Banded cucumber beetle (Coleoptera: Chrysomelidae) resistance in romaine lettuce: understanding latex chemistry

Many plants subjected to herbivore damage exude latex, a rich source of biochemicals, which plays important roles in host plant resistance. Our previous studies showed that fresh latex from Valmaine, a resistant cultivar of romaine lettuce Lactuca sativa L., applied to artificial diet is highly deterrent to feeding by banded cucumber beetle, Diabrotica balteata LeConte, compared to the latex of a closely related susceptible cultivar Tall Guzmaine. The deterrent factor(s) could be extracted from Valmaine latex with water–methanol (20:80). In this study, further fractionation of the methanolic crude extract of Valmaine latex was performed using reverse-phase and ion-exchange solid-phase extraction to isolate the deterrent compounds. Retention of deterrent compounds on anion and cation exchange resin suggested the presence of highly polar compounds with both carboxylic and amine groups in Valmaine latex. Further bioassay-directed fractionation of cation exchange extract using LC/MS indicated the presence of at least 3 major and an unknown number of minor compounds in the bioactive fraction between 3 and 4 min. The m/z 210 out of the 3 major compounds showed strong amino acid characteristics (glutamine and/or glutamic acid) when subjected to further MS ⁿ degradation. Our studies suggest that nitrogenous ingredients of latex play a key role in Valmaine resistance to D. balteata, and latex may be a source of bioactive compounds with a potential use in pest management.     

Romaine lettuce latex deters feeding of banded cucumber beetle: a vehicle for deployment of biochemical defenses

Latex is widely found among plant species and is known to play a defensive role against certain herbivores. Two romaine lettuce, Lactuca sativa L. (Asteraceae) cultivars, 'Valmaine' (resistant) and 'Tall Guzmaine' (susceptible), were selected to study the potential of latex as a defense mechanism against the banded cucumber beetle, Diabrotica balteata LeConte (Coleoptera: Chrysomelidae). Latex from Valmaine strongly inhibited feeding of adult D. balteata compared to latex from Tall Guzmaine when applied to the surface of artificial diet. Beetles consumed significantly less diet from disks treated with Valmaine latex than they consumed from diet treated with Tall Guzmaine latex, in both choice and no-choice tests. In a choice test involving diet disks treated with Valmaine latex from young leaves vs. disks treated with latex from mature leaves, the beetles consumed significantly less diet treated with latex from young leaves. No significant difference in diet consumption was found between diets treated with latex from young and mature leaves of Tall Guzmaine in choice tests. Three solvents of differing polarity (water, methanol, and methylene chloride) were tested to extract deterrent compounds from latex; Valmaine latex extracted with water:methanol (20:80) strongly inhibited beetle feeding when applied to the surface of artificial diet. In no-choice tests, fewer beetles were observed feeding on diet treated with water:methanol (20:80) extract of Valmaine latex than on diet treated with a similar extract of Tall Guzmaine latex, resulting in significantly less consumption of the diet treated with the Valmaine latex extract. These studies suggest that moderately polar chemicals within latex may account for resistance in Valmaine to D. balteata.     

The Impact of Fungal Extracts on Leaf Litter on the Food Preference of Gammarus roeselii

We investigated the effect of methanol and methanol/methylene chloride extracts of the oomycete Pythium sp. JN 1‐b and of the fungi Ascomycete sp. PVSo8, Fusarium sporotrichoides, and Cylindrocarpon sp. 94‐2057 on the food preference of Gammarus roeselii. The preference for leaf discs coated with these extracts compared to uncoated leaf discs was tested in food‐choice assays. Methanol extracts of all strains repelled G. roeselii, and the effect of the extract concentration on relative consumption was strain specific. The repellent effect of these extracts, especially of extracts of Cylindrocarpon sp., decreased when the fungi were grown on leaf extract medium as opposed to synthetic medium containing sucrose. None of the methanol/methylene chloride extracts affected the food preference of the gammarid. We conclude that biologically active compounds were extracted from fungi and an oomycete were soluble in methanol but not in methanol/methylene chloride. Only repellent activity was observed with the extracts, and relative ratios of repellents and attractants might determine the consumption of fungi by G. roeselii. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Association analysis of bacterial leaf spot resistance and SNP markers derived from expressed sequence tags (ESTs) in lettuce (Lactuca sativa L.)

