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Article

The Effect of Biofumigation on the Microbiome Composition in Replanted Soil in a Fruit Tree Nursery

by
Robert Wieczorek
1,*,
Zofia Zydlik
1,
Agnieszka Wolna-Maruwka
2,
Alicja Niewiadomska
2 and
Dariusz Kayzer
3
1
Department of Ornamental Plants, Dendrology and Pomology, Poznan University of Life Sciences, Dąbrowskiego 159, 60-594 Poznan, Poland
2
Department of Soil Science and Microbiology, Poznań University of Life Sciences, ul Szydłowska 50, 60-656 Poznan, Poland
3
Department of Mathematical and Statistical Methods, Wojska Polskiego 28, 60-637 Poznan, Poland
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(10), 2507; https://doi.org/10.3390/agronomy13102507
Submission received: 5 September 2023 / Revised: 21 September 2023 / Accepted: 27 September 2023 / Published: 28 September 2023
(This article belongs to the Special Issue Plant Production and Microorganism Potential in Modern Agro-Ecosystems-2nd Edition)
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Abstract

:
The imbalance of the soil microbiome is a primary indicator of ARD (apple replant disease). Biofumigation is a treatment that enables the restoration of microbiome balance. This study involved an analysis of the taxonomic and functional diversity of bacterial communities in replanted soil (ARD), in replanted soils with forecrops of French marigold (Tagetes patula L.), white mustard (Sinapis alba), and oilseed radish (Raphanus sativus var. oleifera), and in agricultural soil. The biofumigation treatment with phytosanitary plants changed the structure and abundance of the replanted soil microbiome in a fruit tree nursery. The count of operational taxonomic units (OTU) of the Proteobacteria, Bacteroidota, Patescibacteria, Chloroflexi, and Verrucomicrobiota phyla increased, whereas the count of the Firmicutes, Acidobacteriota, and Actinobacteriota phyla decreased. Biofumigation caused an increase in the content of some dominant bacterial genera, such as Flavobacterium, Massila, Sphingomonas, Arenimonas, and Devosia, in the replanted soil. Their presence in the soil may improve the growth of plants, induce their systemic resistance, and thus improve the production properties of soil with ARD. The research results led to the conclusion that the use of phytosanitary plants in nursery production can be an effective alternative to the chemical fumigation of soil.

