Growth and fat formation of Aspergillus oryzae and Aspergillus terreus on enzyme-hydrolyzed sweet potatoes

Two fungi, namely,Aspergillus oryzae andAspergillus terreus, have been grown on enzyme-hydrolyzed sweet potato medium either as such or supplemented with an external source of nitrogen in the form of ammonium carbonate, sodium nitrate, or asparagine. Hydrolysis alone promoted carbohydrate uptake, growth as well as fat formation to a remarkable extent in both fungi.Addition of an external supply of nitrogen to the hydrolyzed media increased further carbohydrate absorption which continued until the potato media were almost completely depleted; on unhydrolysed potato media, appreciable amounts of carbohydrates always remained unutilized. It also accelerated the rate of building up so that the dry mycelium increased by more than 4 times in some cases. Fat formation was consequently remarkably enhanced specially in the presence of ammonium carbonate or asparagine.     

A preliminary study of the applicability of sweet potatoes for mycological fat production

Summary For mycological fat production,Aspergillus nidulans andPenicillium lilacinum were grown on media made up of crushed and boiled sweet potatoes. Mycelial mats containing a fair amount of fat were obtained, namely, 15 % in case ofAspergillus and 24 % in case ofPenicillium. Media containing an external supply of glucose gave rise to increase in percentage fat in mycelium, but the amount of mycelium and consequently the total fat content decreased.Addition of sodium nitrate gave heavier mycelial mats but the total fat content could not be raised.Addition of potassium phosphate or magnesium sulphate or both to the sweet potato medium gave rise to heavier mycelial mats but with lower fat content.     

Bacterial and fungal metabolites of rhizospheric microflora of cotton antagonistic to Rhizoctonia solani Kühn

Summary Growth response ofRhizoctonia solani Kühn, the damping-off fungus, to metabolites of selected antagonistic rhizospheric bacteria and fungi of some Egyptian cotton varieties, namely, two strains ofBacillus subtilis Cohn,Aspergillus terreus Thom, andAspergillus flavus Link produced in culture media containing nitrate- or ammonium-nitrogen sources, proved the potency ofB. subtilis metabolites in inhibitingR. solani mycelial growth whether from nitrate- or ammonium-nitrogen culture media. Metabolite filtrates ofB. subtilis (II) are more potent than those ofB. subtilis (I). Increasing concentration of bacterial metabolite filtrates resulted in a decreased mycelial dry weight ofR. solani. The bacterial inhibitory factor forR. solani mycelial growth is partially affected by heat. Metabolite filtrates ofA. terreus from nitrate-nitrogen are slightly more potent than from ammonium-nitrogen culture media while an opposite relation is evident withA. flavus metabolites. Growth responses ofR. solani to different experimental dilutions of metabolite filtrates ofA. terreus andA. flavus proved the intervention of the nutritive factor in witholding growth of the damping-off fungus.     

Effect of carbon sources upon the mycelial growth of Phytophthora citrophthora

In order to study the effect of carbon originating from different sources on the growth ofP. citrophthora, a modified Christie's formula was used as the basic nutrient medium. Carbon in various forms was then substituted for glucose in equivalent amounts. The fungus grew to a limited extent on glucose as the carbon source, but grew quite well on a maltose medium and less extensively on a sucrose medium. Fungus growth was most profuse on soluble starch, even more so than on potato dextrose and oatmeal medium. No sporangia were found in any of the media.     