Bacterial leaf spot of lettuce, caused by Xanthomonas campestris pv. vitians, is a devastating disease of lettuce worldwide. Since there are no chemicals available for effective control of the disease, host-plant resistance is highly desirable to protect lettuce production. A total of 179 lettuce genotypes consisting of 29 leaf, 15 crisphead, one stem, 21 romaine, and 113 butterhead types were evaluated for response to X. c. vitians. One source of high resistance and five sources of moderate resistance were identified with four being butterhead lettuce and two leaf lettuce. The population genetic structure based on 350 expressed-sequence-tag-derived single nucleotide polymorphism generated two clades: Clade I and Clade II. The butterhead type was genetically distinct from romaine and crisphead types, while the leaf type was found to frequently exchange genes with other types through the history of breeding. Association mapping identified one single nucleotide polymorphism (QGB19C20.yg-1-OP5) associated with disease severity in Q general linear model and Q + K mixed linear model. Two SNP markers (Contig15389-1-OP1 and Contig6039-19-OP1) were associated with the resistance in the leaf lettuce PI 358000-1 which had no disease symptoms. The marker QGB19C20.yg-1-OP5 is in linkage group 2, while both of Contig15389-1-OP1 and Contig6039-19-OP1 are in linkage group 4. The resistant lines and the associated SNPs should be useful to develop resistant cultivars to battle against the devastating disease in lettuce.     

Leaf surface wax layers of Brassicaceae lack feeding stimulants for Phaedon cochleariae

Numerous reports have indicated that glucosinolates are important stimulants for specialist herbivores feeding on Brassicaceae, and that these metabolites might be present on the plant surface and thereby detectable by an alighting insect. We investigated the outermost layer of leaves of two species of Brassicaceae, Brassica napus L. var. ‘Martina’ and Nasturtium officinale R. Br., using two highly selective extraction methods. When the epicuticular wax layer was mechanically removed with gum arabic, no glucosinolates were detectable in the lower and upper leaf surfaces. Extracting the leaf surfaces with a threefold short rinse with chloroform/methanol/water (2 : 1 : 1 vol/vol/vol) led to varying results, depending on the light conditions under which plants had been kept in the period prior to extraction. In plants kept under light, glucosinolates were detectable in a first extraction in minor concentrations, with increasing amounts in a second and third extraction. In plants kept in darkness, glucosinolates were almost absent in the first extraction. We postulate that the polar glucosinolates are washed from the inner leaf tissue through open stomata to the outside during solvent extraction, but are not naturally present in the outermost wax layer. The response of the crucifer specialist Phaedon cochleariae (F.) (Coleoptera: Chrysomelidae) to leaf surfaces of the host plants B. napus and N. officinale and to a glucosinolate was tested. Adults preferred both the adaxial and abaxial leaf surfaces of host plants that had been treated with gum arabic in order to remove the epicuticular waxes over intact surfaces. Waxes may therefore prevent direct contact with the stimulants. Sinigrin (allyl glucosinolate) and/or surface extracts of N. officinale leaves applied on Pisum sativum leaf discs did not evoke feeding, but feeding did occur when total leaf extracts of B. napus or N. officinale were applied on this non‐host. We conclude that glucosinolates might only act as feeding stimulants for P. cochleariae in concert with compounds other than surface waxes.     

Feeding behaviour and performance of different populations of the black currant‐lettuce aphid,Nasonovia ribisnigri,on resistant and susceptible lettuce