1. Introduction

The cultivation of fruit trees is a very specific and demanding procedure because it is a long-term monoculture. This problem also concerns nurseries producing fruit trees. As early as the beginning of the 20th century, researchers found it important to establish nurseries on the soil where such crops had not been grown before. Currently, due to the high specialisation of farms and the lack of new areas, nursery production needs to be done in the same places. This may lead to apple replant disease (ARD). This problem usually occurs in orchards with apple trees [1,2], especially those grown on dwarf rootstocks [3], peach trees [4,5], and cherry trees [6,7]. ARD is increasingly common in plantations with roses [8,9], vines [10,11], asparagus [12], medicinal plants such as Rehmannia glutinosa [13], and some forest tree species [14]. The high incidence of ARD in apple orchards results from the fact that the apple tree is one of the most common orchard species in the world. Due to the intensive fruit production and the emergence of new, more attractive varieties fruit growers increasingly often have to replace their plantings with new ones. Apple replant disease (ARD) has been investigated by many scientists from all over the world [15,16,17,18,19,20,21,22,23]. Research results have shown that when a new orchard is established in place of an old one, trees usually grow worse and the development of small hair roots is impaired, which may result in the death of the roots. In consequence, the growth of the aerial part is strongly reduced, whereas the fruits from these orchards are characterised by low quality [24,25].
ARD is often described as a detrimentally disturbed physiological and morphological response of apple plants to soils that have experienced microbiome changes due to previous apple crops [1] or as a soil microbiome dysbiosis [26,27,28]. Biotic factors are considered to be the main causative agents of this disease. These are fungi (Fusarium, Rhizoctonia, Phytium, Phytophthora spp., and others), bacteria (the Pseudomonas and Bacillus genera and the Actinobacteria phylum) [2], as well as nematodes. According to Manici et al. [29], ARD may primarily be caused by an imbalance in the structure of the soil microbiota and the accumulation of harmful microorganisms. According to Zhao et al. [30], the intensity of ARD in apple orchards is associated with increased soil acidification and the resulting lack of available minerals. Due to the large diversity of causative factors and the complexity of their interactions, it is difficult to effectively reduce the negative effects of ARD [31,32,33].
ARD can be prevented by thermal decontamination within a temperature range of 50–100 °C, which may strongly reduce the total soil microbiota [34], or by gamma radiation [35]. Another option is chemical fumigation, i.e., disinfecting the soil with chemicals. It is considered an effective method, but it is expensive and harmful to the environment. The chemicals used for this type of soil fumigation are toxic. Currently, these are mainly dazomet or sodium methane (both release methyl isothiocyanate) as well as 1,3-dichloropropene/chloropicrin [36,37,38,39].
Due to the non-selective action of chemicals and the deposition of their residues in the soil environment, the abundance of microbiota is reduced, and the time of soil regeneration is usually extended. Therefore, researchers increasingly often talk about the need to reduce the amount of chemical crop protection products used in horticultural production, including nursery production. Anaerobic soil disinfection (ASD) is an alternative to the chemical decontamination of soils with ARD. The method consists of applying a rapidly biodegradable material (organic carbon) into the soil and covering the soil tightly with a transparent film. As a result, soil microorganisms that decompose organic matter consume oxygen completely. Such anaerobic conditions are not lethal for some organisms. However, it is important to note that as a result of the decomposition of organic material, free volatile fatty acids are released, which are toxic to many species of soil organisms, including facultative anaerobes. The ASD method proved to be effective in nurseries with apple trees and cherry trees [40,41]. Another strategy for fighting ARD is to change the biodiversity of the soil environment by introducing composts [42,43].
Biofumigation is a promising method of reducing the negative effects of replantation. It consists of using appropriate forecrops, especially phytosanitary plants, which may reduce the populations of harmful nematodes, bacteria, and pathogenic fungi in the soil. Phytosanitary plants include marigold (Tagetes patula L.), white mustard (Sinapis alba), oil radish (Raphanus sativus var. oleifera), spring rape (Brassica napus), oats (Avena sativa), rye (Secale cereale L.), and asparagus (Asparagus officinalis). Biofumigation is a process that leads to the production of volatile biocidal compounds. Plants of the Brassicaceae family (Sinapis alba, Raphanus sativus) produce secondary metabolites—glucosinolates—after hydrolysis, of which biologically active compounds are formed: isothiocyanates—aliphatic allyl isothiocyanate, aromatic isothiocyanates, 2-phenylethyl isothiocyanate, and benzyl isothiocyanate [44]. The use of fresh biomass is recommended, as this form is particularly rich in glucosinolates. Plants in the Asteraceae Dum. family (primarily Tagetes L.) produce compounds that exhibit, among other things, nematicidal and insecticidal effects, which are the result of metabolites released from the roots of mature plants. These include thiophene compounds such as α- tertienyl [45]. When using phytosanitary plants, it is important to remember that the plant material should be thoroughly crushed and then applied into the soil at a depth of 15–20 cm [46]. Phytosanitary plants produce specific compounds that are released into the soil environment through the roots or through biomass decomposition and thus may cause changes in the soil microbiome [47,48,49].
It was assumed that the biofumigation process based on the use of selected phytosanitary plants would contribute to reducing the abundance of Firmicutes bacteria, which include, among others, bacteria of the genus Bacillus and Clostridium, producing persistent forms in unfavourable environmental conditions for growth, the abundance of the Actinobacteriota type indicating soil dryness, and the Acidobacteriota type indicating soil acidification.
The aim of the study was to understand the structure of bacterial communities in soil with ARD and to assess the direction of changes in the microbiome in replanted soil under the influence of phytosanitary plants—marigold (Tagetes patula L.), white mustard (Sinapis alba), and oil radish (Raphanus sativus var. Oleifera)—in a fruit tree nursery (apple tree).

2. Materials and Methods

2.1. Experiment Design

The experiment was conducted between 2019 and 2021 on stagnic luvisol (according to WRB) in a production nursery in Puszczykowo Zaborze, Poland (52°25′49.10″ N 17°11′34.08″ E). Soil from two different sites was used in the experiment. The soil from the first site had been used in agricultural production. It was optimally prepared for the cultivation of apple trees in a nursery (hereinafter referred to as agricultural soil). The soil from the other site had been used for growing apple trees for three years. It had ARD symptoms (hereinafter referred to as replanted soil). Three different phytosanitary plants were used in the experiment: Tagetes patula L., Sinapis alba, and Raphanus sativus var. oleifer. There were five variants of the experiment: R1—agricultural soil (control variant); R2—replanted soil; R3—replanted soil, with a French marigold forecrop (Tagetes patula L.); R4—replanted soil, with a white mustard forecrop (Sinapis alba); R5—replanted soil, with an oil radish forecrop (Raphanus sativus var. oleifera).
All phytosanitary plants were sown into the soil in the autumn after the apple trees had been dug out. In early spring (March), they were crushed and mixed with the soil. In early May, the soil with the crushed phytosanitary plants was put into containers with a capacity of 7.5 L, and the apple tree strains were planted there. Golden Delicious apple trees on M.9 rootstock obtained from winter grafting were used in the experiment. There were 30 containers in each variant of the experiment.
Before starting the experiment, the physicochemical properties of the soil from both sites were analysed. The analysis showed significant differences in the content of mineral components, humus, and soil pH. The replanted soil had a higher specific weight and significantly lower humus content (Table 1). The content of minerals P, K, Ca, and Mg in the replanted soil was lower than in the agricultural soil. The analysis showed that the replanted soil was characterised by low fertility, and the results indicated the possible occurrence of ARD.
The climatic conditions were characterised on the basis of data from a weather station located 6 km away from the research site. Between 2018 and 2021, the average annual temperature was much higher than the average temperature spanning a long-term period. The amount of rainfall was also much lower (Figure 1).
The analysis of the total rainfall and the average air temperature in individual months showed that in each growing season, there were dry periods, so irrigation was necessary to ensure optimal plant growth. The greatest water shortage occurred in the growing seasons of 2018 and 2019.

