Occurrence of subcutaneous zygomycosis caused by Basidiobolus haptosporus in Brazil

There were described the first three South American cases of subcutaneous zygomycosis caused by B. haptosporus. The patients were children from nearby towns lying just north of 13 ° latitude S. The diagnosis was based on histopathological aspects plus cultural isolation of the fungus.     

First culture-proven gastrointestinal entermophthoromycosis in the United States: A case report and review of the literature

The zygomycoses are fungal infections often occurring in compromised hosts. We report the first culture-proven case of a gastrointestinal infection in the United States by Basidiobolus haptosporus (ranarum). The clinical and histological features are noted in order to distinguish this infection from the more widely reported mucormycosis.     

Gastrointestinal entomophthoramycosis caused by Basidiobolus haptosporus

There was described the first cultural proven case of gastrointestinal entomophthoramycosis caused by B. haptosporus. A review of the literature on gastrointestinal zygomycosis led us to presume that similar clinical reported cases have been also caused by this fungus. A commentary on the designation of the clinical forms of the infections caused by Zygomycetes was also made.Reprints request (Dr. Londero) Tuiuti 1809/201, 97100 Santa Maria, RS, Brazil.     

Occurrence of subcutaneous zygomycosis (Entomophthoromycosis Basidiobolae) caused by Basidiobolus haptosporus with pulmonary involvement

A case of a two-year-old boy with multiple subcutaneous lesions caused by Basidiobolus haptosporus is presented.The child had also a non-toxic familial goiter and clinical and radiological features of a pulmonary illness. The pulmonary manifestations only disappeared with the treatment with potassium iodide. The authors think that the pulmonary lesions must have arisen by direct spread of the fungus from the subcutaneous lesions of the chest.     

A fungal chitinase gene fromRhizopus oligosporus confers antifungal activity to transgenic tobacco

We have studied whether a chitinase involved in cell autolysis of a filamentous fungus,Rhizopus oligosporus, can operate as an antifungal defense system in tobacco. Thechi1 gene was introduced into tobacco by theAgrobacterium tumefaciens leaf disc system. Among 22 transgenic tobacco plants, 2 were selected and their individual homozygous progeny, Tch1-1 and Tch2-1, were studied. Chitinase activity in the extracts of young leaves from Tch1-1 or Tch2-1, in which thechi1 gene product was detected by Western blot analysis, was three- to four-fold higher than that from the control plants. A fungal infection assay on the leaves infected with the discomycete pathogensSclerotinia sclerotiorum andBotrytis cinerea revealed that the symptoms observed with these two were remarkably suppressed as compared with the control leaves.Abbreviations CaMV Cauliflower mosaic virus - Km ^r kanamycin resistant - Km ^s kanamycin sensitive - MS Murashige and Skoog - PCR polymerase chain reaction - PDA potato dextrose agar - PR pathogenesis-related     

Extracellular lipase and proteinase of Basidiobolus haptosporus: Possible role in subcutaneous mycosis

Properties of proteinase(s) and phospholipase A in dialyzed culture filtrate of Basidiobolus haptosporus were studied. Lysolecithin, one of the hydrolytic products of phosphatidyl choline by phospholipase A was prepared and found able to hemolyse human red blood cells and clear rat skin and muscle homogenates. The proteinase was able to digest human serum proteins. The pathogenic mechanism employed by this fungus may involve hydrolysis of lecithin to yield lysolecithin, which destroys cell membranes thereby liberating intracellular contents. The protein components of these contents are then digested by the proteinase(s) yielding amino acids which along with other metabolites serve as nutrients for the growth of the pathogen.     

Role of Humoral Antibodies in Resistance to Mycoplasma pneumoniae Pneumonia in Hamsters

Golden Syrian hamsters adoptively immunized with hyperimmune Mycoplasma pneumoniae rabbit antiserum, immunoglobulin (Ig) M-rich (IgM) fraction, IgG-rich (IgG) fraction, antiserum absorbed with either killed M. pneumoniae or killed Staphylococcus aureus organisms, or antiserum treated with 2-mercaptoethanol (2-ME) were examined for resistance against aerosol infection with virulent M. pneumoniae. Significant resistance to the establishment of infection in the respiratory tract was shown in hamsters pretreated with the untreated antiserum, IgG fraction or 2-ME-treated antiserum, whereas animals pretreated with the IgM fraction and the antisera absorbed with M. pneumoniae or S. aureus organisms were not significantly resistant. Histopathologically, lung lesions were markedly suppressed in animals with high resistance, but were typically pneumonic in animals with low or no resistance. The efficacy of adoptively administered serum preparations was closely related to their antibody titers. The results indicate that humoral antibody plays an important role in protection against experimental M. pneumoniae pneumonia in hamsters, although the participation of the cell-mediated immune response was not determined.     

