Host-associated genetic differentiation and origin of a recent host shift in the generalist parasitic weed Phelipanche ramosa.

Branched broomrape, Phelipanche ramosa (L.) Pomel, is a globally distributed parasitic weed of economic importance. In Europe, where it is native, it can infest several crops, notably tomato, tobacco, and hemp. In western France, it has recently adapted to a new host crop, oilseed rape, causing substantial damage. The aim of this study was to investigate the evolutionary relationships and genetic differentiation among P. ramosa populations infesting different hosts. We collected 1611 P. ramosa samples from 109 fields cultivated with six different crops (oilseed rape, tobacco, hemp, tomato, lentil, and celery) and distributed among six European countries. All samples were genotyped for ten microsatellite loci and a subset of samples was sequenced for two nuclear genes and two chloroplast genes. Genetic differentiation among populations was high (F ST = 0.807) and mainly driven by differentiation among different host crops, with no significant geographic structure. Genetic structure analysis identified up to seven biologically meaningful clusters that matched with host crops of origin. Reconstructed networks of sequence haplotypes and multilocus SSR genotypes showed a large genetic divergence between samples collected on oilseed rape and samples collected on other crops. The phylogeny inferred from DNA sequences placed samples collected from oilseed rape as a basal lineage. Approximate Bayesian Computations were used to compare different evolutionary scenarios of divergence among the three main genetic clusters, associated, respectively, with oilseed rape, tobacco, and hemp as host crops. The best-supported scenario indicated that P. ramosa infesting oilseed rape derived recently from an ancient, unknown lineage. Our results suggest that a more complete description of the genetic diversity of P. ramosa is still needed to uncover the likely source of the recent adaptation to oilseed rape and to anticipate future new host shifts.

Screening for potential mycoherbicides within the endophyte community of Phelipanche ramosa parasitizing tobacco.

Branched broomrape (Phelipanche ramosa (L.) Pomel) is an achlorophyllous root parasitic plant with a wide host range. Its complex management is leading to the abandonment of tobacco or oilseed rape cultivation in the most affected regions in France. Among broomrape regulation factors, soil microorganisms such as fungi seem to be a relevant biocontrol lever. The aim of this work was to detect potential mycoherbicides among fungal endophytic colonizers of P. ramosa parasitizing tobacco. Our hypothesis was that both the inhibitory of broomrape seed germination and the necrotic activities are characteristic of the fungal isolates whatever their taxonomic position. To test this hypothesis, we analysed the taxonomic and functional diversity of fungal isolates of symptomatic P. ramosa collected from infested tobacco-growing regions in France in order to identify one or more fungal strains for future biocontrol. The fungal isolates were characterized using morphological and molecular identification tools and tested for their ability to inhibit the germination of P. ramosa seeds, their necrotic activity on the stems of the pest and their non-pathogenicity to the host plant. We highlighted the specific richness of fungal colonizers associated with symptomatic P. ramosa. Among the 374 collected isolates, nearly 80% belonged to 19 Fusarium species. Eighty-seven isolates representative of this diversity also showed functional diversity by inhibiting seed germination of the parasite. The 20 best-performing isolates showed differences in germination inhibition of P. ramosa at the intraspecific level. Among these 20 fungal isolates, a set of 15 randomly selected isolates was tested for their necrotic activity on the parasite stems. Fusarium venenatum isolates showed dual competence, i.e. germination inhibition and necrotic activity, and were non-pathogenic to tobacco. This led us to discuss the potential mycoherbicidal effect of this fungal species on P. ramosa.

Weeds Harbor an Impressive Diversity of Fungi, Which Offers Possibilities for Biocontrol.

The use of herbicides for weed control is very common, but some of them represent a threat to human health, are environmentally detrimental, and stimulate herbicide resistance. Therefore, using microorganisms as natural herbicides appears as a promising alternative. The mycoflorae colonizing different species of symptomatic and asymptomatic weeds were compared to characterize the possible mycoherbicidal candidates associated with symptomatic weeds. A collection of 475 symptomatic and asymptomatic plants belonging to 23 weed species was established. A metabarcoding approach based on amplification of the internal transcribed spacer (ITS) region combined with high-throughput amplicon sequencing revealed the diversity of fungal communities hosted by these weeds: 542 fungal genera were identified. The variability of the composition of fungal communities revealed a dispersed distribution of taxa governed neither by geographical location nor by the botanical species, suggesting a common core displaying nonspecific interactions with host plants. Beyond this core, specific taxa were more particularly associated with symptomatic plants. Some of these, such as Alternaria, Blumeria, Cercospora, Puccinia, are known pathogens, while others such as Sphaerellopsis, Vishniacozyma, and Filobasidium are not, at least on crops, and constitute new tracks to be followed in the search for mycoherbicidal candidates. IMPORTANCE This approach is original because the diversity of weed-colonizing fungi has rarely been studied before. Furthermore, targeting both the ITS1 and ITS2 regions to characterize the fungal communities (i) highlighted the complementarity of these two regions, (ii) revealed a great diversity of weed-colonizing fungi, and (iii) allowed for the identification of potential mycoherbicides, among which were unexpected genera.

