Phelipanche spp. seedbank by soil solarization and organic supplementation

Scientia Horticulturae 193 (2015) 62–68

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Eradication of Orobanche/Phelipanche spp. seedbank by soil solarization and organic supplementation Rosario Paolo Mauro ∗ , Antonino Lo Monaco, Sara Lombardo, Alessia Restuccia, Giovanni Mauromicale Dipartimento di Agricoltura, Alimentazione e Ambiente (Di3A), Università degli Studi di Catania, via Valdisavoia, 5-95123 Catania, Italy

a r t i c l e

i n f o

Article history: Received 18 February 2015 Received in revised form 19 June 2015 Accepted 24 June 2015 Keywords: Soil solarization Organic amendment Seedbank Orobanche/Phelipanche spp. Tomato yield

a b s t r a c t Broomrapes are holoparasitic weeds responsible for high yield losses in tomato. The efficacy of available means to control them once they have attacked the crop is very limited. Due to this effective and environmental-friendly strategies to prevent these attacks on greenhouse tomato are needed. Both soil solarization and organic supplementation have been proposed to reduce broomrape attacks. However, a setup in their implementation, especially for eradicating broomrape seedbank from highly infested soils, is still needed before they can be widely adopted as a resolutive commercial practice. A set of two experiments was carried out in Southern Italy (37◦ 03 N, 15◦ 18 E, 10 m a.s.l.) to study (i) the effect of repeated solarization (for 1–3 consecutive years) and (ii) a single cycle of soil solarization combined with three levels of organic supplementation (0, 0.35 and 0.70 kg m−2 ), on broomrape seedbank dynamic and fruit yield of greenhouse tomato plants. Soil solarization alone during summer months increased mean maximum soil temperature by about 8.0–13.2 ◦ C (at 5 cm depth) and 4.1–9.3 ◦ C (at 15 cm depth). After one single cycle of soil solarization alone, seedbank mortality accounted for ∼99% of viable seeds, while induced seeds dormancy accounted for the remaining ∼1%. Complete seedbank eradication was achieved after the second year of solarization, while tomato fruit yield, starting from 3.43 kg plant−1 in unheated soil, peaked after the third year of solarization (6.58 kg FW plant−1 ). Organic supplementation prior to solarization further increased the temperature of solarized soil at both 5 and 15 cm soil depths (by up to 4.3 ◦ C, on average) and enhanced the efficacy of solarization against broomrape seedbank. Indeed, total seeds mortality was induced after a single cycle of solarization combined with 0.35 kg m−2 organic supplement, while tomato yield was enhanced up to 0,70 kg m−2 supplement (9.44 kg FW plant−1 ). Our results show that in Mediterranean climatic conditions at least two consecutive years of soil solarization are needed to completely eradicate broomrape seedbank from a highly infested soil. However, the efficacy of this technique may be improved when combined with organic supplementation, with positive effects on the yield of greenhouse tomato. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The genera Orobanche and Phelipanche, commonly known as broomrapes, group achlorophyllous holoparasitic plants that lost their ability for autotrophic life, thus becoming totally dependent on the host plant for nutrient sources (Rispail et al., 2007). These parasitic weeds attack the roots and connect to the vascular tissues of the host plant, through a specialized structure called haustorium (Parker and Riches, 1993; Joel et al., 2007; Pérez-de-Luque et al., 2008). This allows for an intense exchange of substances

∗ Corresponding author. Fax: +39 095 234 449. E-mail address: [email protected] (R.P. Mauro). http://dx.doi.org/10.1016/j.scienta.2015.06.038 0304-4238/© 2015 Elsevier B.V. All rights reserved.

among the two biota, including water, minerals and carbohydrates, which can cause severe damage to the host plant and premature death (Mauromicale et al., 2005a; Joel et al., 2007). Orobanche crenata Forssk. (crenate broomrape) is a widespread holoparisitic weed that inflicts severe damage (yield reduction up to 100%) on most grain and forage legume and other crops as safflower (Carthamus tinctorius L.) and carrot (Daucus carota L.), being widely distributed in the Mediterranean basin and Middle East (Rubiales et al., 2009). The species can only sporadically infest lettuce (Lactuca sativa L.), melon (Cucurbita melo L.), tomato (Solanum lycopersicum L.) and geranium (Pelargonium spp.). The devastating effect of O. crenata Forssk. attacks on legume forces many farmers to delay or abandon their cultivation. Phelipanche ramosa (L.) Pomel (branched broomrape) is the most widespread and damaging of

