AbstractRecent advances in the field of environmental DNA (eDNA) metabarcoding have produced a promising platform for early detection and broad‐spectrum monitoring of plant pests and pathogens. To date, the majority of fungal metabarcoding assays have relied upon universal primers amplifying segments of the ribosomal DNA, to ensure broad taxonomic coverage while studying changes in community composition, or more recently, to screen for presence/absence of target species. In a diagnostics framework however, single, universal molecular barcodes do not discriminate accurately enough for many groups of important fungal phytopathogens at the required species level. Here, a modular, multi‐barcode amplicon sequencing pipeline was established to provide rapid and reliable diagnostics targeting the Ophiostomatales, an order containing economically important phytopathogens vectored by bark and ambrosia beetles (Curculionidae). Using compositionally varied mock communities, we evaluated five barcoding loci: ITS1, ITS2, the large ribosomal sub‐unit (LSU), translation elongation factor 1‐alpha (TEF1α) and calmodulin (CAL), for their ability to provide species‐level resolution. The sensitivity and detection limit of the assay was established by spiking genomic DNA of the exotic Dutch elm disease pathogen, Ophiostoma novo‐ulmi Brasier, into both the controlled mock communities and eDNA sampled from Ips grandicollis (Eichhoff) beetles collected in New South Wales, Australia. The TEF1α barcode successfully detected the Dutch elm pathogen DNA with 100% accuracy down to approx. 5 pg/μl, while the ITS1 barcode had a 100% accuracy down to approx. 0.4 pg/μl within a given sample. Our results demonstrate that a dual‐barcode approach targeting the ITS1 and TEF1α regions simultaneously is sufficient for accurate species level detection and characterisation of the Ophiostomatales and offers confident detection of low‐abundance taxa (relative read abundances of 0.1%–0.01%). Multi‐barcode metabarcoding provides a framework which can quickly and efficiently be adapted to new targets when establishing high throughput diagnostics for post‐border biosecurity surveillance of fungal phytopathogens.

The bird cherry-oat aphid, Rhopalosiphum padi (L.), is an economically significant pest of pasture grasses, the latter being capable of hosting several fungal endophyte–perennial ryegrass symbiota rich in alkaloids and toxic to vertebrates and invertebrates. Measuring aphid feeding behaviour can provide insights into the effectiveness and mode of action of different fungal endophytes. This study investigated the effects of different Epichloë–perennial ryegrass symbiota on the feeding behaviour of R. padi using the electrical penetration graph technique while also assessing the aphid life history. In most cases, endophytes had significant feeding deterrence and paired fecundity and mortality effects. But, in some instances, endophytes with the highest aphid mortality did not significantly deter feeding, suggesting a more complicated scenario of interactions between the relative concentration of metabolites, e.g., host plant defence response metabolites and alkaloids, and/or physical changes to leaf morphology. Overall, this study sheds light on the mode of action of Epichloë endophytes against aphids and highlights the importance of Epichloë–perennial ryegrass symbiota in the management of insect pests such as aphids in pasture-based grazing systems.

Hop cyst nematode, Heterodera humuli Filipjev, 1934 was found in soil samples collected from hop farms in Bushy Park, Tasmania and in Merriang, Victoria, Australia. Morphological and molecular characteristics were consistent with those described for this species in other parts of the world. Novel gene sequences of ITS rRNA, 28S rRNA and CO1 mtDNA generated from cysts were compared against sequences of H. humuli available on the NCBI GenBank database, demonstrating little to no genetic variation between Australian hop cyst nematode populations and those from other regions. This report provides the first molecular sequence data for H. humuli from Australia and reports a significant range extension of this cyst nematode from Tasmania to the Australian mainland.

