Staphylococcus aureus, an important multidrug-resistant pathogen. The antimicrobial peptide nisin is a promising compound for DFI treatment, being effective against S. aureus. However, to avoid the selection of resistant mutants, correct drug therapeutic doses must be established, being also important to understand if nisin subinhibitory concentrations (subMIC) can potentiate resistant genes transfer between clinical isolates or mutations in genes associated with nisin resistance. The mutant selection window (MSW) of nisin was determined for 23 DFI S. aureus isolates; a protocol aiming to prompt vanA horizontal transfer between enterococci to clinical S. aureus was performed; and nisin subMIC effect on resistance evolution was assessed through whole-genome sequencing (WGS) applied to isolates subjected to a MEGA-plate assay. MSW ranged from 5–360 μg/mL for two isolates, from 5–540 μg/mL for three isolates, and from 5–720 μg/mL for one isolate. In the presence of nisin subMIC values, no transconjugants were obtained, indicating that nisin does not seem to promote vanA transfer. Finally, WGS analysis showed that incubation in the presence of nisin subMIC did not promote the occurrence of significant mutations in genes related to nisin resistance, supporting nisin application to DFI treatment.The most prevalent microorganism in diabetic foot infections (DFI) is  Diabetes mellitus . Cl. ClS. aulso MRSA ,50. The vanA was accomplished by a CC5 MSSA not presenting pSK41, which supports the hypothesis that other plasmids could be related to the transfer of the vanA gene.The fact that CC5 strains are repeatedly acquiring resistance to vancomycin is probably related to some predisposition of these strains to HGT . In our vanA transfer was observed. These were interesting results, since it has been described that the presence of subMIC values of antibiotics promotes HGT and the emergence of resistant bacteria [vanA HGT is a complex process, not entirely clarified, that may also be facilitated by some molecules, such as pheromone-inducible surface proteins and specific plasmids [In addition, two matting experiments were performed using the same isolates, in the environmental pressure conditions caused by the presence of nisin and vancomycin, both at subMIC concentrations. None of these experiments allowed the recovery of transconjugants. Similar results were obtained by Cunha et al. , who usebacteria . Howeverplasmids . FurtherS. aureus genome, short-read genome sequencing of the isolates collected during the MEGA-plate assays was performed at BioISI Genomics for variant call analysis  and S. aureus ATCC 29213  strains, grouped according to the MEGA-plate division from which they were collected.S. aureus ATCC 29213 presented far less genetic mutations (with the number of mutated genes ranging between 58 and 78), than the ones derived from the original S. aureus Z25.2 DFI isolate (with the number of mutated genes ranging from 88 to 140). It was not possible to detect a pattern regarding the number of mutated genes and the different assays or the nisin concentration to which they were exposed. However, in the isolates derived from S. aureus ATCC 29213, the number of genes that presented mutations tended to increase in isolates collected from divisions closer to the middle MEGA-plate division, where nisin’s concentration was the highest used in this study (MIC).It was possible to observe that isolates derived from the original S. aureus resistance to nisin. Currently, the glycopeptide resistance-associated two-component system (GraSR) has been identified as a very important two-component system in S. aureus known for being involved in nisin resistance [vraFG operon, which is positioned right downstream of the graXRS genes, ultimately encoding for an ABC transporter that plays a huge role regarding resistance to cationic AMP [mprF gene and other operons [graR, namely an isolate derived from the S. aureus Z25.2 DFI isolate, which was collected in the first assay from a division where nisin’s concentration was at its lowest (2.8125 μg mL−1). Moreover, El Shazely et al. [S. aureus, observed mutations in the vraF gene in strains submitted to subMIC concentrations of the AMP pexiganan, and also in other genes associated with detoxification by efflux, which may be associated with nisin resistance. The analysis performed in this study focused on the evaluation of specific mutations in genes involved in sistance ,53. GraS operons . Additio operons . In thisy et al. , who stunsaS gene, which encodes for a sensor kinase. A study conducted by Arii et al. showed that the exposure of S. aureus to sub-inhibitory concentrations of nisin led to the occurrence of spontaneous mutations in genes associated with this system, which resulted in higher resistance to nisin [Another two-component system of great importance regarding bacitracin and nisin resistance is NsaRS, also known as BraRS ,56,57. Nto nisin , throughvanA transfer, and that nisin subMIC values do not prompt significant mutations in genes related to nisin resistance. As such, nisin seems to be a good candidate to be applied to DFI treatment in the future.In conclusion, results showed that, to avoid the development of resistant mutants, nisin therapeutic doses should be established based on the MSW, that nisin does not seem to promote S. aureus DFI isolates was used. These isolates were previously collected from patients with DFI [S. aureus ATCC 29213 was also included as a control. Each isolate was maintained at −80 °C in buffered peptone water with 20% of glycerol throughout the duration of this study.In this study, a collection of 23 with DFI  and furtwith DFI . AdditioA nisin  with a purity of 2.5% (1000 IU/mg) was used. To obtain a 1000 µg/mL stock solution, 1 g was dissolved in 25 mL of 0.02M HCl  . AfterwaS. aureus DFI isolates under study.A modified version of the protocol described by Sinel et al.  was used8 CFU/mL) was performed and used to inoculate two BHI agar plates. After a 24 h incubation at 37 °C, the bacterial lawn was collected from the two BHI plates and resuspended in 1mL of BHI broth to achieve a bacterial suspension with a concentration of 1010 CFU/mL. To confirm the concentration values of the bacterial suspensions, these were serially diluted for viable cell count. Afterwards, 50 µL of the original 1010 CFU/mL suspensions was inoculated in Mueller–Hinton agar (MHA)  supplemented with the following concentrations of nisin: 5.63, 11.25, 22.5, 45, 90, 180, 360, and 720 µg/mL. These concentrations were selected considering a two-fold increase in the mean MIC value (11.25 µg/mL) that was previously determined [Each isolate was inoculated in brain heart infusion (BHI) agar , and, after a 24 h incubation at 37 °C, a suspension of 0.5 MacFarland . The MPC values of nisin were determined in two independent rounds.DNA extraction was performed according to the protocol described by Mottola et al. . All isovanA gene in the collection of 23 S. aureus DFI isolates under study, using multiplex PCR [vanA (5′ GGG AAA ACG ACA ATT GC 3′) and mecA (5′ TCCAGAT-TACAACTTCACCAGG 3′) were used [Before the horizontal gene transfer protocol, it was necessary to confirm the absence of plex PCR . Two paiere used ,60.vanA primer (0.5 µM), 0.23 μL of the mecA primer (0.4 µM), 12.46 μL of PCR-grade water and 5 μL DNA template (170 ng/μL).The PCR mixture had a final volume of 28.5 μL, including 10 μL of the Supreme NZYTaq 2x Green Master Mix , 0.29 μL of the PCR amplification was completed in a MyCycler Thermal Cycler  using the following conditions: initial denaturation at 94 °C for 4 min; 10 cycles including denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s, and elongation at 72 °C for 45 s; 25 cycles comprising denaturation at 94 °C for 30 s, annealing at 50 °C for 45 s and elongation at 72 °C for 2 min, and a final extension step at 72 °C for 10 min.An electrophoresis gel was performed to perceive the amplified products, using a 1.5% agarose gel  and a buffer stained with GreenSafe  at 90 V for 45 min. A molecular weight marker, NZYDNA ladder VI , was also included. Results were visualized by transillumination .Staphylococcus aureus 01-00694 (mecA positive) and Enterococcus faecium CCUG 36804 (vanA positive), were included in each PCR amplification protocol, as well as a negative control, with no DNA.Two positive control strains, R RifS) E. faecium CCUG 36804 strain as a donor of the vanA gene [S. aureus DFI isolates of our collection. Initially, resistance to rifampicin was induced in the recipients, as described elsewhere [S RifR)To test if nisin selective pressure induced horizontal gene transfer, a protocol adapted from Niederhäusern et al.  was perfanA gene , and as lsewhere , allowinRRifR transconjugants selection. Transconjugants were stored at −80°C in buffered peptone water with 20% glycerol and a PCR analysis was performed to confirm the presence of the vanA gene in these isolates. Moreover, strains with a positive vanA PCR