TY - JOUR
AU - Karpouzas, Dimitrios, G
AB - ABSTRACT Pesticides interact with microorganisms in various ways with the outcome being negative or positive for the soil microbiota. Pesticides' effects on soil microorganisms have been studied extensively in soil but not in other pesticides-exposed microbial habitats like the phyllosphere. We tested the hypothesis that soil and phyllosphere support distinct microbial communities, but exhibit a similar response (accelerated biodegradation or toxicity) to repeated exposure to the fungicide iprodione. Pepper plants received four repeated foliage or soil applications of iprodione, which accelerated its degradation in soil (DT50_1st = 1.23 and DT50_4th = 0.48 days) and on plant leaves (DT50_1st > 365 and DT50_4th = 5.95 days). The composition of the epiphytic and soil bacterial and fungal communities, determined by amplicon sequencing, was significantly altered by iprodione. The archaeal epiphytic and soil communities responded differently; the former showed no response to iprodione. Three iprodione-degrading Paenarthrobacter strains were isolated from soil and phyllosphere. They hydrolyzed iprodione to 3,5-dichloraniline via the formation of 3,5-dichlorophenyl-carboxiamide and 3,5-dichlorophenylurea-acetate, a pathway shared by other soil-derived arthrobacters implying a phylogenetic specialization in iprodione biotransformation. Our results suggest that iprodione-repeated application could affect soil and epiphytic microbial communities with implications for the homeostasis of the plant–soil system and agricultural production. iprodione, phyllosphere, soil, biodegradation, microbial diversity, Paenarthrobacter sp INTRODUCTION Microorganisms are highly responsive to environmental stress conditions. Pesticides applied either in soil or on plant foliage constitute potential environmental stressors for the microbial communities colonizing these habitats. Several studies have explored the responses of the soil microbial communities to pesticides (Itoh et al. 2014; Karas et al. 2018; Gallego et al. 2019). Repeated applications of pesticides lead to the accumulation of pesticide residues in soil when the indigenous microbial community has limited capacity to degrade the given compound. This might impose negative effects on the soil microbial community. For example, nicosulfuron at concentration levels of 0.25–1 μg g−1 significantly reduced the abundance of β-proteobacteria, planctomycetes and actinobacteria, the activity of C- and P-cycling enzymes (Karpouzas et al. 2014a), and the colonization levels and diversity of endomycorrhizal fungi in maize plants (Karpouzas et al. 2014b). In contrast, repeated soil application of pesticide groups like organophosphates (Singh and Walker 2006), carbamates (Karpouzas et al. 1999) and triazines (Krutz et al. 2010) could lead to the proliferation of a small fraction of the soil microbial community that carries specialized catabolic enzymes used for the growth-linked degradation of these pesticides (Itoh et al. 2014; Rousidou et al. 2017). This phenomenon has been termed as accelerated biodegradation and under conducive edaphoclimatic conditions could jeopardize the biological efficacy of pesticides (Suett 1987). In contrast to our good knowledge of the interactions of pesticides with the soil microbial community, we are only just starting to explore the interaction of pesticides with microbial communities in other relevant habitats like plants (Perazzolli et al. 2014) and insects (Cheng et al. 2017). In the phyllosphere, microorganisms are exposed to various environmental constraints (i.e. UV, desiccation, limited nutrients) and potential stressors like pesticides (Vorholt 2012). Zhang et al.(2009a,b) and Gu et al. (2010) first studied the potential effects of synthetic pesticides on the epiphytic microbial community, using Phospholipid Fatty Acid Analysis (PLFAs) and molecular fingerprinting, and observed significant but transient effects. Subsequent studies using amplicon sequencing reported a resilience of the epiphytic microbial community to pesticides (Perazzolli et al. 2014; Ottesen et al. 2015). In contrast, very little is known about the potential of the epiphytic microbial community for accelerated biodegradation of pesticides or the identity of the microorganisms responsible for biodegradation and their relevant degradative genes. In the only relevant study to date, Ning et al. (2012) isolated epiphytic bacteria from rape plants systematically treated with dichlorvos that degraded this organophosphorus insecticide, although the establishment of accelerated biodegradation of dichlorvos on the plant phyllosphere was not explored. The capacity of the epiphytic microorganisms to rapidly degrade foliage-applied pesticides can be beneficial from the environmental and human health perspective, while in its extreme, it could threaten the biological efficacy of foliage-acting pesticides, an aspect largely overlooked. Iprodione is a fungicide used via foliage application or soil drenching for the control of various plant pathogens (Grabke et al. 2014). It has been identified as a potential carcinogen (USEPA 1998) and endocrine-disrupting substance (Blystone et al. 2007). Soil pH is the main factor affecting the dissipation of iprodione in soil with higher degradation rates observed in alkaline soils (Walker 1987). Biodegradation is the main dissipation process of iprodione in soil. Repeated applications of iprodione in soil are known to lead to its accelerated biodegradation (Martin et al. 1990; Mitchell and Cain 1996) and loss of its biological efficacy (Entwistle 1986). Studies in soils exhibiting accelerated biodegradation of the fungicide resulted in the isolation of iprodione-degrading bacteria (Athiel et al. 1995; Campos et al. 2015; Yang et al. 2018). They hydrolyzed iprodione to 3,5-dichloroaniline (3,5-DCA) via the formation of two transient metabolic products: 3,5-dichlorophenyl-carboxiamide (metabolite I) and 3,5-dichlorophenylurea-acetate (metabolite II). On the contrary, soil application of iprodione has been also shown to induce negative effects on the abundance of soil bacteria (Zhang et al. 2017c), on the abundance and diversity of soil fungi (Zhang et al. 2017a) and on microbial processes involved in N cycling (Zhang et al. 2017b, 2018). Recently, Vasileiadis et al. (2018) showed that 3,5-DCA and not iprodione was responsible for the