Exploring the temporal dynamics of a disease suppressive rhizo-microbiome in eggplants.

The rhizosphere microbiome is important for plant health, yet their contributions to disease resistance and assembly dynamics remain unclear. This study employed rhizosphere microbiome transplantation (RMT) to delineate the impact of the rhizosphere microbiome and the immune response of eggplant (Solanum melongena) on resistance to bacterial wilt caused by Ralstonia solanacearum. We first identified disease-suppressive and disease-conducive rhizosphere microbiome in a susceptible tomato recipient. Using a non-destructive rhizobox and 16S rRNA amplicon sequencing, we monitored the dynamics of both microbiome types during the eggplant development. Most differences were observed at the early stage and then diminished over time. The suppressive microbiome maintained a higher proportion of initial community members throughout eggplant development and exhibited stronger deterministic processes in the early stage, underscoring the importance of plant selection in recruiting protective microbes for rhizosphere immunity. Our study sheds light on the development of microbiome-based strategies for plant disease management and resistance breeding.

Attack of the clones: Population genetics reveals clonality of Colletotrichum lupini, the causal agent of lupin anthracnose.

Colletotrichum lupini, the causative agent of lupin anthracnose, affects lupin cultivation worldwide. Understanding its population structure and evolutionary potential is crucial to design successful disease management strategies. The objective of this study was to employ population genetics to investigate the diversity, evolutionary dynamics, and molecular basis of the interaction of this notorious lupin pathogen with its host. A collection of globally representative C. lupini isolates was genotyped through triple digest restriction site-associated DNA sequencing, resulting in a data set of unparalleled resolution. Phylogenetic and structural analysis could distinguish four independent lineages (I-IV). The strong population structure and high overall standardized index of association (r̅d ) indicates that C. lupini reproduces clonally. Different morphologies and virulence patterns on white lupin (Lupinus albus) and Andean lupin (Lupinus mutabilis) were observed between and within clonal lineages. Isolates belonging to lineage II were shown to have a minichromosome that was also partly present in lineage III and IV, but not in lineage I isolates. Variation in the presence of this minichromosome could imply a role in host-pathogen interaction. All four lineages were present in the South American Andes region, which is suggested to be the centre of origin of this species. Only members of lineage II have been found outside South America since the 1990s, indicating it as the current pandemic population. As a seedborne pathogen, C. lupini has mainly spread through infected but symptomless seeds, stressing the importance of phytosanitary measures to prevent future outbreaks of strains that are yet confined to South America.

Genome-wide association study reveals white lupin candidate gene involved in anthracnose resistance.

KEY MESSAGE: GWAS identifies candidate gene controlling resistance to anthracnose disease in white lupin. White lupin (Lupinus albus L.) is a promising grain legume to meet the growing demand for plant-based protein. Its cultivation, however, is severely threatened by anthracnose disease caused by the fungal pathogen Colletotrichum lupini. To dissect the genetic architecture for anthracnose resistance, genotyping by sequencing was performed on white lupin accessions collected from the center of domestication and traditional cultivation regions. GBS resulted in 4611 high-quality single-nucleotide polymorphisms (SNPs) for 181 accessions, which were combined with resistance data observed under controlled conditions to perform a genome-wide association study (GWAS). Obtained disease phenotypes were shown to highly correlate with overall three-year disease assessments under Swiss field conditions (r > 0.8). GWAS results identified two significant SNPs associated with anthracnose resistance on gene Lalb_Chr05_g0216161 encoding a RING zinc-finger E3 ubiquitin ligase which is potentially involved in plant immunity. Population analysis showed a remarkably fast linkage disequilibrium decay, weak population structure and grouping of commercial varieties with landraces, corresponding to the slow domestication history and scarcity of modern breeding efforts in white lupin. Together with 15 highly resistant accessions identified in the resistance assay, our findings show promise for further crop improvement. This study provides the basis for marker-assisted selection, genomic prediction and studies aimed at understanding anthracnose resistance mechanisms in white lupin and contributes to improving breeding programs worldwide.

Untangling the Pea Root Rot Complex Reveals Microbial Markers for Plant Health.

