Mycelial Homogenates Research Articles

A novel indirect ELISA for serodiagnosis of mucormycosis using antigens from Rhizopus arrhizus.

Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection. To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis. We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 μg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample. Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity. The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.

Genetic similarity of four strawberry cultivars in respect to Verticillium wilt susceptibility under in vitro selection

Verticillium wilt caused by pathogenic soil fungus – Vertcillium dahliae Kleb. is one of the most serious strawberry diseases not treated chemically. The strawberry is a species more or less susceptible to Verticillium sp., hence the need to select new genetically resistant cultivars. In this study, susceptibility of four strawberry cultivars to Verticillium wilt was examined under in vitro selection conditions as regards the genetic similarity of selected resistant plants. Microplants of 4 strawberry subclones obtained by in vitro cloning of each cultivar were inoculated in vitro with liquid mycelial homogenate of the pathogen. Development of disease symptoms was observed after 15, 30, 45, 60 and 75 days post inoculation. Results revealed differences of susceptibility to V. dahliae which depended on the subclone. The least susceptible to Verticillium wilt turned out to be ‘Plena’ subclone, followed by the ‘Elsanta’, ‘Feltar’ and ‘Teresa’ subclones. The highest genetic similarity of 59% was found between ‘Elsanta’ and ‘Teresa’ subclones, which after 75 days from inoculation were also characterized by a very similar percentage of resistant plants.

Mycovirus Diversity and Evolution Revealed/Inferred from Recent Studies.

High-throughput virome analyses with various fungi, from cultured or uncultured sources, have led to the discovery of diverse viruses with unique genome structures and even neo-lifestyles. Examples in the former category include splipalmiviruses and ambiviruses. Splipalmiviruses, related to yeast narnaviruses, have multiple positive-sense (+) single-stranded (ss) RNA genomic segments that separately encode the RNA-dependent RNA polymerase motifs, the hallmark of RNA viruses (members of the kingdom Orthornavirae). Ambiviruses appear to have an undivided ssRNA genome of 3∼5 kb with two large open reading frames (ORFs) separated by intergenic regions. Another narna-like virus group has two fully overlapping ORFs on both strands of a genomic segment that span more than 90% of the genome size. New virus lifestyles exhibited by mycoviruses include the yado-kari/yado-nushi nature characterized by the partnership between the (+)ssRNA yadokarivirus and an unrelated dsRNA virus (donor of the capsid for the former) and the hadaka nature of capsidless 10-11 segmented (+)ssRNA accessible by RNase in infected mycelial homogenates. Furthermore, dsRNA polymycoviruses with phylogenetic affinity to (+)ssRNA animal caliciviruses have been shown to be infectious as dsRNA-protein complexes or deproteinized naked dsRNA. Many previous phylogenetic gaps have been filled by recently discovered fungal and other viruses, which haveprovided interesting evolutionary insights. Phylogenetic analyses and the discovery of natural and experimental cross-kingdom infections suggest that horizontal virus transfer may have occurred and continue to occur between fungi and other kingdoms.

Dicer monitoring in a model filamentous fungus host, Cryphonectria parasitica

The ascomycete Cryphonectria parasitica has served as a model filamentous fungus for studying virus host interactions because of its susceptibility to diverse viruses, its genetic manipulability and the availability of many biological and molecular tools. Cryphonectria prasitica is known to activate antiviral RNA silencing upon infection by some viruses via transcriptional up-regulation of key RNA silencing genes. Here, utilizing a newly developed GFP-based reporter system to monitor dicer-like 2 (dcl2) transcript levels, we show different levels of antiviral RNA silencing activation by different viruses. Some viruses such as mycoreovirus 1, a suppressor-lacking mutant of Cryphonectria hypovirus 1 (CHV1-Δp69) and Rosellinia necatrix partitivirus 11 (RnPV11) highly induced RNA silencing, while others such as CHV3, Rosellinia necatrix victorivirus 1 and RnPV19 did not. There was considerable variation in dcl2 induction by different members within the family Hypoviridae with positive-sense single-stranded RNA genomes or Partitiviridae with double-stranded RNA genomes. Northern blotting and an in vitro Dicer assay developed recently by us using mycelial homogenates validated the reporter assay results for several representative virus strains. Taken together, this study represents a development in the monitoring of Dicer activity in virus-infected C. parasitica.