inhibitory effects observed on the ammonia-oxidizing microorganisms in soil and the general microbial activity. Still, we are missing the information about the potential response, toxicity or accelerated biodegradation of the epiphytic microbial community to iprodione exposure. In this study, we explored the hypothesis that although phyllosphere and soil support largely different microbial communities, we expect them to exhibit a similar response to their repeated exposure to a biodegradable pesticide like iprodione. This response could span from accelerated degradation by a fraction of the microbial community to toxicity on members of the microbial community. In this context, a pot experiment with pepper plants repeatedly treated with iprodione, either at the foliage or through soil drenching, was undertaken. Potential accelerated biodegradation of iprodione was evaluated through determination of its degradation at each application in soil and on pepper leaves, while the overall response of the epiphytic and soil bacterial, archaeal and fungal community was determined via 16S rRNA and ITS amplicon sequencing, respectively. Bacteria able to degrade iprodione were isolated via enrichment cultures from both soil and plant leaves and the transformation pathway of iprodione was determined to explore the presence of habitat-specific catabolic traits in iprodione-degrading bacteria isolated from phyllosphere and soil. MATERIALS AND METHODS Chemicals and soil The commercial formulation of iprodione (Rovral® 50%WP) was used for the treatment of pepper plants and soil. Iprodione and 3,5-DCA standards (Pestanal®, purity > 97%) were purchased from Sigma-Aldrich (Taufkirchen, Germany), while 3,5-dichlorophenyl-carboxiamide (metabolite I) and 3,5-dichlorophenylurea-acetate (metabolite II) were synthesized as described before (Campos et al. 2017). The soil used was collected from the top 20 cm of a fallow agricultural field of the Hellenic Agricultural Organization-Demeter in Larissa, Greece (39°63′27″N, 22°36′74″E), with no history of pesticide application for the last 15 years. The soil was homogenized, sieved (2-mm pore size) and stored at 4°C until used. Details about soil sampling and soil properties are given in the Supporting Information. Pot experiment setup Sixty-two pepper plants [Capsicum annuum var. annum(florinis)] at the 3–4 leaf stage were transplanted into 5 L pots filled up with ∼6 kg of soil wet weight. Plants were left to grow in open air (below a net for protection against extreme weather conditions) in the pots (May to July 2017) until flowering, when applications of iprodione were implemented to simulate a realistic application timing of the fungicide. During this period, the plants were watered every day, adjusting the soil moisture content to 50% of its water holding capacity. The first 12 planted pots were treated via soil drenching with 50 mL of an aqueous solution of iprodione (100 mg L−1) aiming to a soil concentration of 1.5 μg g−1, assuming diffusion of the pesticide, applied at the recommended dose rate, to 5-cm depth and a soil bulk density of 1.5 kg L−1. The plants in the second set of 30 pots were sprayed individually with 25 mL of an aqueous solution of iprodione (1500 mg L−1). This dose was selected based on the recommended rate of 300 mg a.i. per 100 L of spraying liquid applied in 40 000 plants per ha. The soil or the foliage of the pepper plants in the remaining 20 pots (2 × 10 pots) were treated with 50 or 25 mL of water without iprodione, respectively, to serve as untreated controls. The same application scheme was repeated four times at 30 days intervals. Immediately after each pesticide application and at regular intervals thereafter triplicate soil (with a cork borer from the root zone in each pot) and leaf samples (each replicate sample was composed of five leaves per plant) were collected from relevant pots and stored at −20°C for analysis of iprodione and 3,5-DCA residues. Similarly, triplicate soil and leaf samples collected at 0 (only on the first application event), 10 and 30 days after each pesticide application were processed for DNA extraction as described below. The leaves collected were well developed and healthy, of the same size and maturity level, located in the upper part of the canopy to minimize the risk of soil transfer. Pesticides residue analysis Iprodione and its transformation products were extracted from soil as described by Campos et al. (2015). The same procedure was followed for the extraction of iprodione and 3,5-DCA from leaves with the only difference that an extra sonication step of 5 min was employed prior to shaking (see the Supporting Information). Analysis of iprodione and its transformation products was performed via HPLC–PDA as described by Campos et al. (2017). DNA extraction from soil and epiphytic microbial biomass Soil DNA extraction was performed from 0.5 g of soil (dry weight) with the PowerSoil® DNA isolation kit (MoBio Laboratories, Carlsbad, USA). DNA extraction from leaves was performed as described by Moulas et al. (2013) (see the Supporting Information). Amplicon sequencing analysis of the soil and epiphytic microbial community The composition of the community of bacteria, archaea and fungi in soil and on plant leaves were determined with amplicon sequencing of the 16S rRNA gene and the ITS region via HiSeq Illumina Rapid Mode 2 × 250 bp paired-end reads. Bacterial and archaeal 16S rRNA genes were amplified with the primer set 515f-806r (Caporaso et al. 2012; Walters et al. 2015) following the protocol of the Earth Microbiome Project (Caporaso et al. 2018). The amplification of ITS was performed with the primers ITS7-ITS4 (White et al. 1990; Ihrmark et al. 2012) following the protocol described by Ihrmark et al.