Chaetomium globosum and Phanerochaete chrysosporium were tested. Both fungi characteristics and changes in morphology, structure and thermal properties were monitored. Biochemical parameters as superoxide dismutase, catalase, soluble protein and malondialdehyde have been determined at different time periods of fungal degradation. The fungi extracellular enzyme activities are slightly decreased in the case of composites containing bioactive compounds. The presence of natural compounds in the PLA-based polymeric system determines an acceleration of fungal degradation and probably the chemical hydrolysis, which further helps the attachment of fungi on the surface of polymeric samples. Significant decreases in average molecular mass of the polymeric samples were observed by fungi action; accompanied by structural changes, increase in crystallinity and decrease of thermal properties and the loss of the physical integrity and finally to degradation and integration of fungal degradation products into environmental medium. It was found that both fungi tested are efficient for PLA-based materials degradation, the most active from them being Chaetomium globosum fungus.The fungal degradation of the complex polymeric systems based on poly(lactic acid) (PLA) and natural bioactive compounds  was studied. Two fungal strains,  The lack of degradability and xenobiotic nature of the synthetic polymeric materials led to high levels of environmental pollution and health hazards. Recalcitrant nature of plastic is a matter of huge environmental concern, and hence new challenges are related to plastic degradation. With the increasing demand for plastics and rising pressure for their safe disposal, biodegradable plastics and plastic biodegradation gained a lot of attention in the last years. Plastics biodegradation is one of the best possible ways to treat these recalcitrant materials in an environment-friendly manner. The best possible routes of biodegradation and their associated social and environmental impacts must be selected . The appThe environmental degradation process and its duration and mechanism strongly depend on the environmental conditions including heat, humidity, pH, oxygen, microbes and so on. High-temperature environments with a rich microorganisms presence facilitate degradation. Poly(lactic acid) (PLA) biodegradation is a very controversial problem because it requires temperature around 60 °C (PLA glass transition temperature) which is not reached in normal soil conditions. In a regular room environment, PLA will resist for many years. However, PLA biodegrades because its origins are natural, is a bio-based polymer, the monomers being derived from renewable resources  and micrThe knowledge of the biodegradation behavior of polymers is crucial for treating plastic wastes and the serious energy crisis. The analysis of biochemical processes involved in PLA biodegradation is a key factor for exploring the high efficient methods of PLA biodegradation. PLA degradation occurs mainly through scission of ester bonds into carboxylic acid and alcohol. In addition, polymer degradation is induced by a range of factors in nature, such as oxidation, photodegradation, thermolysis, hydrolysis, biodegradation or enzymolysis ,10. DuriAmycolatopsis, Saccharothrix, Lentzea, Kibdelosporangium, Streptoalloteichus, Pseudonocardiaceae) which accelerate PLA biodegradation in the natural soil microcosms [Bacillus, Pseudomonas, Stenotrophomonas, etc.) and various fungi [Bacillus subtilis [Pseudomonas geniculata—WS3 is recommended for accelerating PLA degradation [Therefore, the researches on pure isolation of PLA-degrading microorganisms have been raised in recent years. Currently, multiple types of microbes that are able to degrade PLA have been isolated from soil or water. They mostly are actinomycetes  able to assimilate d,l-lactic acid in liquid cultures. Moreover, fungal degradation of PLA had also been studied by using Tritirachium album (ATCC 22563) in a liquid culture [A few studies reported in the scientific literature deal with the fungal degradation of PLA. There are 14 filamentous fungal strains  was a pioneer in the colonization and attack of polycaprolactone (PCL) resulting in significant mass losses. C. globosum is a saprophytic basidiomycete fungus with high capability on degrading plant materials having cellulolytic enzyme systems [Chaetomium globosum is a well-known mesophilic member of the mold family Chaetomiaceae. This saprophytic fungus primarily resides on plants, soil, straw and dung [Phanerochaete chrysosporium is a model of ligninolytic fungus which has been studied for a long time [Phanerochaete chrysosporium white rot fungus was tested for its ability to biodegrade poly(lactic acid)-based materials gamma irradiated or nitrogen plasma activated and chitosan-surface grafted [Trichoderma viride fungus, in liquid medium and controlled laboratory conditions was also studied [Recent scientific studies on the microbial degradation of natural and synthetic molecules show the potential of fungal application on cleaning technologies. It was showed by Vivi et al.  that fro systems . Chaetomand dung . The basong time . The pro grafted . The bio studied . In bothC. globosum and Phanerochaete chrysosporium fungi on the degradation of PLA-based complex materials containing 20 wt% polyethylene glycol (PEG) as plasticizer, 3–6 wt% chitosan (CS) and 0.5 weight percent (wt%) rosemary extract (R). It is expected that the bioactive compounds CS and R to influence the fungal degradation, as they exhibit antimicrobial, antioxidant activities and show a high potential application for food preservation and for therapeutic beneficial use, because they “contain active ingredients parts of plants, or other plant materials, or combinations” [In order to gain valuable insights into the biodegradation of complex polymeric systems, the present paper aimed to develop a comparative study of the action of nations” ,28.The biodegradation was evaluated from two points of view: following the influence of the polymeric materials on biochemical enzymes specific for fungal metabolism and also by changes in physical-chemical properties of the bio-based polymeric materials as changes in average molecular weight, morphology, structure and thermal behavior.Chaetomium globosum (CG) and Phanerochaete chrysosporium (PC) fungi were PLA and polyethylene glycol (PEG)-plasticized PLA as reference samples, and biocomposites containing 0.5 wt% powdered rosemary ethanolic extract (R), 3 wt% and 6 wt% chitosan (CS) and both additives—PLA-based materials subjected to biodegradation under the action of Chaetomium globosum and Phanerochaete chrysosporium fungi purchased from the Institut Scientifique de Sante Publique, Belgium, preserved in the laboratory conditions have been used in this study. Peptone, yeast extract, malt extract, agar-agar and Coomassie Brilliant Blue G250 (Fluka Switzerland), nitro blue tetrazolium, riboflavin, o-dianisidine and hydrogen peroxide , sulfuric acid, perchloric acid, 2-thiobarbituric acid, bovine serum albumin and other chemicals  were of analytical grade ones. High purity double distilled water was employed for preparation of solutions.Detailed experimental conditions have been described in a previous paper . ChaetomC. globosum was grown on agarized Haynes medium  [P. chrysosporium fungus was cultivated with solid Sabouraud agar growth medium , keeping them for 7 days at 28 ± 0.1 °C and stored at 4 °C. The discs of 0.8 cm in diameter from both fungi cultures were inoculated into 100 ml Haynes liquid or Sabouraud liquid media with 7 days old cultures of fungi, respectively using Erlenmeyer flasks. The biodegradation test was performed in controlled laboratory conditions for 14 days using the polymeric samples cut into 20 × 20 mm specimens, thickness 3 ± 0.1 mm. Samples were incubated at 28 ± 0.1 °C in the Incucell room and grown to stationary phase. The both controls were represented by the mycelium grown in the absence of polymeric samples. d water) , while PThe surface morphology of the films before and after fungal biodegradation was examined by scanning electron microscopy (SEM), using a QUANTA 200 scanning electronic microscope , at an accelerating voltage of 10 and 20 kV. The surfaces of PLA-based samples were examined as such  and SEM micrographs being recorded at different magnifications (given on the images). This technique allows to directly performing morphological and surface characterization as well as their three-dimensional shape of morphological features, the presence of aggregates, voids, etc. SEM is a useful tool for evaluating the changes that occur in the microstructure of polymers during degradation/ageing.C. globosum or P. chrysosporium mycelium (0.5 g) were added 2 mL of 0.1 M phosphate buffer solution (pH 7.5) and were subsequently centrifuged at 11,300 rpm, at 4 °C. The superoxide dismutase, catalase, soluble protein and malondialdehyde, as biochemical parameters were determined on fungus mycelium after different inoculation period as 7 and 14 days. Superoxide dismutase (SOD) activity was obtained from light absorbance at 560 nm wavelength by adapted colorimetric Winterbourn method, while the catalase (CAT) activity through the method described by Sinha (1972) [To a (1972) . Lipid pa (1972) . All resa (1972)  and grap−1 wavenumber range, at a 4 cm−1 resolution by performing 136 scans. The ATR module is equipped with a 45° ZnSe crystal (penetration thickness is around 100 µm). For each sample, the evaluations were made on the average spectrum obtained from three recordings. Spectrum processing was performed using OPUS and ORIGIN programs. To determine the structural changes occurred during fungal degradation of the PLA-based biocomposites, the attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) technique was used. The background and samples spectra were recorded with a Bruker VERTEX 70 spectrometer  in the 600 to 4000 cm3) and the experiments were achieved