were submitted for DNA Sanger sequencing by STABVIDA® , to confirm vanA gene transference.After performing a 0.5 MacFarland suspension for each isolate, 500 μL of the donor and 500 μL of each one of the recipients were added to 5 mL of non-supplemented TSB  and incubated at 35 °C for 18 h. After incubation, 1 mL of the bacterial suspension was added to 5 mL of TSB and further incubated for 6 h at 37 °C. Afterwards, 2 mL of each suspension was inoculated in TSA  and incubated for 5 h at 37 °C on a shaker, to promote mating. Then, the plates were incubated at 37 °C for 24 h, after which the bacterial suspension that remained at the surface of the agar plates was removed and inoculated in 5 mL of TSB. After an incubation period of 12 h at 37 °C, 100 μL of the suspension was inoculated in mannitol salt agar  supplemented with 64 μg/mL of rifampicin and 8 μg/mL of vancomycin  to allow VanThis first mating round was performed with non-supplemented TSB, as control. Then, a second mating round was performed in the presence of nisin, with all media used being supplemented with nisin at subMIC (5.63 μg/mL). A third mating round was also performed as a control, in the presence of a subMIC value of vancomycin (0.28 μg/mL), selected based on the previous MIC determination .traE (5′ ACA AAT GCG TAC TAC AGA CCC TAA ACG A 3′), synthesized by STABVIDA®  [To evaluate the presence of the pSK41-like plasmid in the 23 clinical isolates under study, a PCR protocol was performed, using a pair of primers targeting ortugal) ,62.traE primer (0.4 uM), 34.2 μL of PCR-grade water, and 5 μL DNA template (170 ng/μL). PCR amplification was completed in a MyCycler Thermal Cycler  using the following conditions: initial denaturation at 94 °C for 2 min; 30 cycles comprising denaturation at 95 °C for 15 s, annealing at 53 °C for 90 s and elongation at 72 °C for 90 s, and a final extension step at 72 °C for 7 min. An electrophoresis gel was performed to perceive the amplified products, using a 1.5% agarose gel  and a buffer stained with GreenSafe  at 90 V for 45 min. A molecular weight marker, NZYDNA ladder VII , was also included. Results were visualized by transillumination .The PCR mixture had a final volume of 50 μL, including 10 μL of the Supreme NZYTaq 2x Green Master Mix , 0.4 μL of Staphylococcus aureus RN4220 (pGO1 positive), gently provided by Dr. Alex O’Neill from the University of Leeds, was included in the PCR amplification protocol, as well as a negative control, with no DNA [A positive control strain, h no DNA .Three assays were performed using the microbial evolution and growth arena (MEGA)-plate described by Baym et al. , using aS. aureus ATCC 29213 (with 108 CFU/mL) was inoculated on the left extremity of the MEGA-plate, and a suspension of a selected DFI isolate, S. aureus Z.25.2, was inoculated on the right extremity, in an area of 1 cm of thickness. The plates were incubated at 37 °C for a period of 15 days and, each day, the growth of the staphylococci was accessed and registered via time-lapse photography twice a day using a Canon® M50 camera, in order to evaluate the bacterial multiplication progress through time ; and S. aureus ATCC 29213 (genome assembly: ASM126771v2), was performed using Bowtie2 v.2.4.1 [S. aureus Newman strain because, contrary to the Z25.2 strain, the Newman strain is fully sequenced and annotated. Variant calling was performed using Mpileup (Samtools v 1.10) [S. aureus Z.25.2 and S. aureus ATCC 29213) and the isolates collected in the assays were compared using VCFTools v0.1.16 [All Isolates obtained were analyzed by whole-genome sequencing using Illumina MiSeq paired-end short read sequencing at Biosystems and Integrative Sciences Institute (BioISI) Genomics. It was obtained between 0.6 and 1.3 million reads, corresponding to a sequencing depth between 34 and 105 times the length of the  v0.11.9 . Trimmom v0.11.9 . The ali v 1.10) . The var v0.1.16 . ENSEMBL v0.1.16 , within  v0.1.16 ,73.


The CXC chemokines belong to a unique family of chemotactic cytokines that influence the initiation, progression, and clinical outcome of many tumor types. Herein, we investigated the association of the CXC-chemokine ligand 3 (CXCL3) with tumor progression and an unfavorable prognosis for colorectal cancer (CRC).  The quantitative real-time polymerase chain reaction was used to explore the expression of CXCL3 in CRC tissue, adjacent tissue, and plasma. The usefulness of plasma levels of CXCL3 for the diagnosis of CRC was evaluated by receiver operating characteristic curve analysis. Pearson's correlation analysis assessed relationships among plasma CXCL3, cancer tissue CXCL3, and plasma carcinoembryonic antigen (CEA). Kaplan–Meier analysis was used to assess the survival of CRC patients with high and low expression levels of CXCL3. Survival differences were compared by log-rank test. P < 0.001). Compared to healthy controls, CRC patient plasma CXCL3 levels were higher (P < 0.001). The area under the curve was 0.81 with a sensitivity of 0.71 and specificity of 0.82, distinguishing CRC from other tumor types. Plasma CXCL3 was positively related to CXCL3 in cancer tissue , and also to plasma CEA . Plasma CXCL3 was also related to tumor size (P = 0.034), staging (P < 0.001), tumor stage (P = 0.003), differentiation (P = 0.001), and lymph node metastasis (P = 0.007), but not to sex (P = 0.853), age (P = 0.691), tumor site (P = 1.347), or distant metastasis (P = 1.218).  Initial analysis of the GSE156720 dataset identified CXCL3 as the most enriched CXCL gene in CRC patients. Higher CXCL3 levels were detected in CRC tissue than in adjacent tissue ( CXCL3 levels were increased in CRC patients, with plasma CXCL3 levels associated with tumor progression and an unfavorable CRC prognosis. The results of this study suggest that plasma CXCL3 may be a novel diagnostic and prognostic biomarker for CRC. Colorectal cancer (CRC) is one of the most common cancers worldwide, with approximately 1 million new cases diagnosed each year . In the This study was conducted in Xuzhou Central Hospital and was approved by the Research Ethics Committee of Xuzhou Central Hospital. A total of 59 CRC patients  underwent surgery at Xuzhou Central Hospital from January 2018 to June 2020. CRC cancer and adjacent tissue (5 cm removed from the cancer tissue) were excised and digested with collagenase for 15 minutes to prepare cell suspensions that were stored in liquid nitrogen. 169 patients  with nonoperative CRC and 219 healthy subjects  were also evaluated. Plasma samples were collected from all patients and control subjects. Patients had no other underlying medical history and did not receive radiotherapy, chemotherapy, or immunotherapy. All patients signed informed consent before sampling. All tissue and plasma samples were kept at -80 °C before assessment. Plasma levels of the classical tumor marker CEA (carcinoembryonic antigen) were assessed using the CMIA (chemiluminescent microparticle immunoassay) method on ARCHITECT 8200 ci .Total cells from tissue samples or plasma were collected and resuspended in lysis buffer  kept in ice for 5 minutes. And then, the fraction was subjected to protease treatment for 20 minutes at 37 °C by adding an equal volume of proteinase K solution , and RNA was purified using the QIAzol Lysis Reagent . The RNA was subjected to DNase treatment; cDNA was synthesized using the QuantiTect Reverse Transcription Kit  according to the manufacturer's protocol.β-actin was used as a reference gene. The PCR primer for CXCL3 was F: 5′-CGCCCAAACCGAAGTCATAG-3′ R: 5′-GCTCCCCTTGTTCAGTATCTTTT-3′ and β-actin F: 5′-AATATGTGTGTCGCGGGATG-3′, R: 5′- CTCCTTAATGTCACGCACGCACGA-3′. The reaction conditions were: 95 °C, 15 s; 60 °C, 30 s; 74 °C, 30 s; and 72 °C, 20 s, with 40 cycles of amplification. All experiments were repeated at least three times. The analysis was performed using the 2ΔΔCt− method, with β-actin as the endogenous control.qRT-PCR was performed using a Bio-Rad My cycle  according to the manufacturer's instructions. t-test, the rate was compared by Χ2 test, and the data not conforming to normal distribution were compared by Wilcoxon sign rank-sum test. Pearson's correlation analysis was used to assess the relative expression levels of CXCL3 and CEA in plasma and CRC tissues. The CRC diagnostic value of plasma CXCL3 was estimated by receiver operating characteristic curve analysis (ROC). P < 0.05 was considered to be statistically significant.Statistical analysis was conducted using GraphPad prism 8.0. The data conforming to normal distribution were expressed by mean ± standard deviation. The baseline mean between the two groups was compared by independent sample 2 (CC/AT) ≥ 1), while CXCL12, CXCL13, and CXCL14 were poorly expressed in CRC tissue (mean log2 (CC/AT) ≤ 1). CXCL3 was the most enriched (mean log2 (CC/AT) = 3.96) (P < 0.001) The relative mRNA expression of CXCLs in CRC was analyzed with the public GEO dataset, GSE156720. CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, and