lines used for F₁ hybrid seed production: line A, containing the Tm-1 gene responsible for tobacco mosaic virus tolerance introgressed from the wild species Lycopersicon hirsutum, and line B, a tobacco mosaic virus sensitive line. Hybridization patterns led to distinct rDNA maps with two size classes, 10.4 and 10.7 kb, in line A and a single, 8.9-kb class in line B. Size differences were located in the intergenie sequence (IGS). A highly specific 54-bp TaqI fragment was cloned from the line A IGS and used in dot blot experiments to probe total DNA from line A, line B, and their F₁ hybrid. It proved capable of discriminating B from A and the hybrid. This probe could thus serve to screen inbreds in commercial seed lots where line A is used as male. This fragment showed 80–90% sequence homology with the 53-bp subrepeats previously characterized in a region of the tomato IGS close to the 25S rRNA gene. Preliminary comparison of rDNA in line A and several wild related species indicated that the L. hirsutum H₂ genotype was the closest to line A. rDNA variations between line A and this wild genotype could be explained by recombination during the introgression process involving numerous backcrosses or by an important intraspecific polymorphism. Our results strongly suggest that Tm-1 and the rDNA were introgressed together into tomato from L. hirsutum through linkage drag.     

Weed species in tomato production and their role as alternate hosts of Tomato spotted wilt virus and its vector Frankliniella occidentalis

Tomato spotted wilt virus (TSWV) is an important plant virus that infects a wide range of hosts including weeds making its management difficult. A survey was undertaken to establish the occurrence of weed species in tomato production systems in Kenya and their role as hosts of TSWV and its vectors. Selected weed species were further evaluated for their reaction to TSWV, transmission efficiency by Frankliniella occidentalis and ability to support thrips reproduction. Of the 43 weed species identified in the field, 29 species had been reported as hosts of TSWV, two were non‐hosts and 11 had no record of their status. Among the more common species, Amaranthus hybridus, Solanum nigrum, Tagetes minuta and Datura stramonium were susceptible to the virus and supported high levels of thrips reproduction. The TSWV could not be transmitted to Galinsoga parviflora and Sonchus oleraceus by F. occidentalis despite them being highly susceptible in mechanical transmission tests. There was a significant correlation between feeding damage and number of larvae of F. occidentalis on different weeds. Occurrence of weeds that support thrips reproduction and are good hosts of TSWV is a clear indicator of their role in epidemiology and the importance of their management for disease control.