Post-border surveillance for forestry’s high priority pests and pathogens is conducted routinely through established programs focused on the main points-of-entry and across the major plantation growing regions. Currently, most diagnostic protocols used to identify fungal phytopathogens sampled during these surveys rely on traditional methods, such as morphological examination and DNA barcoding techniques. This stepwise process from isolation to species identification is often regarded as slow, expensive, and limited due to the need for disease manifestation and/or comprehensive expertise for rapid and accurate detection. In this study, we applied a recently validated high-throughput, dual-marker amplicon sequencing approach on insect surveillance traps from across Australia to assess its performance for the targeted surveillance of the Ophiostomatales , an order of fungi comprising notable phytopathogens which are vectored by bark beetles. By using a recently validated assay we were able to confidently characterize a range of Ophiostomatalean taxa known to be present in Australia, while reporting eight first detections from environmental DNA. Our study demonstrates the value of targeted multi-barcode amplicon sequencing for high-throughput screening of fungi caught in post-border surveillance traps, in addition to emphasizing research priorities that require further investigation before such methods can be implemented routinely for biosecurity.

Cyst nematodes of the genus Heterodera are a major group of sedentary plant parasites causing a significant economic impact, restricting production and market access globally (Moens et al. 2018). The ryegrass cyst nematode Heterodera mani is in the Avenae group and is found predominantly in pastures and grasslands in Europe, California, and South Africa. It was first described by Mathews (1971) from Northern Ireland. Known hosts are grasses (family Poaceae), principally Lolium perenne (perennial ryegrass), but also Dactylis glomerata (cat grass) and Festuca pratensis (meadow fescue) (Subbotin et al. 2010). Mowat (1974) reported that H. mani causes negligible damage to the yield of L. perenne in pot trials; however, Maas & Brinkman (1982) determined that it may cause significant damage to spring and autumn-sown perennial ryegrass in field conditions. During a routine examination for potato cyst nematode from a farm near Mawbanna in north-west Tasmania, Australia, several pale to dark brown Heterodera cysts were extracted that were lemon shaped with the presence of a small vulval cone at the posterior end and a distinct neck. The J2 (n=20) stylet length ranged from 24-26 µm with round knobs deeply concave anteriorly, hyaline tail length was 37-42 µm, true tail length ranged from 59-68 µm and total body length varied from 526-559 µm. All the above characters match those described for H. mani (Subbotin et al. 2010). To verify this identification, DNA was extracted from five individual J2 juveniles from a single cyst using QIAamp DNA micro kit (Qiagen®), and two gene regions amplified: internal transcribed spacer region of ribosomal RNA (ITS-rRNA) with primer pair AB28 and TW81 and cytochrome oxidase 1 (CO1) with primer pair JB3 and JB5 (Bowles et al. 1992; Curran et al. 1994; Derycke et al. 2005). One PCR reaction contained 10 µM (1 µl each) of each primer, 12.5 µl of OneTaq® DNA Polymerase and 5 µl of DNA template with a final volume of 25 µl. PCR products were sent for purification and Sanger sequencing at Macrogen (Seoul, Rep. of Korea). All resulting sequences were trimmed, aligned, and analysed using Geneious Prime® 2022.0.1 (www.geneious.com). Five ITS sequences (accessions ON402852-ON402856) and five CO1 sequences (accessions ON402857-ON402861) were submitted to GenBank. These ITS sequences were very similar to each other and exhibited 99.16-100% similarity with that of H. mani isolate from Hamminkeln, Germany (AY148377) (Subbotin et al. 2018). The CO1 sequences exhibited 98.96-100% similarity with that of H. mani isolate from Washington, USA (MG523097) (Subbotin et al. 2003). Obtained sequences were mapped to reference sequences downloaded from NCBI GenBank and maximum likelihood phylogenetic trees were calculated. Due to the lack of further living nematode material, pot experiments were not performed. Such experiments are not feasible in Tasmania currently and transfer of live nematode material to the Australian mainland presents logistic and legal issues. However, morphological and molecular evidence for species determination of H. mani was unequivocal and contributes to the list of cyst nematode species present in Australia. This is the first detection of H. mani in Australia and is a range extension of the species from North America, Africa, and Europe to Australia. The nematode may cause damage to perennial ryegrass in Australia, however, impact on yield still needs to be investigated.