Plant health is recognised as a key element to ensure global food security. While plant breeding has substantially improved crop resistance against individual pathogens, it showed limited success for diseases caused by the interaction of multiple pathogens such as root rot in pea (Pisum sativum L.). To untangle the causal agents of the pea root rot complex and determine the role of the plant genotype in shaping its own detrimental or beneficial microbiome, fungal and oomycete root rot pathogens, as well as previously identified beneficials, i.e., arbuscular mycorrhizal fungi (AMF) and Clonostachys rosea, were qPCR quantified in diseased roots of eight differently resistant pea genotypes grown in four agricultural soils under controlled conditions. We found that soil and pea genotype significantly determined the microbial compositions in diseased pea roots. Despite significant genotype x soil interactions and distinct soil-dependent pathogen complexes, our data revealed key microbial taxa that were associated with plant fitness. Our study indicates the potential of fungal and oomycete markers for plant health and serves as a precedent for other complex plant pathosystems. Such microbial markers can be used to complement plant phenotype- and genotype-based selection strategies to improve disease resistance in one of the world's most important pulse crops of the world.

A qPCR Assay for the Fast Detection and Quantification of Colletotrichum lupini.

White lupin (Lupinus albus) represents an important legume crop in Europe and other parts of the world due to its high protein content and potential for low-input agriculture. However, most cultivars are susceptible to anthracnose caused by Colletotrichum lupini, a seed- and air-borne fungal pathogen that causes severe yield losses. The aim of this work was to develop a C. lupini-specific quantitative real-time TaqMan PCR assay that allows for quick and reliable detection and quantification of the pathogen in infected seed and plant material. Quantification of C. lupini DNA in dry seeds allowed us to distinguish infected and certified (non-infected) seed batches with DNA loads corresponding to the disease score index and yield of the mother plants. Additionally, C. lupini DNA could be detected in infected lupin shoots and close to the infection site, thereby allowing us to study the disease cycle of this hemibiotrophic pathogen. This qPCR assay provides a useful diagnostic tool to determine anthracnose infection levels of white lupin seeds and will facilitate the use of seed health assessments as a strategy to reduce the primary infection source and spread of this disease.

A Novel Real Time PCR Method for the Detection and Quantification of Didymella pinodella in Symptomatic and Asymptomatic Plant Hosts.

Didymella pinodella is the major pathogen of the pea root rot complex in Europe. This wide host range pathogen often asymptomatically colonizes its hosts, making the control strategies challenging. We developed a real-time PCR assay for the detection and quantification of D. pinodella based on the TEF-1 alpha gene sequence alignments. The assay was tested for specificity on a 54-isolate panel representing 35 fungal species and further validated in symptomatic and asymptomatic pea and wheat roots from greenhouse tests. The assay was highly consistent across separate qPCR reactions and had a quantification/detection limit of 3.1 pg of target DNA per reaction in plant tissue. Cross-reactions were observed with DNA extracts of five Didymella species. The risk of cross contamination, however, is low as the non-targets have not been associated with pea previously and they were amplified with at least 1000-fold lower sensitivity. Greenhouse inoculation tests revealed a high correlation between the pathogen DNA quantities in pea roots and pea root rot severity and biomass reduction. The assay also detected D. pinodella in asymptomatic wheat roots, which, despite the absence of visible root rot symptoms, caused wheat biomass reduction. This study provides new insights into the complex life style of D. pinodella and can assist in better understanding the pathogen survival and spread in the environment.

A High-Throughput Phenotyping Tool to Identify Field-Relevant Anthracnose Resistance in White Lupin.

The seed- and air-borne pathogen Colletotrichum lupini, the causal agent of lupin anthracnose, is the most important disease in white lupin (Lupinus albus) worldwide and can cause total yield loss. The aims of this study were to establish a reliable high-throughput phenotyping tool to identify anthracnose resistance in white lupin germplasm and to evaluate a genomic prediction model, accounting for previously reported resistance quantitative trait loci, on a set of independent lupin genotypes. Phenotyping under controlled conditions, performing stem inoculation on seedlings, showed to be applicable for high throughput, and its disease score strongly correlated with field plot disease assessments (r = 0.95, P < 0.0001) and yield (r = -0.64, P = 0.035). Traditional one-row field disease phenotyping showed no significant correlation with field plot disease assessments (r = 0.31, P = 0.34) and yield (r = -0.45, P = 0.17). Genomically predicted resistance values showed no correlation with values observed under controlled or field conditions, and the parental lines of the recombinant inbred line population used for constructing the prediction model exhibited a resistance pattern opposite to that displayed in the original (Australian) environment used for model construction. Differing environmental conditions, inoculation procedures, or population structure may account for this result. Phenotyping a diverse set of 40 white lupin accessions under controlled conditions revealed eight accessions with improved resistance to anthracnose. The standardized area under the disease progress curves (sAUDPC) ranged from 2.1 to 2.8, compared with the susceptible reference accession with a sAUDPC of 3.85. These accessions can be incorporated into white lupin breeding programs. In conclusion, our data support stem inoculation-based disease phenotyping under controlled conditions as a time-effective approach to identify field-relevant resistance, which can now be applied to further identify sources of resistance and their underlying genetics.