Allelopathic interference of sweet potato with cogongrass and relevant species

Cogongrass (Imperata cylindria) is an invasive weed and harmful to ecological systems and agricultural production in many countries. It was found that plant extracts and root exudates of sweet potato (Ipomoea batatas) exhibit allelopathic potential and inhibit the growth of cogongrass to a greater extent than either barnyardgrass (Echinochloa crus-galli), Indian goose-grass (Eleushine indica), or lettuce (Lactuca sativa) in bioassays. Greenhouse trials indicated that sweet potato soil reduced the emergence of the noxious weed by 50 %, yet exhibited either weaker inhibition or the promotion of barnyardgrass, Bidens (Bidens pilosa), and Leucaena (Leucaena leucocephala), while the desired growth of upland rice (Oryza sativa) was not affected. In cogongrass fields, the incorporation of 1–2 tons aboveground parts and cultivation of sweet potato provided 80–85 % weed control. On the other hand, the reduction of congograss in fields may be offset by the alternate invasion of B. pilosa which multiplied its biomass by 2–6 times with sweet potato amended soils. The findings of this study indicate that sweet potato is an effective crop in the biologic management of the invasive cogongrass in agricultural fields, thus the interactive mechanism between sweet potato and the invasive weed demands further investigation. Ecologically, this study highlights the specificity of allelopathic interactions between cogongrass and sweet potato that is helpful to minimize the disturbance from infestation of this invasive weed against native species and crops.     

Interaction of Ditylenchus dipsaci and Meloidogyne hapla on Resistant and Susceptible Plant Species

Numbers ofDitylenchus dipsaci or Meloidogyne hapla invading Ranger alfalfa, Tender crop bean, Stone Improved tomato, AH-14 sugarbeet, Yellow sweet clover, and Wasatch wheat from single inoculations were not significantly different from numbers by invasion of combined inoculations. D. dipsaci was recovered only from shoot and M. hapla only from root tissue. Combined inoculations did not affect reproduction of either D. dipsaci or M. hapla. D. dipsaci suppressed shoot growth of all species at 15-30 C, and M. hapla suppressed shoot growth of tomato, sugarbeet, and sweet clover at 20, 25, and 30 C. There was a positive correlation (P < 0.05) between shoot and root growth suppression by D. dipsaci on all cultivars except wheat at 20 C and tomato at 30 C. M. hapla suppressed (P < 0.05) root growth of sugarbeet at 20-50 C and wheat at 30 C. Growth suppression was synergistic in combined inoculations of sweet clover shoot growth at 15 C and root growth at 20-30 C, wheat root growth at 15 and 20 C, and tomato root growth at 15-30 C (P < 0.05) D. dipsaci invasions caused mortality of alfalfa and sweet clover at 15-30 C and sugarbeet at 20-30 C. Mortality rates of alfalfa and sweet clover increased synergistically (P < 0.05) from combined inoculations.     

Studies on the Germination of Pine Pollen (Pinus mugo) in vitro. II. Effects of Different Ions

Studies on the initial germination of pollen of Pinus mugo showed no significant influence of ions on O₂ uptake and uptake of ³²P-labelled phosphate. At the onset of tube growth O₂ uptake decreased in the absence of calcium. In inorganic media tube growth and ³²P uptake were reduced in the absence of calcium or boric acid. In the absence of calcium a requirement for magnesium was observed. When the medium was deprived of polyvalent ions with EDTA, growth and ³²P uptake ceased. The presence of calcium in the medium was found to be essential for the maintenance of the structural and functional integrity of the cell membranne. — The ion requirement was more pronounced when tube growth was stimulated with sucrose. Calcium, magnesium, boric acid, and nitrate (as nitrogen source) were essential constitutents of the medium. The stimulation due to calcium required either magnesium or boric acid. — A density effect was observed which can be related to diffusible substances from the pollen into the medium. This was not observed when calcium and magnesium were present in the medium. The phenomenon is explained as an enrichment of the medium with diffusible substances from non-germinated dead pollen. — Germination and the tube growth were found to be greatly dependent on a short period of equilibration of pollen at room temperature before sowing.     

