Evidence for Sulfhydryl Involvement in Regulation of Phytoalexin Accumulation in Trifolium repens Callus Tissue Cultures

White clover (Trifolium repens L.) callus tissue cultures accumulated the phytoalexin medicarpin after treatment with sulfhydryl reagents. After 24-hour exposures to sulfhydryl reagents, maximum obtainable levels of medicarpin, determined by high performance liquid chromatography analysis, were found with 50 millimolar N-ethyl maleimide, 25 millimolar HgCl₂, 2 millimolar p-chloromercuribenzoic acid, and 0.5 millimolar iodoacetamide. Increased medicarpin levels were also observed in callus treated with p-chloromercuribenzene sulfonic acid, but the highest concentration tested (11.8 millimolar) did not produce the maximum response. After sulfhydryl treatment, medicarpin levels were unchanged for 4 to 6 hours, but steadily increased thereafter with maximum accumulation occurring by 48 to 50 hours for p-chloromercuribenzoic acid, p-chloromercuribenzene sulfonic acid, and HgCl₂ treated callus. Medicarpin levels did not increase in iodoacetamide-treated callus until 8 hours after sulfhydryl exposure, and medicarpin levels were still increasing linearly after 50 hours. Three other metabolic inhibitors, KCN, NaF, and Na₃AsO₄, did not exhibit elicitor activity, indicating cell death was not a factor in the response. Pretreatment of callus with 20 millimolar dithiothreitol followed by 40 millimolar N-ethyl maleimide did not produce the phytoalexin response. Preincubation with dithiothreitol also prevented elicitor activity of HgCl₂ and p-chloromercuribenzene sulfonic acid. These results suggested that dithiothreitol pretreatment somehow prevented sulfhydryl groups within the cell from reacting with the test compounds. These experiments established that the integrity of sulfhydryl groups is important in regulating phytoalexin accumulation in callus cells.     

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10⁵ and 10⁷ spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 × 10⁷ to 2 × 10⁷ spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.     

Spore Load of Ascosphaera Species on Emerging Adults of the Alfalfa Leafcutting Bee, Megachile rotundata

The spore load of Ascosphaera species spores on larval chalkbrood cadavers and newly emergent adults of the alfalfa leafcutting bee, Megachile rotundata, was determined. The spore content of chalkbrood cadavers ranged from 3 × 10⁶ to 5 × 10⁸. Adults emerging through zero to nine cadavers carried spores on all body parts examined by scanning electron microscopy. Estimates of the total number of spores obtained from a series of adult washes ranged from 9 × 10⁴ to 8 × 10⁷. Some adult males which emerged through no cadavers carried 10⁴ to 10⁵ spores, indicating that nesting materials might also have been contaminated. However, the control of chalkbrood in commercial bee populations may not be accomplished simply by providing clean nesting materials as adults may still emerge through diseased larvae.     

Heat Resistance Correlated with DNA Content in Bacillus megaterium Spores

Two subpopulations of Bacillus megaterium spores (1.360 and 1.355 g/ml) were obtained by density gradient centrifugation. The heavier spores had a higher thermoresistance (e.g., D₈₀ = 186 versus 81 min) and a higher DNA content (1.25 × 10⁻¹⁴ versus 0.65 × 10⁻¹⁴ g per spore, apparently corresponding to digenomic versus monogenomic spores). No appreciable differences were found in the mineral and dipicolinic acid contents or in the inactivation kinetics of the two subpopulations. The implications of the findings are discussed with regard to mechanisms of heat resistance and of inactivation.     

Recovery of Spores from Thermophilic Dairy Bacilli and Effects of Their Surface Characteristics on Attachment to Different Surfaces

Spores from four Geobacillus spp. were isolated from a milk powder manufacturing line in New Zealand. Liquid sporulation media produced spore yields of ~10⁷ spores ml⁻¹; spores were purified using a two-phase system created with polyethylene glycol 4000 and 3 M phosphate buffer. The zeta potentials of the spores from the four isolates ranged from −10 to −20 mV at neutral pH, with an isoelectric point between pH 3 and 4. Through contact angle measurements, spores were found to be hydrophilic and had relative hydrophobicity values of 10 to 40%, as measured by the microbial adhesion to hexadecane assay. The most hydrophilic spore isolate with the smallest negative charge attached in the highest numbers to Thermanox and stainless steel (1 × 10⁴ spores cm⁻²), with fewer spores attaching to glass (3 × 10³ spores cm⁻²). However, spores produced by the other three strains attached in similar numbers (P > 0.05) to all substrata (~1 × 10³ spores cm⁻²), indicating that there was no simple relationship between individual physicochemical interactions and spore adherence. Therefore, surface modifications which limit the attachment of one strain may not be effective for all stains, and control regimens need to be devised with reference to the characteristics of the particular strains of concern.     

