Occurrence of fumonisins in asparagus (asparagus officinalis L.) and garlic (allium sativum L.) from Germany

Fusarium proliferatum is able to produce fumonisins and is considered a pathogen of many economically important plants (e.g. corn, rice, asparagus) [1]. The occurrence of fumonisin FB₁ inF. proliferatum infected asparagus spears from Germany was investigated using a liquid chromatography/electrospray ionization-mass spectrometry (LC-ESI-MS) method with isotopically labeled fumonisin FB₁-d₆ as internal standard. Asparagus samples were harvested in July 2000 and screened forFusarium species. AltogetherF. oxysporum, F. proliferatum and F. sambucinum were isolated from the spears. The samples infected with F.proliferatum were subsequently analyzed for fumonisins. FB₁ was detected in 9 of the 10 samples in amounts ranging from 36.4 ng/g to 4513.7 ng/g (based on dry weight). Fumonisins FB₂ and FB₃ were found in six samples in lower concentrations. In asparagus spears of June 2002 we could findF. proliferatum in 6% of the samples, however no fumonisins were detectable. Furthermore the capability of producing FB₁ by the fungus in garlic bulbs was investigated. Therefore garlic was cultured inF. proliferatum contaminated soil and the bulbs were screened for infection with F.proliferatum and for the occurrence of fumonisins by LC-MS. F.proliferatum was detectable in the garlic tissue and all samples contained FB₁ (26.0 ng/g to 94.6 ng/g). This is the first report of the natural occurrence of FB₁ in German asparagus spears and furthermore our findings suggest a potential for natural contamination of garlic bulbs with fumonisins. For detailed results and methods see Ref. [2].     

Fusarium asiaticum: an Emerging Pathogen Jeopardizing Postharvest Asparagus Spears

Asparagus spears are usually vulnerable to pathogenic micro‐organisms. In this study, 217 pathogens were isolated from symptomatic asparagus, and one highly virulent fungus (designated EXAP‐08) isolated from the rotted asparagus spears in cold storage was characterized in detail. Koch's postulates were checked through pathogenicity tests, indicating that EXAP‐08 infection could cause reproducible rot symptoms similar to those observed on naturally infected asparagus spears, and the pathogenicity of EXAP‐08 was also relatively higher than other Fusarium pathogens, especially at 4°C. Through morphological and molecular identification, EXAP‐08 was characterized as Fusarium asiaticum. This identification was further confirmed by phylogenetic analysis with the Histone gene H3 of EXAP‐08 and other Fusarium species. EXAP‐08 also belongs to 3A‐DON (3‐acetyl‐4‐deoxynivalenol) chemo‐type, and the mycotoxin was detected during the infection of plant, implying the potential risks of mycotoxin contamination in fresh crops infected by this pathogen. Thus, this emerging pathogen threatening edible safety of asparagus spears should deserve particular quarantine inspection in the future.     

Identification and Characterization of an mRNA Encoding a Proline-Rich Protein that Rapidly Declines in Abundance in the Tips of Harvested Asparagus Spears

We previously isolated a cDNA clone, pTIP13, whose homologousmRNA rapidly declined in abundance in the tips of harvestedasparagus (Asparagus officinalis L.) spears [King and Davies(1992) Plant Physiol. 100: 1661]. In order to identify factorsregulating the postharvest deterioration of asparagus, we havenow sequenced the pTIP13 cDNA, derived the encoded amino acidsequence and determined the cellular location of pTIP13 mRNAby in situ hybridization. pTIP13 encodes a derived protein thatis rich in proline (22.3%), but also has a high content of lysine(15.2%) and threonine (14.1%). The proline residues are locatedin motifs at the amino-terminal region of the protein. The carboxyl-terminalregion of the derived protein has a high leucine content andshares >64% amino acid identity with derived proteins identifiedfrom cDNA clones to cell wall protein precursor mRNAs obtainedfrom soybean hypocotyls, alfalfa roots, and tomato fruit. GenomicSouthern analysis suggests that pTIP13 is encoded by a single-copygene in asparagus. pTIP13 mRNA was localized to specific celltypes in the young bracts of the asparagus spear tip. The resultsprovide new information on the complexity of tissue responsesin the tips of asparagus spears following harvest. (Received February 5, 1996; Accepted May 16, 1996)     

