Customized plant microbiome engineering for food security.

Plant microbiomes play a vital role in promoting plant growth and resilience to cope with environmental stresses. Plant microbiome engineering holds significant promise to increase crop yields, but there is uncertainty about how this can best be achieved. We propose a step-by-step approach involving customized direct and indirect methods to condition soils and to match plants and microbiomes. Although three approaches, namely the development of (i) 'plant- and microbe-friendly' soils, (ii) 'microbe-friendly' plants, and (iii) 'plant-friendly' microbiomes, have been successfully tested in isolation, we propose that the combination of all three may lead to a step-change towards higher and more stable crop yields. This review aims to provide knowledge, future directions, and practical guidance to achieve this goal via customized plant microbiome engineering.

Rhizosphere bacteria induce programmed cell death defence genes and signalling in chilli pepper.

AIM: To understand how beneficial bacteria assist chilli plants (Capsicum annuum) in defence against biotrophic or hemibiotrophic pathogens. METHOD AND RESULTS: We quantified marker genes of plant defence pathways in Phytophthora capsici-infected chilli pepper treated with anti-oomycete plant growth-promoting rhizobacteria, Bacillus amyloliquefaciens, Bacillus velezensis and Acinetobacter sp. Plants displayed strong resistance, and the pathogen load in the roots was significantly lower in infected plants treated with bacterial biocontrol agents at all time points tested (1, 2 and 7 days after pathogen inoculation, p < 0.05). Gene expression profiling revealed that P. capsici infection in the absence of beneficial bacteria led to the upregulation of a wide array of defence genes. The addition of biocontrol bacteria modulated defence by further enhancing genes involved in programmed cell death, such as CaLOX1, CaPAL1, CaChitIV and CaPTI1, while suppressing others CaLRR1, a negative regulator of cell death. CONCLUSIONS: Our results suggest that the bacteria exerted a combined effect by directly antagonizing the pathogen and enhancing the expression of key plant defence genes, including those involved in cell death, causing resistance at early stages of infection by this hemibiotrophic pathogen.

Transmission of Blood Disease in Banana.

Banana Blood disease is a bacterial wilt caused by Ralstonia syzygii subsp. celebesensis and is an economically important disease in Indonesia and Malaysia. Transmission of this pathogen is hypothesized to occur through insects mechanically transferring bacteria from diseased to healthy banana inflorescences and other pathways involving pruning tools, water movement, and root-to-root contact. This study demonstrates that the ooze from the infected male bell and the sap from various symptomatic plant parts are infective, and the cut surfaces of a bunch peduncle, petiole, corm, pseudostem, and the rachis act as infection courts for R. syzygii subsp. celebesensis. In addition, evidence is provided that R. syzygii subsp. celebesensis is highly tool transmissible, that the bacterium can be transferred from the roots of a diseased plant to the roots of a healthy plant and transferred from the mother plant to the sucker. We provide evidence that local dispersal of Blood disease occurs predominantly through mechanical transmission by insects, birds, bats, or human activities from diseased to healthy banana plants and that long-distance dispersal occurs through the movement of contaminated planting material. Disease management strategies to prevent crop losses associated with this emerging disease are discussed based on our findings.

Simple solution to preserve plant samples for microbiome analyses.

Culture-independent survey techniques are fundamental tools when assessing plant microbiomes. These methods rely on DNA that is carefully preserved after collecting samples to achieve meaningful results. Immediately freezing samples to -80°C after collection is considered one of the most robust methods for preserving samples before DNA extraction but is often impractical. Preservation solutions can solve this problem, but commercially available products are expensive, and there is limited data comparing their efficacy with other preservation methods. In this study, we compared the impact of three methods of sample preservation on plant microbiome surveys: (1) RNAlater, a proprietary preservative, (2) a home-made salt-saturated dimethyl sulphoxide preservation solution (DESS), and (3) freezing at -80°C. DESS-preserved samples, stored at room temperature for up to four weeks, did not show any significant differences to samples frozen at -80°C, while RNAlater inflated bacterial alpha diversity. Preservation treatments did not distinctively influence fungal alpha diversity. Our results demonstrate that DESS is a versatile and inexpensive preservative of DNA in plant material for diversity analyses of fungi and bacteria.

Diagnostics of Banana Blood Disease.

