Soil fungal communities as indicators for replanting new peach orchards in intensively cultivated areas

Europ. J. Agronomy 33 (2010) 188–196

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Soil fungal communities as indicators for replanting new peach orchards in intensively cultivated areas Luisa M. Manici ∗ , Francesco Caputo C.R.A. – Research Centre for Industrial Crops, Via di Corticella 133, Bologna, Italy

a r t i c l e

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Article history: Received 8 January 2010 Received in revised form 12 May 2010 Accepted 19 May 2010 Keywords: DGGE: Diversity index Greenhouse bioassay 18S rDNA Replant problem

a b s t r a c t Italy and Spain are Europe’s main peach-producing countries. Since the mid-90s, however, peach-growing areas have decreased in Italy thus causing its loss of European leadership. One of the reasons for the decline is the presence of replant disorders in peach orchards. As soil-borne pathogens are one of the biotic components of aetiology, a study was performed in an intensively cultivated area of southern Italy, to investigate the use of soil fungi as indicators of soil health when planting new peach orchards. Soil sampling was carried out in five replanted peach orchards and five horticultural sites in the “Piana del Sele”, a peach and vegetable-growing valley in the Campania region. Three different soil fungi communities were recorded: soil-inhabiting filamentous fungi; endophytic root fungi, based on cultural methods; and uncultured soil fungi, based on nucleotide sequences of internal transcribed spacers (ITS1 and ITS2) and rDNA gene fragments. Soil health was evaluated with a greenhouse bioassay, using young plants of peach rootstock GF677. Diversity and composition of fungal communities were compared using PAST, a software which includes several functions commonly used in paleoecology. Each peach tree orchard was characterized by a significantly higher diversity score than horticultural crops. The two cultivation systems differed widely for species composition suggesting that vegetative cover and cultivation practices strongly affected fungal community composition. The bioassay to evaluate soil health showed a significant growth reduction in horticultural sites compared to peach sites. This finding was supported by the high abundance of Cylindrocarpon destructans and Thielviopsis basicola, two aggressive root rot agents, within endophytic fungal communities in horticultural sites. In this survey, greatest fungal diversity corresponds to best plant growth. From a practical point of view, when deciding to replant a new fruit tree orchards in intensively cultivated areas, a greenhouse bioassay with the target crop, combined with a quantitative analysis of endophytic root fungal communities, may help in deciding the best soil use. © 2010 Elsevier B.V. All rights reserved.

1. Introduction European Mediterranean countries possess the most intensive cultivation systems, such as orchards, vineyards and vegetables. Two-thirds of fresh vegetables grown in EU-15 (Austria, Belgium, Denmark, Finland, France, Germany, Greece, Ireland, Italy, Luxembourg, The Netherlands, Portugal, Spain, Sweden and the United Kingdom) come from Italy, Spain and France. Europe’s fruit trees, including citrus and stone fruits, are concentrated in the five Mediterranean countries, Spain, Italy, France, Portugal and Greece which account for 93% of the community’s fruit-growing areas. Italy as recently as 19 years ago was the main peach-producing country in Europe followed by Spain, Greece and France. Nevertheless there has been a decline in the renewal of Italian orchards over the last 19 years compared to the rest of Europe and large-scale grubbing-

∗ Corresponding author. Tel.: +39 051 6316839; fax: +39 051 374857. E-mail address: [email protected] (L.M. Manici). 1161-0301/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.eja.2010.05.005

up cut peach-growing areas by 23% between 1996 and 2001 (Vidal and Ribaille, 2001). These findings reflect the difficulties affecting this commercially important crop for Italy. The Mediterranean climate (cool winters, intensive rainfall in spring and constant high temperatures during the summer) and the intensive cultivations in southern European countries led to a rapid decline in organic matter content of cultivated soils. The main consequence of this progressive SOM (soil organic matter) depletion is microbial biomass reduction and loss of microbial diversity, with reduction of soil function (Bastida et al., 2006). Microbial diversity is one of the best indicators of soil health (Winding and Nielsen, 2002) and represents one of the main components of soil suppressiveness (Garbeva et al., 2004). This can be defined as the potential biological ability of soil to deal with soil-borne pathogens (Borneman and Becker, 2007). The replant problems affecting peach and all fruit tree crops of high economic value for southern Europe are strongly related to land degradation problems. The role of soil-borne pathogens in fruit tree ‘replant disease’ was more fully explained in studies carried out in the 1980s with

