Shifts in the rhizobiome during consecutive in planta enrichment for phosphate-solubilizing bacteria differentially affect maize P status

Summary Phosphorus (P) is despite its omnipresence in soils often unavailable for plants. Rhizobacteria able to solubilize P are therefore crucial to avoid P deficiency. Selection for phosphate‐solubilizing bacteria (PSB) is frequently done in vitro; however, rhizosphere competence is herein overlooked. Therefore, we developed an in planta enrichment concept enabling simultaneous microbial selection for P‐solubilization and rhizosphere competence. We used an ecologically relevant combination of iron‐ and aluminium phosphate to select for PSB in maize (Zea mays L.). In each consecutive enrichment, plant roots were inoculated with rhizobacterial suspensions from plants that had grown in substrate with insoluble P. To assess the plants’ P statuses, non‐destructive multispectral imaging was used for quantifying anthocyanins, a proxy for maize’s P status. After the third consecutive enrichment, plants supplied with insoluble P and inoculated with rhizobacterial suspensions showed a P status similar to plants supplied with soluble P. A parallel metabarcoding approach uncovered that the improved P status in the third enrichment coincided with a shift in the rhizobiome towards bacteria with plant growth‐promoting and P‐solubilizing capacities. Finally, further consecutive enrichment led to a functional relapse hallmarked by plants with a low P status and a second shift in the rhizobiome at the level of Azospirillaceae and Rhizobiaceae

At the scene of the crime : new insights into the role of weakly pathogenic members of the fusarium head blight disease complex

Plant diseases are often caused by a consortium of pathogens competing with one another to gain a foothold in the infection niche. Nevertheless, studies are often limited to a single pathogen on its host. In Europe, fusarium head blight (FHB) of wheat is caused by multipleFusariumspecies, includingFusariumgraminearumandF. poae. Here, we combined a time series of (co)inoculations, monitored by multispectral imaging, transcriptional, and mycotoxin analyses, to study the temporal interaction between both species and wheat. Our results showed coinoculation ofF. graminearumandF. poaeinhibited symptom development but did not alter mycotoxin accumulation compared to a single inoculation withF. graminearum. In contrast, preinoculation ofF. poaereduced both FHB symptoms and mycotoxin levels compared to a singleF. graminearuminfection. Interestingly,F. poaeexhibited increased growth in dual infections, demonstrating that this weak pathogen takes advantage of its co-occurrence withF. graminearum. Quantitative reverse transcription PCR revealed thatF. poaeinducesLOXandICSgene expression in wheat. We hypothesize that the early induction of salicylic and jasmonic acid-related defences byF. poaehampers a subsequentF. graminearuminfection. This study is the first to report on the defence mechanisms of the plant involved in a tripartite interaction between two species of a disease complex and their host

Presence of the weakly pathogenic Fusarium poae in the Fusarium head blight disease complex hampers biocontrol and chemical control of the virulent Fusarium graminearum pathogen

Fusarium head blight (FHB) in wheat (Triticum aestivum L.) is caused by a consortium of mutually interacting Fusarium species. In the field, the weakly pathogenic F. poae often thrives on the infection sites of the virulent F. graminearum. In this ecological context, we investigated the efficacy of chemical and biocontrol agents against F. graminearum in wheat ears. For this purpose, one fungicide comprising prothioconazole + spiroxamine and two bacterial biocontrol strains, Streptomyces rimosus LMG 19352 and Rhodococcus sp. R-43120 were tested for their efficacy to reduce FHB symptoms and mycotoxin (deoxynivalenol, DON) production by F. graminearum in presence or absence of F. poae. Results showed that the fungicide and both actinobacterial strains reduced FHB symptoms and concomitant DON levels in wheat ears inoculated with F. graminearum. Where Streptomyces rimosus appeared to have direct antagonistic effects, Rhodococcus and the fungicide mediated suppression of F. graminearum was linked to the archetypal salicylic acid and jasmonic acid defense pathways that involve the activation of LOX1, LOX2 and ICS. Remarkably, this chemical- and biocontrol efficacy was significantly reduced when F. poae was co-inoculated with F. graminearum. This reduced efficacy was linked to a suppression of the plant’s intrinsic defense system and increased levels of DON. In conclusion, our study shows that control strategies against the virulent F. graminearum in the disease complex causing FHB are hampered by the presence of the weakly pathogenic F. poae. This study provides generic insights in the complexity of control strategies against plant diseases caused by multiple pathogens

