Yarrowia lipolytica increased the activities of disease defense related enzymes and anti-fungal compounds in asparagus (Asparagus officinalis).

Fungal diseases pose significant threats to the production of asparagus, resulting in economic losses and decreased crop quality. The potential of the yeast Yarrowia lipolytica as a biocontrol agent against Fusarium proliferatum, a common pathogen of asparagus, was investigated in this study. The effects of Y. lipolytica treatment on decay incidence, disease index, and activities of major disease defense-related enzymes were investigated. In addition, we examined the levels of antifungal compounds such as total phenols, flavonoids, and lignin in asparagus plants exposed to Y. lipolytica. The results showed that Y. lipolytica treatment significantly reduced decay incidence and disease index caused by F. proliferatum when compared to the control group. Furthermore, Y. lipolytica-treated plants showed increased activity of disease defense-related enzymes, indicating that defense responses were activated. The activities of all evaluated enzymes were significantly higher in Y. lipolytica-treated asparagus, indicating an improved ability to combat fungal pathogens. Furthermore, Y. lipolytica treatment increased the content of antifungal compounds such as total phenols, flavonoids, and lignin, which are known to possess antimicrobial properties. These findings highlight the potential of Y. lipolytica as a biocontrol agent for fungal diseases in asparagus crops. The ability of Y. lipolytica to reduce disease incidence, boost disease defense-related enzymes, and increase antifungal compound content provides valuable insights into its efficacy as a natural and sustainable approach to disease management. However, further investigations are needed to optimize application methods and determine its efficacy under field conditions.

Comparative Metabolome and Transcriptome Analyses of Susceptible Asparagus officinalis and Resistant Wild A. kiusianus Reveal Insights into Stem Blight Disease Resistance.

Phomopsis asparagi is one of the most serious fungal pathogens, which causes stem blight disease in Asparagus officinalis (AO), adversely affecting its production worldwide. Recently, the development of novel asparagus varieties using wild Asparagus genetic resources with natural P. asparagi resistance has become a priority in Japan due to the lack of resistant commercial AO cultivars. In this study, comparative metabolome and transcriptome analyses of susceptible AO and resistant wild Asparagus kiusianus (AK) 24 and 48 h postinoculated (AOI_24 hpi, AOI_48 hpi, AKI_24 hpi and AKI_48 hpi, respectively) with P. asparagi were conducted to gain insights into metabolic and expression changes associated with AK species. Following infection, the resistant wild AK showed rapid metabolic changes with increased levels of flavonoids and steroidal saponins and decreased asparagusic acid glucose ester content, compared with the susceptible AO plants. Transcriptome data revealed a total of 21 differentially expressed genes (DEGs) as the core gene set that displayed upregulation in the resistant AK versus susceptible AO after infection with P. asparagi. Kyoto Encyclopedia of Genes and Genomes pathway analysis of these DEGs identified 11 significantly enriched pathways, including flavonoid biosynthesis and primary metabolite metabolism, in addition to plant signaling and defense-related pathways. In addition, comparative single-nucleotide polymorphism and Indel distributions in susceptible AO and resistant AK plants were evaluated using the latest AO reference genome Aspof.V1. The data generated in this study are important resources for advancing Asparagus breeding programs and for investigations of genetic linkage mapping, phylogenetic diversity and plant defense-related genes.

Effect of a fungus, Hypoxylon spp., on endophytes in the roots of Asparagus.

The fungal isolate Hypoxylon spp. (Sj18) was isolated from the root of pecan. It might have effects on the plant's stress tolerance and endophytic community. Inoculation experiments were carried out on the roots of Asparagus with normal and inactivated Sj18, and the diversity and community structure of endophytes in the root of inoculated Asparagus were studied. It was found that Sj18 fungi affected the endophytic community of Asparagus roots. From being a low-abundance genus, the salt-tolerant bacterium Halomonas became the dominant genus. In order to verify that Sj18 can improve salt tolerance, Arabidopsis thaliana was inoculated with Sj18 in a salt tolerance test. The result showed that A. thaliana grew better in a high salt environment after inoculation with Sj18. Sj18 changed the microbe diversity, community composition and structure of endophytes in the roots of Asparagus, which increased the bacterial diversity. A total of 16 phyla and 184 genera of bacteria were detected. However, the diversity of fungi decreased.

