Bacterial diversity was explored among field samples and cultured isolates from coral reefs within the Veracruz Reef System. Bacterioplankton and bacteriobenthos were characterized by pyrosequencing 16S rRNA genes. Identified sequences belonged to the kingdom Bacteria and classified into 33 phyla. Proteobacteria (likely SAR11 clade) dominated in collective field samples, whereas Firmicutes were the most abundant taxa among cultured isolates. Bioinformatic sorting of sequences to family level revealed 223 bacterial families. Pseudomonadaceae, Exiguobacteraceae and Bacillaceae were dominant among cultured isolates. Vibrionaceae, Alteromonadaceae, and Flavobacteriaceae dominated in reef-associated sediments, whereas Rickettsiaceae and Synechoccaceae were more highly represented in the water column. Bacterial communities from sediments were more diverse than from the water column. This study reveals cryptic bacterial diversity among microenvironmental components of marine microbial reef communities subject to differential influence of anthropogenic stressors. Such investigations are critical for constructing scenarios of environmentally induced shifts in bacterial biodiversity and species composition. Coral reefs are among the best known examples of ecosystems subject to rapid loss of biodiversity within the last several decades . As arguOne of the main goals in biodiversity research on microbes has been to understand their functional role and impact within and among ecosystems at local, regional, and global scales. Both prokaryotic and eukaryotic microbes play a critical role in primary production and in biogeochemical cycles in the ocean , includiThe Veracruz Reef System (VRS) comprises 23 reefs within a marine national park adjacent to the municipalities of Veracruz, Boca del Rio and Alvarado in the southwestern Gulf of Mexico . The VRSThe multi-source but anthropogenically created stress regime provides a suitable environment for exploring how microbial communities have adapted to these unusual conditions. For some reefs within the VRS the coral coverage has been seriously negatively affected while algal biomass and spatial coverage have been increasing . If evenConservation of function and ecosystem services are essential to maintaining healthy reef systems, but to date there are no published comparative studies about the microorganisms present in the VRS, and the functional role they could play in maintaining diversity and ecosystem stability. Although the close relationship between coral reefs and the presence of a unique microbiota has been documented elsewhere ,16, dataThe main objective of this work was to determine if there are discernable bacterial diversity differences between two adjacent coral reefs of the VRS—one close to the port of Veracruz and thus heavily impacted, and the other more distant and less affected by human activities. We analyzed the diversity of bacteria from field populations collected in different environmental compartments—pelagic water column, sediment, and external mucus from corals—from one conserved reef and compared the composition with a more heavily impacted reef to obtain basic knowledge regarding the microbiota. Although we are aware of the culture selection bias, we also compiled DNA sequencing datasets for bacterial diversity from cultured isolates collected from the VRS for comparison. Pyrosequencing of 16S ribosomal DNA was performed from bacterial samples of communities from both reefs to detect even low-abundance members of the microbial population assemblage and to achieve an appropriate degree of taxonomic resolution. A high taxonomic and phylogenetic diversity of marine bacteria was revealed for the first time from various ecological compartments in a coral reef environment in the southern Gulf of Mexico.This study focused on two coral reefs of the VRS along the Gulf of Mexico coast: Isla Verde Reef (IVR)  located offshore from Veracruz, and Punta Gorda Reef (PGR)  north of the city and more heavily affected by adjacent port activities . Data onThalassia testudinum Banks ex König) (T), and from a mucus-like uncharacterized biofilm upon the respective substrate (B). The natural bacterial community was sampled from the water column (11 m depth) and underlying sediments from the same location at IVR in the rainy season in September 2010, and in March 2012 during a transitional period from dominant northerly winds to the dry season. In addition, during the latter sampling period, mucus slurry (5 mL) was carefully collected with sterile syringes from the surface of the massive starlet coral Siderastrea siderea Ellis et Solander for comparison of bacterial composition from apparently healthy specimens and from corals afflicted with prominent necrotic black patches. The more anthropogenically impacted PGR system was also sampled from the water column (3.4 m depth) for bacterioplankton and bacteriobenthos from sediments in May 2012 in the dry season.The IVR was sampled during the rainy season in August 2009 for bacteria to isolate into culture from the water column (W) (1.5 m depth), sediments (S), seagrass  in an ice chest with frozen gel packs to the laboratory. Water column samples were successively filtered with a vacuum pump  at 20 mbar through 5, 1.2 and 0.22 µm pore size nitrocellulose membranes (47 mm diameter) . Sediments were first washed with 5 L heat-sterilized seawater  and the aqueous slurry was filtered as above for the seawater samples. Filters were cut into four equal quadrants for different experiments: one quadrant each of the 5 µm pore size membranes of both water column and sediment samples were used for bacterial cultures, whereas 1.2 and 0.22 µm membranes were retained for rDNA analysis. DNA samples were archived frozen at −70 °C in 0.22 µm sterile-filtered STET lysis solution  .−1, NaCl 5 g·L−1, yeast extract 5 g·L−1) prepared with seawater of the sampling zone  in 250 mL glass bottles placed upon a shaker platform operated at 100 rpm. All cultures exhibiting growth were transferred onto LB (20 g L−1) agar plates by streaking with a flame-sterilized bacterial loop. Each isolated colony strain was characterized based on the following criteria: colony characteristics , cell shape, Gram-staining and 16S rDNA gene sequence. Bacterial cultures from IVR seawater, sediment, seagrass, and mucus biofilm were grown in LB broth  at time t1 and t2, respectively.Experiments on isolates in broth culture were performed over a broad temperature range of 1 to 75 °C with intermediate intervals of 33, 40, 60, 65, and 70 °C to determine temperature tolerance and growth characteristics. Generation times were calculated by spectrophotometry  from the change in optical density measured at 650 nm. Calculated parameters for growth kinetics were specific growth rate (µ) and duplication time (dt) determined through the culture growth cycle , accordiTotal DNA was extracted and prepared for pyrosequencing for comparison of the bacterial taxonomic composition of diverse ecological compartments from two different coral reefs of the VRS. DNA was recovered from the isolated cultured strains and from the 1.2 and 0.22 µm-filtered natural seawater and sediment samples, and mucus slurry from corals, to construct the DNA library. g  at 5 °C for 10 min to pellet cells. Pellets were re-suspended in 500 µL STET lysis solution for 15 min, then transferred to a 1.5 mL Eppendorf microfuge tube with 100 µL of lysozyme, mixed by pipetting, and incubated at 37 °C for 45 min. The reaction was stopped at 97 °C by 50 s immersion in a hot water bath. Samples were centrifuged  at 16,000× g for 12 min at 4 °C. The supernatant was pipetted into a new Eppendorf tube and protein was precipitated with 500 µL phenol by gentle mixing. Each sample was centrifuged as above, and the supernatant was transferred to a new 1.5 mL Eppendorf tube. Tubes were placed in ice bath for 5–10 min after adding 50 µL sterile 4M LiCl, then centrifuged at 16,000× g for 12 min at 4 °C, after which each supernatant was transferred into a new 1000 µL Eppendorf tube. Samples were precipitated with 500 µL isopropanol at room temperature for 5 min. Samples were centrifuged as described and supernatants were discarded. Then 100 µL of 80% ice-cold ethanol was added and decanted immediately, after which the DNA sample adhered to the vial wall was dried at room temperature for 2 h. Finally, the samples were re-suspended in 50 µL sterile nuclease-free water.DNA extraction followed well established methods ,19, withEcoR1 + HindIII restriction enzymes. DNA was quantified at 260 nm in a spectrophotometer  and stored at −20 °C until further analysis.Size and DNA quality were verified by electrophoresis (110 V) in a 1% agarose gel using lambda phage as reference ladder and digested with ca. 1.5 kb) were purified with a Wizard purification kit , and resuspended in heat-sterilized water prepared with a Millipore RiOs-Di 3 water purification system  to a final concentration of 500 ng µL−1. The 16S rRNA gene recovered from each strain was checked for purity with a NanoDrop spectrophotometer . The PCR was run in a BioRad thermal cycler  with bacteria-specific primers 27F and 1492R, as described for coral-associated bacteria . The conThe amplified product was sequenced by the Sanger method  at the Biotechnology Institute, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico (IBT). Sequences were edited and analyzed with 4Peaks software  by processing with the 4Peak 1.7.1 program, using the fragments considered in the program-calculated threshold. Identification and comparison of the sequences was conducted by Basic Local Alignment Search Tool (BLAST) and Ribosomal Database Project (RDP) searches. TM Gel SpinTM DNA extraction kit . Once purified, the products were quantified by Quant-it PicoGreen assay  after pooling the samples at a concentration of 240 ng µL−1 in an equimolar amount. For quality control and to search for fragments around 500 bp, the pool was checked with a Bioanalyzer chip  with a high sensitivity kit.The 16S rRNA was amplified with the universal primer 27F, which included the primer B adaptor for pyrosequencing on the 5’ end. The 1492R included the sequencing primer A and an 8 bp barcode error-correcting Hamming sequence on the 5’ end, slightly modified as previously described , whereas the majority of the mesophiles were G(-). In particular, the