Blood disease in bananas caused by Ralstonia syzygii subsp. celebesensis is a bacterial wilt disease that causes major yield losses of banana in Indonesia and peninsular Malaysia. The disease has significantly increased its geographic distribution in the past decade. Diagnostic methods are an important component of disease management in vegetatively propagated crops such as banana to constrain incursions of plant pathogens. Therefore, the objectives of this study were (i) to design and rigorously validate a novel banana Blood disease (BBD) real-time PCR assay with a high level of specificity and sensitivity of detection and (ii) to validate published PCR-based diagnostic methods targeting the intergenic region in the megaplasmid ("121 assay" with primer set 121) or the phage tail protein-coding sequence in the bacterial chromosome ("Kubota assay" and "BDB2400 assay" with primer set BDB2400). Assay validation included 339 samples (174 Blood disease bacteria, 51 bacteria associated with banana plants, 51 members of the Ralstonia solanacearum species complex, and 63 samples from symptomatic and healthy plant material). Validation parameters were analytical specificity (inclusivity and exclusivity), selectivity, limit of detection, accuracy, and ruggedness. The 121 assay and our newly developed BBD real-time PCR assay detected all R. syzygii subsp. celebesensis strains with no cross-specificity during validation. Two different PCR assays using the primer set BDB2400 lacked specificity and selectivity. This study reveals that our novel BBD real-time PCR assay and the conventional PCR 121 assay are reliable methods for Blood disease diagnostics, as they comply with all tested validation parameters.

Susceptibility of the Banana Inflorescence to Blood Disease.

The bacterium Ralstonia syzygii subsp. celebesensis causes Blood disease of banana, a vascular wilt of economic significance in Indonesia and Malaysia. Blood disease has expanded its geographic range in the last 20 years and is an emerging threat to Southeast Asian banana production. Many aspects of the disease cycle and biology are not well understood, including the ability of different parts of the female and male inflorescence of banana to act as infection courts. This study confirms that the banana varieties of Cavendish, and Kepok 'Kuning' are susceptible to Blood disease and that an inoculum concentration of 102 CFU/ml of R. syzygii subsp. celebesensis is adequate to initiate disease after pseudostem inoculation. Data show that infection occurs through both the male and female parts of a banana inflorescence and the rachis when snapped to remove the male bell. The infection courts are the female flowers, the male bell bract scar, the male bell flower cushion, the snapped rachis, and deflowered fingers. The location of these infection courts concurs with the dye studies demonstrating that dye externally applied to these plants parts enters the plant vascular system. Thus, the hypothesis is supported that infection of R. syzygii subsp. celebesensis occurs through open xylem vessels of the male and female parts of the banana inflorescence.

Geographic Expansion of Banana Blood Disease in Southeast Asia.

Blood disease in bananas caused by Ralstonia syzygii subsp. celebesensis is a bacterial wilt causing significant crop losses in Indonesia and Malaysia. Disease symptoms include wilting of the plant and red-brown vascular staining, internal rot, and discoloration of green banana fruit. There is no known varietal resistance to this disease in the Musa genus, although variation in susceptibility has been observed, with the popular Indonesian cooking banana variety Kepok being highly susceptible. This study established the current geographic distribution of Blood disease in Indonesia and confirmed the pathogenicity of isolates by Koch's postulates. The long-distance distribution of the disease followed an arbitrary pattern indicative of human-assisted movement of infected banana materials. In contrast, local or short-distance spread radiated from a single infection source, indicative of dispersal by insects and possibly contaminated tools, water, or soil. The rapid expansion of its geographical range makes Blood disease an emerging threat to banana production in Southeast Asia and beyond.

Plant-produced bacteriocins inhibit plant pathogens and confer disease resistance in tomato.