When crops are bred for resistance to herbivores, these herbivores are under strong selection pressure to overcome this resistance, which may result in the emergence of virulent biotypes. This is a growing problem for crop species attacked by aphids. The Nr‐gene in lettuce confers near‐complete resistance against the black currant‐lettuce aphid, Nasonovia ribisnigri (Mosely) (Hemiptera: Aphididae). Since 2007, populations of N. ribisnigri have been reported in several locations in Europe to infest resistant lettuce varieties that possess the Nr‐gene. The objective of this study was to analyse the behaviour and level of virulence of several N. ribisnigri populations observed to have colonized Nr‐locus‐containing lettuce lines. We analysed the stylet penetration and feeding behaviour, and the performance of these N. ribisnigri populations on resistant and susceptible lettuce lines. Large variation in the degree of virulence to the Nr‐locus‐containing lettuce lines was found among populations of the Nr:1 biotype. The German population was highly virulent on the Nr‐containing resistant lettuce lines, and showed similar feeding behaviour and performance on both the susceptible and resistant lettuces. The French population from Paris was the second most virulent, though reproduction on the resistant lines was reduced. The French population from Perpignan and a population from Belgium, however, showed reduced performance and feeding rate on the resistant compared to the susceptible lettuces. The lettuce background in which the Nr‐gene is expressed influences the level of resistance to the various Nr:1 aphid populations, because the performance and feeding behaviour differed between the aphids on the cultivars (romaine lettuce) compared to the near‐isogenic lines (butterhead/iceberg lettuce). This study also shows that being able to feed on a plant not automatically implies that a population can successfully develop on that plant, because aphids showed phloem ingestion during the 8‐h recording period on resistant lettuce, but were not able to survive and reproduce on the same lettuce line.     

Feeding behavior and performance of Nasonovia ribisnigri on grafts,detached leaves,and leaf disks of resistant and susceptible lettuce

Aphids are dependent on the phloem sap of plants as their only source of nutrients. Host‐plant resistance in lettuce, Lactuca sativa L. (Asteraceae), mediated by the Nr gene is used to control the lettuce aphid Nasonovia ribisnigri (Mosely) (Hemiptera: Aphididae). The resistance is located in the phloem; however, the exact mechanism of resistance is unknown. In this study, we investigated whether the resistance factor (or factors) is synthesized in the root or in the shoot. The feeding behavior and performance of avirulent N. ribisnigri were studied on grafts of resistant and susceptible lettuce. In addition, the persistence of resistance in excised lettuce tissue was measured, by studying the feeding behavior and performance of N. ribisnigri on detached leaves and leaf disks of resistant lettuce. It appears that the resistance factor encoded by the Nr gene is produced in the shoots: aphid feeding was reduced on resistant shoots grafted on susceptible roots, whereas aphids were able to feed on grafts of susceptible shoots on resistant roots. Partial loss of resistance was observed after detachment of leaves and excision of leaf disks from resistant plants. Aphids fed longer on excised resistant plant tissue compared with intact resistant plants; however, compared with excised plant tissue of the susceptible cultivar, the time spent on feeding was shorter, indicating resistance was not completely lost. Our findings caution against the use of excised leaf material for aphid resistance bioassays.     

Oviposition‐induced plant cues: do they arrest Trichogramma wasps during host location?

Plants can defend themselves against herbivorous insects before the larvae hatch from eggs and start feeding. One of these preventive defence strategies is to produce plant volatiles, in response to egg deposition, which attract egg parasitoids that subsequently kill the herbivore eggs. Here, we studied whether egg deposition by Pieris brassicae L. (Lepidoptera: Pieridae) induces Brussels sprouts plants to produce cues that attract or arrest Trichogramma brassicae Bezdeko (Hymenoptera: Trichogrammatidae). Olfactometer bioassays revealed that odours from plants with eggs did not attract or arrest parasitoids. However, contact bioassays showed that T. brassicae females were arrested on egg‐free leaf squares excised from leaves with 72 h‐old egg masses, which are highly suitable for parasitisation. We tested the hypothesis that this arresting activity is due to scales and chemicals deposited by the butterflies during oviposition and which are thus present on the leaf surface in the vicinity of the eggs. Indeed, leaf squares excised from egg‐free leaves, but contaminated with butterfly deposits, arrested the wasps when the squares were tested 1 day after contamination. However, squares from egg‐free leaves with 72 h‐old butterfly deposits had no arresting activity. Thus, we exclude that the arresting activity of the leaf area near 72 h‐old egg masses was elicited by cues from scales and other butterfly deposits. We suggest that egg deposition of P. brassicae induces a change in the leaf surface chemicals in leaves with egg masses. A systemic induction extending to an egg‐free leaf neighbouring an egg‐carrying leaf could not be detected. Our data suggest that a local, oviposition‐induced change of leaf surface chemicals arrests T. brassicae in the vicinity of host eggs.     