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soil fumigants. In fact, the significant increase of young fruit tree plant growth in fumigated plots in comparison to non-fumigated plots suggested that soil micro-organisms play an important role in growth reduction generally classified as “replant disorders” (Browne et al., 2006; May and Abawi, 1981). The main agents of replant disease are several soil-borne fungal pathogens which can vary according to soil type, cropping practices, crops, etc. They are generally defined as “root rot complex”. Among the fungal pathogen agents of fruit tree root rot disorders figure several species belonging to Cylindrocarpon sp., Pythium sp. Rhizoctonia sp. and Phytophthora sp. (Hine, 1961; May and Abawi, 1978; Mircetich, 1971; Petit and Gubler, 2005; Savory, 1966). Diversity of root fungal flora has been found to be negatively correlated with disease incidence of crops, because crop rotation increases the diversity of root soil fungi and reduces the inoculum of soil-borne pathogens selected by monoculture (Manici and Caputo, 2009; Nitta, 1991). Fungi represent the greater part of soil microbial biomass in volume (Lin and Brookes, 1999), play an important role in macro-aggregate formation (Bossuyt et al., 2001) and their C assimilation efficiency is markedly higher than that of bacteria (Bailey et al., 2002). However, contrarily to bacteria, fungi have not yet been widely investigated as soil biological indicators in agricultural soils. This is mainly due to the difficulties concerning isolation and enumeration of fungi and to the limited availability of fungal strain reference sequences in DNA databases and the identification accuracy of reference strains enclosed in these databases (Vilgalys, 2003). A study of soil fungal communities in two intensively cultivation systems (continuous horticultural crops, replant fruit tree orchards) in a cultivated area of southern Italy (Piana del Sele, Salerno) was performed using analysis tools typically used in ecological studies. The aims of this study were: (i) to evaluate the impact of different continuous cultivation systems on soil fungal diversity; (ii) to analyse the impact of vegetative cover on fungal community composition; and (iii) to investigate the potential use of soil fungi as indicators when replanting new peach orchards. 2. Material and methods 2.1. Site description and soil sampling The study was performed in the Italian agricultural ‘Piana del Sele’ area, near Salerno, in Campania (Lat 40◦ 36 N, Long 15◦ 4 E), on plots chosen for homogeneity of pedo-climatic conditions, with SOM varying from 1.4 to 2.5%. The sampling time was early April 2007. The experimental sites were located in a area of 20 km in diameter; they were five replanted peach orchards (peach sites) and five fields intensively cultivated with vegetables (horticultural sites) near to the orchards. The peach sites were all orchards, no less than 8 years of age. They were characterized by three replants over the last 30-years; sod system with permanent vegetative cover; high amount of animal manure amendment at pre-plant; periodic animal manure amendments to maintain the SOM level, which varied from 1.5 to 2.3%. Soil samples were collected from each orchard at 50 cm from the trunks of five randomly chosen trees. Several soil cores (diameter 6 cm) to a depth of 25 cm were collected at the four cardinal points around each tree. The soil samples collected from each orchard were mixed to obtain an homogeneous sample. The five horticultural sites were lately cultivated with fennel, lettuces, cauliflower; 1–2 cycles in the winter-spring period, summer fallow for the last 35 years; a cereal crop at 5-year intervals; periodic organic amendment in commercial formulations; and cropping practices based on chemical fertilization. The SOM varied from 1.4 to 2.3%. Six soil cores (6 cm diameter) to a depth of 25 cm were collected in five 2-m areas on the diagonal of each field and mixed to obtain a homogeneous sample.

