Persistence Of Spores Research Articles

Sporulation of arbuscular mycorrhizal fungi in organic-rich patches following host excision

Soil organic matter is known to directly influence nutrient capture and affect the growth of arbuscular mycorrhizal fungi (AMF). The effect of organic-rich patches in soil on subsequent sporulation and the persistence of spores will have important implications for sustainable agricultural practices particularly as AMF spores are likely to play a significant role in soil carbon dynamics and ecosystem productivity. We have used a mesh-exclusion approach to quantify the temporal dynamics of AMF sporulation in organic-rich patches following host shoot excision. Wheat plants were grown in pots, filled with sterile sand inoculated with Glomus intraradices, Glomus mosseae or a non-mycorrhizal control. Each pot contained three 25 cm 3 mesh bags (35 μm), filled with either farmyard manure (FYM), soil or sand. At 6 weeks post-emergence the host shoot was excised and AMF spores were quantified in each of the mesh bags and again at 24, 52, 66 and 80 days after host excision. Spore numbers were far higher in FYM than in patches of soil, although by 24 days after shoot excision the number of spores had dramatically decreased in patches of both FYM and soil; however this was followed by a significant increase in spore numbers of G. intraradices in the FYM patches. Extractable P, NO 3 − and NH 4 + in the mesh bags were all significantly higher in the FYM patches than in the patches of soil, although there were no differences between the control and the mycorrhizal treatments. However, the concentration of soluble phenolics in the FYM colonised by both of the AMF species significantly decreased between 0 and 24 days after host excision compared to the control. This finding provides novel insights into the nature of asymbiotic sporulation in organic-rich patches and has important implications for sustainable agricultural practices, including FYM application enhancing the ‘mycorrhizal potential’ of soil following a harvest, by increasing the amount of early colonisation in the next crop.

Spore Persistence and Likelihood of Aeroallergenicity of Entomopathogenic Fungi Used for Mosquito Control

Entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, are being researched as alternatives to chemical adulticides to control mosquito vectors of malaria and dengue. Two cited concerns of fungal control include conidial viability and risks fungal entomopathogens pose to human health. We measured spore viability of 10 fungal isolates over 26 weeks and found a range of persistence. Three B. bassiana isolates maintained > or = 50% viability 14 weeks after application. No M. anisopliae isolate persisted longer than 1 week. To help assess risk of conidia as potential human allergens, we measured airborne conidia in enclosed environments after simulated biopesticide treatment of M. anisopliae. Conidia were detectable immediately after treatment, with concentrations of ~7000/m(3), decreasing over 48 hours to 500 conidia/m(3). At most, Metarhizium conidia comprised 2% of total visible particulate matter, falling to 0.1% by 2 days. The implications for viability of biological control of adult mosquitoes are discussed.

Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines

Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 3′ border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were undetected after 8 wk whereas viable spores still could be detected at 17 wk.

Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines

Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 3' border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were undetected after 8 wk whereas viable spores still could be detected at 17 wk.

Intracellular fate and immunogenicity of B. subtilis spores

To support our work on the development of bacterial spores as oral vaccines we examined the immunogenicity and intracellular fate of Bacillus subtilis endospores in a murine model. Mice dosed orally with spores developed systemic IgG and mucosal sIgA responses. Analysis of IgG subclasses revealed a predominance of the IgG2a subclass during the early stages of immunisation. Analysis of cytokine mRNA in GALT and lymphoid organs showed early induction of IFN-γ, a Th1 cytokine, as well as the pro-inflammatory cytokine TNF-α. Significant levels of IgG antibodies were produced against vegetative bacilli following dosing with spores. This showed that spores could germinate in the GI tract. In vitro studies detailing the intracellular fate and persistence of spores in a macrophage-like cell line (RAW264.7) demonstrated that spores could germinate efficiently in macrophages, initiate gene expression as well as inducing pro-inflammatory cytokines.

Sex slows down the accumulation of deleterious mutations in the homothallic fungus Aspergillus nidulans.

