The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline

Biological Control xxx (2016) xxx–xxx

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The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline L.M. Manici a,⇑, F. Caputo a, F. Nicoletti a, F. Leteo b, G. Campanelli b a b

Council for Agricultural Research and Economics, Agriculture and Environment Research Center (CREA – AA), Via di Corticella 133, 40128 Bologna, Italy Council for Agricultural Research and Economics, Horticulture and Floriculture Research Center (CREA – OF), Via Salaria 1, 63030 Monsapolo del Tronto (Ascoli Piceno), Italy

h i g h l i g h t s
The impact of two cover crops on next vegetable growth was investigated.
Hairy vetch and barley harbored differing root colonizing fungal communities.
Hairy vetch and barley did not affect differently root fungi in following crops.
Tomato and zucchini showed the best growth response after hairy vetch.
Vetch and barley induced a diverse rhizosphere bacterial composition in vegetables.

a r t i c l e

i n f o

Article history: Received 22 May 2016 Revised 24 October 2016 Accepted 8 November 2016 Available online xxxx Keywords: Organic agriculture Rhizosphere bacteria Root-colonizing fungi 16S rDNA PCR-DGGE Root rot

a b s t r a c t Crop decline is a progressive soil health reduction commonly associated to the specialized cultivations. As a series of biotic factors are involved in this phenomenon, it can be controlled with essentially agronomic methods, the impact of winter cover crops on early growth at subsequent vegetable crops was investigated in a two-year field study. Barley and hairy vetch were incorporated into the soil following two mechanical terminations of cover crops (green manure and green mulching). Immediately after, tomato and zucchini seedlings were transplanted and grown for 28 days. The above-ground biomass of the vegetables was taken as indicator of crop response to pre-plant treatments, whilst root colonizing fungi and rhizosphere bacteria were the two analyzed soil microbial components. Root-colonizing fungi were evaluated using culture-based methods, bacteria were analyzed by amplification of rhizosphere soil DNA with 16S rDNA, then processed with PCR-DGGE. Tomato and zucchini growth response after vetch was always significantly higher than after barley regardless of mechanical termination. Rhizosphere bacterial communities differed significantly between cover crops and this differences was maintained also in the subsequent vegetable crops. Root-colonizing fungi differed between barley and vetch, although they shared most species (70%) such as Pythium spp., binucleate Rhizoctonia AG-A and several Fusarium spp. The latter three fungal groups were also found most abundant in tomato and zucchini roots. Pathogenicity test showed that tomato was more susceptible than zucchini to Pythium, Rhizoctonia solani, Fusarium oxysporum and Fusarium spp. and that their relationship with vegetable plants varied from pathogenic to neutral, up to mutualistic in the case of Rhizoctonia AG-A. These findings indicate difficulty to identify specific biotic agents responsible of crop decline. Tomato and zucchini showed an undoubted growth improvement after legume; however, a certain specificity of Cylindrocarpon-like fungi and Phoma spp. to hairy vetch suggests that, when exploiting benefit of this cover crop for mitigating yield decline of vegetable crops, legume should alternate cereal and other botanically distant genera in rotation. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Soil-borne fungal pathogens can cause yield losses in herbaceous and tree fruit crops and forest trees; but vegetable crops ⇑ Corresponding author. E-mail address: [email protected] (L.M. Manici).

are commonly considered the most susceptible to those pathogens, especially in the early stages of growth (Koike et al., 2006). Yield losses due to soil borne diseases have been estimated accounting for 10% in vegetable crops in 2009 in U.S. (Sherm, 2012), however, up to 60% yield losses have been recorded in specific crops such onion (Schwartz, 2011). The biotic causal agents of crop yield decline are difficult to estimate because the resulting lack of vigor

http://dx.doi.org/10.1016/j.biocontrol.2016.11.003 1049-9644/Ó 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: Manici, L.M., et al. The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline. Biological Control (2016), http://dx.doi.org/10.1016/j.biocontrol.2016.11.003

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L.M. Manici et al. / Biological Control xxx (2016) xxx–xxx

