Molecular diversity of arbuscular mycorrhizal fungi colonising arable crops

Authors


*Corresponding author. Present address: Soil-Plant Dynamics Unit, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK. Tel.: +44 (1382) 562731; Fax: +44 (1382) 568502; E-mail: t.daniell@scri.sari.ac.uk

Abstract

We used differences in small subunit ribosomal RNA genes to identify groups of arbuscular mycorrhizal fungi that are active in the colonisation of plant roots growing in arable fields around North Yorkshire, UK. Root samples were collected from four arable fields and four crop species, fungal sequences were amplified from individual plants by the polymerase chain reaction using primers NS31 and AM1. The products were cloned and 303 clones were classified by their restriction pattern with HinfI or RsaI; 72 were subsequently sequenced. Colonisation was dominated by Glomus species with a preponderance of only two sequence types, which are closely related. There is evidence for seasonal variation in colonisation in terms of both level of colonisation and sequence types present. Fungal diversity was much lower than that previously reported for a nearby woodland.

1. Introduction

Arbuscular mycorrhizas (AM) are an intimate association between the majority of plants (around 80% of families) and fungi of the order Glomales. This order comprises five recognised families: the Glomaceae (with the single genus Glomus), Acaulosporaceae (Acaulospora and Entrophospora) and Gigasporaceae (Gigaspora and Scutellospora) [1], plus the recently described Archaeosporaceae and Paraglomaceae [2]. There are around 150 known species of AM fungi [3]. This association between plant and fungus has been postulated to have been essential for the colonisation of land by plants because of its role in nutrient cycling in soil, particularly of phosphorus [4].

AM fungi are thought to have a number of other beneficial functions within the symbiosis, including protection of the plant from pathogens and drought [5,6]. There is increasing evidence that individual species can be functionally distinct [7–9].

Most studies on AM fungal function have concentrated on phosphorus uptake, particularly in situations of low phosphorus availability (for examples see [1]). Arable crops, which are often heavily fertilised and mostly have highly branched root systems, are less likely to rely on AM fungi for phosphorus nutrition. However, the AM symbiosis is multifunctional and it has been hypothesised that plants with highly branched root systems rely on the fungal partner increasingly for protection against pathogen attack [6]. Previous work has shown that arable crops can be colonised extensively by AM fungi with a suggestion, despite the relatively large root area of these crops, that the symbiosis may be important for phosphorus nutrition [10]. Understanding the diversity of the AM fungi in crop plant root systems is therefore a prerequisite for effective management in sustainable agricultural systems.

Identification of species in the Glomales has relied extensively on the morphology of spores collected from soil. However, it is impossible to correlate spore information to specific colonisation events in plant roots as spores are typically produced on external hyphae that are easily detached from the root, and the level of spore production does not reflect the abundance of the species in roots [11–13]. Since the fungi are obligate symbionts, it is the fungi colonising roots that are likely to be of most relevance to function. In general, however, detailed prior knowledge of the species present is needed before taxa can be distinguished below the genus level by morphological methods based on staining roots [14–16]. It is now possible, using molecular methods, to identify groups of fungi at far higher resolution than can be achieved using morphological methods. These methods rely on a number of target sequences, mainly within the ribosomal gene cluster [17–21]. The DNA primer AM1 was designed by Helgason et al. [19] to match all small subunit ribosomal RNA (SSU) sequences known at that time from the Glomales, but to differ from corresponding plant SSU sequences. It has become apparent that the AM1 primer excludes a number of fungal types from previously unrecognised groups, the Archaeosporaceae and Paraglomaceae identified by Morton and Redecker [2,22,23]. Thus, AM1 can be used in combination with the general eukaryotic primer NS31 [17] in a polymerase chain reaction (PCR) to amplify sequences from the three traditional families of Glomales from colonised roots. This approach has been used to study the natural symbionts of bluebell, Hyacinthoides non-scripta[21], and in a preliminary comparison of AM fungal diversity in woodland and arable plants [19], in which a subset of the samples presented in this paper, May and June 1997, was used.

