Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

The effect of alkanes on the physiology and metabolism of the entomopathogenic fungus,Isaria fumosorosea

The influence of different alkanes on spore morphology, glyoxlate pathway enzyme activities, total lipid contents and fatty acid composition of Isaria fumosorosea were investigated under laboratory conditions. Fungal spores grown on different alkanes showed higher germination and mycelial growth when compared to control. A strong induction of glyoxlate cycle enzymes in cell-free extracts was observed for cells grown on different alkanes when compared to glucose and control. Higher activities of glyoxlate cycle enzymes were observed for cells grown on alkanes when compared to other treatments. Even numbered fatty acids accounted for the majority of fatty acid production with a significant increase in relative amounts of linoleic acid and palmatic acid observed for conidia grown on alkanes. These results indicate that addition of alkanes to culture media can be a tool to pre-induce metabolic adaptations that can facilitate successful infection of insect host by entomopathogenic fungi.     

Morphological development of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Application of neural network and cluster analysis for characterization of mycelial morphology

Background  

Although the citric acid fermentation by Aspergillus niger is one of the most important industrial microbial processes and various aspects of the fermentation appear in a very large number of publications since the 1950s, the effect of the spore inoculum level on fungal morphology is a rather neglected area. The aim of the presented investigations was to quantify the effects of changing spore inoculum level on the resulting mycelial morphology and to investigate the physiology that underlines the phenomena. Batch fermentations were carried out in a stirred tank bioreactor, which were inoculated directly with spores in concentrations ranging from 10⁴ to 10⁹ spores per ml. Morphological features, evaluated by digital image analysis, were classified using an artificial neural network (ANN), which considered four main object types: globular and elongated pellets, clumps and free mycelial trees. The significance of the particular morphological features and their combination was determined by cluster analysis.     

Differential expression of desaturases and changes in fatty acid composition during sporangiospore germination and development in Mucor rouxii

Polyunsaturated fatty acids (PUFAs), namely, oleic (C18:1), linoleic (C18:2), and gamma-linolenic acid (C18:3), constituted the majority in the total fatty acid content (44%) of sporangiospores of Mucor rouxii. At 30 degrees C, the germination begins within 1h at which time spore swelling occurs, followed by germ tube emergence within 3-4h. Throughout germination, an increase in gamma-linolenic acid (GLA) was observed and its content was highest at germ tube emergence. It took longer for sporangiospores of M. rouxii to germinate at sub-optimal temperatures (15 and 35 degrees C). However, the content of GLA was higher at the germ tube initiation than at the mycelial stage at all temperatures, suggesting the association of GLA and germination of sporangiospores. This finding was substantially confirmed by differential expression of delta9-, delta12-, and delta6-desaturase genes measured during spore germination. The expression of three desaturase genes parallels the pattern of GLA synthesis. By using RT-PCR techniques to follow gene expression, we found that mRNA of delta12- and delta6-desaturase genes were translated as soon as the spores were introduced into a fresh medium while the mRNA of delta9-desaturase gene could not be detected until 2h after introduction. A sharp increase in mRNA of delta6-desaturase genes correlated well with an increase in GLA content at germ tube emergence (4h). These results demonstrated that changes in fatty acid composition of sporangiospore of M. rouxii and differential expression of desaturase genes occurred during germination, and that extensive changes in GLA synthesis associated with some events in germination process.     

Influence of inocula and grains on sclerotia biomass and carotenoid yield of<Emphasis Type="Italic"> Penicillium</Emphasis> sp. PT95 during solid-state fermentation

Various inocula and grains were evaluated for carotenoid production by solid-state fermentation using Penicillium sp. PT95. Millet medium was more effective in both sclerotia growth and carotenoid production than other grain media. An inoculum in the form of sclerotia yielded higher sclerotia biomass compared to either a spore inoculum or a mycelial pellet inoculum. Adding wheat bran to grain medium favored the formation of sclerotia. However, neither the inoculum type nor addition of wheat bran resulted in a significant change in the carotenoid content of sclerotia. Among grain media supplemented with wheat bran (wheat bran:grain =1:4 w/w, dry basis), a medium consisting of rice and wheat bran gave the highest sclerotia biomass (15.10 g/100 g grain), a medium consisting of buckwheat and wheat bran gave the highest content of carotenoid in sclerotia (0.826 mg/g dry sclerotia), and a medium consisting of millet and wheat bran gave the highest carotenoid yield (11.457 mg/100 g grain).     