R.P. Mauro et al. / Scientia Horticulturae 193 (2015) 62–68

broomrape species, affecting some 2.6 Mha of solanaceous crops [mainly tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.), tomato and eggplant (S. melongena L.)] across the Mediterranean Basin, North Africa and Asia (Qasem, 1998; Zehhar et al., 2002; Boari and Vurro, 2004). In Italy, it is responsible for significant yield losses in tobacco, cabbage [Brassica oleracea (L.) Alef. conv. capitata L.] and both field- and greenhouse-grown tomato (Diana and Castelli, 1994; Boari and Vurro, 2004; Mauromicale et al., 2005b). The parasite has recently been recognized as a production threat of oilseed rape in Western France (Gibot-Leclerc et al., 2004) and has made its first appearance in South Australia, Central America and the United States (Boari and Vurro, 2004; Brault et al., 2007; Joel et al., 2007). Infestation can cause total yield loss in tomato, tobacco and potato (Qasum, 1998; Goldwasser and Kleifeld, 2004; Haidar et al., 2003). Together with the harmful effects on food production, broomrapes have a number of characteristics which make difficult to eradicate their presence on a farm scale. Among these, the ability to release numerous dust-like and long-living seeds into the soil (up to ∼4 million seeds m−2 in the top soil layer), along with their behavioral specificity in response to chemical signals released by the host plants, play a pivotal role in broomrape capacity to become a persistent treat in many grown areas worldwide (López-Granados and García-Torres, 1996; Parker and Riches, 1993; Joel et al., 2007; Bouwmeester et al., 2007). Tomato is a widely consumed vegetable crop throughout the world, with an estimated production of 159 Mt from more than 4.8 Mha cropland (Faostat, 2012). In the coastal regions of Mediterranean Basin it is the primary field and greenhouse vegetable crop. In Italy, where the crop represents a pivotal resource for the agricultural economy, it is grown on 104 Kha generating 6.0 Mt of fruit (Tognoni and Serra, 2003). Since the phase-out of the main soil fumigants such as methyl bromide, chloropicrin or 1,3dichloropropene, the control of tomato weeds and soil-borne pests (SBPs), especially in the greenhouse context, has largely been based on the use of soil fumigants such as metam sodium, metam potassium or dazomet. However, because of their toxic side-effects on the environment, they could be phased out in the future (Lombardo et al., 2012). Since this is expected to potentially exacerbate a number of problems related to tomato cultivation, including soil infestation by branched broomrape, there is an urgent need to develop efficient and environmental-friendly strategies aimed at eradicating, on a farm scale, the presence of broomrapes (Mauromicale et al., 2008). In the Mediterranean Basin, soil solarization has proven to be a valid alternative to conventional means of SBPs control for a number of crops (Katan and DeVay, 1991). This technique, by modifying the thermal regime of the top soil layer, makes the substrate hot enough to devitalize weeds seeds and nematodes, along with plant-pathogenic fungi and bacteria (Candido et al., 2011; Ozores-Hampton et al., 2012; Vitale et al., 2011, 2012). Beyond the thermal effect, soil solarization causes physical, chemical and microbiological modifications within the soil, so avoiding the creation of a biological vacuum, stimulating root growth, increasing crop yield and improving the product quality in a number of vegetable crops (Stapleton and DeVay, 1984; Chellemi and Rosskopf, 2004; Camprubi et al., 2007). All these effects are improved when soil solarization is combined with organic fertilization (biofumigation) (Matthiessen and Kirkegaard, 2006), because of the higher temperatures reached within the soil (Simmons et al., 2013) along with the release of volatile compounds (Gamliel and Stapleton, 1993; Klein et al., 2007), increased minerals availability and improved crops yield performances (Scopa et al., 2009; Mauromicale et al., 2010, 2011). In addition, it has been demonstrated that manure fertilization increases the killing effects of soil solarization against O. crenata Forssk. seeds (Haidar and Sidahmed, 2000). However, up to now information still lacks about the possibilities to implement such techniques to develop,