Coexistence of sexual and asexual reproduction within the same individual is an intriguing problem, especially when it concerns homothallic haplonts, like the fungus Aspergillus nidulans. In this fungus asexual and sexual offspring have largely identical genotypes. This genetic model organism is an ideal tool to measure possible fitness effects of sex (compared to asex) resulting from causes other than recombination. In this article we show that slightly deleterious mutations accumulate at a lower rate in the sexual pathway than in the asexual pathway. This secondary sex advantage may contribute to the persistence of sexual spores in this fungus. We propose that this advantage results from intra-organismal selection of the fittest gametes or zygotes, which is more stringent in the costly sexual pathway.

INCIDENCE OF BACILLUS ANTHRACIS IN SOIL

Interest in anthrax has increased recently due to its use in bioterrorism attacks. Bacillus anthracis, the causative agent of anthrax, is genetically similar to other Bacillus spp. that occur in the environment, and is known to persist in soil for years in the form of spores. In many soils, naturally occurring anthrax infections tend to occur during dry periods following a wet period. The “incubator theory” suggests that spores are concentrated in low-lying areas during rainfall events and animals are subsequently exposed to contaminated soil during foraging in dry periods. It is currently believed that B. anthracis spores require a host for germination, and thus do not undergo proliferation cycles in the soil. There is a potential for B. anthracis spores to be transported as an aerosol, but human infection due to inhalation of spores is unlikely given the high minimum infectious dose required to cause disease. Historically, naturally occurring anthrax infections in humans have most commonly occurred due to contact with diseased animals or animal products. However, B. anthracis has been developed as a biological weapon with accidental and intentional releases resulting in human death. With the development of an anthrax vaccine, anthrax outbreaks have generally been controlled in developed countries; however, anthrax is still a major problem in many parts of the world. The advent of molecular techniques has enhanced the detection of B. anthracis spores, which is now possible in less than 1 h. However, due to the persistence of spores, it is difficult to eliminate B. anthracis contamination from the environment. There remains a need for additional research on anthrax in several areas including evaluation of the conditions that favor B. anthracis survival in soil, determination of whether B. anthracis undergoes a growth cycle in soil, and determination of the potential for transfer of B. anthracis virulence genes to other soil microorganisms.

Bacterial endospores and their significance in stress resistance.

In terms of resistance to extreme environmental stresses, the bacterial spore represents a pinnacle of evolution. Spores are highly resistant to a wide variety of physical stresses such as: wet and dry heat, UV and gamma radiation, oxidizing agents, chemicals, and extremes of both vacuum and ultrahigh hydrostatic pressure. Some of the molecular mechanisms underlying spore resistance properties have been elucidated in the laboratory, and involve both: (i) protection of vital spore macromolecules during dormancy, and (ii) repair of damaged macromolecules during germination. Our group has recently become interested in testing if the laboratory model of spore UV resistance is relevant to spore persistence in the environment. We have constructed a number of Bacillus subtilis strains which are defective in various DNA repair systems and spore structural components. Using spores of these strains, we have been exploring: (i) the types of damage induced in DNA by the UV-B and UV-A components of sunlight; (ii) the relative contribution of the major spore DNA repair systems to spore solar radiation resistance; and (iii) the role of spore structural components such as the spore coats and dipicolinic acid (DPA) in attenuation of the lethal and mutagenic effects of solar UV. The current data are reviewed with the ultimate goal of obtaining a complete model describing spore persistence and longevity in the terrestrial solar UV radiation environment.

Increased Persistence of Beauveria bassiana Spore Viability Under High Ultraviolet-blocking Greenhouse Plastic

A field study was conducted to assess the effect of various commercially available polyethylene plastic greenhouse coverings on the persistence of viable spores of the microbial insecticide Beauveria bassiana. Selected coverings blocked the transmission of UV light with wavelengths of 360 nm and below or 380 nm and below. Two coverings also contained an infrared blocking component. A commercial formulation of B. bassiana was applied for 3 consecutive weeks to plants growing in the plastic covered hoop houses. The percentage of viable spores was calculated up to 13 days after the final application. The persistence of viable B. bassiana spores was significantly longer under the plastic that blocked a greater portion of the UV spectrum (<380 nm) than the plastics that only blocked UV wavelengths below 360 nm. One week after application, percentage of spore germination was at least twice as high under the <380 nm blocking plastic compared to <360 nm blocking plastics.

