Pongtharin Lotrakul

First Report of Tomato yellow leaf curl virus in Louisiana

Tomato yellow leaf curl virus (TYLCV) is a begomovirus (Geminiviridae) that causes a serious disease of tomato throughout the world. In 1997, the strain from Israel of TYLCV (TYLCV-IS) was found infecting tomatoes in Florida for the first time in the United States (1). During late spring of 2000, approximately 90% of the tomato plants (Lycopersicon esculentum) in a farm near New Orleans exhibited severe stunting, leaf cupping, and chlorosis. Symptoms were similar to those caused by TYLCV. Whiteflies (Bemisia tabaci biotype B) were present in the field but in relatively low numbers. The effect on yield reduction varied from negligible (late infections) to 100% (early infections). Six selected plants showing symptoms were assayed by polymerase chain reaction (PCR) using begomovirus-specific primers. Capsicum frutescens infected with an isolate of Texas pepper virus from Costa Rica was used as positive control. DNA was extracted using Plant DNAzol Reagent (GIBCO BRL). PCR was conducted using degenerate primers AV494/AC1048 that amplify the core coat protein region of most begomoviruses (2). PCR yielded a DNA fragment of approximately 550 bp, suggesting that a begomovirus was associated with the disease. The amplified DNA of one field isolate was cloned and the nucleotide (nt) sequence determined. Sequence comparisons with other begomoviruses in the GenBank Database indicated that the Louisiana isolate shared 100% nt identity with TYLCV-IS (GenBank Accession X76319). Successful transmission (100%) to Bonny Best tomato were obtained with four groups of 10 whiteflies each (B. tabaci biotype B) that fed on TYLCV-IS infected tomato plants. Acquisition and transmission feedings were for 2 days. In all cases, the virus was diagnosed by the ability to reproduce typical TYLCV-like symptoms in tomato and PCR. The virus was also successfully graft-transmitted to tomato cv. Bonny Best, Nicotiana benthamiana, and tomatillo (Physalis ixocarpa) using scions from tomato plants infected with a whitefly transmitted virus isolate. This is the first report of TYLCV-IS in Louisiana. References: (1) J. E. Polston et al. Plant Dis. 83:984-988, 1999. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288-1293, 1996.

Polycyclic aromatic hydrocarbons (PAHs) degradation by laccase from a tropical white rot fungus Ganoderma lucidum

Laccase enzyme was produced from an isolate of the white rot fungus, Ganoderma lucidum Chaaim-001 BCU. The enzyme was subsequently evaluated for its degradative ability towards sixteen types of polycyclic aromatic hydrocarbons (PAHs). The G. lucidum laccase degraded antracene completely with or without a redox mediator (2 mM 1-hydroxybenzotriazole) and also degraded benzo[a]pyrene, fluorine, acenapthene, acenaphthylene and benzo[a]anthracene up to 100.0, 98.6, 95.4, 90.1 and 85.3 %, respectively, when the mediator was present. In the absence of the mediator, the ability to degrade these compounds dropped to 71.71, 62.9, 80.49, 85.85 and 9.14% respectively. Compared to the laccase enzyme from Trametes vesicolor, G. lucidum laccase appeared to retain more of its capability to degrade these PAHs when the mediator was absent.

Occurrence of a Strain of Texas pepper virus in Tabasco and Habanero Pepper in Costa Rica