The aim of the work presented in this paper is to identify the main AM species involved in root colonisation of several arable crop plants and to determine whether their distribution reflects the host species (wheat, maize, barley and peas) or the location.

2. Materials and methods

2.1 Sampling

Four sampling sites were used, all in North Yorkshire: Rand Grange, Bedale (Ordnance Survey grid reference SE 256 891; soil type predominantly fine loamy soil of the East Keswick 1 series, pH 7.4), ADAS High Mowthorpe (SE 882 697; typical argillic brown earth of the Wold series, pH 7.3) and two neighbouring fields at Mount Pleasant Farm, Escrick (SE 652 418; typical sandy gley soil of the Everingham series, pH 7.2; and SE 656 420; gleyic brown sand of the Kexby series, pH 7.2). Sites were monitored from May 1997 to July 1998, during which time all of the sites went through one crop rotation. One of the sites at Escrick (SE 652 418) was left fallow in the second season and was therefore excluded from sampling during the second rotation. Four crop species were cultivated during the experimental period: winter wheat (Triticum aestivum), barley (Hordeum vulgare), maize (Zea mays) and peas (Pisum sativum). Peas and maize were sown in the spring, whereas the barley and wheat were autumn-sown (Table 1). Four plants were sampled at random from each field site at each time point and taken to the laboratory, root fragments were washed free of soil, dried in tissue paper and stored frozen at −20°C.

Table 1.  Root samples used for analysis of AM fungal diversitya
SiteCrop speciesNumber of roots showing successful amplification and cloningb,c
  May '97June '97Sept. '97Dec. '97Feb. '98Mar. '98Apr. '98June '98July '98
  1. a The vertical black line represents a crop rotation.

  2. b Four root samples analysed at each time point.

  3. c Samples from May and June 1997 were included in Helgason et al. [19].

BedaleZea mays04       
 Triticum aestivum  2002432
High MowthorpeTriticum aestivum242103332
 Hordeum vulgare  3304342
Escrick 1Pisum sativum24       
 Triticum aestivum  1001340
Escrick 2Triticum aestivum44       
Percentage of plants with successful amplification and cloning (n=16)501005025063818838

2.2 Molecular analysis

DNA was extracted from a subsample (approx. 0.5 g) of the stored roots using a modification of the potassium ethyl xanthate method [24], replacing the first chloroform extraction with an additional phenol/chloroform extraction. Partial fungal SSU DNA fragments (∼550 bp) were amplified by PCR from the total extracted DNA using Pfu DNA polymerase (Stratagene), and primers NS31 [17] and AM1 [19]. Reactions (50 μl) were performed on a Gradient 96 Robocycler (Stratagene) as described by Helgason et al. [21], using 0.2 mM dNTPs, 10 pmol of each primer and the supplied reaction buffer. PCR conditions were: nine cycles of 94°C for 1 min, 58°C for 1 min and 72°C for 2 min, followed by 20 cycles of 94°C for 0.5 min, 58°C for 1 min and 72°C for 3 min, and a final additional 7 min at 72°C. Samples yielding no product were tested for inhibition by repeating the PCR in the presence of an aliquot of the positive control DNA (extracted from Agaricus bisporus) and, where necessary, samples were further purified by the use of a 100-kDa microfilter (Microcon, Millipore) to remove low molecular mass inhibitory compounds. Products were cloned using a PCR Script kit (Stratagene) according to the manufacturer's instructions. Resulting putative positives were screened using standard T3/T7 amplification. Up to 10 positives from each root sample were digested independently with HinfI and/or RsaI, according to the manufacturer's instructions, to identify restriction fragment length polymorphism (RFLP) classes. Examples of each class were purified using a QIAquick PCR purification kit and sequenced, on both strands, using an ABI 377 sequencer and the dye terminator cycle sequencing kit with AmpliTaqFS DNA polymerase (ABI Perkin-Elmer) and T3 and T7 as sequencing primers. The RFLP classes have been described previously from woodland and arable systems [19]. Sequences relating to pyrenomycete fungi were occasionally amplified, and were recognised by their RFLP pattern and excluded from further study.

