Mycologia
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DOI: 10.3852/mycologia.98.1.16
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Mycologia, 98(1), 2006, pp. 16-22.
© 2006 by The Mycological Society of America

Fungal spore diversity of arbuscular mycorrhizal fungi associated with spring wheat: effects of tillage


S. Schalamuk
S. Velazquez
H. Chidichimo
M. Cabello 1

     Instituto de Botánica Spegazzini, Avenida 53 Nu. 477, 1900 La Plata, Argentina

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

We investigated the influence of tilling, N fertilization and crop stage on arbuscular mycorrhizae (AM) fungal species diversity in a wheat monoculture in the Pampa region of Argentina. Glomalean spores were isolated by wet sieving and decanting from conventionally tilled and nontilled soils cropped with wheat with or without N fertilization, at three phenological stages of the crop (tilling, flowering and grain filling) and fallow. Morphological characterization yielded at least 24 AM fungi taxa in the field samples, belonging to six genera of AMF: Acaulospora Archaeospora, Entrophospora, Gigaspora, Glomus and Scutellospora. Tilling and fertilization treatments did not result in decreased spore biodiversity. Wheat phenology influenced AM communities, with highest spore biodiversity during grain filling.

Key words: AMF, biodiversity, conventional tillage, Glomeromycota, no-tillage, wheat phenological stages


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Arbuscular mycorrhizae fungi, belonging to the phylum Glomeromycota (Schüßler et al 2001Go), are obligate symbionts and form associations with about 80% of plant species (Trappe 1987Go). Arbuscular mycorrhizal associations are the most frequent symbioses found in nature because of their broad association with plants and cosmopolitan distribution (Harley and Smith 1983Go).

Agricultural practices such as tillage and fertilization can affect the structure of AMF communities; tilling can reduce either AMF spore density (Kabir et al 1998Go) or AMF colonization of crops (McGonigle et al 1990Go). The soil environment, plant physiological conditions and mycorrhizae can be greatly changed through different tillage or fertilization systems. Sieverding (1991)Go found that in agroecosystems with monocultures, conventional tillage, high application of soluble phosphate, nitrogen and pesticides the number of fungal species decreases more than 50% in comparison to native ecosystems. No-tillage systems often are characterized by the accumulation of crop residues on the soil surface, leading to greater carbon, nitrogen and surface water compared to conventional tillage (Doran and Linn 1994Go). We hypothesize that changes in soil management can make crop species almost independent of AM fungi or eliminate certain AM fungal species because: (i) they do not tolerate the new edaphic conditions; (ii) they are not able to infect the host plant under these conditions; or (iii) they are not able to compete with other AMF fungal species that have become dominant due to the new growth conditions (Sieverding 1991Go). Kurle and Pfleger (1994)Go hypothesized that when more intensive tillage systems are used, resulting in greater soil disturbance, it is likely that AM fungal species, which sporulate more heavily, would be favored. Species that are more dependent on the mycelial network and on hyphal remnants in root fragments for carry-over would be at a relative disadvantage because of disruption of the network and accelerated decomposition of roots.

Mycorrhizal communities are site specific and each AMF species can be affected in several ways by different agricultural management practices, so generalization is difficult. The effect of fertilizers on AMF diversity has been studied in different agroecological conditions (Johnson 1993Go, Sieverding 1991Go). Johnson et al (2003)Go have pointed out differences among AMF taxa in their strategies for the acquisition of nutrients. AMF colonization in roots change across different phenological stages of wheat (Mohammad et al 1998Go, Schalamuk et al 2004Go). Several studies have found temporal variation in the diversity of mycorrhizal communities of natural ecosystems (Lee and Koske 1994Go, Merryweather and Fitter 1998Go, Eom et al 2000Go). However it is still unclear how AMF communities vary with different phenological stages of crops. Seasonal patterns of AM fungal sporulation have been investigated in the grasslands of Argentina (Lugo and Cabello 2002Go) but there is little information about AMF in agricultural systems in our country (Menén-dez et al 2001Go).

Doran and Parkin (1994)Go suggested that new strategies based on microbiology should be designed to promote long-term soil sustainability in agroeco-systems. We hypothesized that the agricultural practices of tillage and fertilization would affect the composition of the AM fungal community. To test this hypothesis we studied experimental wheat monocultures that had experienced different levels of N fertilization and tillage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
The field experiment was carried out at the agricultural experimental station Ing. Agr. Hirshhorn, Faculty of Agriculture, University of La Plata, on a long-term field tillage experiment. The previous crops had been: 1993 soybean; 1994 corn; and since 1995 wheat. The soil examined was an Argiudoll with light internal drainage deficiency and this composition (top 20 cm layer): 3.9–4.1% organic matter; 0.179–0.191% N; 10.1–11.3 ppm P. The field study performed in 2001 and 2002 was composed of two tillage treatments: no-tillage (NT) and conventional tillage (CT)—autumn plowed and disked before planting. The nitrogen fertilization was evaluated by taking into account the location of the fertilization plots in the previous years. The fertilization treatments were: (i) control without nitrogen (N0) and (ii) 90 kg/ha N at the time of planting (N90), which is the amount of fertilizer applied for wheat cultivation in this area (Maddonni et al 2003Go). Urea was used as N fertilizer, which was broadcast applied. The treatments were arranged in a randomized, split-plot design and replicated three times. To study the AM fungal spore diversity, samples were collected during three crop stages: tilling, flowering, grain filling and fallow for 2 y. Five to six entire wheat individuals and their rhizospheric soil were collected from each treatment (CTN0, CTN90, NTN0, NTN90) from each replicate.