2.2. Soil Analyses

The composition of the soil microbiome was analysed in samples collected in September of each year of the research period. A soil sample weighing 30 g was collected with a laboratory spatula from each container in the variant. They were mixed, and an aggregate sample with a total weight of 900 g was obtained.

2.2.1. Identification of Soil Microorganisms—DNA Extraction

Total DNA was extracted from 500 mg of each sample with a Genomic Mini AX Soil kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s instructions. The extracted DNA was quantified with a Quant-iT HS dsDNA assay kit (Invitrogen, Carlsbad, CA, USA) on a Qubit2 fluorometer, and 2 μL of extracts were examined on a 0.8% agarose gel.

2.2.2. PCR Amplification

The metagenomic analysis was based on the hypervariable region V3–V4 of the 16S rRNA gene. Specific primers (341F and 785R) were used for the amplification of this region and to prepare libraries. A PCR was conducted with a Q5 Hot Start High-Fidelity DNA Polymerase kit (NEB Inc., Ipswich, MA, USA). The reaction conditions were maintained according to the manufacturer’s specifications. An Illumina MiSeq PE300 sequencer (Genomed S.A., Warsaw, Poland) in 2 × 250 bp paired-end (PE) technology with a v2 Illumina chemistry kit was used for sequencing. The reactions were conducted according to the Illumina V3–V4 16S RNA amplification protocol (Illumina, San Diego, CA, USA). The data were analysed automatically with the MiSeq and in the Illumina BaseSpace cloud environment according to the 16S Metagenomics protocol (ver. 1.0.1). The libraries were prepared in an analogous way to the attached Illumina protocol.

2.3. Statistical and Bioinformatics Analyses

The data were subjected to a conventional analysis of variance with the STATISTICA® 10 software (StatSoft, Krakow, Poland). Venn diagrams were used to present the similarities and differences in the genus composition of experimental variants, representing the relative abundance of bacteria according to the type of forecrops used. Differences in the mean abundance of bacteria between the soils, in which, before establishing the apple tree nursery, the forecrops of French marigold (Tagetes patula L.), white mustard (Sinapis alba), and oil radish (Raphanus sativus var. oleifera) had been used, and the soil after agricultural crops were calculated and visualised. The datasets were also subjected to principal component analysis (PCA), which showed the relationships between the experimental variants and the relative abundance of the phylum composition of bacteria to the type of forecrops used [50].

3. Results and Discussion

The obtained results of research on the bacterial microbiome of replanted soils subjected to biofumigation with selected phytosanitary plants confirmed the thesis of reducing the population of bacteria indicating poor soil condition (Firmicutes, Actinobacteriota, and Acidobacteriota) in favour of increasing the population indicating its revitalisation (Proteobacteriota, Bacteroidota, Patescibacteria, and Chloroflexi).

