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April 21, 2020  |  

Genome analysis and genetic transformation of a water surface-floating microalga Chlorococcum sp. FFG039.

Microalgal harvesting and dewatering are the main bottlenecks that need to be overcome to tap the potential of microalgae for production of valuable compounds. Water surface-floating microalgae form robust biofilms, float on the water surface along with gas bubbles entrapped under the biofilms, and have great potential to overcome these bottlenecks. However, little is known about the molecular mechanisms involved in the water surface-floating phenotype. In the present study, we analysed the genome sequence of a water surface-floating microalga Chlorococcum sp. FFG039, with a next generation sequencing technique to elucidate the underlying mechanisms. Comparative genomics study with Chlorococcum sp. FFG039 and other non-floating green microalgae revealed some of the unique gene families belonging to this floating microalga, which may be involved in biofilm formation. Furthermore, genetic transformation of this microalga was achieved with an electroporation method. The genome information and transformation techniques presented in this study will be useful to obtain molecular insights into the water surface-floating phenotype of Chlorococcum sp. FFG039.


April 21, 2020  |  

Modulation of metabolome and bacterial community in whole crop corn silage by inoculating homofermentative Lactobacillus plantarum and heterofermentative Lactobacillus buchneri.

The present study investigated the species level based microbial community and metabolome in corn silage inoculated with or without homofermentative Lactobacillus plantarum and heterofermentative Lactobacillus buchneri using the PacBio SMRT Sequencing and time-of-flight mass spectrometry (GC-TOF/MS). Chopped whole crop corn was treated with (1) deionized water (control), (2) Lactobacillus plantarum, or (3) Lactobacillus buchneri. The chopped whole crop corn was ensiled in vacuum-sealed polyethylene bags containing 300 g of fresh forge for 90 days, with three replicates for each treatment. The results showed that a total of 979 substances were detected, and 316 different metabolites were identified. Some metabolites with antimicrobial activity were detected in whole crop corn silage, such as catechol, 3-phenyllactic acid, 4-hydroxybenzoic acid, azelaic acid, 3,4-dihydroxybenzoic acid and 4-hydroxycinnamic acid. Catechol, pyrogallol and ferulic acid with antioxidant property, 4-hydroxybutyrate with nervine activity, and linoleic acid with cholesterol lowering effects, were detected in present study. In addition, a flavoring agent of myristic acid and a depression mitigation substance of phenylethylamine were also found in this study. Samples treated with inoculants presented more biofunctional metabolites of organic acids, amino acids and phenolic acids than untreated samples. The Lactobacillus species covered over 98% after ensiling, and were mainly comprised by the L. acetotolerans, L. silagei, L. parafarraginis, L. buchneri and L. odoratitofui. As compared to the control silage, inoculation of L. plantarum increased the relative abundances of L. acetotolerans, L. buchneri and L. parafarraginis, and a considerable decline in the proportion of L. silagei was observed; whereas an obvious decrease in L. acetotolerans and increases in L. odoratitofui and L. farciminis were observed in the L. buchneri inoculated silage. Therefore, inoculation of L. plantarum and L. buchneri regulated the microbial composition and metabolome of the corn silage with different behaviors. The present results indicated that profiling of silage microbiome and metabolome might improve our current understanding of the biological process underlying silage formation.


April 21, 2020  |  

Complete Assembly of the Genome of an Acidovorax citrulli Strain Reveals a Naturally Occurring Plasmid in This Species.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit crop production worldwide. Based on genetic and phenotypic properties, A. citrulli strains are divided into two major groups: group I strains have been generally isolated from melon and other non-watermelon cucurbits, while group II strains are closely associated with watermelon. In a previous study, we reported the genome of the group I model strain, M6. At that time, the M6 genome was sequenced by MiSeq Illumina technology, with reads assembled into 139 contigs. Here, we report the assembly of the M6 genome following sequencing with PacBio technology. This approach not only allowed full assembly of the M6 genome, but it also revealed the occurrence of a ~53 kb plasmid. The M6 plasmid, named pACM6, was further confirmed by plasmid extraction, Southern-blot analysis of restricted fragments and obtention of M6-derivative cured strains. pACM6 occurs at low copy numbers (average of ~4.1 ± 1.3 chromosome equivalents) in A. citrulli M6 and contains 63 open reading frames (ORFs), most of which (55.6%) encoding hypothetical proteins. The plasmid contains several genes encoding type IV secretion components, and typical plasmid-borne genes involved in plasmid maintenance, replication and transfer. The plasmid also carries an operon encoding homologs of a Fic-VbhA toxin-antitoxin (TA) module. Transcriptome data from A. citrulli M6 revealed that, under the tested conditions, the genes encoding the components of this TA system are among the highest expressed genes in pACM6. Whether this TA module plays a role in pACM6 maintenance is still to be determined. Leaf infiltration and seed transmission assays revealed that, under tested conditions, the loss of pACM6 did not affect the virulence of A. citrulli M6. We also show that pACM6 or similar plasmids are present in several group I strains, but absent in all tested group II strains of A. citrulli.


