Sensitivity of the Alternaria infectoria species-group 1 to cycloheximide

Aims:  A possibility of using cycloheximide tolerance and/or sensitivity as an additional diagnostic tool for distinguishing morphologically related species within common small-spored Alternaria has been tested during this study.
Methods and Results:  A total of 33 strains from four Alternaria species-groups, namely Alternaria alternata , Alternaria arborescens , Alternaria infectoria and Alternaria tenuissima were tested for their growth response to 100 μg m⁻¹ cycloheximide in potato carrot agar. All A. infectoria strains were completely inhibited, showing no growth at all even after prolonged incubation. In contrast, all other strains representing the remnant three species exhibited a high resistance to this antibiotic.
Conclusions:  Cycloheximide sensitivity represents a further important physiological character for distinguishing A. infectoria from the three similar species.
Significance and Impact of the Study:  The relevance of these findings corresponds with the potential ability of the Alternaria species produce mycotoxins. Cycloheximide may be in future used in the design of selective media for the isolation of some potentially toxigenic food-borne Alternaria species such as A. alternata , A. tenuissima and/or A. arborescens , for example in screening cereals for toxigenic Alternaria spp. and for their direct separation from nontoxigenic representatives of A. infectoria species-group.     

Interaction of Galactosaminoglycan with Neurospora Conidia

The inactivation of Neurospora crassa conidia by galactosaminoglycan isolated from cultures of this organism was followed by measuring colony-forming ability and ability to take up radiolabeled metabolites. When kinetic data on the loss of transport function and on killing were analyzed by use of target theory, it appeared that few "hits" are required for inactivation. However, studies with radio-labeled galactosaminoglycan mucopolysaccharides showed that cells receiving a single lethal hit have approximately 10(5) galactosaminoglycan molecules bound to them.     

A polyphasic approach to the taxonomy of the Alternaria infectoria species–group

Different taxa in the species–group of Alternaria infectoria (teleomorph Lewia spp.) are often isolated from various cereals including barley, maize and wheat grain, ornamental plants and skin lesions from animals and humans. In the present study we made a polyphasic characterization of 39 strains morphologically identifiable as belonging to the A. infectoria species–group together with 12 strains belonging to closely related species: Alternaria malorum (syn. Cladosporium malorum), Chalastospora cetera (syn. Alternaria cetera) and Embellisia abundans. Morphological examination separated the 51 strains in three groups based on conidial appearance and arrangement: the A. infectoria species–group, E. abundans and a group containing C. cetera and A. malorum. The metabolite analyses on three different media showed two clusters, one containing all 39 A. infectoria species–group strains and one containing 10 strains of E. abundans, C. cetera and A. malorum. One E. abundans strain and one A. malorum strain were not included due to insufficient metabolite production. The separation of the A. infectoria species–group from E. abundans, C. cetera and A. malorum resulted mainly from the ability to produce altertoxins and novae-zelandins. The metabolite analyses also showed that all 51 strains were able to produce infectopyrones. The metabolite profiles of C. cetera and A. malorum were very similar with several metabolites of unknown structure in common. This is the first time that E. abundans, C. cetera and A. malorum have been reported as producers of infectopyrones. Sequence analyses of the internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase (gpd) and translocation elongation factor 1α (tef-1α) showed two clades: one with the 39 strains from the A. infectoria species–group and one with the 12 strains of E. abundans, C. cetera and A. malorum. The polyphasic approach in this study suggests that A. malorum var. polymorpha and the eight A. malorum strains do not belong in Alternaria, but in Chalastospora, however, as several distinct species. Splits Tree alignment of gpd sequences of 38 strains belonging to the A. infectoria species–group indicates that only three strains showed signs of recombination, while the remaining strains appeared to be clonal. Long term incubation at 7 °C in the dark showed that 12 out of 33 tested strains from the A. infectoria species–group were able to produce proascomata in axenic culture, but with no mature ascospores after 6 months. These findings suggest that Lewia/A. infectoria species–group must, at least in part, be homothallic. The results presented in this study show that ITS, tef-1α and gpd do not reflect ecology, secondary metabolism or morphology of the A. infectoria species–group and that molecular cladification and phylogeny cannot predict pathogenicity, host specificity or mycotoxin production.     

