A unique midgut-associated bacterial community hosted by the cave beetle \u3cem\u3eCansiliella servadeii\u3c/em\u3e (Coleoptera: Leptodirini) reveals parallel phylogenetic divergences from universal gut-specific ancestors

Background Cansiliella servadeii (Coleoptera) is an endemic troglobite living in deep carbonate caves in North-Eastern Italy. The beetle constantly moves and browses in its preferred habitat (consisting in flowing water and moonmilk, a soft speleothem colonized by microorganisms) self-preens to convey material from elytra, legs, and antennae towards the mouth. We investigated its inner and outer microbiota using microscopy and DNA-based approaches. Results Abundant microbial cell masses were observed on the external appendages. Cansiliella’s midgut is fully colonized by live microbes and culture-independent analyses yielded nearly 30 different 16S phylotypes that have no overlap with the community composition of the moonmilk. Many of the lineages, dominated by Gram positive groups, share very low similarity to database sequences. However for most cases, notwithstanding their very limited relatedness with existing records, phylotypes could be assigned to bacterial clades that had been retrieved from insect or other animals’ digestive traits. Conclusions Results suggest a history of remote separation from a common ancestor that harboured a set of gut-specific bacteria whose functions are supposedly critical for host physiology. The phylogenetic and coevolutionary implications of the parallel occurrences of these prokaryotic guilds appear to apply throughout a broad spectrum of animal diversity. Their persistence and conservation underlies a possibly critical role of precise bacterial assemblages in animal-bacteria interactions

Edophytes Watching: combining molecular and microscopy approches to isolate, identify, tag, and monitor fungi and bacteria inside plants

