Simon Cadiou
email: simon.cadiou@chu-rennes.fr

Omar Al Tabaa

Chi-Duc Nguyen

Marine Faccin

Raphaël Guillin

Matthieu Revest

Pascal Guggenbuhl

Eric Houvenagel

Edouard Pertuiset

Guillaume Coiffier

Cadiou Simon

; Al Tabaa Omar

; Nguyen

; Faccin

Guillin Raphaël

; Revest

Guggenbuhl Pascal

Houvenagel Eric

; Pertuiset

Back pain following instillations of BCG for superficial bladder cancer is not a reactive complication review of 30 Mycobacterium bovis BCG vertebral osteomyelitis cases

Keywords: Bladder cancer, BCG-instillation, Mycobacterium bovis, vertebral osteomyelitis, Interferon-γ release assay

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mycobacterium tuberculosis is the most frequent pathogen of vertebral osteomyelitis worldwide [START_REF] Gouliouris | Spondylodiscitis: update on diagnosis and management[END_REF], and represent half of osteoarticular tuberculous infections [START_REF] Tuli | General principles of osteoarticular tuberculosis[END_REF]. In the past decades in developed countries, the incidence of tuberculosis in spinal infections decreased, involving 6% of vertebral osteomyelitis [START_REF] Doutchi | Changing trends in the epidemiology of vertebral osteomyelitis in Marseille, France[END_REF].

Bacillus Calmette-Guérin (BCG) is a strain obtained from Mycobacterium bovis, used for vaccination and immunotherapy for bladder cancer since 1976 [START_REF] Morales | Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors[END_REF]. Complications of bladder instillations occur in about 5% or less of patients [START_REF] Lamm | Efficacy and safety of bacille Calmette-Guérin immunotherapy in superficial bladder cancer[END_REF], mainly with local urinary adverse effects such as pollakiuria, cystitis or haematuria.

Systemic reactions with spinal involvement such as Reactive Arthritis (ReA) [START_REF] Bernini | Reactive arthritis induced by intravesical BCG therapy for bladder cancer: our clinical experience and systematic review of the literature[END_REF] can appears in weeks following instillations. Two principal clinical patterns were described: symmetric or asymmetric polyarthritis (involving small or large and upper-lower joints are more common than asymmetric mono-oligoarthritis the large joints of lower limbs (especially, knee and ankle). Actually, ReA after BCG bladder instillations cannot be fully integrated in the spondyloarthritis concept, because clinical patterns are less correlated with HLA B27 status, if compared with ReA from enteric and urogenital infections, and the sacro-iliac or spinal involvement are rarer [START_REF] Bernini | Reactive arthritis induced by intravesical BCG therapy for bladder cancer: our clinical experience and systematic review of the literature[END_REF].

However, ReA after BCG bladder instillations are not the only osteoarticular events described in literature, and true infections such as mycobacterial vertebral osteomyelitis have also been described. Therefore, we performed a literature review of 27 cases previously published , and we had three original French cases.

Original cases

A. Case 1

A 65-year-old male was admitted to Pontoise hospital in January 2014 for low back pain which lasted for three months. His medical history included weaned smoking, hypertension, and a non-infiltrating urothelial carcinoma of the bladder. He was treated from December 2012 to March 2013 with six BCG instillations. Low back pain began rapidly, 6 months after the end of BCG instillation without any other triggering factor. He had lost eight kilos in three months and developed night sweats. A first X-ray showed moderate osteoarthritis of the spine. In December 2013, Magnetic Resonance Imaging (MRI) was performed. An inflammatory signal of L3 and L2 vertebral bodies and L2-L3 discs was found, with slight soft tissue reaction. Metastatic bone involvement was first suspected.

The physical examination on his admission in the rheumatology unit showed limited multidirectional lumbar mobility of the lumbar spine, without neurological deficit. The rest of the medical examination was normal. C-reactive protein (CRP) was high, at 123 mg/L. A PET-scan was performed. It showed an intense uptake of L2 and L3 vertebral bodies. Finally, an L3 bone biopsy was performed showing non-specific osteitis without granuloma. Acid-fast bacillus (AFB) testing and bacterial cultures including Lowenstein and Coletsos were negative. in February 2014, a second MRI demonstrated a typical image of L2-L3 spondylodiscitis with pre-and paravertebral abscesses. A new biopsy did not show histological abnormalities, but two samples were positive for Staphylococcus epidermidis. Antibiotics were started, without biological improvement. During hospitalisation, the patient became confused and developed pyramidal signs, without neurological deficit. A new MRI demonstrated vertebral fracture of L2 with retreat of the posterior wall, mild epidural involvement, partial compression of cauda equina, associated with a right psoas abscess (Figure 1).

At needle aspiration of the psoas abscess direct examination for acid-fast bacilli (AFB) was positive, A 4-drug anti-tuberculosis therapy was then initiated. PCR identified M. tuberculosis complex (MBTC), and cultures revealed six weeks later M. bovis, finally identified as M. bovis BCG by the French national reference center for tuberculosis. Pyrazinamide was discontinued. Surgical treatment was performed due to neurological signs and vertebral instability, consisting of L2 vertebrectomy, posterior laminectomy of L3 and T11-L5 arthrodesis. Ethambutol was stopped after two months. Isoniazid and rifampicin were continued for a total duration of three years because of persisting images in the psoas on MRI. Clinical, biological and radiological outcome was good. Thirteen months after discontinuing treatments, the patient was considered to be cured.

B. Case 2

A 73-year-old man was hospitalised in Rennes University Hospital in July 2017 for low back pain for 11 weeks. His medical history only reported a urothelial carcinoma, treated with transurethral resection of bladder tumor (TURBT) and 9 instillations of BCG, ended in December 2016. He was a retired veterinarian. Low back pain onset was reported as brutal, triggered by a bending effort. Secondarily, he developed anorexia, weight loss and asthenia.

Before his hospitalisation, he was examined with a CT-scan showing an osteolysis of L1 vertebral body. He was referred to his oncologist and had a PET-scan as part of a metastatic hypothesis. It showed an uptake of L1 and L2 vertebral bodies (Figure 2), with possible epiduritis. For further explorations, he had lumbar spine MRI two months after the first symptoms (Figure 2). A typical vertebral osteomyelitis of L1 and L2 vertebral bodies was found, with centro-somatic erosion suggesting a Mycobacterium infection.

The physical examination at his admission only showed limited multidirectional lumbar mobility, and no neurological impairment. Laboratory analysis showed a normal white blood cell (WBC) count, and a high CRP level of 70 mg/L. A first discovertebral puncture was negative on direct examination, MBTC PCR was negative, and cultures were sterile after seven days. A puncture of the right psoas abscess showed AFB on direct examination. MBTC was identified in a culture six days later. The search for mutation of rpoB that confers resistance to rifampicin was negative, and an anti-tuberculosis quadritherapy was started. M. bovis BCG was finally identified by PCR and pyrazinamide was stopped. In June 2018, the patient had an excellent evolution, with a good tolerance of his nine months of triple therapy.

C. Case 3

A 77-year-old man, with a history of gastric adenocarcinoma (1996), urothelial carcinoma (2011) and chronic alcoholism, was hospitalised in August 2017 in the rheumatology department of the Saint Philibert hospital (Lomme) for insidious onset of low back pain a few weeks earlier. He had been diagnosed with a bladder papillary urothelial carcinoma (pTa high-grade) in 2011, treated by transurethral resection in July 2011, followed by intravesical BCG therapy from September 2011 to August 2014. He reported inflammatory back pain evolving since 2016, without radiculalgia. There was no weight loss. He experienced fever at 38.2°C, associated with an increase of CRP levels of 126 mg/L.

Spine MRI showed an L4-L5 vertebral osteomyelitis with fluid collection developed in the epidural space, sheathing the right L5 and S1 roots. A discovertebral biopsy was performed, showing AFB on direct examination and the MTBC was detected using real-time PCR. Other tests were unremarkable (negative HIV serology, CD4 cell count was normal, no hypogammaglobulinemia, no hypocomplementemia). The thoracoabdomino-pelvic CT scan did not find another tuberculous localisation. Blood cultures were sterile and the interferon-gamma release assays were negative. The culture was sterile. The patient did not report any contact with tuberculosis. Antituberculosis therapy with isoniazid, rifampicin and ethambutol was introduced. Evolution was favourable after 12 months of treatment, with a decrease in pain and fever, and a decrease in acute phase reactants. At the end of follow-up, he was just diagnosed with lung cancer.

Methods

Based on PubMed research on the 1 st January 2019, we used to identify Mycobacterium bovis BCG vertebral osteomyelitis the following keywords: "osteomyelitis AND BCG AND bladder", "spondylodiscitis AND BCG AND bladder", "spondylitis AND BCG AND bladder". Seventy-five articles had been identified. We also used the keywords "aneurysm AND BCG AND bladder", and looked for vertebral osteomyelitis due to overlap with M. bovis BCG infectious aneurysms, identifying 42 articles.

A total of 117 articles had been identified, and 24 cases had been included. Three other articles had been identified through previous reviews (Figure 3). We had three original French cases.

Review

Since 1992, 25 cases of M. bovis BCG vertebral osteomyelitis following instillations for bladder cancer have been reported in English literature and two cases in non-English literature [START_REF] Stahl | Rare, but significant complication after BCG instillation therapy after urothelial carcinoma[END_REF][START_REF] Lara-Oya | Spondilodyscitis with medullary and spinal abscess caused by Bacillus Calmette-Guérin (BCG)][END_REF]. Other articles with M. bovis BCG infection and spinal pain have been published [START_REF] Bornet | Tuberculous aneurysm of the femoral artery: a complication of bacille Calmette-Guérin vaccine immunotherapy--a case report[END_REF][START_REF] Nam | Infected Aortic Aneurysm caused by Mycobacterium bovis after Intravesical Bacillus Calmette-Guérin Treatment for Bladder Cancer[END_REF][START_REF] Leeman | Psoas abscess after bacille Calmette-Guérin instillations causing iliac artery contained rupture[END_REF], but they were not consistent with a vertebral osteomyelitis diagnosis. The frequency is possibly underestimated knowing the possible difficulties in documenting such an infection, as in two other publications [START_REF] Białecki | Spinal tuberculosis with severe neurological symptoms as a complication of intravesical BCG therapy for carcinoma of the bladder[END_REF][START_REF] Sugita | Tuberculous spondylitis after intravesical BCG instillation: a case report[END_REF].