3.1. Bacterial Phyla

The metapopulation analysis based on the analysis of the 16S rRNA sequence showed that the previous use of the soil in the nursery, the phytosanitary plants used in the experiment, and the years of research influenced the number of operational taxonomic units of bacteria in the soil (Table 2). Next-generation sequencing is an increasingly popular and extremely sensitive method of determining similarities and differences within the soil microbiome. This fact was confirmed by the results of our research (Table 2, Figure 2, Figure 3 and Figure 4) and the data provided in reference publications [51,52,53]. Depending on the soil site and the year of the research, there were 20–34 bacterial phyla and 378–554 genera identified (Table 2). Due to the large number of operational taxonomic units (OTUs), only those with an average share of more than 1% were shown in Figure 2, Figure 3 and Figure 4.
Throughout the study period, the following bacterial phyla were dominant in the soil: Proteobacteria (the relative abundance ranged from 33.23% to 60.08%), Firmicutes, Actinobacteriota, Acidobacteriota, Chloroflexi, and Verrucomicrobiota (Figure 2, Figure 3 and Figure 4). Depending on the year of the soil metagenomic analyses, the following phyla were also dominant: Bacteroidetes, Planctomycetes, Tenericutes, Spirochaetes, Chlamydiae, Cyanobacteria, Gemmatimonadota, Bacteroidota, and Fatescibacteria (Figure 2, Figure 3 and Figure 4). Mahnkopp-Dirks et al. [54] found that Proteobacteria were the dominant phylum in both the ARD soil and unaffected soil (up to 83.7% of the OTU content).
Fierer et al. [55] proposed the concept of bacterial classification, in which Proteobacteria were described as fast-growing copiotrophs, i.e., microorganisms developing in environments with high carbon availability, whose abundance is closely correlated with the degree of carbon mineralisation in the soil. Our experiment showed that, regardless of the year of the study, the most intensive proliferation of Proteobacteria was observed in the soil in variant R3, slightly weaker—in the replanted soil with the white mustard forecrop (Sinapis alba) (R4), and then in variant R5 (Figure 2, Figure 3 and Figure 4). The lowest percentage of the OTU content was found in the replanted soil (R2) in 2020. A year earlier, despite several attempts to isolate the bacterial DNA, it was impossible to obtain research material due to the degree of soil sterilisation.
In 2019, regardless of the experimental variant, Firmicutes bacteria were the most dominant phylum (Figure 2). In the following years of the research, the count of Actinobacteriota increased and was greater than the counts of other bacterial phyla (Figure 3 and Figure 4). The intensive growth and development of Actinobacteriota were particularly noticeable in variant R2. According to Swędrzyńska and Małecka-Jankowiak [56] as well as Niewiadomska et al. [57], Actinobacteriota are a saprophytic group of actinobacteria that quickly adapt to unfavourable environmental conditions, such as desiccation. Therefore, they actively decompose organic matter when the soil moisture is low.
Figure 2, Figure 3 and Figure 4 show relative differences between the dominant types of bacteria in the control variant (R1) and the other experimental variants, expressed as a percentage of sequence. The analysis of the research results showed that regardless of the year of the study, the OTU content of the Proteobacteria phylum in all experimental variants was lower than in the control variant (R1). However, it is necessary to stress the fact that the difference in the content of OTUs of the Proteobacteria phylum in the soils subjected to induced biofumigation with the phytosanitary plants—Tagetes patula L. (R3), Sinapis alba (R4), and Raphanus sativus var. oleifera (R5)—in relation to the agricultural soil was significantly lower than the difference observed in the soil with ARD (Figure 5, Figure 6 and Figure 7). Apart from that, in 2020, the application of French marigold (R3-R1) caused a significant increase in the content in OTUs of the Proteobacteria phylum (Figure 6). Thus, it can be concluded that the application of phytosanitary plants to the soil with ARD causes an increase in the content of OTUs belonging to the Proteobacteria phylum. This conclusion is similar to the findings of the authors of studies on ARD soil recultivation by adding compost [58,59] or by exposure to gamma radiation [53].
A similar trend was also observed for the Firmicutes phylum. The metagenomic analysis of the soil conducted between 2019 and 2021 showed that the sequence content of seven bacterial phyla in the soil samples was lower than in the control variant. However, these differences were not observed in all experimental variants (Figure 5, Figure 6 and Figure 7). They were ranked as follows according to the frequency of their occurrence in the soil: Bacteroidota > Acidobacteriota > Actinobacteriota = Gemmatimonadota > Patescibacteria.
The Verrucomicrobiota and Chloroflexi bacterial phyla, and especially Planctomycetota and Cyanobacteria, were isolated less often from the agricultural soil (R1), especially when compared with the replanted soil subjected to fumigation. The Chloroflexi phylum encompasses nitrifying bacteria developing in anaerobic or microaerophilic conditions. They can survive in intensively changing, extreme conditions. The incidence of the Chloroflexi phylum in soils with ARD after biofumigation was higher than in the control soil. This phenomenon can be explained by the fact that the development of the community of these bacteria is based on the use of cellular compounds from dead microorganisms and their metabolites, which is typical of soils with ARD [60]. A similar dominance of this group of microorganisms was observed by Tang et al. [61], who used rice straw and biochar to effectively improve soil quality. According to the information provided in reference publications, Cyanobacteria is an important phylum indicating the reconstruction of soils. They are credited with an important role in fixing atmospheric nitrogen and the synthesis of exopolysaccharides, which increase soil fertility and water retention and improve its structure and stability [62]. In our study, fumigation induced by the use of French marigold (R3) increased the content of OTUs in the Cyanobacteria phylum by 83% as compared with the soil with ARD and the agricultural soil.
The aforementioned changes in the qualitative and quantitative composition of the bacterial microbiome caused by the previous use of soil and the effect of phytosanitary plants enabled the identification of microorganisms, which can be regarded as bioindicators of soil fertility. According to Fierer et al. [55], in order to better understand the soil regeneration process, it is important to know both the communities of microorganisms inhabiting the soil and their interrelationships. The principal component analysis (PCA) revealed the relationships between the different types of soil bacteria in the experimental variants during the three years of the research (Figure 8). It showed that soil biodiversity ranged from 71.19% to 94.28%. It also showed that the relationships between the different types of bacteria were related to the year of the study. Regardless of the experimental variant, in the first year of the study, the analysis revealed a clear correlation between the percentage of taxonomic sequences of the Verrucomicrobiota, Bacteroidota, Cyanobacteria, and Tenericutes phyla and between Actinobacteriota and Planctomycetota. A similar relationship between Verrucomicrobiota and Bacteroidota was also observed in the second year of the study (Figure 8). However, in 2020 and 2021, there was a correlation between the percentage of OTUs in the Proteobacteria phylum and Bacteroidota, which was in line with the results of the study by Fazi et al. [63].

