Streptomyces rhizophilus Causes Potato Common Scab Disease.

Common scab (CS) caused by Streptomyces spp. is a significant soilborne potato disease that results in tremendous economic losses globally. Identification of CS-associated species of the genus Streptomyces can enhance understanding of the genetic variation of these bacterial species and is necessary for the control of this epidemic disease. The present study isolated Streptomyces strain 6-2-1(1) from scabby potatoes in Keshan County, Heilongjiang Province, China. PCR analysis confirmed that the strain harbored the characteristic Streptomyces pathogenicity island (PAI) genes (txtA, txtAB, nec1, and tomA). Pathogenicity assays proved that the strain caused typical scab lesions on potato tuber surfaces and necrosis on radish seedlings and potato slices. Subsequently, the strain was systemically characterized at morphological, physiological, biochemical, and phylogenetic levels. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 6-2-1(1) shared 99.86% sequence similarity with Streptomyces rhizophilus JR-41T, isolated initially from bamboo in rhizospheric soil in Korea. PCR amplification followed by Sanger sequencing of the 16S rRNA gene of 164 scabby potato samples collected in Heilongjiang Province from 2019 to 2020 demonstrated that approximately 2% of the tested samples were infected with S. rhizophilus. Taken together, these results demonstrate that S. rhizophilus is capable of causing potato CS disease and may pose a potential challenge to potato production in Heilongjiang Province of China.

Comparison of the Distinct, Host-Specific Response of Three Solanaceae Hosts Induced by Phytophthora infestans.

Three Solanaceae hosts (TSHs), S. tuberosum, N. benthamiana and S. lycopersicum, represent the three major phylogenetic clades of Solanaceae plants infected by Phytophthora infestans, which causes late blight, one of the most devastating diseases seriously affecting crop production. However, details regarding how different Solanaceae hosts respond to P. infestans are lacking. Here, we conducted RNA-seq to analyze the transcriptomic data from the TSHs at 12 and 24 h post P. infestans inoculation to capture early expression effects. Macroscopic and microscopic observations showed faster infection processes in S. tuberosum than in N. benthamiana and S. lycopersicum under the same conditions. Analysis of the number of genes and their level of expression indicated that distinct response models were adopted by the TSHs in response to P. infestans. The host-specific infection process led to overlapping but distinct in GO terms and KEGG pathways enriched for differentially expressed genes; many were tightly linked to the immune response in the TSHs. S. tuberosum showed the fastest response and strongest accumulation of reactive oxygen species compared with N. benthamiana and S. lycopersicum, which also had similarities and differences in hormone regulation. Collectively, our study provides an important reference for a better understanding of late blight response mechanisms of different Solanaceae host interactions.

Comparative Genomic and Physiological Analyses of a Superoxide Dismutase Mimetic (SODm-123) for Its Ability to Respond to Oxidative Stress in Tomato Plants.

Superoxide dismutases (SODs) are a group of enzymes that have a crucial role in controlling oxidative stress in plants. Here, we synthesized an environmentally friendly SOD mimic, SODm-123, from L-aspartic acid and manganese oxide. SODm-123 showed similar enzymatic activity to Mn-SOD. To gain insights into the role of SODm-123 in oxidative stress tolerance, a series of experiments were conducted to assess the physiological and molecular responses of tomato plants when treated with SODm-123. The results showed that the levels of O2-• and H2O2 in tomato cells were affected by SODm-123 treatment, indicating that SODm-123 can control oxidative stress like Mn-SOD. The results also exhibited that SODm-123 increased the contents of photosynthetic pigments. However, it was noted that SODm-123 resulted in a reduction in the content of soluble sugar and MDA. These results indicate that SODm-123 promoted the efficiency of photosynthesis by regulating the content of H2O2. To further investigate the role of SODm-123 in controlling oxidative stress, a transcriptome analysis was used to identify differentially expressed genes (DEGs) associated with SODm-123 treatment. The results indicated that SODm-123 treatment resulted in 341 differentially expressed genes (DEGs) in treated tomato leaves at 96 h after treatment. Kyoto encyclopedia of genes and genomes (KEGG) revealed that DEGs were involved in pathways such as photosynthetic pigment biosynthesis, ABC transporters, sugar metabolism, and MAPK signaling, which further confirmed a positive role of SODm-123 in improving stress tolerance in plants. Overall, the results of this study suggest that SODm-123 promotes the growth and development of tomato seedlings and therefore can be used as a potential growth-promoting agent for plants.

Genome-scale analyses and characteristics of putative pathogenicity genes of Stagonosporopsis cucurbitacearum, a pumpkin gummy stem blight fungus.