(2012). All samples were initially amplified (28 cycles) using the domain-specific primers mentioned above, followed by a PCR (7 cycles) using primers carrying sample-associated indices for performing the multiplex sequencing. Primers and PCR conditions are listed in Tables S1 and S2 (Supporting Information), respectively. Sequences were demultiplexed with Flexbar v3.0 (Dodt et al. 2012) and the reads were quality trimmed with Trimmomatic v0.32 (Bolger, Lohse and Usadel 2014). The resulting read pairs were assembled with FLASH v1.2.8 (Magoc and Salzberg 2011). The remaining tasks were carried out with the lOTUs v1.58 perl wrapper (Hildebrand et al. 2014). OTU calling at 97% identities was performed with the UPARSE v10.0.240 software (Edgar 2013). Chimeric sequences were identified with the UCHIME v4.2 software (Edgar et al. 2011) using the RDP Gold database vMicrobiomeutil-r20110519 for bacteria and the UNITE ITS2 v985.20150311 reference database (Nilsson et al. 2015) for fungi. Sequence classification was performed with Lambda v0.9.1(Hauswedell, Singer and Reinert 2014) against the Silva v128 database (Yilmaz et al. 2014) for bacteria and the UNITE ITS v7_99_20150302 database (Kõljalg et al. 2013) for fungi (see details in the Supporting Information). Isolation and characterization of iprodione-degrading bacteria Enrichment cultures and isolation At 30 days after the fourth application of iprodione, soil and leaf samples from the iprodione-treated pots were collected. Three soil samples (50 g) were collected from the root zone of each pot and they were composited to a uniform soil sample used as starting inoculum in soil enrichments. Similarly, three to five intact and fully developed leaves were collected from each of the pots that had received repeated iprodione applications and they were bulked up in one sample. The isolation of iprodione-degrading bacteria from soil and leaf samples was done by enrichment cultures in mineral salts medium (MSM), MSM+NH4Cl (MSMN) and MSM+sodium citrate (MSM+SC), all amended with iprodione (10 mg L−1). In those media, iprodione constituted the sole C and N, C or N source, respectively. Growth media were prepared as previously described (Campos et al. 2015; Perruchon et al. 2015) and they were supplemented with iprodione by addition of appropriate amounts of a filter-sterilized Dimethyl sulfoxide (DMSO) stock solution (10 000 mg L−1). The DMSO percentage in the growth media was <0.2%, which according to preliminary tests did not impair bacterial growth. For the isolation of iprodione-degrading bacteria from plant leaves, 5 g of pepper leaves were fully immersed in TE buffer and 0.01% Tween 80. The samples were vortexed for 30 s, agitated for 15 min in an orbital shaker at 200 rpm and placed in an ultrasonic bath for 3 min. They were then vortexed (30 s) and shaken for 5 min before centrifugation at 8000 × g for 7 min. The supernatant was discarded, and the microbial pellet was redissolved in 2 mL of sterilized ddH2O, which was used as inoculum for the enrichment cultures. Enrichment cultures in the three selective media were inoculated with 0.5 mL of the epiphytic microbial pellet or 0.5 g of soil and further proceeded as described by Perruchon et al. (2015) (see the Supporting Information). At the point where >50% degradation of iprodione in the fourth enrichment cycle had occurred, serial dilutions were prepared and spread on iprodione-amended (20 mg L−1) MSM, MSMN and MSM+SC agar plates prepared as described by Karpouzas and Walker (2000). The plates were incubated for 4–5 days at 25°C and 120 well-separated colonies (20 per medium × matrix combination) were picked up and tested for their degrading ability in the corresponding liquid medium. Cultures exhibiting >50% iprodione in 6 days were considered as positive and they were plated on LB and the respective selective media agar plates to check purity. The bacteria that appeared as pure in plates went through another cycle of single colony testing of their degradation capacity before being processed for DNA extraction. Identification of iprodione-degrading bacteria DNA extraction from the bacterial isolates was performed with the NucleoSpin® Tissue kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). The primer pair 8f-1512r, which amplifies the near full size of the 16S rRNA gene (1504 bp) (Felske et al. 1997), was used for the identification of the isolated bacteria as described previously (Perruchon et al. 2015). The near full-length 16S rRNA sequence was subjected to phylogenetic analysis as described by Campos et al. (2015) and the phylogenetic tree was prepared using Seaview4 (Gouy et al. 2010). The 16S rRNA sequences of the iprodione-degrading strains were deposited in the GenBank under the accession numbers MK386866 to MK386885. Characterization of the transformation pathway of iprodione by the isolated bacteria Triplicate flasks of MSM amended with iprodione (10 mg L−1) were inoculated with fresh cultures of the selected isolated bacterial strains grown to the late logarithmic phase (OD600 = 0.1 corresponding to ∼2 × 107 cells mL−1). Triplicate non-inoculated flasks for each medium were also prepared as abiotic controls. All samples were incubated on a shaking platform at 25°C. The degradation of iprodione and the formation of metabolite I, metabolite II and 3,5-DCA were measured immediately after inoculation and at regular intervals thereafter by High Performance Liquid Chromatography – Photodiode Array Detection (HPLC–PDA) as described by Campos et al. (2017). In parallel, we determined the proliferation of the two Paenarthrobacter strains along with the degradation of iprodione via q-PCR. Samples (2 mL) were collected from all bacterial cultures at regular intervals and used for DNA extraction with the Nucleospin Tissue kit (MACHEREY-NAGEL GmbH & Co. KG, Germany). DNA was quantified by the Qubit® 2.0 Fluorometer (Life Technologies, Paisley, UK). A set of primers Paen_F and Paen_R designed to amplify a 292-bp fragment of the 16S rRNA gene of the Paenarthrobacter strains was used. Details of primers (design, quality control and sequences), q-PCR conditions, reagents, efficiency and copy number determination are given in the Supporting Information. Data analysis Pesticides degradation kinetics The degradation data of iprodione were fitted to four kinetic models as suggested by the FOCUS working group (FOCUS 2006). The goodness of fit per model was assessed using a χ2 test (<15%, for a = 0.05), visual inspection and the distribution of residuals. All kinetic analyses were performed on the R software with the mkin package. Significant differences (P < 0.05%) between the degradation rates (kdeg) of the repeated applications of iprodione in soil and on plant leaves were determined with the student's t-test as described in the Supporting Information. Statistical analysis of microbial diversity data The OTU matrices of bacteria, archaea and fungi were used to assess the impact of iprodione and 3,5-DCA on the α- and β-diversity. The impact of iprodione on the α-diversity was determined