Evaluating Mechanisms of Soil Microbiome Suppression of Striga Infection in Sorghum.

The root parasitic weed Striga hermonthica has a devastating effect on sorghum and other cereal crops in Sub-Saharan Africa. Available Striga management strategies are rarely sufficient or not widely accessible or affordable. Identification of soil- or plant-associated microorganisms that interfere in the Striga infection cycle holds potential for development of complementary biological control measures. Such inoculants should be preferably based on microbes native to the regions of their application. We developed a method to assess microbiome-based soil suppressiveness to Striga with a minimal amount of field-collected soil. We previously used this method to identify the mechanisms of microbe-mediated suppression of Striga infection and to test individual microbial strains. Here, we present protocols to assess the functional potential of the soil microbiome and individual bacterial taxa that adversely affect Striga parasitism in sorghum via three major known suppression mechanisms. These methods can be further extended to other Striga hosts and other root parasitic weeds. Key features • This protocol provides a detailed description of the methods used in Kawa et al. [1]. • This protocol is optimized to assess soil suppressiveness to Striga infection by using natural field-collected soil and the same soil sterilized by gamma-radiation. • This protocol is optimized to test bacterial (and not fungal) isolates. • This protocol can be easily extended to other host-parasite-microbiome systems.

The soil microbiome modulates the sorghum root metabolome and cellular traits with a concomitant reduction of Striga infection.

Sorghum bicolor is among the most important cereals globally and a staple crop for smallholder farmers in sub-Saharan Africa. Approximately 20% of sorghum yield is lost annually in Africa due to infestation with the root parasitic weed Striga hermonthica. Existing Striga management strategies are not singularly effective and integrated approaches are needed. Here, we demonstrate the functional potential of the soil microbiome to suppress Striga infection in sorghum. We associate this suppression with microbiome-mediated induction of root endodermal suberization and aerenchyma formation and with depletion of haustorium-inducing factors, compounds required for the initial stages of Striga infection. We further identify specific bacterial taxa that trigger the observed Striga-suppressive traits. Collectively, our study describes the importance of the soil microbiome in the early stages of root infection by Striga and pinpoints mechanisms of Striga suppression. These findings open avenues to broaden the effectiveness of integrated Striga management practices.

Back to the roots: defining the core microbiome of Sorghum bicolor in agricultural field soils from the centre of origin.

Sorghum is a major staple crop in sub-Saharan Africa with yields severely impacted by biotic and abiotic factors. Here, we analysed the taxonomic diversity and biogeographical distribution of bacterial taxa of 48 agricultural fields along a transect of approximately 2000 km across the Ethiopian sorghum belt, the centre of origin of sorghum. The ultimate goal is to identify-yet-unexplored-beneficial plant-microbe associations. Based on bulk soil bacterial communities and DArT-SNP analyses of 59 sorghum accessions, we selected three microbiologically distinct field soils and 12 sorghum genotypes, including commercial varieties, wild relatives, and farmer-preferred landraces. The results showed a core rhizosphere microbiome of 2125 amplicon sequence variants (ASVs), belonging to eight bacterial families consistently found across the three soil types and the 12 sorghum genotypes. Integration of the rhizosphere bacterial community analysis with DArT-SNP sorghum genotyping revealed the association of differentially abundant ASVs with sorghum genotypic traits, including the distinct recruitment of Pseudomonadaceae by the stay-green, drought-tolerant, and wild sorghum genotypes. Collectively, these results provide new insights into the core and accessory bacterial taxa in the sorghum rhizosphere in the centre of origin, setting a baseline for targeted isolation and functional characterization of putative beneficial rhizobacteria.

An improved strategy to analyse strigolactones in complex sample matrices using UHPLC-MS/MS.