Bacteriocins are a diverse group of bacterial antimicrobial peptides (AMPs) that represent potential replacements for current antibiotics due to their novel modes of action. At present, production costs are a key constraint to the use of bacteriocins and other AMPs. Here, we report the production of bacteriocins in planta - a potentially scalable and cost-effective approach for AMP production. Nine bacteriocin genes with three different modes of action and minimal or no post-translational modifications were synthesized, cloned and used to transform Arabidopsis thaliana. To confirm bacteriocin functionality and the potential to use these plants as biofactories, Arabidopsis T3 crude leaf extracts were subjected to inhibition assays against the bacterial pathogens Clavibacter michiganensis subsp. michiganensis (Cmm) and Pseudomonas syringae pv. tomato DC3000 (Pst). Six and seven of nine extracts significantly inhibited Cmm and Pst, respectively. Three bacteriocin genes (plantaricin, enteriocin, and leucocin) were then selected for over-expression in tomato (Solanum lycopersicum). In vitro plant pathogen inhibition assays of T0, T1 and T2 transgenic tomato leaf extracts confirmed antimicrobial activity against both pathogens for all three generations of plants, indicating their potential use as stable biopesticide biofactories. Plantaricin and leucocin-expressing T2 tomato plants were resistant to Cmm, and leucocin-expressing T2 plants were resistant to Pst. This study highlights that plants can be used as biofactories for AMP production and that the expression of bacteriocins in planta may offer new opportunities for disease control in agriculture.

Sample Preparation for Culture-Independent Profiling and Isolation of Phyllosphere Bacteria to Identify Potential Biopesticides.

Studying the plant phyllosphere to understand inhibition patterns to the growth of fungal foliar pathogens by using the Arabidopsis thaliana pathosystem offers unique opportunities for evaluating strategies for plant protection against foliar diseases. The wide array of bacteria inhabiting the phylloplane of plants has been researched to a much lesser extent compared to the bacteria in the rhizosphere. This difference is especially evident as bacteria derived from the aerial section of plants are rarely used in formulations of foliage sprays against pathogens and pests. In this chapter we outline easy and reliable methods for sample preparation to profile phyllosphere bacteria using high throughput amplicon sequencing and isolate/characterize potentially beneficial phyllosphere bacteria from Arabidopsis thaliana that inhibit in vitro the growth of foliar pathogens such as Alternaria brassicicola. The use of the described methods for profiling and screening phyllosphere bacteria may provide tangible progress on the discovery of new potential biological control agents against agriculturally important pathogens.

Evidence for the plant recruitment of beneficial microbes to suppress soil-borne pathogens.

An emerging experimental framework suggests that plants under biotic stress may actively seek help from soil microbes, but empirical evidence underlying such a 'cry for help' strategy is limited. We used integrated microbial community profiling, pathogen and plant transcriptive gene quantification and culture-based methods to systematically investigate a three-way interaction between the wheat plant, wheat-associated microbiomes and Fusarium pseudograminearum (Fp). A clear enrichment of a dominant bacterium, Stenotrophomonas rhizophila (SR80), was observed in both the rhizosphere and root endosphere of Fp-infected wheat. SR80 reached 3.7 × 107 cells g-1 in the rhizosphere and accounted for up to 11.4% of the microbes in the root endosphere. Its abundance had a positive linear correlation with the pathogen load at base stems and expression of multiple defence genes in top leaves. Upon re-introduction in soils, SR80 enhanced plant growth, both the below-ground and above-ground, and induced strong disease resistance by boosting plant defence in the above-ground plant parts, but only when the pathogen was present. Together, the bacterium SR80 seems to have acted as an early warning system for plant defence. This work provides novel evidence for the potential protection of plants against pathogens by an enriched beneficial microbe via modulation of the plant immune system.

Draft Genome of the Macadamia Husk Spot Pathogen, Pseudocercospora macadamiae.

Pseudocercospora macadamiae causes husk spot in macadamia in Australia. Lack of genomic resources for this pathogen has restricted acquiring knowledge on the mechanism of disease development, spread, and its role in fruit abscission. To address this gap, we sequenced the genome of P. macadamiae. The sequence was de novo assembled into a draft genome of 40 Mb, which is comparable to closely related species in the family Mycosphaerellaceae. The draft genome comprises 212 scaffolds, of which 99 scaffolds are over 50 kb. The genome has a 49% GC content and is predicted to contain 15,430 protein-coding genes. This draft genome sequence is the first for P. macadamiae and represents a valuable resource for understanding genome evolution and plant disease resistance.

Soil bacterial diffusible and volatile organic compounds inhibit Phytophthora capsici and promote plant growth.