Imaging mass spectrometry: principle and application

Imaging mass spectrometry (IMS) is two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules, which does not need either separation or purification of target molecules, and enables us to monitor not only the identification of unknown molecules but also the localization of numerous molecules simultaneously. Among the ionization techniques, matrix assisted laser desorption/ionization (MALDI) is one of the most generally used for IMS, which allows the analysis of numerous biomolecules ranging over wide molecular weights. Proper selection and preparation of matrix is essential for successful imaging using IMS. Tandem mass spectrometry, which is referred to MSⁿ, enables the structural analysis of a molecule detected by the first step of IMS. Applications of IMS were initially developed for studying proteins or peptides. At present, however, targets of IMS research have expanded to the imaging of small endogenous metabolites such as lipids, exogenous drug pharmacokinetics, exploring new disease markers, and other new scientific fields. We hope that this new technology will open a new era for biophysics.     

Cell wall biochemistry and biomechanics of harvested white asparagus shoots as affected by temperature

The effects of temperature on the dynamics of changes in shoot mechanical properties, cell wall components, relevant soluble sugars and respiration activity of harvested white asparagus spears were investigated during a 7-day storage period. All functional cell wall components of asparagus spears increased closely temperature dependent. The content of soluble glucose declined with a similar temporal dynamics and to a comparable degree, indicating a major carbon flow of this storage sugar into cell walls (60–70%). Irrespective of temperature, the contents of stored soluble fructose and sucrose remained more or less constant. Lower temperatures reduced cell wall development but do not significantly affect the relative carbon flow from storage sugars into cell walls or maintenance respiration. Compared with cell walls, maintenance respiration is by far the smaller carbon sink in stored asparagus spears. Temperature differentially affects the absolute amount and the relative contribution of the different cell wall components and the temporal dynamics of changes in structural carbohydrate and lignin content. At higher temperatures, secondary cell wall thickening resulted mainly from a large increase in cellulose content. The pronounced increase in the fractions of cellulose and especially lignin may stress the important role of lignin in cell wall strengthening. While the fraction of cell wall proteins decreased, those of hemicellulose and the pectic components were not influenced.     

Spargelstangenuntersuchungen zur Haupterntezeit auf Infektionen mitFusarium spp. und Kontaminationen mit Fumonisin B1

Asparagus spears collected from a total of six commercial plantings in Austria during the main harvest periods in May and June of 2003 and 2004 were examined for endophytic colonization byFusarium spp., particularlyF. proliferatum. Potentially toxigenic fungi such asF. proliferatum were isolated and identified by morphological characteristics using light microscopy. Fumonisin B₁ inF. proliferatum-infected asparagus spears was detected with IAS-HPLC-FLD or HPLC-MS/MS. The identity of endophytic fungi colonizing of a total of 816 individual spears was determined. The incidence of infection byF. proliferatum and otherFusarium spp. was highly dependent on location and sampling date. The dominantFusarium species among the endophytic microflora wasF. oxysporum. Other frequently isolated species includedF. proliferatum, F. sambucinum, F. culmorum, F. avenaceum andF. equiseti. The incidence ofF. proliferatum-infected asparagus spears was less than 10% at four of the six sampling locations. At the two remaining locations, 20–47% of the spears examined were infected withF. proliferatum. Further exploration of FB₁ generation in asparagus is required because the low levels of FB₁ (10–50 (μg/kg) detected in harvested spears in 2003 and 2004 cannot be explained by the results of this study.

