Effects of non-chemical soil fumigant treatments on root colonisation with arbuscular mycorrhizal fungi and strawberry fruit production

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Crop Protection 55 (2014) 35e41

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Crop Protection

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Effects of non-chemical soil fumigant treatments on root colonisationwith arbuscular mycorrhizal fungi and strawberry fruit production

Darinka Koron a,*, Silva Sonjak b, Marjana Regvar b

aAgricultural Institute of Slovenia, Department of Fruit and Vine Growing, Hacquetova ulica 17, SI-1000 Ljubljana, SloveniabUniversity of Ljubljana, Biotechnical Faculty, Department of Biology, Ve�cna pot 111, SI-1000 Ljubljana, Slovenia

a r t i c l e i n f o

Article history:Received 12 November 2012Received in revised form16 September 2013Accepted 18 September 2013

Keywords:Arbuscular mycorrhizal fungi (AMF)Biofumigant plantsGlomusTemporal temperature gradient gelelectrophoresis (TTGE)

Abbreviations: AMF, arbuscular mycorrhizal fungi;septate endophyte; Es, Eruca sativa; HPLC, high-perraphy; Sa, Sinapis alba; TTGE, temporal temperature g* Corresponding author. Tel.: þ386 1 280 5142; fax

E-mail address: [email protected] (D. Koron).

0261-2194/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.cropro.2013.09.009

a b s t r a c t

The effects of biofumigation and soil heating on arbuscular mycorrhizal fungi (AMF) colonisation,strawberry growth and strawberry yield in pot experiments compared with untreated soil and chemicalfumigation with dazomet were tested. Three different Brassicaceae species (Brassica juncea, Eruca sativa,Sinapis alba) were used as biofumigant plant green manure and soil heating was applied to simulate soilsolarisation. Half of the plants were inoculated with indigenous arbuscular mycorrhizal fungi inoculum.With one exception (E. sativa) among the uninoculated plants, the treatments significantly decreasedthe mycorrhizal colonisation parameters compared with the untreated control. Dazomet displayed thegreatest inhibitory effects on AMF establishment. In addition, the intensity and number of bands cor-responding to Glomus spp. obtained with temporal temperature-gradient gel electrophoresis werelower for strawberry plants from biofumigant treatments than from the control. For the inoculatedplants, there were almost no significant differences among the mycorrhizal colonisation parameters.The mass of leaves for the uninoculated and inoculated plants was higher for almost all non-chemicalsoil fumigant treatments compared with the control, except for heating of the uninoculated treatments.The number of strawberry fruits for the uninoculated biofumigant treatments was the highest, beinghigher than the values observed for the heating treatments, the chemical disinfection treatments andthe control. There were no significant differences among the inoculated treatments. Biofumigation withBrassicaceae species resulted in higher soil organic matter and mineral nutrients and had a relativelysmall effect on AMF colonisation (F% ¼ 59.0, 80.3, 47.3 for Bj, Es and Sa, respectively) compared withuninoculated controls (F% ¼ 84.3). Despite the reduced AMF colonisation, biofumigation resulted in ahigher fruit number and mass of leaves. Therefore, it represents a non-chemical soil fumigation methodthat should be applied in sustainable strawberry production.

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1. Introduction

Strawberry (Fragaria � ananassa Duch.) is one of the mostintensively produced fruit crop. However, yields often suffer fromsoil-borne fungal pathogens (Mass, 1998). Therefore, chemicaldisinfection of the soil is widely used by growers. Methyl bromidewas the most frequently used chemical disinfectant in the past(Martin and Bull, 2002; Shaw and Larson,1999). In 2005, its usewasprohibited due to its depletion of the ozone layer (U.S.Environmental Protection Agency, 1999; EC No, 2037/2000).

Bj, Brassica juncea; DSE, darkformance liquid chromatog-radient gel electrophoresis.: þ386 1 280 5255.

All rights reserved.

Therefore, methyl bromide has been replaced by less harmfulchemicals, such as 1,3-dichloropropene, which was phased out in2009 in the European Union, chloropicrin (which will be phasedout in 2013), dazomet, and others (Ajwa and Trout, 2004; García-Méndez et al., 2008; Mark and Cassells, 1999).

Therefore, the use of non-chemical control approaches has beenencouraged as a sustainable alternative to chemical methods inagricultural plant defence. These approaches include various cul-tural practices (e.g., solarisation, biofumigation), induction of plantdefence responses and application of biological control agents(Linderman, 1994; Charron and Sams, 1999; Fageria et al., 2005;Alabouvette et al., 2006). For soil solarisation, the soil is coveredby a transparent plastic foil, which facilitates heating by solar ra-diation to temperatures that are detrimental to soilborne patho-gens (Pinkerton et al., 2002). Biofumigation is used to suppresssoilborne pests by taking advantage of toxic compounds that can bereleased from soil-incorporated tissues of plants. The biofumigant

D. Koron et al. / Crop Protection 55 (2014) 35e4136

plants of the Brassicaceae family are characterised by high gluco-sinolate content. Glucosinolates are released into the soil, wherethey can influence microbial growth (Lazzeri and Manici, 2001;Seigies and Pritts, 2006). Isothiocyanates (glucosinolate hydrolysisproducts) are the most common Brassicaceae volatiles that havebiofumigant properties (Gardiner et al., 1999; Schreiner and Koide,1993), and they are chemically changed to the active degradationproducts of synthetic fumigants like dazomet, and other methyl-isothiocyanate generating active ingredients (Mattner et al.,2008). In addition, the incorporation of Brassicaceae residues cansignificantly modify the biological, physical and chemical proper-ties of the soil. These plants act as green manure and thus enhancethe fertilising effects of the soil and consequently affect the soilmicrobial community (Lazzeri and Manici, 2001; Seigies and Pritts,2006). It has been demonstrated that the incorporation of differentBrassicaceae biofumigant plants into the soil can decrease thepopulations of pathogenic fungi (Charron and Sams, 1999; Lazzeriand Manici, 2001; Martin and Bull, 2002).

