Immuno-identification of cultures of fungi pathogenic to man

Because isolates of the fungal pathogensBlastomyces dermatitidis. Coccidioides immitis, Histoplasma capsulatum, andParacoccidioides brasiliensis frequently vary widely in gross and microscopic features and are often difficult or impossible to convert to their tissue forms, a simple diagnostic procedure not dependent upon sporulation is needed to identify them specifically and rapidly. The exoantigen technique has been found to meet this need effectively. On the basis of studies with 166Histoplasma spp. isolates, 128C. immitis isolates, 59B. dermatitidis isolates, 30P. brasiliensis isolates, and 181 saprophytes, we determined that the exoantigen test is valuable for the presumptive identification of the four fungal pathogens studied. All of the positive reactions have correlated with the cultural, histologic, or other available laboratory data, and we are unaware of any false positive or flase negative reactions.     

Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution

Dual biological control, of both insect pests and plant pathogens, has been reported for the fungal entomopathogens, Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) and Lecanicillium spp. (Ascomycota: Hypocreales). However, the primary mechanisms of plant disease suppression are different for these fungi. Beauveria spp. produce an array of bioactive metabolites, and have been reported to limit growth of fungal plant pathogens in vitro. In plant assays, B. bassiana has been reported to reduce diseases caused by soilborne plant pathogens, such as Pythium, Rhizoctonia, and Fusarium. Evidence has accumulated that B. bassiana can endophytically colonize a wide array of plant species, both monocots and dicots. B. bassiana also induced systemic resistance when endophytically colonized cotton seedlings were challenged with a bacterial plant pathogen on foliage. Species of Lecanicillium are known to reduce disease caused by powdery mildew as well as various rust fungi. Endophytic colonization has been reported for Lecanicillium spp., and it has been suggested that induced systemic resistance may be active against powdery mildew. However, mycoparasitism is the primary mechanism employed by Lecanicillium spp. against plant pathogens. Comparisons of Beauveria and Lecanicillium are made with Trichoderma, a fungus used for biological control of plant pathogens and insects. For T. harzianum Rifai (Ascomycota: Hypocreales), it has been shown that some fungal traits that are important for insect pathogenicity are also involved in biocontrol of phytopathogens.     

Tinea capitis in the adult: Two case studies

Ascites fluid from hybridoma cell lines producing monoclonal antibodies to an exoantigen of Microsporum canis was assayed for its precipitating properties. Three types of monoclonal antibodies were used for serological analysis of heterologous mycelial cultures. Based on specific antigenic determinants, the fungal strains showed different immunodiffusion reactivity within the species. The monoclonal antibodies also proved to be effective reagents for serotyping heterogeneous species. They may also have potential applications in epidemiology.     

In vitro Antagonism of Rhizobacteria Isolated from Coffea arabica L. against Emerging Fungal Coffee Pathogens

In vitro antagonistic effects of rhizobacteria associated with Coffea arabica L. against some fungal coffee pathogens were studied. The aims were to screen indigenous coffee‐associated isolates for their inherent antagonistic potential against major coffee wilt diseases induced by Fusarium spp. Antagonistic effects, siderophore, HCN and lytic enzyme production were determined on standard solid media. Chemical methods were employed to categorize the major types of siderophores. From a total of 212 rhizobacterial isolates tested, over 10 % (all Pseudomonas and Bacillus spp.) exhibited remarkable inhibition against Fusarium spp. One isolate AUPB24 (P. chlororaphis) showed maximum inhibition of mycelial growth against all fungal pathogens tested, whereas other isolates were mostly inhibitory to F. stilboides and F. oxysporum. The isolate AUBB20 (B. subtilis) was most antagonistic to F. xylarioides. Of the rhizobacterial isolates tested, 67 % produced siderophores and 35 % produced HCN. Many strains (all Pseudomonas spp.) produced siderophores of the hydroxamate type and only a small proportion produced those of the catecholate type. Few antagonists showed chitinase activity. The production of siderophores and HCN by Pseudomonas spp., lipase and protease by all antagonists and β‐1,3‐glucanase by several Bacillus spp. could be considered the major mechanisms involved in the inhibition of fungal growth. The in vitro results provide the first evidence of an antagonistic effect of coffee‐associated rhizobacteria against the emerging fungal coffee pathogens F. stilboides and F. xylarioides and indicate the potential of both bacterial groups for biological control of coffee wilt diseases.     