A. Clinical characteristics

We have collected information from 30 cases (27 literature cases and our 3 new cases), and we have listed the main features in Table 1. Diagnostic characteristics are resumed in Table 2. Case 3 was included in our review, given the recently proven usefulness of interferon-γ release assay in vertebral osteomyelitis infection with Mycobacterium tuberculosis [START_REF] Choi | Diagnostic usefulness of the QuantiFERON-TB gold in-tube test (QFT-GIT) for tuberculous vertebral osteomyelitis[END_REF], and its recommendation by the IDSA (Infectious Diseases Society of America) [START_REF] Berbari | 2015 Infectious Diseases Society of America (IDSA) Clinical Practice Guidelines for the Diagnosis and Treatment of Native Vertebral Osteomyelitis in Adults[END_REF].

We have estimated from these articles the period of time between the first and the last BCG instillation and the diagnosis (i.e. bacteriological diagnosis). When the dates mentioned in the article were not sufficiently detailed, we considered the first and the last instillation time-lap as the same. The median time between diagnosis and first or last instillation was 22.5 ± 28.5 months and 14 ± 25.5 months, respectively. At mean, patients received 9.4 ± 4.6 instillations of BCG. There was not enough information about the BCG strains to establish a difference between them. Seven of 30 cases had a cancer in their medical past history. No one had received immunosuppressive therapy between instillation and vertebral osteomyelitis diagnosis. Neoplasia was considered as cured, in remission [START_REF] Josephson | A case of Pott's disease with epidural abscess and probable cerebral tuberculoma following Bacillus Calmette-Guérin therapy for superficial bladder cancer[END_REF], or localized [START_REF] Civen | Vertebral osteomyelitis after intravesical administration of bacille Calmette-Guérin[END_REF][START_REF] Samadian | Mycobacterium bovis vertebral osteomyelitis and discitis with adjacent mycotic abdominal aortic aneurysm caused by intravesical BCG therapy: a case report in an elderly gentleman[END_REF]. No patients had received DMARDs for rheumatic condition, and one had psoriasis without musculoskeltal disorder.

All M. bovis BCG vertebral osteomyelitis cases occurred in men of 73.4 ± 8.7-year-old. Twenty-six cases involved two contiguous vertebrae, and two cases one vertebra. Half of infections affected the thoracic spine (from T1-T2 to T12-L1), and the other half affected the lumbar spine (from L1-L2 to L5-S1). No cervical osteomyelitis was reported. Back pain was almost always present (97%). The median of back pain duration at diagnosis was 5 (1-9) months, and the median period of time between the last instillation and back pain was 5 (0-16.5) months. No peripheral skeletal involvement had been reported.

Clinically, fever was reported only in 20% of cases, 75% developed anorexia and/or weight loss and/or asthenia. Sensitivo-motor deficit was present at diagnosis in 42% of cases.

On imaging (CT scan and/or MRI) 70% had epiduritis or epidural abscesses (Figure 1), and 47% had paravertebral/psoas abscesses (Figure 1 and2). Other infectious locations occurred in nine cases, mostly abdominal aortic aneurysms.

Biologically, 77% of patients had CRP > 5 mg/l, and 21% an elevated WBC count. The tuberculin test was negative for 5 out of 6 patients, and the seven IFN-gamma tests were all negative (only one patient receive both tests, both negative). Biopsy showed a granuloma in 60% of cases, of which 56% was necrotic. Mycobacterium bovis BCG was almost always identified through cultures.

The direct exam was positive in 55% of bacteriological samples. All molecular tests and PCR were positive. Bacteriological identification was done through CT-guided biopsy with 79% positivity, and through surgical biopsy or abscess puncture which were always positive. When CT-guided biopsies were negative, diagnosis was made with surgical biopsy in two cases, and with abscess puncture in two cases. Abscess puncture was the only procedure used in four cases.

B. Treatment and outcomes

Frequently, the classical anti-tuberculosis therapy was started, and then Pyrazinamide was stopped when BCG was identified due to BCG natural resistance [START_REF] Scorpio | Rapid differentiation of bovine and human tubercle bacilli based on a characteristic mutation in the bovine pyrazinamidase gene[END_REF]. Isoniazid, rifampicin and ethambutol were normally used throughout the treatment period. Nevertheless, antibiotic modalities were heterogeneous (Table 1). Treatment duration, known for 20 cases, was 12.1 ± 7.0 months.

The outcome was known for 14 patients with 18.5 months of mean follow up: 12 cases were considered cured, and 2 died from cardiovascular causes without vertebral osteomyelitis symptoms. Fifty percent had one or more cases of spine surgery, (ranging from one to three surgeries). Among them, five patients had spinal arthrodesis during the follow-up: three for neurological worsening, one for persistent root pain, and one for vertebral instability.

C. Comparison of M. bovis BCG with M. tuberculosis

Compared to vertebral osteomyelitis due to M. tuberculosis [START_REF] Batirel | The course of spinal tuberculosis (Pott disease): results of the multinational, multicentre Backbone-2 study[END_REF], M. bovis BCG vertebral osteomyelitis seems to involved an older population with a male predominance, probably due to the bladder cancer population. However, we noticed that all cases in this review were men, while 74.1% of non-muscle-invasive bladder carcinoma in the United States are male [START_REF] Seo | Demographic and Survivorship Disparities in Non-muscle-invasive Bladder Cancer in the United States[END_REF]. About clinical presentation, lumbar and thoracic involvement of BCG vertebral osteomyelitis were consistent with literature, but no cases of nonadjacent multiple-level involvement were reported with BCG after bladder instillation. Neurological complications at diagnosis and psoas abscesses prevalence seems to be higher in BCG than tuberculosis.

In contrast of tuberculosis, there was no other sites of M. bovis BCG vertebral osteomyelitis infection, whereas vertebral Pott's disease is frequently associated to pulmonary tuberculosis at diagnosis. We assumed that's it's probably due to the gateway of Mycobacteria: through airway contamination leading to pulmonary tuberculosis, and through catheterization leading to local urinary reaction or systemic reaction which are sometimes treated with antituberculosis treatment, decreasing risk of other infection.

D. Pathogenesis discussion

BCG instillations are a cornerstone for superficial bladder carcinoma treatment, and they have changed the prognosis of this disease [START_REF] Morales | Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors[END_REF][START_REF] Lamm | Efficacy and safety of bacille Calmette-Guérin immunotherapy in superficial bladder cancer[END_REF]. Septic side effects are rare, between 0.4% and 1/15,000 according to Lamm [START_REF] Lamm | Efficacy and safety of bacille Calmette-Guérin immunotherapy in superficial bladder cancer[END_REF], and the benefit of the treatment remains higher than the risk of such events. However, other distant infections are described in the literature, mostly vascular aneurysms in a recent literature review [START_REF] Higashi | Mycobacterium bovis-induced Aneurysm after Intravesical Bacillus Calmette-Guérin Therapy: A Case Study and Literature Review[END_REF], but also disseminated infections also called BCGitis [START_REF] Geerdes-Fenge | BCGitis with Involvement of Lung, Liver and Bone Marrow after Immunotherapy of Urothelial Cancer][END_REF], small-vessel central nervous system vasculitis [START_REF] Parent | The first case of bacillus Calmette-Guérin-induced smallvessel central nervous system vasculitis[END_REF] and even nosocomial infections, including vertebral osteomyelitis [START_REF] Gupte | Mycobacterium bovis BCG spinal osteomyelitis in a patient with bladder cancer without a history of BCG instillation[END_REF]. Some risk factors of distant infections could be suggested: a traumatic catheterisation during instillation [START_REF] Lamm | Efficacy and safety of bacille Calmette-Guérin immunotherapy in superficial bladder cancer[END_REF], haematuria, instillations in the few days around an urological trauma [START_REF] Mackel | Mycobacterial Osteomyelitis of the Spine Following Intravesical BCG Therapy for Bladder Cancer[END_REF] (pelvic radiation, surgery, bladder resection), arterial catheterisation [START_REF] Laberge | Diagnosis please. Case 9: mycotic pseudoaneurysm of the abdominal aorta in association with mycobacterial psoas abscess--a complication of BCG therapy[END_REF], and general immunosuppressive factor such as HIV infection.

Pathogenesis of M. bovis BCG-associated infections following instillations can be supported in three ways. First, through the pelvic lymphatic drainage system, particularly for psoas abscesses. Even if iliopsoas abscess pathogenesis is not clear, haematogenous or lymphatic spread is commonly admitted [START_REF] Shields | Iliopsoas abscess -A review and update on the literature[END_REF]. Such abscesses can be linked to BCG spread from bladder to the lymph nodes, particularly those closely related to the iliopsoas muscle, such as the common iliac and paravertebral lymph nodes. However, an exclusive lymphatic pathogenesis underlies an infection by contiguity only with vertebrae which have an anatomical relationship with iliopsoas, i.e. T12 to L4. Secondarily, as recently suggested [START_REF] Mackel | Mycobacterial Osteomyelitis of the Spine Following Intravesical BCG Therapy for Bladder Cancer[END_REF], through the vertebral venous plexus (known as Batson's venous plexus). Once again, a venous spread of BCG does not explain thoracic osteomyelitis. Finally, through an arterial route: Higashi et al. [START_REF] Higashi | Mycobacterium bovis-induced Aneurysm after Intravesical Bacillus Calmette-Guérin Therapy: A Case Study and Literature Review[END_REF] suggested a septic aneurysm pathogenesis from vasa vasorum, or directly by adhesion on atherosclerotic degeneration in a review of 29 cases (including three common cases of patients with vertebral osteomyelitis and infectious aneurysm). Considering these vascular infections and the pathogenesis of haematogenous spread in vertebral osteomyelitis, arterial dissemination seems to be the principal way, as suggested by Civen et al. [START_REF] Civen | Vertebral osteomyelitis after intravesical administration of bacille Calmette-Guérin[END_REF] who described a positive blood culture of BCG. However, there seems to be an association between infectious aneurysm and vertebral osteomyelitis: among the 17 cases including L1 to L4 vertebral involvement (i.e., vertebrae having a direct anatomical relationship with the abdominal aorta), there were five out of six cases of infectious aneurysm, and only one thoracic osteomyelitis was associated with infectious aneurysm. This higher frequency of aneurysms in lumbar osteomyelitis suggests pathogenesis other than the arterial route for aneurysms. Finally, a simultaneous infectious aneurysm may also seem to be associated with cardiovascular risk, because of a more frequent cardiovascular medical history (Table 1).