Occurrence of azoxystrobin‐resistant isolates in Passalora fulva, the pathogen of tomato leaf mould disease

Since 2007, serious damage to tomato from leaf mould caused by Passalora fulva has frequently been observed in commercial greenhouses in Gifu Prefecture, Japan. One of the factors relating to this damage was suspected to be a decrease in azoxystrobin sensitivity of the pathogen. Biological and molecular studies were conducted to characterize fungicide resistance. In in vitro sensitivity tests using mycelial homogenate placed on fungicide‐amended medium, the minimum inhibitory concentrations (MIC) of azoxystrobin for mycelial growth of the isolates divided into two ranges, 0.031–0.5 mg L−1 and 8–32 mg L−1. Isolates with MICs within the two ranges were considered as sensitive and resistant, respectively, to azoxystrobin because, in in vivo tests, the percentage protection conferred by this fungicide (100 mg a.i. L−1) against these isolates was 89.7–100% and 4.5–31.1%, respectively. Resistant isolates had a replacement of phenylalanine with leucine at codon 129 (F129L) in cytochrome b. Forty‐five percent of the 271 isolates collected from 63 tomato greenhouses from 2007 to 2008 were resistant to azoxystrobin. In many greenhouses where the isolation frequency of resistant isolates was 80% or more, azoxystrobin had been used twice per crop for approximately 6 years. In 2012, 27% of the 405 isolates collected were resistant to azoxystrobin, and there was a marked difference in the frequency of occurrence of resistant isolates in the field populations between the three locations sampled. The occurrence of azoxystrobin‐resistant P. fulva isolates (F129L mutants) inflicted considerable damage on greenhouse tomatoes.

Oxidative Stress in Experimental Rodent Corneas Infected With Aflatoxigenic and Nonaflatoxigenic Aspergillus flavus

To seek a possible association between aflatoxigenicity and oxidative stress in keratitis caused by Aspergillus flavus in an experimental rodent model. Wistar rats were divided into 3 groups of 6 each. Group 1 served as mock-inoculated controls. Experimental fungal keratitis was induced in group 2 and group 3 rats using aflatoxigenic and nonaflatoxigenic A. flavus conidial suspensions, respectively, and clinical features were scored for 5 days after inoculation. At this time, animals were killed, and test corneas were excised and examined histologically. Expression of IL-1β and TNF-α genes was sought in excised corneas. Levels of malondialdehyde (MDA) and reduced glutathione (GSH) and activities of key antioxidant enzymes were measured in excised corneas and fungal mycelial homogenates. Antioxidant enzyme isoforms were sought in mycelial homogenates by native polyacrylamide gel electrophoresis. Mean levels of MDA and GSH and mean activities of catalase, superoxide dismutase, and glutathione peroxidase were significantly (P < 0.05) higher in a mycelial homogenate of aflatoxigenic A. flavus than in the nonaflatoxigenic mycelial homogenate. Increased numbers of well-stained isoforms were detected in aflatoxigenic mycelial homogenates. Significantly (P < 0.05) higher expression profiles of IL-1β and TNF-α genes, MDA and GSH levels, and antioxidant enzyme activities were noted in group 2 rat corneas than in group 3 rat corneas. Clinical and histological scores suggested a more severe keratitis in group 2 rat corneas than in group 1 and group 3 rat corneas. Aflatoxigenicity is associated with more intense oxidative stress in experimental A. flavus keratitis.

Involvement of jasmonic acid signalling in bacterial wilt disease resistance induced by biocontrol agent Pythium oligandrum in tomato

When the biocontrol agent Pythium oligandrum (PO) colonizes the rhizosphere, it suppresses bacterial wilt disease in tomato (Solanum lycopersicum cv. Micro‐Tom) caused by Ralstonia solanacearum, and a homogenate of its mycelia exhibits elicitor activity, inducing an ethylene (ET)‐dependent defence response in Micro‐Tom. Since salicylic acid (SA) and jasmonic acid (JA) play an important role in plant defence responses to pathogens, the involvement of SA‐ and JA‐dependent signal transduction pathways in resistance to R. solanacearum was investigated in tomato roots treated with a mycelial homogenate of PO. Bacterial wilt disease was also suppressed in tomato cv. Moneymaker treated with the PO homogenate. However, the SA‐inducible PR‐1(P6) gene was not up‐regulated in either Micro‐Tom or Moneymaker. SA did not accumulate in homogenate‐treated roots in comparison with distilled water‐treated controls, even 24 h after inoculation. Induced resistance against R. solanacearum was not compromised in SA‐non‐accumulating NahG transgenic plants treated with the PO homogenate. On the other hand, the expression of the JA‐responsive gene for the basic PR‐6 protein was induced in both tomato cultivars treated with the PO homogenate. Furthermore, quantitative disease assays showed that the induced resistance against R. solanacearum was compromized in PO homogenate‐treated jai1‐1 mutant plants defective in JA signalling. These results indicated that the JA‐dependent signalling pathway is required for PO‐induced resistance against R. solanacearum in tomato.