Fusarium oxysporum (Fo) is ubiquitous in soil and forms a species complex of pathogenic and putatively non-pathogenic strains. Pathogenic strains cause disease in over 150 plant species. Fusarium oxysporum f. sp. ciceris (Foc) is a major fungal pathogen causing Fusarium wilt in chickpeas (Cicer arietinum). In some countries such as Australia, Foc is a high-priority pest of biosecurity concern. Specific, sensitive, robust and rapid diagnostic assays are essential for effective disease management on the farm and serve as an effective biosecurity control measure. We developed and validated a novel and highly specific PCR and a LAMP assay for detecting the Indian Foc race 1 based on a putative effector gene uniquely present in its genome. These assays were assessed against 39 Fo formae speciales and found to be specific, only amplifying the target species, in a portable real-time fluorometer (Genie III) and qPCR machine in under 13 min with an anneal derivative temperature ranging from 87.7 to 88.3 °C. The LAMP assay is sensitive to low levels of target DNA (> 0.009 ng/µl). The expected PCR product size is 143 bp. The LAMP assay developed in this study was simple, fast, sensitive and specific and could be explored for other Foc races due to the uniqueness of this marker to the Foc genome.

Research into understanding the structure, composition and vertical transmission of crop seed microbiomes has intensified, although there is much less research into the seed microbiomes of crop wild relatives. Our previous study showed that the standard seed storage procedures (e.g., seed drying and storage temperature) can influence the seed microbiome of domesticated Glycine max. In this study, we characterized the seed microbiota of Glycine clandestina, a perennial wild relative of soybean (G. max (L.) Merr.) to expand our understanding about the effect of other storage procedures such as the periodic regeneration of seed stocks to bulk up seed numbers and secure viability on the seed microbiome of said seed. The G. clandestina microbiota was analysed from Generation 1 (G1) and Generation 2 (G2) seed and from mature plant organs grown in two different soil treatments T (treatment [native soil + potting mix]) and C (control [potting mix only]). Our dataset showed that soil microbiota had a strong influence on next generation seed microbiota, with an increased contribution of root microbiota by 90% and seed transmissibility by 36.3% in G2 (T) seed. Interestingly, the G2 seed microbiota primarily consisted of an initially low abundance of taxa present in G1 seed. Overall, our results indicate that seed regeneration can affect the seed microbiome composition and using native soil from the location of the source plant can enhance the conservation of the native seed microbiota.

Global seed vaults are important, as they conserve plant genetic resources for future breeding to improve crop yield and quality and to overcome biotic and abiotic stresses. However, little is known about the impact of standard storage procedures, such as seed drying and cold storage on the seed bacterial community, and the ability to recover seed-associated bacteria after storage. In this study, soybean [Glycine max (L.) Merr.] seeds were analyzed to characterize changes in the bacterial community composition and culturability under varying storage conditions. The G. max bacterial microbiome was analyzed from undried seed, dried seed, and seed stored for 0, 3, 6, and 14months. Storage temperatures consisted of −20°C, 4°C, and room temperature (RT), with −20°C being commonly used in seed storage vaults globally. The seed microbiome of G. max was dominated by Gammaproteobacteria under all conditions. Undried seed was dominated by Pantoea (33.9%) and Pseudomonas (51.1%); however, following drying, the abundance of Pseudomonas declined significantly (0.9%), Pantoea increased significantly (73.6%), and four genera previously identified including Pajaroellobacter, Nesterenkonia, env.OPS_17, and Acidibacter were undetectable. Subsequent storage at RT, 4, or −20°C maintained high-abundance Genera at the majority of time points, although RT caused greater fluctuations in abundances. For many of the low-abundance Genera, storage at −20°C resulted in their gradual disappearance, whereas storage at 4°C or RT resulted in their more rapid disappearance. The changes in seed bacterial composition were reflected by cultured bacterial taxa obtained from the stored G. max seed. The main taxa were largely culturable and had similar relative abundance, while many, but not all, of the low-abundance taxa were also culturable. Overall, these results indicate that the initial seed drying affects the seed bacterial composition, suggesting that microbial isolation prior to seed drying is recommended to conserve these microbes. The standard seed storage condition of −20°C is most suitable for conservation of the bacterial seed microbiome, as this storage temperature slows down the loss of seed bacterial diversity over longer time periods, particularly low-abundance taxa.

