Fusarium crown and root rot (FCRR), probably the most important disease of asparagus in the world (Schreuder et al., 1995; Elmer et al., 1996; Blok & Bollen, 1997), has a complex etiology, with several Fusarium spp. associated with the roots of asparagus, involved as causal agents of the disease (Blok & Bollen, 1995; Elmer, 2001).

The most important species in Spain are: Fusarium oxysporum f. sp. asparagi (Foa), F. proliferatum (Fp), F. solani (Fs) and F. verticillioides (syn. F. moniliforme) (Tello et al., 1985; Seifert et al., 2003; Corpas-Hervias et al., 2006; Wang & Jeffries, 2006). The FCRR of asparagus most frequently affects adult plants, but also devastates seedlings and young plantations. The distribution and importance of these Fusaria depend on the geographic area, but the symptoms caused are similar. These include vascular staining and rotting of roots, rhizomes and stems; necrotic lesions on the surface of the root and stem; reduced plant size; yellowing and senescence of stems and crowns and, in the most severe cases, death of the plant (Johnston et al., 1979; Elmer, 2001). Consequently, crop yield declines slowly, because of the lower production of individual diseased plants and by the decrease in plant density due to the death of the most affected ones.

This disease is difficult to control due to, among other factors, the long-term survival of the pathogen in the soil and its easy propagation by planting material (Elmer, 2001; Corpas-Hervias et al., 2006). Furthermore, chemical treatments are frequently ineffective or provide short-term protection (Elmer, 2001). Moreover, the use of methyl bromide, the most effective fumigant for the suppression of soil pathogens, is no longer allowed, because its destructive effect on the ozone layer in the stratosphere that protects life on Earth from harmful radiations (Gamliel et al., 2000; Katan, 2000; Basallote-Ureba et al., 2010).

Soil solarization is a non-chemical method of soil disinfestation that increases the soil temperature by retaining the energy of solar radiation using transparent polyethylene sheets, under suitable climatic conditions, to reduce pathogen populations and disease incidence. The multiple mechanisms involved determine the thermal inactivation of the pathogen due to increased soil temperature (Katan et al., 1976), or the weakening of pathogen propagules that become more susceptible to competition or antagonistic activity of the indigenous soil microflora (Stapleton, 2000). Soil solarization, either alone or combined with organic amendments, and soil flooding, are effective in controlling many soilborne plant pathogens (Katan, 1981; Blok et al., 2000; Klein et al., 2011; Melero-Vara et al., 2011). In addition, soil solarization frequently enhances plant growth by improving soil structure, releasing nutrients (Chen et al., 1991) and stimulating plant growth promoting rhizobacteria (Gamliel & Stapleton, 1993).

Soil organic amendments (OAs) including animal manures, such as poultry manure (PM), and pellet of PM (PPM), composts (e.g., olive residue compost, ORC) and green manures, may suppress or reduce many soilborne plant pathogens (Gamliel & Stapleton, 1993; Abbasi et al., 2002, 2008; Noble & Coventry, 2005; Bonanomi et al., 2007; García-Ruíz et al., 2009; Avilés et al., 2011; Borrego-Benjumea et al., 2014a,b). Organic amendments affect aeration and soil structure, drainage, water holding capacity, nutrient availability and microbial ecology of soil (Davey, 1996). The suppression of pathogens by incorporation of OAs in the soil depends on several mechanisms. A factor involved in the survival of the pathogen is the production of toxic compounds, such as volatile fatty acids (acetic, propionic and isobutyric acids) during the microbial degradation of manure. The mechanism of suppression of microsclerotia and other fungal propagules arisen by nitrogen-rich amendments, such as PM or its pelletized form, is due to the production of ammonia and nitrous acid following degradation of the amendments by microorganisms (Tenuta & Lazarovits, 2002). Another possible mechanism of action of the OAs, such as composts, is the decrease in disease severity due to significant increases of microbial populations present in the amendment (Tsao & Oster, 1981; Conn & Lazarovits, 1999). Thus, the use of compost on asparagus fields enhanced the soil microbial biomass, as well as crop yield and spear number (Ngouajio & Counts, 2012). This fact leads to greater competition for nutrients in the soil and ecological niches, increasing the effect of antagonistic microorganisms, antibiosis, microbial production of lytic enzymes, fatty acid degradation, parasitism, changes in nutrient availability and induction of host resistance (Tsao & Oster, 1981; Hoitink & Boehm, 1999; Borrero et al., 2006; Avilés et al., 2011).