thermophilic isolates were similar in morphology and physiological characteristics, with four exceptions  , seagrass (ef (IVR) , which tMost thermophilic isolates exhibited bacillary-form with cells ranging from 0.5–3 μm length and about 0.6 μm diameter. Greater morphological variation was found among the mesophilic colonies, which showed both bacillary and coccoid forms of cells in equal proportion. Growth calculated from change in optical density over a broad range of NaCl concentrations (0.5–2 M) indicated halophilic tendencies for all isolates.Maximum permissive isolation temperatures were 70 and 75 °C from biofilms, and from water and sediment, respectively, at 11 m depth at IVR. Two strains, IVR-W-10-51 from the water column and IVR-S-10-1 from sediment were at first assumed to be psychrophilic because they were isolated at 1 °C but both grew rapidly at 33 °C and generated sufficient biomass for phylogenetic analysis within 15 h. Aeribacillus pallidus, but the RDP species similarity does not confirm that the sequences are identical , with an average of 640 sequences per sample (median = 277). The number of distinct sequences retrieved varied widely among samples from different compartments between the two reef systems: from IVR-NW-10, 129 sequences and 100 genotypes; IVR-NS-10, 1113 sequences and 277 genotypes; IVR-NW-12, 3962 sequences and 2780 genotypes; IVR-NS-12, 410 sequences and 86 genotypes; PGR-NW-12, 849 sequences and 638 genotypes; and PGR-NS-12, 1320 sequences and 519 genotypes. As expected, comparison of distinct sequences recovered from a combined mix of all bacterial cultured isolates (IVR-CMix) from IVR revealed a highly reduced number (119 sequences and 80 genotypes) relative to the natural population.All sequences were classified into 33 bacterial phyla , wherebyIn general, based upon the total number of taxa represented among different major phylogenetic groups, bacterial samples recovered from sediments were more diverse than those from the water column at both reefs. For example, in sediments from PGR, only one phylum (Lentisphaerae) was absent from the total list, but it was present in sediments from IVR. In contrast, the phylum Lentisphaerae was absent from all water column samples.Pseudomonas Migula; the main identified genera were Jeotgalicoccus Yoon et al., Exiguobacterium Collins et al., Bacillus Ehrenb, and Pseudomonas Migula , and both reefs shared the Synechococcophycideae group within the water column. In the sediments, after α-Proteobacteria, the class γ-Proteobacteria was the second most represented taxonomic group (10–18%) at both reefs. In contrast, according to the pyrosequencing analysis , the claAlteromonadales was the most abundant order in sediment samples at IVR (11.3–11.7%), but was absent from PGR, where most taxa found at the latter reef belonged to the order Flavobacteriales (16.1%). In the water column, the order Synechococcales was the most abundant (23.3%) at IVR in the transition between high northerly winds and the dry season, whereas Rickettsiales dominated (26%) in the dry season. This latter order was also a major component found at PGR in the dry season, representing 11.3%, along with similar percentages for Rhodobacterales (11.2%) and Lentisphaerales (11.3%). The high percentage of Synechococcales was not found at PGR; this group was absent from PGR and was replaced by Oceanospirillales as co-dominant order .In total, 223 families of bacteria were identified from the two reef systems, with samples from the water column containing lower bacterial family diversity than from sediments. QIIME analysis showed that in general sediments recovered from IVR in September during the rainy season exhibited more taxonomic diversity (maximum 119 families at IVR-NS-10) than from the March transitional period to the dry season from PGR, representing 98 families, and IVR with 52 families . FlavobaThe three water column samples collected in different years during alternative seasons or from different reefs all shared the presence of Halomonadaceae (from 4.7% to 8.2%). In the dry season transition, members of Mamiellaceae were found in PGR samples but at higher abundance in the water column (13.5%) versus only 2.3% in sediments, and this low abundance trend was also reflected from IVR sediments (3.1%). In the same season, both reefs shared taxa belonging to the Rickettsiales  as the most abundant order. In the rainy season, members of this order (20.9%) were also present at IVR in the water column, following the high relative abundance of Synechoccaceae (23.3%). Vibrionaceae were predominant in the microbiota of sediment from both reefs, although the two reefs exhibited markedly different microbial taxonomic composition. The three different sediment samples shared the presence of the Lentisphaeraceae family (ranged from 0.5% to 4.4%) . The VibComparison of the family-level bacterial community associated with apparently diseased (IV1E-12) versus healthy (IV1S-12) coral indicated a heavy dominance of Vibrionaceae but a lower proportion of Desulfovibrionanceae and Rhodobacteraceae in healthy relative to diseased coral . No linkThe Shannon-Wiener diversity index (1) confirms the data presentation in The low success rate of isolation and growth of monoclonal bacterial isolates from diverse marine ecosystems has long been recognized as a critical limitation in valid comparisons of genetic diversity among populations and from different geographical locations . Many baExtrapolation of the responses of bacterial cultures to abiotic environmental factors  to the natural environment has also proven difficult, particularly with respect to seasonal fluctuations. Most marine bacteria from coral reefs do not exhibit favorable growth when cultured in nutrient-rich media , particu3 + NO2 + NH4) was shown to vary by approximately 50% and dissolved inorganic phosphate (PO4) by three-fold throughout the year [Environmental data suggest that bacterial community composition of the VRS may not be strictly selected for oligotrophic conditions. Although low nutrient concentrations have been considered characteristic of the VRS, dissolved inorganic nitrogen  and a transitional period between the northerly winds and the dry season; samples from PGR collected in the dry season (May 2012) also exhibited roughly similar bacterioplankton composition. Furthermore, there was a consistent pattern in bacterial composition from the sediments at both reefs, with α- and γ-proteobacteria dominating at both reefs during the three seasonal comparisons, but at the family level there were significant differences in abundances within the sediments between IVR and PGR.3 s−1, whereas this river is, in general, characterized by weak discharge (1.9 × 106 m3 year−1) during most of the year [6 m3 year−1 [−1; (2) the rainy season (June to October), when river discharges increase to >30 m3 s−1, with low wind intensities (approximately 50%) alternating between southerlies and northerlies; and (3) the dry season (October to March), with total river discharges < 30 m3 s−1, and when northerlies blow at least 70% of the time at velocities < 5 m s−1. During March there are variable transitional conditions, typically lasting up to 20 days [Environmental evidence suggests that terrestrial run-off may be a more important seasonal determinant of bacterial diversity and distribution in the VRS than seasonal water temperature shifts alone. Variability of the water temperature and salinity in the VRS is mainly determined by the discharges of the Jamapa and Papaloapan rivers. During the rainy season, the Jamapa River discharges up to 180 m 20 days . This se 20 days .2, pH) collected during sampling  have been shown to be due to pH changes, particularly affecting some pH-susceptible groups, such as γ-proteobacteriaant taxa . In the Among selective environmental factors, salinity has been reported to have the strongest impact at phylogenetic levels; with a rise of salinity α- and γ-proteobacteria can increase, contrasting with a decrease of β-proteobacteria with a decrease of salinity . BacteriCommunity composition of marine bacteria can respond to local environmental conditions, such as salinity and temperature effects on biofilms , but theJeotgalicoccus halotolerans Yoon et al. and J. psychrophilus Yoon et al. representing more extreme environments, have been isolated from Korean fermented marine products  [Pseudomonas, are frequently isolated from coastal waters [Exiguobacterium have been previously reported [Bacteria from surface waters were the focus of our studies on cultured isolates because these populations are expected to be subject to most rapid environmental changes . This leh sauce) . The γ-pl waters ,44. Heatreported , but in The presence of thermophilic microorganisms in coral reefs is significant because these nominally mesophilic environments can reach maximum temperatures even exceeding 30 °C. On the other hand, cultivable thermophilic microorganisms, specifically Archaea, have been reported in non-thermophilic coastal zones, including superficial waters of the Pacific Ocean, temperate waters in the North Atlantic, the Mediterranean and even the Antarctic . In Mexico, the VRS is influenced by the contribution of rivers from hydrothermal zones, like Río Pescados that crosses the hydrothermal system El Carrizal, and is associated with the Pico de Orizaba volcanic system at 120 km from the port of Veracruz. Hence at least some of these bacteria found within the VRS could have a remote origin, probably from submarine hydrothermal upwellings, and the bacterioplankton could have been dispersed by marine currents. Geobacillus and Aeribacillus which have been described as aerobic or facultative anaerobic neutrophilic bacilli [Most thermophilic isolates from the VRS belonged to the genera  bacilli . These a bacilli ,49 and a bacilli . Five phPelagibacter ubique Rappé et al. [Prochlorococcus Chisholm et al. or Synechococcus Nägeli [P. ubique typically grows under oligotrophic conditions, whereas growth attempts with isolates in the laboratory were conducted upon highly nutrient-enriched LB media. Constraints on the culture of cyanobacteria such as Prochlorococcus are likely since experiments were not conducted under the necessary growth conditions for such photosynthetic organisms.Despite the wide distribution and abundance of proteobacteria and cyanobacteria