Agrobacterium tumefaciens-mediated transformation of a hevein-like gene into asparagus leads to stem wilt resistance.

Asparagus stem wilt, is a significant and devastating disease, typically leading to extensive economic losses in the asparagus industry. To obtain transgenic plants resistant to stem wilt, the hevein-like gene, providing broad spectrum bacterial resistance was inserted into the asparagus genome through Agrobacterium tumefaciens-mediated transformation. The optimal genetic transformation system for asparagus was as follows: pre-culture of embryos for 2 days, inoculation using a bacterial titre of OD600 = 0.6, infection time 10 min and co-culturing for 4 days using an Acetosyringone concentration of 200 µmol/L. Highest transformation frequencies reached 21% and ten transgenic asparagus seedlings carrying the hevein-like gene were identified by polymerase chain reaction. Moreover, integration of the hevein-like gene in the T1 generation of transgenic plants was confirmed by southern blot hybridization. Analysis showed that resistance to stem wilt was enhanced significantly in the transgenic plants, in comparison to non- transgenic plants. The results provide additional data for genetic improvement and are of importance for the development of new disease-resistant asparagus varieties.

Effect of ultrasound treatment on microbial inhibition and quality maintenance of green asparagus during cold storage.

The study focused on inhibiting microorganism and improving preservation of green asparagus (Asparagus officinalis L.) during cold storage. Green asparagus is treated with ultrasound (US), acetic acid and gibberellin acid (AG) separately as well as combination (US + AG) and then stored at 4 °C for 20 days. Microorganism, physicochemical qualities and sensory characteristics were monitored at regular intervals. Results showed that the US treatment significantly (p < 0.05) reduced the total number of colonies, mold and yeast merely in the 12th and 16th day of storage, while the US + AG treatment not only achieved an effective decontamination (up to 2 log reduction) of green asparagus throughout the storage, but also retained the physicochemical characteristics to a higher level in comparison to other treatments. The US + AG treatment exhibited lower weight loss, higher levels of total soluble solid (TSS), ascorbic acid, chlorophyll content and total phenolic content (TPC), as well as kept better sensory attributes. Moreover, the US + AG treatment significantly inhibited the activities of phenylalanine ammonia lyase (PAL) and peroxidase (POD) (p < 0.05), suppressing the biosynthesis of lignin. These results suggested that the US + AG treatment could be a potential strategy to preserve quality of green asparagus during cold storage.

Comparative transcriptome analysis of the garden asparagus (Asparagus officinalis L.) reveals the molecular mechanism for growth with arbuscular mycorrhizal fungi under salinity stress.

Soil salinity is one of the most abiotic stress factors that severely affects the growth and development of many plants, which can ultimately threaten crop yield. Arbuscular mycorrhiza fungi (AMF) has been proven to be effective in mitigating salinity stress by symbiosis in many crops. Asparagus officinalis are perennial plants grown in saline-alkaline soil, however, limited information on their molecular mechanisms has restricted efficient application of AMF to garden asparagus under salinity stress. In this study, we conducted a transcriptome analysis on the leaves of garden asparagus to identify gene expression under salinity stress. Seedlings were grown in 4 treatments, including non-inoculated AMF using distilled water (NI), inoculated AMF using distilled water (AMF), non-inoculated with salinity stress (NI + S), and inoculated with salinity stress (AMF + S). A total of 6019 novel genes were obtained based on the reference-guided assembly of the garden asparagus transcriptome. Results revealed that 455 differentially expressed genes (DEGs) were identified when comparing NI + S to AMF + S. However, among the up-regulated DEGs, 41 DEGs were down-regulated, while 242 DEGs had no differences in their expression levels when comparing NI to NI + S. These DEGs' expression patterns may be key induced by AMF under salinity stress. Additionally, the GO and KEGG enrichment analyses of 455 DEGs revealed that these genes mainly participate in the improvement of the internal environment in plant cells, nitrogen metabolic-related processes, and possible photoprotection mechanisms. These findings provide insight into enhanced salinity stress adaptation by AMF inoculation, as well as salt-tolerant candidate genes for further functional analyses.