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on farm scale, a strategy for eradicating broomrape seedbank from the soil. The goal of the present work was therefore, to evaluate two different strategies aimed at reclaiming a greenhouse soil, heavily infested by broomrape seeds: the first one based on repeated cycles of soil solarization (for up to three consecutive years), and a second one based a single cycle of soil solarization combined with different levels of organic supplementation. In particular we evaluated the effects of the treatments under study on broomrape viability and germination capability, as well as on yield and main fruit characteristics of greenhouse tomato. 2. Materials and methods 2.1. Site, climate and soil Two greenhouse experiments were conducted over the period 2004/2005–2006/2007, on the coastal plain South of Siracusa (Sicily, South Italy; 37◦ 03 N, 15◦ 18 E, 10 m a.s.l.), in an area where previous solarization experiments were conducted (Mauromicale et al., 2008; Lombardo et al., 2012). The soil was a moderately deep Calcixerollic Xerochrepts (USDA, 1975) naturally infested with crenate and branched broomrapes. Before solarization, the soil characteristics were the following: clay 15.5%, silt 29.1%, sand 55.4%, organic matter 2.0%, pH 7.6, total nitrogen 1.8‰, available phosphorus 100 ppm, exchangeable potassium 680 ppm. The experimental field had been cultivated in a potato-lettucewatermelon rotation for 10 years, covered in plastic and used for greenhouse tomato production for the last year. The local climate is semi-arid/Mediterranean, with mild and wet winters, and hot, dry summers. The mean 30-year maximum summer monthly temperatures are 29.6 ◦ C (June), 32.5 ◦ C (July), 31.6 ◦ C (August) and 27.3 ◦ C (September) (Servizio idrografico, 1959-1998). 2.2. Experiment A The experiment was conducted from 2004–2005 to 2006–2007 season, in a randomized blocks experimental design with three replications, in order to assess the effects of repeated solarization treatments on broomrape seedbank viability and germination, tomato fruit yield and fruit characteristics. The solarization treatments were: non-solarized soil (S0 ), one-year solarization (during 2006 summer, S1 ); two-year solarization (during 2005 and 2006 summer, S2 ); three-year solarization (solarized from 2004 to 2006 summer, S3 ). During late spring of each year, the soil was ploughed and leveled, in order to provide a uniform soil surface; one day before mulching, all plots were irrigated to field capacity, in order to maximize the effects of soil heating (Katan, 1981). Solarization treatment, based on a plot size of 3.45 × 15 m, was achieved in each year, by covering bare soil with a 20 ␮m thick transparent polyethylene film [≥88% total visible transmittance, 20% infrared (IR) absorption] (Agriplast, Ragusa, Italy), from mid July to mid September (∼60 days per each year). Plastic sheets were stretched close to the soil surface and then soil-anchored at the edges. At the end of the solarization period, the sheets were carefully removed, limiting as much as possible any soil disturbance. 2.3. Experiment B This experiment was performed during the 2006–2007 growing season, in order to check the effects of organic supplementation on the efficacy of soil solarization on broomrapes seedbank, tomato fruit yield and fruit characteristics. Three levels of organic supplementation [0 (OS0 ), 0.35 (OS350 ) and 0.70 (OS700 ) kg m−2 ] were incorporated into the soil two days before solarization. A non solarized, non-supplemented control was included in the experiment. A randomized block design, with three replications per treatment,

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based on a 3.45 × 15 m plot size, was used. The commercially formulated product Humoscam® (SCAM s.r.l., Modena – Italy), containing organic-C 29.0%, humic-C 13.0%, humic-acid 22.0% and C/N 14.5, was used for supplementation. This was uniformly applied over the surface and incorporated into the top 20 cm of soil using a rotovator. All the other operations related to soil solarization were carried out as described for the experiment A.

2.4. Soil temperature measurement During the solarization period of both experiments, the soil temperature was recorded every 30 min, at 5 and 15 cm below the soil surface, using thermistors within a wire probe (HI 762W) buried in the center of the plots, and connected to a portable digital HI 98,840 microprocessor (Hanna Instruments, Padova, Italy).

2.8. Seedbank viability The seed viability test was carried out by slightly modifying the procedure suggested by Khalaf 1991. The seeds were surfacesterilized in sodium hypochlorite solution (6% chlorine) for 5 min, and then repeatedly washed in distilled water. After the surfacesterilizing treatment, the seeds were oven-dried at 30 ◦ C (WTB Binder Labortechnik, Tuttlingen, Germany) for 2 h and successively imbibed on Whatman paper No.1 in Petri dishes of 6 cm diameter. Then 3 mL of a 1% solution of 2,3,5-triphenyl tetrazolium chloride (TTC), prepared following International Seed Testing Association (1996) rules, was added to each Petri dish, wrapped in aluminum foil and incubated at 25 ◦ C for 72 h. Red or pink seeds were considered to be viable, whereas, those without pigmentation were considered dead (modified from Linke, 1987). All the observations were performed using a MS5 Leica stereomicroscope (Leica Microsystems, Wetzlar, Germany).