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Soil samples were maintained at 18 ◦ C for 5 days to be dried before processed, while samples for DNA extraction were frozen at −20 ◦ C immediately after sampling. 2.2. Soil-inhabiting fungal communities 25 g of soil samples, previously sieved with a 2 mm mesh, were dissolved for 10 min in 225 ml of sterile water with a magnetic stirrer. The soil suspensions were serial diluted and 1 ml of 10−2 and 10−3 dilutions was quickly mixed in a 250 ml flask with 49 ml of water agar at 40 ◦ C + 3 g l−1 ox-gall and 200 mg l−1 streptomycin sulphate melted, distributed in five Petri dishes (9 cm diameter) and incubated for 48 h at 24 ± 2 ◦ C under natural light. Colonies were counted by visual observation and expressed as colony forming units (CFU) g−1 of air-dried soil. The composition of the fungal communities was recorded by visual observation under natural light from transparent agar disks, including the germinated fungal propagules within the soil suspension. Each form of germinated propagule observed was first identified with a symbol, then counted. Finally, a representative number of colonies (from 10 to 30 according with the abundance of propagules belonging to the symbol) for each symbol was transferred to potato dextrose agar (PDA) + 200 mg l−1 of streptomycin sulphate on Petri dishes (4.5 cm diameter) and incubated for 6 days at 22 ± 2 ◦ C under near-UV light, for further identification. Fungal isolates were identified on the basis of macroscopic and microscopic morphological characteristics using the relevant taxonomic keys (Ellis, 1971; Nelson et al., 1983; Samson and van Reenen-Hoekstra, 1988; Watanabe, 2002). When, more then one species corresponded to the same symbol within the groups of representative of colonies, the groups were split to obtain the relative frequency or abundance of all species of each fungal community. The relative frequency, or abundance, of each species was expressed as a percentage of the total CFU for each community. The nucleotide sequences of internal transcribed spacers (ITS1 and ITS2) and rDNA gene fragments (18S rDNA, 5.8S rDNA, 28S rDNA) of the more representative strains were submitted to GenBank database. These sequences were obtained as follows: the fungal isolates were grown in potato sucrose broth (PSB) for 3–5 days at 25 ◦ C. Fungal mycelium was harvested by filtration on filter paper and stored at −20 ◦ C prior to DNA isolation. The frozen mycelium was then crushed using a bead beater (Mixer Mill, MM 300, Qiagen, Valencia, CA, USA) and tungsten carbide beads. Total genomic DNA extraction was performed with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. The PCR amplification was carried out using primers ITS1 and ITS4 (White et al., 1990). PCR products were purified with ExoSAP-IT reaction (Amersham Biosciences, Uppsala, Sweden), and the purified DNA obtained was then sequenced by the Bio Molecular Research Service (C.R.I.B.I. Università di Padova) by cycle sequencing using the BigDye sequencing kit and an automated DNA sequencer (Applied Biosystem, Foster City, CA, USA) with ITS4 as primer. 2.3. Root endophytic fungal communities Commercial in vitro plants of rootstock GF677, the most commonly used peach tree rootstock in Italy, were transferred to clean peat from sterile agar media and grown in a greenhouse under mist for 15 days at 24 ± 2 ◦ C under 14/10 day/night hours photoperiod. Then, conditioned plants were transplanted to 45-hole (7 cm deep, diameter 5 cm) thermo-formed trays filled with sieved soil (4 mm) from each of the 10 sites of the study (five peach sites and five horticultural sites). The trial was arranged in a randomized block design with three replicates of 15 plants each (45 plants per each of 10 sites). In vitro plants were grown at 24 ± 2 ◦ C under 15/9 day/night hours photoperiod in a greenhouse for 70 days, then they were har-

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vested and the plant height of each plant, considering the above ground part, was recorded. Endophytic fungal communities were evaluated as follows: roots of young rootstock plants were washed under running water, disinfected for 1 min in 1% sodium hypochlorite, and rinsed twice with sterile water. Six root segments were taken from each plant; two root tissue segments (0.2–0.3 cm) were excised from the distal, middle and proximal parts of the root of each plant. Root segments were then placed on water agar and incubated for 4 days at 24 ± 2 ◦ C under natural light. The relative colonization frequency was recorded by dividing the number of root segments on which fungal hyphae grew by the number of root segments incubated for each site (270 explants for site: 90 for each three replicates). The relative frequency of each species was evaluated by transferring each colony to potato sucrose agar (PSA) + 200 mg ml−1 of streptomycin sulphate, and identified as per the composition of inhabiting communities. 2.4. Soil fungal communities by denaturing gradient gel electrophoresis (DGGE) analysis 1 g bulk soil was preliminary sonicated for 15 min, then DNA was extracted from 0.5 g with the Ultra Clean Soil DNA Kit (Mo Bio Laboratories, Solana Beach, CA). A 0.5 g portion of soil was processed according to the protocol indicated by the manufacturer. Yield and fragmentation of extracted DNA were checked by agarose gel electrophoresis (1.5%, w/v). Band sizes were determined with 1 kb DNA ladders (Invitrogen, Carlsbad, CA) molecular size marker and agarose gel was stained with Gel Red TM (Biotium, Hayward, CA, USA) and visualized under UV light. Amplification prior of DGGE analysis was performed using a nested PCR approach with primer pairs ITS1F/ITS4 and ITS1FGC/ITS2 (Anderson and Carney, 2004). The first amplification was performed with ITS1F-ITS4 as previously described by Gardes and Bruns (1993). PCR reactions were performed with approximately 100 ng of soil DNA, using a Thermocycler PCR Gradient (Biometra biomedizinische Analytik GmbH, Goettingen, Germany) with 25 ␮l reaction volumes containing approximately 100 ng of template DNA, 10 pmol of each primer, 1.7 mM MgCl2 , 10× buffer (200 mM Tris–HCl pH 8.4, 500 mM KCl), 200 ␮M dNTPs mix and 2.5 U Taq polymerase (Invitrogen, Carlsbad, CA, USA). Amplicons were diluted 1:10 in sterile water and 1 ␮l of each dilution used as template for the second amplification with primer ITS1F-CG/ITS2 using identical PCR conditions to those described above. The analysis was repeated twice to confirm the results. Band sizes were determined with a low DNA mass ladders (Invitrogen) molecular size marker. Yield and fragmentation of PCR products were checked by agarose gel electrophoresis (2%), stained with Gel Red TM (Biotium) and visualized under UV light. DGGE analysis was performed with a DCode Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, USA). PCR samples (200–300 ng) were loaded on 8% polyacrylamide gels in a 1× TAE buffer with a denaturing gradient ranging from 20 to 50%. One hundred percent of denaturant corresponds to 7 M urea and 40% formamide. The gradient gel was cast using the Gradient Delivery System Model 475 (Bio-Rad Laboratories). Electrophoresis was run at a constant voltage of 65 V at 65 ◦ C. A PCR product volume of 200–300 ng was loaded on DGGE. The DGGE analysis was repeated three times to confirm the pattern. After electrophoresis for 16 h, the gel was stained with Gel RedTM (Biotium) at 10,000× dilution in 1× TAE for 30 min and photographed using a UV illuminator alpha imager (Alpha Innotech, San Leandro, CA, USA). DGGE bands were excised from the denaturing gels, each band was then diluted in 250 ␮l of sterile water, vortexed and incubated overnight at 4 ◦ C. The suspension was centrifuged for 30 s at 500 × g, and 10 ␮l of the resulting suspension was re-amplified

