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Specific detection of the toxigenic species Fusarium proliferatum and F. oxysporum from asparagus plants using primers based on calmodulin gene sequences.

Fusarium proliferatum and Fusarium oxysporum are the causal agents of a destructive disease of asparagus called Fusarium crown and root rot. F. proliferatum from asparagus produces fumonisin B1 and B2, which have been detected as natural contaminants in infected asparagus plants. Polymerase chain reaction (PCR) assays were developed for the rapid identification of F. proliferatum and F. oxysporum in asparagus plants. The primer pairs are based on calmodulin gene sequences. The PCR products from F. proliferatum and F. oxysporum were 526 and 534 bp long, respectively. The assays were successfully applied to identify both species from the vegetative part of the plants.

Asparagus Plant↗

A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus.

In North America, asparagus (Asparagus officinalis) production suffers from a crown and root rot disease mainly caused by Fusarium oxysporum f. sp. asparagi and F. proliferatum. Many other Fusarium species are also found in asparagus fields, whereas accurate detection and identification of these organisms, especially when processing numerous samples, is usually difficult and time consuming. In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess Fusarium species diversity in asparagus plant samples. Fusarium-specific PCR primers targeting a partial region of the translation elongation factor-1 alpha (EF-1 alpha) gene were designed, and their specificity was tested against genomic DNA extracted from a large collection of closely and distantly related organisms isolated from multiple environments. Amplicons of 450 bp were obtained from all Fusarium isolates, while no PCR product was obtained from non-Fusarium organisms. The ability of DGGE to discriminate between Fusarium taxa was tested over 19 different Fusarium species represented by 39 isolates, including most species previously reported from asparagus fields worldwide. The technique was effective to visually discriminate between the majority of Fusarium species and/or isolates tested in pure culture, while a further sequencing step permitted to distinguish between the few species showing similar migration patterns. Total genomic DNA was extracted from field-grown asparagus plants naturally infested with different Fusarium species, submitted to PCR amplification, DGGE analysis and sequencing. The two to four bands observed for each plant sample were all affiliated with F. oxysporum, F. proliferatum or F. solani, clearly supporting the reliability, sensitivity and specificity of this approach for the study of Fusarium diversity from asparagus plants samples.

Asparagus Plant↗

An Agrobacterium-transformed cell culture from the monocot Asparagus officinalis.

Cultured stem fragments from the monocotyledonous plant Asparagus officinalis infected by the oncogenic bacterium Agrobacterium tumefaciens developed tumorous proliferations. This tissue was propagated in vitro on hormone-free culture medium. The T-DNA-encoded markers nopaline and agrocinopine were unambiguously detected in these tissues. The data demonstrate that stable T-DNA transfer as well as expression of T-DNA genes is possible in at least some monocotyledonous plants. This opens new possibilities for plant genetic engineering using the Ti plasmid as a gene vector.

Journal Article↗

Changes in communities of Fusarium and arbuscular mycorrhizal fungi as related to different asparagus cultural factors.

Asparagus (Asparagus officinalis) is a high-value perennial vegetable crop that has shown a marked decline in productivity after many years of continuous harvesting. This decline is caused by an increase in both abiotic (autotoxicity, harvesting pressure) and biotic stresses [fungal infections, mainly Fusarium crown and root rot (FCRR)]. To gain insight into disease development and possible mitigation strategies, we studied the effects of harvesting, time in the growing season, and field age on FCRR development, Fusarium species composition, and arbuscular mycorrhizal fungi (AMF) communities in both a controlled field experiment and an ecological survey of commercial fields. In one experiment, a 3-year-old asparagus field was subdivided into plots that were harvested or not and sampled throughout the growing season to assess short-term dominant Fusarium species shifts. In addition, diseased and healthy asparagus plants sampled from six commercial fields in the same geographical region were used to assess Fusarium and AMF communities in relation to different parameters. Fusarium and AMF communities were described by using a polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) approach, and results were analyzed by mainly correspondence analysis and canonical correspondence analysis. Results showed that dominant Fusarium taxa assemblages changed throughout the growing season. Harvested plots had significantly more FCRR symptomatic plants at the end of the growing season, but this effect was not related with any trend in Fusarium community structure. Sampling site and plant age significantly influenced AMF community structure, whereas only sampling site consistently influenced the Fusarium community. Diseased and healthy plants harbored similar Fusarium and AMF communities. Shifts in Fusarium community might not be responsible for different disease incidence because they are ubiquitous regardless of plant health status or harvesting regime. The different incidence noted might rather be related to plant physiology, antagonist microbial communities, or soil parameters.