CXCL12 were highly expressed in CRC tissue compared to adjacent nontumor tissue (AT) (mean log = 3.96) . The mRNP < 0.001) for patients with CRC : 0.77~ 0.86) with a sensitivity of 0.71 and a specificity of 0.82, and the cutoff value of 15.5. When plasma CXCL3 and CEA values were combined for the diagnosis of CRC, the AUC increased to 0.85 (95% CI: 0.81~ 0.89), and the sensitivity and specificity were 0.74 and 0.96, respectively, indicating that CXCL3 added a significant advantage for a diagnosis of CRC ) were enrolled in this study. Plasma CXCL3 levels were measured for CRC patients and HC. Compared to HC (8.82 ± 6.79 pg/ml), preoperative plasma CXCL3 levels were significantly elevated , r = 0.50, P < 0.01)  .P = 0.034, P < 0.001, P = 0.003, P = 0.001, P = 0.007, P = 0.853, P = 0.691, P = 1.347P = 1.218, β = 0.773, P = 0.042), tumor stage , and differentiation  were found to be independent factors of plasma CXCL3 levels  were found to be independent factors of overall survival.Kaplan–Meier analysis demonstrated an overall survival of patients in the high CXCL3 expression group to be shorter than in the low expression group . Compared to healthy controls, the plasma level of CXCL3 in patients with CRC was substantially higher (P < 0.01). Further, with an AUC of 0.81 (95% CI: 0.77~ 0.86) and a sensitivity of 0.71, and a specificity of 0.82, the ideal cutoff value was 15.5. When plasma CXCL3 and CEA values were combined for the diagnosis of CRC, the AUC increased to 0.85 (95% CI: 0.81~ 0.89) and the sensitivity and specificity to 0.74 and 0.96, respectively, indicating that CXCL3 was advantageous for the diagnosis of CRC. As such, a combination of CXCL3 and CEA was sensitive and specific for CRC diagnosis, providing reliable data for early clinical diagnosis of CRC. Spearman's rank correlation analysis showed that the level of CXCL3 in plasma of CRC patients was positively related to the expression of CXCL3 in cancer tissue . Further, the level of CXCL3 in plasma of CRC patients was positively related to CEA (r = 0.50), with the plasma expression of CXCL3 in CRC related to tumor size, staging, tumor stage, differentiation, and lymph node metastasis, but not to sex, age, tumor site, or distant metastasis. Kaplan–Meier analysis demonstrated overall survival of patients with high CXCL3 expression to be shorter than that of patients with low expression (P < 0.01).This study demonstrated the expression of CXCL3 in CRC cancer tissue to be significantly higher than in matched adjacent tissue (There are limitations to this study. First, results may be confounded by variations in clinical case screening and differences in treatment. Second, cases were selected from only one hospital and may not represent other local or national sites. Third, the study was cross-sectional and requires confirmation of the role of CXCL3 in the pathogenesis of CRC cell differentiation, invasion, metastasis, clinical treatment, and prognosis. Such confirmation will be the purpose of our subsequent studies.In summary, this study analyzed the expression of CXCL3 in cancer tissue and plasma of CRC patients and found relationships among CXCL3 and patient clinic pathological characteristics. Also, a potential clinical diagnostic value for the measurement of plasma CXCL3 in CRC patients was found. The area under the curve analysis allowed for a better understanding of the CRC diagnostic value of CXCL3. It is important to note that the onset of CRC is insidious and that patients are often referred to a doctor at an advanced disease stage, limiting treatment options. Therefore, there is an urgent need for an early diagnostic marker with high sensitivity and specificity as a means by which to improve clinical diagnosis. That need will be the focus of our future work.


The genotypes/crosses showed greater genotypic variance (GV) than phenotypic variance (PV) for number of pods/plant, pod length, number of seeds/pod, number of aborted ovules/pod, fresh pod weight, seeds weight/pod, and seeds yield/plant. All studied variables showed high heritability (H%) in genotypes/crosses, despite the exception of seeds weight/pod, which ranged from 29.14 in ‘D331’ to 83.7 in F2 of Col x Com1. F2 plants and their parents’ genotypes showed greater H%. Cr7 x AI developed the most H%, 99.04% for number of pods/plant. D331 x Com1 and Cr7 x AI exhibited moderate H% for fresh pod weight in F1, but all other crosses had high H%. F1 and F2 crosses yielded moderate to high GCV and PCV for number of seeds/pod. Variations in parental genotypes and crossings reflect genetic diversity and the possibility of selection. Crossing with ‘AI,’ and ‘Com1’ genotypes enhanced the performance of the other varieties, ‘Col’, ‘D331’ and ‘Cr7’. Cr7 x Com1 and D331 x AI were selected as the most promising crosses for cowpea breeding programs.Cowpea, in addition to being a food and feed crop, plays a key role in sustainable farming. The present study’s goal is to develop new high-yielding cowpea varieties. A Field experiment was carried out across 3 summer seasons and the breeding program included 28 distinct cowpea varieties, out of which five potential parents were selected for this investigation. Local cultivars, i.e., Cream 7 ‘Cr7’, Dokki 331 ‘D331’, Commercial 1 ‘Com1’, and introduced cultivars, i.e., Colossus ‘Col’ and Asian Introduction ‘AI’ were utilized to produce six crosses in two generations apart; F Vigna unguiculata L. Walp.) is a diploid species (2n = 22), and it is classified as one of the most pertinent food sources in Africa’s arid and semi-arid regions . Cowpea is a leguminous crop with a massive tendency to increase legume production; it is a member of the Phaseoleae (L.) tribe, a family of Leguminosae, and a self-pollinated dicotyledonous crop plant . Cowpea cultivars are classified into five species: unguiculata, sesquipedalis, textles, melanophtalmus, and biflora. Crop seeds have a high calcium and iron content, as well as carbohydrates and protein, yet they are low in fat . Local commercial cultivars, i.e., Cream 7 cv. ‘Cr7’, Dokki 331 cv. ‘D331’, and introduced cultivars, i.e., Colossus cv. ‘Col’ and Asian Introduction ‘AI’. Another cultivar, Commercial 1 ‘Com1’, was collected from the local market in El-Minia governorate for its seed quality characteristics. Twenty-eight cowpea varieties, including the current genotypes, were investigated for over 8 seasons in a comprehensive study undertaken by the author. Meanwhile, these genotypes were selected because of genetic variation in morphological, floral, pod, and seed traits , whereas the plot area was 14 m2. The inner three ridges were used for sampling, and the two outer ridges were left as guard ridges. The genotypes and crosses were organized using a Randomized Complete Block Design (RCBD). Each genotype was represented by a single plot, which was repeated three times. During the three seasons, seeds were sown at a rate of 2 seeds per hole, with a spacing of 25 cm between holes. During harvesting, crosses plants were sampled, and data was collected. All other agricultural practices were in accordance with commercial production guidelines.In this experiment, crossing among five cowpea genotypes, i.e., ‘Cr7’, ‘D331’, ‘Col’, ‘AI’, and ‘Com1’, was done to produce six crosses, which were studied in advanced generations of FAt harvesting time, plants and pods were chosen at random, and the morphological, floral, pods, seeds, and yield traits were examined.Thirty plants were randomly selected from each plot of each genotype/cross, and the average shoot length, number of branches/plant, and stem diameter were investigated.The average length and diameter and number of peduncles/plant and flower length were examined in thirty plants which were randomly harvested from each genotype/cross.The average number of pods/plant, seed weight, seed width, and seed length were calculated for thirty plants for each genotype/cross. Meanwhile, the average pod length, pod diameter, pod weight, number of seeds/pod, and number of aborted ovules/pod of ten pods were measured.The weight of the seeds per plant were measured in thirty plants randomly selected from each plot for each parental genotype and cross.The genotypic and phenotypic variance were estimated by using the following equations:Whereas:MSt = Mean sum of squares for trait of genotype/cross.MSe = Mean sum of squares for error of genotype/cross.r = Number of replications.Whereas:2p = Phenotypic variance for each trait of genotype/cross.σ2g = Genotypic variance for each trait of genotype/cross.σ2e = Environmental variation among the tested traits of genotype/cross.