and, later, poor yields may be mistakenly ascribed to abiotic stress (cold, water stress) or insufficient plant nutrition rather than the presence of root disease (Laemmlen, 2001). Vegetable and fruit tree crops, which account for 10.7% of the organic area in EU-15 (the European Union of 15 member states before 1 May 2004), are higher in the organic sector than in EU agriculture as a whole, given that the demand for fruit and vegetables for the organic market is the highest (Stephen and Willer, 2014). Pest and pathogen control in organic agriculture is mainly based on prevention with cropping practices aimed at controlling through the suppression mechanisms (Bugg and Pickett, 1998). Italy, is the EU member state with the largest area invested with organic vegetable (almost 23,405 ha) followed by Germany (18,000 ha), France and Spain. Theoretically, an organic cropping system should have the capacity to suppress root diseases; however, organic vegetable farms in Italy are mostly located near urban areas and are small-medium sized, thus produce vegetables in rotation on the same land. In this context, such cropping systems are subject to a gradual increase of soil-borne pathogens and a decrease of microbial balance which are responsible of progressive growth reduction generally defined as ‘‘crop decline”. (PérezBrandán et al., 2014a). The complex of soil borne fungal pathogens causing crop decline varies according to the agro-environment and the target crops; moreover, crop susceptibility decreases from early to ripening growth stages (Jarosz and Davelos, 1995). The uneven distribution of soil-borne fungal pathogens across fields and difficult in diagnosis make overall difficult a direct correlation with crop losses (Dixon and Tilston, 2010). For this reason, plant growth parameters such as plant biomass are commonly used as indicator of disease severity or soil health (Johnson, 1993; Gram et al., 2016). One of the most frequent agronomic practices adopted to mitigate crop decline in organic farms devoted to vegetable production of Central and Northern Italy, is the enrichment of crop sequence with winter cover crops, which improves functional diversity (Lamb et al., 2011), reduces soil erosion, preserves soil organic matter content and soil biomass, increases nutrient supply and N leaching control (Struik and Bonciarelli, 1997). Barley (Hordeum vulgare L.) and hairy vetch (Vicia villosa Rhot.) are the most commonly adopted winter cover crops in Italy and worldwide. However, unlike to their impact on nutrient supply and soil erosion N which has been largely investigated (Cicek et al., 2014), little is known on how they can influence rhizosphere microbial communities and any associated effect on the crop health. Therefore, a two-year study on plant-soil microbial interaction was performed in an organic experimental field for vegetable production in central Italy to evaluate whether barley and hairy vetch can affect rhizospheric bacteria and root-colonizing fungi of subsequent vegetable crops. Two different mechanical termination methods for incorporating cover crops into the soil, were added to the experimental set. Indeed, cropping practices have a great impact in organic agriculture, where plant residues turnover guarantees most of nutrient supply.

The experimental field was located near Monsanpolo del Tronto, in the coastal area of the Marche Region in Central Italy (latitude 42° 530 N, longitude 13° 480 E) with Mediterranean climate. The soil taxonomy classification was Fine-loamy mixed thermic. Soil organic matter content accounted for 1.6% with a pH 8.2 in the experimental field at the beginning of this trial in spring 2014. This field was under organic management for 14 years; during this period, six vegetable crops (tomato, zucchini, fennel, melon, cauliflower and bean/lentil) were cultivated over a 4-year rotation. Plant vigor decline in post plant period of tomato and zucchini was observed in the last years; moreover, tomato and zucchini gave an variable performance in the last 6 years, indicating a non-specific crop decline. 2.2. Field trial description The impact of two winter cover crops (barley and hairy vetch) and two termination techniques (green manure and green mulching obtained by flattening plants with roller crimper) was evaluated on two subsequent vegetable crops: tomato and zucchini. The same factorial experiment was repeated in 2014 and 2015 (Fig. 1). Barley cv. Trasimeno and hairy vetch cv. Minnie were sown in late October and late September respectively on two main plots

2. Material and methods 2.1. Experimental site description A two-year trial was carried out in a long term organic experimental field. The study was limited to the growing period immediately after transplant which represents one the most critical stages of plants in horticulture due to the high susceptibility of seedlings to soil-borne pathogens.

Fig. 1. Map of experimental design where tomato and zucchini growth response to 2 previous cover crops (hairy vetch and barley) and 2 techniques of cover crop termination (green manure and roller crimping) were evaluated. The same experimental design was two years repeated. s Zucchini; r Tomato.

Please cite this article in press as: Manici, L.M., et al. The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline. Biological Control (2016), http://dx.doi.org/10.1016/j.biocontrol.2016.11.003

L.M. Manici et al. / Biological Control xxx (2016) xxx–xxx

of 352 m2. Just before termination, which was performed on April 30 in both years, a total of 60-plants (3 replicates of 20 plant each), were sampled diagonally from each plot as described below for tomato and zucchini Then, plots each plot was split into two sub-plots of 165 m2 (8  22 m) and the following mechanical terminations were applied (Fig. 1): i. green manure (GM), a common practice for plant residue soil incorporation in which cover crops were chopped whilst green and ploughed with a disk plough to a depth of 25 cm; ii. ii. roller crimping (RC), a – mechanical termination applied in conservation agriculture in which cover crops are mechanically killed to obtain a green surface mulching (Kornecki et al., 2009). Rows for seedling transplants were produced by disking to a depth of 25 cm immediately after flattening. Biomass production corresponded to 66.5 and 59.3 t ha1 for vetch, and 59.3 and 34.7 t ha1 for barley in 2014 and 2015 respectively. Seven days after cover crop termination, seedlings were transplanted by hand in 22 m single rows 1.5 m spaced, to obtain three replicates of zucchini and three of tomato per each sub-plot (Fig. 1). Each tomato (Solanum lycopersicum L. cv. SAAB CRA) replicate were formed by eight plants 15 cm spaced on the row, those of zucchini (Cucurbita melopepo L., cv. Zuboda) by four plants each transplanted at 30-cm spacing. A total of 48 plants of zucchini and 96 of tomato were grown and subsequently analyzed both in 2014 and in 2015. Before transplanting the vegetables, 40 kg ha1 of nitrogen (N) was applied in doses of 13.3 kg ha1 of pelleted animal manure with 3% of N. During the trial period no other treatment was applied, apart from supplemental irrigation according to each crop’s water requirement. Vegetables were grown for a 28-day period, which corresponded approximately to the vegetative growth phase of the crops. Then, the tomato and zucchini replicated samples were carefully explanted from each subplot and kept separated. Plants of each sample were divided into below and above ground part, the above ground parts were dried overnight at 105 °C to record dry matter production which was assumed as indicator of crop health, whilst roots were processed for evaluating root colonizing fungi as below described. 2.3. Root-colonizing fungi and rhizosphere bacteria Root-colonizing fungi and rhizosphere bacterial communities were evaluated in both cover crops and in subsequent vegetables. 2.3.1. Root-colonizing fungi Roots were washed under running water for 30 min, disinfected for 2 min in 1.5% sodium hypochlorite, rinsed twice with sterile water, dried under sterile air flow for 15 min then root section root segment excised with a scalpel (0.3–0.4 mm each) were placed on water agar (20 g l1 Agar Technical No. 3 Oxoid LTD, Basingstoke, UK) in 9 cm diameter Petri dishes and incubated for 4 days at 22 ± 2 °C under natural light. The total number of root sections evaluated per treatment was: 360 in both barley and hairy vetch (from a total of 60 plants each), 576 root sections from both zucchini and tomato (from 48 and 96 plants respectively). Fungal infection was evaluated by dividing the number of sections from which fungal hypha grew on water agar by the total number of sections of each replicate. After 4 day incubation, the fungal hyphae grown from root segments on water agar were transferred to Potato Sucrose Agar (PSA) and incubated from 6 to 10 days at 22 ± 2 °C to obtain single pure colonies. Those colonies

