Production and characterization of extracellular liamocins produced from fungal strains of Aureobasidium spp.

Liamocins, a group of high-density glycolipids, are only produced by certain strains of the yeast-like fungi in the genus Aureobasidium. Until now, few studies have focused on the surfactant properties of liamocins produced from the highly diverse tropical strains of Aureobasidium. Therefore, the aims of this research were to screen the liamocin production from tropical strains of Aureobasidium spp. and to characterize their surfactant properties. A total of 41 strains of Thai Aureobasidium spp. were screened for their ability to produce liamocins, and the products were detected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thin-layer chromatography. Of those strains, 30 strains of Aureobasidium spp. tested were found to produce liamocins with yields ranging from 0.53 to 10.60 g/l. The nature of all crude liamocins was heterogeneous, with different compositions and ratios depending on the yeast strain. These liamocins exhibited relatively high emulsifying activity against vegetable oils tested, with an emulsification index of around 40-50%; the emulsion stability of some liamocins was up to 30 days. The obtained critical micelle concentration values were varied, with those ​​of liamocins produced from A. pullulans, A. melanogenum and A. thailandense falling in ranges from 7.70 to 119.78, 10.73 to > 1,000, and 68.56 to > 1,000 mg/l, respectively. The emulsification activity of liamocins was higher than that of the analytical grade rhamnolipids. These compounds showed strong surface tension reduction in a sodium chloride concentration range of 2-12% (w/v), pH values between 3 and 7, and temperatures between 4 and 121 °C. This is the first report of liamocins produced by A. thailandense.

Xylooligosaccharides produced from sugarcane leaf arabinoxylan using xylanase from Aureobasidium pullulans NRRL 58523 and its prebiotic activity toward Lactobacillus spp.

In an attempt to enhance the value of sugarcane leaf, xylan was extracted and used for xylooligosaccharide (XO) production via enzymatic hydrolysis using xylanase from the black yeast Aureobasidium pullulans. The xylan was extracted from sugarcane leaf using alkali extraction according to the response surface methodology. The highest xylan yield (99.42 ± 4.05 % recovery) was obtained using 14.32 % (w/v) NaOH, 13.25:1 liquid: solid ratio, at 121 °C and 15 lb.in2 for 32 min. Sugar composition and FTIR spectrum analyses confirmed its structure as arabinoxylan. The extracted arabinoxylan had a relatively high molecular weight compared to previous studies. Crude endoxylanase from A. pullulans NRRL 58523 was selected for enzymatic hydrolysis of the xylan. The enzyme hydrolyzed well at 50 °C, pH 4.0 and was relatively stable under this condition (87.38 ± 1.26 % of the activity remained after 60 h). XOs, especially xylobiose and xylotriose, were obtained at the maximum yield of 237.51 ± 17.69 mg/g xylan via endoxylanase hydrolysis under the optimum conditions (50 °C, pH 4.0, 65.31 U/g xylan, 53 h). XOs exhibited species-specific prebiotic activity toward three strains of Lactobacillus spp. but not toward Bifidobacterium spp.

Production and characterization of thermostable acidophilic ß-mannanase from Aureobasidium pullulans NRRL 58524 and its potential in mannooligosaccharide production from spent coffee ground galactomannan.

The maximum yield of the crude mannanase from Aureobasidium pullulans NRRL 58524 was 8.42 ± 0.18 U mL-1 when cultured for 72 h in the optimized medium containing 3% (w v-1) defatted spent coffee grounds (SCG) and 0.67% (w v-1) ammonium sulphate. Two forms of mannanase were observed in the crude enzyme and the principal mannanase was enriched to apparent homogeneity via sequential filtration and anion exchange chromatography. The molecular mass of the enzyme was approximately 63 kDa as determined by SDS-PAGE. The enriched mannanase was active at high temperatures (45-75 °C) and a pH range from 3 to 6 with the maximum activity at 55 °C and pH 4.0. The enzyme was relatively thermostable with more than 75% of its initial activity remained after a 12 h incubation at 55 °C. The half-lives of the enriched mannanase were over 8 and 6 h at 60 and 65 °C, respectively. The enzyme was not adversely affected by chelator and most ions tested. This enzyme could hydrolyze both glucomannan and galactomannan and exhibited limited catalytic activity on beta-glucan. When the crude mannanase was used to hydrolyze galactomannan extracted from SCG, the maximum yield of reducing sugars mainly comprising of mannobiose (16.27 ± 0.84 mg 100 mg-1), and mannotriose (2.85 ± 0.20 mg 100 mg-1) was obtained at 58.22 ± 2.04 mg 100 mg-1 dry weight, under optimized condition (84.87 U g-1 mannanase, 41 h 34 min incubation at 55 °C and pH 4.0). These results suggested the prospect of the enzyme in mannan hydrolysis and mannooligosaccharide production at a larger scale. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03301-4.