Solid Substrate Production of Alternaria alternata f. sp. sphenocleae Conidia

Sphenoclea zeylanica (gooseweed), a major weed of paddy rice in Southeast Asia, is one of the targets in a biological weed control research program in the Philippines. A fungal pathogen, Alternaria alternata f. sp. sphenocleae , is being evaluated as a biological control agent for this weed. The feasibility of solid substrate fermentation for the mass production of A. alternata f. sp. sphenocleae has been examined. Conidia production and virulence of A. alternata f. sp. sphenocleae were affected by temperature, light, and incubation period. Abundant conidia were produced under continuous light on seeds of sorghum, hard red spring wheat, and barley at 28 o C. The greatest number of conidia was produced on sorghum seed followed by barley and oats seeds at 28 o C exposed to near-ultraviolet (NUV). More conidia were produced at 28 o C under NUV light on sorghum, barley, oats, and hard red spring wheat seeds, cornmeal, and polished rice grains than on the other substrates. Less conidia were produced on these substrates under light. At 28 o C, large numbers of virulent conidia were produced on sorghum seeds after 4 weeks of incubation under either constant light or dark. A mix of equal quantities of sorghum seeds and water (w/v) maximized conidial production. Conidia produced on sorghum seeds had a shelf life of at least 12 months when stored in production flasks under room conditions (24 ±2 o C). The use of sorghum seeds as a solid substrate for production of A. alternata f. sp. sphenocleae could be a feasible method to produce conidia in a village co-operative or cottage industry type scenario in Southeast Asia.     

Subcutaneous phaeohyphomycosis due to Alternaria infectoria in a renal transplant patient: Surgical treatment with no long-term relapse

BackgroundPhaeohyphomycosis can be caused by a number of different species, being the most common Alternaria alternata and Alternaria infectoria. The biggest risk factor for the development of the infection is immunosuppression.AimsWe present the case of a 64-year-old male renal transplant patient who came to hospital for presenting a tumour in the Achilles region which had been gradually growing in size.MethodsA skin biopsy was taken for histological study and culture of fungi and mycobacteria. Blood tests and imaging studies were performed.ResultsHistopathology study and cultures identified A. infectoria as the causal agent. Imaging studies ruled out internal foci of infection. The lesion was surgically removed with no signs of recurrence after 24 months of follow-up.ConclusionsThere are no treatment guidelines at present for cutaneous and subcutaneous Alternaria spp. infections. Various systemic antifungals have been used, either in combination with surgical removal or alone, with varying results. Surgery alone could be useful in the treatment of solitary, localised lesions in transplant patients in whom there are difficulties in controlling immunosuppression.     

Phaeohyphomycosis caused by Alternaria infectoria presenting as multiple vegetating lesions in a renal transplant patient

The genus Alternaria is one of the most common black moulds and appears to be increasing as a causative agent of subcutaneous phaeohyphomycosis, particularly among immunosuppressed patients. A 53-year-old patient who had received a kidney transplant presented with multiple verrucous lesions on the distal extremities. Positive histopathology and cultures, in addition to rDNA ITS region sequencing, identified the fungal isolate as Alternaria infectoria. Oral itraconazole was administered for 10 months. A follow-up at 15 months demonstrated no signs of infection. Clinical manifestations of cutaneous alternariosis vary significantly and only a few cases have been described in the literature. Although optimal treatment options remain controversial, this case of phaeohyphomycosis was successfully treated with itraconazole monotherapy.     

Effects of Nutrition on Desiccation Tolerance and Virulence of Colletotrichum truncatum and Alternaria alternata Conidia

The promising mycoherbicides Colletotrichum truncatum and Alternaria alternata were grown respectively in liquid and solid semi-defined media. C. truncatum conidia produced in a medium with a C:N ratio of 5:1 showed higher desiccation tolerance (survival during storage) at 15% relative humidity and 25°C, greater germination on the host leaf and greater disease expression on Sesbania exaltata than those produced in media with C:N ratios of 15:1 or 40:1. Similar results were obtained with conidia of A. alternata produced on a medium with a C:N ratio of 15:1. Conidia washed with 0.9% (w/v) NaCl produced higher tolerance to desiccation, and greater disease incitement, than unwashed conidia of C. truncatum or conidia washed with water. In contrast, washing had no positive effect on desiccation tolerance in A. alternata .     

