Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures

Conidia of Trichoderma harzianum produced from either solid or liquid fermentation must be dried to prevent spoilage by microbial contamination, and to induce dormancy for formulation development and prolonged self-life. Drying conidia of Trichoderma spp. in large scale production remains the major constraint because conidia lose viability during the drying process at elevated temperatures. Moreover, caking must be avoided during drying because heat generated by milling conidial chunks will kill conidia. It is ideal to dry conidia into a flow-able powder for further formulation development. A method was developed for microencapsulation of Trichoderma conidia with sugar through spray drying. Microencapsulation with sugars, such as sucrose, molasses or glycerol, significantly (P < 0.05) increased the survival percentages of conidia after drying. Microencapsulation of conidia with 2% sucrose solution resulted in the highest survival percentage when compared with other sucrose concentrations and had about 7.5 × 10¹⁰ cfu in each gram of dried conidia, and 3.4 mg of sucrose added to each gram of dried conidia. The optimal inlet/outlet temperature setting was 60/31 °C for spray drying and microencapsulation. The particle size of microencapsulated conidia balls ranged from 10 to 25 μm. The spray dried biomass of T. harzianum was a flow-able powder with over 99% conidia, which could be used in a variety of formulation developments from seed coatings to sprayable formulations.     

First results from conservation studies of chlorophyllous spores of the Royal fern (Osmunda regalis, Osmundaceae)

Pteridophytes spore banks are a promising ex situ conservation tool used to increase the chances of survival of ferns, in fact that large quantities of germplasm with high genetic variation can be conserved in a small space with low economic and technical costs. However, methods to maintain the viability of chlorophyllous spores during storage are less understood.The aim of this study was to investigate the influence of long term storage on the viability of Royal Fern spores, which were stored under different conditions derived from various combinations of temperature and degrees of hydration. Survival and germination tests were performed after 1 and 28 months of storage. Our results showed the highest survival percentages for spores stored under Normal humidity at subzero temperatures (T = ? ?20 °C). These spores received no pre-treatment, dehydration, or cryoprotectants, which resulted in fast germination and gametophyte development which seemed to be stimulated by low temperatures.     

Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of growth substrate on conidial survival and virulence against mosquitoes

Metarhizium anisopliae was grown on six complex mycological media and on three types of rice at three moisture levels to determine the effect of growth substrate on conidial yield, viability, and virulence against mosquitoes immediately after spore maturation and after the storage of conidia at four different temperature-relative humidity (RH) combinations over a 1-year period. Conidial yields varied with the mycological media, but the viability and virulence of conidia against mosquitoes produced on all substrates were similar when spores were stored under the same conditions. The storage conditions were more critical to spore survival and virulence than the substrate upon which conidia were produced. The comparison of rice types for conidial production indicated that conidial yield, viability, and virulence to mosquitoes were more dependent upon the moisture level during growth and on the storage conditions that upon the rice used. The best storage conditions among those tested for the retention of both spore viability and virulence against mosquitoes were 19°C–97% RH and 4°C–0% RH.     

Effects of wetting on the persistence of quiescent conidia of Isaria fumosorosea

Quiescent conidia of Isaria fumosorosea were submitted to various wetting–drying cycles under different regimes of temperature and air humidity. Germination and viability of conidia collected on cultures freshly host-passed (P2) were not affected at 25 °C during five cycles at increasing wet phase duration (2–12 h per daily cycle) under any moisture conditions (13–86% RH). Infectivity levels remained stable, but mortality was slightly postponed. In vitro-cultured inocula (P5) were significantly affected after only one cycle at higher air humidity (75 and 86% RH) and temperature (35 and 40 °C). The persistence of I. fumosorosea conidia suspended in water soluble extracts of leaf surfaces (corn and cabbage) confirmed the better persistence of P2 conidia and the relatively higher detrimental effect of lower air humidity conditions when combined with moderate temperatures. Quiescent conidia deposited in situ on potted plants of cabbage showed a higher persistence on wet foliage and on foliage submitted to wetting–drying cycles, than on dry foliage. These results underline that constraints prevailing in targeted environments and ecological fitness of fungal isolates have to be taken into account for assessing microbial control strategies.     