3.2. Bacterial Genera

Due to the fact that next-generation sequencing resulted in a relatively large number of sequences of bacterial genera, only the most numerous of them (>1%) were shown in Figure 9, Figure 10 and Figure 11. In 2019, the dominant genera were: Pseudomonas (0.86–25.24%), Bacillus (3.76–27.76%), Clostridium (2.59–18.12%), and Cohnella (0.42–8.42%)—a highly cellulolytic bacterial genus belonging to the Paenibacillaceae family (Figure 9). The metagenomic analysis of the soil showed that in the next two years of the research, the most common bacterial genera in all experimental combinations were Rhodanobacter (1.63–8.66%) and Sphingomonas (2.80–5.01%), as well as Gaillales uncul. (2.64–7.12%) in 2020 and Cellulosimicrobium (8.69–25.34%) in 2021 (Figure 10 and Figure 11).
The percentage of operational taxonomic units (OTUs) of individual bacterial genera depended on the experimental variant. In the first year of the study, the highest counts of bacteria of the Pseudomonas and Clostridium genera were found in the agricultural soil (R1), whereas the lowest were found in the soil in variant R5 (Figure 9). In the next two years of research (Figure 10 and Figure 11), it was mainly the Rhodanobacter genus of the Gammaproteobacteria class, Xanthomonadales order, and Xanthomonadaceae family that occurred more often in the variant with replanted soil (R2). This genus of bacteria is considered an indicator of soils degraded by agriculture. Wolińska et al. [64] selected the Rhodanobacter genus as a metagenomic analysis indicator characteristic of soils degraded by agriculture. According to the researchers, these bacteria are resistant to agricultural practices. They can be classified as oligotrophs with low nutritional requirements. This fact may account for the high content of their OTUs found in variants R1 and R2 in our study. The forecrop of phytosanitary plants (variants R3, R4, and R5) resulted in a lower count of bacteria of the Rhodanobacter genus (Figure 10). In 2020 and 2021, there was a smaller count of bacteria of the Gaiellales genus in the replanted soil (Figure 10 and Figure 11). According to the data provided in reference publications, this genus plays a key role in the soil because it inhibits root rot caused by fungi of the Fusarium genus [65]. Moreover, according to Wu et al. [66], if the soil conditions are unfavourable for plant growth, this genus can adjust its metabolism so as to promote plant growth by increasing the availability of nutrients.
In 2020 and 2021, the presence of other resistant types of bacteria inhabiting the replanted soils with ARD was observed, i.e., Peanibacillus and Chitinophagaceae (Figure 10 and Figure 11). According to the data provided in the reference publications, the former genus has all possible characteristics of plant growth-promoting rhizobacteria (PGPR), which can improve plant growth by induction of immunity, production of growth hormones, sharing of phosphorus, etc.). On the other hand, some species of PGPR cause diseases in honeybees. This has a negative influence on nurseries, which cannot produce high-quality trees with high yields [67].
Another genus found in the replanted soil (R2) was Chitinophagaceae_uncul. These bacteria are credited with an important role in the decomposition of organic carbon. They increase the intensity of its mineralisation, which is a phenomenon characteristic of agriculturally degraded soils [68]. Bacteria of the Chitinophagaceae_uncul genus were also identified in the soils after induced biofumigation, but their count was lower than in the replanted soil without the forecrop of phytosanitary plants (Figure 9 and Figure 10).
In our study, apart from the bacterial genera resistant to soil degradation, there were also genera that did not react to soil dysfunction caused by ARD. The number of their OTUs in the soil in all variants of the experiment was similar. In 2020 and 2021, it was the Sphingomonas genus whose content in all variants of the experiment was similar (Figure 10 and Figure 11). According to scientific publications, species belonging to this genus have multifaceted functions, ranging from the remediation of environmental pollution to the production of phytohormones, gibberellins, and indole acetic acid, which have indirect mutualistic effects on plants. Some species of this genus improve plant growth under soil stress, such as drought, salinity, or the content of heavy metals [69].
Other genera of microorganisms that occurred in identical counts in various combinations were Devosia and Pseudolabrys in 2020 (Figure 10) and Chujaibacter and uncultivated bacteria of the Gemmatimonadaceae family in 2021 (Figure 11). The presence of these bacterial genera in agriculturally degraded soils and soils with ARD was also confirmed in the available reference publications. The Devosia genus is well known for its dominance in soil habitats contaminated with various toxins, and it is best characterised by its bioremediation potential. In addition, the authors of studies on this genus of microorganisms stress their genomic plasticity to ensure adaptation, bioremediation, and the potential to use a wide range of substrates in degraded soils [70].
In 2020, the genus found in the soil collected from all experimental variants was Pseudolabrys bacteria (Figure 10), whereas in 2021 it was Chujaibacter (Figure 11). These are bacteria of the Nitrobacteriaceae family that are involved in the nitrification process [71]. They are also characterised by very high resistance to negative environmental factors and by increased succession with decreasing soil pH, which is characteristic of soils with ARD [72].
In 2020 and 2021, Flavobacterium, a sensitive bacterial genus, was detected in the replanted soil. Its smallest count was found in the replanted soil (R2), where it amounted to 0.82% in 2020 and 0.31% in 2021. After the application of the phytosanitary plants (variants R3, R4, and R5), the abundance of these bacteria in 2020 and 2021, respectively, increased to 1.08% and 0.46%, 1.61% and 1.68%, and 2.55% and 0.80% OTU (Figure 12). Flavobacterium bacteria are potential inhibitors of pathogens in root ecosystems. Apart from that, selected representatives of this genus are treated as plant growth-promoting rhizobacteria (PGPR) [73]. If ARD occurs, these microorganisms interact antagonistically with nematodes, thus alleviating the symptoms of soil disease [74]. The results of our experiment concerning the influence of French marigold, white mustard, and oilseed radish on the Flavobacterium genus were in line with the findings of other scientific publications. Hanschen and Winkelmann [75] observed that induced fumigation, e.g., by using Brassica juncea and Sinapis alba, increased the count of plant growth-promoting bacteria while inhibiting ARD.
In 2020, Massilia, which has similar characteristics and properties to Flavobacterium, was identified as an additional genus of sensitive bacteria in the replanted soil [76] (Figure 12). In 2021, apart from Flavobacterium, Saccharimonadales was another genus of ARD-sensitive bacteria. Like the Massilia genus, these microorganisms stimulate the growth of plants and play an important role in providing them with phosphorus and other nutrients [49].
It is likely that the increase in the content of beneficial bacteria in the soils with ARD after induced fumigation (variants R3–R5) was caused by the phytosanitary plants, which produced bioactive compounds (including alpha-terthienyl). In consequence, they limited the development of pathogens such as Rhizoctonia solani and Fusarium solani and thus contributed to the succession of the abovementioned genera of beneficial bacteria [54].
The results of our experiment presented in the form of Venn diagrams confirmed the influence of the previous method of soil use and the research period on the structure of the bacterial microbiome (Figure 13). The presence of all taxa within a particular taxonomic category and the research period were taken into account. As a result, 482–512 genera common to all variants were selected. For example, the following bacterial genera were identified in all experimental variants: Pseudomonas, Bacillus, Arthrobacter, Streptomyces, Chujaibacter, Sphingomonas, Flavobacterium, and Devosia.
The number of bacterial sequences identified in our study provides grounds for the conclusion that the cultivation of the phytosanitary plants contributed to the change in the qualitative composition of the soil microbiome. The highest number of unique taxa in the experimental variants was observed in 2019 in variant R5 (Table 3). These were 28 bacterial genera, which included saprophytic and plant growth-promoting species, as well as plant pathogens. In the consecutive years of the analyses, the number of unique genera in variants R5, R3, and R4 decreased significantly. During the analyses, the lowest number of unique bacterial taxa was found in the control variant, especially in the second and third years of the study, when the Amycolatopsis genus of the Pseudonocardiacea family was identified (Table 4 and Table 5). This genus includes species recognised as biocontrol factors, which play an important role in destroying plant pathogens and in the bioremediation process [77,78].

