Transgenic Soybean for Production of Thermostable α-Amylase.

Alpha-amylases are crucial hydrolase enzymes which have been widely used in food, feed, fermentation, and pharmaceutical industries. Methods for low-cost production of α-amylases are highly desirable. Soybean seed, functioning as a bioreactor, offers an excellent platform for the mass production of recombinant proteins for its ability to synthesize substantial quantities of proteins. In this study, we generated and characterized transgenic soybeans expressing the α-amylase AmyS from Bacillus stearothermophilus. The α-amylase expression cassettes were constructed for seed specific expression by utilizing the promoters of three different soybean storage peptides and transformed into soybean via Agrobacterium-mediated transformation. The event with the highest amylase activity reached 601 U/mg of seed flour (one unit is defined as the amount of enzyme that generates 1 micromole reducing ends per min from starch at 65 °C in pH 5.5 sodium acetate buffer). The optimum pH, optimum temperature, and the enzymatic kinetics of the soybean expressed enzyme are similar to that of the E. coli expressed enzyme. However, the soybean expressed α-amylase is glycosylated, exhibiting enhanced thermostability and storage stability. Soybean AmyS retains over 80% activity after 100 min at 75 °C, and the transgenic seeds exhibit no significant activity loss after one year of storage at room temperature. The accumulated AmyS in the transgenic seeds represents approximately 15% of the total seed protein, or about 4% of the dry seed weight. The specific activity of the transgenic soybean seed flour is comparable to many commercial α-amylase enzyme products in current markets, suggesting that the soybean flour may be directly used for various applications without the need for extraction and purification.

First report of black spot on Ophiopogon japonicus caused by Alternaria alternata in Zhejiang Province, China.

Ophiopogon japonicus (Linn. f.) Ker-Gawl, a traditional Chinese medicinal plant, is widely cultured in China. The root of O. japonicus, is used as the main ingredient in many presriptions. It is rich in chemical components for steroidal saponins, homoisoflavonoids and polysaccharides, which have various pharmacological activities, such as cardiovascular protection, anti-inflammation and anti-diabetes (Chen. et al. 2016). In May and July for 2018 and 2019, the symptoms of black spot on O. japonicus were observed with an incidence of 40% in Cixi County, Zhejiang Province, China. The pathogen mainly infected leaves causing severe black spots, which resulted in a 28% yield loss per acre. At the early stage of the disease, the tip of the leaf began to turn yellow, then the discoloration gradually spread to the base of the leaf and finally the whole leaf turned reddish brown with visible black spot. Symptomatic leaves were cut into small pieces (1.0 cm × 1.0 cm) and disinfected successively by submersion in 75% ethanol for 30s and 1% NaClO for 30s under aseptic conditions. After rinsing with sterile water three times and air drying, segments were placed on potato dextrose agar (PDA), and incubated at 28 ℃ in dark for a week. Then, pathogen on the PDA were transferred onto potato carrot agar (PCA), and incubated at 23 ℃ under the condition of alternation of day (12 h) and night (12 h) for a week. Colonies on PDA were dark gray in the center surrounded by white to gray on the upper side, and black with white margins on the back of the plate. Colonies on PCA were grayish with sparse hyphae. The conidia were obclavate or ellipsoid, pale brown, with 3~8 transverse septa and 1~4 longitudinal septa. Conidiophores were septate, arising singly, and measured (17.0~81.0) × (8.0~23.5) µm, Most conidia had a conical or columnar beak, approximately (0~23.5) × (2.5~9.0) µm in size. According to morphological and cultural characteristics, these isolates were preliminarily identified as Alternaria alternata. A. alternata is one of the most typical plant pathogen, more than 95% of which facultatively parasitize on plants, causing disease in numerous crops. To further confirm identification of pathogens, the internal transcribed spacer region (ITS), translation elongation factor 1-α gene (EF-1α), RNA polymerase Ⅱ second largest subunit (RPB2), major allergen Alt a 1 gene (Alt a 1), Histon 3 gene (His) and plasma membrane ATPase (ATP)were amplified with primer pairs ITS1/ITS4, EF1-728F/EF1-986R, RPB2-7cr/RPB2-5f2, Alt-for/Alt-rev, His 3-F/His 3-R, ATP-F/ATP-R (Lawrence D.P. et al. 2013; Hong, S.G., et al. 2005). BLASTN analysis of NCBI using ITS (Accession NO. MW989987), Alt a1 (Accession NO. MW995953), EF-1α (Accession NO.MW995955), ATP (Accession NO.MW995957), His (Accession NO. MW995954) and RPB2 (Accession NO. MW995956) showed 100%, 100%, 97%, 99%, 99% and 97% identity to A. alternata MN249500.1, MN304714.1, MK637432.1, MK804115.1, MK460236.1, MK605888.1, respectively. To verify pathogenicity, healthy plants (1-year-old) of O. japonicus in ten pots were spray-inoculated with conidial suspension (1 × 106 conidia/ml). Ten plants, which were treated with sterile water, were used as the control. All plants were maintained in a climatic chamber (26 ± 1 ℃, 70-80% relative humidity and a photoperiod of 16:8 [L: D] h). Fourteen days later, all inoculated plants showed typical symptoms of black spot identical to those observed in the fields. Control plants remained symptomless and healthy. The pathogenicity analysis was repeated three times. Pathogens re-isolated from symptomatic plants were identified as A. alternata by morphology observation and sequence analysis. To our knowledge, this is the first report of black spot caused by A. alternata on O. japonicus in Zhejiang, China.

First report of root rot on Dendrobium officinale caused by Fusarium incarnatum-equiseti species complex in Zhejiang Province, China.

