Synergistic effect of organic and inorganic fertilization on the soil inoculum density of the soilborne pathogens Verticillium dahliae and Phytophthora spp. under open-field conditions

Abstract

Background

The increasing demand of food causes an excessive exploitation of agricultural lands, often inducing phenomena of soil sickness accompanied by the development of soilborne diseases. The use of residual biomasses together with inorganic fertilizers can be considered a good agricultural practice for controlling the inoculum density of soilborne phytopathogens since soil conditioners can release inorganic nitrogen, polyphenols and fatty acids that, especially in vitro, have demonstrated various degree of suppressiveness against such pathogens. Further, soil organic amendments can also modify the population of soil culturable bacteria and fungi that, in turn, can affect the soilborne diseases in several ways. With this study, the authors aim to evaluate the impact of the synergistic application of different biomasses and inorganic fertilizers on the soil inoculum density of Verticillium dahliae and Phytophthora spp. during two potato cycles under open-field conditions. The biomasses used for the fertilization of the potato crop were olive pomace residues (OPR), composts from municipal solid wastes (CMW), spent mushroom compost (SMC), and livestock manure-based compost (BRX).

Results

The inoculum density of Verticillium dahliae appeared inhibited by BRX due to its low C/N ratio that caused a quicker release of inorganic nitrogen with respect to the others soil conditioners. In contrast, OPR was conducive to the aforementioned soilborne pathogen since that biomass was characterized by a very high percentage of unsaturated fatty acids that, rather, stimulate the inoculum density of V. dahliae. Finally, polyphenols did not influence the same pathogen because they apparently turned into no toxic compounds very quickly. The inoculum density of Phytophthora spp. was reduced equally by all the biomasses used in combination with the inorganic fertilizers, regardless of their composition and quantity, mainly because of the development of general microbial suppression. Therefore, the chemical characteristics of the soil conditioners apparently did not affect the inoculum density of Phytophthora spp.

Conclusions

The results of this work underline the behavioral diversity of the different pathogens towards the different means adopted. Phytophthora spp. are sensitive to any kind of biomasses combined with inorganic fertilizers while the inoculum density of Verticillium dahliae should be reduced using soil conditioners characterized by low C/N ratio and low quantity of unsaturated fatty acids.

Background

Soilborne diseases are responsible to the reduction of yield and quality of crops and contribute to the development of the soil sickness [1]. Since the use of chemicals (fumigants and fungicides) has been restricted, other sustainable approaches are necessary to control the soilborne pathogens and guarantee high yield and quality of crops [2].

Organic amendments have been tested to control many soilborne pathogens such as Rhizoctonia solani [3, 4], Verticillium dahliae [5, 6], Fusarium spp. [7, 8], Phytophthora spp. [9], Pythium spp. [9, 10], Sclerotinia spp. [4], Chilosi et al. [11]) and Sclerotium cepivorum [12]. However, organic soil conditioners can be suppressive to some pathogens and conducive to others. Vestberg et al. [13] tested 21 composts against the strawberry crown rot caused by Phytophthora cactorum and the cucumber wilt disease caused by Pythium spp., and found that only 7 composts showed suppressiveness capacity towards these pathogens. Mazzola et al. [14] reported an increase of Pythium-related disease on apple after the application of Brassica napus seed meal at different rates, and a decrease of Rhizoctonia spp. with the lowest dose of the same treatment. Gilardi et al. [15] tested two Brassica carinata residues, Brassica juncea-based biofumigation, compost, chicken manure and cattle manure towards Fusarium oxysporum on lettuce crop and found that Brassica carinata and compost reduced the pathogen, while the green manure of Brassica juncea, cattle and chicken manure provided only its partial control. Scheuerell et al. [16] tested 36 composts against Pythium ultimum and Pythium irregulare on cucumber and against R. solani on cabbage and found disease suppression in 49% of cases but the sole R. solani was stimulated in 14% of trials. Bonanomi et al. [17] evaluated the effects of several biomasses on soilborne pathogens and found that compost was the most suppressive material, with more than 50% of cases; peat was suppressive only in 4% of treatments, while crop residues were suppressive and conducive in 45% and 28% of the trials, respectively. Chilosi et al. [11] used a green compost mixed with peat to evaluate its suppressiveness towards many soil pathogens and found a significant reduction of root rot by Sclerotinia sclerotiorum, no effect toward Phytophthora nicotianae and an increase of disease produced by Rhizoctonia solani. Koivunen et al. [18] conducted a study on the persistence of Botrytis cinerea in organic field soils and found that this pathogen was suppressed by the addition of manure-based compost but not by yard waste compost. Further, the lower dose of compost and the addition of crab meal, i.e., chitin, enhanced the suppressiveness towards the pathogen.