Outbreaks of gummy stem blight (GSB), an emerging seed pumpkin disease, have increased in number and have become more widespread in recent years. Previously we reported that Stagonosporopsis cucurbitacearum (Sc.) is the dominant fungal cause of pumpkin seedling GSB in Northeast China, where it has greatly reduced crop yields in that region. Here, high-throughput whole-genome sequencing and assembly of the Sc. genome were conducted toward revealing pathogenic molecular regulatory mechanisms involved in fungal growth and development. Zq-1 as representative Sc. strain, DNA of Zq-1was prepared for genomic sequencing, we obtained 5.24 Gb of high-quality genomic sequence data via PacBio RS II sequencing. After sequence data was processed to filter out low quality reads, a hierarchical genome-assembly process was employed that generated a genome sequence of 35.28 Mb in size. A total of 9844 genes were predicted, including 237 non-coding RNAs, 1024 genes encoding proteins with signal peptides, 2066 transmembrane proteins and 756 secretory proteins.Transcriptional identification revealed 54 differentially expressed secretory proteins. Concurrently, 605, 130 and 2869 proteins were matched in the proprietary databases Carbohydrate-Active EnZymes database (CAZyme), Transporter Classification Database (TCDB) and Pathogen-Host Interactions database (PHI), respectively. And 96 and 36 DEGs were identified form PHI database and CAZyme database, respectively. In addition, contig00011.93 was an up-regulated DEG involving ATP-binding cassette metabolism in the procession of infection. In order to test relevance of gene predictions to GSB, DEGs with potential pathogenic relevance were revealed through transcriptome data analysis of Sc. strains pre- and post-infection of pumpkin. Interestingly, Sc. and Leptosphaeria maculans (Lm.) exhibited relatively similar with genome lengths, numbers of protein-coding genes and other characteristics. This work provides a foundation for future exploration of additional Sc. gene functions toward the development of more effective GSB control strategies.

Genetic Dissection of Germinability under Low Temperature by Building a Resequencing Linkage Map in japonica Rice.

Among all cereals, rice is highly sensitive to cold stress, especially at the germination stage, which adversely impacts its germination ability, seed vigor, crop stand establishment, and, ultimately, grain yield. The dissection of novel quantitative trait loci (QTLs) or genes conferring a low-temperature germination (LTG) ability can significantly accelerate cold-tolerant rice breeding to ensure the wide application of rice cultivation through the direct seeding method. In this study, we identified 11 QTLs for LTG using 144 recombinant inbred lines (RILs) derived from a cross between a cold-tolerant variety, Lijiangxintuanheigu (LTH), and a cold-sensitive variety, Shennong265 (SN265). By resequencing two parents and RIL lines, a high-density bin map, including 2,828 bin markers, was constructed using 123,859 single-nucleotide polymorphisms (SNPs) between two parents. The total genetic distance corresponding to all 12 chromosome linkage maps was 2,840.12 cm. Adjacent markers were marked by an average genetic distance of 1.01 cm, corresponding to a 128.80 kb physical distance. Eight and three QTL alleles had positive effects inherited from LTH and SN265, respectively. Moreover, a pleiotropic QTL was identified for a higher number of erected panicles and a higher grain number on Chr-9 near the previously cloned DEP1 gene. Among the LTG QTLs, qLTG3 and qLTG7b were also located at relatively small genetic intervals that define two known LTG genes, qLTG3-1 and OsSAP16. Sequencing comparisons between the two parents demonstrated that LTH possesses qLTG3-1 and OsSAP16 genes, and SN-265 owns the DEP1 gene. These comparison results strengthen the accuracy and mapping resolution power of the bin map and population. Later, fine mapping was done for qLTG6 at 45.80 kb through four key homozygous recombinant lines derived from a population with 1569 segregating plants. Finally, LOC_Os06g01320 was identified as the most possible candidate gene for qLTG6, which contains a missense mutation and a 32-bp deletion/insertion at the promoter between the two parents. LTH was observed to have lower expression levels in comparison with SN265 and was commonly detected at low temperatures. In conclusion, these results strengthen our understanding of the impacts of cold temperature stress on seed vigor and germination abilities and help improve the mechanisms of rice breeding programs to breed cold-tolerant varieties.

QTL Mapping of Fiber-Related Traits Based on a High-Density Genetic Map in Flax (Linum usitatissimum L.).