Dendrobium officinale Kimura et Migo is a rare and valuable Chinese herb cultivated in Zhejiang and Yunnan Provinces, China, which is known for its functions as an anti-neoplastic and for lowering the blood sugar (Cheng et al., 2019). In September and October of 2018 and 2019, symptoms of root rot on D. officinale were observed with an incidence of 15-20% in Wuyi County, Zhejiang Province, China. The pathogen mainly infected roots causing severe root rot, which resulted in significant economic losses. At the early stage of this disease, the stalk turned brown, then the whole plant rotted from bottom to top within a few days. Symptomatic roots were cut into small pieces (1.0 cm × 1.0 cm) and disinfected successively by submersion in 75% ethanol for 30 s and 1% NaClO for 30 s under aseptic conditions. After rinsing with sterile water three times and air drying, segments were placed on potato dextrose agar (PDA). After incubation at 25 °C for 5 d in the dark, white to pale cream colored colonies were produced. The average mycelial growth rate was 15.2-18.5 mm day-1 at 25 ℃. Macroconidia were falciform with three to five septa and (18.0-32.0)×(3.0-5.0) µm in size. Microconidia were fusiform with two to three septa (7.0-10.0)×(2.1-3.0) µm. Based on morphological characteristics of macroconidia, and microconidia, isolates were identified as Fusarium incarnatum-equiseti species complex (span style="font-family:'Times New Roman'; font-size:12pt">FIESC) (Avila et al., 2019). The internal transcribed spacer (ITS) region, translation elongation factor (EF-1α), RNA polymerase largest subunit (RPB1), and RNA polymerase second largest subunit (RPB2) gene were amplified and sequenced respectively using ITS1/ITS4, EF1/EF2, Fa/G2R and 5f2/7cr primers (O'Donnell et al., 2010). BLASTN analysis of FUSARIUM-ID using ITS (Accession NO. MW172977), EF-1α (Accession NO. MW172978, RPB1(Accession NO. MW172979), and RPB2(Accession NO. MW172980) showed 99.8%, 100%, 99.74%, and 98.63% identity to FIESC isolates NRRL43619, NRRL34059, NRRL32864, and NRRL32175, respectively. To verify pathogenicity, ten 1-year-old healthy D. officinale plants were used for inoculation tests. One milliliter of a conidial suspension (106 conidia ml-1) was pipetted onto the soil around the base of D. officinale plants per pot. Ten plants, which were treated with sterile water, were used as the control. All plants were maintained in a climatic chamber (26 ± 1 ℃, 70-80% relative humidity and a photoperiod of 16:8 [L: D] h). Seven days later, all inoculated plants showed typical symptoms of root rot identical to those observed in the fields. Control plants remained symptomless and healthy. The pathogenicity analysis was repeated three times. Pathogens re-isolated from symptomatic plants were identified as FIESC species by morphology observation and sequence analysis. To our knowledge, this is the first report of root rot caused by FIESC species on D. officinale in Zhejiang, China.

Comparative Analysis of the Diversity of the Microbial Communities between Non-Fertilized and Fertilized Eggs of Brown Planthopper, Nilaparvata lugens Stål.

Yeast-like symbionts (YLSs), harbored in the abdominal fat body of brown planthoppers (BPHs), Nilaparvata lugens Stål, play an important role in the growth, development, and reproduction of their host. However, little is known about the diversity of the symbiotic fungal YLSs that are harbored in the eggs of BPHs and the difference between fertilized eggs and non-fertilized eggs. Here, we investigate the fungal community compositions of non-fertilized and fertilized eggs of BPHs and identified the YLSs in the hemolymph by qPCR. A total of seven phyla, 126 genera, and 158 species were obtained from all samples, and Ascomycota and Basidiomycota were the most predominant phyla in the non-fertilized and fertilized eggs. The richness index indicated that microbial diversity in the non-fertilized and fertilized eggs exhibited a profound difference. In addition, 11 strains were only identified in the fertilized eggs, and these strains provide new insights into the constitution of species in YLSs. The difference of Pichia guilliermondii in the female hemolymph indicated that fertilization affected the diversity in the eggs by changing the YLSs in the hemolymph. Our research provides a comprehensive understanding of YLS species and their abundance in the eggs of BPHs, and it primarily explores how the changes of YLSs in the hemolymph lead to this difference.

Substantial improvement of toyocamycin production in Streptomyces diastatochromogenes by cumulative drug-resistance mutations.

Toyocamycin is a member of the nucleoside antibiotic family and has been recognized as a promising fungicide for the control of plant diseases. However, low productivity of toyocamycin remains an important bottleneck in its industrial production. Therefore, dramatic improvements of strains for overproduction of toyocamycin are of great interest in applied microbiology research. In this study, we sequentially selected for mutations for multiple drug resistance to promote the overproduction of toyocamycin by Streptomyces diastatochromogenes 1628. The triple mutant strain, SD3145 (str str par), was obtained through sequential screenings. This strain showed an enhanced capacity to produce toyocamycin (1500 mg/L), 24-fold higher than the wild type in GYM liquid medium. This dramatic overproduction was attributed at least partially to the acquisition of an rsmG mutation and increased gene expression of toyA, which encodes a LuxR-family transcriptional regulator for toyocamycin biosynthesis. The expression of toyF and toyG, probably directly involved in toyocamycin biosynthesis, was also enhanced, contributing to toyocamycin overproduction. By addition of a small amount of scandium (ScCl3·6H2O), the mutant strain, SD3145, produced more toyocamycin (2664 mg/L) in TPM medium, which was the highest toyocamycin level produced in shake-flask fermentation by a streptomycete so far. We demonstrated that introduction of combined drug resistance mutations into S. diastatochromogenes 1628 resulted in an obvious increase in the toyocamycin production. The triple mutant strain, SD3145, generated in our study could be useful for improvement of industrial production of toyocamycin.