Induction of transient ethylene and reduction in severity of tomato bacterial wilt by Pythium oligandrum

Pythium oligandrum (PO) is a mycoparasite on a wide range of fungi and suppresses diseases caused by fungal pathogens when colonizing the rhizosphere. PO and its cell wall proteins (CWPs) have elicitor activity that induces defence responses in plants. The potential of a mycelial homogenate of PO to suppress bacterial diseases was studied in roots of tomato (Lycopersicon esculentum cv. Micro‐Tom) inoculated with Ralstonia solanacearum. PO‐treated plants showed enhanced resistance to R. solanacearum and reduction in severity of wilt symptoms. As ethylene often acts as one of the signal molecules for induced resistance, its production following treatment of tomato roots with the mycelial homogenate or CWP of PO was measured. The level of ethylene in PO‐ and CWP‐treated plants was transiently elevated six‐ to 11‐fold at 4–8 h after treatment, followed by high expression of three basic ethylene‐inducible defence‐related genes (PR‐2b, PR‐3b and PR‐5b). Analysis of PR‐5b gene expression in the leaves of PO‐ and CWP‐treated plants suggested that PR gene expression was induced systemically. The expression of LeERF2 and LeETR4, which confer an ethylene‐dependent signalling pathway, was also significantly accelerated by such treatments. These results indicate that PO has the potential to control bacterial wilt disease and that CWP may play an important role in the induction of resistance to R. solanacearum accompanying the activation of the ethylene‐dependent signalling pathway.

Detection of Tilletia caries, Causal Agent of Common Bunt of Wheat, by ELISA and PCR

AbstractThe paper reports about the development and evaluation of two methods, a PCR‐based assay and an enzyme‐linked immunosorbent assay (ELISA), for the detection of the common bunt fungus Tilletia caries (syn. T. tritici) in young wheat plants. Using the published primer pair Tcar2A/Tcar2B for polymerase chain reaction (PCR) with DNA from axenic cultures of T. caries or from T. caries‐infected plants, we obtained a single band after electrophoresis of the amplification products. By PCR the bunt pathogen could be detected in shoots (EC 12) as well as in leaves (EC 13–14) of infected plants. Immunological detection was performed using a double antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) with biotinylated detection antibodies. The antibodies were obtained after injection of mycelial homogenates of axenic cultures of T. caries into rabbits. The detection limit was 16 pg DNA per 100 mg plant fresh weight for the PCR and 7 ng/ml fungal protein for the ELISA, respectively. Except for the closely related T. controversa, no cross‐reactions with other fungi were observed with both methods. While it was possible to detect teliospores of T. caries by PCR, the ELISA did not react with spore extracts. Analysis by ELISA of shoots of individual plants grown from inoculated seeds revealed that at EC 10 all plants were infected. There was, however, a large variability in the amount of T. caries present in the plants. This observation and reports in the literature indicate quantitative differences in the degree of colonization of the tissue between individual plants even in a given variety. Regarding the use of modern diagnostics to assist in the development of resistant varieties we therefore suggest that for the wheat –T. caries pathosystem the non‐quantitative PCR‐assay employed here is less suited than the ELISA that allows precise quantification of the amount of fungal antigen present in the plant. However, to routinely employ the ELISA in resistance breeding further development work is needed.

Biosynthesis and degradation of glycerides in external mycelium of Glomus mosseae

The activities of enzymes involved in the glyceride metabolism of Glomus mosseae external mycelium are reported. Total mycelial homogenates were incubated with radiolabeled triolein and palmitic acid for various times under different conditions. The results obtained demonstrate the capacity of G. mosseae external mycelium to synthesize and hydrolyze its own acylglycerides. Neutral lipid biosynthesis progressively increased along with root colonization. Incorporation of [14C]-palmitate was mainly into triacylglycerols and as a minor fraction into diacylglycerols. The activity of palmitoyl-CoA ligase in external mycelium also increased in parallel with mycorrhiza development. The hydrolysis of triacylglycerols was very low at the beginning of colonization and then increased. However, lipase activity was lower than that of acyl-CoA ligase even at late stages of colonization. Thus, triacylglycerol biosynthesis apparently prevails over degradation during G. mosseae mycelium development in the period examined.