Antioxidant and UV-Blocking Properties of a Carboxymethyl Cellulose-Lignin Composite Film Produced from Oil Palm Empty Fruit Bunch.

Oil palm empty fruit bunch (EFB) pulp with the highest cellulose content of 83.42% was obtained from an optimized process of acid pretreatment (0.5% v/v H2SO4), alkaline extraction (15% w/w NaOH), and hydrogen peroxide bleaching (10% w/v H2O2), respectively. The EFB cellulose was carboxymethylated, and the obtained carboxymethyl cellulose (CMC) was readily water-soluble (81.32%). The EFB CMC was blended with glycerol and cast into a composite film. Lignin that precipitated from the EFB black liquor was also incorporated into the film at different concentrations, and its effect on the UV-blocking properties of the film was determined. Interestingly, the EFB CMC film without lignin addition completely blocked UV-B transmittance. The incorporation of lignin at all concentrations significantly enhanced the UV-A blocking and other physical properties of the film, including the surface roughness, thickness, and thermal stability, although the tensile strength and water vapor permeability were not significantly affected. Complete UV-A and UV-B blocking were observed when lignin was added at 0.2% (w/v), and the film also exhibited the highest antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals with an half-maximal inhibitory concentration (IC50) value of 3.87 mg mL-1.

Toxicity evaluation and biodegradation of phenanthrene by laccase from Trametes polyzona PBURU 12.

The newly isolated Trametes polyzona PBURU 12 demonstrated a high tolerance and potential for the degradation of phenanthrene. The fungal isolate was able to tolerate 100 ppm of phenanthrene with 45% relative growth. The crude laccase produced by Trametes polyzona PBURU 12 was able to degrade phenanthrene by up to 98% within 24 h. The degradation metabolites showed the absence of toxic compounds. Microbial viability tests using E. coli and B. subtilis revealed that the treated phenanthrene was less toxic than untreated phenanthrene. Phytotoxicity and genotoxicity tests, using Vigna radiata and Allium cepa, indicated that the treated phenanthrene was less toxic to the plants. No mutagenic activity was found in the Ames test. The crude laccase from Trametes polyzona PBURU 12 was demonstrated as a potential tool for the biodegradation of PAHs (phenanthrene), with low toxic effects after the degradation.

Production of cutinase from Fusarium falciforme and its application for hydrophilicity improvement of polyethylene terephthalate fabric.

Among 23 isolates of cutinase-producing fungi from Thailand, one strain of Fusarium falciforme PBURU-T5 exhibited the greatest cutinase activity (3.36 ± 0.12 U ml-1) against p-nitrophenyl butyrate. This strain was found to produce an inducible cutinase when cultivated in the liquid mineral medium containing cutin from papaya peel as the sole carbon source. By optimizing the production condition based on the central composite experimental design, the maximal cutinase activity up to 4.82 ± 0.18 U ml-1 was attained under the condition: 0.4% (w/v) papaya cutin as the carbon source, 0.3% (w/v) peptone as the nitrogen source, incubation temperature at 30 °C for 4 days, and initial pH 7.0. The crude enzyme was optimally active at 35 °C and pH 9.0 which was suitable for textile industrial application. The treatment with the crude PBURU-T5 cutinase (100 U g-1 dry weight of fabric) could enhance the wetting time, water adsorption and moisture regain of polyethylene terephthalate fabric up to 1.9-, 1.2- and 1.3-fold, respectively, comparing with the conventional 1M NaOH treatment. The increment of these fabric properties by enzymatic treatment could facilitate the dyeing process and enhance the fabric softness. Thus, F. falciforme PBURU-T5 is the promising source of cutinase for the modification of the PET fabric surface.

Genome sequence and phylogenetic analysis of a novel comovirus from tabasco pepper (Capsicum frutescens).