Asparagus Plant↗

Biotransformation of alpha- and 6beta-santonin by fungus and plant cell cultures.

One fungus, Abisidia coerulea IFO 4011, and suspended cell cultures of one plant, Asparagus officinalis, were employed to bioconvert alpha- and 6beta-santonin. Incubation of alpha-santonin with the cell cultures of the fungus afforded two products, 11beta-hydroxy-alpha-santonin (1, in 76.5% yield) and 8alpha-hydroxy-alpha-santonin (2, in 2.0% yield). And from 6beta-santonin, four major products (3, 4, 5 and 6) and four minor products (7, 8, 9 and 10) were obtained, including 8alpha-hydroxylated products in trace yields. Very interestingly, a skeletal rearrangement occurred and a guaiane product (13) formed in a very low yield when alpha-santonin incubating with A.officinalis cell cultures, while not in the case of 6beta-santonin as substrate. Among the obtained 15 products, 2, 7, 8, 9, 10 and 12 are new compounds. The fact of 8alpha hydroxylation of santonin enables the formation of 8,12-eudesmanolide instead of 6,12-eudesmanolide and some useful modification at C-8 position. In addition, these reactions would provide evidence for the biogenesis between different types of eudesmane and/or guaiane compounds in the plants in nature.

Asparagus Plant↗

Host-plant finding by the asparagus fly, Plioreocepta poeciloptera (Diptera: Tephritidae), a monophagous, monovoltine tephritid.

The role of various olfactory and visual stimuli was studied in host-plant finding by the asparagus fly Plioreocepta poeciloptera (Schrank), a monophagous monovoltine tephritid causing serious damage to asparagus spears. Volatiles released by asparagus plants were extracted by diethyl ether after cryotrapping concentration, and identified by gas chromatography-mass spectrometry. Twelve of the 13 compounds identified were tested using electroantennography to measure the response of the fly. Behavioural response was analysed using two different flight tunnels according to circadian rhythm, age and sex of adults, presence of the plant and of different coloured lures, presence of a male congener, or exposure to four pure asparagus odour compounds that elicited responses in electroantennography, i.e. hexanal, (E)-2-hexenal, (Z)-2-hexen-1-ol and decanal. Data showed that males locate the host plant more quickly than females. Females are attracted mainly by the blend of plant odour and male pheromone. Both sexes respond to a complex of stimuli only during the afternoon. These findings will be helpful in developing new and effective approaches to control this pest insect.

Animals↗

Analysis of habituated embryogenic lines in Asparagus officinalis L.: growth characteristics, hormone content and ploidy level of calli and regenerated plants.

Habituated asparagus embryogenic lines derived from eleven genotypes were maintained on hormone-free medium and grew actively through secondary embryogenesis. Secondary embryos were of single cell origin and emerged from the transversal division of some epidermal or subepidermal cotyledonary cells of primary embryos. The intensity of secondary embryogenesis was found to be variable between embryogenic lines. Plants regenerated from three of these lines have been previously demonstrated to carry a mutation whose phenotype was the direct appearance of somatic embryos on apices or nodes cultured on hormone-free medium. Habituated lines of embryogenic calli and various tissues of embryogenic mutant and wild type plants were analysed for their hormonal content in ABA, IAA, iP, Z and their metabolites ABA-GE, iPA, iMP, ZR. No significant difference was found between different embryogenic lines, except the level of iPA, or between cladophyll or apex cultures of mutant and wild type plants. Flow cytometry analyses indicated only 34% of the embryogenic lines were diploid, most of the others being tetraploid, but 62% of regenerated plants from these lines were diploid. This indicated the process of maturation and conversion selected diploid embryos in the embryogenic lines.

Journal Article↗

T-DNA organization in tumor cultures and transgenic plants of the monocotyledon Asparagus officinalis.

Asparagus officinalis was the first monocotyledonous plant from which hormone-independent and opine-producing crown gall tissue could be isolated. We confirm by DNA hybridization that tumor lines obtained after infection of this plant by Agrobacterium strains harboring wild-type nopaline and octopine tumor-inducing (Ti) plasmids are stably transformed and contain transferred DNA (T-DNA) segments identical to the T-DNA found in dicotyledonous plants. We have also infected Asparagus with a nononcogenic T-DNA vector that carries a chimeric aminoglycoside phosphotransferase [NOS-APH(3')II] gene and selected transformed tissues on kanamycin-containing medium. The transformed status of these tissues was then confirmed by DNA hybridization. From these calli we regenerated kanamycin-resistant shoots that were subsequently rooted. Thus we report the isolation of transgenic monocotyledonous plants engineered via the Agrobacterium vector system.

Journal Article↗

Plant regeneration after long-term callus culture in clones of Asparagus officinalis L.