Mycobacterium bovis BCG identification is crucial in M. bovis BCG associated osteomyelitis. Culture is the first step, but a specific identification has to be done. Oldest cases used high-pressure liquid chromatography (HPLC), which has been nowadays replaced by molecular technics. Multiplex PCR targeting specific regions of Mycobacterium spp. genomes can now differentiate with a total specificity [START_REF] Quan | Development of one-tube multiplex polymerase chain reaction (PCR) for detecting Mycobacterium bovis[END_REF] M. bovis BCG from the others mycobacteria. It means that, when vertebral osteomyelitis is suspected in a patient with medical history of BCG instillations for bladder carcinoma, AFB on direct examination or positive culture for MTBC must have the biologist carry out the precise identification of the strain by mutliplex PCR, including BCG detection, the therapeutic impact being significant.

Other related events following BCG instillations were described, such as ReA [START_REF] Bernini | Reactive arthritis induced by intravesical BCG therapy for bladder cancer: our clinical experience and systematic review of the literature[END_REF] with a HLA-B27 (or B51 in the Japanese population) association, suggesting a Th17 response. On the other hand, the anti-tumour effect of BCG therapy is based on a Th1 response [START_REF] Luo | Th1 cytokine-secreting recombinant Mycobacterium bovis bacillus Calmette-Guérin and prospective use in immunotherapy of bladder cancer[END_REF] as well as the anti-infectious response. Tumour recurrence could not be evaluated in this review but occurred in eight patients. Based on purely immunological considerations, we assume that there could be a link between tumour recurrence and these signalling pathways. M. bovis BCG associated infections could be correlated to a lack of Th1 lymphocyte polarisation, and thus an increased risk of recurrence, making M. bovis BCG-associated infections a risk factor for the inefficacy of BCG instillations. In the same way, Th17 polarisation observed in ReA could reduce a Th1 response (because of the exclusive nature of the polarisation [START_REF] Das | Transforming growth factor beta is dispensable for the molecular orchestration of Th17 cell differentiation[END_REF]). Such assumptions need more specific studies to evaluate correlations between tumour recurrence, infections and immunological reactions.

In conclusion, when vertebral osteomyelitis is suspected in a man with a medical history of bladder carcinoma treated with BCG instillations, M. bovis BCG osteomyelitis must be sought even if the last instillation was months before. A direct exam showing AFB and positive cultures followed by multiplex PCR are used for bacteriological diagnosis. The presence of aneurysms at osteomyelitis diagnosis should be investigated because of its potential life-threatening complications. The treatment is not codified, but the pyrazinamide resistance must be known. The duration of anti-tuberculosis therapy is heterogeneous, and individual adaptation has to be done, regards to the septic severity, neurological deficit, or treatment adverse effects.

Figure 2 .

 2 Figure 2. (a) MRI Axial T1 with Fat Sat sequence and Gadolinium injection showing epiduritis with partial cauda equina compression, and voluminous right psoas abscesses (b) MRI Sagittal T2 with Fat Sat, showing hypersignal of L1 and L2 vertebral body with a vertebral fracture of L2 and retreat of posterior wall.

Figure 3 .

 3 Figure 3. (a) 18F-FDG PET-CT showing FDG uptake on L1-L2 vertebral bodies (b) MRI Axial T1-weighted with contrast showed hypersignal of L2 body and a well circumscribed right psoas abscess with Gadolinium enhancement, without epiduritis. (c) MRI Sagittal STIR sequence of the lumbar spine showed hypersignal of L1 and L2 vertebral bodies, with hypersignal in the L1-L2 disc confirming the discitis. White arrow: centro-somatic erosion suggesting a Mycobacterium infection.

Figure 1 .

 1 Figure 1. (a) MRI Axial T1 with Fat Sat sequence and Gadolinium injection showing epiduritis with partial cauda equina compression, and voluminous right psoas abscesses (b) MRI Sagittal T2 with Fat Sat, showing hypersignal of L1 and L2 vertebral body with a vertebral fracture of L2 and retreat of posterior wall.

Figure 1

 1 Figure 1Click here to access/download;Figure;Figure 1_BCG_Submission_CR.docx

Figure 2 .

 2 Figure 2. (a)18F-FDG PET-CT showing FDG uptake on L1-L2 vertebral bodies (b) MRI Axial T1-weighted with contrast showed hypersignal of L2 body and a well circumscribed right psoas abscess with Gadolinium enhancement, without epiduritis. (c) MRI Sagittal STIR sequence of the lumbar spine showed hypersignal of L1 and L2 vertebral bodies, with hypersignal in the L1-L2 disc confirming the discitis. White arrow: centro-somatic erosion suggesting a Mycobacterium infection 60 .

Figure 2

 2 Figure 2 Click here to access/download;Figure;Figure 2_BCG_Submission_CR.docx

Figure

  Figure 3. Flow chart of literature review

Figure 2

 2 Figure 2 Click here to access/download;Figure;Figure 3_submission_CR.docx

Table 1 .

 1 Clinical, biological, bacteriological and imaging features of 30 reported cases of M. bovis BCG vertebral osteomyelitis after instillations of BCG for bladder cancer.

	Results

SD: standard deviation; IQR: interquartile range; ESR: erythrocyte sedimentation rate; WBC: white blood cell count; CRP: C-reactive protein.

Table 1

 1 Click here to access/download;Table;Table1_CR.docx

Table 2 .

 2 Description of 30 cases of M. bovis BCG vertebral osteomyelitis after instillations of BCG for bladder cancer.

# Periods of time are the same when the dates mentioned in the article were not enough detailed; HBP Hyper Blood Pressure; NR: not reported; AAA: abdominal aortic aneurysm; INH: isoniazid; RIF: rifampicin; ETH: ethambutol;

  3. Flow chart of literature review

	Identification	PubMed search with "osteomyelitis AND BCG AND bladder", "spondylodiscitis AND BCG AND bladder", "spondylodiscitis AND BCG AND bladder", "aneurysm AND BCG AND
		bladder" = 141
			24 duplicates
	Screening	117 after duplicates removing	93 results excluded:
			-44 other infections
			-37 of reactive arthritis
			-3 recommendations
			-5 letters to editor
			-2 other reasons
			-1 vertebral osteomyelitis
			without BCG instillation 46
		24 cases of M. bovis BCG	-1 vertebral abscess 36
		vertebral osteomyelitis in
		literature
		Other sources:
	Included	-2 results from a previous review 31 -1 from article
		references 15
		-3 new cases
		30 cases reviewed

Disclosure of interest

The authors declare that they have no competing interest.


domain: sdv sde

text:
Achouak Wafa

Danis Abrouk

Julien Guyonnet

Mohamed Barakat

Philippe Ortet

Laurent Simon

Catherine Lerondelle

Thierry Heulin

Feth El Zahar Haichar
email: fethelzaharhaichar@univ-lyon1.fr

Plant hosts control microbial denitrification activity

Keywords: Rhizosphere, Denitrification, Root, Root-adhering soil, Active microbiota, Root exudates, Nitrogen uptake

In the rhizosphere, complex and dynamic interactions occur between plants and microbial networks that are primarily mediated by root exudation. Plants exude various metabolites that may influence the rhizosphere microbiota. However, few studies have sought to understand the role of root exudation in shaping the functional capacities of the microbiota. In this study, we aim to determine the impact of plants on the diversity of active microbiota and their ability to denitrify via root exudates. For that purpose, we grew four plant species, Triticum aestivum, Brassica napus, Medicago truncatula and Arabidopsis thaliana, separately in the same soil. We extracted RNA from the root-adhering soil and the root tissues, and we analysed the bacterial diversity by using 16S rRNA metabarcoding. We measured denitrification activity and denitrification gene expression (nirK and nirS) from each root-adhering soil sample and the root tissues using gas chromatography and quantitative PCR, respectively. We demonstrated that plant species shape denitrification activity and modulate the diversity of the active microbiota through root exudation. We observed a positive effect of T. aestivum and A. thaliana on denitrification activity and nirK gene expression on the root systems. Together, our results underscore the potential power of host plants in controlling microbial activities.

INTRODUCTION

The ability to secrete a wide range of compounds into the rhizosphere is one of the most remarkable metabolic features of plant roots with approximately 5 to 21% of the total photosynthetically fixed carbon being transferred into the rhizosphere through root exudates [START_REF] Whipps | Carbon economy[END_REF][START_REF] Marschner | Mineral nutrition of higher plants[END_REF][START_REF] Nguyen | Rhizodeposition of organic C by plants: mechanisms and controls[END_REF]. These compounds are metabolized by soil-borne microorganisms as carbon and energy sources providing the basis for the establishment of plant-microorganism interactions that benefit plant growth by increasing the availability of mineral nutrients, production of phytohormones, degradation of phytotoxic compounds and suppression of soilborne pathogens [START_REF] Bais | The role of root exudates in rhizosphere interactions with plants and other organisms[END_REF]Philippot et al., 2013;[START_REF] Haichar | Root exudates mediated interactions belowground[END_REF]. This demonstrates the importance of studying the functional properties of the soil microbiota and the manner in which plant species influence bacterial diversity and microbial activities.