Metabolism of the phytoalexins medicarpin and maackiain by Fusarium solani

Non-inhibitory concentrations of the pterocarpan phytoalexin medicarpin were completely metabolized by isolates of Fusarium solani f. sp. pisi, f. sp. cucurbitae, f. sp. phaseoli and two other F. solani isolates genetically related to f. sp. pisi during 24 hr of growth in liquid medium. The major metabolic products accumulated without significant further degradation. Medicarpin was modified at one of three adjacent carbon atoms to form either an isoflavanone derivative, a 1a-hydroxydienone derivative or 6a-hydroxymedicarpin. Whereas each isolate degraded medicarpin to one or more metabolises, the isolates varied as to which metabolise they produced. Maackiain, another pterocarpan phytoalexin, was also metabolized by all the isolates to products analogous to those formed from medicarpin. The ability to metabolize medicarpin and maackiain was not always associated with the ability to metabolize pisatin and phaseollin, two other pterocarpan phytoalexins that were degraded by several of the isolates. Tolerance of medicarpin and maackiain was similarly not always associated with tolerance to pisatin.     

Interactions between Bacillus thuringiensis subsp. israelensis and Fathead Minnows, Pimephales promelas Rafinesque, under Laboratory Conditions

Interactions between Bacillus thuringiensis subsp. israelensis and fathead minnows, Pimephales promelas, were studied in laboratory exposures to two commercial formulations, Vectobac-G and Mosquito Attack. Mortality among fatheads exposed to 2.0 × 10⁶ to 6.5 × 10⁶ CFU/ml with both formulations was attributed to severe dissolved oxygen depletion due to formulation ingredients rather than to direct toxicity from the parasporal crystal. No adverse effects were observed at 6.4 × 10⁵ CFU/ml and below. Fathead minnows rapidly accumulated high numbers of spores with 1 h of exposure to 2.2 × 10⁵ CFU of Mosquito Attack per ml, producing whole-body counts of 4.0 × 10⁶ CFU per fish. Comparison of counts on gastrointestinal tract samples and whole-body samples and high numbers of spores in feces indicated that ingestion was the major route of exposure. B. thuringiensis subsp. israelensis spore counts decreased rapidly after transfer of fish to clean water, with a drop of over 3 orders of magnitude in 1 day. Spores were rarely detected in fish after 8 days but were detectable in feces for over 2 weeks. These findings suggest that fish could influence the dissemination of B. thuringiensis subsp. israelensis, and possibly other microbial agents, in the aquatic environment.     

Identification by Quantitative Carrier Test of Surrogate Spore-Forming Bacteria To Assess Sporicidal Chemicals for Use against Bacillus anthracis

The spores of six strains of Bacillus anthracis (four virulent and two avirulent) were compared with those of four other types of spore-forming bacteria for their resistance to four liquid chemical sporicides (sodium hypochlorite at 5,000 ppm available chlorine, 70,000 ppm accelerated H₂O₂, 1,000 ppm chlorine dioxide, and 3,000 ppm peracetic acid). All test bacteria were grown in a 1:10 dilution of Columbia broth (with manganese) incubated at 37°C for 72 h. The spore suspensions, heat treated at 80°C for 10 min to rid them of any viable vegetative cells, contained 1 × 10⁸ to 3 × 10⁸ CFU/ml. The second tier of the quantitative carrier test (QCT-2), a standard of ASTM International, was used to assess for sporicidal activity, with disks (1 cm in diameter) of brushed and magnetized stainless steel as spore carriers. Each carrier, with 10 μl (≥10⁶ CFU) of the test spore suspension in a soil load, was dried and then overlaid with 50 μl of the sporicide being evaluated. The contact time at room temperature ranged from 5 to 20 min, and the arbitrarily set criterion for acceptable sporicidal activity was a reduction of ≥10⁶ in viable spore count. Each test was repeated at least three times. In the final analysis, the spores of Bacillus licheniformis (ATCC 14580^T) and Bacillus subtilis (ATCC 6051^T) proved to be generally more resistant than the spores of the strains of B. anthracis tested. The use of one or both of the safe and easy-to-handle surrogates identified here should help in developing safer and more-effective sporicides and also in evaluating the field effectiveness of existing and newer formulations in the decontamination of objects and surfaces suspected of B. anthracis contamination.     