2.5. Tomato management practices after soil solarization 2.9. Germination bioassays In both experiments, at the end of solarization ∼5 week-old tomato plants (cv. ‘Ikram’, Syngenta, Italy) were transplanted in a 0.4 × 1.15 m format (corresponding to 2.17 plant m−2 ), and grown from mid-September until the end of the cycle (June). The greenhouses hosting the crops had a steel tubular structure and lateral windows along the sides, and were covered soon after solarization with a 200 ␮m-thick ethylene vinyl acetate (EVA) film, with a total visible transmittance ≥86%. The same commercial crop management was applied, providing the crop with 24.4 g N, 18.5 g P2 O5 , 26.2 g K2 O, 27.0 g MgO and 0.4 g Fe per plant, and administering drip irrigation when accumulated daily evaporation reached 25 mm. Pest control was performed by applying Imidachloprid (128 g a.i. ha−1 ), Bacillus thuringiensis Berliner formulates (150 g a.i. ha−1 ) and Bupimirate (59.5 g a.i. ha−1 ), when needed, while bumblebees were introduced into the greenhouse to maximize pollination. No additional heat, light or CO2 were provided. Fruit harvesting continued from January to June, recording the number and the weight of red-ripe stage fruits.

To compare viability with germination, an artificial germination stimulant, GR24 (an analogue of strigol), was used to promote germination of broomrape seeds (Johnson et al., 1981). The stimulant solution was prepared by dissolving 10 mg of the synthetic germination stimulant GR24 (Johnson et al., 1981) in 10 mL of acetone, and diluting 1000-fold with a 0.3 mM buffer of 2-(N-morpholino) ethanesulfonic acid (MES), pH 6.1. For the germination test, seeds were placed in a 9 cm Petri dish on one piece of filter paper (Whatman #3), wetted with 5 mL of stimulant solution, wrapped with aluminium foil to provide absolute darkness and incubated in controlled growth chambers MLR-351H (SANYO Electric Co., Japan, Ltd.) at the temperature of 18 ± 1 ◦ C. Emergence was recorded under a MS5 Leica stereomicroscope at 3-day intervals until no more germination occurred. When the germ tube was at least as long as its width (90–130 ␮m), seeds were considered to have germinated and were removed. 2.10. Statistical Analysis

2.6. Soil samples collection Soil samples were collected 1 day after soil solarization, along the diagonals of each plot. Nine soil cores per plot (each of 0.75 dm3 ) were collected with a metallic probe up to 15 cm depth. All soil cores were divided in two subsamples (0–5 and 5–15 cm depth) and stored in paper bags under laboratory conditions (20 ± 2 ◦ C), until tests were performed. A total of 216 soil samples (4 soil solarization treatments × 9 sampling points per plot × 2 depth × 3 replicates) were obtained in both experiments.

When appropriate, Levene’s test was used in both experiments to test for homoscedasticity, while differences among treatments were determined by applying a one-way analysis of variance (ANOVA), related to the experimental design adopted in the field. Percentage data were Bliss’ transformed before the ANOVA (untransformed data are reported and discussed) while multiple mean comparisons were performed through Fisher’s protected least significant difference (LSD) test (at least for P ≤ 0.05). 3. Results

2.7. Seedbank extraction

3.1. Experiment A

Each soil sample was carefully mixed in laboratory and 100 g were used for the seedbank analysis. Before seed extraction, soil samples from each core were pre-treated and mixed with 5 g of sodium hexametaphosphate solution for 20 min, to disperse readily the colloid matrix and facilitate the subsequent washing stages. The suspension was passed through a series of sieves with different mesh (from 5 to 300 ␮m). The O. crenata Forssk. and P. ramosa (L.) Pomel seed was extracted from the soils, without distinguishing between genera, by a sieving/floatation technique described by Benvenuti et al. (2001), thought immersion in a saturated sodium iodide solution. Indeed, it has been demonstrated that the sieving floatation method provides very good estimates of the relative density of broomrapes in the seedbank.

3.1.1. Soil temperature during solarization Soil solarization increased soil temperatures at both depths and in all monitored growing seasons (Table 1). At 5 cm depth, over the experimental period the absolute maximum temperature in solarized plots never dropped below 52.7 ◦ C, while mean minimum and maximum soil temperature ranged between 34.6 and 38.7 ◦ C, and between 47.0 and 50.9 ◦ C, respectively. On the average of 2004–2006 period, this led to an average soil thermal increase equal to 10.9 (mean maximum temperature) and 4.1 ◦ C (mean minimum temperature), as compared to non-solarized soil (Table 1). At 15 cm depth, although thermal differences were less evident, solarized soil showed the highest mean maximum and minimum soil temperatures, with a three-season average thermal increase, as

R.P. Mauro et al. / Scientia Horticulturae 193 (2015) 62–68

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Table 1 Absolute and average (in brackets) maximum (max) and minimum (min) temperature (T) in solarized and non-solarized soil recorded during the solarization periods (2004–2006) at two soil depths. Soil depth