Denitrification is one of the microbial activities that occurs in the plant rhizosphere. Denitrification is the microbial process of the nitrogen cycle that allows the return of fixed nitrogen (reduction of atmospheric N2) to the atmosphere. The reduction of soluble NO3 -or NO2 -into gas (NO, N 2 O or N 2 ) by denitrification is catalysed by metalloenzymes: nitrate reductases (Nar and Nap), nitrite reductases (NirK and NirS), nitric oxide reductases (cNor and qNor) and nitrous oxide reductase (Nos) [START_REF] Zumft | Cell biology and molecular basis of denitrification[END_REF][START_REF] Philippot | Use of functional genes to quantify denitrifiers in the environment[END_REF]. This stepwise reduction of nitrate is an alternative respiration pathway occurring in the case of oxygen depletion by phylogenetically diverse microorganisms in a wide range of ecosystems [START_REF] Zumft | Cell biology and molecular basis of denitrification[END_REF]. Most denitrifiers belong to various subclasses of bacteria, although the ability to denitrify has also been found in some archaea and fungi (Philippot, 2002a). In addition, co-occurrences of denitrification genes do not appear to be randomly distributed among taxonomic groups (Graf et al., 2014).

Denitrification represents a significant loss of nitrogen in soils (25-90%) (van der [START_REF] Van Der Salm | N leaching across European forests: Derivation and validation of empirical relationships using data from intensive monitoring plots[END_REF][START_REF] Radersma | Assessing denitrification and N leaching in a field with organic amendments[END_REF], and this microbial process therefore affects the availability of nitrogen to plants. This process contributes to N 2 O emission, a powerful greenhouse gas (300-fold more heat-trapping capacity than CO 2 , per molecule) and the single most important ozone-depleting agent known [START_REF] Ravishankara | Nitrous oxide (N2O): The dominant ozone-depleting substance emitted in the 21st century[END_REF][START_REF] Coskun | Nitrogen transformations in modern agriculture and the role of biological nitrification inhibition[END_REF].

The major factors regulating denitrification can be modulated in the rhizosphere: nitrate concentration (via absorption by plants) and oxygen partial pressure (via root respiration and the surrounding root moisture) are decreased, while the C availability (via rhizodeposition) is generally increased [START_REF] Tiedje | Ecology of denitrification and dissimilatory nitrate reduction to ammonium[END_REF][START_REF] Mounier | Influence of maize mucilage on the diversity and activity of the denitrifying community[END_REF]Langarica-Fuentes et al., 1918). The impact of the rhizosphere on denitrifying activity has already been reported [START_REF] Mahmood | Denitrification with and without maize plants (Zea mays L.) under irrigated field conditions[END_REF][START_REF] Mounier | Influence of maize mucilage on the diversity and activity of the denitrifying community[END_REF][START_REF] Patra | Effects of grazing on microbial functional groups involved in soil dynamics[END_REF][START_REF] Henry | Disentangling the rhizosphere effect on nitrate reducers and denitrifiers: insight into the role of root exudates[END_REF]. For example, Philippot et al. (2002b) observed that the presence of maize roots led to a change in the structure of the nitrate-reducing community. However, little is known about how denitrification is regulated in the rhizosphere. Determining the role of host plant on denitrification process modulation is of real importance in understanding the regulation of denitrification in the rhizosphere.

The aim of this study was to determine the impact of four plant species, wheat (Triticum aestivum), rape (Brassica napus), barrel clover (Medicago truncatula) and Arabidopsis thaliana (ecotype Colombia), cultivated in the same soil on i) the diversity of the bacterial community by metabarcoding the 16S rRNA gene genes, and on ii) the activity of the denitrifying community in the root-adhering soil (RAS) and on the root system by evaluating the denitrification activity and nirK and nirS gene expression.

MATERIAL AND METHODS

Plant growth

A laboratory experiment was performed with winter wheat (T. aestivum L. cv. Taldor), rape (B. napus cv. Drakkar), A. thaliana (ecotype Colombia) and barrel clover (M. truncatula, ecotype A17) on a calcareous silty-clay soil collected from the upper 20 cm layer of the agricultural site located in the Aix en Provence region. The pH was 8.2 and it contained 5.7% sand, 46.7% silt, 47.6% clay, 1.0% CaCO 3 , 1.8% organic C and 0.18% organic N. The soil was sieved (1-mm mesh size), air-dried, and the water holding capacity (WHC) measured according to [START_REF] Bouyoucos | A new, simple, and rapide method for determining the moisture equivalent of soils and the role of soil colloids on this moisture equivalent[END_REF] based on the pulling out of water from soil by suction or vacuum forces. The WHC was determined in triplicates and was about 7.82%. 150 g dry soil was placed into polypropylene cylindrical pots. Soil moisture was maintained at 75% of WHC by adding the necessary amount of water. Seeds were sterilized according to [START_REF] Achouak | Phenotypic variation of Pseudomonas brassicacearum as a plant root-colonization strategy[END_REF] and one seed was planted per pot. Sixteen pots of each plant were grown in a growth chamber. 4 pots (4 repetitions) per plant were dedicated to denitrification activity measurements (on the RAS and the root system), 3 pots (3 repetitions) per plant were dedicated to molecular analysis (16S rRNA diversity from the RAS and the root system) and 9 pots per plant were dedicated to isotope labelling experiment.

Sixteen pots with soil but without plants (bulk soil treatment) were used: 4 pots (4 repetitions) were dedicated to denitrification activity measurements, 3 pots (3 repetitions) were dedicated to molecular analysis (16S rRNA diversity) and 9 pots were dedicated to isotope labelling experiment. Plants and bulk soil pots were incubated in a growth chamber with a day-night period about 12/12, respectively; light intensity was 400 mmol photon m -2 .s -1 and maximum daily temperature ranged from 20 to 22 °C. Soil moisture was manually controlled.

Plant harvesting

After 5 weeks of plant growth, plants were collected as follows: the root system of 3 replicates of each plant were separated from its root-adhering soil (RAS) and 2 g of RAS from each plant were frozen in liquid N 2 immediately and stored at -80 °C for molecular analysis. The root systems were washed with water to remove adhering soil particles, frozen in liquid N 2 and stored at -80 °C. The bulk soil microcosms (4 replicates) were also collected for microbial activities measurements and 2 g were frozen in liquid N 2 immediately and stored at -80 °C for molecular analysis.

The entire plant from 4 replicates for each plant was separated from the RAS and both, the whole plant and the RAS of the planted microcosm and soil of the unplanted microcosm were used to measure denitrification activity.

The remaining 9 replicates for each plant and the bulk soil treatment were used for isotope labelling experiments.

Measurements of microbial denitrification activities Denitrification Enzyme Assay without carbon addition

We measured the denitrification activity of microbial communities inhabiting the root system in each plant rhizosphere in 4 replicates according to [START_REF] Guyonnet | The effects of plant nutritional strategy on soil microbial denitrification activity through rhizosphere primary metabolites[END_REF]. The entire plant with the root system, rinsed with water to remove soil particles, was placed in a 150 ml airtight plasma-flask sealed by a rubber stopper. In each flask, air was removed and replaced with a He/C2H2 mixture (90/10 v/v) to create anoxic conditions and inhibit N2O-reductase. Potassium nitrate (50 µg of N-KNO3 g -1 of fresh root or dried soil) was added to each vial to provide microbial communities with nitrate source. The amount of N2O during incubation at 28 °C was measured each 4 h for 48 h. The slope of the linear regression was used to estimate anaerobic respiration (denitrification enzyme assay without the addition of carbon) to estimate the produced N2O (g -1 .h -1 ). N2O was measured with a gas chromatograph coupled to a micro-catharometer detector (µGC-R3000, SRA instruments, Marcy l'Etoile, France).

Denitrification enzyme activity (DEA) of plant roots and soils

We measured the denitrification enzyme activity (DEA) in soil, according to [START_REF] Bardon | Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites[END_REF]. Ten grams of fresh soil sample (RAS from each plant and bulk soil) were placed in a 150 ml airtight plasma-vial sealed with a rubber stopper. In each flask, air was removed and replaced with a mixture of He/C2H2 (90/10 v/v) to create anoxic conditions and inhibit N2O-reductase. A nutritive solution (0.5 ml) containing glucose (0.5 mg of C-glucose.g -1 of dried soil), glutamic acid (0.5 mg of C-glutamic acid g -1 of dried soil) and potassium nitrate (50 µg of N-KNO3 g - 1 of dried soil) was added to the soil. The amount of N2O during incubation at 28 °C was measured each hour for 5 h. The slope of the linear regression was used to estimate anaerobic respiration (denitrification) by measuring the production of N2O (g -1 .h -1 ) using a gas chromatograph coupled to a micro-catharometer detector (µGC-R3000, SRA instruments, Marcy l'Etoile, France).