in the marine environment, neither Pelagibacterales (SAR11), including é et al. ,52, nor s Nägeli ,51,53 weDelftia acidovorans (den Dooren de Jong) Wen et al. but with a poor match (85% similarity). Furthermore, D. acidovorans has been reported as a G(−) bacillus [Delftia Wen et al. In this study, both mesophilic and thermophilic bacterial isolates were associated mainly to Firmicutes and Proteobacteria, and clustered into the classes Bacilli, and β- and γ-proteobacteria . Althougbacillus , whereasThe microbiome of coral surface mucus includes bacteria that may comprise an important component of the microbial food web. The bacterial component exhibits a dynamic relationship with overall microbial communities of coral ,55, contBacillus as the most frequent genus [Proteobacteria was the dominant phylum represented in environmental samples from the water column and sediments at both reefs of the VRS. According to previous metagenomic studies, Proteobacteria tend to dominate the bacterioplankton and sediments of reef habitats, including deep cold-water coral reef systems in Norway . In the nt genus . Findingnt genus .Geobacillus are commonly found in geothermal environments and are widely distributed in soil, hot springs, contaminated food and ocean sediments [Geobacillus isolates are typically able to grow at 30–75 °C [Geobacillus and were first assigned to G. pallidus  Banat et al. on the basis of 16S sequences; in the final analysis, the strains were reclassified as Aeribacillus sp. [Members of the genus ediments ,49. With30–75 °C ,49 and o30–75 °C . The strllus sp.  with a dLysinibacillus Ahmed et al. is a G(+) bacterium found globally in both soil samples and fish tissues [Lysinibacillus and marine Pseudomonaceae, respectively. tissues . The theNeisseria Trevisan and Burkholderia Yabuuchi et al. Although most β-proteobacteria have been reported as mesophilic, there are thermophilic strains (most of them chemolithotrophic), such as Thiomonas thermosulfata Moreira et Amils, Thermothrix azorensis Odintsova et al., T. thiopara Caldwell et al., Tepidimonas ignava Moreira et al., Caldimonas manganoxidans Takeda et al., Tepidiphilus margaritifer Manaia et al., and Hydrogenophilus hirschii Hayashi et al. isolated from hot springs at Yellowstone Park in western USA [T. margaritifer, originate from hydrothermal systems, an environment unlike that of the origin of our isolates from the biofilm covering seagrass.The β-proteobacteria are common in coastal ecosystems, with chemolithotrophic degradative potential; this group also includes some higher animal pathogens such as tern USA . These ttern USA , as did Kosmotoga arenicorallina Nunoura et al. and Sulfurivirga caldicuralii Takai et al., flourishing around a coral reef in the south of Japan; this locality, however, is associated with a shallow marine hydrothermal system [Bacillus from hydrothermal vents has been detected years ago [Geobacillus, the homologous genus. In the Gulf of California near Guaymas, Mexico, Teske et al. [Geobacillus in the VRS, since this system receives freshwater inflow from the Pescados River associated with the hot spring El Carrizal. There are reports of thermophilic bacteria, namely the anaerobic l system ,63. The ears ago , but thee et al.  found poMost Firmicutes are G(+) and Proteobacteria are G(−), coinciding with VRS strains, except for the proteobacterium IVR-T-12-23 that stained G(+), and a few other Firmicutes that were G(−). These isolates are possible new species but are not strains known to originate directly from human or soil sources.Coral reefs are well-described marine environments, where healthy microbial community dynamics often characterized by low nutrient concentrations but high recycling and regeneration rates. Oligotrophic systems such as coral reefs are suggested to have a relatively low microbial diversity . On the All the sequences from the VRS were classified into 33 phyla, yielding the Proteobacteria cluster as dominant in all environmental samples. Similar results have been reported for large oceanic areas, where the Proteobacteria clade (SAR11) represented up to 35% of the prokaryotic picoplankton diversity in surface water, with highest species abundance in the North Atlantic .The studied reef system is adjacent to the Atlantic Ocean, from where the reef-associated water masses are derived . AdvectiFirmicutes has been reported as a characteristic group in some coral holobionts , but thiProchlorococcus and Synechococcus [After Proteobacteria, the water column of the world oceans tends to be dominated by members of the phylum Cyanobacteria, with high abundances of photosynthetic coccoid picoplankton such as hococcus ,51,53. AAt the family level, the diversity dominance patterns differed between seasons, even if the sequences were derived from the same reef, and within the reef among habitats. At IVR, the Synechococcaceae (23.3%) was the main group in the water column in September, whereas dominance shifted to unclassified Rickettsiales (26%) in March. In contrast, in sediments, the samples taken in September were dominated by Vibrionaceae (10.2%) and those from March by Alteromonadaceae (9%). PGR was sampled only in the dry season, with unclassified Rickettsiales as the most abundant group in the water column (17%), but with higher representation by Flavobacteriaceae (15.8%) in sediments.Prochlorococcus was more abundant than Synechococcus. This is consistent with previous findings, where Prochlorococcus was dominant in the tropical Atlantic. At the same time, α-proteobacteria (SAR11) have also been reported as a dominant group in surface waters [In general, at the genus level within the VRS, e waters , in agree waters . The Bacteroidetes was the third most abundant group represented in the water column and sediment samples of both reefs. This bacterial contingent could be related to organic matter processing, particularly in association with known eukaryotic harmful algal blooms in the study area ,70, but The Flavobacteriaceae and Halomonadaceae are considered to be moderately halophilic ; both meMost of the halotolerant bacterial isolates from the VRS belong to Firmicutes and Bacteroidetes. Although none of the isolated clades were detected in environmental samples, and vice versa, this does not necessarily mean that they are absent. Pyrosequencing studies of bacteria from hypersaline water report Firmicutes abundances of 15% in systems with a salinity gradient up to 6.4% .Members of the phylum Actinobacteria have been previously isolated from the marine environment ; this phVibrio Pacini, Thalassomonas Macián et al. or Serratia Bizio spp.) belong to the γ-Proteobacteria [Worldwide there are more than 20 reported coral diseases, but only six isolated agents responsible for them have been identified . Most cobacteria , also a bacteria ,74. Hortbacteria  found thbacteria .Vibrio shiloi Kushmaro et al.), bleaching and lysis , aspergillosis (Aspergillus sydowii (Bainier et Sartory) Thom et Church), white band disease , white plague disease , white pox disease (Serratia marcescens Bizio), yellow band disease  and black band disease, apparently associated with a microorganismal consortium [There is no published information on the interactive role of microorganisms and environmental conditions regarding the health status of corals in the VRS. Furthermore, there are no previous taxonomic or phylogenetic studies about the normal microflora found in this system, even when the close relationship among certain microorganisms and coral diseases has been established, such as for bleaching , whereas healthy corals contained 52.16% Proteobacteria. The profile distinction concurs with the finding shown in the current study   common in the Caribbean  but of usidereal , the gennt study  that assVibrio pathogen because Vibrionaceae are commonly present (3.8–10.2%) in the sediments of this study. Nevertheless, even though potential coral pathogenic species may be present in both reefs, it does not necessarily mean that the corals are infected with such pathogens, because bacteria that live in/on the coral holobiont are not the same strains as those of the surrounding water [The VRS sediments may host a t 3.8–10.% in the ng water . FurtherAlthough both reefs are close to the coast and near sewage discharge sites for the city, we did not find any member of the Enterobacteriales, as would be characteristic of fecal contamination. The current and wind patterns, among other factors, have an important role in bacterial dispersion and likely account for the absence of coliforms in the IVR and PGR samples.The earliest publications on marine microbial populations report a clear diversity difference between bacterioplankton and bacteriobenthos recovered from the water column and sediment samples, respectively . For exaThe bacterial communities in VRS sediments and the water column were distinct, even if they belonged to the same reef, but very similar between reefs if they originated from the same habitat. The fact that the two studied reefs belong to the same subzone within the park  perhaps The dominant taxonomic groups  in the water column and sediments of both studied reefs of the VRS were consistent with the groups previously found to be dominant in marine coastal environments in the northeastern Gulf of Mexico . In thisThe dominance by γ-proteobacteria in the bacteriobenthos in the studied sediment samples of the VRS could also be related to historically low levels of oil pollution transported to the reefs, but we did not explore the degradative capacity of γ-proteobacterial isolates nor determine the spectrum and concentration of exogenous hydrocarbons within the VRS. Such screening studies of both the water column and sediments of reef systems in the Gulf of Mexico would be required to demonstrate hydrocarbon degrading capabilities of the bacterial community, as well as to establish relevant shifts in the biodiversity profile as a consequence of recent or historical exposure to oil contamination. Many common sequences of 16S rRNA were found between cultured isolates and those recovered directly from environmental samples from the VRS, particularly among the Firmicutes and Proteobacteria. Nevertheless, the many distinct sequences revealed deep differences between abundances among isolates and environmental samples at lower taxonomic levels . In any case, the lower diversity of the cultured isolates compared with natural bacterial communities reflects the well-known “genetics of survivors” limitation due to the failure to provide a broad spectrum of permissible growth conditions in culture. The bacterial composition of sediments is more diverse than found in the water column. The diversity variations observed between the water column and sediments can be attributed to the different conditions that exist in these habitats, nutrients, exposure to wind-driven mixing and tidal currents, and the presence or absence of suitable substrates for adhesion. Most significantly, in spite of the expected high impact of anthropogenic stressors on the VRS, there is no discernable evidence from the diversity patterns of either cascading disequilibrium in the composition of the microbial contingent or pathogenic loading to indicate an unhealthy reef community. The bacterial communities within the water column and sediments are rather diverse and represent a generally balanced cell abundance and taxonomic representation in their respective compartments. This tends to indicate that reef response to seasonal and anthropogenic influences at least at the studied sites of the VRS is rather robust and resilient. The stability and compositional diversity of bacterial communities as a reflection of coral reef health should always be considered as a critical element for management decisions for environmental policies.


