Septoglomus nigrum, a new arbuscular mycorrhizal fungus from France, Germany and Switzerland

A new arbuscular mycorrhizal fungus, Septoglomus nigrum, was found in several agricultural field sites in France, Germany and Switzerland, especially in extensively to intensively managed natural meadows and pastures and in extensively managed cropping systems. The fungus was propagated in trap pots and single species cultures on Lolium perenne, Trifolium pratense, Plantago lanceolata and Hieracium pilosella. It differentiates black spores with triple-layered walls, 95–175× 90–170 μm in diameter, formed singly in soils or rarely in roots. Phylogenetically, it forms a distinct clade close to S. altomontanum and S. africanum, which can morphologically be distinguished from spores of S. nigrum by the characteristics of the spore wall and by the color, size and shape of the subtending hyphae. An identification key is provided that differentiates all species so far described in Septoglomus.


Introduction
In 2011, a new genus in the Glomeraceae, Septoglomus Sieverd. et al., was described based on concomitant spore morphological and molecular phylogenetic analyses (Oehl et al. 2011). Only a few species were transferred to the new genus, namely the type species S. constrictum, and S. africanum, S. deserticola, and S. xanthium (Trappe 1977, Trappe et al. 1984, Błaszkowski et al. 2004. During the following years, several new species emerged in the genus Septoglomus that were morphologically similar to the type species but phylogenetically different (Błaszkowski et al. 2013a, 2013b, Goto et al. 2013, Palenzuela et al. 2013, Symanczik et al. 2014).
Already in 2003, a black spored Glomus sp. strain BR3 resembling Glomus constrictum was mentioned in the literature (Oehl et al. 2003), which forms exclusively black spores singly in soils or in loose clusters, in between an enormously dense network of hyaline mycelia hyphae. We detected this fungus repeatedly also in other grass-and cropland sites in France, Germany and Switzerland. The objective of the present study was to describe this fungus as sp. nov. based on both, morphological and phylogenetic analyses and to evaluate its biogeographical distribution in Central Europe. An identification key is included that differentiates all Septoglomus species hitherto described.

Material and Methods
Study sites, soil sampling: During the last 18 years, soil samples were repeatedly taken in agricultural field sites in France, Germany and Switzerland. The sites comprise extensive meadows, and organic and conventional cropping sites of a wide range of soil pH, organic carbon and available P contents subjected to no-tillage, reduced tillage or conventional soil tillage, but with almost permanent plant cover throughout the years. The soil types ranged from Leptosols and Regosols to Cambisols and Luvisols. Samples were generally taken at 0-10 cm soil depth, as described in Oehl et al. (2003). Selected chemical soil characteristics were determined as also described in Oehl et al. (2003). A summary of selected isolation sites is given in Table 1.
AM fungal trap and monosporic cultures: AM fungal trap cultures and monosporic cultures were established as described in Oehl et al. (2003). The field soil for trap cultures from that the type was isolated, had been collected in Niederösch (Canton Bern, Switzerland). The cultures were established in April 2009 and maintained for two vegetation periods until December 2010 using Hieracium pilosella L., Lolium perenne L., Plantago lanceolata L. and Trifolium pratense L. as plant hosts in pot cultures. Thereafter the trap culture substrates were harvested and air-dried. In May 2011, spores of the new species were isolated from the substrates and inoculated for the establishment of monosporic cultures. Single spores were placed in several pipette tips (one mL) filled with Loess soil (Oehl et al. 2003, Tchabi et al. 2010. Hieracium pilosella was seeded on the surface of the pipette tip substrate, and exactly above each spore that was placed at about 5 mm depth with a Pasteur pipette (Oehl et al. 2003, Tchabi et al. 2010. The pipette tips were placed in a rack, and the bottom of the tip reached into water so that the soil was watered by capillary forces of the soil. After 5 weeks, the pipette tips with the inoculated plant were directly transplanted into bigger pots (350 mL) filled with sterile soil substrate, after cutting the tips horizontally in two halves to ease the root growth from the pipette tips into the pots. The cultures were maintained for 14 months before checking for mycorrhizal root colonization and spore formation. In one of the initiated monosporic cultures, single spores, small spore clusters and sporocarps of different sizes, and mycorrhizal roots were found and analyzed applying morphological and molecular analyses. The successful cultures were deposited in the Swiss collection of arbuscular mycorrhizal fungi (SAF) at Agroscope in Zürich (Switzerland) under the accession number SAF86, among other cultures from the same and other farming sites (e.g. SAF47 from Vogtsburg, SAF84-85 from Niederösch, SAF136-138 from Frick, SAF171-175 from Rubigen).
Morphological analyses: Spores of the new fungus were separated from the soil samples, trap cultures and monosporic culture by a wet sieving process as described by Sieverding (1991). The described morphological spore characteristics and their subcellular structures are based on observations of specimens mounted in polyvinyl alcohollactic acid-glycerol (PVLG; Koske & Tessier 1983), Melzer's reagent, in a mixture of PVLG and Melzer's reagent (Brundrett et al. 1994), a mixture of lactic acid to water at 1:1, and in water (Spain 1990). The terminology of the spore structure basically is that presented in Błaszkowski (2012) and Oehl et al. (2015) for species with glomoid spore formation. Photographs were taken with a digital camera (Leika DFC 295) on a compound microscope (Leitz Laborlux S) using Leica Application Suite Version V 4.1 software. Specimens mounted in PVLG and a (1:1) mixture of PVLG and Melzer's reagent were deposited at Z+ZT (ETH Zurich, Switzerland).