5

Treatment

Tmax

Soil depth (cm) Tmin

◦

15 Tmax

Tmin

C

2004 Non solarized soil Solarized soil

49.4 (39.4) 55.2 (50.9)

28.7 (32.2) 28.7 (32.2)

44.1 (37.0) 50.2 (46.3)

29.9 (32.9) 32.6 (35.5)

Non solarized soil Solarized soil

42.5 (39.0) 53.2 (47.0)

31.4 (33.5) 35.6 (38.7)

41.9 (38.3) 46.3 (41.9)

31.5 (33.6) 36.9 (40.3)

Non solarized soil Solarized soil

39.5 (35.8) 52.7 (49.0)

28.3 (30.5) 32.4 (35.2)

34.8 (32.7) 45.6 (42.8)

30.2 (31.9) 35.4 (37.7)

2005

2006

Table 2 Seedbank, seed viability and germination at two soil depths as affected by solarization treatment. Different letters within each column indicate significance at Fisher’s protected LSD test (P ≤ 0.05). Soil depth

5

Soil depth (cm)

Treatment

Total Viable Seedbank (seeds m−2 )

Germinable Seedbank (%)§

Dormant

Total Viable Seedbank (seeds m−2 )

Germinable Seedbank (%)§

Dormant

S0 S1 S2 S3 F

7816 a 116 b 0 0

3830 a 8b 0 0

10.2 b 100 a 0 0

22176 a 495 b 0 0

10201 a 45 b 0 0

***

***

***

***

87.5 0 0 0 –

12.5 b 100 a 0 0

***

89.8 0 0 0 –

15

***

SS1 : solarized during 2006 summer; S2 : solarized during 2005 and 2006 summer; S3 : solarized during 2004, 2005 and 2006 summer. § Percentage referred to viable seedbank.

compared to non-solarized soil, equal to 7.7 and 5.0 ◦ C, respectively (Table 1). 3.1.2. Broomrape seedbank Solarization treatments exerted effects on all the measured broomrape variables and at both soil depths (Table 2). Compared to the non-solarized test, at 5 cm depth the S1 treatment significantly reduced both soil seedbank abundance (from 7816 to 116 seeds m−2 ) and seeds viability (from 3830 to 8 seeds m−2 ), while seeds dormancy increased from 10.2 to 100%. All these variables were reduced to zero under both S2 and S3 treatments (Table 2). A similar response of broomrape seedbank to solarization was recorded at 15 cm soil depth, since both soil seedbank and seed viability were significantly reduced passing from S0 to S1 (by 98 and 99%, respectively), while seedbank dormancy increased from 12.5 to 100%. All these variables were nullified at S2 and S3 (Table 2). 3.1.3. Tomato fruit yield and its components Tomato fruit yield and yield components were significantly affected by solarization treatments (Table 3). By increasing the number of solarization cycles, fruit yield passed from 3.43 (S0 ) to 5.54 kg FW plant−1 (on the average of S1 and S2 ), then it peaked at 6.58 kg FW plant−1 (S3 ). Overall fruit yield increased by 92%

between among the extreme treatments. The number of fruit per plant was significantly affected by soil solarization too, since it rose from 42.0 (S0 ) to 59.4 fruit plant−1 (S1 ), then increased further up to 73.5 fruits plant−1 (S3 ), for an overall 75% increase recorded between S0 and S3 . By contrast, the fruit average weight, passed from 81.7 (S0 ) to 88.7 g (S1 , +8.3%), did not showed any significant response to the additional cycles of soil solarization (Table 3). 3.2. Experiment B 3.2.1. Soil temperature during solarization At both soil depths the incorporation of organic supplementation increased soil temperature during solarization period (Table 4). As compared to OS0 , organic supplementation increased both mean maximum and minimum soil temperature at 5 cm depth by 1.8 and 1.3 ◦ C (OS350 ), and by 3.6 and 1.7 ◦ C (OS700 ). At 15 depth, similar thermal differences were recorded, so in all supplemented plots both soil mean maximum and minimum temperature never dropped down to 45 and 36 ◦ C, respectively. 3.2.2. Broomrape seedbank Organic fertilization improved the effects of soil solarization with regard to both broomrape seedbank consistence and seed

Table 3 Tomato fruit yield, number of fruits per plant and fruit average weight as affected by solarization treatment. Different letters within each column indicate significance at Fisher’s protected LSD test (P ≤ 0.05). Q: quadratic. Variable Treatment

Fruit yield (kg FW plant−1 )

Number of fruits (n plant−1 )

Fruit unitary weight (g)

S0 S1 S2 S3 F Trend

3.43 c 5.27 b 5.80 b 6.58 a

42.0 c 59.4 b 65.6 ab 73.5 a

81.7 b 88.7 a 88.4 a 89.5 a

***

***

*

Q*

Q **

Q*

S0 : non solarized soil; S1 : solarized during 2006 summer; S2 : solarized during 2005 and 2006 summer; S3 : solarized during 2004–2006 summer.