using a flow rate of 1.0 mL/min at 30 °C temperature. The RI/VI detector was calibrated with PS standards (580-1350000 DA) having narrow molecular weight distribution. Fragments of the samples were dissolved in HPLC grade chloroform (CHCl3) (0.02 g/mL CHCl3) and stirred at 25 °C, for 1 h, using a heater-stirrer. Diluted solutions were filtered using a 0.2 Teflon filter to extract insoluble fraction, before injecting the solute through the PLgel columns. Weight average molecular mass (Mw), number average molecular weight (Mn), as well as the polydispersity index (Mw/Mn) and viscosity were determined, using the PARSEC Chromatography software. The mean of at least two injections are given.Average molecular weights of the samples before and after incubation with fungi were determined by Gel Permeation Chromatography instrument WGE SEC-3010 multi-detection system , consisting of a pump, two PLgel columns , and dual detector RI/VI (Refractometer/Viscometer) WGE SEC-3010. The working solutions were prepared in chloroform . The degree of crystallinity (Xc) of the PLA and its composites was calculated by dividing the melting enthalpy of the sample by ΔHm = 93.7 J/g, which is equilibrium enthalpy of a PLA sample with 100% crystallinity (PLA 100%). An overall accuracy of ±0.5 °C in temperature and ±1% in enthalpy was estimated [For Differential Scanning Calorimetry a Shimadzu 60 Plus instrument  was used to examine the thermal behavior of PLA-based samples. The samples were sealed in aluminum pans. Differential scanning calorimetry (DSC) analysis was conducted under nitrogen flow (20 mL/min) within a temperature range of 20 and 250 °C with a heating rate of 10 °C/min. Calibration was performed using an indium standard (stimated .2 flow (20 mL/min). Thermogravimetry was performed by using a Perkin Elmer TGA 8000  device to evaluate the thermal degradation process of the samples. The temperature program was set from 30 to 700 °C with a heating rate of 10 °C/min under NThe PLA-composite samples were characterized before and after each exposure period (7 and 14 days) in fungal media.The presence of biodegradable polysaccharide, chitosan, into PLA-based composites hastened the disintegration rate; the composite samples exposed to liquid incubation medium were completely disintegrated after 7 days. Is noticed that the presence of PEG plasticizer in PLA matrix leads to an accelerated degradation of the polyester, the visual inspection highlighting the changing color from pale yellow  to white and a marked increase of brittleness (after 14 days the samples become difficult to handle)—Chaetomium globosum fungus undergoes a more pronounced change of the initial features.The occurred changes in the optical properties of the samples may be due to the modification of their crystallinity and/or water absorption and to biodegradation process. Based on visual examination it can be noted that the samples exposed to Chaetomium globosum fungus. The presence of the rosemary powder into PLA matrix composition, besides the adhesion of the fungi to the surface, causes cracks into the sample.The SEM photomicrographs  show thaChaetomium globosum and Phanerochaete chrysosporium fungi were covered by cracks of varying diameters which eventually created holes. Larger cracks are observed when the samples were incubated with Chaetomium globosum, resulting in loss of integrity of the sample. These findings may suggest that PLA-based samples served as the source of carbon and energy for the studied fungi. As revealed by SEM images in Dense network of fractures was particularly visible for the samples containing PEG; this may be due to the hydrophilic nature of the plasticizer, which makes the samples more accessible for fungal attack. PLA/PEG and PLA/PEG/R samples after 14 days incubation with dm) and before (m0) and were expressed as percentages.Weight losses are widely applied in the biodegradation tests, although no precise proof of evaluation can be obtained . In the Chaetomium globosum and Phanerochaete chrysosporium, the values represent an average of three experiments. Measurements of mass loss can be problematic due to moisture absorption or difficult recovery of disintegrated material. In Chaetomium globosum. The PLA-based composites samples exhibited relatively high rates of weight loss, 43% within 14 days. In the presence of the rosemary extract alone, the weight loss is smaller mainly after 7 days fungal degradation because of its high content in phenolic compounds with antioxidant activity [Chaetomium globosum shows average percentage of weight loss higher than the ones exposed to Phanerochaete chrysosporium. Similar results were reported also by Geweely and Ouf [It is known that native PLA has a slow rate of disintegration and the microorganisms that degrade PLA are rarely found in nature . Among tactivity . The PEG and Ouf  testing Both the characteristics of the fungi and their activities on polymer degradation depend on enzymes action, therefore, biochemical studies are necessary to elucidate the fungal bioactivity which gives information about their role in polymer biodegradation. Cells continuously produce free radicals and reactive oxygen species (ROS) as part of their metabolic processes, but in case of their overproduction the oxidative stress occurs. Current knowledge has shown that ROS play a key role as an agent in normal cell signal transduction and cell cycling .Hancock et al. (2001)  demonstrP. chrysosporium and C. globosum fungi  a,b.Effect of the PLA-based samples on superoxide dismutase activity. Superoxide dismutase (SOD) (EC.1.15.1.1) is considered as a key enzyme in cells, a first line of defense against oxidative damage induced by various environmental factors. The SOD catalyzes the dismutation of two molecules of superoxide anion (2O2) and molecular oxygen (O2), consequently rendering the potentially harmful superoxide anion less hazardous one [Phanerochaete chrysosporium determine at 7 days after incubation a diminution of SOD activity  while PLA sample the higher activity (7%) compared with control. At another studied time interval, 14 days after inoculation, the trend of SOD activity was the same, for both species of fungi but on a different scale values  comparatively with control. On the other hand, in C. globosum mycelium, SOD activity was very different, PLA/PEG/R sample determined the maximum activity (stimulation rate 20%) while PLA/PEG/3CS samples evidenced a lowest activity 15% compared with the control.dous one . The polactivity a. The sae values b. Thus, Effect of the PLA-based samples on catalase activity. Catalase (CAT) (EC 1.11.1.6) is a common antioxidant enzyme present almost in all living tissues that utilize oxygen which catalyzes the degradation or reduction of hydrogen peroxide (H2O2) to water and molecular oxygen, consequently completing the detoxification process imitated by SOD [2O2 molecules to H2O and O2 per minute, and stress conditions reduce the rate of protein turnover [d by SOD . Among aturnover .P. chrysosporium mycelium a more intense decrease (41%) was recorded at PLA/PEG/6CS/R sample. The CAT activity was diminished by introducing the polymeric samples in culture media of C. globosum and the lowest value being registered at PLA/PEG/3CS/R sample. In the 14 day after inoculation  is one of the most known secondary products of lipid peroxidation, and it can be used as one of the most frequent biomarker of cell membrane injury [e injury .The ROS-induced peroxidation of the lipid membranes is a reflection of stress-induced damage at the cellular level .P. chysosporium media displayed a diminution of MDA content, compared to the control—P. chysosporium fungus. Thereby, as light enhanced level of lipid peroxidation, as indicated by the MDA content, was observed after 14 days in P. chysosporium mycelium in response to presence of the polymeric samples. Thus, it would indicate a decreased oxidative stress as effect of polymeric samples. However, only PLA/PEG/3CS and PLA/PEG/6CS has determined the least amount of MDA measured, with 10% and 8%, respectively  was observed in the case of PLA/PEG/6CS/R sample. Regarding the content of MDA measured after 14 days of inoculation , brominated high impact polystyrene, and polyurethanes have been shown to be biodegradable in a variety of circumstances  despite a strong predisposition suggesting that many of these polymers were recalcitrant to the effects of biodegradation .The average molecular weight of the PLA-based materials before and after fungal degradation was determined by GPC technique, using a refractive index detector. The representative chromatograms are given in For all samples, it appears a clear tendency to decrease of molecular weight after fungal degradation both for PLA and PLA/R. For PEG-plasticized samples the main peak on chromatograms is split in two with increasing proportion of fractions with low molecular weight. This can be due to loss of PEG which has a low molecular weight and also to degradation. Similar trend was found also in variation of the intrinsic viscosity.Chaetomium globosum and Phanerochaete chrysosporium fungi are shown in It is well accepted  that theC=O, with a maximum at 1749 cm−1 (stretching) and at 1266 cm−1 (bending). The absorption bands of native PLA are also noticed in the spectrum of the fungal-biodegraded PLA but slightly shifted to higher wavenumbers and with changes in intensities, and new bands also appears. The spectra of neat PLA, PLA/R, PLA/PEG and PLA/PEG/R samples after being exposed to fungi had a shift in the bands corresponding to C=O group to higher wavenumbers from 1749 cm−1 to 1754 cm−1 depicting a reduction in their molecular weights. In the case of biodegraded neat PLA sample new bands appear, at 3323, 1652, 1546, 920 cm−1, for Chaetomium globosum fungus and at 3498, 1654, 1631, 1523, 921 cm−1 for PLA exposed to Phanerochaete chrysosporium. The mentioned bands above are assigned to -OH stretching, amide C=O stretching, N-H bending and helical backbone vibrations with the CH3 rocking modes, respectively. In the case of all biodegraded PLA-based samples, with both fungi, arises in the FTIR spectra a new band at 920–921 cm−1 that is characteristic to crystalline phase  [−1. This finding indicates that exposure of PLA-based samples to fungal environment determines an increase in crystallinity, which may be assigned to changes in the amorphous/crystalline (more stable to degradation) fraction ratio, being known that the degradation firstly occurs in amorphous region, or to the reorganization of the remaining undegraded chains with lower molecular length because of the molecular weight decrease, which can crystallize [The general band assignments for PLA-based samples was done according to literature data ,54,55 anrystals) , being astallize ,58.