Occurrence and distribution of soil Fusarium species under wheat crop in zero tillage

The presence of Fusarium species in cultivated soils is commonly associated with plant debris and plant roots. Fusarium species are also soil saprophytes. The aim of this study was to examine the occurrence and distribution of soil Fusarium spp. at different soil depths in a zero tillage system after the wheat was harvested. Soil samples were obtained at three depths (0-5 cm, 5-10 cm and 10-20 cm) from five crop rotations: I, conservationist agriculture (wheatsorghum-soybean); II, mixed agriculture/livestock with pastures, without using winter or summer forages (wheatsorghum-soybean-canola-pastures); III, winter agriculture in depth limited soils (wheat-canola-barley-late soybean); IV, mixed with annual forage (wheat-oat/Vicia-sunflower); V, intensive agriculture (wheat-barley-canola, with alternation of soybean or late soybean). One hundred twenty two isolates of Fusarium were obtained and identified as F. equiseti, F. merismoides, F. oxysporum, F. scirpi and F. solani. The most prevalent species was F. oxysporum, which was observed in all sequences and depths. The Tukey’s test showed that the relative frequency of F. oxysporum under intensive agricultural management was higher than in mixed traditional ones. The first 5 cm of soil showed statistically significant differences (p = 0.05) with respect to 5-10 cm and 10-20 cm depths. The ANOVA test for the relative frequency of the other species as F. equiseti, F. merismoides, F. scirpi and F. solani, did not show statistically significant differences (p ≤ 0.05). We did not f ind significant differences (p ≤ 0.05) in the effect of crop rotations and depth on Shannon, Simpson indexes and species richness. Therefore we conclude that the different sequences and the sampling depth did not affect the alpha diversity of Fusarium community in this system. Additional key words: crop rotation; cultivated soil; soil depth; species diversity. * Corresponding author: vmoreno@faa.unicen.edu.ar Received: 10-05-12. Accepted: 29-01-13. This work has a Supplementary Table that do not appear in the printed article but that accompany the paper online. Abbreviations used: AM (arbuscular mychorriza); CLA (carnation leaf agar); Fr (relative frequency); PCR (polymerase chain reaction); PDA (potato dextrose agar); SNA (Spezieller Nährstoffarmer Agar); VAM (vesicular arbuscular mychorriza). ✝ Angélica M. Aramabarri passed away after this article was accepted for publication. We dedicate this article to her memory. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2013 11(1), 72-79 Available online at www.inia.es/sjar ISSN: 1695-971-X http://dx.doi.org/10.5424/2013111-3081 eISSN: 2171-9292


