Aspartic proteinase from the seeds of figleaf gourd (Cucurbita ficifolia)*.
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Immunoglobulins anti-endonuclease 32 kDa inhibit DNA synthesis. We observed that low concentrations of IgGs (about 50 micrograms IgG per 1 x 10(6) cell nuclei) temporary inhibit DNA synthesis. This inhibition concerns only the synthesis of DNA bound to the nuclear matrix (associated with isolated nuclear matrix). Preincubation of cell nuclei of White bush with IgG generates longer DNA fragments than in controls. Involvement of the 32 kDa endonuclease or an endonuclease-65 kDa protein complex from the nuclear matrix in replication or structural organisation of replication is considered.
The nuclear matrices of plant cell nuclei display intrinsic nuclease activity which consists in nicking supercoiled DNA. A cDNA encoding a 32 kDa endonuclease has been cloned and sequenced. The nucleotide and deduced amino-acid sequences show high homology to known 14-3-3 protein sequences from other sources. The amino-acid sequence shows agreement with consensus sequences for potential phosphorylation by protein kinase A and C and for calcium, lipid and membrane-binding sites. The nucleotide-binding site is also present within the conserved part of the sequence. By Northern blot analysis, the differential expression of the corresponding mRNA was detected; it was the strongest in sink tissues. The endonuclease activity found on DNA-polyacrylamide gel electrophoresis coincided with mRNA content and was the highest in tuber.
Phloem-specific proteins (P proteins) are particularly useful markers to investigate long-distance trafficking of macromolecules in plants. In this study, genus-specific molecular probes were used in combination with intergeneric grafts to reveal the presence of a pool of translocatable P protein subunits. Immunoblot analyses demonstrated that Cucurbita spp P proteins PP1 and PP2 are translocated from Cucurbita maxima stocks and accumulate in Cucumis sativus scions. Cucurbita maxima or Cucurbita ficifolia PP1 and PP2 mRNAs were not detected in Cucumis sativus scions by either RNA gel blot analysis or reverse transcription-polymerase chain reaction, indicating that the proteins, rather than transcripts, are translocated. Tissue prints of the Cucumis sativus scion, using antibodies raised against Cucurbita maxima PP1 or PP2, detected both proteins in the fascicular phloem of the stem at points distal to the graft union and in the petiole of a developing leaf, suggesting that the proteins move within the assimilate stream toward sink tissues. Cucurbita maxima PP1 was immunolocalized by light microscopy in sieve elements of the extrafascicular phloem of Cucumis sativus scions, whereas Cucurbita maxima PP2 was detected in both sieve elements and companion cells.
Antibiosis and resistance of six Cucurbita and two Lagenaria accessions to the carmine spider mite, Tetranychus cinnabarinus Boisduval, were evaluated in the laboratory. Significant differences among accessions were observed three days after the inoculation of detached leaf discs. The Lagenaria accessions, Slawi and Sus, proved to be the most resistant to mites, with average populations of mite eggs, 87 and 95%, respectively less than that of the susceptible C. pepo accession, Orangetti. The Cucurbita accessions, Tace, Brava, Tetsukabuto, Phoenix and TZ-148 had mite egg totals 4, 9, 13, 26 and 40%, respectively, less than those of accession Orangetti. The Sus accession of Lagenaria was resistant to T. cinnabarinus from the four-leaf stage until fruit set in laboratory and field tests. Grafting the susceptible Brava onto Sus rootstock increased the resistance of the scion to the same level as that of non-grafted Sus. Grafting the susceptible Cucumis melo Noy Yizre'el on resistant or susceptible rootstocks of Cucurbita and Lagenaria accessions did not affect its susceptibility to T. cinnabarinus. The results indicate that resistance to T. cinnabarinus can be transferred by grafting from Lagenaria stocks to Cucurbita scions but not in the opposite direction.
