Microbial reduction of ethyl acetoacetate to ethyl (R)-3-hydroxybutyrate in an ionic liquid containing system

A hydrophilic ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF₄) was successfully employed as co-solvent for asymmetric bioreduction of ethyl acetoacetate (EOB) to ethyl (R)-3-hydroxybutyrate (R-EHB) catalyzed by Pichia membranaefaciens Hansen ZJPH07 cells. The results demonstrated that the addition of [BMIM]BF₄ in reaction system can markedly reduce the substrate inhibition and moderately improve the enantioselectivity compared to that in monophasic aqueous system. Among different alcohols and carbohydrates tried as co-substrate, glucose was a proper electron donor. Although isopropanol gave the best enantioselectivity with the highest yield, S-enantiomer was obtained. To optimize the bioreduction, some reaction parameters for the biosynthesis of R-EHB in this IL-containing system were investigated, such as temperature, buffer pH, shaking speed, substrate concentration, wet cells concentration and reaction time. Under the optimum conditions, best conversion of 77.8% and product enantiomeric excess (e.e.) of 73.0% were obtained. A comparative study was performed either in the presence or in the absence of [BMIM]BF₄, higher reaction yield (77.8% versus 68.5%) and product e.e. (73.0% versus 65.1%) were observed in IL-containing system with 0.55 M of the substrate, but 0.35 M of substrate concentration for the reduction in aqueous system without the addition of [BMIM]BF₄.     

Variability in Indian isolates of Sclerotium rolfsii

Variability among 26 isolates of Sclerotium rolfsii collected from various hosts/soil samples and localities in India is reported. The isolates varied in colony morphology, mycelial growth rate, sclerotium formation, teleomorph production and sclerotial size and color. Out of 26 isolates, only 4 produced the teleomorph stage on Cyperus rotundus rhizome meal agar medium. Mycelial incompatibility among the isolates was also seen, and out of 325 combinations, only 29 combinations (8.9%) showed compatible reactions. Based on mycelial compatibility, 13 vegetative incompatibility groups (VCG) were identified among the isolates. HPLC analysis of the ethyl acetate fraction of culture filtrates of the isolates revealed 10-22 peaks. Six peaks were identified as gallic, oxalic, ferulic, indole-3-acetic acid (IAA), chlorogenic, and cinnamic acids. Oxalic, IAA, and cinnamic acids were present in the culture filtrates of all the isolates in varying amounts. The other three phenolic acids were not detected in some of the isolates. A comparative HPLC analysis of sclerotial exudate, sclerotia, mycelia, and culture filtrates of two S. rolfsii isolates (leaf spot- and collar rot-causing) producing different symptoms on their respective hosts revealed variation in the content of phenolic acids, IAA, and oxalic acid.     

The respiration of sclerotia of Sclerotium rolfsii

Summary The respiration of sclerotia ofS. rolfsii was investigated using the Warburg constant-volume respirometer to measure oxygen uptake. The effects of age of sclerotia, pH, and temperature were studied. Sclerotia produced on prune agar were ideal for respirometric studies, being uniformly round and of approximately equal size. On a dry weight basis, the respiration rates of sclerotia were considerably less than those of vegetative mycelium. Sclerotia showed a decrease in respiration with increasing age. This was accompanied by morphological changes in the outer hyphal rind of the sclerotium during maturation. The respiration rate of sclerotia was approximately the same at 30° and 40° C, but was significantly lower at 45° C. Respiration of sclerotia was not markedly affected by normally encountered hydrogen-ion concentrations. However, a pH of 8.0 markedly repressed oxygen uptake. Sclerotia produced in rye grain cultures were chemically analyzed. The nitrogen content was 4.7 %, the petroleum-ether-soluble lipid content was 0.7 %, and the crude glycogen content was 14.2 % of the oven dry weight of the sclerotia.Contribution No. 345 from The Department of Botany. Portion of a thesis presented by the senior author in partial fullfillment for the M.S. degree.     

Sclerotium rolfsii: Status in cellulase research

Abstract Microbial degradation of native cellulose to glucose is catalysed by cellulases which refers to a group of enzymes acting in concert. The extracellular enzyme systems of Trichoderma reesei, Sporotrichum pulverulentum, Aspergillus niger and Sclerotium rolfsii have been examined more extensively than other microbial sources. The objective of this review is to present a comparative study of the research on cellulase and hemicellulase enzymes from S. rolfsii .     

