Monitoring populations of the carrot fly, Psila rosae, with sticky and water traps

The relative, effectiveness of Rebell®, small cylinder, large cylinder, windmill and water traps, the five types of trap used currently for monitoring populations of the carrot fly, Psila rosae, was assessed in nine field experiments, three in south west Lancashire, four in the Fens (Suffolk, Norfolk, Cambridgeshire), one in East Suffolk and one at Wellesbourne, Warwickshire. Regression analysis of the numbers of flies caught on each type of trap against the numbers caught on the Rebell® trap indicated that each trap samples a constant proportion of the fly population relative to the other traps. Therefore, provided the fly population was sufficiently large for insects to be caught on the least effective traps, any of the five traps would monitor adequately fluctuations in carrot fly populations. However, the Rebell® trap caught 4–17 times as many flies/trap and 5–7 times as many flieshnit area of trap as any of the other traps tested.
Operators considered the Rebell® trap to be the easiest to use. However, it was more expensive than any of the other traps tested.     

Behavioural responses of the carrot fly larva, Psila rosae, to carrot root volatiles

ABSTRACT. The behaviour of the carrot fly larva, Psila rosae F. (Diptera, Psilidae), was investigated in the presence of six concentrations of thirty-four volatiles associated with carrot root and of trans -methyl-iso-eugenol, a constituent of carrot herb oil. Bomyl acetate, 2,4-dimethyl styrene, α-ionone, SbT-ionone and biphenyl were the most consistently preferred compounds. Klinotaxis and klinokinesis were the orientation responses near the stimulus source. Trans -2-nonenal was the most consistently avoided compound. Extracts derived by steam distillation of carrot roots and by concentration of vapour from roots were resolved, by gas liquid chromatography, into forty-five and twenty-two major constituents, respectively. The five preferred compounds are micro-components of these extracts, collectively representing 1% and 0.6% of root extract oil and vapour, respectively. It is suggested that these secondary plant constituents are specific host finding cues for the larva, and that frara-2-nonenal is a carrot defence compound.     

Development of carrot inbreds with resistance to carrot fly using a single seed descent programme

Two carrot cultivars, ‘Sytan’ and ‘Long Chantenay’, representing commercially important carrot types and selected for their partial resistance to the carrot fly (Psila rosae) were crossed as the basis for a single seed descent programme. The resulting F₁ progeny were mass pollinated to produce an F₂ generation and approximately 2000 plants were raised from this segregating family in the glasshouse in 1981. By careful choice of sowing date and glasshouse temperatures it was possible to stimulate the plants to flower within 10 months. Individual king umbels were enclosed within bags and pollinated with blowflies. Resulting seed was sown in pots in the following August and the process of seed production repeated in a 12 month cycle. Each inbred line was selfed in this way over three generations until the F₅ stage. Stocks of seed were then multiplied. A total of 753 inbreds were produced by 1987. The vigour of each inbred was evaluated in glasshouse tests and the level of chlorogenic acid was determined by a fluorescence technique. The most vigorous lines with the lowest levels of chlorogenic acid were tested in field experiments against carrot fly at Wellesbourne. Nine inbreds with promising agronomic quality and moderate resistance to carrot fly were selected and seeded. These nine lines were submitted to seed companies with the aim of developing new cultivars of carrot.     

Field and laboratory studies on the effects of temperature on the development of the carrot fly (Psila rosae F.)

