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Introduction

Earthworms are keystone organisms in the environment, and together with ants and termites are denominated "ecosystem engineers" (LAVELLE et al., 1997). The living habit of these organisms can modify soil chemical, physical, and biological characteristics (BROWN et al., 2000). Moreover, these invertebrates also interact with other organisms; several studies have reported positive effects of earthworms on plant growth (GROENIGEN et al., 2014). However, the importance of earthworms in diseased plants demonstrates numerous interactions whose results are dependent on the invertebrate species, plant and pathogen considered (ELMER, 2009; LAFONT et al., 2007).

Earthworms are distributed throughout Europe (RUTGERS et al., 2016). Lumbricus terrestris is an important earthworm species found on the continent, mainly in temperate regions and living in natural and agricultural sites. Data regarding soil invertebrate communities in Spain are scarce, but L. terrestris in agricultural systems, including strawberry fields has been previously studied (KUKKONEN et al., 2006).

The strawberry (Fragaria × ananassa) is a fruit appreciated worldwide. Global production is around 8.1 million Mg with Asia responsible for 48% of this total. In 2014, Spain produced 291,870 Mg of strawberries (7,790 ha), 44% more than Poland, the second biggest producer in Europe (FAO, 2017). Several diseases can reduce strawberry productivity, but Fusarium wilt has received special attention. Fusarium oxysporum is a globally distributed soil pathogen with several host-plants (KOIKE; GORDON, 2015). The damage caused by this microorganism is variable and dependent upon several conditions, but losses of 60% in strawberry fields have been reported in Australia (GOLZAR et al., 2007). The difficulties of Fusarium wilt control are similar to those found with other soil pathogens (high costs and inefficient control) and represent a barrier to increasing agricultural productivity (STRAUSS; KLUEPFEL, 2015). Studies with alternative management to control soil pathogens have increased in recent years, probably due to increased interest in sustainable agriculture.

The employment of good agricultural practices, such decreased soil disturbance and reductions in pesticide application, has increased earthworm populations in agricultural fields around the world, but little is known about the effects of these belowground invertebrates on strawberry plants (CRITTENDEN et al., 2014). Thus, the aim of this study was to evaluate the effects of interactions between L. terrestris and F. oxysporum f. sp. fragariae on strawberry growth and production in Utrera city, Spain.

Material and Methods

Study description

The greenhouse experiment was carried at the University of Seville School of Agricultural Engineering (USSAE), Utrera city, Spain. Strawberry plants were used in a factorial experiment with a completely randomized design, with four treatments and five replicates. The treatments were: control (absence of F. oxysporum f. sp. fragariae and earthworms); T1 (absence of F. oxysporum f. sp. fragariae, two L. terrestris per pot); T2 (Inoculum of F. oxysporum f. sp. fragariae, absence of L. terrestris); T3 (Inoculum of F. oxysporum f. sp. fragariae and two L. terrestris per pot).

The microcosms utilized were plastic pots with 2.8-L capacity. Each one received a mixture (Mix) of the substrate (Kekkilä Brown 025W), sand, and soil (1:1:1 proportion). Afterward, chemical analysis was performed, which showed the following characteristics: pH_water 7.09; C_org 34.4 g dm^-3; water soluble P 14.91 mg kg^-1; Ca²⁺ 0.24 cmol_c kg^-1; Mg²⁺ 0.18 cmol_c kg^-1; K⁺ 0.16 cmol_c kg^-1; Na⁺ 0.08 cmol_c kg^-1; Fe 10.29 mg kg^-1; Mn 1.42 mg kg^-1; Cu 0.34 mg kg^-1; Zn 1.90 mg kg^-1 (AUSTRALIAN STANDARD, 1993).

The earthworm species used in this study was Lumbricus terrestris, collected in a wheat crop under a no-tillage system in Alcalá de Guadaíra, Seville city. Adults (clitellum present) were identified using the external morphology according to Moreno (2004). Before inoculation, the earthworms were placed in pots containing the same substrate mixture for seven days to allow acclimation and cleansing of the intestinal tract. Strawberries (rooted cuttings) were planted in pots containing 3.0 Kg of Mix, and the earthworms were inoculated in the experimental units ten days after the planting. Prior to inoculation, earthworm weights were measured (grams per individual) to allow maintenance of a similar biomass between the replicates.