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Table 4 Effect of organic supplementation on soil temperature (T) during the solarization period. Absolute and average (in brackets) values of maximum (max) and minimum (min) soil temperature at two soil depths are reported. Soil depth (5 cm)

Soil depth (15 cm)

Treatment

Tmax (◦ C)

Tmin (◦ C)

Tmax (◦ C)

Tmin (◦ C)

OS0 OS350 OS700

52.4 (49.0) 54.3 (50.8) 56.7 (52.6)

27.0 (33.4) 28.0 (34.7) 29.0 (35.1)

46.0 (43.4) 47.9 (45.3) 50.3 (46.9)

27.0 (34.3) 28.4 (36.1) 28.9 (36.2)

OS0 : solarized and non supplemented soil; OS350 : solarized soil plus 350 g m−2 organic supplementation; OS700 : solarized soil plus 700 g m−2 organic supplementation. Table 5 Evaluated seedbank, seed viability and germination at two soil depths of solarized soil as affected by organic supplementation. Different letters within each column indicate significance at Fisher’s protected LSD test (P ≤ 0.05). Soil depth (5 cm)

Soil depth (15 cm)

Treatment

Total Viable Seedbank (seeds m−2 )

Germinable Dormant Seedbank (%)§

Total Viable Seedbank (seeds m−2 )

Germinable Dormant Seedbank (%)§

Test OS0 OS350 OS700 F

7855 a 123 b 0 0

3807 a 11 b 0 0

11.1 b 100 a 0 0

22098 a 506 b 0 0

10184 a 47 b 0 0

***

***

88.9 0 0 0 –

***

***

***

87.9 0 0 0 –

12.1 b 100 a 0 0 ***

Test: non solarized and non supplemented soil; OS0 : solarized and non supplemented soil; OS350 : solarized soil plus 350 g m−2 organic supplementation; OS700 : solarized soil plus 700 g m−2 organic supplementation. § Percentage referred to viable seedbank. Table 6 Tomato fruit yield, number of fruits per plant and fruit average weight as affected by organic supplementation. Different letters within each column indicate significance at Fisher’s protected LSD test (P ≤ 0.05). Variable Treatment

Fruit yield (kg FW plant−1 )

Number of fruit (n plant−1 )

Fruit weight (g)

OS0 OS350 OS700 F Trend

6.46 c 7.23 b 9.44 a

64.5 c 70.8 b 83.3 a

100.2 b 102.1 b 113.3 a

***

***

**

Q ***

Q ***

Q ***

OS0 : solarized and non supplemented soil; OS350 : solarized soil plus 350 g m−2 organic supplementation; OS700 : solarized soil plus 700 g m−2 organic supplementation.

viability. On the average of both soil depths, OS0 decreased seedbank abundance and viability (by about 98 and 99%, respectively), while increased seedbank dormancy up to 100%. Differently, these variables were reduced to zero passing to OS350 and OS700 (Table 5). 3.2.3. Tomato fruit yield and its components Tomato fruit yield, number of fruits per plant and fruit weight were all significantly affected by organic fertilization (Table 6). Indeed, compared to OS0 (6.46 kg FW plant−1 ), fruit yield increased up to 7.23 and 9.44 kg FW plant−1 at OS350 and OS700 , respectively, showing a trend significantly related to the quadratic component of the polynomial regression (Table 6). A similar response was recorded for the number of fruits per plant, which, starting from 64.5 fruits plant−1 (OS0 ), showed a significant increase at OS350 (70.8 fruits plant−1 , +10%) and subsequently at OS700 (83.3 fruits plant−1 , +29%) (Table 6). By contrast, the fruit average weight was responsive only to the maximum level of organic fertilization, as it rose from 101.2 g (on the average of OS0 and OS350 ) to 113.3 g at OS700 (+9%) (Table 6). 4. Discussion Successful strategy to control parasitic weeds such as broomrapes, might be to adopt an integrated approach (Rispail et al., 2007; Pérez-de-Luque et al., 2008), with multiple objectives including the eradication of the parasitic seedbank from the soil. Soil solariza-

