Biofertilizer Endophytes of Cannabis

Abstract

The present invention relates to novel endophytes of plants of the Cannabaceae family, particularly biofertilizer Enterobacter sp. endophytes, and also to plants and parts thereof infected therewith, and related methods, including methods for conferring biofertilization to plants and for selecting a biofertilizer endophyte of a plant of the Cannabaceae family.

Claims

1-39. (canceled)

40. A substantially purified or isolated Enterobacter sp. endophyte of a plant of the Cannabaceae family; wherein the endophyte is capable of conferring a biofertilizer phenotype to the plant or part thereof from which it is substantially purified or isolated and/or is capable of conferring a biofertilizer phenotype to a plant or part thereof to which the bacteria is inoculated.

41. An endophyte according to claim 40, wherein the biofertilizer phenotype is enhanced growth of the plant under conditions of below-normal nitrogen levels as compared to a plant that is absent of the endophyte.

42. An endophyte according to claim 40, wherein the biofertilizer phenotype is associated with expression of a nitrogen fixation (nif) gene cluster.

43. An endophyte according to claim 40, wherein one or more of the following applies: i) the biofertilizer phenotype is associated with up-regulation of one or more of nifA, nifB, nifF and nifL; ii) the biofertilizer phenotype is associated with down-regulation of one or more of nifD, nifH, nifJ and nifK; and iii) the biofertilizer phenotype is associated with enhanced growth of a plant or part thereof wherein the plant part is a root or a shoot.

44. An endophyte according to claim 43, wherein enhanced growth of the root length is between about 2%-30% longer relative to an uninoculated control plant, at least 7 days after inoculation.

45. An endophyte according to claim 40, wherein the endophyte is isolated from roots of the plant.

46. An endophyte according to claim 40, wherein the plant of the Cannabaceae family is a Cannabis sativa species plant.

47. An endophyte according to claim 40, wherein the endophyte is a strain denoted EB-008, EB-016 and/or EB-018, as deposited with the National Measurement Institute of 1/153 Bertie St, Port Melbourne, Victoria 3207 Australia on 24 Nov. 2020 with accession numbers V20/025721, V20/025724, and V20/025726, respectively.

48. A plant or part thereof inoculated with one or more endophytes according to claim 40; wherein the endophyte confers a biofertilizer phenotype to the plant or part thereof.

49. A plant according to claim 48, wherein the biofertilizer phenotype is enhanced growth of the plant under conditions of below normal nitrogen levels as compared with a plant that is absent of the endophyte.

50. A plant according to claim 48, wherein the biofertilizer phenotype is associated with enhanced growth of a plant or part thereof, and wherein the plant part is a root or a shoot.

51. A plant according to claim 50, wherein enhanced growth of the root length is between about 2%-30% longer relative to an uninoculated control plant, at least 7 days after inoculation.

52. A plant according to claim 48, wherein the endophyte is inoculated into a plant or part thereof that is free of that endophyte and is stably infected with said endophyte.

53. A method for selecting a biofertilizer endophyte of a plant of the Cannabaceae family, said method comprising a) substantially purifying or isolating one or more endophytes; b) subjecting said one or more endophytes to microbiome profiling; c) analysing the transcriptome of said one or more endophytes, preferably via sequencing, to identify expression of one or more genes associated with nitrogen fixation; and d) selecting an endophyte which is capable of conferring a biofertilizer phenotype to the plant from which it is substantially purified or isolated and/or is capable of conferring a biofertilizer phenotype to a plant or part thereof to which it is inoculated.

54. A method according to claim 53, wherein the step of substantially purifying or isolating one or more endophytes includes: a. providing one or more samples of said plant or part thereof; b. preparing an extract(s) from said sample(s); and c. growing bacterial colonies from said extract(s).

55. A method according to claim 53, wherein the step of subjecting the endophyte to microbiome profiling includes generating sequence data by metagenomics sequencing.

56. A method according to claim 55, wherein metagenomics sequencing is conducted using primers directed to a 16S rRNA gene and PNA PCR blockers.

57. A method according to claim 54, wherein the sample of plant material is selected from one or more ofthe group consisting offlowers, flowerbracts, leaves, petioles, roots and stem.

58. A method according to claim 53, wherein one or more of the following applies: i) the biofertilizer phenotype is enhanced growth of the plant under conditions of below-normal nitrogen levels; ii) the biofertilizer phenotype is associated with up-regulation of one or more of nifA, nifB, nifF and nifL; and iii) the biofertilizer phenotype is associated with down-regulation of one or more of nifD, nifH, nifJ and nifK.