Seasonal deviation effects foliar endophyte assemblage and diversity in Asparagus racemosus and Hemidesmus indicus.

BACKGROUND: Fungal endophytes are the living symbionts which cause no apparent damage to the host tissue. The distribution pattern of these endophytes within a host plant is mediated by environmental factors. This study was carried out to explore the fungal endophyte community and their distribution pattern in Asparagus racemosus and Hemidesmus indicus growing in the study area. RESULTS: Foliar endophytes were isolated for 2 years from A. racemosus and H. indicus at four different seasons (June-August, September-November, December-February, March-May). A total of 5400 (675/season/year) leaf segments harbored 38 fungal species belonging to 17 genera, 12 miscellaneous mycelia sterile from 968 isolates and 13 had yeast like growth. In A. racemosus, Acremonium strictum and Phomopsis sp.1, were dominant with overall relative colonization densities (RCD) of 7.11% and 5.44% respectively, followed by Colletotrichum sp.3 and Colletotrichum sp.1 of 4.89% and 4.83% respectively. In H. indicus the dominant species was A. strictum having higher overall RCD of 5.06%, followed by Fusarium moniliforme and Colletotrichum sp.2 with RCD of 3.83% and 3%, respectively. Further the overall colonization and isolation rates were higher during the wet periods (September-November) in both A. racemosus (92.22% and 95.11%) and H. indicus (82% and 77.11%). CONCLUSION: Study samples treated with 0.2% HgCl2 and 75% EtOH for 30 s and 1 min, respectively, confirmed most favorable method of isolation of the endophytes. Owing to high mean isolation and colonization rates, September-November season proved to be the optimal season for endophyte isolation in both the study plants. Assessing the bioactive potential of these endophytes, may lead to the isolation of novel natural products and metabolites.

Evaluation of the TOM-CAST Forecasting Model in Asparagus for Management of Stemphylium Leaf Spot in Ontario, Canada.

In the last 5 years, asparagus acreage in Canada has increased by over 25%. Stemphylium leaf spot, caused by Stemphylium vesicarium, has emerged as the predominant foliar pathogen of asparagus. Typically, contact fungicides are applied every 14 days; however, regardless of the number of applications, growers are not achieving adequate control of the disease. The TOM-CAST forecasting model is used widely in Michigan asparagus fields, but it has never been assessed for suitability in Ontario or in the popular cultivar, Guelph Millennium. Six field trials were conducted in 2012 and 2013 to evaluate the TOM-CAST forecasting model in two asparagus cultivars. The fungicides chlorothalonil or azoxystrobin/difenoconazole were applied according to the forecasting model or on a 14-day interval. The effectiveness of the forecasting model differed between sites and cultivars. Even though TOM-CAST is used in all cultivars in Michigan, TOM-CAST was not effective on Guelph Millennium. In the cultivar Jersey Giant, however, TOM-CAST with a 20 disease severity value spray interval improved control of Stemphylium leaf spot without increasing the number of sprays, compared with a 14-day treatment. The results in Guelph Millennium differed between sites. At one site, TOM-CAST maintained similar levels of Stemphylium leaf spot, but increased the number of applications, compared with a 14-day application interval. Of more concern, none of the fungicide treatments differed greatly from the untreated control at the other site. Our results show that forecasting models need to be validated locally in asparagus cultivars relevant to production today.

Measurement of Fructose-Asparagine Concentrations in Human and Animal Foods.