April 21, 2020  |  

Denitrifying Bacteria Active in Woodchip Bioreactors at Low-Temperature Conditions.

Woodchip bioreactor technology removes nitrate from agricultural subsurface drainage by using denitrifying microorganisms. Although woodchip bioreactors have demonstrated success in many field locations, low water temperature can significantly limit bioreactor efficiency and performance. To improve bioreactor performance, it is important to identify the microbes responsible for nitrate removal at low temperature conditions. Therefore, in this study, we identified and characterized denitrifiers active at low-temperature conditions by using culture-independent and -dependent approaches. By comparative 16S rRNA (gene) analysis and culture isolation technique, Pseudomonas spp., Polaromonas spp., and Cellulomonas spp. were identified as being important bacteria responsible for denitrification in woodchip bioreactor microcosms at relatively low temperature conditions (15°C). Genome analysis of Cellulomonas sp. strain WB94 confirmed the presence of nitrite reductase gene nirK. Transcription levels of this nirK were significantly higher in the denitrifying microcosms than in the non-denitrifying microcosms. Strain WB94 was also capable of degrading cellulose and other complex polysaccharides. Taken together, our results suggest that Cellulomonas sp. denitrifiers could degrade woodchips to provide carbon source and electron donors to themselves and other denitrifiers in woodchip bioreactors at low-temperature conditions. By inoculating these denitrifiers (i.e., bioaugmentation), it might be possible to increase the nitrate removal rate of woodchip bioreactors at low-temperature conditions.


April 21, 2020  |  

Closing the Yield Gap for Cannabis: A Meta-Analysis of Factors Determining Cannabis Yield.

Until recently, the commercial production of Cannabis sativa was restricted to varieties that yielded high-quality fiber while producing low levels of the psychoactive cannabinoid tetrahydrocannabinol (THC). In the last few years, a number of jurisdictions have legalized the production of medical and/or recreational cannabis with higher levels of THC, and other jurisdictions seem poised to follow suit. Consequently, demand for industrial-scale production of high yield cannabis with consistent cannabinoid profiles is expected to increase. In this paper we highlight that currently, projected annual production of cannabis is based largely on facility size, not yield per square meter. This meta-analysis of cannabis yields reported in scientific literature aimed to identify the main factors contributing to cannabis yield per plant, per square meter, and per W of lighting electricity. In line with previous research we found that variety, plant density, light intensity and fertilization influence cannabis yield and cannabinoid content; we also identified pot size, light type and duration of the flowering period as predictors of yield and THC accumulation. We provide insight into the critical role of light intensity, quality, and photoperiod in determining cannabis yields, with particular focus on the potential for light-emitting diodes (LEDs) to improve growth and reduce energy requirements. We propose that the vast amount of genomics data currently available for cannabis can be used to better understand the effect of genotype on yield. Finally, we describe diversification that is likely to emerge in cannabis growing systems and examine the potential role of plant-growth promoting rhizobacteria (PGPR) for growth promotion, regulation of cannabinoid biosynthesis, and biocontrol.


April 21, 2020  |  

Comprehensive characterization of T-DNA integration induced chromosomal rearrangement in a birch T-DNA mutant.

Integration of T-DNA into plant genomes via Agrobacterium may interrupt gene structure and generate numerous mutants. The T-DNA caused mutants are valuable materials for understanding T-DNA integration model in plant research. T-DNA integration in plants is complex and still largely unknown. In this work, we reported that multiple T-DNA fragments caused chromosomal translocation and deletion in a birch (Betula platyphylla × B. pendula) T-DNA mutant yl.We performed PacBio genome resequencing for yl and the result revealed that two ends of a T-DNA can be integrated into plant genome independently because the two ends can be linked to different chromosomes and cause chromosomal translocation. We also found that these T-DNA were connected into tandem fragment regardless of direction before integrating into plant genome. In addition, the integration of T-DNA in yl genome also caused several chromosomal fragments deletion. We then summarized three cases for T-DNA integration model in the yl genome. (1) A T-DNA fragment is linked to the two ends of a double-stranded break (DSB); (2) Only one end of a T-DNA fragment is linked to a DSB; (3) A T-DNA fragment is linked to the ends of different DSBs. All the observations in the yl genome supported the DSB repair model.In this study, we showed a comprehensive genome analysis of a T-DNA mutant and provide a new insight into T-DNA integration in plants. These findings would be helpful for the analysis of T-DNA mutants with special phenotypes.


April 21, 2020  |  

Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses.

Geminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses.Our results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus.Our study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.


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