Strawberry plants are generally colonised by mutualistic fungi:the arbuscular mycorrhizal fungi (AMF). A functional symbiosisenhances strawberry plant growth (Malusa et al., 2006; Vestberget al., 2004) and increases resistance against pathogens instrawberries and other crops (Filion et al., 2003; Matsubara et al.,2004; Smith and Read, 1997). Thus, the use of current soil disin-fection methods, including chemical control, biofumigant plantsand solarisation, can affect the soil microbial population. How-ever, little is known about the effects of these approaches onstrawberry plant interactions with AMF. Therefore, the main aimof this study was to assess the effects of selected non-chemical soilfumigant treatments (biofumigation and heating) on AMF colo-nisation of strawberry plants and on strawberry plant growth andfruit production compared with untreated control plants andplants subjected to chemical fumigation. The diversity of the AMFassociated with the strawberry roots was determined, and thespecies were identified using temporal temperature-gradient gelelectrophoresis (TTGE) molecular technique and sequencing(Cornejo et al., 2004). In addition, the effects of indigenous AMFinoculum from a strawberry field on growth conditions andstrawberry production were tested.

2. Materials and methods

2.1. Plant material, experimental design and management

The pot experiment was conducted in a greenhouse at theAgricultural Institute of Slovenia on strawberry plantsFragaria � annanasa Duch. var. ‘Marmolada’ obtained by micro-propagation and acclimatised in a greenhouse at day/night tem-peratures of 22 �C/18 �C with a 14-h photoperiod for one monthbefore transplantation in December 2003 (Berljak et al., 2003). Theplants were transplanted into pots filled with treated soil andgrown under greenhouse conditions with natural light at temper-atures maintained above 20 �C for one year. The plants werewatered manually according to their needs (approximately 3 l/pot/month).

Pots were arranged in a completely randomised experimentaldesignwith 15 replications (pots) and two factors: inoculationwithAMF and soil treatment methods. The AMF inoculation groupincluded uninoculated or inoculated soil. Inoculum was producedby growing the AMF for 4months inmaize (Zeamays) as host plantsin soil collected from the organic strawberry field (AgriculturalInstitute of Slovenia). Inoculum potential was characterised usingthe level of AMF root colonisation of the host plant, where themycorrhizal frequency (F%), mycorrhizal intensity (M%), and

arbuscular density (A%) were assessed according to Trouvelot et al.(1986) (F% ¼ 96.7; M% ¼ 20.9; A% ¼ 10.9) (Regvar et al., 2001).

The soil treatments included the following: 1. Indian mustard,Brassica juncea (L.) Czern. & Coss, ‘Negro Caballo’ (Bj); 2. arugula,Eruca sativaMiller, unknown variety (Es); 3. White mustard, Sinapisalba L., ‘Asta’ (Sa); 4. heating; 5. The chemical disinfectant dazometand 6. untreated control.

The Brassicaceae plants were sown on 22 April (50 for Bj and Saand 70 plants/m2 for Es) and grown in the Agricultural InstituteExperimental Field at Brdo pri Lukovici, Slovenia (latitude 47� 560

1900 N e longitude 11� 360 4700 E e altitude 350 m a.s.l.; soil char-acteristics in Section 2.2) for 44 days. The above-ground parts werecollected just before flowering (11 June) and frozen at �20 �C. Theaverage mass of plants in the field was 4.1 for Bj, 3.2 for Es and2.3 kg/m2 for Sa. For all treatments, soil from the experimentalfield was used. For the uninoculated first three treatments withBrassicaceae plants, 25 g of frozen and ground-up plants wereadded to 250 g of soil. Pots of uninoculated treatments 4, 5 and 6were filled with 275 g of heated, disinfected or untreated soil. Inthe inoculated treatments, 100 g of soil was replaced with AMFinoculum. For the dry heat treatment, the soil was incubated at37 �C for 200 h (Inkubator I 50, Kambi�c, Slovenia) to simulate thetemperature regime of solarisation using transparent foil. In apreliminary soil disinfection field experiment, this regime wasdetermined to be most effective under our climate conditions. Forthe chemical fumigation, the soil was manually treated withdazomet at 50 g m�2 of soil, according to the manufacturer’s in-structions (Basamid�, BASF, AG, Germany) and Lazzeri and Manici(2001). All of the treatments and the substrates were prepared 12days before the strawberry plantlets transplanting, except for thetreatment with dazomet, which was prepared two months inadvance.

2.2. Soil and Brassicaceae plants analyses

The texture of soil used in experiment was silt loam (24.2% clay,8.8% sand, 18.9% coarse silt, 48.1% fine silt) (Saxton et al., 1986). Thesupply of mineral nutrients in the soil, except P, was good accordingto the Slovene national soil classification (Miheli�c et al., 2010). Thesoil parameters were measured before the experiment in July 2003(pre-treatment) and after the experiment in October 2004 (post-treatment; ten months of strawberry growth in soil without addedAMF inoculum). The organic matter content was determined ac-cording to ISO 14235:1998 Soil quality e determination of organiccarbon by sulphochromic oxidation; the pH was determined ac-cording to ISO 10390:2005 Soil quality e determination of pH; andthe total nitrogenwas determined according to ISO 11261:1995 Soilquality e determination of total nitrogen-modified Kjeldahlmethod. The amount of available phosphorus (P2O5) and potassium(K2O) in the soil was determined using the EgnereRiehmeDomi-ngo method (Egner et al., 1960).