via calculation of the diversity indices richness (S), Fisher Alpha, Inverse Simpson, Shannon (Jost 2006) and Pielou's evenness (Pielou 1975). The data per habitat were subjected to two-way ANOVA and post-hoc tests to determine the impact of iprodione and time (main factors) on the α-diversity of bacteria, archaea and fungi and also on the relative abundance of the major bacterial, archaeal and fungal taxa. Differential abundance tests for identifying taxa and OTUs that were responsive to iprodione treatment were performed using the Fisher's exact test for P-values of 0.05 as adjusted according to the Benjamini–Hotchberg algorithm (Benjamini and Hochberg 1995). To enhance the statistical test sensitivity, only the differentially abundant OTUs were used for downstream multivariate tests that provided the variance portion of these subcommunities that coincided with the experimental treatments. Detrended correspondence analysis was performed, and Canonical correspondence analysis (CCA) was preferred over redundancy analysis (RDA) if the first axis value was higher than 3 standard deviations (Lepš and Šmilauer 2003). Spearman's correlation tests between the measured concentrations of iprodione and 3,5-DCA in soil and on plant leaves and the sequence counts of bacterial, fungal genera and archaeal classes were carried out to assess possible effects of either the parent compound or 3,5-DCA on the microbial community members. All statistical analyses were performed with the R v3.5.2 software (R Core Team 2017). The data were submitted to Sequence Read Archive of NCBI with bioproject accession number PRJNA513949. RESULTS Degradation of iprodione in soil and phyllosphere The degradation patterns of iprodione in soil and on plant leaves are presented in Fig. 1. In all cases, the degradation of iprodione was best described by single first order (SFO) kinetics (Table 1). Iprodione showed a rapid degradation in soil observed even from the first application (DT50 = 1.24 days) (Table 1). Its degradation rate remained constantly high in the second (DT50 = 1.23 d), third (DT50 = 1.14 d) and fourth application (DT50 = 0.45 d). On plant leaves, no degradation of iprodione was observed during the 30 days after the first application of iprodione (DT50 extrapolated > 365 days). However, a significant increase (P< 0.05) in its degradation was evident in the second, third and fourth application with DT50 values of 15.1, 11.5 and 5.95 days, respectively (Table 1). The degradation of iprodione in soil was accompanied by the transient formation of 3,5-DCA (Fig. 1A). In contrast, on pepper leaves, no 3,5-DCA or any of the other transformation products considered (metabolites I and II) were detected during iprodione degradation (Fig. 1B). Figure 1. Open in new tabDownload slide The dissipation patterns of the four successive applications of iprodione (•) and the formation and dissipation of its main metabolic product 3,5-DCA (▓) in soil (A) and plant leaves (B). Each value is the mean of three replicates with error bars representing the standard deviation of the mean. Figure 1. Open in new tabDownload slide The dissipation patterns of the four successive applications of iprodione (•) and the formation and dissipation of its main metabolic product 3,5-DCA (▓) in soil (A) and plant leaves (B). Each value is the mean of three replicates with error bars representing the standard deviation of the mean. Table 1. The kinetic parameters describing the degradation of iprodione in soil and leaves of pepper plants calculated by fitting the data to the SFO kinetics model. Habitat . Application number . DT50 (days) . χ2 (%) . Soil 1 1.24 6.7 2 1.23 7.1 3 1.14 7.7 4 0.45 14.3 Leaves 1 >365 1.3 2 15.10 7.2 3 11.50 20.6 4 5.95 14.3 Habitat . Application number . DT50 (days) . χ2 (%) . Soil 1 1.24 6.7 2 1.23 7.1 3 1.14 7.7 4 0.45 14.3 Leaves 1 >365 1.3 2 15.10 7.2 3 11.50 20.6 4 5.95 14.3 Open in new tab Table 1. The kinetic parameters describing the degradation of iprodione in soil and leaves of pepper plants calculated by fitting the data to the SFO kinetics model. Habitat . Application number . DT50 (days) . χ2 (%) . Soil 1 1.24 6.7 2 1.23 7.1 3 1.14 7.7 4 0.45 14.3 Leaves 1 >365 1.3 2 15.10 7.2 3 11.50 20.6 4 5.95 14.3 Habitat . Application number . DT50 (days) . χ2 (%) . Soil 1 1.24 6.7 2 1.23 7.1 3 1.14 7.7 4 0.45 14.3 Leaves 1 >365 1.3 2 15.10 7.2 3 11.50 20.6 4 5.95 14.3 Open in new tab The impact of iprodione on the microbial community The composition of the soil and epiphytic microbial community In total, 1596 046 quality sequences for bacteria and archaea (9959–59 201 and 10 132–45 180 sequences per sample in soil and leaves, respectively) and 1200 925 for fungi (9137–29 835 and 8344–35 455 sequences per sample in soil and leaves, respectively) were obtained. These were assigned to 4872 OTUs for bacteria and archaea, and to 4560 OTUs for fungi. Rarefaction curves reached a plateau in all samples suggesting that our sequencing effort adequately covered the diversity of epiphytic and soil bacteria, archaea and fungi (Figure S2, Supporting Information). This is further supported by the Good's coverage (Good 1953) values for bacteria, archaea and fungi, which were 98.3 ± 0.0%, 92.6 ± 0.1% and 99.7 + 0.0%, respectively (Table S3, Supporting Information). Soil and plant leaves supported distinct bacterial, fungal and archaeal communities (Fig. 2). The epiphytic bacterial community was dominated by Proteobacteria (mostly γ-, α- and β-Proteobacteria), which constituted on average >50% of the total bacterial community, followed by Actinobacteria (mostly of the class of Rubrobacter) and Bacilli (Fig. 2A). In contrast, the soil bacterial community showed a more even composition with high abundance of Actinobacteria (Rubrobacteria, Thermoleophilia), followed by Proteobacteria (γ- and α-Proteobacteria) and Bacilli (Fig. 2A). The Soil Crenarchaeotic Group (SCG) prevailed in the phyllosphere at the earlier sampling dates, while Aenigmarchaeota, Eyryarchaeota and Bathyarchaeota were abundant only sporadically and their relative abundance did not follow a temporal or treatment trend (Fig. 2B). The soil archaeal community was dominated by SCG, while Thermoplasmata were detected at low abundances throughout the experimental duration in all samples (Fig. 2B). Τhe epiphytic fungal community was dominated by Ascomycetes (mainly Dothideomycetes, Sordariomycetes, Microbotryomycetes), and