Enhanced catharanthine production in catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors.

Chemical and fungal elicitors were added to Catharanthus roseus cell suspension cultures so as to improve the production of indole alkaloids. A synergistic effect on alkaloid accumulation was observed in C. roseus cell cultures when treated with some combined elicitors of fungal preparations and chemicals. Among them, the combination of tetramethyl amminium bromide and Aspergillum niger mycelial homogenate gave the highest ajmalicine yield (63 mg l(-1)) and an improved catharanthine accumulation (17 mg l(-1)). The combined elicitors of malate and sodium alginate resulted in the highest catharanthine yield (26 mg l(-1)) and a high ajmalicine accumulation (41 mg l(-1)) in the cell cultures. Based on the synergistic effect of malate and sodium alginate, a process with enhanced catharanthine production in Catharanthus roseus cell cultures was developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg l(-1), 32 mg l(-1) and 22 mg l(-1) catharanthine yield were obtained in 500-ml flasks, 1000-ml flasks and in a 20-l airlift bioreactor, respectively. Upon malate-alginate combining treatments, peroxidase, catalase and superoxide dismutase activities decreased in elicited cells but phenylalanine ammonia lyase and lipoxygenase activities increased dramatically. That suggests a typical defense responses took place in the combined elicitors-treated cell cultures. Furthermore, the combined elicitors also caused a significant increase of malondialdehyde level in cell cultures, which suggests a serious lipid peroxidation occurred in the elicited cell cultures. Comparison of these results suggests that malate and alginate combining treatment also stimulates defense responses, such as lipid peroxidation, in all C. roseus culture processes and this may mediate the indole alkaloid production via jasmonate pathway.

In vitro testing of strawberry resistance to Verticillium dahliae and Phytophthora cactorum

The effects of culture filtrates and homogenates of Verticillium dahliae and Phytophthora cactorum on in vitro growth and development of strawberry shoots of cvs. Elsanta, Real and Senga Sengana were investigated. The filtrates of fungal cultures as well as filtrates of homogenated mycelia did not affect strawberry shoot growth and development, whereas inoculation with mycelia of V. dahliae and P. cactorum resulted in leaf yellowing, growth inhibition, inhibition of root formation, and after several days, the death of infected plants. However, fungal growth on the media that were used was irregular and the infection rate of individual plants was very variable. Mycelial homogenates affected growth and development of strawberry shoots in a similar way to that of an intact mycelium. Because the homogenate was distributed evenly in the culturing medium, the effect was uniform and thus possible to quantitate. It was also possible to differentiate a pathogen’s inoculum potential by adequate dilution of the homogenate. The best effect was obtained with V. dahliae. Fungal homogenate inhibited root formation and shoot growth, caused leaf necrosis and death of the plants after 7–8 weeks. The reaction of the cultivars tested was similar to their sensitivity to verticillium wilt in the field. P. cactorum affected in vitro strawberry shoot growth and development in a similar way to V. dahliae, but the effect was not cultivar-dependent. Preliminary tests showed that the method developed is useful for selection of somaclonal variants resistant/tolerant to infection with these pathogens.

Selection of phage antibodies to surface epitopes of Phytophthora infestans

Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were isolated from a diverse phage library displaying single-chain Fv (scFv) antibody fragments. The library was subpanned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated. Binding of monoclonal phage antibodies was demonstrated by enzyme-linked immunosorbent assay (ELISA) and diversity was established by BstNI restriction enzyme digest patterns. Antibodies were subcloned as fusions at the C-terminus of maltose binding protein (MBP) and expressed as soluble proteins in Escherichia coli. These antibody fusion proteins bound to P. infestans germlings and to mycelial homogenates from various Phytophthora species. The binding activities to mycelial homogenates of fungal species not belonging to the order Peronosporales were substantially lower. Several phage-displayed scFvs were used in conjunction with fluorescently labelled antiphage antibody to visualise the distribution of their cognate epitopes on the surface of the germlings. The combination of procedures developed here with Phytophthora demonstrates the potential of phage antibody technology in isolating antibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions.

