Bioprotectant Endophytes of Cannabis

Abstract

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

Claims

1-35. (canceled)

36. A substantially purified or isolated Pseudomonas sp. endophyte of a plant of the Cannabaceae family, wherein said endophyte is capable of conferring a bioprotection phenotype to the plant or part thereof from which it is substantially purified or isolated and/or is capable of conferring a bioprotection phenotype to a plant or part thereof to which it is inoculated.

37. An endophyte according to claim 36, wherein the bioprotection phenotype is improved resistance to pests and/or diseases.

38. An endophyte according to claim 37, wherein said disease is infection by a fungal pathogen.

39. An endophyte according to claim 38, wherein said fungal pathogen is a Botrytis sp. fungus or a Fusarium sp. fungus.

40. An endophyte according to claim 36, wherein one or more of the following applies: i) said endophyte is capable of presenting with an in vitro resistance to a pathogenic Botrytis cinerea species strain of at least about 75% against no-endophyte control, and/or of at least about 20% greater resistance than a Paenibacillus pabuli species bacterial strain; ii) said endophyte is capable of presenting with an in vitro resistance to a pathogenic Fusarium proliferatum species strain of at least about 45% against no-endophyte control, and/or of at least about 30% greater resistance than a Paenibacillus pabuli species bacterial strain; and iii) said endophyte is positive for one or more of the fragin, arylpolyene (APE), bacteriocin, NAGGN, betalactone, cupriachelin, non-ribosomal peptide (NRP) siderophore, TIPKS (e.g. entolysin), siderophor, hserlactone, pyoverdin, crochelin and NRP+polyketide biosynthesis gene clusters.

41. An endophyte according to claim 40, wherein one or both of the following apply: i) said endophyte is positive for one or more of the APE Vf (arylpolyene), fengycin (betalactone) and syringomycin (hserlactone) biosynthesis gene clusters; and ii) said endophyte is positive for the syringomycin biosynthesis gene cluster.

42. An endophyte according to claim 36, wherein said endophyte is negative for one or more secondary metabolite biosynthesis gene clusters commonly associated with human toxicity.

43. An endophyte according to claim 42, wherein a secondary metabolite biosynthesis gene cluster commonly associated with human toxicity and for which the endophyte is negative is the pyocyanin gene cluster.

44. An endophyte according to claim 36, wherein said plant or part thereof to which it is capable of conferring a bioprotection phenotype is one which is or may be infected by a Botrytis sp. Fungus and/or a Fusarium sp. Fungus and wherein said plant or part thereof to which it is capable of conferring a bioprotection phenotype is of the Vitis, Cannabaceae, Fragaria, Rubus, Vaccinium, Ribes, Solanum, Brassica, Phaseolus and/or Lactuca families.

45. An endophyte according to claim 44, wherein said plant or part thereof to which it is capable of conferring a bioprotection phenotype is of the Vitis and/or Cannabaceae family.

46. An endophyte according to claim 36, wherein said endophyte is capable of conferring in planta resistance to a plant of the Vitis vinifera species to a pathogenic Botrytis cinerea species strain of a pathogen infection score at most about 2.2, against no-endophyte control.

47. An endophyte according to claim 36, wherein said plant of the Cannabaceae family is a Cannabis saliva, Cannabis indica or Cannabis ruderalis species plant.

48. An endophyte according to claim 36, wherein said endophyte is substantially purified or isolated from a flower, flower bract, leaf, petiole, stem or root of the plant of the Cannabaceae family.

49. An endophyte according to claim 48, wherein said endophytes is substantially purified or isolated from a root.

50. An endophyte according to claim 36, wherein said endophyte is a Pseudomonas corrugata species strain.

51. An endophyte according to claim 36, wherein said endophyte is a strain denoted EB-010, EB-013, EB-017 and/or EB-117 as deposited with The National Measurement Institute on 24 Nov. 2020 with accession numbers V20/025722, V20/025723, V20/025725 and V20/025727, respectively.

52. A plant or part thereof inoculated with one or more endophytes according to claim 36 and exhibiting a bioprotection phenotype conferred to the plant by the endophyte.

53. A method for conferring a bioprotection phenotype to a plant or part thereof, said method including inoculating the plant or part thereof with an endophyte according to claim 36.