−1 arising from C–O, C–O–C stretches and C–O–H bends vibrations of PEG in PLA. After fungal degradation, first band mentioned is shifted at higher wavenumbers, while the second one is displaced at lower wavenumbers, meaning that oxygen containing bonds suffers changes by fungal action. Furthermore, for the initial samples containing PEG (PLA/PEG and PLA/PEG/R) a characteristic band is observed at 842–843 cm−1 that is assigned to C–O stretching, and CH2 rocking. After incubation with fungi this band disappears indicating that the addition of the hydrophilic plasticizer leads to an increased susceptibility to biodegradation of the PLA, acting as zones of adhesion for fungi.For PLA/PEG sample the bands assigned to C–O stretching mode were merged in the very broad envelope centered on 1182 and 1101 cm−1  indicating that binding interaction occurred between PLA and PEG. After incubation with fungi this band suffers a sharp decrease in intensity, revealing that the chemical integrity of the blends is affected. Specific to the samples incubated with the fungi was found also the splitting of the band at around 1453 cm−1, attributed to methyl C-H asymmetric bend, which is recently associated with the oxidation and degradation of polymeric materials [FTIR spectra of samples before biodegradation showed the absorbance of CH-COC bonding increased with PEG addition into PLA matrix at frequency 1270 cmaterials . Tg), melt temperature (Tm), crystallization temperature (Tcc), enthalpy of endothermal melting (ΔHm) and degree of crystallization (Xc) of samples obtained by DSC technique, before and after fungal degradation were determined.The glass transition temperature (The DSC curves of PLA and PLA/PEG based samples before and after biodegradation (PLA/14d/CG and PLA/14d/PC) in the presence of the two fungi, PC and CG, were shown in Tg values slightly decrease, Tcc was not more present in DSC curves, while melting peak is split into two melting peaks at 148 °C and 157 °C for PLA/14d/CG and at 150 and 157 °C for PLA/14d/PC, respectively. The two melting peaks observed may result from the melting of two different crystals of PLA which was broken after the action of fungi as the GPC results also proved by decrease of average molecular weight. The shorter chains resulted after degradation can easily reorganize in different crystallites type and this is evident by the two melting peaks in DSC curves and significant increase in crystallinity index Xc from 11.05% to 32.76% and to 35.70% for PLA/14d/CG and PLA/14d/PC, respectively. A similar melting behavior of PLA was reported by Su et al. [Chaetomium globosum was also studied by Ding et al. [As depicted in u et al.  and alsou et al. . Biodegrg et al. . The funTg = 44.4 °C and a complex melting peak with two shoulders at 128.9 °C and 144.7 °C and average Tm = 138.9 °C—Xc significantly increases up to 48%–58%.By plasticizing of PLA with PEG, the thermal characteristics resulted in a decrease of Xc. The biodegraded matrices, PLA/R/14d/CG and PLA/R/14d/PC presented a melting peak split in two distinct melting peaks which may be ascribed to recrystallization of the fragments resulted under fungi action. A similar behavior was found also by Musuc et al. in the case of addition of rosemary to polyethylene films and irradiated [The thermal behavior of the PLA/R and PLA/PEG/R samples is similar with those of PLA and PLA/PEG ones, respectively. Finally, the fungal degradation led to increase of the melting in two steps and to the increase in radiated .The effect of the fungi on the structure was evidenced by the disappearance of the shoulder and the determined thermal events are shifted indicating presence of smaller molecules which crystalize leading to higher crystallinity. Backes et al.  observedThe thermal behavior at high temperatures of PLA matrix before and after biodegradation in the presence of CG and PC fungi was represented in PLA sample decomposed in a single step between 279–383 °C, with a mass loss of 98%, results being confirmed by previously reported study . PLA samThe plasticized PLA with PEG showed even lower thermal stability by comparison with PLA—onset = 282 °C, the DTG peak temperature was found to the same value of 362 °C; Tf of PLA/R was found also slightly higher of 390 °C and the degraded mass being of 94%. A small second degradation step was detected at higher temperature over 390 °C which could be assigned to the degradation of the products resulted from first step of degradation. The two fungi used for biodegradation of PLA/R matrices seemed to act in a similar manner on this sample, as no significant differences were observed.Addition of rosemary extract to PLA matrices slightly increased the thermal stability, TThe TG/DTG curves of the PEG-plasticized PLA with incorporated rosemary extract show two degradation steps—Chaetomium globosum and Phanerochaete chrysosporium, were tested for their ability to biodegrade complex polymeric systems based on PLA and natural bioactive compounds . The fungi extracellular enzyme activities  are slightly decreased in the case of composites containing natural additives. The presence of natural compounds in the PLA-based polymeric systems determines an acceleration of fungal degradation and probably the chemical hydrolysis, which further helps the attachment of fungi to the resulting polymeric fragments and consequently the acceleration of biodegradation. Significant decreases in average molecular mass of the polymeric samples were observed by fungi action; accompanied by structural changes, increase in crystallinity and decrease of thermal properties, the loss of the physical integrity and finally to degradation with integration in environmental medium. Such findings are in accordance with other reports on microbial degradation of polymeric materials which offer new emerging technological opportunities to treat the enormous pollution problems arising by the use of polymeric products. Degrading fungi (some found in soil), as Aspergillus tubingensis [Chaetomium globosum.Two fungal strains, ingensis , showed


Phaseolus vulgaris L.) in association with rhizobia is often characterized as poor compared to other legumes, and nitrogen fertilizers are commonly used in bean production to achieve high yields, which in general inhibits nitrogen fixation. In addition, plants cannot take up all the nitrogen applied to the soil as a fertilizer leading to runoff and groundwater contamination. The overall objective of this work is to reduce use of nitrogen fertilizer in common bean production. This would be a major advance in profitability for the common bean industry in Canada and would significantly improve the ecological footprint of the crop. In the current work, 22 bean genotypes [including recombinant inbred lines (RILs) from the Mist × Sanilac population and a non-nodulating mutant (R99)] were screened for their capacity to fix atmospheric nitrogen under four nitrogen regimes. The genotypes were evaluated in replicated field trials on N-poor soils over three years for the percent nitrogen derived from atmosphere (%Ndfa), yield, and a number of yield-related traits. Bean genotypes differed for all analyzed traits, and the level of nitrogen significantly affected most of the traits, including %Ndfa and yield in all three years. In contrast, application of rhizobia significantly affected only few traits, and the effect was inconsistent among the years. Nitrogen application reduced symbiotic nitrogen fixation (SNF) to various degrees in different bean genotypes. This variation suggests that SNF in common bean can be improved through breeding and selection for the ability of bean genotypes to fix nitrogen in the presence of reduced fertilizer levels. Moreover, genotypes like RIL_38, RIL_119, and RIL_131, being both high yielding and good nitrogen fixers, have potential for simultaneous improvement of both traits. However, breeding advancement might be slow due to an inconsistent correlation between these traits.The nitrogen fixing ability of common bean ( Phaseolus vulgaris L.) is the most important legume crop used for direct human consumption. It is low in fat but rich in proteins, slowly metabolizable starch, minerals, and fibers. Beans are staple foods in many regions of South America and Africa (2) and convert it to the usable NH3 form (Common (dry) bean  and high N (100 kg ha−1) and two rhizobia strains], However, there are differences in the nodulation sensitivity to nitrogen levels among legumes . A numbeIn addition to the genetic background, environmental factors such as drought , salinit15N enriched isotope dilution and the 15N natural abundance methods have been widely used for SNF determination. In the isotope dilution technique, enriched 15N-labeled fertilizer is applied to both legume and reference crops, while in the natural abundance method the naturally occurring difference in atom% 15N of soil available nitrogen and atmospheric nitrogen is used to estimate SNF. In both methods, differences in dilution of 15N between the legume and the reference crop are used to calculate SNF. Although it is considered as the most accurate method for estimating the amount of nitrogen