A virus isolate from tabasco pepper (Capsicum frutescens) has been reported as a strain of the comovirus Andean potato mottle virus (APMoV). Using the replicative intermediate viral dsRNA, the pepper virus strain was sequenced by Illumina MiSeq. The viral genome was de novo assembled resulting in two RNAs with lengths of 6028 and 3646 nt. Nucleotide sequence analysis indicated that they corresponded to the RNA-1 and RNA-2 of a novel comovirus which we tentatively named pepper mild mosaic virus (PepMMV). Predictions of the open reading frame (ORF) of RNA-1 resulted in a single ORF of 5871 nt with five cistrons typical of comoviruses, cofactor proteinase, helicase, viral protein genome-linked, 3C-like proteinase (Pro), and RNA-dependent RNA polymerase (RdRP). Similarly, sequence analysis of RNA-2 resulted in a single ORF of 3009 nt with two cistrons typical of comoviruses: movement protein and coat protein (large coat protein and small coat proteins). In pairwise amino acid sequence alignments using the Pro-Pol protein, PepMMV shared the closest identities with broad bean true mosaic virus and cowpea mosaic virus, 56% and 53.9% respectively. In contrast, in alignments of the amino acid sequence of the coat protein (small and large coat proteins) PepMMV shared the closest identities to APMoV and red clover mottle virus, 54% and 40.9% respectively. A phylogenetic tree constructed using the conserved domains for the Pro-Pol from all members of the family Secoviridae confirmed the comovirus nature of the virus. Phylogenetic and sequence analyses supports proposing PepMMV as a new species of the genus Comovirus.

Purification and Characterization of Cellulase from Obligate Halophilic Aspergillus flavus (TISTR 3637) and Its Prospects for Bioethanol Production.

A cellulase from the extreme obligate halophilic fungus, Aspergillus flavus, isolated from a man-made solar saltern in Phetchaburi, Thailand, was purified by ammonium sulfate precipitation and using Sephadex G-100 gel filtration column chromatography. The cellulase was found to be approximately 55 kDa by SDS-PAGE. Using CMC as a substrate, the specific activity of the cellulase was 62.9 units (U) mg-1 with Vmax and Km values of 37.87 mol min-1 mg-1 and 3.02 mg mL-1, respectively. Characterization of the enzyme revealed it to be an extremozyme, having an optimum activity at pH 10, 60 °C, and 200 g L-1 of NaCl. The enzyme activity was not significantly altered by the addition of divalent metal cations at 2 mM and neither did ß-mercaptoethanol, while EDTA was found strongly inhibiting the cellulase. Compared with commercial cellulase, the purified cellulase from A. flavus was more active in the extremity of conditions, especially at pH 10, 60 °C, and 150 g L-1 NaCl, whereas the commercial cellulase had a very low activity.

Fusigen Reduces Intracellular Reactive Oxygen Species and Nitric Oxide Levels.

BACKGROUND/AIM: Oxidative stress caused by the production of excessive cellular reactive oxygen species (ROS) and high levels of nitric oxide contribute to several human pathologies. This study aimed to examine the anti-oxidant effects of fusigen, a compound produced from Aureobasidium melanogenum. MATERIALS AND METHODS: Extracts of A. melanogenum were selected as a source for the isolation of fusigen. The anti-oxidant, nitric oxide suppression, as well as the free radical scavenging activities of fusigen were tested in BEAS-2B human bronchial epithelial cell line (BEAS-2B cells) and human dermal papilla cells (DP cells) using specific fluorescence dyes and flow cytometry analysis. Cell viability was determined by the MTT assay. RESULTS: Fusigen did not exert cytotoxicity in the human normal BEAS-2B and DP cells at concentrations up to 100 µM. Fusigen decreased basal levels of cellular ROS, as well as the levels of ROS induced by hydrogen peroxide and ferrous ion enrichment. ROS decreasing effect was confirmed in DP cells. In addition, fusigen treatment suppressed intracellular NO levels in both BEAS-2B and DP cells. CONCLUSION: The optimal process of production of purified fusigen from A. melanogenum was determined. Fusigen exhibited a low cytotoxic effect and the potential to suppress ROS and NO. These results demonstrated that fusigen may be used for the treatment or prevention of human diseases.

Enzymatic hydrolysis of tropical weed xylans using xylanase from Aureobasidium melanogenum PBUAP46 for xylooligosaccharide production.