Differential plant response to inoculation with two VA mycorrhizal fungi isolated from a low-pH soil

Sericea lespedaza and ladino clover were inoculated withAcaulospora laevis andGigaspora margarita VA mycorrhizal fungi, both isolated from a local soil having a pH of 4.4. Plants were grown in a greenhouse in fumigated (methyl bromide) soil with four rates of applied P. This soil had a pH of 5.1 for theG. margarita experiment and a 5.8 for theA. laevis experiment. Neither plant species responded to theG. margarita isolate in terms of mycorrhizal infection of roots, top growth, or elemental uptake. TheA. laevis isolate caused increased growth of Ladino clover at the lowest rate of P application and increased growth of sericea lespedeza at the two lowest P application rates. Shoot tissue concentrations of P for both plant species were greater at the two lowest rates of P application when inoculated withA. laevis. Inoculation withA. laevis also resulted in different VAM fungal root colonization patterns between the two plant species as a function of P application rate. Roots of both plants had high infection rates (near 70%) for the two lowest P application rates but sericea lespedeza declined to 40 and 6% at the next two P application rates, respectively, whereas infection in Ladino clover was 74 and 41% at the next two P application rates, respectively. The results of this study support the concept that plant host-VAM fungal endophyte interactions are extremely variable and that characterization of individual combinations must be done if types and magnitudes of responses are to be defined.     

Larval Mortality Factors of <Emphasis Type="Italic">Spodoptera Littoralis</Emphasis> in the Azores

Mortality among larval developmental stages of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), was determined by weekly sampling on weeds in a pasture on São Miguel Island (Azores, Portugal), from August to December, over a 3-year period (1999–2001). In all the years surveyed, larvae of S. littoralis usually appeared in pastures after the third week of August, with higher abundances in September and the beginning of October. Three different factors causing larval mortality were identified: one fungal pathogen, Furia virescens (Thaxter) Humber (Zygomycetes: Entomophthoraceae), two nucleopolyhedroviruses and one larval parasitoid, Meteorus communis (Cresson) (Hymenoptera: Braconidae). The percentages of dead larvae infected by virus or fungus were significantly higher than the other causes of mortality, regardless of the year. Furthermore, the percentage of larvae that died due to virus contamination was generally higher than the percentage of larvae infected by fungus. Significant correlations between the environmental factors and the percentage of larvae infected by virus or by fungus, were only observed during 2001. In 2001, the prevalence of fungal infection was negatively correlated with that of viral infection although prevalences of these two agents were positively correlated in both 1999 and 2000. These results show that virus and fungus are potential biological control agents for S. littoralis in Azores.     

Mycovirus effect on the endophytic establishment of the entomopathogenic fungus Tolypocladium cylindrosporum in tomato and bean plants

Two endophytic strains of the entomopathogenic fungus Tolypocladium cylindrosporum, originally isolated from the grass Festuca rubra, were artificially inoculated in tomato and bean plants. Strains 11-1L and 11-0BR were isolated from asymptomatic leaf fragments of both plant species at 3, 7, 14, 21, and 35 days after their inoculation. The percentage of leaf fragments infected by the fungus in inoculated leaves decreased at each sampling time, and no systemic colonization of the plants occurred. The two T. cylindrosporum strains tested were isogenic, differing in the infection by the victorivirus TcV1, harboured by strain 11-1L, but not by 11-0BR. The percentage of infected leaf fragments in leaves inoculated with the virus infected strain was greater in bean than in tomato plants, while the virus-free strain was more successful in tomato than in bean plants. This result suggests that the mycovirus infection can affect the adaptation of T. cylindrosporum to particular host plants.     

The effects of dsRNA mycoviruses on growth and murine virulence of Aspergillus fumigatus

Some isolates of the opportunistic human pathogenic fungus Aspergillus fumigatus are known to be infected with mycoviruses. The dsRNA genomes of two of these mycoviruses, which include a chrysovirus and a partitivirus, have been completely sequenced and an RT-PCR assay for the viruses has been developed. Through curing virus-infected A. fumigatus isolates by cycloheximide treatment and transfecting virus-free isolates with purified virus, as checked by RT-PCR, isogenic virus-free and virus-infected lines of the fungus were generated whose phenotypes and growth have been directly compared. Mycovirus infection of A. fumigatus with either the chrysovirus or the partitivirus resulted in significant aberrant phenotypic alterations and attenuation of growth of the fungus but had no effect on susceptibility to common antifungals. Chrysovirus infection of A. fumigatus caused no significant alterations to murine pathogenicity.     