2.3 Phylogenetic analysis

CLUSTALX [25] was used for multiple sequence alignment and phylogenetic analysis. Clustal uses the neighbor-joining algorithm [26] with the Kimura two-parameter model. Alignments were adjusted manually using GeneDoc [27] and trees were drawn using Treeview [28].

2.4 Morphological analysis

Where there was no detectable amplification, subsamples of roots were stained with acid fuchsin and analysed by the method of Merryweather et al. [29] in order to confirm the apparent lack of colonisation.

3. Results

3.1 Seasonal and growth stage variation in AM colonisation

Table 1 summarises the success in cloning of AM fungal sequences from the root samples, which reflects the colonisation pattern of AM fungi. A total of 40 clones were sampled from each time point and the number of positive clones recovered gives a crude reflection of colonisation. Some plants became colonised rapidly after germination. For example, maize plants sampled from Bedale were colonised before growth stage 19 (nine or more leaves unfolded [30]), pea plants sampled from Escrick 1 were colonised before growth stage 101 (first node leaf fully unfolded with one pair of leaflets and no tendril [31]) and AM fungi were present in wheat plants sampled before growth stage 12 or 13 (two or three leaves unfolded [30]) from all sites in September 1997.

Colonisation of autumn-sown crops does not appear to be sustained over winter, as AM fungi were detected only at High Mowthorpe in December 1997 and not from any site in February 1998. Lack of colonisation in the samples from February 1998 was confirmed by microscopy of stained roots (data not shown). The data suggest that plants are re-colonised in spring (March 1998), with the symbiosis reaching a peak in April/June 1998 as plants near maturity, although it appears that the level of active colonisation declines as the crops approach harvest (July 1998).

3.2 Phylogenetic analysis

Eight SSU RFLP groups were detected in these samples, and phylogenetic analysis of representative sequences shows that they cover the three traditional families of the Glomales (Fig. 1). Most sequences appear to represent Glomus species. In general, sequences that share the same RFLP pattern form a monophyletic clade. The exceptions are Glo1A (Glo1B forms a distinct clade within it) and Glo10 (Glo8 is internal). Two closely related sequence types (Glo1A and Glo1B) include known sequences from the database, Glomus mosseae and Glomus geosporum respectively. Other than these two, the majority of sequence groups fall reliably into one of the three families of the Glomales but do not cluster closely with any known sequence. All but two of the sequence types amplified and cloned fall within the genus Glomus, with one group representing Acaulospora and one Scutellospora.

Figure 1.

A neighbor-joining phylogenetic tree showing AM fungal sequences isolated from roots of arable crops. Bootstrap values are shown where they exceed 75% (1000 replicates). Group identifiers (for example Glo1A) relate to the RFLP types discussed in the text and by Helgason et al. [19]. Clone identifiers relate to site (B=Bedale, E=Escrick and H=High Mowthorpe), crop species (B=barley, M=maize, P=pea and W=wheat) and clone number. Multiple clones with identical sequences are represented by * or †. Clone identifiers for these are as follows: * Acau1: BW3 and HB7-8; Glo8: HW 16 and 18; Glo10: HW13-14, EP7 and BW2; Glo4: BW1, HB4 and EW15 (AF074353); Glo1A: EW10 and 11; Glo1B: EP3-5 (5=AF074357), BM8-10 (9=AF074358) and BW4. † Glo4: HB4 and BM14; Glo1A: BM 2-5, EW 1-9 (2=AF074359); HW 1-7 and EP 1-2 (1=AF074360). Accession numbers are given for sequences published previously. All new sequences have been submitted to the EMBL database (accession numbers AJ309396–AJ309465).