Spore production in solid-state fermentation of rice by Clonostachys rosea,a biopesticide for gray mold of strawberries

Gray mold caused by Botrytis cinerea is an important disease of strawberry. Clonostachys rosea is a mycoparasite of B. cinerea that reduces fruit losses when used as a biocontrol agent. Since spore production by C. rosea has not been optimized, we investigated factors affecting sporulation under aseptic conditions on white rice grains. The greatest spore production in glass flasks, 3.4 × 10⁹ spores/g-dry-matter (gDM), occurred with an initial moisture content of 46% (w/w wet basis), inoculated with 1 × 10⁶ spores/gDM and hand shaken every 15 days. However, a lower inoculum density (9 × 10³ spores/gDM) and no shaking also gave acceptable sporulation. In plastic bags 1.1 × 10⁸ spores/gDM were produced in 15 days, suggesting that larger scale production may be feasible: with this spore content, 24 m² of incubator space would produce sufficient spores for the continued treatment of 1 ha of strawberry plants.     

Stayin' alive: survival of mycorrhizal fungal propagules from 6-yr-old forest soil

Spores and sclerotia are the main propagules that allow fungi to persist through unfavorable conditions and disperse into new environments. Despite their importance, very little is known about their longevity and dormancy, especially in ectomycorrhizal fungi. To assess the viability of ectomycorrhizal fungal spores in forest soil, we collected and buried non-sterile forest soil, in pots, in the field distant from an inoculum source. After 6 yr, a subset of this soil was assayed for viable spores by baiting the fungi with Bishop pine (Pinus muricata) seedlings. Our results show that the three most frequent colonizers in year 1 continued to colonize significant percentages of seedlings in year 6: Wilcoxina mikolae (77 %), Rhizopogon vulgaris (13 %) and Suillus brevipes (9 %). While three species that colonized low percentages of seedlings in year 1, Suillus pungens (1 %), Rhizopogon salebrosus (2 %), and Thelephora terrestris (5 %) were not recovered in year 6. Laccaria proxima, a species not seen in year 1, was recovered on a single seedling in year 6. This is the first report of long-term survival of S. brevipes and W. mikolae. Our results reveal a more complete picture of ectomycorrhizal fungal spore longevity in soil spore banks.     

Heat activation and germination–promotion of Bacillus subtilis spores by infrared irradiation

The influence of infrared (IR) radiation on the viability and heat-activation of Bacillus subtilis spores, suspended in phosphate-buffered saline, was investigated. Two types of IR heaters with different spectral distributions were used. Near-infrared (NIR) and far-infrared (FIR) heaters with main wavelengths of approximately 1 μm and 3–6 μm, respectively, were utilized. Although both irradiation treatments decreased the number of B. subtilis colonies at a bulk temperature of approximately 75 °C, the mode of action was clearly different. In the case of the NIR heater, the number of colony-counts decreased gradually. In contrast, use of the FIR heater resulted in heat activation of the spores during the early stage of irradiation at a low bulk temperature (40–60 °C) over several minutes, followed by a decrease in the number of colonies. Consequently, FIR irradiation inactivated 90% of B. subtilis spores more effectively as compared to NIR irradiation for 20 min with a suspension volume of 20 ml and irradiation energy of 7.57 kW m^?2. Spore exposure to FIR irradiation accelerated their germination rate in nutrient broth; however, this was not true for treatment with the NIR heater. The absorption IR spectrum of B. subtilis spores indicated that FIR radiation was absorbed easily by the spore cell components and might activate the bioactive substances involved in germination. Even at the same irradiation energy, the influence of infrared radiation on spore germination was dependent on the IR spectral distribution. Bacterial spores undergoing germination lose their resistance to stressors, such as heat, chemicals and ultraviolet rays. FIR heating promotes heat activation and germination, thereby producing vegetative cells that are more susceptible to other killing methods, enabling the killing of bacterial spores at lower stress without product damage.     