The total amount of glucosinolates in the plant dry mass wasdetermined for the Bj, Es and Sa plants. Frozen plant samples werelyophilised, ground and analysed according to ISO 10633-1:1995Oilseed residues e determination of glucosinolates content e Part1: Method using high-performance liquid chromatography (HPLC).A Waters HPLC system was used (Milford, Massachusetts, USA),with a 600E pump, a 717 plus autosampler, and 996 diode-arraydetection.

2.3. Determination of strawberry root colonisation and plantgrowth

Root fungal colonisation was examined after seven weeks ofgrowth for 10 randomly selected plants from the control and from

D. Koron et al. / Crop Protection 55 (2014) 35e41 37

each treatment, without or with AMF inoculum. The roots obtainedfrom the pots were stored in Bless fixative (63% ethanol, 2.8%formaldehyde, and 4% acetic acid) until used. After storage, theroots were thoroughly washed with tap and distilled water, clearedin 10% KOH, and stained with 0.05% Trypan blue (Phillips andHayman, 1970). Root colonisation was estimated according toTrouvelot et al. (1986) using a Reichert 310 light microscope(Reichert, Inc., Depew, NY, USA) at a magnification of �100. Thirtyroot fragments per plant specimen were examined for mycorrhizalfrequency (F%), mycorrhizal intensity (M%) and arbuscular density(A%). The root fragments were also examined for the presence ofcharacteristic structures of dark septate endophyte (DSE) micro-sclerotia. The percentages of plants with these structures wererecorded. The mass of strawberry leaves was determined afterpicking in August 2004. The plant yields were determined byrecording the total number of fruits per plant produced in the firstfruiting season after their transplantation, over a period of sevenmonths (July 2004).

2.4. TTGE analysis and fungal identification

The roots of two of the most colonised strawberry plants fromthe control pots and of two plants from the treatment pots withoutmycorrhizal inoculum but with the highest colonisation frequencywere selected for further DNA analysis after 10months of growth inthe year 2004. The roots of each of these specimens were washedthoroughly with tap and sterile water, dried with clean tissue,randomly sampled (ca. 150 mg), ground up in liquid nitrogen, andstored at �20 �C until use. The total DNA was extracted from theroots using GenElute� Plant Genomic DNA miniprep kits (SigmaeAldrich, St. Luis, Mo., USA), following the manufacturer’srecommendations.

The fungi associated with the roots were analysed using thePCReTTGE technique, as described by Cornejo et al. (2004), toseparate the ca. 250-bp-long region of the small subunit ribo-somal DNA (SSU-rDNA) of most of the AMF, as obtained bynested PCRs. In the first PCR, a 1330-bp SSU-rDNA fragment wasamplified using crude DNA extract and the MH2 and MH4primers (Vandenkoornhuyse and Leyval, 1998). In the secondnested PCR, the NS31 universal eukaryotic primer (Simon et al.,1992) and the AM1 AMF-specific primer (Helgason et al., 1998)were used. The product of this PCR was then amplified with theGC-NS31 and Glo1 primers (Cornejo et al., 2004) to obtain DNAfor application on a TTGE polyacrylamide gel. The TTGE electro-phoresis was run as described by Cornejo et al. (2004). The mainsingle bands were excised from the TTGE gel, the DNA re-amplified with the NS31/Glo1 primer pair (no GC clamp) andthe PCR products separated electrophoretically. The expectedbands were excised, purified and cloned. One positive clone wasselected for sequencing. The sequences obtained were comparedto the available sequences of the National Center for

Table 1Analyses of the soil parameters before the non-chemical soil fumigant treatment (pre-tr

Treatment pH P2O5 (mg/100 g soil) K

Pre-treatment 6.0 � 0.3 4.1 � 0.1 2

Post-treatmentBj 5.9 � 0.1 b 7.9 � 0.1 c 2Es 5.8 � 0.1 bc 11.0 � 0.1 a 2Sa 6.2 � 0.1 a 11.0 � 0.1 a 2Heating 5.2 � 0.1 d 6.9 � 0.3 d 1Dazomet 5.1 � 0.0 d 5.8 � 0.1 eControl 5.5 � 0.1 c 8.4 � 0.1 b 1

Bj e Brassica juncea; Es e Eruca sativa; Sa e Sinapis alba.

Biotechnology Information (NCBI) using the BLAST-n programme(Altschul et al., 1990, 1997), and they were deposited in GenBank;their accession numbers are given in Table 3. The PCR conditions,volumes, reagents, kits, and equipment used were as described inSonjak et al. (2009).

2.5. Statistical analyses

The effects of the different fumigation treatments on root AMFcolonisation, strawberry plant growth and fruit yield data werestatistically analysed in a complete randomised factorial designwith two factors: soil treatments (six levels: Bj, Es, Sa, heating,dazomet and control) and AMF inoculation (two levels: uninocu-lated and inoculated) with 10 replications (single plant) for AMFcolonisation and 15 replications (single plant) for growth and yieldparameters. To improve the residual error distribution, an arc-sinsquare-root transformation was applied to the percent AMF colo-nisation data. Means and SEs back-transformed to the original scaleare reported. We used the STATGRAPHICS Centurion XV.II pro-gramme (StatPoit, Inc., 2006) for analysis of variance (ANOVA).Significant differences were determined according to Duncan’sMultiple Range Test (p � 0.05).