Other studies involving biochar, a C-rich mineral product of biomass pyrolysis, showed reduction of FCRR of asparagus following biochar applications at 10 to 30% (v/v) in Foa infested soil (Matsubara et al., 2002) and at 1.5 and 3.0% (w/w) in infested soil by Foa and Fp (Elmer & Pignatello, 2011), associated with root colonization by arbuscular mycorrhizae, though the reduction was more effective with the higher rate of amendment.

A new method of biological soil disinfestation was also proposed to control soilborne pathogens, which combine the addition of OAs to irrigation and air-tight plastic tarping leading to soil anaerobiosis. The amendment of soil with fresh broccoli or grass (3.4 to 4 kg m²) followed by tarping for 15 weeks was an effective method against a wide range of soilborne pathogens, including Foa, where toxic volatile products, including isothiocyanates formed by the hydrolysis of glucosinolates achieved by the enzyme myrosinase, both occurring in cruciferous plants, and confined under the tarp, contributed to the inactivation of fungal propagules, whereas precluding thermal inactivation (Blok et al., 2000). Analogous results were found with F. oxysporum f. sp. lycopersici (Fol) after addition of a number of plant biomass sources such as Brassica spp., wheat bran, rice straw, rice bran, or other organic substances such as molasses (1 to 2 kg/m² of organic material), followed by irrigation and tarping for three weeks to induce reducing soil conditions (Shinmura, 2004).

This work has been conducted in the framework of organic farming using different by-products from the local agricultural industry (i.e., poultry manure, pelleted poultry manure and olive residue compost) as organic amendments, thus promoting more sustainable agriculture by reusing them, to aim disease control for the pathosystem Fusarium pp./asparagus. The objectives were to determine the efficacy of those organic amendments combined with two temperatures and several incubation periods, on populations of Fo, Fp and Fs from Spain as well as the impact of these combined treatments on the FCRR symptoms development and on the fresh weight of asparagus plants. This amplifies the results already obtained with Canadian isolates of Fo and Fs (Borrego-Benjumea et al., 2014a) with Spanish isolates including one of F. proliferatum, the amendment with poultry manure and incubation simulating the conditions in which soil solarization is carried out in southern Spain (periods extended up to 30-45 days, at 30 and 35ºC).

The application of organic amendments to the infested substrates combined with temperature and period of incubation considerably reduced the viability of fungal propagules of the three isolates of Fusarium (Fo5, Fp3 and Fs2) studied. This agrees with the loss of viability of several soil-borne plant pathogenic fungi, including Foa, when amendment of soils with animal and plant residues combined with soil solarization was performed (Gamliel & Stapleton, 1993; Blok et al., 2000; Abbasi et al., 2002, 2008; Borrego-Benjumea et al., 2014b).

The most effective amendment in declining the viability of Fo5 and Fs2 was pelleted poultry manure (PPM-1 and 2%), with a more pronounced effect for the higher application rate, reaching undetectable levels in the case of Fs2 after 45 days incubation at 35ºC. This demonstrated a larger effect of temperature for Fs2 than for Fo5 and seems concomitant with sustained increase of total bacteria populations and overall moderate increases of pH and N content in PPM as compared to the other amendments tested (Borrego-Benjumea et al., 2014b). As a matter of fact, the N-richest amendments, with a low C/N ratio, which release and accumulate higher amounts of volatile toxic compounds such as NH₄ and HNO₂ in the soil during organic matter decomposition, are able to eliminate many soil-borne fungi (Tsao & Oster, 1981; Tenuta & Lazarovits, 2002; Borrego-Benjumea et al., 2014a). However, amendments with high C/N ratio may be effective in the control of these pathogens because those compounds can stimulate microbial activity, enhancing depletion of N availability, and, consequently, impairing the pathogen infection process (Snyder et al., 1959).

