Christina Papazlatani

Soil fumigants constitute a major tool for the control of soil borne fungal plant pathogens in protected crops. Dimethyl disulfide (DMDS) is a novel soil fumigant used either alone or in combination with other fumigants for the control of soil borne pests and diseases. In a commercial greenhouse for tomato production, we evaluated the impact of DMDS, comparatively to the alternative fumigant metam sodium, on the population of the dominant fungal plant pathogens in the study soil via q-PCR. Prior to soil fumigation, estimation of the fungal diversity in the studied soil via clone libraries identified Fusarium oxysporum and Rhizoctonia solani as the most abundant soil borne plant pathogens, while Cladosporium spp., known as opportunistic airborne tomato pathogens, were the most dominant fungi and based on this their dynamics upon fumigation was also studied. DMDS, at two dose rates, induced a drastic reduction in the population of F. oxysporum and R. solani, which lasted for the whole cultivation season. On the contrary, metam sodium exhibited an inhibitory effect on F. oxysporum that was alleviated at 120 d post fumigation. Both DMDS and metam sodium induced only a temporal reduction in the soil population of Cladosporium sp. which recovered by 60 days post fumigation. Our data suggest that DMDS even at the low dose rate (56.4 g m−2) could drastically reduce the population of the major soil borne tomato pathogens F. oxysporum and R. solani. Establishment of population thresholds as determined by q-PCR could represent a valuable tool for the estimation of the risk for disease severity and crop yield losses.

Agro-food industries that use pesticides constitute significant point sources for the contamination of natural water resources. Despite that, little is known about the treatment of their pesticide-contaminated effluents. Biobeds could be a possible solution for the depuration of these effluents. In this context, we explored the degradation and adsorption of pesticides used in seed-coating (carboxin (CBX), metalaxyl-M (MET-M), fluxapyroxad (FLX), fludioxonil (FLD)), bulb-dipping (chlorothalonil (CHT), thiabendazole (TBZ), FLD) and fruit-packaging activities (FLD) in a biomixture, used as biobed packing material, and in soil. The degradation of pesticides was tested individually and in mixtures relevant to their industrial use, while FLD was also tested at different concentrations (10, 20, and 150 mg kg−1) representing its use in the different industries. CBX, FLD, and CHT, when applied individually, and all other pesticides when applied in mixtures, degraded more rapidly in biomixture than in soil. In most cases pesticides application in mixtures retarded their degradation. This was more pronounced in soil than in biomixture, especially for MET-M and FLD. CHT had the most prominent inhibitory effect on the degradation of TBZ and FLD. FLD degradation showed a dose-dependent pattern (DT50 42.4 days at 10 mg kg−1 and 107.6 days at 150 mg kg−1). All pesticides showed higher adsorption affinity in the biomixture (Kf = 3.23–123.3 g mL−1) compared to soil (Kf = 1.15–31.2 g mL−1). We provide initial evidence for the potential of the tested biomixture to remove pesticides contained in effluents produced by different agro-industrial activities. Tests in full-scale biobeds packed with this biomixture will unravel their full depuration potential for the treatment of these agro-industrial effluents.

The impact of DMPP (3,4-dimethylpyrazole phosphate), applied at two doses (low: recommended for agronomic use; high: > 100 × the recommended), on the function, diversity, and dynamics of target microorganisms (ammonia-oxidizing microorganisms, AOM), functionally associated microorganisms (nitrite-oxidizing bacteria (NOB) and denitrifiers), and total prokaryotic and fungal microbial communities was assessed in two loamy soils, mainly differing in pH (acidic vs. alkaline), in a 35-day microcosm study. This was achieved via monitoring inorganic N-pools, potential nitrification (PN) rates, amoA gene and transcripts abundance, the abundance of other phylogenetic marker genes (nxrB, narG, nirS, nirK, nosZ, 16S rRNA, 18S rRNA), and amplicon sequencing of amoA, 16S rRNA, and ITS. Overall, DMPP was more persistent in the acidic soil. Its low dose successfully inhibited nitrification in the alkaline but not in the acidic soil, where effective inhibition was observed only at the high dose. This was mainly attributed to the consistently higher activity of DMPP towards ammonia-oxidizing bacteria (AOB) prevailing in the alkaline soil, unlike ammonia-oxidizing archaea (AOA) whose abundance and transcriptional activity was reduced only by the high dose. DMPP, at the high dose, reduced the abundance of Nitrobacter but not Nitrospira NOB, while its low dose increased the abundance of denitrifying bacteria, prokaryotic, and fungal populations in the alkaline soil. Amplicon sequencing revealed that DMPP imposed significant changes in the composition of the prokaryotic, fungal, and AOB communities in both soils, unlike AOA which were less responsive. These were associated with dose-dependent changes in the abundance of bacteria and fungi known to control key soil functions implying possible effects for the soil ecosystem homeostasis. Our study paves the way for a more comprehensive analysis of the effects of NIs on the soil microbial community, beyond the current focus on target AOM.