59. A method for conferring a biofertilizer phenotype to a plant or part thereof, said method including inoculating the plant or part thereof with an endophyte according to claim 40.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0091] FIG. 1 represents an Average Nucleotide Identity (ANI) analysis of novel bacterial strains EB-008, EB-016 and EB-018 with genome sequences of E. soli and E. asburiae isolates from NCBI.

[0092] FIG. 2 represents a pan-genome (Roary) of novel bacterial strains EB-008, EB-016 and EB-018 with genome sequences of E. soli and E. asburiae isolates from NCBI.

[0093] FIG. 3 represents nif gene clusters in the novel bacterial strains EB-018, EB-008, EB-016 identified using BLASTn.

[0094] FIG. 4 represents transcriptomic analysis of the expression of the nif gene clusters in two novel bacterial strains (EB-016 and EB-018).

[0095] FIG. 5 represents the average shoot and root length of tomato seedlings inoculated with novel bacterial strains (EB-008, EB-016, EB-018) and E. coli after 10 days growth on Burks medium (no nitrogen) and MS medium (normal nitrogen).

[0096] FIG. 6 represents the distribution of novel bacterial strain EB-016 throughout a mature medicinal cannabis plant (FFlower, FLFlower Bract, O-LOld Leaf, Y-LYoung Leaf, O-POld Petiole, Y-PYoung Petiole, STEMStem, RRoot). The microbiome profile was established by mapping 16S metagenomic reads from the above organs to the 16S sequence of EB-016.

DETAILED DESCRIPTION OF EMBODIMENTS

[0097] In the following examples it is demonstrated that three novel plant associated Enterobacter sp. bacterial strains EB-008, EB-016 and EB-018 were isolated from medicinal cannabis (Cannabis sativa) plants. Each of the novel strains display the ability to increase the growth plants when grown without nitrogen and with normal nitrogen levels. The genomes of the three novel bacterial strains have been sequenced and represent a novel Enterobacter species. Analysis of the genome sequences showed all three bacterial strains have a nitrogen fixation gene cluster, which was transcriptionally active when strains were grown without nitrogen and with normal nitrogen levels. The three novel strains were ubiquitously distributed throughout organs of medicinal cannabis but was most concentrated in the roots.

Example 1Isolation of Bacterial Strains

[0098] Leaves, petioles, stems, flowers and roots were harvested from four different chemotypes (lines) (Cannbio2, 3, 4, 5) of mature cannabis plants. Plants were grown in a greenhouse in pots containing two different substrates: standard potting mix and coconut matting/Jiffy. Root tissues were washed in sterile distilled water to remove soil particles and all the harvested tissues were cut into approximately 1 cm.sup.2 pieces. The plant tissues and organs belonging to different Cannbio lines were separately placed in micro collection tubes and submerged in sufficient Phosphate Buffered Saline (PBS) to completely cover the plant tissue. Plant tissues were ground using a Qiagen TissueLyser II, for 1 minute at 30 Hertz. A 10 l aliquot of the macerate was added to 90 l of PBS. Subsequent 1 in 10 dilutions of the 10.sup.1 suspension were used to create additional 10.sup.2 to 10.sup.4 suspensions. Once the suspensions were well mixed, 50 l aliquots of each suspension were plated onto Reasoners 2 Agar (R2A) for growth of bacteria. Dilutions that provided a good separation of bacterial colonies were subsequently used for isolation of individual bacterial colonies through re-streaking of single bacterial colonies from the dilution plates onto single R2A plates to establish a pure bacterial colony.

[0099] Around 126 bacterial strains were obtained from mature plants grown in standard potting mix.

[0100] The novel bacterial strains EB-008 and EB-018 were collected from roots of medicinal cannabis plants Cannbio 59 and EB016 from Cannbio 3.