Decreases in severity of root symptoms in asparagus were much more pronounced in Fo5- and Fs2-infested substrate amended, respectively, with PM and PPM, and PM and ORC at the higher rates, but the effect varied with incubation temperature. Disease reduction after application of different organic amendments (including poultry manure) on soils infested with F. oxysporum, F. solani and F. equiseti, F. oxysporum f. sp. cumini and F. oxysporum f. sp. spinaciae were also reported by Escuadra & Amemiya (2008), Israel et al. (2011) and Martínez et al. (2011). On the other hand, the lower severity of root symptoms caused by isolate Fp3, as compared to Fo5 and Fs2 (except for incubation of substrate amended with PPM-2% at 30ºC) is likely due to poor survival of F. proliferatum in bare soils, as this species does not produce chlamydospores; therefore the initial inoculum density was lower for this than for the two other species tested (Elmer et al., 1996; Elmer, 2001). This is in agreement with previous results (Reid et al., 2002) that showed a low root severity (20%) using F. proliferatum at 1.3 × 10⁴ CFU/g (similar to that in our experiments). For the other treatments, however, we found much lower root severity values than the un-amended control.

The increase of root severity and the negative effect on plant fresh weight observed, in plants established in Fp3- and Fs2-infested substrates amended with PPM-2%, and incubated for 30-45 days regardless of temperature, suggests a phytotoxic effect of this PPM rate. A similar effect was also observed when high concentrations of PPM were applied to pots with substrate infested with F. oxysporum f.sp. dianthi and incubated at high temperature before transplanting rooted cuttings of carnation (Nava-Juárez, 2013), but these phytotoxic symptoms were absent from plants growing in greenhouse on soil to which equivalent dosages of PPM were applied (Melero-Vara et al., 2011). The reason is likely that volatile compounds generated by PPM-2%, mainly ammonia, develop more quickly at 30ºC (Lazarovits, 2001; Tenuta & Lazarovits, 2002; Borrego-Benjumea et al., 2014a). To this regard it is interesting the comparison of N content and electrical conductivity between PPM and PM, as those for PPM are, respectively, almost double and 4-times those of PM (Table 1).

However, substrates infested with Fo5 and then treated with PPM-2%, resulted in the higher plant weight as compared to the un-amended infested control, mainly with 45 days of incubation at 30ºC. At these conditions, all amendments, except PMM-2%, had a positive effect on the vigor of plants growing on Fp3- and Fs2-infested substrate. This is in agreement with previous results reported on increased plant growth of asparagus `Jersey Giant´ inoculated with one Canadian isolate of F. oxysporum and `Jersey Giant´ and `Grande´ inoculated with another isolate of F. solani also from Canada (Borrego-Benjumea et al., 2014b).

For the three pathogens studied, low and high severity values frequently correlated with high and low plant weight values respectively, especially for PM and PPM amendments. Nevertheless this general trend showed variability, according to the type and dosage of organic amendment, in relation to temperature and period of incubation. Therefore these effects were predominant for a mode of action related to toxic compounds release, whereas effects on soil biology and chemistry seem to occur more likely with ORC than with the other amendments tested.

Summarizing, asparagus disease levels decreased and plant weight increased when the integration of organic amendment, temperature and incubation periods used resulted in reducing inoculum viability, which corresponded in most cases with long periods of incubation. However, additional field experiments are required before implementation of our results. From these studies we are able to conclude that different organic amendments are suitable to FCRR management and do not cause phytotoxicity problems (except PPM-2%) to asparagus plants growing in nurseries and fields, such as has been reported for the control of Fusarium wilt of carnation.