3.3 RFLP group analysis

Table 2 shows the colonisation patterns of each set of samples. Clone numbers, as detected by RFLP, demonstrate the overall dominance of Glomus sequence groups, particularly Glo1A (62% of all clones isolated) and Glo1B (20%). No other sequence group contributed more than 10% of clones. The single Acaulospora sequence group was present early in the colonisation cycle, in samples where the two major Glomus sequence types were not dominant. The two rarest sequence groups (Glo6 and Scut1), represented by one and two clones respectively, were observed at only one site (Escrick 1). A maximum of four groups were observed in any one root system.

Table 2.  Number of clones of each sequence group obtained from each sample of roots
DateAM typeSite
  BedaleHigh MowthorpeEscrick 1Escrick 2
Crop species Zea maysTriticum aestivumTriticum aestivumHordeum vulgarePisum sativumTriticum aestivumTriticum aestivum
May '97Glo1A0 2 0 20
 Glo1B0 1 0 1
 Acau10 0 1 0
 Scut10 0 1 0
June '97Glo1A8 19 7 15
 Glo1B5 1 12 0
 Glo43 0 0 1
 Glo60 0 1 0
 Glo101 0 0 0
Sept. '97Glo1A 127 0 
 Glo1B 151 0 
 Glo4 104 0 
 Acau1 205 1 
 Scut1 000 1 
Dec. '97Glo1A 046 0 
 Glo1B 022 0 
 Glo4 001 0 
 Glo10 003 0 
Mar. '98Glo1A 5515 1 
 Glo1B 512 0 
 Glo4 100 0 
Apr. '98Glo1A 4711 11 
 Glo1B 943 1 
 Glo4 010 1 
June '98Glo1A 6416 10 
 Glo1B 810 0 
 Glo4 000 2 
 Glo10 232 1 
 Glo8 130 3 
July '98Glo1A 311 0 
 Glo10 010 0 

Differences in colonisation patterns were suggested between peas and wheat at Escrick 1, where these crops were grown in successive years, and Escrick 2, where wheat was grown in year 1. At these sites wheat appears predominantly colonised by Glo1A while Glo1B was more abundant in pea roots. The majority of clones from Escrick 2 and High Mowthorpe were also Glo1A, whereas at Bedale, the two major groups appeared co-dominant on both maize and wheat.

3.4 Variation in diversity

The number of clones analysed from each sample was too low to allow the calculation of meaningful diversity indices, so Shannon diversity indices were calculated for data grouped across time points and field locations. There was little difference in diversity between field sites in the second rotation: Bedale 1.19, High Mowthorpe wheat 1.19, barley 0.98, Escrick 1 1.14. Diversity indices across one season (1997/98), combining data from all sites, showed large differences: September 1997 1.45, December 1997 1.12, February 1998 no colonisation, March 1998 0.67, April 1998 0.78, June 1998 1.49 and July 1998 0.45.

4. Discussion

These data represent a temporal survey of AM colonisation at different arable sites using a molecular method that allows an accurate identification of the fungal sequence types and hence the diversity of the fungi involved. This work confirms our earlier finding, using a subset of the presented data from 1997, that the diversity of AM fungi in arable fields (when combined over four sites) is low compared to that in a nearby woodland [19]. There are also marked differences in the sequence types detected at the arable and the woodland sites. They have only one Glomus species in common, Glo8, which is a rare type in both systems. The single representatives of the other two families (Acau1 and Scut1) are rare in the arable system but are common sequence types in the woodland. Interestingly, Acau1 is the dominant sequence type in the woodland during July, at a time when the plants are well established. This is in contrast to the arable system when this type is found only early in the crop cycle.