Introduction
The Fusarium species are widely distributed in different soil types commonly associated with plant debris and roots.Fusarium spp.also exhibits a saprophytic activity in the soil, which can be attributed to the capacity of Fusarium species to grow on a wide range of substrates and their efficient dispersion mechanisms (Burgess, 1981).It is also cited the endophyte condition of this fungus (Pitt & Hocking, 1999).In agricultural systems, the importance of this fungus is strongly associated with several diseases that cause losses on crop yields and mycotoxin contamination of grains.
In Argentina, over the last 30 years conventional tillage has been replaced by zero-tillage and minimum tillage systems.These systems maintain and/or improve the quality of natural resources in the agricultural production process (Acevedo & Silva, 2003).The zero-tillage system brings important qualitative and quantitative changes in soil environment: the stubble cover, less disturbance with moisture and temperature conditions completely different from those in conventional tillage.The advantages offered by zero-tillage, such as soil conservation, increased efficiency in water use and fuel economy in the machines, are sometimes confron-ted with disadvantages such as the high nutrient requirements at the time to install the practice, the need to replace planters and the potential threat of necrotrophic pathogens found in the crop stubble conditions that favor its saprophytic phase (Ivancovich, 1992;Fernández et al., 2008).
The interaction of the tillage system with Fusarium infection has been a matter of particular interest, especially at wheat and maize growing areas (Fernández et al., 2008).Different researches suggest that the diversity of Fusarium community has a strong relationship with crop rotation (Steinkellner & Langer, 2004;Luque et al., 2005;Wakelin et al., 2008).
Within zero tillage systems the top up layer breakdown brings carbon and organic forms of nitrogen on soil surface, producing the increase of bacterial and fungal biomass in the first 10 cm of soil.Fusarium spp. is a fungus with the ability to survive in this layer of soil.Fusarium spp.can survive as chlamisdospore in the soil, therefore the soil may be infected, and when environmental conditions are favorable for the fungus a disease development on plants may occur.Rupe et al. (1997) observed that the density of F. solani was lower when sorghum [Sorghum bicolor (L.) Moench] or wheat (Triticum aestivum L.) was incorporated in the soybean [Glicine max (L.)] system.The same effect was observed for root rot on green bean (Hall & Philips, 1992).However, some authors considered that the limited soil tillage increases the abundance of Fusarium sp. in the soil (Wakelin et al., 2008).Luque et al. (2005) observed that the addition of maize stubble increases the diversity of Fusarium species.Some reports suggested the high influence of soil depth on the amount of Fusarium propagules in the soil (Rodríguez-Molina et al., 2000;Yi et al., 2002;Steinkellner & Langer, 2004).
In Argentina, many reports evaluate the effects of tillage systems on fungal pathogens populations, arbuscular (AM) and vesicular-arbuscular mychorriza (VAM) or entomopathogenic fungi (Bonel & Morrás, 2000;Luque et al., 2005;Nesci et al., 2006;Schalamuck et al., 2006Schalamuck et al., , 2007;;Gómez et al., 2007;Lori et al., 2009).The studies available in Argentina establish relationships among Fusarium spp.populations and stubble crops or plant roots (Luque et al., 1999;Weber et al., 2001;Fernández et al., 2008).To our knowledge, comparative studies about the effects of agricultural managements on soil Fusarium community are scarce.Nesci et al. (2006) observed that the Fusarium community was significantly different comparing conventional with zero tillage.Steinkellner & Langer (2004) observed that the soil Fusarium spp.frequency was affected by sampling year, tillage and cultivated crop.Wakelin et al. (2008) using molecular tools proved that the community of Fusarium spp. is strongly related with the agricultural management.
The aim of this study was to examine the occurrence and distribution of soil Fusarium spp. at different sampling depths, when the wheat was harvested under a zero tillage crop system.

Origin of samples
The experiment was established in 1997 in the Barrow Experimental Station (38°19' 25'' S; 60°14' 33''W), Tres Arroyos, Buenos Aires Province, Argentina, with a long history of zero tillage.The soil is a typical petrocalcic argiudol, Serie Tres Arroyos, depth ranging between 50 and 55 cm, clay loam texture.The agroclimatic conditions of the experimental place and characteristics of soils in the rotation systems are shown in Table 1 and Table 2 The plots were arranged in complete blocks and the treatments (rotation systems) randomized, using three replicates and 420 m 2 plot area (14 m × 30 m).Sequences consisted of f ive different crop rotations: I, conservationist agriculture (wheat-sorghum-soybean); II, mixed agriculture/livestock with pastures, without using winter or summer forages (wheat-sorghumsoybean-canola-pastures); III, winter agriculture in depth limited soils (wheat-canola-barley-late soybean); IV, mixed with annual forage (wheat-oat/Vicia sativasunflower); V, intensive agriculture (wheat-barley-canola, with alternation of soybean or late soybean).The cultivars used were barley Quilmes Ayelen; canola SW 2836; soybean A 4613 RG; late soybean A 3726 RG; oat Bonaerense Maja; sorghum DK 61 T; wheat BIOINTA 2001 and sunflower DK 3920.
The sequences reflected what happens in regional production systems.The two mixed production situations (II and IV), both with grazing animals, make a difference in various physical soil parameters compared with agricultural sequences.The three remaining situations (I, III and V) are exclusively agricultural.Sequence I is held by many "traditional" farmers, who generally own the land with one crop per year.Sequence V appeared in recent years by hand of companies that lease the land, with the intention of intensifying the rotation (double cropping).Sequence III has been the most volatile of all.It started with a sequence of one crop per year in the first cycle (regular rotation in the area of limited soils), but the arrival of soybean made it appropriate to be included as a double crop in those years when soil moisture conditions made soybean planting possible, on the basis of winter crops that are more secure in such soils.
The soil samples were extracted with a hydraulic borer to a depth of 20 cm, and each one was divided in 0-5 cm, 5-10 cm and 10-20 cm fractions, when the wheat cultivar was harvested (December 2009).