Callus growth and plant regeneration from long-term callus cultures were studied in two elite clones of Asparagus officinalis cv. Argenteuil, to establish a suitable protocol for a prospective in vitro selection program. Callus initiation and growth was evaluated on MS medium with 3% sucrose, 0.9% agar, 1 mg x l(-1) kinetin, and three levels of 2,4-D. The highest callus relative growth was obtained on medium with 1.5 mg x l(-1) 2,4-D and 1 mg x l(-1) kinetin. Shoot primordia (SP) induction from > 18-months-old calluses was evaluated on several media; the highest percentage of SP induction (89%) and average number of SP per callus (8.6) were obtained with clone "265" on MS medium with 5 mg x l(-1) 2iP, 1 mg x l(-1) IAA, 3% sucrose and 0.9% agar. The highest percentage of root induction (100%) was achieved with clone '265' on MS medium with 0.1 mg x l(-1) kinetin, 0.1 mg x l(-1) NAA, 1.32 mg x l(-1) ancymidol, 7% glucose and 0.8% agar. Important medium x genotype interactions were detected, pointing to the need of adjusting this and other in vitro protocols for specific asparagus genotypes.

2,4-Dichlorophenoxyacetic Acid↗

Chemical constituents from Asparagus dumosus.

The isolation and characterization of calonysterone (1), blechnoside B (2), 20-hydroxyecdysone and isovanillin from the whole plant of Asparagus dumosus are being reported for the first time from this source.

Humans↗

Immunoprotection by botanical drugs in cancer chemotherapy.

Most of the synthetic chemotherapeutic agents available today are immunosuppressants, cytotoxic, and exert variety of side effects that are particularly evident in cancer chemotherapy. Botanical based immunomodulators are often employed as supportive or adjuvant therapy to overcome the undesired effects of cytotoxic chemotherapeutic agents and to restore normal health. Total extract, polar and non-polar extracts, and their formulations, prepared from medicinal plants mentioned in Ayurveda, namely, Withania somnifera (Linn Dunal) (Solanaceae), Tinospora cordifolia (Miers) (Menispermaceae), and Asparagus racemosus (Willd.) (Liliaceae), exhibited various immunopharmacological activities in cyclophosphamide (CP)-treated mouse ascitic sarcoma. Treatment of ascitic sarcoma-bearing mice with a formulation of total extracts of Withania somnifera and Tinospora cordifolia (80:20) and alkaloid-free polar fraction of Withania somnifera resulted in protection towards CP-induced myelo- and immunoprotection as evident by significant increase in white cell counts and hemagglutinating and hemolytic antibody titers. Treatment with these candidate drugs will be important in development of supportive treatment with cancer chemotherapy.

Adjuvants, Immunologic↗

Steroidal saponins from Asparagus dumosus.

A new steroidal saponin, dumoside, characterized as (20S)-3 beta, 16 beta-dihydroxy pregn-5-ene-22-carboxylic acid (22, 16)-lactone-3-O-beta-chacotrioside, was isolated from the whole plant of Asparagus dumosus Baker and the structure was deduced from spectral data. In addition to dumoside three more steroidal saponins characterized as 3 beta-dihydroxy pregn-5,16-dien-20-one 3-O-beta-chacotrioside, 3 beta, 22 alpha, 26-trihydroxyfurost-5-ene-3-O-beta-chacotrioside-26-O- beta-D-glucopyranoside and its corresponding 22 alpha-O methoxy analogue were also isolated for the first time from this source. The structures have been identified with the help of FAB-MS, 1H NMR, 13C NMR and extensive 2D NMR spectroscopy, as well as comparison with reported spectroscopic data.

Carbohydrate Conformation↗

Field response of some asparagus varieties to rust, Fusarium crown root rot, and violet root rot.