Molecular analyses:
All the isolated spores, derived from the monosporic pure culture (see above) established on H. pilosella, were surface-sterilized (Mosse 1962) using chloramine T (2%), streptomycin (0.02%) and Tween 20 (2-5 drops in 25 mL final volume). Crude extracts were obtained by crushing 5-6 spores from a loose spore clusters with a sterile disposable micropestle in 23 μL milli-Q water, as described by Palenzuela et al. (2013). Direct PCR of these crude extracts were performed in an automated thermal cycler (Gene Amp PCR System 2400, Perkin-Elmer, Foster City, CA, USA) with a pureTaq Ready-To-Go PCR Bead (Amersham Biosciences Europe GmbH, Germany) following manufacturer's instructions with 0.4 μM concentration of each primer. A two-step PCR was conducted to amplify the ribosomal fragment consisting of partial SSU, ITS1, 5.8S, ITS2 and partial LSU rDNA using the primers SSUmAf/LSUmAr and SSUmCf/LSUmBr consecutively according to Krüger et al. (2009). PCR products from the second round of amplifications (~1500 bp) were separated electrophoretically on 1.2% agarose gels, stained with Gel Red™ (Biotium Inc., Hayward, CA, USA) and viewed by UV illumination. The band of the expected size was excised with a scalpel and isolated from the gel with the QIAEX II Gel Extraction kit (QIAGEN, USA) following the manufacturer's protocol, cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA, USA), and transformed into One Shot® TOP10 chemically competent Escherichia coli (Invitrogen, Carlsbad, CA, USA). Recombinant colonies were selected by blue/white screening and the presence of inserts detected by PCR amplification with GoTaq® Green Master Mix (Promega) using universal forward and reverse M13 vector primers. After isolation from transformed cells, plasmids were sequenced on both strands with M13R/T7 primers using the BigDye Terminator kit 3.1v (Applied Biosystems). The products were analyzed on an automated DNA sequencer (Perkin-Elmer ABI Prism 373). Just the partial LSU rDNA fragment was sequenced satisfactorily. Sequence data were compared to gene libraries (EMBL and GenBank) using BLAST (Altschul et al. 1990). The new sequences were deposited in the EMBL database under the accession numbers MK234700 and MK234701.

Phylogenetic analyses:
The AM fungal sequences (partial LSU of the rDNA) obtained were aligned with other related glomeromycotan sequences from GenBank in ClustalX (Larkin et al. 2007).

MycoBank MB 828735
Etymology: nigrum referring to the black spores of the new species, found both in field soils and in trap and single species cultures, when observed under stereo-microscopes.