Fungal isolation and identification
Each soil sample (n = 45; 5 sequences × 3 replicates × 3 depths) was washed according to the modified methodology of Parkinson & Williams (1961).Fifty soil particles of each soil sample were placed on Petri dishes containing potato dextrose agar (2% PDA) amended with 250 mg chloramphenicol L -1 to suppress bacterial growth.Plates were incubated for 5 days in a controlled chamber at 25°C under 12-h light/dark conditions, fungal colonies morphologically similar to those of Fusarium were taken with a sterile claw and subcultured in Petri dishes with carnation leaf agar (CLA) (Fisher et al., 1982) and PDA tubes (16 mm × 150 mm).Plates were incubated for 6 to 15 days in a controlled chamber at 25°C under 12-h light/dark conditions.The Fusarium species were morphologically identified according to the taxonomic keys proposed by Nelson et al. (1983) and Leslie & Summerell (2006).
The isolates identified as Fusarium spp., were cultivated in Spezieller Nährstoffarmer Agar (SNA) (Nirenberg, 1976)  To avoid miss-identif ications at morphological level, 47 monosporic representative isolates selected at random (43 isolates of F. oxysporum, one of F. equiseti and three of F. solani), were cultured in Petri dishes with 2% PDA for 7 days at 25°C under 12-h light/dark conditions.The mycelia were harvested and the genomic DNA was extracted using the cetyltrimethylammnonium bromide (CTAB) protocol described by Stenglein & Balatti (2006).The DNA quality was examined by electrophoresis in 0.8% (w/v) agarose gels containing GelRed TM (Biotium, Hayward, USA) at 80 V in 1X Trisborate-EDTA buffer for 3 h at room temperature.The DNA was visualized under UV light.DNA concentrations were calculated using a fluorometer (Qubit TM -Invitrogen, Buenos Aires, Argentina).
Polymerase chain reaction (PCR) analyses (XP Termal Cycler, BIOR Technology CO, Hangzhou, China) using available species-specific primers for F. oxysporum (Mishra et al., 2003) and F. equiseti (Jurado et al., 2005) were made and compared with positive controls.In order to confirm the identity of F. solani isolations, the elongation factor 1-α (EF-1α) was amplified (O'Donnell et al., 1998;Geiser et al., 2004).The fragments were purified by AccuPrep ® Gel Purification Kit (Bioneer Corporation, California, USA).DNA sequencing, from both the sense and antisense ends of the fragments, were carried out using Big Dye Terminator version 3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) in an Applied Biosystems Sequencer (ABI/Hitachi Genetic Analyzer 3130).The sequences were compared with the NCBI database using BLASTN (Altschul et al., 1990).
Products from PCR reactions were examined by electrophoresis in 1.5% (w/v) agarose gels containing GelRed TM (Biotium, Hayward, USA) at 80 V in 1 X Trisborate-EDTA buffer for 1 h at room temperature.Fragments were visualized under UV light.The size of the DNA fragments were estimated by comparing the DNA bands with a 100 bp DNA ladder (Genbiotech SRL, Buenos Aires, Argentina).

Community diversity
The isolation frequency (Fr) was calculated according to Marasas et al. (1988) and the alpha-diversity through the Shannon (H'), Simpson (J) indexes and species richness (S), according to Magurran (1988) using the software PRIMER 5 (PIMER-E Ltd, UK, 2001).To detect the effect of crop rotation and depth on the mean of alpha-diversity parameters a variance analysis (ANOVA) was made using a free version of the INFOSTAT software (Di Rienzo et al., 2010).