Stabilisation of some of the protein synthesis components in the thermophilic fungus,Humicola lanuginosa

The thermal stabilities of tRNA from the thermophilic fungus,Humicola lanuginose were compared with that from the mesophilic yeast,Candida utilis, by measuring the increase in the optical density with temperature. tRNAs from both the species were stable in the presence of millimolar quantities of magnesium chloride upto 50°C, the optimum growth temperature of the fungus. Aminoacyl tRNA synthetases were maximally active at 40°C under thein vitro assay conditions. They were fractionated and one species of valine tRNA synthetase was purified to homogeneity. The purified enzyme was protected against inactivation to varying degrees when preincubated with the substrates valine, tRNA and ATP as well as spermine. Protein turnover studies showed that the rate of turnover was higher at higher temperatures. It was concluded from these results that the protein synthesizing machinery of this fungus has no intrinsic stability but it is stabilised by intracellular factors. Higher rate of protein turnover also plays a role for growth at higher temperature.     

Fungal associations of roots of dominant and sub-dominant plants in high-alpine vegetation systems with special reference to mycorrhiza

Summary Types of root infection were analysed in healthy dominant and sub-dominant plants of zonal and azonal vegetation above the timberline in the Central and Northern Calcareous Alps of Austria. In the open nival zone vegetation, infection by fungi of the Rhizoctonia type was predominant, vesicular-arbuscular mycorrhizal infection, which was mostly of the fine endophyte (Glomus tenuis) type, being light and mainly restricted to grasses in closed vegetation patches. More extensive Glomus tenuis infection was found in the alpine grass heath, but in Carex, Rhizoctonia was again the most important fungus. The ericaceous plants of the dwarf shrub heath have typical ericoid infection, but quantitative analysis reveals a decrease of infection intensity with increase of altitude. The possible function of the various types of root infection are discussed, and the status of Rhizoctonia as a possible mycorrhizal fungus is considered.     

Antifungal Activity of the Essential Oil of Basil (Ocimum basilicum)

The antifungal and fungicidal effects of two chemotypes of basil (Ocimum basilicum) oil and its major individual components were studied in a series of in vitro and in vivo experiments. Mycelial growth of the plant pathogenic fungus Botrytis fabae was reduced significantly by both the methyl chavicol chemotype oil and the linalol chemotype oil, and the major individual components of the oils all reduced fungal growth, with methyl chavicol, linalol, eugenol and eucalyptol reducing growth significantly. Combining the pure oil components in the same proportions as found in the whole oil led to very similar reductions in fungal growth, suggesting that the antifungal effects of the whole oils were due primarily to the major components. When the fungus was exposed to the oils in liquid culture, growth was reduced by concentrations considerably smaller than those used in the Petri dish studies. Botrytis fabae and the rust fungus Uromyces fabae were also controlled in vivo, with the whole oils of both chemotypes, as well as pure methyl chavicol and linalol, reducing infection of broad bean leaves significantly. Most effective control of fungal infection was achieved if the treatments were applied 3 h postinoculation.     

Susceptibility to a mouse acquired immunodeficiency syndrome is influenced by theH-2

The development of a mouse acquired immunodeficiency syndrome (MAIDS) induced following LP-BM5 MuLV infection depends on host genetic factors. Susceptible mice, such as C57BL/6J mice, develop a profound impairment of lymphoproliferative response to mitogens and hyperplasia of lymphoid organs and succumb to infection within 6 months. These changes do not occur in resistant mice, such as A/J mice. Resistance to MAIDS is a dominant trait since (C57BL/6JxA/J)F₁ hybrid mice did not develop any immune dysfunctions following infection. Genetic regulation of the trait of resistance/susceptibility to MAIDS was determined in AXB/BXA recombinant inbred (RI) mouse strains (derived from resistant A/J and susceptible C57BL/6J progenitors). Two different criteria were used to determine their resistance or susceptibility to developing MAIDS: the gross pathologic evaluation of lymphoid organs at 13–15 weeks of infection, and survival. RI mouse strains segregated into two non-overlapping groups. The first group did not develop any significant pathology, and these mouse strains were considered as resistant to MAIDS. The second group showed the virus-induced pathological changes as well as an immunological dysfunction as seen in C57BL/6J progenitor mice, and these strains were thus considered as susceptible to MAIDS. This bimodal strain distribution pattern of resistance/susceptibility to MAIDS among the RI strains suggests that this phenotype is controlled by a single gene. Linkage analysis with other allelic markers showed a strong association between resistance/susceptibility to MAIDS and theH-2 complex. Possession of theH-2 ^b haplotype derived from C57BL/6J mice was associated with susceptibility to MAIDS, while theH-2 ^a haplotype conferred resistance to the disease. This finding was confirmed by demonstrating thatH-2 ^a congenics on the susceptible C57BL/10 background were as resistant to MAIDS as A/J mice which donated theH-2 ^a locus. Gene(s) within theH-2 complex thus represent the major regulatory mechanism of resistance/susceptibility to MAIDS.     