using primers ITS1F-GC and ITS2 as described above. PCR products were loaded on DGGE and run with the same conditions described above to confirm the original position. When the position was confirmed, samples were sequenced using ITS1F primer as described above. The maximum similarity of sequences was checked on the Web at the NCBI by comparing sequences with the nucleotide collection database using the nucleotide BLAST program. 2.5. Pathogenicity test The pathogenicity test was performed for 10 fungal isolates (five Cylindrocarpon destructans (Zins.) Scholten and five Thielaviopsis basicola (Berkeley & Broome, Ferraris)) as follows: in vitro plants of rootstock GF677, handled as in the greenhouse bioassay, were transplanted to pots (5 cm diameter × 7 cm deep) with artificially infested peat. Fungal isolates were inoculated using 5/1 (v/v) peat/sand-meal inoculum (Nene et al., 1981). Eight plants for each strain were grown in the greenhouse for 50 days after transplanting at 24–28 ◦ C under 15/9 day/night hours photoperiod. The control was represented by eight plants grown on un-inoculated peat. The growth score (GS) was assessed by multiplying plant height (of the above ground part) by plant health score (0: dead plant; 0.5: chlorosis of the above ground part combined with root necrosis and discoloration; 0.8: root discoloration and necrosis; 1: healthy plant). 2.6. Data analysis The frequency of each species was expressed as a percentage of the total number of CFU recorded for each population. Data were transformed to Log 10 (X + 1) and were subjected to diversity analysis using PAST program software for data analysis in paleoecology (Hammer et al., 2001), available on the web (http://folk.uio.no/ohammer/past/). To compare diversity of fungal communities of peach and horticultural cropping systems, abundance data of five peach and horticultural sites were pooled to obtain two samples for comparison. To increase the robustness of the analysis, eight diversity indices (Harper, 1999) were compared, four of them were more biased toward richness and four toward evenness. The Berger–Parker diversity (1/d), an index retrieved from Berger–Parker dominance (d) was used also as indicator of disturbance in a biological environment (Berger and Parker, 1970; Shaw et al., 1983). The diversity indices were compared using two different randomization procedures: bootstrapping and permutation. In both cases the small probability indicates a significant difference in diversity index between the two samples (Hammer et al., 2009). The diversity comparison findings were confirmed by diversity profile, a graphical test which uses the exponential of the Renyi index (Mayoral, 1998), which depends upon a parameter alpha, where ˛ = 0 corresponds to total species number (Richness); ˛ = 1 is an index proportional to the Shannon index; while ˛ = 2 is proportional to Simpson diversity index (biased toward evenness) (Magurran, 1988). Composition of both soil-inhabiting and root endophytic fungal communities was analyzed by multivariate analysis using PAST. Similarity analysis was performed on abundance data of fungal communities fungi in the 10 sites under study (peach sites: P1–P5 and horticultural sites: H1–H5), while the analysis of DGGE fingerprinting was performed on presence–absence data matrix. Similarity analysis was performed using Cluster analysis with a Bray Curtis distance measure, UMPGA algorithm using Arithmetic averages Unweighted Pair-Group Method using Arithmetic averages and 1000 bootstrap replicates.

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Table 1 Diversity indices (Harper, 1999) for soil-inhabiting fungal communities in peach and vegetable cultivation systems compared using two different randomization procedures: bootstrapping and permutation. Diversity indices R

Taxa (S) Shannon HR SimpsonE MenhinickR MargalefR Equitability JE Fisher alphaR Berger–Parker (1/d)b E

Peach orchard

Horticultural crop

Sign.a

35 3.23 0.95 0.98 4.75 0.91 6.65 8.55

18 2.73 0.92 0.57 2.47 0.94 3.13 6.69

** ** ** ** ** ** ** **

E

Diversity index mostly biased toward evenness. Diversity index mostly biased toward richness. a Significance according to randomization procedures: bootstrapping and permutation. **P > 0.01, *P > 0.05. b d = Berger–Parker dominance.