Spinosad and phloxine B are two more environmentally friendly alternative toxicants to malathion for use in bait sprays for tephritid fruit fly suppression or eradication programs. Laboratory tests were conducted to assess the relative toxicity of these two toxicants for melon fly, Bactrocera cucurbitae Coquillett; oriental fruit fly, Bactrocera dorsalis Hendel; and Mediterranean fruit fly, Ceratitis capitata (Wiedemann) females. Field tests also were conducted with all three species to compare these toxicants outdoors under higher light and temperature conditions. In laboratory tests, spinosad was effective at much lower concentrations with LC50 values at 5 h of 9.16, 9.03, and 4.30 compared with 250.0, 562.1, and 658.9 for phloxine B (27, 62, and 153 times higher) for these three species, respectively. At 16 ppm spinosad, LT50 values were lower for all three species (significantly lower for C. capitata and B. dorsalis) than 630 ppm phloxine B LT50 values. At 6.3 ppm spinosad, the LT50 value for C. capitata (3.94) was still significantly less than the 630 ppm phloxine B LT50 value (6.33). For all species, the 100 ppm spinosad concentrations gave LT50 values of < 2 h. In comparison among species, C. capitata was significantly more sensitive to spinosad than were B. cucurbitae or B. dorsalis, whereas B. cucurbitae was significantly more sensitive to phloxine B than were C. capitata or B. dorsalis. LC50 values were reduced for both toxicants in outdoor tests, with greater reductions for phloxine B than for spinosad for B. dorsalis and B. cucurbitae. Fly behavior, though, is likely to keep flies from being exposed to maximum possible outdoor light intensities. Comparable levels of population suppression for any of the three species tested here will require a much higher concentration of phloxine B than spinosad in the bait.
The possibility of proving the undeclared addition of pumpkin puree in apricot nectars and jams has been investigated by using the phenol compound fingerprint and sensory evaluation. The cheaper pumpkin admixtures in apricot nectars and jams could not be detected by the sensory evaluation, particularly if present in quantities of <15%. The lower admixtures of pumpkin puree in apricot nectars and jams could be detected by the presence of syringic acid, a phenolic compound characteristic of the investigated pumpkins (Cucurbita pepo cv. Gleisdorff and Table Gold, Cucurbita maxima cv. Turkinja, and Cucurbita moschata cv. Argenta). Syringic acid was isolated from pumpkin puree and determined by using HPLC with diode array detection. By using the phenolic profile, undeclared pumpkin admixture (> or =5%) in the apricot nectars and jams could be proven.
Reanalysis and direct accelerator mass spectrometry radiocarbon dating of the cucurbit assemblage from Coxcatlan Cave provide information on the timing and sequence of the initial appearance of three domesticated plants in the Tehuacán Valley (Puebla, Mexico) and allow reassessment of the overall temporal context of plant domestication in Mexico. Cucurbita pepo is the earliest documented domesticate in the cave, dating to 7,920 calibrated calendrical (cal) years B.P. The bottle gourd (Lagenaria siceraria) is dated at 7,200 cal years B.P. Cucurbita argyrosperma does not appear until 2,065 cal years B.P. The earlier identification of Cucurbita moschata specimens is not confirmed. Seventy-one radiocarbon dates, including 23 accelerator mass spectrometry dates on cucurbits, provide ample evidence of postdepositional vertical displacement of organic materials in the western half of Coxcatlan Cave, but they also indicate that the eastern half of the cave was largely undisturbed.
Function of the Drosophila melanogaster hobo transposon in tephritid species was tested in transient embryonic excision assays. Wild-type and mutant strains of Anastrepha suspensa, Bactrocera dorsalis, B. cucurbitae, Ceratitis capitata, and Toxotrypana curvicauda all supported hobo excision or deletion both in the presence and absence of co-injected hobo transposase, indicating a permissive state for hobo mobility and the existence of endogenous systems capable of mobilizing hobo. In several strains hobo helper reduced excision. Excision depended on hobo sequences in the indicator plasmid, though almost all excisions were imprecise and the mobilizing systems appear mechanistically different from hobo. hobo-related sequences were identified in all species except T. curvicauda. Parsimony analysis yielded a subgroup including the B. cucurbitae and C. capitata sequences along with hobo and Hermes, and a separate, more divergent subgroup including the A. suspensa and B. dorsalis sequences. All of the sequences exist as multiple genomic elements, and a deleted form of the B. cucurbitae element exists in B. dorsalis. The hobo-related sequences are probably members of the hAT transposon family with some evolving from distant ancestor elements, while others may have originated from more recent horizontal transfers.
