Stigmas of holoparasitic Phelipanche arenaria (Orobanchaceae) - a suitable ephemeric flower habitat for development unique microbiome

Abstract

Background: Microbial communities have occasionally been observed in part of the ephemeric reproductive structure of floral stigmas, but their prevalence, phylogenetic diversity and ecological roles are understudied. This report describes the first study of bacterial and fungal communities in immature and mature stigma tissue of the endangered holoparasitic plant Phelipanche arenaria. Culture-dependent methods coupled with next-generation sequencing indicated that a small surface of the flower stigma was an unexpectedly rich and diverse microhabitat for colonization of microbial. We also compared the enzymatic activity of the bacterial communities between immature and mature stigmas samples.

Results: Using high-throughput sequencing methods, we identified and classified 39 to over 51 OTUs per sample for bacterial OTUs represented by Pantoea agglomerans and P. ananatis, comprising 50.6%, followed by Pseudomonas, Luteibacter spp., Sphingomonas spp. with 17% of total frequency. The bacterial profile of immature stigmas of P. arenaria contained unique microorganisms (21 of the most numerous OTUs) that were not confirmed in mature stigmas. However, the enzymatic activity of bacteria in mature stigmas of P. arenaria showed more activity than observed in immature stigmas. In the fungal profile, we recorded even 80 OTUs in mature stigmas, consisting of Capnodiales 45.03% of the total abundance with 28.27% of frequency was created by Alternaria eichhorniae (10.55%), Mycosphaerella tassiana (9.69%), and Aureobasidium pullulans (8.03%). Additionally, numerous putative plant growth-promoting bacteria, fungal pathogens and pathogen-antagonistic yeasts were also detected.

Conclusions: Our study uncovered that P. arenaria stigmas host diverse bacterial and fungal communities. These microorganisms are well known and have been described as beneficial for biotechnological and environmental applications (e.g., production of different enzymes and antimicrobial compounds). This research provided valuable insight into the parasitic plant-microbe interactions.

Keywords: 16S rRNA gene; Environment; Flower microbiome; Internal transcribed spacer (ITS); Next-generation sequencing; Parasitic plants.

Fig. 1

A phylogenetic tree based on 16S rRNA sequences demonstrated for the identified strains of bacteria compared to other strains. The bar indicates sequence divergence. The black rectangle marks bacteria that colonized immature stigmas from closed flowers (green dots) and mature stigmas from opened flowers (red dots). The potentially pathogenic (down arrow) and beneficial effect (up arrow) of microorganisms on plants were presented. PAB – identified strains of bacteria

Fig. 2

A phylogenetic tree based on ITS sequences demonstrated for the identified strains of fungi compared to other strains. The bar indicates sequence divergence. The black rectangle marks fungi that colonized mature stigmas from opened flowers (red dots). The potentially pathogenic (down arrow) and beneficial effect (up arrow) of microorganisms on plants were presented

Fig. 3

Selected colonies of culturable bacteria and fungi. a orange colonies of Sphingomonas aquatilis (Rose Bengal Agar), b bacterial isolates grown on the Trypticase Soy Agar, dominant: 1. Pantoea agglomerans. 2. Pseudomonas lurida. 3. Stenotrophomonas maltophilia, c Beauveria bassiana grown on Yeast Extract–Peptone–Glycerol Agar, with Stenotrophomonas maltophilia bacteria nearby, d Fusarium avenaceum (on the left) and Cladosporium sp. (on the right) on the Rose Bengal Agar

Fig. 4

Agglomerative Hierarchy Clustering analysis (Bray‒Curtis method) of distance between phyla and order representing microbial communities inhabiting Phelipanche arenaria (PA) stigmas. Immature stigmas from closed flowers (2–4) and mature stigmas from opened flowers (1) (fungi on the right, bacteria on the left)

Fig. 5

Principal Component Analysis (PCA) of the data on microbial communities and diversity indices (a) and the data of biochemical potential of identified microorganisms vs. diversity indices (b) of the immature (PA2-PA4) and mature stigma (PA1) Phelipanche arenaria samples. The lines represent the correlation coefficient between the principal component scores and each of the factors. a Set I included: Paenibacillus spp., Rhizobiaceae, Xanthomonadaceae, Pseudomonas fragi, Pantoea ananatis, Hannaella coprosmae, Chaetosphaeronema sp., Alternaria eichhorniae, Mycosphaerellaceae, Gibberella tricincta, Filobasidium wieringae, Sarocladium strictum, Aureobasidium pullulans, Mycosphaerella tassiana, Pleosporales, Dothideomycetes, Ascomycota, Capnodiales, Fungi, Phoma sp.. Set II included: Sphingomonas spp., Methylobacterium-Methylorubrum sp., Evenness-b, Xylophilus sp., Chryseobacterium spp, Massilia sp., Pseudarthrobacter sp., Diversity-b, Duganella sp., Nocardioides sp., Microbacteriaceae. Set III included: Pseudomonas tolaasii, Aeromicrobium spp., Luteibacter spp., Variovorax sp., Aureimonas spp. b Set I included: N-acetyl-ß-glucosaminidase, α-galactosidase, valine arylamidase, leucine arylamidase, malic acid. Set II included: potassium nitrate (nitrate reduction), Naphtol-AS-BI-phosphohydrolase, D-glucose (fermentation), potassium gluconate, ß-galactosidase and evenness indices of fungi. Set III included: cystine arylamidase, phosphatase alkaline and α-glucosidase

Fig. 6

Heatmap Cluster Analysis of the normalized data: potential biochemical activity expressed by microbial communities between immature and mature stigmas samples of Phelipanche arenaria. The increase in the relative content of the potential biochemical activity is represented by a transition from green to black to red, as shown in the legend

Fig. 7

Studied holoparasitic Phelipanche arenaria. a general habit, (b, c) opened flower with whitish stigma, phot. R. Piwowarczyk; (d-g ZOOM micrographs of two-lobed stigma of P. arenaria with numerous papillae covered with a viscous secretion and above the pistil with glandular trichomes, phot. K. Zubek. Scale bars: c, d, f, g 1 mm; e 0.5 mm

Fig. 8

Experiment scheme of analysis microbial communities of stigmas in Phelipanche arenaria