Comparative analysis of peach and pear fruit volatiles attractive to the oriental fruit moth,Cydia molesta

Cydia molesta is an important pest of peach and pear fruits late in growing season. We identified and quantified volatiles from immature and mature fruits of peach and pear using gas chromatography-mass spectrometry. Antennally, active compounds were identified by gas chromatography-electroantennogram detection and were further tested in the field. Consistent electroantennographic activity was obtained for nine compounds from headspace collections of the mature fruits of two peach and two pear varieties. Esters were the major components, but qualitative and quantitative differences were found among four odor profiles. Blends mimicking pear fruit volatiles were more attractive to both sexes than blends mimicking peach fruit volatiles in both orchards. Our finding indicated that mixtures mimicking peach and pear fruit volatiles attracted both females and males of C. molesta, and these host volatiles may also be involved in the host switch behavior from peach to pear in mid-late season.     

Electrophysiological and behavioral response of the oriental fruit moth <Emphasis Type="Italic">Grapholita</Emphasis><Emphasis Type="Italic">molesta</Emphasis> (Busck, 1916) (Lepidoptera: Tortricidae) to the volatiles of apple fruits at different stages of development

The oriental fruit moth, Grapholita molesta, occurs in Southern Brazil throughout the year, and migrates from peach to apple orchards. Because moths rely on volatile organic compounds (VOCs) during the host-location process, variations in the emission of these compounds during fruit maturation can influence the time of infestation and preference of the moths for a particular genotype. The aim of this work was to identify VOCs emitted by the apples “Eva” and “Gala” at different stages of development and to determine the behavioral and electrophysiological responses of G. molesta to these compounds. For this purpose, VOCs from immature, maturing, and mature fruits of both cultivars were collected and analyzed by gas chromatography and gas chromatography-mass spectroscopy. The response of the antennae of virgin males and females and mated females to volatiles released by the three fruit stages was registered by gas chromatography coupled to an electroantennography. A dual-choice behavioral test for the different combinations of insect groups and fruit stage was also performed. Amongst the volatiles released by mature fruits, twelve compounds elicited a response. The antennae of the oriental fruit moth did respond to isoamyl hexanoate and α-farnesene emitted by “Eva” maturing fruits. In general, virgin females did not respond to volatiles in olfactometer bioassays and mated females were attracted to volatiles released by mature fruits. Our results show that the variation in the emission during the maturation of fruits can influence the orientation of G. molesta.     

Response of female Cydia molesta (Lepidoptera: Tortricidae) to plant derived volatiles

Peach shoot volatiles were attractive to mated female oriental fruit moth, Cydia molesta (Busck), in a dual choice arena. No preference was observed between leaf odours from the principle host plant, peach, and the secondary host plant, apple. Twenty-two compounds were identified in headspace volatiles of peach shoots using gas chromatography-mass spectrometry. Green leaf volatiles accounted for more than 50% of the total emitted volatiles. A bioassay-assisted fractionation using different sorbent polymers indicated an attractant effect of compounds with a chain length of 6-8 carbon atoms. The major compounds of this fraction were tested either singly or in combinations for behavioural response of females. Significant bioactivity was found for a three-component mixture of (Z)-3-hexen-1-yl acetate, (Z)-3-hexen-1-ol and benzaldehyde in a 4:1:1 ratio. This synthetic mixture elicited a similar attractant effect as the full natural blend from peach shoots as well as the bioactive fraction.     

Olfactory response of a predatory mirid to herbivore induced plant volatiles: multiple herbivory vs. single herbivory

Plants infested with a single herbivore species can attract natural enemies through the emission of herbivore‐induced plant volatiles (HIPVs). However, under natural conditions plants are often attacked by more than one herbivore species. We investigated the olfactory response of a generalist predators Macrolophus caliginosus to pepper infested with two‐spotted spider mites, Tetranychus urticae, or green peach aphid, Myzus persicae, vs. plants infested with both herbivore species in a Y‐tube olfactometer set up. In addition, the constituents of volatile blends from plants exposed to multiple or single herbivory were identified by gas chromatography‐mass spectrometry (GC‐MS). The mirid bugs showed a stronger response to volatiles emitted from plants simultaneously infested with spider mites and aphids than to those emitted from plants infested by just one herbivore, irrespective of the species. Combined with results from previous studies under similar conditions we infer that this was a reaction to herbivore induced plant volatiles. The GC‐MS analysis showed that single herbivory induced the release of 22 additional compounds as compared with the volatiles emitted from clean plants. Quantitative analyses revealed that the amount of volatile blends emitted from pepper infested by both herbivores was significantly higher than that from pepper infested by a single herbivore. Moreover, two unique substances were tentatively identified (with a probability of 94% and 91%, respectively) in volatiles emitted by multiple herbivory damaged plants: α‐zingiberene and dodecyl acetate.     