Induction of Medicarpin Biosynthesis in Ladino Clover Callus by p-Chloromercuribenzoic Acid Is Reversed by Dithiothreitol

Treatment of Ladino clover (Trifolium repens L.) callus with 1 millimolar p-chloromercuribenzoic acid (PCMBA) stimulated the biosynthesis of the phytoalexin medicarpin. Increased medicarpin concentration was detected at 6 hours, and maximum level was achieved by 36 hours. Dithiothreitol (DTT), added to callus at 2, 6, 10, and 18 hours following addition of PCMBA, partially counteracted the effect of PCMBA in the cells. The greatest effect on reversal of medicarpin accumulation was found at the earlier times for DTT addition. Compared to a 24-hour PCMBA control, final medicarpin concentrations were 9 to 19%, 37 to 45%, and 92 to 105% of the control value when DTT was added at 0.5, 1 or 2 hours, 3 or 4 hours, and 6, 8, 10, or 12 hours, respectively, after PCMBA addition. The results indicate that stimulation of medicarpin biosynthesis is reversible, but once activated, the pathway is not shut down by removal of elicitor.     

Stress Responses in Alfalfa (Medicago sativa L.) : V. Constitutive and Elicitor-Induced Accumulation of Isoflavonoid Conjugates in Cell Suspension Cultures

The isoflavonoid conjugates medicarpin-3-O-glucoside-6″-O-malonate (MGM), afrormosin-7-O-glucoside (AG), and afrormosin-7-O-glucoside-6″-O-malonate (AGM) were isolated and characterized from cell suspension cultures of alfalfa (Medicago sativa L.), where they were the major constitutive secondary metabolites. They were also found in alfalfa roots but not in other parts of the plant. The phytoalexin medicarpin accumulated rapidly in suspension cultured cells treated with elicitor from Colletotrichum lindemuthianum, and this was subsequently accompanied by an increase in the levels of MGM. In contrast, net accumulation of afrormosin conjugates was not affected by elicitor treatment. Labeling studies with [¹⁴C]phenylalanine indicated that afrormosin conjugates were the major de novo synthesized isoflavonoid products in unelicited cells. During elicitation, [¹⁴C]phenylalanine was incorporated predominantly into medicarpin, although a significant proportion of the newly synthesized medicarpin was also conjugated. Treatment of ¹⁴C-labeled, elicited cells with l-α-aminooxy-β-phenylpropionic acid, a potent inhibitor of PAL activity in vivo, resulted in the initial appearance of labeled medicarpin of very low specific activity, suggesting that the phytoalexin could be released from a preformed conjugate under these conditions. Our data draw attention to the involvement of isoflavone hydroxylases during the constitutive and elicitor-induced accumulation of isoflavonoids and their conjugates in alfalfa cell cultures.     

Aphidicolin Inhibition of DNA Synthesis and Germination in Spores of Anemia phyllitidis L. Sw

Aphidicolin inhibits DNA synthesis and nuclear division in spores of Anemia phyllitidis. In spite of blocked DNA replication, spores germinate under continuous dark conditions, if induced by addition of 5 × 10⁻⁵ grams per milliliter gibberellic acid. Differentiation of aphidicolin-treated prothallia indicate the existence of a prepattern in the dry spore which is realized independent of cell division during early events of spore germination.     

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

A single-chain Fv (scFv) antibody was developed and applied for efficient and specific detection of Bacillus anthracis spores. The antibody was isolated from a phage display library prepared from spleens of mice immunized with a water-soluble extract of the outer membrane of the B. anthracis spore (exosporium). The library (7 × 10⁶ PFU) was biopanned against live, native B. anthracis ATCC Δ14185 spores suspended in solution, resulting in the isolation of a unique soluble scFv antibody. The antibody was affinity purified and its affinity constant (3 × 10⁸ ± 1 × 10⁸ M⁻¹) determined via flow cytometry (FCM). Preliminary characterization of scFv specificity indicated that the scFv antibody does not cross-react with representatives of some phylogenetically related Bacillus spores. The potential use of scFv antibodies in detection platforms was demonstrated by the successful application of the soluble purified scFv antibody in enzyme-linked immunosorbent assays, immunofluorescence assays, and FCM.     

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30°C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30°C for Glomus caledonius or at 35°C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 μg of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 μg/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40°C for 80 min, 55°C for 10 min, and 60°C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 × 10⁸ ergs cm⁻² from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.     

Stress Responses in Alfalfa (Medicago sativa L.): I. Induction of Phenylpropanoid Biosynthesis and Hydrolytic Enzymes in Elicitor-Treated Cell Suspension Cultures

Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum within 24 hours after exposure to an elicitor preparation from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by increases in the extractable activities of the isoflavonoid biosynthetic enzymes l-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A-ligase, chalcone synthase, chalcone isomerase, and isoflavone O-methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine ammonia-lyase activity but did not induce medicarpin accumulation, whereas reduced glutathione was totally inactive as an elicitor in this system. The fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes, caffeic acid O-methyltransferase and coniferyl alcohol dehydrogenase, but did not induce appreciable increases in the activities of the hydrolytic enzymes chitinase and 1,3-β-d-glucanase. The results are discussed in relation to the activation of isoflavonoid biosynthesis in other legumes and the development of the alfalfa cell culture system as a model for studying the enzymology and molecular biology of plant defense expression.     