Antioxidant and Antibacterial Activities of Crude Extracts and Essential Oils of Syzygium cumini Leaves

This research highlights the chemical composition, antioxidant and antibacterial activities of essential oils and various crude extracts (using methanol and methylene chloride) from Syzygium cumini leaves. Essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS).The abundant constituents of the oils were: α-pinene (32.32%), β-pinene (12.44%), trans-caryophyllene (11.19%), 1, 3, 6-octatriene (8.41%), delta-3-carene (5.55%), α-caryophyllene (4.36%), and α-limonene (3.42%).The antioxidant activities of all extracts were examined using two complementary methods, namely diphenylpicrylhydrazyl (DPPH) and ferric reducing power (FRAP). In both methods, the methanol extract exhibited a higher activity than methylene chloride and essential oil extracts. A higher content of both total phenolics and flavonoids were found in the methanolic extract compared with other extracts. Furthermore, the methanol extract had higher antibacterial activity compared to methylene chloride and the essential oil extracts. Due to their antioxidant and antibacterial properties, the leaf extracts from S. cumini may be used as natural preservative ingredients in food and/or pharmaceutical industries.     

Behavioural responses of the Colorado potato beetle to trichomes and leaf surface chemicals of Solanum tarijense

Wild Solanum species constitute a source of resistance to several pests and diseases of potato. Several species of wild tuber‐bearing potato have been identified as resistant to the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), including Solanum tarijense Hawkes (Solanaceae). Our objective was to determine the mechanism of resistance of S. tarijense to the Colorado potato beetle and, because the resistance is limited to the adult stage of the insect, to study the host selection behaviour on resistant plants. In the field, Colorado potato beetles demonstrated a unique behaviour when in contact with S. tarijense, abandoning the plant by falling to the ground after a few minutes. The abundant trichomes on the leaves of S. tarijense induced the falling behaviour. However, on S. tarijense feeding remained low even after the trichomes were mechanically removed. Observations demonstrated that the normal sequence of behaviour leading to feeding was interrupted before adult beetles fed on S. tarijense leaves. Feeding experiments using volatile and non‐volatile fractions of leaf surface extracts identified a phagodeterrent effect of the volatile fraction. Our results contrast with a similar evaluation of the mode of resistance of Solanum berthaultii Hawkes, a close relative of S. tarijense, on which some feeding occurred and adults did not show falling behaviour. This study presents information on S. tarijense as a new source of resistance to the Colorado potato beetle that can be used for potato breeding.     

Pod surface exudates of wild relatives of pigeonpea influence the feeding preference of the pod borer, Helicoverpa armigera

Wild relatives of crops are an important source of resistance genes against insect pests. However, it is important to identify the accessions of wild relatives of crops with different mechanisms of resistance to broaden the basis and increase the levels of resistance to insect pests. Therefore, we studied the feeding behavior of pod borer, Helicoverpa armigera, which is the most damaging pest of pigeonpea, in relation to biochemical characteristics of the pod surface exudates in a diverse array of germplasm accessions belonging to 12 species of pigeonpea wild relatives. Feeding by H. armigera larvae was significantly lower on the unwashed or water-, methanol-, or hexane-washed pods of Canajus sericeus, C. scarabaeoides, Flemingia bracteata, F. stricta, and Rhynchosia aurea than those of C. acutifolius, C. albicans, C. cajanifolius, C. lineatus, D. ferruginea, P. scariosa, R. bracteata, and the cultivated pigeonpea, C. cajan genotypes, ICPL 87, and ICPL 332, although there were a few exceptions. The methanol-washed pods of wild relatives were less preferred for feeding by the H. armigera larvae than the unwashed pods, but the hexane-washed pods were preferred more than the unwashed pods. The results suggested that methanol extracted the phagostimulants from the pod surface, while hexane removed the antifeedants. The high-performance liquid chromatography (HPLC) finger printing of methanol and hexane pod surface extracts showed qualitative and quantitative differences in compounds present on the pod surface of different wild relatives of pigeonpea. Some of the peaks in HPLC profiles were associated with feeding preference of the third-instar larvae of H. armigera. There was considerable diversity in wild relatives of pigeonpea as revealed by principal component analysis based on HPLC fingerprints of pod surface extracts in methanol and hexane, and H. armigera feeding on the pods. Wild pigeonpea accessions with low amounts of phagostimulants and high amounts of antifeedants may be used for introgression of resistance genes into the cultivated pigeonpea to develop varieties with broad-based resistance to H. armigera. There is considerable diversity among the wild relatives of pigeonpea, and the accessions with resistance to pod borer. These can be used to broaden the basis and increase the levels of resistance to H. armigera.     