Interaction of Synthetic Dipeptide Bestim with Mouse Thymocytes and Macrophages

Tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. [³H]bestim was found to bind with high affinity to mouse peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM) and also plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM respectively). The specific bonding of [³H]bestim with macrophages and thymocytes was inhibited by unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM respectively). Treatment of the macrophages and thymocytes with trypsin led to their loss of capacity to bind [³H]bestim. Bestim at concentrations range of 0.1–1000 nМ reduced the adenylate cyclase activity in macrophage and thymocyte membranes.     

土拉弗朗西斯菌与巨噬细胞膜的早期相互作用

评估土拉弗朗西斯菌LVS在感染鼠巨噬细胞早期与细胞膜的相互作用。用表达GFP的土拉弗朗西斯菌LVS感染鼠巨噬细胞1774A1。结合单抗的小窝蛋白-1或转铁蛋白受体-1分别用键合了Alexa594的羊抗鼠二抗显色。土拉弗朗西斯菌疫苗株LVS可以诱导宿主细胞膜伸出伪足,将细菌吸收进入巨噬细胞。分布在细胞膜上的小窝蛋白-1和转铁蛋白受体-1参与巨噬细胞对弗朗西斯菌的摄入。这些发现说明,弗朗西斯菌进入巨噬细胞需要细胞膜微结构域和小窝蛋白;在感染早期转铁蛋白受体-1参与了细菌的摄入,这可能与弗朗西斯菌获取铁以利在胞内生存有关。     

RASSF6 Expression in Adipocytes Is Down-Regulated by Interaction with Macrophages

Macrophage infiltration into adipose tissue is associated with obesity and the crosstalk between adipocytes and infiltrated macrophages has been investigated as an important pathological phenomenon during adipose tissue inflammation. Here, we sought to identify adipocyte mRNAs that are regulated by interaction with infiltrated macrophages in vivo. An anti-inflammatory vitamin, vitamin B6, suppressed macrophage infiltration into white adipose tissue and altered mRNA expression. We identified >3500 genes whose expression is significantly altered during the development of obesity in db/db mice, and compared them to the adipose tissue mRNA expression profile of mice supplemented with vitamin B6. We identified PTX3 and MMP3 as candidate genes regulated by macrophage infiltration. PTX3 and MMP3 mRNA expression in 3T3-L1 adipocytes was up-regulated by activated RAW264.7 cells and these mRNA levels were positively correlated with macrophage number in adipose tissue in vivo. Next, we screened adipose genes down-regulated by the interaction with macrophages, and isolated RASSF6 (Ras association domain family 6). RASSF6 mRNA in adipocytes was decreased by culture medium conditioned by activated RAW264.7 cells, and RASSF6 mRNA level was negatively correlated with macrophage number in adipose tissue, suggesting that adipocyte RASSF6 mRNA expression is down-regulated by infiltrated macrophages in vivo. Finally, this study also showed that decreased RASSF6 expression up-regulates mRNA expression of several genes, such as CD44 and high mobility group protein HMGA2. These data provide novel insights into the biological significance of interactions between adipocytes and macrophages in adipose tissue during the development of obesity.     

Conidia of Alternaria in the atmosphere of the city of Cordoba, Spain in relation to meteorological parameters

In this study, we have analyzed the presence of conidia belonging to different species of the genus Alternaria in the atmosphere of the city of Cordoba, using a Hirst sampler. The results show that spores of this genus are present all year, with a clear seasonal pattern which shows two peaks, one in spring and the other in fall. A total of 26,822 conidia/m³ have been sampled, which implies a daily mean of 74.3 conidia/m³. Statistical analyses comparing the data with meteorological parameters show a positive correlation with maximum, minimum and mean temperatures, and a negative correlation with rain. Nevertheless, meteorological parameters seem to affect the number of conidia differently according to the season of the year. Regression analyses carried out in order to obtain a predictive pattern show that the best fit is between the 7-day running mean of the number of conidia and a week’s accumulated mean temperature. Received: 8 April 1998 / Accepted: 25 February 1999     