The food-borne bacterial pathogen, Salmonella enterica, can utilize fructose-asparagine (F-Asn) as its sole carbon and nitrogen source. F-Asn is the product of an Amadori rearrangement following the nonenzymatic condensation of glucose and asparagine. Heating converts F-Asn via complex Maillard reactions to a variety of molecules that contribute to the color, taste, and aroma of heated foods. Among these end derivatives is acrylamide, which is present in some foods, especially in fried potatoes. The F-Asn utilization pathway in Salmonella, specifically FraB, is a potential drug target because inhibition of this enzyme would lead to intoxication of Salmonella in the presence of F-Asn. However, F-Asn would need to be packaged with the FraB inhibitor or available in human foods. To determine if there are foods that have sufficient F-Asn, we measured F-Asn concentrations in a variety of human and animal foods. The 400 pmol/mg F-Asn found in mouse chow is sufficient to intoxicate a Salmonella fraB mutant in mouse models of salmonellosis, and several human foods were found to have F-Asn at this level or higher (fresh apricots, lettuce, asparagus, and canned peaches). Much higher concentrations (11 000-35 000 pmol/mg dry weight) were found in heat-dried apricots, apples, and asparagus. This report reveals possible origins of F-Asn as a nutrient source for Salmonella and identifies foods that could be used together with a FraB inhibitor as a therapeutic agent for Salmonella.

Generation and Screening of T-DNA Insertion Mutants Mediated by Agrobacterium tumefaciens in the Garden Asparagus Stem Blight Pathogen Phomopsis asparagi.

The garden asparagus stem blight caused by filamentous fungus Phomopsis asparagi exposes a serious threat on asparagus production globally. However, to present, we understand poorly about the molecular mechanisms of fungal pathogenicity. To facilitate functional genomics research of P. asparagi, here we developed a highly efficient and stable Agrobacterium tumefaciens-mediated transformation approach which yielded 150-200 transformants per 1 × 106 conidia. Our results indicated that 25 °C, acetosyringone concentration of 150 µmol/L, and 72 h were recommended as optimal co-cultivation conditions for the transformation. Using this transformation approach, we constructed a T-DNA insertion mutant library containing 1253 strains. Twenty randomly selected T-DNA insertion mutants were able to grow on 0.2 × PDA selective media after five successive subcultures without selective pressure, indicating that the exogenous T-DNA was stably integrated into the P. asparagi genome. We confirmed several randomly selected mutants using PCR with primers specific to the hph gene. Southern blots suggested that three out of the five selected mutants have a single T-DNA insertion. Interestingly, multiple mutant candidates with growth defects were obtained from the growth assay. Moreover, several mutants were selected for further analysis on the T-DNA flanking sequences through TAIL-PCR analysis. A sequence comparison of total junction fragments implied that the insertion of T-DNA within P. asparagi genome appeared to be a random event. The transformation technology and genetic resources developed here will facilitate studies of pathogenic mechanisms in this devastating filamentous fungal pathogen of garden asparagus.

Optimal Fermentation Conditions of Hyaluronidase Inhibition Activity on Asparagus cochinchinensis Merrill by Weissella cibaria.

This study was conducted to evaluate the hyaluronidase (HAase) inhibition activity of Asparagus cochinchinesis (AC) extracts following fermentation by Weissella cibaria through response surface methodology. To optimize the HAase inhibition activity, a central composite design was introduced based on four variables: the concentration of AC extract (X1: 1-5%), amount of starter culture (X2: 1-5%), pH (X3: 4-8), and fermentation time (X4: 0-10 days). The experimental data were fitted to quadratic regression equations, the accuracy of the equations was analyzed by ANOVA, and the regression coefficients for the surface quadratic model of HAase inhibition activity in the fermented AC extract were estimated by the F test and the corresponding p values. The HAase inhibition activity indicated that fermentation time was most significant among the parameters within the conditions tested. To validate the model, two different conditions among those generated by the Design Expert program were selected. Under both conditions, predicted and experimental data agreed well. Moreover, the content of protodioscin (a well-known compound related to anti-inflammation activity) was elevated after fermentation of the AC extract at the optimized fermentation condition.