Basidiomycetes (mostly Tremellomycetes) were detected at a lower abundance (Fig. 2C). Ascomycetes (Sordariomycetes, Dothideomycetes, Eurotiomycetes, Pezizomycetes) also prevailed in soil, while Basidiomycetes (Agaricomycetes, Tremellomycetes) were less abundant (Fig. 2C). Significant temporal patterns on the relative abundance of certain bacterial, archaeal and fungal taxa were observed only in the phyllosphere regardless of iprodione treatment: (i) γ- and α-Proteobacteria relative abundance showed complementary patterns during the experimental duration. The former showed significantly higher (P< 0.05) and lower relative abundance (P < 0.05) after the 2nd and 4th applications and after the 1st and 3rd application, respectively. An exact opposite pattern was evident for α-Proteobacteria. (ii) The relative abundance of Tremellomycetes and Microbotryomycetes increased with time (P < 0.001). (iii) The relative abundance of SCG decreased with time (P< 0.05) (Fig. 2). Figure 2. Open in new tabDownload slide The relative abundance of the major classes of bacteria (A), archaea (B) and fungi (C) in the phyllosphere and soil of pepper plants repeatedly treated or not treated (control) with iprodione. Captions below each bar represent the number of application (1 to 4) followed by the sampling day per application (0, 10 or 30 days). The values presented at each time are the average of three biological replicates. Within each microbial taxon per treatment, statistical differences between time points are denoted with lower case letters. Figure 2. Open in new tabDownload slide The relative abundance of the major classes of bacteria (A), archaea (B) and fungi (C) in the phyllosphere and soil of pepper plants repeatedly treated or not treated (control) with iprodione. Captions below each bar represent the number of application (1 to 4) followed by the sampling day per application (0, 10 or 30 days). The values presented at each time are the average of three biological replicates. Within each microbial taxon per treatment, statistical differences between time points are denoted with lower case letters. Effects of iprodione on the diversity of the microbial community Iprodione did not induce significant effects on the α-diversity of bacteria, archaea and fungi in soil as shown by the different diversity indices (Figure S1, Supporting Information), whereas foliage applications of iprodione induced significant effects on the α-diversity of fungi (increase of Simpson index P < 0.05) and archaea (increase of Pielou's evenness index, P < 0.05) (Figure S1, Supporting Information). CCA or RDA explored the effect of iprodione on the β-diversity in the two studied habitats and identified OTUs that increased in relative abundance in the presence or in the absence of iprodione exposure. The fungicide had a significant (P < 0.001) treatment-wise effect on the structures of bacterial community members in both habitats (Fig. 3A and B). In soil, OTUs belonging to α-Proteobacteria (Rhizobium, Rubellimicrobium, Microvirga, Altererythrobacter), Gemmatimonadetes(Longimicrobium), Chloroflexi and Blastococcus increased in relative abundance in the samples treated with iprodione (Fig. 3A). In the phyllosphere, OTUs belonging to Bacteroidetes (Mucilaginibacter, Spirosoma) Enterococcus and Entomoplasmateles showed increasing abundance in the iprodione-treated samples, whereas OTUs belonging to Actinobacteria (Corynebacterium, Arthrobacter, Pseudonocardia), Staphylococcus and Escherichia–Shigella showed increased abundance in the phyllosphere of plants not treated with iprodione (Fig. 3B). When the impact of iprodione on the β-diversity of archaea was investigated, RDA (Fig. 3C) and CCA (Fig. 3D), revealed a significant effect (P < 0.01) only in soil, where several OTUs (135, 751, 1042) affiliated to Canditatus Nitrososphaera showed increased abundance in the non-treated samples (Fig. 3C). Iprodione induced significant treatment-wise changes (P< 0.001) in the β-diversity of fungi in both studied habitats (Fig. 3E and F). OTUs belonging to Dothideomycetes(Lasiosphaeriaceae), Sordariomycetes (Fusarium, Nectriaceae, Clonostachys), Basidiomycetes (Entoloma) and Chytridiomycetes(Spizellomyces) increased in relative abundance in the soil samples treated with iprodione, in contrast to OTUs belonging to Cladosporium and Aureobasidium, which showed increased abundance in the samples not treated with iprodione (Fig. 3E). In the phyllosphere, we observed OTUs belonging to Saccharomycetes, Sordariomycetes(Nigrospora), Mucorales, Chytridiomycetes(Rhizophlyctidales) and Basidiomycetes(Puccinia) that showed increased abundance in iprodione-treated plants, compared to OTUs belonging to Agaricomycetes(Hypholoma, Parasola) and Taphrinomycetes(Taphrina), which flourished in the samples not treated with iprodione (Fig. 3F). Figure 3. Open in new tabDownload slide Multivariate analysis (CCA or RDA depending on the outcome of the first axis or detrended correspondence analysis) of the bacterial (A, B), archaeal (C, D)and fungal (E, F)OTU matrix in soil (A, C, E) and in the phyllosphere (B, D, F). The tested model was that of the community structure (bacterial/fungal/archaeal) being a function of the iprodione application with the coefficient of determination providing the model shared variance and the P-value indicating the null hypothesis probability (i.e. no effect). Arrows indicate the OTU gradients among samples as linearly regressed to the sample scores (i.e. OTUs are more abundant in the samples of their arrow directions). Due to the fact that the tested parameter is one, only a single axis (X-axis) is canonical (contains the constrained variance) and the second axis (Y-axis) is that of the first principal component or the first correspondence analysis axis. Figure 3. Open in new tabDownload slide Multivariate analysis (CCA or RDA depending on the outcome of the first axis or detrended correspondence analysis) of the bacterial (A, B), archaeal (C, D)and fungal (E, F)OTU matrix in soil (A, C, E) and in the phyllosphere (B, D, F). The tested model was that of the community structure (bacterial/fungal/archaeal) being a function of the iprodione application with the coefficient of determination providing the model shared variance and the P-value indicating the null hypothesis probability (i.e. no effect). Arrows indicate the OTU gradients among samples as linearly regressed to the