Potential of Anaerobic Digestates in Suppressing Soil-Borne Plant Disease

Abstract

The use of anaerobically digested slurries (ADSs) is promising strategies in resource management and agricultural production. ADSs has a potential as alternatives to chemical fertilizer. ADSs from various source materials suppressed the growth of some plant pathogenic fungi in vitro. ADS filtrates did not suppress them, indicating the effect was caused not by water-soluble substances in ADSs. In pot experiment, the efficacy of ADS from dairy cow manure (AD) for Fusarium wilt of spinach was assessed. Applying 8% (w/w) of AD significantly reduced pathogen density in soil and promoted the growth of spinach. Inoculation of a bacterial isolate AD-3 from AD, which showed high suppressiveness against Fusarium spp. in vitro, effectively controlled the disease. Based on the results, AD-3 strain is related to the disease control ability of AD that belonged to Bacillus velezensis. ADSs can supply not only plant nutrients but also antagonistic microbes. For crop production, ADSs application would be effective to infected soil. It was effective for improving ADS handling that ADS was absorbed to foamed glass and dried at 60°C. To apply ADS to farmland for crop production, these findings are promising for sustainable agriculture.

1. Introduction

Fusarium wilt disease, caused by Fusarium oxysporum f. sp. spinaciae (Fos), is a serious soil-borne disease. F. oxysporum has high host specificity and is responsible for severe damage to economically important plants [1]. Chemical fungicides or soil fumigation is commonly used to control the disease. However, it is needed to develop alternatives to these conventional controls for sustainable agriculture. Organic amendments can be used to improve soil quality and manage soil-borne diseases [2]. Among organic amendments, compost has been studied the most commonly. Composting is a well-known method for recycling organic waste, and the final by-product can be used in agriculture [3]. There have been reported that compost has suppressive effects against soil-borne fungal diseases [4, 5]. Anaerobic digestion is another countermeasure for the treatment of agro-industrial waste and the organic parts of source-separated household waste. Anaerobic digestion produces biogas as a source of renewable energy and anaerobically digested slurry (ADS) that can be used as liquid fertilizer [6, 7]. Organic wastes from agriculture and industry, and the organic parts of source-separated household wastes are considered as biomass and the most dominant future renewable energy sources. Organic waste materials could be specific important resources because these sources do not compete with food crops in agricultural land usage. Anaerobic treatment is suitable for their utilization. The treatment is an attractive solution since producing biogas can be used as renewable energy and anaerobically digested slurry (ADS) can be applied to farmland as organic amendment. Scientific reports of anaerobic treatment, especially biogas, have increased rapidly in terms of renewable energy technology [8]. In general, ADS has a high concentration of plant nutrients such as nitrogen (N), phosphorus (P), and potassium (K), which are available in a suitable form for plants to absorb, meaning that ADS can be used as liquid fertilizer. Normally, it needs appropriate handling before discharging to river water because of high content of nutrients such as N, P, and K. This means, on the other hand, it has a potential as alternatives to chemical fertilizer. When considering land application of ADSs, risks and impacts for agricultural production should be concerned as the chemical composition including phytotoxic compounds and heavy metals is different among ADSs, depending on their primarily source materials [9, 10]. Therefore, the chemical composition of ADSs should be accurately assessed before being used as fertilizer. In Europe, there is the application rate of ADS recommending its use based on specific regulations and guidelines [11, 12]. There are some studies reported that ADS is a valuable alternative to fertilizer in agriculture [13, 14]. The self-sufficiency of fertilizer must be increased for sustainable agriculture and food security. The use of ADS has mainly focused on a function as nutrient availability, crop productivity, and reusing organic waste [15, 16, 17]. The role of ADS in plant disease control has been studied. For example, Tao reported that ADS sourced from pig manure suppressed the growth of plant pathogenic fungi in vitro [18], and Amari reported that the application of ADS to soil has been shown to suppress several plant diseases including Ralstonia spp. [19] and Fusarium spp. [19], and Cao founded the suppressive effect on Phytophthora spp. [20]. These reports focused on physicochemical properties as factors for plant disease control. Microorganisms in ADS may also have a suppressive effect. However, the disease control ability of organic amendments and ADS nutrient contents might be varied depending on the source materials. There are few findings indicating disease suppression of ADSs. Furthermore, it still has two difficulties for its handling and storage. For handling, ADS is a liquid, but it contains plant residues or sediments, and it is difficult to spray to farmland like liquid fertilizer; some attachment or special application agricultural machinery is needed. Hence, there are microbes in ADSs, which continuously digest remaining organic materials in ADSs, and methane gas emission has been continuing. Therefore, it is difficult to keep ADS in plastic bottles or plastic bags like chemical fertilizer. This made farmers difficult to use ADSs. In Japan, ADS is generally treated at attached wastewater treatment facility and released into river water. The first objective was to assess how the ADS generated from different source materials is suppressing F. oxysporum f. sp. spinaciae (Fos). We isolated bacteria from ADS and tested the effects of these against Fos in vitro. The second objective was to verify the suppressive effect of a selected bacterial isolate against Fusarium wilt of spinach in vivo. The third objective was to demonstrate the applicability of ADS not only antagonism against Fos but also against another plant pathogens. Furthermore, relatively new and old ADSs were used for the applicability experiment. The fourth objective was to improve the handling of ADS for agricultural use, ADS was absorbed to porous materials, and the antagonism of its material against seven kinds of plant pathogen was investigated. Furthermore, heat dry temperature effect of ADS absorbed material was investigated whether drying process reduces the suppression.