derived from nitrogen fixation and nitrogen from fertilizer and soil, the 15N dilution method has some limitations, including a requirement for accessing labeled fertilizer and the difficulty in establishing a stable and uniform 15N enrichment of soil mineral nitrogen in space and time. Alternatively, the 15N abundance method can be easily utilized in field settings but analyses, performed either by GC–MS or optical emission spectrometry, may be expensive.The nitrogen fixing ability of legumes can be analyzed using various methods and different tissues. In general, the amount of fixed nitrogen depends on the method and/or reference crop (cultivar) used for its determination . The 15N15N natural abundance assessment method applied to mature seeds a convenient way of comparing the relative capacities of genotypes to fix nitrogen during a growing season  gene pool] common bean genotypes, including 17 recombinant inbred lines (RILs) from a Mist × Sanilac mapping population, two parental genotypes (Sanilac and Mist) and a non-nodulating line R99 Figure 1Two additional genotypes were included in the 2017 and 2018 trials. OAC Rico is a backcross-derived anthracnose-resistant cultivar developed at University of Guelph from the cross Ex Rico 23 × Narda, tested as OAC 81-4 and registered in 1983. It is determinate cultivar similar in agronomic performance to Ex Rico 23 . Hi_N_Li°39′N, longitude 80°25′W, elevation 376.4 m; 2,680 CHU }, ON, C−1 (N)], the sub plot was rhizobia [two levels: with (R) and without rhizobia (NoR)], and the genotype (20) was set as a sub sub plot. To simplify field operations, rhizobium was set as a main plot and nitrogen as a sub plot in the 2017 (field 2) and 2018 (field 3) trials (experiment 2). In addition, two new genotypes (OAC Rico and Hi_N_Line) were included, and 22 genotypes were evaluated in the second experiment. Trials were machine planted in 4-row, 6-m-long plots with 36-cm row spacing and 350 seeds per plot .Experiments were set in a split-split-plot design (SSPD) with four replications (same field). Within each replication, randomization was done at three levels: main plot (randomization 1), sub plot (randomization 2) and sub sub plot (randomization 3). In 2016 , the main plot was nitrogen  in 2017 and two applications in 2018  were necessary to control pests/diseases in the bean fields.P-K fertilizers .http://climate.weather.gc.ca/climateData/dailydata_e.html and/climate_normals/) , and Canadian climate normals and averages data for the 1981–2010 period were collected from the nearest station   was used to randomly collect 10–16 soil samples at 0–9 inch (22.86 cm) depths across all areas of each nitrogen treatment (N and NoN) within each replication to create a bulk sample. After hand mixing, representative samples were sent for analysis to the SGS Agri-Food Laboratories Inc., Guelph, ON. The soil was analyzed only in 2017 and 2018 trials and did not include testing for the presence of indigenous rhizobia.In the 2016 pilot study (experiment 1), plot-based above ground crop data were collected for five traits, including maturity, harvestability, yield, carbon isotope discrimination, and percent nitrogen derived from atmosphere in seeds. Four additional traits  were evaluated in experiment 2 conducted in 2017 and 2018 .Days to flowering  was reported as a number of days from planting to 50% of plants in a plot with at least one fully opened flower. Days to maturity  was calculated as a number of days from planting to physiological maturity.−1). Hundred seed weight (SW) was the weight of 100 threshed seeds, expressed in g and adjusted to 18% moisture content. Plant height (PH) was measured from the soil surface to the tip of the main stem at the mid-pod filling stage and expressed in cm. Harvestability (HR) was determined at maturity as a suitability (standability) of plants in plot for mechanical harvesting using a scale of 1 to 5 [where 1 represents erect plants suitable for combine harvesting) and 5 are prostrate plants (not suitable for machine harvesting)].Yield (YD) was calculated from the seed weight measured for each plot and adjusted to 18% moisture content (kg haRelative leaf chlorophyll content (SPAD) was measured as greenness of the plants using a portable non-destructive SPAD  chlorophyll meter  on eight fully expanded young leaves in a plot  at mid-pod filling stage [63 days after planting (DAP)] and expressed as SPAD values .15N natural abundance method  was weighed into tin capsules . The closed and compressed capsules were placed in 96-well microplates and sent to the Agriculture and Agri-Food Canada, Lethbridge Research Centre  for analysis. Data were collected for δ15N, total N and δ13C using a Finnigan Delta V Plus  Isotope Ratio Mass Spectrometer (IRMS) equipped with a Flash 2000 Elemental Analyzer  and Conflo IV  interface between the elemental analyzer and the IRMS.SNF was estimated as percent nitrogen derived from atmosphere (%Ndfa) in seed samples using the e method  and R99 The %Ndfa values were calculated using the equation:where:15Nref.plant is the d15N in a non-nodulating reference genotype (R99); to reduce impact of any natural 15N variation in soil, treatment-based d15N values for R99 were used in all calculations; δ15Nfix.plant is the d15N in the analyzed common bean samples.δB is the d15N of the beans grown in a nitrogen-free medium (entire nitrogen was obtained from the nitrogen fixation); B = −1.988 .13C, ‰), as an indicator of water use efficiency  in the atmospheric air and δp is isotopic ratio of 13C to 12C in the seed sample.δ13C and SPAD).Abbreviations will be used only for the trait names consisting of more than three words  procedure in SAS  v.9.4 software . Treatmewhere:ijkl = measurement of outcome variableYµ = overall meani = random effect of replication (Rep)Repj = fixed effect of factor R (rhizobia)–main plotRij = random interaction between the Rep and the factor R (main plot factor) –error term for factor R (main plot error)RepRk = fixed effect of factor N (nitrogen) – sub plotNjk = fixed interaction between factor R and factor NRNijk = random interaction between the Rep and the factor N (sub plot factor) and the factor R (main plot factor) –error term for factor N and the interaction between factors R (main plot factor) and N (sub plot error)RepRNl = fixed effect of factor G (genotype)–sub sub plotGjl = fixed interaction between factor R (rhizobia) and factor G (genotype)RGkl = fixed interaction between factor N (nitrogen) and factor G (genotype)NGjkl = fixed interaction between factor R (rhizobia), factor N (nitrogen) and factor G (genotype)RNGijkl = residual error EFor experiment 2, a combined analysis over two years (2017 and 2018) was also computed. In the combined analysis, year, replication (year), and interactions between replications and rhizobia and among replications, rhizobia and nitrogen were evaluated as random effects using ‘random’ and ‘covtest’ statements. When necessary, ‘nobound’ option was used to remove boundary constraints on covariances (0), which allows their estimates to be negative. A few outliers were removed, and data were re-analyzed. Line R99 was used as a non-nodulating reference genotype in %Ndfa calculations and was removed from the analysis of that trait. If significant treatment differences were detected, pairwise multiple comparisons (LSmeans) were made using a Tukey–Kramer method . Residual analysis diagnostic plots were used to check assumptions for Gaussian distribution  and find “the most suitable remedy in case, violations were identified” .The relationships among traits (raw data and LSmeans) were analyzed by Pearson’s correlation in SAS. Similar to To identify high yielding genotypes with good nitrogen fixing capabilities, yield and %Ndfa data were plotted against each other for each treatment and experiment . The nitrate levels in the N samples were higher than those measured for the NoN samples in both years; 2.4-fold in 2017 and 1.9-fold in 2018, respectively  and N , and they can be considered to have been drought-stress environments , the weather conditions at ERS varied considerably in three experimental years  for harvestability and nitrogen*genotype (N*G) and R*N*G, respectively for %Ndfa] indicated that genotypes reacted differently (%Ndfa and harvestability) to four nitrogen/rhizobia treatments in 2016. Beans under the R treatment had significantly higher values for %Ndfa (41.5 vs. 18.0%) but yielded less and had higher δ13C values  compared to the beans under the N treatment. The addition of mineral nitrogen, alone (N treatment) or in combination with rhizobia (RN treatment), as well as rhizobia inoculation (R treatment) significantly increased harvestability  compared to the control ] (vs. 19.4% N treatment). In comparison, RIL_46 was the best nitrogen fixer under the N treatment; it had the highest %Ndfa (23.2%) and relatively high yield . Cultivar Sanilac also had a high value for %Ndfa (23.2%) but had the lowest yield  among the genotypes in 2016  treatments for all analyzed traits  of 22 bean genotypes [20 genotypes evaluated in 2016 plus OAC Rico  and a black genotype Hi_N_Line] were evaluated at the ERS on low nitrogen soil with four nitrogen/rhizobia treatments in 2017 and 2018. The year was considered a random effect Table 5.vs. 45.5%) and SPAD but highest harvestability score  and seed weight compared to the control (C treatment). They also flowered and matured later; the plants were taller and yielded more  when compared to the control. In general, beans under the RN treatment performed similar to the bean plants under the N treatment. They had higher values for %Ndfa (25.4 vs. 20.4%) and SPAD but were lower yielding. The addition of rhizobia (R treatment) did not have significant effects on beans. However, there were significant differences in performance between the beans grown under the added mineral nitrogen fertilizer or seed inoculation with rhizobia. The beans under the R treatment had significantly higher values for %Ndfa (42.6 vs. 20.4%), matured earlier (97 vs 99 days), were shorter (47 vs. 50 cm), and had better harvestability (2.1 vs. 2.4) but yielded less  compared to the beans under the N treatment ] (vs. 26.2% N treatment) but was still among the best nitrogen fixers under the N treatment   consistently inhibited the level of nitrogen fixation  in all bean genotypes. However, reduction was variable among the treatments and genotypes. The average reduction was the highest in the N treatment . Nitrogen fixation under the RN treatment was reduced by 44.4% [range −29.9% (RIL_119) to −54.6% (RIL_24)]. In general, rhizobia inoculation (R treatment) did not affect %Ndfa, which was similar to that in the control treatment. The differences in the %Ndfa values for the genotype mean between the control and rhizobia inoculated samples was reduced by 6.2% in the 2017–2018 trials. The R induced change ranged from an increase of 8.9% in RIL_70 to a decrease of −20.2% in RIL_46  and LSmeans in combined 2017–2018 trials. The %Ndfa was significantly positively associated with SPAD and seed weight but was not related to maturity (the possible length of nitrogen fixation cycle). Yield was positively correlated with maturity and plant height and negatively with harvestability. There was also significant positive correlation between flowering and maturity, and both traits were positively associated with plant height. However, the δ017–2018 Table 8.A principal component analysis (PCA), based on the correlation matrix, was used to reduce the dimensionality of the data. Using the Kaiser criterion , nine prCumulatively, the first two PCs explained 54.1% of the trait’s variability. Averaged over two years, PC1 had large positive associations with yield, flowering, maturity, and plant height. PC2 had large positive associations with seed weight and SPAD Figure 2Although genotypes were not divided into distinct clusters, the non-nodulating mutant line R99 and black Hi_N_Line were positioned furthest from the rest of the beans. In addition, cultivars Mist  and Sanilac  were placed in separate quadrants. Four out of seven N-dependent RILs  were placed in the same quadrant and close to Sanilac Figure 2In 2016, four (C treatments) to seven (R treatment) genotypes displayed high yields with a high nitrogen fixing capacities (%Ndfa)  and treatment sum (Trmt sum = C + R + N + RN) rank  identified RIL_38 as the best performer in the 2016 pilot study as well as in the 2017–2018 experiments Table 10th in 2016. RIL_119 ranked fourth at the treatment sum level in 2017–2018 and was first under the R treatment. This line was ninth in the 2016 trial. Three genotypes  had similar treatment sum ranks  in 2017–2018. Among them, RIL_113 was sixth in the 2017–2018 experiment but ranked high third in the 2016 pilot study. RIL_122 was ranked similarly in two experiments, seventh in the 2017–2018 study and fifth in 2016 pilot study. However, this line ranked first in the R treatment in 2016. RIL_136 was more variable; it ranked as fifth in 2017–2018 but was 10th in the 2016 study , the cultivar Sanilac [N parent (high yield under nitrogen fertilizer)] and RIL_70 placed at the bottom of the genotype rank list, having low yields and low nitrogen fixing capabilities both, at the experiment level as well as treatment sum level ] ranked 12um level Table 10The bean breeding program at the University of Guelph initiated a long term study with the goal of reducing inputs and making bean crops less dependent on nitrogen fertilizers. The results of the current work confirmed the existence of variation in nitrogen fixation (%Ndfa) among the beans from the Sanilac × Mist cross. The genotype effect was significant for all traits, including yield and %Ndfa in both experiments and is consistent with reports in the literature for a strong genetic influence on these traits. For example, The significant differences identified among 22 genotypes for %Ndfa, yield and yield-related traits, both at the experiment level as well as the levels of individual treatments, suggested that these genotypes may provide a good genetic basis for breeding better nitrogen fixing beans. The general correspondence of the genotypic trends in nitrogen fixing abilities and responses to nitrogen between the previous study by 13C, SPAD (less depending on cumulative effects compared to the others), flowering, and maturity] that were analyzed in this study were in common with the previously reported work that was performed with the whole (140) Mist × Sanilac RIL population (13C r = 0.16* and SPAD r = 0.17*) except yield, and %Ndfa correlated with maturity (r = 0.19*). No correlation was observed between yield and any of the traits measured. Overall, the associations that were detected in the previous study were different from those we found in the current study, except for a positive association between flowering and maturity in the 2017–2018 experiment (r = 0.51*). Among five traits  that were evaluated in both experiments of the current study, only significant positive correlation between yield and maturity was in common for the two trials. The differences are likely a reflection of the strong effects that environment has in conditioning these traits. The weather conditions, especially rainfall distribution at ERS, were different during the bean growing season in two experiments. Year 2016 received the most rainfall [above the 30-year precipitation average for this site (363 mm)] with the precipitation peaks in July and August. On the other hand, both 2017 and 2018 could be considered as drought-stress environments  is in agreement with Some of the correlations observed in the current work have also been identified in other studies. For example, the significant positive association between δ13C in experiments with 36 Mesoamerican (bush type) beans in both irrigated and drought trials over two years. Phenotypic correlation among traits is influenced both by genotype and environment. Bradyrhizobium inoculated peanuts (five genotypes) under two water conditions. Moreover, relatively high heritability for SPAD estimated in maize ] was identified in the 2017–2018 trials. This correlation may indicate the rate of nitrogen fixation, as suggested in soybean , and agrin maize  and confin maize  suggeste−1) delayed flowering and maturity, increased yield, but inhibited nitrogen fixation (%Ndfa) in 22 common bean genotypes evaluated in the current study over three years. These are common responses well documented in literature. Numerous studies have shown that nitrogen fixation is inhibited by the addition of nitrogen fertilizer. In soybean, −1 nitrogen fertilizer by 47% in 2016 and 55% in 2017–2018. Similar differences in SNF ability and genotypic response were reported by −1) in bean (with or without inoculation) at all experimental sites. In addition, −1 nitrogen significantly increased the number of pods per plant, number of seeds per pod and grain yields. Although the current study utilized the pre-planting nitrogen recommendation for Ontario beans of 100 kg ha−1 (The addition of nitrogen fertilizer (100 kg ha kg ha−1  and a tr−1 and R. etli) and found that both rhizobia and nitrogen increased total yield of snap beans for 18 and 42%, respectively.Seed inoculation of 22 bean genotypes with rhizobia resulted in minor to no effects on yield and nitrogen fixation (measured as %Ndfa) in the current study. This agreed with other studies reporting that inoculation with rhizobia in field experiments rarely increased yield of beans and gave no significant positive effects in nodulation and biomass production . HoweverRhizobium leguminosarum biovar viceae, Rhizobium leguminosarum biovar phaseoli and Bradyrhizobium sp.) was used. The poor response of common bean to rhizobia inoculation observed in this study could be attributed to failure by the strains that were introduced to adapt to the environmental conditions. However, the soil was not analyzed for the presence and effectiveness of indigenous rhizobia, and any competition among the strains cannot be ruled out.Common bean can be considered as a promiscuous host since a number of rhizobia species are capable of nodulating bean and a diversity of bean-rhizobia interactions exist . PositivThe effects and interactions observed among bean genotypes, rhizobia strains, nitrogen fertilizer, and environment have been characterized to be of great importance. In general, selection of high yielding genotypes with good ability for nitrogen fixation (%Ndfa) has been slow in beans. Selection for this trait is hampered by low and/or inconsistent correlations between the yield and %Ndfa, as reported previously  and confThe sensitivity of legume–rhizobia relationships to nitrogen fertilizers varies among species  and, as The current study identified a few high yielding genotypes with good nitrogen fixing capabilities (measured as %Ndfa). Genotypes like RIL_38 , Mist (consistently high yielding), and Hi_N_Line (high %Ndfa black bean) were identified in this study as good candidates for breeding high yielding and/or superior nitrogen fixers. Classification of the bean genotypes based on all treatment rank (C + R + N + RN) for the combined yield and %Ndfa values identified RIL_38 and RIL_131 as the best performers, both in the 2016 pilot study as well as in the 2017−2018 combined experiment. RIL_38 was the best in combining high yield with greater %Ndfa in all treatments in both experiments. A few additional lines also ranked consistently high. RIL_113 ranked as the third in 2016 and sixth in 2017–2018 while RIL_119 ranked as the fourth in 2017–2018 and ninth in 2016.