     

An approach to enhancing coverage of the urinary metabonome using liquid chromatography-ion mobility-mass spectrometry

The potential of drift tube ion mobility (IM) spectrometry in combination with high performance liquid chromatography (LC) and mass spectrometry (MS) for the metabonomic analysis of rat urine is reported. The combined LC-IM-MS approach using quadrupole/time-of-flight mass spectrometry with electrospray ionisation, uses gas-phase analyte characterisation based on both mass-to-charge (m/z) ratio and relative gas-phase mobility (drift time) following LC separation. The technique allowed the acquisition of nested data sets, with mass spectra acquired at regular intervals (65 micros) during each IMS separation (approximately 13 ms) and several IMS spectra acquired during the elution of a single LC peak, without increasing the overall analysis time compared to LC-MS. Preliminary results indicate that spectral quality is improved when using LC-IM-MS, compared to direct injection IM-MS, for which significant ion suppression effects were observed in the electrospray ion source. The use of reversed-phase LC employing fast gradient elution reduced sample preparation to a minimum, whilst maintaining the potential for high throughput analysis. Data mining allowed information on specific analytes to be extracted from the complex metabonomic data set. LC-IM-MS based approaches may have a useful role in metabonomic analyses by introducing an additional discriminatory dimension of ion mobility (drift time).     

Proteomic analysis of formalin-fixed paraffin-embedded tissue by MALDI imaging mass spectrometry

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE-TMAs using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE-TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis.     

Long-term effects of asparagus virus 2 infection on growth and productivity in asparagus

An asparagus field trial was established with clonal plants to determine the long-term effects of asparagus virus 2 (AV2) infection on spear production. Yield data, analysed by ANOVA, showed that AV2 infection caused significant (P < 0.05) decreases in spear yield which became more pronounced as the trial progressed. Mean marketable spear yields were reduced by 14%, 28%, 20%, 48% and 57% and reject spear yields were increased by 93%, 105%, 207%, 352% and 167%, during harvest years 1–5 respectively. Marketable spear yields from AV2–free plants increased annually to yr 5, but for AV2–infected plants, yields increased to yr 3 and decreased annually thereafter. Spears from AV2–infected plants were thinner than those from AV2–free plants, resulting in more reject thin spears by 109%, 88%, 220%, 499% and 216% during harvest yr 1–5, respectively. Further, data collected in yr 4 and 5 showed that AV2 infection had caused a 31 % reduction in mean spear diameter and reductions of 27% and 22% respectively, in diameter and height of fern stalks. Clearly, plants with smaller fern stalks were less able to accumulate carbohydrate reserves and therefore produced fewer, smaller spears and fern stalks the following spring. This may result in annual cycles of diminishing productivity in which the size and number of spears and fern stalks decrease with each successive year. The type and timespan of symptoms caused by AV2 infection in this trial are similar to those reported for asparagus decline syndrome and therefore it is likely that AV2 infection is a factor contributing to asparagus decline.     

Identification of Protein Markers Specific for Papillary Renal Cell Carcinoma Using Imaging Mass Spectrometry

Since the emergence of proteomics methods, many proteins specific for renal cell carcinoma (RCC) have been identified. Despite their usefulness for the specific diagnosis of RCC, such proteins do not provide spatial information on the diseased tissue. Therefore, the identification of cancer-specific proteins that include information on their specific location is needed. Recently, matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) based imaging mass spectrometry (IMS) has emerged as a new tool for the analysis of spatial distribution as well as identification of either proteins or small molecules in tissues. In this report, surgical tissue sections of papillary RCC were analyzed using MALDI-IMS. Statistical analysis revealed several discriminative cancer-specific m/z-species between normal and diseased tissues. Among these m/z-species, two particular proteins, S100A11 and ferritin light chain, which are specific for papillary RCC cancer regions, were successfully identified using LC-MS/MS following protein extraction from independent RCC samples. The expressions of S100A11 and ferritin light chain were further validated by immunohistochemistry of human tissues and tissue microarrays (TMAs) of RCC. In conclusion, MALDI-IMS followed by LC-MS/MS analysis in human tissue identified that S100A11 and ferritin light chain are differentially expressed proteins in papillary RCC cancer regions.     

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.     