Production of gamma-linolenic acid by Mucor circinelloides and Mucor rouxii with acetic acid as carbon substrate

Summary The production of gamma-linolenic acid (GLA) by Mucor circinelloides CBS 203.28 and M. rouxii CBS 416.77 in fed-batch cultures operated in pH-stat mode with acetic acid as carbon substrate and titrant compared favourably with the performance of M. circinelloides in batch culture on glucose. On acetic acid M. circinelloides accumulated up to 39.8 mg GLA/g biomass, with a crude oil content of 28% containing 91% neutral lipids. The GLA content of the neutral lipid fraction was 15.6%.     

Gamma-linolenic acid production of Mucor rouxii by solid-state fermentation using agricultural by-products

Aims:  This study aims to maximize the yield of gamma-linolenic acid by a filamentous fungus, Mucor rouxii , using low cost production by solid-state fermentation.
Methods and Results:  We optimized substrate types and culture conditions including inoculum size and temperature. The optimal growth of M. rouxii was found in the cultures inoculated with 5 × 10⁵ spores g⁻¹ substrate. The fungal cells grew well on rice bran and soy bean meal, whereas a lower biomass was found in the cultures grown on polished rice, broken rice and spent malt grain. The GLA content was highly accumulated in rice bran ferment and its maximal content of about 6 g kg⁻¹ fermented mass was observed in the 5th-day culture grown at 30°C. However, the GLA content in the rice bran ferment was not enhanced by low temperature.
Conclusions:  The GLA production of M. rouxii could be enhanced by optimizing the agricultural by-product substrates and culture condition.
Significance and Impact of the Study:  Low cost GLA production process was achieved, and fermented product containing GLA can be incorporated into feed additives without further oil extraction to alternate the expensive plant oils.     

Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans

In the Ascomycete fungus Aspergillus nidulans, the ratio of conidia (asexual spores) to ascospores (sexual spores) is affected by linoleic acid moieties including endogenous sporogenic factors called psi factors. Deletion of odeA (Delta odeA), encoding a Delta-12 desaturase that converts oleic acid to linoleic acid, resulted in a strain depleted of polyunsaturated fatty acids (18:2 and 18:3) but increased in oleic acid (18:1) and total percent fatty acid content. Linoleic acid-derived psi factors were absent in this strain but oleic acid-derived psi factors were increased relative to wild type. The Delta odeA strain was reduced in conidial production and mycelial growth; these effects were most noticeable when cultures were grown at 26 degrees C in the dark. Under these environmental conditions, the Delta odeA strain was delayed in ascospore production but produced more ascospores than wild type over time. This suggests a role for oleic acid-derived psi factors in affecting the asexual to sexual spore ratio in A. nidulans. Fatty acid composition and spore development were also affected by veA, a gene previously shown to control light driven conidial and ascospore development. Taken together our results indicate an interaction between veA and odeA alleles for fatty acid metabolism and spore development in A. nidulans.     

Cyclone‐based spore trapping,quantitative real‐time polymerase chain reaction and high resolution melting analysis for monitoring airborne inoculum of Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases in rice worldwide. We aimed to develop an integrated approach for convenient collection, quantification and characterisation of M. oryzae spores (airborne inoculum) in the field. We developed an easy‐to‐use cyclone‐based spore trap (the AirSampler) and a standard procedure for handling a small amount of airborne spores. Using a specific primer pair or a probe designed for the single‐copy gene mif23, SYBR Green and TaqMan assays could quantify 10 and 4 copy numbers, respectively, of M. oryzae DNA. During 2012 and 2013, the AirSampler and SYBR Green quantitative real‐time polymerase chain reaction were used to monitor temporal dynamics of M. oryzae spores in nursery fields of rice showing symptoms of blast disease. During four cropping seasons, the new techniques could detect M. oryzae spores before the appearance of rice blast symptoms. The amount of spores was low in the early season, then increased, with high fluctuations during the mid‐season and decreased to low levels at the heading stage in the late season. To improve the handling and storage of spore samples, we tested the effect of different treatments on the preservation of spore DNA. DNA loss was reduced with samples protected from ultraviolet B radiation, suspended in CTAB buffer, kept at room temperature or 4°C and used for DNA extraction in 2 weeks. Finally, we demonstrated that the high resolution melting analysis could be used for rapid determination of A, D, A + D and C alleles of the avirulence gene pex31 (Avr‐Pik/kp/km) in M. oryzae.     