4. Conclusions

The use of metagenomics (functional analysis of genetic material isolated from the soil) as a tool for assessing soil biodiversity in the nursery after replantation proved to be a sensitive and precise method of assessment of the soil microbiome in the nursery. The analyses of the microbiome composition showed that biofumigation with phytosanitary plants—French marigold (Tagetes patula L.), white mustard (Sinapis alba), and oil radish (Raphanus sativus var. oleifera)—changed the structure and count of bacteria in the replanted soil in the fruit tree nursery. The phytosanitary plants increased the abundance of operational taxonomic units (OTU) of the Proteobacteria, Bacteroidota, Patescibacteria, Chloroflexi, Fatescibacteria, and Verrucomicrobiota phyla, but decreased the abundance of the Firmicutes, Acidobacteriota, and Actinobacteriota phyla. The biofumigation also increased the content of some dominant bacterial genera in the replanted soil, such as Flavobacterium, Massila, Sphingomonas, Arenimonas, and Devosia. These genera are considered crucial in promoting plant growth and inducing plant systemic immunity, which may indicate the regeneration of replanted soil.
Studies have shown that regardless of the species of phytosanitary plants used, there was an increase in the abundance of beneficial microbiomes. In practice, when planning production plantings, it is recommended to use a one-year break during which phytosanitary plants will be cultivated.