σThe PCV and GCV expressed as percentages were calculated as suggested by Heritability in broad sense meaning was estimated as the ratio of genetic variance to the phenotypic variance as reported by It was categorized according to Genetic advance as a percent of mean (GAM) was estimated and categorized as reported by Whereas:K = 2.06 at 5% selection intensity.H = Heritability.P = Phenotypic standard deviation.Meanwhile, GAM was categorized to three classes: less than 10% (Low), 10-20% (Moderate) and more than 20% (High).1 and F2 were germinated in Petri dishes with two layers of moist filter paper at room temperature for 48 hrs. Roots with a length of 1-2 cm were cut and fixed for 24 hrs. in a newly produced farmer’s fixative solution . The fixed roots were stored in 70% ethyl alcohol in the refrigerator at 4°C until analysis. The fixed roots were rinsed with distilled water, then hydrolyzed in 1 N HCl at 60°C for 10 min before being washed again. Mitotic investigations were conducted using the aceto-carmine squash preparation. For each genotype, almost 1000 cells were investigated (consisting of ten seeds). Images were captured with an Olympus BX51 microscope and a C-4040 zoom digital camera whenever possible. Mitotic index, phase index, and chromosomal aberrations were recorded for each genotype, and mitotic index was computed using Cowpea seeds from the studied plant materials which included five parental genotypes and six crossings in FPercentage of abnormality of each stage of mitosis was counted for each slide.Data obtained from this study were subjected to analysis using SAS, version 9.3 . Differences among cowpea genotypes were tested by an analysis of variance (ANOVA), and mean significant differences were tested by the Least Significant Difference (LSD) test at the 0.05 level of significance.1 and F2 generation crosses, as shown in 1 plants that were taller than F2 plants. F1 of Cr7 x AI cross had the shortest shoot length with a mean value of 122.7 cm, while D331 x AI cross had the largest shoot length with a mean value of 234.0 cm and exceeded the better parent. On the other hand, D331 x AI cross had the shortest shoot length of all F2 plants (133.3 cm), while Col x Com1 cross was superior and had the longest shoot (193.7 cm). Furthermore, the parental cultivar ‘D331’ had the highest number of branches per plant (7.4 branches) with a range of 4.0–12 branches, whilst ‘AI’ was the least branching with a mean value of 2.9 and a range of 2.0–4.0 branches. In comparison, F1 of Col x Com1 cross and F2 of Cr7 x AI cross produced the highest values of 9.0 and 5.5, with a range of 7.0–11 and 3–9 branches, respectively . At the same time, the stem diameter of the obtained crosses ranged from 0.6 cm for cross Cr7 x AI with an average of 0.7 cm to 2.1 cm for cross Col x Com1 with an average of 2.0 cm in F1s, when it varied from 0.9 cm for Cr 7 x AI, Cr7 x Com 1, Col x AI, and D331 x AI to 2.4 cm for D331 x Com1 with an average of 1.6 cm in F2s. The data obtained, as given in 1 cross’s Col x Com1 (2.0 cm).Morphological variability in shoot length, number of branches/plant, and stem diameter was noted among Fectively Table 2.1 and F2 of Cr7 x Com1 cross outperformed the other crosses, with a mean of 51.9 and 40.2 cm and a range of 31.0-66.7 cm and 20.4-62.6 cm, respectively. Nevertheless, this cross (Cr7 x Com1) produced a taller peduncle than the better parent, ‘D331’ cv., which had a peduncle length of 33.2 cm. The peduncle diameter of the parental genotypes, on the other hand, ranged from 0.3 to 0.4 cm, and better parents were ‘Col’ cv. and ‘Com1’ cv. The peduncle diameter of F1 plants ranged from 0.2 cm in Cr7 x AI cross to 1.1 cm in Col x Com1 cross. F2 of Col x Com1 cross produced the largest peduncle diameter, with an average of 0.5 cm and a range of 0.4 to 0.7 cm. These crosses outperformed the better parents, as indicated in In terms of peduncle length, F1s had more peduncles than the crosses in F2s. F1 of Cr7 x Com1 cross produced the most peduncles (63.4) with a range of 24–167, outnumbering all other crosses and parental genotypes, while F2 of D331 x AI cross produced the fewest, still more than the least parent ‘AI’. Flower length also differed between parental genotypes and the six produced crosses in F1 and F2 generations. In general, when compared to other genotypes and crosses, ‘AI’ had the longest flower length. In contrast to the previously mentioned characteristics, F2 crosses had longer flower lengths than F1 crosses. The mean flower length in F1 and F2 crosses varied widely, from 2.3 cm for Col x AI cross to 2.6 cm for Cr7 x Com1 and Col x AI crosses. None of the tested crosses outperformed the flower length of the better genotype ‘AI’ (2.7 cm). On the other hand, F1 cross between the parental genotypes ‘D331’ and ‘Com1’ had the shortest flower length (1.9 cm) as shown in The better parental genotype, ‘Cr7’ cv., produced the most peduncles (48.9) with a range of 30–74, whereas ‘AI’ produced the fewest peduncles (18.2) with a range of 10–23. In terms of the developed six crosses, the crosses in F1 and F2 generations showed considerable variations in pods and seed traits . The paternal parents, ‘AI’ and ‘Com1’ improved the pod traits in the six crosses. According to the approximated pod length values, F1 of the crossing between ‘D331’ cv. and ‘AI’ and F2 of the crossing between ‘Col’ and ‘AI’ showed the longest pod length . However, none of the crosses exceeded the better parent, ‘AI’, which had the tallest pod with an average of 34.2 cm and a range of 24.0 to 42.0 cm , while the F2 cross of Col x Com1 yielded the same result. The largest number of aborted ovules were found in ‘AI’ and F1 of Cr7 x AI cross, with 8.6 and 7.8, respectively . On the other hand, ‘Cr7’ cv. and F2 of Cr7 x Com1 cross had the lowest value (0.7 cm in seed length). At the same time in this trial, the mean seed width of the six crossings produced increased from 0.4 to 0.5 in F1s and F2s. In comparison to the other crosses, F1 and F2 of Col x Com1 cross generated the greatest values of 0.5 cm, but the crosses did not surpass the better parent, ‘Col’ cv. which had 0.6 cm in seed width . 1 of Cr7 x AI cross to 2.0 g for F1 of Col x Com1 cross. However, in F2, the seed weight ranged from 0.9 g for D331 x Com1 cross to 2.2 g for Col x Com1 cross. Meanwhile, Col x Com1 cross was superior in the two generations but still weighted less than the better parents, ‘Com1’ cv. (2.7 g) and ‘Col’ cv. (2.6 g) as clear in 1 and F2 generations of crossings, as well as the parental genotypes, indicated significant variation. In parental genotypes, ‘Com1’ and ‘Col’ cv. yielded higher seed weight than the others, with mean values of 24.7 and 23.1 g/plant, respectively, while ‘AI’ produced the least of all genotypes and examined crosses. Crosses in F1 had a higher seed weight than crosses in F2, with the exception of Cr7 x AI cross, which had the lowest seed weight of all the crosses. D331 x AI cross exceeded the parental genotypes as well as the other crosses in F1 with an average of 80.7 g/plant and a range of 55.4-106.1 g/plant. Col x AI had heavier seeds in F2s than other crosses with an average of 27.7 g/plant and a range from 10.2-59.0 g/plant, which was still greater than the better parents , genotypic variance (GV), phenotypic coefficient of variance (PCV %), genotypic coefficient of variance (GCV %), heritability (H %), and genetic advance mean (GAM) of number of pods/plant, pod length, number of seeds/pod, number of aborted ovules/pod, fresh pod weight, seeds weight/pod and seeds yield/plant of the five parental genotypes and six crosses in F1 and F2 were evaluated. Other morphological and floral parameters, such as shoot length, number of branches/plant, peduncle length, and number of peduncles/plant, were also genetically analyzed for parental genotypes and crosses as clear in 1 and F2, the genotypic variance (GV) was greater than the phenotypic variance (PV) in the five parents and the six crossings. Simultaneously, GCV % had greater impacts than PCV % in the studied characteristics. All parents and six crossings in F1 and F2 had high heritability values. GAM ranges were found to be broad for all parents and crosses in F1 and F2. Heritability values for the parental genotypes and F1 and F2 crossings ranged from 76.68% to 96.96%, with a high H% for pod length. With the exception of ‘D331’ cv., which showed low values for PCV %, GCV %, and GAM, all parental genotypes had moderate PCV % and GCV %, whereas the six crossings in F1 and F2 had high GAM values for the trait, as shown in The genetic analysis revealed significant differences between the parental genotypes Table 5 1 and F2 crosses  was obtained from Col x AI cross in F1 and ‘AI’ genotype, whereas the lowest value (2.39%) was obtained from Cr7 x Com1 cross. Except for the Col x AI cross in F1 and the ‘AI’ genotype, which had the same value (4.53%), and Col x AI cross in F2 (4.31%), the percentages of MI were almost same with in all genotypes/crosses. On the other hand, prophase index data revealed a significant difference across all studied parents and crossings. Furthermore, ‘Col’ cv. exhibited the greatest percentage of prophase index (44.60%), with a substantial rise above almost all other genotypes. ‘Com 1’ cv., on the other hand, had the lowest proportion (18.34%). Nevertheless, as shown in 1, exhibited the greatest prophase index of 36.84% and 33.46%, respectively, when compared to the other produced crossings in F1 and F2. Metaphase index values were often greater than previous phases. Among all genotypes, D331 x AI F1 cross had the highest metaphase index (66.23%), whereas ‘Col’ cv. produced the lowest (39.84%). Despite the fact that Col x AI cross in F2 (26.22%), which was greater than its F1 value (19.16%), the anaphase and telophase indexes appeared at a low frequency compared to other phases.The mean proportions of phase and mitotic index were measured in root meristem cells from the five parents and six Frossings Table 7.1 and ‘Cr7’ cv. provided the highest values of total mitotic abnormalities , while the two crosses in F2, Col x AI and D331 x AI recorded the lowest values . The percentage of remained genotypes with abnormalities varied from 1.43% to 5.57%. 