Viability Of Fungal Spores Research Articles

Aggressiveness of Alternaria alternata (Fr.) Keiss for influence of sunflower hybrids and cultivation technologies

Phytopathogenic aggressiveness is the ability of a fungus to reproduce in the tissues of host plants, which is manifested in different growth rates of mycelium, intensity of spore formation, viability of fungal spores, duration of development cycle and its resistance to extreme conditions. Varieties and hybrids of crops have a significant effect on changing the aggressiveness of phytopathogenic micromycetes. Therefore, the aim of the study was to determine the aggressiveness of the fungus Alternaria alternata (Fr.) Keiss under the influence of sunflower hybrids grown in different technologies. The influence of sunflower hybrids on the change of aggression was studied using the methods given in the methodological recommendations of M.O. Lemezi. The aggressiveness of A. alternata under the influence of sunflower hybrids grown by different technologies was analyzed. The following indicators were used for evaluation: viability of phytopathogenic micromycete spores, intensity of its spore formation and score of sunflower seedlings by the studied fungus. And also determined the degree of aggressiveness and pathogenicity of the fungus A. alternata. According to the results of the study, a high dependence of the aggressiveness of the studied micromycete on the biological properties of sunflower hybrids of different breeding origin was established. The Dushko hybrid, compared to the Oliver hybrid, both in traditional and organic cultivation technologies, can significantly reduce the aggressiveness of the phytopathogenic fungus A. alternata. This indicator should be used for ecological research of sunflower hybrids as a factor of biological pollution of the agrocenosis, that will allow to managing the stability of agroecosystems and obtain environmentally friendly and safe crop products.

Essential oils for the postharvest control of blue mold and quality of 'Fuji' apples

Abstract: The objective of this work was to evaluate the effect of rosemary, cinnamon, citronella grass, and clove essential oils on the in vitro growth of Penicillium expansum, on the diameter of blue mold lesions, and on the physical and chemical attributes of 'Fuji' apples stored under refrigeration for different periods of time. The compositions of the essential oils were determined, and their effects on the growth inhibition, number, and viability of fungal spores in vitro were evaluated at 0, 100, and 1,000 μL L-1 oil concentrations. At postharvest, evaluations were performed for fruit treated with 0, 50, 100, and 500 μL L-1 essential oil and kept under refrigeration for 30 days, and for fruit treated with 0, 100, and 500 μL L-1 oil and kept under refrigeration for two days. The essential oils - eucalyptol (rosemary), eugenol (cinnamon), citronellal (citronella), and eugenol (clove) - reduce the growth, number, and viability of P. expansum spores 24 hours after the induction to germination, as well as the diameter of blue mold lesions in apples, except for citronella oil at 500 μL L-1 after 30 days of cold storage. There are no changes in the physicochemical attributes of apples, treated with different concentrations of the oils after refrigeration for five months, followed by seven days at room temperature.

Viability of Fungal Spores Isolated from Sorghum Grains Sampled from the Field, Market and Different Storage Facilities in the Six Agro-ecological Zones of Nigeria

Viability of Fungal Spores Isolated from Sorghum Grains Sampled from the Field, Market and Different Storage Facilities in the Six Agro-ecological Zones of Nigeria

A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity

Long-lasting viable fungal spores are one of the important aspects in emergence, spread and disease development of pathogenic fungi. We developed a rapid and miniaturized system using Alamar Blue (resazurin dye; 7-hydroxy-3H-phenoxazin-3-one 10-oxide) for assessing fungal spore viability, using the ascomycete Leptosphaeria maculans (causing blackleg disease on canola) as a ‘model pathogen’. The assay is dependent on the metabolic activity of viable fungal spores to convert the dark blue of resazurin (maximum absorbance 605 nm) into the pink colour of resorufin (maximum absorbance 573 nm). The Alamar Blue assay uses an optimised micro-titre based format that was far superior for determining fungal spore viability in comparison with current conventional techniques including trypan blue staining, a TC10 cellometer cell counter, or by assessing germination of the spores under the microscope. This new assay was also more rapid and reproducible than current conventional tests to detect viable spores. Viable spores could be reliably detected within two hours. The successful application of the Alamar Blue assay to measure fungal spore viability in the current study has important benefits for biosecurity operations relating to faster and more reliable confirmation of viability of potential invasive exotic fungal pathogens and in minimising any consequent disease outbreaks. The effectiveness of the Alamar Blue assay was confirmed by successfully determining the relative retention times of viable L. maculans ascospores across a range of different potential spore-carrier materials, including steel, fabric, wood, paper, rubber and leather, over a time period of eight months. To further confirm the wide applicability of the Alamar Blue assay, it was successfully applied to detect viable spores of fungal pathogens of diverse taxonomic groups, including Kabatiella caulivora, Magnaporthe oryzae and Puccinia striiformis f.sp. tritici, and also of the yeast Saccharomyces cerevisiae.

