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April 21, 2020  |  

Depiction of secondary metabolites and antifungal activity of Bacillus velezensis DTU001.

For a safe and sustainable environment, effective microbes as biocontrol agents are in high demand. We have isolated a new Bacillus velezensis strain DTU001, investigated its antifungal spectrum, sequenced its genome, and uncovered the production of lipopeptides in HPLC-HRMS analysis. To test the antifungal efficacy, extracts of B. velezensis DTU001 was tested against a range of twenty human or plant pathogenic fungi. We demonstrate that inhibitory potential of B. velezensis DTU001 against selected fungi is superior in comparison to single lipopeptide, either iturin or fengycin. The isolate showed analogous biofilm formation to other closely related Bacilli. To further support the biocontrol properties of the isolate, coculture with Candida albicans demonstrated that B. velezensis DTU001 exhibited excellent antiproliferation effect against C. albicans. In summary, the described isolate is a potential antifungal agent with a broad antifungal spectrum that might assist our aims to avoid hazardous pathogenic fungi and provide alternative to toxicity caused by chemicals.


April 21, 2020  |  

The mitochondrial genome analysis of Isaria tenuipes (Hypocreales: Cordycipitaceae)

The mitochondrial genome of Isaria tenuipes, strain TTZ2017-3, was sequenced on the Illumina Hiseq 4000 and the PacBio Sequel Sequencer and annotated. The genome is 66703bp in length, encoding 15 conserved protein-coding genes (PCGs) including ribosomal protein S3, two rRNA genes and 26 tRNA genes. The nucleotide composition of I. tenuipes mitochondrial genome was 39.1% of A, 35.6% of T, 11.2% of C, 14.2% of G, 74.7% of AþT content. Phylogenetic analysis with other Hypocreales species revealed that I. tenuipes was more closely related to Cordyceps militaris, separated from Lecanicillium muscarium, Paecilomyces hepialid, and Beauveria species with Cordyceps teleomorph. This study provided valuable information on the gene contents of the mitochondrial genome and would facilitate the study of function and evolution of Isaria.


April 21, 2020  |  

Complete genome sequence of a marine-sediment-derived bacterial strain Bacillus velezensis SH-B74, a cyclic lipopeptides producer and a biopesticide.

A marine-sediment sample-derived strain Bacillus velezensis SH-B74 has the capacity to produce cyclic lipopeptides (CLPs), and these CLPs secreted by the strain show biological activities against various pests under both in vitro and in planta conditions, such evidence has supported that the strain SH-B74 is a biopesticide. To get a better insight into the mechanisms on the control of the pesticides by the strain, a genome sequencing project has been applied to the genomic DNA of the strain SH-B74. The results show that the strain SH-B74 has a chromosome size of 4,042,190 bp, with a GC content of 46.5%, in addition, the strain contains a 61,634 bp plasmid pSH-B74, with a GC content of 40.8%. Data from bioinformatic analysis reveal that the strain SH-B74 has genes with the capacity to increase environmental adaptation, promote the rhizosphere fitnesses and secrete a spectrum of antibiotics, including nonribosomal peptide synthetases (NRPSs)-derived CLPs bacillopeptin, plipastatin, and surfactin. The presence of CLPs in the bacterial cultures of the strain SH-B74 was confirmed further by LC-MS analysis. Thus, genome sequencing and analyses together with chemical analysis reveal the promising perspectives of the strain SH-B74 that are of spectacular importance to its trait as a plant beneficial microbe to be used in agriculture practices.


April 21, 2020  |  

Valinomycin, produced by Streptomyces sp. S8, a key antifungal metabolite in large patch disease suppressiveness.

Large patch disease, caused by Rhizoctonia solani AG2-2, is the most devastating disease in Zoysiagrass (Zoysia japonica). Current large patch disease control strategies rely primarily upon the use of chemical pesticides. Streptomyces sp. S8 is known to possess exceptional antagonistic properties that could potentially suppress the large patch pathogen found at turfgrass plantations. This study aims to demonstrate the feasibility of using the strain as a biological control mechanism. Sequencing of the S8 strain genome revealed a valinomycin biosynthesis gene cluster. This cluster is composed of the vlm1 and vlm2 genes, which are known to produce antifungal compounds. In order to verify this finding for the large patch pathogen, a valinomycin biosynthesis knockout mutant was created via the CRISPR/Cas9 system. The mutant lost antifungal activity against the large patch pathogen. Consequently, it is anticipated that eco-friendly microbial preparations derived from the S8 strain can be utilized to biologically control large patch disease.