Photoprotection of Beauveria bassiana: Testing simple formulations for control of the coffee berry borer

The entomopathogenic fungus, Beauveria bassiana is considered to be one of the few natural enemies available for use against the coffee berry borer. In an attempt to enhance the efficacy of this pathogen, a range of concentrations of 22 substances was tested in simple laboratory tests using natural sunlight or a UV light source. Unprotected B. bassiana spores were almost completely inactivated by exposure to 60 min of direct sunlight or 20 s of UV light of 302 nm wavelength. Seven of the 22 substances tested showed little or no photoprotective properties and eight of the substances appeared directly detrimental to spore germination. Of the remainder, sucrose, yeast, yeast extract, uric acid, casein, and molasses had limited photoprotective properties. The most effective substances tested were egg albumen and skimmed milk powder which could extend the persistence of B. bassiana spores by a factor of almost three. A mixture of 3% (w/v) albumen and 4% (w/v) milk powder gave the highest degree of spore protection per unit cost. Young coffee plants sprayed with this mixture did not suffer any significant phytotoxic effects. A field trial, involving two applications of spores with or without the milk and albumen mixture, failed to show that improved spore persistence resulted in increased coffee berry borer control. Very lowlevels of pest infestation observed in field plots together with unusual, unfavourable weather conditions may have accounted for this unexpected result.

Isolation of a Toxin B–Deficient Mutant Strain ofClostridium difficilein a Case of RecurrentC. difficile–Associated Diarrhea

Clostridium difficile-associated diarrhea (CDAD) recurs in approximately 15%-20% of patients after discontinuation of metronidazole or vancomycin therapy. Most recurrences are believed to be endogenous relapses due to the persistence of spores. However, there is evidence that reinfection with a different strain is a cause of recurrence. We report the case of a patient with a history of multiple episodes of C. difficile colitis. The patient, a 56-year-old female, has had 5 years of repeated recurrences, each shortly after discontinuing vancomycin therapy. During the course of these episodes, three isolates were cultured from her stools at different times. These isolates were analyzed for the presence of toxin A and B gene sequences and genotyped by means of arbitrarily primed polymerase chain reaction (AP-PCR). The original two isolates contained the toxin A and B genes, as determined by PCR, and were of the same AP-PCR type. During her last relapse, a C. difficile strain lacking at least a portion of the toxin B gene was isolated. AP-PCR analysis of this isolate showed a different DNA banding pattern from that of the previous isolates. A vancomycin susceptibility assay revealed a slight decrease in vancomycin activity as compared with that against the prior isolate. This case demonstrates two unique features: (1) recurrent infections can be due to reinfections and (2) toxin B mutants can possibly cause CDAD. This study also raises concerns about long-term vancomycin use and the development of resistance of C. difficile to vancomycin.

Persistence and nontarget effects of Bacillusthuringiensis in soil: a review

This review presents information on the occurrence, persistence and nontarget effects of Bacillusthuringiensis Berliner (B.t.) in soil, with particular emphasis on forest ecosystems. Both field and laboratory studies have confirmed that B.t. is able to survive for several years after spraying, but studies on long-term persistence and possible accumulation of spores in soils are inadequate. The ecological role of B.t. in the soil ecosystem is poorly understood; we do not even know where and under what conditions B.t. multiplies in nature. Information on the effects of B.t. on soil microflora is contradictory, with studies variously suggesting that B.t. caused an increase, a decrease, or did not affect indigenous bacterial populations. Toxicity of B.t. to species of several invertebrate taxa (Acarina, Nematoda, Collembola, Annelida, Hymenoptera) inhabiting the soil has been demonstrated, but only rarely is it possible to relate dosage information to field situations, and in many cases the B.t. subspecies tested are not currently used for pest control in North America. There is an urgent need for further research to elucidate the relationships between B.t. and the natural soil microflora and fauna.