2, had the greatest frequencies of chromosomal bridges  compared to the other parental genotypes and crosses studied. Almost all cowpea genotypes tested positive for the outside chromosome. It was found in high frequency in D331 x Com1 cross in F1 and ‘Cr7’ cv.  with considerable differences with all other genotypes. In terms of chromosomal stickiness %, the aforementioned genotypes ‘Cr7’ cv. followed by D331 x Com1 cross in F1 displayed the greatest values . Laggard chromosomes were only detected in five genotypes with low frequencies: ‘AI’, Cr7 x AI F1 cross, ‘Com1’ cv., D331 x Com1 F1 cross, and Cr7 x Com1 F2 cross. Micronuclei had the lowest frequency of all mitotic aberrations detected in this experiment. It was found in only Cr7 x AI F1 cross and ‘Com1’ cv., and at extremely low levels .via its crossings with introduced cultivars, such as ‘Col’ cv. and ‘AI’, to improve the performance of local commercial cultivars, such as ‘Cr7’ cv., ‘D331’ cv., and ‘Com1’. Plant breeders attempt to develop varieties that reduce the genotype’s adverse climatic interactions, varieties that can control their developmental processes so that high yields of high-quality food are produced  for pod length and number of seeds/pod. Nevertheless, the five parents showed high genotypic coefficient of variance (GCV %), phenotypic coefficient of variance (PCV %), H %, and GAM of the number of aborted ovules/pod. Except for the F1 crosses, D331 x Com1 and Cr7 x AI, which had moderate values for H % for pod weight, all crossings in F1 and F2 exhibited high values for H % for the trait. All F1 and F2 crosses, on the other hand, produced moderate to high PCV % and GCV % of seeds weight/pod. These differences show variability as a result of additive and non-additive gene effects, emphasizing the possibility of developing novel varieties or hybrids  to total phenotypic variance (PV) in cowpea for selected agronomically significant variables. Other studies  found a  hybrids . CarvalhV. unguiculata and some allied wild species  was found in all cowpea genotypes and crosses studied. The majority of the genotypes/crosses had significantly different mitotic index (MI) overall numbers. Few genotypes showed a high prevalence of total mitotic chromosomal abnormalities. Mitotic abnormalities such as lagging chromosomes, chromosomal bridges, outside chromosomes, stickiness, and micronuclei were seen in all genotypes. The chromosomal number found in this study agrees with previous findings of 2n = 22 for  species . By inve species . The mea species . This st1 and F2, the study indicated that there is a substantial degree of genotypic variability across the variables investigated in cowpea parental genotypes. Genotypic variance was greater than phenotypic variance for the number of pods/plant, pod length, number of seeds/pod, number of aborted ovules/pod, fresh pod weight, seeds weight/pod, and seeds yield/plant. Except for seeds weight/pod in ‘D331’ cv. and F2 of Col x Com1, genotypes/crosses showed high H % for all variables tested. F2 plants had a greater H % than F1 plants and the genotypes of their parents. The produced crosses, Cr7 x Com1 and D331 x AI, have the potential for future genetic breeding research and are especially promising for yield and yield component selection. This might allow cowpea producers in Egypt and other similar regions to engage in strategic breeding and breeding trait modification.Six crosses were developed in two generations apart using five different parental genotypes: ‘AI’, ‘Col’, ‘Com1’, ‘Cr7’, and ‘D331’. When examined under different crossings in FThe original contributions presented in the study are included in the article/HZ: conceptualization, data curation, formal analysis, investigation, methodology, project administration, software, supervision, validation, visualization, writing - original draft, and writing - review and editing. KR: data curation, formal analysis, investigation, methodology, visualization, writing - original draft, and writing - review and editing. All authors contributed to the article and approved the submitted version.The authors would like to express their gratitude to the Department of Genetics, Faculty of Agriculture, Minia University, El-Minia, Egypt, for technical assistance with cytological examination of cowpea genotypes.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.


Toxic elements have a negative impact on health, especially among infants and young children. Even low levels of exposure can impair the normal growth and development of children. In young children, all organs and metabolic processes are insufficiently developed, making them particularly vulnerable to the effects of toxic elements. The aim of this study is to estimate the concentration of toxic elements in products consumed by infants and young children. The health risk of young children due to consumption of ready-made products potentially contaminated with As (arsenic), Cd (cadmium), Hg (mercury), and Pb (lead) was also assessed. A total of 397 samples  were analyzed for the content of toxic elements. Inductively coupled plasma mass spectrometry (ICP-MS) was used to assess As, Cd, and Pb concentration. The determination of Hg was performed by atomic absorption spectrometry (AAS). In order to estimate children’s exposure to toxic elements, the content of indicators was also assessed: estimated daily intake (EDI), estimated weekly intake (EWI), provisional tolerable weekly intake (PTWI), provisional tolerable monthly intake (PTMI), the benchmark dose lower confidence limit (BMDL), target hazard quotient (THQ), hazard index (HI), and cancer risk (CR). The average content of As, Cd, Hg, and Pb for all ready-made products for children is: 1.411 ± 0.248 µg/kg, 2.077 ± 0.154 µg/kg, 3.161 ± 0.159 µg/kg, and 9.265 ± 0.443 µg/kg, respectively. The highest content As was found in wafer/crisps (84.71 µg/kg); in the case of Cd, dinners with fish (20.15 µg/kg); for Hg, dinners with poultry (37.25 µg/kg); and for Pb, fruit mousse (138.99 µg/kg). The results showed that 4.53% of the samples attempted to exceed Pb, and 1.5% exceeded levels of Hg. The highest value of THQ was made in the case of drinks, for Cd and Pb in mousses for children, and Hg for dairy products. The THQ, BMDL, and PTWI ratios were not exceeded. The analyzed ready-to-eat products for children aged 0.5–3 years may contain toxic elements, but most of them appear to be harmless to health. Toxic elements are found in the human environment, and, as such, they can have a negative effect on health. Their consumption is especially dangerous in the early years of life, as this can affect children’s development, even at low exposure levels. Among infants and young children, changes in the structure and function of the main organs occur very quickly, making them a group more susceptible to the negative effects of toxic elements . InfantsThe inorganic form of arsenic (As) is listed by the IARC (Group 1) as a carcinogen . ExposurCadmium (Cd) is also on the IARC list of carcinogenic pollutants (Group 1) . As withMercury (Hg) is not classified in the IARC list for carcinogenicity (Group 3) . Hg enteLead (Pb) is recognized as “possibly carcinogenic to humans Group 2B)” in the International Agency of Research on Cancer (IARC) list of carcinogenic contaminants B” in the. ExposurIn the European Union (EU), products for infants and young children are subject to specific standards regarding the composition, nutritional value, and contamination of food. The regulation requires food producers to evaluate toxic elements in products intended for infants and young children. According to the regulation of the European Commission, the permissible concentrations of toxic elements in children’s products are more restrictive compared to conventional food .In Poland and in Europe, there are no extensive publications assessing the content of toxic elements in children’s products. Studies from outside of Europe show that the content of As and Cd are exceeded in 75% and 14% of samples tested; therefore, there is a need to evaluate food for this age group .The