Occurrence of cyst nematode Heterodera daverti Wouts and Sturhan 1978 (Wouts & Sturhan 1978) is reported from Australia for the first time. Cysts were recovered from soil samples collected from a hop farm in Merriang, Victoria. Morphological and morphometric characteristics of cysts and second stage juveniles match those described for this species reported from other parts of the world. Identification was further supported by molecular evidence through comparison of newly generated sequences of ITS rRNA, CO1 mtDNA and 28S rRNA gene regions with sequences previously available on NCBI GenBank.

Background Plant microbiome composition has been demonstrated to change during the domestication of wild plants and it is suggested that this has resulted in loss of plant beneficial microbes. Recently, the seed microbiome of native plants was demonstrated to harbour a more diverse microbiota and shared a common core microbiome with modern cultivars. In this study the composition of the seed-associated bacteria of Glycine clandestina is compared to seed-associated bacteria of Glycine max (soybean). Results The seed microbiome of the native legume Glycine clandestina (crop wild relative; cwr) was more diverse than that of the domesticated Glycine max and was dominated by the bacterial class Gammaproteobacteria. Both the plant species (cwr vs domesticated) and individual seed accessions were identified as the main driver for this diversity and composition of the microbiota of all Glycine seed lots, with the effect of factor "plant species" exceeded that of "geographical location". A core microbiome was identified between the two Glycine species. A high percentage of the Glycine microbiome was unculturable [G. clandestina (80.8%) and G. max (75.5%)] with only bacteria of a high relative abundance being culturable under the conditions of this study. Conclusion Our results provided novel insights into the structure and diversity of the native Glycine clandestina seed microbiome and how it compares to that of the domesticated crop Glycine max. Beyond that, it also increased our knowledge of the key microbial taxa associated with the core Glycine spp. microbiome, both wild and domesticated. The investigation of this commonality and diversity is a valuable and essential tool in understanding the use of native Glycine spp. for the discovery of new microbes that would be of benefit to domesticated Glycine max cultivars or any other economically important crops. This study has isolated microbes from a crop wild relative that are now available for testing in G. max for beneficial phenotypes.

Research into the bacterial component of the seed microbiome has been intensifying, with the aim of understanding its structure and potential for exploitation. We previously studied the intergenerational seed microbiome of one cultivar of perennial ryegrass with and without one strain of the commercially deployed fungal endophyte Epichloë festucae var. lolii. The work described here expands on our previous study by exploring the bacterial seed microbiome of different commercial cultivar/Epichloë festucae var. lolii combinations in collections of single seeds from the harvest year 2016. In this dataset, a cultivar effect could be seen between the seed microbiomes from cultivars Alto and Trojan. The bacterial component of the seed microbiome from pooled seeds from a single cultivar/E. festucae var. lolii combination harvested from 13 seed production farms around Canterbury in the year 2018 was also studied. This dataset allows the effect of different production locations on the bacterial seed microbiome to be examined. By comparing the two sets of data, bacteria from the genera Pantoea, Pseudomonas, Duganella, Massilia, and an unknown Enterobacteriaceae were observed to be in common. This core bacterial microbiome was stable over time but could be affected by supplemental taxa derived from the growth environment of the parental plant; differing microbiomes were seen between different seed production farms. By comparison to a collection of bacterial isolates, we demonstrated that many of the members of the core microbiome were culturable. This allows for the possibility of exploiting these microbes in the future.