Results
Fungal growth was observed on 983 soil particles from 2,250 analyzed ones.One hundred and twenty two isolates of Fusarium spp.were obtained.The 35.53% of isolates were obtained from soils with the conservationist agriculture rotation system (I), 27.27% from mixed agriculture/livestock with pastures (without using winter or summer forages) (II), 15.70 % from intensive agriculture (V), 12.40% from winter agriculture in depth limited soils (III), and 9.09% from mixed agriculture with annual forage (IV).
The Fusarium spp.species identified were: The number of Fusarium species isolated per sample varied from 1 to 3. The most prevalent species was F. oxysporum.This species was observed in all sequences and sampled depths.ANOVA analyses showed significant differences among sequences and depths for F. oxysporum Fr (Table 3).Tukey's test showed that F. oxysporum Fr under intensive agriculture (V) was significantly higher than mixed with annual forage (IV) (Fig. 1a).The first 5 cm of soil had a statistically significant higher F. oxysporum Fr index with respect to 5-10 cm and 10-20 cm depths (Fig. 1b).
Other Fusarium species were observed in lower percentages compared with F. oxysporum.The effect of sequences and depths on these species did not show statistically significant differences (Table 3).F. scirpi was isolated from soil particles from the first 5 cm of soil in winter agriculture in depth limited soils (III) and intensive agriculture (V) crop rotation systems; F. equiseti was obtained from the first 5 cm of soil in sequence V and from 10-20 cm of sequence III; F. merismoides was only obtained from 5-10 cm of sequence III and from 10-20 cm in sequence IV.F. solani was observed in the first 5 cm II and III, from 5-10 cm in IV and from 10-20 cm of II.
The ANOVA analysis for the H', J indexes and S did not show significant differences between crop rotation systems and sample depth for all species, including F. oxysporum (Table 4).