To obtain the inoculum of Fusarium oxysporum f. sp. fragariae, we utilized contaminated strawberry plants from Huelva province. The fungal isolates were identified by Borrero et al. (2017) using molecular techniques proposed by Suga et al. (2013). After inoculation, the plants were stored in the vegetable sanitation laboratory of USSAE. To acquire a satisfactory population of F. oxysporum f. sp. fragariae, the material was replicated in 50 Petri dishes using AMAP culture media (medium composed of 10 g L^-1 Agar, 10 g L^-1 Malt extract, 2 g L^-1 Asparagine and 0.5 g L^-1 Peter's foliar feed 27-15-12/N-P₂O₅-K₂O + micronutrients) (BORRERO et al., 2009) at 25 ºC for seven days. Next, water was used to homogenize the suspension, then 0.1 mL was transferred to a Neubauer chamber to determine cell concentration. The inoculations of F. oxysporum f. sp. fragariae was performed in four periods (Table 1), and the pathogen dosages were inoculated following dilution in water (soil field capacity). The fertirrigation was realized daily, intercalating the application of Peter's foliar feed (1.0 g L^-1), urea (0.3 g L^-1) and magnesium sulfate (0.7 g L^-1 of MgSO₄ 7H₂O) with 0.7 g of calcium chlorate (CaCl₂ 2H₂O) + 0.19 g of Sequestrene^® 138 Fe G 100 6% in the other.

Variables measured

To evaluate production, mature fruits were collected from each pot once a week, and the fresh weight measured using a digital balance (decimal precision). The accumulated production was calculated by adding the productivity obtained in one week plus the production of the previous week. At the end of the experiment, the disease severity was evaluated and was given a score based on a symptom severity scale where: 0 = plant well-developed, with no disease symptoms; 1 = 1-25% plants with wilted or dead leaves; 2 = 26 - 50% plants with wilted or dead leaves; 3 = 51-75% plants with wilted or dead leaves; 4= 76-100% plants with wilted or dead leaves; 5 = plant death (FANG et al., 2011). Disease ratings were made relative to control plants. In addition, the density of F. oxysporum f. sp. fragariae was determined according to Borrero et al. (2012) with some modifications, were utilized 5 g of soil re-suspended in 90 mL of pyrophosphate (TUITERT et al., 1998). After, the strawberry plants were retired (cut above the soil), dried at 60 ºC and weighed (digital balance) to obtain the dry shoot matter. Using a composite sample of five leaves, leaf nutrient content was determined. The elements measured were: P (MURPHY; RILEY, 1962), total N (Elemental analyzer CNS - 2000), K, S, Ca, Mg, Na, Fe, Mn, Zn, Cu, and B, using an ICE 3500 atomic absorption spectrometer.

Statistical analysis

Strawberry production, disease severity, fresh shoot weight, and leaf nutrient content were submitted to Shapiro-Wilk test, and when necessary (non-normal expected distribution), data were transformed using the equation "$\sqrt[3]{\mathit{x}}$". Analysis of variance (ANOVA) was performed using Tukey's HSD test (p<0.05). The programs utilized were Statistica^® 10.0 (STATSOFT, INC., 2011) and SigmaPlot^® 11.0 (SYSTAT SOFTWARE, INC., 2008).

Results and Discussion

The inoculation of F. oxysporum f. sp. fragariae was effective, and the inoculum density in the plants revealed means between 6,896 and 12,037 colony forming units (CFU) g^-1 for both inoculation treatments. Populations were superior to control, where we found only 1,602 CFU g^-1. The values of disease severity observed were 40.16% for T2 (without earthworms), which were significantly superior to T3 (two L. terrestris), which were 28.22%. Some results related to strawberry production did not show differences among the treatments (first two months).

Strawberry production data did not show an interaction between earthworms and F. oxysporum f. sp. fragariae, indicating that L. terrestris was unable to reduce the negative effects of Fusarium wilt on fruit production. However, positive effects of earthworm presence in the absence of F. oxysporum f. sp. fragariae were observed. The influence of treatments in strawberry production occurred in the last two experimental months (Figure 1). In the period from April 8 to May 13, the production had the same distribution pattern, with T1 (two L. terrestris) superior to T2 (F. oxysporum f. sp. fragariae inoculation, Figure 1a, 1b). By May 20, inoculation with two L. terrestris resulted in production of 388.64 g (Figure 1c), while T2 and T3 produced a total of 325.07 and 352.09 g of strawberries, respectively. Considering the data distribution in the last four evaluations, it is possible to identify the formation of three data groups: T1, with final strawberry production (accumulated at 24/06) of 485.12 g pot^-1; control with 440.91 g pot^-1 and the groups with F. oxysporum f. sp. fragariae inoculations, T3 (366.82 g pot^-1) and T2 (343.64 g pot^-1). The observed increases in T1 in relation to control represent around 1,000 kg per hectare (considering 24,000 strawberry plants hectare^-1).

Several surveys have reported positive effects of earthworms on plant growth (LAOSSI et al., 2010; PASHANASI et al., 1996; PUGA-FREITAS et al., 2012). This effect can occur in different ways, with increased available nitrogen probably the main factor in this phenomenon, due to the high uptake by the plants and typical low availability in the soil (AGREN et al., 2012). In a meta-analysis, Groenigen et al. (2014) separated the different effects of earthworms on plant productivity, and showed the importance of these invertebrates for release of some forms of nitrogen in the soil (NO₃^- and NH₄⁺), by comparing experiments with the presence and absence of legume plants. However, the effect of earthworms on soil fertility is dependent on many conditions. Clause et al. (2014) showed an interaction between different species of these invertebrates and soils, and found increases in the total nitrogen in L. terrestris casts.