3. Results

3.1. Pre- and post-treatment soil analyses

The pre-treatment soil supply of mineral nutrients, except P2O5,was good according to the national classification (optimum forplant available P2O5 is 13e15 mg/100 g of soil and for K2O is 20e30mg/100 g of soil) (Miheli�c et al., 2010) (Table 1). After tenmonthsof strawberry growth (post-treatment), the amount of P2O5 and thepercentages of organic matter were higher in all soil treatments,with P2O5 especially higher in the Es and Sa treatments. The levelsof total nitrogen, K2O and the pH were more comparable. In theheating, dazomet and control treatments, the amount of K2Odecreased, and the percentages of total nitrogen in the heating,dazomet and control treatments were lower than before theexperiment (pre-treatment).

3.2. Brassicaceae plants analyses

The total glucosinolates in the dry mass of the biofumigantplants was 16.8 mmol g�1 for the Bj plants, 16.6 mmol g�1 for the Esplants and 6.4 mmol g�1 for the Sa plants. The amount of bio-fumigant residues in the soil was evaluated on the basis of Lazzeriand Manici (2001). We added 91 g of each biofumigant plant per kgof soil or 78.9 mg GSL per kg of soil to the Bj treatment, 75.2 mg GSLper kg of soil to the Es treatment and 28.6 mg GSL per kg of soil tothe Sa treatment.

eatment) and after the experiment (post-treatment).

2O (mg/100 g soil) Total nitrogen (%) Organic matter (%)

5.9 � 0.1 0.27 � 0.01 3.4 � 0.1

9.0 � 0.3 a 0.28 � 0.01 ab 5.6 � 0.1 b5.0 � 0.3 b 0.27 � 0.01 b 5.3 � 0.1 c5.0 � 0.7 b 0.30 � 0.00 a 6.3 � 0.1 a0.0 � 0.5 d 0.25 � 0.00 b 4.1 � 0.1 e7.5 � 0.1 e 0.25 � 0.00 b 4.3 � 0.1 e3.0 � 0.1 c 0.25 � 0.01 b 4.8 � 0.1 d

D. Koron et al. / Crop Protection 55 (2014) 35e4138

3.3. Determination of strawberry root colonisation and plantgrowth rates

Uninoculated strawberry plants from all of the treatments were,to some extent, colonised with indigenous AMF (Fig. 1). The colo-nisation levels of uninoculated control plants were 84.3, 9.5 and 7.6for F%, M% and A%, respectively. The most prominent decrease inindigenous AMF was observed for the dazomet treatment, wherethe average F% reached only 1.3, and theM% and A% were both near

Fig. 1. Effects of different soil treatments on inoculated and uninoculated strawberry plantcolonisation (M%), arbuscular density (A%), mass of the leaves and number of fruits. The leinoculated series. The data are the averages � SE (n ¼ 10). Bj e Brassica juncea; Es e Eruca

zero. For the biofumigant plants, treatment with Es resulted in thehighest F% (80.3), whereas M% and A% were not significantlydifferent from the other two treatments.

Inoculation with indigenous AMF significantly improved the F%,M% and A% of the inoculated strawberry plants in comparison withuninoculated plants (Fig. 1). The differences between both mainfactors (uninoculated and inoculated strawberry plants) are highlystatistically significant at p > 0.001 (Table 2). F% within the inoc-ulated treatments is relatively similar, whereas the same was not

s, as compared with their respective control, on AMF frequency (F%), intensity of AMFtters indicate significant differences between the treatments within inoculated or un-sativa; Sa e Sinapis alba.

Table 2Effects of treatments (Bj, Es, Sa, Heating, Dazomet, Control), inoculation with AMF(uninoculated treatments, inoculated treatments) and their interaction on the AMFfrequency (F%), intensity of AMF colonisation (M%), arbuscular density (A%), biomassof leaves and number of fruits.

Source of variation F (%) M (%) A (%) Mass of leaves(g/plant)

Fruits (number/plant)

Treatment (T) *** *** *** *** ***Inoculation (AMF) *** *** *** *** NST � AMF *** ** ** * ***

NS ¼ nonsignificant; *, **, ***Significance at 0.05, 0.01 and 0.001, respectively.

Fig. 2. Representative TTGE gel of two control samples (C1, C2) and two samplestreated with Eruca sativa (Es1, Es2). The numbers 1e10 indicate the bands sequenced(see Table 2).

D. Koron et al. / Crop Protection 55 (2014) 35e41 39

true for M% and A%. Root fragments were moderately colonisedwith DSE microsclerotia. Within the uninoculated treatments, onlyone plant (n ¼ 10) of the Es treatment had DSE microsclerotia,whereas in the series with the AMF inoculum and the Es and Bjtreatments, there were four plants with DSE microsclerotia. All ofthe plants were generally healthy. The greatest leaf mass was ob-tained with the biofumigant plant treatments with inoculated anduninoculated strawberry plants. Lower biomass, but still higherthan that observed with the control, was obtained with the dazo-met treatment (Fig. 1). However, this was not reflected by the fruitnumber. Instead, for the uninoculated strawberry plants, the Bj andSa treatments displayed the highest fruit production, and AMFinoculation significantly improved the fruit production of thecontrol treatments to the point that they matched the numbers ofthe Es and Sa treatments of the uninoculated plants. Interestingly,fruit production of the dazomet treatment was also significantlyimproved by AMF inoculation.

Similar results as here presented were obtained also in twoadditional, equally designed but incomplete (some treatmentsmissing) experiments performed in different years.