Wildly Growing Asparagus (Asparagus officinalis L.) Hosts Pathogenic Fusarium Species and Accumulates Their Mycotoxins.

Asparagus officinalis L. is an important crop in many European countries, likely infected by a number of Fusarium species. Most of them produce mycotoxins in plant tissues, thus affecting the physiology of the host plant. However, there is lack of information on Fusarium communities in wild asparagus, where they would definitely have considerable environmental significance. Therefore, the main scientific aim of this study was to identify the Fusarium species and quantify their typical mycotoxins present in wild asparagus plants collected at four time points of the season. Forty-four Fusarium strains of eight species--Fusarium acuminatum, Fusarium avenaceum, Fusarium culmorum, Fusarium equiseti, Fusarium oxysporum, Fusarium proliferatum, Fusarium sporotrichioides, and Fusarium tricinctum--were isolated from nine wild asparagus plants in 2013 season. It is the first report of F. sporotrichioides isolated from this particular host. Fumonisin B1 was the most abundant mycotoxin, and the highest concentrations of fumonisins B1-B3 and beauvericin were found in the spears collected in May. Moniliformin and enniatins were quantified at lower concentrations. Mycotoxins synthesized by individual strains obtained from infected asparagus tissues were assessed using in vitro cultures on sterile rice grain. Most of the F. sporotrichioides strains synthesized HT-2 toxin and F. equiseti strains were found to be effective zearalenone producers.

Changes in microbial diversity of brined green asparagus upon treatment with high hydrostatic pressure.

The application of high hydrostatic pressure (HHP, 600MPa, 8 min) on brined green asparagus and the changes in bacterial diversity after treatments and during storage at 4 °C (30 days) or 22 °C (10 days) were studied. HHP treatments reduced viable cell counts by 3.6 log cycles. The residual surviving population did not increase during storage at 4 °C. However, bacterial counts significantly increased at 22 °C by day 3, leading to rapid spoilage. The microbiota of green asparagus was composed mainly by Proteobacteria (mainly Pantoea and Pseudomonas), followed by Firmicutes (mainly Lactococcus and Enterococcus) and to a less extent Bacteroidetes and Actinobacteria. During chill storage of untreated asparagus, the relative abundance of Proteobacteria as well as Enterococcus and Lactococcus decreased while Lactobacillus increased. During storage of untreated asparagus at 22 °C, the abundance of Bacteroidetes decreased while Proteobacteria increased during late storage. The HHP treatment determined a reduction of the Proteobacteria both early after treatment and during chill storage. In the HHP treated samples stored at 22 °C, the relative abundance of Pseudomonas rapidly decreased at day 1, with an increase of Bacteroidetes. This was followed by a marked increase in Enterobacteriaceae (Escherichia) simultaneously with increase in viable counts and spoilage. Results from the study indicate that the effect of HHP treatments on the viability ofmicrobial populations in foods also has an impact on the dynamics of microbial populations during the storage of the treated foods.

Innovative Approach to the Accumulation of Rubrosterone by Fermentation of Asparagus filicinus with Fusarium oxysporum.

Rubrosterone, possessing various remarkable bioactivities, is an insect-molting C19-steroid. However, only very small amounts are available for biological tests due to its limited content from plant sources. Fungi of genus Fusarium have been reported to have the ability to convert C27-steroids into C19-steroids. In this study, Asparagus filicinus, containing a high content of 20-hydroxyecdysone, was utilized to accumulate rubrosterone through solid fermentation by Fusarium oxysporum. The results showed that F. oxysporum had the ability to facilitate the complete biotransformation of 20-hydroxyecdysone to rubrosterone by solid-state fermentation. The present method could be an innovative and efficient approach to accumulate rubrosterone with an outstanding conversion ratio.

Quantitative Analysis of the Migration and Accumulation of Bacillus subtilis in Asparagus officinalis.