Genotypic Diversity among Brazilian Isolates of Sclerotium rolfsii

Thirty isolates of Sclerotium rolfsii Sacc. from different hosts and regions of Brazil were studied in relation to morphology, mycelial compatibility, analysis of genomic DNA through random amplified polymorphic DNA (RAPD), variation within the nuclear rDNA [internal transcribed spacers (ITS)] and sequencing of ITS fragments. There was considerable variability among isolates in relation to the number, size and location of sclerotia on the medium surface. Thirteen mycelial compatibility groups (MCG) were identified among 23 isolates. Seven isolates were only self‐compatible. With the exception of group 3, where all the isolates came from soybean, there was no apparent correlation between group and isolate origin. On the basis of RAPD profiles, 11 haplotypes (A to K) were identified. There was an association between the RAPD groups and MCG. Haplotypes A, B, D, G, I and K belonged to MCG groups 1, 2, 3, 4, 5 and 6, respectively. All other RAPD haplotypes contained incompatible isolates. Polymerase chain reaction (PCR) amplification with primers 4R and 5F amplified two fragments containing ITS1, ITS2 and 5.8 S rDNA sequences, that were present in all isolates, with molecular sizes of 739 and 715 bp. Restriction analysis of PCR products showed that the two fragments had sequence divergency which is referred to as ‘ITS types’. Four arbitrarily chosen soybean isolates (2, 6, 7 and 23) and two non‐soybean isolates (11 and 22) were used to investigate the variation within the ITS sequence and its role in the phylogeny. The strict consensus of nine most‐parsimonious trees inferred from the data set which included six isolates of S. rolfsii, four of which have two different ‘ITS types’, showed three well‐supported groupings. The neighbour‐joining tree inferred from the data set also showed three major clades as did the parsimony tree. The major difference was that in the neighbour‐joining tree the ‘ITS type’ 11 was resolved and grouped in one clade. These results show that the ‘ITS types’ within isolates are almost always phylogenetically distinct. There was no clear correlation between ITS‐based phylogeny and isolate origin.     

Production of scleroglucan from Sclerotium rolfsii MTCC 2156

Scleroglucan, a neutral homopolysaccaride consisting of a linear chain of beta-D-(1-3)-glucopyranosyl and beta-D-(1-6)-glucopyranosyl groups, was produced by pure culture fermentation from Sclerotium rolfsii MTCC 2156 by submerged culture. Fermentation process was optimized in two steps. In the first step, one-factor-at-a-time method was used to investigate the effects of medium constituents such as carbon and nitrogen sources. In the second step, concentration of medium components was optimized using an L16-orthogonal array method. In all, 10 different carbon sources and eight different nitrogen sources were evaluated. Maximum yield of 16.58 g/l was obtained in a medium containing sucrose as a carbon source and sodium nitrate and yeast extract as nitrogen source.     

Activities of glycosidases from Sclerotium rolfsii in non-aqueous media

The rates of hydrolysis by b-D-glucosidase, b-D-xylosidase and a-L-arabinofuranosidase isolated from Sclerotium rolfsii were increased 17 to 220 fold in organic solvents as compared to aqueous system with the highest rates occurring in chlorinated hydrocarbons. The molecular weight, log P, Hildebrand solubility parameter and dielectric constant of the solvents correlated with the activities of glycosidases.     

Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii

A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-β-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-β-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control.     

Sclerotium rolfsii management in tomato using Aframomum melegueta,Ocimum gratissimum and Cymbopogon citratus

Tomato (Lycopersicon esculentum Mill.) is a popular fruit vegetable in Nigeria but production is low due to diseases, among the various disease problems associated with tomato production is southern blight caused by Sclerotium rolfsii. This study was carried out to evaluate the efficacy of Aframomum melegueta, Ocimum gratissimum and Cymbopogon citratus in vitro and in vivo. Infected tomato stems were obtained from the experimental plots of National Horticultural Research Institute, Nigeria. Cultural and morphological identification of the organism was done with the use of microscope. A. melegueta seeds, fresh leaves of O. gratissimum and C. citratus were used. The experiment was replicated three times, radial growth (mm) was measured for 5?days at an interval of 24?h. The in vivo experiment was conducted using the same extracts in a screen house and the experiment was laid out in triplicates in completely randomised design with the synthetic fungicide as control. Data collected were subjected to analysis of variance and significant means were separated using Fisher’s Least Significant Difference at P???0.05. The results showed that the extracts differed significantly (P???0.05) in their potential to inhibit the growth of S. rolfsii. The highest reduction (1.27?mm) in radial growth was obtained with 5% O. gratissimum extract while the least (4.63?cm) was recorded with the control. Disease severity and fruit yield varied among the treatments. The highest yield (170.6?g) was obtained on plants treated with 5% O. gratissimum extract and this was comparable with the yield (155.8?g) obtained on plants treated with funguforce.     