sample scores (i.e. OTUs are more abundant in the samples of their arrow directions). Due to the fact that the tested parameter is one, only a single axis (X-axis) is canonical (contains the constrained variance) and the second axis (Y-axis) is that of the first principal component or the first correspondence analysis axis. Further Spearman's correlation tests identified significant correlations between iprodione and 3,5-DCA concentrations and the abundance of bacterial and fungal genera, and archaeal classes, obtained from amplicon sequencing (Figure S3, Supporting Information). Hence, 3,5-DCA concentrations in soil were negatively correlated with Sphingomonas and positively correlated with Thermoplasmatales. Iprodione concentrations on the phyllosphere were positively correlated with fungi belonging to the genera of Coniosporium, ChalastosporaandAlternaria, and negatively correlated with fungi of the genera Sordaria, Rhodotorula and Bensingtonia. Isolation of iprodione-degrading bacteria from soil and phyllosphere The transformation of iprodione in the enrichment cultures inoculated with soil and epiphytic microbial pellet was rapid in all media, while a slower degradation of iprodione was observed in the non-inoculated samples throughout the enrichment cultures (Figure S4, Supporting Information). From the 120 colonies screened for iprodione degradation in the corresponding media, two colonies (TA1 and TA2) obtained from the MSM+iprodione soil enrichment cultures and three colonies obtained from the MSM+iprodione (LP1, LP8) and MSM+SC (LP13) leaf enrichment cultures achieved >90% degradation in 6 days, compared to each medium control. Further subculturing and purification tests resulted in the isolation of three pure cultures named TA1.6, TA1.8 and LP13.7, which were composed of the same colony morphotype. Phylogenetic analysis based on the sequences of the 16S rRNA gene showed that all three isolates were closely associated and belonged to the genus Paenarthrobacter with highest match to a P. nitroguajacolicus strain (>99%) (Fig. 4). Sequencing alignment of the full-length 16S rRNA gene showed that the leaf isolate LP13.7 differed by the soil isolates TA1.6 and TA1.8 in 1 and 2 bp, respectively, while the two soil isolates showed variation in three nucleotides. Figure 4. Open in new tabDownload slide Phylogenetic analysis of the iprodione-degrading isolates TA1.6, TA1.8 and LP13.7 based on the complete 16S rRNA gene sequence. One thousand bootstrap replicates were run with PhyML following the GTRGAMMAI (General Time Reversible with GAMma rate heterogeneity and considering Invariable sites) model. The bootstrap support is expressed in a scale from 0 to 100. The NCBI accession numbers of each bacterium are indicated. Figure 4. Open in new tabDownload slide Phylogenetic analysis of the iprodione-degrading isolates TA1.6, TA1.8 and LP13.7 based on the complete 16S rRNA gene sequence. One thousand bootstrap replicates were run with PhyML following the GTRGAMMAI (General Time Reversible with GAMma rate heterogeneity and considering Invariable sites) model. The bootstrap support is expressed in a scale from 0 to 100. The NCBI accession numbers of each bacterium are indicated. Transformation of iprodione by the isolated bacteria TA1.8 (soil-derived) transformed iprodione within 69 h with a DT50 of 19.8 h, as calculated by fitting the SFO kinetic model to the degradation data. The transformation of iprodione was accompanied by the transient formation of 60 and 40 nmol mL−1 of metabolites I and II, respectively, at 48 h. These were further transformed to 3,5-DCA, which showed a peak concentration at 69 h and partially degraded thereafter (Fig. 5A). A similar transformation pattern was evident for LP13.7 (phyllosphere-derived) where the rapid degradation of iprodione (DT50 = 15.2 h) was accompanied by the transient formation of metabolite I and metabolite II, the latter at concentrations exceeding the 100 nmol mL−1 (Fig. 5B). Metabolite II was further transformed to 3,5-DCA, which peaked between 36 and 48 days and degraded partially until the end of the study. Q-PCR analysis revealed that the rapid degradation of iprodione was accompanied by the proliferation of both bacterial strains from 36 h to maximum abundance at 117 h (Figure S5, Supporting Information). Figure 5. Open in new tabDownload slide The degradation of iprodione (▓) and the formation and degradation of metabolite I (▲), metabolite II (•) and 3,5-dichloraniline (3,5-DCA) (○) by isolates TA1.8 (A) and LP13.7 (B) in MSM. The degradation of iprodione in non-inoculated controls is also presented (□, dashed line). Each value is the mean of three replicates with error bars representing the standard deviation of the mean. Figure 5. Open in new tabDownload slide The degradation of iprodione (▓) and the formation and degradation of metabolite I (▲), metabolite II (•) and 3,5-dichloraniline (3,5-DCA) (○) by isolates TA1.8 (A) and LP13.7 (B) in MSM. The degradation of iprodione in non-inoculated controls is also presented (□, dashed line). Each value is the mean of three replicates with error bars representing the standard deviation of the mean. DISCUSSION Repeated applications of iprodione in soil and on plant leaves resulted in rapid degradation of iprodione in both habitats with a more distinct accelerated degradation pattern observed on plant leaves. Previous studies have also reported a rapid degradation of iprodione in soils repeatedly treated with the fungicide in the laboratory and in soils from fields with history of fungicide exposure (Walker 1987; Martin et al. 1990; Mercadier et al. 1996). However, accelerated degradation of pesticides on the plant phyllosphere has not been reported before. The documented vulnerability of iprodione to accelerated biodegradation in soil coupled with the degradation pattern observed in the phyllosphere of pepper plants suggest that the epiphytic microbial community is equally capable to degrade iprodione in an accelerated mode. Amplicon sequencing analysis showed that phyllosphere and soil samples supported distinct microbial communities, in accordance with previous studies in rice (Knief et al. 2012), populus (Cregger et al. 2018) and the evergreen shrub Scaevola taccada (Amend et al. 2019). The epiphytic bacterial community was dominated by Sphingomonadales, Methylobacteriaceae and Pseudomonadaceae, in line with specific functional traits