Economic injury level and crop loss assessment for Tuta absoluta (Lepidoptera: Gelechiidae) on different tomato cultivars

Specifying the relationship between pests and their host plants in terms of damage is one of the basic concepts of integrated pest management (IPM) programmes. The crop loss and economic injury level (EIL) of Tuta absoluta (Lepidoptera: Gelechiidae) were determined on different tomato cultivars using gain threshold (field experiment) and regression (semi‐field experiment) methods. By assessing the crop loss in 2015 and 2016, four out of seven infested tomato cultivars had a significant yield loss in terms of weight of total fruits versus the control under the open‐field conditions. However, the total number of tomato cultivars had no significant difference to control treatment. The semi‐field experiment included six treatments (0, 2, 4, 6, 8 and 10 gravid females/plant) and examined two tomato cultivars. The tomato cages with between 6 and 10 gravid females/plant showed the highest number and weight of damaged fruits. Based on pesticide control costs, the market value of tomato and the control efficacy, we determined the EIL for T. absoluta. In the field experiment, the EIL value for T. absoluta on “Petomech,” “Cal JN3,” “Rio Grande” and “Early Urbana Y” cultivars was 4.15, 4.47, 4.70 and 5.04 larvae per plant, respectively. Based on regression equations, we calculated the EIL values for T. absoluta on “Cal JN3” and “Early Urbana Y” to be 4.75 and 5.44 larvae/plant, respectively. Crop loss assessment and EIL are necessary components of cost‐effective IPM programs and can be effective tools for making decisions about the application of pesticides against T. absoluta.     

Mutation in the ace‐1 gene of the tomato leaf miner (Tuta absoluta) associated with organophosphates resistance

The tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae), is a major invasive pest that has spread throughout many countries in the Mediterranean basin and parts of Asia over the last decade. The control of T. absoluta has relied heavily on the use of chemical insecticides, a strategy that has led to the evolution of resistance. In this study, biological and molecular methods were used to determine the susceptibility of five strains of T. absoluta to the organophosphate chlorpyrifos and to investigate the molecular mechanisms underlying resistance to this class of insecticides. High levels of resistance to chlorpyrifos were observed in all five strains tested. Cloning and sequencing of the gene encoding the organophosphate target site, ace‐1, of T. absoluta revealed the presence of an alanine to serine substitution at a position that has been previously linked with organophosphate resistance across a range of different insect and mite species. The presence of this mutation at high frequency in T. absoluta populations originating from various countries further supports the suggestion that the rapid expansion of this species is, in part, mediated by the resistance of this pest to chemical insecticides.     

Light‐dependent regulation of ascorbate in tomato by a monodehydroascorbate reductase localized in peroxisomes and the cytosol

Ascorbate is a powerful antioxidant in plants, and its levels are an important quality criteria in commercial species. Factors influencing these levels include environmental variations, particularly light, and the genetic control of its biosynthesis, recycling and degradation. One of the genes involved in the recycling pathway encodes a monodehydroascorbate reductase (MDHAR), an enzyme catalysing reduction of the oxidized radical of ascorbate, monodehydroascorbate, to ascorbate. In plants, MDHAR belongs to a multigene family. Here, we report the presence of an MDHAR isoform in both the cytosol and peroxisomes and show that this enzyme negatively regulates ascorbate levels in Solanum lycopersicum (tomato). Transgenic lines overexpressing MDHAR show a decrease in ascorbate levels in leaves, whereas lines where MDHAR is silenced show an increase in these levels in both fruits and leaves. Furthermore, the intensity of these differences is light dependent. The unexpected effect of this MDHAR on ascorbate levels cannot be explained by changes in the expression of Smirnoff–Wheeler pathway genes, or the activity of enzymes involved in degradation (ascorbate peroxidase) or recycling of ascorbate (dehydroascorbate reductase and glutathione reductase), suggesting a previously unidentified mechanism regulating ascorbate levels.     

Predation and reproduction of the generalist predator Nabis pseudoferus preying on Tuta absoluta

The damsel bug, Nabis pseudoferus Remane (Hemiptera: Namidae), is a generalist predator of small arthropods, including key insect pests of vegetable crops. In this study, we characterized the predation and development of various N. pseudoferus life stages when preying on the eggs and first- and fourth-instar nymphs of the invasive South American tomato pinworm, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). These findings were compared to those of the bug’s common prey, cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae). Of the life stages tested, females showed the highest predation rate on all tested prey, due to their high longevity, large body, and great energy requirements for reproduction. The predator’s oviposition rate was clearly influenced by prey type, and was highest when feeding on fourth instars of T. absoluta. Considering the zoophytophagy of most of the life stages of other mirid species of tomato, and the lower propensity of N. pseudoferus to feed on plants, these results show that this species can be considered as a key indigenous natural enemy for sustainable pest control packages against T. absoluta in newly invaded areas.     

Next‐generation sequencing (NGS)‐based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next‐generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty‐five genes belonging to carotenoids and folate metabolism were PCR‐amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600‐bp amplicons were directly sequenced in a non‐overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128‐fold pooling. An evaluation of six different software programs (camba , crisp , gatk unified genotyper , lofreq , snver and vipr ) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.     