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126

AIMS: Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126. METHODS AND RESULTS: Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0.6 mol l-1 MgSO4 as osmotic stabilizer was designed to produce approx. 2 x 109 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0.6 mol l-1 sucrose agar media. CONCLUSIONS: The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.     

Acquired resistance of Alternaria solani and Sclerotium rolfsii to Polyoxin-D

Acquired resistance to the antibiotic polyoxin-D was studied in two phytopathogenic fungi, Alternaria solani Sorauer, and Sclerotium rolfsii Sacc. The ED₅₀ value of the antibiotic for A. solani was 100 μg/ml and S., rolfsii 200 μg/ml. A. solani and S. rolfsii could be trained to tolerate concentrations upto 1.000 μg/ml and 2.200 μg/ml respectively. The acquired resistance in both cases was not lost on continued subculturing in fungicide-free, media. On transfer to fungicide-free media, Polyoxinresistant strains of both the fungi showed faster growth rate and appreciable reduction in sporulation compared to the original strains. The adapted strain of A. solani showed cross-resistance to Cycloheximide and Difolatan but not to Hinosan and Bayleton.     

Integrated Control of Sclerotium rolfsii on Groundnut in South Africa

Sclerotium rolfsii Sacc. causes disease of numerous crop plants worldwide, including groundnuts. Control of this pathogen is difficult as it produces sclerotia which overwinter in the soil to emerge as inoculum and cause disease the following season. Various chemical, biological and cultural control strategies have been suggested and implemented, some of which have reduced disease incidence in the field. No studies have yet been undertaken in South Africa to control this disease on groundnut, either chemically, biologically or by cultural practices. In this study, several strategies were investigated for the control of S. rolfsii on groundnuts. Difenoconazole was identified as a fungicide that could possibly be applied in combination with Trichoderma harzianum, a biological antagonist of S. rolfsii, above carbendazim and flusilazole, and chlorothalonil. Difenoconazole significantly reduced the growth rate of S. rolfsii but not of T. harzianum. The cultivation of infected fields with an inversion plough significantly reduced infection of groundnuts by S. rolfsii and also improved the quality of the produce, while yield was not increased. Lower plant density increased the incidence of disease in an infected field, and is therefore not considered to be a viable form of cultural control.     

Trichoderma koningii as a potential parasite of sclerotia of Sclerotium rolfsii

Three different inoculum forms of Trichoderma koningii were tested in vitro for their ability to parasitize the sclerotia of Sclerotium rolfsii. Tests were conducted under two temperature regimes and three incubation periods. Wheat bran was proved to be the most potent inoculum form of the antagonist in reducing 0the viability of the sclerotia. Microscopical observations revealed the presence of hyphae, chlamydospores and conidia of T. koningii in the medullar tissues of the sclerotia. This is the first report of the effect of different inoculum forms of T. koningii on the sclerotia of S. rolfsii and of propagule (chlamydospores and conidia) formation of the antagonist inside the sclerotia of S. rolfsii.     

Biological control of tomato collar rot induced by Sclerotium rolfsii using Trichoderma species isolated in Bangladesh

This study evaluated three Trichoderma strains (T. harzianum TR05, T. virens TR06 and T. asperellum TR08) originating from Bangladesh as potential biological control agents against collar rot of tomato under greenhouse conditions. After seed treatment with TR05, a disease incidence of collar rot (5.36%) was lower than for TR06 (34.2%) and TR08 (20.8%). Germination percentage of tomato was highest for TR05 (90.3%). In soil treatment, inoculation with TR08 resulted in the lowest disease incidence (9.78%), and the disease incidence was statistically no different from that for TR05 (16.4%). Thus, TR05 and TR08 have potential as biological control agents of collar rot in tomato.