The maximum yield of xylanase from Aureobasidium melanogenum PBUAP46 was 5.19 ± 0.08 U ml-1 when cultured in a production medium containing 3.89% (w/v) rice straw and 0.75% (w/v) NaNO3 as carbon and nitrogen sources, respectively, for 72 h. This enzyme catalyzed well and was relatively stable at pH 7.0 and room temperature (28 ± 2 °C). The produced xylanase was used to hydrolyze xylans from four tropical weeds, whereupon it was found that the highest amounts of reducing sugars in the xylan hydrolysates of cogon grass (Imperata cylindrical), Napier grass (Pennisetum purpureum), and vetiver grass (Vetiveria zizanioides) were at 20.44 ± 0.84, 17.50 ± 0.29, and 19.44 ± 0.40 mg 100 mg xylan-1, respectively, but it was not detectable in water hyacinth (Eichhornia crassipes) hydrolysate. The highest combined amount of xylobiose and xylotriose was obtained from vetiver grass; thus, it was selected for further optimization. After optimization, xylanase digestion of vetiver grass xylan at 27.94 U g xylan-1 for 92 h 19 min gave the highest amount of reducing sugars (23.65 ± 1.34 mg 100 mg xylan-1), which were principally xylobiose and xylotriose. The enriched XOs exhibited a prebiotic property, significantly stimulating the growth of Lactobacillus brevis and L. casei by a factor of up to 3.5- and 6.5-fold, respectively, compared to glucose.

Enzymatic Hydrolysis of Black Liquor Xylan by a Novel Xylose-Tolerant, Thermostable ß-Xylosidase from a Tropical Strain of Aureobasidium pullulans CBS 135684.

From three cell-associated ß-xylosidases produced by Aureobasidium pullulans CBS 135684, the principal enzyme was enriched to apparent homogeneity and found to be active at high temperatures (60-70 °C) over a pH range of 5-9 with a specific activity of 163.3 units (U) mg-1. The enzyme was thermostable, retaining over 80% of its initial activity after a 12-h incubation at 60 °C, with half-lives of 38, 22, and 10 h at 60, 65, and 70 °C, respectively. Moreover, it was tolerant to xylose inhibition with a K i value of 18 mM. The K m and V max values against p-nitrophenyl-ß-d-xylopyranoside were 5.57 ± 0.27 mM and 137.0 ± 4.8 µmol min-1 mg-1 protein, respectively. When combining this ß-xylosidase with xylanase from the same A. pullulans strain, the rate of black liquor xylan hydrolysis was significantly improved by up to 1.6-fold. The maximum xylose yield (0.812 ± 0.015 g g-1 dry weight) was obtained from a reaction mixture containing 10% (w/v) black liquor xylan, 6 U g-1 ß-xylosidase and 16 U g-1 xylanase after incubation for 4 h at 70 °C and pH 6.0.

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 (ß-L-malic acid), heavy oil liamocins, siderophore, and aubasidan-like ß-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.

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 Vmax and Km 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-Fe(2+) or Zn(2+)) 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.

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.

Effect of polyols on thermostability of xylanase from a tropical isolate of Aureobasidium pullulans and its application in prebleaching of rice straw pulp.

In an attempt to find a thermostable xylanase enzyme for potential application in the pretreatment prior to H2O2 bleaching of paper pulp for industry, an extracellular xylanase from Aureobasidium pullulans CBS 135684 was purified 17.3-fold to apparent homogeneity with a recovery yield of 13.7%. Its molecular mass was approximately 72 kDa as determined by SDS-PAGE. The optimal pH and temperature for activity of the purified enzyme were pH 6.0 and 70°C, respectively. The enzyme was relatively stable at 50°C, retaining more than half of its original activity after 3-h incubation. The thermostability of the enzyme was improved by the addition of 0.75 mM sorbitol prolonging the enzyme's activity up to 10-fold at 70°C. When the potential of using the enzyme in pretreatment of rice straw pulp prior to bleaching was evaluated, the greatest efficiency was obtained in a mixture containing xylanase and sorbitol. Treatment of the rice straw pulp with xylanase prior to treatment with 10% (v/v) H2O2 and production of hand sheets increased the ISO sheet brightness by 13.5% and increased the tensile and tear strengths of the pulp by up to 1.16 and 1.71-fold, respectively, compared with pulps treated with H2O2 alone. The results suggested the potential application of the enzyme before the bleaching process of paper pulp when the maintenance of high temperature and enzyme stability are desirable.

Effects of sugar and amino acid supplementation on Aureobasidium pullulans NRRL 58536 antifungal activity against four Aspergillus species.