−1 in the N treatment and 3,511 kg ha−1 in the non-fertilized C treatment) and 12% in 2017–2018 , respectively was identified in RIL_38. At the same time, %Ndfa reduction of 56.5% (43.7% in the C treatment and 19.0% in the N treatment) in 2016 and 51.1% (50.7% in the C treatment and 24.8% in the N treatment) in 2017–2018, respectively was observed. Similar response was observed in the second ranking line, RIL_131 suggesting that nitrogen fixation (%Ndfa) in these genotypes was less inhibited by the addition of nitrogen fertilizer.High yield is still the major objective in most bean breading programs. Under the fertilized conditions, yield increase of 8.4% in 2016 , but the effect was variable among genotypes and years, confirming importance of both genotype and environment in expression of these traits.This work identified genotypes that can be used in the selection of beans with less sensitivity to nitrogen fertilizer. Four genotypes can be grown either in low or high nitrogen environments. Among them, line RIL_38 was the most stable, showing consistently high yields and good nitrogen fixation capacities (%Ndfa). In some years, a few genotypes performed better in low nitrogen soils than in high nitrogen soil. However, their performance was inconsistent. The cultivar Mist and a Hi_N_Line were identified as good genotypes for breeding high yielding or superior nitrogen fixing cultivars, respectively. Collectively, the study identified genotypes that have the potential for reducing inputs and making bean crops less dependent on nitrogen fertilizers.Additional studies with these lines may lead to an understanding of the phenomenon of nitrogen fixation suppression by nitrogen fertilizer. In addition, lines like RIL_38 would be the most suitable genotype to use in a reduced input production system due to its stable high nitrogen fixation (%Ndfa) and yield with and without nitrogen and/or rhizobia. However, the suitability of these lines for breeding high yielding, nitrate insensitive, efficient nitrogen fixing bean cultivars, needs to be confirmed by additional testing with different rhizobia strains and nitrogen treatments, including low nitrogen soil levels and with a knowledge of the indigenous rhizobia in the soils. The results of the current work indicate that selection under low and/or high soil nitrogen environments may result in the development of high yielding lines with good nitrogen fixing capabilities.The datasets generated for this study are available on request to the corresponding author.KPP designed the project. KPP, THS, and YR conceived and planned the work. THS and LS set up experiments and performed field work. Crop data were collected by LS and YR. FM and BH were responsible for the IRMS seed analysis. YR analyzed the data and wrote first draft of the manuscript. KPP revised and proofread the manuscript.This project is a part of the Food From Thought research program at the University of Guelph. The research was undertaken in part thanks to generous funding from the Canada First Research Excellence Fund. Current study was also funded by the Ontario Bean Growers, Agriculture and Agri-Food Canada, the Ontario Ministry for Food and Rural Affairs and the Ontario Ministry for Research and Innovation. 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.


We report an extremely rare case of combined classical and periodontal Ehlers−Danlos syndrome (EDS) with early severe periodontitis and a generalized lack of attached gingiva. A German family with classical EDS was investigated by physical and dental evaluation and exome and Sanger sequencing. Due to the specific periodontal phenotype in the affected child, an additional diagnosis of periodontal EDS was suspected. Physical and genetic examination of two affected and three unaffected family members revealed a family diagnosis of classical EDS with a heterozygous mutation in COL5A1 . Additional to the major clinical criteria for classical EDS—generalized joint hypermobility, hyperelastic skin, and atrophic scarring —the child aged four years presented with generalized alveolar bone loss up to 80%, early loss of two lower incisors, severe gingival recession, and generalized lack of attached gingiva. Due to these clinical findings, an additional diagnosis of periodontal EDS was suspected. Further genetic analysis revealed the novel missense mutation c.658T>G (p.Cys220Gly) in C1R in a heterozygous state. Early severe periodontitis in association with generalized lack of attached gingiva is pathognomonic for periodontal EDS and led to the right clinical and genetic diagnosis in the present case. The Ehlers–Danlos syndromes (EDSs) comprise a clinically and genetically heterogeneous group of connective tissue disorders characterized by variable combinations of joint hypermobility, skin hyperextensibility, connective tissue fragility, and other manifestations. In 2017 the international EDS consortium published a revised classification system, “The 2017 International Classification of the Ehlers–Danlos Syndromes” . In contCOL5A1 and COL5A2) in more than 90% of patients; rarely specific mutations in the genes encoding type I collagen (COL1A1 and COL1A2) are detected.Classical EDS (cEDS) is inherited in an autosomal-dominant pattern and is characterized by the major criteria hyperextensibility of the skin, atrophic scarring, and generalized joint hypermobility . AffecteC1R and C1S) must be detected. pEDS mutations cause constitutive intracellular activation of C1s (and C1r) serine proteases resulting in C4 cleavage and local complement cascade activation  did not display any signs of EDS. The maternal grandfather [II:2], 59 years old, was reported to have a similar phenotype as the mother see . UnfortuDue to premature loss of two lower incisors at four years of age, the proband was referred to the Clinic for Oral and Maxillofacial Surgery of the University Hospital Münster for a special consultation on “Rare Diseases with Oral Manifestations”. The mother reported that the teeth had fallen out without root resorption. Clinical examination showed a primary dentition with missing teeth 71 and 81. In all four quadrants, CPITN was grade I revealing gingival inflammation and probing pocket depths ≤3 mm. Furcation involvement grade III was diagnosed for deciduous molars 54, 64, 74, and 84. Lack of attached gingiva was diagnosed, and severe gingival recession up to 6 mm was found for all primary teeth except the lower second molars Figure 4B). Radiologically, generalized alveolar bone loss of up to 80% was diagnosed . Due to early severe periodontal destruction and lack of attached gingiva periodontal EDS was suspected. No periodontal destruction was observed at the mother  and the Illumina HiSeq2500system  was performed in the index patient. This analysis revealed a heterozygous single nucleotide deletion c.1502del in exon 12 of the C1R and C1S was performed by PCR amplification and Sanger sequencing, using standard methods. This analysis revealed the novel C1R missense variant c.658T>G in a heterozygous state. This variant is predicted to replace a highly conserved cysteine residue with glycine in the “R” subunit of the C1r protein, denoted p.(Cys220Gly). It is not listed in population databases such as gnomAD and is not listed in the disease variant databases ClinVar or HGMD. The analysis software MutationTaster, fathmm, Mutation Assessor, SIFT, fathmm-MKL coding, LRT, and PROVEAN consistently consider this variant as pathogenic. By Sanger sequencing, this mutation was excluded in the parents and thus confirmed to be de novo in our index. On these premises, as well as on pathophysiological considerations, we classify the variant as likely pathogenic, confirming the clinical diagnosis of periodontal EDS.Six months later, due to periodontal involvement and lack of attached gingiva, it was hypothesized that the proband could also be affected by periodontal EDS. Molecular analysis of the genes COL5A1 in a German family, combined with periodontal EDS caused by a de novo C1R in an almost five-year-old girl. This is the second case of two concomitant types of EDS in one person, the first being a family with periodontal and vascular EDS published in 2018 [Here we report a unique case of classical EDS with a frameshift mutation in  in 2018 . Both reTotally one hundred eight individuals with molecularly confirmed periodontal EDS have been reported until now, with early severe periodontitis diagnosed in 99% of adult individuals ,14,15,16In contrast to these data, severe (inflammatory) periodontitis has never been reported in any case of molecularly confirmed classical EDS up to now . The usuFor individuals affected by EDS, it is quite important to distinguish between these clinical nuances. Classical and periodontal EDS are two specific entities with different treatment needs, especially in early childhood. With classical EDS, only symptomatic treatment of the skin and of joint-related symptoms is possible. Physiotherapy may slow down joint degeneration and reduce pain. In severe cases, joints can be stabilized surgically or by specific orthoses. Most patients need pain therapy, either permanently or occasionally. In the case of slowly healing wounds or scarring , plastic surgery may be needed. With periodontal EDS, treatment in childhood mainly focuses on dental prophylaxis consisting of strict dental hygiene to stop biofilm-associated periodontal hyperinflammation and subsequent bone and tooth loss. In general, dentists play an important role in therapy and early diagnoses of underlying diseases based on periodontally relevant manifestations .Many other inflammatory conditions like mast cell activation disorder, ankylosing spondylitis, rheumatoid or inflammatory arthritis may occur with EDS, particularly together with hypermobile EDS . AlthougOur case highlights the need to have an open mind with regard to the possibility of two (or more) independent monogenic conditions in the same individual. This should be considered if clinical features in a genetically defined disease do not fully fit the known spectrum of phenotypes. Severe periodontitis with early-onset is the hallmark of pEDS, but there is no evidence that it is also part of the clinical phenotype of other forms of EDS. Clinical cohort studies on different types of EDS are needed to define specific dental and periodontal manifestations.