Climate change is predicted to increase the incidence and severity of drought conditions, posing a significant challenge for agriculture globally. Plant microbiomes have been demonstrated to aid crop species in the mitigation of drought stress. The study investigated the differences between the seed microbiomes of drought tolerant and drought susceptible wheat lines. Furthermore, it highlighted and quantified the degree of drought tolerance conferred by specific microbes isolated from drought tolerant wheat seed microbiomes. Metagenomic and culture-based methods were used to profile and characterise the seed microbiome composition of drought tolerant and drought susceptible wheat lines under rainfed and drought conditions. Isolates from certain genera were enriched by drought tolerant wheat lines when placed under drought stress. Wheat inoculated with isolates from these targeted genera, such as Curtobacterium flaccumfaciens ( Cf D3-25) and Arthrobacter sp. ( Ar sp. D4-14) demonstrated the ability to promote growth under drought conditions. This study indicates seed microbiomes from genetically distinct wheat lines enrich for beneficial bacteria in ways that are both line-specific and responsive to environmental stress. As such, seed from stress-phenotyped lines represent an invaluable resource for the identification of beneficial microbes with plant growth promoting activity that could improve commercial crop production.

Asexual Epichloë fungi are strictly seed-transmitted endophytic symbionts of cool-season grasses and spend their entire life cycle within the host plant. Endophyte infection can confer protective benefits to its host through the production of bioprotective compounds. Inversely, plants provide nourishment and shelter to the resident endophyte in return. Current understanding of the changes in global gene expression of asexual Epichloë endophytes during the early stages of host-endophyte symbiotum is limited. A time-course study using a deep RNA-sequencing approach was performed at six stages of germination, using seeds infected with one of three endophyte strains belonging to different representative taxa. Analysis of the most abundantly expressed endophyte genes identified that most were predicted to have a role in stress and defence responses. The number of differentially expressed genes observed at early time points was greater than those detected at later time points, suggesting an active transcriptional reprogramming of endophytes at the onset of seed germination. Gene ontology enrichment analysis revealed dynamic changes in global gene expression consistent with the developmental processes of symbiotic relationships. Expression of pathway genes for biosynthesis of key secondary metabolites was studied comprehensively and fuzzy clustering identified some unique expression patterns. Furthermore, comparisons of the transcriptomes from three endophyte strains in planta identified genes unique to each strain, including genes predicted to be associated with secondary metabolism. Findings from this study highlight the importance of better understanding the unique properties of individual endophyte strains and will serve as an excellent resource for future studies of host-endophyte interactions.

A strain of Podosphaera clandestina has been highlighted as a priority pest threat to the Australian cherry industry. Australia currently has no records of powdery mildew on cherry (Prunus avium). P. clandestina is reported to cause disease on a range of Rosaceae genera including Crataegus and Prunus; in Australia, P. clandestina has only been recorded on Crataegus. A recent species revision identified Podosphaera cerasi on P. avium as a separate species from P. clandestina. Therefore, a revision of which powdery mildew species is present in Australia on Crataegus is required to inform Australian plant biosecurity. Reference collection specimens from the Victorian Plant Pathology Herbarium (VPRI) recorded as Podosphaera spp. collected between 1889 to 2008 on cherry and three other host plant genera from Australia and overseas were sampled for DNA extraction and next-generation sequencing (NGS). Sequence data from preserved specimens were successfully mapped to internal transcribed spacer (ITS) sequences of P. clandestina in the strict sense, P. cerasi, and Podosphaera prunicola, and chloroplast matK sequences were used to identify plant hosts. Australian specimens on Crataegus hosts were P. clandestina in the strict sense and specimens on Prunus from the USA were identified as P. cerasi and P. prunicola. The outcome of this study confirmed the powdery mildew on Australian Crataegus specimens to be P. clandestina and none of the cherry powdery mildews (Podosphaera pruni-avium, P. cerasi, or P. prunicola) are present on Australian specimens in the VPRI collection, which suggests they are not present in Australia.