Imazalil (IMZ) is an imidazole fungicide commonly used by fruit-packaging plants (FPPs) to control fungal infections during storage. Its application leads to the production of pesticide-contaminated wastewaters, which, according to the European Commission, need to be treated on site. Considering the lack of efficient treatment methods, biodepuration systems inoculated with tailored-made inocula specialized on the removal of such persistent fungicides appear as an appropriate solution. However, nothing is known about the biodegradation of IMZ. We aimed to isolate and characterize microorganisms able to degrade the recalcitrant fungicide IMZ and eventually to test their removal efficiency under near practical bioengineering conditions. Enrichment cultures from a soil receiving regular discharges of effluents from a FPP, led to the isolation of a Cladosporium herbarum strain, which showed no pathogenicity on fruits, a trait essential for its biotechnological exploitation in FPPs. The fungus was able to degrade up to 100 mg L−1 of IMZ. However, its degrading capacity and growth was reduced at increasing IMZ concentrations in a dose-dependent manner, suggesting the involvement of a detoxification rather than an energy-gain mechanism in the dissipation of IMZ. The isolate could tolerate and gradually degrade the fungicides fludioxonil (FLD) and thiabendazole (TBZ), also used in FPPs and expected to coincide alongside IMZ in FPP effluents. The capacity of the isolate to remove IMZ in a practical context was evaluated in a benchtop immobilized-cell bioreactor fed with artificial IMZ-contaminated wastewater (200 mg L−1). The fungal strain established in the reactor, completely dominated the fungal community and effectively removed >96% of IMZ. The bioreactor also supported a diverse bacterial community composed of Sphingomonadales, Burkholderiales and Pseudomonadales. Our study reports the isolation of the first IMZ-degrading microorganism with high efficiency to remove IMZ from agro-industrial effluents under bioengineering conditions.

Agro-food processing industries generate large amounts of pesticide-contaminated effluents that pose a significant environmental threat if managed improperly. Biopurification systems like biobeds could be utilized for the depuration of these effluents although direct evidence for their efficiency are still lacking. We employed a column leaching experiment with pilot biobeds to (i) assess the depuration potential of biobeds against fungicide-contaminated effluents from seed-producing (carboxin, metalaxyl-M, fluxapyroxad), bulb-handling (thiabendazole, fludioxonil and chlorothalonil) and fruit-packaging (fludioxonil, imazalil) industries, (ii) to monitor microbial succession via amplicon sequencing and (iii) to determine the presence and dynamics of mobile genetic elements like intl1, IS1071, IncP-1 and IncP-1ε often associated with the transposition of pesticide-degrading genes. Biobeds could effectively retain (adsorbed but extractable with organic solvents) and dissipate (degraded and/or not extractable with organic solvents) the fungicides that were contained in the agro-industrial effluents with 93.1–99.98% removal efficiency in all cases. Lipophilic substances like fluxapyroxad were mostly retained in the biobed while more polar substances like metalaxyl-M and carboxin were mostly dissipated or showed higher leaching potential like metalaxyl-M. Biobeds supported a bacterial and fungal community that was not affected by fungicide application but showed clear temporal patterns in the different biobed horizons. This was most probably driven by the establishment of microaerophilic conditions upon water saturation of biobeds, as supported by the significant increase in the abundance of facultative or strict anaerobes like Chloroflexi/Anaerolinae, Acidibacter and Myxococcota. Wastewater application did not affect the dynamics of mobile genetic elements in biobeds whose abundance (intl1, IS1071, IncP-1ε) showed significant increases with time. Our findings suggest that biobeds could effectively decontaminate fungicide-contaminated effluents produced by agro-food industries and support a rather resilient microbial community.