Itaconic Acid Production by Filamentous Fungi in Starch-Rich Industrial Residues

Several fungi and starch-rich industrial residues were screened for itaconic acid (IA) production. Out of 15 strains, only three fungal strains were found to produce IA, which was confirmed by HPLC and GC–MS analysis. These strains were identified as Aspergillus terreus strains C1 and C2, and Ustilago maydis strain C3 by sequencing of 18S rRNA gene and internal transcribed spacer regions. Cis-aconitate decarboxylase (cad) gene, which encodes a key enzyme in IA production in A. terreus, was characterized from strains C1 and C2. C1 and C2 cad gene sequences showed about 96% similarity to the only available GenBank sequence of A. terreus cad gene. 3-D structure and cis-aconitic acid binding pocket of Cad enzyme were predicted by structural modeling. Rice, corn and potato starch wastes were screened for IA production. These materials were enzymatically hydrolyzed under experimentally optimized conditions resulting in the highest glucose production of 230 mg/mL from 20% potato waste. On comparing the production potential of selected strains with different wastes, the best IA production was achieved with strain C1 (255.7 mg/L) using potato waste. Elemental composition as well as batch-to-batch variation in waste substrates were analyzed. The difference in IA production from two different batches of potato waste was found to inversely correlate with their phosphorus content, which indicated that A. terreus produced IA under phosphate limiting condition. The potato waste hydrolysate was deionized to remove inhibitory ions like phosphate, resulting in improved IA production of 4.1 g/L by C1 strain, which is commercially competitive.     

Cell-free synthesis of silver nanoparticles in spent media of different Aspergillus species

The recovery and valorization of metals and rare earth metals from wastewater are of great importance to prevent environmental pollution and recover valuable resources. Certain bacterial and fungal species are capable of removing metal ions from the environment by facilitating their reduction and precipitation. Even though the phenomenon is well documented, little is known about the mechanism. Therefore, we systematically investigated the influence of nitrogen sources, cultivation time, biomass, and protein concentration on silver reduction capacities of cell-free cultivation media (spent media) of Aspergillus niger, A. terreus, and A. oryzae. The spent medium of A. niger showed the highest silver reduction capacities with up to 15 μmol per milliliter spent medium when ammonium was used as the sole N-source. Silver ion reduction in the spent medium was not driven by enzymes and did not correlate with biomass concentration. Nearly full reduction capacity was reached after 2 days of incubation, long before the cessation of growth and onset of the stationary phase. The size of silver nanoparticles formed in the spent medium of A. niger was influenced by the nitrogen source, with silver nanoparticles formed in nitrate or ammonium-containing medium having an average diameter of 32 and 6 nm, respectively.     

Effect of enhanced CaCl2, MgSO4, and KH2PO4 on improved in vitro growth of potato

Potato (Solanum tuberosum L.) is a major global food crop. Contemporary potato production largely utilizes micropropagation to produce healthy seed potatoes. The micropropagation of potatoes is widely achieved through nodal explants using the conventional Murashige and Skoog (MS) medium. Currently, effective culture media that can facilitate rapid propagation are increasingly required for new cultivars that have been developed to possess improved traits. In this study, we evaluated the effect of enhanced meso nutrients (CaCl₂.2H₂O, MgSO₄, and KH₂PO₄) in MS medium on the growth of S. tuberosum. The cultivars used in this study were representative of Japanese, European, and Peruvian lines. Enhanced meso nutrients improved the overall quality of all cultivars, as indicated by longer shoots and larger leaves with dark color, compared with MS medium only. Shoots grown on enhanced mesos were approximately 1.5 times longer than on MS medium. Quantitative ion analysis revealed that plantlets with improved shoot length and leaf quality in most cultivars had increased calcium, magnesium, potassium, and phosphorus uptake than plantlets on MS medium. The results suggest that the reduced iron uptake on 3.0×MS, compared with 2.0× or 2.5×MS mesos, reduced plant growth. This study revealed for the first time that mesos concentrations higher than MS medium concentrations, complemented by enhanced calcium, magnesium, potassium, phosphorus, and iron uptake, play a significant role in improving the in vitro growth of potato.     

Isolated Phosphate-Solubilizing Soil Bacteria Promotes In vitro Growth of Solanum tuberosum L.