2. Materials and methods

2.1 Sampling and characteristics of ADSs

In this study, five ADSs were generated from different source materials: dairy cow manure (AD), sewage sludge (AS), food garbage (AF), pigmanure + foodgarbage (APF), and sewagesludge + nightsoilsludge + foodgarbage (ASNF). Mesophilic fermentation of anaerobic biological treatment in facilities occurred at $35°\mathrm{C}.$ All digestates were taken from a methane fermentation tank running in continuous mode without dehydrating, and the slurry was used for experiments. After sampling, the slurry was stored in 20-L plastic tanks at $4°\mathrm{C}$ refrigerator. The details of each ADS source materials rates are presented in Table 1.

Sample1	Rate of source materials (%)
Name	Dairy cow manure	Sewage sludge	Food garbage	Pig manure	Night soil sludge	Treatment temperature	HRT (days)	Microbial immobilization method	Pre-Treatment	Facility location
AD	100					35–36	45	Anaerobic fluidized bed process	Solid liquid separation	Tochigi Pref.
AS		100				35–36	30	Anaerobic contact process	Gravitation enrichment	Yamagata Pref.
AF			100			35–36	20	Anaerobic fluidized bed process	Acid fermentation	Hokkaido
APF			10	90		35–36	30	Anaerobic fluidized bed process	Crush	Okayama Pref.
ASNF		74	9		17	35–36	30	Anaerobic fluidized bed process	Acid fermentation	Fukuoka Pref.

Table 1.

Source material composition, processing conditions, and facility location for five anaerobically digested slurries (ADSs).

¹

ADSs generated from different source materials. AD, dairy cow manure; AS, sewage sludge; AF, food garbage; APF, pig manure + food garbage; ASNF, sewage sludge + night soil sludge + food garbage

2.2 Analysis of chemical properties of ADSs

The following parameters were determined in the fresh digestate samples: pH, analyzed by multi-function water quality meter (MM-60R TOADKK Corp.) followed the methods of analysis of feeds and feed additives [21]; dry matter content (DM) after drying the digestate sample at $105°\mathrm{C}$ for 24 h followed the method of Japanese Industrial standards method (JIS) JIS K 0102 14.2 [22]; the volatile solids, which reflect the OM content, by loss on ignition at $500°\mathrm{C}$ for 24 h followed the method of JIS K 0102 14.4 [22]. The total organic carbon (TOC) was analyzed as liquid samples using an automatic analyzer TOC-LCSH system (Shimadzu Corp.) followed the method of JIS K 0102 22 [22], total nitrogen (TN) followed the testing method for fertilizer 4.1.1.b [23], and total carbon (TC) followed the testing method for fertilizer 4.11.1.b [23] were measured in dried samples by NCH-22 (Sumika Chemical Analysis Service, Ltd.). C/N was calculated by the division of carbon by nitrogen. Ammonia and chlorine were analyzed by ion chromatograph LC-20 AD sp., detector CDD-10A, columns; IC-C4 for cation and IC-SA2 for anion (Shimadzu Corp.). After HNO3 digestion, the following elements were determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES, iCAP 6200, Thermo Fisher Scientific) followed the testing method for fertilizer [23]: P, K, S, Na, Ca, Mg, Fe, Cu, Mn, Al, Zn, Pb, Cd, Cr, and Ni. Hg was analyzed by cold-vapor atomic absorption spectrometry followed the testing method for fertilizer 5.12.1 [23]. Lignin and cellulose were analyzed according to acid detergent method [21], and CEL/LIGN was calculated by the division of cellulose by lignin.

2.3 Antifungal activity of raw and filtrate ADSs

The plant pathogen F. oxysporum f. sp. spinaciae (Fos, MAFF: 103059) was used. Fos was cultured on potato-dextrose agar (PDA) (Nihon pharmaceutical Co., Ltd., Tokyo, Japan) in petri dishes (90×15 mm) at $25°\mathrm{C}$ for 14 d and used as inocula. Raw and filtrate ADSs were used for the assay. Filtrate ADSs were prepared as follows: 10 mL of each raw ADS was centrifuged at 3000 rpm for 20 min, and the supernatant was filtered through a 0.2 μm disposable membrane filter (Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The fungal colony was taken as a small colony disk using a 5-mm cork borer, and one side was placed on a PDA plate. Sterilized filter paper disks (5 mm) were placed in 10 mL of each raw and filtered ADS for 10 min to absorb it, and then placed on the other side of the PDA plates. A pathogen-only plate was used as a control. All treatments had five replicates. All plates were placed in incubator for 18 d at $25°\mathrm{C}$ in the dark. After incubation, two plates were randomly selected from each treatment. Photos were taken of the plates, and the digital images were used to measure the diameter of any colonies developed from the mycelial disk, using ImageJ software (version 1.52, NIH, Bethesda, USA). The experiment was repeated twice.

2.4 Isolation of bacteria from five ADSs

The five ADSs were serially diluted with sterilized water, and the dilutions were spread onto nutrient agar (NA, Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). After incubation for 2 to 4 d at $25°\mathrm{C}$ in the dark, bacterial colonies that appeared on the plates were transferred on NA plates, and single colony isolates were obtained. These isolates were preserved in solution (10 g of skim milk and 1.5 g sodium L-glutamate monohydrate⁻¹ 100 mL distilled water) at $‐20°\mathrm{C}$ until use.

2.5 Antifungal activity of bacterial isolates from ADSs

Bacterial isolates from the five ADSs described in section above were used for confrontation assays. Small bacterial colony disks were removed from cultures on NA plates after 24–48 h by 5-mm cork borer and were placed on one side on PDA plates. Fos fungal colonies were also taken as a small colony disk in the same manner. A pathogen-only plate was used as a control. All treatments had five replicates. All plates were placed in incubator for 18 d at $25°\mathrm{C}$ in the dark. After incubation, two plates were randomly selected from each treatment. Photographs were taken, and the digital images were used to measure the diameter of mycelial colonies developed from a mycelial disk, using ImageJ software (version 1.52, NIH, Bethesda, MD, USA).