HT proteins contribute to S‐RNase‐independent pollen rejection in Solanum

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self‐incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S‐RNase and HT protein function in a pistil‐side IRB that causes rejection of pollen from self‐compatible (SC) red/orange‐fruited species in the tomato clade. However, S‐RNase‐independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S‐RNase‐independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S‐RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S‐RNase, and then used RNAi to test whether they also function in S‐RNase‐independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S‐RNase‐dependent and S‐RNase‐independent pollen rejection. The results support the view that overall compatibility results from multiple pollen–pistil interactions with additive effects.     

microRNA159‐targeted SlGAMYB transcription factors are required for fruit set in tomato

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e. auxin and gibberellins) in controlling fruit set after pollination and fertilization, the role of microRNA‐based regulation during ovary development and fruit set is still poorly understood. Here we show that the microRNA159/GAMYB1 and ‐2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159‐overexpressing tomato cv. Micro‐Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159‐overexpressing plants resulted in misregulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA‐like genes and the miR167/SlARF8a module. Similarly to SlMIR159‐overexpressing plants, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159‐targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set.     

The role of a class III gibberellin 2‐oxidase in tomato internode elongation

A network of environmental inputs and internal signaling controls plant growth, development and organ elongation. In particular, the growth‐promoting hormone gibberellin (GA) has been shown to play a significant role in organ elongation. The use of tomato as a model organism to study elongation presents an opportunity to study the genetic control of internode‐specific elongation in a eudicot species with a sympodial growth habit and substantial internodes that can and do respond to external stimuli. To investigate internode elongation, a mutant with an elongated hypocotyl and internodes but wild‐type petioles was identified through a forward genetic screen. In addition to stem‐specific elongation, this mutant, named tomato internode elongated ‐1 (tie‐1) is more sensitive to the GA biosynthetic inhibitor paclobutrazol and has altered levels of intermediate and bioactive GAs compared with wild‐type plants. The mutation responsible for the internode elongation phenotype was mapped to GA2oxidase 7, a class III GA 2‐oxidase in the GA biosynthetic pathway, through a bulked segregant analysis and bioinformatic pipeline, and confirmed by transgenic complementation. Furthermore, bacterially expressed recombinant TIE protein was shown to have bona fide GA 2‐oxidase activity. These results define a critical role for this gene in internode elongation and are significant because they further the understanding of the role of GA biosynthetic genes in organ‐specific elongation.     

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.     

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.     

Evaluation of Tomato Hybrids Carrying Ty‐1 and Ty‐2 Loci to Japanese Monopartite Begomovirus Species

Tobacco leaf curl Japan virus, Honeysuckle yellow vein mosaic virus and Tomato yellow leaf curl virus are three begomoviruses that infect tomato crops in Japan. Tomato infection by begomoviruses has increased in Japan after the development of a high level of resistance to certain insecticides in some populations of the vector B. tabaci biotypes ‘B and Q’. Ty‐1 and Ty‐2 homozygous tomato hybrids were evaluated for reaction to monopartite begomovirus species in Japan by Agrobacterium‐mediated inoculation. Test plants were evaluated by a disease assessment scale (DAS), varying from 1 = no symptoms to 4 = severe symptoms, and systemic infection was evaluated by polymerase chain reaction (PCR), using specific begomovirus primers for each virus. Ty‐1 hybrids showed tolerance to HYVMV and with a large number of plants being neither virus‐free nor symptom‐free. The response of Ty‐1 hybrids was also resistant to moderately resistant against TbLCJV. The response of Ty‐2 hybrids was resistant to highly resistant against the three monopartite begomoviruses, when compared with susceptible plants.     

Potential of a tomato MAGIC population to decipher the genetic control of quantitative traits and detect causal variants in the resequencing era

Identification of the polymorphisms controlling quantitative traits remains a challenge for plant geneticists. Multiparent advanced generation intercross (MAGIC) populations offer an alternative to traditional linkage or association mapping populations by increasing the precision of quantitative trait loci (QTL) mapping. Here, we present the first tomato MAGIC population and highlight its potential for the valorization of intraspecific variation, QTL mapping and causal polymorphism identification. The population was developed by crossing eight founder lines, selected to include a wide range of genetic diversity, whose genomes have been previously resequenced. We selected 1536 SNPs among the 4 million available to enhance haplotype prediction and recombination detection in the population. The linkage map obtained showed an 87% increase in recombination frequencies compared to biparental populations. The prediction of the haplotype origin was possible for 89% of the MAGIC line genomes, allowing QTL detection at the haplotype level. We grew the population in two greenhouse trials and detected QTLs for fruit weight. We mapped three stable QTLs and six specific of a location. Finally, we showed the potential of the MAGIC population when coupled with whole genome sequencing of founder lines to detect candidate SNPs underlying the QTLs. For a previously cloned QTL on chromosome 3, we used the predicted allelic effect of each founder and their genome sequences to select putative causal polymorphisms in the supporting interval. The number of candidate polymorphisms was reduced from 12 284 (in 800 genes) to 96 (in 54 genes), including the actual causal polymorphism. This population represents a new permanent resource for the tomato genetics community.     