The effectiveness of soil organic conditioners in the reduction of soil pathogens is linked to some conditions such as the plant–pathogen relationship, the rate of application, the degree of maturity of composted amendments or the decomposition stage of not composted organic residues [19], and the colonization of compost by specific groups of antagonistic microorganisms (Chilosi et al. [11, 20]. In addition, the composition of the biomasses used as soil conditioners can influence their effects against soilborne pathogens. The abundance of chitins or chitin-derived C compounds in some matrices stimulates the proliferation of chitinolytic agents responsible to the degradation of fungal pathogen cell walls and to reduction of their pathogenicity, while labile C substrates, such as simple sugars and cellulose, can reduce the suppressiveness capacity of composts [21, 22]. The pH of the biomasses can influence the availability of nutrients and the activity of microorganisms, in this sense, in more alkaline conditions pathogens such as fungi lose the competition with bacteria and through a biocontrol mechanism their pathogenicity is reduced [23, 24]. A higher electrical conductivity (EC) has also been shown to reduce sporulation and virulence of pathogens [24]. The kind of nitrogen, especially ammonium and nitrate, present in inorganic fertilizers or released from the mineralization of biomasses can influence plant disease incidence [25]. Dixon [26] reported that the use of calcium cyanamide suppresses soil-borne pathogens encouraging the development of beneficial microorganisms due to the slow release of N and the ready availability of calcium. Tenuta and Lazarovits [27] and Postma et al. [28] found that the application of biomasses characterized by a high N content was effective in the eradication of Verticillium microsclerotia. Rousk and Bååth [29] found a higher suppression of nematodes and pathogen fungi with the application of alfa residues, characterized by a low C/N ratio, with respect to barley straw showing high C/N ratio and this result was ascribed to a favored bacterial growth and activity. Lazarovits et al. [30] found that the addition of organic amendments rich in N reduced the populations of plant pathogens, and strongly increased the populations of soil bacteria. Other studies have demonstrated that some short chain saturated fatty acids (FA) show inhibitory effects on several plant pathogens [30,31,33]. Even polyphenols have antimicrobial effects towards different bacteria, yeast, and fungi [34] and may be released during the decomposition of organic amendments or produced by microorganisms implied in the same decomposition, even if they are easily broken down by specific bacteria, yeasts and fungi in a short time and incorporated into the soil humic fraction [35].

The soil culturable microorganisms represent only a small part of the microbial community, but they are involved in the active and ongoing processes and are sensitive to soil management [36]. In addition, they include important genera of bacteria and fungi (e.g., Bacillus, Pseudomonas, Streptomyces, Trichoderma) that feature plant growth promoting activities, produce secondary metabolites useful for crop protection, show antagonistic features [37]. According to the review by Bonanomi et al. [38], total culturable bacteria and, with a similar pattern but a lower extent, total culturable fungi, appear the best microbiological parameters in terms of suppression indices with all kind of organic amendments and soil pathogens, and their increase is strictly related to the level and the transformation processes of the organic matter [39]. In particular, fluorescent pseudomonads, sporigenous bacteria and Trichoderma spp. show the highest percentage of positive correlation (73, 60% and 56%, respectively), with no cases of negative correlation with suppressiveness.