3

were first identified based on macroscopic features; then Deuteromycetes were identified on microscopic features using the most common keys for identification of filamentous fungi (CAB, 1990; Nelson et al., 1983; Watanabe, 2002). Cylindrocarpon-like isolates were identified based on micro and macro taxonomic features using recent literature on the revised taxonomy of Cylindrocarpon anamorph species complex (Cabral et al., 2012). Conversely, Rhizoctonia and Pythium spp. isolates were identified using molecular techniques. DNA of each isolate or representative isolates in case of large populations was obtained from pure fungal colonies as described in Manici and Bonora (2007). Total genomic DNA extraction was performed with a DNeasy Plant Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions; PCR amplification of rDNA gene fragments was carried out using primers ITS1F/ ITS4 and ITS1/ITS4 (White et al., 1990; Gardes and Bruns, 1993) for Rhizoctonia and Pythium respectively, following amplification conditions described in Manici and Bonora (2007). PCR products were purified with ExoSAP-IT reaction (Amersham Biosciences, Uppsala, Sweden) and the purified DNA obtained was then directly sequenced at the Bio Molecular Research Service (CRIBI Padua University, Italy) using the abovementioned forward and reverse primers. Fungal identification was performed by submitting nucleotide sequences to the nucleotide BLAST program available on the web at the National Centre for Biotechnology Information (NCBI). A total of eleven sequences of Rhizoctonia and Pythium isolates were enclosed to GenBank. References isolates of fungal species recovered in this study were enclosed in the working culture collection of CREA Bologna research center, available on web (http://www.cracin.it/Micoteca_web/pdf/Micoteca.pdf). Data were expressed as percentage and organized as species/abundance matrix. 2.3.2. Rhizosphere bacteria Immediately after plant samples were harvested, rhizosphere soil adhering to roots was sampled after having gently shaken the roots of each replicate consisting of 8 plant in tomato and 4 plants in zucchini; whilst rhizospheric soil samples were taken from replicates of 10 plants in barley and hairy vetch. This allowed to remove soil which was not tightly adhering to the roots and obtain rhizospheric soil from each replicate. A subsample of 25 g of soil was taken, air dried at room temperature for 12 h and stored in 50 ml sterile vials at 80 °C. DNA extraction was performed from one gram of rhizospheric soil which was preliminary sonicated for 15 min, then soil DNA was extracted from 0.50 g of soil using a Power Soil DNA kit (Mo Bio Laboratories, Inc. Carrlsbad, CA, USA) according to manufacturer’s instructions. PCR-DGGE fingerprinting was performed with two sets of primers 63f (CAG GCC TAA CAC ATG CAA GTC), and 518r (ATT ACC GCG GCT GCT GG), (El Fantroussi et al., 1999). A 40-nucleotide GC clamp was inserted to the 50 end of the forward primer for DGGE analysis to prevent complete melting of PCR products during the runs (Muyzer et al., 1993). All PCRs in this study were performed using a Taq polymerase kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and conducted in a TGradient Thermal Cycler (Biometra GmbH, Göttingen, Germany). A PCR was conducted as an initial test to assess purity and integrity of the extracted DNA. The PCR mixture used contained 1  buffer (20 mM Tris pH 8.4, 50 mM KCl), 2.0 mM MgCl2, 200 lM of each dNTP, 1.25 U of Taq polymerase (Invitrogen), and 10 pmol of each primer, 100 ng of total DNA and sterile water to a final volume of 50 ll. Samples were amplified as follows: 94 °C 3 min 94 °C 45 s; 60 °C 30 s; 72 °C 1 min, 20 cycles, and 10 cycles 94 °C 45 s; 55 °C 30 s; 72 °C 1 min, with a final extension at 72 °C for 5 min. DGGE was performed as described by Muyzer et al. (1993), using a D-code system (Bio-Rad Laboratories, Hercules, CA, USA).