Evaluation of administering Duddingtonia flagrans through Complete Feed Block for Controlling Haemonchus contortus in Sheep

In this experiment, a device was developed and evaluated to deliver nematophagous fungus Duddingtonia flagrans to sheep by using local fungal isolate. Sterilized barley grains supporting the fungal growth were incorporated into straw based complete feed blocks (CFB). The viability of fungal spores was assessed for the biological control of ovine Haemoncus contortus. Two groups (fungus-treated and control without fungus), each consisting of five Garole x Malpura lambs (about 6–9 months old) which were artificially infected with 6000 Haemonchus contortus larvae per lamb. The treated group received fungus incorporated CFB (400g block per lamb delivering 6 million chlamydospores) daily during 7 weeks. Fungal chlamydospores survived well in CFB with very little moisture (11.8%) when stored at room temperature. Mean larval development was significantly (P<0.001) lower compared to control from day 1 to the end of the feeding. Larval recovery from the grass samples started from 2nd week of the experiment, and it increased markedly on the plot grazed by the control group reaching as high as 4000 L3/kg DM compared to less than 600 L3/kg DM in the plot grazed by experimental group fed on CFB with fungal spores. No significant difference was observed in the number of eggs per gram of faeces between both the groups. However, significant (P<0.001) reduction in developed larvae on coprocultures was seen for fungal spore incorporated group. A low level of larval counts were maintained in the plot grazed by fungus treated group throughout the experiment. The study demonstrated that CFB feeding might prove a potential means of delivery of D. flagrans to sheep for successful biological control of H. contortus.

Biopesticidal Formulation Of Beauveria Bassiana Effective Against Larvae Of Helicoverpa Armigera

The present study was to emphasize entomopathogens in pest management of cash crops over chemical pesticides, optimization of media for growth of Beauveria bassiana and bioassay of different formulations for their efficacy as marketable and easily applicable biopesticide. Beauveria bassiana (Order: Hypocreales, Family: Cordycipitaceae) popularly known as white muscardine entomogenous fungi was isolated from soyabean fields of Misrod, Bhopal. Media for optimal growth of fungus were standardized. Its different formulations viz. carrier based powder formulation, oil based formulation and bentonite oil based formulation using homogenizers were prepared. These formulations were bioassayed against Helicoverpa armigera (Order: Lepidoptera, Family: Noctuidae), the most destructive pest in soyabean. Bentonite based liquid formulation was observed to be most effective as determined by measuring larval mortality as well as viability of fungal spores and ease of applicability.

Abundance and viability of fungal spores along a forestry gradient – responses to habitat loss and isolation?

Regional variation in spore deposition and viability was studied for two fungi, Fomitopsis rosea (Alb. &amp; Schwein.: Fr.) P. Karst. and Phlebia centrifuga P. Karst., both confined to old‐growth spruce forests in the boreal zone. Seven regions in Sweden were studied along a north‐south transect in which the historical impact from forestry increases and the amount old forests decreases towards the south. The two southernmost regions were located outside the distribution border of the species. Spore deposition was measured species specifically as heterokaryotisation of homokaryotic mycelia growing on wood discs. There was a significant decline in spore deposition towards the south for both species. F. rosea deposited an average amount of 111 spores m−2 24 h−1 in the northernmost region compared to less than 1 spore in the four southernmost regions. The corresponding values for P. centrifuga were 27 spores m−2 24 h−1 in the north compared to less than 2 spores in the 4 southernmost regions. No deposition was found south of the distribution borders. The viability of spores from local populations within each region was measured as germination success on nutrient media. Individual fruiting bodies from large populations in the north generally produced spores with higher germinability than fruiting bodies from geographically isolated populations in the central and southern regions. However, there was a high variation among the southern populations. Our data suggest that some populations in mid‐ and south Sweden may suffer from negative genetic effects, possibly associated with fragmentation and loss of habitat. Thus, the combination of low spore deposition and low germinability of spores may be a threat to the long‐term persistence of F. rosea and P. centrifuga in southern Sweden. Several other species may experience the same situation, especially when considering the severe decline of dead wood in Swedish forests.

Effect of sampling time on the culturability of airborne fungi and bacteria sampled by filtration.