of members of these groups that support their epiphytic fitness like the efficient intracellular uptake of sugars (Sphingomonas), the assimilation of methanol released on plant phyllosphere (Methylobacteriaceae) and the motility to access nutrients (Pseudomonas) (Delmotte et al. 2009; Knief et al. 2012; Ryffel et al. 2016). Actinobacteria were the most abundant taxa in soil, as reported previously (Papadopoulou et al. 2018). The epiphytic and soil fungal communities were dominated by Ascomycetes and Basidiomycetes with different classes prevailing in the two habitats. Dothideomycetes and Tremellomycetes prevailed on plant leaves, in line with previous studies also reporting dominance of these fungal taxa on the phyllosphere of various plants (Bálint et al. 2015; Yang et al. 2016; Gdanetz and Trail 2017). Sordariomycetes and Agaricomycetes dominated in soil in accordance with their capacity to efficiently exploit nutrients available in the root zone (Hussain et al. 2011; Simoes et al. 2015; Wang et al. 2018). The SCG was dominant in the soil archaeal community followed by Thermoplasmata, in accordance with previous reports (Vasileiadis et al. 2013; Chroňáková et al. 2015). In contrast the epiphytic archaeal community was more diverse and comprised of the SCG, Aenigmarcheota and Eyryarcheota. Previous studies have suggested that archaea are under-represented in the phyllosphere (Knief et al. 2012; Müller et al. 2015), hence their epiphytic communities have not been extensively explored. Recently, Taffner et al. (2019) verified the epiphytic dominance of the SCG and Eyryarcheota on the phyllosphere of Eruca sativa; however, the factors shaping epiphytic communities of archaea remain unknown. The composition of the bacterial and fungal epiphytic and soil communities was significantly altered by the application of iprodione, in contrast to archaea whose β-diversity was significantly altered by iprodione only in soil. Recent studies using amplicon sequencing showed that iprodione, either repeatedly applied in soil (Zhang et al. 2017b,c) or used at increasing dose rates (Vasileiadis et al. 2018) induced significant changes on the β-diversity of soil bacteria and fungi. Additionally, our study provides first evidence for the response of the epiphytic microbial communities, including archaea, to pesticides exposure. We further identified OTUs that increased in relative abundance in the presence or absence of iprodione. Hence, iprodione application favored epiphytic microorganisms that are (i) involved in biomass decomposition like Mucilaginibacter (Pankratov et al. 2007), Saccharomycetes, Mucorales, Rhizophlyctidales (Letcher et al. 2008; Hoffmann et al. 2013), (ii) potential human pathogens like the lactic acid bacterium Enterococcus (Lebreton et al. 2013), often observed in plant phyllosphere (Vokou et al. 2012), and plant pathogens like Nigrospora (Wang et al. 2017) and Puccinia (Abbasi, Goodwin and Scholler 2005) and (iii) insect symbionts like Entomoplasmatales (Kautz et al. 2013). The stimulation of plant pathogenic fungi belonging to Nigrospora and Puccinia, that are within the spectrum of activity of iprodione (Mueller, Jeffers and Buck 2005), might be associated with its accelerated biodegradation on plant leaves compromising its biological efficacy. In contrast, in the absence of iprodione, we observed increased abundance of OTUs assigned to (i) potential human and plant pathogens like Staphylococcus, Escherichia–Shigella and Corynebacterium, Taphrina, respectively (Tsai et al. 2014; Chattaway et al. 2017; Oliveira et al. 2017; Richardson et al. 2018) and (ii) organic matter decomposers like Parasola and Hypholoma (Nagy et al. 2009). In soil, iprodione treatment favored OTUs of α-Proteobacteria belonging to Rhizobiales, Erythrobacteraceae, Methylobacteraceae, in line with findings by Zhang et al. (2017c), who also reported an increase in the abundance of OTUs belonging to the same α-Proteobacterial taxa in soil after four repeated applications of iprodione. This could be attributed to their involvement in growth-linked degradation of iprodione or more likely to their capacity to tolerate iprodione and occupy soil niches liberated from competition upon toxicity of iprodione. Iprodione also favored fungal OTUs that (i) are involved in cellulose decomposition like Spizellomyces (Letcher et al. 2008), (ii) are mycoparasitic like Clonostachys (Salamone, Gundersen and Inglis 2018) and (iii) belong to taxa rich in plant pathogens like Fusarium and Nectriaceae (Lombard et al. 2015), in line with the limited fungicidal activity of iprodione against Fusaria (Smiley and Craven 1979). In contrast, in the absence of iprodione exposure fungal OTUs associated with saprotrophic fungi like Cladosporium (Bensch et al. 2012) and Aureobasidium (Zalar et al. 2008) were favored. An observation worth noting was the high abundance of OTUs belonging to Candidatus Nitrososphaera, an ubiquitous soil ammonia-oxidizing crenarchaeon (Tourna et al. 2011), in the untreated soil samples denoting a potential toxicity of iprodione. Similarly, Vasileiadis et al. (2018) demonstrated a significant negative correlation between iprodione soil concentrations and the abundance of OTUs belonging to the lineage Nitrososphaerales where Candidatus Nitrososphaera belongs. Overall the application of iprodione significantly affected, positively or negatively, the abundance of OTUs assigned to microbial groups with important role for the homeostasis of the plant–soil ecosystem, which should be reconsidered in the context of the One Health system approach (Destoumieux-Garzon et al. 2018). Apart from pesticide-driven effects, we observed clear succession in the abundance of certain bacterial and fungal taxa in the phyllosphere but not in soil. Compared to soil, the leaf surface is directly exposed to extreme air temperatures, UV radiation, wind and precipitation, which could drastically affect the composition of the epiphytic community (Copeland et al. 2015; Hamonts et al. 2018). An observation worth noting is the compensatory relationship between α- and γ-Proteobacteria in the phyllosphere, regardless of iprodione treatment. Similar observations were reported in the phyllosphere of perennial biofuel crops and were attributed to nutrient availability regulated by the plant development stage (Grady et al. 2018). Enrichment cultures from soil and phyllosphere samples repeatedly