Biotic interactions through diffusible and volatile organic compounds (VOCs) are frequent in nature. Soil bacteria are well-known producers of a wide range of volatile compounds (both organic and inorganic) with various biologically relevant activities. Since the last decade, they have been identified as natural biocontrol agents. Volatiles are airborne chemicals, which when released by bacteria, can trigger plant responses such as defence and growth promotion. In this study, we tested whether diffusible and volatile organic compounds (VOCs) produced by soil bacterial isolates exert anti-oomycete and plant growth-promoting effects. We also investigated the effects of inoculation with VOC-producing bacteria on the growth and development of Capsicum annuum and Arabidopsis thaliana seedlings. Our results demonstrate that organic VOCs emitted by bacterial antagonists negatively influence mycelial growth of the soil-borne phytopathogenic oomycete Phytophthora capsici by 35% in vitro. The bacteria showed plant growth promoting effects by stimulating biomass production, primary root growth and root hair development. Additionally, we provide evidence to suggest that these activities were deployed by the emission of either diffusible organic compounds or VOCs. Bacterial VOC profiles were obtained through solid phase microextraction (SPME) and analysis by gas chromatography coupled with mass spectrometry (GC-MS). This elucidated the main volatiles emitted by the isolates, which covered a wide range of aldehydes, alcohols, esters, carboxylic acids, and ketones. Collectively, twenty-five VOCs were identified to be produced by three bacteria; some being species-specific. Our data show that bacterial volatiles inhibits P. capsici in vitro and modulate both plant growth promotion and root system development. These results confirm the significance of soil bacteria and highlights that ways of harnessing them to improve plant growth, and as a biocontrol agent for soil-borne oomycetes through their volatile emissions deserve further investigation.

Molecular Diagnostics of Banana Fusarium Wilt Targeting Secreted-in-Xylem Genes.

Fusarium wilt is currently spreading in banana growing regions around the world leading to substantial losses. The disease is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), which is further classified into distinct races according to the banana varieties that they infect. Cavendish banana is resistant to Foc race 1, to which the popular Gros Michel subgroup succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial subgroup worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to cultivars of Cavendish, and other commonly grown cultivars. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana cultivars. Implementation of quarantine and containment measures depends on early detection of the pathogen through reliable diagnostics. In this study, we tested the hypothesis that secreted in xylem (SIX) genes, which currently comprise the only known family of effectors in F. oxysporum, contain polymorphisms to allow the design of molecular diagnostic assays that distinguish races and relevant VCGs of Foc. We present specific and reproducible diagnostic assays based on conventional PCR targeting SIX genes, using as templates DNA extracted from pure Foc cultures. Sets of primers specifically amplify regions of: SIX6 in Foc race 1, SIX1 gene in TR4, SIX8 in subtropical race 4, SIX9/SIX10 in Foc VCG 0121, and SIX13 in Foc VCG 0122. These assays include simplex and duplex PCRs, with additional restriction digestion steps applied to amplification products of genes SIX1 and SIX13. Assay validations were conducted to a high international standard including the use of 250 Fusarium spp. isolates representing 16 distinct Fusarium species, 59 isolates of F. oxysporum, and 21 different vegetative compatibility groups (VCGs). Tested parameters included inter and intraspecific analytical specificity, sensitivity, robustness, repeatability, and reproducibility. The resulting suite of assays is able to reliably and accurately detect R1, STR4, and TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas.

Soil amendments with ethylene precursor alleviate negative impacts of salinity on soil microbial properties and productivity.

Some microbes enhance stress tolerance in plants by minimizing plant ethylene levels via degradation of its immediate precursor, 1-aminocyclopropane-1-carboxylate (ACC), in the rhizosphere. In return, ACC is used by these microbes as a source of nitrogen. This mutualistic relationship between plants and microbes may be used to promote soil properties in stressful environments. In this study, we tested the hypothesis that amendments of ACC in soils reshape the structure of soil microbiome and alleviate the negative impacts of salinity on soil properties. We treated non-saline and artificially-developed saline soils with ACC in different concentrations for 14 days. The structure of soil microbiome, soil microbial properties and productivity were examined. Our results revealed that microbial composition of bacteria, archaea and fungi in saline soils was affected by ACC amendments; whereas community composition in non-saline soils was not affected. The amendments of ACC could not fully counteract the negative effects of salinity on soil microbial activities and productivity, but increased the abundance of ACC deaminase-encoding gene (acdS), enhanced soil microbial respiration, enzymatic activity, nitrogen and carbon cycling potentials and Arabidopsis biomass in saline soils. Collectively, our study indicates that ACC amendments in soils could efficiently ameliorate salinity impacts on soil properties and plant biomass production.