R

Seriation, a simple ordination method for presence/absence data, was applied to data matrix of 10 communities belonging to peach (P1–P5) and horticultural (H1–H5) cropping systems. Seriation provides an absence–presence matrix obtained according to the algorithm described by Brower and Kyle (1988), which is commonly adopted in paleoecology for stratigraphical or environmental ordering of taxa (species) and locality (Hammer et al., 2009). The application of this method allows easier analysis of species occurrence and distribution of soil-inhabiting and endophytic fungal communities in two cropping systems. Plant growth on soil samples from peach and horticultural sites and infection frequency by endophytic fungi (ArcSin transformed) were subjected to one-way analysis of variance using the Statgraphic program, ver. XV (Statpoint Inc., Harndon, VA, USA). 3. Results 3.1. Soil-inhabiting fungal communities The diversity of soil-inhabiting filamentous fungal communities in peach orchards was significantly higher than that of horticultural crops (Table 1); the indices biased toward richness as well as toward evenness were significantly higher in peach orchards. The higher diversity in peach orchards was confirmed by the graphic diversity profile test, in fact the profile of peach orchards was higher and did not intersect that of horticultural crops (Fig. 1).

Fig. 1. Diversity profile of soil-inhabiting fungal communities performed on pooled data of peach tree (five sites) and horticultural crops (five sites). ˛ = 0 corresponds to total species number, ˛ = 1 is an index proportional to the Shannon index, a diversity index biased toward richness, while ˛ = 2 is proportional to Simpson index, a diversity index biased toward evenness.

Fig. 2. Similarity of 10 soil-inhabiting fungal communities (five peach and five horticultural sites), Cluster analysis obtained using UPGMA algorithm and 1000 bootstrap replicates.

Cluster analysis showed wide differences in community compositions, in peach and horticultural sites, with two separate clusters with low similarity (28%) (Fig. 2). Out of a total of 41 species, the two cropping systems had in common only 13 species, but only Fusarium oxysporum (Slecht.), Fusarium solani (Mart,) Appel & Wollenw, Humicola grisea and Aspergillus prasiticus Speare were more widely recovered (Table 2). Several species of Penicillium, Aspergillus, and Fusarium were recovered from peach soils while they were poorly represented in horticultural soils (Table 2). Fusarium acuminatum Ell. & Ev., F. solani and Mortierella sp. were present in all peach sites (Table 2). F. acuminatum was one of the most abundant species in all peach sites, but it was not recovered from any of the horticultural sites (Table 2). The GenBank submission number of isolates representative of the more abundant fungal species of soil-inhabiting fungal communities is reported in Table 3. 3.2. Root endophytic fungal communities There was no difference in the infection frequency (P = 0.1452) of endophytic fungal communities on the rootstock GF677 on soil from peach (71.1%) and horticultural sites (63.4%). The diversity indices of endophytic fungal communities in peach orchards was significantly higher than that of horticultural soils (Table 4), however in this case one of the indices biased toward evenness, the Equitability J, did not significantly differ, suggesting that in this case the evenness of peach and horticultural systems was closer than it was for soil-inhabiting fungal communities. This was confirmed by the pattern of the diversity profiles; in fact, in correspondence to ˛ = 2 the profile of peach and horticultural crops were closer than that observed for soil-inhabiting fungi, but the difference in the ˛ = 2 index was sufficient to maintain separate the profiles (Fig. 3), allowing comparison of diversity (Hammer et al., 2009). The close values of the indices biased on evenness suggest that in this case the cultivation system affects richness more than evenness. Cluster analysis divided peach and horticultural sites in two separate clusters differing by more than 45% (Fig. 4). Out of a total number of 26 fungal species isolated from roots of peach rootstock grown on 10 soil samples, only eight were shared by the communities of both cultivation systems (Table 4). These included: Rhizoctonia sp. (Thanatephorus sp), Chaetomium funicola

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Table 2 Composition of soil-inhabiting fungal communities as free propagules in peach (P) and horticultural (H) sites. Absence/presence matrix of species in rows and localities in columns (maintained fixed) concentrated along the diagonal according to seriation method using the algorithm described by Brower and Kyle (1988).

Fig. 4. Similarity of 10 root endophytic fungal communities (five peach and five horticultural sites); Cluster analysis obtained using UPGMA algorithm and 1000 bootstrap replicates.