A viral disease causing severe leaf malformation and yellow mottle on Tabasco (Capsicum frutescens) and Habanero (C. chinense) pepper plants was observed in 1997 on farms in southwestern Costa Rica. Whiteflies (Bemisia tabaci) were present on affected farms and transmitted the putative virus. Total DNA was extracted from a whitefly-transmitted isolate, and polymerase chain reaction (PCR) was performed using degenerate primers. The expected PCR product (550 bp) was obtained, suggesting the presence of a geminivirus. This was confirmed by Southern analysis using a geminivirus-specific probe. The virus was mechanically transmitted from pepper to pepper. Electron microscopy of ultrathin sections from infected Tabasco pepper plants revealed fibrillar rings and viruslike particles in the nucleus of the vascular parenchyma cells. The sequence of DNA A was obtained from three overlapping PCR fragments amplified using three pairs of degenerate primers; PAL1v1978/PAR1c496, PCRc1/AV494, and PCRv181/ AC1048. The complete sequence of DNA A of this begomovirus consisted of 2,619 bp (GenBank accession number: AF149227) containing four open reading frames (ORF). The nucleotide sequence of the virus was 92.3% identical to DNA A of the Tamaulipas strain of Texas pepper virus (TPV-TAM). Phylogenetic analyses using AC1 and AV1 nucleotide sequences also indicated a close relationship between this virus and TPV. Based on the biological characteristics, the high percentage of nucleotide and derived amino acid sequence identities, and phyloge-netic analyses, we concluded that this virus is a distinct strain of TPV, and designated it as the Costa Rica strain. This is the first report of TPV in Costa Rica.

Purification, Characterization, and Potential of Saline Waste Water Remediation of a Polyextremophilic α-Amylase from an Obligate Halophilic Aspergillus gracilis

An obligate halophilic Aspergillus gracilis which was isolated from a hypersaline man-made saltern from Thailand was screened for its potential of producing extracellular α-amylase in the previous studies. In this study the α-amylase was extracted and purified by the help of column chromatography using Sephadex G-100 column. Presence of amylase was verified by SDS-PAGE analysis, showing a single band of approximately 35 kDa. The specific activity of the enzyme was found to be 131.02 U/mg. The Lineweaver-Burk plot showed the V max and K m values of 8.36 U/mg and 6.33 mg/mL, respectively. The enzyme was found to have the best activity at 5 pH, 60°C, and 30% of NaCl concentration, showing its polyextremophilic nature. The use of various additives did not show much variation in the activity of enzyme, showing its resilience against inhibitors. The enzyme, when tested for its use for synthetic waste water remediation by comparing its activity with commercial amylase in different salt concentrations showed that the α-amylase from A. gracilis was having better performance at increasing salt concentrations than the commercial one. This shows its potential to be applied in saline waste water and other low water activity effluents for bioremediation.

Purification and Characterization of a Polyextremophilic α-Amylase from an Obligate Halophilic Aspergillus penicillioides Isolate and Its Potential for Souse with Detergents

An extracellular α-amylase from the obligate halophilic Aspergillus penicillioides TISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 U·mg−1 and V max⁡ and K m values of 1.05 µmol·min−1·mg−1 and 5.41 mg·mL−1, respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80°C, and 300 g·L−1 NaCl. The addition of CaCl2 at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe2+ or Zn2+) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 g·L−1. Accordingly, it has a good potential for use as an α-amylase in a low water activity (high salt concentration) and at high pH and temperatures.

Biological and Molecular Properties of a Begomovirus from Dicliptera sexangularis

ABSTRACT Sixangle foldwing, Dicliptera sexangularis (Acanthaceae), showing severe yellow mottle and leaf distortion symptoms was collected from the shoreline of Calusa Island (Lee County, FL). The putative virus was transmitted from infected D. sexangularis to healthy seedlings by mechanical, whitefly (Bemisia tabaci biotype B), and graft-inoculations. Different forms of geminivirus-like DNAs were detected in total DNA extracted from infected plants by Southern blot hybridization analyses using DNA-A and -B of Bean golden mosaic virus (BGMV) from Guatemala as probes. Preliminary polymerase chain reaction experiments and sequence comparisons indicated that the virus was a distinct bipartite begomovirus. The virus was designated Dicliptera yellow mottle virus (DiYMV). Replicative dsDNAs of DiYMV were extracted, digested with selected restriction enzymes, and cloned into a plasmid vector. Both DNA-A and -B were sequenced and compared with those of other begomoviruses. Phylogenetic analyses using AV1, AC1, and BV1 nucleotide sequences indicated that DiYMV has a close relationship with the New World begomoviruses, especially those distributed in the nearby geographic areas of the Florida coast and the Caribbean Basin. However, different percent nucleotide sequence identities and phylogenetic relationships were detected when different open reading frames (ORFs) of DiYMV were compared with their counterparts from begomoviruses from the Caribbean Basin. Based on phylogenetic analyses of the AC1 and BV1 ORFs, DiYMV was closely related to BGMV type II isolates, whereas sequence comparisons of the common region and the AC4-derived amino acid sequences indicated its close relationship with Potato yellow mosaic virus from Venezuela.