Binding of virus-like particles of Norwalk virus to romaine lettuce veins

Noroviruses (NoV) annually cause millions of cases of gastrointestinal disease in the United States. NoV are associated with raw shellfish outbreaks, particularly oysters, which are thought to bioaccumulate NoV particles during the filter-feeding process. NoV outbreaks, however, have also been known to occur from other common-source food-borne vehicles, such as lettuce, frozen raspberries, and salad. In this study, we evaluated romaine lettuce as a potential vehicle for NoV transmission by testing the binding and distribution of NoV to the surface of romaine. Recombinant Norwalk virus-like particles (rNVLP) applied to the surface of romaine lettuce localized as large clusters primarily on the leaf veins. An extract of romaine lettuce leaves in phosphate-buffered saline (PBS) (romaine extract [RE]) bound rNVLP in a dose-dependent manner. RE did not bind rNVLP by histo-blood group antigens (HBGA), nor was RE competitive with rNVLP binding to porcine gastric mucin. These results suggested that non-HBGA molecules in RE bind rNVLP by a binding site(s) that is different from the defined binding pocket on the virion. Extracts of cilantro, iceberg lettuce, spinach, and celery also bound rNVLP. Samples of each of the vegetables spiked with rNVLP and tested with anti-NVLP antibody revealed by confocal microscopy the presence of rNVLP not only on the veins of cilantro but also throughout the surface of iceberg lettuce.     

Stylet activity of cowpea aphid (Homoptera: Aphididae) on leaf extracts of resistant and susceptible cowpea cultivars

Electrical penetration graph recordings using direct current (DC-EPGs) were used to analyze aspects of the probing behavior of cowpea aphid,Aphis craccivora Koch, on intact plants and on hexane, ethyl acetate, and methanol extracts of leaves of aphid-resistant (ICV-12) and aphid-susceptible (ICV-1) cultivars of cowpeaVigna unguiculata (L.) Walp. In one set of experiments, recordings were done on plants with or without parafilm wrapping, or on plants painted with raw leaf juice and extracts of the two cultivars. In another study, recordings were done on leaf extracts homogenized in water or in 0.5M sucrose solution and then placed in parafilm membrane sachets. Electrodes were inserted into soil mix for the experiments on potted plants or into extract fractions and raw juice for the membrane feeding experiments on leaf extracts in parafilm sachets. Waveform signals were recorded from resistance fluctuations from interactions between aphids and substrates, and electromotive forces generated within each preparation. ICV-12 plants with or without parafilm wrapping, and ethyl acetate extracts and raw juice of that cultivar significantly (P≤0.05) reduced stylet penetration behavior. Thus, antixenosis as manifested by disruption of aphid stylet activity on host substrates, appeared to be a governing modality of aphid resistance in ICV-12.     

Predator avoidance in phytophagous mites: response to present danger depends on alternative host quality

We studied whether volatiles released by putative host plants affect the antipredator response of an herbivorous mite, Tetranychus urticae, when the patch was invaded by Phytoseiulus persimilis. Tetranychus urticae laid a lower number of eggs on tomato leaves than on lima bean leaves, suggesting that lima bean is a preferred host food source for T. urticae. In addition, T. urticae preferred lima bean plant volatiles to tomato plant volatiles in a Y-tube olfactometer test. To investigate the antipredator response of T. urticae, we examined the migration of T. urticae from a lima bean leaf disc to a neighbouring leaf disc (either a tomato or lima bean leaf disc) when ten predators were introduced into the original lima bean disc. A Parafilm bridge allowed for migration between the leaf discs. No migrations occurred between leaf discs when there were no predators introduced to the original leaf disc. However, when predators were introduced migrations did occur. When the neighbouring leaf disc was upwind of the original disc, the migration rate of the mite from original lima bean leaf disc to a neighbouring tomato leaf disc was significantly lower than that to a neighbouring lima bean leaf disc. By contrast, when the neighbouring leaf disc was downwind of the original leaf disc, there was no difference in the migration rates between lima bean leaf discs and tomato leaf discs. The number of T. urticae killed by P. persimilis for each treatment was not different, and this clearly shows that the danger was the same in all treatments regardless of the decision made by T. urticae. From these results, we conclude that T. urticae change their antipredator response by evaluating the difference in host plant volatiles in the patch they inhabit.     