Ames, Fusarium spp., Macrophomina phaseolina (Tassi) Goid. and C. destructans (Table 5).While F. acuminatum resulted the most abundant species of soil-inhabiting fungal communities in peach soils and was one of the most abundant species of the endophytic fungal communites recovered in plants grown on peach sites (Table 5), it was not recorded in any root samples grown on horticultural soils. C. destructans and T. basicola, two widespread agents of root rot Table 3 GenBank number of the fungal strains representative of some of the most abundant species of soil-inhabiting and root endophytic fungal communities. Species

Origin

Strain

GenBank no.

Aspergillus parasiticus Verticillium tenerum Fusarium equiseti Fusarium acuminatum Fusarium acuminatum Fusarium oxysporum Humicola grisea. Myrothecium verrucaria Macrophomina phaseolina Cylindrocarpon destructans Cylindrocarpon destructans Cylindrocarpon destructans Thielaviopsis basicola Thielaviopsis basicola

Soil-inhabiting Soil-inhabiting Soil-inhabiting Soil-inhabiting Soil-inhabiting Soil-inhabiting Soil-inhabiting Soil-inhabiting Root endophyte Root endophyte Root endophyte Root endophyte Root endophyte Root endophyte

ASPpar1 VERten2 Fequ9 Facu2 Facu1 FOX52 Hum1 MYRver2 MAC166 Cyl 19 Cyl 17 Cyl 10 Thie 4 Thie 2

GQ131879 GQ131880 GQ131881 GQ131883 GQ131882 GQ131884 GQ131885 GQ131886 GQ131887 GQ131874 GQ131875 GQ131876 GQ131877 GQ131878

Table 4 Diversity indices (Harper, 1999) of endophytic fungal communities evaluated on rootstock GF677 grown for two months on soil samples coming from peach orchard and horticultural cropping systems. The comparison was performed with two different randomization procedures: bootstrapping and permutation. Diversity indices R

Taxa (S) Shannon HR SimpsonE MenhinickR MargalefR Equitability JE Fisher alphaR Berger–Parker(1/d)b E Fig. 3. Diversity profile of root endophytic fungal communities performed on pooled data of peach tree (five sites) and horticultural crops (five sites). ˛ = 0 corresponds to total species number, ˛ = 1 is an index proportional to the Shannon index, a diversity index biased toward richness, while ˛ = 2 is proportional to Simpson index, a diversity index biased toward evenness.

E

Peach orchard

Horticultural crop

Sign.a

21 2.42 0.87 0.76 3.01 0.79 3.99 5.28

13 1.97 0.82 0.53 1.87 0.77 2.34 3.73

** ** ** ** ** n.s. ** **

Diversity index mostly biased toward evenness. Diversity index mostly biased toward richness. a Significance according to randomization procedures: bootstrapping and permutation. **P > 0.01, *P > 0.05. b d = Berger–Parker dominance. R

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Table 5 Composition of root endophytic fungal communities in peach (P) and horticultural (H) sites. Absence/presence matrix of species in rows and localities in columns (maintained fixed), concentrated along the diagonal according to seriation method using the algorithm described by Brower and Kyle (1988).

complex, colonized respectively 12 and 3% of roots in plants grown on soil samples from horticultural sites. C. destructans was isolated with lowest frequency (<1.5%) in rootstocks grown on peach soil while T. basicola was never recovered in these soil samples. Rootstock plant growth on soil samples differed significantly (P < 0.01) between the two cultivation systems. Plants grown on horticultural soil (average of plant height 17.6 cm, s.d. 1.673) showed a growth reduction of 28% compared to those grown on peach soil (average of plant height 24.4 cm, s.d. 1.517). Visual inspection of roots did not show clear signs of necrosis, but only slight root discoloration. 3.3. Denaturing gradient gel electrophoresis (DGGE) analysis The DGGE profile gave 8–10 bands for each site, four of them were shared by all sites (Fig. 5). Cluster analysis performed on presence–absence data matrix of DGGE profile gave an high similarity (68%) between peach (P1–P5) and horticultural sites (H1–H5) (Fig. 5). The bands excised from samples of the peach cropping system corresponded to Macrophomina (3g, 5f, 7f, corresponding respectively to the GenBank accessions GQ131521, GQ131522, GQ131523), one of the most abundant species on endophytic fungal communities of peach soils. The three bands excised from horticultural soils corresponded respectively to an uncultured ascomycete (9g: GQ131524), F. oxysporum (11f: GQ131526) and to T. basicola (12a: GQ131525) (Fig. 5), two fungal species isolated from roots of peach rootstock grown on those soils. 3.4. Pathogenicity test All the isolates tested caused stunting (Fig. 6). For both C. destructans and T. basicola growth reduction was due to the reduction in stem internodes (Fig. 7). Pathogenicity was classified as high for isolates with a GS score lower than 5 (Cyl 9, Thie 3 and Thie 4), medium for those scoring 5–10 (four isolates of C. destructans and two of T. basicola), while the isolate Thie 7, which caused a growth reduc-