Please cite this article in press as: Manici, L.M., et al. The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline. Biological Control (2016), http://dx.doi.org/10.1016/j.biocontrol.2016.11.003

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L.M. Manici et al. / Biological Control xxx (2016) xxx–xxx

PCR samples (200–250 ng) were loaded on 6% polyacrylamide gels in a 1 TAE buffer with a denaturation gradient ranging from 40% to 80%. Electrophoresis was run at a constant voltage 75 V at 60 °C. The DGGE analysis was repeated three times to confirm the pattern. After electrophoresis for 16 h, the gel was stained with GelRedTM (Biotium Inc, Hayward, CA, USA) at 10,000 dilution in 1 TAE for 30 min, washed in water for 20 min, and photographed using an Alpha Image UV illuminator (Alpha Innotech, San Leandro, CA, USA). Data were organized as presence/absence matrix. 2.4. Growth response of tomato and zucchini to 8 soil-borne fungal species A total of 22 fungal strains isolated from both vegetables and cover crops were subjected to an in-pot assay for evaluating growth response of tomato and zucchini to the root colonization by each fungal isolates, they represented the eight (Table 1) most abundant fungal species isolated from roots in the 2-years trial. Inoculum of each fungal isolate was obtained starting from 7day-old colonies grown in 9 cm dia. petri dishes on PSA. Each of the 500 ml autoclavable plastic jars, containing 270 g sand, 30 g corn meal, 60 ml water, autoclaved twice at 121 °C for 20 min were inoculated with six 4 mm disks of fungal colonies and incubated at 24 ± 2 °C for 3 weeks before inoculation. Peat was inoculated with each isolate at a ratio of 5:1 (v:v) peat vs sand-corn meal inoculum. For each fungal isolate, eight 10  10  10 cm pots containing infested peat were planted with one-week old zucchini and tomato seedlings. A control treatment of 8 plants (grown on peat amended with sterile sand-corn meal media) was included. Plants were grown for three weeks in a greenhouse at 24 ± 2 °C under natural light; then they were collected and processed as follows: the above ground part of each treatment was dried overnight at 105 °C, while root fungal infection of roots by each inoculated species was confirmed as above described in the evaluation of fungal root colonization of plant samples from field trial. Data were expressed as percentage of dry matter production on inoculated peat compared to un-inoculated control. 2.5. Data analysis Quantitative data. Statistical analysis was performed using Statgraphics centurion software (2005 STATPOINT Inc. Virginia, USA). Plant growth measurements were subjected to one way and two way Analysis of variance and to mean separation tests using Fisher’s least significant difference (LSD) procedure, at a 95% confidence level. All percentage data were ln (x + 1) transformed before statistical analysis. Species/abundance data matrices of fungal communities and presence/absence data matrices of bacteria were subjected to mul-

tivariate analysis using PAST program ver. 3.12 software for data analysis in paleoecology (Hammer et al., 2001), available on the web http://folk.uio.no/ohammer/past/. Two way multivariate analysis of variance (ANOVA) was performed using the Bray-Curtis distance for species/abundance (fungi) and Euclidean distance respectively for presence/absence data (bacteria). Bray Curtis distances was adopted in the principal component analysis (PCA) of fungal communities inferred between-groups using the ‘‘convex hulls” option to define a bi-plot area corresponding to each treatment (cover crop). Eigenvalues for the first two components derived from the PCA were considered significant when they were larger than the average of all eigenvalues obtained (Ramette, 2007). An analysis of similarity (ANOSIM) test using 9999 permutations was applied to evaluate distance between rhizosphere bacterial communities using Euclidean distance. Chao 2 diversity and standard deviation obtained with bootstrapping procedures were used to perform graphical comparison of presence/absence data from DGGE fingerprinting of bacterial communities. Graphical comparison of the number of taxa and Shannon index, obtained by grouping replicates in one column for treatment, was adopted for fungi using a 95% confidence interval computed with a bootstrap procedures (9999) through the plot option of diversity menu in Past program.

3. Results 3.1. Plant growth of vegetable crops Tomato and zucchini growth response was significantly affected by the previous cover crop in both 2014 and in 2015 (Table 2). Hairy vetch in all cases induced a significantly higher biomass production than barley in the subsequent vegetable crops (Table 2). Mechanical termination technique impacted significantly in only one out of the two trial years (Table 2); in every case, green manure always gave the highest dry matter production (Table 2). Average temperature during April and May 2014 and 2015 was very similar; rainfall and relative humidity were higher in 2014 than 2015, however this difference was not considered in analyzing data because irrigation was performed according to crop requirement and no extreme rain events occurred.