54. A method for selecting a bioprotective endophyte of a plant of the Cannabaceae family, said method comprising: a. substantially purifying or isolating one or more endophytes; and b. subjecting said one or more endophytes to one or more of: i. an in vitro bioprotection activity assay; ii. an in planta bioprotection activity assay; and iii. an analysis for a secondary metabolite biosynthesis gene cluster associated with the production of biocidal compounds, and based thereon, selecting an endophyte which is capable of conferring a bioprotection phenotype to the plant from which it is substantially purified or isolated and/or is capable of conferring a bioprotection phenotype to a plant or part thereof to which it is inoculated and wherein a selected endophyte is a Pseudomonas sp. endophyte.

55. A method according to claim 54, further including subjecting said endophyte colonies or selected endophyte(s) to genetic analysis to identify the endophyte species.

56. A method according to claim 54, wherein a selected endophyte is substantially purified or isolated from the roots a plant of the Cannabaceae family.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0068] FIG. 1 depicts the 16S amplicon sequence of novel bacterial strain EB-010 (SEQ ID NO: 1).

[0069] FIG. 2 depicts the 16S amplicon sequence of novel bacterial strain EB-117 (SEQ ID NO: 2).

[0070] FIG. 3 depicts the 16S amplicon sequence of novel bacterial strain EB-017 (SEQ ID NO: 3).

[0071] FIG. 4 depicts the 16S amplicon sequence of novel bacterial strain EB-013 (SEQ ID NO: 4).

[0072] FIG. 5 depicts a phylogenetic tree based on an Average Nucleotide Identity (ANI) analysis of novel bacterial strains EB-117, EB-017, EB-013 and EB-010 and genome sequences of P. corrugata isolates from NCBI.

[0073] FIG. 6 depicts a phylogenetic tree based on Pan-genome analysis (Roary) of novel bacterial strains EB-117, EB-017, EB-013 and EB-010 and genome sequences of P. corrugata isolates from NCBI.

[0074] FIG. 7 contains photographs of grapes used in in planta bioprotection assays and provides a pictorial representation of disease inhibition ratings of from 1 to 5 based on percent lesion size caused by Botrytis cinereal pathogen infection.

[0075] FIG. 8 is a microbiome profile presented as a bar graph plotting the distribution of novel bacterial strain EB-010 in a mature medicinal Cannabis plant (FFlower, FLFlower Bract, O-LOld Leaf, Y-LYoung Leaf, O-POld Petiole, Y-PYoung Petiole, STEMStem, R Root), established by mapping 16S metagenomic reads from these plant organs to the 16S sequence of EB-010.

DETAILED DESCRIPTION OF EMBODIMENTS

[0076] In the following examples it is demonstrated that four novel Pseudomonas corrugata bacterial strains EB-117, EB-017, EB-013, EB-010 were isolated from medicinal Cannabis (Cannabis sativa) plants. They each display the ability to inhibit the growth of plant fungal pathogens in in vitro and in planta assays. The genomes of the four novel bacterial strains have been sequenced and are shown to be related to Pseudomonas corrugata. Analysis of the genome sequence has shown that all four bacterial strains have secondary metabolite gene clusters with known bioprotectant compounds, while they had no cluster associated with any human health effects.

Example 1Isolation of Bacterial Strains

[0077] Leaves, petioles, stems, flowers and roots were harvested from four different chemotypes (lines) (CannBio 2, 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-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. Around 126 bacterial strains were obtained from mature plants grown in standard potting mix.

[0078] The novel bacterial strains EB-010, EB-013, EB-017 and EB-117 were collected from roots of medicinal Cannabis plants Cannbio 4, Cannbio 3, Cannbio 2 and Cannbio 5, respectively.

Example 2Identification of Novel Bacterial Strains

[0079] Proteomics

[0080] Bacterial isolates and the MALDI-TOF quality control Escherichia coli 25992 strain (from 80 C. storage) were streaked on to R2A agar plates and incubated at 22 C. for 48 hours in the dark. A single colony was picked from each culture and streaked on to a fresh R2A plate and incubated at 22 C. for an additional 48 hours in the dark.