It is noteworthy that almost all the aforementioned researches have been conducted in vitro and not in less predictable and more complex open-field conditions where other advanced investigation techniques, such as the next generation sequencing, show a low reliability [40]. Therefore, with the present study, the authors aimed to evaluate the effects of different biomasses, abundant in Mediterranean region and therefore easily available, on the soil inoculum density (ID) of Verticillium dahliae and Phytophthora spp. during two potato crop cycles inserted in a wider crop rotation. In particular, the authors investigated how the selected biomasses influenced the soilborne pathogens ID through their composition and input of polyphenol and fatty acids, their impact on the soil culturable bacteria and fungi and their effects on the mineral forms of nitrogen.

Methods

Site description, treatments, and soil sampling

The trend of the soil ID of Verticillium dahliae and Phytophthora spp. was investigated in open field at a private farm located in Polignano a Mare (Bari), Italy. Since the farm produces many vegetables, the authors investigated the two crop cycles of potato of the following crop rotation: spinach, potato, head cabbage, potato, celery leaves.

The experimental field was divided into 15 randomized plots (5 × 5 m), deriving from three replicates of the following treatments: (1) olive pomace residues (OPR, 50 t ha⁻¹) arising from the mechanical extraction of olive oil of two phase mills; (2) compost deriving from composting of the organic fraction of municipal solid and pruning residues (CMW, 20 t ha⁻¹); (3) spent mushroom compost (SMC, 30 t ha⁻¹); (4) compost deriving from composting of cow, horse and poultry manures (BRX, 1.5 t ha⁻¹); (5) control without any application of soil conditioners (CON). Only the potato crop was treated with the previous organic soil amendments, while details of the entire crop rotation are reported in Table 1. The amendments have been incorporated into the soil by rotavation at about 20 cm soil depth. The dose of each amendment incorporated was established according to the local good agricultural practices, except for BRX that was incorporated at the label dose. Further details of the experimental site, amendments and treatments are reported elsewhere [1].

Each plot was sampled for chemical analyses by collecting five soil cores at a depth of 20 cm using an auger and a W sampling scheme. Analogously, each plot was sampled at a depth where V. dahliae and Phytophthora spp. may be more abundant (8–15 cm) for quantifying their ID, keeping samples in refrigerator at 5 °C for 3 days maximum till microbial analyses.

Table 1 Fertilization and sampling scheme of the experiment

Chemical analyses of organic amendments and soils

Soil chemical analyses were performed in accordance with the methods by Page et al. [41]. The lipidic fraction of the biomasses was extracted using petroleum ether as a solvent in a Soxhlet apparatus and quantified. The fatty acids (FA) composition was determined as follows: the extracted phase was solubilized using 250 µL of H₂SO₄ 1% v/v methanol solution and put in 1.5 mL centrifuge tubes. Samples were heated at 50 °C overnight. Two hundred and fifty µL of heptane were then added to each sample and mixed. After stratification, the upper phase was collected in amber glass vials and used for gas chromatographic analysis. A HP 5890 gas chromatographer equipped with a HP 88 column 100 m × 0.25 mm id × 0.2 film thickness was used. Fatty acid methyl esters were identified by external standards injection and expressed as percentage of the lipidic fraction [42].

The total polyphenols content of each soil conditioner was determined using the slightly modified Folin–Ciocalteu method described by Gambacorta et al. [43]. The amount of total phenols was expressed as gallic acid equivalents (GAE, mg gallic acid kg⁻¹ sample) through the calibration curve of gallic acid.

Microbial analyses

The population of the soil culturable fungi and bacteria was determined according to the slightly modified protocol reported by De Mastro et al. [36]. In details, 10 g of each soil sample were suspended in 90 mL of sterile phosphate buffer (0.1 M, pH 6.8) and shaken vigorously at 270 rpm for 1 h. Then, 100 μL from several tenfold serial dilutions (10^–1–10^–6) of each sample were spread onto 5 plates of (i) 0.1% tryptic soy agar (TSA) for total bacterial counts and (ii) potato dextrose agar (PDA) with 50 mg chlortetracycline and 1 mL L⁻¹ tergitol for total fungal counts. Bacterial and fungal plates were incubated at 28 °C and 25 °C, respectively, for a week, and colonies were counted and expressed as log (CFU + 1) g⁻¹ of dry soil (CFU = Colony Forming Unit).