2.6 Pot experiment

2.6.1 Preparation of Fos inoculum

Fos inoculum was prepared on potato-sucrose broth (200 g potato and 20 g sucrose $L^{-1}$ distilled water) at $25°\mathrm{C}$ by shaking the culture at 110 rpm for 5 d. The resulting spore suspension was filtered through double gauze to remove the hyphae and was then transferred into sterile 50-mL plastic tubes, centrifuged at 2000 rpm for 3 min, and then, the supernatants were discarded. The Fos inoculum was prepared with distilled water and adjusted to $1.0\times 10^6\text{to}1.0\times 10^7\mathrm{bud}‐\text{cells}{\mathrm{mL}}^{-1}$ using a hemocytometer (AS ONE Corp., Osaka, Japan).

2.6.2 Preparation of AD-3

As mentioned above, ADS had several effective bacteria against Fos among ADSs. Of the several bacteria in AD, AD-3, which suppressed Fos growth the most, was used for pot experiments. AD-3 cells were grown on NA plates for 24 h, and the culture was grown on nutrient broth media (5 g meat extract, 5 g NaCl, and 10 g peptone $L^{-1}$ distilled water) at $25°\mathrm{C}$ on a constant rotary shaker at 100 rpm for 48 h. The suspension was transferred in sterile 50-mL plastic tubes, centrifuged at 3000 rpm for 10 min, and the supernatants were discarded (repeated twice). The AD-3 cell pellet was dissolved in sterilized distilled water. The AD-3 suspension was adjusted to an optical density of 1.0 at 600 nm using a spectrophotometer (Thermo Electron Corp., Waltham, MA, USA).

2.6.3 Pot assay of AD-3

The experiment was performed in pots (10.5×9 cm) containing the equivalent of 200 g of dried black loam soil. The soil was sieved through 2 mm mesh, and soil pH was adjusted to within the range 6.9 to 7.1 using hydrate lime. The amount of hydrate lime was calculated according to the Arrhenius equation. The final rates of N, ${\mathrm{P}}_2{\mathrm{O}}_5,$ and ${\mathrm{K}}_2\mathrm{O}$ were adjusted to $80\mathrm{kg}{\mathrm{ha}}^{-1}$ using ammonium sulfate, fused phosphate, and potassium chloride. Treatments of the pot experiment were as follows: F, only Fos-inoculated soil; F + AD – 3, soil amended with Fos and AD-3; and unamended, non-pathogen control. To prepare Fos-infected soil, the bud-cell suspension of Fos was inoculated into the soil to give a final $\text{concentration\hspace{0.17em}of}1.0\times 10^5\mathrm{bud}‐\text{cells}{\mathrm{g}}^{-1}$ dry soil, and pots were incubated for 5 d at $25°\mathrm{C}$ in the dark. AD-3 suspension was added to the infected soil to give a final concentration of $1.0\times 10^6{\mathit{\text{CFUg}}}^{-1}$ dry soil. All pots were incubated for 10 d at $25°\mathrm{C}$ in the dark. During the incubation, soil moisture was maintained at 60% water-holding capacity (WHC) by spraying with distilled water. As for spinach cultivar, “Okame” (Spinacia oleracea L.; Takii Seed, Kyoto, Japan) with high susceptibility to Fos [24] was used. After incubation, spinach seeds were sown in each pot and the plants were grown in an incubator (day/night: $25/22°\mathrm{C},$ 12/12 h). All pots were irrigated daily to keep the soil moisture at 60% WHC. A total of 60 pots were prepared; 30 pots (15 pots of F and 15 pots of F + AD – 3) were used to estimate the density of Fos in the soil. Soil was sampled from three pots that were randomly selected per treatment at 0, 7, 14, 21, and 28 d after sowing, and the density of Fos was measured using Komada selective medium [25]. The other 30 pots (10 pots per treatment) were used to evaluate disease severity at 28 d after sowing. The disease symptoms were categorized using a disease index of 0, no infection; 1, 1–30% of leaves wilted; 2, 31–60% of leaves wilted; 3, 61–90% of leaves wilted; 4, more than 90% of leaves wilted or dead. Disease severity (DS) was calculated according to the following formula:

$$\mathit{DS}=\sum \mathit{nd}100/4T$$E1

where n = number of plants in each disease index, d = disease index, and T = total number of plants by treatments. The inhibitory effect (IE) of AD-3, as a percentage reduction in disease severity, was calculated according to the following formula:

$$\mathit{IE}\left(\%\right)=\left(100-\mathit{DSF}+\mathit{AD}-3\right)/\mathit{\text{DSFx}}100$$E2

where DSF + AD – 3 = disease severity of F + AD – 3, DSF = disease severity of F. The pot experiments were repeated twice (Experiment 1, Exp 1; Experiment 2, Exp 2). In total, disease severity for 27 plants (T = 9) in Exp 1 and 21 plants (T = 7) in Exp 2 was investigated. R v. 3.5.3 software was used for statistical analyses. The antagonistic activity of ADSs and bacterial isolates against Fos in vitro were analyzed with Tukey’s HSD test following one-way analysis of variance (ANOVA). The effect of inoculation of AD-3 on Fos density was analyzed using a t-test. The data for disease severity were arcsine transformed in advance, and Tukey’s HSD test following one-way analysis of variance (ANOVA) was performed to reveal the efficacy of AD-3 against Fusarium wilt of spinach (p<0.05).

2.7 Genome sequencing

DNA extraction from cells was performed using the Promega Maxwell RSC PureFood GMO Kit (Promega Corporation, Madison, WI, USA). Genome sequencing was performed with the GridION X5 (Oxford Nanopore Technologies, Oxford, UK) followed by preparation of the genome library using a Rapid Barcoding Sequencing Kit (SQK-RBK004) (Oxford Nanopore Technologies, Oxford, UK). Read sequences were assembled using Unicycler (version0.4.8) [26].