April 21, 2020  |  

The golden death bacillus Chryseobacterium nematophagum is a novel matrix digesting pathogen of nematodes.

Nematodes represent important pathogens of humans and farmed animals and cause significant health and economic impacts. The control of nematodes is primarily carried out by applying a limited number of anthelmintic compounds, for which there is now widespread resistance being reported. There is a current unmet need to develop novel control measures including the identification and characterisation of natural pathogens of nematodes.Nematode killing bacilli were isolated from a rotten fruit in association with wild free-living nematodes. These bacteria belong to the Chryseobacterium genus (golden bacteria) and represent a new species named Chryseobacterium nematophagum. These bacilli are oxidase-positive, flexirubin-pigmented, gram-negative rods that exhibit gelatinase activity. Caenorhabditis elegans are attracted to and eat these bacteria. Within 3 h of ingestion, however, the bacilli have degraded the anterior pharyngeal chitinous lining and entered the body cavity, ultimately killing the host. Within 24?h, the internal contents of the worms are digested followed by the final digestion of the remaining cuticle over a 2-3-day period. These bacteria will also infect and kill bacterivorous free-living (L1-L3) stages of all tested parasitic nematodes including the important veterinary Trichostrongylids such as Haemonchus contortus and Ostertagia ostertagi. The bacteria exhibit potent collagen-digesting properties, and genome sequencing has identified novel metalloprotease, collagenase and chitinase enzymes representing potential virulence factors.Chryseobacterium nematophagum is a newly discovered pathogen of nematodes that rapidly kills environmental stages of a wide range of key nematode parasites. These bacilli exhibit a unique invasion process, entering the body via the anterior pharynx through the specific degradation of extracellular matrices. This bacterial pathogen represents a prospective biological control agent for important nematode parasites.


April 21, 2020  |  

Comprehensive analysis of full genome sequence and Bd-milRNA/target mRNAs to discover the mechanism of hypovirulence in Botryosphaeria dothidea strains on pear infection with BdCV1 and BdPV1

Pear ring rot disease, mainly caused by Botryosphaeria dothidea, is widespread in most pear and apple-growing regions. Mycoviruses are used for biocontrol, especially in fruit tree disease. BdCV1 (Botryosphaeria dothidea chrysovirus 1) and BdPV1 (Botryosphaeria dothidea partitivirus 1) influence the biological characteristics of B. dothidea strains. BdCV1 is a potential candidate for the control of fungal disease. Therefore, it is vital to explore interactions between B. dothidea and mycovirus to clarify the pathogenic mechanisms of B. dothidea and hypovirulence of B. dothidea in pear. A high-quality full-length genome sequence of the B. dothidea LW-Hubei isolate was obtained using Single Molecule Real-Time sequencing. It has high repeat sequence with 9.3% and DNA methylation existence in the genome. The 46.34?Mb genomes contained 14,091 predicted genes, which of 13,135 were annotated. B. dothidea was predicted to express 3833 secreted proteins. In bioinformatics analysis, 351 CAZy members, 552 transporters, 128 kinases, and 1096 proteins associated with plant-host interaction (PHI) were identified. RNA-silencing components including two endoribonuclease Dicer, four argonaute (Ago) and three RNA-dependent RNA polymerase (RdRp) molecules were identified and expressed in response to mycovirus infection. Horizontal transfer of the LW-C and LW-P strains indicated that BdCV1 induced host gene silencing in LW-C to suppress BdPV1 transmission. To investigate the role of RNA-silencing in B. dothidea defense, we constructed four small RNA libraries and sequenced B. dothidea micro-like RNAs (Bd-milRNAs) produced in response to BdCV1 and BdPV1 infection. Among these, 167 conserved and 68 candidate novel Bd-milRNAs were identified, of which 161 conserved and 20 novel Bd-milRNA were differentially expressed. WEGO analysis revealed involvement of the differentially expressed Bd-milRNA-targeted genes in metabolic process, catalytic activity, cell process and response to stress or stimulus. BdCV1 had a greater effect on the phenotype, virulence, conidiomata, vertical and horizontal transmission ability, and mycelia cellular structure biological characteristics of B. dothidea strains than BdPV1 and virus-free strains. The results obtained in this study indicate that mycovirus regulates biological processes in B. dothidea through the combined interaction of antiviral defense mediated by RNA-silencing and milRNA-mediated regulation of target gene mRNA expression.