Table 3

3.4. Diversity and identification of mycorrhizal fungi

In the experimental series without the AMF inoculum, in addi-tion to the control, the highest colonisation frequencies were ob-tained for the strawberry plants from the Es treatment. Todetermine the most frequent indigenous root colonisers of thesestrawberry plants, the roots of the two most-colonised plants fromboth the control and the Es treatments were analysed for thepresence of AMF using TTGE and sequencing. For each sample, up tofive clearly visible TTGE bands were obtained. The numbers andintensities of the bands were lower for the Es samples. The verystrong bands obtained for the control samples were absent in the Essamples (Fig. 2, bands 3, 7).

The sequencing of the bands confirmed the presence of Glomusspecies. As revealed by BLAST analysis, most of the sequences hadthe highest similarity with uncultured Glomus sp., and only onesequence had 99% similarity with Glomus fasciculatum (Table 3).

GenBank accession numbers of sequences obtained and closest GenBank matches.

Bandnumbera

GenBankaccessionumber

Closest GenBank match Similarity %(E value)

1 JF895998 EU332711 Glomus sp. 99 (9e�117)Y17640 Glomus fasciculatum 99 (5e�114)

2 JF895999 AM946834 Uncultured Glomus 98 (2e�113)3 JF896000 AM946834 Uncultured Glomus 98 (2e�113)4 JF896001 EU123460 Uncultured Glomus 97 (4e�110)5 JF896002 EU123454 Uncultured Glomus 99 (4e�115)6 JF896003 AM946834 Uncultured Glomus 98 (9e�112)7 JF896004 EU123454 Uncultured Glomus 99 (9e�117)8 JF896005 EU573762 Uncultured Glomus 99 (4e�115)9 JF896006 EU573765 Uncultured Glomus 98 (9e�112)10 JF896007 AJ852534 Glomus sp. 99 (4e�115)

a See Fig. 2.

4. Discussion

All of the strawberry plant samples examined, regardless of thetreatments, were to some extent colonised by AMF. As 11.8% ofexamined plants also contained microsclerotia, the F% values ob-tained can be partly ascribed to the presence of DSE fungi, whichare widespread organisms that can form mutualistic, mycorrhiza-like associations with their host plants (Jumpponen, 2001). Thefungal colonisation levels were dramatically reduced in the roots ofthe uninoculated plants when dazomet was applied, reaching av-erages of 1.3 F% and M% and A% values of 0.0. These results are inagreement with a study of Mark and Cassells (1999), where theydemonstrated that after treatment with dazomet, no mycorrhizal

colonisation by indigenous AMF occurred in commercial straw-berries. Dazomet is fungicidal for a broad spectrum of fungi,including most of the important strawberry pathogens. As has beenshown for in addition, a similar effect has been shown for methylbromide:chloropicrin (McGraw and Hendrix, 1986). In contrast todazomet, soil heating or biofumigation of the soil only moderatelyreduced root AMF colonisation, as compared with the control. Thisis in agreement with studies comparing the effect of solarisationand fumigation with metam sodium and methyl bromide on AMF.Solarisation did not reduce AMF colonisation, but fumigationsignificantly reduced AMF colonisation (Schreiner et al., 2001).

According to Garland et al. (2011), the incorporation of non-mycorrhizal cover crop hosts, as compared to other summercover crops and a no-cover crop control treatments, did notsignificantly affect the percent mycorrhizal colonisation in thestrawberry roots. All treatments reduced the AMF colonisationparameters in our study, with the exception of the F% values for theroots of the Es-treated strawberry plants. These effects did notappear to be related to the total glucosinolate content of the bio-fumigant plants used. However, a greater specific activity of one ormore of the glucosinolate degradation products in amixture cannotbe excluded, as some indole glucosinolates might be involved in

D. Koron et al. / Crop Protection 55 (2014) 35e4140

preventing AMF formation in plant species (Vierheilig et al., 2000).As the glucosinolate contents and diversity are known to changeduring plant ontogenesis (Pongrac et al., 2008), the developmentalstage of the biofumigant plants might be one of the factors influ-encing AMF colonisation after treatment. Furthermore, freezing ofglucosinolates containing plants results in an increase of isothio-cyanate release into soil by two orders of magnitude (Morra andKirkegaard, 2002) and thus enhances the impact of the Brassica-ceae green manure on mycorhizal development. In addition to thebiocidal activity against Phytium, an increase in the total fungal soilpopulation was observed when soil was treated with selectedbiofumigant and non-biofumigant plants (Lazzeri and Manici,2001). These authors concluded that soil fungal population devel-opment is affected by the addition of fresh organic matter. Brassi-caceae greenmanure has many potential beneficial effects: not onlythe suppression of pathogens and weeds but also improvements inthe soil structure and health. Brassicaceae can increase the watercapacity of the soil, the organic and mineral content, and the mi-crobial diversity (Matthiessen and Kirkegaard, 2006). Althoughculturable soil bacteria populations have been demonstrated to beunaffected by the incorporation of certain Brassicaceae species, thisdoes not appear to be the case for fungi (Mattner et al., 2008). OurTTGE analysis of strawberry plant roots growing in soil with Esconfirmed a decrease in the AMF population when compared withthe control, in addition to decreased AMF colonisation parametersin the treatments with biofumigant plants. As our sequencingconfirmed, the TTGE bands corresponded to Glomus species.Accordingly, we can conclude that these biofumigant plants caninhibit the establishment and growth of particular Glomus species.