Plant breeding for intercropping is lagging because most varieties currently available in the market are selected for sole cropping systems. The present study analyzed the response of durum wheat (12 varieties) and faba bean (3 varieties) in pure and mixed cropping. Field trials were conducted in 2019 and 2020. The performance of each variety in mixed and pure cropping was evaluated using both univariate and multivariate analyses of the grain yield and land equivalent ratio (LER). For durum wheat, grain protein content was also evaluated. Durum wheat varieties were characterized by good performance in both years, whereas faba bean varieties were more affected by the growing season, suggesting that much breeding effort is warranted to improve the latter as a pure and mixed crop. Moreover, the relative performance of all varieties was affected by their combination in mixed cropping, as evaluated based on the ratio (LERratio) between LER for wheat (LERw) and LER for faba bean (LERfb). To further evaluate the overall performance of wheat and faba bean in mixed cropping, total yield, LERtotal (LERw + LERfb), and ln(LERratio) were subjected to principal component and cluster analyses. The first principal component combined the total yield and LERtotal in a single index of the overall performance of each mixed crop combination. The second principal component, based on ln(LERratio), highlighted the relative performance of varieties in each mixed crop combination. The proposed multivariate approach can be applied in the breeding programs for intercropping to identify variety combinations based on crop performance and the relative importance of the proportion of cereal and legume grains in the total harvest. Triticum aestivum L.), durum wheat (Triticum turgidum ssp. durum Desf. Husn.), and barley (Hordeum vulgare L.) as cereals intercropped with faba bean (Vicia faba L.) and pea (Pisum sativum L.) as grain legumes (–2) than for grain legumes . Typically, in intercropping, the cereal–legume combinations are grown as mixed crops, with the plants of the two crops planted as a mixture in the field without a specific row arrangement  emissions and N losses, ultimately lowering the inputs in more sustainable agricultural systems (Orobanche spp.), which represent the major hurdles to increase grain legume cultivation, particularly in the Mediterranean countries . Durum wheat is mainly cultivated for human consumption, whereas small-seed faba beans are primarily used as a protein concentrate in feedstock  compare the performance of mixed crop combinations with that of sole crops, (2) compare the effects of mixed and sole cropping on durum wheat grain protein content, and (3) develop an approach based on multivariate analyses  for the characterization of the overall performance of durum wheat–faba bean mixed crop combinations. Although based on commercial varieties, the results of the present study provide information that could be applied to gather a comprehensive evaluation of durum wheat-faba bean combinations in breeding programs for intercropping.Two field trials were set at the experimental station of the Università Politecnica delle Marche  on December 10, 2018  and January 22, 2020 . Regarding crop rotation, the preceding crops were sunflower and barley for trials 1 and 2, respectively. The high level of precipitation  register–1), and high (55 g kg–1) active calcium carbonate content, moderate (11.9 g kg–1) and low (9.5 g kg–1) available P, high available K (305 and 295 g kg–1), low (16.9 g kg–1), and moderate (20.6 g kg–1) organic matter content, and moderate total N content (1.20 and 1.15 g kg–1), respectively.Both field trials were conducted in silty–clayey soils, with the relative sand, silt, and clay content of 11.7, 42.4, and 45.9% in trials 1 and 17.4, 43.3, and 39.3% in trial 2, respectively. Soils in trials 1 and 2 were characterized by similarly high pH (8.13 and 8.14), very high  and mixed crops (90 kg N⋅ha–1) because the amount of N fertilizer  was set based on durum wheat seed density. Pure faba bean crops were not fertilized, according to the local farming practices.Different levels of N fertilization were applied to durum wheat pure  spaced 15 cm apart.A total of 12 durum wheat and three faba bean varieties, cultivated as pure crops in central Italy and representing a wide range of varieties in terms of grain yield and quality, were included in the field trials to assess their responses to mixed cropping . A totalSince a preliminary trial performed in 2017 suggested that the 50:50 replacement ratio was suboptimal, mixed crops were sown at a seed density ratio of 50 and 65% for durum wheat and faba bean, respectively . A factorial design was applied to evaluate 36 and 24 mixed crop combinations in 2019 and 2020, respectively. In July 2019 and 2020, each plot was harvested using a Wintersteiger Delta combine harvester for field experimental trials.–1) of pure and mixed crops was measured. Wheat and faba bean grains from mixed cropping were separated via sieving to determine the yield (Mg ha–1) of each crop. Moreover, durum wheat grain protein content (%) was evaluated.The total grain yield  of each crop and total LER were calculated as follows :w) = Durum wheat yield as a mixed crop/durum wheat yield as a pure cropLER for wheat (LERfb) = Faba bean yield as a mixed crop/faba bean yield as a pure cropLER for faba bean  = LERw + LERfbTotal LER  represents the relative performance of a specific variety in terms of its performance as a pure crop, the LERratio = LERw/LERfb was used as an index of the relationship between the relative performances of the two crops in each mixed crop combination. The LERratio recalls the competitive ratio (CR) proposed by Intercropping is considered to present better land-use efficiency than sole cropping when LERratio was as follows: LERratio = 1 indicates that LERw = LERfb, therefore both crops show equal relative performance in mixed cropping. In contrast, LERratio > 1, that is LERw > LERfb, indicates that durum wheat performed better than faba bean in mixed cropping, or vice versa if LERratio < 1, that is LERw < LERfb.The rationale behind the use of LERratio were log-transformed  to obtain ln(LERratio). The ln-transformation was needed to overcome the heteroscedasticity of residual errors due to the different ranges of variation of raw LERratio data when LERratio > 1 or when LERratio < 1 (0 < LERratio < 1). Moreover, after ln-transformation, complementary situations of cereal and legume performance in mixed cropping share the same absolute ln(LERratio) but with the opposite sign. For instance, if the performance of durum wheat is two times higher than that of faba bean, LERratio = 2/1 = 2; if the performance of faba bean is two times higher than that of durum wheat, LERratio = 1/2 = 0.5. Following ln-transformation, the ln(LERratio) values are ln(2) = + 0.693 and ln(0.5) = −0.693. After ANOVA and mean comparisons based on ln-transformed data, the ln(LERratio) means were subsequently transformed to LERratio ratios using the exponential (exp) function.For data analysis, the raw data of LERAnalysis of variance (ANOVA) was performed on the data of only Chiaro di Torrelama and Prothabat69 as faba bean varieties since they were included in both years. Different ANOVA fixed models were applied based on the results of the Shapiro–Wilk and Bartlett’s tests, applied to assess the normal distribution of residuals and homogeneity of variances, respectively.