September 22, 2019  |  

ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm.

Evolution of pest resistance threatens the benefits of genetically engineered crops that produce Bacillus thuringiensis (Bt) insecticidal proteins. Strategies intended to delay pest resistance are most effective when implemented proactively. Accordingly, researchers have selected for and analyzed resistance to Bt toxins in many laboratory strains of pests before resistance evolves in the field, but the utility of this approach depends on the largely untested assumption that laboratory- and field-selected resistance to Bt toxins are similar. Here we compared the genetic basis of resistance to Bt toxin Cry2Ab, which is widely deployed in transgenic crops, between laboratory- and field-selected populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that resistance to Cry2Ab is associated with mutations disrupting the same ATP-binding cassette transporter gene (PgABCA2) in a laboratory-selected strain from Arizona, USA, and in field-selected populations from India. The most common mutation, loss of exon 6 caused by alternative splicing, occurred in resistant larvae from both locations. Together with previous data, the results imply that mutations in the same gene confer Bt resistance in laboratory- and field-selected strains and suggest that focusing on ABCA2 genes may help to accelerate progress in monitoring and managing resistance to Cry2Ab.


September 22, 2019  |  

The bacterial microbiome of Dermacentor andersoni ticks influences pathogen susceptibility.

Ticks are of medical importance owing to their ability to transmit pathogens to humans and animals. The Rocky Mountain wood tick, Dermacentor andersoni, is a vector of a number of pathogens, including Anaplasma marginale, which is the most widespread tick-borne pathogen of livestock. Although ticks host pathogenic bacteria, they also harbor bacterial endosymbionts that have a role in tick physiology, survival, as well as pathogen acquisition and transmission. The goal of this study was to characterize the bacterial microbiome and examine the impact of microbiome disruption on pathogen susceptibility. The bacterial microbiome of two populations of D. andersoni with historically different susceptibilities to A. marginale was characterized. In this study, the microbiome was disrupted and then ticks were exposed to A. marginale or Francisella novicida to determine whether the microbiome correlated with pathogen susceptibility. Our study showed that an increase in proportion and quantity of Rickettsia bellii in the microbiome was negatively correlated to A. marginale levels in ticks. Furthermore, a decrease in Francisella endosymbionts was associated with lower F. novicida infection levels, demonstrating a positive pathogen-endosymbiont relationship. We demonstrate that endosymbionts and pathogens have varying interactions, and suggest that microbiome manipulation may provide a possible method for biocontrol by decreasing pathogen susceptibility of ticks.


September 22, 2019  |  

Differential expression analysis of olfactory genes based on a combination of sequencing platforms and behavioral investigations in Aphidius gifuensis.

Aphidius gifuensis Ashmead is a dominant endoparasitoid of aphids, such as Myzus persicae and Sitobion avenae, and plays an important role in controlling aphids in various habitats, including tobacco plants and wheat in China. A. gifuensis has been successfully applied for the biological control of aphids, especially M. persicae, in green houses and fields in China. The corresponding parasites, as well as its mate-searching behaviors, are subjects of considerable interest. Previous A. gifuensis transcriptome studies have relied on short-read next-generation sequencing (NGS), and the vast majority of the resulting isotigs do not represent full-length cDNA. Here, we employed a combination of NGS and single-molecule real-time (SMRT) sequencing of virgin females (VFs), mated females (MFs), virgin males (VMs), and mated males (MMs) to comprehensively study the A. gifuensis transcriptome. Behavioral responses to the aphid alarm pheromone (E-ß-farnesene, EBF) as well as to A. gifuensis of the opposite sex were also studied. VMs were found to be attracted by female wasps and MFs were repelled by male wasps, whereas MMs and VFs did not respond to the opposite sex. In addition, VFs, MFs, and MMs were attracted by EBF, while VMs did not respond. According to these results, we performed a personalized differential gene expression analysis of olfactory gene sets (66 odorant receptors, 25 inotropic receptors, 16 odorant-binding proteins, and 12 chemosensory proteins) in virgin and mated A. gifuensis of both sexes, and identified 13 candidate genes whose expression levels were highly consistent with behavioral test results, suggesting potential functions for these genes in pheromone perception.