Occurrence of Fungal Pathogens Associated with Grapevine Nurseries and the Decline of Young Vines in Spain

The occurrence of fungal grapevine trunk pathogens associated with grapevine nurseries and the decline of young vines in Spain was determined in extensive surveys conducted in nurseries and vineyards with young plants. The presence of Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Cylindrocarpon spp., Botryosphaeria obtusa and Botryosphaeria spp. was detected in all the surveys that were carried out. This study provides evidence for the presence of these pathogens in Spanish grapevine nurseries. Cylindrocarpon spp., B. obtusa and Botryosphaeria spp., were isolated very early in the planting material production process, nevertheless, P. aleophilum and P. chlamydospora were not detected until the rootstock–scion combinations were planted in the field to develop shoots and roots during summer. The occurrence of trunk disease pathogens in symptomatic young vineyards was also high, suggesting that infected plant material might have been used. Phaeomoniella chlamydospora and P. aleophilum were the main species associated with the decline of young vines, followed by Botryosphaeria spp., Cylindrocarpon spp. and B. obtusa. This research confirmed the importance of fungal grapevine trunk pathogens associated with the decline of young vineyards in Spain and suggested the connection between the presence of these fungi in the nurseries and the disease in the fields.     

Delayed-type Hypersensitivity Response to Crude and Fractionated Antigens from Fonsecaea pedrosoi CMMI 1 Grown in Different Culture Media

Chromoblastomycosis is a subcutaneous fungal disease caused by dematiaceous fungi, especially by Fonsecaea pedrosoi, regarded as its major causative agent in Brazil. In recent years there has been a decline in the use of skin testing for delayed-type hypersensitivity (DTH) in epidemiological surveys of fungal infections, mainly because of the unpredictability of positive reactions and lack of specificity of the antigens used. The aim of the present study was to assess delayed-type skin tests in guinea pigs experimentally infected with F. pedrosoi using exoantigens prepared from two culture filtrates. Sixteen adult male guinea pigs were inoculated intratesticularly with fungal cells and submitted to sensitivity assays 4 weeks after inoculation. They received an intradermal injection with crude and fractionated antigens from Alviano’s and Smith’s cultures, and were assessed 24 and 48 h thereafter. Except for one animal, all of them had positive indurations after 48 h. There were no statistical differences between the measurements at 24 and 48 h for each exoantigen used, neither among the induration measurements at 48 h when different preparations were compared. Our results suggest that a delayed-type skin test using antigens produced in synthetic media may be useful for the assessment of primary exposure to chromoblastomycosis.     

Saponin Glycosylation in Cereals

Triterpene saponins are glycosylated plant secondary metabolites that are common in dicotyledonous species but rare in monocots. These compounds are antimicrobial and are important for plant defence. They also have a variety of pharmaceutical and industrial applications. Oat (Avena spp.) produces triterpene saponins called avenacins, specialised metabolites that are not represented in other cereals. The avenacins are synthesised and stored in roots and provide protection against soil-borne fungal pathogens. Glycosylation is often critical for bioactivity of saponins. We are using a combination of mutant analysis, database mining, expression profiling and functional characterisation to identify glycosyltransferases required for addition of sugar units to avenacins. By identifying the molecular machinery required for avenacin biosynthesis we hope to understand the broader elements of the evolution of specialised metabolic pathways.     

Fungi associated with storage rots of cocoyams (Colocasia spp.) in Nsukka,Nigeria

Cocoyam (Colocasia spp.) corms and cormels showing spoilage symptoms were collected from many stores in Nsukka locality and examined for rot and associated fungal pathogens. Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum, and F. solani were recovered from rotten cocoyams. The representative isolates of these species caused cocoyam rot in pathogenicity tests. The rot due to A. niger, B. theobromae and C. rolfsii was extensive resulting in complete maceration of cocoyam tissue. Potassium sorbate (0.1 mg/ml) protected cocoyams from fungal rot with the exception of C. rolfsii.     