Plant growth-promoting bacteria can improve host plant traits including nutrient uptake and metabolism and tolerance to biotic and abiotic stresses. Understanding the molecular basis of plant–bacteria interactions using dual RNA-seq analyses provides key knowledge of both host and bacteria simultaneously, leading to future enhancements of beneficial interactions. In this study, dual RNA-seq analyses were performed to provide insights into the early-stage interactions between barley seedlings and three novel bacterial strains (two Paenibacillus sp. strains and one Erwinia gerundensis strain) isolated from the perennial ryegrass seed microbiome. Differentially expressed bacterial and barley genes/transcripts involved in plant–bacteria interactions were identified, with varying species- and strain-specific responses. Overall, transcriptome profiles suggested that all three strains improved stress response, signal transduction, and nutrient uptake and metabolism of barley seedlings. Results also suggested potential improvements in seedling root growth via repressing ethylene biosynthesis in roots. Bacterial secondary metabolite gene clusters producing compounds that are potentially associated with interactions with the barley endophytic microbiome and associated with stress tolerance of plants under nutrient limiting conditions were also identified. The results of this study provided the molecular basis of plant growth-promoting activities of three novel bacterial strains in barley, laid a solid foundation for the future development of these three bacterial strains as biofertilisers, and identified key differences between bacterial strains of the same species in their responses to plants.

The Podosphaera tridactyla species complex is highly variable morphologically and causes powdery mildew on a wide range of Prunus species, including stone fruit. A taxonomic revision of the Po. tridactyla species complex in 2020 identified 12 species, seven of which were newly characterised. In order to clarify which species of this complex are present in Australia, next generation sequencing was used to isolate the fungal ITS+28S and host matK chloroplast gene regions from 56 powdery mildew specimens of stone fruit and ornamental Prunus species accessioned as Po. tridactyla or Oidium sp. in Australian reference collections. The specimens were collected in Australia, Switzerland, Italy and Korea and were collected from 1953 to 2018. Host species were confirmed using matK phylogenetic analysis, which identified that four had been misidentified as Prunus but were actually Malusprunifolia. Podosphaera species were identified using ITS+28S phylogenetic analysis, recognising three Podosphaera species on stone fruit and related ornamental Prunus hosts in Australia. These were Po.pannosa, the rose powdery mildew, and two species in the Po. tridactyla species complex: Po. ampla, which was the predominant species, and a previously unidentified species from peach, which we describe here as Po. cunningtonii.

Background The fungal pathogen Fusarium oxysporum f.sp. pisi (Fop) causes Fusarium wilt in peas. There are four races globally: 1, 2, 5 and 6 and all of these races are present in Australia. Molecular infection mechanisms have been studied in a few other F. oxysporum formae speciales; however, there has been no transcriptomic Fop-pea pathosystem study. Results A transcriptomic study was carried out to understand the molecular pathogenicity differences between the races. Transcriptome analysis at 20 days post-inoculation revealed differences in the differentially expressed genes (DEGs) in the Fop races potentially involved in fungal pathogenicity variations. Most of the DEGs in all the races were engaged in transportation, metabolism, oxidation-reduction, translation, biosynthetic processes, signal transduction, proteolysis, among others. Race 5 expressed the most virulence-associated genes. Most genes encoding for plant cell wall degrading enzymes, CAZymes and effector-like proteins were expressed in race 2. Race 6 expressed the least number of genes at this time point. Conclusion Fop races deploy various factors and complex strategies to mitigate host defences to facilitate colonisation. This investigation provides an overview of the putative pathogenicity genes in different Fop races during the necrotrophic stage of infection. These genes need to be functionally characterised to confirm their pathogenicity/virulence roles and the race-specific genes can be further explored for molecular characterisation.