Early Interaction of Rhinoviruses with Host Cells

The rate of attachment of type 2 virions to suspensions of HeLa cells is much greater than that of type 14, but the number of receptor sites per cell is similar for each type. The receptor sites may be partly saturated with excess virions; attachment is greatly reduced after about 10(4) particles have been taken up per cell. A lack of saturation of type 14 receptors by excess type 2 indicates that their receptor sites are separate on the cell surface. Excess of type 2 blocks attachment of type 1A, however, and excess of type 14 blocks type 51. Attachment of the human rhinoviruses is temperature-dependent with a Q(10) of 2.7. The eclipse reaction is also temperature-dependent. At 34.5 C, the irreversible eclipse of cell-associated rhinovirus type 2 requires only a few minutes, whereas the rate of eclipse of cell-associated type 14 is considerably slower. The eclipse product of type 2 rhinovirus has been recovered from infected cells. It sediments at about 90% of the rate of the infective virions and is missing virus polypeptide 4 (the smallest of the capsid polypeptides). Upon being subjected to CsCl gradient centrifugation, virus polypeptide 2 is also lost but the product still contains ribonucleic acid and bands at about 1.45 g/cc.     

The Interaction of Acanthamoeba spp. with Activated Macrophages and with Macrophage Cell Lines

Acanthamoeba spp. are free-living amebae associated with amebic keratitis and chronic granulomatous amebic encephalitis. The present studies were undertaken to compare the pathogenicity of three species of Acanthamoeba in B₆C₃F₁ mice after intranasal challenge with Acanthamoeba-induced cytopathogenicity for different macrophage populations. The ability of murine macrophage cell lines and activated murine peritoneal macrophages to lyse Acanthamoeba has been assessed by coincubating macrophages with ³H-uridine labeled amebae. Conversely, destruction of macrophages by Acanthamoeba was determined by measuring the release of chro-mium-51 from radiolabeled macrophages. Acanthamoeba culbensoni , which is highly pathogenic for mice, destroys macrophage cultures in vitro. Activated primary peritoneal macrophages were more resistant to Acanthamoeba -mediated destruction than macrophage cell lines activated in vitro. Activated macrophages were capable of limited destruction of Acanthamoeba polyphaga and Acanthamoeba castellanii. Acanthamoeba -specific antibodies increased the amebicidal activity of activated macrophages. Macrophage-mediated destruction was by contact-dependent cytolysis and by ingestion of amebae. Conditioned medium obtained from macrophage cultures after treatment with lipopolysaccharide and interferon gamma was neither cytolytic nor cytostatic for Acanthamoeba spp. Purified recombinant cytokines including tumor necrosis factor α. interleukin 1α, and interleukin 1β, alone or in combination, were not cytolytic for Acanthamoeba trophozoites.     

Characterization of Alternaria isolates from the infectoria species-group and a new taxon from Arrhenatherum,Pseudoalternaria arrhenatheria sp. nov.

The infectoria species-group within the genus Alternaria was originally conceived by Simmons in 1993 and was based upon common morphological characteristics that included the development of conidial chains with primary, secondary, and tertiary branching resulting in substantial three-dimensional complexity. These characters can overlap to varying degrees with numerous taxa in another Alternaria group, the alternata species-group, making species-group differentiation difficult. However, members of the infectoria species-group are also distinguished from other small-spored Alternaria species based upon colony characteristics that typically include white or nearly white floccose colonies on DRYES medium and clumps of sporulation islands on low sugar media such as V8 agar, PCA, and weak PDA. In addition, the infectoria species-group contains representatives that are known to produce teleomorphs (Lewia), whereas the members of the alternata species-group and other Alternaria species-groups are strictly asexual. In this study, an assemblage of isolates recovered from varied hosts from the west coast of the United States were examined based upon morphological characters and compared to previously described members of the infectoria species-group. These isolates and members of the infectoria species-group typically produce arachnoid vegetative hyphae with multiple primary conidiophores, whereas other small-spored Alternaria species produce primary conidiophores predominately directly from the agar surface. Additionally, molecular phylogenetic analyses resolved these isolates and members of the infectoria species-group as distinctly nested amongst other sexual taxa in Allewia (Embellisia anamorph) and Macrospora (Nimbya anamorph) and phylogenetically distant to asexual lineages of Alternaria. One taxon among these isolates was novel and clustered with the asexual A. rosae in a distinct clade basal to all other members of the infectoria species-group. A new genus is proposed, Pseudoalternaria gen. nov. and a new taxon is described, Pseudoalternaria arrhenatheria sp. nov.. Moreover, a second taxon is reclassified, Pseudoalternaria rosae comb. nov.