aim of the study was to assess the safety of ready-to-eat products for children aged 0.5–3 years in terms of the content of toxic elements. In addition, the health risk of infants and young children as a result of exposure to toxic elements in ready-to-eat food was assessed and compared with the applicable standards.The research material consisted of 397 samples. The aim was to collect as many different categories as possible to obtain representative groups. The selection of samples reflects the range of products on the Polish market. The samples were purchased in brick-and-mortar stores  in north-eastern Poland, as well as in Polish online stores between December 2020 and September 2021. We have selected the most accessible and most consumed products by children aged 0.5–3 years old. The products were sourced from leading baby food producers, such as Nestle, Nutricia, Hipp, Humana, Holle, and Helpa. Most of the analyzed products can be consumed by children from 6 months of age, while products such as biscuits, cookies, crips and fruit bars should be consumed after 12 months of age. Conventional products for the majority of consumers were also analyzed, such as cheese, milk desserts, and yoghurts, since they are often eaten by children.3 . The reagent was added according to the recommendations of the producer. Closed-loop microwave mineralization took place in four phases: first phase with a duration of 10 min, at a temperature of 170 °C, pressure 20 atm, power 90%; second phase with a duration of 10 min, at a temperature of 190 °C, pressure 30 atm, power 90%; third phase with a duration of 40 min, temperature 210 °C, pressure 40 atm, power 90%; and fourth phase with a cooling duration of 18 min, temperature 50 °C, pressure 40 atm, power 0%. After mineralization, the samples were quantitatively transferred to polypropylene vessels and diluted 10 times.All products were homogenized in a grinder or ground in a mortar, weighed , and then placed in mineralization vessels made of polytetrafluoroethylene. The next step was the addition of 4 mL of concentrated 69% HNOIn accordance with the regulations of the European Commission ,17, indiInductively coupled plasma mass spectrometry  with a kinetic discrimination chamber (KED) was used for As analysis, while Cd and Pb analysis was performed in standard mode. To correct for polyatomic interference in this configuration, kinetic energy discrimination and collisions were applied. The limit of detection (LOD) was determined by producing 10 independent blank measurements. The LOD values were as follows: for As: 0.019 µg/kg; for Cd: 0.017 µg/kg; for Pb: 0.16 µg/kg, taking LOD as a three-fold standard deviation from the mean value of the sample concentration.Hg analysis was determined without the initial mineralization process. Determination of Hg was performed by atomic absorption spectrometry (AAS) using the amalgamation technique . The samples were weighed (sample weight 0.018–0.023 g), placed in a nickel cuvette, and analyzed. In the first step, the sample was dried and ashed with oxygen at 600 °C. Hg vapors were collected by the amalgamator. In the last step, the element was released from the amalgamator and measured by atomic absorption spectrometry at 254 nm. The LOD for each sample was 0.003 ng/kg.Before starting the analyses, the certified reference material (CRM) was marked to verify the accuracy of the method. In the case of baby dinners, it was Simulated diet D ; for porridges and snacks—corn flour ; for dairy products—skim milk powder (Community Bureau of Reference BCR). A total of six independent samples were performed for each CRM. To estimate the exposure to toxic elements in a short time, indicators such as estimated daily intake (EDI), estimated weekly intake (EWI), provisional tolerable weekly intake (PTWI), provisional tolerable monthly intake (PTMI) were used. Long-term exposure to toxic elements was estimated using the following indicators: the benchmark dose lower confidence limit (BMDL), target hazard quotient (THQ), hazard index (HI), and cancer risk (CR) .Currently, the application of the PTWI index for Pb and As, previously recommended by EFSA in 2002, has been withdrawn. A more appropriate indicator is the BMDL, which is used to compare the coverage of the benchmark dose lower confidence limit .In order to determine the dose of the oral exposure of toxic elements, the EDI values were calculated in accordance with the equation:n Poland . StandarThe PTWI value was also calculated according to the equation:g BW/day ,20.The BMDL index was also determined according to the equation:BMDL values for As are 3 µg/kg/BW/day, with a benchmark dose lower confidence of 0.5% (BMDL0.5) according to FAO/WHO standards. For Pb, it is 0.5–6 µg/kg BW/day. PTWI values which are above the levels set by the standards may pose a health hazard. The results are presented as a percentage of the reference value .The THQ index is also determined to produce adverse health effects due to the properties of the toxic elements, according to the equation:(US EPA) . If the (US EPA) .−4, it refers to an increased carcinogenic risk. The THQ and CR values for Hg were not calculated since, according to the US EPA position, it is not considered a carcinogenic element [Cancer risk (CR) is used to determine the carcinogenic risk for each toxic square, calculated according to the equation: element .p < 0.05, p < 0.01, and p < 0.001 were adopted.The obtained results were analyzed using the Statistica software . The Shapiro–Wilk test was performed, and the distribution was found to be non-normal. Non-parametric Mann–Whitney U tests and the Kruskal–Wallis ANOVA test were used to analyze the concentrations of toxic elements in various product groups. The results were summarized using the median and quartiles. To compare the results of our own research with other authors, the tables contain the mean, along with the standard deviation, as well as the maximum and minimum values. In order to assess significance, the values The average content of As in all samples was 1.41 ± 0.25 µg/kg, while in 69 samples, no As was recorded. These were mainly products without the addition of cereals , and the highest value was determined in a fruit bar with the addition of rice (84.71 µg/kg). Among all of the categories, the lowest As content was found in fruit and cereal mousses (0.16 ± 0.09 µg/kg) and dairy products (0.10 ± 0.16 µg/kg). In turn, the highest As value was recorded in porridges (2.30 ± 0.33 µg/kg), and, in particular, in gluten-free porridges (4.31 ± 0.72 µg/kg). High concentrations were also obtained in snacks “for the hand” (2.92 ± 10.77 µg/kg), and especially in wafers/chips (4.88 ± 15.34 µg/kg). In accordance with the level of the European Commission , the maxThe mean concentration of Cd in all the tested samples was 2.08 ± 0.15 µg/kg. The lowest concentration of Cd in products was 0.14 µg/kg, recorded in juices for children . The highest Cd concentration was 20.15 µg/kg, and was in a ready-to-eat dinner for children after 6 months of age, consisting mainly of vegetables and salmon. Taking into account the product categories, the lowest Cd values were recorded in dairy products (0.95 ± 2.56 µg/kg) and especially in the yoghurt subgroup (0.45 ± 0.78 µg/kg), mousses (1.39 ± 2.31 µg/kg), and also in fruit juices (0.66 ± 2.01 µg/kg). The highest levels of Cd were obtained in drinks for children (3.39 ± 1.30 µg/kg), especially in fruit drinks and water (4.17 ± 5.98 µg/kg) and snacks “for the hand” (3.09 ± 1.69 µg/kg), especially in the waffle/crisps subcategory (3.51 ± 3.38 µg/kg). According to the European Commission Regulation (EU) 2021/1323 of 10 August 2021 amending Regulation (EC) No 1881/2006 as regards maximum levels of Cd in certain foodstuffs, the maximum level of Cd is 100 µg/kg for cereal, 40 µg/kg for baby food, 20 µg/kg for fruits and vegetables, 20 µg/kg for juices, and 50 µg/kg for meat and fish [The average Hg level in all samples was 3.16 ± 0.16 µg/kg, The lowest value was recorded in the fruit and cereal bar  at 0.005 µg/kg, while the highest Hg value was 37.25 µg/kg in a lunch based on vegetables, rice, and hake. Among all categories, the lowest amounts of Hg were found in snacks “for the hand” (2.36 ± 1.69 µg/kg) and in the subcategory of vegetable mousses (2.26 ± 0.83 µg/kg). The highest amounts of Hg were recorded in porridges (4.20 ± 0.35 µg/kg), in particular in gluten-free (4.89 ± 0.69 µg/kg) and gluten (4.86 ± 0.60 µg/kg) porridges; in the subcategories, the highest levels were recorded in fish dinners (as much as 9.213 ± 0.571 µg/kg) and fruit and vegetable mousses (5.748 ± 1.118 µg/kg). In accordance with European Commission Regulation (EU) 2018/73 of 16 January 2018 amending Annexes II and III to Regulation (EC) No 396/2005 of the European Parliament and of the Council, regarding the maximum residue levels for Hg compounds in or on certain products, the maximum allowable level of Hg is: 5 µg/kg for fish, 10 µg/kg for cereal, baby