This work aims at investigating plant-endophyte interactions under many different standpoints. We took into consideration bacterial as well as fungal microorganisms. Regarding the fungi, endophytic species occurring in orchid plants either as possible mycorrhizal partner as well as other internal colonizers were studied. We analyzed three different species of wild orchids found in the Euganean Hills area, North-Eastern Italy, namely Orchis militaris, Spiranthes spiralis and Orchis purpurea, which are mostly regarded as species in endangered status and for which little is known on the nature and presence of symbionts. An approach involving both molecular methods and microscopy techniques was used. Fungal isolation was performed from surface sterilized roots to obtain pure mycelia. A molecular approach allowed us to amplify the Internal transcribed spacer (ITS) region, starting both from root portions of the orchid plants and from pure cultured mycelia. This genetic region varies relatively little within species but dramatically between species and is easy to amplify because of its high copy number. In addition relatively few primers sets are needed due to the highly conserved SSU and LSU flanking regions. Different amplicons were obtained and analyzed from each species, at first upon their different ARDRA profiles obtained by enzymatic digestions. Representative cases were sequenced and results were examined by BLAST. Fungi of mycorrhizal nature and an additional series of endophytic ones, which are an important component of fungal biodiversity, were found. With different microscopy approaches (Fluorescence, Confocal and Transmission Electron Microscopy) we localized fungi within plant tissues and investigated their features. The hyphal septa found in Orchis militaris and Spiranthes spiralis samples were of basidiomycete type, which was confirmed by the results obtained from DNA extraction, ITS amplification and sequencing. In parallel bacterial endophytes were considered, starting from a previous work in which the coexistence of rhizobia with diverse, endophytic bacterial taxa within nodules of wild legume plants had been demonstrated, using molecular and microscopy-based approaches. In this work in order to co-localize the relevant endophytes inside plant tissues, different fluorescent proteins were used as markers for the different kinds of bacteria. Bacterial strains tagged with GFP were obtained using the pUTgfr2X plasmid, a delivery system for a mini-Tn5 trasposon, expressing kanamycin resistance and the GFP protein. While to obtain bacteria tagged with the rfp gene, a replacement of the gfp with a rfp gene was made starting from plasmid pRL765gfp, obtaining pRL765rfp. Both pRL765rfp and pUT gfp2X vectors were used to incorporate the GFP or RFP cassettes into the chromosome of R. leguminosarum bv. trifolii. In both cases plasmids were introduced by biparental mating. Pseudomonas sp. Hs1::gfp from a wild type Pseudomonas sp. isolated from wild legume nodules was obtained introducing pUTgfp2X by biparental mating. In parallel, to tag an Enterobacter agglomerans also isolated from legumes, pRL765rfp was introduced by electroporation. The four bacterial strains constructed were used to inoculate seedlings of Trifolium repens in nodulation tests. Tagged bacteria were localized on the surface and within plant tissues using Confocal microscopy. Subsequently Pseudomonas sp. Hs1::gfp and Enterobacter agglomerans pRL765rfp were used as co-inoculant strains during nodulation tests performed with seeds of wild legume plants from Sardinia. Their ability to be true endophytes was investigated using jointly standard colony isolation methods and direct PCR amplification of prokaryotic DNA from nodules and other tissues. We found that Pseudomonas sp. Hs1::gfp, upon root inoculation was able to invade one of the wild species of legumes (Tetragonolobus purpureus) and be traslocated to its aerial portions.Questo lavoro ha come obbiettivo, lo studio con diversi approcci delle interazioni tra piante ed endofiti, sono perciò stati esaminati sia microrganismi batterici che fungini. Per quanto riguarda i microrganismi fungini, sono state studiate le specie endofitiche riscontrabili in piante di orchidea, sia come possibili partner micorrizici che come generici colonizzatori interni. Abbiamo analizzato tre diverse specie di orchidee selvatiche, considerate in pericolo, ritrovate nell’area dei Colli Euganei (Orchis militaris, Spiranthes spiralis e Orchis purpurea) per le quali poco si conosce riguardo alla presenza ed alla natura dei simbionti. Per questo studio è stato scelto un approccio che prevedeva l’utilizzo sia di metodologie molecolari che di tecniche di microscopia; inoltre per ottenere miceli fungini in coltura pura si è provveduto a isolarli dalle radici delle piante sterilizzate in superficie. L’approccio molecolare ci ha permesso di amplificare la regione ITS, partendo sia da miceli in coltura pura che direttamente da porzioni di radici. La regione ITS è una regione facilmente amplificabile con un numero relativamente esiguo di primers, per il suo alto numero di copie e per l’alta conservazione delle regioni che la fiancheggiano. Ulteriormente questa regione ci permette di ottenere interessanti informazioni, in quanto varia relativamente poco all’interno delle specie, ma molto tra specie diverse. Per ogni specie di orchidea analizzata sono stati ottenuti diversi ampliconi, che sono stati differenziati in base ai lori diversi profili ARDRA in seguito a digestione enzimatica. I casi più rappresentativi sono stati sequenziati e i risultati sono stati analizzati su piattaforma BLAST. Mediante analisi di omologie di sequenze sono stati identificati alcuni funghi di natura micorrizica e una serie di altri funghi endofitici, che risultano essere una componente importante della biodiversità fungina all’interno dei tessuti delle piante. Diversi tipi di microscopia: a fluorescenza, confocale ed elettronica a trasmissione sono stati usati per localizzare gli stessi funghi all’interno dei tessuti e analizzarne le caratteristiche. I setti (dolipori) ritrovati nelle ife fungine all’ interno dei campioni di Orchis militaris Spiranthes spiralis erano caratteristici dei basidiomiceti. Queste osservazioni hanno perciò permesso di confermare i risultai ottenuti dalle indagini molecolari. Partendo da un precedente lavoro nel quale è stata dimostrata la coesistenza di rizobi e altri batteri endofitici all’interno di noduli di leguminose selvatiche, per questo progetto si sono considerati anche gli endofiti di tipo batterico. In particolare per localizzare gli endofiti all’interno dei tessuti delle piante si è deciso di utilizzare dei marker fluorescenti diversi per tipi differenti di batteri endofitici e rhizobi. In alcuni ceppi batterici è stato inserito il gene codificante la proteina GFP usando un plasmide pUTgfr2X. Questo sistema trasporta un mini trasposone-Tn5, che esprime oltre alla proteina fluorescente anche la resistenza alla kanamicina. Per ottenere i ceppi marcati con RFP si è dovuto manipolare il plasmide pRL765gfp sostituendo il gene codificante la GFP con quello per la RFP, ricavando così un nuovo plasmide chiamato pRL765rfp. Per quanto riguarda i rizobi sia pRL765rfp che pUTgfp2X sono stati introdotti per coniugazione in R. leguminosarum bv. trifolii, così che i geni codificanti le proteine fluorescenti si integrassero nel cromosoma. Considerando invece le specie endofitiche diverse dai rizobi, un ceppo di Pseudomonas sp. Hs1::gfp è stato ottenuto introducendo per coniugazione in Pseudomonas sp. wt il plasmide pUTgfp2X. Pseudomonas sp. wt era stato precedentemente isolato da noduli di leguminose selvatiche così come Enterobacter agglomerans. Quest’ultimo ceppo (Enterobacter agglomerans) è stato marcato con la RFP introducendo il plasmide pRL765rfp per elettroporazione. I quattro ceppi batterici ottenuti sono stati utilizzati per inoculare piantine di Trifolium repens nei test di nodulazione, utilizzando il microscopio confocale è stato possibile localizzare i batteri sulla superficie delle radici o all’interno delle stesse Successivamente gli stessi endofiti marcati, utilizzati nei test di nodulazione su T. repens (Pseudomonas sp. Hs1::gfp e Enterobacter agglomerans pRL765rfp), sono stati co-inoculati in plantule di leguminose selvatiche della Sardegna per investigare la loro abilità di essere dei veri endofiti. Per questa ultima parte del lavoro si sono utilizzate comuni tecniche di isolamento e amplificazione del DNA dei procarioti tramite PCR. Pseudomonas sp. Hs1::gfp in particolare è in grado di colonizzare una delle specie analizzate (Tetragonolobus purpureus) e di essere traslocato nelle parti aeree

