Evaluation of the possible persistence of potential human pathogenic bacteria in olive orchards irrigated with treated urban wastewater

Sofo, Adriano; Mininni, Alba Nicoletta; Fausto, Catia; Scagliola, Marina; Crecchio, Carmine; Xiloyannis, Cristos; Dichio, Bartolomeo

doi:10.1016/j.scitotenv.2018.12.264

Cited by 22 publications

(26 citation statements)

References 16 publications

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“…However, while Actinobacteria and Proteobacteria bacteria were present in all three olive genotypes, only "Picual" contained Firmicutes and Bacteroidetes, and only "Acebuche" contained bacteria from the Deinococcus-Thermus phylum. These results are in accordance with those obtained previously following similar approaches from olive leaves and xylem sap (Müller et al, 2015;Fausto et al, 2018;Sofo et al, 2019) as well as from other woody plants, like grapes, oak, poplar, and Pinus (Uroz et al, 2010;Bonito et al, 2014;Cregger et al, 2018;Deyett and Rolshausen, 2019). Interestingly, our results were more in agreement with those obtained by Deyett and Rolshausen (2019) that found similar proportions for Proteobacteria and Actinobacteria on grapevine xylem sap than those on olives obtained by Fausto et al (2018), when using a similar xylem sap extraction method with a Scholander pressure chamber.…”

Section: Discussionsupporting

confidence: 93%

“…Although several studies on woody plant species have revealed the power of using culture-independent approaches for revealing previously unappreciated microbial diversity on xylem samples (Müller et al, 2015;Deyett et al, 2017;Fausto et al, 2018;Deyett and Rolshausen, 2019;Sofo et al, 2019), our study is unique in that culture-dependent and -independent methods were compared directly and simultaneously (i.e., applied to the same samples) in different genotypes of the host plant using two different extraction methods, to perform a quantitative and qualitative assessment of the xylem bacterial microbiome. Although knowing the specific limitations of each of the culture-dependent and culture-independent approaches, and the short number of olive genotypes and samples evaluated, our study is relevant in that it paves the way for future studies on the identification and characterization of olive xylem sap endophytic bacteria that could be involved in plant defense by acting as biocontrol agents against diverse xylem-inhabiting pathogens and by promoting plant health and growth promotion of olive plants.…”

Section: Discussionmentioning

confidence: 99%

“…With olive, most microbiome studies have focused on determining the microbial composition of the rhizosphere (Mercado-Blanco et al, 2004;Berg and Hallmann, 2006;Mendes et al, 2007;Aranda et al, 2011;Prieto et al, 2011;Montes-Borrego et al, 2013;Caliz et al, 2015;Gómez-Lama Cabanás et al, 2018), whereas only a few studies have focused on the endosphere or xylem microbiomes (Müller et al, 2015;Fausto et al, 2018;Sofo et al, 2019). Unraveling the microbiome of the olive xylem sap should provide a better understanding of the microbes that systemically move throughout the plant or are horizontally transmitted from plant to plant during vegetative propagation.…”

Section: Introductionmentioning

confidence: 99%

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Culture-Dependent and Culture-Independent Characterization of the Olive Xylem Microbiota: Effect of Sap Extraction Methods

et al. 2020

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Microbial endophytes are well known to protect host plants against pathogens, thus representing a promising strategy for the control of xylem-colonizing pathogens. To date, the vast majority of microbial communities inhabiting the olive xylem are unknown; therefore, this work pursues the characterization of the xylem-limited microbiome and determines whether the culture isolation medium, olive genotype, and the plant material used to analyze it can have an effect on the bacterial populations retrieved. Macerated xylem tissue and xylem sap extracted with the Scholander chamber from olive branches obtained from two cultivated and a wild olive genotypes were analyzed using culturedependent and-independent approaches. In the culture-dependent approach using four solid culture media, a total of 261 bacterial isolates were identified after performing Sanger sequencing of 16S rRNA. Culturable bacteria clustered into 34 genera, with some effect of culture media for bacterial isolation. The cultivated bacteria belonged to four phyla and the most abundant genera included Frigoribacterium (18.8%), Methylobacterium (16.4%), and Sphingomonas (14.6%). On the other hand, in the culture-independent approach conducted using Illumina MiSeq 16S rRNA amplicon sequencing [next-generation sequencing (NGS)] of the xylem extracts, we identified a total of 48 operational taxonomic units (OTUs) belonging to five phyla, being Sphingomonas (30.1%), Hymenobacter (24.1%) and Methylobacterium (22.4%) the most representative genera (>76% of reads). In addition, the results indicated significant differences in the bacterial communities detected in the xylem sap depending on the genotype of the olive tree studied and, to a minor extent, on the type of sap extraction method used. Among the total genera identified using NGS, 14 (41.2%) were recovered in the culture collection, whereas 20 (58.8%) in the culture collection were not captured by the NGS approach. Some of the xylem-inhabiting bacteria isolated are known biocontrol agents of plant pathogens, whereas for others little information is known and are first reported for olive. Consequently, the potential role of these bacteria in conferring olive tree protection against xylem pathogens should be explored in future research.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Culture-Dependent and Culture-Independent Characterization of the Olive Xylem Microbiota: Effect of Sap Extraction Methods

