Effects of Rhizobium Species Living with the Dark Septate Endophytic Fungus Veronaeopsis simplex on Organic Substrate Utilization by the Host

Bacteria harbored in/on the hyphae of the dark septate endophyte, Veronaeopsis simplex Y34, were identified as a single Rhizobium species by molecular analyses of bacterial 16S rRNA genes, and were successfully isolated from the endophyte. The Rhizobium-cured fungus was prepared thoroughly by an antibiotic treatment, thereby allowing an examination of their effects on organic substrate utilization. Assays with Biolog® FF microplates revealed that the respiration potential for 52.6% of the tested compounds were significantly different between Rhizobium-harboring and -cured fungal hosts, indicating that organic substrate utilization by V. simplex Y34 was significantly influenced by the presence of the associated Rhizobium sp. VsBac-Y9.


Fungal isolates
In this study, we used Veronaeopsis simplex CBS 588.66 isolated from the litter below Acacia karroo in South Africa; V. simplex Y34 isolated from forest soil in Yakushima, Kakoshima, and V. simplex IBAK45 isolated from forest soil in Ami, Ibaraki Japan. All these isolates were incubated on half-strength cornmeal-malt-yeast (1/2 CMMY) agar plate at 23°C.

Preliminary treatment and microscopic observation
We firstly removed loosely-associated bacteria with V. simplex using a modified van Tieghem method, as described by Sato et al., 2010 (7). The bacterial presence/absence inside and outside the hyphae of original and van Tieghem-treated V. simplex isolates was confirmed by staining using a LIVE/DEAD BacLight TM Bacterial Viability Kit (Thermo Fisher Scientific, Waltham, MA, USA) and observation by fluorescence in situ hybridization (FISH) with a universal bacterial probe EUB338, as previously described (3,5). At the same time, an endobacteria-harboring fungus Mortieralla elongata FMR23-6 I-B1 was observed as a positive control for confirming the presence of endohyphal bacteria (5). Microscopic observation was conducted under blue and green light using a fluorescence microscope (BX51, Olympus, Tokyo, Japan) equipped with a complementary metal-oxide-semiconductor (CMOS) camera (Xi7, Canon, Tokyo, Japan).

DNA extraction, PCR amplification, T-RFLP, and clone library construction
The V. simplex isolates treated by the van Tieghem method were incubated on 1/2 CMMY agar for 6 weeks at 23°C. DNA was extracted from the recovered mycelia using a PrepMan ® Ultra Sample Preparation Reagent (Thermo Fisher Scientific, Waltham, MA, USA) with a minor modification. PCR amplification of bacterial 16S rRNA genes for T-RFLP analysis was performed in a 30 µL reaction mixture containing 0.1 µg of template DNA, 5 pmol of primers (Qarc-10F and 926R), 1 × Ex buffer, 75 pmol of dNTPs, and 1.5 U of Ex Taq polymerase (Takara Bio, Shiga, Japan) in a thermal cycler (Bio-Rad, Hercules, CA, USA) using the following thermal conditions: 5 min at 95°C, then 25 cycles of 95°C for 30 s, 54°C for 45 s, and 72°C for 90 s (7). The fluorescently labeled PCR products were purified using a Qiagen PCR Purification Kit (Qiagen, Hilden, Germany), and eluted in a final volume of 40 µl. Aliquots (10 µl) of the purified 16S rRNA gene products were separately digested with 1 U of HaeIII, HhaI, and MspI (Takara Bio) for 2 h, according to the manufacturer's instructions. The fragments were purified using the Qiagen Gel Extraction Kit. The precise lengths of terminal restriction fragments (T-RFs) were determined using a 3130xl PE Applied Biosystems Automated DNA Sequencer (Applied Biosystems, Foster City, CA, USA) according to the method of Nishizawa et al. 2008 (4).
The T-RFLP data were imported into GeneMapper software (version 3.7, Applied Biosystems). The peaks were identified using 30 as the peak height cut-off value and 30-700 bases as the size range. The raw T-RFLP data sets were uploaded to T-REX software (2), which was used to denoise (one standard deviation using peak height) and align peaks (clustering threshold of 0.5, at most one peak per profile), and create a data matrix (samples vs. aligned T-RFs). The values in the data matrix represented the relative abundance of the T-RFs.
On the other hand, the un-fluorescently labeled PCR products amplified using primer set of 10F-926R were purified using a Qiagen PCR purification Kit (Qiagen, Hilden, Germany), then ligated into a pGEM-T-easy vector (Promega, Madison, WI, USA) and used to transform into Escherichia coli DH5α competent cells (TOYOBO, Osaka, Japan). The transformants were screened as described previously (5).
Clone sequences were determined using a Sanger sequencer (3130xl Applied Biosystems Automated DNA Sequencer). The DNA sequences were assembled and trimmed using GENETYX-MAC (version 18, Software Development, Tokyo, Japan). The clone sequences were deposited in NCBI/DDBJ/EMBL with accession No.