Evaluation of the overall process on bioethanol production from miscanthus hydrolysates obtained by dilute acid pretreatment

In this study, dilute sulfuric acid pretreatment was performed to improve the sugars recovery from Korean Miscanthus straw. The effect of pretreatment conditions on solubilized xylose was fundamentally investigated for the efficient removal of xylan. The optimal conditions were determined using a statistical method, and were shown to be a temperature of 121.6°C, an acid concentration of 1.1%, and a reaction time of 12.8 min. The combined severity factor was shown to be 1.1 under the optimum conditions. Following the pretreatment, the solubilized xylose in liquid fraction was found to be 71.2%, and about 72.6% of the solid was recovered. After enzymatic hydrolysis, about 86.4% glucose conversion was achieved when the pretreated biomass was used as a substrate, with the conversion being improved 4-fold compared with the control (untreated). The hydrolysates, approximately 10 g/L glucose, were applied to the fermentation of Saccharomyces cerevisiae K35, and the ethanol yield was about 96%. The overall process was evaluated based on the material balance, and the results show that approximately 172 g bioethanol can be produced when 1,000 g Miscanthus straw is loaded into the process.

The current status of Aureobasidium pullulans in biotechnology

Different strains of the saprophytic yeast-like fungus Aureobasidium pullulans (Ascomycota: Dothideales) exhibit different biochemical characteristics, while their ubiquitous occurrence across diverse habitats and environmental conditions makes them an easily accessible source for biotechnological exploitation. They are useful in agricultural and industrial applications. Their antagonistic activities against postharvest pathogens make them suitable bioagents for the postharvest preservation of fruits and vegetables, while they possess antimicrobial activities against bacteria and fungi. Additionally, A. pullulans appears to be a potent source of single-cell protein. Many strains of A. pullulans harbor a wide range of industrially important enzymes, while the trademark exopolysaccharide pullulan that they produce has been extensively studied and is currently used in many applications. They also produce poly (β-l-malic acid), heavy oil liamocins, siderophore, and aubasidan-like β-glucan which are of interest for future applications. Ongoing studies suggest that A. pullulans holds many more interesting properties capable of further potential biotechnological applications.

Comparative Study of Physical Factors and Microbial Diversity of Four Man-Made Extreme Ecosystems

Ten soil samples were collected from four man-made extreme ecosystems, located adjacent to each other, at Ban Laem district, Phetchaburi province, Thailand. The area is used for sea salt extraction produced in artificial solar salterns. Soil analysis was performed which revealed the extreme conditions in terms of salinity, moisture content, organic matter and nitrogen content. Selective medium was provided for the isolation of microorganisms. Bacteria were found to be the most dominating group of microorganisms followed by fungi, actinomycetes and algae. Morphological and biochemical identification were performed to determine the genus of each microorganism. An actinomycete genus: Streptomyces, an algal genus: Dunaliella, three bacterial genera: Halobacillus, Halobacterium and Halomonas, and a fungal genus: Aspergillus, were the most frequently found microbes in these extreme man-made ecosystems. Interesting inter and intra relationships were observed between physical factors and microbial diversity. Except pH and salinity all physical factors were found to have positive correlation effect on microbial numbers. Actinomycetes was found to favor the fungal populations in man-made extreme ecosystems.