Exploring antifungal activities of acetone extract of selected Indian medicinal plants against human dermal fungal pathogens

A broad spectrum of medicinal plants was used as traditional remedies for various infectious diseases. Fungal infectious diseases have a significant impact on public health. Fungi cause more prevalent infections in immunocompromised individuals mainly patients undergoing transplantation related therapies, and malignant cancer treatments. The present study aimed to investigate the in vitro antifungal effects of the traditional medicinal plants used in India against the fungal pathogens associated with dermal infections. Indian medicinal plants (Acalypha indica, Lawsonia inermis Allium sativum and Citrus limon) extract (acetone/crude) were tested for their antifungal effects against five fungal species isolated from skin scrapings of fungal infected patients were identified as including Alternaria spp., Curvularia spp., Fusarium spp., Trichophyton spp. and Geotrichum spp. using well diffusion test and the broth micro dilution method. All plant extracts have shown to have antifungal efficacy against dermal pathogens. Particularly, Allium sativum extract revealed a strong antifungal effect against all fungal isolates with the minimum fungicidal concentration (MFC) of 50–100 μg/mL. Strong antifungal activity against Curvularia spp., Trichophyton spp., and Geotrichum spp. was also observed for the extracts of Acalypha indica, and Lawsonia inermis with MFCs of 50–800 μg/mL respectively. The extracts of Citrus limon showed an effective antifungal activity against most of the fungal strains tested with the MFCs of 50–800 μg/mL. Our research demonstrated the strong evidence of conventional plants extracts against clinical fungal pathogens with the most promising option of employing natural-drugs for the treatment of skin infections. Furthermore, in-depth analysis of identifying the compounds responsible for the antifungal activity that could offer alternatives way to develop new natural antifungal therapeutics for combating resistant recurrent infections.     

The Relationship Between Gorgonian Coral (Cnidaria: Gorgonacea) Diseases and African Dust Storms

The number of reports of coral diseases has increased throughout the world in the last 20 years. Aspergillosis, which primarily affects Gorgonia ventalina and G. flabellum, is one of the few diseases to be characterized. This disease is caused by Aspergillus sydowii, a terrestrial fungus with a worldwide distribution. Upon infection, colonies may lose tissue, and ultimately, mortality may occur if the infection is not sequestered. The spores of A. sydowii are <5 m, small enough to be easily picked up by winds and dispersed over great distances. Aspergillosis is prevalent in the Caribbean, and it appears that this primarily terrestrial fungus has adapted to a marine environment. It has been proposed that dust storms originating in Africa may be one way in which potential coral pathogens are distributed and deposited into the marine environments of the Caribbean. To test the hypothesis that African dust storms transport and deposit pathogens, we collected air samples from both dust storms and periods of non-dust in St. John, U.S. Virgin Islands. Because we focused on fungal pathogens and used A. sydowii as a model, we isolated and cultured fungi on various types of media. Fungi including Aspergillus spp. were isolated from air samples taken from dust events and non-dust events. Twenty-three separate cultures and seven genera were isolated from dust event samples whereas eight cultures from five genera were isolated from non-dust air samples. Three isolates from the Virgin Islands dust event samples morphologically identified as Aspergillus spp. produced signs of aspergillosis in seafans, and the original pathogens were re-isolated from those diseased seafans fulfilling Koch's Postulates. This research supports the hypothesis that African dust storms transport across the Atlantic Ocean and deposit potential coral pathogens in the Caribbean.     

Moths that Vector a Plant Pathogen also Transport Endophytic Fungi and Mycoparasitic Antagonists

Claviceps paspali, a common fungal pathogen of Paspalum grasses, attracts moth vectors by producing sugary exudates in the grass florets it infects. These exudates also support mycoparasitic Fusarium species that may negatively influence C. paspali fitness. We examined the potential for moths on which C. paspali depends to also transmit mycoparasitic Fusarium and fungal endophytes, which inhabit asymptomatic plant tissue and may influence host susceptibility to pathogens. We quantified infections by C. paspali, Fusarium spp., and endophytic fungi associated with Paspalum spp. at focal sites in the southeastern USA and used data from the nuclear internal transcribed spacer (ITS rDNA) to compare communities of plant-associated and moth-borne fungi. ITS sequences of moth-borne fungi were identical to reference sequences of mycoparasitic Fusarium heterosporum and to three distinct endophytic fungi isolated from Paspalum species. Our results demonstrate an unexpected overlap of fungal communities between disparate locations and among plant species and plant tissues, and suggest an unexpected role of moths, which vector a plant pathogen, to transmit other guilds of fungi. In turn, the potential for insects to transmit plant pathogens as well as mycoparasites and endophytic fungi suggests complex interactions underlying a commonly observed grass–pathogen system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A rapid and specific method for the immunological identification of mycelial form cultures ofParacoccidioides brasiliensis

We developed two simple methods for extracting specific, cell-free, soluble antigens of the mold form ofParacoccidioides brasiliensis. Detection of these antigens by a microimmunodiffusion test permits the rapid and accurate identification of cultures ofP. brasiliensis. ThirtyP. brasiliensis isolates treated by these techniques produced specific exoantigens detectable by the immunodiffusion test. None of the other 78 fungal pathogens or saprophytes tested produced identical exoantigens. Personnel in any diagnostic laboratory who want a rapid and specific method for identifying or confirming suspected isolates ofP. brasiliensis can use the simple procedures described.     