RNA extraction and cDNA synthesis

RNA was extracted from the RAS and root system from each plant rhizosphere in triplicates using an RNA "Power Soil isolation" kit (MO BIO) that produced total non-degraded RNA according to the manufacturer's recommendations. DNA was removed from RNA extracted from RAS and root tissues by using RNeasy Mini Kit (Qiagen) according to the manufacturer's recommendations. 16S rRNA gene amplifications (300 bp) were carried out using the treated RNA as a template to ensure that DNA was completely removed. One µg of RNA from each RAS and root system sample retrieved from each plant was transcribed into complementary DNA (cDNA) using the SuperScript III Reverse Transcriptase (Life Technologies) according to the manufacturer's protocol and stored at -20 °C.

nirK and nirS genes expression

Denitrifier abundance was estimated by real-time quantitative reverse transcription PCR (qRT-PCR) targeting the nirK and nirS genes encoding the copper and cd 1 nitrite reductases, respectively. For nirK, the amplification was performed using the primers nirK876 (5'-ATYGGCGGVCAYGGCGA-3') and nirK1040 (5'-GCCTCGATCAGRTTRTGGTT-3') [START_REF] Henry | Quantification of denitrifying bacteria in soils by nirK gene targeted real-time PCR[END_REF]. The 20 µl final reaction volume contained SYBRgreen PCR Master Mix (QuantiTect SYBRgreen PCR kit, Qiagen, Courtaboeuf, France), 1µM of each primer, 400 ng of T4gp32 (MPbiomedicals, Illkvich, France) and from 1 to 3 µl of cDNA according to 16S rRNA gene normalisation. Thermal cycling was as follow: 15min at 95°C; 6 cycles of 95°C for 15s, 63°C for 30s with a touchdown of -1°C by cycle, 72°C for 30s; 40 cycles of 95°C for 15s, 58°C for 30s and 72°C for 30s. For nirS, the amplification was performed using the primers nirSCd3aF (5'-AACGYSAAGGARACSGG-3') and nirSR3cd

(5'-GASTTCGGRTGSGTCTTSAYGAA-3') (Throbäck et al, 2004). The 25 µl final reaction volume contained SYBRgreen PCR Master Mix (QuantiTect SYBRgreen PCR kit, Qiagen, Courtaboeuf, France), 1µM of each primer, 400 ng of T4gp32 (MPbiomedicals, Illkvich, France) and from 1 to 3 µl of cDNA according to 16S rRNA gene normalisation. Thermal cycling was as follow: 15min at 95°C; 6 cycles of 95°C for 15s, 59°C for 30s with a touchdown of -1°C by cycle, 72°C for 30s and 80°C for 30s; 40 cycles of 95°C for 15s, 54°C for 30s and 72°C for 30s and 80°C for 30s. The standard curves for nirK and nirS qPCR were generated by amplifying 10-fold dilutions (10 7 -10 2 ) of a linearized plasmid containing the nirK gene of Sinorhizobium meliloti 1021 and nirS gene of Pseudomonas stutzeri Zobell DNA (GenArt, Invitrogen, Lifetechnologies, Regensburg, Germany). Melting curves analysis confirmed the specificity of amplification and amplification efficiencies for nirK and nirS genes were higher than 90%.

Sequencing of 16S rDNA gene

For each plant, 10 µL of cDNA obtained from RNA extracted from the root system and the RAS from each plant in triplicates were sent to FASTERIS (Switzerland) for sequencing using MiSeq Illumina technology. V3-V4 domain of 16S rRNA was amplified with tagged primers 16S Fwd primer 3'-CCTACGGGNGGCWGCAG-5' and 16S Rev primer 3'-GACTACHVGGGTATCTAATCC-5'. Tagged primers structure was 5'-N2-4X6Pn -3', with "N2-4" were random bases, "X6" were 6-bases tag and "Pn" was specific primer. Amplification conditions were 3 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at 55 °C, 90 s at 72 °C, and 5 min at 72 °C.

Isotope labelling and analysis of plant and soil material

After 5 weeks of plant growth, all pots (9 pots per plant and 9 pots of bulk soil) received the same amount of two forms of nitrogen: ammonium (NH4 + ) and nitrate (NO3 -), and where the nitrogen was either 14 N or 15 N. 3 replicates from each plant and bulk soil treatment received 15 N labelled ammonium and unlabelled nitrate ( 15 NH4 + + NO3 -) and 3 others replicates received unlabelled ammonium and 15 N labelled nitrate (NH4 + + 15 NO3 -). The remaining 3 replicates per plant and bulk soil treatment received unlabelled nitrogen (NH4 + + NO3 -) in order to measure the natural abundance of 15 N. The four plant species and the unplanted soil underwent three treatments in triplicates resulting in 45 pots.

In total, 0.24 µg N.g -1 dry soil was added with 50 % of each nitrogen form. Five millilitres of each solution were added to each pot in 1 ml aliquots with a single injection homogeneously distributed over the soil surface. Each aliquot was injected with a syringe with 21-gauge needle that was slowly removed to ensure uniform distribution throughout the profile. After 12 h of incubation, the shoots of all pots were cut off and stored at -80 °C. The root systems and their root-adhering soil fractions were subsequently separated. The roots were rinsed with tap water.

All roots and RAS samples were stored at -80 °C. The 12 h incubation time was used in accordance with previous studies, which showed maximal uptake of label around this time [START_REF] Streeter | Amino acids as a nitrogen source in temperate upland grasslands: the use of dual labelled (13C, 15N) glycine to test for direct uptake by dominant grasses[END_REF][START_REF] Bardgett | Soil microbes compete effectively with plants for organic-nitrogen inputs to temperate grasslands[END_REF][START_REF] Weigelt | Inter-specific variability in organic nitrogen uptake of three temperate grassland species[END_REF].

After freeze-drying, 2.5 mg of crushed plant tissue and 10 mg of soil were used to measure nitrogen concentration and the 15 N/ 14 N ratio using an isotopic ratio mass spectrometer (Isoprime 100, Isoprime Ltd, Manchester, UK) coupled in continuous flow with an elemental analyser (FlashEA 1112 Thermo Electron, Milan, Italy). N concentration data were calibrated using aspartic acid. For the 15 N/ 14 N measurements, a two-point normalization of the data was performed using international reference material IAEA-305a and IAEA-311 [28]. The 15 N/ 14 N ratios were expressed as atomic fraction (i.e. atomic %, Coplen, 2011):

x( 15 N) = ( 15 N/ 14 Nsample) / (1 + 15 N/ 14 Nsample)

Values of atom fraction and concentration of N (in %) were used to calculate uptake of 15 N on a per unit mass basis (mg excess of 15 N per gram). Mean values of 15 N abundance of the unlabelled control plants were used as reference for 15 N excess.

Bioinformatics analysis Sequence processing and data analysis

Sequence data were processed and analysis of high-throughput community sequencing data was performed with QIIME version 1.8 [START_REF] Caporaso | QIIME allows analysis of high-throughput community sequencing data[END_REF]. Sequences were trimmed of barcodes and primers, and then short sequences (< 200 bp), sequences with ambiguous base calls, and sequences with homopolymer runs exceeding 6 bp were removed. Operational Taxonomic Units (OTUs) were then defined by clustering at 97% similarity. Final OTUs were taxonomically classified using Blast [START_REF] Altschul | Basic local alignment search tool[END_REF] and compiled into each taxonomic level into both "counts" and "percentage" files. OTU tables were rarefied at the lowest sequencing depth to control for differences in sequencing depth. A total of 7,185,297 valid reads and 392,892 OTUs were obtained from the 27 samples through sequencing analysis (3 root samples and 3 root-associated soil samples per plant species (4 species) and 3 bulk soil samples).

Alpha diversity analysis was done using the Phyloseq R package version 1.20.0 [START_REF] Mcmurdie | phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data[END_REF]. Species richness (observed richness) and species evenness (Inverse Simpson index) were estimated by multiple subsampling with replacement (n = 10 3 ) to the minimum libraries sizes to standardize the sequencing effort.

Core microbiota

The core microbiota was identified using QIIME [START_REF] Caporaso | QIIME allows analysis of high-throughput community sequencing data[END_REF] and was determined by plotting OTU abundance in the core at 5% intervals (from 50% to 100% of samples). We defined the core microbiota of each plant as the OTUs present in 100% of samples. Determination of a core microbiota was accomplished by comparing all samples from each plant across the two compartments (RAS and root). Any taxa found to be ubiquitous across all samples were then defined to be part of the core microbiota of the compartment. From these data Venn diagrams were constructed using the R package: VennDiagram, to show common and unique OTUs within the four plant species (T. aestivum, B. napus, M. truncatula and A. thaliana).

Network analysis

Network inference was made by computing all Spearman's rank correlations between all OTUs of the "family" taxonomic level. P-value was adjusted using the procedure of [START_REF] Benjamini | Controlling the False Discovery Rate: A Practical and powerful approach to multiple testing[END_REF] to finally consider only significant correlation (Spearman correlation coefficient |r| > 0.8 and P-adj < 0.05). The same procedure was used to compute the correlation between each OTU and each compartment (root system and root-adhering soil).

Each network was visualized, analysed and different metrics (Table S1) (i.e. degree, diameter, average path length, average clustering coefficient, modularity) were calculated using the Gephi open-source software [START_REF] Bastian | Gephi: an open source software for exploring and manipulating networks[END_REF].

We analysed OTUs interactions by comparing the high and low DAE graphs for the R and RAS compartments.

By computing intersection, union or difference of this graphs, we can retrieve edges present or not in our DAE condition of interest. The graphical analysis was done using the igraph R package.

Statistical analysis

All results are presented as means (± standard error). For each plant, a one-way analysis of variance (ANOVA) and post-hoc Tukey HSD were performed to test the effect of root exudation on measured variables. Before analysis, Shapiro and Bartlett tests were performed to ensure conformity with the assumptions of normality and homogeneity of variances. Effects with p < 0.05 are referred to as significant.

In order to test the significance between microbiota inhabiting the RAS and those colonizing the root tissues on all plants a non-parametric permutation-based multivariate analysis of variance (PERMANOVA, vegan R package, Anderson 2001) on abundance based (Bray-Curtis) dissimilarity matrix was performed. All statistical analyses were carried out using R statistical software 3.1.0 (R Development Core Team, 2008).

Data deposition

The sequence data generated in this study was deposited at EMBL-ENA public database (http://www.ebi.ac.uk/ena/data/view/PRJEB25281).

RESULTS

Denitrifying enzyme activity in plant root systems

To investigate the impact of the root exudates produced by each plant species on denitrifying activity, we measured the emission of N2O after nitrate amendment only on each plant root system, without adding any carbon source (Fig. 1A). The denitrification activity of the microbiota colonizing the root system of each plant species differs significantly from one another. The denitrification activity was higher on the root system of T. aestivum and A. thaliana (5.2 and 4.6 g N-N2O h -1 .g -1 dried roots, respectively) and very low on the root system of M. truncatula and B. napus (0.6 and 0.08 g N-N2O h -1 .g -1 dried roots, respectively). Interestingly, B. napus root exudates appear to inhibit denitrification activity in the root system or counterselect denitrifying microorganisms.