et al. 2020

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“…comparing the four primer pairs, results indicated that primer pair 799F-1193R recovered the highest number of bacterial OTUs (242) and displayed a low coamplification rate of organellar rRNA gene; although for several of the samples a 6 removal of unspecific bands by agarose gel purification was needed prior to library 6 sequencing. The Actinobacteria, Firmicutes and Proteobacteria phyla have been 6 described as the most abundant in olive xylem sap by other authors (Sofo et al, 2019;6 Anguita-Maeso et al, 2020). In our study, although the same most abundant phyla, were 6 detected by all PCRs; some phyla were detected exclusively by some PCR primers.…”

mentioning

confidence: 87%

Evaluation of established methods for DNA extraction and primer pairs targeting 16S rRNA gene for bacterial microbiome profiling of olive xylem sap

Haro

Anguita-Maeso

Metsis³

et al. 2020

Preprint

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Next Generation Sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number of factors can affect the accuracy of microbial community assessment, such as the DNA extraction method, the hypervariable region of 16S rRNA gene targeted or the PCR primers used for amplification. The aim of this study was to assess the influence of commercially available DNA extraction kits and different primer pairs to provide a nonbiased vision of the composition of bacterial communities present in olive xylem sap. For that purpose, branches from ‘Picual’ and ‘Arbequina’ olive cultivars were used for xylem sap extraction using a Scholander chamber device. The DNA extraction protocol significantly affected xylem sap bacterial community assessment. That resulted in significant differences in alpha (Richness) and beta diversity (UNIFRAC distances) metrics among DNA extraction protocols, with the 12 DNA extraction kits evaluated being clustered in four groups behaving differently. Although the core number of taxa detected by all DNA extraction kits included four phyla, seven classes, 12 orders, and 16 or 21 families, and 12 or 14 genera when using the Greengenes or Silva database for taxonomic assignation, respectively, some taxa, particularly those identified at low frequency, were detected by some DNA extraction kits only. The most accurate depiction of a bacterial mock community artificially inoculated on sap samples was generated when using the PowerPlant DNA extraction Kit, the combination of 799F/1193R primers amplifying the hypervariable V5-V7 region and the Silva 132 database for taxonomic assignation. The DESeq2 analysis displayed significant differences among genera abundance between the different PCR primer pairs tested. Thus, Enterobacter, Granulicatella, Prevotella and Brevibacterium presented a significant higher abundance in all PCR protocols when compared with primer pair 799F/1193R, while the opposite was true for Pseudomonas and Pectobacterium. The methodological approach followed in this study can be useful to optimize plant-associated microbiome analysis, especially when exploring new plant niches. Some of the DNA extraction kits and PCR primers selected in this study will contribute to better characterize bacterial communities inhabiting within the xylem sap of olives or other woody crop species.

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“…Particularly, under Mediterranean climates, a sustainable soil management aimed at increasing soil organic carbon and microbiological fertility is of key importance. In these environments, the need for a new approach in orchard management is urgent for improving or maintaining soil quality, health, and fertility (Sofo et al, 2019a). Particularly, in fruit crops, the positive influence of sustainable management systems on soil physical, biochemical, and microbiological characteristics has been described (Sofo et al, 2019b).…”

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confidence: 99%

Microbial ecology in sustainable fruit growing: Genetic, functional, and metabolic responses

et al. 2020

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Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

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Evaluation of the possible persistence of potential human pathogenic bacteria in olive orchards irrigated with treated urban wastewater

Cited by 22 publications

References 16 publications

Culture-Dependent and Culture-Independent Characterization of the Olive Xylem Microbiota: Effect of Sap Extraction Methods

Culture-Dependent and Culture-Independent Characterization of the Olive Xylem Microbiota: Effect of Sap Extraction Methods

Evaluation of established methods for DNA extraction and primer pairs targeting 16S rRNA gene for bacterial microbiome profiling of olive xylem sap

Microbial ecology in sustainable fruit growing: Genetic, functional, and metabolic responses

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