The thermal requirements for the pre-oviposition period, egg, larval, pupal and adult stages of the carrot fly were measured under field conditions and at a range of constant temperatures in the laboratory. In the laboratory, the pre-oviposition period lasted from 4 days at 24^oC to 28 days at 9^oC. In general, female carrot flies laid about 20–40 eggs in each batch. Once the first eggs had been laid, subsequent batches were laid after an average of 3 days at 24^oC to 7 days at 11.5^oC. The numbers of days required for egg, larval and pupal development ranged from 5, 31 and 24 days respectively at 21.5^oC to 25, 145 and 84 days respectively at 9^oC. Under laboratory conditions, complete development from egg to adult required from 60 days at 21.5^oC to 254 days at 9^oC. Newly-formed carrot fly pupae were exposed to temperatures of 22–30^oC for various 5–10 day periods during pupal development. Exposure to temperatures of 24^oC and 26^oC caused some, and exposure to 28^oC and 30^oC caused all, of the pupae to delay development. Pupae were sensitive to high temperatures only for approximately 4–10 days after pupation. Under field conditions between mid-May and early September, full carrot fly development (egg-adult) took 84–100 days. The numbers of day-degrees required (base temperatures of 2^oC and 4^oC) for carrot fly development in the laboratory and in the field were similar for egg hatching but not for the pre-oviposition period or for egg-adult development. The thermal requirement for fly development in the field varied between inoculation dates, fewest day-degrees being required when development was rapid.     

Further investigations of the host range of the carrot fly, Psila rosae (F.)

Techniques were developed for testing umbelliferous species against naturally-occurring populations of carrot fly in the field. The most efficient technique involved exposure of plants to either first or second generations of the insect in the field, followed by caging of infested plots and trapping of emerged flies in water in yellow water dishes. In a series of field experiments between 1981 and 1989 inclusive, a total of 132 umbelliferous species and sub-species were tested against carrot fly. Seventy-eight of these proved to be new hosts, 27 were confirmed as hosts and 27 failed to support any carrot flies. Six non-umbelliferous plant species failed to be colonised by carrot fly. Sources of variability in investigations of the host range of insects are discussed.     

Carrot fly cultivar preferences: some influencing factors

Abstract. 1. Factors affecting cultivar preferences by the carrot fly Psila rosae F. were examined in choice tests using foliage from carrot lines representing extremes of resistance and susceptibility to attack.
2. Flies were offered a choice to oviposit on foliage, artificial substrates impregnated with leaf surface extracts or enclosed in vapours of different cultivars to establish the importance of contact and olfactory stimuli. Susceptible cv. Danvers Half Long 126 foliage was preferred over that of resistant cvs. Clause's Sytan Original and Tip-Top. But, whereas the Sytan leaf surface extract was as effective as that of Danvers, the surface extract of Tip-Top was very much inferior as an oviposition stimulant. By contrast, Sytan and Tip-Top foliar vapours were both more attractive to the fly than that from Danvers.
3. Headspace vapours over Sytan foliage evoked significantly higher electroantennogram responses than those from Danvers.
4. Cold-trapped foliar volatiles of Sytan and Danvers were analysed by gas chromatography linked with antennographic detection. Foliage of Sytan released, among other chemo-stimulants, higher levels of host plant attractants, i.e. green leaf aldehydes and the phenylpropanoids, trans-asarone and trans-methylisoeugenol.
5. These findings indicate that olfactory and contact chemostimuli may be involved in preferences shown by the carrot fly for carrot cultivars, but these factors alone do not fully account for the preferences observed.     

Evidence for simply inherited dominant resistance to Meloidogyne javanica in carrot

Root-knot nematodes (Meloidogyne spp.) are serious pests of carrot (Daucus carota L.) worldwide. While soil treatment with nematicides is the primary means for managing nematodes in carrot, there is a need to identify and introduce host plant resistance for crop improvement. This study was conducted to determine the inheritance of resistance to root-galling and reproduction by M. javanica (Treub) Chitwood in a selection (BR-1252) of carrot variety Brasilia. F₂, F₃, F₄, and BC₁ progenies from the cross BR-1252×B6274 (a susceptible inbred line) were screened in pot tests for reaction to M. javanica. The observed reactions based on galling and egg production on fibrous roots gave segregation patterns in all tests that were consistent with relatively simply inherited dominant resistance. Field testing in progress indicates that this resistance is very effective against both M. javanica and M. incognita. A single gene model fits the observed data acceptably well in F₃ generations. However, the range of 3% to 51% susceptible plants in segregating F₃ families and 1% to 47% in segregating F₄ families is much wider than the 25% expected with a single-gene model, and linked duplicate factors in the coupling phase could also explain the observed segregation patterns. The variation in percentage susceptibility among these families did not clearly cluster into three expected categories (25% S, 20.25% S, and 0.25% S for a 10-cM linkage distance, or 25% S, 16% S and 1% S for 20 cM), but it did tend to occur over the same range. Thus a 10-cM to 20-cM-linked duplicate factor model cannot be dismissed at this time. Egg production data in the F₂, F₃, and F₄ families provided evidence for slightly lower resistance expression in the heterozygous condition. Thus, while overall expressed in a dominant fashion, the resistance does exhibit some allelic dosage response. Received: 14 June 1999 / Accepted: 7 July 1999     