–1), including both durum wheat and faba bean varieties, was analyzed using the following ANOVA model:Pure crop yield ;ρj(i) = blocks  nested within the year; βk = pure crop effect ; αβik = year × pure crop interaction; and εijk = residual error.where y–1), total yield (Mg ha–1), and ln(LERratio) were analyzed using the following ANOVA model, including year as the main effect and its first- and second-order interactions:For mixed crops, the durum wheat yield ; ρj(i) = blocks  nested within the year; βk = wheat ; γl = faba bean ; βγkl = wheat × faba bean interaction; αβik = year × wheat interaction; αγil = year × faba bean interaction; αβγikl = year × wheat × faba bean interaction; and εijkl = residual error.where yFor the faba bean yield in mixed cropping, the 2019 and 2020 data were separately analyzed using the following ANOVA model:ijk = faba bean grain yield; μ = overall mean; ρi = block effect ; αj = wheat;βk = faba bean ; αβjk = wheat × faba bean interaction; and εijk = residual error.where yFor durum wheat grain protein content, the data from 2019 and 2020 were separately analyzed because of the highly significant heteroscedasticity of residual errors. The cropping system, including pure and mixed crop combinations with the two faba bean varieties, was included as the main effect in the following ANOVA model:ijk = wheat grain protein content; μ = overall mean; ρi = block effect ; αj = wheat; βk = cropping system ; αβjk = wheat × cropping system interaction; and εijk = residual error.where ytotal values.Tukey’s honestly significant difference (HSD) test was applied for multiple comparisons among means for the main effects, and pairwise comparisons (with Bonferroni correction) were used for interactions. Moreover, confidence intervals were calculated to test significant differences from one of the mean LERtotal, and ln(LERratio). Specifically, principal component analysis (PCA) was performed using Pearson’s correlation matrix . Claudio was the best performing durum wheat variety, and its grain yield was significantly higher than that of most other varieties; Aureo was the lowest yielding variety. Pairwise contrasts (P < 0.001). Only Nazareno and Aureo produced significantly (P < 0.05) higher mean yield in 2020 than in 2019. Therefore, in pure cropping, faba bean varieties were more influenced by the growing season than durum wheat varieties, reflecting the well-known yield instability of faba bean  were significant. As expected, mean grain yield across years was significantly higher for most durum wheat varieties than for faba bean varieties , with a ontrasts  showed taba bean .P = 0.13) and the wheat × faba bean interaction (P = 0.07) were not significant . All the remaining sources of variation were highly significant (P < 0.001). Therefore, particular attention was paid to the second-order interaction (wheat × faba bean × year) and The year main effect  revealed significant main effects, but it was only in 2020 that the wheat × faba bean interaction was significant.–1) and 2020 (1.58 vs. 1.38 Mg ha–1), suggesting different general mixing abilities of the two faba bean varieties. Moreover, the performance of both faba bean varieties was poorer in 2020 than in 2019, reflecting the same trends observed in pure cropping. Regarding the main effect of durum wheat varieties on faba bean yield, in 2019, mixed cropping with four varieties, namely Nazareno, Natur, Achille, and Odisseo, showed significantly higher faba bean yields than most other combinations  between durum wheat and faba bean yield, suggesting that the average performance of faba bean significantly varied according to the durum wheat variety included as a combination crop. Therefore, the average yield of faba bean has increased as the yield of wheat decreased, suggesting some level of balance due to the interaction between the two crops in mixed cropping. In contrast, in 2020, no correlation between durum wheat and faba bean yield was detected, as a consequence of the lower average performance of faba bean. Interestingly, the mean faba bean yield in mixed cropping with Natur was the highest in both years, suggesting that Natur could be considered as the least competitive variety for faba bean in the set of durum wheat varieties evaluated.In 2019, there was a highly significant and negative correlation  was significantly higher than that of mixed crops including Chiaro di Torrelama (5.25 Mg ha–1). Therefore, including the faba bean variety with the lowest performance in mixed cropping resulted in a higher mean total yield because of the increased performance of durum wheat.The mean total yield of mixed crops including Prothabat69 . Regarding the average effects of the two faba bean varieties, the mean ln(LERratio) values were positive (0.410) and negative (−0.112) for Prothabat69 and Chiaro di Torrelama, respectively, and the difference was highly significant. Therefore, the average relative performance of the durum wheat was significantly better than faba bean in mixed cropping with Prothabat69, whereas Chiaro di Torrelama showed, on average, a better performance than durum wheat in mixed cropping.The ANOVA (model 2) revealed highly significant wheat and faba bean main effects and significant wheat × faba bean × year interactions (ratio) values of the durum wheat varieties are presented in ratio) values. Three varieties, namely San Carlo, Rangodur, and Aureo, were ranked as intermediate, while Tirex (the highest) and Natur (the lowest) showed significantly different extreme means.The results of multiple comparisons among overall mean ln(LERratio) values were negative and positive for almost all mixed crop combinations including Chiaro di Torrelama and Prothabat69, respectively. The negative ln(LERratio) values for Chiaro di Torrelama indicate that in 2019, LERfb was higher than LERw, whereas a contrasting trend was noted for Prothabat69. Therefore, in 2019, Chiaro di Torrelama showed a better performance than Prothabat69 in mixed cropping with most durum wheat varieties. However, the performance of Chiaro di Torrelama in 2020 was much poorer than that in 2019. Consequently, the mean ln(LERratio) values were positive for most mixed crop combinations with both faba bean varieties (ratio) values with Chiaro di Torrelama in 2020, suggesting that in less favorable growing seasons for faba bean, these two durum wheat varieties were characterized by the lowest competitive ability against the best performing faba bean variety.Regarding the two-way interaction , in 2019arieties . Only Acratio) values confirmed that the relative performance of cereal and legume crops is an important parameter when assessing the effectiveness of durum wheat–faba bean mixed cropping.Overall, the analysis of ln. Multiple comparisons among cropping systems revealed that in both years, on average, mixed cropping increased the protein content of durum wheat , and thetotal, and ln(LERratio). The combined results of the PCA and CA, including eigenvalues and eigenvectors, are presented in To further analyze the information obtained through univariate analyses, a more comprehensive approach based on PCA, followed by CA, was applied. For this analysis, the most important variables, summarizing different features of mixed cropping performance, were considered: total yield, LERP < 0.001) and explained 93.6% of the total variation. PC1 was related to the total yield and LERtotal, and both variables showed positive PC1 eigenvector coefficients. Therefore, the higher the PC1 score, the higher the overall mixed crop performance as a combination of the total yield and LERtotal. Moreover, PC1 effectively highlighted the different performances of mixed crops in the 2 years; the PC1 scores were mainly positive and negative for the mixed crops evaluated in 2019 and 2020, respectively.The first two principal components (PC1 and PC2) were highly significant  was not correlated with either total yield  or LERtotal , but it was important for PC2, explaining 33.35% of the total variance. For PC2, high positive, intermediate, and low negative scores were related to mixed crops with a better performance of durum wheat than of faba bean, a similar performance of the two crops, and better performance of faba bean than of durum wheat, respectively.The ln(LERratio) in the characterization of mixed crop combinations was further confirmed using CA, which identified main clusters of mixed crops within each year, with only a few exceptions (The effectiveness of ln(LERceptions . In 2019ratio) values were always positive, ranging between 0.41  and 0.85 . These results indicate that in the mixed crop combinations of cluster A, durum wheat varieties overcame Prothabat69 in relative performance, and all mixed crop combinations, except the one including Claudio in 2019 , showed LERtotal values significantly higher than one. Interestingly, based on CA, the mixed crop combinations of Claudio–Prothabat69 evaluated in 2019 and 2020 formed a sub-cluster within cluster A  values of −0.27 (LERw = 0.76 × LERfb) and 0.17 (LERw = 1.18 × LERfb), respectively. Therefore, cluster B included mixed crop combinations that showed a more balanced LERw/LERfb ratio in 2019.Cluster B showed intermediate PC2 scores. The lowest and highest PC2 scores were recorded for Claudio–Chiaro di Torrelama and Odisseo–Prothabat69, with ln(LERratio) values ranged between −0.68  and −0.50 .Cluster C was characterized by mixed crop combinations, including only Chiaro di Torrelama, and showed low negative PC2 scores because the relative performance of the faba bean variety was higher than the durum wheat varieties. The ln were identified by CA . The PC1–1, respectively, and their LERtotal values were not significantly different from 1. Interestingly, cluster F showed negative PC2 scores and, similar to cluster C, included mixed crops combinations with only Chiaro di Torrelama as the faba bean companion crop.The main difference between clusters D and E was related to the total yield, which was mostly higher and lower than 5 Mg haFinally, the durum wheat variety Aureo warrants specific attention. In fact, PCA revealed that this variety acted as an outgroup in 2019 when cultivated in combination with Chiaro di Torrelama as well as in 2020 when cultivated in combination with Prothabat69. Aureo is a well-known low-yielding but high-quality variety. As a mixed crop, Aureo showed the poorest performance with both faba bean varieties in 2019 and with Prothabat69 in 2020. Its PC1 scores were low and, based on the PC2 score, this variety showed a better competitive ability against Prothabat69 than against Chiaro di Torrelama in both years. These results suggest that Aureo should be selected as a mixed crop with caution, as its performance is more closely linked to the companion faba bean variety and growing season than the other durum wheat varieties.The faba bean variety Rumbo was included in 2019 alone and results regarding this variety are summarized as The present study examined the durum wheat–faba bean mixed cropping system using a set of varieties that are commonly cultivated in central Italy as pure crops. Two years of field trials provided information that could be usefully extended to future breeding for mixed cropping.Durum wheat is a very important cereal in Italy and the Mediterranean Basin, this crop has been subjected to intense breeding, a wide range of varieties is currently available for farmers , and as Due to the good performance as a pure crop, farmers could not positively look at the replacement of pure durum wheat with mixed crops. Therefore, a proper and effective strategy, aimed at the valorization in the final products of the advantages related to agroecological and grain quality aspects, should be applied to make farmers choose to intercrop as an effective alternative practice