挥发性香气物质和乙烯生产在"湖景蜜露"桃果实发育过程中的变化

以"湖景蜜露"水蜜桃(Prunus persica L.)为试材,检测了果实从未成熟到成熟发育过程中乙烯生成、呼吸速率及挥发性香气性物质的变化;同时对果实大小、果皮色泽、果肉硬度、可溶性固形物、可滴定酸进行了测定;对与果实乙烯产生密切相关的1-氨基环丙烷-1-羧酸(ACC)含量、ACC合成酶活性、ACC氧化酶活性也进行了测定.结果表明,随果实成熟度的增加,果实大小、果皮L*值、可溶性固形物含量增加,而果实硬度、果皮h°值、可滴定酸含量减少.在未成熟的果实中,C6的醛类(反式-2-己烯醛)和醇类(顺式-3-己烯醇)是主要的成分;乙烯生成量很低;呼吸速率较高.到跃变阶段C6～C12的内酯类物质明显增加,尤其是γ和δ-内酯类成为果实主要的香气挥发性物质.推测果实乙烯、呼吸作用等基本的生理变化可能调节着内酯类物质的生成.在乙烯跃变上升时果肉中ACC氧化酶的活性下降,ACC含量和ACC合成酶活力的变化与乙烯生成量变化的趋势一致.根据以上结果可以认为桃果实主要的香气挥发性物质的形成与乙烯、呼吸跃变的开始密切相关.香气物质形成速率动态变化可能是桃果实发育过程中成熟度的另一个生理学指标.     

华北大黑鳃金龟对桃树挥发物的电生理反应

【目的】华北大黑鳃金龟Holotrichia oblita是一种分布范围广,寄主种类多的农业害虫。本研究鉴定了桃树顶空挥发物中对华北大黑鳃金龟雌虫具有触角电生理活性的化合物,并探索桃树挥发物活性成分的剂量对华北大黑鳃金龟雌、雄虫触角电生理反应的影响。【方法】采用顶空吸附法收集桃树枝叶挥发物,用气相色谱 触角电位联用技术(GC-EAD)和气相色谱 质谱联用技术(GC-MS)鉴定有电生理活性的桃树挥发物成分,最后用触角电位(electroantennograph, EAG)技术测试这些挥发物在0.01,0.1, 1, 10和100 μg剂量下引起的华北大黑鳃金龟雌、雄成虫EAG反应。【结果】桃树挥发物中能引起华北大黑鳃金龟雌成虫触角电生理反应的成分有7种,包括Z-β-罗勒烯、顺-3-己烯乙酸酯、顺-3-己烯醇、壬醛、水杨酸甲酯以及2种未知化合物。所测试的对华北大黑鳃金龟雌成虫触角具有电生理活性的5种桃树挥发物成分在各剂量下都能引起雌、雄成虫明显的EAG反应。雌、雄虫对同种物质的EAG反应随着剂量的增加而有增大的趋势,并且雌虫的EAG反应显著高于雄虫。【结论】桃树挥发物中,罗勒烯、顺-3-己烯乙酸酯、顺-3-己烯醇、壬醛、水杨酸甲酯均能引起华北大黑鳃金龟雌、雄成虫的剂量依赖性的触角电生理反应,而且存在显著的性别差异。可利用这些化合物开展行为反应和田间诱集试验,进一步验证其对华北大黑鳃金龟的引诱活性。     

Synergism between aromatic compounds and green leaf volatiles derived from the host plant underlies female attraction in the oriental fruit moth