The inoculum density (ID) of V. dahliae microsclerotia was estimated according to the protocol of Harris et al. [44], with some modifications. Briefly, soil samples were thoroughly mixed, air dried at room temperature for 30–40 days, carefully crumbled, and sieved with 2-mm openings. Twenty-five grams of soil were placed in 250 mL flasks and made up to 100 mL volume with sterile distilled water. Then, samples were agitated on a gyratory shaker, at 240 rpm for one hour, and the suspension was washed under running tap water through nested sieves with 160-μm and 25-μm openings. The residue collected on the 25-μm sieve was washed into a 50-ml beaker to obtain a final volume of 20 mL. Aliquots of 2-mL soil suspension under continuous stirring were individually distributed, by using a wide orifice pipette, on the surface of 10 Petri dishes containing a semiselective medium [45]. The inoculated petri dishes were incubated in dark room and at room temperature for 10–14 days. Then, the surface of incubated selective medium was gently washed under quite stream of fresh water to remove soil particles. All washed plates were incubated again at room temperature for 15–20 days. The ID of V. dahliae, determined by recording the colonies arising from microsclerotia in each plate under a 20 × magnification stereomicroscope, was calculated as number of microsclerotia per gram of dried soil (mscl g⁻¹).

The ID of Phytophthora spp. was assessed using a selective medium (Masago et al. [46]) with minor modifications. Briefly, 10 g of 2-mm mesh sieved soil were suspended in 90 mL of water in an Erlenmeyer flask, and vigorously shaken, using a magnetic stirrer. One-milliliter aliquots of soil suspension were transferred with a wide orifice pipette to the surface of 6 Petri dishes containing the selective medium and spread by shaking the dish. After 24 h of incubation at 20 °C, soil was removed from the plates with a gentle stream of running water and incubated again at 20 °C for additional three days. Colonies showing the morphology of Phytophthora spp. were then counted to give the ID expressed as propagules per gram (ppg) of dry soil. Soil moisture was determined by desiccating 20 g of soil for 24 h at 110 °C.

Statistical analysis

Each treatment was performed in three biological replicates. Experimental data were tested against the normal distribution using the Shapiro–Wilk’s test together with their homoscedasticity by means of the Levene test, then it was performed the analysis of variance (ANOVA) followed by the Tukey HSD test.

Results

Chemical results

Table 2 shows the main soil parameters at the beginning of the trial. Noteworthy are the textural class (silt clay loam), the relatively low organic carbon content and the high concentration of available phosphorous.

Table 2 Main soil properties at the beginning of the trial

No significant difference was observed in the soil N-NH4⁺ concentration at the end of each potato crop regardless of the specific treatment (Table 3) although the quantity of N added with each amendment differed substantially [1]. In contrast, the soil nitrate content changed significantly among treatments: in fact, the BRX amended plots showed the highest values and OPR and CON the lowest ones (Table 3).

Table 3 Ammonium and nitrate content in soils at different sampling times

Table 4 shows the content of polyphenols in each soil conditioner and the quantity supplied to the soil through the treatments. Considering the quantity of soil conditioner used, OPR added the highest amount of polyphenols to the soil, i.e., about 6.5 and 12 kg ha⁻¹ in the first and the second year of trial, respectively. Conversely, BRX added the lowest amount, about 0.4 and 0.45 kg ha⁻¹ the first and the second year of trial, respectively, even if that soil conditioner was the richest in polyphenols content among all the biomasses tested.

Table 4 Polyphenols content in the different organic matrices added to soil at each treatment