Assessment of Elasticity and Topography of Aspergillus nidulans Spores via Atomic Force Microscopy

Previous studies have described both surface morphology and adhesive properties of fungal spores, but little information is currently available on their mechanical properties. In this study, atomic force microscopy (AFM) was used to investigate both surface topography and micromechanical properties of Aspergillus nidulans spores. To assess the influence of proteins covering the spore surface, wild-type spores were compared with spores from isogenic rodA⁺ and rodA⁻ strains. Tapping-mode AFM images of wild-type and rodA⁺ spores in air showed characteristic “rodlet” protein structures covering a granular spore surface. In comparison, rodA⁻ spores were rodlet free but showed a granular surface structure similar to that of the wild-type and rodA⁺ spores. Rodlets were removed from rodA⁺ spores by sonication, uncovering the underlying granular layer. Both rodlet-covered and rodlet-free spores were subjected to nanoindentation measurements, conducted in air, which showed the stiffnesses to be 110 ± 10, 120 ± 10, and 300 ± 20 N/m and the elastic moduli to be 6.6 ± 0.4, 7.0 ± 0.7, and 22 ± 2 GPa for wild-type, rodA⁺ and rodA⁻ spores, respectively. These results imply the rodlet layer is significantly softer than the underlying portion of the cell wall.     

Changes in the profile of flavonoid accumulation in Medicago truncatula leaves during infection with fungal pathogen Phoma medicaginis

Medicago truncatula is a model species for the study of the unique secondary metabolism in legumes. LC/MS/MS analysis was used to identify and profile flavonoid glycoconjugates and free aglycones in leaves of M. truncatula (ecotype R108-1) infected with the fungal pathogen Phoma medicaginis. Use of a high resolution analyzer with a collision induced dissociation tandem mass spectrometer (CID MS/MS) permitted structural elucidation of target secondary metabolites and four new acylated flavone glycosides have been identified. Changes in the phytoalexin medicarpin and its isoflavone precursors were quantitatively monitored at various time points after fungal spore application. Application of spores induced disease symptoms in the leaves of infected plants and resulted in an increase in the medicarpin precursors formononetin 7-O-glucoside and malonylated formononetin 7-O-glucoside between one and three days post-infection. Relative concentrations of medicarpin were highest five days post-infection. The rapid increase of these molecules was clearly positively correlated to the infection process as certain of them were absent in uninfected leaves, suggesting that the relative rate of their synthesis is tightly related with the infection process.     

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereus Spores

Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 10⁴ B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.     

Detection and Quantification of Airborne Conidia of Fusarium circinatum, the Causal Agent of Pine Pitch Canker, from Two California Sites by Using a Real-Time PCR Approach Combined with a Simple Spore Trapping Method

Pinus radiata (Monterey pine), a tree native to coastal California and Mexico, is widely planted worldwide for timber production. A major threat to Monterey pine plantations is the fungal disease pine pitch canker, caused by Fusarium circinatum (Hypocreales). We present a novel trapping approach using filter paper in combination with a rapid molecular method to detect the presence of inoculum in the air. The assay is also useful for diagnosing the presence of the pathogen on plants. The test is based on the F. circinatum specific primer pair CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment in the intergenic spacer region of the nuclear ribosomal operon. Real-time PCR was used to calculate the number of fungal spores present in each reaction mixture by comparing the threshold cycle (Ct) of unknown spore samples to the Ct values of standards with known amounts of F. circinatum spores. The filter paper method allows prolonged and more sensitive spore sampling in the field compared to traditional traps using petri dishes filled with selective medium. A field test at two sites in coastal California infested with pine pitch canker was carried out during the summer and fall of 2002. Spore counts were in the range of ca. 1 × 10³ to ca. 7 × 10⁵/m², with the highest spore counts in the fall, suggesting a seasonal fluctuation.     

Force Required to Detach Conidia of Helminthosporium maydis

The force required to break the conidium-conidiophore attachment in Helminthosporium maydis was measured by centrifugation and by a small jet of air. The force which removed half the spores from their stalks was found by both methods to be about 1 × 10⁻² dynes. The corresponding speed of the air jet was about 10 m/sec. Although spores are removed over a range of applied force, most of this spread is accounted for by the variation in spore size. Therefore, the apparent strength of the attachment of conidia to conidiophores, though relatively large, is surprisingly uniform.