Air sampling of bioaerosols by filtration may be preferable for many epidemiological studies because the methods can be used to collect personal samples for a full work-shift. There is some concern, however, that the viability of fungal spores and bacterial cells might be compromised by sampling for as long as a full shift. This study was designed to determine the effect of sampling up to 6 h on the viability (measured by culture) of airborne fungi and bacteria at composting facilities. Six side-by-side samples were collected in two locations at each of three composting facilities for 1 h at 2 l/m on polycarbonate filters. Two samples in each set were then capped while clean, HEPA-filtered air was drawn across two others for an additional 2 h and across the last two for an additional 5 h. Filters were washed and the samples were analyzed for culturable bacteria and fungi, and for total bacteria and fungi by microscopic counting. Concentrations ranged from 1.7 x 10(3) to 6.2 x 10(7) c.f.u./m3 of culturable fungi and 1.17 x 10(4) to 1.0 x 10(6) c.f.u./m3 of culturable bacteria. In linear models that included duration of sampling, location, and the interaction of location and sample duration, neither sample duration nor the interaction term were significant predictors of the logs of the concentrations of culturable fungi or bacteria or of the ratio of the logs of the culturable concentrations to total concentrations for fungi or bacteria. This suggests that increased sampling time does not affect the viability of the organisms commonly found in the air at composting facilities.

Development of a rapid and highly sensitive biochemical method for the measurement of fungal spore viability. An alternative to the CFU method

1 A biochemical method, based on dehydrogenase activity (DHA) measurement, has been developed as an alternative to colony forming unit (CFU) enumeration, for assessing the viability of fungal spores. In viable cells, a tetrazolium salt (MTT) is reduced to a coloured formazan (MTTf) by cellular dehydrogenase enzymes. From the colorimetric assay developed by Mosmann for mammalian cells, the procedure has been adapted and optimised using P. digitatum spores as a model. Propan-2-ol has been selected as the best solvent to extract the MTTf from the spores. The sensitivity of the method has been considerably increased by determining the optimal conditions of incubation for the MTT reduction by spores: temperature at 50°C and pH 8. Using the assay, a correlation has been established between DHA and the number of viable spores as measured by the CFU method (10 5 CFU = 1.14 DHA -26; r 2 = 0.94). This standardised procedure allows the viability of P. digitatum spores to be estimated with accuracy and reliability. Moreover, the extension of this assay to other strains has been demonstrated ( Aspergillus niger and Metarhizium flavoviride).

Protocol for assessing the viability of Sphaerotheca fuliginea conidia by tetrazolium chloride (INT) reduction

The use of tetrazolium salt reduction for assessing the viability of fungal spores is briefly reviewed, and a protocol utilizing 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) salts is described for the vital staining of spores of the cucurbit powdery mildew fungus, Sphaerotheca fuliginea. This protocol is validated against some established methods of assessing spore viability in fungi (i.e. germination assay and Evans Blue exclusion) and indicates that Evans Blue staining is an unreliable indicator of viability as conidia rapidly become permeable to Evans Blue during immersion. Sensitivity of conidia to immersion appears to be related to spore maturation stage as Evans Blue inclusion by conidia still attached 1o the conidiophore is greater than in free conidia. !n contrast INT is readily taken up by both viable and non-viable fungal spores. Whereas oxidised INT is colourless and non-fluorescent, respiratory reduction of INT within viable spores results in the intracellular deposition of formazan crystals. INT-formazan is bright red in colour and has detectable fluorescence between 515 and 565 nm when illuminated with near-UV light. Variability in INT-formazan staining was related to spore developmental stage, with free conidia staining more rapidly and intensely than those still attached to the conidiophore. Fluorescing INT-formazan in viable spores incubated at 20°C can be detected as early as one hour after incubation providing background autofluorescence is low, with fluorescent detection optimal by 6 hours and detectable increases in fluorescence ceasing by 12 hours. In contrast, detection of INT-formazan under transmitted light only becomes optimal by 12 hours, with detectable increases up to 20 hours. Since a time-course analysis of Evans Blue exclusion by immersed spores found that membrane integrity began to be compromised by 12 hours, INT viability staining is suitable for powdery mildew spores that are intolerant of longer periods of liquid immersion.

Estimating the Viability of Vesicular-Arbuscular Mycorrhizae Fungal Spores Using Tetrazolium Salts as Vital Stains

The tetrazolium salts 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride were evaluated as vital stains for estimating the viability of fungal spores of Glomus mosseae and Glomus clarum. Respiring fungal spores accumulated tetrazolium bromide-formazan and tetrazolium chloride-formazan in the cytoplasm as a visually distinct color reaction. Estimates, however, of fungal spore viability based on tetrazolium bromide staining were inconsistent with viability estimates based on a bioassay using single spores of G. clarum. Inconsistencies were due largely to the ambiguous interpretation of the significance of black spores. In contrast, color reactions in tetrazolium chloride were relatively stable and consistent with bioassay estimates of viability. Generally, incubation in 1 mg ml-1 tetrazolium chloride for 48 to 72 h was sufficient to ensure maximum color reaction. Viable spores were distinguishable from those which were unable to colonize a host plant on the basis of the intensity of color reaction in tetrazolium chloride. We conclude that tetrazolium chloride staining can be used as a rapid indicator of vesicular-arbuscular mycorrhizae fungal spore viability.