Effect of cryopreservation and microencapsulation of lactic acid bacterium Enterococcus faecium MC13 for long-term storage

The aim of this work was to investigate the effect of cryoprotectants on the survival of probiotic bacterium Enterococcus faecium MC13 during freeze drying and storage. The maximum relative cell viabilities were observed when cells were freeze dried and stored at −20 °C, which is optimum temperature for the preservation of E. faecium. At all storage temperatures, trehalose was found to be retaining the highest relative cell viability than other cryoprotectants. In addition, alginate–chitosan capsules were produced to encapsulate E. faecium with the aim of enhancing survival of probiotic cells and keeping the probiotic during exposure to the harsh gastro-intestinal conditions. Encapsulation of probiotic into alginate–chitosan capsules found to be retaining higher survival of probiotic cells (4.342 ± 0.26 Log CFU mL⁻¹) at −20 °C for six months. Microencapsulated cells were resistant to simulated gastric (pH 2.0) and intestinal fluids (pH 7.5), resulting in significantly enhanced survival when compared with free cells. During in vivo treatment, capsules were broken and probiotic cells were directly released into the intestinal tract of rat. This result showed that microencapsulation of E. faecium MC13 with alginate and a chitosan coating offers an effective means of delivery of viable cells to the colon and maintains their survival during the adverse gastro-intestinal conditions.     

A new two-phase kinetic model of sporulation of Clonostachys rosea in a new solid-state fermentation reactor

A new two-phase kinetic model of sporulation of Clonostachys rosea in a new solid-state fermentation (SSF) reactor was proposed. The model including exponential and logistic models was applied to study the simultaneous effect of temperature, initial moisture content, medium thickness and surface porosity of the plastic membrane on C. rosea sporulation. The model fits experimental data very well and allows accurate predictions of spore production. The maximum spore production achieved 3.360 × 10¹⁰ (spores/gDM), about 10 times greater than that in traditional SSF reactor(data not shown). The new reactor can provide two times sporulation surface area. Moisture content can be adjusted by changing the surface porosity to meet the spore production. Two mixings carried out during fermentation makes medium loose and results in a mass of new sporulation surface area. Therefore, the new SSF reactor would have great potential for application in bulk spore production of fungal biocontrol agents.     

Conversion of cassava flour to fuel ethanol by sequential solid state and submerged cultures

A process for conversion of cassava flour to ethanol was developed. This involved direct inoculation of Aspergillus awamori spores into a cassava flour paste and incubation for some period during which hydrolytic enzymes are produced (solid state culture or koji production) and subsequent addition of water and yeast cells, during which there is simultaneous hydrolysis and ethanol production (submerged culture). When cassava flour alone was used for the solid state phase, the paste was very sticky, making mixing and aeration difficult. However, addition of rice bran improved the texture and enzyme production. The optima rice bran concentration, spore inoculum concentration, and duration of solid state culture before submerged culture were 20%, 6.16 × 10⁶ spores/100 g, and 2 days, respectively. Under these optimum conditions, a high ethanol concentration of 120 g/L and ethanol yield of 0.309 g-ethanol/g-cassava flour were obtained. This ethanol yield corresponds to 0.44 g-ethanol/g-cassava starch.     

Fungal surface remodelling visualized by atomic force microscopy

Most fungal growth is localized to the tips of hyphae, however, early stages of spore germination and the growth of certain morphological mutant strains exhibit non-polarized expansion. We used atomic force microscopy (AFM) to document changes in Aspergillus nidulans wall surfaces during non-polarized growth: spore germination, and growth in a strain containing the hypA1 temperature sensitive morphogenesis defect. We compared wall surface structures of both wild-type and mutant A. nidulans following growth at 28 ° and 42 °C, the latter being the restrictive temperature for hypA1. There was no appreciable difference in surface ultrastructure between wild-type and hypA1 spores, or hyphal walls grown at 28 °C. When dry mature A. nidulans conidia were wetted they lost their hydrophobin coat, indicating an intermediate stage between dormancy and swelling. The surface structure of hypA1 germlings grown at 42 °C was less organized than wild-type hyphae grown under the same conditions, and had a larger range of subunit sizes. AFM images of hyphal wall surface changes following a shift in growth temperature from restrictive (42 °C) to permissive (28 °C), showed a gradient of sizes for wall surface features similar to the trend observed for wild-type cells at branch points. Changes associated with the hyphal wall structure for A. nidulans hypA1 offer insight into the events associated with fungal germination, and wall remodelling.     