2.8 Genome analysis

A genome sequence was annotated using DFAST (version 1.1.6, https://dfast.ddbj.nig.ac.jp/) [27]. AntiSMASH (version 5.0, https://antismash.secondarymetabolites.org/) [28] was used for the prediction of secondary metabolite gene clusters. A 16S rDNA sequence on the genome was analyzed to identify the strain using EzBioCloud (https://www.ezbiocloud.net/) [29]. Phylogenetic relationships were analyzed on the basis of 16S rDNA sequences using MEGA x (version 10.2.6) [30].

2.9 Pot experiment of AD

The pot experiment was conducted in pots (10.5 × 9 cm), each containing 200 g of dried black loam soil under incubator conditions at $25°\mathrm{C}.$ The bud-cell suspension of Fos (1.0 × 10⁵ bud-cells/g dry soil) was inoculated into black loam soil, and 8% (w/w) of AD was applied to non-infested and Fos-infested soil 10 days before sowing the spinach seeds. The pot experiment had four treatments, including the control: (1) Fos, pathogen-only control; (2) Fos + AD, soil amended with Fos and 8% (w/w) of AD; (3) AD, soil amended with 8% (w/w) of AD; (4) Ctrl, control with no amendments. Each treatment was replicated for 10 plants. All treatments of N, P₂O₅, and K₂O were adjusted as the equivalent of 80 kg ha⁻¹, and recommended fertilizer application rate. For AD-amended treatment, 8% (w/w) of AD was applied not to exceed the rate.

2.10 ADS applicable experiment against seven plant pathogens

The plant pathogens as shown in Table 2 were used for testing the ADS applicability. Plant pathogen was cultured on potato dextrose agar (PDA) (Nihon pharmaceutical co., Ltd., Tokyo, Japan) in petri dishes (90×15 mm) at suitable temperature for each plant pathogen as shown in Table 2 and used as inocula. Sterilized filter paper disks (7 mm) were placed in 10 mL of ADS for 10 min to absorb it and then placed on the other side of the PDA plates. A pathogen-only plate was used as a control. All treatments had five replicates. All plates were incubated for suitable days as shown in Table 2 in the dark. After incubation, three plates were randomly selected from each plant pathogen. Photos were taken of the plates, and clear inhibition zone were judged by eyes. Two FW ADSs where its facility is in Kanagawa Prefecture were used to examine the applicability of fresh and old ADS, one is 3 month past after the 30 days HRT and sampling, and the other is 5 month past after the 30 days HRT and sampling. Mesophilic fermentation of anaerobic biological treatment in facility occurred at $35°\mathrm{C},$ and pre-treatment was crush method. After sampling, the slurry was stored in 20-L plastic tanks at $4°\mathrm{C}.$

	Plant pathogen name	MAFF No.	Incubation temperature $\left(°\mathrm{C}\right)$	Incubation period (days)
1	Pyrenochaeta lycopersici	238,906	25	75
2	Setophoma terrestris	243,290	25	90
3	Phialophora gregata f. sp. Adzukicola	241,056	20	150
4	Helicobasidium mompa Nobuj. Tanaka	305,915	20	100
5	Pyricularia oryzae	101,418	25	120
6	Fusarium graminearum	240,353	20	30
7	F. oxysporum Schlechtendahl f. sp. fragariae Winks et Y.N. Williams	744,009	25	14

Table 2.

Tested plant pathogen list for applicability experiment.

2.11 Supporting material effect on the antifungal activity of ADS absorbed material

To improve ADS handling, ADS was absorbed to four industrial waste materials each; wood ash, steel-making slag, activated carbon, foamed glass diameter 3 to 5 mm, and soaked to ADS for 1 hour and the mixed ratio was as shown in Table 3. As a result of 4.1. newer ADS was used for the following experiments. The absorbed material was weighed as dried ADS equivalent to 23 mg each and put on one side of PDA media, and then, the fungal colony was removed as a small colony disk using a 7-mm cork borer and was placed on the other side of the PDA plate. A pathogen-only plate was used as a control. Tested plant pathogens was F. oxysporum Schlechtendahl f. sp. fragariae Winks et Y.N. Williams. All treatments had five replicates. Plant pathogen plates were incubated for 14 days $25°\mathrm{C}$ in the dark. After the incubation period, three plates were randomly selected from each. Photos were taken of the plates and the inhibitory effect was judged by eyes.

Mixed ratio %(w/w)	Carrier material name
	Wood ash	Steel-making slag	Activated carbon	Foamed glasses
Carrier material ratio	80	92.5	45	75
ADS ratio	20	7.5	55	25

Table 3.

Mixed percentage of carrier material and ADS.

2.12 Dry heat temperature effect on the antifungal activity of ADS adsorbed material

Adsorbed AF-ADS materials were weighed 2 g on aluminum foil and dried at 60, 80, 100, and $150°\mathrm{C}$ in dry heat oven for 3 h each. After cooled to room temperature in a desiccator dried materials and non-heated material were weighed as dried AF-ADS equivalent to 23 mg each and put on one side of PDA media, and then, the fungal colony was removed as a small colony disk using a 7-mm cork borer and was placed on the other side of the PDA plate. F. oxysporum Schlechtendahl f. sp. fragariae Winks et Y.N. Williams was selected as inocula since its mycelia growth rate is high. As a control a pathogen-only plate was used. All treatments had five replicates. All plates were incubated for 14 d at $25°\mathrm{C}$ in the dark. After incubation, three plates were randomly selected from each treatment. Photos were taken of the plates, and the inhibitory effect was judged by eyes.

2.13 Bacteria and archaebacteria flora analysis for succession confirmation

In order to support the result of mentioned above, bacteria and archaebacteria flora were analyzed to determine whether ADS was adsorbed to the carrier material. The ADS absorbed foamed glass material was produced in the same manner as mixed ratio in Table 3 and dried at $60°\mathrm{C}$ for 1 h. AF-ADS and the AF-ADS absorbed foamed glass, and foamed glass were analyzed by a next-generation sequencer (Qiime) targeting 16S rRNA.