Bioaccumulation of Microcystins in Lettuce

The contamination of lettuce (Lactuca sativa L.) by water‐borne crude extracts of the cyanobacterium microcystin‐producing Microcystis aeruginosa (Kützing) Kützing was investigated. The aim of the study was to determine whether bioaccumulation of microcystins occurs in lettuce foliar tissue when sprayed with solutions containing microcystins at concentrations observed in aquatic systems (0.62 to 12.5 μg · L^?1). Microcystins were found in lettuce foliar tissues (8.31 to 177.8 μg per Kg of fresh weight) at all concentrations of crude extracts. Spraying with water containing microcystins and cyanobacteria may contaminate lettuce at levels higher than the daily intake of microcystins recommended by the World Health Organization (WHO), underscoring the need to monitor such food exposure pathways by public authorities.     

Cabbage waxes affect Trissolcus brochymenae response to short‐range synomones

We show that induced synomones, emitted as a consequence of Murgantia histrionica activity on Brassica oleracea, are adsorbed by the epicuticular waxes of leaves and perceived by the egg parasitoid Trissolcus brochymenae. Leaves were exposed to M. histrionica females placed on the abaxial leaf surface. After 24 h, the leaves were treated mechanically using gum arabic, or chemically using chloroform, on the adaxial surface, and finally the adaxial surface was assayed with T. brochymenae by two‐choice tests in a closed arena. Wasp females responded to mechanically dewaxed cabbage leaf portions with feeding punctures and footprints (Ff) and with feeding punctures, oviposition and footprints (FOf), showing no effect of wax removal. In contrast, the removal of the epicuticular waxes from leaf portions close to FOf, and from leaves with oviposition and footprints (Of), determined the lack of responses by T. brochymenae. Solvent extracts of different treatments were bioassayed, but only FOf triggered parasitoid response. Thus the detection of oviposition‐induced synomones by the parasitoid depends on their adsorption by the epicuticular waxes. Mechanical wax removal from leaf portions contaminated with host footprints (f) also determined a lack of wasp responses, suggesting that the footprints might trigger the induction of a “footprint‐induced synomone” adsorbed onto the epicuticular waxes and exploited by the parasitoid. Leaf portions with the abaxial lamina previously dewaxed and then contaminated by footprints (D+f) of M. histrionica did not affect the parasitoid response, indicating that the abaxial epicuticular waxes are not directly involved in the chemicals induced by M. histrionica footprints.     

Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1

Background  

Lettuce (Lactuca saliva L.) is susceptible to dieback, a soilborne disease caused by two viruses from the family Tombusviridae. Susceptibility to dieback is widespread in romaine and leaf-type lettuce, while modern iceberg cultivars are resistant to this disease. Resistance in iceberg cultivars is conferred by Tvr1 - a single, dominant gene that provides durable resistance. This study describes fine mapping of the resistance gene, analysis of nucleotide polymorphism and linkage disequilibrium in the Tvr1 region, and development of molecular markers for marker-assisted selection.     