Research was carried out to evaluate the behaviour of some asparagus genotypes against three most important fungal diseases: 1) asparagus rust caused by Puccinia asparagi D.C.; 2) Fusarium crown and root rot caused by Fusarium oxysporum (Schlecht.) f.sp. asparagi (Cohen & Heald) and Fusarium proliferatum (Matstush.) Nirenberg; 3) violet root rot caused by Rhizoctonia violacea Tul. The object of this research was also to found an eventual correlation between the plant susceptibility to asparagus rust and the sensibility to Fusarium crown root rot and violet root rot attacks. Resistant genotypes to rust should be less susceptible to attacks from F. oxysporum f.sp. asparagi, F. proliferatum and R. violacea, a fungal complex causing the plant decline. Asparagus genotypes were compared in a randomized complete block experiment design, replicated four times, in order to search that ones showing the best behaviour to escape the diseases. Phytopathological observations were carried out on November when the control plots showed 100% infected plants. The pathogens were isolated and identified. The diseased plants were registered. According to symptom evaluation scales, all the plants were grouped into infection classes, calculating frequency and McKinney index. Wishing to learn something about the infection trend of F. oxysporum f.sp. asparagi or R. violacea in relation to P. asparagi attack, the relative curvilinear regressions were calculated. The Italian cultivars "Marte" and "Grande" showed significantly the best behaviour in terms of resistance to asparagus rust, exhibiting 37% and 42% of diseased plants. The McKinney index was 9.1% and 15.6%, respectively. The susceptible plots showed 100% of infected plants and different McKinney index: 46% for "Eros", about 60% for "H 519", "Atlas" and "Golia", over 70% for the remainder. "Marte" and "Grande" showed good tolerance to F. oxysporum f.sp. asparagi and to R. violacea exhibiting up to 100% of healthy plants. The regression between plants affected by asparagus rust and those diseased by Fusarium crown root rot showed a linear equation with a regression coefficient b = 1.186 and a correlation coefficient R2 = 0.98. The regression between infection caused by rust and that caused by violet root rot exhibited a regression coefficient b = 1.03 and a coefficient of correlation R2 = 0.9. "Marte" and "Grande" exhibited the best behaviour against the rust attacks. Plants without rust were tolerant to pathogens causing plant decline.

Asparagus Plant↗

Cryoprotection in plant tissues related to reduced volume expansion of cryoprotectant solution.

To clarify the mechanism of reduced volume expansion-related cryoprotection changes in solution volume during freezing using several types of cryoprotectant were investigated. The effect of each cryoprotectant solution on the survival of asparagus nodal segments cooled slowly (0.5 degrees C/min) to -40 degrees C was also examined. The ratio of the volume at -40 degrees C to the volume at +20 degrees C was used as an index for expansion, calculated as a ratio of the density at +20 degrees C to the density at -40 degrees C. Distilled, deionized water showed the largest volume change at a ratio of 1.094. The ratio gradually decreased with an increase in the molar concentration of cryoprotectant, with the magnitude of the change dependent on the nature of the cryoprotectant. Raffinose was the most effective in reducing volume expansion when compared with other cryoprotectants at a same concentration. Raffinose exhibited greatest cryoprotection in asparagus tissue at 0.6 M where the solution became saturated. Dimethyl sulfoxide (Me2SO) at 1.69 M had the largest effect on cryoprotecting asparagus tissue. Furthermore, Me2SO was also the most effective in reducing volume expansion among the group of cryoprotectants permeable to the plasma membrane. It is concluded that cryoprotection in tissues was closely related to reduced volume expansion especially at low concentration (< or = 1.0 M). Cryoprotectants of impermeable sugar group lost their cryoprotective effect at > 1.0 M, which may due to severe dehydration and cell damage occurred in hypertonic solution. Useful cryoprotectants should be furnished with high ability of reducing volume expansion during freezing as well as low toxicity and high permeability for cells.

Asparagus Plant↗

A new FISH protocol with increased sensitivity for physical mapping with short probes in plants.

Fluorescence in situ hybridization (FISH) is a well-established technique used for the detection of specific DNA regions, that has been applied to interphase nuclei, pachytene and metaphase chromosomes as well as to extended DNA fibres. This technique allows the physical mapping of specific DNA sequences both on individual chromosomes and extended fibres. A new FISH protocol is described here that enhances the sensitivity of the method. Probes for small unique DNA sequences of less than 2 kb give high signal-to-noise ratio with this method, and can be visualized easily by means of conventional fluorescence microscopy.

Asparagus Plant↗

An endo-polygalacturonase (PG) of Fusarium moniliforme escaping inhibition by plant polygalacturonase-inhibiting proteins (PGIPs) provides new insights into the PG-PGIP interaction.

Polygalacturonase-inhibiting proteins (PGIPs) are plant defence molecules inhibiting the activity of fungal endo-polygalacturonases (endo-PGs). We found that soybean and bean PGIPs inhibited the endo-PG activity produced by the isolate FC-10 of Fusarium moniliforme but not the enzyme activity produced by the isolate PD of F. moniliforme. The bean PGIP proved to be ineffective against all the PG isoforms produced by the PD isolate. Deduced amino acid sequence comparison between PGs from PD, FC-10 and 62264 isolates identified the structural regions of the enzyme possibly related to its resistance to PGIP inhibition. These include one region at the N-terminal portion of the enzyme and a few single amino acid substitutions along the entire sequence, two of which surrounding the active site.

Amino Acid Sequence↗