Isolation and characterization of colchicine-resistant cell lines of carrot

Colchicine changes plant cell shape by disrupting cortical microtubules. This change in cell shape involves the loss of cell rigidity and, subsequently, an increase in cell volume. Dimethylsulfoxide prevents the colchicine-stimulated cell enlargement but cannot maintain the cell shape. We have isolated colchicine-resistant cell lines, col-4 and col-3, which can maintain their cell shape in colchicine at 10⁻⁴ and 10⁻³ M , respectively. Both col-4 and col-3 accumulate a low level of tubulins when grown in colchicine while the wild-type cells do not. Hence the ability to accumulate tubulins correlates with the ability to maintain cell shape. The mechanism of colchicine-resistance of col-4 is not clear but may be associated with the expression of 5 proteins with molecular masses of 64, 45, 29, 28, and 26 kDa. Col-3 cells were isolated from col-4 and presumably shared this mechanism of resistance since they also express these 5 proteins. However, col-3 cells have an additional defect resulting in reduced colchicine uptake.     

Relationship between root volatiles of some carrot cultivars and their resistance to the carrot fly, Psila rosae

Field experiments investigated the resistance of some carrot cultivars to Psila rosae. In addition, headspace vapour and steam distillate from the roots of resistant and susceptible varieties were compared by gas-liquid chromatography. The field data confirmed that resistance may operate by decreasing the numbers of eggs laid indicating a nonpreference by the female Psila. Root resistance to the larva was also confirmed but the mechanism was unclear. A new finding was that root resistance is independent of the effect of egg laying, some cultivars evincing one or the other effect and some such as Regulus Imperial displaying both. It was clear that root resistance to the larva is the crucial prerequisite in breeding resistant varieties.One consistent difference was detected by the chemical comparisons: intact roots of resistant varieties released substantially less volatiles. Specifically, Regulus released almost five times less of the volatiles already shown to positively influence host-finding behaviour by the larva.

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Résumé La résistance à P. rosae de quelques cultivars de carotte a été étudiée en plein champ. Parallèlement, les substances volatiles diffusées et celles extraites par la vapeur des racines de variétés résistantes et sensibles, ont été comparées en chromatographie gaz-liquide (GLC). Les résultats en champ ont confirmé que la résistance peut être due à une diminution du nombre d'oeufs pondus, révélant une absence d'attractivité pour les femelles de P. rosae. La résistance des racines aux larves a été aussi confirmée, mais les raisons n'en étaient pas claires. Un aspect nouveau est que la résistance des racines est indépendante de l'effet de la ponte, quelques cultivars présentant l'un ou l'autreeffet et certains, comme Regulus Imperial, manifestant les deux. Il est net que la résistance racinaire aux larves est la condition essentielle pour la sélection de variétés résistantes. Une différence importante a été mise en évidence par les comparaisons chimiques: les racines intactes de variétés résistantes libèrent nettement moins de substances volatiles. Précisément, Regulus a libéré 5 fois moins de substances volatiles déjà connues comme influençant positivement le comportement de découverte de l'hôte par la larve.