ImShot: An Open-Source Software for Probabilistic Identification of Proteins In Situ and Visualization of Proteomics Data

Imaging mass spectrometry (IMS) has developed into a powerful tool allowing label-free detection of numerous biomolecules in situ. In contrast to shotgun proteomics, proteins/peptides can be detected directly from biological tissues and correlated to its morphology leading to a gain of crucial clinical information. However, direct identification of the detected molecules is currently challenging for MALDI–IMS, thereby compelling researchers to use complementary techniques and resource intensive experimental setups. Despite these strategies, sufficient information could not be extracted because of lack of an optimum data combination strategy/software. Here, we introduce a new open-source software ImShot that aims at identifying peptides obtained in MALDI–IMS. This is achieved by combining information from IMS and shotgun proteomics (LC–MS) measurements of serial sections of the same tissue. The software takes advantage of a two-group comparison to determine the search space of IMS masses after deisotoping the corresponding spectra. Ambiguity in annotations of IMS peptides is eliminated by introduction of a novel scoring system that identifies the most likely parent protein of a detected peptide in the corresponding IMS dataset. Thanks to its modular structure, the software can also handle LC–MS data separately and display interactive enrichment plots and enriched Gene Ontology terms or cellular pathways. The software has been built as a desktop application with a conveniently designed graphic user interface to provide users with a seamless experience in data analysis. ImShot can run on all the three major desktop operating systems and is freely available under Massachusetts Institute of Technology license.     

Impact of a Transposon Insertion in phzF2 on the Specialized Metabolite Production and Interkingdom Interactions of Pseudomonas aeruginosa

In microbiology, gene disruption and subsequent experiments often center on phenotypic changes caused by one class of specialized metabolites (quorum sensors, virulence factors, or natural products), disregarding global downstream metabolic effects. With the recent development of mass spectrometry-based methods and technologies for microbial metabolomics investigations, it is now possible to visualize global production of diverse classes of microbial specialized metabolites simultaneously. Using imaging mass spectrometry (IMS) applied to the analysis of microbiology experiments, we can observe the effects of mutations, knockouts, insertions, and complementation on the interactive metabolome. In this study, a combination of IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the impact on specialized metabolite production of a transposon insertion into a Pseudomonas aeruginosa phenazine biosynthetic gene, phzF2. The disruption of phenazine biosynthesis led to broad changes in specialized metabolite production, including loss of pyoverdine production. This shift in specialized metabolite production significantly alters the metabolic outcome of an interaction with Aspergillus fumigatus by influencing triacetylfusarinine production.     

A multimodal metabolomics approach using imaging mass spectrometry and liquid chromatography-tandem mass spectrometry for spatially characterizing monoterpene indole alkaloids secreted from roots

Plants release specialized (secondary) metabolites from their roots to communicate with other organisms, including soil microorganisms. The spatial behavior of such metabolites around these roots can help us understand roles for the communication; however, currently, they are unclear because soil-based studies are complex. Here, we established a multimodal metabolomics approach using imaging mass spectrometry (IMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially assign metabolites under laboratory conditions using agar. In a case study using Catharanthus roseus, we showed that 58 nitrogen (N)-containing metabolites are released from the roots into the agar. For the metabolite assignment, we used ¹⁵N-labeled and non-labeled LC-MS/MS data, previously reported. Four metabolite ions were identified using authentic standard compounds as derived from monoterpene indole alkaloids (MIAs) such as ajmalicine, catharanthine, serpentine, and yohimbine. An alkaloid network analysis using dot products and spinglass methods characterized five clusters to which the 58 ions belong. The analysis clustered ions from the indolic skeleton-type MIAs to a cluster, suggesting that other communities may represent distinct metabolite groups. For future chemical assignments of the serpentine community, key fragmentation patterns were characterized using the ¹⁵N-labeled and non-labeled MS/MS spectra.     

Strongly acidic auxin indole-3-methanesulfonic Acid: synthesis of [C]indole-3-methanesulfonic Acid and studies of its chromatographic, spectral, and biological properties

A radiochemical synthesis is described for [¹⁴C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [¹⁴C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles.     