2.14 Applicability experiment of antifungal activity of AF-ADS absorbed material

AF-ADS was absorbed to two industrial waste materials: wood ash and foamed glasses diameter 3 to 5 mm for 1 h and mixed as shown in Table 3, and dried at $60°\mathrm{C}$ for 1 h in dry heat oven. After cooled to room temperature in a desiccator, dried materials and non-heated material were weighed as dried ADS equivalent to 23 mg each and put on one side of PDA media, and then, the fungal colony was removed as a small colony disk using a 7-mm cork borer and was placed on the other side of the PDA plate. A pathogen-only plate was used as a control. Tested plant pathogens are shown in Table 2. All treatments had five replicates. Each plant pathogen plates were incubated for suitable days as shown in Table 2 in the dark. After incubation, three plates were randomly selected from each plant pathogen. Photos were taken of the plates and the digital images were used to measure the diameter of any colonies developed from the mycelial disk, using Image J software.

3. Results

3.1 Chemical properties of ADSs

Application of organic amendments to the soil would improve their physical, chemical, and biological properties. The variability of these properties depends on characteristics of ADSs, reflecting the initial biomass inputs. The chemical properties of ADSs used in this study are showed in Table 4. Carbon and nitrogen are the important components (Michalzik et al. [51]; Jenkinson et al. [52]) and their relative ratio will affect agronomic use of amendments (Havlin et al. [53]). C/N ratio of slurries was found to be variable between 5 and 19. AD which feedstock was mainly dairy cow manure showed highest C/N ratio because dairy cows are ruminants, and its manure contains low ratio of easily degradable carbon and relatively higher ratio of persistent carbon. ASNF, APF, and AF showed greater mineral nitrogen fractions (43–52% total N to the total N fraction), suggesting that those could be used as fertilizer (Tambone et al. [12]; Paavola and Rintala [54]). In contrast, AD, AS, and AFPS had displayed lower mineral fraction (27, 35, and 36% each to the total N), and especially AD showed the lowest caused by lower degradability of dairy manure digestate. This indicated these ADSs have a high potential of stability as organic amendment (Nkoa [55]). The P and K are also essential nutrients as fertilizer. AS contained a higher amount of total-P that could be used as P fertilizer. Total-K was highly contained in AD, while lower in AS, ASNF, and AFPS. For biochemical fractionation, cellulose/lignin ratio indicates the degree of humification of the organic materials (Nkoa [55]), and a value of 0.5 has been suggested as boundary value between fresh and mature wastes (Komlis and Ham [56]). According to the value, APF and AFPS were comparably fresh digestates. Na that is contained in food garbage compost may affect plant growth (Hargreaves et al., [57]). The value of salt contained in food waste could be up to 8% (DM) for composting and the compost can be applied to soil 10 t ha⁻¹y⁻¹ without phytotoxic effects (Jin et al. [49]). Applying this value to digestates, upper limit for soil application can be calculated 80 g m⁻², which means digestate can contain 6240 mg L⁻¹ Na as upper limit concentration for 50 t ha⁻¹ digestate application. Digestates ASNF, AF, APF, and AFPS containing food garbage as its substrates had sufficiently lower than the calculated value. Considering the land application of amendments derived from organic wastes, the risk of soil contamination by phytotoxic compounds (Boydston et al. [50]; Gough and Carlstorm [58]) and heavy metals (Alburquerque et al. [9]; Wong et al. [59]) should be concerned. A value of pH is an indicator to determine harmless of ADSs, since it controls the behavior of heavy metals (Kapanen and Itavaara [60]). All the ADSs showed a safety value from 7.4 to 7.9. Composition of heavy metals in ADSs showed quite low heavy metal contents comparing with upper limit based on Japanese Fertilizers Regulation Act and recommended limit of Japanese Central Union of Agricultural Co-operatives. Although Mn is an essential component for plant growth, excess Mn in the soil would interfere with activities of the other mineral components such as Ca, Mg, Fe, and P (Clark [61]). The concentration of Mn in digestates can as high as 50–55 ppm (Sahm [44]), and all the ADSs contained sufficiently lower value of Mn. Besides heavy metals, micronutrients such as Cu and Zn also have risks for agricultural soil once in excess in the soil. Both nutrients are generally used as additives in livestock feeds to promote livestock growth and prevent the livestock disease (Nicholson et al. [62]; Alburquerque et al. [9]). The concentration of Cu and Zn in AD, APF, and AFPS was lower, and thereby, these ADSs could be used for land application.