Blends of volatile compounds emitted by host plants are known to mediate the attraction of gravid female herbivores to oviposition sites, but the role of individual odor components is still little understood. We characterized the olfactory response of mated female Cydia (Grapholita) molesta (Busck) (Lepidoptera: Tortricidae) to synthetic mixtures of compounds emitted by peach shoot, a key host plant of this herbivore, and investigated the role of important constituents of bioactive mixtures in moth attraction. Relative ratios of constituents of the mixtures corresponded to the natural ratio of volatile compounds collected in the plant's headspace. A significant attractant effect was found for a comparatively complex 10‐compound mixture that included four green leaf volatiles [(Z)‐3‐hexen‐1‐ol, 1‐hexanol, (E)‐2‐hexenal, and (Z)‐3‐hexen‐1‐yl acetate], five aromatics (benzaldehyde, methyl salicylate, methyl benzoate, benzonitrile, and phenylacetonitrile), and a carboxylic acid (valeric acid). Using a subtraction approach, the number of compounds was progressively decreased, resulting in a bioactive 5‐compound mixture composed of two constituents, green leaf volatiles and aromatic compounds. Further evaluations revealed that benzaldehyde and benzonitrile must be present in association with three distinct green leaf volatiles to produce an attractant effect on the female moths. This 5‐compound mixture was as attractive as natural peach shoot volatiles, which are known to comprise over 20 compounds. Results are discussed in light of the documented synergistic effect between the three general green leaf volatiles and the two specific aromatic compounds.     

Antagonist effects of non-host fruit volatiles on discrimination of host fruit by Rhagoletis flies infesting apple (Malus pumila), hawthorn (Crataegus spp.), and flowering dogwood (Cornus florida)

In previous flight‐tunnel tests Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) flies originating from domestic apple (Malus pumila), hawthorn (Crataegus spp.), and flowering dogwood (Cornus florida), displayed greater numbers of upwind flights to blends of volatiles identified from their natal fruit compared to non‐natal fruit. Here, we show that when certain non‐host volatiles were added to the host blend, significantly fewer apple, hawthorn, and dogwood flies exhibited sustained upwind flight to the source. Specifically, the upwind flight of apple flies to the apple blend was significantly antagonized by the addition of the hawthorn or dogwood blends, the addition of 3‐methylbutan‐1‐ol alone (a key volatile for hawthorn and dogwood flies), or the combination of 3‐methylbutan‐1‐ol and another key dogwood volatile, 1‐octen‐3‐ol. Similarly, the upwind flight of dogwood and hawthorn flies to their respective natal blends was antagonized by the addition of the apple blend or the key apple volatile butyl hexanoate. Experiments were also conducted to determine whether non‐natal fruit volatiles could disrupt the close‐range flight response of flies to the visual stimulus of fruit alone, represented by an odorless red sphere. Tests with apple‐origin flies showed that when the hawthorn blend, the dogwood blend, or the key antagonist volatiles from each (3‐methylbutan‐1‐ol and 1‐octen‐3‐ol) were added to a red sphere fruit mimic, significantly lower proportions of flies were captured, compared with captures when no odor was present. Our results support the hypothesis that agonist and antagonist properties of fruit volatiles can play an important role in host recognition/discrimination by Rhagoletis flies.     

Postharvest temperature influences volatile lactone production via regulation of acyl-CoA oxidases in peach fruit

The biosynthesis of volatile compounds in plants is affected by environmental conditions. Lactones are considered to be peach‐like aroma volatiles; however, no enzymes or genes associated with their biosynthesis have been characterized. White‐fleshed (cv. Hujingmilu) and yellow‐fleshed (cv. Jinxiu) melting peach (Prunus persica L. Batsch) fruit were used as materials in two successive seasons and responses measured to four different temperature treatments. Five major lactones accumulated during postharvest peach fruit ripening at 20 °C. Peach fruit at 5 °C, which induces chilling injury (CI), had the lowest lactone content during subsequent shelf life after removal, while 0 °C and a low‐temperature conditioning (LTC) treatment alleviated development of CI and maintained significantly higher lactone contents. Expression of PpACX1 and activity of acyl‐CoA oxidase (ACX) with C16‐CoA tended to increase during postharvest ripening both at 20 °C and during shelf life after removal from cold storage when no CI was developed. There was a positive correlation between ACX and lactones in peach fruit postharvest. Changes in lactone production in response to temperatures are suggested to be a consequence of altered expression of PpACX1 and long‐chain ACX activity.     