Bacillus subtilis B96-II is a broad-spectrum biological control strain. It effectively suppresses soil-borne fungal diseases in vegetables. A green fluorescence protein (GFP) was expressed in B96-II to detect migration of B96-II into the root and stem of asparagus. The GFP-tagged B96-II (B96-II-GFP) strain exhibited bright green fluorescence under a fluorescence microscope. GFP was stable and had no apparent effects on the growth of the strain. Asparagus plants were planted in the soil inoculated with B96-II-GFP. Our results showed that B96-II-GFP was detected in both the root and stem 15, 30, and 45 days after the asparagus seedlings were planted. B96-II-GFP was also detected in leaves but at a lower concentration. The highest concentration was detected in 15 days, and the number of bacteria decreased subsequently irrespective of duration of growth or sampling period. The highest concentration of B96-II-GFP was present in the root base suggesting that the root base served as the hub of bacterial migration from the soil to the stem.

Effect of chitosan and its derivatives as antifungal and preservative agents on postharvest green asparagus.

The antifungal activity and effect of high-molecular weight chitosan (H-chitosan), low-molecular weight chitosan (L-chitosan) and carboxymethyl chitosan (C-chitosan) coatings on postharvest green asparagus were evaluated. L-chitosan and H-chitosan efficiently inhibited the radial growth of Fusarium concentricum separated from postharvest green asparagus at 4 mg/ml, which appeared to be more effective in inhibiting spore germination and germ tube elongation than that of C-chitosan. Notably, spore germination was totally inhibited by L-chitosan and H-chitosan at 0.05 mg/ml. Coated asparagus did not show any apparent sign of phytotoxicity and maintained good quality over 28 days of cold storage, according to the weight loss and general quality aspects. Present results inferred that chitosan could act as an attractive preservative agent for postharvest green asparagus owing to its antifungal activity and its ability to stimulate some defense responses during storage.

Characterization of Fusarium isolates from asparagus fields in southwestern Ontario and influence of soil organic amendments on Fusarium crown and root rot.

Fusarium crown and root rot (FCRR) of asparagus has a complex etiology with several soilborne Fusarium spp. as causal agents. Ninety-three Fusarium isolates, obtained from plant and soil samples collected from commercial asparagus fields in southwestern Ontario with a history of FCRR, were identified as Fusarium oxysporum (65.5%), F. proliferatum (18.3%), F. solani (6.4%), F. acuminatum (6.4%), and F. redolens (3.2%) based on morphological or cultural characteristics and polymerase chain reaction (PCR) analysis with species-specific primers. The intersimple-sequence repeat PCR analysis of the field isolates revealed considerable variability among the isolates belonging to different Fusarium spp. In the in vitro pathogenicity screening tests, 50% of the field isolates were pathogenic to asparagus, and 22% of the isolates caused the most severe symptoms on asparagus. The management of FCRR with soil organic amendments of pelleted poultry manure (PPM), olive residue compost, and fish emulsion was evaluated in a greenhouse using three asparagus cultivars of different susceptibility in soils infested with two of the pathogenic isolates (F. oxysporum Fo-1.5 and F. solani Fs-1.12). Lower FCRR symptom severity and higher plant weights were observed for most treatments on 'Jersey Giant' and 'Grande' but not on 'Mary Washington'. On all three cultivars, 1% PPM consistently reduced FCRR severity by 42 to 96% and increased plant weights by 77 to 152% compared with the Fusarium control treatment. Populations of Fusarium and total bacteria were enumerated after 1, 3, 7, and 14 days of soil amendment. In amended soils, the population of Fusarium spp. gradually decreased while the population of total culturable bacteria increased. These results indicate that soil organic amendments, especially PPM, can decrease disease severity and promote plant growth, possibly by decreasing pathogen population and enhancing bacterial activity in the soil.

Genetic variation of Fusarium oxysporum isolates forming fumonisin B(1) and moniliformin.