The highest mass of leaves and the highest number of fruitswere obtained for the strawberry plants growing in the soil withbiofumigant plants. As the AMF colonisation parameters in thebiofumigant treatments were lower than in the control treatment,this effect could be attributed to changes in the soil nutrients andorganic matter content that arise from the incorporation of bio-fumigant plants into the soil where they function as green manure(Table 1) (Fageria et al., 2005; Seigies and Pritts, 2006). However,Garland et al. (2011) observed that cover crop and mycorrhizaltreatment did not significantly affect average strawberry weight,marketable yield or total yield nor were there any interactionsamong treatments. As Sa is commonly used in crop rotation inSlovenia, it was selected as the most efficient among the threebiofumigant species tested. Es appears less interesting for straw-berry fruit production because of the lower biomass produced. Bjwas selected for this experiment because of its high glucosinolatecontent; however, it is not a common crop grown in Slovenia. In ourexperiment, plants treated with Sa displayed the highest mass ofthe leaves, which was accompanied by high fruit production. Dry Saplant material contained much lower, half of Bj and Es amount oftotal glucosinolates, however, detrimental effect of its treatment onAMF was not significantly lower. It is possible that if trials wereperformed in soils with a high soil-borne fungi presence (AMF, DSEand pathogens) these differences would be more evident also incase of plant production. Nonetheless incorporation of biofumigantplants into the soil should be encouraged, as this method canprovide an increase in strawberry fruit production. Based on ourresults Sinapis alba does seem to be themost promising of the threebiofumigant species tested.

For the strawberry plants grown in the heat-treated soil, boththe mass of the leaves and the fruit production were comparable tothose in the uninoculated control, whereas for the plants from thedazomet-treated soil, a higher leaf biomass was obtained, alongwith fruit levels that did not differ significantly from the control. Ina study where different chemicals were comparedwith solarisationand untreated control, treatment with dazomet resulted in a very

similar yield to the control (Rieger et al., 2001). The beneficial ef-fects of dazomet result from its suppression of soil pathogens;however, as dazomet is a severe fumigation substance, it inhibitsAMF and other soil microorganisms as well and can consequentlyreduce strawberry production. Similarly, of all of the soil treat-ments, the lowest crop yield was obtainedwith themethyl bromideplus chloropicrin treatment, and no association between vegetativevigour and fruit production was observed in a study of strawberryfruit production under field conditions (Seigies and Pritts, 2006).These results do not support the use of chemical fumigation infarming, which is aimed at suppression of soil pathogens to in-crease crop yield. Under field conditions, however, the plants aremore readily exposed to pathogens, and thus the beneficial effectsof chemical fumigation might be more pronounced.

Fungal inoculation of strawberry plants might produce differingresponses in terms of plant growth and fruit production. Theapplication of commercially available or native AMF inoculants tostrawberry plants resulted in higher root colonisation levels andleaf area but did not result in increased yield (Garland et al., 2011).Different AMF can either enhance or reduce the growth of straw-berry shoots and roots (Taylor and Harrier, 2001; Vestberg et al.,2004). Inoculation with indigenous fungal populations that comefrom long-term funguseplant compatibility selection usually pro-vides the most satisfying results, due to the opportunity to findcompatible symbionts and to enhance the mutualistic effects withtwo ormore symbionts, rather than just with one. At the same time,the chances of negative interactions between the introduced AMFare diminished. In addition, indigenous AMF are presumably bestsuited to the prevailing environmental conditions, which willpresumably be one of the reasons for their long-lasting persistenceat a site (Regvar et al., 2003). In our experimental set-up, the dif-ferences between uninoculated and inoculated strawberry plantswere highly statistically significant in all AMF parameters, reflect-ing the facts that the AMF inoculations of the treatments withindigenous fungal populations containing several Glomus speciesfrom the strawberry field reduced the deleterious effects of soilfumigation treatments. Due to the green-manure effect of thesebiofumigant plants, the inoculated strawberry plants did notdisplay significantly better yield than the uninoculated plants inthese treatments. In general, Sa had an influence on soil parameters(available phosphorous, total nitrogen and organicmatter), onmassof leaves and number of fruits, but Es had a less detrimental effecton AMF colonisation and also positive influence on strawberrygrowth and yield.

In conclusion, replanting without crop rotation can result indecreased strawberry plant vigour, growth and yield. The use ofdazomet was demonstrated here to have positive influences onplant growth, although it inhibited plant production and it greatlyinhibited colonisation with AMF, which is an important part ofsustainable agricultural systems. Thus, dazomet should be avoided,or alternatively, be applied with the subsequent addition of AMFinoculum, although this would consequently increase the price ofproduction.

Heating as a simulation of solarisation can provide a good bal-ance between AMF sustainability and plant yield through its effectson soil organic matter decomposition and the related nutrientavailability, as well as for suppression of pathogens. Therefore,heating is a potential alternative to chemical fumigationtreatments.

The most productive results were obtained using biofumigantplants for soil fumigation. Bj is the biofumigant plant that is mostoften cited, although Sa is a commonly used green-manure crop inSlovenia. The soil treatments with each of these three biofumigantplants tested revealed moderate inhibitory effects on strawberryplan AMF colonisation, whereas they increased the plant growth

D. Koron et al. / Crop Protection 55 (2014) 35e41 41

and fruit production, especially for the Bj and Sa treatments. Theseresults indicate that the use of biofumigant plants should beencouraged in organic strawberry production and should be anintegral part of the production as a crop-rotation plant and as greenmanure to improve strawberry production and to maintain thefertility of the soil and the diversity of microorganisms in therhizosphere. When compared with chemical soil fumigationtreatments, the application of biofumigant plants might be moreexpensive, but this treatment represents a sustainable and publiclyacceptable alternative and should thus be considered in contem-porary strawberry crop production.