QTL mapping of aroma compounds analysed by headspace solid-phase microextraction gas chromatography in the apple progeny ‘Discovery’ × ‘Prima’

Improving fruit quality of apple varieties is an important but complex breeding goal. Flavour is among the key factors of apple fruit quality but in spite of the analytical and biochemical knowledge about volatiles little is known about the genetic and molecular bases of apple aroma. The aim of this study was to use a saturated molecular linkage map of apple to identify QTLs for aroma compounds such as alcohols, esters and terpenes, but also for a number of unidentified volatile compounds (non-targeted analysis approach). Two parental genetic maps were constructed for the apple cultivars ‘Discovery’ and ‘Prima’ by using mainly AFLP and SSR markers. ‘Discovery’ and ‘Prima’ showed very different volatile patterns, and ‘Discovery’ mostly had the higher volatile concentrations in comparison with the Vf-scab resistant ‘Prima’ which has its origin in the small-fruited apple species Malus floribunda. About 50 putative QTLs for a total of 27 different apple fruit volatiles were detected through interval mapping by using genotypic data of 150 F₁ individuals of the mapping population ‘C3’ together with phenotypic data obtained by head-space solid phase microextraction gas chromatography. QTLs for volatile compounds putatively involved in apple aroma were found on 12 out of the 17 apple chromosomes, but they were not evenly dispersed. QTLs were mainly clustered on linkage groups LG 2, 3 and 9. In a first attempt, a LOX (lipoxygenase) candidate gene, putatively involved in volatile metabolism, was mapped on LG 9, genetically associated with a cluster of QTLs for ester-type volatiles. Implications for aroma breeding in apple are discussed.     

Identification of a specific isoform of tomato lipoxygenase (TomloxC) involved in the generation of fatty acid-derived flavor compounds

There are at least five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE) present in tomato (Lycopersicon esculentum Mill.) fruit, but their role in generation of fruit flavor volatiles has been unclear. To assess the physiological role of TomloxC in the generation of volatile C6 aldehyde and alcohol flavor compounds, we produced transgenic tomato plants with greatly reduced TomloxC using sense and antisense constructs under control of the cauliflower mosaic virus 35S promoter. The expression level of the TomloxC mRNA in some transgenic plants was selectively reduced by gene silencing or antisense inhibition to between 1% and 5% of the wild-type controls, but the expression levels of mRNAs for the four other isoforms were unaffected. The specific depletion of TomloxC in transgenic tomatoes led to a marked reduction in the levels of known flavor volatiles, including hexanal, hexenal, and hexenol, to as little as 1.5% of those of wild-type controls following maceration of ripening fruit. Addition of linoleic or linolenic acid to fruit homogenates significantly increased the levels of flavor volatiles, but the increase with the TomloxC-depleted transgenic fruit extracts was much lower than with the wild-type control. Confocal imaging of tobacco (Nicotiana tabacum) leaf cells expressing a TomloxC-GFP fusion confirmed a chloroplast localization of the protein. Together, these results suggest that TomloxC is a chloroplast-targeted lipoxygenase isoform that can use both linoleic and linolenic acids as substrates to generate volatile C6 flavor compounds. The roles of the other lipoxygenase isoforms are discussed.     

Chemical signatures affecting host choice in the Eucalyptus herbivore,Gonipterus sp. (Curculionidae: Coleoptera)