Interactions between earthworms and microorganisms in organic-matter breakdown

The digestive system of earthworms consists of a pharynx, oesophagus and gizzard (‘reception zone’) followed by an anterior intestine that secretes enzymes and a posterior intestine that absorbs nutrients. During progress through this digestive system there is a dramatic increase in numbers of micro-organisms of up to 1000 times. There is experimental evidence that microorganisms provide food for earthworms. Bacteria are of minor importance in the diet, algae are of moderate importance; protozoa and fungi are major sources of nutrients. Worms, produced under sterile conditions, could live on individual cultures of certain bacteria, fungi and protozoa, but grew best on various mixtures of microorganisms. Symbiotic interactions between earthworms and microorganisms break down and fragment organic matter progressively, finally incorporating it into water-stable aggregates. The mineral nutrients in earthworm casts and lining earthworm burrows are in a form readily available to plants. There is evidence that interactions between earthworms and microorganisms not only provide these available nutrients, but stimulate plant growth indirectly in other ways. There are some studies that show that earthworms can disperse pathogenic microorganisms and influence the viability of fungal spores and nematode cysts. Earthworms have complex interactions with microorganisms that can lessen or increase plant disease attack.

Biochemical Determination of the Viability of Fungal Spores and Hyphae

Germination is commonly used to monitor spore viability, but many fungal spores do not germinate readily. Rapid viability tests are needed. Methods were developed to monitor viability, based on enzyme activity and ATP content of fungal spores and hyphae. ATP content, measured by the luciferin-luciferase system, was much higher in living spores than in dead spores. Hydration was an additional aid in differentiating the ATP content of viable spores compared with dead spores. Approximately 40 Mg of spores were required to measure the ATP level. Preparation from 25 Mg of living spores or 100-150 Mg fresh hyphae contained sufficient phosphatase or glucosidase to give a positive fluorescence test for hydrolysis of methylumbelliferyl-phosphate or methylumbelliferyl-glucoside at 37 C in 2 h. Dead spores and hyphae did not fluoresce. These methods were used to test the viability of: Tilletia controversa, T caries, Ustilago scitaminea, Puccinia striiformis, Pisolithus tinctorius, Rhizopogon colossus, and Elaphomyces granulatus spores, and hyphae of T caries, Rhizoctonia solani, Hymenochaete tabacina, Phellinus weirii, Haematostereum sanguinolentum and Gaeumannomyces graminis var. tritici. These techniques should be useful in determining quickly the effects of fumigants and fungicides on fungal spores and hyphae.

Biochemical Determination of the Viability of Fungal Spores and Hyphae

Germination is commonly used to monitor spore viability, but many fungal spores do not germinate readily. Rapid viability tests are needed. Methods were developed to monitor viability, based on enzyme activity and ATP content of fungal spores and hyphae. ATP content, measured by the luciferin-luciferase system, was much higher in living spores than in dead spores. Hydration was an additional aid in differentiating the ATP content of viable spores compared with dead spores. Approximately 40 μg of spores were required to measure the ATP level. Preparation from 25 μg of living spores or 100–150 μg fresh hyphae contained sufficient phosphatase or glucosidase to give a positive fluorescence test for hydrolysis of methylumbelliferyl-phosphate or methylumbelliferyl-glucoside at 37 C in 2 h. Dead spores and hyphae did not fluoresce. These methods were used to test the viability of: Tilletia controversa, T. caries. Ustilago scitaminea, Puccinia striiformis, Pisolithus tinctorius, Rhizopogon colossus, and Elaphomyces granulatus spores, and hyphae of T. caries, Rhizoctonia solani, Hymenochaete tabacina, Phellinus weirii, Haematostereum sanguinolentum and Gaeumannomyces graminis var. tritici. These techniques should be useful in determining quickly the effects of fumigants and fungicides on fungal spores and hyphae.

Influence of Incubation Temperature and of Storage on the Viability of Fungal Spores damaged by Phenol

THE evidence for an improved revival of damaged bacteria on incubation at temperatures below the optimal for the undamaged species applies mainly to irradiated cells (see, for example, refs. 1 and 2), but optimal revival at 27°–29° C of E. coli damaged by phenol has also been reported3,4. The storage of irradiated bacteria and mammalian cells at sub-optimal growth temperature for some hours leads to an increase in the number of survivors5. We find that storage has a beneficial effect, but not a lower optimal incubation temperature with fungal spores damaged by phenol.