Parameter	AD	AS	AF	APF	ASNF	Value range
DM (%)	2.6	2.1	1.8	1.2	1.8	1.5	—	45.7 [31, 32, 33, 34, 35]
Organic matter (% DM)	37	62	33	7	51	36	—	75.4 [33, 34, 36, 37]
Total N (kg Mg^−1 DM)	11	15	19	14	16	31	—	140 [36, 37, 38]
Total N (kg Mg^−1 FM)	2.9	3.1	3.5	1.8	2.9	1.2	—	15 [39]
NH4-N (% FM)	0.08	0.11	0.15	0.09	0.13	1.5	—	6.8 [31, 32]
NH4-N (% DM)	0.03	0.05	0.08	0.07	0.07			—
NH4+ share on total N (%)	27	35	43	52	45	35	—	81 [35, 37]
TC (% DM)	21.2	21.4	8.8	17.2	15.9	36	—	45 [37]
C/N	19	15	5	12	10	2	—	24 [35, 36, 38]
TOC	1300	400	620	220	530			—
Soluble C/N	1.7	0.4	0.4	0.2	0.4	0.55	—	6 [40]
Total P (% DM)	1.5	3.7	1.4	1.2	2	0.6	—	1.7 [33, 34, 37]
Total P (kg Mg^−1 FM)	0.4	0.8	0.2	0.2	0.4	0.4	—	2.6 [41]
Total K (% DM)	1.8	0.4	2.7	1.5	1.8	1.9	—	4.3 [39, 41]
Total K (kg Mg^−1 FM)	0.5	0.1	0.5	0.2	0.3	0.4	—	11.5 [41]
Total Ca (kg Mg^−1 FM)	0.8	0.4	0.7	0.1	0.4	1	—	2.3 [37, 41]
Total Mg (kg Mg^−1 FM)	0.3	0.1	0.1	0.1	0.1	0.3	—	0.7 [37]
Total S (kg Mg^−1 FM)	0.1	0.3	0.2	0	0.2	0.2	—	0.6 [42, 43]
Si (kg Mg^−1 FM)	1.7	1.5	0.63	0.16	0.75			—
Mn (kg Mg^−1 FM)	6.7	6.8	2.1	0.6	3.1	50	—	55 [44]
Fe (kg Mg^−1 FM)	39	410	140	9	240			—
Na (kg Mg^−1 FM)	280	85	1200	120	600			—
Cl (kg Mg^−1 FM)	760	77	1900	220	930			—
Zn (kg Mg^−1 DM)	54	71	11	16	56			<1800 [45, 46]
As (kg Mg^−1 DM)	0.1	0.8	0.9	0.1	0.9			<50 [45, 46]
Cd (kg Mg^−1 DM)	ND	ND	ND	ND	ND			<5 [45, 46]
Ni (kg Mg^−1 DM)	0.4	1.4	0.6	ND	1.1			<300 [45, 46]
Cr (kg Mg^−1 DM)	ND	ND	ND	ND	ND			<500 [45, 46]
Hg (kg Mg^−1 DM)	0	0	0	ND	0			<2 [45, 46]
Pb (kg Mg^−1 DM)	ND	1.4	ND	ND	0.6			<100 [45, 46]
Cu (kg Mg^−1 DM)	3.8	20.5	2.2	3.2	11.1			<600 [45, 46]
Al (kg Mg^−1 FM)	16	180	10	3	120			—
CEL/LIGN	0.44	0.51	0.36	0.82	0.38	0.22	—	1.71 [43, 44, 47, 48, 49]
pH	7.6	7.4	7.9	7.8	7.8	7.3	—	9 [36, 38, 50]

Table 4.

Chemical properties of ADSs.

Source: ADSs generated from different source materials. AD, dairy cow manure; AS, sewage sludge; AF, food garbage; APF, pig manure + food garbage; ASNF, sewage sludge + night soil sludge + food garbage.

3.2 Antagonistic activities of raw and filtrate ADSs in vitro

The five raw ADSs (AD, AF, AS, APF, and ASNF) significantly suppressed mycelial growth when compared with the control (Table 5). Of these, AD, AS, AF, and ASNF produced a clear inhibition zone. In contrast to the raw ADSs, the filtrate ADSs did not suppress mycelial growth (Table 5).

Sample*	Mycelial growth (mm)**
	Raw		Filtrate
AD	56.1	b	85.6	NS
AS	55.9	b	85.2
AF	60.8	b	85.5
APF	57.6	b	86.3
ASNF	53.5	b	86.4
Control	82.9	a	83.3

Table 5.

Antifungal activity of raw and filtrate anaerobically digested slurries (ADSs) against Fos, indicated as mycelial growth by co-culture test.

*

ADSs generated from different source materials. AD, dairy cow manure; AS, sewage sludge; AF, food garbage; APF, pig manure + food garbage; ASNF, sewage sludge + night soil sludge + food garbage.

**

The same letter indicates no significant difference based on Tukey’s HSD (p < 0.05; n = 4). NS indicates not significant.

3.3 Antagonistic activities of raw and filtrate ADSs in vitro

Overall, 32 strains were isolated from the five ADSs. In descending order, nine isolates were obtained from AD, and named AD-3, AD-6, and AD-8 significantly suppressed the growth of Fos (Figure 1). Seven isolates were obtained from AF, and named AF-1, AF-3, and AF-5 significantly suppressed the growth of Fos (Figure 1). Six isolates each were obtained from APF and ASNF, and named APF-1 and ASNF-4 significantly suppressed the growth of Fos (Figure 1). Although four isolates were obtained from AS, no isolates suppressed the growth of Fos (Figure 1).

Figure 1.

Antifungal activity of bacterial isolates from five anaerobically digested slurries (ADSs) against F. oxysporum f. sp. spinaciae (Fos) indicated as mycelial growth (bars ± SD) by co-culture test. AD, dairy cow manure; AS, sewage sludge; AF, food garbage; APF, pig manure+food garbage; ASNF, sewage sludge + night soil sludge + food garbage. SD, standard deviation of the mean. Bars with the same letter are not significantly different based on Tukey’s HSD test (P<0.05; n=3).

3.4 Effects of AD-3 on Fusarium wilt of spinach

The pot experiments were repeated twice (Exp 1 and Exp 2) and the results were similar. Disease severity with F + AD-3 was significantly (P < 0.05) lower than that with F (Table 6). The inhibitory effect of F + AD-3, as a relative reduction percentage in disease severity compared with that of F, was 64.3% (Exp 1) and 44.1% (Exp 2). As for Fos density in soil, there were no significant differences between F and F + AD-3 in either experiment (Figure 2). Fos density during cultivation was in the range of 4.3–4.8 ${\mathit{\text{CFUg}}}^{-1}$ dry soil (Exp 1) and 3.9–4.4 $\mathrm{CFU}{g}^{-1}$ dry soil (Exp 2).

Treatmenta	Relative plant growthb
Fos	0.76 ± 0.24
Fos + AD	0.99 ± 0.40
AD	1.23 ± 0.36
Ctrl	1.00 ± 0.25

Table 6.