Table 5 reports the lipidic content of each soil conditioner, the percentage of FA of the lipidic fraction, the quantity of lipids and fatty acids added to soil during the trial. The highest quantity of FA has been supplied by OPR (about 2.2 and 1.9 t ha⁻¹ the first and second year, respectively) since this biomass is still characterized by a certain amount of olive oil and was used in high quantity (50 t ha⁻¹). In contrast, SMC showed the lowest content of lipidic fraction (on average 0.24%) and the lowest percentage of FA (on average 35% of the lipidic fraction), therefore, even if it was added in relatively high quantity (30 t ha⁻¹), the amount of FA provided was the lowest (about 10 and 13 kg ha⁻¹ the first and the second year, respectively). The FA composition among biomasses varied: OPR presented the lowest number of FA, mainly oleic (about 63% of all FA), palmitic (17%), linoleic (9%) and vaccenic (2%), whereas BRX showed the highest number of FA, with oleic (33%), palmitic (26%), stearic (14%) and linoleic (5%) being the most represented acids. CMW and SMC contained almost the double FA than OPR but their composition was different, the former showed mostly oleic (25%), palmitic (19%), stearic (6%), elaidic (4%), myristic (3.6%), caprylic (2.8%) and lauric (2.5%) acids, whereas the latter was composed mainly by palmitic (7.8%), caproic (5%), stearic (3%) and oleic (3%) acids, with the remaining ones showing roughly similar concentration.

Table 5 Content of lipidic fraction and fatty acids in organic matrices and their quantity added with each treatment

Total culturable bacteria and fungi

Figure 1 shows the trend of soil culturable bacteria (SCB) and fungi (SCF), during the trial. One month after the first organic amendment (T1), control plots had the lowest SCB population with respect to the treated soils, showing the effect of the biomasses on these microorganisms. After that, OPR was the only soil conditioner that induced always a significantly higher SCB population, probably due to the largest amount applied over the course of the two crop cycles (Fig. 1a). A similar trend was observed for the SCF population: at T1, control and BRX plots showed the lowest SCF population, but in the following sampling point OPR induced the highest population level of SCF, with significantly different value than the control (Fig. 1b).

Fig. 1

Effect of organic amendments on the population level of soil culturable bacteria (a) and fungi (b). Bars represent the standard error of the mean (n = 3). Mean values marked with different letters are significantly different according to the Tukey’s HSD test (p < 0.05). See Table 1 for details of the reading points

Inoculum density of soil pathogens

The ID of V. dahliae during the trial is reported in Fig. 2. At the beginning of the experiment (T0), before the incorporation of the biomasses, the ID was similar among all plots. One month later, the number of microsclerotia increased significantly in plots treated with OPR remaining lower and similar in all other treatments and CON. At the end of the first potato cycle (T3), OPR plots showed the highest value of V. dahliae microsclerotia while BRX was the treatment with the lowest content of the pathogen. At the end of the second potato cycle (T7), OPR and CON plots resulted in the highest number of microsclerotia with respect to the other treatments, and the same trend was observed at the last sampling too. Two months later the synergistic application of a calcium cyanamide-based fertilizer (400 kg ha⁻¹) and BRX (800 kg ha⁻¹) to all plots (T8, Table 1), the ID was still significantly lower in BRX plots with respect to OPR and CON.

Fig. 2

Effect of organic amendments on the soil inoculum density of Verticillium dahliae (mscl g⁻¹ dry soil). Bars represent the standard error of the mean (n = 3). Mean values marked with different letters are significantly different according to the Tukey’s HSD test (p < 0.05). See Table 1 for details of the reading points

The BRX application, above all, was the treatment that markedly reduced the ID of V. dahliae, whereas OPR was the worst, due to the similar results with CON, rather it was even conducive at the beginning of the trial, one month after the first application of the biomasses (T1). The CMW and SMC treatments showed roughly the same trend, with results closer to BRX than OPR.

A significant negative correlation has been found between V. dahliae ID and soil nitrate concentration (r = − 0.991, p = 0.001) or biomass pH (r = − 0.975; p = 0.02), while the EC of each organic matrix was also negatively correlated to the ID of the pathogen, but not significantly (r = − 0.827; p = 0.173). Significant positive correlations have been found between the total polyphenols or the quantity of lipids supplied with each soil conditioner and the ID of V. dahliae (r = 0.994, p = 0.006 and r = 0.991, p = 0.009, respectively).