Complete Genome Sequence of Achromobacter spanius UQ283, a Soilborne Isolate Exhibiting Plant Growth-Promoting Properties.

Achromobacter spanius UQ283 is a soilborne bacterium found to exhibit plant growth-promoting and disease-suppressing attributes in several plant species. Accordingly, we used long-read sequencing to determine its complete genome sequence. The assembled genome will aid in understanding the multifaceted interactions between plant growth-promoting rhizobacteria, pathogens, and plants.

Identification of Soil Bacterial Isolates Suppressing Different Phytophthora spp. and Promoting Plant Growth.

Bacterial isolates obtained from the rhizosphere of Arabidopsis and a plantless compost potting mix was screened for anti-oomycete activity against Phytophthora capsici, Phytophthora citricola, Phytophthora palmivora, and Phytophthora cinnamomi. Three out of 48 isolates exhibited more than 65% inhibition against all tested Phytophthora species and were selected for further studies. These strains, named UQ154, UQ156, and UQ202, are closely related to Bacillus amyloliquefaciens, Bacillus velezensis, and Acinetobacter sp., respectively, based on 16S rDNA sequence analysis. The isolates were evaluated for their ability to fix nitrogen, solubilize phosphate, as well as for siderophore, indoleacetic acid, cell wall degrading enzymes and biofilm production. Their plant growth promoting activities were evaluated by measuring their effect on the germination percentage, root and shoot length, and seedling vigor of lettuce plants. All of these traits were significantly enhanced in plants grown from seeds inoculated with the isolates compared with control plants. Moreover, bacteria-inoculated P. capsici-infected chili plants exhibited improved productivity based on CO2 assimilation rates. Both real-time quantitative PCR and disease severity index revealed significant decreases in pathogen load in infected chili root tissues when plants were previously inoculated with the isolates. Biocontrol activity may result from the secretion of diketopiperazines as identified by Gas chromatography-mass spectrometry analysis of bacterial cultures' extracts. Collectively, this work demonstrates the potential of bacterial isolates to control Phytophthora infection and promote plant growth. They can, therefore be considered as candidate microbial biofertilizers and biopesticides.

Complete Nucleotide Sequence of Australian Tomato spotted wilt virus Isolate TSWV-QLD2.

The complete nucleotide (nt) sequence of an Australian isolate of Tomato spotted wilt virus was determined by deep RNA sequencing and deep small RNA sequencing. The tripartite genome consists of an 8,914-nt L segment, a 4,851-nt M segment, and a 2,987-nt S segment.

An Optimized Transient Dual Luciferase Assay for Quantifying MicroRNA Directed Repression of Targeted Sequences.

Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence.

Jasmonic acid signalling and the plant holobiont.

The plant holobiont - which is the plant and its associated microbiome - is increasingly viewed as an evolving entity. Some interacting microbes that compose the microbiome assist plants in combating pathogens and herbivorous insects. However, knowledge of the factors that influence the microbiome in the context of defence signalling pathways is still in its infancy. Recent research reported that changes in jasmonic acid (JA) and salicylic acid signalling affects the root microbiome of Arabidopsis thaliana. This review aims to present the hypothesis that the JA pathway represents a novel mechanism for microbiome engineering for improved holobiont fitness in agricultural systems.

Effects of jasmonic acid signalling on the wheat microbiome differ between body sites.

Jasmonic acid (JA) signalling helps plants to defend themselves against necrotrophic pathogens and herbivorous insects and has been shown to influence the root microbiome of Arabidopsis thaliana. In this study, we determined whether JA signalling influences the diversity and functioning of the wheat (Triticum aestivum) microbiome and whether these effects are specific to particular parts of the plant. Activation of the JA pathway was achieved via exogenous application of methyl jasmonate and was confirmed by significant increases in the abundance of 10 JA-signalling-related gene transcripts. Phylogenetic marker gene sequencing revealed that JA signalling reduced the diversity and changed the composition of root endophytic but not shoot endophytic or rhizosphere bacterial communities. The total enzymatic activity and substrate utilisation profiles of rhizosphere bacterial communities were not affected by JA signalling. Our findings indicate that the effects of JA signalling on the wheat microbiome are specific to individual plant compartments.