Co-localizing symbiont and endophytic bacteria in legumes by tagging with different fluorescent proteins.

Bacterial endophytes can promote plant growth and yield, suppress pathogens, solubilize nutrients or contribute to nitrogen uptake in plants. The use of fluorescent reporters is nowadays a key tool for studying microbe-plant interactions. In a previous work we assessed the presence of different endophytes in addition to rhizobia inside root nodules of wild legume plants and showed that rhizobia share nodules with a variety of different co-infecting taxa (Muresu et al., 2008). In the present report we explore the plant-endophyte relationships attempting the co-localization of endophytes (Pseudomonas sp., Enterobacter agglomerans) and rhizobia (Rhizobium leguminosarum bv. trifolii) by the introduction of different fluorescent proteins as bacterial markers for the different kinds of bacteria. This would help their localization throughout the plant and in particular the distinction of rhizobia from other endophytes coinfecting root nodules. Green fluorescent protein (GFP) and red fluorescent protein (RFP) markers were integrated into the bacterial chromosome. For the marked constructs, a suicide plasmid, carrying the gfp gene in a transposable element was mobilized into the endophytic species. To obtain rfp-tagged bacteria, a replacement of gfp with rfp was made starting from the pRL765gfp plasmid. The use of dual fluorescence markers will allow to co-localize different bacterial taxa within plant tissues and will enable to plan different innovative applications in the field of symbiosis, biocontrol and other endophytic plantmicrobe interactions

Analysis, determination and cultivation of endophytic fungi associated with the orchid Spiranthes spiralis.

Mycorrhizas can sustain plants in most habitats through mutualistic interactions with fungi. The presence of these in internalized location can be due to true symbiotic relationships or to different levels of endophytism. In the case of orchids the fungal assistance is crucial throughout the seedling stage and only established plants begin to return organic carbon to the symbiont. Fungi from orchids are often difficult to isolate and their cultivation depends on seasonal and physiological conditions. The submediterranean species Spiranthes spiralis (L.) Chevall. is the latest-blooming native orchid in western Europe. Its rosettes appear in summer to die off in the following spring, while stalks flower in September (Willems and Dorland, 2000). The putative mycorrhizal fungi were isolated in culture from the surfacesterilized Spiranthes spiralis roots and their taxonomical identity was assessed by molecular techniques upon amplification of diagnostic ribosomal DNA regions. In parallel, DNA was isolated directly from the root tissues for comparison. Selective PCR amplification using ITS1-ITS4, ITS1F-ITS4 primers was carried out. Additionally, using fluorescence and confocal microscopy on acridine orange-stained freehand sections we observed a diffuse cortical colonization by intracellular hyphae. Their further ultrastructural details were resolved by electron microscopy

Fungi associated with the southern Eurasian orchid Spiranthes spiralis (L.) Chevall

The hitherto unknown relationships between the European orchid Spiranthes spiralis (L.) Chevall and its internally associated fungi were explored by a combined approach involving microscopy-based investigations at a morpho-histological level as well as by molecular analyses of the identity of the eukaryotic endophytes present in the root tissue of the plant. We found that this orchid which is currently reported to have a vulnerable status in northern Italy, can host and interact with at least nine types of fungi. Some of these fungi show similarity to mycorrhizal genera found in orchids such as the Ceratobasidium-Rhizoctonia group. Other fungi found are from the genera Davidiella (Ascomycota), Leptosphaeria (Ascomycota), Alternaria (Ascomycota), and Malassezia (Basidiomycota), some of which until have not previously been reported to have an endophytic relationship with plants. The repeated occurrence of often pathogenic fungi such as Fusarium oxysporum, Bionectria ochroleuca, and Alternaria sp., within healthy specimens of this orchid suggests a tempered interaction with species that are sometimes deleterious to non-orchid plants. The fact is reminiscent of the symbiotic compromise established by orchids with fungi of the rhizoctonia group