The presence of earthworms increases microbial activity in soil, an effect that occurs during gut passage, casting, and burrowing (BROWN et al., 2000). When casts are excreted, the high activity of microorganisms elevates the rates of soil organic matter (SOM) mineralization, contributing to the release some nutrients, such as nitrogen and phosphorus (SIMEK; PIZL, 2010). Due to the anoxic environment of the casts, bacterial populations are dominant, and the availability of the reduced form of nitrogen (NH₄⁺) is higher than oxidized forms (CHAPUIS-LARDY et al., 2010). Considering the high uptake of nitrogen by strawberry plants and the importance of this element in strawberry production, it is probable the increased availability of this nutrient is utilized for fruit production (ANDRIOLO et al., 2011), minimizing the effects of earthworms on the plant growth observed in other studies when compared with this experiment. Other mechanisms also can explain the positive effects of earthworms on plant productivity. As an example, these invertebrates can stimulate plant symbionts such as arbuscular mycorrhizal fungi (which improve nutrient uptake) and plant growth-promoting bacteria (GORMSEN et al., 2004).

When analyzing the effects of L. terrestris inoculation on dry shoot matter (Figure 2) we observed that the presence of earthworms did not influence the negative effects of F. oxysporum f. sp. fragariae on strawberry growth. Instead, the inoculation of the pathogen alone or with earthworms produced decreased dry matter, with values of 3.68 and 4.09 for T3 and T2, respectively.

The lack of reduction in the damage caused by F. oxysporum f. sp. fragariae (related to fruit production) following the earthworm inoculation in this experiment was not expected, as normally the positive effects of these invertebrates in the soil negate the negative consequences of plants pathogens (DEMETRIO et al., 2017; LAFONT et al., 2007). For example, Stephens et al. (1993) found a reduction of Rhizoctonia solani severity in wheat plants in the presence of Aporrectodea trapezoides. The authors also observed an increase in the shoot dry weight, an effect attributed to an increase in nitrogen content in the soil and possible ingestion of hyphae fungi, which can reduce pathogen populations and roots infections. Lafont et al. (2007) studied the potential of the earthworm Pontoscolex corethrurus as a control for the root-knot nematode Radopholus similis in banana plants and do not observe direct effects on pathogen severity, but their results revealed superior plant growth when earthworms were inoculated. Elmer (2009) evaluated the effects of L. terrestris and F. oxysporum on different plant species and verified a 58% increase in tomato plant weight in the treatment with earthworms. However, the increases in the ammonium/nitrate ratio that usually occurs when earthworms are inoculated also can exacerbate the negative effects of Fusarium wilt in plants (BORRERO et al., 2012). According to Woltz and Jones (1973), increased ammonium content reduced soil pH, improving the development of F. oxysporum.

The concentration of nutrients in strawberry leaves was similar among the treatments, and only Ca, S, Mn, and Cu showed statistical differences (Table 2). The values obtained for Ca were higher in T3 (2.02 %) than control (1.67 %), but the content of S was greater in T1 (0.22 %) compared with T2 (0.18 %). The concentrations of Mn were 42.45 and 57.19 mg kg^-1 for control and T2, respectively. Cu was higher in the treatments with L. terrestris inoculation in relation to control.

The previously cited effects of earthworms on SOM mineralization can increase the availability of these metals, due to the release of elements before being adsorbed in organic-metal interactions, increasing the uptake by strawberry plants. Additionally, the production of CaCO₃ in the calciferous glands can change pH in the casts, also modifying the availability of some metals (EDWARDS, 2004; SHUMAN, 1999). Cheng and Wong (2002) observed an increase of some fractions of extractable Zn in the soil when Pheretima sp. were inoculated. Similar results are cited by Wen et al. (2004) for Cr, Zn, and Cu in the presence of earthworms. Although the foliar content of Mn had changed in our study between the treatments, this micronutrient normally presents higher variations in strawberry plants, with values around 30-350 mg kg^-1 (MILLS; BENTON JONES, 1996). On the other hand, the increase in Mn content may have resulted from a physiological response of the plant to pathogen attack, since this element acts as an enzymatic activator of many enzymes, such as dehydrogenases, transferases, hydroxylases and decarboxylases (BURNELL, 1988), related to the immunophysiological defense system of the plant (ELMER; DATNOFF, 2014).

Conclusion

In this work, the earthworm Lumbricus terrestris was unable to suppress the negative effects of Fusarium oxysporum f. sp. fragariae on strawberry production and shoot dry matter. However, in the treatments without pathogen inoculation, the presence of earthworms increased plant productivity.

Acknowledgments

Thanks to the Coordination for the Improvement of Higher Level Personnel (CAPES) for the post-doctoral opportunity in Seville University, Spain.

References

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Author notes

*Author for correspondence

IR