Fig. 5. DGGE pattern of fungal communities of 10 soil samples from peach and horticultural soils, generated by separation of amplicons obtained with ITS1Fand ITS2 primers. Marks indicate the excised bands from which sequences were determined. Cluster analysis of presence–absence data matrix was performed maintaining fixed the site order (P1–P5) and (H1–H5). Bootstrap analysis carried out on 1000 repetition counts. Bands 3g, 5f, 7f, corresponding respectively to the GenBank accessions GQ131521, GQ131522, GQ131523; bands 9g, 11f, 12a, corresponding respectively to the GenBank accession GQ131524, GQ131526 and GQ13152

tion of <20% as compared to control, was defined as non-pathogenic (Fig. 6). Three representative strains of C. destructans Cyl 19, Cyl 17 and Cyl 10 and two of T. basicola were submitted to GenBank (Table 3). All C. destructans and T. basicola strains were inserted in the COLMIA culture collection (on the web http://www.colmia.it/), funded by the Italian Ministry of Politics for Agriculture, Food and Forests (MIPAAF). 4. Discussion On the basis of the comparison of diversity indices of soilinhabiting and root endophytic fungal communities, it would appear that the cultivation systems deeply affect diversity. Although the plots for the peach and horticultural cultivation systems were chosen in a relatively limited area and organic amendments to maintain SOM were applied as common practice in both agricultural systems, the management of fruit tree orchards (sod system with permanent vegetative cover, periodic amendment with animal manure) seems to have favoured the diversity of soil-inhabiting fungal communities as compared to horticultural crops, where the rotation of few crops, control of natural weeds, periodic tillage and summer fallows may have reduced fungal diversity. The Berger–Parker diversity (1/d) of horticultural crops which was significantly lower than that of peach orchards (Table 1), suggested that soil-inhabiting fungal communities of horticultutal sites were characterized by a higher state of disturbance.

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Fig. 6. Pathogenicity test of five isolates of C. desctructans and five of T. basicola. Growth score of micropropagated plants of rootstock GF677 grown on inoculated soil compared to control. GS = plant height × plant health score (0: dead plant; 0.5: chlorosis combined with root necrosis and discoloration; 0.8: root discoloration and necrosis; 1: healthy plant).

On the basis of the findings using soil-inhabiting fungi as indicators, the orchards showed a significantly higher diversity according to indices biased on richness and evenness (Table 1). Diversity analysis of root endophytic fungi confirmed these findings (Table 4). There were large differences in fungal community composition between the cultivation systems: Cluster analysis displayed horticultural and peach sites in separate groups, differing by more than

Fig. 7. Symptoms of internode reduction observed on young rootstock plants grown on soil artificially inoculated with C. destructans and T. basicola isolates.

70% as regards soil-inhabiting fungi and by 45% for endophytic fungi recorded in young peach rootstocks (Fig. 2). Communities of soilinhabiting fungi from peach sites, were characterized by several species of Fusarium, Penicillium, Aspergillus and Trichoderma genera (Fig. 2), as well as significantly higher diversity. Penicillium, Aspergillus and Trichoderma genera are known to play an important role in suppressiveness, by killing weakened fungal pathogens and preventing re-infestation (Chet and Elad, 1983; Papavizas and Lewis, 1988). Therefore the richness of those genera in peach soils could represent a component of the highest health status of soil observed on basis of better growth of rootstock plants on these samples in the bioassay greenhouse test. Among the Fusarium spp. recovered from peach soils, F. acuminatum, one of the most abundant species in all soil samples from peach orchards, is characterized by antibiotic activity towards a wide number of fungal species and by the highest ability to colonize soils (Logrieco et al., 2008). Moreover, in this study F. acuminatum showed a good ability to colonize peach rootstocks, that supports the antagonistic features already observed for this species which may have had a role in protecting roots of peach rootstock. On the contrary F. oxysporum and F. solani, were the predominat Fusarium species of soil-inhabiting fungal communities in horticultural sites; in these sites Aspergillus and Penicillium spp. were occasionally isolated, except for A. parasiticus which was isolated in three sites in peach as well as in horticultural system (Table 2). Most fungal species isolated by culture methods were saprophytic fungi belonging mainly to ascomycetes (Tables 2 and 5); Zygomycota and Oomicota (Chromista) were poorly represented. The largest presence of ascomycetes may be explained by their role in undecomposed plant material degradation. In fact, they are known to be the group of fungi most involved in the initial steps of decomposition of leaf litter, namely the loss of soluble components and the following holocellulose decomposition (Osono and Takeda, 2002; Osono, 2006). Permanent vegetative cover in peach orchards and crop residues of the horticultural cropping system, represent the typical undecomposed leaf litter rich in herbaceous material, therefore they could have influenced the composition of fungal communities in the cultivated layer. According to the findings of the DGGE analysis the difference of diversity and composition of fungal communities between the two cultivation systems was lower than that obtained with cultural methods. As tillage strongly affects soil fungal biomass (van der Wal et al., 2006), the highest number of DGGE bands has until now been found in undisturbated soils such forest (Anderson et al., 2003); while in cultivated soils, the number of bands recovered with the same method does not differ widely from that found in our study (Oros-Sichler et al., 2006). DGGE is a potential good methods to evaluate soil fungal communities, and semi-nested PCR method represents an improvement, providing higher reproducibility thank to a higher concentration of specific amplicons. However, DGGE and other molecular methods applied on soil fungi still have several weaknesses mainly due to the relatively low concentration of fungal DNA. Therefore the molecular method was used in this study as an additional trial, while the complete qualitative analysis of soil fungi was performed on the communities of culturable fungi. All the excised bands, with the exception of 9g, corresponded to culturable fungi already recovered among root endophytic fungal communities. The most representative bands of the DGGE pattern corresponded to that of some endophytic root fungi isolated from roots, such as M. phaseolina, T. basicola and F. oxysporum; that may suggest a great abundance of those fungal species in soil. As regards the soil health of peach orchards, there was a correspondence between the highest fungal diversity in peach orchards (Table 1) and the highest growth (measured in height) of the