The capacity of four bacterial strains isolated from productive soil potato fields to solubilize tricalcium phosphate on Pikovskaya agar or in a liquid medium was evaluated. A bacterial strain was selected to evaluate in vitro capacity of plant-growth promotion on Solanum tuberosum L. culture. Bacterial strain A3 showed the highest value of phosphate solubilization, reaching a 20 mm-diameter halo and a concentration of 350 mg/l on agar and in a liquid medium, respectively. Bacterial strain A3 was identified by 16S rDNA analysis as Bacillus pumilus with 98% identity; therefore, it is the first report for Bacillus pumilus as phosphate solubilizer. Plant-growth promotion assayed by in vitro culture of potato microplants showed that the addition of bacterial strain A3 increased root and stems length after 28 days. It significantly increased stem length by 79.3%, and duplicated the fresh weight of control microplants. In this paper, results reported regarding phosphorus solubilization and growth promotion under in vitro conditions represent a step forward in the use of innocuous bacterial strain biofertilizer on potato field cultures.Key words: Bacillus sp., phosphorus soluble, Pikovskaya agar, potato rhizosphere, plant growth promoting rhizobacteria     

Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.)

Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l⁻¹ 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l⁻¹ hygromycin and 200 mg l⁻¹ cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato.     

Extracellular proteinase spectrum ofAspergillus terreus grown on various substrates

The level of proteinase activity and the ratio of proteinases I and II, secreted byAspergillus terreus, a cellulase producer, was followed during its growth on media containing various carbon sources. Correlation was found between the level of proteinase secretion and the rate of change of the cellulase complex spectrum. The extracellular proteolytic system ofA. terreus was presented mainly by proteinase II (metalloproteinase) during cultivation under conditions favoring fast accumulation of low-molar mass cellulases. The results indicate that proteinase II could be responsible for the limited proteolysis of high-molar mass cellulases ofA. terreus into smaller enzymes of the cellulolytic complex, thus changing their substrate specificity.     

Effect of carbon and nitrogen sources on neutral proteinase production byPseudomonas aeruginosa

A strain ofPseudomonas aeruginosa from soil produced large quantitaties of extracellular neutral proteinase and could utilize several organic substances as carbon and nitrogen sources for enzyme production. The growth media required the presence of a high amount of phosphate when glucose was the carbon source. The intermediates of citric-acid cycle acids supported the proteinase production more than any other carbon sources. However, complex nitrogenous substances supported enzyme production more efficiently. Higher concentration of casamino acids suppressed the proteinase synthesis.     

Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)⁻¹, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.     

Sweet potato: A novel substrate for pullulan production by Aureobasidium pullulans

A strain Aureobasidium pullulans AP329, was used for the production of pullulan by employing hydrolysed sweet potato as cultivation media. Hydrolysis with α-amylase alone resulted in the lowest yields of pullulan. In contrast continuous hydrolysis with pullulanase and the β-amylase in sweet potato itself gave higher yields, but prolonged hydrolysis with amyloglucosidase decreased the yield. The maximum pullulan yield (29.43 g/l) was achieved at the dextrose equivalent value of 45 and pH of 5.5 for 96 h. As a substitute of sucrose, hydrolysed sweet potato was found to be hopeful and the yield of pullulan was higher than that of glucose and sucrose. The molecular weight of pullulan obtained from hydrolysed sweet potato media was much higher than that of sucrose and glucose media. Results of this work indicated that sweet potato was a promising substrate for the economical production of pullulan.     