food, fruit and vegetables, meat, and milk, and 20 µg/kg for juices [The mean concentration of Pb in all samples was 9.27 ± 0.44 µg/kg, with the lowest recorded concentration being 0.46 µg/kg in children’s juice . The highest Pb value was 138.99 µg/kg, found in a mousse . Among all categories, the lowest Pb level was recorded in drinks for children (1.14 ± 0.98 µg/kg). The highest concentrations of Pb were found in snacks “for the hand” (12.80 ± 7.56 µg/kg), porridges (8.09 ± 0.70 µg/kg), mousses for children (7.97 ± 0.02 µg/kg), especially fruit-based mousses (9.40 ± 4.06 µg/kg), and in children’s dinners (7.55 ± 0.41 µg/kg). According to the regulations of the European Commission, the maximum permissible level of Pb is: 10 µg/kg for meat, 20 µg/kg for cereal, baby food and milk, 30 µg/kg for fish, 50 µg/kg for juices, and 100 µg/kg for fruit and vegetables [Detailed content of toxic elements in individual groups and subgroups is presented in There were 18 exceedances in the content of the daily Pb limit (4.53%). The highest Pb content was recorded in drinks (seven samples) and baby snacks (six samples). The lowest Pb exceedances were recorded in dairy products (one sample), mousses (one sample), and dinners (one sample).p < 0.01). Differences were also noted in porridges and drinks (p < 0.01), mousses and drinks (p < 0.01), baby snacks and drinks (p < 0.01), dairy and drinks (p < 0.01), and groups: poultry dinners and fish dinners (p < 0.001), beef dinners and fish dinners (p < 0.001), beef dinners and fish dinners (p < 0.001), pork dinners and fish dinners (p < 0.001), rabbit dinners and fish dinners (p < 0.001), vegetarian dinners and fish dinners (p < 0.001), and milk porridge and gluten-free cereal (p < 0.001).p < 0.05), as well as in the pork dinners and vegetarian dinners subgroup (p < 0.05). For the Pb content, one difference was found between fruit drinks and water–fruit juices (p < 0.01). In this study, no statistically significant differences in the Cd content were found.There were also statistically significant differences in the Hg content in mousses and dairy . The estimated daily consumption was the lowest in dairy products in the case of As , while in the case of Cd, this was in dairy products  and porridges . Baby dinners showed the highest value of EDI and EWI in the case of Pb , while in the case of Hg, the highest value was found in mousses . The lowest EDI value for Hg and Pb was determined in porridges . In the case of the PTWI and BMDL indicators, the specified limits were not exceeded .Baby dinners have the highest EDI and EWI values for As and Cd (EDIexceeded . The PTW−7 ± 6.18 × 10−7). In the case of As, the highest value of the index was 1.41 × 10−7 ± 3.04 × 10−7, and these levels were found in baby dinners. In the case of Cd, the highest THQ was recorded in porridges for children, and THQ was 1.31 × 10−8 ± 2.01 × 10−8. In the case of Hg, it was 1.01 × 10−7 ± 1.19 × 10−7, found in dinners for children. In the case of Pb, it was porridges 1.01 × 10−7 ± 1.19 × 10−7. The norms of THQ values were not exceeded in the tested assortment, therefore there is no increased risk of non-carcinogens effect. The CR index was not exceeded, so the research shows that the risk of developing cancer as a result of consuming the analyzed products is low.The estimated THQ and CR ratios for the analyzed toxic elements are presented in The analyzed products were also divided according to the intended use of the given products in terms of age category: for children 6–12 months, for children under 12 months, and products without an age declaration. The highest average contents of As, Cd, and Hg were recorded in the group of products intended for children aged 6–12 months . In the case of Pb, it was in the group of products without age declaration (10.95 ± 7.02 µg/kg). The highest mean content of As was recorded in the group of products for children 6–12 months (2.01 ± 3.67 µg/kg), and the lowest for children under 12 months (1.01 ± 2.54 µg/kg). The highest mean Cd content was observed in the group of products for children aged 6–12 months (2.22 ± 3.14 µg/kg), and the lowest in the group of products without declaration (1.98 ± 3.23 µg/kg), although these differences were small ; in the group of 6–12 months and in the first group without age declaration (p < 0.001); in the group of products above 12 months month and the product group without age declaration (p < 0.001). In the case of As, statistical significance was noted in the groups of products for children aged 6–12 months and over 12 months of age (p < 0.01); and in the group of 6–12 months in the group without age declaration (p < 0.001). The content of Cd differed statistically significantly in the group of products intended for children 6–12 months, the group without age declaration (p < 0.001); and in the group over 12 months of age, and the group without age declaration (p < 0.01).The most statistically significant differences were recorded in the case of Pb. The Pb content differed statistically significantly in the group of products intended for children aged 6–12 months and over 12 months  showed that Cd was found in 57% of samples. Out of 91 trials of baby food, 14% exceeded the acceptable limits for Cd consumption . The stuIn our study, the maximum allowable content was exceeded for Hg in 1.5% of samples analyzed. In the study by Guerin et al., (2018), the Hg content of 291 products for infants and young children, as well as conventional foods consumed by this group, on the French market was determined. This element was detected in 7.6% of all samples, while none of the products exceeded the permitted Hg limit . Higher In our study, the maximum allowable content was exceeded for Pb in 4.5% of samples. The Gardener et al., (2019) study assessed the content of Pb in products consumed by infants and young children . Pb was found in 37% of samples . In the Our results indicate that baby food may be safe, and the estimated health risk indicators did not show an increased health risk in terms of exposure of toxic elements in ready-made products for children. However, it is worrying that some products have exceeded the daily limit for Pb (18 samples) and Hg (6 samples). In Poland, around 60% of parents use convenience food, which should cause concern, especially in children, who often base their nutrition on ready-to-eat foods .According to our results, it is worth paying attention to ready-to-eat products containing fish. In the study, children’s meals containing salmon had the highest levels of Cd (20.15 µg/kg), and those containing hake had the highest levels of Hg (37.25 µg/kg). Among the assortment of lunches intended for children over 1 year of age was a ready-made tuna dinner that should not be eaten at all in this age group. Some fish products are especially recommended for children , while, due to the large accumulation of pollutants harmful to health, perch, shark, swordfish, and tuna are not recommended. The consumption of fish by children brings many health benefits, so we should not exclude them in the diet of children, while parents should pay attention to the selection of fish species that do not pose a health risk .In our study, cereal products  contained high amounts of As . Cereals for children and snacks were characterized by one of the higher concentrations of toxic elements .This allows us to conclude that cereal products easily accumulate toxic elements and, if consumed in large amounts, may pose a health hazard to infants and young children.An unexpected observation is the high contribution of Cd in the group of beverages for children (4.17 ± 5.98 µg/kg), and the exceedance of the daily limit of Pb content in beverages . This may result from contamination of the water used for the production of beverages or flavored waters. In Poland, there is an obligation to regularly test water for, e.g., toxic elements by provincial sanitary and epidemiological stations. This water has the correct parameters, while the problem may be an outdated sewage system located, for example, close to a factory, from which harmful substances, including toxic elements, may be released. The range of ready-to-eat products for children aged 0.5–3 years is a source of toxic elements, but most of the assessed products shown do not pose a health risk. In our study, the estimated health risk indicators did not show an increased health risk in terms of exposure to toxic elements in ready-made products for children. In some samples, Hg and Pb concentrations were exceeded, which may suggest that these products are not completely safe for children. There is a need for monitoring in ready-to-eat products intended for children aged 0.5–3 years. Particular attention should be paid to children’s consumption of fish-based products, cereal products, snacks, and flavored drinks and waters. These product groups should be subject to special control with regard to the health safety of children already at the production stage.