Discussion
It is known that Fusarium spp. is a cosmopolitan genus and a natural soil fungus.In agricultural soils, Fusarium spp. is a typical genus as Penicillium spp.and Trichoderma spp.among others.The results of this work agree with previous reports from other authors, who observed that Fusarium spp. is one of the most abundant fungi in agricultural soils and grasslands (Thorn, 1997;Nesci et al., 2006;Samaniego-Gaxiola & Madinaveitia-Chew, 2007).The presence of Fusarium spp. in agricultural soils could impact on crops health and conse-quently on the amount of human and animal food.Wakelin et al. (2008) suggested that the incorporation of stubble into the soil could produce increase of Fusarium spp.populations.Fusarium is a cellulolytic fungus and the stubble incorporation could increase fungal development and favour the breakdown of C and N source incorporated into the soil (Collins et al., 1990).
The diversity of Fusarium spp.species depended on crop rotations.Wakelin et al. (2008) showed that the diversity of Fusarium spp.community under stubbleburnt was less than stubble-retained systems.In the same study they observed, under glasshouse conditions, that the origin of stubble-plant affects significantly the Fusarium community structure.
In this study, in a long-term zero tillage soil, we identified five Fusarium species: F. equiseti, F. merismoides, F. oxysporum, F. scirpi and F. solani.Bateman & Murray (2001) in the UK met the same Fusarium species from wheat-field soils.In the same way, Steinkellner & Langer (2004)  Total 44 1 Sequence: I, II, III, IV and V (see text). 2 Depth: 0-5 cm, 5-10 cm, 10-20 cm.ns: non-significant differences.*** F value significant at p ≤ 0.05.F. merismoides and F. solani as the most frequent species in agricultural soils under different tillages in Ansfelden, Upper Austria.Moreover, Marasas et al. (1988) observed that F. oxysporum, F. equisetii and F. solani were the most prevalent species obtained from plant debris in South African agricultural soils.
The distribution of the five Fusarium species was different as regards agricultural sequences and depths.However, these differences were statistically significant only for F. oxysporum (Table 2).We observed the highest Fr in the first 5 cm of soil and the same result was found in Coronel Suárez (Buenos Aires province, Argentina) by Cabello (1986a).The highest value of Fr (8%) for F. oxysporum was observed in the sequence of intensive agriculture, a sequence of crops actually suggested by agricultural companies who rent the land (Forjan & Manso, 2010, 2011).The lowest value of Fr (2%) for F. oxysporum was observed in the crop rotation system mixed with annual forage.This result could be explained by the presence of grassing-cattle and the low availability of stubble in this crop rotation compared with other rotation systems.In the same way, Wakelin et al. (2008) observed the presence of F. oxysporum across rotations.Chehri (2011) detected F. oxysporum in wheat soils with higher percentage than in other crops.Moreover, F. oxysporum is considered a cosmopolitan species (Edel et al., 1997).Jeschke et al. (1990) showed F. oxysporum as the principal Fusarium species obtained from different altitudinal zones in Southern Africa.The high density of F. oxysporum in agricultural soils is a matter of study, because this fungus develops pathogenic and non-pathogenic isolates (Burgess, 1981).This feature could lead to the selection of non-pathogenic isolates of F. oxysporum to be used as biocontrol agents (Paulizt et al., 1987;Postma & Rattink, 1992).
In this work, F. scirpi was isolated from soil particles of the upper soil layer (0-5 cm) in the crop rotation sys-tems of soils with limitations (III) and of intensive agriculture (V).Both sequences present a high agricultural use due to double-cropping (late soybean that is usually sowed at the harvesting time of wheat or another winter crop).Chehri (2011) also observed F. scirpi in agricultural soils of Iran.F. scirpi has been usually/ commonly present soils of arid and semiarid environments (Leslie & Summerell, 2006).
The isolates of F. equiseti were obtained from winter agriculture in soils with limitations (III) and with intensive agriculture crop rotation systems (V).Similar results were observed by Marasas et al. (1988) and Fernández et al. (2008) who showed that the presence of F. acuminatum, F. equiseti and F. poae could be related with the incorporation of oilseed crops in the field sequence.F. equiseti was isolated from those sequences that presented combination of cereals (wheat/barley) with an oilseed crop.The change in the structure of fungal community could be produced by the presence of oilseed crops in the rotation that increases the N supply (Douglas et al., 1980).
In our study, F. merismoides was obtained only from 5-10 cm soil layer from the rotation of winter agriculture in soil with limitations (III) and from 10-20 cm soil layer of the traditional mixed rotation with annual forage (IV).Jeschke et al. (1990) observed low frequency of F. merismoides in soil samples collected at different altitudes in Southern Africa.Leslie & Summerell (2006) suggested that F. merismoides is a fungal saprophyte of soil, but it has the potential to cause some plant diseases if the environmental conditions are appropriate.Bateman & Murray (2001) observed that F. merismoides frequency increases in wheat field soil under dry conditions, suggesting that the presence of a mucilaginous matrix produced by this fungal species may favor its survival and ability to compete in dry conditions (Louis & Cooke, 1983).
We also detected isolates of F. solani in the 0-5 cm and 10-20 cm soil layers of II and in the 0.5 cm soil layer of III, and in the 10-20 cm soil layer in mixed agriculture with annual forage.Rupe et al. (1997) observed that the presence of sorghum and wheat in the sequence decreases the density of F. solani compared with the sequence with soybean and/or fescue.However, we observed F. solani in the sequences with soybean and wheat-oat/vicia-sunflower. The knowledge about the distribution of F. solani in crop rotations is important, because this pathogen is one of the causal agents of Soybean Sudden Death Syndrome (SDS).Cabello (1986a)  Total 44 1,2 See Table 3. ns: non-significant differences.
first centimeters of the Natraucol soil in Coronel Suárez, Buenos Aires province, without agricultural practices but with stubble of grasses and other herbaceous dicotyledonous species.Moreover, Marasas et al. (1988) found that the frequency of F. solani is higher in agricultural than in uncultivated soils.The same authors observed F. solani in soil samples collected at different altitudes in Southern Africa.We observed a different distribution and abundance of Fusarium species related to crop rotation systems and soil depth, however the ANOVA did not show significant differences for H', J and S richness indexes (Table 4).Therefore, we could not conclude that the alpha diversity of Fusarium community is affected by crop sequences and depths.However, Maina et al. (2009) observed significant differences among soil depth for S index in a Kenya soil from Taita Taveta district.Wakelin et al. (2008), using PCR analyses and denaturalizing gradient gel electrophoresis (DGGE), observed a strong relationship between the Fusarium community structure and crop sequences.
Several aspects (rotation, biodiversity, fertilization, biotechnology and soil nutrient balance) involved in zero tillage can produce a number of changes on fungal populations with particular ecological importance.Understanding the response of soil Fusarium spp.community to different agroecosystem managements is crucial to analyze the agricultural practices.

Figure 1 .
Figure 1.Frequency (Fr) values of Fusarium oxysporum populations in crop rotation systems (a) or soil depth (b).Different letters indicate significant differences between groups (p ≤ 0.05).
. The determination of P, NO 3

Table 2 .
and conserved in the fungal collection of the Laboratory of Functional Biology and Biotechno-Soil characteristics at experimental place (Barrow Experimental Station, Tres Arroyos, Buenos Aires Province)

Table 3 .
identified F. oxysporum, F. equisetii, Mean square values from the ANOVA analysis of the amount of Fusarium species

Table 4 .
observed a high frequency of F. solani in the Soil Fusarium population under wheat crop in zero tillage 77 Mean square values from the ANOVA analysis of H', J indexes and S of Fusarium community