AMF communities were studied by spore extraction from soil. Hence 100 g soil (dry weight) of each subsample was wet-sieved, following the methodology proposed by Gerdemann and Nicolson (1963)Go. Samples were centrifuged in sucrose gradient (Walker et al 1982Go). Quantification was carried out in 9 cm diam Petri dishes with a gridline of 1 cm per side under a stereoscopic microscope at 50 x (Lugo and Cabello 2002Go). Ten divisions were counted and related to the total number of spores by using the method modified by McKenney and Lindsey (1987)Go. For taxonomic identification fungal spores were mounted onto slides with PVA (Omar et al 1979Go) with or without Melzer reagent (Morton 1988Go). Specimens were compared with original species descriptions and reference isolates described by the International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi. In most cases identification of the samples was assessed by the observation of morphological features of the spores obtained after sieving and decanting. These observations were corroborated with observations of freshly formed AMF spores in trap cultures. Many of these species were isolated in monospecific cultures and incorporated to the germplasm bank, AMF living culture collection at Spegazzini Institute, La Plata, Argentina.

The frequency of occurrence of each AMF species was computed with the formula: Xi/X0 x 100, where Xi = the population density for an individual species and X0 = the total population. The frequency of occurrence of each species was used to calculate the Shannon-Weaver biodiversity index (H) species richness (S) and evenness (E), according to Magurran (1988)Go. Differences in AM diversity among treatments were determined with multifactor ANOVA with least significant difference (LSD) test. The percentages of each family of Glomeromycota were arcsin transformed and compared with ANOVA and LSD test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
We identified 24 AM fungal taxa belonging to six genera of AMF: Acaulospora, Archaeospora, Entrophos-pora, Gigaspora, Glomus and Scutellospora in samples from the study field. The AMF species recorded are shown (TABLE IGo).


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  		TABLE I. AM fungal species isolated in the field experiment

 
At all stages of the crop the dominant species were Acaulospora excavata, A. mellea, Glomus claroideum, G. clarum, G. etunicatum, G. mosseae and Scutellospora aff. dipapillosa. The biodiversity index showed significant interaction between tillage and year (P = 0.02), hence the data of the 2 y is shown separately (FIG. 1Go). In 2001 the biodiversity index was higher in conventional tillage (P = 0.004), whereas no significant differences were found in 2002 between both tillage systems. In 2002 many species that had not been present in 2001 appeared: A. birreticulata, A. delicata, E. infrequens and Scutellospora sp. No significant differences were found for the biodiversity index between the fertilized and nonfertilized treatments (FIG. 2Go).


Figure 1
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  		FIG. 1. Total biodiversity index calculated for CT 2001, NT 2001 and CT 2002, and NT 2002. CT: Conventional tillage NT: No-tillage. Data are means of three replicates and all the wheat phenological stages evaluated and fertilization plots. Error bars: SE. The same letter above bars indicates that the values do not differ significantly as determined by one-way ANOVA and LSD (P < 0.05) test.

 

Figure 2
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  		FIG. 2. Total biodiversity index calculated for N0 2001, N90 2001, N0 2002 and N90 2002. N0: control without nitrogen fertilizer N90: nutrient addition plots. Error bars: SE. (Same notations as in FIG. 1Go)

 
Significant changes in biodiversity index along the different wheat phenological stages (P = 0.002) are shown (FIG. 3Go). The index increased from tilling to grain filling, and decreased in the fallow. In 2001 AMF biodiversity of the tilling stage was higher in the conventional tillage treatments than in the no-tillage ones (data not shown). The same year diversity did not show significant differences for flowering and grain filling, and in the fallow the conventional tillage without N fertilizer was found to have significantly lower diversity than the rest of the treatments. In 2002 the results were quite different; diversity reached significantly higher levels in the no-tillage treatment without fertilization at flowering stage and in the conventional tillage treatment with nitrogen fertilization at the grain filling stage. During the 2002 fallow period no significant differences among the treatments were recorded.