Quantitative investigations into the elimination of in vitro-obtained spores of the non-pathogenic Clostridium butyricum strain CNRZ 528, and their persistence in organs of different species following intravenous spore administration

Before the Clostridium tumour assay can be applied to the diagnosis of cancer, we sought to investigate - within the framework of a biopharmaceutical safety test - the organ persistence of test spores of Clostridium butyricum CNRZ 528. We found that non-pathogenic spores obtained in vitro, like pathogenic native spores, escape phagocytosis in various organs up until about 2 years, as tested by anaerobic cultures. The elimination of spores depended on the species of animal, the spore dose and the organs investigated. In rabbits, one week after injection, we recovered clostridial spores from blood and spleen cultures more rarely than from liver and lung. The half-life of blood clearance in patients was one day or, at half the spore dose, two days. That deep tissues of healthy animals are not normally sterile bacame evident in rabbits after sporadic isolation and characterization of non-administered saccharolytic and proteolytic clostridial species. During a 10-year observation period, the rate of obtainment of viable spores by in vitro cultures lessened; however, for administration of the spores in clinical phase I and phase II studies, the spore quality was acceptable.

Intranasal amphotericin B reduces the frequency of invasive aspergillosis in neutropenic patients

To retrospectively study the prophylaxis of invasive aspergillosis in neutropenic patients and to relate the frequency of this fungal disease to any causal or modifying factors that could be identified. Between 1977 and 1988, 130 patients underwent 158 intensive treatment episodes to control acute leukemia, lymphoma, and aplastic anemia, and the frequency of complicating aspergillus infection was determined. Proven invasive aspergillus infections occurred in 22 cases, 12 of which were fatal. Invasive aspergillosis was suspected in a further 16 cases and all these patients recovered with amphotericin B treatment. Colonization by Aspergillus in the absence of clinically significant infection was seen in 31 treatment episodes. Invasive aspergillosis involved mainly the upper and lower respiratory tract and skin. Control of the infection was closely related to the control of the underlying disease, with subsequent return of normal marrow function and resolution of neutropenia. The incidence of aspergillus infection has decreased dramatically since 1985, most probably due to the introduction of intranasal amphotericin B. This occurred despite the persistence of aspergillus spores in the hematology ward air during the 1986 to 1988 period. Intranasal aerosolized amphotericin B may protect against invasive aspergillosis, even when neutropenic patients are cared for in conventional wards without HEPA filtration.

Intranasal amphotericin B reduces the frequency of invasive aspergillosis in neutropenic patients

purpose: To retrospectively study the prophylaxis of invasive aspergillosis in neutropenic patients and to relate the frequency of this fungal disease to any causal or modifying factors that could be identified patients and methods: Between 1977 and 1988, 130 patients underwent 158 intensive treatment episodes to control acute leukemia, lymphoma, and aplastic anemia, and the frequency of complicating aspergillus infection was determined. results: Proven invasive aspergillus infections occurred in 22 cases, 12 of which were fatal. Invasive aspergillosis was suspected in a further 16 cases and all these patients recovered with amphotericin B treatment. Colonization by Aspergillus in the absence of clinically significant infection was seen in 31 treatment episodes. Invasive aspergillosis involved mainly the upper and lower respiratory tract and skin. Control of the infection was closely related to the control of the underlying disease, with subsequent return of normal marrow function and resolution of neutropenia. The incidence of aspergillus infection has decreased dramatically since 1985, most probably due to the introduction of intranasal amphotericin B. This occurred despite the persistence of aspergillus spores in the hematology ward air during the 1986 to 1988 period. conclusion: Intranasal aerosolized amphotericin B may protect against invasive aspergillosis, even when neutropenic patients are cared for in conventional wards without HEPA filtration.

Persistence and recycling of Bacillus sphaericus 2362 spores in Culex quinquefasciatus breeding sites in West Africa

A flowable concentrate of Bacillus sphaericus strain 2362 was applied at 10 g/m2 against Culex quinquefasciatus mosquito larvae in cesspools. Complete control of larvae was maintained during 5 to 6 weeks, due to a very low settling of B. sphaericus spores, and was related to the presence of at least 100 to 500 spores per ml in upper water layers. Larval cadavers sedimented within 48 h after treatment. B. sphaericus was shown to recycle in dead larvae but not in mud. Spore persistence exceeded 5 months in bottom mud and the concentration of persistent spores was higher in cesspools, the bottom of which was cemented. Depth, temperature, pH, dissolved oxygen and suspended matter content of the water remained relatively constant throughout 4 months. In laboratory experiments, the final amounts of spores recycled in larvae was not influenced by spore concentration in water or by detergent, but it was affected by organic matter. Projected costs of B. sphaericus formulation indicates that its use even at high dosages, would be more cost effective than the use of chemical insecticides, especially where c. quinquefasciatus is resistant to these latter. A new strategy for controlling this vector could be deployed, using B. sphaericus and insect growth regulators in alternation.