The ophiostomatoid fungi are an assemblage of ascomycetes which are arguably best-known for their associations with bark and ambrosia beetles ( Curculonidae ) and blue stain (sap stain) of many economically important tree species. These fungi are considered a significant threat to coniferous forests, which has resulted in numerous studies characterising the diversity of bark beetles and their ophiostomatoid associates globally. The diversity of ophiostomatoid fungi present in Australian pine plantations, however, remains largely undetermined. The aims of this study were therefore to reconsider the diversity of ophiostomatoid fungi associated with Pinus in Australia, and to establish the baseline of expected taxa found within these plantation ecosystems. To achieve this, we reviewed Australian plant pathogen reference collections, and analysed samples collected during forest health surveillance programs from the major pine growing regions in south-eastern Australia. In total, 135 ophiostomatoid isolates (15 from reference collections and 120 collected during the current study) were assessed using morphological identification and ITS screening which putatively distinguished 15 taxonomic groups. Whole genome sequencing (WGS) of representative isolates from each taxon was performed to obtain high-quality sequence data for multi-locus phylogenetic analysis. Our results revealed a greater than expected diversity, expanding the status of ophiostomatoid fungi associated with Pinus in Australia to include 14 species from six genera in the Ophiostomatales and a single species residing in the Microascales . While most of these were already known to science, our study includes seven first records for Australia and the description of one new species, Graphilbum ipis-grandicollis sp. nov .. This study also provides an early example of whole genome sequencing (WGS) approaches replacing traditional PCR-based methods for taxonomic surveys. This not only allowed for robust multi-locus sequence extraction during taxonomic assessment, but also permitted the rapid establishment of a curated genomic database for ophiostomatoid fungi which will continue to aid in the development of improved diagnostic resources and capabilities for Australian biosecurity.

Paenibacillus species are Gram-positive bacteria that have been isolated from a diverse array of plant species and soils, with some species exhibiting plant growth-promoting (PGP) activities. Here we report two strains (S02 and S25) of a novel Paenibacillus sp. that were isolated from perennial ryegrass ( Lolium perenne ) seeds. Comparative genomics analyses showed this novel species was closely related to P. polymyxa . Genomic analyses revealed that strains S02 and S25 possess PGP genes associated with biological nitrogen fixation, phosphate solubilisation and assimilation, as well as auxin production and transportation. Moreover, secondary metabolite gene cluster analyses identified 13 clusters that are shared by both strains and three clusters unique to S25. In vitro assays demonstrated strong bioprotection activity against phytopathogens ( Colletotrichum graminicola and Fusarium verticillioides ), particularly for strain S02. A transcriptomics analysis evaluating nitrogen fixation activity showed both strains carry an expressed nif operon, but strain S02 was more active than strain S25 in nitrogen-free media. Another transcriptomics analysis evaluating the interaction of strains with F. verticillioides showed strain S02 had increased expression of core genes of secondary metabolite clusters (fusaricidin, paenilan, tridecaptin and polymyxin) when F. verticillioides was present and absent, compared to S25. Such bioactivities make strain S02 a promising candidate to be developed as a combined biofertiliser/bioprotectant.

Paenibacillus species are Gram-positive bacteria that have been isolated from a diverse array of plant species and soils, with some species exhibiting plant growth-promoting (PGP) activities. Here we report a novel Paenibacillus sp. (strains S02 and S25) that was isolated from perennial ryegrass ( Lolium perenne ) seeds. Comparative genomics analyses showed this novel species was closely related to P. polymyxa . Genomic analyses revealed that strains S02 and S25 possess PGP genes associated with biological nitrogen fixation, phosphate solubilisation and assimilation, as well as auxin production and transportation. Moreover, secondary metabolite gene cluster analyses identified 13 clusters that are shared by both strains and three clusters unique to S25. In vitro assays demonstrated strong bioprotection activity against phytopathogens ( Colletotrichum graminicola and Fusarium verticillioides ), particularly for strain S02. A transcriptomics analysis evaluating nitrogen fixation activity showed both strains carry an expressed nif operon, but strain S02 was more active than strain S25 in nitrogen-free media. Another transcriptomics analysis evaluating the interaction of strains with F. verticillioides showed strain S02 had increased expression of core genes of secondary metabolite clusters (fusaricidin, paenilan, tridecaptin and polymyxin) when F. verticillioides was present and absent, compared to S25. Such bioactivities make strain S02 a promising candidate to be developed as a combined biofertiliser/bioprotectant.