BACKGROUND: Strigolactones represent the most recently described group of plant hormones involved in many aspects of plant growth regulation. Simultaneously, root exuded strigolactones mediate rhizosphere signaling towards beneficial arbuscular mycorrhizal fungi, but also attract parasitic plants. The seed germination of parasitic plants induced by host strigolactones leads to serious agricultural problems worldwide. More insight in these signaling molecules is hampered by their extremely low concentrations in complex soil and plant tissue matrices, as well as their instability. So far, the combination of tailored isolation-that would replace current unspecific, time-consuming and labour-intensive processing of large samples-and a highly sensitive method for the simultaneous profiling of a broad spectrum of strigolactones has not been reported. RESULTS: Depending on the sample matrix, two different strategies for the rapid extraction of the seven structurally similar strigolactones and highly efficient single-step pre-concentration on polymeric RP SPE sorbent were developed and validated. Compared to conventional methods, controlled temperature during the extraction and the addition of an organic modifier (acetonitrile, acetone) to the extraction solvent helped to tailor strigolactone isolation from low initial amounts of root tissue (150 mg fresh weight, FW) and root exudate (20 ml), which improved both strigolactone stability and sample purity. We have designed an efficient UHPLC separation with sensitive MS/MS detection for simultaneous analysis of seven natural strigolactones including their biosynthetic precursors-carlactone and carlactonoic acid. In combination with the optimized UHPLC-MS/MS method, attomolar detection limits were achieved. The new method allowed successful profiling of seven strigolactones in small exudate and root tissue samples of four different agriculturally important plant species-sorghum, rice, pea and tomato. CONCLUSION: The established method provides efficient strigolactone extraction with aqueous mixtures of less nucleophilic organic solvents from small root tissue and root exudate samples, in combination with rapid single-step pre-concentration. This method improves strigolactone stability and eliminates the co-extraction and signal of matrix-associated contaminants during the final UHPLC-MS/MS analysis with an electrospray interface, which dramatically increases the overall sensitivity of the analysis. We show that the method can be applied to a variety of plant species.

Rhizobacterial community structure differences among sorghum cultivars in different growth stages and soils.

Plant genotype selects the rhizosphere microbiome. The success of plant-microbe interactions is dependent on factors that directly or indirectly influence the plant rhizosphere microbial composition. We investigated the rhizosphere bacterial community composition of seven different sorghum cultivars in two different soil types (abandoned (CF) and agricultural (VD)). The rhizosphere bacterial community was evaluated at four different plant growth stages: emergence of the second (day 10) and third leaves (day 20), the transition between the vegetative and reproductive stages (day 35), and the emergence of the last visible leaf (day 50). At early stages (days 10 and 20), the sorghum rhizosphere bacterial community composition was mainly driven by soil type, whereas at late stages (days 35 and 50), the bacterial community composition was also affected by the sorghum genotype. Although this effect of sorghum genotype was small, different sorghum cultivars assembled significantly different bacterial community compositions. In CF soil, the striga-resistant cultivar had significantly higher relative abundances of Acidobacteria GP1, Burkholderia, Cupriavidus (Burkholderiaceae), Acidovorax and Albidiferax (Comamonadaceae) than the other six cultivars. This study is the first to simultaneously investigate the contributions of plant genotype, plant growth stage and soil type in shaping sorghum rhizosphere bacterial community composition.

Mutation in sorghum LOW GERMINATION STIMULANT 1 alters strigolactones and causes Striga resistance.

Striga is a major biotic constraint to sorghum production in semiarid tropical Africa and Asia. Genetic resistance to this parasitic weed is the most economically feasible control measure. Mutant alleles at the LGS1 (LOW GERMINATION STIMULANT 1) locus drastically reduce Striga germination stimulant activity. We provide evidence that the responsible gene at LGS1 codes for an enzyme annotated as a sulfotransferase and show that functional loss of this gene results in a change of the dominant strigolactone (SL) in root exudates from 5-deoxystrigol, a highly active Striga germination stimulant, to orobanchol, an SL with opposite stereochemistry. Orobanchol, although not previously reported in sorghum, functions in the multiple SL roles required for normal growth and environmental responsiveness but does not stimulate germination of Striga This work describes the identification of a gene regulating Striga resistance and the underlying protective chemistry resulting from mutation.

Random mutagenesis of the nucleotide-binding domain of NRC1 (NB-LRR Required for Hypersensitive Response-Associated Cell Death-1), a downstream signalling nucleotide-binding, leucine-rich repeat (NB-LRR) protein, identifies gain-of-function mutations in the nucleotide-binding pocket.

Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins confer immunity to pathogens possessing the corresponding avirulence proteins. Activation of NB-LRR proteins is often associated with induction of the hypersensitive response (HR), a form of programmed cell death. NRC1 (NB-LRR Required for HR-Associated Cell Death-1) is a tomato (Solanum lycopersicum) NB-LRR protein that participates in the signalling cascade leading to resistance to the pathogens Cladosporium fulvum and Verticillium dahliae. To identify mutations in NRC1 that cause increased signalling activity, we generated a random library of NRC1 variants mutated in their nucleotide-binding domain and screened them for the ability to induce an elicitor-independent HR in Nicotiana tabacum. Screening of 1920 clones retrieved 11 gain-of-function mutants, with 10 of them caused by a single amino acid substitution. All substitutions are located in or very close to highly conserved motifs within the nucleotide-binding domain, suggesting modulation of the signalling activity of NRC1. Three-dimensional modelling of the nucleotide-binding domain of NRC1 revealed that the targeted residues are centred around the bound nucleotide. Our mutational approach has generated a wide set of novel gain-of-function mutations in NRC1 and provides insight into how the activity of this NB-LRR is regulated.