[0081] The MALDI BioTyper (Bruker Daltonics) system was used in initial bacterial identification. A standard dried droplet method was followed for sample preparation for MALDI-TOF analysis of all bacterial cultures. To provide biological and analytical replicates, n=2 single colonies were spotted twice for each isolate and a total of three MALDI-TOF runs were performed with freshly prepared 48 hour-old cultures to provide technical replicates. Similarly, the Bruker bacterial test standard (BTS) was spotted (1 L aliquot each) as replicates for MALDI-TOF instrument calibration purposes.

[0082] Individual mass spectra profiles of all samples were acquired on a Bruker MALDI-TOF mass spectrophotometer with flexControl v 3.3 software using the standard MBT-AutoX method. The equipment was calibrated using the BTS before every sample analysis, which comprised of eight calibration points and covered a mass range of between 1960 to 20,000 Da. The generated spectra were compared to a reference library, as well as the standard database supplied by Bruker using standard software. Based on the analogy between observed and referenced data, a score value was produced. Isolates with a cut-off score value of 22.0 were identified to the species level and isolates with a cut-off score of 22.3 were identified to the species level with significant accuracy.

[0083] The cut-off score values and initial species identification for the four novel bacterial isolates are provided in Table 1.

TABLE-US-00001 TABLE 1 Identification of novel bacterial strains using MALDI-TOF MS Isolate ID Organism (Best Match) Score EB-010 Pseudomonas corrugata 2.379 EB-013 Pseudomonas corrugata 2.431 EB-017 Pseudomonas corrugata 2.473 EB-117 Pseudomonas chlororaphis 2.106

[0084] Genomics

[0085] The genomes of novel bacterial strains EB-117, EB-017, EB-013, EB-010 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). To enable full genome assembly, long reads were generated for the four 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 230 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 Mk1B 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 base-calling using ONT's Albacore software (Version 2.3.1) with default parameters. 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 the sequencing run, while Porechop (Version 0.2.3, https://github.com/rrwick/Porechop) was used to remove adapter sequences from the reads. Reads that were shorter than 300 bp were removed and the worst 5% of reads (based on quality) were discarded using Filtlong (Version 0.2.0, https://github.com/rrwick/Filtlong).

[0086] The whole genome sequence of the four novel bacterial strains were assembled using Unicycler (Wick et al. 2017). MinION reads were mainly used to resolve repeat regions in the genome. 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).

[0087] A complete circular chromosome sequence was produced for the four novel bacterial strains. The genome size for the novel bacterial strains EB-117, EB-017, EB-013, EB-010 were 6,624,132 bp, 6,657,479 bp, 6,539,460 bp and 6,487,017 bp, respectively (Table 2).

TABLE-US-00002 TABLE 2 Summary of properties of the final genome sequence assembly Genome Plasmid GC content Strain ID size (bp) Size (bp) (%) Coverage EB-117 6,624,132 135,326 60.1 385.0 EB-017 6,657,479 15,831 60.3 604.7 EB-013 6,539,460 60.3 604.9 EB-010 6,487,017 20,489 60.4 177.9

[0088] Novel bacterial strains EB-117, EB-017 and EB-010 also contained a plasmid, ranging in size from 15,831 bp to 135,326 bp. The percent GC content was around 60%. The 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. The number of genes for the novel bacterial strains EB-117, EB-017, EB-013, EB-010 were 6090, 7768, 7528, and 5829 genes, respectively (Table 3).

TABLE-US-00003 TABLE 3 Summary of genome coding regions No. of No. of No. of No. of Strain ID tRNA tmRNA rRNA No. of CDS genes EB-117 69 1 16 6004 6090 EB-017 72 1 17 7678 7768 EB-013 70 1 16 7441 7528 EB-010 70 1 16 5742 5829

[0089] A phylogenetic analysis of the novel bacterial strains EB-117, EB-017, EB-013, EB-010 was undertaken by sequence homology comparison of the 16S rRNA gene regions extracted from whole genome sequence of each bacteria (FIGS. 1-4). 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-117, EB-017, EB-013, EB-010 based on 16S sequences were Pseudomonas corrugata (Tables 4 and 5)