MICROORGANISMS AS POTENTIAL SOURCES OF NOVEL DIPHENOL OXIDASE INHIBITORS

Culture filtrates and mycelial homogenates from over 100 microorganisms were tested for their ability to inhibit catecholase and laccase (o- and p-diphenol oxidase) activity using simple, rapid, colorimetric assays in microtiter plates or polarographic assays using an O2-electrode. Of the organisms screened, 56 produced inhibition of catecholase activity and 34 gave rise to laccase inhibition, with most of the latter group showing inhibition of both enzymes. The role of this enzyme inhibitor system in plant pathogenicity is also discussed.

1-Octen-3-ol and 13-hydroperoxylinoleate are products of distinct pathways in the oxidative breakdown of linoleic acid by Pleurotus pulmonarius

The oxidative breakdown of linoleic acid leading to the formation of the mushroom flavor alcohol 1-octen-3-ol by mycelial homogenate of the edible mushroom Pleurotus pulmonarius grown in submerged culture was studied. The compounds 13-hydroperoxy- cis-9, trans-11-octadecadienoic acid (13-HPOD) and 10-oxo- trans-8-decenoic acid (10-oxo-acid) were found to be the major nonvolatile metabolites associated with the enzymatic cleavage of linoleic acid to 1-octen-3-ol. Whereas 1-octen-3-ol and 10-oxo-acid were produced at all linoleic acid concentrations studied (up to 8 mM), 13-HPOD was absent at linoleic acid concentrations below 1 mM but became the major nonvolatile product of the enzymatic oxidation at concentrations above 1 mM. Despite its accumulation, 13-HPOD was found not to be the precursor of 1-octen-3-ol when it was supplied as the substrate instead of linoleic acid. Periodic addition of linoleic acid during the course of the reaction maintained 1-octen-3-ol formation at a constant rate while 13-HPOD accumulated, suggesting that 1-octen-3-ol formation may be limited by either competitive product inhibition or substrate availability. These results suggest the involvement of two different lipoxygenases in 1-octen-3-ol and 13-HPOD formation. Mild heat treatment of the pellets before homogenization and incubation completely inhibited 1-octen-3-ol formation and resulted in the accumulation of an additional HPOD, possibly a 1-octen-3-ol precursor. It would appear, therefore, that 13-HPOD accumulation takes place in parallel with 1-octen-3-ol and 10-oxo-acid biosynthesis, suggesting that these are two distinct biosynthetic pathways catalyzed by two different lipoxygenases.

Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.

Treatment of opium poppy (Papaver somniferum L.) cell cultures with autoclaved mycelial homogenates of Botrytis sp. resulted in the accumulation of sanguinarine. Elicitor treatment also caused a rapid and transient induction in the activity of tyrosine/dopa decarboxylase (TYDC, EC 4.1.1.25), which catalyzes the conversion of L-tyrosine and L-dopa to tyramine and dopamine, respectively, the first steps in sanguinarine biosynthesis. TYDC genes were differentially expressed in response to elicitor treatment. TYDC1-like mRNA levels were induced rapidly but declined to near baseline levels within 5 h. In contrast, TYDC2-like transcript levels increased more slowly but were sustained for an extended period. Induction of TYDC mRNAs preceded that of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) mRNAs. An elicitor preparation from Pythium aphanidermatum was less effective in the induction of TYDC mRNA levels and alkaloid accumulation; however, both elicitors equally induced accumulation of PAL transcripts. In contrast, treatment with methyl jasmonate resulted in an induction of TYDC but not PAL mRNAs. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and the protein kinase inhibitor staurosporine partially blocked the fungal elicitor-induced accumulation of sanguinarine. However, only staurosporine and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, blocked the induction of TYDC1-like transcript levels, but they did not block the induction of TYDC2-like or PAL transcript levels. These data suggest that activation mechanisms for PAL, TYDC, and some later sanguinarine biosynthetic enzymes are uncoupled.

In vitro susceptibility of fungi to acyclic inhibitors of 2,3-oxidosqualene cyclases.