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plants as observed with the bioassay. Furthermore, as well as a significantly higher growth of rootstocks in soil samples from peach orchards, the lack of root rot symptoms in the young peach rootstocks suggests that the peach orchards, in the intensively cultivated area selected for this study, were not affected by replant disorder problems. This was confirmed by the peach tree growers in this area who have not observed replant problems in young peach orchards; they merely reported isolated attacks of white fungi. Indeed, no known root rot agent species was isolated from roots of young plants grown on soil samples from peach sites (Browne et al., 2006; Jaffee et al., 1982; May and Abawi, 1981), except for C. desctructans, recorded with lowest abundance in three sites. The most abundant species of endophytic fungi in peach sites were F. acuminatum and M. phaseolina. The first one, as observed above, is a highly antagonistic species and its presence could be related to the better plant growth observed in the peach soil as compared to horticultural soil. The second one is the polyphagous fungus M. phaseolina, known to be a weak or secondary pathogen, that can be economically important for herbaceous crops, such as sunflower or soybean, under drought conditions (Dhiangra and Sinclair, 1978); but it has also been reported as a pathogen of forestry and fruit tree saplings (Butin, 1995). For these reasons, its presence in the orchards of southern Italy should be monitored. The relative growth reduction observed in plants grown on soil samples from horticultural sites, was probably due to the high abundance of two important root rot agents in those soils: C. destructans and T. basicola known as agents of the root rot complex in a wide number of herbaceous and fruit tree crops (Braiford, 1993; Shew and Mayer, 1993). Both these pathogens have already been reported as agents of fruit tree root rot (Braun, 1995; Browne et al., 2006; Hoestra, 1965, 1988; May and Abawi, 1981). After the greenhouse bioassay, the necrosis or discoloration of roots could not be observed by visual inspection, however, the pathogenicity test of C. destructans and T. basicola isolates made it possible to clearly distinguish the biotic components of “replant disorders” observed in the greenhouse bioassay of this study: strong reduction in internode elongation and variability of aggressiveness of those pathogens (Fig. 7). In fact, for long time the term “replant disorder” has been used in reference to replant problems resulting from biotic and abiotic causes. The role of soil-borne pathogens in a etiology of fruit tree replant problems was only clearly seen after chemical fumigation trials carried out in the United States (May and Abawi, 1981). These results were further confirmed in 2000 by several authors, who reported a significant increase in plant growth in fumigated soil (Browne et al., 2006; Trout et al., 2002). The greenhouse bioassay has been widely used for the replant problem of fruit tree orchards (May et al., 1994; Neilsen et al., 1991; Nitta, 1991) and symptoms observed on peach have already been reproduced with C. destructans and Pythium isolates by Braun (1995). However the analysis of diversity and composition of soil fungal communities combined with findings of plant growth in greenhouse bioassay makes it possible to reach further conclusions: (i) in non-fumigated soils, the cropping system with a higher diversity was characterized by greater soil health; (ii) intensive crop management such as the horticultural sites considered in this study, can increase the abundance of aggressive soil-borne pathogens such C desctructan and T. basicola naturally present in soils; (iii) the type of undecomposed plant material (natural vegetation cover for orchards, crop residue in horticultural soils) which more frequently incorporates the organic material in cultivated soil layer, strongly affects the composition of soil fungal communities; and (iv) C. destructans and T. basicola, increased by continuous horticultural crops, can be pathogenic, with variable aggressiveness, toward the new peach orchards.

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