Author Contributions

Conceptualisation, R.W., Z.Z., A.W.-M. and A.N.; methodology, R.W., A.W.-M. and A.N.; software, R.W. and D.K.; validation, R.W., Z.Z. and A.W.-M.; formal analysis, R.W., Z.Z., A.W.-M. and A.N.; investigation, R.W., A.W.-M. and A.N.; resources, R.W.; data curation, R.W., A.W.-M. and A.N.; writing—original draft preparation, R.W., Z.Z., A.W.-M., A.N. and D.K.; writing—review and editing, R.W., Z.Z., A.W.-M., A.N. and D.K.; visualisation, R.W., A.W.-M. and D.K.; supervision, Z.Z. and A.W.-M.; funding acquisition, R.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Not available.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The course of temperature and rainfall between 2018 and 2021.
Figure 1. The course of temperature and rainfall between 2018 and 2021.
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Figure 2. Relative abundance of dominant phyla of bacteria in 2019. The classifications with less than 1% abundance are gathered into the category “Other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Figure 2. Relative abundance of dominant phyla of bacteria in 2019. The classifications with less than 1% abundance are gathered into the category “Other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
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Figure 3. Relative abundance of dominant phyla of bacteria in 2020. The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Figure 3. Relative abundance of dominant phyla of bacteria in 2020. The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
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Figure 4. Relative abundance of dominant phyla of bacteria in 2021. The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Figure 4. Relative abundance of dominant phyla of bacteria in 2021. The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
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Figure 5. Relative abundance of dominant classes of bacteria phyla in 2019 (A—R3 via R1; B—R4 via R1; C—R5 via R1). The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut; “yellow” means negative difference; “blue” means positive difference).
Figure 5. Relative abundance of dominant classes of bacteria phyla in 2019 (A—R3 via R1; B—R4 via R1; C—R5 via R1). The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut; “yellow” means negative difference; “blue” means positive difference).
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Figure 6. Relative abundance of dominant classes of bacteria phyla in 2020 (A—R2 via R1; B—R3 via R1; C—R4 via R1; D—R5 via R1). The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut; “yellow” means negative difference; “blue” means positive difference).
Figure 6. Relative abundance of dominant classes of bacteria phyla in 2020 (A—R2 via R1; B—R3 via R1; C—R4 via R1; D—R5 via R1). The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut; “yellow” means negative difference; “blue” means positive difference).
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Figure 7. Relative abundance of dominant classes of bacteria phyla in 2020 (A—R2 via R1; B—R3 via R1; C—R4 via R1; D—R5 via R1). The classifications with less than 1% abundance are gathered into the category “other”. (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut; “yellow” means negative difference; “blue” means positive difference.
Figure 7. Relative abundance of dominant classes of bacteria phyla in 2020 (A—R2 via R1; B—R3 via R1; C—R4 via R1; D—R5 via R1). The classifications with less than 1% abundance are gathered into the category “other”. (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut; “yellow” means negative difference; “blue” means positive difference.
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Agronomy 13 02507 g008aAgronomy 13 02507 g008b
Figure 8. Principal component analysis of the relative abundance of dominant phyla of bacteria in the different soils (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Figure 8. Principal component analysis of the relative abundance of dominant phyla of bacteria in the different soils (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Agronomy 13 02507 g008aAgronomy 13 02507 g008b
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Figure 9. Relative abundance of the dominant (rodzaj) genus of bacteria in 2019. The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Figure 9. Relative abundance of the dominant (rodzaj) genus of bacteria in 2019. The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
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Figure 10. Relative abundance of the dominant genus of bacteria in 2020. The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Figure 10. Relative abundance of the dominant genus of bacteria in 2020. The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
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Figure 11. Relative abundance of the dominant genus of bacteria in 2021. The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Figure 11. Relative abundance of the dominant genus of bacteria in 2021. The classifications with less than 1% abundance are gathered into the category “other” (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