Effects of prefermented cereal-derived substrates (ground barley and rye bran) enriched with fungal γ-linolenic acid on rumen fermentation parameters and lipid metabolism in vitro

Aims: To increase rumen output of γ‐linolenic acid (GLA), we used two cereal‐derived substrates, ground barley (GB) and rye bran (RB), enriched with fungal GLA as components of feed rations. We examined their effects on rumen fermentation patterns, lipid metabolism and the ciliated protozoan population in an artificial rumen. Methods and Results: Four diets consisting of meadow hay (MH) plus unfermented (GB or RB) or prefermented (GB ? TE or RB ? TE) cereal‐derived substrates were fermented in an artificial rumen with ovine rumen inoculum. The cereal‐derived substrates were prefermented with the fungus Thamnidium elegans (TE) by fungal solid‐state fermentation. The diets with TE increased the rumen input of dietary GLA (mg day^?1) from 0 to 21 (GB ? TE) or 26 (RB ? TE). Both experimental diets increased the rumen output of GLA (P < 0·001). Adverse effects on the ciliate population were observed. Both diets also had an effect on the fatty acids profile. Fermentation patterns were also affected with MH + RB ? TE. Conclusion: Cereal‐derived substrates enriched with GLA effectively enhanced the output of GLA in artificial rumen. Significance and Impact of the Study: The ability of the fungal strain T. elegans to grow and utilize various agro‐industrial substrates might be useful in developing potential new animal diets enriched in GLA.     

Mass production of spores of lactic acid‐producing Rhizopus oryzae NBRC 5384 on agar plate

Mass production of sporangiospores (spores) of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) on potato‐dextrose‐agar medium was studied aiming at starting its L (+)‐lactic acid fermentation directly from spore inoculation. Various parameters including harvest time, sowed spore density, size of agar plate, height of air space, and incubation mode of plate (agar‐on‐bottom or agar‐on‐top) were studied. Ordinarily used shallow Petri dishes were found out to be unsuitable for the full growth of R. oryzae sporangiophores. In a very wide range of the sowed spore density, the smaller it was, the greater the number of the harvested spores was. It was also interesting to find out that R. oryzae grown downward vertically with a deep air space in an agar‐on‐top mode gave larger amount of spores than in an agar‐on‐bottom mode at 30°C for 7‐day cultivation. Scale‐up of the agar plate culture from 26.4 to 292 cm² was studied, resulting in the proportional relationship between the number of the harvested spores/plate and the plate area in the deep Petri dishes. The number of plates of 50 cm in diameter needed for 100 m³ industrial submerged fermentation started directly from 2 × 10⁵ spores/mL inoculum size was estimated as about 6, from which it was inferred that such a fermentation would be feasible. Designing a 50 cm plate and a method of spreading and collecting the spores were suggested. Bioprocess technological significance of the “full‐scale industrial submerged fermentation started directly from spore inoculation omitting pre‐culture” has been discussed. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:876–881, 2013     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Preparation of fungal starter culture in liquid fluidized bed reactor

Summary Pregerminated Trichoderma reesei (Rut C-30) spores were grown on corncob particles in a liquid fluidized bed reactor (LFBR). Hyphal mass covered particles were recovered from the top of the reactor in 24 h and used as starters for solid substrate fermentation. The starter from LFBR gave better biomass production than spore or mycelial inoculum.     

Acid proteinases production by humicola lutea cells immobilized in polyhydroxyethylmethacrylate gel

The spores of Humicola lutea entrapped in polyhydroxyethylmethacrylate gel were precultivated in production medium for mycelial formation. The immobilized mycelium was reused in batch mode for acid proteinases production. The influence of precultivation time, initial inoculum gel volume, and gel particle size on the enzyme activity and proteinases production half-life were studied. After 70 h precultivation of the entrapped spores (10 ml initial inoculum volume, 12–27 mm³ gel particle size) maximum proteinases activity of 100–140% (compared with free cells) was registered in 15 reaction cycles. Under the same condition the half-life time was 18 cycles, while for the free cells it was 5 cycles. The main advantage of the polyhydroxyethylmethacylate immobilized H. lutea was the long acid proteinases production half-life at a low concentration of outgrowing cells in the medium.     