Diagnosis:
Differing from Septoglomus constrictum in forming black, triple layered and smaller spores (90-175 μm) instead of dark brown to brown black spores with bi-layered walls, and generally larger than 175 μm in diam.
Type: Holotype, deposited at Z+ZT (accession ZT Myc 59679), derived from a monosporic culture established on the host plant Hieracium pilosella in the greenhouse of the Swiss collection for Arbuscular mycorrhizal fungi (SAF) at the Institute for Sustainability Sciences, Agroscope, in Zürich, Switzerland. Spore for the culture originated from an extensively managed grassland site in Niederösch (Kanton Bern, Switzerland; 47°06'56''N; 7°36'32''E). Collector was F. Oehl and collection date 15.6.2012. Isotypes (ZT Myc 59680) and paratypes from other sites in France, Germany and Switzerland were also deposited at Z+ZT. Living cultures of the fungus are currently maintained at SAF under the accession numbers SAF86 and SAF175.
Subtending hyphae (SH) of spores often recurved to rarely straight, constricted to rarely cylindrical, 11.0-17.3 μm broad and 15-70 μm long, with a wall thickening toward the spore base, generally forming a plug-like, straight septum at the spore base, 2.0-5.1 μm Figs 1-5. Septoglomus nigrum 1. The dark spores of S. nigrum are formed in trap cultures frequently after > 1 year of cultivation. 2-3. Mature spores are regularly dark brown black to black. 4. Segment of a young, developing spore becoming coffee-brown, while formation of a septum at spore base has started. Spore wall with three layers (SWL1-3). 5. Mature, black spore of S. nigrum. Rarely all three spore wall layers are present on mature spores, as SWL1 is evanescent and short-lived, while SWL2 is semi-persistent, but might also lack completely in older spores.
thick. Pore (and the closing septum) at base 6.2-9.1 μm broad. SH wall triple-layered, as continuous with the triple-layered spore wall. Outer wall layer usually evanescent and often missing in fully developed spores, second layer 1.2-1.8 μm, and third layer tapering from 4.0-7.5 to 1.8-3.5 μm within 50 μm distance to the spore base.  Distribution: A summary of locations where the new species were collected is given in Table 1. The fungus seems to be frequently occurring in Central Europe, with grasslands, and no-tillage to reduced tillage or other low-input cropping systems as preferred habitats when compared to intensively managed, periodically ploughed habitats (Oehl et al. 2003; Table 1).

Molecular analyses:
Phylogenetic analyses of the partial LSU rDNA reveal that the sequences of the new species group with environmental sequences obtained from soil (KC411094) and roots from green needle grass (EU380052), switch grass (EU380116), and Ixeris repens (AB670113). The nearest species phylogenetically to S. nigrum are S. altomontanum and S. africanum (Fig. 6).

Discussion
Septoglomus nigrum can easily be distinguished from all other species in the genus by the combination of spore color, size and the structure of the spore and subtending hyphal walls. The morphologically most similar species are S. constrictum (Trappe 1977) and S. altomontanum (Palenzuela et al. 2013). However, S. constrictum has larger (150-330 μm), brown to black brown spores, and S. altomontanum has stronger, cylindrical to funnel-shaped subtending hyphae, which are regularly slightly lighter in colour (dark yellow-brown to reddish brown) than the spores. Spores and subtending hyphae of S. nigrum are generally completely black, and the subtending hyphae at spore base are regularly constricted to rarely cylindrical. Phylogenetically, S. nigrum is closest to S. altomontanum and S. africanum, but rather distant to S. constrictum.
In the old genus Glomus, there are several other species whose dark brown to black spores might resemble S. nigrum, such as Glomus ambisporum and Glomus tenebrosum (Oehl et al. 2011). However, both these species usually form spores in sporocarps, which is not known for Septoglomus species. Moreover, G. tenebrosum forms larger spores (200-270 μm;Berch and Fortin 1983) than S. nigrum, and G. ambisporum forms triple-layered spores with a dark brown to black, laminate middle layer, and a membranous, thin inner-most layer (Smith and Schenck 1985), while in S. nigrum the middle layer is subhyaline to light brown, semi-persistent, and the innermost layer is black and laminate.
Some Septoglomus spp. are not totally separated in the tree (S. nakheelum, S. furcatum, S. constrictum and some others). The LSU rDNA is known to be a good marker to identify species and classify them from genera to phyla (Moore et al. 2011), but in Glomeromycota a more variable region (ITS) produce better results to separate species. Unfortunately, it was not possible to obtain the ITS rDNA fragment of S. nigrum, but even with a more conserved marker (partial LSU rDNA), we demonstrated phylogenetically that this fungus is a new taxon.
The new species is quite frequent in Central European agricultural sites, such as extensively to intensively managed meadows and pastures, or cropping systems with plant cover almost all year long. The species is sensitive to periodic soil cultivation and when soils are left bare over months (Oehl et al. 2003), but seems to be relatively insensitive to high fertilization or nutrient availability levels (Table 1). Currently, it is difficult to evaluate the biogeographical distribution of S. nigrum on larger scales, concerning e.g. soil, vegetation, or climatic gradients, as in the past the new species, and several other Septoglomus spp. might have been often confused with S. constrictum. On the other hand, there was a fast progress on the taxonomy and systematic classification of this genus in the last decade (e.g. Błaszkowski et al. 2013aBłaszkowski et al. , b, 2014. This will certainly lead us to a better understanding of the geographical distribution and the functional importance of this genus, its type species S. constrictum and all the other members, including S. nigrum, in natural and man-made ecosystems around the globe.