Synergism between fungal enzymes and bacterial antibiotics may enhance biocontrol

The interactions between biocontrol fungi and bacteria may play a key role in the natural process of biocontrol, although the molecular mechanisms involved are still largely unknown. Synergism can occur when different agents are applied together, and cell wall degrading enzymes (CWDEs) produced by fungi can increase the efficacy of bacteria. Pseudomonas spp. produce membrane-disrupting lipodepsipeptides (LDPs) syringotoxins (SP) and syringomycins (SR). SR are considered responsible for the antimicrobial activity, and SP for the phytotoxicity. CWDEs of Trichoderma spp. synergistically increased the toxicity of SP_25-A or SR_E purified from P. syringae against fungal pathogens. For instance, the fungal enzymes made Botrytis cinerea and other phytopathogenic fungi, normally resistant to SP_25-A alone, more susceptible to this antibiotic. Pseudomonas produced CWDEs in culture conditions that allow the synthesis of the LDPs. Purified bacterial enzymes and metabolites were also synergistic against fungal pathogens, although this mixture was less powerful than the combination with the Trichoderma CWDEs. The positive interaction between LDPs and CWDEs may be part of the biocontrol mechanism in some Pseudomonas strains, and co-induction of different antifungal compounds in both biocontrol bacteria and fungi may occur. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biocontrol of aerial plant diseases in agriculture and horticulture: current approaches and future prospects

Until recently, the majority of research on the biological control of aerial plant diseases was focused on control of bacterial pathogens. Such research led to the commercialization of the biocontrol agent Pseudomonas fluorescens A506, as BlightBan A506™, for control of fire blight of pear. In contrast, chemical fungicides typically have provided adequate control of most foliar fungal pathogens. However, fungicide resistance problems, concerns regarding pesticide residues and revocation of registration of certain widely used fungicides have led to increased activity in the development of biocontrol agents of foliar fungal pathogens. Much of this activity has centered around the use of Trichoderma spp and Gliocladium spp to control Botrytis cinerea on grape and strawberry. The biocontrol agent Trichoderma harzianum T39 is commercially available in Israel, as Trichodex ™, for control of grey mold in grapes and may soon be registered for use in the US. Also targeted primarily against a foliar disease of grapes, in this case powdery mildew caused by Uncinula necator, is the biocontrol agent Ampelomyces quisqualis AQ10, marketed as AQ^{10  TM} biofungicide. Another promising development in the area of foliar disease control, though one which is not yet commercialized, is the use of rhizobacteria as seed treatments to induce systemic resistance in the host plant, a strategy which can protect the plant against a range of bacterial and fungal pathogens. Received 06 February 1997/ Accepted in revised form 05 June 1997     

Antigenic studies on Rhizopus microsporus,Rh. rhizopodiformis,progeny and intermediates (Rh. chinensis)

Fourteen isolates belonging to Rhizopus microsporus, Rh. rhizopodiformis, the progeny of Rh. microsporus x Rh. rhizopodiformis and an intermediate species, Rh. chinensis, were serologically tested either by a modification of the exoantigen technique of Kaufman & Standard (1978) or by the more analytical cross immunoelectrophoresis technique with intermediate gel. Common antigenic determinants in the isolates studied indicated that the species, their progeny and the intermediate species Rh. chinensis, are antigenically very closely related.Through absorbtions, a specific reference antiserum for Rh. microsporus was obtained. With this absorbed monospecific antiserum, using the exoantigen test, the identity of the isolates in study, determined after their general morphology as Rh. microsporus, was confirmed. For comparative purposes, the use of reversed phase high performance liquid chromatography for the investigation of fungal isolates is also reported.     