3.2. Interaction between cover crops and soil microbial communities 3.2.1. Root colonizing fungi Average fungal colonization was 53 and 50% in barley and 63 and 65% in vetch, in 2014 and 2015 respectively. Fungal communities of barley and hairy vetch did not differ for number of taxa, while the Shannon index was highest in hairy vetch in 2014 and

Table 1 Fungal species evaluated for plant growth response.

a b c

Fungal species

Isolates number

Isolate codeb

Fusarium incarnatum Fusarium equiseti Pythium ultimum Fusarium oxysporum Pythium spp.a Fusarium crockwellense Rhizoctonia solani AG-4 bnRhizoctonia AG-A

2 2 4 5 2 2 3 2

F_sem17-18 F_eq21-23 Pyt_ult36-37-38-39 F_oxy66-67-71-72-73 P_diss01; P_syl02 F_crock04-05 Rh249-250-258-260 Rh258-260-

Plant growth response in bioassayc Zucchini (%)

Tomato (%)

87 a 97 a 100 a 100 a 100 a 96 a 90 a 105 a

26 a 30 a 36 a 60 ab 70 ab 80 abc 88 bc 142 c

Pythium dissotocum and Pythium sylvaticum. Code of isolates as included in the working collection of CRA-CIN, available at: http://www.cracin.it/Micoteca_web/pdf/Micoteca.pdf. Plant growth response on artificially infested soil expressed as percentage of dry matter produced compared to that of the un-inoculated control.

Please cite this article in press as: Manici, L.M., et al. The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline. Biological Control (2016), http://dx.doi.org/10.1016/j.biocontrol.2016.11.003

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L.M. Manici et al. / Biological Control xxx (2016) xxx–xxx Table 2 Two way and mean separation test of above ground dry matter production per plant in tomato and zucchini 28 days after transplant.

a b c

ANOVA

df

Cover crop M. termination Cover X M. termination

1 1 8

Tomato

Zucchini

2014

2015

2014

1015

0.0001 <0.05 ns

<0.001 ns ns

<0.001 ns ns

<0.001 <0.01 <0.05

Mean separation testa

Dry matter/plant (g)b

Dry matter/plant (g)

Dry matter/plant (g)

Dry matter/plant (g)

Barley Hairy vetch Rolling/crimping Green manure

2.3 5.9 3.4 4.8

1.3 3.2 2.2 2.5

8.7 bc 18.0 a 8.6 a 9.1 a

2.0 B 12.8 a 5.5 b 9.3 a

Gran mean

5.15

14.4

13.23

bc a b a

b a a a

3.25

Mean separation test using 95% Fisher’s least significant difference (LSD) procedure. Average of 8 plants per replicate. Paired numbers in the same column that do not share a common letter differ significantly.

in barley in 2015. The indicated that diversity of root-colonizing fungi did not substantially differ in cover crops. Composition of root endophytic fungal communities in barley and hairy vetch differed significantly (P < 0.01) in each of the two-year trials. However, barley and hairy vetch shared about 70% of identified species. Rhizoctonia spp. (mostly binucleate Rhizoctonia sp., while Rizoctonia solani AG-4 was occasionally isolated only from vetch), Pythium spp., Fusarium oxysporum, F. incarnatum and, F. equiseti were the most frequently occurring and shared species. Indeed, although principal component analysis (PCA) displayed communities of barley and hairy vetch in opposite quadrants (Fig. 2a and b), most of the vectors in both years were short (Fig. 2a) or perpendicular to the XY plane in the 3D space, or displayed along the X-axis (Fig. 2b). This suggested that the majority of the abovementioned species were shared by all the samples. Whereas, the longest vectors of Pythium spp. F. equiseti and Trichoderma viride oriented toward barley and those of Phoma medicaginis, F. crockwellense oriented toward hairy vetch in 2014 (Fig. 2a), indicated that these species had a greater impact on differentiation between root endophytic fungal communities of two cover crops. Conversely, in 2015 (Fig. 2b) Cylindrocarpon-like species (Dactylonectria torresensis, Ilyonectria robusta, Thelonectria veuillotiana) and F. solani were the variables which most affected the fungal community on hairy vetch, whereas Cephalosporium graminum, Epicoccum nigrum and other fungal species commonly recovered on grain cereals such as Bipolaris sorokiniana were the variables which most characterized the root fungal community of barley (Fig. 2b).

3.2.2. Rhizospheric bacteria Diversity of bacterial communities in barley and hairy vetch rhizosphere never significantly differ. Conversely, bacterial community composition differed at P < 0.01 and P < 0.05 in 2014 and 2015 respectively showing that host plants were able to affect composition of rhizosphere bacteria even when grown in the same field.

3.3. Interaction between vegetable plants and soil microbial communities 3.3.1. Root colonizing fungi Root fungal colonization frequency did not differ significantly between treatments, the average infection frequency in tomato was 32 and 21%, in zucchini 42 and 48% in 2014 and 2015 respectively. Endophytic fungal communities of tomato and zucchini roots differed significantly according to previous cover crop in