September 22, 2019  |  

Complete genome sequence of Paenibacillus polymyxa YC0136, a plant growth–promoting rhizobacterium isolated from tobacco rhizosphere.

Paenibacillus polymyxa strain YC0136 is a plant growth-promoting rhizobacterium with antimicrobial activity, which was isolated from tobacco rhizosphere. Here, we report the complete genome sequence of P. polymyxa YC0136. Several genes with antifungal and antibacterial activity were discovered. Copyright © 2017 Liu et al.


September 22, 2019  |  

Identification of putative coffee rust mycoparasites using single molecule DNA sequencing of infected pustules.

The interaction of crop pests with their natural enemies is a fundament to their control. Natural enemies of fungal pathogens of crops are poorly known relative to those of insect pests despite the diversity of fungal pathogens and their economic importance. Currently, many regions across Latin America are experiencing unprecedented epidemics of coffee rust (Hemileia vastatrix). Identification of natural enemies of coffee rust could aid in developing management strategies or in pinpointing species that could be used for biocontrol. Here we characterize fungal communities associated with coffee rust lesions by single molecule DNA sequencing of fungal ribosomal RNA barcodes from leaf discs (˜28 mm(2)) containing rust lesions and control discs with no rust lesions. The leaf disc communities were hyper-diverse in fungi, with up to 57 taxa per control disc, and the diversity was only slightly reduced in rust-infected discs. However, geography had a greater influence on the fungal community than whether the disk was infected by coffee rust. Through comparisons between control and rust-infected leaf discs, as well as taxonomic criteria, we identified 15 putative mycoparasitic fungi. These fungi are concentrated in fungal family Cordycipitaceae and order Tremellales. These data emphasize the complexity of fungal diversity of unknown ecological function within a leaf that might influence plant disease epidemics or lead to the development of species for biocontrol of fungal disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


September 22, 2019  |  

Prey range and genome evolution of Halobacteriovorax marinus predatory bacteria from an estuary

Halobacteriovorax strains are saltwater-adapted predatory bacteria that attack Gram-negative bacteria and may play an important role in shaping microbial communities. To understand how Halobacteriovorax strains impact ecosystems and develop them as biocontrol agents, it is important to characterize variation in predation phenotypes and investigate Halobacteriovorax genome evolution. We isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island using Vibrio from the same site as prey. Small, fast-moving, attack-phase BE01 cells attach to and invade prey cells, consistent with the intraperiplasmic predation strategy of the H. marinus type strain, SJ. BE01 is a prey generalist, forming plaques on Vibrio strains from the estuary, Pseudomonas from soil, and Escherichia coli. Genome analysis revealed extremely high conservation of gene order and amino acid sequences between BE01 and SJ, suggesting strong selective pressure to maintain the genome in this H. marinus lineage. Despite this, we identified two regions of gene content difference that likely resulted from horizontal gene transfer. Analysis of modal codon usage frequencies supports the hypothesis that these regions were acquired from bacteria with different codon usage biases than H. marinus. In one of these regions, BE01 and SJ carry different genes associated with mobile genetic elements. Acquired functions in BE01 include the dnd operon, which encodes a pathway for DNA modification, and a suite of genes involved in membrane synthesis and regulation of gene expression that was likely acquired from another Halobacteriovorax lineage. This analysis provides further evidence that horizontal gene transfer plays an important role in genome evolution in predatory bacteria. IMPORTANCE Predatory bacteria attack and digest other bacteria and therefore may play a role in shaping microbial communities. To investigate phenotypic and genotypic variation in saltwater-adapted predatory bacteria, we isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island, assayed whether it could attack different prey bacteria, and sequenced and analyzed its genome. We found that BE01 is a prey generalist, attacking bacteria from different phylogenetic groups and environments. Gene order and amino acid sequences are highly conserved between BE01 and the H. marinus type strain, SJ. By comparative genomics, we detected two regions of gene content difference that likely occurred via horizontal gene transfer events. Acquired genes encode functions such as modification of DNA, membrane synthesis and regulation of gene expression. Understanding genome evolution and variation in predation phenotypes among predatory bacteria will inform their development as biocontrol agents and clarify how they impact microbial communities.