TABLE-US-00004 TABLE 4 BLASTn hit against database nr; Pseudomonas corrugata 16S ribosomal RNA gene, partial sequence Strain Query E- % ID Coverage Value Identity Species Accession EB-117 100% 0 100% Pseudomonas NR117826.1 corrugata EB-017 100% 0 99.79% Pseudomonas NR117826.1 corrugata EB-013 100% 0 99.59% Pseudomonas NR117826.1 corrugata EB-010 100% 0 100% Pseudomonas NR117826.1 corrugata

TABLE-US-00005 TABLE 5 BLASTn hit against database 16S ribosomal RNA; Pseudomonas corrugata strain BS3649 16S ribosomal RNA gene, genome assembly Strain Query E- % ID Coverage Value Identity Species Accession EB- 100% 0 99.96% Pseudomonas corrugata LT629798.1 117 EB- 100% 0 99.76% Pseudomonas corrugata LT629798.1 017 EB- 100% 0 99.72% Pseudomonas corrugata LT629798.1 013 EB- 100% 0 99.94% Pseudomonas corrugata LT629798.1 010

[0090] Six P. corrugata genome sequences and one P. chlororaphis genome sequence that were publicly available on NCBI were acquired and used for average nucleotide identity (ANI, no P. chlororaphis strain) calculation and pan-genome/comparative genome sequence analysis alongside P. corrugata novel bacterial strains EB-117, EB-017, EB-013 and EB-010. ANI values were calculated using the Pyani package and a phylogenetic tree was generated. (Pritchard L. 2016). In the phylogenetic tree EB-013 and EB-017 formed a clade, while EB-117 and GCF_000522485.1 formed a clade, and EB-010 and GCF_001708425.1 formed a clade (FIG. 5). The four novel bacterial strains EB-117, EB-017, EB-013 and EB-010 had >99% average nucleotide identity to each other and P. corrugata isolates. Based on an ANI species cut of 95-96%, it is evident that isolates EB-117, EB-017, EB-013 and EB-010 are representative isolates of P. corrugata.

[0091] Prokka (Seemann 2014) annotated novel bacterial genomes were provided to Roary (Page et al. 2015) and a total of 625 genes that are shared by all eleven strains were identified. 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 v1.4.4 (Rambaut A. 2018). The novel bacterial strains EB-117, EB-017, EB-013 and EB-010 clustered together with, P. corrugata reference isolates, and separate from the P. chlororaphis strain, confirming these novel bacterial strains belong to the species P. corrugata (FIG. 6).

Example 3Bioprotection Activity (In Vitro) of Novel Bacterial Strains

[0092] In vitro bioassays were established to test the bioactivity of the novel bacterial strains EB-010, EB-013, EB-017 and EB-117 against two major fungal pathogens of medicinal Cannabis Botrytis cinerea (VPRI 42964) and Fusarium proliferatum (VPRI 42958). An unrelated bacterial strain X (Paenibacillus pabuli) was used as a negative control. The two fungal pathogens were isolated from infected medicinal Cannabis plants obtained from the National Collection of Fungi (Herbarium VPRI) and the Agriculture Victoria Research collection. Bacterial isolates were tested for in vitro antagonism towards fungal pathogens by following the standard co-inoculation technique with three inoculation methods (dual culture one species streak, dual culture mix species streak and four spot drop-inoculation) on NA plates (BD Biosciences). For both the dual culture one species streak and dual culture mix species streak, a 6 mm6 mm agar plug of actively growing mycelia from the pathogen was placed at the centre of one side of the plate. Nutrient agar plates were incubated for 24-48 hours or until the fungi started establishing on the plate at 24 C. in the dark. Once the fungal culture established in NA plate each bacterial strain was freshly streaked on opposite side of the same plate in such a way that smear of bacterial colony completely cover the half of the plate, and the plate was then incubated at 26 C. for 7-14 days in the dark.

[0093] For the four-spot drop-inoculation method each bacterial strain was drop-inoculated (20 L) onto four equidistant points on a Nutrient Agar (BD Biosciences) plate, which was then incubated overnight at 28 C. A 6 mm6 mm agar plug of actively growing mycelia from the pathogen was placed at the centre of the plate. All the bioassays were incubated for 7-14 days at 28 C. in the dark, and then the diameter of the fungal colony on the plate was recorded. For each treatment three plates were prepared as biological triplicates. OriginPro 2018 (Version b9.5.1.195) was used to carry out One-way ANOVA and Tukey Test to detect the presence of any significant difference (p0.05) between treatments.