In the last two decades, the zebrafish has emerged as an important model species for heart regeneration studies. Various approaches to model loss of cardiac myocytes and myocardial infarction in the zebrafish have been devised, and have included resection, genetic ablation, and cryoinjury. However, to date, the response of the zebrafish ventricle to cautery injury has not been reported. Here, we describe a simple and reproducible method using cautery injury via a modified nichrome inoculating needle as a probe to model myocardial infarction in the zebrafish ventricle. Using light and electron microscopy, we show that cardiac cautery injury is attended by significant inflammatory cell infiltration, accumulation of collagen in the injured area, and the reconstitution of the ventricular myocardium. Additionally, we document the ablation of cardiac nerve fibers, and report that the re-innervation of the injured zebrafish ventricle is protracted, compared to other repair processes that accompany the regeneration of the cauterized ventricle. Taken together, our study demonstrates that cautery injury is a simple and effective means for generating necrotic tissue and eliciting a remodeling and regenerative response in the zebrafish heart. This approach may serve as an important tool in the methods toolbox for regeneration studies in the zebrafish. Cardiovascular diseases remain the number one cause of death in the economically developed world . They coThe heart’s response to MI modeled in the adult mouse is attended by a non-regenerative repair response. The infarcted region proceeds through sequential and temporally overlapping processes of tissue necrosis, inflammation, and granulation tissue formation, ultimately evolving into a complex and dynamic scar ,12. The Myocardial infarction has been modeled by a variety of approaches. In mice, necrosis and loss of myocardial tissue is induced primarily through permanent coronary occlusion, occlusion and reperfusion injury, cauterization, or cryoinjury ,22,23,24Devario. cf aequipinnatus) and goldfish using cautery injury to produce a significant amount of necrotic myocardial tissue [Our lab previously studied cardiac regeneration in the giant danio (l tissue . Howevern = 142). Adult zebrafish were raised in 3 or 10 L tanks (at a maximum of three fish per liter) in an Aquatic Habitat system  at a temperature of 28.5 °C and were kept on 14/10 h day/night light cycles. System water was maintained at a pH of 7.00 and hardness of 800 µS, with automated water exchange of 10% per day. Adult zebrafish were fed primarily Zeigler Adult Zebrafish Diet  twice a day and supplemented twice a week with brine shrimp hatched from artemia cysts  or frozen Omega Super brine shrimp . Experimental procedures were approved by the DePauw University Institutional Animal Care and Use Committee on 16 July 2015 (Project identification Code: ZebrafishStudyProtoc-Heart-Rev4BF). For these experiments, zebrafish aged 6 months to 1 year old, measuring an average of 28.5 mm in standard length and 482.9 mg in wet body weight, were used. Surgeries were performed in cohorts averaging seven to eight experimental fish at a time, with a survival rate ranging from 80 to 100%, for an overall average of 87.3%  for 30 s to a minute, then fit supine into a slit cut in a sponge block such that the ventral aspect of the fish was facing up under a Leica ZOOM 2000 dissecting microscope . Using a pair of Dumont #5 fine-point tweezers , the ventral scales were removed; then the skin was perforated, and the pectoral muscles were gently dissected midline, and held open with the forceps tips to create an approximately 3 mm opening that exposed the silvery pericardium. Following the gentle dissection of the pericardial membrane, the heart ventricle and its apex were identified. During the procedure, a plastic Pasteur pipette was used to wet the sponge around the fish with system water at 30 s to 1 min intervals to prevent dehydration.The custom nichrome wire probe was immediately introduced into the open flame of a Bunsen burner for 4 to 6 s then brought to close proximity to the heart, allowing for cooling of the probe for 4 to 5 s. The tip of the heated probe was left hovering close over the apex of the beating ventricle and was moved closer during ventricular contraction (downward motion) so that the beating ventricle made contact with the heated probe during relaxation (upward motion). Before removal of the probe from the apical ventricular surface, the probe was allowed to cool further for 4 to 5 s. Following this procedure, the fish were returned to a new tank with system water. Next, a plastic Pasteur pipette was used to aerate the water and generate water flow for 45 s to a minute over the anesthetized fish to speed up recovery from the anesthetic. For analysis, adult zebrafish were euthanized at 24 h, 3, 7, 14, 30, 60, and 120 days post-cautery  using 0.2% buffered MS tricaine . Body weight and standard length of the fish were measured once all body and opercular movements had stopped. The hearts were removed by first severing the atrium at the sinus venosus–atrial junction. Then, using the forceps tips, the aorta was pinched at a segment proximal to the bulbus and gently pulled to excise the ventricle.Excised hearts were transferred into 1X PBS at room temperature and allowed to beat to evacuate luminal blood. Hearts were immediately imaged under brightfield microscopy using a Leica S6D dissecting microscope equipped with a Leica MC170 HD camera . Following imaging, the hearts were immediately fixed in 4% paraformaldehyde (PFA) in 1X PBS for further processing. Gross estimation of injury was performed by displaying the brightfield images of whole hearts on a Dell 17-inch monitor overlaid with a point-counting grid with 280 equidistant point intercepts. Injured area fraction was calculated based on the fraction of point intercepts landing randomly on the cauterized and clotted, or repairing, area to the total number of point intercepts covering the sagittal projection of the ventricular field.For scanning electron microscopy imaging, the euthanized zebrafish were first injected ventrally, near the base of the pectoral fin, with 50 microliters of heparin sulfate 1000 USO units per mL . The injected heparin was allowed to circulate for one minute. The atrium and aorta were severed and the hearts were left to beat for another minute to allow for clearing of the luminal blood. The hearts were then fixed in 4% PFA and kept at 4 °C overnight. Next the hearts were sectioned sagittally using a clean microtome knife to produce two equal halves. For dehydration, the sectioned hearts were transferred to a covered 24-well plate  and were subjected to three exchanges of 100% ethyl alcohol in a fume hood. Next, the alcohol was substituted with increasing concentrations of hexamethyldisilizane  in ETOH, from 33.3, 66.6, and 100%, three times, for 30 min, for each concentration. Following the exchanges in 100% HMDS, the hearts were left uncovered in the 24-well plate to dry overnight. The samples were next coated with 80% palladium–20% gold using an MCM-100 ion sputter coater  and imaged on a NanoImages SNE-4500M . The estimation of myocardial tissue recovery was performed using the point intercept method described above. Myocardial tissue area fraction was calculated based on point intercepts landing randomly on trabeculae and compact myocardium (excluding amorphous necrotic and remodeling tissues) and point intercepts covering the entire ventricle.For TEM, zebrafish hearts were fixed in 2.5% fresh glutaraldehyde in 100 mM cacodylate buffer overnight at 4 °C, then washed in cacodylate buffer. Next, the hearts were post-fixed in 1% tannic acid, then 1% osmium tetroxide, dehydrated in acetone, and embedded in Embed-812 resin . Two-micron thick sagittal sections were cut and stained with toluidine blue for light microscopy analysis. Ultrathin sections (100 nm thick) were mounted on single-slot or 200-mesh copper or nickel grids and imaged on a Tecnai G2 Spirit BioTWIN electron microscope .Adult hearts were fixed in ice-cold 4% paraformaldehyde for 3 h at room temperature, or overnight at 4 °C. The hearts were then cryoprotected in 30% sucrose and placed in 13 mm diameter aluminum seal cups , filled with freezing medium  and stored at −80 °C before sectioning. Sagittally sectioned 10 μm slices were obtained using a Leica