September 22, 2019  |  

Genome and secretome analysis of Pochonia chlamydosporia provide new insight into egg-parasitic mechanisms.

Pochonia chlamydosporia infects eggs and females of economically important plant-parasitic nematodes. The fungal isolates parasitizing different nematodes are genetically distinct. To understand their intraspecific genetic differentiation, parasitic mechanisms, and adaptive evolution, we assembled seven putative chromosomes of P. chlamydosporia strain 170 isolated from root-knot nematode eggs (~44?Mb, including 7.19% of transposable elements) and compared them with the genome of the strain 123 (~41?Mb) isolated from cereal cyst nematode. We focus on secretomes of the fungus, which play important roles in pathogenicity and fungus-host/environment interactions, and identified 1,750 secreted proteins, with a high proportion of carboxypeptidases, subtilisins, and chitinases. We analyzed the phylogenies of these genes and predicted new pathogenic molecules. By comparative transcriptome analysis, we found that secreted proteins involved in responses to nutrient stress are mainly comprised of proteases and glycoside hydrolases. Moreover, 32 secreted proteins undergoing positive selection and 71 duplicated gene pairs encoding secreted proteins are identified. Two duplicated pairs encoding secreted glycosyl hydrolases (GH30), which may be related to fungal endophytic process and lost in many insect-pathogenic fungi but exist in nematophagous fungi, are putatively acquired from bacteria by horizontal gene transfer. The results help understanding genetic origins and evolution of parasitism-related genes.


September 22, 2019  |  

Complete genome sequence of Lactobacillus pentosus SLC13, isolated from mustard pickles, a potential probiotic strain with antimicrobial activity against foodborne pathogenic microorganisms.

Lactobacillus pentosus SLC13 is a high exopolysaccharide (EPS)-producing strain with broad-spectrum antimicrobial activity and the ability to grow in simulated gastrointestinal conditions. SLC13 was isolated from mustard pickles in Taiwan for potential probiotic applications. To better understand the molecular base for its antimicrobial activity and high EPS production, entire genome of SLC13 was determined by PacBio SMRT sequencing.L. pentosus SLC13 contains a genome with a 3,520,510-bp chromosome and a 62,498-bp plasmid. GC content of the complete genome was 46.5% and that of plasmid pSLC13 was 41.3%. Sequences were annotated at the RAST prokaryotic genome annotation server, and the results showed that the genome contained 3172 coding sequences and 82 RNA genes. Seventy-six protein-coding sequences were identified on the plasmid pSLC13. A plantaricin gene cluster, which is responsible for bacteriosins biosynthesis and could be associated with its broad-spectrum antimicrobial activity, was identified based on comparative genomic analysis. Two gene clusters involved in EPS production were also identified.This genomic sequence might contribute to a future application of this strain as probiotic in productive livestock potentially inhibiting competing and pathogenic organisms.


September 22, 2019  |  

Complete genome sequence of Pseudomonas Parafulva PRS09-11288, a biocontrol strain produces the antibiotic phenazine-1-carboxylic acid.

Rhizoctonia solani is a plant pathogenic fungus, which can infect a wide range of economic crops including rice. In this case, biological control of this pathogen is one of the fundmental way to effectively control this pathogen. The Pseudomonas parafulva strain PRS09-11288 was isolated from rice rhizosphere and shows biocontrol ability against R. solani. Here, we analyzed the P. parafulva genome, which is ~?4.7 Mb, with 4310 coding sequences, 76 tRNAs, and 7 rRNAs. Genome analysis identified a phenazine biosynthetic pathway, which can produce antibiotic phenazine-1-carboxylic acid (PCA). This compound is responsible for biocontrol ability against R. solani Kühn, which is one of the most serious fungus disease on rice. Analysis of the phenazine biosynthesis gene mutant, ?phzF, which is very important in this pathway, confirmed the relationship between the pathway and PCA production using LC-MS profiles. The annotated full genome sequence of this strain sheds light on the role of P. parafulva PRS09-11288 as a biocontrol bacterium.


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