Denitrifying enzyme activity on the root-adhering soil

To determine the potential rate of the denitrification of microorganisms present on the root-adhering soil, the DEA of the RAS fractions retrieved from each plant rhizosphere was measured. The DEA of T. aestivum, B. napus and M. truncatula are significantly different from the unplanted soil (Fig. 1B), indicating a rhizosphere effect.

However, the DEA of A. thaliana is significantly lower than that of the unplanted soil and the rhizosphere of the other plants. In addition, T. aestivum, B. napus and M. truncatula do not differ significantly from each other (Fig. 1B).

Preferential uptake of soil nitrogen forms by plant species: NO3 -and/or NH4 +

In shoot tissues, the 15 N content after the separate addition of NO3 -or NH4 + for each plant species reveals a significant preferential uptake of NO3 -by T. aestivum, B. napus and A. thaliana plantlets (Fig. 2A). Conversely, none of the forms of N tested are preferentially absorbed by M. truncatula in our experimental conditions. B. napus had a significantly greater uptake capacity for NO3 -than the other plant species. The patterns of the root tissue contents in 15 N show the same trend as the shoot tissues with a significant preferential uptake of NO3 -by T. aestivum and B. napus (Fig. 2B). As with the shoot tissues, M. truncatula uptakes NO3 -or NH4 + forms of nitrogen.

Diversity of active microbiota inhabiting each plant rhizosphere

To investigate the impact of the root exudates produced by each plant species on the selection of active microbiota, the RNA extracted from the root-adhering soil and the root system was reverse transcribed and analysed using 16S rRNA gene sequencing. An average of 7,185,297 sequences were generated and utilized for analysis. The rarefaction curves, displaying the observed OTUs richness as a function of the sequencing effort, indicated that the sequencing depth has almost been reached to completely capture the diversity present in the bulk soil, RAS and root tissues of all the plants (Fig. S1). The richness and diversity of species (Inverted Simpson) were significantly increased in the RAS compared to the bulk soil for each plant, except for M. truncatula, and decreased in the root compartment compared to the RAS fraction, especially for A. thaliana and B. napus (Fig. S2).

Several phyla were present in different amounts between the two compartments studied (RAS and root system) and between the plants (Fig. 3A). In addition, globally on all plants, significant differences (p. value = 0.001) were observed between microbiota inhabiting the RAS and those colonising the root system. The general trend is a very low difference between microbial diversity in the RAS fraction of all the plantlets compared to the one of the reservoir (bulk soil) at the phyla level. Another general trend is an important increase in the Bacteroidetes abundance (five-fold in the rhizosphere of T. aestivum, B. napus and M. truncatula) and of the OD1 phylum (from x15 to x150 in the four rhizosphere samples) (Additional File 1). In the root tissues, Chloroflexi was more abundant on the root system of T. aestivum (8%) than the other plants (3-5%), while Bacteroidetes was more abundant on the root system of B. napus (15%) and T. aestivum (13%) compared to the other plants (9-10%) (Fig. 3A, Additional File 1). Verrucomicrobia was highly enriched on the root system of B. napus (8%) compared to the other plants (1-3%). Firmicutes and Acidobacteria were less abundant on the plant root systems (1-2% and 1-3% respectively), except on the root tissues of A. thaliana where they were slightly more abundant (3% and 5% respectively) (Fig. 3A, Additional File 1). The relative abundance of Crenarchaeota was increased on the root system of A. thaliana (0.3%) compared to that of the other plants (less than 0.07%). For the RAS fractions, few differences in phyla abundance were observed between the plant species. However, Bacteroidetes was more abundant in the RAS of A. thaliana (6%) than in those of T. aestivum, B. napus and M. truncatula (2-3%).

Planctomycetes was counter-selected in the RAS fraction of A. thaliana (9%) compared to other plant rhizospheres and bulk soil (15-17%) (Fig. 3A, Additional File 1). Some phyla have been more enriched on the RAS than on the root tissues and vice versa independently of the plant species (Fig. 3A, Additional File 1). For example, this is the case for Actinobacteria, Gemmatimonadetes, Plantomycetes and Crenarchaeota, which were more abundant in the RAS of B. napus, T. aestivum and M. truncatula compared to their root systems (from two-fold to ten-fold increases). Despite the high abundance of Proteobacteria in all the plant rhizospheres, they were more abundant on the root tissues (44-52%) than in the RAS (34-39%).

To examine a potential role of the plant species on the root-associating bacterial assemblage, we compared the bacterial profiles obtained from T. aestivum, B. napus, M. truncatula and A. thaliana root compartments (Fig. 3B).

Within the root OTUs (rOTUs) community, only four OTUs were enriched in the root systems of all the plants studied, while 21, 106, 74 and 103 OTUs were significantly enriched in the root systems of A. thaliana, M. truncatula, T. aestivum, and B. napus, respectively (Fig. 3B, Additional File 2). The common and unique OTUs retrieved from the root system of each plant are shown in Additional File 2.

To examine the impact of the plant species on shaping the bacterial community inhabiting the root-adhering soil (RAS), we compared the bacterial profiles obtained in the root-adhering soil of T. aestivum, B. napus, M. truncatula and A. thaliana (Fig. 3C). We identified 24 root-adhering soil OTUs (rasOTUs) shared by the four plant species. These shared OTUs can be regarded as the core microbiome of the four rhizospheres. Unique rasOTUs corresponding to the bacterial community specifically inhabiting each plant species were also observed with 130, 65, 133 and 1683 OTUs found in the rhizosphere of A. thaliana, M. truncatula, T. aestivum, and B. napus, respectively. Common and unique OTUs retrieved from the plant root-adhering soils are shown in Additional File 3.

Network description

To explore co-occurrences between the bacterial phyla colonizing the root system and inhabiting the rhizosphere (RAS fractions) of T. aestivum, B. napus, M. truncatula and A. thaliana, we used a network inference based on strong and significant correlations (using non-parametric Spearman's) (Fig. 4, Table S1). The number of positive correlations (co-occurrences) was higher than the number of negative correlations (co-exclusions) in the rhizosphere of B. napus and A. thaliana and equal for the two others (T. aestivum, M. truncatula) (Table S1).

The bacterial network of OTUs from the root system (rOTU) and the RAS (rasOTUs) compartments of B. napus is highly connected (Fig. 4). The structure of the network demonstrates densely connected groups of nodes, forming a clustered topology. Comparing the properties of the calculated networks, we observed that the B. napus network contains a significantly higher percentage of negative (220) and positive (171) edges than the other plants (Table S1). OTUs considered to be keystone species belonged primarily to different genera within the phyla of Proteobacteria, Planctomycetes and Actinobacteria (Fig. 4).

The T. aestivum bacterial network that associates OTUs from the root system (rOTU) and its RAS (rasOTUs) is also highly connected (Fig. 4) with 105 positive edges and 110 negative edges (Table S1). The OTUs considered to be keystone species (depicted as nodes with larger sizes in the network) belonged primarily to different genera within the phyla of Proteobacteria, Bacteroidetes and Actinobacteria (Fig. 4).

For the bacterial networks of M. truncatula and A. thaliana, we noticed a clear separation between the rOTUs and rasOTUs with a very weak connection between them. More bacterial phyla colonizing RAS co-occur in the rhizosphere of M. truncatula compared to the root system (Fig. 4). For A. thaliana, the co-occurring bacterial phyla display an equal distribution in the RAS and the root system, and very few co-occur on the root tissues.

Finally, co-exclusions and co-occurrences found between certain rOTUs and/or rasOTUs retrieved from each plant are shown in Additional File 4. Overall, regardless of the plant studied, we noticed more co-exclusion and less cooccurrence relationships among the significant interactions (Fig. 4 and Table S1). Among several positive interactions observed, we noted strong co-occurrence relationships for some members of Firmicutes

thaliana. A positive correlation was also observed between Rhizobiaceae and Chloroflexi and between

Chlamydiales and Planctomycetales on the root system of T. aestivum and M. truncatula, respectively (Additional File 4). Among several negative interactions observed, we noted a co-exclusion relationship for A. thaliana for the OTUs of the Acidobacteria (Chloracidobacteria) found on the RAS with the OTUs of the Firmicutes (Paenibacillaceae) found on the root system (Additional File 4). In the rhizosphere of T. aestivum, the OTUs from Gemmatimonadetes were negatively correlated with those of the Opitutales (Verrucomicrobia). For example, in the rhizosphere of B.

nirS and nirK transcript levels in the rhizosphere microbiota

To gain additional insights on the role of root exudates in regulating denitrification, we measured the expression of the nirK and nirS genes on the root system and in the root-adhering soil of each plant species (Fig. 5). The number of nirK (encoding a copper-containing nitrite reductase) transcripts from the denitrifiers was higher on the root system of A. thaliana and T. aestivum (2.3 X 10 4 and 2.8 X 10 4 nirK cDNA copies per g of dried root respectively), in contrast with a lower expression on the root system of B. napus (0.9 X 10 4 nirK cDNA copies per g of dried root) and the almost complete absence of nirK expression on the root system of M. truncatula (Fig. 5A).

The nirS gene that encodes a cytochrome cd1-containing nitrite reductase was detected only on the root system of M. truncatula (1.7 X 10 4 nirS cDNA copies per g of dried root).

Given the level of expression of the nirK gene in the root-adhering soil, no significant difference was found between the RAS of T. aestivum and M. truncatula compared to the bulk soil. In contrast, the expression of nirK was significantly decreased in the RAS of A. thaliana and B. napus compared to the bulk soil (Fig. 5B). As on the root system, the expression of nirK is the lowest in the RAS of the B. napus plantlets (Fig. 5A and5B). No expression of nirS was detected in the RAS fraction of the four plant species and in the bulk soil. In summary, only nirK (not nirS) was expressed on the root system of T. aestivum and A. thaliana and, to a lesser extent, on the one of B. napus and repressed in the presence of M. truncatula. nirK was repressed in the RAS of A. thaliana and B. napus and unchanged in that of T. aestivum and M. truncatula. Surprisingly, nirS expression was detected only on the root system of M. truncatula.