It is well known that herbivorous insects respond to host plant volatiles. Yet details of how these insects perceive the complex profile of volatiles from different potential host plants have not been studied for most insects. Gonipterus spp. are important pests of Eucalyptus worldwide, but differ in their preference for different species of this host. In this study, we consider whether host volatiles affect the host choice for a Gonipterus sp. and we characterize the response of the female insect to the volatile profiles from these hosts in an electro-antennographic experiment. We sampled volatiles from freshly damaged leaves of three Eucalyptus species and analysed the profiles by gas chromatography coupled to electro-antennography (GC-EAD) and gas chromatography coupled to mass spectrometry. Female weevils gave a mixed range of electro-physiological responses to volatile puffs from leaves of different tree species. This suggests that differences in volatile profiles of different trees play a role in how these beetles discriminate between potential hosts. GC-EAD analysis showed that responses were as complex as the volatile chemical compositions of the leaves. A number of these chemicals were identified, and responses were mostly due to general green leaf volatiles. This was also evident from the fact that the insects showed a markedly greater response to the total volatile profile from freshly damaged leaves for all species. The females of the Gonipterus sp. can therefore detect damaged leaves, which may indicate host quality. Host specificity information is further expected to lie in the relative differences in emission ratios and synergism between different host chemical compounds, rather than specific individual compounds.     

Development and mapping of peach candidate genes involved in fruit quality and their transferability and potential use in other Rosaceae species

The goal of the present study was to identify candidate genes (CGs) involved in fruit quality in peach that can be transferred to other Rosaceae species. Two cDNA libraries from fruit of the “Fantasia” peach cultivar, constructed at two stages of development, were used to generate a set of expressed sequence tag sequences. A total of 1,730 peach unigenes were obtained after clustering. Sequences and corresponding annotations were stored in a relational database and are available through a web interface. Fifty-nine CGs involved in fruit growth and development or fruit quality at maturity, focusing on sweetness, acidity, and phenolic compound content, were selected according to their annotation. Fifty-five primer pairs, designed from peach CG sequences and giving PCR products in peach, were tested in strawberry and 36 gave amplified products. Eight CGs were mapped in peach, 14 in strawberry, four in both species and confirmed the pattern of synteny already proposed using comparative mapping. In peach, the CGs are located in three linkage groups (3, 5, 7), and in strawberry they are distributed in all seven Fragaria linkage groups. Colocalization between some of these CGs and quantitative trait loci for fruit quality traits were identified and are awaiting confirmation in further analyses.     

Stimuli affecting selection of oviposition sites by female peach twig borer, Anarsia lineatella Zeller (Lepidoptera: Gelechiidae)

In laboratory and field experiments, stimuli were tested that might affect oviposition decisions by female peach twig borer moths, Anarsia lineatella Zeller (Lepidoptera: Gelechiidae). When given a choice between immature green peach fruits, green mature peach fruits and soft-ripe peach fruits, the latter received the fewest eggs. Fuzzy halves of peach fruits received ten times more eggs then shaved hairless halves. Volatiles from both almond and peach shoots induced more oviposition by females than by control stimuli. Similarly, volatiles from immature green peach fruits, mature green or mature hard-ripe peach fruits induced more oviposition than their respective control stimuli. In a choice experiment, volatiles from immature peach fruit stimulated three times more oviposition than those from soft-ripe peach fruit. Discrimination against mature soft-ripe peach fruits as potential oviposition sites may lie in the phenology of A. lineatella and host peach fruits. Larval development to the pupal stage takes 15–27 days. Therefore, any eggs laid on a ripe fruit 14 days before it falls from the tree will not likely develop into adult insects because developing larvae will only reach third or fourth instar before the fruit is decomposed, and only first and second instar larvae can overwinter.     

New high-quality peach (Prunus persica L. Batsch) genome assembly to analyze the molecular evolutionary mechanism of volatile compounds in peach fruits

Peach (Prunus persica L. Batsch) is an economically important fruit crop worldwide. Although a high-quality peach genome has previously been published, Sanger sequencing was used for its assembly, which generated short contigs. Here, we report a chromosome-level genome assembly and sequence analysis of Chinese Cling, an important founder cultivar for peach breeding programs worldwide. The assembled genome contained 247.33 Mb with a contig N50 of 4.13 Mb and a scaffold N50 of 29.68 Mb, representing 99.8% of the estimated genome. Comparisons between this genome and the recently published one (Lovell peach) uncovered 685 407 single nucleotide polymorphisms, 162 655 insertions and deletions, and 16 248 structural variants. Gene family analysis highlighted the contraction of the gene families involved in flavone, flavonol, flavonoid, and monoterpenoid biosynthesis. Subsequently, the volatile compounds of 256 peach varieties were quantitated in mature fruits in 2015 and 2016 to perform a genome-wide association analysis. A comparison with the identified domestication genomic regions allowed us to identify 25 quantitative trait loci, associated with seven volatile compounds, in the domestication region, which is consistent with the differences in volatile compounds between wild and cultivated peaches. Finally, a gene encoding terpene synthase, located within a previously reported quantitative trait loci region, was identified to be associated with linalool synthesis. Such findings highlight the importance of this new assembly for the analysis of evolutionary mechanisms and gene identification in peach species. Furthermore, this high-quality peach genome provides valuable information for future fruit improvement.     