Isolation of the V. simplex-associated bacteria and identification of the bacterial isolate VsBac-Y9
Isolation of the V. simplex-associated bacteria was performed according to a previously described method (3). In brief, the V. simplex isolates Y34, IBAK45 and CBS 588.66 were cultivated on 1/2 CMMY agar plates for 7 days at 23°C. Cultivated fungal mycelia were homogenized by sterilized stainless crusher on a shaking mixer for 5 min and centrifuged at 1800 × g for 10 min. The supernatant was filtered through 8-µm-and 3-µm-pore membrane filters to remove fragmented hyphae and sporangiospores. Aliquots of the filtered suspension were spread on Difico TM nutrient broth agar and incubated for 7 days at 30°C. The bacterial growth was only confirmed on the NB agar plate inoculated with the aliquots of isolate Y34. After a purification step, a single colony was transferred onto a fresh NB agar plate, and the isolate was named as 'VsBac-Y9'.
Genomic DNA of VsBac-Y9 was extracted using a lysozyme buffer method, and a most-complete 16S rRNA gene sequence was obtained by a PCR-based protocol using the genomic DNA as template (5). The sequence was determined and analyzed as the above-described methods. The sequences were deposited in NCBI/DDBJ/EMBL with accession No. MG251442. In addition, the partial 16S rRNA gene of VsBac-Y9 was analyzed by 16S rRNA gene-targeting T-RFLP to deduce the abundance of VsBac-Y9 in the bacterial community associated with the hyphae of V. simplex isolates, according to above mentioned method.

Phylogenetic analysis
Determined sequences of 16S rRNA gene clones were compared with similar nucleotide sequences retrieved from the NCBI/DDBJ/EMBL databases using the BLAST program. Multiple alignments with/without clone sequences were performed with Molecular Evolutionary Genetics Analysis (MEGA ver. 7.0.26). Evolutionary distances were calculated by Kimura 2-parameter method, and neighbor-joining trees were constructed with 1000-replicate bootstrap test.

Preparation of axenic fungal culture and Biolog fiamentous fungus (FF) microplate assays
For the V. simplex isolates associated with Rhizobium spp., we eliminated the bacteria harbored in the hyphae by antibiotic treatment of mycelia in a mix solution of 50 µg mL -1 ciprofloxacin, 50 µg mL -1 kanamycin, 100 µg mL -1 ampicillin, and 100 µg mL -1 chloramphenicol, for 24 hours at 23°C. The treated mycelia were then grown on 1/2 CMMY agar for 7 days at 23°C to check whether the bacteria were present, by PCR amplification of 16S rRNA gene as described above. Only van Tieghem-treated Y34 (Rhizobium-harboring V. simplex Y34) was cured of the associated bacteria, and defined as cured V. simplex Y34. The Rhizobium-harboring and -cured V. simplex Y34 were used to examine organic substrate utilization using a Biolog FF MicroPlate (BiOLOG, Hayward, CA, USA), as in previous study (8) with a minor modification. In brief, V. simplex Y34 associated with/without Rhizobium species were pre-incubated on a 1/2 CMMY agar covered with a piece of sterile cellophane for 7 days at 23°C, then the mycelia grown on the cellophane were recovered, avoiding the agar itself, and transferred into a 75 ml sterile blender containing 20 ml of BioLOG FF-IF broth to prepare a suspension of hyphal fragments. After centrifuging at 3000 g for 5 min,