to durum wheat pure crop. However, further studies are necessary on the effects of intercropping on other important durum wheat quality traits, such as yellowness and gluten index .P < 0.01) was found between durum wheat yield as pure and in mixed cropping, but pure crop yield was not correlated with LERw. Therefore, for the set of varieties evaluated here, the performance in pure crop could not be considered as an index of durum wheat performance in mixed cropping. For example, the variety Claudio ranked among the highest yielding ones in 2019 and 2020 both as pure and in mixed cropping. However, in 2019 the LERtotal values of mixed crops including Claudio were the lowest. These results were due to its low LERw value (LERw = 0.46) in combination with Chiaro di Torre Lama and its high LERw value (LERw = 0.69) with Prothabat69 that was followed by very low performance of this faba bean variety (LERfb = 0.36). Differently, in 2020, both combinations including Claudio were the best-performing ones in terms of the LERtotal, because the high LERw value compensated for the low general performance of faba bean. These results suggested that a specific relationship between the durum wheat and the faba bean variety, as also affected by environmental conditions, determined the overall performance of each mixed crop combination. This trend was further confirmed by the negative correlation found in 2019, followed by no correlation in 2020, between durum wheat and faba bean yields in mixed cropping.In both years a highly significant correlation  shifted to cluster B or C when Chiaro di Torrelama replaced Prothabat69 as a companion crop. Therefore, the relative performance of these durum wheat varieties decreased in combination with Chiaro di Torrelama. The same result was observed for durum wheat varieties that, in combination with Prothabat69, were included in cluster B but shifted to cluster C when Chiaro di Torrelama was the companion crop. Interestingly, this trend was retained also in 2020, although the range of variation of PC2 score was lower than that detected in 2019.Overall, the univariate (ANOVA) and multivariate (PCA and CA) analyses provided complementary information for interpreting the performance of durum wheat–faba bean mixed cropping. The average performance of each variety in mixed cropping could provide information on the general mixing ability, but the negative correlation between durum wheat and faba bean yield in 2019 and the overall clustering of mixed crop combinations over years suggested that specific mixing ability should not be disregarded. Therefore, the multivariate analysis of grain yield, LER, and LERThe multivariate approach highlighted the clear differentiation between mixed crops based on the combinations of durum wheat and the faba bean varieties. Therefore, the principal component scores could be applied as indices of selection within breeding programs aimed to simultaneously improve both cereal and legume performance. This approach could allow the identification of the best combinations that could be considered as an alternative to durum wheat pure crop, faba bean pure crop, or both. Of course, the interpretation of PC scores could vary based on the features of the genotypes of durum wheat and faba bean under evaluation.The present study involved durum wheat, which is probably the most important cereal in the Mediterranean area, and it is involved in a market that asks the farmers to combine high yield with high-quality parameters. All varieties included in the present study not only reflected these needs but also represented a good sample to test their mixing ability with the aim of gathering information on genetic variability that could be available for future breeding programs. The evaluation of this representative set of durum wheat varieties highlighted that efforts are needed to select new durum wheat genotypes because selection for pure crops did not reflect their performance in mixed cropping.Moreover, although a restricted number of faba bean varieties was included, results of PCA and CA suggested that different combinations of durum wheat and faba bean varieties could result in different relative performances of the cereal and the legume crops. The faba bean choice deeply influenced the performance of durum wheat and, consequently, of the whole mixed crop combination, as better highlighted by the multivariate rather than univariate analysis. Therefore, the analysis at the mixed crop combination level is an important feature to be considered in further breeding programs for durum wheat-faba bean mixed cropping. However, breeding efforts for faba bean in mixed cropping must also be addressed to reduce the instability of performance over years. For this purpose, a wider range of faba bean varieties available in the EU Common Catalog of Plant Varieties, together with further germplasm accessions, could be evaluated and included in specifically targeted breeding programs for mixed cropping.Vice versa, durum wheat-faba bean mixed cropping is a real opportunity as an alternative to faba bean pure cropping, especially in low input conventional or organic farming. However, the more constant performance of faba bean varieties in mixed cropping is requested, and therefore breeding programs for this grain legume should also involve multiyear trials together with selection carried out under mixed cropping with durum wheat genotypes.In conclusion, consideration is necessary about climate change. In the last years, agriculture has been facing the effects of strong year-to-year variation in environmental conditions that deeply influenced crop performance. The 2 years involved in the present study reflected the variability of crop performance under very contrasting growing seasons, confirming, based on the set of varieties available in the market, the much higher resilience of durum wheat than faba bean. Therefore, our results highlighted the need for a much more intense breeding work for faba bean than for durum wheat to let mixed cropping be more positively evaluated by farmers as an alternative to durum wheat pure crops. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Both authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Triticum turgidum L. subsp. durum (Desf.) Husn) is a staple crop of the Mediterranean countries, where more frequent waterlogging events are predicted due to climate change. However, few investigations have been conducted on the physiological and agronomic responses of this crop to waterlogging. The present study provides a comprehensive evaluation of the effects of two waterlogging durations  on two durum wheat cultivars . An integrated analysis of an array of physiological, biochemical, biometric, and yield parameters was performed at the end of the waterlogging events, during recovery, and at physiological maturity. Results established that effects on durum wheat varied depending on waterlogging duration. This stress imposed at tillering impaired photosynthetic activity of leaves and determined oxidative injury of the roots. The physiological damages could not be fully recovered, subsequently slowing down tiller formation and crop growth, and depressing the final grain yield. Furthermore, differences in waterlogging tolerance between cultivars were discovered. Our results demonstrate that in durum wheat, the energy maintenance, the cytosolic ion homeostasis, and the ROS control and detoxification can be useful physiological and biochemical parameters to consider for the waterlogging tolerance of genotypes, with regard to sustaining biomass production and grain yield.Durum wheat ( Triticum turgidum L. subsp. durum (Desf.) Husn) is one of the oldest cultivated cereals and plays a pivotal role in global food security. Although the necessity of wheat grain will increase by 60%, its production might decline by 29% because of the environmental stresses driven by climate change [Durum wheat (e change . Durum we change . Specifie change .2) concentration decreases rapidly, while the carbon dioxide (CO2) and ethylene concentrations increase in the root environment [2 influx is the main cause of injury to the roots and to the shoots they support [2 deprivation. From a physiological point of view, excess water accumulation in the root zone can induce osmotic stress and disrupt cell ion homeostasis. To cope with such stressful conditions, plants tend to accumulate compounds called osmoprotectans , as their accumulation may decrease the osmotic potential [2 levels and limit carbon fixation [2O2), superoxide radicals, and hydrogen radicals which impair mesophyll conductance [In rain-fed situations, flooding happens when more rain falls than the soil can absorb, or the atmosphere can evaporate. In central Italy, excess water is likely to occur from October to April but is more expected during the winter months (from January to February), due to lower transpiration and evaporation rates of the crop. Therefore, durum wheat is more prone to excess water during the tillering stage, which is critical for tiller production and spikelet initiation . Based oironment . A slowe support . The plaotential ,10. Nextfixation ,12. Addiductance , and harductance ,15.2 deficiency caused by waterlogging has been demonstrated to prematurely induce leaf senescence, reduce root and shoot growth, and constrain spike development, thus decreasing the final grain yield of the crop [In durum wheat, Othe crop . Howeverthe crop  and barlthe crop . Recoverthe crop . Neverththe crop ,17, withTo fill the gap of knowledge about the mechanism(s) of the response of durum wheat to waterlogging and to relate the physiological responses of leaves and roots to the growth, recovery ability, and final grain yield of the crop, the present research aimed to investigate the effects of different waterlogging durations  at tillering on the growth and the grain yield of durum wheat, as well as identify the main physiological traits involved in the response of roots, shoots, and leaves.As the cultivar choice may represent a key factor in coping with waterlogging ,19, we cMore specifically, our objectives were to assess the mechanism of response of the two durum wheat genotypes to 14 and 35 days of waterlogging at tillering, evaluating: (i) the immediate impairment of root and shoot growth and related physiological and biochemical parameters, as