Automated multispectral imaging as a tool for the in planta selection of rhizosphere competent bacterial biostimulants

Driven by ecological awareness on excessive use of agrochemicals, research and industry have turned to plant growth promoting rhizobacteria (PGPR) that exhibit significant plant growth promoting and/or biocontrol activities. Most adopted selection procedures for PGPR exclusively consist of the in vitro isolation and selection of bacterial strains with desired agronomic traits, which are only tested on a host plant in the last screening steps. As rhizosphere competence and activity are not accounted for in the initial in vitro selection, promising candidates often fail to perform in planta. Therefore, we developed an in planta enrichment platform coupled to a phenotyping platform which simultaneously selects for biostimulant traits and rhizosphere competence. The platform was utilized to select for phosphate solubilizing bacteria (PSB) on maize (Zea mays L.). As maize exhibits phosphorous deficiency through the accumulation of anthocyanin, we calculated the modified Anthocyanin Reflectance Index (mARI) through the phenotyping platform encompassing an automated multispectral camera, enabling a non-destructive assessment of the phosphorous deficiency status of maize plants. Through a cyclic in planta inoculation approach under phosphorous limiting conditions, several microbial consortia were generated. When inoculated onto maize plants grown in the presence of insoluble phosphorous, some consortia induced a comparable anthocyanin phenotype and phosphorous content to maize plants grown on full nutrient solution. This platform has shown the potential to effectively generate bacterial biostimulants, from which now individual strains can be isolated for further assessment. Currently we are expanding the use of the phenotyping platform to other nutrient deficiencies such as nitrogen, zinc, manganese and iron, and exploring its use for the selection of biocontrol strains against plant pathogens

Shaping the rhizosphere : can in planta rhizobiome selection for phosphate solubilizing bacteria outperform classical in vitro selection methods?

Phosphorus is, next to nitrogen, one of the key nutrients for plant growth and development. As the majority of P in soils is bound to recalcitrant organophosphates, adsorbed on clay minerals or fixed in metal complexes, plants are often suffering of P-deficiency. The rising ecological awareness on the excessive use of chemical (P-)fertilizers led to the quest for highly efficient, beneficial phosphate solubilizing microorganisms. It was at the previous Biostimulants World Congress (4th BWC, 2019) that we highlighted pitfalls of the commonly used in vitro isolation and selection procedures, in which a major concern was the possible lack of rhizosphere competence of the bacterial isolates or consortia. As an alternative, we presented an in planta enrichment platform for the successful selection of rhizosphere competent, phosphate solubilizing bacteria, in which plants were fully relieved of P-stress. In this follow-up research, a metabarcoding analysis was performed to assess the bacterial composition of the sequentially enriched consortia. Trends in plant P-content were similar to trends in abundance of Proteobacteria, Verrucomicrobia and Actinobacteria. Additionally, a total of 250 bacterial isolates were selected in vitro from the enriched consortia, evaluated for their phosphate solubilizing traits (acid production, siderophore production, growth on selective media), their phosphate solubilizing capacity in liquid medium and their root colonizing ability (motility assay). Metabarcoding analysis and in vitro experiments were compared side by side to evaluate the added value of the in planta enrichment platform over the classical in vitro selection method

In planta selection of rhizosphere competent biostimulant microorganisms through an automated plant phenomics platform

Driven by ecological awareness on excessive use of chemical fertilizers, research and industry are aiming to develop biostimulant inoculants harboring plant growth-promoting bacteria (PGPR). However, most adopted selection procedures for PGPR consist of the initial exclusive in vitro isolation and selection of bacterial strains, which are only subsequently assayed in the host plant rhizosphere. As rhizospheric competence and activity are not accounted for in the initial in vitro selection, promising candidates often fail to perform in planta. In order to obtain superior candidates for biostimulant products, we developed an in planta enrichment platform with simultaneous selection for the biostimulant trait and rhizosphere competence. The platform was implemented to select for phosphate solubilizing bacteria (PSB) on maize (Zea mays L.). As maize exhibits phosphorous deficiency through the accumulation of anthocyanin, a phenotyping platform encompassing a multispectral camera was used to capture anthocyanin accumulation to monitor the plant’s phosphorous status in a nondestructive manner. Through a cyclic approach in which plants were consecutively exposed to insoluble phosphorous, we succeeded to enrich the rhizosphere for a PSB consortium by the third enrichment cycle. Inoculated maize grown under phosphorous limitation showed a significant improvement over non-inoculated maize and harbored comparable phenotypes and phosphorous contents to maize grown on full nutrient solution. This platform has shown the potential to generate efficient in planta consortia, from which now individual strains could be isolated for further assessment. Furthermore, the platform can be implemented for other plant growth promoting traits such as nitrogen use efficiency and iron uptake