DISCUSSION

Plant species shape denitrification activity through root exudation

The measurement of the denitrification activity of the root system without additional carbon (Fig. 1) as conducted in a previous study [START_REF] Guyonnet | The effects of plant nutritional strategy on soil microbial denitrification activity through rhizosphere primary metabolites[END_REF], likely displayed higher activity than in the root-adhering soil where carbon source was added. This result suggests the role of the root exudates of each plant species in the management of the denitrification activity. This plant intervention might be conducted by selecting beneficial microorganisms involved in plant nutrition and protection against pathogens, which may happen to denitrify. The host plant may also produce compounds that could control the expression of the genes involved in denitrification. Figure 1 shows a higher denitrification activity on the root system of T. aestivum and A. thaliana, which is consistent with the high expression of the bacterial nirK gene for these two host species (Fig. 5). This suggests a potential positive effect of the root exudates from T. aestivum and A. thaliana to select bacterial populations able to denitrify or to activate the expression of denitrification genes. The very low level of denitrification activity on the root system of B. napus is consistent with a lower level of expression of the nirK gene compared to that of the root system of T. aestivum and A. thaliana. These results suggest that the plantlets of B. napus may not recruit bacterial populations able to denitrify or may alter the expression of denitrification genes on the root system probably via root exudation. In addition, by measuring the preferential uptake of B. napus for nitrate and ammonium, our results demonstrated a greater avidity of B. napus for nitrogen uptake primarily in the form of nitrate compared to the other plants (Fig. 2), suggesting potential competition for nitrate with denitrifiers as previously observed for other plants [START_REF] Kuzyakov | Competition between roots and microorganisms for nitrogen: mechanisms and ecological relevance[END_REF][START_REF] Bardon | Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites[END_REF]. Denitrification inhibition, named Biological Denitrification Inhibition (BDI), has already been observed in Fallopia sp. and has been defined as the ability of the plant to release secondary metabolites such as procyanidins that inhibit denitrifiers, and therefore enables the plant to recover nitrate for its growth [START_REF] Bardon | Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites[END_REF][START_REF] Bardon | Mechanism of biological denitrification inhibition (BDI) : procyanidins induce an allosteric transition of the membrane-bound NO3reductase through membrane alteration[END_REF][START_REF] Bardon | Biological denitrification inhibition (BDI) with procyanidins induces modification of root traits, growth and N status in Fallopia x bohemica[END_REF]. B. napus may perform BDI, because the presence of procyanidins has already been demonstrated on its roots [START_REF] Wronka | Localization of phenolic compounds in the root cap Columella of six-year-old dry seeds of Brassica napus during imbibition and germination[END_REF][START_REF] Nesi | The promoter of the Arabidopsis thaliana BAN gene is active in proanthocyanidinaccumulating cells of the Brassica napus seed coat[END_REF]. On the root system of M. truncatula, the nitrogen uptake was very low regardless of its form, suggesting that unlike B. napus, competition for nitrogen with denitrifiers is not expected. However, the low denitrification rate primarily achieved by denitrifying bacteria harbouring the nirS gene indicates a counter-selection of the populations harbouring the nirK gene. However, we did find that a high level of denitrification activity on the legume RAS positively correlated with the nirK gene expression (Fig. 1). Several studies have investigated the effect of legume cultivation on nitrogen cycle processes, since legumes associated with nitrogen-fixing bacteria can be used as a substitute for mineral fertilizers [START_REF] Philippot | Ecology of denitrifying prokaryotes in agricultural soil[END_REF]. Researchers reported high denitrification rates with legume rhizospheres compared to other plants [START_REF] Svensson | Soil denitrification in three cropping systems characterized by differences in nitrogen and carbon supply[END_REF][START_REF] Kilian | Enhanced denitrification in plots of N2 fixing faba beans compared to plots of a non-fixing legume and non-legumes[END_REF] which is consistent with our data. Although, several studies have reported that denitrification is very common in rhizobia and that many strains can denitrify both as nodule bacteroids and as free-living bacteria [START_REF] Philippot | Ecology of denitrifying prokaryotes in agricultural soil[END_REF]. In our study, either M. truncatula root-associated Rhizobia (bacteroids or free-living bacteria) are not involved in the denitrification process, since nirK gene expression was not detected (Fig. 5, Additional File 2), or it can be assumed that only the nirS copy is transcribed, since it was recently suggested that certain rhizobia may possess Cu-type (nirK) and cd1-type (nirS) nitrite reductase genes (Sánchez and Minamisawa, 2018).

Our results provided evidence that the denitrification activity of the microbial communities inhabiting the rhizosphere (root-adhering soil) of T. aestivum was significantly higher than that of the unplanted soil, where less carbon is available (Fig. 1). Similarly, the measurement of the denitrification rate of cereals, such as barley grown in pots [START_REF] Klemedtsson | Microbial nitrogen transformations in the root environment of barley[END_REF][START_REF] Metz | Detection methods for the expression of the dissimilatory copper-containing nitrite reductase gene (DnirK) in environmental samples[END_REF] and maize in field conditions [START_REF] Mahmood | Denitrification with and without maize plants (Zea mays L.) under irrigated field conditions[END_REF], also revealed high denitrification activity compared to the unplanted soil. However, even though denitrification is primarily conducted by bacteria, we cannot exclude the role of fungi and archaea as previous studies have already shown their capacity to denitrify (Philippot, 2002a[START_REF] Maeda | N2O production, a widespread trait in fungi[END_REF]. In addition, the nirK and nirS primers used in this study are not universal enough to survey all the soil denitrifiers, since they were designed based on a limited number of sequences, primarily from laboratory strains. Further studies developing new nirK and nirS primers and targeting denitrification genes expression from fungi and Archaea are needed to confirm our hypothesis.

Core and specific active microbiota among plant species

Metabarcoding of 16S rRNA revealed the enrichment of members of Chloroflexi on the root system of T. aestivum, Bacteroidetes and Verrucomicrobia on that of B. napus and Firmicute and Acidobacteria on that of A. thaliana at the root system level (Figure 3A, Additional File 1). These results are consistent with previous data using a stableisotope probing (SIP) approach on B. napus [START_REF] Gkarmiri | Identifying the active microbiome associated with roots and rhizosphere soil of oilseed rape[END_REF], A. thaliana [START_REF] Bulgarelli | Revealing structure and assembly cues for Arabidopsis rootinhabiting bacterial microbiota[END_REF][START_REF] Lundberg | Defining the core Arabidopsis thaliana root microbiome[END_REF] and wheat [START_REF] Wang | Single cell stable isotope probing in microbiology using Raman microspectroscopy[END_REF].

Comparison of the microbiota inhabiting the root-adhering soil revealed differences in the phyla abundance with high abundance, for example, Bacteroidetes in the rhizosphere of A. thaliana (Figure 3A, Additional File 1), which is consistent with previous research by [START_REF] Bulgarelli | Revealing structure and assembly cues for Arabidopsis rootinhabiting bacterial microbiota[END_REF] on this model plant. Remarkably, the rhizosphere of A. thaliana was enriched with the Crenarchaeota phylum, which is consistent with the results of [START_REF] Bressan | Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots[END_REF]. This phylum, considered to be the most abundant ammonia-oxidizing phylum in soil ecosystems [START_REF] Leininger | Archaea predominate among ammonia-oxidizing prokaryotes in soils[END_REF], has been found to be involved in root exudate assimilation in the rhizosphere of A. thaliana using an SIP approach [START_REF] Bressan | Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots[END_REF].

The comparison of the microbial assemblages revealed differences and similarities in the composition of the microbiota inhabiting the roots and the rhizosphere retrieved from the four plant species (Fig. 3B andC). First, we demonstrated that four rOTUs belonging to members of the families Caulobacteraceae, Comamonadaceae, Chitinophagaceae and Bacillaceae were enriched in the root systems of all the plants studied. These rOTUs can be considered generalists, since they are associated with the root system of all plants studied as determined by [START_REF] Guyonnet | The effects of plant nutritional strategy on soil microbial denitrification activity through rhizosphere primary metabolites[END_REF]. Second, some clear differences were observed between the microbiota from the four plant root systems (Fig. 3B andC). For example, some members of the Cytophagaceae and the Chitinophagaceae family are specifically selected by A. thaliana (Additional File 3), while members of the families Opitutaceae, Verrucomicrobiacea, Solibacterales and Ellin 6075 were specifically associated with B. napus. In contrast, the enrichment of the members of the Microbacteriaceae family appears to be a distinct feature of the microbiota of the T. aestivum roots. In a recent study by [START_REF] Tomasek | Increased denitrification rates associated with shifts in prokaryotic community composition caused by varying hydrologic connectivity[END_REF], a positive correlation between the abundance of Cytophagaceae, Chitinophagaceae and Microbacteriaceae and denitrification activity was observed, suggesting that these families potentially play an important role in denitrification at the root of A. thaliana and T. aestivum.

For microbiota colonizing the root-adhering soil, we identified 24 rasOTUs common to all the plants studied (Additional File 3), suggesting that these OTUs represent generalist bacteria, while those retrieved only from a single plant species are considered to be specialist bacteria [START_REF] Guyonnet | The effects of plant nutritional strategy on soil microbial denitrification activity through rhizosphere primary metabolites[END_REF].

By comparing the root microbiota with the RAS microbiota, it appears that the root compartment is more selective than the soil surrounding the root system, indicating the specific recognition and nutritional selection of bacterial communities on the root tissues prior to the release of nutrients into the rhizosphere [START_REF] Guyonnet | The effects of plant nutritional strategy on soil microbial denitrification activity through rhizosphere primary metabolites[END_REF][START_REF] Haichar | Stable isotope probing of bacterial community structure and gene expression in the rhizosphere of Arabidopsis thaliana[END_REF][START_REF] Haichar | Host plant specific control of 2,4-diacetylphloroglucinol production in the rhizosphere[END_REF].