[0094] The four novel bacterial strains inhibited the growth of both pathogens, indicating that they have broad spectrum biocidal activity, unlike strain X. Novel bacterial strain EB-017 significantly inhibited the growth of Botrytis cinerea with the highest percentage inhibition (82.30%) in comparison to strain X (Paenibacillus pabuli) (58.33%). Novel bacterial strains EB-010, EB-013 and EB-117 also significantly inhibited the growth of Botrytis cinerea in comparison to strain X (Paenibacillus pabuli) (82.20%, 79.50% and 78.33%, respectively cf. 58.33% for control). Novel bacterial strain EB-010 significantly inhibited the growth of Fusarium proliferatum with highest percentage inhibition (53.5%) in comparison to strain X (Paenibacillus pabuli) (16.3%). Novel bacterial strains EB-013, EB-017 and EB-117 also significantly inhibited the growth of Fusarium proliferatum in comparison to strain X (Paenibacillus pabuli) (51.2%, 51.2% and 48.8%, respectively cf. 16.3% for control). These results are presented in Table 6 (superscript .sup.a represents statistical significance).

TABLE-US-00006 TABLE 6 Bioprotection bioassay indicating the percentage inhibition (versus the control) of the Pseudomonas sp. novel bacterial strains EB-010, EB-013 and EB-017 against plant pathogenic fungi, Botrytis cinerea and Fusarium proliferatum. Pathogen ID EB-010 EB-013 EB-017 EB-117 strain X Botrytis cinereal 82.20.sup.a 79.50.sup.a 82.30.sup.a 78.33.sup.a 58.33.sup.b (VPRI 42964) Fusarium proliferatum 53.5.sup.a 51.2.sup.a 51.2.sup.a 48.8.sup.a 16.3.sup.b (VPRI 42958)

Example 4Bioprotection Activity (in Planta) of Novel Bacterial StrainsGrapes

[0095] A table grape (Vitis vinifera) assay was established to evaluate the in planta bioprotection activity of novel bacterial strains EB-010, EB-013, EB-017 and EB-117 against the fungal phytopathogen Botrytis cinerea (VPRI 42964). The bacterial strains were cultured in nutrient broth (BD Bioscience) and incubated overnight at 28 C. in a shaking incubator (200 rpm). Next day, table grapes (ripe white seedless table grapes) were surface sterilised with 70% alcohol for 30 seconds, 1% sodium hypochlorite (NaOCl) for 1 minute, then rinsed with sterile distilled water (SDW) three times. Surface sterilised fruits were air dried inside the laminar floor or wiped with clean paper towels to remove the excess water.

[0096] Each bacterial strain was spray inoculated onto table grapes to coat the whole fruit. Six fruit were tested per bacterial isolate. Negative control fruit were coated with nutrient broth medium, and positive control fruit were left without inoculating the bacteria. The inoculated table grapes were kept on plastic trays elevated inside sealed moist incubation chambers, which consisted of moistened paper towelling lining the base of rectangular disposable food containers and incubated overnight at 26 C. in the dark.

[0097] To make conidial suspensions of the pathogenic fungus, 10 mL of SDW was added to 7-15 day-old cultures, the mycelia were scraped with a sterile glass rod and the suspension filtered through muslin cloth. The concentration of spore suspension was adjusted to 10.sup.6 conidia/mL. Bacteria-treated, overnight-incubated fruits were inoculated by adding 5 L of the spore suspension from each isolate onto the upper surface of the fruit; or a 6 mm6 mm agar plug of actively growing mycelia from the pathogen was placed at the centre of the fruit. All the fruits were inoculated with pathogen by both wound and non-wound methods. The wound method involved pricking the fruit surface with a sterilised needle after adding the spore suspension/mycelial plug. Control fruits were treated with 5 L of SDW and the experiment was carried out seven times.