Acknowledgements

This study was supported by the Slovenian Research Agency inits funding of the research project “Alternative soil disinfectionmethods using solarisation and plant glucosinolates” (L4-6294) andthe research programme “Biology of plants” (P1-0212) and COST8.38 “Managing arbuscular mycorrhizal fungi for improving soilquality and plant health in agriculture”.

References

Ajwa, A.H., Trout, T., 2004. Drip application of alternative fumigants to methylbromide for strawberry production. HortScience 39, 1707e1715.

Alabouvette, C., Olivain, C., Steinberg, C., 2006. Biological control of plant diseases:the European situation. Eur. J. Plant Pathol. 114, 329e341.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic localalignment search tool. J. Mol. Biol. 215, 403e410.

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J.,1997. Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs. Nucl. Acid Res. 25, 3389e3402.

Berljak, J., Marn, M., Koron, D., 2003. In vitro plant regeneration from somatic tissueof strawberry Fragaria � ananassa Duch. Acta Biol. Slov. 46, 47e50.

Charron, C.S., Sams, C.E., 1999. Inhibition of Pythium ultimum and Rhizoctonia solaniby shredded leaves of Brassica species. J. Am. Soc. Hort. Sci. 124, 462e467.

Cornejo, P., Azcón-Aguilar, C., Barea, J.M., Ferrol, N., 2004. Temporal temperaturegradient gel electrophoresis (TTGE) as a tool for the characterization of arbus-cular mycorrhizal fungi. FEMS Microbiol. Lett. 241, 265e270.

Egner, H., Riehm, H., Domingo, W.R., 1960. Untersuchungen über die chemischeBodenanalyse als Grundlage für die Beurteilung des Nährstoffzustandes derBöden. II. In: Chemische Extraktionsmethoden zur Phosphor- und Kaliumbes-timmung. Kgl. Lantbruskögsk, Bd. 26, pp. 199e215.

Fageria, N.K., Baligar, V.C., Bailey, B.A., 2005. Role of cover crops in improving soiland row crop productivity. Commun. Soil Sci. Plant Anal. 36, 2733e2757.

Filion, M., St-Arnaud, M., Jabaji-Hare, S.H., 2003. Quantification of Fusarium solani f.sp. phaseoli in mycorrhizal bean plants and surrounding mycorrhizosphere soilusing real-time polymerase chain reaction and direct isolation on selectivemedia. Phytopathology 93, 229e235.

García-Méndez, E., García-Sinovas, D., Becerril, M., De Cal, A., Melgarejo, P., Martí-nez-Treceno, A., Fennimore, S.A., Soria, C., Medina, J.J., López-Aranda, M., 2008.Chemical alternatives to methyl bromide for weed control and runner plantproduction in strawberry nurseries. HortScience 43, 177e182.

Gardiner, J.B., Morra, M.J., Eberlein, C.V., Brown, P.D., Borek, V., 1999. Allelochemicalsreleased in soil following incorporation of rapeseed (Brassica napus) greenmanures. J. Agric. Food Chem. 47, 3837e3842.

Garland, B.C., Schroeder-Moreno, M.S., Fernandez, G.E., Creamer, G.N., 2011. Influ-ence of summer cover crops and mycorrhizal fungi on strawberry production inthe southeastern United States. HortScience 46, 985e992.

Helgason, T., Daniell, T.J., Husband, R., Fitter, A.H., Young, J.P.W., 1998. Ploughing upthe wood-wide web? Nature 394, 431.

Jumpponen, A., 2001. Dark septate endophytes e are they mycorrhizal? Mycorrhiza11, 207e211.

Lazzeri, L., Manici, L.M., 2001. Allelopathic effect of glucosinolate-containing plantgreen manure on Pythium sp. and total fungal population in soil. HortScience36, 1283e1289.

Linderman, R.G., 1994. Role of VAM fungi in biocontrol. In: Pfleger, F.L., Linderman, R.G.(Eds.), Mycorrhizae and Plant Health. APS Press, St. Paul, pp. 1e26.

Malusa, E., Sas-Paszt, L., Popi�nska, W., �Zurawicz, E., 2006. The effect of a substratecontaining arbuscular mycorrhizal fungi and rhizosphere microorganisms(Trichoderma, Bacillus, Pseudomonas and Streptomyces) and foliar fertilization ongrowth response and rhizosphere pH on three strawberry cultivars. Int. J. FruitSci. 6, 25e41.

Mark, G.L., Cassells, A.C., 1999. The effect of dazomet and fosetyl-aluminium onindigenous and introduced arbuscular mycorrhizal fungi in commercialstrawberry production. Plant Soil 209, 253e261.

Martin, F.N., Bull, C.T., 2002. Biological approaches for control of root pathogens ofstrawberry. Phytopathology 92, 1356e1362.

Mass, J.L., 1998. Compendium of Strawberry Diseases, second ed. APS Press, St. Paul.Matsubara, Y., Hirano, I., Sassa, D., Koshikawa, K., 2004. Increased tolerance to

fusarium wilt in mycorrhizal strawberry plants raised by capillary wateringmethods. Environ. Control Biol. 42, 185e191.

Matthiessen, J.N., Kirkegaard, J.A., 2006. Biofumigation and enhanced biodegrada-tion: opportunity and challenge in soil-borne pest and disease management.Crit. Rev. Plant Sci. 25, 235e265.

Mattner, S.W., Porter, I.J., Gounder, R.K., Shanks, A.L., Wren, D.J., Allen, D., 2008.Factors that impact on the ability of biofumigants to suppress fungal pathogensand weeds of strawberry. Crop Prot. 27, 1165e1173.