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Figure 12. Principal component analysis of the relative abundance of dominant phyla of bacteria in the different soils (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Figure 12. Principal component analysis of the relative abundance of dominant phyla of bacteria in the different soils (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
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Figure 13. Venn diagram of overlapping bacterial communities (phyla) (A—2019; B—2020; C—2021). (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut.); the numbers indicate the number of unique bacterial sequences.
Figure 13. Venn diagram of overlapping bacterial communities (phyla) (A—2019; B—2020; C—2021). (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut.); the numbers indicate the number of unique bacterial sequences.
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Table 1. The chemical analysis of the soil before starting the experiment (R1—agricultural soil; R2—replanted soil).
Table 1. The chemical analysis of the soil before starting the experiment (R1—agricultural soil; R2—replanted soil).
Properties of the SoilR1R2
pH (H2O)7.65.8
Bulk density (g dm−3)16001830
Salinity (g Na Cl dm−3)0.230.23
Humus content (%)4.881.70
Mineral content (mg dm−3): N-NO3<3.9<3.9
P12730
K22989
Ca1333240
Mg18838
Cl<21.3<21.3
Table 2. Number of bacterial taxonomic units according to experimental combinations (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Table 2. Number of bacterial taxonomic units according to experimental combinations (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Taxonomic UnitsR1R2R3 R4 R5
2019 year
Phylum20-222323
Class44-485047
Order87-969396
Family180-201197205
Genus378-441407470
Species456-591532704
2020 year
Phylum3032313231
Class8489899085
Order190201216224207
Family280294320338317
Genus457499554510513
Species8329061007935940
2021 year
Phylum3230343330
Class8076798579
Order180170195200189
Family272255302301280
Genus480458552553505
Species838774953942988
Table 3. Unique bacterial taxa in individual experimental variants in 2019. (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Table 3. Unique bacterial taxa in individual experimental variants in 2019. (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
R1R2R3R4R5
Number of Unique Taxa
10091828
Anaerococcus
Dorea
Eggerthella
Enhydrobacter
Fructobacillus
Mitsuokella
Paraprevotella
Sarcina
Thiocystis
Verminephrobacter		_Chlamydia
Desulfonatronovibrio
Desulfotalea
Flectobacillus
Helcococcus
Muricauda
Roseateles
Sinomonas
Spirochaeta		Citricoccus
Cryobacterium
Dehalobacterium
Desulfomicrobium
Entomoplasma
Fusobacterium
Jeotgalicoccus
Kibdelosporangium
Kytococcus
Octadecabacter
Odoribacter
Peptostreptococcus
Roseococcus
Saccharomonospora
Salinivibrio
Sporanaerobacter
Thermococcus
Xenococcus		Antarctobacter
Aureispira
Bulleidia
Butyricimonas
Coprococcus
Haliscomenobacter
Lachnobacterium
Limnothrix
Luteococcus
Nevskia
Parabacteroides
Porphyromonas
Propionigenium
Pseudanabaena
Psychroflexus
Rhodothalassium
Roseburia
Roseiflexus
Roseivivax
Salinimicrobium
Snowella
Streptomonospora
Sulfuricurvum
Sulfuritalea
Teredinibacter
Terriglobus
Thermacetogenium
Zobellia
Table 4. Unique bacterial taxa in individual experimental variants in 2020. (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Table 4. Unique bacterial taxa in individual experimental variants in 2020. (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
R1R2R3R4R5
Number of Unique Taxa
14543
AmycolatopsisHaliscomenobacter
Novibacillus
f_Enterobacteriaceae;
Other
Hafnia-
Obesumbacterium		f_Nostocaceae; Other
Archangium
Aetherobacter
f_Polyangiaceae;g_
uncultured
Leptospira		Vicingus
Blastopirellula
Chitinimona
f_Parachlamydiaceae;
g_uncultured		o_Babeliales;
Other;
Lactococcus
OM60(NOR5)_clade
Table 5. Unique bacterial taxa in individual experimental variants in 2021. (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
Table 5. Unique bacterial taxa in individual experimental variants in 2021. (R1—agricultural soil; R2—replanted soil; R3—replanted soil with Tagetes patula L. foregut; R4—replanted soil with Sinapis alba foregut; R5—replanted soil with Raphanus sativus var. oleifera foregut).
R1R2R3R4R5
Number of Unique Taxa
112160
AmycolatopsisRahnella1f_Myxococcaceae;Other
Candidatus_Falkowbacteria		Paludibacter
WCHB1-32
Vitellibacter
Bacteriovorax
Pseudarcobacter
Candidatus_Megaira
Tolumonas
Idiomarina
Shewanella
Noviherbaspirillum
Candidatus_Accumulibacter
f_Methylococcaceae;g_uncultured
Methylophaga
Halomonas
Alkanindiges
f_Criblamydiaceae;g_uncultured		-
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Wieczorek, R.; Zydlik, Z.; Wolna-Maruwka, A.; Niewiadomska, A.; Kayzer, D. The Effect of Biofumigation on the Microbiome Composition in Replanted Soil in a Fruit Tree Nursery. Agronomy 2023, 13, 2507. https://doi.org/10.3390/agronomy13102507

AMA Style

Wieczorek R, Zydlik Z, Wolna-Maruwka A, Niewiadomska A, Kayzer D. The Effect of Biofumigation on the Microbiome Composition in Replanted Soil in a Fruit Tree Nursery. Agronomy. 2023; 13(10):2507. https://doi.org/10.3390/agronomy13102507

Chicago/Turabian Style

Wieczorek, Robert, Zofia Zydlik, Agnieszka Wolna-Maruwka, Alicja Niewiadomska, and Dariusz Kayzer. 2023. "The Effect of Biofumigation on the Microbiome Composition in Replanted Soil in a Fruit Tree Nursery" Agronomy 13, no. 10: 2507. https://doi.org/10.3390/agronomy13102507

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