Example 2Identification of Novel Bacterial Strains

Genomics

[0101] The genomes of novel bacterial strains EB-008, EB-016 and EB-018 were sequenced. These novel bacterial strains were retrieved from the glycerol collection stored at 80 C. by streaking on NA plates. Single colonies from these plates were grown overnight in Nutrient Broth and pelleted. These pellets were used for genomic DNA extraction using the bacteria protocol of Wizard Genomic DNA Purification Kit (A1120, Promega). DNA sequencing libraries were generated for Illumina sequencing using the Illumina Nextera XT DNA library prep protocol. All libraries were sequenced using an Illumina HiSeq platform. Raw reads from the sequencer were filtered to remove any adapter and index sequences as well as low quality bases using fastp (Chen, Zhou, et al., 2018) quality controller software for fastq files. To enable full genome assembly, long reads were generated for the three novel bacterial strains by sequencing DNA using Oxford Nanopore Technologies (ONT) MinION platform. The DNA from the Wizard Genomic DNA Purification Kit was first assessed with the genomic assay on Agilent 2200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA) for integrity (average molecular weight 30 Kb). The sequencing library was prepared using an in-house protocol modified from the official protocols for transposases-based library preparation kits (SQK-RAD004/SQK-RBK004, ONT, Oxford, UK). All libraries were sequenced on a MinION Mk1 B platform (MIN-101B) with R9.4 flow cells (FLO-MIN106) and under the control of MinKNOW software. After the sequencing run finished, the fast5 files that contain raw read signals were transferred to a separate, high performance computing Linux server for local basecalling using ONT's Albacore software (Version 2.3.1) with default parameters. For libraries prepared with the barcoding kit (SQK-RBK004), barcode demultiplexing was achieved during basecalling. The sequencing summary file produced by Albacore was processed by the R script minion qc (https://github.com/roblanf/minion_qc) and NanoPlot (De Coster et al. 2018) to assess the quality of each sequencing run, while Porechop (Version 0.2.3, https://github.com/rrwick/Porechop) was used to remove adapter sequences from the reads. Reads which were shorter than 300 bp were removed and the worst 5% of reads (based on quality) were discarded by using Filtlong (Version 0.2.0, https://github.com/rrwick/Filtlong).

[0102] The whole genome sequence of the three novel bacterial strains were assembled using Unicycler (Wick et al. 2017). Unicycler performed hybrid assembly when both Illumina reads, and MinION reads were available. MinION reads were mainly used to resolve repeat regions in the genome, whereas Illumina reads were used by Pilon (Walker et al. 2014) to correct small base-level errors. Multiple rounds of Racon (Vaser et al. 2017) polishing were then carried out to generate consensus sequences. Assembly graphs were visualised by using Bandage (Wick et al. 2015).

[0103] A complete circular chromosome sequence was produced for the three novel bacterial strains. The genome size for the novel bacterial strains EB-008, EB-016 and EB-018 were 4,929,453 bp, 5,017,261 bp and 5,112,947 bp respectively (Table 1).

TABLE-US-00001 TABLE 1 Summary of properties of the final genome sequence assembly Strain Genome size GC content Coverage Coverage ID (bp) (%) Illumina reads ONT MinION EB-008 4,929,453 53.2 160.6x 461.9x EB-016 5,017,261 53.2 65.5x 645.6x EB-018 5,112,947 52.9 92.3x 105.1x

[0104] The percent GC content ranged from 52%-53%. These novel bacterial strains were annotated by Prokka (Seemann 2014) with a custom, genus-specific protein database to predict genes and corresponding functions, which were then screened manually to identify specific traits.

[0105] The number of genes for the novel bacterial strains EB-008, EB-016 and EB-018 were 6,268, 6,562 and 4,828 genes respectively (Table 2).

TABLE-US-00002 TABLE 2 Summary of genome coding regions No. of No. of No. of No. of No. of Strain ID tRNA tmRNA rRNA CDS gene EB-008 84 1 22 6161 6268 EB-016 83 1 22 6456 6562 EB-018 83 1 22 4722 4828

[0106] A phylogenetic analysis of the novel bacterial strains EB-008, EB-016 and EB-018 was undertaken by sequence homology comparison of the 16S rRNA gene regions extracted from whole genome sequence of each bacteria (Table 3, SEQ ID NOs: 1, 2 and 3).

TABLE-US-00003 TABLE 3 16s rRNA gene regions identified within Enterobacter strains EB-008, EB-016 and EB-018 Nucleic acid Enterobacter strain Gene SEQ ID NO: EB008 16s rRNA 1 EB016 16s rRNA 2 EB018 16s rRNA 3

[0107] The sequences were aligned by BLASTn on NCBI against the non-redundant nucleotide database and the 16S ribosomal RNA database. The preliminary taxonomic identification of the novel bacterial strains EB-008, EB-016 and EB-018 were Enterobacter sp. closely related to E. soli (Tables 4 and 5).