2014 but not in 2015, whereas they never differed according to the mechanical termination (Table 3). In 2014, the tomato plants shared 6 out of 15 fungi (40%), which had been isolated from previous cover crops: Pythium spp. Rhizoctonia (Rhizoctonia solani AG-4 and binucleate Rhizoctonia AG-A and AG-G), Fusarium oxysporum, F. incarnatum, F. equiseti, F. solani. Zucchini shared 8 out of 18 fungal species (44%) isolated from previous cover crops (Pythium spp., Rhizoctonia spp., F. oxysporum, F. incarnatum, F. equiseti, F. solani, F. crockwellense and F. avenaceum). In actual fact, tomato and zucchini shared most root-colonizing species (89%): Pythium spp., Rhizoctonia (bnRhizoctonia AG-A and AG K and R. solani AG-4), F. oxysporum and F. equiseti occurred with an average abundance of 16–18% each, followed by F. solani (11%). In 2015, only five fungal species were isolated from tomato roots, they were: Pythium spp., Rhizoctonia spp., F. oxysporum, F. equiseti, F. solani; all these species had also been harbored previously by both barley and hairy vetch. To the contrary, zucchini were 90% colonized by Pythium spp (70% P. ultimum, 30% P. sylvaticum and Pythium spp.) regardless of previous cover crops and mechanical termination. Sequences of five Rhizoctonia (four bnRhizoctonia AG-A, one R. solani AG-4) and six Pythium spp. (four P. ultimum, one P. dissotocum and one P. sylvaticum) isolates were submitted to GenBank with the following accession numbers: KU052605- KU052615. 3.3.2. Rhizospheric bacteria Chao 2 diversity index of rhizosphere bacterial communities in tomato and zucchini did not differ, while their composition differed significantly according to the preceding cover crops in 2014 and in 2015 (Table 3) suggesting that barley and hairy vetch affected the rhizosphere bacterial composition of subsequent tomato and zucchini crops. Mechanical terminations significantly (P < 0.05) affected bacterial composition only in 2015 (Table 3). Although bacterial communities always differed significantly for factor cover crops, dissimilarities between bacterial communities following barley and hairy vetch in tomato were lower than in zucchini. Indeed the R value accounted for 0.34 and 0.24 in 2014 and 2015 respectively, according to ANOSIM tests (where R = 0 corresponds to total similarity; R = 1 total dissimilarity); while in zucchini larger differences were recorded, namely R = 0.46 and 0.47 (P < 0.05) in 2014 and 2015 respectively. 3.4. Growth response of tomato and zucchini to 8 soil-borne fungal species Plant response to the inoculants in bioassay varied from plant death up to plant growth promotion. The relationship accounting

Please cite this article in press as: Manici, L.M., et al. The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline. Biological Control (2016), http://dx.doi.org/10.1016/j.biocontrol.2016.11.003

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L.M. Manici et al. / Biological Control xxx (2016) xxx–xxx

Fig. 2. (a and b) Principal component analysis (PCA) inferred from species/abundance data matrix of fungal communities colonizing roots of barley and hairy vetch in 2014 (a) and in 2015 (b) Symbol legend: Hairy vetch s; Barley j.

for dry matter production of less than ±10% compared to a noninoculated control was considered neutral; those lower and higher than 10% were considered pathogenic and mutualistic plant-fungal relationships respectively. In all cases, the inoculated species were re-isolated from roots of the relevant treatment.

Zucchini showed a generally lower susceptibility than tomato. Their growth response to inoculants did not differ significantly (Table 1); in most cases, inoculated seedlings did not exceed the control of ±10% in biomass production, (Fig. 3). As the plant response to isolates of the same species varied greatly, plant

Please cite this article in press as: Manici, L.M., et al. The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline. Biological Control (2016), http://dx.doi.org/10.1016/j.biocontrol.2016.11.003

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L.M. Manici et al. / Biological Control xxx (2016) xxx–xxx

Table 3 Difference in rhizosphere microbial composition (root colonizing fungi and rhizobacteria) of tomato and zucchini based on previous cover crop and their mechanical termination. Root colonizing fungi

Tomato 2014 **

Cover cropa M. terminationb

a b ** *

*

2015

ns

2014

2015

2014

*

**

*

**

ns

*

ns

*

Tomato

Cover crop M. termination

Zucchini 2014

ns ns

ns

Rhizosphere bacteria

2015

ns ns

Zucchini 2015

barley and hairy vetch preceding crops. Green manure and rolling-crimping. P < 0.01. P < 0.05.

Box-and-Whisker Plot Zucchini

a

F. crockw. F. equiseti F. oxysporum F. incarnatum Pythium spp.

P. ultimum R. solani AG-4 Rhizoc. AG-A 0 20 40 60 80 100 120 140 160 180 Dry matter production vs non-inoculated control (%) Tomato

a

F. crockw. F. equiseti F. oxysporum F. incarnatum Pythium spp. P. ultimum R. solani AG-4

Rhizoc. AG-A 0

20

40

60

80

100 120 140 160 180

Dry matter production vs non-inoculated control (%) Fig. 3. Box-and-Whisker Plot showing zucchini and tomato growth response to 22 artificially inoculated fungal isolates belonging to eight species (Table 1). Plant response is expressed as percentage of dry matter produced on inoculated soil as compared to non-inoculated control. a: The black line shows the dry matter production corresponding to control.