Tributyl phosphate biodegradation to butanol and phosphate and utilization by a novel bacterial isolate,Sphingobium sp. strain RSMS

A Sphingobium sp. strain isolated from radioactive solid waste management site (RSMS) completely degraded 7.98 g/L of tributyl phosphate (TBP) from TBP containing suspensions in 3 days. It also completely degraded 20 mM dibutyl phosphate (DBP) within 2 days. The strain tolerated high levels of TBP and showed excellent stability with respect to TBP degradation over several repeated subcultures. On solid minimal media or Luria Bertani media supplemented with TBP, the RSMS strain showed a clear zone of TBP degradation around the colony. Gas chromatography and spectrophotometry analyses identified DBP as the intermediate and butanol and phosphate as the products of TBP biodegradation. The RSMS strain utilized both TBP and DBP as the sole source of carbon and phosphorous for its growth. The butanol released was completely utilized by the strain as a carbon source thereby leaving no toxic residue in the medium. Degradation of TBP or DBP was found to be suppressed by high concentration of glucose which also inhibited TBP or DBP dependent growth. The results highlight the potential of Sphingobium sp. strain RSMS for bioremediation of TBP and for further molecular investigation.     

Effects of single and mixed inoculation with two arbuscular mycorrhizal fungi in two different levels of phosphorus supply on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers

Aims

This study aimed to determine the effect of arbuscular mycorrhizal (AM) fungi and phosphorus (P) supply levels on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers.

Methods

Two commercial AM fungal isolates of Glomus intraradices (IFP Glintra) and Glomus mosseae (IFP Glm) which differ in their life cycles were used. Sweet potato plants were grown in a horizontal split-root system that consisted of two root compartments. A root-free fungal compartment that allowed the quantification of mycelial development was inserted into each root compartment. The two root compartments were inoculated either with the same or with different AM isolates, or remained free of mycorrhizal propagules. Each fungal treatment was carried out in two P supply levels.

Results

In the low P supply level, mycorrhizal colonization significantly increased β-carotene concentrations in sweet potato tubers compared with the non-mycorrhizal plants. Glomus intraradices appeared to be more efficient in increasing β-carotene concentrations than G. mosseae. Dual inoculation of the root system with the two mycorrhizal fungi did not result in a higher increase in tuber β-carotene concentrations than inoculation with the single isolates. Improved P nutrition led to higher plant tuber biomass but was not associated with increased β-carotene concentrations.

Conclusions

The results indicate a remarkable potential of mycorrhizal fungi to improve β-carotene concentrations in sweet potato tubers in low P fertilized soils. These results also suggest that β-carotene metabolism in sweet potato tubers might be specifically activated by root mycorrhizal colonization.     

Biocontrol Activity of Aspergillus terreus ANU-301 against Two Distinct Plant Diseases,Tomato Fusarium Wilt and Potato Soft Rot

To screen antagonistic fungi against plant pathogens, dual culture assay (DCA) and culture filtrate assay (CFA) were performed with unknown soil-born fungi. Among the different fungi isolated and screened from the soil, fungal isolate ANU-301 successfully inhibited growth of different plant pathogenic fungi, Colletotrichum acutatum, Alternaria alternata, and Fusarium oxysporum, in DCA and CFA. Morphological characteristics and rDNA internal transcribed spacer sequence analysis identified ANU-301 as Aspergillus terreus. Inoculation of tomato plants with Fusarium oxysporum f. sp. lycopersici (FOL) induced severe wilting symptom; however, co-inoculation with ANU-301 significantly enhanced resistance of tomato plants against FOL. In addition, culture filtrate (CF) of ANU-301 not only showed bacterial growth inhibition activity against Dickeya chrysanthemi (Dc), but also demonstrated protective effect in potato tuber against soft rot disease. Gas chromatography-tandem mass spectrometry analysis of CF of ANU-301 identified 2,4-bis(1-methyl-1-phenylethyl)-phenol (MPP) as the most abundant compound. MPP inhibited growth of Dc, but not of FOL, in a dose-dependent manner, and protected potato tuber from the soft rot disease induced by Dc. In conclusion, Aspergillus terreus ANU-301 could be used and further tested as a potential biological control agent.     

Lovastatin Biosynthesis by Aspergillus terreus in a Chemically Defined Medium

Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.