well as water status; (ii) the ability to recover from the end of waterlogging up to maturity; and (iii) the final grain yield.During the experiment , the total rainfall was 672 mm spread over about 80 rainy days and was mainly concentrated in the period from December to February , as is tEmilio Lepido started tillering at the same time of Svevo; the two cultivars also reached flowering simultaneously . WaterloTOT) and calcium ion (Ca2+), leaf and root malondialdehyde (MDA), and shoot-to-root biomass ratio values were higher in Emilio Lepido than in Svevo, while the maximum quantum efficiency of the photosystem II (PSII) photochemistry (Fv/Fm), chlorophyll a/b ratio (Chl a/b), de-epoxidation state (DEPS), leaf and root hydrogen peroxide (H2O2), leaf potassium ion (K+), root Ca2+, and shoot and root biomass levels were higher in Svevo (data not shown). Fourteen DOW reduced the CO2 assimilation rate (A) and stomatal conductance (gs) only in Svevo , whereas 35 DOW reduced these parameters regardless of the cultivar  increased only in Svevo at 35 DOW  only decreased in Emilio Lepido at 35 DOW  was reduced by 35 DOW  were similarly reduced in both cultivars by 14 DOW (−27%) and even more by 35 DOW . Conversely, root MDA levels were increased by 14 DOW in both cultivars  and by 35 DOW in both cultivars , whereas it was more decreased in Emilio Lepido than in Svevo by 35 DOW  and only in Emilio Lepido at 35 DOW , whereas it was similarly reduced by 35 DOW in both cultivars , whereas it decreased only in Svevo with 35 DOW −60%; a. Shoot p ≤ 0.001). Among the Cans, the first two  accounted for 90.5% of the total variability  and gs increased in both cultivars subjected to both WL14 and WL35, by about 35%. Ci increased in both cultivars subjected only with WL35 (+7%), ΦPSII and qP were higher in Emilio Lepido subjected to WL35 (+14 and +11%), and qNP was lower in Emilio Lepido subjected to WL35 (−26%), as well as in Svevo subjected to both WL14 and WL35 . Nevertheless, similarly between cultivars, WUEin was lower in plants subjected to WL35 (−18%).Few studies have evaluated the impact of waterlogging on durum wheat ,22, and 2 assimilation rate was similarly impaired between cultivars by the longer 35 DOW. These photosynthetic impairments were clearly due to stomatal limitations , suggesting an isohydric behavior of both cultivars [i did not accumulate. The interpretation of a minor occurrence of non-stomatal limitations of photosynthesis was supported by the absence of PSII photodamage , as well as by the slight reduction in qP similarly reported between cultivars and for different waterlogging durations. No waterlogging effects on Fv/Fm  was interesting and unexpected. Water-use efficiency is largely used in the selection of cultivars with high capacity of adaption and high yield in crop breeding projects [Our results confirmed that a large variation in wheat responses to waterlogging exists, depending on different durations of stress conditions and on diverse genotypic sensitivity . Photosyultivars , while ms marker ) have als marker . The incprojects ,26.PSII), together with an activation of the dissipation of the excess excitation energy as heat , were observed only in Emilio Lepido at 35 DOW . This also confirms that this cultivar was not able to tolerate oxygen deprivation so long .Our findings indicate that Svevo likely adopted a better strategy to regulate the use of water in an attempt to cope with the longer waterlogging duration. Actually, a reduction in PSII performance  adopted by the crop to maintain turgor and cell volume under such detrimental conditions. The importance of osmotic adjustment to improve drought tolerance in plants is notorious [As paradoxical as it may sound, waterlogging often reduces water availability to plants ; this protorious ; the preotorious . Overall2 fixation. In some conditions, disturbance to cation homeostasis  and the possible damage of leaves from ROS or phytotoxins  might also contribute to this [TOT and CarTOT) which play a crucial role in light harvesting for photosynthesis. The degradation of chlorophyll and carotenoids was already reported in plants exposed to waterlogging, e.g., as shown in [v/Fm, and also confirmed by the lack of accumulation of leaf MDA (one of the major indicators of cell membrane damage) [During waterlogging, factors such as decreases in chlorophyll or other components of the photosynthetic apparatus, as a result of nitrogen deficiency and/or negative feedback from carbohydrate accumulation, have been reported as possible causes of reduced CO to this ,27,32. Tshown in , as wellshown in ,35, signshown in  were not damage) . This ap2O2 occurred in the roots of waterlogged durum wheat. This outcome confirms the importance of evaluating the belowground responses as well to fully elucidate the effects of waterlogging on plants. Increased lipid peroxidation was reported in the roots of both cultivars subjected to 14 DOW, although an accumulation of root H2O2 occurred only in Emilio Lepido. Although root MDA accumulation was not reported at 35 DOW, a strong accumulation of H2O2 occurred in the roots of both Emilio Lepido and Svevo subjected to longer periods of waterlogging . Excessive MDA accumulation commonly indicates cell membrane damage, which leads to a series of negative physiological and biochemical events, including reduced photosynthesis [2O2 production is one of the hallmarks of the low oxygen stress signal [2O2 accumulation reported between cultivars and waterlogging durations undoubtedly needs and suggests further research (the lack of root MDA increase at 35 DOW was particularly unexpected). However, this phenomenon was likely due to the activation/depression of enzymatic and non-enzymatic antioxidants, adopted by plants to regulate the stress response and signaling [Although it has been largely reported that roots are the plant organs mostly affected by waterlogging ,27, the ynthesis . Increass signal , as wells signal ,41. The ignaling . Among aignaling .2O2 accumulation due to genotype and duration also appeared to be linked to specific regulations of membrane transporters, which were investigated at both the leaf and root levels. Membrane transporters are known to play a crucial role in mediating adaptive responses to oxygen deprivation and waterlogging, especially at the root level [+ uptake is commonly and markedly reduced [+ homeostasis and K+ channel functionality was named as an essential component of plant acclimation to hypoxia [2+ concentration in plant cells [+ content were fully in accordance with the above-mentioned reductions in the CO2 assimilation rate observed only in Emilio Lepido at 14 DOW and in both cultivars at 35 DOW, whereas leaf K+ content decreased in both cultivars, regardless of waterlogging duration . An elevation in Ca2+ content was instead observed only in leaf tissue, specifically in Svevo at 14 DOW and in Emilio Lepido at 35 DOW, indicating that waterlogging disturbed not only the mineral uptake, but also the transport of ions to the aerial organs that might have impaired the stomatal conductance and negatively affected the CO2 fixation, translocation, and utilization of assimilates. Our findings corroborate those of [2+- and K+-permeable cation channels at the plasma membrane. Thereby they also mediate Ca2+-based signaling events and K+ ion leakage. These outcomes not only confirm the importance of cation homeostasis in waterlogging response but also the higher physiological sensitivity of Svevo reported at 14 DOW and the inability of both cultivars (Emilio Lepido results more sensitive in terms of WUEin and PSII performance) to tolerate the longer oxygen deprivation .Variations in lipid peroxidation and Hot level . Specifi reduced ,44, so t hypoxia . Conversnt cells ,46. In ant cells . The prethose of , which fin and the preservation of PSII performance was not successful in terms of biomass production. The root biomass of the two cultivars was similarly impaired by 35 DOW, confirming that root dry weights significantly decrease with waterlogging longer than 20 days [The contrasting responses in physiological, water status, and biochemical parameters were only partially confirmed by the biometric measurements. The different reactions between cultivars at 14 DOW were not accordingly highlighted by biomass production, as the number of culms, shoot, and root biomass and the shoot-to-root biomass ratio were similarly affected in both Emilio Lepido and Svevo. In particular, waterlogging induced different biomass distribution regardless of the cultivar. Conversely, number of culms and the amount of shoot biomass indicated a higher sensitivity of Svevo at 35 DOW, suggesting that the strategy adopted by this cultivar in terms of WUE 20 days . Thus, t 20 days .in was reduced in both cultivars previously subjected to 35 DOW); instead it was due to a mismatch between the developmental stages of the control and the waterlogged plants, i.e., the controls were closer to maturity and thus lowered the photosynthetic process. We can thus infer that the plant growth had been slowed down by prolonged water excess, as similarly demonstrated in other winter cereals by [Although the detrimental effects due to waterlogging events on photosynthesis and PSII performance were no longer detectable at recovery, this phenomenon was not due to an ability of durum wheat to recover its optimal physiological functioning , corroborating previous results for waterlogging imposed at tillering in durum wheat that displayed differences yield losses related to waterlogging duration [ DOW, corFrom an agronomic point of view, plant tolerance to waterlogging involves the maintenance of a relatively high grain yield under waterlogged conditions relative to non-waterlogged conditions. Accordingly, our findings clearly showed that Emilio Lepido was more tolerant to waterlogging, whereas Svevo was more sensitive even with a waterlogging duration shorter than 20 days. To the best of our knowledge, any other durum wheat cultivar from Claudio and Svevo has been investigated for agronomic waterlogging tolerance ,10. HoweThe research was carried out from December 2020 to June 2021 at the field station of the Department of Agriculture, Food and Environment of the University of Pisa, Italy . The climate of the area is hot in summer Mediterranean conditions (Csa), with mean annual maximum and minimum daily air temperatures of 20.2 and 9.5 °C, respectively, and a mean rainfall of 971 mm per year. The daily air minimum and maximum temperatures and rainfall were recorded throughout the entire period of the research by an automatic meteorological station located close to the experimental site.