A genetic map is an important and valuable tool for quantitative trait locus (QTL) mapping, marker-assisted selection (MAS)-based breeding, and reference-assisted chromosome assembly. In this study, 112 F2 plants from a cross between Linum usitatissimum L. "DIANE" and "NY17" and parent plants were subjected to high-throughput sequencing and specific-locus amplified fragment (SLAF) library construction. After preprocessing, 61.64 Gb of raw data containing 253.71 Mb paired-end reads, each 101 bp in length, were obtained. A total of 192,797 SLAFs were identified, of which 23,115 were polymorphic, with a polymorphism rate of 11.99%. Finally, 2,339 SLAFs were organized into a linkage map consisting of 15 linkage groups (LGs). The total length of the genetic map was 1483.25 centimorgans (cM) and the average distance between adjacent markers was 0.63 cM. Combined with flax chromosome-scale pseudomolecules, 12 QTLs associating with 6 flax fiber-related traits were mapped on the chromosomal scaffolds. This high-density genetic map of flax should serve as a foundation for flax fine QTL mapping, draft genome assembly, and MAS-guided breeding. Ultimately, the genomic regions identified in this research could potentially be valuable for improving flax fiber cultivars, as well as for identification of candidate genes involved in flax fiber formation processes. SIGNIFICANCE STATEMENT: A high-density genetic map of flax was constructed, and QTLs were identified on the sequence scaffolds to be interrelated with fiber-related traits. The results of this study will not only provide a platform for gene/QTL fine mapping, map-based gene isolation, and molecular breeding for flax, but also provide a reference to help position sequence scaffolds on the physical map and assist in the process of assembling the flax genome sequence.

Application of cavitation system to accelerate aqueous enzymatic extraction of seed oil from Cucurbita pepo L. and evaluation of hypoglycemic effect.

Cavitation-accelerated aqueous enzymatic extraction (CAEE) of seed oil from Cucurbita pepo was performed. An enzyme cocktail comprised of cellulose, pectinase and proteinase can work synergistically in releasing the oil. The CAEE extraction conditions were optimized by a Plackett-Burman design followed by a central composite methodology. A maximal extraction yield of 58.06% was achieved under optimal conditions of vacuum degree -0.07, enzyme amount 1.05% and extraction time 69min. As compared to soxhlet extraction (SE)-derived oil, CAEE-derived oil exhibited similar physical properties and better oxidation stability. In addition, chemical composition analyzing showed that the content of linoleic acid obtained by CAEE (47.67%) was higher than that of SE (44.51%). Moreover, the IC50 of oil obtained by CAEE and SE, as measured by α-amylase inhibition assay, were 40.68µg/mL and 45.46µg/mL. All results suggest that CAEE represents an excellent alternative protocol for production of oil from oil-bearing materials.

Microwave-assisted aqueous enzymatic extraction of oil from pumpkin seeds and evaluation of its physicochemical properties, fatty acid compositions and antioxidant activities.

Microwave-assisted aqueous enzymatic extraction (MAAEE) of pumpkin seed oil was performed in this study. An enzyme cocktail comprised of cellulase, pectinase and proteinase (w/w/w) was found to be the most effective in releasing oils. The highest oil recovery of 64.17% was achieved under optimal conditions of enzyme concentration (1.4%, w/w), temperature (44°C), time (66 min) and irradiation power (419W). Moreover, there were no significant variations in physicochemical properties of MAAEE-extracted oil (MAAEEO) and Soxhlet-extracted oil (SEO), but MAAEEO exhibited better oxidation stability. Additionally, MAAEEO had a higher content of linoleic acid (57.33%) than SEO (53.72%), and it showed stronger antioxidant activities with the IC50 values 123.93 and 152.84, mg/mL, according to DPPH radical scavenging assay and ß-carotene/linoleic acid bleaching test. SEM results illustrated the destruction of cell walls and membranes by MAAEE. MAAEE is, therefore, a promising and environmental-friendly technique for oil extraction in the food industry.

Bacillus subtilis subspecies virginiana, a new subspecies of antitermitic compound-producing endophytic bacteria isolated from Juniperus virginiana.

Termites are worldwide pests causing considerable damage to agriculture, forestry and buildings. Although physical and chemical methods have been tried to eliminate termite populations, they have the limitations such as low effectiveness, high-toxicity residue, environmentally harmful and high cost. Therefore, it has attracted much attention to develop highly effective, low-toxic, long residual period, environmentally friendly and low-cost termiticidals. Here, we report the characterization and antitermitic activities of a new antitermitic compound-producing endophytic bacterium HUB-I-47 isolated from eastern red-cedar, Juniperus virginiana L. The morphological, physiochemical characteristics of strain HUB-I-47 and its 16S rDNA sequences, and the antitermitic compound were analyzed by gas chromatography-mass spectrometry were studied. We found that the morphology of HUB-I-47 was very similar to that of Bacillus subtilis but presented some differences in shape and cell size. Growth evaluation showed that the lowest, highest, and optimum growth temperatures of HUB-I-47 were 12, 47, and 31 degrees C, respectively, which were different from those of reference strains. The 16S rDNA sequence analysis revealed a high similarity of 99% to those of B. subtilis. Based on these analyses, we named strain HUB-I-47 as B. subtilis subsp. virginiana D. P. Zhou, K. Zhao, J. Liu et W. X. Ping, subsp. nov. This is the first report on the analysis of antitermitic compounds from endophytic bacteria. Our study identified a new resource of antitermitic compounds through endophytic bacteria fermentation.