The use of intergenic spacer (IGS) fragments of plant ribosomal DNA (rDNA) for the differentiation between genera and species is tested by cross-hybridization experiments with different IGS probes of two Cucurbitaceae, Cucurbita pepo (zucchini) and Cucumis sativus (cucumber). Hybridization with cloned fragments of different parts of the IGS of ribosomal DNA exhibit a different degree of conservation within and between the Cucurbitaceae genera. In general, Cucurbita species seem to be closer related to each other than the Cucumis species. A repetitive element of the external transcribed spacer (ETS) shows a more genus-specific pattern, reacting only with the respective genera; the region preceding the ETS is conserved between the Cucurbita species but also cross-hybridizes weakly with the Cucumis species. A GC-rich element of the Cucumis sativus IGS ("Cfo-cluster") is present in small amounts in Cucumis melo (melon) and even less represented in other genera of the Cucurbitaceae.
Methyl eugenol (4-allyl-1,2-dimethoxybenzene-carboxylate) and cue-lure [4-(p-acetoxyphenyl)-2-butanone] are highly attractive kairomone lures to oriental fruit fly, Bactrocera dorsalis (Hendel), and melon fly, B. cucurbitae (Coquillett), respectively. Plastic bucket traps were evaluated as dispensers for methyl eugenol and cue-lure for suppression of the 2 fruit flies in Hawaii. Methyl eugenol and cue-lure mixtures were compared with pure methyl eugenol or cue-lure over 4 seasons. B. dorsalis captures differed significantly with treatment and season. B. dorsalis captures with 100% methyl-eugenol were significantly greater than all other treatments (25, 50, and 75%). B. cucurbitae captures also differed significantly with treatment but not with season. Captures with 100, 75, and 50% cue-lure were not significantly different. Bucket traps baited with cue-lure (+ malathion) and weathered under Hawaiian climatic conditions were attractive to B. cucurbitae up to 8 wk. Two methyl eugenol dispensers (canec disks and Min-U-Gel) were compared with bucket traps. Dispensers (methyl eugenol + malathion) were weathered for 2-16 wk under Hawaiian climatic conditions and bioassayed during summer and winter. Initially, captures of B. dorsalis were not significantly different for the 3 dispensers. Bucket traps and canec disks were most resistant to weather, remaining attractive to B. dorsalis flies up to 16 wk. Min-U-Gel was least resistant, losing attractiveness to B. dorsalis flies within 2 wk. On the basis of performance, bucket traps and canec disks were equally long-lived up to 14 wk; thereafter, bucket traps were slightly more attractive during winter. Canec disks were cheapest, but on the basis of possible environmental concerns, bucket traps may be the best all-around choice for areawide suppression of fruit flies.
Spinosad was evaluated in Hawaii as a replacement for organophosphate insecticides (naled, dichlorvos [DDVP], and malathion) in methyl eugenol and cue-lure bucket traps to attract and kill oriental fruit fly, Bactrocera dorsalis Hendel, and melon fly, B. cucurbitae Coquillett, respectively. In the first and second methyl eugenol trials with B. dorsalis, naled was in the highest rated group for all evaluation periods (at 5, 10, 15, and 20 wk). Spinosad was equal to naled at 5 and 10 wk during both trials 1 and 2, and compared favorably with malathion during trial 2. During the first cue-lure trial with B. cucurbitae, naled and malathion were in the top rated group at 5, 10, 15, and 20 wk. Spinosad was equal to naled at 5 wk. During the second cue-lure trial, spinosad and naled were both in the top rated group at 10, 15, and 20 wk. Use of male lure traps with methyl eugenol or cue-lure had no effect on attraction of females into test areas. Our results suggest that spinosad, although not as persistent as naled or malathion, is safer to handle and a more environmentally friendly substitute for organophosphate insecticides in methyl eugenol and cue-lure traps for use in B. dorsalis and B. cucurbitae areawide integrated pest management programs in Hawaii.