Effect of Placement and Sequence of Inoculation with Fusarium oxysporum on its Interaction with Meloidogyne arenaria on Subterranean Clover

The effect of the placement of inoculum of Fusarium oxysporum at two soil depths, and the sequences of inoculations with Meloidogyne arenaria and Fusarium oxysporum on root growth and development of root disease in Trifolium subterraneum L. (subterranean clover) were investigated. The timing of infection and the proximity of root tips of the host root system to infection by M. arenaria and F. oxysporum appeared to be the major determining factors of root growth and of disease development in plants exposed to the pathogens. Immediate contact of roots with F. oxysporum (where the fungus was placed at seed level of 10 mm depth) appeared to result in more severe effects on roots in the presence of the nematode than later infection by the fungus placed at 30 mm depth. The production of galls by the nematode and early infection by F. oxysporum at 10 mm depth resulted in a severe inhibition of root growth, particularly of the lateral roots. But no such growth inhibition was evident when F. oxysporum and M. arenaria were introduced together at the lower depth of 30 mm. The lowest density of M. arenaria inoculum was sufficient to cause severe root rot if F. oxysporum was present at the host seed level. With the fungus at 30 mm depth, however, the expression of root rot appeared to be influenced by the inoculum level of the nematode. In sequential inoculation with F. oxysporum or M. arenaria, the organism added 2 weeks later had little or no effect on root development. The first organism (M. arenaria or F. oxysporum) to infect the germinated seedlings was the main cause of root growth inhibition. The organism that came into contact with the roots 2 weeks later had little or no effect on the roots. Concurrent infection by F. oxysporum and M. arenaria resulted in less M. arenaria gall production on the tap root system than those added with the nematode alone or in advance of the fungus.     

Aberrant alterations of the expressions and S-nitrosylation of calmodulin and the downstream factors in the brains of the rodents during scrapie infection

The aberrant alterations of calmodulin (CaM) and its downstream substrates have been reported in some neurodegenerative diseases, but rarely described in prion disease. In this study, the potential changes of Ca²⁺/CaM and its associated agents in the brains of scrapie agent 263K-infected hamsters and the prion infected cell line SMB-S15 were evaluated by various methodologies. We found that the level of CaM in the brains of 263K-infected hamsters started to increase at early stage and maintained at high level till terminal stage. The increased CaM mainly accumulated in the regions of cortex, thalamus and cerebellum of 263K-infected hamsters and well localization of CaM with NeuN positive cells. However, the related kinases such as total and phosphorylated forms of CaMKII and CaMKIV, as well as the downstream proteins such as CREB and BDNF in the brain of 263K-infected hamsters were decreased. Further analysis showed a remarkable increase of S-nitrosylated (SNO) form of CaM in the brains of 263K-infected hamsters. Dynamic analysis of S-nitrosylated CaM showed the SNO form of CaM abnormally increases in a time-dependent manner during prion infection. Compared with that of the normal partner cell line SMB-PS, the CaM level in SMB-S15 cells was increased, meanwhile, the downstream proteins, such as CaMKII, p-CaMKII, CREB, as well as BDNF, were also increased, especially in the nucleic fraction. No SNO-CaM was detected in the cell lines SMB-S15 and SMB-PS. Our data indicate an aberrant increase of CaM during prion infection in vivo and in vitro.     

Leptographium pini-densiflorae sp. nov. from Japanese red pine

ALeptographium species was isolated from deadPinus densiflora at six sites in Japan. The fungus is morphologically most similar toL. lundbergii but could be distinguished from that species by its short stipes, primary branches of conidiophores, and conidia with a rounded to sub-truncate base. In addition, the colony morphology, growth rate and tolerance to the antibiotic cycloheximide of theLeptographium species andL. lundbergii differed markedly. Here we describe the fungus as a new species,Leptographium pini-densiflorae. Contribution No. 144, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Efficient gene targeting in the filamentous fungusAlternaria alternata

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1^–) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy^R) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1^– transformants accounted for only 5% of Hy^R transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1^–. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.     

Transformation of a filamentous fungus<Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> using<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1×10⁶ conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.