The primer combination NS31–AM1 amplifies sequences from all three well-established families of the Glomales, i.e. the Glomaceae, the Acaulosporaceae and the Gigasporaceae. However, the phylogenetic depth of the AM fungi has recently been considerably extended by the discovery that certain species have SSU sequences that are much more divergent and form several new clades outside those of the three traditional families [22]. The sequence of Acaulospora gerdemanii for example, is closer to that of Geosiphon pyriforme, a non-AM fungus that forms a symbiosis with a cyanobacterium [32]. Two new families have been suggested, the Archaeosporaceae and the Paraglomaceae, for these fungi [2], which have in the past been considered to belong to the genera Glomus or Acaulospora on the basis of their morphology. Examination of the sequences and experimental tests (our unpublished observations) have shown that the NS31–AM1 primers will amplify some, but not all, of the SSU sequences reported from these groups. It is therefore possible that there are additional AM fungi present in the roots we have examined but not detected by our methodology. At present there is no method available that will reliably detect and identify all the currently known AM fungi while discriminating against other fungi, and indeed such a method may not be feasible given the very deep phylogeny that the AM fungi are now known to encompass. Our methodology reliably detects the three classical AM fungal families, so our study should be understood as a survey of this clade.

The absence of detectable AM colonisation over winter, confirmed by both DNA studies and microscopy, indicates seasonal variation in colonisation of the arable crops. This observation agrees with direct morphological observation of seasonal changes in both gross and specific colonisation. For example, changes in the types of AM fungi colonising plants have recently been documented for bluebell (H. non-scripta) [13].

In addition to the seasonal variation in colonisation, there are changes in the diversity of the fungi, with higher diversity at early and late time points within a crop rotation. The dominance of a small number of Glomus sequence types observed in this study may reflect a steady state for arable systems. Dramatic shifts in the population structure of AM fungi were observed in experiments in which undisturbed grassland was converted to arable cropping land [33]: rotated crops became dominated by Glomus species. The molecular methods applied here enabled us to demonstrate that the two dominant sequence types we found in arable crops are closely related, but distinct, in terms of their SSU sequence. The dominant sequence type Glo1A is closely related to a database sequence for G. geosporum whereas Glo1B is closely related or identical to sequences for G. mosseae. The dominance of these two types suggests that they possess an ability to survive under agricultural conditions that may be unfavourable for the majority of AM fungi.

One possible reason why Glomus species are dominant in such a disturbed system is related to differences in propagative units between the glomalean families. For example, it has been suggested that the Gigasporaceae are only capable of propagation via spore dispersal or infection from an intact mycelium [34]. In contrast, the Glomaceae are also capable of colonising via fragments of mycelium [35]. Furthermore, Giovannetti et al. [36] have shown that Glomus (but not Gigaspora or Scutellospora) readily forms anastomoses between mycelia and might therefore have the ability to re-establish an interconnected network after mechanical disruption. Such differences can explain the dominance of the Glomaceae over members of the Gigasporaceae in an environment with repetitive severe physical disturbance, for example ploughing between crop cycles. Physical disturbance has been shown to have a significant impact on the AM fungal populations [37]. Other aspects of modern agricultural practice that have been shown to have significant effects on AM populations include chemical applications (such as fertilisers and pesticides) and repeated monoculture [38–40].

The Glomales have traditionally been held to be non-specific but there is evidence suggesting that, while there may not be host specificity per se, partners may show preference [9,33,41]. Although clone numbers are low, our data for roots taken from the two Escrick sites provide some support for this idea. Escrick 1 was sown with peas in the first year and wheat in the second, while Escrick 2 had wheat in the first year; the data suggest that the colonisation pattern depends on the crop plant. Peas seem to be preferentially colonised by Glo1B, whereas wheat grown at the same time in an adjacent field, or in the same soil in the following year, is predominantly colonised by Glo1A. We cannot discount a combination of an inoculum potential difference and annual seasonal variation, but the most parsimonious explanation is that this observation is due to a real difference in colonisation preference between host species. Further experimental work is required before any suggested colonisation preference can be confirmed.

The data presented in this paper demonstrate the impact modern agricultural practice can have on a key functional group in the soil. This study extends and confirms the finding by Helgason et al. [19] that AM fungal diversity is low in arable crops. The causes and consequences of this low diversity remain to be established.

Acknowledgments

This project was funded by EU Contract BIO 4 CT96-0027 (IMPACT II). We thank Melvin Peacock, Andrew Bainbridge and ADAS for allowing access to field sites and Allen Mould for support with sequencing.

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