Amulet C-L (cue-lure) and Amulet ME (methyl eugenol) molded paper fiber "attract-and-kill" dispensers containing fipronil were tested under Hawaiian weather conditions against Bactrocera cucurbitae (Coquillett) (melon fly) and Bactrocera dorsalis (Hendel) (oriental fruit fly), respectively. In paired tests (fresh versus weathered), C-L dispensers were effective for at least 77 d, whereas ME dispensers were effective for at least 21 d. Thus, C-L dispensers exceeded, whereas ME dispensers did not meet the label interval replacement recommendation of 60 d. Addition of 4 ml of ME to 56-d-old ME dispensers restored attraction and kill for an additional 21 d. This result suggested the fipronil added at manufacture was still effective. By enclosing and weathering ME dispensers inside small plastic bucket traps, longevity of ME dispensers was extended up to 56 d. Fipronil ME and C-L dispensers also were compared, inside bucket traps, to other toxicants: spinosad, naled, DDVP, malathion, and permethrin. Against B. dorsalis, fipronil ME dispensers compared favorably only up to 3 wk. Against B. cucurbitae, fipronil C-L dispensers compared favorably for at least 15 wk. Our results suggest that fipronil C-L dispensers can potentially be used in Hawaii; however, fipronil ME dispensers need to be modified or protected from the effects of weathering to extend longevity and meet label specifications. Nonetheless, Amulet C-L and ME dispensers are novel prepackaged formulations containing C-L or ME and fipronil that are more convenient and safer to handle than current liquid insecticide formulations used for areawide suppression of B. dorsalis and B. cucurbitae in Hawaii.
Attraction and feeding responses of oriental fruit fly, Bactrocera dorsalis (Hendel), and melon fly, Bactrocera cucurbitae (Coquillett), were determined for different protein baits. In separate choice attraction assays for each species, significantly more flies arrived at stations with bait than water, but no differences existed among baits of GF-120 Fruit Fly Bait, GF-120 NF Naturalyte Fruit Fly Bait, Provesta 621 autolyzed yeast extract, and Mazoferm E802. In comparison with B. dorsalis, B. cucurbitae had 2.8 times more responders and a 4.8 times better discrimination between baits and water. In a second attraction assay with only B. dorsalis, volume of bait was negatively correlated to numbers of flies alighting on the bait. Feeding assays for both species demonstrated that time spent feeding and duration on a leaf were both significantly affected by bait type. B. dorsalis fed the longest on Provesta 621, with significantly less feeding on the other baits, and with all baits resulting in more feeding than water. The longest feeding times for B. cucurbitae resulted with Mazoferm E802 and Provesta 621, and all baits except GF-120 NF resulted in eliciting a significantly longer feeding duration than water. In separate toxicology assays for each species, significantly higher mortality resulted from bait formulations containing spinosad compared with blank baits, but no differences existed between GF-120 and GF-120 NF formulations. The differences are discussed between the two Bactrocera species primarily in regard to bait preference, extent of response, and previous work on laboratory flies.
Experiments were conducted to assess the bioavailability of polyclycic aromatic hydrocarbons (PAHs) in soil from a Manufactured Gas Plant site. Three plant species were cultivated for four consecutive growing cycles (28 days each) in soil contaminated with 36.3 microg/g total PAH. During the first growth period, Cucurbita pepo ssp. pepo (zucchini) tissues contained significantly greater quantities of PAHs than did Cucumis sativus (cucumber) and Cucurbita pepo ssp. ovifera (squash). During the first growth cycle, zucchini plants accumulated up to 5.47 times more total PAH than did the other plants, including up to three orders of magnitude greater levels of the six ring PAHs. Over growth cycles 2-4, PAH accumulation by zucchini decreased by 85%, whereas the uptake of the contaminants by cucumber and squash remained relatively constant. Over all four growth cycles, the removal of PAHs by zucchini was still twice that of the other species. Two earthworm species accumulated significantly different amounts of PAH from the soil; Eisenia foetida and Lumbricus terrestris contained 0.204 and 0.084 microg/g total PAH, respectively, but neither species accumulated measurable quantities 5 or 6 ring PAHs. Lastly, in abiotic desorption experiments with an aqueous phase of synthetically prepared organic acid solutions, the release of 3 and 4 ring PAHs from soil was unaffected by the treatments but the desorption of 5-6 ring constituents was increased by up to two orders of magnitude. The data show that not only is the accumulation of weathered PAHs species-specific but also that the bioavailability of individual PAH constituents is highly variable.