Genetic Variability of Phytopathogenic Fusarium proliferatum Associated with Crown Rot in Asparagus officinalis

Fusarium proliferatum (teleomorph: Gibberella intermedia ) is a causal agent of crown rot of Asparagus officinalis and is one potential fumonisin-producing species within the genus Fusarium . It colonizes roots and crowns of asparagus plants, but could also be isolated from symptomless asparagus spears. Fusarium proliferatum isolates obtained from perennial asparagus plantings from Austria and Germany were included in a study on detectability and variability of two essential genes of the fumonisin-gene cluster. Genetic fingerprinting of 45 isolates revealed 14 different fingerprint groups, indicating genetic heterogenicity of F. proliferatum . Most isolates differentiated into three main fingerprint clusters, but no association was found between fingerprint group and origin of the isolates. By gene-specific PCR it was shown that, in 25 isolates tested, both initial genes of the fumonisin biosynthetic pathway – FUM1 , encoding a polyketide synthase and FUM8 , a gene for a putative aminoacyl transferase – were detectable. This suggests that these isolates were able to produce fumonisins and could contribute to the detected contamination in originating asparagus spears with this mycotoxin. Thus, early detection of FUM -genes in F. proliferatum -colonized asparagus may be suited to prevent uptake of fumonisin contaminated food with the human diet. Restriction fragment length polymorphism analysis (PCR-RFLP) of the amplified FUM gene fragments revealed little sequence variability, suggesting a conserved structure of these genes within this species. However, sequence analysis confirmed intraspecific nucleotide polymorphisms of these genes.     

Increased Expression of Phosphatidylcholine (16:0/18:1) and (16:0/18:2) in Thyroid Papillary Cancer

A good prognosis can be expected for most, but not all, cases of thyroid papillary cancer. Numerous molecular studies have demonstrated beneficial treatment and prognostic factors in various molecular markers. Whereas most previous reports have focused on genomics and proteomics, few have focused on lipidomics. With the advent of mass spectrometry (MS), it has become possible to identify many types of molecules, and this analytical tool has become critical in the field of omics. Recently, imaging mass spectrometry (IMS) was developed. After a simple pretreatment process, IMS can be used to examine tissue sections on glass slides with location information.Here, we conducted an IMS analysis of seven cases of thyroid papillary cancer by comparison of cancerous with normal tissues, focusing on the distribution of phospholipids. We identified that phosphatidylcholine (16:0/18:1) and (16:0/18:2) and sphingomyelin (d18:0/16:1) are significantly higher in thyroid papillary cancer than in normal thyroid tissue as determined by tandem mass (MS/MS) analysis. These distributional differences may be associated with the biological behavior of thyroid papillary cancer.     

Intracellular Proteases of Bacillus subtilis

The purpose of this study was to clarify the relationship between various cultivation conditions and the amounts of the rutin (RT) and protodioscin (PD) in asparagus spears. Green and white spears were grown in open culture and under two different blanching conditions. Although RT was detected only in the green spears, PD was detected mainly in white spears produced by covering with soil. The RT and PD contents of cladophylls grown in an open field and in a closed cultivation system were also investigated, and the closed system resulted in cladophylls with low RT and high PD, unlike the open field.     

Fusarium Species Colonizing Spears and Forming Mycotoxins in Field Samples of Asparagus from Germany and Poland

The occurrence of Fusarium spp. and associated mycotoxins in asparagus spears was evaluated in Poland in 2002 and 2003 and in Germany in 2002. Spears of two cultivars, Eposs and Gijnlim, were collected from two locations in Poland, Swidwowiec and Poznan, on sandy and sandy loam soil, respectively. Fusarium oxysporum and F. proliferatum were detected at an average incidence of 38.3% and 15.8% in the spear sections sampled, respectively. In stands of 11 (tested) cultivars of asparagus sampled in Germany on sandy soil, the same species dominated, however, they were less frequent than in Poland (26.6% and 5.6% of the spears infected with F. oxysporum and F. proliferatum, respectively). Chemical analyses revealed that fumonisin B₁ (FB₁) and moniliformin (MON) were present in some of the spears sampled in Poland. FB₁ was not found and MON was not assessed in spears sampled in Germany in 2002, but F. proliferatum was able to form the toxin in vitro in the range from 101.4 up to 205.8 μg/kg maize kernel substrate. Asparagus samples in Poland contained FB₁ at up to 5.6 μg/kg spear fresh weight. The highest MON concentration (1350 μg/kg) was detected in cultivar Eposs in Marcelin, Poland, in 2002. MON and FB₁ were found in spears infected by both F. oxysporum and F. proliferatum, however, only the latter fungus was able to synthesize both toxins.