Effects of application of AD on spinach growth in plant dry weight. Values are expressed as means ± SD (n = 10).

^a

Fos, inoculation of Fos alone; Fos + AD, soil amended with Fos and 8% (w/w) of AD; AD, soil amended with 8% (w/w) of AD; Ctrl: control.

^b

Relative plant growth was calculated based on plant dry weight of control treated as 1.

Figure 2.

Density of F. oxysporum f. sp. spinaciae (Fos) (plots ± SD) in soil after seedling emergence. SD, standard deviation of the means. Each plot represents the average of three replicates (pots). The experiment was repeated twice (Exp 1 and Exp 2).

3.5 Identification of AD-3

The morphological analysis showed that the strain AD-3 was a Gram-positive, rod-shaped bacterium. Based on the 16S rRNA gene sequence analysis, the closest species to strain AD-3 was B. velezensis, showing 99.8% similarity. Additionally, phylogenetic analysis of the 16S rDNA indicated that strain AD-3 was positioned in the same group as other B. velezensis strains (Figure 3).

Figure 3.

Phylogenetic tree based on 16S rDNA gene sequences of AD-3 strain and related species using neighbor-joining analysis. Bacillus licheniformis NBRC12200 served as an outgroup. Scale bar refers to a phylogenetic distance of 0.002 nucleotide substitutions per site. Bootstrap values were obtained based on 1000 replications.

3.6 Efficacy of AD on Fusarium wilt of spinach

Application of organic amendments contributes to reduce the addition of chemical fertilizer and improve soil productivity and crop performance. Additionally, that has been found to cause controlling soil-borne diseases [63]. In this study, the effects of AD on Fusarium wilt disease incidence and plant growth were assessed. At the end of the bioassay, the plant dry weight of Fos + AD was 1.3 times higher than that of Fos treatment. Furthermore, AD-treated non-infested soil also showed 1.2 times better plant growth compared to the control (Table 6). Thus, application of AD showed the effectiveness of plant growth in both the Fos-infested soil and the non-infested soil. AD application showed significantly positive effect on spinach growth (Table 7). The factors of suppressive effect of AD and the existence of the strain AD-3 in AD might be related. Even N, ${\mathrm{P}}_2{\mathrm{O}}_5,{\mathrm{K}}_2\mathrm{O}$ levels were same to the other treatment. However, the number of AD-3 derived from 8% (w/w) of AD was estimated to be less than $1.0\times {10}^4$ CFU/g dry soil. In addition, AD contained some bacterial candidates suppressing Fos, as shown in Figure 1. The application of antagonistic microbes with organic amendments enhanced the disease control ability more efficiently than did the use of a single strain alone [64, 65]. Combining the results, we concluded the efficacy of application of AD for Fusarium wilt disease control was not only by a single microbe but also by the microbial community in AD.

Factor	Df	Sum Sq	Mean Sq	F value	Pr(>F)
Fos	1	0.559	0.5593	4.914	0.033	*
AD	1	0.522	0.5221	4.587	0.039	*
Fos x AD	1	0	0	0	0.996
Residuals	36	4.097	0.1138

Table 7.

Application effects of AD and Fos on spinach growth in plant dry weight * significantly different two-way analysis of variance (P<0.05).

*

Signif. code: 0.05.

3.7 ADS applicable experiment against seven plant pathogens

The relatively new and old AF ADSs significantly suppressed mycelial growth when compared with the control (Figure 4). Both AF-ADSs produced a clear inhibition zone against all tested plant pathogens. ADS suppressive effect had kept for at least 7 months.

Figure 4.

Antifungal activity of relatively new and old ADSs against seven plant pathogens as mycelial growth by co-culture test.

3.8 Effect of carrier material to antifungal activity of ADS adsorbed materials

Figure 5 shows that carrier material wood ash and foamed glass produced clear zone showing antifungal activity. Therefore, these two materials were used as carrier material.

Figure 5.

Effect of carrier material to antifungal activity of ADS adsorbed materials against F.oxysporum Schlechtendahl f. sp. fragariae winks et Y.N. Williams.

3.9 Dry heat temperature effect on the plant pathogen suppression of ADS adsorbed material

Comparing to control non-heated, 60, 80, and $100°\mathrm{C}$ produced a clear inhibition zone. In contrast, dry heat temperature $150°\mathrm{C}$ did not suppress mycelial growth (Figure 6). Dry heat temperature until $100°\mathrm{C}$ did not reduce absorbed ADS material suppressive effect. Effective microbes could survive until $100°\mathrm{C}$. From the view of energy cost and plant pathogen suppression, $60°\mathrm{C}$ was reasonable and effective.

Figure 6.

Dry heat temperature effect on the plant pathogen suppression of ADS adsorbed material.

3.10 Bacteria and archaebacteria flora analysis for succession confirmation

Figure 7 shows the results of bacterial flora analysis using a next-generation sequencer (Qiime) targeting 16S rRNA. As shown in Figure 7, when the bacterial flora of AF-ADS and the ADS absorbed foamed glass were compared, the AF-ADS absorbed foamed glass was found to inherit most of the flora of the AF-ADS. On the other hand, foamed glass bacterial flora was different from that of AF-ADS and AF-ADS absorbed foamed glass. It was clarified that the bacteria contained in the AF-ADS were adsorbed to the carrier material almost as they were.

Figure 7.

Bacteria and archaebacteria flora analysis for succession confirmation of AF-ADS absorbed foamed glass.

3.11 Antifungal activity of ADS adsorbed materials against seven kinds of plant pathogen, indicated as relative mycelial

The two ADS absorbed materials significantly suppressed mycelial growth when compared with the control (Table 8). Both absorbed ADSs produced a clear inhibition zone against all tested plant pathogens. However, there was the physical condition difference. Wood ash could not keep the foam and be muddy after mixed with ADS. On the other hand, foamed glasses could keep the structure and easy to handle.