Heritable Variation in Pea for Resistance Against a Root Rot Complex and Its Characterization by Amplicon Sequencing.

Soil-borne pathogens cause severe root rot of pea (Pisum sativum L.) and are a major constraint to pea cultivation worldwide. Resistance against individual pathogen species is often ineffective in the field where multiple pathogens form a pea root rot complex (PRRC) and conjointly infect pea plants. On the other hand, various beneficial plant-microbe interactions are known that offer opportunities to strengthen plant health. To account for the whole rhizosphere microbiome in the assessment of root rot resistance in pea, an infested soil-based resistance screening assay was established. The infested soil originated from a field that showed severe pea root rot in the past. Initially, amplicon sequencing was employed to characterize the fungal microbiome of diseased pea roots grown in the infested soil. The amplicon sequencing evidenced a diverse fungal community in the roots including pea pathogens Fusarium oxysporum, F. solani, Didymella sp., and Rhizoctonia solani and antagonists such as Clonostachys rosea and several mycorrhizal species. The screening system allowed for a reproducible assessment of disease parameters among 261 pea cultivars, breeding lines, and landraces grown for 21 days under controlled conditions. A sterile soil control treatment was used to calculate relative shoot and root biomass in order to compare growth performance of pea lines with highly different growth morphologies. Broad sense heritability was calculated from linear mixed model estimated variance components for all traits. Emergence on the infested soil showed high (H 2 = 0.89), root rot index (H 2 = 0.43), and relative shoot dry weight (H 2 = 0.51) medium heritability. The resistance screening allowed for a reproducible distinction between PRRC susceptible and resistant pea lines. The combined assessment of root rot index and relative shoot dry weight allowed to identify resistant (low root rot index) and tolerant pea lines (low relative shoot dry weight at moderate to high root rot index). We conclude that relative shoot dry weight is a valuable trait to select disease tolerant pea lines. Subsequently, the resistance ranking was verified in an on-farm experiment with a subset of pea lines. We found a significant correlation (r s = 0.73, p = 0.03) between the controlled conditions and the resistance ranking in a field with high PRRC infestation. The screening system allows to predict PRRC resistance for a given field site and offers a tool for selection at the seedling stage in breeding nurseries. Using the complexity of the infested field soil, the screening system provides opportunities to study plant resistance in the light of diverse plant-microbe interactions occurring in the rhizosphere.

miCROPe 2019 - emerging research priorities towards microbe-assisted crop production.

The miCROPe 2019 symposium, which took place from 2 to 5 December 2019 in Vienna, Austria, has unified researchers and industry from around the world to discuss opportunities, challenges and needs of microbe-assisted crop production. There is broad consensus that microorganisms-with their abilities to alleviate biotic and abiotic stresses and to improve plant nutrition-offer countless opportunities to enhance plant productivity and to ameliorate agricultural sustainability. However, microbe-assisted cultivation approaches face challenges that need to be addressed before a breakthrough of such technologies can be expected. Following up on the miCROPe symposium and a linked satellite workshop on breeding for beneficial plant-microbe interactions, we carved out research priorities towards successful implementation of microbiome knowledge for modern agriculture. These include (i) to solve context dependency for microbial inoculation approaches and (ii) to identify the genetic determinants to allow breeding for beneficial plant-microbiome interactions. With the combination of emerging third generation sequencing technologies and new causal research approaches, we now stand at the crossroad of utilising microbe-assisted crop production as a reliable and sustainable agronomic practice.

Advances in Breeding for Mixed Cropping - Incomplete Factorials and the Producer/Associate Concept.

Mixed cropping has been suggested as a resource-efficient approach to meet high produce demands while maintaining biodiversity and minimizing environmental impact. Current breeding programs do not select for enhanced general mixing ability (GMA) and neglect biological interactions within species mixtures. Clear concepts and efficient experimental designs, adapted to breeding for mixed cropping and encoded into appropriate statistical models, are lacking. Thus, a model framework for GMA and SMA (specific mixing ability) was established. Results of a simulation study showed that an incomplete factorial design combines advantages of two commonly used full factorials, and enables to estimate GMA, SMA, and their variances in a resource-efficient way. This model was extended to the Producer (Pr) and Associate (As) concept to exploit additional information based on fraction yields. It was shown that the Pr/As concept allows to characterize genotypes for their contribution to total mixture yield, and, when relating to plant traits, allows to describe biological interaction functions (BIF) in a mixed crop. Incomplete factorial designs show the potential to drastically improve genetic gain by testing an increased number of genotypes using the same amount of resources. The Pr/As concept can further be employed to maximize GMA in an informed and efficient way. The BIF of a trait can be used to optimize species ratios at harvest as well as to extend our understanding of competitive and facilitative interactions in a mixed plant community. This study provides an integrative methodological framework to promote breeding for mixed cropping.