growth response was shown with Box-and-Whisker Plots, in which box represents the interval covered by the middle 50% of values and a vertical line is drawn at the median (Fig. 3). F. oxysporum, P. ultimum and R. solani AG-4 varied from pathogenic, to beneficial, including one F. oxysporum isolate which was represented by an outside point in the Box-and-Whisker Plot (Fig. 3). This means that biomass production of plants inoculated with this F. oxysporum isolate was more than 3 times the interquartile range above the relevant box (Fig. 3). Although F. incarnatum showed a growth response varying from damping off to no growth reduction, it resulted in slightly pathogenicity to zucchini giving an average

growth reduction lower than 20% compared to the control (Fig. 3). bnRhizoctonia AG-A and Pythium spp. (P. dissotocum and P. sylvaticum) showed a neutral (Fig. 3) to beneficial relationship with zucchini suggesting that these species should not be considered pathogenic. Tomato growth response significantly differed (P = 0.05) between eight fungal species (Table 1). Unlike zucchini, tomato was susceptible to most of the tested species. All Fusarium spp., Pythium spp., P. ultimum and R solani AG-4 varied from a pathogenic to neutral relationship with plants (Fig. 3). Only bnRhizoctonia promoted the growth of tomato by inducing an increase of dry matter of up to 80% (Table 1, Fig. 3). Disease severity in tomato was noticeably variable within species; P. ultimum and Pythium spp. caused damping off; R solani AG-4 caused collar rot, while F. oxysporum and F. incarnatum produced root necrosis and general reduction of root development.

4. Discussion Tomato and zucchini growth response in post plant period was always better after hairy vetch than barley. Mechanical termination only affected plant response in a few cases where direct incorporation into the soil of plant residues (green manure) always gave better performance than flattening cover crops for obtaining a green mulching (roller crimping). The tomato and zucchini growth response after hairy vetch was in all cases at least twice that observed after barley. This difference was six times higher in 2015, when zucchini were highly infected by P. ultimum regardless of the preceding crops. However, root colonizing fungal communities and their infection level in tomato and zucchini did not actually differ according to preceding cover crop while the rhizosphere bacterial communities largely differed. Although numerous studies have shown that composition and abundance of bacterial populations in rhizosphere are affected by rhizodeposits released by roots through specific interactions (Philippot et al., 2013), findings of this study also show that rhizosphere bacterial communities in tomato and zucchini were significantly affected by the preceding cover crops in early growth stages. The highest growth response of tomato and zucchini to hairy vetch seems principally due to an increased ability of plants to escape the growth reduction caused by root necrosis deriving not only from a greater N availability, but also from bacterial growth promotion associated with legume crops as already observed in other studies (Akhtar and Siddiqui, 2009; Pérez-Montaño et al., 2014). Beneficial interactions between Rhizobium spp. and plant growth promoting (PGP) bacteria occurring in Leguminosae and non-Leguminosae crops, such as Bacillus, Pseudomonas, Azospirillum, Klebsiella spp. etc, is so well-known that their associations are

Please cite this article in press as: Manici, L.M., et al. The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline. Biological Control (2016), http://dx.doi.org/10.1016/j.biocontrol.2016.11.003

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being extensively exploited in the production of commercial biofertilizers (Akhtar and Siddiqui, 2009). Therefore, the theory that a legume cover crop such as hairy vetch induce multi-functional beneficial bacterial changes in a subsequent crop fits the findings of this study. On the other hand, a recent study by Cicek et al. (2014) on the impact of legume cover crops on organic wheat production shows that the presence of legume cover crops increased wheat N uptake at stem elongation, maturity and yield, even when the biomass production of cover crops was modest. This may suggest that functionalities deriving from legume-soil microbial interaction and differing from the N-benefits of legume, might mitigate the detrimental impact of soil-borne fungal pathogens in subsequent vegetable crops. Winter cover crops and subsequent vegetables shared the most abundant root-colonizing fungal species such as binucleate Rhizoctonia AG-A, P. ultimum, F. incarnatum, F. equiseti, F. oxysporum. Hairy vetch harbored the potentially pathogens Phoma medicaginis, Phoma spp. and Cylindrocarpon-like species (Cabral et al., 2012; Djebali, 2012) which were not recovered from any other crop, thus showing a partial specificity toward these potential soil-borne pathogens. Indeed, the group of Cylindrocarpon-like fungi is one most frequently associated with yield decline or replant problems (Agustí-Brisach et al., 2016; Manici and Caputo, 2010; Petit and Gubler, 2005). Conversely, barley showed the highest abundance of Pythium spp. in 2014. However, no related increase of Pythium was found in tomato and zucchini roots grown after barley, as well as Phoma spp. and Cylindrocarpon-like fungi were never recovered after vetch. Therefore, the main effect of cover crops on soil fungal communities inhabiting the horticultural cropping system appears to be due to an increase of diversity deriving from the ability to harbor a series of fungal saprophytes, such as Cephalosporium gramineum, and Humicola grisea, Trichoderma viride or specific weak pathogens, such as the Bipolaris sorokiniana agent of common root rot and spot blotch on barley, none of which occurred in vegetable crops. The possibility to reduce impact of soil-borne pathogen in post plant phase of vegetable crops using cover crops was estimated on growth response due to difficulties in diagnosis of early root attacks. Indeed, root tip infection stops root development causing plant growth delay even in the absence of severe necrosis (Gunawardena and Hawes, 2002). However, the pathogenicity test with fungal isolates originating from the experimental field gave some new elements for understanding the impact of ‘‘root rot complex” in specialized horticultural systems. The greater tomato susceptibility to inoculated fungi than zucchini suggests that it is possible to estimate a significant degree of vegetable susceptibility to ‘‘root rot disease”. Thus, classification of vegetable susceptibility could help organic farmers when setting the best long-term rotation plan and to modify it when yield and quality losses caused by fungal attack on roots have been observed in the most recent cycles. Though it has been long known that soil-borne fungi may colonize roots and express a different relationship depending on plant host and/or environmental conditions (Schulz et al., 1999), this research suggests that when vegetables return with high frequency on the same land, some pathogenic fungal species can be selected. According to plant response observed in the pathogenicity test, P. ultimum, R. solani AG-4, F. incarnatum and F. equiseti can be classified as pathogenic fungal species inhabiting the experimental field. To the contrary, the neutral or beneficial relationship with plants shown by bn Rhizoctonia AG-A showing was consistent with the potential role of this endophyte in promoting plant growth (Sneh, 1996). Moreover, binucleate Rhizoctonia widespread presence in soils suggests further investigation into its role in soil suppressiveness not only as a growth promoter, but also as a competitor of root rot fungal agents in colonizing the young roots