Uncovering new insights and misconceptions on the effectiveness of phosphate solubilizing rhizobacteria in plants : a meta-analysis

As the awareness on the ecological impact of chemical phosphate fertilizers grows, research turns to sustainable alternatives such as the implementation of phosphate solubilizing bacteria (PSB), which make largely immobile phosphorous reserves in soils available for uptake by plants. In this review, we introduce the mechanisms by which plants facilitate P-uptake and illustrate how PSB improve the bioavailability of this nutrient. Next, the effectiveness of PSB on increasing plant biomass and P-uptake is assessed using a meta-analysis approach. Our review demonstrates that improved P-uptake does not always translate in improved plant height and biomass. We show that the effect of PSB on plants does not provide an added benefit when using bacterial consortia compared to single strains. Moreover, the commonly reported species for P-solubilization, Bacillus spp. and Pseudomonas spp., are outperformed by the scarcely implemented Burkholderia spp. Despite the similar responses to PSB in monocots and eudicots, species responsiveness to PSB varies within both clades. Remarkably, the meta-analysis challenges the common belief that PSB are less effective under field conditions compared to greenhouse conditions. This review provides innovative insights and identifies key questions for future research on PSB to promote their implementation in agriculture

Innovative rhizosphere-based enrichment under P-limitation selects for bacterial isolates with high-performance P-solubilizing traits

With the growing awareness on the ecological impact of chemical phosphate fertilizers, research concerning the use of phosphate solubilizing bacteria (PSB) as a sustainable alternative for, or addition to these fertilizers is of paramount importance. In previous research, we successfully implemented a plant-based enrichment technique for PSB, which simultaneously selected for the rhizosphere competence and phosphate solubilizing characteristics of bacterial suspensions

Uncovering Genomic Features and Biosynthetic Gene Clusters in Endophytic Bacteria from Roots of the Medicinal Plant Alkanna tinctoria Tausch as a Strategy To Identify Novel Biocontrol Bacteria

ABSTRACT The world’s population is increasing at a rate not seen in the past. Agriculture, providing food for this increasing population, is reaching its boundaries of space and natural resources. In addition, changing legislation and increased ecological awareness are forcing agriculture to reduce its environmental impact. This entails the replacement of agrochemicals with nature-based solutions. In this regard, the search for effective biocontrol agents that protect crops from pathogens is in the spotlight. In this study, we have investigated the biocontrol activity of endophytic bacteria isolated from the medicinal plant Alkanna tinctoria Tausch. To do so, an extensive collection of bacterial strains was initially genome sequenced and in silico screened for features related to plant stimulation and biocontrol. Based on this information, a selection of bacteria was tested in vitro for antifungal activity using direct antagonism in a plate assay and in planta with a detached-leaf assay. Bacterial strains were tested individually and in combinations to assess the best-performing treatments. The results revealed that many bacteria could produce metabolites that efficiently inhibit the proliferation of several fungi, especially Fusarium graminearum. Among these, Pseudomonas sp. strain R-71838 showed a strong antifungal effect, in both dual-culture and in planta assays, making it the most promising candidate for biocontrol application. Using microbes from medicinal plants, this study highlights the opportunities of using genomic information to speed up the screening of a taxonomically diverse set of bacteria with biocontrol properties. IMPORTANCE Phytopathogenic fungi are a major threat to global food production. The most common management practice to prevent plant infections involves the intensive use of fungicides. However, with the growing awareness of the ecological and human impacts of chemicals, there is a need for alternative strategies, such as the use of bacterial biocontrol agents. Limitations in the design of bacterial biocontrol included the need for labor-intensive and time-consuming experiments to test a wide diversity of strains and the lack of reproducibility of their activity against pathogens. Here, we show that genomic information is an effective tool to select bacteria of interest quickly. Also, we highlight that the strain Pseudomonas sp. R-71838 produced a reproducible antifungal effect both in vitro and in planta. These findings build a foundation for designing a biocontrol strategy based on Pseudomonas sp. R-71838