Effects of different inoculum densities of Trichoderma harzianum and Trichoderma viride against Meloidogyne javanica on tomato

A greenhouse experiment was conducted to evaluate the effects of different inoculum densities of two Saudi isolates of Trichoderma harzianum and Trichoderma viride against Meloidogyne javanica on tomato. Four densities (10⁴, 10⁶, 10⁸ and 10¹⁰ spores/g of soil) of each fungus were used. The results indicate that all four inoculum densities of the two Trichoderma species suppressed the nematode reproduction and root galling; and increased the growth of tomato plants, compared to controls. Efficacy of both fungi increased as their inoculum densities increased. Generally, efficacy of T. harzianum was better than that of T. viride, especially at the highest used density (10¹⁰ spore/g soil) which resulted in the best control.     

Soil microbial biomass influence on growth and biocontrol efficacy of Trichoderma harzianum

Hyphal growth and biocontrol efficacy of Trichoderma harzianum may depend on its interactions with biotic components of the soil environment. Effects of soil microbial biomass on growth and biocontrol efficacy of the green fluorescent protein transformant T. harzianum ThzID1-M3 were investigated using different levels of soil microbial biomass (153, 328, or 517 μg biomass carbon/g of dry soil). Hyphal growth of T. harzianum was significantly inhibited in soil containing 328 or 517 μg biomass carbon/g of dry soil compared with soil containing 153 μg biomass carbon/g. However, when ThzID1-M3 was added to soil as an alginate pellet formulation, recoverable populations of ThzID1-M3 varied, with the highest populations in soil containing 517 μg biomass carbon/g. When sclerotia of Sclerotinia sclerotiorum were added to soils (10 sclerotia per 150 g soil) with ThzID1-M3 (20 pellets per 150 g soil), colonization of sclerotia by ThzID1-M3 was significantly lower in the soil containing the highest level of biomass. Addition of alginate pellets of ThzID1-M3 to soils (10 pellets per 50 g) resulted in increased indigenous microbial populations (total fungi, bacteria, fluorescent Pseudomonas spp., and actinomycetes). Our results suggest that higher levels of microbial soil biomass result in increased interactions between introduced T. harzianum and soil microorganisms, and further that microbial competition in soil favors a shift from hyphal growth to sporulation in T. harzianum, potentially reducing its biocontrol efficacy.     

Experimental infection of red dwarf honeybee,Apis florea,with Nosema ceranae

This research is the first record of the infection of Apis florea by Nosema ceranae, a newly identified pathogen of honeybee in Thailand which was initially isolated from A. florea workers. Each Nosema free-bee was fed 2 μl of 50% (w/v) sucrose solution containing 0, 10,000 20,000 or 40,000 Nosema spores/bee. The survival rates of treated bees were significantly lower compared to control bees. Infectivity was not statistically different among the three spore concentrations, whereas no infection was found in control bees. Protein content of control bee hypopharyngeal glands 14 days post inoculation (p.i) was significantly higher (21.47 ± 0.17 mg/bee) compared to all treatments. The infection ratio of bees treated with 40,000 spores/bee increased with time after inoculation. These results suggest that N. ceranae has a significant negative effect on honeybee hypopharyngeal gland protein production and contributes to their shortened life span.     

Determination of coleopteran insects associated with spore dispersal of Cryptoporus volvatus (Polyporaceae: Basidiomycota) in Korea

The veiled polypore, Cryptoporus volvatus, is distributed widely in North America and East Asia and is believed to have a mutualistic relationship with coleopteran species—the fungus providing food and shelter in basidiocarps and beetle helping disperse spores.Seventy fresh basidiocarps of C. volvatus were collected from the Japanese red pine (Pinus densiflora) in the spring season of 2013 from two sites in Korea. A total of 251 insects (101 adult and 150 larvae) were collected from the inside of basidiocarps and identified using morphology and mitochondrial cytochrome c oxidase subunit I (COI) sequences. Six species belonging to five coleopteran families were identified. The number of spores attached to the bodies of adult insects was counted and average spore counts for each of the six species ranged between 1.0 × 10⁴ and 5.2 × 10⁵ spores/individual. Across localities, three species were shared (Aethina suturalis, Trogossita japonica and Parabolitophagus felix) and carried spores at high densities on their bodies, making them more likely to aid in spore dispersal.     