tion, due to its limiting action on a wide range of SBPs, may have a central role in planning such strategies. In the present experiment, soil solarization during summer months increased mean maximum soil temperature by 8.0–13.2 ◦ C (at 5 cm depth) and 4.1–9.3 ◦ C (at 15 cm depth). At a depth of 5 cm, mean maximum temperature in solarized soil exceeded 47 ◦ C, above the threshold of 42–44 ◦ C required to nullify the germination of broomrape seeds (Mauromicale et al., 2001). Such increase in soil temperature is adequate to control many SBPs, and affects the activity, ecology and population dynamics of the whole soil biota, hence its multifunctional effects in horticultural ecosystems (Gamliel et al., 2000; Gelsomino and Cacco, 2006). In our experiment after a single cycle of soil solarization, the broomrape seedbank germination was entirely suppressed at both soil depths, and no shoot emergence was recorded during the following cropping cycle (data not shown). Several experiments demonstrated the effectiveness of this technique in suppressing emergence in a number of broomrape species (Saueborn et al., 1989; Abu-Irmaileh, 1991; Mauromicale et al., 2005a), with sometimes superior results than those obtained with soil fumigants (Lombardo et al., 2012). This is an important result, especially in terms of avoiding the environmental impact from soil fumigation. Indeed, a substantial proportion of fumigants used can be lost to the soil, so contaminating ground water moreover, certain molecules, in particular halocarbons such as chloropicrin and 1,3-dichloropropene, tend to break down into molecules involved in ozone depletion (Molina and Rowlands, 1974; Wang et al., 2001). The results presented here demonstrate that two mechanisms were involved in suppressing broomrape shoots emergence after soil solarization: a main mechanism, responsible for lethal effects in the majority of the viable seedbank, and a secondary, responsible for physiological effects (induced dormancy) in the remaining seeds. After one single cycle of soil solarization, the first mechanism accounted for ∼99% elimination of viable seedbank, while the second one accounted for the remaining ∼1%. As a consequence, incomplete seedbank eradication was possible after a single cycle of soil solarization, resulting in the presence of ∼53 broomrape seeds m−2 within the top 15 cm soil layer, albeit in a dormant state. Such result merits consideration to avoid potential phenomena of soil reinfestation, since broomrape seeds exhibit an annual dormancy—non-dormancy cycle (López-

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Granados and García-Torres, 1996), whose germination can be recovered in response to root exudates and to plant growth regulators (Song et al., 2006). This implies that at least two consecutive cycles of soil solarization will be needed in Mediterranean environments to completely eradicate the broomrape seedbank from a highly infested soil. Indeed, the complete absence of seedbank recorded within the soil after the second cycle of soil solarization could be explained by induced damage to the seed coat, leaving them unprotected from deterioration caused by soil microbial attacks. From an agronomical viewpoint, over a short period (three consecutive years), repeated soil solarization progressively increased the yield of tomato plants, indicating a certain dose-response pattern, consistent with the quadratic component of polynomial regression. Although a certain fruit weight increase was recorded, this increase was linked to an increased number of fruits (and clusters) per plant. This would point to a physiological effect of solarization on the growth and development of tomato plants, which was mirrored in an increased yield rate of the crop. As suggested by Gruenzweig et al., (1993) this response, known as increased plant growth (IPG), is primarily triggered by signals from the solarized soil through the roots, i.e., the only organ connecting the soil with the foliage. Although in our experiment it was difficult to ascertain which type of mechanism underlies such root-mediated response, the observed dose–response pattern among repeated solarization and yield suggests that soil nutritional modifications induced by soil heating could be involved in this phenomenon. Indeed, an increased release of nutrients (inorganic N, available P and K and other cations due to accelerated organic matter decay) due to soil solarization, and a subsequent improvement of plant growth and yield increase were reported both under field-scale (Stapleton et al., 1985; Gelsomino et al., 2006; Mauromicale et al., 2011) and growth chamber simulated solarization (Grünzweig et al., 1999). Additionally, the increased N availability might be attributed to the rapid heat-induced mineralization of the microbial N pool from declining microbial populations, since soil temperatures near 50 ◦ C were considered lethal for most mesophilic microbes dominating the majority of soils world-wide (Stapleton, 1996; Gelsomino et al., 2006). Indeed, long lasting (up to 17 months) increases of N availability after soil solarization have been reported (Stevens et al., 1991), a condition which could create more favorable soil fertility for the subsequent tomato crop, hence supporting sustained growth and developmental processes during the juvenile stages. Such hypothesis was partially confirmed by Experiment B, in which 0.70 kg m−2 organic supplementation prior to solarization increased the yield of tomato crop markedly up to 9.44 kg FW plant−1 (equivalent to ∼205 t ha−1 fruit yield), a 75% yield above the long-term average for greenhouse grown tomatoes in Italy (Istat, 2014). In this case, such yield increase was due to a greater number of fruits plant−1 and to an increased weight of tomato fruits. It is well known that incorporating organic materials can improve the nutrient status of the soil and promote higher levels of microbial population and activity (Gelsomino et al., 2006). In Mediterranean greenhouse tomato crops it has been demonstrated that organic supplementation prior to solarization promotes a long lasting increase (up to 9 months) in total N, exchangeable K2 O and available P2 O5 content within the soil, thereby enhancing growth and developmental processes and yield, through a more favorable source-sink balance of the plant (Mauromicale et al., 2010). In our experiment, organic supplementation showed a strong synergistic effect with soil solarization in affecting the viability of broomrape seedbank, so that this was annulled already by combining 0.35 kg m−2 of organic supplementation and a single cycle of soil solarization. Such effect can be attributable in part to the increased thermal regime of solarized soil recorded in response to organic supplementation. However, it must