Insights to plant-microbe interactions provide opportunities to improve resistance breeding against root diseases in grain legumes.

Root and foot diseases severely impede grain legume cultivation worldwide. Breeding lines with resistance against individual pathogens exist, but these resistances are often overcome by the interaction of multiple pathogens in field situations. Novel tools allow to decipher plant-microbiome interactions in unprecedented detail and provide insights into resistance mechanisms that consider both simultaneous attacks of various pathogens and the interplay with beneficial microbes. Although it has become clear that plant-associated microbes play a key role in plant health, a systematic picture of how and to what extent plants can shape their own detrimental or beneficial microbiome remains to be drawn. There is increasing evidence for the existence of genetic variation in the regulation of plant-microbe interactions that can be exploited by plant breeders. We propose to consider the entire plant holobiont in resistance breeding strategies in order to unravel hidden parts of complex defence mechanisms. This review summarizes (a) the current knowledge of resistance against soil-borne pathogens in grain legumes, (b) evidence for genetic variation for rhizosphere-related traits, (c) the role of root exudation in microbe-mediated disease resistance and elaborates (d) how these traits can be incorporated in resistance breeding programmes.

Ecological studies of the bio-inoculant Trichoderma hamatum LU592 in the root system of Pinus radiata.

The plant health- and growth-promoting biological inoculant (bio-inoculant) Trichoderma hamatum LU592 was transformed with the constitutively expressed green fluorescent protein (gfp) and hygromycin B resistance (hph) genes to specifically monitor the isolate in the root system of Pinus radiata within a strong indigenous Trichoderma population. A modified dilution plating technique was developed to allow the determination of the mycelia proportion of total propagule levels. LU592 was shown to colonize the rhizosphere most effectively when 10(5)  spores per pot were applied compared with inoculum concentrations of 10(3) and 10(7)  spores per pot. LU592 extended its zone of activity beyond the rhizosphere to at least 1 cm away from the root surface. A positive relationship was shown between P. radiata root maturation and the spatial and temporal proliferation of LU592 in the root system. A steep increase in mycelia levels and proportion of penetrated root segments was observed after 12 weeks. This study reinforces the value of genetic markers for use in ecological studies of filamentous fungi. However, despite isolate-specific recovery of the introduced isolate, it was shown that total propagule counts do not always correlate with the amount of viable mycelium present in the root system. Therefore, it is proposed that the differentiation of mycelia from spores and root penetration is used as more accurate measures of fungal activity.

Understanding Trichoderma in the root system of Pinus radiata: associations between rhizosphere colonisation and growth promotion for commercially grown seedlings.

Two Trichoderma isolates (T. hamatum LU592 and T. atroviride LU132) were tested for their ability to promote the growth and health of commercially grown Pinus radiata seedlings. The colonisation behaviour of the two isolates was investigated to relate rhizosphere competence and root penetration to subsequent effects on plant performance. Trichoderma hamatum LU592 was shown to enhance several plant health and growth parameters. The isolate significantly reduced seedling mortality by up to 29%, and promoted the growth of shoots (e.g. height by up to 16%) and roots (e.g. dry weight by up to 31%). The introduction of LU592 as either seed coat or spray application equally improved seedling health and growth demonstrating the suitability of both application methods for pine nursery situations. However, clear differences in rhizosphere colonisation and root penetration between the two application methods highlighted the need for more research on the impact of inoculum densities. When spray-applied, LU592 was found to be the predominant Trichoderma strain in the plant root system, including bulk potting mix, rhizosphere and endorhizosphere. In contrast, T. atroviride LU132 was shown to colonise the root system poorly, and no biological impact on P. radiata seedlings was detected. This is the first report to demonstrate rhizosphere competence as a useful indicator for determining Trichoderma bio-inoculants for P. radiata. High indigenous Trichoderma populations with similar population dynamics to the introduced strains revealed the limitations of the dilution plating technique, but this constraint was alleviated to some extent by the use of techniques for morphological and molecular identification of the introduced isolates.