Improvement of Laccase Production by Thielavia terrestris Co3Bag1. Enhancing the Bio-Catalytic Performance of the Native Thermophilic TtLacA via Immobilization in Copper Alginate Gel Beads.

A 32-fold increase in laccase activity production by the thermophilic biomass-degrading fungus T. terrestris Co3Bag1 was achieved when the microorganism was grown on a modified medium containing fructose, sodium nitrate, and copper. A 70 kDa laccase (TtLacA), produced under the above conditions, was purified, immobilized in copper alginate gel beads, and characterized. TtLacA, both free and immobilized enzymes, exhibited optimal activity at pH 3.0, at a temperature of 65 and 70 °C, respectively, although both displayed 70% of activity from 40 to 70 °C. Free and immobilized enzymes retained at least 80% of relative activity in the pH range from 3 to 4.6. Immobilized TtLacA manifested a 2.3-fold higher thermal stability than the free form of the enzyme at 60 and 70 °C. Immobilized TtLacA retained 95% initial activity for six consecutive reuse cycles at 60 °C, and also retained 86% of initial activity after 12 days of storage at 4 °C. Based on the biochemical features, thermophilic TtLacA may be an efficient enzyme for dye decolorization and other industrial applications at high temperatures or acidic conditions. This work represents the first report about the immobilization and biochemical characterization of a thermophilic laccase from a member of the genus Thielavia.

First Report of Erysiphe betae Causing Powdery Mildew on Chard (Beta vulgaris var. cicla) in Mexico.

Chard (Beta vulgaris var. cicla; Chenopodiaceae) is a vegetable native to the Mediterranean, widely cultivated for its nutritional properties. In June 2020, an outbreak of powdery mildew was detected in a commercial crop of chard in San Martín Texmelucan, Puebla (19°14'37.1"N; 98°27'12.5"W), Mexico. The disease was present in 86% of the plants (n=400) and the pathogen was found to cover up to 95% of the surface of the leaves. Initially, small whitish patches were observed on both sides of the leaves. Subsequently, the patches grew rapidly to cover most of the leaf surface and premature senescence of infected leaves was observed. The signs of the pathogen were observed as abundant whitish masses of conidia. Microscopic analysis of the fungus showed amphigenous mycelia with lobed hyphal appressoria. Conidiophores (n=30) were simple and erect, 93133 × 7.58.5 µm. Foot cells (n=30) were cylindrical, predominately straight, and rarely somewhat curved at the base, 30.036.5 µm, followed by a longer cell and two shorter cells, and the conidium. Conidia (n=100) were hyaline, ellipsoid-ovoid, 3745 × 1416 µm. Germ tubes (n =30) were terminal, short (0.52.0 times the conidial width) and stout. Conidial appressoria (n=30) were mostly lobed, showing from 2-6 lobes. Chasmothecia were not found. The morphological characteristics observed correspond to previous descriptions of Erysiphe betae by Braun and Cook et al. (2012). A voucher specimen (accession no. UACH450) was deposited in the Department of Agricultural Parasitology Herbarium at the Chapingo Autonomous University. To confirm identification, DNA was extracted from the fungus, and the internal transcribed spacer (ITS) and the 28S gene region of rDNA from one sample were amplified by PCR, using the primers ITS1/ITS4 (White et al. 1990) and PM3 (Takamatsu and Kano 2001)/TW14 (Mori et al. 2000). The sequences obtained from our specimen were registered to the GenBank under the accession numbers ON157053 and ON157047 for ITS and LSU, respectively. Our sequences shared 100% identity for ITS (KX574674) and 99.8% for LSU (OM033348 and OM368494) with sequences of E. betae in BLAST'n search. Based on phylogenetic analysis using the Maximum Likelihood method including a published ITS + 28S dataset for Erysiphe species, the isolate UACH450 was grouped into a clade with E. betae. Takamatsu et al. (2015) found that E. betae, E. malvae and E. heraclei are phylogenetically indistinguishable (they form the E. heraclei species complex), nevertheless, E. malavae infects Lavatera and Malva (Malvaceae), E. heraclei predominately forms on hosts of Apiaceae and E. betae is commonly found on Beta and Chenopodium (Chenopodiaceae) (Braun and Cook 2012). Pathogenicity was verified by spraying a suspension of conidia (1107 conidia/ml) onto the leaves of six healthy chard plants and six plants were sprayed with sterile distilled water to serve as controls. All plants were maintained at temperatures from 28 2 °C and relative humidity of 802 %. All inoculated leaves developed powdery mildew symptoms after 14 days, whereas the control plants remained symptomless. The pathogenicity test was performed twice, observing the same results. The recovered pathogen showed the same morphological characteristics as the inoculated pathogen, thus fulfilling Koch's postulates. To our knowledge, this is the first report of Erysiphe betae causing powdery mildew on Beta vulgaris var. cicla in Mexico. This pathogen has been previously reported in Iraq (Amano, 1986) and Greece (Vakalounakis and Kavroulakis, 2017) on Beta vulgaris var. cicla. Also, Erysiphe betae has been reported in Mexico on Chenopodium and throughout the world on sugar beet (Farr and Rossman, 2022). This pathogen is a major issue as it can completely cover the leaves of the diseased plants, making them difficult to market.

Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

Taxonomic identification of the thermotolerant and fast-growing fungus Lichtheimia ramosa H71D and biochemical characterization of the thermophilic xylanase LrXynA.

The zygomycete fungus Lichtheimia ramosa H71D, isolated from sugarcane bagasse compost, was identified by applying phylogenetic analysis based on the DNA sequence of the Internal Transcribed Spacer (ITS), and subsequent secondary structure analysis of ITS2. L. ramosa H71D was able to grow over a wide range of temperatures (25-45 °C), manifesting optimal growth at 37 °C. A 64 kDa xylanase (named LrXynA) was purified from the culture supernatant of L. ramosa H71D grown on 2% carboxymethylcellulose (CMC), as the only carbon source. LrXynA displayed optimal activity at pH 6 and temperature of 65 °C. The enzyme retained more than 50% of its maximal activity over a broad range of pH values (4.5-7.5). Enzyme half-life (t½) times at 55, 65 and 75 °C were 80, 25, and 8 min, respectively. LrXynA showed higher affinity (k M of 2.87 mg/mL) and catalytic efficiency (k cat /k M of 0.651 mg s/mL) towards Beechwood xylan in comparison to other substrates such as Birchwood xylan, Oat-spelt xylan, CMC, Avicel and Solka floc. The predominant final products from LrXynA-mediated hydrolysis of Beechwood xylan were xylobiose and xylotriose, suggesting that the enzyme is an endo-ß-1,4 xylanase. Scanning electron microscopy (SEM) imaging of sugar cane bagasse (SCB) treated with LrXynA, alone or in combination with commercial cellulases, showed a positive effect on the hydrolysis of SCB. To our knowledge, this is the first report focusing on the biochemical and functional characterization of an endo-ß-1,4 xylanase from the thermotolerant and fast-growing fungus Lichtheimia ramosa.