Is induction ability of seed germination of Phelipanche ramosa phylogenetically structured among hosts? A case study on Fabaceae species.

Phelipanche ramosa is a major root-holoparasitic damaging weed characterized by a broad host range, including numerous Fabaceae species. In France, the agricultural threat posed by P. ramosa has increased over two decades due to the appearance of a genetically differentiated pathovar presenting a clear host specificity for oilseed rape. The new pathovar has led to a massive expansion of P. ramosa in oilseed rape fields. The germination rate of P. ramosa seeds is currently known to vary among P. ramosa pathovars and host species. However, only a few studies have investigated whether phylogenetic relatedness among potential host species is a predictor of the ability of these species to induce the seed germination of parasitic weeds by testing for phylogenetic signal. We focused on a set of 12 Fabaceae species and we assessed the rate of induction of seed germination by these species for two pathovars based on in vitro co-cultivation experiments. All Fabaceae species tested induced the germination of P. ramosa seeds. The germination rate of P. ramosa seeds varied between Fabaceae species and tribes studied, while pathovars appeared non-influential. Considering oilseed rape as a reference species, we also highlighted a significant phylogenetic signal. Phylogenetically related species therefore showed more similar rates of induction of seed germination than species drawn at random from a phylogenetic tree. In in vitro conditions, only Lotus corniculatus induced a significantly higher germination rate than oilseed rape, and could potentially be used as a catch crop after confirmation of these results under field conditions.

Trophic Relationships between the Parasitic Plant Species Phelipanche ramosa (L.) and Different Hosts Depending on Host Phenological Stage and Host Growth Rate.

Phelipanche ramosa (L.) Pomel (branched broomrape) is a holoparasitic plant that reproduces on crops and also on weeds, which contributes to increase the parasite seed bank in fields. This parasite extracts all its nutrients at the host's expense so that host-parasite trophic relationships are crucial to determine host and parasite growth. This study quantified the intensity with which P. ramosa draws assimilates from its host and analyzed whether it varied with host species, host phenological stage and host growth rate. A greenhouse experiment was conducted on three host species: the crop species Brassica napus (L.) (oilseed rape) and two weed species, Capsella bursa-pastoris (L.) Medik. and Geranium dissectum (L.). Plants were grown with or without P. ramosa and under three light levels to modulate host growth rate. The proportion of host biomass loss due to parasitism by P. ramosa differed between host species (at host fructification, biomass loss ranged from 34 to 84%). B. napus and C. bursa-pastoris displayed a similar response to P. ramosa, probably because they belong to the same botanical family. The sensitivity to P. ramosa in each host species could be related to the precocity of P. ramosa development on them. Host compartments could be ranked as a function of their sensitivity to parasitism, with the reproductive compartment being the most severely affected, followed by stems and roots. The proportion of biomass allocated to leaves was not reduced by parasitism. The proportion of pathosystem biomass allocated to the parasite depended on host species. It generally increased with host stage progression but was constant across light induced-host growth rate, showing that P. ramosa adapts its growth to host biomass production. The rank order of host species in terms of sink strength differed from that in terms of host sensitivity. Finally, for B. napus, the biomass of individual parasite shoots decreased with increasing their number per host plant, regardless of host growth rate. Results will be incorporated into a mechanistic model in order to analyze the effect of parasitic plant species on weed community assembly and to design new cropping systems for controlling P. ramosa.

Development of microsatellite markers in the branched broomrape Phelipanche ramosa L. (Pomel) and evidence for host-associated genetic divergence.

Phelipanche ramosa is a parasitic plant that infects numerous crops worldwide. In Western Europe it recently expanded to a new host crop, oilseed rape, in which it can cause severe yield losses. We developed 13 microsatellite markers for P. ramosa using next-generation 454 sequencing data. The polymorphism at each locus was assessed in a sample of 96 individuals collected in France within 6 fields cultivated with tobacco, hemp or oilseed rape. Two loci were monomorphic. At the other 11 loci, the number of alleles and the expected heterozygosity ranged from 3 to 6 and from 0.31 to 0.60, respectively. Genetic diversity within each cultivated field was very low. The host crop from which individuals were collected was the key factor structuring genetic variation. Individuals collected on oilseed rape were strongly differentiated from individuals collected on hemp or tobacco, which suggests that P. ramosa infecting oilseed rape forms a genetically diverged race. The microsatellites we developed will be useful for population genetics studies and for elucidating host-associated genetic divergence in P. ramosa.