−1 total nitrogen (Kjeldahl method), 4.5 mg kg−1 available P (Olsen method), and 68.9 mg kg−1 available K (BaCl2-TEA method). The crop was sown on 15 December 2020 within the optimum sowing time for winter cereal production in central Italy. After emergence, the seedlings were thinned to eight plants per pot, corresponding to 400 plants m–2. Phosphorus and potassium were applied pre-planting as triple mineral phosphate and potassium sulfate, at the rates of 150 kg ha−1 of P2O5 and K2O. Nitrogen was applied at the rate of 150 kg N ha−1, and split into three applications at sowing, at pseudo-stem erection (BBCH 30), and at first node detectable (BBCH 31) as urea, in the following proportions: 30–60–60 kg N ha–1. The rate of mineral N supply was the recommended value for optimal durum wheat production in central Italy, and the adopted splitting management was proved to be an optimal mineral fertilization practice to ensure both production quantity and quality in the Mediterranean climate [The experimental design consisted of two durum wheat cultivars exposed to 14 and 35 days of waterlogging (DOW) at the tillering stage, compared to well-drained controls. We used the two commercial cultivars Svevo and Emilio Lepido. Svevo is a very early maturing cultivar that was released in 1996 from the genealogy CIMMYT line/Zenit and is high yielding. Emilio Lepido is a more modern cultivar, released in 2011 from the genealogy Orobel/Arcobaleno/Svevo, which matures early and is resistant to cold temperatures. Both have a good resistance to lodging. Plants were grown in 16-L pots made from polyvinyl chloride (PVC) tubes (80 cm long and 16 cm in diameter) fitted with a PVC base. A 30 mm diameter hole was drilled in the bottom of each pot, which was fitted with a 0.9 mm mesh to contain roots and substrate loss. Pots were filled with a sandy-loam soil collected from an adjacent field that was previously cultivated with rapeseed. Main soil properties were: 55.3% sand (2 mm < ∅ < 0.05 mm), 33.8% silt (0.05 mm < ∅ < 0.002 mm), 10.9% clay (<0.002 mm), 7.6 pH, 0.7 g kg climate  to deterFor each cultivar, at waterlogging imposition (0 DOW–24 February 2021) three replicate pots were harvested to determine biomass and physiological characteristics before waterlogging imposition. At the end of each period of waterlogging—that is after two and five weeks (14 and 35 DOW)—all plants of three waterlogged pots and three well-drained pots (controls) were measured for physiological and biochemical parameters (they were performed on the second and third upper and fully expanded leaves). Then three pots per cultivar were moved from the container filled with water to drained conditions. These pots (WL pots to be measured at maturity) were supplied with the scheduled top-dressing N fertilization and kept in drained conditions until plants reached maturity. Control pots received N at the same time of the waterlogged pots. Additional measurements of physiological and water status parameters were carried out during the recovery period, at 70 days after the beginning of waterlogging , to assess the water status and the physiological activities of control and waterlogged plants. At maturity, three waterlogged and three control pots for each cultivar were harvested to assess final biomass and grain yield production.2 assimilation rate (A), stomatal conductance (gs), and intercellular CO2 concentration (Ci) were determined using a LI-6400 portable photosynthesis system equipped with a 2 × 3 cm chamber and a 6400-02B LED light source , operating at 400 ppm of CO2 concentration, 25 ± 2 °C of leaf temperature, 45 ± 5% of RH, 1.8 ± 0.2 kPa of VPD and saturating light conditions (1500 µmol m−2 s−1 PAR). Intrinsic water-use efficiency (WUEin) was calculated as A/gs.The COv/Fm), the PSII-operating efficiency in light conditions (ΦPSII), the photochemical quenching (qP), and the non-photochemical quenching (qN) were determined by a PAM-2000 chlorophyll a fluorometer , set as reported by [After a 40 min dark-adaptation of leaves (same used for leaf gas-exchange measurements), the maximum quantum efficiency of the photosystem II (PSII) photochemistry . Relative water content was calculated as (FW-DW)/(TW-DW) × 100, where FW is the fresh weight, TW is the turgid weight after rehydrating samples for 24 h, and DW is the dry weight after oven-drying leaves at 60 °C until constant weight.Water status parameters were determined at mid-day, according to . Leaf wa® SRT 15 aseptic filters. The pigments were eluted using 100% solvent A  for the first 14 min to elute xanthophylls , followed by a 1.5 min linear gradient to 100% solvent B , which was pumped for 14.5 min to elute chlorophyll b (Chl b) and chlorophyll a (Chl a) and β-carotene (β-car), followed by 2 min linear gradient to 100% solvent A. The flow rate was 1 mL min−1. The column was allowed to re-equilibrate in 100% solvent A for 1 min before the next injection. The pigments were detected by their absorbance at 445 nm. To quantify the pigment content, known amounts (0.003–0.5 mg mL−1) of pure standards  were injected into the UHPLC system, and an equation correlating the peak area to pigment concentration was formulated. Chromatographic data were processed and recorded by Chromeleon Chromatography Management System software, version 7.2.10–2019 (Thermo Scientific). Total chlorophyll content (ChlTOT) was calculated as Chl a + Chl b. Total carotenoid content (CarTOT) was calculated as Neo + Vio + Ant + Lut + Zea + β-car, while the xanthophyll cycle pigment content (VAZ) was calculated as Vaz + Ant + Zea. The de-epoxidation state (DEPS) was calculated as (Ant + Zea)/VAZ.Leaf pigments were determined by ultra-high performance liquid chromatography (UHPLC) using a Dionex UltiMate 3000 system equipped with an Acclaim 120 C18 column  maintained into a Dionex TCC-100 column oven at 30 °C, and a Dionex UVD 170U detector . Samples were incubated at 95 °C for 30 min and centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was measured for absorbances at 440, 532, and 600 nm, using a fluorescence/absorbance microplate reader . The amount of malondialdehyde (MDA) was calculated as 106 × ((A − B)/157,000), where A = (Abs 532 + TBA − Abs 600 + TBA) − (Abs 532-TBA − Abs 600-TBA) and B = (Abs 440 + TBA − Abs 600 + TBA) × 0.0571.Lipid peroxidation was measured by the thiobarbituric acid reactive substances (TBARS) method, according to . Briefly2O2 was determined with the above-reported fluorescence/absorbance microplate reader at 530 and 590 nm for the excitation and emission of resorufin fluorescence, respectively.Hydrogen peroxide content was measured using the Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit , according to . After e+ and Ca2+ contents were determined by Ion Chromatography . According to [g for 10 min. After filtration through 0.2 μm Minisart® SRT 15 aseptic filters, supernatants were eluted with 20 mM methanesulfonic acid at 1 mL min−1.Leaf and root Krding to , 12.5 mgAt all harvesting times , subsequently to the above-mentioned physiological measurements, plants were manually cut at ground level. After the shoots were removed, the roots were recovered from the soil by gently washing them with a soft water flow. Washing water was additionally filtered through a fine mesh to prevent root loss. Root sub-samples were used for the biochemical analyses after recording their fresh weight. At maturity (BBCH 99), the shoots were partitioned into culms, leaves, and spikes and spikes separated into kernels, and chaff, and the roots were recovered and measured with the same above-reported procedure.Biomass of roots, vegetative above-ground plant parts (VAP) and grain yield were determined. For dry weight (DW) determination of all plant parts, the samples were oven-dried at 65 °C to achieve a constant weight.p ≤ 0.05. Statistical analyses were run in JMP 13.2.0 .The Shapiro-Wilk test was used to evaluate the normal distribution of data and homogeneity of variances was tested through Levene’s tests, prior to analyses. The effects of cultivar, waterlogging, and their interaction on the investigated parameters were assessed by a two-way analysis of variance (ANOVA), using Tukey’s test as the post hoc test. Statistically significant effects were considered for A discriminant analysis was applied to the full set of parameters collected at the end of waterlogging treatments to select those that best discriminated among cultivars (Emilio Lepido and Svevo), waterlogging treatment (control and waterlogged), and waterlogging duration (14 and 35 days).2O2 accumulation in the root systems; and (iii) finally depressed the grain yield, due to slowed-down tiller formation and crop growth. Additionally, our results showed that genotypic differences in waterlogging tolerance of durum wheat are present. The two genotypes differed not only in their immediate responses to waterlogging but also in the recovery of growth once the soil was drained. Consequently, the final grain yields of the two cultivars were affected differently. As a matter of fact, one cultivar (Emilio Lepido) was more tolerant to waterlogging than the other (Svevo).In conclusion, our pioneering study demonstrated that waterlogging imposed to durum wheat at tillering (i) impaired photosynthetic activity, mainly due to stomatal limitations, pigment degradations and altered cation homeostasis; (ii) determined oxidative damage and HTherefore, our results suggest that the waterlogging tolerance of durum wheat can be achieved by pyramiding the numerous physiological, water status, and biochemical parameters that confer efficient key processes such as energy maintenance, cytosolic ion homeostasis, and ROS control and detoxification, and consequently ensure satisfying biomass production and yield.Additional research is obviously required to evaluate the responses of present and other cultivars of durum wheat to waterlogging, under several environmental and cropping conditions. This would give a better picture of overall crop performance and allow further endorsement of our present results.