Figure 3
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  		FIG. 3. Total diversity index calculated for tilling, flowering, grain filling and fallow. Error bars: SE. (Same notations as in FIG. 1Go)

 
The frequency of occurrence for the members of the families Acaulosporaceae, Gigasporaceae and Glo-meraceae for all the treatments and phenological stages analyzed are provided (FIG. 4Go). The percentage of species belonging to Glomeraceae for both years was significantly higher in no-tillage (65.55%) than in conventional tillage (56.87%) (P = 0.024), and the contribution of this family was higher in 2001 (P = 0.008). In both years the increase of Glomeraceae in no-tillage appeared to be detrimental to Acaulospor-aceae and Gigasporaceae. The contribution of each family at tilling did not differ markedly in the two years. However, in flowering, differences were notable due to the increase of Acaulosporaceae in 2002.


Figure 4
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  		FIG. 4. Frequency of occurrence for the members of the families Acaulosporaceae, Gigasporaceae and Glomeraceae over a 2 y sampling period at different phenological stages of wheat crop and fallow. CTN0: conventional tillage without nitrogen fertilizer; CTN90; conventional tillage with nitrogen fertilizer. NTN0: no-tillage without nitrogen fertilizer; NTN90 no-tillage with nitrogen fertilizer.

 
During tilling the species most represented in all treatments was G. etunicatum. This trend was not so strong in flowering when G. claroideum, G. clarum, G. coronatum and G. etunicatum predominated. The contribution of G. mosseae to diversity was particularly high in conventional tillage fertilized with N during fallow in 2001–2002.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
In the field experiment studied, agricultural practices such as tilling and fertilizing were not detrimental to AM biodiversity. We observed 24 species of Glomer-omycota, a high number compared to those obtained by Jansa et al (2002)Go and Menéndez et al (2001)Go in arable soil. An important result is that biodiversity was higher in tilled plots than in untilled ones in 2001. This pattern is opposite to what was expected because Hendrix et al (1990)Go and Altieri (1999) pointed out that tillage leads to a reduction in the soil diversity and Jansa et al (2002)Go found no significant effect of tillage on the diversity indices of the AMF community. Moreover the contribution of species belonging to Glomeraceae increased in no-tillage plots to the detriment of Acaulosporaceae and Gigasporaceae, contradicting Jansa et al (2002)Go, who found that, in an intensively used agricultural soil under long-term (13 years) reduced tillage management, the presence of certain AMF species, especially those which do not belong to Glomus spp., had a tendency to increase. The occurrence of one species sporulating at the expense of others may be regulated by such factors as interspecific competition, spatial restriction and/or edaphic factors (Gemma et al 1989Go). Allen et al (2003)Go have pointed out that AMF root colonization is mediated by interspecific fungal interactions, such as competition, antagonism and dominance. Results from other studies (Sylvia 1986Go, Koske 1987Go) suggest that individual AMF species compete for resources through a combination of strategies resulting in the maintenance of a diverse AMF community. Therefore a possible explanation for the patterns found in our experiment is the competitive interaction among AMF species. Brundrett et al (1999)Go and Klironomos and Hart (2002)Go found differences among AMF genera in their life-history characteristics and suggest that the mycelium is of major importance as propagule for some Glomus species. Taking these affirmations into consideration we hypothesize that the lack of hyphal network disruption in no-tillage could have favored a competitive advantage of Glomeraceae species.

On the other hand Johnson (1993)Go, Egerton-Waburton and Allen (2000)Go and Johnson et al (2003)Go have found differences in species composition with N enrichment. However in our experiment those differences were not found, probably due to the low amount of fertilizer applied for wheat cultivation in Argentina (Maddonni et al 2003Go).

Biodiversity showed larger differences over the crop cycle than those arising from the different treatments. The increase in biodiversity coincides with the end of the host growing cycle, showing a decrease of this index in fallow treatments. Temporal variation in the community may reflect changes in the functional role of the symbiosis during the season (Rosendahl and Stukenbrock 2004Go), and the changes in the bio-diversity during the crop cycle show the importance of sampling in different moments of the year to reveal AMF biodiversity. The H index was lower in fallows than those indices at different crop phenological stages. During fallow times we observed an increase of Glomus spp. such as Glomus claroideum, G. coronatum, G. etunicatum and G. mosseae (fallow 2001–2002) and G. etunicatum (fallow 2002–2003). Thus a low bio-diversity with predominance of Glomus spp. was observed during fallow and in no-tillage treatments. A higher dominance of Glomus species in fallow also was found by Troeh and Loynachan (2003)Go. Because fallow samples were taken before tillage, many plant species were growing and hosting AM fungi. The decline of biodiversity indices in fallow shows that competition among AM fungi and interspecific fungal interactions appeared to be more important than vegetative characteristics on determining the composition of the AMF community, a fact that is supported by Gemma et al (1989)Go.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr B. Roy and anonymous reviewers for the critical review of the manuscript. Schala-muk S. and Velázquez S. are recipients of scholarships from Comisión de Investigaciones Científicas de la Prov. de Buenos Aires (C.I.C). Cabello M. and Chidichimo H. are researchers from C.I.C. A grant from CIC supported this research.


   FOOTNOTES
 
Accepted for publication November 10, 2005.

1 Corresponding author. E-mail: mcabello{at}netverk.com.ar


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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