As the bacterial community colonizing the root system differs according to the plant species, the differences in denitrification activity could also be linked to the selection of certain denitrifying bacteria that are more or less effective for each plant through root exudates. Similarly, the diversity of the bacterial community inhabiting the root-adhering soil could also explain the denitrification activity profiles. Futures studies targeting the diversity of denitrifiers using denitrification genes, such as nirK and nirS, are needed. As described above, the current challenge is to design optimal nirK and nirS universal primer pairs to target entire denitrifier communities in the environment as already suggested by [START_REF] Bonilla-Rosso | Design and evaluation of primers targeting genes encoding NO-forming nitrite reductases: implications for ecological inference of denitrifying communities[END_REF].

Microbial interaction networks

Positive correlations between microbial populations suggest the occurrence of a mutualistic interaction and coexistence, while negative correlations might suggest the presence of host-competitive exclusion or a predation relationship between the microorganisms [START_REF] Steele | Marine bacterial, archaeal and protistan association networks reveal ecological linkages[END_REF], Barbéran et al., 2011[START_REF] Ju | Bacterial assembly and temporal dynamics in activated sludge of a full-scale municipal wastewater treatment plant[END_REF]. A closer look at the bacterial networks shows that several associations confirm or reveal interesting ecological patterns for the taxa that have not been as well studied as was also shown by Barbéran et al. (2011) (Fig. 4, Additional File 4). This is the case for the Crenarchaeotal OTU, which is particularly remarkable because of our poor understanding of the ecological niches occupied by this taxon, even though it has been proposed that related Crenarchaeota are ubiquitous in the soil (Bates et al., 2010;Barbéran et al., 2011) and that they may have an important role in the nitrogen cycle as ammonia oxidizers [START_REF] Simon | Crenarchaeota colonize terrestrial plant roots[END_REF][START_REF] Leininger | Archaea predominate among ammonia-oxidizing prokaryotes in soils[END_REF]Barbéran et al., 2011). In this study, taxa related to Crenarchaeota OTUs co-occurred with the Alphaproteobacteria class (Ellin329 order) and Acidobacteria in the RAS of B. napus and with Chloroflexi (Anaerollinea class) in the RAS of M. truncatula. The co-occurrence between the Crenarchaeota OTUs is probably involved in the nitrogen cycling with the OTUs of the families Acidobacteria, Alphaproteobacteria and Chloroflexi, which are probably also involved in carbon cycling [START_REF] Ward | Three genomes from the phylum Acidobacteria provide insight into the lifestyles of these microorganisms in soils[END_REF][START_REF] Hug | Community genomic analyses constrain the distribution of metabolic traits across the Chloroflexi phylum and indicate roles in sediment carbon cycling[END_REF][START_REF] Brauer | A Novel Isolate And Widespread Abundance Of The Candidate Alphaproteobacterial Order (Ellin 329), In Southern Appalachian Peatlands[END_REF], highlight the link between these two cycles and the importance of each for a better understanding of the other.

It is interesting to note that Nitrospirales and Solirubrobacterales co-occur only in the RAS of T. aestivum and B. napus displaying high DEA, suggesting a syntrophic relationship, in which ammonium released by Solirubrobacterales during the organic metabolism of N (Tu et al., 2017) could serve as a substrate for Nitrospira to achieve nitrification [START_REF] Daims | Complete nitrification by Nitrospira bacteria[END_REF] and provide nitrate to denitrifiers. Similar interactions might also be suggested between Candidatus nitrosphaera participating to nitrification process [START_REF] Spang | The genome of the ammonia-oxidizing Candidatus Nitrososphaera gargensis: insights into metabolic versatility and environmental adaptations[END_REF] and Rhodospirillales known to perform denitrification as described by [START_REF] Saarenheimo | Functional gene pyrosequencing reveals core proteobacterial denitrifiers in boreal lakes[END_REF].

CONCLUSION

Collectively our results have shown that plant species shape denitrification activity and modulate the diversity of active microbiota through root exudation. As expected, a positive effect of the root exudates on denitrification activity was observed on the root system of A. thaliana and T. aestivum, while B. napus appears to alter denitrification activity on the root system through root exudates. This denitrification inhibition is probably due to competition between B. napus and the denitrifiers for nitrate, since nitrate is preferentially utilized by B. napus. Some OTUs were associated with all the plants studied and are considered generalists, while others are specifically associated with plant species and are considered to be specialists. Network analysis indicated that Nitrospirales and Solirubrobacterales were positively correlated with high denitrification activity in the RAS of T. aestivum and B. napus, while a positive correlation was observed between Cytophagaceae, Chitinophagaceae and Microbacteriaceae and a high DEA at the roots of A. thaliana and T. aestivum. (ring of the cercal) corresponds to the phylum, while the size of the nodes is proportional to their abundance. The red and blue lines specify significant positive and negative correlations (Spearman correlations ≥ 0.8 or ≤-0.8, P.adj < 0.05) between two nodes, respectively. Full circles with green or brown colour correspondent to OTUs more abundant on the root system and the root-adhering soil respectively. Empty circles correspondent to OTUs with the same abundance between the root system and the root-adhering soil compartments.

FIGURES, TABLE, ADDITIONAL FILES

Additional files:

Table S1. Global network statistics for bacterial association networks from the rhizosphere of wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula), and Arabidopsis thaliana. Red edges correspondent to positive correlations (co-occurrences) and blue one to negative correlations (co-exclusions).

(

  Paenibacillaceae) with Acidobacteria and Actinobacteria (Frankiaceae) in the RAS of B. napus (Additional File 4). A positive correlation was also observed between Nitrospiraceae and Actinobacteria in the RAS of B. napus and A. thaliana and between Nitrospiraceae and Planctomycetes in the RAS of T. aestivum, B. napus and A.

  napus, the OTUs of the Chloroflexi correlate negatively with the OTUs of the Comamonadaceae. By comparing OTUs interactions under high and low DEA conditions in the R and RAS compartments of the studied plant, we have identified a few OTUs that co-occur only in the RAS of T. aestivum and A. thaliana, with high DEA, while, other OTUs co-occur only in the R of T. aestivum and B. napus with high DEA (Additional File 5), suggesting a potential role for these OTUs in denitrification. This is the case, for example, of the co-occurrence on the RAS of T. aestivum and B. napus between OTUs of Nitrospira and Solirubrobacterales and Rhodospirillales and Candidatus nitrososphaera or between OTUs of Acidobacteria and Verrucomicrobia on the R of T. aestivum and A. thaliana (Additional File 5).

Figure 1 .

 1 Figure 1. Denitrification activities of microbiota colonising the root system and inhabiting the root-adhering soil of wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula), and Arabidopsis thaliana plantlets. (A) Impact of root exudates on denitrifying activity of microbial community colonising the root system. Denitrification activity (g N-N2O h -1 .g -1 dried roots) was measured in triplicates after the addition of nitrate source only. (B) Denitrification enzyme activity (DEA) of soil samples amended with nitrate (50 µg of N-KNO3 g -1 of dried soil) and carbon sources (0.5 mg of C-glucose and 0.5 mg of C-glutamic acid g -1 of dried soil) measured in triplicates. Letters show which means differed between treatments (Tukey's test; a = 0.05). Vertical bars: Means

Figure 2 .

 2 Figure 2. Nitrogen uptake of ammonium and nitrate for aboveground (A) and belowground (B) tissues 12 h after 15 N labelling (NO3 -or NH4 + ) of wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula), and Arabidopsis thaliana plantlets. Vertical bars: Means ± Standard Errors. Means significantly different from 15 N-NO3 -: *, P < 0.05. Nitrogen uptake was not measured belowground for A. thaliana due to low amount of roots.

Figure 3 .

 3 Figure 3. Analysis of bacterial diversity. (A) Distribution of the 15 th majors bacterial phyla (abundance in %) among the root system and the root-adhering soil of wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula), and Arabidopsis thaliana plantlets. A Venn diagram showing shared and unique bacterial OTUs at 100% identity among bacterial community colonising the root system (B) and those inhabiting the root-adhering soil (C) retrieved from the rhizosphere of wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula), and Arabidopsis thaliana.

Figure 4 .

 4 Figure 4. Significant co-occurrence and co-exclusion relationships among the microbiota inhabiting the rootadhering soil (in green) and colonising the root system (in brown) of wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula), and Arabidopsis thaliana plantlets. The colour of nodes

Figure 5 .

 5 Figure 5. Predictive functional metagenome composition. Heatmap of the normalized relative abundances of the predicted functional categories (level 3) of the microbiota colonising the bulk soil, the root system and the rootadhering soil (RAS) of wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula)and Arabidopsis thaliana plantlets.

Figure 6 .

 6 Figure 6. Quantification of nirK and nirS genes transcripts from RNA extracted from the root system (A) and the root-adhering soil (B) from wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula) and Arabidopsis thaliana plantlets by real-time RT-PCR. Letters show which means differed between treatments (Tukey's test; a = 0.05). Vertical bars: Means ± Standard Errors.

Figure S1 :

 S1 Figure S1: Rarefaction curves demonstrating species richness of microbiota retrieved from the root system (R) and root-adhering soil (RAS) compartment from wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula), and Arabidopsis thaliana plantlets.

Figure S2 :

 S2 Figure S2: Species Richness and diversity (Inverted Simpson) of microbiota associated with the bulk soil (BS), the root-adhering soil (RAS) and the root system (R) of wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula), and Arabidopsis thaliana plantlets.

ACKNOWLEDGEMENTS

This work was supported by the French national programme « EC2CO-MicrobiEn, CNRS », (RhizoDen Project).

We thank Elise Lacroix for her help in plant culture and harvesting. Plant culture was performed at the greenhouse platform (FR41, University Lyon1). We thank J. Gervaix for his help on nitrogen form uptake experiments. The authors gratefully thank the editor Prof. Wietse de Boer and reviewers for improving our manuscript.

The authors declare no conflict of interest.

CONFLICT OF INTEREST

Additional File 1: Abundance in percentage (%) of the 15 more abundant phyla (used in Figure 3) retrieved from the bulk soil and the root system (R) and the root-adhering soil (RAS) of wheat (Triticum aestivum), rapeseed (Brassica napus), barrel clover (Medicago truncatula), and Arabidopsis thaliana plantlets.

Additional