Cultured cell extracts from ten tropical strains of Aureobasidium pullulans were screened for antifungal activity against four pathogenic Aspergillus species (Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, and Aspergillus terreus) using the well diffusion and conidial germination inhibition assays. The crude cell extract from A. pullulans NRRL 58536 resulted in the greatest fungicidal activity against all four Aspergillus species and so was selected for further investigation into enhancing the production of antifungal activity through optimization of the culture medium, carbon source (sucrose and glucose) and amino acid (phenylalanine, proline, and leucine) supplementation. Sucrose did not support the production of any detectable antifungal activity, while glucose did with the greatest antifungal activity against all four Aspergillus species being produced in cells grown in medium containing 2.5 % (w/v) glucose. With respect to the amino acid supplements, variable trends between the different Aspergillus species and amino acid combinations were observed, with the greatest antifungal activities being obtained when grown with phenylalanine plus leucine supplementation for activity against A. flavus, proline plus leucine for A. terreus, and phenylalanine plus proline and leucine for A. niger and A. fumigatus. Thin layer chromatography, spectrophotometry, high-performance liquid chromatography, (1)H-nuclear magnetic resonance, and MALDI-TOF mass spectrometry analyses were all consistent with the main component of the A. pullulans NRRL 58536 extracts being aureobasidins.

α-Amylase activity during pullulan production and α-amylase gene analyses of Aureobasidium pullulans.

Aureobasidium pullulans is the source of commercially produced pullulan, a high molecular weight polysaccharide that is used in the manufacture of edible films. It has been proposed that α-amylase decreases the molecular weight of pullulan in late cultures. Based on a recent phylogenetic analysis, five representative strains were chosen to study the relationship between α-amylase and pullulan production. In sucrose-grown cultures, pullulan yields increased over time while the molecular weight of pullulan generally decreased. However, no α-amylase activity was detected in these cultures. Low levels of α-amylase were present in starch-grown culture, but pullulan analysis was complicated by residual starch. To facilitate further studies on the role of α-amylase in the reduction of pullulan molecular weight, the α-amylase gene from A. pullulans NRRL Y-12974 was cloned and characterized. The coding region of the complete α-amylase gene contains 2,247 bp, including 7 introns and 8 exons. The putative mRNA was 1,878 bp long, encoding an α-amylase of 625 amino acid residues. Southern blot analysis indicated that there was only one copy of this gene in the genome. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the gene was transcribed in both sucrose- and starch-grown cultures. It is possible that very low levels of α-amylase attack the minor maltotetraose subunits of pullulan and cause the reduction of molecular weight.

Multilocus phylogenetic analyses, pullulan production and xylanase activity of tropical isolates of Aureobasidium pullulans.

Aureobasidium pullulans is the source of the commercially valuable polysaccharide pullulan and the enzyme xylanase. Isolates are typically off-white to pale pink or black on solid media, while some tropical isolates have been described as 'color variants' with bright pigments of red, yellow or purple. We sequenced 5 loci (internal transcribed spacer, intergenic spacer 1, translation elongation factor-1 alpha, beta tubulin, and RNA polymerase II) from 45 new isolates from Thailand. Based on the phylogenetic analyses, isolates were classified into 12 clades. Each clade showed different colors on different culture media including two clades with 'color variants' and some clades exhibited high levels of pullulan production or xylanase activity. Colony characteristics do not correlate perfectly with DNA sequence phylogeny or the physiological characters, but DNA sequence differences rapidly identify isolates with genetic novelty.

Whitefly transmission of sweet potato viruses.

Three genera of plant viruses, Begomovirus (Geminiviridae), Crinivirus (Closteroviridae) and Ipomovirus (Potyviridae), contain members that infect sweet potato (Ipomoea batatas) and are transmitted by whiteflies. The begomoviruses, Sweet potato leaf curl virus (SPLCV) and Ipomoea leaf curl virus (ILCV), and the ipomovirus Sweet potato mild mottle virus are transmitted by Bemisia tabaci, the sweet potato whitefly. The crinivirus, Sweet potato chlorotic stunt virus (SPCSV), is transmitted by B. tabaci and Trialeurodes abutilonea, the bandedwinged whitefly. Transmission experiments were done with three of these viruses using laboratory-reared whiteflies and 2-day acquisition and transmission feeding periods. SPLCV and ILCV were transmitted from single and double infections by B. tabaci at rates of 5-10%. Transmission rates for SPLCV by B. tabaci were 15-20%. T. abutilonea transmitted SPCSV at a rate of ca. 3% but did not transmit ILCV or SPLCV.

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.