Electrophysiological and behavioral identification of a volatile blend involved in host location of female strawberry sap beetle,Stelidota geminata

The strawberry sap beetle (SSB), Stelidota geminata (Say) (Coleoptera: Nitidulidae), is a serious direct pest of strawberries in the northeastern USA. To date, however, no food or pheromone‐based attractants for SSB have been identified. A combination of solid‐phase microextraction, adsorbent sampling, gas chromatography coupled with electroantennographic detection (GC–EAD), and gas chromatography–mass spectrometry was used to identify volatile compounds from strawberry fruit acting as behavioral attractants for female SSB. Consistent EAD activity was obtained for 16 ester compounds. In Y‐tube olfactometer bioassays, the blend of these 16 compounds mixed at a ratio observed from strawberry headspace was significantly more attractive to adult female SSB than a control. Female SSB showed no difference in response levels between the 16‐component blend and a strawberry volatile adsorbent extract. Our data indicate that the combination of the ethyl acetate with some or all of the remaining 15 compounds is necessary for this ester blend to be attractive to female SSB. Previously identified host volatiles found for other species of sap beetles included mostly alcohols associated with over‐ripe fruit rather than the esters identified from ripe strawberries for SSB. A highly attractive synthetic food odor will be useful for developing new management options for SSB.     

Response of female oriental fruit moth to volatiles from apple and peach trees at three phenological stages

The attractiveness of peach ( Prunus persica L. Batsch) and apple ( Malus domestica L. Borkh.) (both Rosaceae) tissue to gravid female oriental fruit moth, Grapholita (=  Cydia ) molesta (Busck) (Lepidoptera: Tortricidae), was assessed at three distinct stages throughout the growing season using a dual-choice bioassay. Plant material offered to the female moths consisted of a plant shoot in early spring, before fruit became available. Later, it consisted of a leaf-bearing twig and a fruit, either alone or in combination. The level of attraction of the female moths to the various plant tissues varied substantially over time and according to the plant species. Before fruit became available, female moths were significantly attracted to peach as well as to apple shoots. During the early fruit growth stage, moths were attracted to a leaf-bearing twig originating from a peach tree, but not to that from an apple tree. In peach, it was the vegetative tissue that accounted for the attraction, whereas in apple, it was the reproductive tissue (a developing fruit). During the late fruit growth stage, both peach fruit and apple fruit were highly attractive, whereas a twig with leaves from either an apple or a peach tree was neither attractive nor repellent to the female moths. This changing female olfactory response to volatiles emitted by vegetative tissue and fruits from the two host plant species with progressing season is discussed with respect to the biology and the dispersal of this moth species.     

Metabolic engineering of aroma components in fruits

Plants have the ability to produce a diversity of volatile metabolites, which attract pollinators and seed dispersers and strengthen plant defense responses. Selection by plant breeders of traits such as rapid growth and yield leads, in many cases, to the loss of flavor and aroma quality in crops. How the aroma can be improved without affecting other fruit attributes is a major unsolved issue. Significant advances in metabolic engineering directed at improving the set of volatiles that the fruits emit has been aided by the characterization of enzymes involved in the biosynthesis of flavor and aroma compounds in some fruits. However, before this technology can be successfully applied to modulate the production of volatiles in different crops, further basic research is needed on the mechanisms that lead to the production of these compounds in plants. Here we review the biosynthesis and function of volatile compounds in plants, and the attempts that have been made to manipulate fruit aroma biosynthesis by metabolic engineering. In addition, we discuss the possibilities that molecular breeding offers for aroma enhancement and the implications of the latest advances in biotechnological modification of fruit flavor and aroma.