     

Evidence for phosphorylation of tubulin in carrot suspension cells

Two-dimensional gels of phosphoproteins from carrot ( Daucus carota L. var. Juwarot) suspension cells labeled in vivo or in vitro revealed phosphoproteins that comigrate with carrot tubulin. A polyclonal antiserum to hibiscus tubulin immunoprecipitated an in vivo labeled phosphoprotein of 50 kDa. Cell-free extracts of carrot suspension cells phosphorylated both purified carrot and bovine brain tubulins in the presence of gamma-labeled adenosine triphosphate. This tubulin phosphorylating activity was reduced 2-fold in extracts from globular stage embryos and approximately 10-fold in extracts from heart/torpedo stage embryos. These data suggest that carrot cells phosphorylate tubulin, and that tubulin phosphorylating activity may be developmentally regulated     

Introgression and persistence of cultivar alleles in wild carrot (Daucus carota) populations in the United States

Premise

Cultivated species and their wild relatives often hybridize in the wild, and the hybrids can survive and reproduce in some environments. However, it is unclear whether cultivar alleles are permanently incorporated into the wild genomes or whether they are purged by natural selection. This question is key to accurately assessing the risk of escape and spread of cultivar genes into wild populations.

Methods

We used genomic data and population genomic methods to study hybridization and introgression between cultivated and wild carrot (Daucus carota) in the United States. We used single nucleotide polymorphisms (SNPs) obtained via genotyping by sequencing for 450 wild individuals from 29 wild georeferenced populations in seven states and 144 cultivars from the United States, Europe, and Asia.

Results

Cultivated and wild carrot formed two genetically differentiated groups, and evidence of crop–wild admixture was detected in several but not all wild carrot populations in the United States. Two regions were identified where cultivar alleles were present in wild carrots: California and Nantucket Island (Massachusetts). Surprisingly, there was no evidence of introgression in some populations with a long-known history of sympatry with the crop, suggesting that post-hybridization barriers might prevent introgression in some areas.

Conclusions

Our results provide support for the introgression and long-term persistence of cultivar alleles in wild carrots populations. We thus anticipate that the release of genetically engineered (GE) cultivars would lead to the introduction and spread of GE alleles in wild carrot populations.     

Changes in peroxidase activity and in peroxidase isozymes in carrot callus

Activity of soluble peroxidase and its isozyme patterns in carrot ( Daucus carota L.) callus after excision and transfer to fresh Murashige and Skoog (MS) culture medium was investigated. The activity decreased markedly until day 1 and then increased gradually during days 7–10 at room temperature. The rapid initial decrease of the activity was also observed at low temperature (2°C) as well as in the presence of cycloheximide. However, the subsequent increase in the peroxidase activity after day I was slower at low temperature than at room temperature, and was not detected in the presence of cycloheximide. Activity of catalase decreased slightly within 4 days and cycloheximide enhanced the decrease of the activity. Two cationic and one anionic peroxidase isozymes disappeared or decreased markedly within the first day and one cationic and anionic peroxidase recovered 3–6 days after excision.     

An evaluation of gene expression during somatic embryogenesis of two temperature-sensitive carrot variants unable to complete embryo development

The synthesis of putative stage-specific polypeptides during somatic embryogenesis of the carrot ( Daucus carota L. cv. Danvers) was investigated in the temperature-sensitive variants OB-2 and OB-3. These variants undergo normal embryo development to produce mature plantlets at the permissive temperature (24°C), but are arrested at the oblong stage to form elongated embryos without cotyledons at the restrictive temperature (33°C). Using two-dimensional polyacrylamide gel electrophoresis of in vivo labelled polypeptides, the patterns of stage-specific polypeptides in both lines were compared in: (1) oblong embryos grown at continuous 24°C vs oblong embryos exposed to 33°C during their temperature-sensitive period (i.e. embryos of identical morphology but different developmental fates); and (2) heartshaped embryos grown at constant 24°C vs enlarged oblong embryos exposed to 33°C during their temperature-sensitive period (i.e. embryos of the same age but different morphologies). The 22 putative stage-specific, polypeptides observed in this study fall into four classes: (1) line-specific, (2) age-specific, (3) unsynchronized, and (4) synchronized polypeptides. Only the last class, which consists of 4 polypeptides, exhibits synthesis patterns which are consistent with the polypeptides being causally involved in somatic embryo development. It is concluded that stage-specific behavior as assayed by PAGE analyses of simple 'present or absent' comparisons is insufficient to identify most of the polypeptides that may be relevant for somatic embryogenesis.     