McGraw, A.-C., Hendrix, J.W., 1986. Influence of soil fumigation and source ofstrawberry plants on population densities of spores and infective propagules ofendogonaceous mycorrhizal fungi. Plant Soil 94, 425e434.

Miheli�c, R., �Cop, J., Jak�se, M., �Stampar, F., Majer, D., Tojnko, S., Vr�si�c, St, 2010.Guidelines for the Professional Approach Fertilization. Ministry for Agricultureand the Environment, Slovenia, pp. 91e114.

Morra, M.J., Kirkegaard, J.A., 2002. Isothiocyanate release from soil-incorporatedBrassica tissues. Soil Biol. Biochem. 34, 1683e1690.

Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots andstaining parasitic and vesicularearbuscular mycorrhizal fungi for rapidassessment of infection. Trans. Brit. Mycol. Soc. 55, 158e161.

Pinkerton, J.N., Ivors, K.L., Reeser, P.W., Bristow, P.R., Window, G.E., 2002. The use ofsoil solarization for the management of soil-borne plant pathogens in straw-berry and red raspberry production. Plant Dis. 6, 645e651.

Pongrac, P., Vogel-Miku�s, K., Regvar, M., Tolrà, R., Poschenrieder, C., Barceló, J., 2008.Glucosinolate profiles change during the life cycle and mycorrhizal colonizationin a Cd/Zn hyperaccumulator Thlaspi praecox (Brassicaceae). J. Chem. Ecol. 34,1038e1044.

Regvar, M., Groznik, N., Goljav�s�cek, K., Gogala, N., 2001. Diversity of arbuscularmycorrhizal fungi at various differentially managed ecosystem in Slovenia. ActaBiol. Slov. 44 (3), 27e34.

Regvar, M., Vogel-Miku�s, K., �Severkar, T., 2003. Effect of AMF inoculums from fieldisolates on the yield of green pepper, parsley, carrot and tomato. Folia Geobot38, 223e234.

Rieger, M., Krewer, G., Lewis, P., 2001. Solarization and chemical alternatives tomethyl bromide for preplant soil treatment of strawberries. Hort Technol. 11,258e264.

Saxton, K.E., Rawls, W.J., Romberger, J.S., Papendick, R.I., 1986. Estimating general-ized soilewater characteristics from texture. Soil Sci. Soc. Am. J. 50 (4), 1031e1036.

Schreiner, R.P., Koide, R.T., 1993. Mustards, mustard oils and mycorrhizas. NewPhytol. 123, 107e113.

Schreiner, R.P., Ivors, K.L., Pinkerton, J.N., 2001. Soil solarization reduces arbus-cular mycorrhizal fungi as a consequence of weed suppression. Mycorrhiza 11,273e277.

Seigies, A.T., Pritts, M., 2006. Cover crop rotations alter soil microbiology and reducereplant disorders in strawberry. HortScience 41, 1303e1308.

Shaw, D.V., Larson, K.D., 1999. A meta-analysis of strawberry yield response to pre-plant soil fumigation with combinations of methyl bromideechloropicrin andfour alternative systems. HortScience 34, 839e845.

Simon, L., Lalonde, M., Bruns, T.D., 1992. Specific amplification of 18S fungal ribo-somal genes from vesicularearbuscular endomycorrhizal fungi colonizing roots.Appl. Environ. Microbiol. 58, 291e295.

Smith, S.E., Read, D.J., 1997. Mycorrhizal Symbiosis, second ed. Academic Press, SanDiego.

Sonjak, S., Udovi�c, M., Wraber, T., Likar, M., Regvar, M., 2009. Diversity of halo-phytes and identification of arbuscular mycorrhizal fungi colonising their rootsin an abandoned and sustained part of Se�covlje salterns. Soil Biol. Biochem. 41,1847e1856.

Taylor, J., Harrier, L.A., 2001. A comparison of development and mineral nutrition ofmicropropagated Fragaria � ananassa cv. Elvira (strawberry) when colonised bynine species of arbuscular mycorrhizal fungi. Appl. Soil Ecol. 18, 205e215.

Trouvelot, A., Kough, J.L., Gianinazzi-Pearson, V., 1986. Mesure du taux demycorhization VA d’un système radiculaire. Recherche de méthodes d’estima-tion ayant une signification fonctionnelle. In: Gianinazzi-Pearson, V.,Gianinazzi, S. (Eds.), Physiological and Genetical Aspects of Mycorrhizae. INRAPress, Paris, pp. 217e221.

United States Environmental Protection Agency, 1999. Protection of stratosphericozone: incorporation of Montreal protocol adjustment for a 1999 interimreduction in Class I, Group VI controlled substances. Fed. Registr. 64, 29240e29245.

Vandenkoornhuyse, P., Leyval, C., 1998. SSU rDNA sequencing and PCR-fingerprinting reveal genetic variation within Glomus mosseae. Mycologia 90,791e797.

Vestberg, M., Kukkonen, S., Saari, K., Parikka, P., Huttunen, J., Tainio, L., Devos, N.,Weekers, F., Kevers, C., Thonart, P., Lemoine, M.C., Cordier, C., Alabouvette, C.,Gianinazzi, S., 2004. Microbial inoculation for improving the growth and healthof micropropagated strawberry. Appl. Soil Ecol. 27, 243e258.

Vierheilig, H., Bennet, R., Kiddle, G., Kaldorf, M., Ludwig-Müller, J., 2000. Differencesin glucosinolate patterns and arbuscular mycorrhizal status of glucosinolate-containing plant species. New Phytol. 146, 343e352.