TABLE-US-00004 TABLE 4 BLASTn hit against database nr; Enterobacter sp. strain LSRC69 16S ribosomal RNA gene, partial sequence Query E- % Coverage Value Identity Species Accession EB-008 100% 0 99.58% Enterobacter sp. JF772075.1 EB-016 100% 0 99.72% Enterobacter sp. JF772075.1 EB-018 100% 0 99.27% Enterobacter sp. JF772075.1

TABLE-US-00005 TABLE 5 BLASTn hit against database 16S ribosomal RNA; Enterobacter soli strain LF7 16S ribosomal RNA gene, partial sequence Query E- % Coverage Value Identity Species Accession EB-008 100% 0 99.36% Enterobacter soli NR_117547.1 EB-016 100% 0 99.50% Enterobacter soli NR_117547.1 EB-018 100% 0 99.16% Enterobacter soli NR_117547.1

[0108] Three E. soli genome sequences and Two E. asburiae genome sequences that are publicly available on NCBI were acquired and used for average nucleotide identity (ANI) calculation (FIG. 1) and pan-genome/comparative genome sequence analysis alongside novel bacterial strains EB-008, EB-016 and EB-018 (FIG. 2). ANI values were calculated using the Pyani package (Pritchard L. 2016 https://github.com/widdowquinn/pyani) and three novel bacterial strains EB-008, EB-016 and EB-018 had >99% average nucleotide identity to each other while 94% identity to E. soli and <87% to E. asburiae isolates. Based on an ANI species cut of 95-96%, it is evident that isolates EB-008, EB-016 and EB-018 are representative of a novel Enterobacter species.

[0109] Prokka annotated novel bacterial genomes were provided to Roary (Page et al. 2015) and a total of 1327 genes that are shared by all eight strains were identified by running Roary. PRANK (Lytynoja 2014) was then used to perform a codon aware alignment and visualization of phylogenetic tree derived from core gene alignment was produced with FigTree (version 1.4.4 https://github.com/rambaut/figtree/releases). The novel bacterial strains EB-008, EB-016 and EB-018 clustered tightly together, suggesting a close phylogenetic relationship between these bacterial strains. Moreover, this cluster was separated from other Enterobacter species used in the analysis including E. soli with strong local support value (100%). This separation supports that these three bacterial strains are from a novel Enterobacter species, but closely related to E. soli.

Example 3Genome Sequence Features Supporting the Biofertilizer Niche of the Novel Bacterial Strains

Nif Gene Clusters

[0110] The genome sequences of the four novel bacterial strains EB-008, EB-016 and EB-018 were assessed for the presence of features associated with biofertilization. The annotated genome sequences were assessed for the presence of the nif gene cluster (nifA, nifB, nifD, nifF, nifH, nifJ, nifK, nifL, nifS, nifW) by aligning the genome sequences against nif genes using BLASTn. The nif gene cluster was identified in all three genomes (Table 6, SEQ ID NOs: 4-16).

TABLE-US-00006 TABLE 6 nif genes identified within Enterobacter strains EB-008, EB-016 and EB-018 Nucleic acid Gene Enterobacter strain SEQ ID NO: nifA EB008, EB016 4 nifA EB018 5 nifB EB008, EB016, EB018 6 nifD EB008, EB016, EB018 7 nifF EB008, EB016, EB018 8 nifH EB008, EB016 9 nifH EB018 10 nifJ EB008, EB016 11 nifJ EB018 12 nifK EB008, EB016, EB018 13 nifL EB008, EB016, EB018 14 nifS EB008, EB016, EB018 15 nifW EB008, EB016, EB018 16

[0111] Annotated genome sequences were passed through BLAST Ring Image Generator (BRIG) (Alikhan, Petty, et al., 2011) and presence of nif gene cluster in all three genomes were graphically interpreted, and found to be localised in different regions throughout the genomes. (FIG. 3).

Example 4Transcriptomics Supporting the Biofertilizer Niche of the Novel Bacterial Strains