Identification of the root nodule symbiont of the rare legume species Hedysarum confertum Desf. (Hedysarum humile L.) in its Italian relictual site

Hedysarum confertum Desf. (Fabaceae) represents a particular rarity in the Italian flora, signalled as late as in 1931 and nowadays still occurring only in few isolated stations in the central Apennine mountains. Within a framework of loss prevention of relevant botanical resources and landscape protection, we aimed at investigating possible factors limiting the distribution of this taxon and its potential to colonize suitable habitats. For these reasons we verified the existence of symbiotic relationships between H. confertum and soil bacteria assessing their identity and physiology. The plant was found to form histologically complete root nodules which are regularly invaded by bacteria. However, bacterial isolation in pure culture and cultivation ex planta appears prevented by a state of non-culturability. To assess the taxonomical position of nodule occupants the problem was circumvented by direct PCR amplification of 16S ribosomal RNA gene and nucleotide sequencing which revealed that H. confertum hosts bacteria of the genus Mesorhizobium, and that their ribosomal sequence has undergone a higher-than-average degree of divergence from the mesorhizobia nodulating other legumes in different countries. These differences suggest a possible link between the non-culturability of the symbiont and the isolated relictual status of its host plant

Use of antioxidant treatments to enhance culturability of rhizobial and non-rhizobial endophytes from nodules and other plant tissues

When rescuing either symbionts or other endophytic bacteria from internal plant tissues, including root nodules, and attempting their isolation and culturing on laboratory media, failure to obtain colonies is often experienced. This phenomenon applies particularly to the rhizobia from wild legume species which frequently result in non-culturable status (1), possibly as a consequence of a biochemical stress inherent to the isolation procedure. Other taxa can co-occupy nodules along with rhizobia, mostly belonging to the Bacillaceae, Pseudomonadaceae and Enterobacteriaceae families, which appear much less sensitive to the putative stress as their isolation from nodules, is apparently not impaired. We hypothesized that the limited culturability of rhizobia, compared to that of other types of nodule invaders, could be related to an oxidative stress caused by the histological disruption of plant tissues when surface-sterilized nodules are squashed for bacterial isolation. In order to test this hypothesis we isolated bacteria from nodules of two mediterranean wild legumes (Hedysarum spinosissimum and Tetragonolobus purpureus) by squashing nodules either in control phosphate buffer saline (PBS) or in PBS supplemented with scavenging systems apt to prevent damage from reactive oxygen species. The treatments included: an antioxidant cocktail (Glutathione, Sodium Ascorbate and EDTA) or an enzyme cocktail (Catalase, Peroxidase, Superoxide Dismutase). The mixtures were tested either as modified squashing buffers or as additions to the plates on which bacteria were spread after squashing in control PBS. The latter option allowed to distinguish whether the oxidative stress were due to immediate damage from plant-derived reactions or as a consequence of the transfer in carbon-rich agar media from the plate culturing stage. Some of the combinations yielded orders of magnitude-different increases of culturability in terms of colony counts and also allowed the cultivation of otherwise undetectable taxa. The coexistence of up to three different rhizobiaceae within the same nodules was observed, two of which of supposedly non-symbiotic nature (unable to re-induce nodule formation in axenic nodulation tests). Isolates were subsequently tested for tolerance to H2O2 in liquid cultures, which showed differences in comparison with the levels displayed by non-alphaproteobacterial endophytes. Ecological and physiological implications are discussed

Identification of Two Fungal Endophytes Associated with the Endangered Orchid Orchis militaris L.

A survey of the endangered orchid Orchis militaris populations was carried out in north-eastern Italy. The occurrence of fungal root endophytes was investigated by light and electron microscopies and molecular techniques. Two main sites of presence were individuated in the Euganean Hills, differing as to the percentage of flowering individuals and of capsules completing maturity. Fluorescence microscopy revealed an intracellular cortical colonization by hyphal pelotons. Two ITS PCR products co-amplified. Sequencing revealed for the former an identity and a high similarity (99%) with a Tulasnellaceae (Basidiomycota) fungus found within tissues of the same host in independent studies in Hungary and Estonia, suggesting an interesting case of tight specificity throughout the Eurosiberian home range. The second amplicon had 99% similarity with Tetracladium species (Ascomycota) recently demonstrated as potential endophytes. TEM revealed two different hyphal structures. Double fungal colonization appears to occur in Orchis militaris and the possible requirement of a specific fungal partner throws light on the causes of this plant\u2019s rarity and threatened status