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pointed out that the effects of soil solarization against soil-borne pathogens and parasites also depend on the increased concentration of volatile toxic compounds within the soil, and that this effect is amplified by biologically-active molecules released upon a heatdriven decay of organic amendments during solarization (Katan, 1981; Gamliel, 2000; Mauromicale et al., 2010). The results of the present experiment suggests that combining organic amendment and soil solarization, beyond increasing greenhouse tomato yield, could have a pivotal role in developing more efficient farm-scale programs aimed at both eradicating broomrape seedbank and safeguarding long-term soil fertility. On the first aspect, this shows the possibility to halve (from 2 to 1), in Mediterranean conditions, the number of solarization treatments required to eradicate broomrape seedbank, hence to speed-up and economize the remediation of heavily broomrape-infested soils. This becomes important in terms of avoiding long-term negative effects that repeated solarization could cause on soil microbial communities (Mauromicale et al., 2005a) and organic matter content (Gelsomino et al., 2006). This is especially pertinent in Mediterranean climates where agricultural soils are often characterized by low organic matter and hence low N reserves and mineralization potential (Ierna et al., 2012; Mauro et al., 2014). 5. Conclusions Broomrapes eradication from agricultural soils is of great public concern in Mediterranean agriculture, where horticulturalists face growing demands to produce vegetables with high health properties and to mitigate the environmental impact of greenhouse cultivation. In this context soil solarization represents an attractive option that can be used to enhance the yield of greenhouse tomato and reclaim broomrape infested soils. The practice has a environmental friendly profile when compared to soil fumigants, especially in terms of groundwater contamination and soil chemical residues. Therefore, it can represents a cornerstone on which to build integrated defense strategies against SBPs and improve the yield characteristics of greenhouse tomato. Organic supplementation prior to soil solarization represents a management option that can be used to improve the effectiveness of soil solarization against broomrape, by reducing the number of soil solarization treatments needed to eradicate its seedbank from the soil, and to further enhance the yield of greenhouse tomato. This integrated approach is compatible with organic production systems, and its implementation would be advisable in the light of preserving horticultural soils fertility in the long-term period. In this view, additional data are still needed on the residual effects over the years of these combined techniques on pathogens control and crop productivity benefits, in order to encourage its wider application on greenhouse crops. References Abu-Irmaileh, B.E., 1991. Soil solarization controls broomrapes (Orobanche spp.) in host vegetable crops in the Jordan valley. Weed Technol. 5, 575–581. Benvenuti, S., Silvestri, N., Simonelli, G., Macchia, M., Bonari, E., 2001. Valutazione della flora potenziale e della relativa dinamica di infestazione in alcuni sistemi colturali di omosuccessione di mais. Riv. Agro. 35, 23–34. Boari, A., Vurro, M., 2004. Evaluation of Fusarium spp. and other fungi as biological control agents of broomrape (Orobanche ramosa). Biol. Control 30, 212–219. Bouwmeester, H.J., Roux, C., Lopez-Raez, J.A., Bécard, G., 2007. Rhizosphere communication of plants, parasitic plants and AM fungi. Trends Plant Sci. 12, 224–230. Brault, M., Betsou, F., Jeune, B., Tuquet, C., Sallé, G., 2007. Variability of Orobanche ramosa populations in France as revealed by cross infections and molecular markers. Environ. Exp. Bot. 61, 272–280. Camprubi, A., Estaùn, V., El Bakali, M.A., Garcia-Figueres, F., Calvet, C., 2007. Alternative strawberry production using solarization, metham sodium and beneficial soil microbes as plant protection methods. Agron. Sustain. Dev. 27, 179–184.

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