The trend of Phytophthora spp. ID was different from that of V. dahliae. At T0 and T1, no differences were observed among all treatments probably due to the lack of specific host plant to parasitize (the first transplantation occurred three months later) and the low winter temperature that could have affected the surviving rate of the pathogens (Fig. 3). At the end of the first and second crop cycle (T3 and T7), the effects of biomasses on the ID of Phytophthora spp. were evident since the control showed a significantly higher ID than the plots treated with biomasses. No significant correlation was observed between the growth of Phytophthora spp. and each single chemical parameter (r = 0.461, p = 0.539; r = 0.473, p = 0.527; r = − 0.384, p = 0.523; r = − 0.435, p = 0.565; r = − 0.278, p = 0.722 for pH, EC, nitrate, polyphenols and lipids, respectively).

Fig. 3

Effect of organic amendments on soil inoculum density of Phytophthora spp. (ppg g⁻¹ dry soil). Bars represent the standard error of the mean (n = 3). Mean values marked with different letters are significantly different according to the Tukey’s HSD test (p < 0.05). See Table 1 for details of the reading points

Discussion

The higher nitrate content recorded at the end of each potato crop cycle is the results of the fertilization of potato and, since all plots received the same amount of inorganic fertilizers, the differences among treatments were possibly due to the quality of each biomass, rather than the quantity applied, that could have played a role in the mineralization and release of the mineral N. In particular, at the end of the experiment, soil nitrate content increased by about 152% after the amendment with BRX, while it increased only by 26% after the amendment with OPR. This result is apparently associated to the C/N ratio of biomasses, since it can influence their mineralization and the development of the beneficial microbial community [27, 29, 30]. BRX and OPR showed C/N ratio of 12.3 and 92.4, respectively [1], and since a positive correlation between N assimilation into microbial biomass and the C/N ratios of soil conditioners exists [47], the former soil conditioner induced the soil microbial community to immobilize less N and increased the rate of nitrification. The faster mineralization of BRX, as indicated by the higher soil nitrate content (Table 2) at the end of each crop cycle (T3 and T7, Table 1) and the negative correlation between V. dahliae ID and soil nitrate concentration (r = − 0.991, p = 0.001), determined a better control of this pathogen. The reduction of V. dahliae ID observed with the application of BRX may be caused by toxic substances, such as ammonia and nitric acid that can disrupt the membranes of microsclerotia and that are released especially when the C/N ratio of the medium is low [27]. In addition, OPR showed higher V. dahliae ID because that biomass was characterized by the lowest values of pH (5.7–6.3) and EC (0.9–1 dS m⁻¹) among the biomasses studied [1], while suppressiveness is related to alkaline pH and high EC value [24].

Other parameters generally responsible to lower pathogens ID are the amount and composition of SCB and SCF, influenced by the nature of amendments. Enwall et al. [48], Pezzolla et al. [49] and Ye et al. [47] reported that the application of organic amendments influences the soil microbial biomass due to the introduction of exogenous microorganisms and available organic compounds. In particular, the quantity applied and the presence of lignocellulosic residues, deriving from the ground olive kernels, can be the reason for the positive effect of OPR on the SCB and SCF populations (Fig. 1). Our results are in accord with another study reporting an increase of bacterial and fungal populations in the soil after the amendment with OPR [50], and a high soil microbial population increases the competition for nutrients among microorganisms and enhances the antibiotic-producing bacteria [51]. Although the OPR application resulted in higher SCB and SCF, it was the worst biomass in controlling the ID of V. dahliae, suggesting that the control of this pathogen was mainly due to the release of mineral N rather than the stimulation of beneficial microorganisms. With regards to Phytophthora spp., Bonanomi et al. [38] demonstrated that the C/N ratio was not indicative of suppressiveness for those soilborne pathogens, and the microbiostasis was the mechanisms against phytopathogens with propagules less than 200 μm diameter as Pythium spp. and Phytophthora spp. In our study, the higher SCB and SCF registered in the amended plots with respect to the control ones (Fig. 1) could be a marker indicating an increase of antagonistic microbial population able to control Phytophthora spp. and leading to similar effect of the different soil conditioners, as reported elsewhere [9, 20].