[0098] A range of strategies were evaluated to optimise the bioactivity of bacteria, to optimise the pathogen infection, and simulate commercial application. [0099] Bioactive bacteria inoculation techniquespray, drop inoculation or coating (short/long duration) [0100] Single or mixed cultures of bioactive bacteria [0101] Pathogen inoculum sourcespores or mycelia on agar [0102] Woundingwith and without [0103] Order of inoculation (pathogenbioactive bacteria; bioactive bacteriapathogen) [0104] Condition of fruit (age/maturity) [0105] Replication

[0106] The bioprotection activity (inhibition against symptoms development) at the inoculation site were evaluated at 7-14 days after inoculation based on percent lesion size (the size of necrotic zones and the fungal hyphal growth). A disease inhibition rating was assigned to each from between the values of 0 to 5, where 1=strong disease inhibition, to 5=no disease inhibition (FIG. 7).

[0107] The results represent the mean disease inhibition score across all seven trials. Novel bacterial strains EB-010, EB-013 (individual) and the mix of EB-013, EB-017 and EB-117 (combinations of isolates) showed the highest in planta bioprotection activity against the fungal phytopathogen Botrytis cinerea (FIG. 7 and Table 7).

TABLE-US-00007 TABLE 7 Bioprotection bioassay indicating the mean inhibition scores of the novel bacterial strains EB-010, EB-013, EB-017 and EB- 117 against plant pathogenic fungi, Botrytis cinerea. Sample name Species Mean Activity rating EB-017 Pseudomonas corrugata 1.67 ** EB-117 Pseudomonas corrugata 2.17 * EB-025 Pseudomonas libanensis 2.8 * EB-010 Pseudomonas corrugata 0.83 *** EB-013 Pseudomonas corrugata 0.3 *** EB-117, EB-010, EB- Pseudomonas corrugata 2.17 * 013 (mixture) EB-117, EB-017, EB- Pseudomonas corrugata 1.5 ** 013 (mixture)

Example 5Genome Sequence Features Supporting the Bioprotection Niche of the Novel Bacterial Strains

[0108] Secondary Metabolite Biosynthesis Gene Clusters

[0109] The genome sequences of the four novel bacterial strains EB-010, EB-013, EB-017 and EB-117 were assessed for the presence of features associated with bioprotection. The annotated genome sequences were analysed by antiSMASH (Weber et al. 2015) to identify secondary metabolite biosynthesis gene clusters that are commonly associated with the production of biocidal compounds that aid in their defence. Annotated genome sequences were passed through antiSMASH with the following options: clusterblastasfknownclusterblastsubclusterblastsmcogsfull-hmmer. A total of three secondary metabolite gene clusters were identified in the genome sequences of all four novel bacterial strains (Table 8). A total of 11 biosynthetic gene clusters were identified, with novel bacterial strain EB-010 and EB-117 having 9 clusters, while EB-013 had 8 and EB-017 had 7. All isolates contained the cluster associated with syringomycin production, but had no cluster associated with any known human health effects.

TABLE-US-00008 TABLE 8 Secondary metabolite biosynthesis gene clusters in the novel bacterial strains identified using antiSMASH (Weber et al. 2015). Type Most similar known cluster EB-013 Similarity EB-010 Similarity EB-017 Similarity EB-117 Similarity 1 NRPS-like fragin NRP Y 37% Y 37% Y 60% 2 arylpolyene APE Vf Other Y 40% Y 45% Y 45% Y 45% 3 bacteriocin Y Y Y Y 4 NAGGN Y Y Y Y 5 betalactone fengycin NRP Y 13% Y 13% Y 20% Y 13% 6 NRPS cupriachelin NRP:NRP Y 11% siderophore 7 NRPS-like, T1PKS entolysin NRP Y 17% Y 17% 8 siderophore Y Y Y 12% Y 9 hserlactone, NRPS syringomycin NRP Y 100% Y 100% Y 100% Y 100% 10 NPRS pyoverdin NRP Y 1% 11 NRPS crochelin NRP + Polyketide Y 11% Y 7% 8 9 7 9

Example 6Distribution of Novel Bacterial Strains in Medicinal Cannabis Plants

[0110] 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 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). Gydle software suite (https://www.gydle.com/) was used for dereplication, taxonomical assignment and removal of organelle OTUs. Reads were mapped to the 16S sequence of EB-010 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 1000 for flowers, flower bracts, leaves (old and young), petioles (old and young) and stems, while numbers were higher in roots (up to 236,330) (FIG. 8). As such, the novel bacterial strains appear ubiquitously distributed across the medicinal Cannabis plant, but more concentrated in roots than other organs.