Fate of Bacillus sphaericus 1593 and 2362 spores used as larvicides in the aquatic environment.

Dry powders produced from insecticidal Bacillus sphaericus strains 1593 and 2362 were applied against Culex tarsalis and Anopheles franciscanus mosquito larvae in small-plot field trials. Good control of C. tarsalis, but not of A. franciscanus, was produced at 0.1 and 0.2 lb/acre [ca. 0.122 and 0.244 kg/ha]. B. sphaericus spores settled rapidly from upper water layers and accumulated in bottom muds. Control of third/fourth-instar C. tarsalis larvae was maintained through day 4 after testing and was related to the presence of at least 100 spores per ml in the upper water layer 2 days earlier. B. sphaericus was shown to recycle in dead larvae both in the laboratory and in the field, producing an increase of 100- to 1,000-fold in spore numbers. There was no evidence of recycling in treated water nor of significant spore persistence upon reflooding of ponds after a very hot, dry period.

Production and efficiency of protozoa

AbstractDespite the chronic and debilitative nature of the infection they cause, several species of microsporidia and neogregarines offer a good potential as microbial control agents, particularly against insect pests of high economic thresholds. Techniques for mass production of protozoa have usually involved per os, inoculation or injection of the protozoa into their usual or alternate hosts. The spores are harvested subsequently from heavily infected host tissues by grinding, filtration, and differential centrifugation. Although fresh spores are used in most field tests, the spores of many species can be stored with high survival either frozen or in water at low temperatures (0–4°C) for up to several months. Sunlight or ultraviolet (UV) radiation is a serious factor limiting spore persistence. However, the protozoa do not appear to be significantly limiting spore persistence. However, the protozoa do not appears to be significantly more susceptible to UV radiation than other insect pathogens and persistence can be prolonged with UV protectants. Most field tests with protozoa have involved the application of spores in sprays and have usually resulted in a high degree of infection in the target host species. The potential for control of few species has been improved by formulation of spores in to baits, and the potential of other species will likely increase if suitable bait formulation can be devised in the future. One species, Nosema locustae, formulated as a bait, has been successfully used to control grasshoppers on rangelands. Limited laboratory and field studies have also suggested that increased short‐term control might be obtained if candidate protozoan species can be combined with certain insecticides. While recent and increased efforts have been devoted to assess the potential of protozoa as microbial control agents, potential hazards to nontarget organism have been investigated for only three species. Their close relation taxonomically to protozoa pathogenic for mammals will necessitate careful evaluation of the safety of candidate control species for nontarget organisms.

Persistence of Spores of Vairimorpha necatrix on Tobacco, Cotton, and Soybean Foliage12

Vairimorpha necatrix (Kramer) spore suspensions with and without adjuvants were sprayed onto foliage of 3 agricultural crops from which random samples of leaves were subsequently bioassayed for infective spores with 3rd instars of the corn earworm, Heliothis zea (Boddie). Spore persistence, which lasted for up to 2 wk in some treatments, was affected more by spore dosage than by UV light protectants and sticker-spreader adjuvants. Spore survival up to 4 days after application was higher on tobacco than on cotton; and from 7–14 days, survival was higher on soybeans than on tobacco or cotton. Spore persistence under field conditions was sufficient not to in terfere with effectiveness and possible development of V. necatrix as a microbial control agent for the temporary suppression of certain lepidopterous pests.

Über die Wirkung von Bacillus thuringiensis kombiniert mit synthetischem Pyrethroid auf Orgyia antiqua L.

The efficacy of the biopreparations Dipel and Thuricide HP in combination with the pyrethroid Permethrin, and that of Permethrin in the control ofOrgyia antiqua larvae has been tested in the field. The results indicate that these preparations may replace satisfactorily insecticides of the group of chlorated carbohydrates, and organophosphates, in the control ofOrgyia larvae. The persistence of spores of both Dipel and Thuricide was found to be good on the pine needles. The main advantage of the tested preparations is their complete harmlessness to vertebrates. Since they do not contaminate the external environment, they can be used safely in the treatment of large areas of infected forest stands located at the site of important water resources and recreation areas.