In vitro water stress bioassays with the nematophagous fungus Pochonia chlamydosporia: Effects on growth and parasitism

The biocontrol potential of Pochonia chlamydosporia, a fungus with parasitic activity against economically important plant-parasitic nematodes, can be influenced by abiotic factors such as water availability. The objective of this study was to evaluate the effects of different water stress regimes on in vitro growth, sporulation, germination and parasitism of P. chlamydosporia isolates. The osmotic water potential of 1.7% corn meal agar (CMA) was modified by addition of potassium chloride (KCl) or glycerol, and the matric water potential was modified using polyethylene glycol (PEG 8000). The fungus was able to grow over a range of potentials but radial growth rates decreased with the increase of osmotic and matric stress. No growth was observed at −10 MPa on 1.7% CMA amended with glycerol and at −7.1 MPa on medium with PEG 8000 but all isolates were able to resume growth when transferred onto unmodified 1.7% CMA. The production of chlamydospores was repressed in both osmotic and matric modified media. Although the production of conidia increased in medium modified with KCl, the germination rate was lower. Spores/hyphal fragments remained viable in all isolates that were previously inoculated onto media with growth-limiting water potential (−10 MPa on 1.7% CMA amended with glycerol and −10 MPa on medium with PEG 8000). The percentage of viable conidia produced on 1.7% CMA, after inoculation under osmotic or matric stress conditions for 25 days, was over 74.5% in all isolates (osmotic stress) and ranged from 1% (Pc1) to 65.8% (Pc280) (matric stress). The in vitro infection of potato cyst nematodes, Globodera rostochiensis eggs by P. chlamydosporia isolates, grown under these limiting conditions, was studied using a standard bioassay. The percentage of parasitized eggs was significantly higher under osmotic stress except for isolates Pc2 and Pc3. P. chlamydosporia spores/hyphal fragments can remain viable at water potentials limiting for growth, for prolonged periods of time, suggesting that the osmoregulation mechanisms, used to compensate water stress, affect in vitro sporulation and increased pathogenicity. Knowledge on water requirements of P. chlamydosporia enables a better understanding of its survival and growth strategies in the soil environment and could aid the development of effective strategies to increase the production and quality of inoculum, thus contributing to the implementation of biosafe, sustainable management strategies against plant-parasitic nematodes.     

The effects of some storage conditions on viability of Lecanicillium lecanii conidia to whitefly (Homoptera: Trialeurodes vaporariorum)

A study on the survival of Lecanicillium lecanii conidia in storage at room temperature was carried out. Firstly, drying methods of conidia powder were compared. Vacuum-freeze drying (VFD) was more suitable for drying conidia as compared to vacuum drying (VD) at room temperature. Vacuum-freeze drying for 24-h resulted in a water content of 5.4%, and a viability, determined as germination of conidia in 2% glucose solution after16 h, was 90.3% and the infection in greenhouse whitefly, Trialeurodes vaporariorum was about 94.7% at a dose of 1×10⁸ conidia/mL. Secondly, the factors influencing viability of conidia stored at room temperature were evaluated in the laboratory. Temperature was the most critical factor influencing conidial storage stability, among the tested factors affecting survival of conidia stored at room temperature for 6 months. Both conidial germination and infection of hosts decreased with storage temperature increasing from 15 to 35°C, and at 35°C the survival of stored conidia for 6 months was near zero. The moisture content of the conidial powder was another major factor influencing viability of stored conidia at room temperature. Conidial powder dried to about 5% moisture content showed higher viability than non-dried conidial powder. For the carriers, clay and charcoal were more suitable for storage of L. lecanii conidia at room temperature. At a room temperature of 25°C, L. lecanii conidia which were dried to 5% water content and mixed with clay or charcoal could retain about 50% survival after 6 months' storage.     