CM 1900 cryostat . Cryosections from the middle of each heart containing the cauterization site were stained to detect inflammatory activity using the Peroxidase  Leukocyte kit, according to the manufacturer’s protocol . Briefly, the slides were washed gently in running tap water for 15 to 30 s and allowed to air dry. Fifty mL of Trizmal buffer was warmed to 37 °C. Peroxidase indicator reagent and 200 μL of 3% hydrogen peroxide were added to the Trizmal buffer. Slides were incubated in peroxidase indicator reagent solution at room temperature for 30 min in the dark. Slides were then washed in gently running tap water for 15 to 30 s and allowed to air dry. The MPO-stained slides were then counterstained with eosin, dehydrated in an alcohol–xylene, and coverslipped using Permount. MPO-positive cells were identified and counted at 40X magnification in the ventricular apical region in two to three fields and averaged in sections representative of each heart.For collagen visualization, Masson’s trichrome staining and Fast Green/Sirius Red staining were carried out following the manufacturer’s protocol . Briefly, slides were incubated in Bouin’s solution at room temperature overnight and stained with acid hematoxylin solution for 5 min. Next, they were stained in 0.1% Biebrich Scarlet–0.1% Acid Fuchsin in 1% acetic acid, incubated in 10% phosphotungstic–phosphomolybdic acid solution, then in 2.4% Aniline Blue solution. For Fast Green/Sirius Red staining, sections were fixed with pre-warmed Bouins’ solution overnight at room temperature, then stained with 0.1% Fast Green  for 10 min. Following a 2 min incubation in 1% acetic acid, sections were stained with 0.1% Sirius Red  in saturated picric acid for 30 min. Sections were dehydrated in graded alcohol, then coverslipped using Permount mounting media . Volume density of collagen was quantitated using 280-point grid point intercepts, and was obtained by calculating the number of point intercepts randomly intersecting collagen fibers (red/orange) divided by the total number of point intercepts intersecting green-stained myocardial tissue.For wholemount nerve fiber staining, adult hearts were fixed in 4% paraformaldehyde for 3 h at room temperature, or overnight at 4 °C. The hearts were rinsed in 1X PBS and permeabilized overnight in 0.5% Triton X-100 in 1X PBS. Samples were incubated for 48 to 72 h in zn-12 primary antibody  in 3% BSA at 4 °C. Samples were rinsed in 1X PBS before incubation in anti-mouse FITC secondary antibody  for 3 h. For wheat germ agglutinin (WGA) staining, the hearts were incubated at 4 °C for 3 h in in 400 μL of lectin-containing Tris buffer saline  at a final concentration of 2 μg/mL. The hearts were then washed in PBS, and nuclei were stained with Hoechst stain , washed three times in 1X PBS, and imaged. Adult hearts were mounted in glass-bottom tissue culture dishes , then stabilized with 2% low-melting agarose . Whole hearts were imaged at 5X and the apical region at 10X using a Zeiss AxioImager M equipped with an ApoTome.2 for optical sectioning and ZEN imaging software . Image stacks were combined into three-dimensional projections and captured. Images of the apical region were scaled to fit a 280-point grid with equidistant point intercepts. Nerve fiber volume density was calculated by the number of point intercepts randomly intersecting a nerve divided by the total number of point intercepts randomly intersecting the heart.p value of ≤0.05 was considered significant. Analysis and graphing were performed using GraphPad Prism 7 .Collected data are expressed as means and standard error of means (SEM). An ordinary one-way ANOVA followed by Tukey’s post hoc test was used to analyze data on injury sizes in brightfield images and scanning electron micrographs, volume density of nerve fibers, and inflammatory cell accumulation. A Tools, typical setup, and approach to the performance of adult zebrafish ventricular cauterization are illustrated in To gain a better understanding of the internal structural changes that accompanied the response to cauterization, we studied the injured ventricle using scanning electron microscopy (SEM). The non-injured ventricle A displayWe further characterized the cauterized ventricle in 2 μm thick toluidine blue-stained sections and by transmission electron microscopy (TEM). Observations in plastic sections were consistent with the gross ventricular muscle damage observed in brightfield and SEM, providing additional structural details. The uninjured heart displayed the expected organizational pattern of an outer compact and inner trabeculated myocardium A. FollowIn contrast to the lateral edge of the injury at 14 dpc, the junctional region of the medial apical area appears absent or was poorly defined, or the transitional cells were inappropriately localized. As a result, the separation of the compact and spongy heart was less discernible. In that region and, for the most part, cardiac myocytes were separated or displayed loose apposition to each other. Where present, electron-dense and filament-rich EDTCMs were connected to adjacent structurally immature CMs G. DesmosWe studied the inflammatory response in the cauterized heart using myeloperoxidase (MPO) reactivity. The uninjured zebrafish ventricle contained few luminal MPO-positive cells and displayed no other histological indication of inflammation. At 24 hpc, the injured apical zone A containCollagen accumulation is an important component of cardiac remodeling. We studied the extent and distribution of collagen during ventricular repair using Masson’s trichrome. Little collagen was observed in the uninjured ventricle A. HoweveTo determine the effect of apical myocardium cauterization on the ventricular innervation of the zebrafish heart, we studied zn12 immunoreactivity up to 120 dpc. Uninjured adult zebrafish ventricles displayed a relatively parsimonious pattern of nerve fibers A. The znIn this study, we demonstrate that cauterization is an effective and reliable method to create thermal necrotic injury in the adult zebrafish heart, enabling studies of cardiac remodeling and regeneration. Various approaches to ventricular injury have been used to model myocardial infarction. Apical amputation of the ventricle and cryoinjury are the most often used methods to initiate a regenerative response in the zebrafish . CryoinjAstyanax mexicanus [In these studies, the contact-mediated thermal ablation of myocardial structures by the heated nichrome probe was documented through a variety of means, including the observation of the generated clot under brightfield, identification of tissue necrosis in toluidine blue-stained sections, and confirmation of tissue damage and loss of trabecular structures by SEM and TEM. These approaches were also used to document repair and regeneration of the cauterized ventricle. In the limited number of fish species studied thus far, cardiac regenerative ability appears to range from modest to robust. Regeneration appears to be absent in the medaka  and the exicanus , similarexicanus . HoweverA preponderance of evidence supports the idea that in zebrafish, regenerated cardiac tissue relies upon a population of proliferating resident ventricular cardiac myocytes ,45. StudWe have previously described the ultrastructural characteristics of the junctional region between the compact  and spongy trabeculated myocardium in the uninjured zebrafish heart, documenting the structural heterogeneity of cardiac myocytes in that region . In partFinally, our study documents the innervation of the cauterized and regenerated zebrafish myocardium. In vertebrates, homeostatic maintenance of cardiovascular function is predicated on the presence of both extrinsic and intrinsic autonomic innervation of the heart chambers ,53,54. TIn summary, we show thermal cauterization of the zebrafish heart serves as a reliable injury model with which to study cardiac regeneration. With cautery injury, the early repair response, cardiac remodeling, and the medium- to long-term regeneration processes temporally replicate the sequence described in the cryoinjury model. However, a major advantage of this model is the ease with which locally targeted and voluminous necrotic tissue can be generated using basic and inexpensive equipment found in most laboratories. This approach adds an additional and useful tool to the technical toolbox available for the zebrafish.