(González et al., 2006). Finally, F. oxysporum originating from soil of the organic experimental field was characterized by a relationship with the host plant that varies from slightly pathogenic to growth promoting in agreement with the features of the widespread non host-specific populations of this species (Alabouvette et al., 1979; Gordon and Martyn, 1997). The variable relationship with plants shown by F. incarnatum, P. ultimum and R. solani AG-4, which are characterized by large cross pathogenicity (Zhang and Yang, 2000; Ohkura et al., 2009), may indicate a large degree of variability in their specialization toward vegetables crops. Indeed, the media in which soil-borne pathogens are grown, can affect their aggressiveness toward host crops (Weinhold et al., 1969); therefore saprophytic soil-borne fungi can reach a certain degree of specialization toward the recurrent host crop by acquiring a specific ability to metabolize one plant tissue composition as opposed to another (Ohkura et al., 2009). Therefore, although findings suggest that the return of vegetable crops to the same field selected a root rot complex including P. ultimum, R. solani AG-4, and F. incarnatum, the soil biodiversity guaranteed by long term rotation performed in the organic experimental field under study may have mitigated detrimental effect of soil-borne fungal pathogens. The high positive impact of hairy vetch on subsequent vegetables and the cross pathogenicity of the most abundant fungal endophytes made difficult identifying the microbial components involved in the cover crops impact on growth performance of vegetable crops. Certainly, the consistent growth reduction of zucchini in 2015 regardless of the preceding cover crop can be linked to Pythium spp. root infection. Ingram and Cook (1990) demonstrated legume-cereal cross pathogenicity of several Pythium spp., including P. ultimum, isolated from wheat. Moreover, Pythium is strongly affected by soil management, in particular, no tillage and low input agriculture tend to increase its inoculum due to the highly saprophytic behavior of this pathogen, which greatly increases when non-decomposed plant material is available in soil (Manici et al., 2004). Therefore, Pythium spp. inhabiting the organic field could be more easily controlled with indirect actions, such as reduction of abiotic stress or the increase of plant vigor and root development with suitable cover crops as observed in this study, than with strategies aimed at reducing inoculum in soil. Finally, these findings indicate that, when root rot disease is severe, a dominant agent commonly prevails. To the contrary, when higher diversity characterizes communities of root endophytes, the balance between pathogenic fungal/oomycete species can mitigate disease severity by involving mechanisms such as antibiosis, antagonism in colonizing roots, plant growth promotion (Maciá-Vicente et al., 2009) and other functionalities that are provided by soil biodiversity which is one of the main effect of organic agriculture (Stirling et al., 2012). In conclusion, when vegetable transplant is performed immediately after termination of cover crops, fungal colonization of tomato and zucchini did not seem directly influenced by the preceding crop. Conversely, cover crops were able to modify rhizosphere bacterial communities in the next crops. Hairy vetch indisputably gave greater benefits to the subsequent vegetables than barley arising from the expected increased N supply. A more careful evaluation of changes in bacterial composition will be evaluated in the future in an experimental set where an extra N will be added to non-legume plot to equilibrate the N supply at planting time. However, the preference toward hairy vetch by some soilborne pathogens such as Phoma medicaginis and Phoma spp., Cylindrocarpon-like fungi as well as R. solani AG-4, suggests the need for a careful survey into root rot in previous crop cycles and setting crop rotation in which legume cover crop should alternate cereal and other botanically distant genera. In summary, according to findings of this study plant vigor induced by hairy vetch in the

Please cite this article in press as: Manici, L.M., et al. The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline. Biological Control (2016), http://dx.doi.org/10.1016/j.biocontrol.2016.11.003

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Please cite this article in press as: Manici, L.M., et al. The impact of legume and cereal cover crops on rhizosphere microbial communities of subsequent vegetable crops for contrasting crop decline. Biological Control (2016), http://dx.doi.org/10.1016/j.biocontrol.2016.11.003