The content of polyphenols added with the different amendments was higher for OPR with respect to the other soil conditioners, especially with respect to BRX (Table 4). El Abbassi et al. [52] reported many previous studies in which some polyphenols, especially those deriving from cinnamic and benzoic acids from biomasses such as OPR, inhibit different pathogenic fungi, but in our study positive correlation has been found among the ID of V. dahliae and the content of polyphenols added (r = 0.994, p = 0.006). In the open-field conditions, the apparent stimulation of the pathogen can be the result of a fast degradation/mineralization of the in vitro active antifungal polyphenols into new molecules less or no toxic implied in the formation of soil humic substances [35, 53]. In fact, previous studies reported that polyphenols persist temporarily in soils due to the degradation by fungi, bacteria and yeasts [53,54,55,57], and monomers can be adsorbed by the soil colloids, or recombined to form new polyphenols.

In the present study, the biomass that added the higher quantity of lipids was OPR. Its lipidic fraction was mainly constituted by unsaturated FA (about 80%, Table 5), and by a relatively high content of the saturated palmitic acid (about 400 and 350 kg ha⁻¹ added the first and second year, respectively), and that could have promoted the V. dahliae ID. In fact, even if fatty acids are considered capable of inhibiting or disrupting biofilm formation by various microbial pathogen [58] or interfering with fungal sphingolipid biosynthesis [59], several in vitro studies have shown that saturated short chain fatty acids are the most efficient in the suppression of pathogens. Kumar et al. [58] demonstrated that capric acid and undecilic acid, 10 and 11-carbon saturated fatty acids, respectively, killed the pathogen Candida albicans even at low concentrations. Liu et al. [32], in a study conducted in vitro on three phytopathogenic fungi, found an inhibitory effect on spore germination with the addition of caproic, caprylic, or capric acid at very low doses (1 µm L⁻¹), or lauric or palmitic acid at a higher dose and only on some pathogens. In the same study no inhibitory effect was observed with the addition of oleic, linoleic or myristic acid. Undecilic acid, together with the myristic one, were identified as antifungal agents towards a series of pathogens such as Trichoderma, Aspergillus, Candida, Fusarium, Pythium, Rhizoctonia [60]. In contrast, laboratory tests have demonstrated that unsaturated fatty acids stimulated the germination of Pythium ultimum [61], and had a sporogenic effect on Aspergillus spp. [31, 62]. Harman et al. [63] found that palmitic acid stimulated the germination of Fusarium solani together with oleic and linoleic acids. In addition, a previous study [64] has demonstrated that linoleic and oleic acids were substrates utilized by fungi for the production of oxylipins that regulated the fungal development. The significantly higher values of microsclerotia observed in OPR plots can be presumably ascribed to the quality of FA that did not control the pathogen. This behavior was particularly evident at T7, when plots treated with OPR, applied twice during the trial, showed a peak of microsclerotia similar to CON and over seven times higher than BRX. The second soil conditioner in terms of quantity of lipids added was CMW, and its FA were almost equally divided between unsaturated and saturated (Table 4), among the latter ones, the relatively high content of myristic, lauric and caprylic acids could have promoted its good results in controlling V. dahliae.

Conclusions

The application of different biomasses with the same inorganic fertilization affected differently the ID of V. dahliae and Phytophthora spp. in open field. With respect to the former pathogen, the mineral N can be considered the most important parameter for its control, therefore one should select for that aim biomasses characterized by less recalcitrant organic matter and low C/N ratio, since their organic N can mineralize easier.

Soil conditioners characterized by large quantity of unsaturated fatty acids and applied at high rate per hectare can be conducive to V. dahliae and can stimulate the population level of both culturable fungi and bacteria. Soils already infested by V. dahliae or cultivated with crops susceptible to that pathogen should not be amended with this type of biomasses.

The polyphenols content of each soil conditioner apparently does not play an important role in controlling V. dahliae since they should be quickly transformed into no toxic compounds shortly after the amendment.

Phytophthora spp. are always susceptible to the synergistic application of inorganic fertilizers and biomasses regardless of the composition and the quantity of the latter. The reasons could be ascribed to an increase of beneficial microorganisms able to control these pathogens, rather than chemical parameters; however, more studies in open fields are necessary to confirm these conclusions.