Example 7Bioprotection Activity (in Planta) of Novel Bacterial StrainsMedicinal Cannabis

[0111] A medicinal Cannabis (Cannabis sativa) detached leaf assay was established to evaluate the in planta bioprotection activity of novel bacterial strains EB-010, EB-013, EB-017 and EB-117 against the fungal phytopathogen Borytis cinerea (VPRI 42964). An additional isolate was also evaluated, Pseudomonas libanensis (EB-025). The bacterial strains were cultured in nutrient broth (BD Bioscience) and incubated overnight at 28 C. in a shaking incubator (200 rpm). The next day, young leaves harvested from medicinal Cannabis were surface sterilised with 70% ethanol for 30 seconds, 1% sodium hypochlorite (NaOCl) for 1 minute, then rinsed with sterile distilled water (SDW) three times. Surface sterilised leaves were air dried inside the laminar flow and wiped with sterile paper towels to remove the excess water.

[0112] Optical density (OD) measurements were taken for overnight cultures of each bacterial strain using a spectrophotometer (Eppendorf BioPhotometer D30), following which the OD600 value was adjusted to 1 by diluting with nutrient broth medium, if needed. Each bacterial strain was spray inoculated onto leaves to coat the whole leaf using a handheld atomiser. Three leaves were tested per bacterial isolate. Negative control leaves were coated with nutrient broth medium, and positive control leaves were untreated. The inoculated leaves were incubated overnight at 26 C. in the dark in a plastic box (used separate box per bacteria) with wet sterile paper towel, prior to the inoculation of the pathogen.

[0113] A conidial suspensions of the fungal pathogen B. cinerea was prepared by adding 10 mL of SDW to 15 day-old fungal cultures, following which mycelia were scraped from the culture with a sterile glass rod, and the suspension filtered through sterile muslin cloth. The concentration of the spore suspension was adjusted to 10.sup.6 conidia/mL. Leaves were inoculated by adding 5 L of the spore suspension from each isolate onto the upper surface of the leaf. All leaves were wounded as the pathogen was inoculated by pricking the leaf surface with a sterile needle and then adding the spore suspension. Negative control leaves were treated with 5 L of SDW.

[0114] The bioprotection activity (inhibition against fungal growth) was evaluated at 5 days after inoculation of the pathogen. An assessment of fungal growth was based on the size of necrotic zones and the aerial hyphal growth. A disease expression rating was assigned for each between the values of 1 to 5, where 1=no disease (no aerial hyphae, no necrosis), 2=minor disease (no aerial hyphae, necrosis), 3=moderate disease (moderate aerial hyphae, necrosis), 4=severe disease (severe aerial hyphae, necrosis) and 5=extensive disease (extensive aerial hyphae, necrosis). Statistical analysis was performed on disease inhibition rating using OriginPro. The four novel bacterial strains EB-010, EB-013, EB-017 and EB-117 significantly (p<0.05) reduced the growth of B. cinerea infection in detached Cannabis leaves, compared to the fungi only treatment (positive control) and P. libanensis (EB-025) (Table 9). Novel bacterial strains EB-013 was the most active at reducing the growth of B. cinerea, however all P. corrugata isolates were significantly equivalent.

TABLE-US-00009 TABLE 1 Bioprotection detached leaf bioassay indicating the mean disease expression rating of the novel bacterial strains EB-010, EB-013, EB-017 and EB-117 against plant pathogenic fungi, Botrytis cinerea. Disease Expression Sample name Species Rating Activity rating EB-017 Pseudomonas corrugata 1.2 *** EB-117 Pseudomonas corrugata 1.2 *** EB-010 Pseudomonas corrugata 1.2 *** EB-013 Pseudomonas corrugata 1.0 *** EB-025 Pseudomonas libanensis 3 * Fungi only NA 3.8 NA

[0115] 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.

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