Direct effect of biocontrol agents on wilt and root-rot diseases of sesame

In Egypt, sesame cultivation is subject to attack by wilt and root-rot diseases caused by Fusarium oxysporum f.sp. sesami (Zap) Cast. and Macrophomina phaseolina (Maubl) Ashby causing losses in quality and quantity of sesame seed yield. Bacillus subtilis and Trichoderma viride isolates which were isolated from sesame rhizosphere were the most effective to antagonise fungal pathogens, causing high reduction of hyphal fungal growth. Trichoderma viride was found to be mycoparasitic on Fusarium oxysporum f.sp. sesami and M. phaseolina causing morphological atternation of fungal cells and sclerotial formation. In general, Bacillus subtilis, T. viride, avirulent Fusarium oxysporum isolate and Glomus spp. (Amycorrhizae) significantly reduced wilt and root-rot incidence of sesame plants at artificially infested potted soil by each one or two pathogens. Data obtained indicate that Glomus spp significantly reduced wilt and disease severity development on sesame plants followed by T. viride. Meanwhile, avirulent Fusarium oxysporum isolate followed by Glomus spp. were effective against root-rot disease incidence caused by M. phaseolina. Glomus spp. followed by B. subtilis significantly reduced wilt and root-rot disease of sesame plants. All biotic agents significantly reduced F. oxysporum f.sp. sesami and M. phaseolina counts in sesame rhizosphere at the lowest level. Glomus spp. and the avirulent isolate of F. oxysporum eliminated M. phaseolina in sesame rhizosphere. Meanwhile T. viride was the best agent at reducing F. oxysporum at a lower level than other treatments. Application of VA mycorrhizae (Glomus spp.) in fields naturally infested by pathogens significantly reduced wilt and root-rot incidence and it significantly colonised sesame root systems and rhizospheres compared to untreated sesame transplantings.     

Serological Survey of Paracoccidioidomycosis in Sheep

The objective of this study was to evaluate the prevalence of antibodies against Paracoccidioides brasiliensis in sheep from Guarapuava, Paraná State, Brazil. The seroepidemiological study was carried out in 262 sheep. The samples were analyzed by ELISA and immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively. Initially, two sheep were immunized with P. brasiliensis to evaluate whether contact with the fungal cells could induce a humoral immune response against gp43 and exoantigen from P. brasiliensis. Both animals produced antibodies against gp43 and exoantigen, the main antigens used for diagnosis and seroepidemiology of paracoccidioidomycosis. A reactivity of 37% was observed to the P. brasiliensis gp43 antigen by ELISA although no reactivity had been observed by the immunodiffusion test. Sheep under extensive grazing system showed higher frequency of positivity to P. brasiliensis (P ≤ 0.05) than those under intensive and semi-intensive systems. These data suggest that sheep may be a useful epidemiological marker of P. brasiliensis presence in the environment and reinforce that contact with soil is an important risk factor for infection.     

Acquisition,retention and dispersal of soilborne plant pathogenic fungi by fungus gnats and moth flies

Adult fungus gnats and moth flies were experimentally demonstrated to function as potential above‐ground vectors for three soilborne plant pathogens: Verticillium dahliae, Fusarium acuminatum and Thielaviopsis basicola. The adult insects externally acquired the conidia of the pathogens after exposure to the cultures as confirmed by scanning electron microscope photography. The intestinal contents and frass deposits of larvae exposed to fungal cultures contained viable fungal propagules. Internally infested larvae developed into internally infested pupae; however, the emerging adults were free of fungal structures. Because of the maintenance of a high level of inoculum on the external body surface and the ability of these adult insects to fly, they can be a significant factor in the dispersal of soilborne fungi in greenhouse agriculture. The rate of dispersal of T. basicola by adult fungus gnats was 1.78 cm² h^?1 per insect and by adult moth flies was 1.17 cm² h^?1 per insect. The area over which the pathogen was dispersed by the adult insects increased with the increase in exposure time. The study demonstrated that adult insects are efficient distributors of soilborne plant pathogenic fungal propagules.     

Detection of Plant Pathogen Spores of Economic Significance on Pollen Trap Slides

Real‐time polymerase chain reaction (PCR) assays were used to evaluate a small number of samples from a volumetric spore trap normally used for counting pollen grains. Samples from a total of 6 days during July and August 2011 were screened. Pathogen DNA was detected from three of four groups of economically significant plant pathogens for which real‐time PCR assays were available. These were Tilletia spp. on 1 day, Puccinia spp. on 2 days and Fusarium spp. on all 6 days. No amplification of real‐time PCR assays was detected for Phytophthora infestans or P. ramorum. The results indicate that plant pathogens can be detected in air sampling networks, which are remote from arable cropping and deployed for other purposes. This has implications for rapidly identifying periods of pathogen dispersal and improving the accuracy of information on pathogen spore load in the atmosphere.