Fungal interaction between Trichoderma spp. and Pleurotus ostreatus on the enriched solid media with licorice Glycyrrhiza glabra root extract

The aim of this study is to investigate the antifungal activity of mycelia of Pleurotus ostreatus (white oyster mushroom) and licorice (Glycyrrhiza glabra) root extract against three undesirable fungi. They are Trichoderma spp., Trichoderma harzianum I and Trichoderma harzianum II which was tested on PSA (potato sucrose agar) medium enriched with licorice (Glycyrrhiza glabra) root extract (PSA-G media) using three concentrations (0.05, 0.10 and 0.20 g/L) in alone and dual cultures. Trichoderma spp. showed less mycelial growth of 8.75, 9.17 and 9.50 mm/day on PSA-G0.05, PSA-G0.1 and PSA-G0.2 respectively compared with 10.25 mm/day on fresh PSA (control) in dual culture. The best mycelial growth inhibition was recorded on PSA-G0.2 (14.97%) by T. harzianum II in alone culture opposite 63.72% in dual ones. The lower mycelial growth rate of T. harzianum I was 17.75 mm/day on PSA-G0.1 (0.10 g/L). In dual culture, overgrowth time of T. harzianum I had 5 days compared as approx. 6 days in alone culture. Generally, when the concentration of licorice extract increased, the mycelial growth rate of the undesirable fungi decreased. Also, all PSA-G media, especially PSA-G0.2, indicated low growth averages compared with the control (fresh PSA) against the pathogen while this concentration encourages growth of oyster mushroom. Also, this concentration reduced the density of sporulation of green molds; therefore, this concentration can be applied to reduce influence this pathogen in cultivation farm.     

The response of bacterial spores to vacuum treatments. II. Germination and viability studies.

The viability of Bacillus megaterium spores has been determined after exposure to vacuum dehydration at temperatures between 0 and 65 °C, for periods up to 24 hr. A curvilinear relationship has been demonstrated between viability and drying temperature, with minimum viability occuring around 15 °C and increases in viability being shown above 35 °C. In contrast to vegetative bacteria, reequilibration of the dried spores to 2 × 10^?3 or 10 Torr aqueous vapor pressure, and/or subsequent exposure to oxygen had no effect on viability. Dehydration, rehydration and oxygen treatments had no effect on the time for outgrowth of the spores or on the growth rate of the resultant vegetative cells. Physical loss of spores from samples was not demonstrated during any of these treatments. Evidence has been presented for a novel type of spore activation, which occurs during vacuum dehydration at high temperatures, to an extent that is dependent upon drying time. The mechanism of this activation is unlike that of conventional heat or chemical activation but is oxygen independent and unaffected by reequilibration to 2 × 10^?3 or 10 Torr.     

Fine structure of Chloromyxum menticirrhi n. sp. (Myxozoa) infecting the urinary bladder of the marine teleost Menticirrhus americanus (Sciaenidae) in Southern Brazil

A myxosporidian was found in the urinary bladder of the teleost Menticirrhus americanus Linnaeus, 1758 (Sciaenidae) collected from the South Atlantic coast of Brazil. Polysporic amoeboid plasmodia containing sporoblasts, developing pansporoblasts and spores were free in the bladder lumen. The prevalence of infection was 17.64% (15/85). Unfixed spores were spherical to subspherical, on average 10.5 μm long, 9.8 μm wide and 10.1 μm thick (n=25), and fixed spores measured 10.1×9.5×9.7 μm. The two spore valves were of equal size and each possessed prominent sutural lines and about 41 (37–45) surface ridges aligned parallel with the suture line. These ridges gave transverse sections a cog-wheel-like outline. The spores contained four pyriform polar capsules of equal size (3.20×2.0 μm) (n=25) (fixed), each with a polar filament having 3–4 (rarely 5) coils. The binucleate sporoplasm was irregular in shape, with granular matrix and randomly distributed dense bodies. The shape and dimensions of the spore, as well as the number, position and arrangement of the surface ridges, polar capsules and polar filament indicate that this is a new species, herein designated Chloromyxum menticirrhi. The gill, liver, gall bladder and intestine of the host showed no abnormalities.