Effect of culture conditions on growth and sporulation ofBacillus thuringiensis subsp.israelensis and development of media for production of the protein crystal endotoxin

Summary The influence of medium composition on the inoculum and production stages of theBacillus thuringiensis subsp.israelensis bioinsecticide fermentation was investigated. Media which inhibited sporulation were selected for inoculum development stages. Bioinsecticide production media were designed to produce high cell counts and >90% sporulation in a 48h fermentation. Maximum insecticidal activity occurred at the point of maximum bacterial cell lysis/spore release. A process involving two inoculum stages and a 48h production stage in a 40 l fermenter yielded a viable cell count of 6.5 x 10⁹/ml with greater than 95% sporulation. Good correlation existed between spore counts and bioinsecticide activity.     

Infection ability of mycelium and spores of<Emphasis Type="Underline">Microdochium</Emphasis><Emphasis Type="Underline">nivale</Emphasis> (Fr) samuels hallett to<Emphasis Type="Underline">Lolium</Emphasis><Emphasis Type="Underline">Perenne</Emphasis> L.

Mycelium and spores ofMicrodochium nivale /Syn.Fusarium nivale/ were compared according to their ability to infect plants ofLolium perenne. The experiments were carried out according to the “cold chamber” method (Cormack, Lebeau 1956 modified by Pronczuk 1987). Between these two types of inoculum significant differences were found. The spore inoculum did not give any symptoms while the mycelial inoculum incited a severe disease in plants ofLolium perenne during one month of incubation.Under laboratory conditions it was found that the spore cultures ofMicrodochium nivale grew very slowly at 0 – 1°C, whereas their growth at 18 – 20°C was very fast. Growth of the mycelial cultures was not as profoundly affected by temperatures studied as the spore ones.It was concluded that to incite a disease the spore inoculum require longer incubation time than mycelial ones. The mycelial inoculum is more useful for screening of plants for resistsance.     

Filamentous Growth of Mucor rouxii Under Nitrogen

The form of growth of Mucor rouxii (National Regional Research Laboratory 1894) under nitrogen is dependent on inoculum size. With a large inoculum (10⁶ spores inoculated per ml), the morphology consists mainly of swollen spores with some filaments and yeastlike cells. At lower inoculum levels growth is filamentous. The morphology of this strain on incubation under nitrogen and the dependence of the form of growth on inoculum size are similar to those found previously for other strains of M. rouxii.     

Production of two entomopathogenic Aspergillus species and insecticidal activity against the mosquito Culex quinquefasciatus compared to Metarhizium anisopliae

The spore productivity and insecticidal activity of two opportunistic insect pathogenic Aspergillus species (namely: Aspergillus clavatus Desmazieres and Aspergillus flavus Link (Ascomycota: Eurotiales, Trichocomaceae)) were compared to Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin (Ascomycota: Hypocreales, Clavicipitaceae) for mosquito (Diptera: Culicidae) control. The production of aerial spores on wheat bran and white rice was investigated in solid-, semi-solid-, and liquid-state media supplemented with a nutritive solution. Wheat bran-based media increased the spore yield in solid-state from three to sevenfold: A. clavatus produced 48.4?±?5.2 and 15.7?±?1.6?×?10⁸ spores/g, A. flavus produced 22.3?±?4.1 and 3.1?±?2.5?×?10⁸ spores/g, and M. anisopliae produced 39.6?±?6.5 and 13.1?±?2.6?×?10⁸ spores/g of wheat bran or white rice, respectively. A. clavatus, A. flavus and M. anisopliae spores harvested from wheat bran-based solid-state media showed lethal concentrations (LC₅₀) of 1.1, 1.8, and 1.3?×?10⁸ spores/ml against Culex quinquefasciatus Say larvae in 72?h. Because A. clavatus and M. anisopliae displayed similar features when cultured under these conditions, our results suggest that insect pathogenic Aspergillus species may be as productive and virulent against mosquito larvae as a well-recognised entomopathogenic fungus.