[0112] The transcriptomes of novel bacterial strains EB-016 and EB-018 were sequenced under normal nitrogen and no nitrogen conditions. The novel bacterial strains were conditioned for 24 hours on nitrogen-free Burks solid medium (Wilson & Knight 1952) and then transferred into either Burks liquid broth or Murashige and Skoog (MS) broth for 24 hours. Bacterial cell pellets from these cultures were used for total RNA extraction using the TRIzol plus RNA purification Kit (cat No: 12183555, Invitrogen). Subsequently, ribosomal RNA was removed using NEBNext rRNA depletion kit (NEB #E6310, BioLabs New England) and cDNA sequencing libraries were generated for Illumina sequencing using the NEBNext ultra-RNA library prep kit for Illumina (NEB #E7530, BioLabs New England) protocol. All libraries were sequenced using an Illumina MiSeq platform. The raw RNA-Seq reads were filtered to remove any adapter and index sequences as well as low quality bases using fastp (Chen, Zhou, et al., 2018). Reads were mapped to the nif genes of the respective isolate using the Gydle software suite (https://www.gydle.com) to obtain the abundance of reads per gene. The nif gene cluster was transcriptionally active in novel bacterial strains EB-016 and EB-018 with reads detected in nif genes of both strains (FIG. 4). NifJ (electron donor to the nitrogenase) had the highest number reads detected, followed by nifL (negative regulator) and nifA (positive regulator). Comparing the two conditions (no N and normal N), both strains responded similarly with the upregulation of nifA (SEQ ID NOs: 4 and 5), nifB (SEQ ID NO: 6), nifF (SEQ ID NO: 8) and nifL (SEQ ID NO: 14), and the down regulation of nifD (SEQ ID NO: 7), nifH (SEQ ID NOs: 9 and 10), nifJ(SEQ ID NOs: 11 and 12) and nifK(SEQ ID NO: 13) under no nitrogen.

Example 5Biofertiliser Activity (in Planta) of the Enterobacter sp. Novel Bacterial Strains

[0113] To assess direct interactions between the novel bacterial strains and plants, an early seedling growth assay was established in tomato. A total of 4 bacterial strains (EB-008, EB-016, EB-018 and Eschericia coli) were cultured in Burks media overnight. The following day seeds of tomato were surface-sterilised (3% bleach), washed 5 times in sterile distilled water. The seeds were then soaked in the overnight cultures for 4 hours in a shaking incubator. For control seedlings, seeds were soaked in Burks media without bacteria for 4 hours in a shaking incubator. The seeds were transferred to petri plates containing sterile filter paper, the seeds were sprayed with sterile MQ water and allowed to germinate for 7 days. Germinated seedlings were transferred to semi-solid Burk's medium (no nitrogen) and MS medium (normal nitrogen) after and the seedlings allowed to grow for 10 days. The lengths of roots and shoots were measured.

[0114] Seedlings inoculated with the novel bacterial strains were healthy with no disease symptoms recorded on leaves or roots. The length of the shoots inoculated with the novel bacterial strains were 17.0-25.8% longer than the control under normal nitrogen, and 2.8-12.5% under no nitrogen (FIG. 5). The length of the roots inoculated with the novel bacterial strains were 2.4-10.4% longer than the control under normal nitrogen, and 25.0-29.5% under no nitrogen. Statistical analysis was performed using OriginPro. The three novel bacterial strains EB-008, EB-016, and EB-018 resulted in significantly longer roots in low nitrogen media, compared to the control (p=0.05).

Example 6Distribution of Novel Bacterial Strains in Medicinal Cannabis Plants

[0115] For microbiome profiling, flowers, flower bracts, leaves (old and young), petioles (old and young), roots and stem were collected from mature plants. DNA extraction was performed in 96-well plates using the QIAGEN MagAttract 96 DNA Plant Core Kit according to manufacturers' instructions with minor modifications for use with a Biomek FX liquid handling station. The bacterial microbiome was profiled targeting the V4 region (515F and 806R) of the 16S rRNA gene (SEQ ID NOs: 1, 2 and 3) according to the Illumina 16S Metagenomic Sequencing Library Preparation protocol, with minor modifications to include the use of PNA PCR blockers to reduce amplification of 16S rRNA genes sequences derived from the plant chloroplast genome and mitochondrial genome (Wagner et al., 2016). Paired-end sequencing was performed on a MiSeq to generate 2300 bp reads. Sequence data was trimmed and merged using PandaSEQ (removal of low quality reads, 8 bp overlap of read 1 and read 2, removal of primers, final merged read length of 253 bp) (Massela et al., 2012). The Gydle software suite was used for dereplication, taxonomical assignment and removal of organelle OTUs. Reads were mapped (Gydle) to the 16S sequence of EB-016 as a representative of the four novel bacterial strains to determine the distribution of the strains through medicinal cannabis plants. Reads were identified in all organs, with numbers ranging around 1500 for flowers, flower bracts, leaves (old and young), petioles (old and young) and stems, while numbers were higher in roots (up to 150,105) (FIG. 6). As such, the novel bacterial strains appeared ubiquitously distributed across the medicinal cannabis plant, but more concentrated in roots than other organs.

[0116] Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein. In particular, the present invention may be applied to a range of agricultural hosts, not limited to the Poaceae species (such as sugarcane, rice, corn) and Legumes (such as pigeon pea), as well as other horticultural species (such as sweet potato, grapes and tomato).

REFERENCES

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