Oviposition and chemosensory stimulation of the root flies Delia radicum and D. floralis in response to plants and leaf surface extracts from resistant and susceptible Brassica genotypes

In Brassica crops differences in susceptibility to root fly attack can be largely attributed to antixenotic resistance. Plants of four genotypes (two swedes and two kales) with widely differing resistance in field trials, were compared in laboratory choice assays for their susceptibility to oviposition by the root flies Delia radicum (L.) and D. floralis (Fallen) (Diptera, Anthomyiidae). For both species the preference among the genotypes corresponded to the susceptibility of the genotypes in the field. The preference ranking in response to surrogate leaves treated with methanolic surface extracts of the four genotypes was identical to the preference among potted plants, demonstrating that chemical factors on the leaf surface mediate host preference for oviposition in these species.For both species of fly, glucosinolates are major oviposition stimulants and for D. radicum an additional, nonglucosinolate oviposition stimulant, presently called CIF, is known. We describe a procedure for chromatographic separation of glucosinolates from CIF in leaf surface extracts. In oviposition-choice assays with D. radicum, the CIF-fractions of the two swede genotypes applied to surrogate leaves received a 1.8 and 4.6 times higher proportion of eggs than the respective glucosinolate-fractions, confirming the major importance of CIF as an oviposition stimulant. The genotype of swede that was preferred by both fly species in tests with plants and methanolic leaf surface extracts, also stimulated oviposition more in tests with the glucosinolate-fractions or the CIF-fractions derived from the surface extracts, respectively. Thus, glucosinolates and CIF together account for the observed preference among the genotypes and may also be responsible for their susceptibility under field conditions. In the two kale genotypes the preference for plants or surface extracts differed from the preference among the corresponding glucosinolate- and CIF-fractions, indicating that additional, as yet unknown chemical factors may also be involved.For both groups of stimulants tarsal chemoreceptors allow electrophysiological monitoring of glucosinolate- and CIF-activity in fractionated surface extracts. For D. radicum the chemosensory activity of both glucosinolate- and CIF-fractions corresponded to the respective behavioural activity in the oviposition preference tests, suggesting that preference for oviposition among genotypes can be predicted from the electrophysiological activity of their fractions. The chemosensory response of D. floralis, in particular to the CIF-fractions, was less pronounced than the response of D. radicum, indicating interspecific differences in the perception of the major oviposition stimulants. We discuss the potential application of electrophysiological techniques in support of other screening methods used in breeding for root fly resistance in Brassica crops.     

Bioassay of metalaxyl in plant tissue

A method for determining the concentration of metalaxyl in methanolic extracts of plant tissue is described. Extracts were applied to filter papers in Petri dishes and covered with small volumes of lima bean agar. The growth of an isolate of Pythium ultimum sensitive to metalaxyl was related to the quantity of fungicide applied. The bioassay could detect quantities as low as 0·025 μg in the filter paper, a sensitivity which was confirmed by using ¹⁴C-metalaxyl. Whilst untreated extracts of lettuce and brassicas were not fungitoxic to the isolate of P. ultimum employed, this was not the case with tobacco and grape-vine. Hence alternative test organisms may be required before the method can be generally applied to all species.     

In vitro enzymatic chlorophyll catabolism in wheat elicited by cereal aphid feeding

Chlorophyll degradation is a complex phenomenon that often accompanies insect feeding damage to plants. Loss of chlorophyll can be initiated by several reactions, including oxidative bleaching, chlorophyllase activity, and Mg-dechelatase activity. Extracts from the Russian wheat aphid [Diuraphis noxia (Mordvilko)], the bird cherry-oat aphid [Rhopalosiphum padi (L.)], and aphid-infested and uninfested wheat plants were assayed in vitro for activities involved in chlorophyll degradation. Although the initial infestation was the same (10 apterous adults) for both aphid species, D. noxia weight was significantly higher than R. padi after feeding for 12 days. Consequently, D. noxia feeding caused greater fresh leaf weight reduction than R. padi feeding. Chlorophyll degradation assays showed no activity from either D. noxia or R. padi extracts. Plant extract assays showed a significant difference in Mg-dechelatase activity, while no difference was detected in either the chlorophyllase or oxidative bleaching pathways among the aphid-infested or uninfested plant extracts. Diuraphis noxia-infested leaf extracts showed a greater increase of Mg-dechelatase activity than either R. padi-infested or the uninfested plants. The findings suggest that leaf chlorosis elicited by D. noxia feeding is different from the chlorophyll degradation that occurs in natural plant senescence. Aphid-elicited chlorosis might be the result of a Mg-dechelatase-driven catabolism of chlorophyll in challenged wheat seedlings, however, the factor(s) from D. noxia that elicited the increase of Mg-dechelatase activity still remain to be determined.