treated with iprodione resulted in the isolation of phylogenetically close but not identical Paenarthrobacter strains from soil and plant phyllosphere. The genus Paenarthrobacter was recently formed by the reassignment of strains belonging to the Arthrobacter aurescens subgroup (Busse and Busse 2016). It comprises members with high catabolic versatility like the atrazine-, nicotine- and 4-nitroguaiacol-degrading strains P. aurescens TC1 (Mongodin et al. 2006), P. nicotinovorans pAO1 (Mihăşan et al. 2018) and P. nitroguaiacolicus (Kotoučková et al. 2004), respectively, showing remarkable fitness in soil (Mongodin et al. 2006) and plant phyllosphere (Scheublin and Leveau 2013; Scheublin et al. 2014). Our strains clustered together with two other iprodione-degrading strains: the recently isolated iprodione-degrading strain Paenarthrobacter YJN-5 (Yang et al. 2018) and Arthrobacter sp. strain C1 (Campos et al. 2015). The bacteria isolated hydrolyzed iprodione to 3,5-DCA with the intermediate formation of metabolites I and II. This transformation pathway is shared among bacteria isolated from soils (Athiel et al. 1995; Campos et al. 2017; Yang et al. 2018), but it is reported for the first time in bacteria isolated from the plant phyllosphere. The capacity of the isolated bacterium, from the phyllosphere, to transform iprodione to 3,5-DCA contradicts to the lack of detection of this metabolite on plant leaves despite the accelerated degradation of iprodione. This could be most probably attributed to the rapid photodegradation and volatilization of 3,5-DCA on leaves surface once formed (Papantoni, Mahiasson and Nillson 1995; Othmen and Boule 1999). The presence of extremely efficient 3,5-DCA-degrading epiphytic microorganisms that rapidly degrade 3,5-DCA is unlikely, considering the remarkable recalcitrance of 3,5-DCA to microbial degradation (Yao et al. 2011). The consistent presence of iprodione-catabolic traits in Arthrobacter-like bacteria isolated from distant geographic areas suggests a potential phylogenetic specialization of this bacterial genus in the degradation of iprodione, which is not common in the bacterial world. This is further supported by the isolation of phylogenetically related iprodione-degrading Paenarthrobacter strains from soil and plant phyllosphere in our study. The mechanism driving this potential specialization of Arthrobacter-like bacteria to iprodione biodegradation would be further explored using comparative genomics. No OTUs matching the 16S rRNA of our iprodione-degrading isolate were found in the amplicon sequences of the soil and epiphytic bacterial community. Correlation testing showed significant positive correlations between 3,5-DCA and iprodione concentration in soil and plant leaves with the abundance of bacterial, archaeal and fungal genera that have never been reported (i.e. Thermoplasmata, Coniosporium) or scarcely reported (Micromonospora, Alternaria) as pesticide degraders (Lipok et al. 2003; Fuentes et al. 2010). The positive correlation between Alternaria OTUs and iprodione concentrations might be attributed to resistance mechanisms. Alternaria plant pathogens are within the spectrum of fungicidal activity of iprodione (Mukherjee, Gopal and Chaterjee 2003) and resistance to iprodione is ubiquitous among Alternaria strains (McPhee 1980; Ma and Michailides 2004). Although our sequencing effort provided a good coverage of the bacterial diversity in soil and phyllosphere samples, it cannot be excluded that the isolated bacterium remained at low abundance throughout the pot study due to the limited growth supported by the in situ concentrations of iprodione in the soil and the phyllosphere. Gallego et al. (2019) showed that pesticide-degrading bacteria constitute a particularly small fraction of the total bacterial community whose response to repeated pesticide exposure is not often detectable with DNA-based amplicon sequencing approaches and becomes visible only when RNA-based amplicon sequencing targeting the active fraction of the bacterial community is used. Overall, repeated soil and foliage applications of iprodione induced compositional alterations in the soil and the epiphytic bacterial and fungal community. On the one hand, it affected, negatively or positively, microorganisms with critical functional roles for the homeostasis of the plant–soil system. On the other hand, it resulted in the accelerated biodegradation of iprodione, a result not previously reported in plant foliage and whose consequences for the (i) agricultural practice (i.e. loss of pesticide efficacy toward plant pathogens), (ii) environmental quality and (iii) consumers health (pesticides-free environment and products) could be important. Closely related iprodione-degrading bacteria of the genus Paenarthrobacter were isolated from soil and plant leaves repeatedly treated with iprodione, adding to the list of soil arthrobacters degrading iprodione and implying a possible phylogenetic specialization in the degradation of this compound. Further studies will aim to (i) disentangle the mechanism driving the development of pesticide accelerated biodegradation in the plant phyllosphere and (ii) explore the arsenal of genes carried by the isolated bacteria with a putative role in the transformation of iprodione using comparative genomics and transcriptomics. FUNDING AK was supported by a Ph.D. scholarship from the State Scholarship Foundation of Greece with resources of the EP ‘Development of Human Resources, Education and Life-long Learning 2014–2020’ and co-funded by the European Social Fund and the Greek State. Conflicts of interests. None declared. REFERENCES Abbasi M , Goodwin SB, Scholler M. Taxonomy, phylogeny, and distribution of Puccinia graminis, the black stem rust: new insights based on rDNA sequence data . Mycoscience . 2005 ; 46 : 241 – 7 . Google Scholar Crossref Search ADS WorldCat Amend AS , Cobian GM, Laruson AJ et al. . Phytobiomes are compositionally nested from the ground up . 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TI - The response of soil and phyllosphere microbial communities to repeated application of the fungicide iprodione: accelerated biodegradation or toxicity?
JF - FEMS Microbiology Ecology
DO - 10.1093/femsec/fiaa056
DA - 2020-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-response-of-soil-and-phyllosphere-microbial-communities-to-WO0YKD4RM4
VL - 96
IS - 6
DP - DeepDyve
ER -