In the present study we determine the antifungal properties of two acyclic inhibitors of 2,3-oxidosqualene cyclases: 22,23-epoxy-2-aza-2,3-dihydrosqualene (EAS) and azasqualene alcohol (ASA). Fungistatic and fungicidal activity towards dermatophytes and other fungi involved in cutaneous and systemic infections was tested (48 isolates from 10 species). The tests were carried out by inoculating 10 microliters of mycelial homogenate in 1 ml of Sabouraud glucose liquid medium containing serial dilutions of 100 to 0.25 micrograms ml-1 of the substance. For each isolate, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of both compounds were determined. EAS was more active (MIC range 1.5-25 micrograms ml-1) than ASA (MIC range 3-50 micrograms ml-1). At the highest concentration tested, EAS also showed fungicidal action towards some isolates of Trichophyton mentagrophytes, T. terrestre, Epidermophyton floccosum, Microsporum canis and Scopulariopsis brumptii. The most sensitive species was T. mentagrophytes, the most resistant T. rubrum.

Defence related reactions of seedling roots of Norway spruce to infection by Heterobasidion annosum (Fr.) Bref.

Norway spruce seedlings were cultivated in sterile conditions. Roots were infected with a concentration series of germinating conidiospores of Heterobasidion annosum (101–106 ml−1). In other experiments, roots were treated with either mycelial preparations of H. annosum, other wood inhabiting fungi, with protein fractions of culture filtrates of H. annosum, or with chemical elicitors. Successive steps observed during infection were necrosis, formation of phenolics and increasing lignification of cortex and endodermis, colonization of meristem and finally of the stele. High spore concentrations caused necrosis and invasion within 48–72 h; these processes were delayed at low spore concentrations. Lyophilized culture filtrates of H. annosum caused a greater hypersensitive response than protein fractions but less than NaCl, Polygalacturonic acid or ethephon. Mycelial homogenate from nine other wood inhabiting fungi (saprophytes/parasites) induced a hypersensitive response to various extents but this was not correlated to their degree of cross-reactivity with a polyclonal antibody to H. annosum [enzyme-linked immunosorbent assay (ELISA)]. Peroxidase activity increased (two-threefold) in roots infected with H. annosum and one acidic isozyme was considered responsible for the increase in peroxidase. Using immunohistochemical and enzyme staining, peroxidase was found mainly in the cortical/endodermal regions of roots. Cytochemical labelling using anti-peroxidase and immunogold demonstrated increased peroxidase activity in cell walls, papillae and uninvaded middle lamellar cell corners of infected roots.

Production of conidia of Alternaria cassiae with alginate pellets

Abstract Formulations of alginate-encapsulated mycelia are used to generate spores for mycoherbicidal application to weed-infested fields and for bulk production of spore-based products. Spore yield of such formulations is a primary determinant of product efficacy. A number of parameters of the alginate process were studied to develop an optimal alginate formulation for field application of Alternaria cassiae , a mycoherbicide for sicklepod ( Cassia obtusifolia ). The composition of the fermentation medium and of the filler used in formulation and the fermentation time were important variables. The addition of nutrients to the mycelial homogenate after fermentation increased sporulation but the amount and ratio of nutrients in the fermentation medium had a greater influence on spore yield from pellets. Optimal sporulation resulted from mycelia produced during a 60- to 70-h fermentation in 2.4% dehydrated potato dextrose broth and 14% V-8 vegetable juice and entrapped in pellets containing corn cob grits as the filler.

Immunoblot Analyses of the Serologic Response to Aspergillus fumigatus Antigens in Experimental Invasive Aspergillosis

The antibody response to Aspergillus fumigatus proteins was studied by the immunoblot technique in a rabbit model of invasive aspergillosis. Components of an A. fumigatus mycelial homogenate homogenate unbound by concanavalin A-Sepharose 4B chromatography were fractionated using SDS-PAGE and transferred to nitrocellulose papers. The protein fraction was probed with serial sera obtained from immunosuppressed or nonimmunosuppressed rabbits inoculated intravenously with saline or graded inocula of A. fumigatus conidia. Seroconversion against antigens of 41, 54, and 71 kDa was demonstrated in 7 (50%), 3 (21%), and 3 (21%) of 14 infected animals that survived greater than or equal to 10 days. Antibodies against the three antigens were detected in 4 (12.5%), 19 (59%), and 14 (44%) of 32 rabbits before immunosuppression or infection. Two-dimensional immunoblotting revealed that the 41-, 54-, and 71-kDa antigens were derived in denaturing conditions from a single component resolved in nondenaturing polyacrylamide gel electrophoresis.