Semi‐field evaluation of a granulovirus and Bacillus thuringiensis ssp. kurstaki for season‐long control of the potato tuber moth,Phthorimaea operculella

There are few insecticidal options for potato tuber moth (PTM), Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae), control late in the growing season. We evaluated the PTM granulovirus (PoGV) and Bacillus thuringiensis Berliner ssp. kurstaki (Btk) for season‐long control of PTM on potato foliage in 2006 and 2007. Compared to untreated controls, 10 weekly applications of PoGV (10¹³ occlusion bodies/ha) reduced PTM populations in replicated 1‐m³ field cages by 86–96% on pre‐harvest foliage and 90–97% on tubers added to cages shortly before harvest. Infection rates of 82–95% of L4 larvae by PoGV were noted within individual larval cohorts. Equivalently timed Btk treatments (1.12 kg product/ha) were significantly less effective at population suppression, with a 36–76% reduction in larvae recovered from tubers added to cages. A PoGV/Btk alternation was significantly more effective than Btk alone and as effective as PoGV in 2007, but not in 2006. There was some evidence that reduced rate PoGV treatments (10% rate or 50% application frequency) were less effective than the standard program. There were no treatment effects on percentage of tubers growing in the ground that were infested at harvest, which remained comparatively low at ≤8.1%. Bioassays were conducted to evaluate the residual activities of foliar deposits. Early‐season applications were highly effective for the first 24 h (≥93% mortality) with a steady decline in activity over 10 days. A second application, applied later in the season, showed similar patterns, although in this case Btk was less persistent than PoGV, whereas both agents provided significant larval mortality compared with controls over 14 days. Both PoGV and Btk provide alternatives to manage field infestations of PTM prior to harvest, thus reducing the risk of tuber infestations in storage.     

Identification of a distinct strain of Cotton leaf curl Gezira virus infecting tomato in Oman

Tomato (Solanum lycopersicum) plants exhibiting yellowing, curling and stunting symptoms were identified in fields of the Tawoos Agricultural Systems, in Al‐Batinah in Oman. Cloning and sequencing of restriction endonuclease digested rolling circle amplified viral DNA identified a cotton begomovirus (family Geminiviridae) associated with the symptomatic tomato plants. Detailed analysis of complete sequences showed the virus to be a previously unknown strain of Cotton leaf curl Gezira virus (CLCuGeV) in association with the betasatellite Tomato leaf curl betasatellite (ToLCB). The new CLCuGeV strain, for which the name “Al Batinah” strain is suggested, has the greatest levels of sequence identity (91.9%) to an isolate of CLCuGeV recently reported from the neighbouring United Arab Emirates. Additionally, CLCuGeV‐Al Batinah was shown to have a recombinant origin with sequences donated by an African cassava mosaic virus‐like parent. This is the first identification of this Malvaceae‐adapted begomovirus in tomato. Although ToLCB is common in Oman, being one of only two betasatellites identified there so far, this is the first identification of this betasatellite with CLCuGeV. The significance of these findings is discussed.     

A farnesyl pyrophosphate synthase gene expressed in pollen functions in S‐RNase‐independent unilateral incompatibility

Multiple independent and overlapping pollen rejection pathways contribute to unilateral interspecific incompatibility (UI). In crosses between tomato species, pollen rejection usually occurs when the female parent is self‐incompatible (SI) and the male parent self‐compatible (SC) (the ‘SI × SC rule’). Additional, as yet unknown, UI mechanisms are independent of self‐incompatibility and contribute to UI between SC species or populations. We identified a major quantitative trait locus on chromosome 10 (ui10.1) which affects pollen‐side UI responses in crosses between cultivated tomato, Solanum lycopersicum, and Solanum pennelliiLA0716, both of which are SC and lack S‐RNase, the pistil determinant of S‐specificity in Solanaceae. Here we show that ui10.1 is a farnesyl pyrophosphate synthase gene (FPS2) expressed in pollen. Expression is about 18‐fold higher in pollen of S. pennellii than in S. lycopersicum. Pollen with the hypomorphic S. lycopersicum allele is selectively eliminated on pistils of the F₁ hybrid, leading to transmission ratio distortion in the F₂ progeny. CRISPR/Cas9‐generated knockout mutants (fps2) in S. pennelliiLA0716 are self‐sterile due to pollen rejection, but mutant pollen is fully functional on pistils of S. lycopersicum. F₂ progeny of S. lycopersicum × S. pennellii (fps2) show reversed transmission ratio distortion due to selective elimination of pollen bearing the knockout allele. Overexpression of FPS2 in S. lycopersicum pollen rescues the pollen elimination phenotype. FPS2‐based pollen selectivity does not involve S‐RNase and has not been previously linked to UI. Our results point to an entirely new mechanism of interspecific pollen rejection in plants.