Thirty single-spore isolates of a toxigenic fungus, Fusarium oxysporum, were isolated from asparagus spears and identified by species-specific polymerase chain reaction (PCR) and translation elongation factor 1-α (TEF) sequence analysis. In the examined sets of F. oxysporum isolates, the DNA sequences of mating type genes (MAT) were identified. The distribution of MAT idiomorph may suggest that MAT1-2 is a predominant mating type in the F. oxysporum population. F. oxysporum is mainly recognised as a producer of moniliformin-the highly toxic secondary metabolite. Moniliformin content was determined by high-performance liquid chromatography (HPLC) analysis in the range 0.05-1,007.47 µg g(-1) (mean 115.93 µg g(-1)) but, also, fumonisin B(1) was detected, in the concentration range 0.01-0.91 µg g(-1) (mean 0.19 µg g(-1)). There was no association between mating types and the mycotoxins biosynthesis level. Additionally, a significant intra-species genetic diversity was revealed and molecular markers associated with toxins biosynthesis were identified.

Application of PCR-denaturing-gradient gel electrophoresis (DGGE) method to examine microbial community structure in asparagus fields with growth inhibition due to continuous cropping.

Growth inhibition due to continuous cropping of asparagus is a major problem; the yield of asparagus in replanted fields is low compared to that in new fields, and missing plants occur among young seedlings. Although soil-borne disease and allelochemicals are considered to be involved in this effect, this is still controversial. We aimed to develop a technique for the biological field diagnosis of growth inhibition due to continuous cropping. Therefore, in this study, fungal community structure and Fusarium community structure in continuously cropped fields of asparagus were analyzed by polymerase chain reaction/denaturing-gradient gel electrophoresis (PCR-DGGE). Soil samples were collected from the Aizu region of Fukushima Prefecture, Japan. Soil samples were taken from both continuously cropped fields of asparagus with growth inhibition and healthy neighboring fields of asparagus. The soil samples were collected from the fields of 5 sets in 2008 and 4 sets in 2009. We were able to distinguish between pathogenic and non-pathogenic Fusarium by using Alfie1 and Alfie2GC as the second PCR primers and PCR-DGGE. Fungal community structure was not greatly involved in the growth inhibition of asparagus due to continuous cropping. By contrast, the band ratios of Fusarium oxysporum f. sp. asparagi in growth-inhibited fields were higher than those in neighboring healthy fields. In addition, there was a positive correlation between the band ratios of Fusarium oxysporum f. sp. asparagi and the ratios of missing asparagus plants. We showed the potential of biological field diagnosis of growth inhibition due to continuous cropping of asparagus using PCR-DGGE.

Ambispora granatensis, a new arbuscular mycorrhizal fungus, associated with Asparagus officinalis in Andalucia (Spain).

A new dimorphic fungal species in the arbuscular mycorrhiza-forming Glomeromycota, Ambispora granatensis, was isolated from an agricultural site in the province of Granada (Andalucía, Spain) growing in the rhizosphere of Asparagus officinalis. It was propagated in pot cultures with Trifolium pratense and Sorghum vulgare. The fungus also colonized Ri T-DNA transformed Daucus carota roots but did not form spores in these root organ cultures. The spores of the acaulosporoid morph are 90-150 µm diam and hyaline to white to pale yellow. They have three walls and a papillae-like rough irregular surface on the outer surface of the outer wall. The irregular surface might become difficult to detect within a few hours in lactic acid-based mountings but are clearly visible in water. The structural central wall layer of the outer wall is only 0.8-1.5 µm thick. The glomoid spores are formed singly or in small, loose spore clusters of 2-10 spores. They are hyaline to pale yellow, (25)40-70 µm diam and have a bilayered spore wall without ornamentation. Nearly full length sequences of the 18S and the ITS regions of the ribosomal gene place the new fungus in a separate clade next to Ambispora fennica and Ambispora gerdemannii. The acaulosporoid spores of the new fungus can be distinguished easily from all other spores in genus Ambispora by the conspicuous thin outer wall.