Low external pH replaces 2,4-D in maintaining and multiplying 2,4-D-initiated embryogenic cells of carrot

A mixed culture comprised of both embryonic globules and nonembryogenic callus. was derived from seedling hypocotyls of Daucus carota cv. Scarlet Nantes on 2,4-D-containing medium using well-established methods. Then the mixed cultures were transferred to, and serially subcultured on, a hormone-free medium near pH 4. The medium contained 1 m M NH+ as the sole nitrogen source. When cultured in this way, embryonic globules were able to multiply without development into later embryo stages Nonembryogenic callus did not survive. Continuous culture of embryonic globules on this low pH hormone-free medium yielded cultures consisting entirely of preglobular stage proembryos (PGSPs). PGSP cultures have been maintained as such with continuous multiplication for nearly 2 years without loss of embryogenic potential. These hormone-free-maintained PGSPs continue their development to later embryo stages when cultured on the same hormone-free medium buffered at pH 5.8. We show that hormone-free medium near pH 4 can replace 2.4-D in its ability to sustain multiplication of 2,4-D-initiated embryogenic cells of carrot at an acceptable growth rate without their development into later embryo stages. This procedure provides selective conditions that do not permit the growth of nonembryogenic cells while providing an adequate environment for embryogenic cell proliferation and should prove invaluable in studying habituation.     

Cavity spot of carrot (Daucus carota)

Cavity spot disease of carrot (Daucus carota) has been one of the intractable problems for both growers and scientists. Carrots are rejected at grading with one or two visible lesions, and when disease incidence passes a relatively low threshold it becomes uneconomic to harvest crops. For the scientist, there has been considerable pressure to produce both information on the cause of the disease and a cure. Many putative causes have been advanced over the years, but these were almost always contradicted by subsequent work. The first solid indication of involvement of a pathogen was when three different fungicides with activity against Oomycete fungi all reduced disease. Very quickly the causal agents Pythium violae and Pythium sulcatum were isolated from cavity spot lesions and Koch's postulates satisfied. The species are not typical of the more common pythia, having slow growth at normal temperatures, which means that in the context of isolation work, plates may be overgrown by other species before they are seen. Metalaxyl fungicide was identified as the most effective in controlling cavity spot caused by P. violae, but P. sulcatum is naturally tolerant of the fungicide. Recently, metalaxyl has been shown to be subject to enhanced microbial degradation. This phenomenon has been associated with failure to control cavity spot. No other fungicide has been shown to be consistently effective in the field, and none has been registered for disease control. For the future, this means that control of cavity spot can not be based solely on fungicidal control. Other, complementary strategies are necessary for reducing disease. Calcium carbonate is known to have significant effects on cavity spot, probably by inducing a soil microflora inhibitory to filamentous fungi. Management of agronomic aspects such as irrigation, soil cultivation and the length of time for which crops are grown may all be used, while carrot cultivars with some field resistance may be beneficial. However, one of the most significant factors is disease avoidance by not selecting fields with high inoculum levels. One serology‐based risk assessment test has been produced and commercialised, and molecular probes which could be the basis of more sensitive tests are available for both pathogens. The potential for disease reduction via a management strategy combining several key components is discussed.     

Chitinase profiles in mature carrot (Daucus carota) roots and purification and characterization of a novel isoform

The profile of chaitinases (EC 3.2.1.14) in mature carrot ( Daucus carota L. cv. Eagle) roots was studied. Multiple chitinase bands (8–10) were observed in native and sodium dodecylsulfate-denaturing polyacrylamide gels. The molecular masses of these chitinases were estimated to be from 20 000 to 40 000. One major chitinase was purified and found to be an acidic protein with pI at 4.3 and a molecular mass of 39 500. The optimum pH for enzymatic activity was around 5 and the optimum temperature was 25°C. The enzyme was stable at pHs below 8 and temperatures below 60°C. The protein did not have a chitin-binding domain, but showed some similarity to the amino acid composition of tobacco class I chitinase. The N-terminal amino acid sequence did not resemble any of the described classes of chitimases. This chitinase did not possess lysozyme activity and showed antifungal activity when tested against Trichoderma sp.