Characterization and expression profile analysis of a new cDNA encoding taxadiene synthase from Taxus media.

A full-length cDNA encoding taxadiene synthase (designated as TmTXS), which catalyzes the first committed step in the Taxol biosynthetic pathway, was isolated from young leaves of Taxus media by rapid amplification of cDNA ends (RACE). The full-length cDNA of TmTXS had a 2586 bp open reading frame (ORF) encoding a protein of 862 amino acid residues. The deduced protein had isoelectric point (pI) of 5.32 and a calculated molecular weight of about 98 kDa, similar to previously cloned diterpene cyclases from other Taxus species such as T. brevifolia and T. chinenisis. Sequence comparison analysis showed that TmTXS had high similarity with other members of terpene synthase family of plant origin. Tissue expression pattern analysis revealed that TmTXS expressed strongly in leaves, weak in stems and no expression could be detected in fruits. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in Taxol biosynthetic pathway in different tissues of Taxus plants. Phylogenetic tree analysis showed that TmTXS had closest relationship with taxadiene synthase from T. baccata followed by those from T. chinenisis and T. brevifolia. Expression profiles revealed by RT-PCR under different chemical elicitor treatments such as methyl jasmonate (MJ), silver nitrate (SN) and ammonium ceric sulphate (ACS) were also compared for the first time, and the results revealed that expression of TmTXS was all induced by the tested three treatments and the induction effect by MJ was the strongest, implying that TmTXS was high elicitor responsive.

Cloning and expression analysis of a water stress-induced gene from Brassica oleracea.

Plant growth and productivity are greatly affected by water stress, such as drought and salinity. Here we report on the cloning and expression analysis of a water stress-induced gene from Brassica oleracea (designated as BoWS, GenBank accession number AY571333) by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoWS consisted of 594 bp and contained a 285 bp open reading frame (ORF) encoding a 95-amino-acid protein. The deduced protein had a calculated molecular mass of 10.53 kDa and an isoelectric point of 6.93. The sequence similarity and comparative analysis showed that BoWS was 84% identical to Arabidopsis thaliana putative water stress-induced protein (GenBank accession number AAM67282). Southern blot analysis indicated that BoWS was a low-copy gene. Northern blot analysis revealed that the expression of BoWS was upregulated by abscisic acid (ABA), mannitol, NaCl, drought, salicylic acid (SA) and hydrogen peroxide (H(2)O(2)). Our results indicate that BoWS is extremely related to the water-deficit stress in B. oleracea.

Preference of simple sequence repeats in coding and non-coding regions of Arabidopsis thaliana.

MOTIVATION: Simple sequence repeats or microsatellites have been found abundantly in many genomes. However, the significance of distribution preference has not been completely understood. Completion of the Arabidopsis genome sequencing allows us to better understand and characterize microsatellites. RESULTS: Microsatellite distribution was more abundant in 5'-flanking regions of genes compared with that expected in the whole genome, with an over-representation of AG and AAG repeats; there were clear differences from distributions in 3'-flanks and coding fractions, where triplet frequencies evidently corresponded to codon usage. We identified 1140 full-length genes that contained at least one locus of AG or AAG repeats in their upstream sequences, and whose functional characteristics were significantly associated with the repeats. This observation indicates that selective pressure markedly differed in the three transcribed regions, with positive selection of AG and AAG repeats in 5'-flanks close to those genes whose products are preferentially involved in transcription.

Molecular cloning of a new lectin gene from Z. grandiflora.

Using RNA extracted from Zephyrathes grandiflora young leaves and primers designed according to the conservative regions of Amaryllidaceae lectins, the full-length cDNA of Z. grandiflora agglutinin (ZGA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of ZGA was 934 bp and contained a 576 bp open reading frame (ORF) encoding a 191 amino acid protein. Through comparative analysis of zga gene and its deduced amino acid sequence with those of other Amaryllidaceae species, it was found that zga encoded a precursor lectin with signal peptide. Molecular modeling suggested that ZGA was a mannose-binding lectin with three mannose-binding sites like lectins from other Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that zga belonged to a multi-copy gene family like those of many other Amaryllidaceae species such as Galanthus nivalis and Clivia miniata.