	Treatment						IE2
	F		F + AD-3		Untreated
Exp 11	77.8	a	27.8	b	0	c	64.3
Exp 2	95.8	a	53.6	b	0	c	44.1

Table 8.

Disease severity (DS) of Fusarium wilt of spinach and inhibitory effect (IE) 28 d after sowling.

¹

The pot experiment was repeated twice (Exp 1 and Exp 2).

²

Values followed by the same letter with in a row are not significantly different according to Tukey’s HSD test P < 0.05; n = 9 (Exp 1) and n = 7 (Exp 2).

4. Discussion

The effect of the strain AD-3, isolated from AD, on Fusarium wilt disease incidence was assessed. In this study, approximately $1.0\mathrm{x}10^4\mathrm{CFU}{g}^{-1}$ dry soil was made and inoculated with AD-3 to achieve a concentration of $1.0\mathrm{x}10^6\mathrm{CFU}{g}^{-1}$ dry soil. The pathogen density of the infected soil was mostly consistent with previous studies examining the effect of antagonistic bacteria against Fusarium diseases [66, 67]. AD-3 inoculation into Fos-infected soil effectively reduced disease severity (by 64.3 and 44.1% in the two experiments). Thus, strain AD-3 effectively suppressed Fusarium wilt of spinach. Strain AD-3 belongs to the B. velezensis group. Recently, B. velezensis was reclassified as a synonym of several species including Bacillus amyloliquefaciens subsp.plantarum, Bacillus methylotrophicus, and Bacillus oryzicola [68, 69]. B. velezensis has been frequently isolated from soil [70, 71], rivers [72], and fermented food [73], and is accepted as a safe biological resource [73]. In this study, AD-3 was isolated from ADS sourced from dairy manure. Many strains of B. velezensis showed biocontrol effects against plant pathogens and have been applied for controlling common diseases of tomato, cucumber, lettuce, and wheat [74, 75, 76, 77]. Our results revealed that AD-3 had the ability to control spinach Fusarium wilt. B. velezensis FZB42, which is close to the strain AD-3, which has gene clusters associated with the synthesis of secondary metabolites according to antimicrobial activity [69, 78, 79]. The strain AD-3 had a gene cluster related to surfactin synthesis. This result corresponds with those of Palazzini et al. [80]. Surfactin is an important lipopeptide in the suppression of plant disease [81]. Yokota et al. reported that although lipopeptides produced from B. subtilis suppressed Fusarium yellows, slight pathogen density reduction was shown [82]. We obtained similar results; inoculation with AD-3 significantly suppressed Fusarium wilt of spinach though a reduction in pathogen density was not observed. Five different source ADSs suppressed the growth of Fos in vitro. There are several reports about suppressive factors that affect plant pathogens. For example, Amari et al. reported that confrontation assay in vitro showed that ammonia and acetic acid in the slurry were the main factors influencing disease suppression [19]. Tao et al. reported that the supernatant of centrifuged ADS had a lower suppressive effect than raw ADS [18] and our results support this finding: Filtrate ADSs did not suppress Fos growth in vitro (Tables 2 and 9). Several antagonistic bacteria were isolated from ADSs (AD, AF, APF, and ASNF), except AS. Based on the results, the bacteria presence is important for the inhibitory effect of ADSs. The application of antagonistic microbes with organic amendment could provide more effective plant disease control than the use of a single strain alone [64, 65]. ADS showed its applicability of antagonism effects not only Fos but also the other tested seven plant pathogens and showed positive effect on plant growth. To utilize ADSs in crop production, further study is needed to assess the effects and dynamics of AD-3 when AD is applied to infected soil. To improve the ADS handling, ADS absorbed to foamed glass as porous material was the most effective among tested support materials: wood ash, steel-making slag, activated carbon, and foamed glass. ADS absorbed foamed glass was found to inherit most of the flora of the ADS. After foamed glass soaked into ADS for 1 h, and dried at $60°\mathrm{C}$ for 1 h, the material maintained its antifungal activity. To apply ADS to farmland for crop production, these findings are promising for sustainable agriculture.

Plant pathogen name	Wood ash		Foamed glasses
P. lycopersici	41	b	49	b
S.terrestris	56	b	65	b
P. gregata f. sp. adzukicola	18	a	11	a
H. mompa Nobuj. Tanaka	59	b	74	c
P. oryzae	23	a	16	a
F. graminearum	82	c	84	c
F. oxysporum Schlechtendahl f. sp. fragariae Winks et Y.N. Williams	57	b	76	c

Table 9.

Antifungal activity of anaerobically digested slurries (ADSs) against plant pathogens, indicated as relative mycelial growth to pathogen growth only by co-culture test.

5. Conclusions

The use of ADS has mainly focused on a function as nutrient availability, crop productivity, and reusing organic waste. Thus, far reports focused on physicochemical properties as factors for plant disease control. We investigated that microorganisms in ADS also have a suppressive effect. The use of ADS for plant disease control has not been well studied compared with other types of organic amendments such as compost. Filtrate ADSs did not suppress Fos growth in vitro. Several antagonistic bacteria were isolated from ADSs (AD, AF, APF, and ASNF), except AS. Based on the results, the presence of bacteria is important for the inhibitory effect of ADSs. ADSs can supply not only plant nutrients but also antagonistic microbes. For crop production, ADSs application would be effective to infected soil. It was effective for improving ADS handling that ADS was absorbed to foamed glass and dried at $60°\mathrm{C}$. To apply ADS to farmland for crop production, these findings are promising for sustainable agriculture.

Acknowledgments

I am grateful to Tomomi Sugiyama for collaboration of this work. Funding: This research was supported by grants from Japan Association for Livestock New Technology.

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Conflict of interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.