Bacillus sp. PCH94 (EGaseBL) with thermo-alkali stable properties. The BLEGase gene was cloned in pET-28b(+) and expressed in Escherichia coli BL21(DE3) cells. The recombinant protein was purified 94-fold with a yield of 67.8%. The biochemical characterization revealed an active enzyme at a wide pH (4.0–10.0) and temperature (4–100°C). It showed a Km and Vmax of 1.10 mg/ml and 208.24 IU/mg, respectively, using β-glucan as a substrate. The EGaseBL showed dual activities for endoglucanase (134.17 IU/mg) and exoglucanase (28.76 IU/mg), assayed using substrates β-glucan and Avicel, respectively. The enzyme is highly stable in neutral and alkaline pH and showed a half-life of 11.29 h, and 8.31 h in pH 7.0 and 9.0, respectively. The enzyme is also compatible with commercial detergents  of the Indian market and retained > 85% enzyme activity. Concisely, robustness, extreme functionality, and detergent compatibility endorse EGaseBL as a potential bioresource for the detergent industry, in addition to its implications for the bioethanol industry.Present study reports a novel and robust GH9 processive endoglucanase β-1,4-glucanase from Highlights– Cloning, expression, and purification of putative novel GH9 family β-1,4-glucanase.– Processive endoglucanase with CBM3 domain and bi-functional (endo/exo) activity.– Broad pH-temperature active and stable enzyme.– Compatible with commercial detergent powders. The global detergent market is growing at a CAGR of 6.3% after an estimated value of 154.34 billion in 2021.Endoglucanases (EGs) are required in various industries like biorefinery, paper, and pulp, and detergent for applications such as biomass conversion, biopulping, deinking, and biopolishing, respectively . The celThermomonospora fusca was reported in 1993  and a carbohydrate-binding module CBM; . The CBM in 1993 , follower genera . The bi-r genera . The PEGr genera .Bacillus sp. PCH94 from compost in the Western Himalayas and studied its biomass hydrolysis potential on damaged rice grain waste  for NcoI (Forward primer 5′-CATG CCATGG GC ATG AAA GCG CTT TGT TTG GC-3′) and XhoI (Reverse primer 5′-CCG CTCGAG GTA ACC GGG CTC ATG TCC GAA-3′). The PCR to amplify the gene was carried out under the following conditions: initial denaturation at 95°C for 2 min; 35 cycles of 95°C for 30s; 58°C for 30s; 72°C for 2 min; and a final extension at 72°C for 7 min. The amplified gene product was purified using the FavorPrep PCR purification kit  as per the manufacturer’s instructions. The final BLEGase gene product and pET-28b(+) vector were further subjected to double restriction digestion with Fast digest NcoI and XhoI restriction enzymes. The digested products were purified and ligated using T4 DNA ligase. The ligated product was transformed into competent BL21 (DE3) cells using the heat-shock method. The colonies were isolated on LB plates with 50 μg/ml kanamycin, and positive transformants were screened through the colony PCR technique. The desired cloned gene in positive transformants was also confirmed through T7 promoter-based sequencing in ABI PRISM™ 3,130 × l Genetic Analyzer .The BLEGase gene was carried out under optimized conditions, i.e., 1.0 ml of overnight grown seed culture was inoculated in a 50 ml LB broth containing Kanamycin (50 μg/ml) and incubated at 37°C/200 rpm. The culture was grown until OD600 reached 0.4–0.6, supplemented with 0.1 mM Isopropyl β-D-1-Thiogalactopyranoside (IPTG), and transferred to 28°C/200 rpm for 24 h. The cells were harvested, centrifuged, and the pellet was re-suspended in lysis buffer containing Tris–HCl buffer (25 mM/pH 8.0), 300 mM of NaCl, and 5 mM imidazole. The cells were homogenized using an ultrasonic sonicator (Ultrasonic Processor SKL-150D) with 10s on/off, 5 cycles. After sonication, the lysed cells solution was centrifuged at 12000 g for 10 min at 4°C, and cell-free supernatant (CFS) was used as a crude enzyme for further studies. The expression of the desired protein was visualized on a 10% SDS-PAGE.The expression of BL enzyme was determined using the DNSA method for reducing sugars (v/v). The enzyme-specific activity of β-1,4-glucanase was defined as μ moles of reducing sugar released from β-glucan per minute per mg of the enzyme under the assay conditions. Each reaction with its controls was carried out in triplicates in all the experiments. The observed values were subjected to standard deviation (SD), and ± 2 SD was considered significant.The enzyme activity of the EGaseg sugars . The reaBL protein was expressed and produced in 1.0 liter LB broth media. The HisPur Cobalt superflow resin was used for the purification of recombinant EGaseBL. The chromatography column was equilibrated using a basic buffer containing 25 mM Tris–HCl pH 8.0, 300 mM NaCl, and 5 mM imidazole. The CFS was filtered through a 0.45 μM syringe filter and loaded on the pre-equilibrated column. The CFS was further subjected to IMAC  resin-based purification using HisPur™ cobalt Superflow Agarose. The CFS was allowed to pass through the gravity column twice for the maximum binding. The column was washed twice with the basic buffer using 10 column volumes to remove non-specific proteins. The desired EGaseBL protein bound to the resin was eluted by using elution buffer, i.e., Tris–HCl pH 8.0, 300 mM NaCl, and 150 mM imidazole. The total amount of protein was estimated by using the Bradford assay , potassium phosphate (pH 6.0–7.0), Tris–HCl (pH 8.0–10.0), and sodium carbonate–bicarbonate (pH 9.0–10.0). The reaction conditions were as follows: temperature: 50°C, time: 60 min, and substrate: 1.0% (w/v) barley β-glucan. The buffer system with the highest specific activity was considered the optimum pH for the reaction. Furthermore, the optimum reaction temperature was assessed under optimized pH in 60 min, and 1.0% (w/v) barley β-glucan substrate. The reactions were incubated at a temperature range of 4–100°C. The enzyme concentration in the reaction was optimized under optimum pH and temperature conditions. The enzyme concentration was varied from 0.1 to 5.0 μg per reaction and incubated for 30 min. Finally, the incubation time was optimized from a varying range from 5 to 30 min under previously optimized conditions.The one factor at a time (OFAT) scheme was used to study the biochemical characteristics of purified EGaseKm) and maximum reaction velocity (Vmax) were estimated on EGaseBL activity on β-glucan under optimized reaction conditions. The reactions were carried out at 60°C/ pH 7.0 buffer (25 mM Potassium Phosphate). The substrate concentration varied from 0.05 to 0.5% to a constant enzyme concentration. The Michaelis–Menten equation and double-reciprocal Lineweaver–Burk plots were used for calculating Km and Vmax. The multi-substrate-specificity of EGaseBL was also studied on β-glucan, Avicel, filter paper, cellobiose, beechwood xylan, and starch.The kinetic parameters like Michaelis constant (t1/2) of the enzyme at different pH levels was calculated. Similarly, to study the half-life of EGaseBL at different temperatures, the enzyme was incubated at 50, 60, 70, and 80°C.The purified enzyme was incubated in various buffer systems for 5 h to investigate the effect of pH on enzyme activity. The three different buffers (25 mM), i.e., Citrate buffer pH 5.0, Potassium Phosphate buffer pH 7.0, and sodium carbonate–bicarbonate pH 9.0, were used. The enzyme reaction was performed every hour, and the half-life  is 28.88, classifying this protein as stable. The protein is extracellular in nature, as predicted by SignalP server. Further protein sequence analysis revealed that the protein belongs to the Glycosyl hydrolase-9 (GH9) family as predicted from the SWISS-MODEL server  gene was amplified from the genomic DNA of Bacillus sp. PCH94 using gene-specific primers. The pET-28b(+)-EGaseBL construct was transformed in the expression host E. coli BL21(DE3) (BL was observed after 36 h at 28°C. The cell-free supernatant’s (CFS’s) quantitative estimation of β-1,4-glucanase activity showed the specific activity of 0.205 IU/mg on the β-glucan substrate. The use of affinity protein purification resulted in a single-step 94-fold purification of protein with a yield of 67.8%. Under unoptimized enzyme reaction conditions, the purified protein showed 19.35 IU/mg specific activity  . The posactivity . The purvity  and a temperature range of 30–100°C  and Vmax (50.09 IU/mg) on CMC as a substrate  of 0.80, 11.29, and 8.31 h was observed at pH of 5.0, 7.0, and 9.0  of 14.41 h, 8.63 h, 27 min, and > 5 min was observed at 50, 60, 70, and 80°C, respectively  was cloned, expressed, and purified. The sequence analysis, presence of CBM module, and bi-functional activity confirm the processive nature of the GH9 family EGaseBL. The recombinant protein was highly purified to 94 fold with a recovery of 67.7%. Also, the physicochemical characterization revealed the robust properties of EGaseBL activity and stability in broad pH and temperature ranges. The enzyme revealed remarkable stability (> 85%) in commercial detergents for 30 min. The compatibility and stability of EGaseBL in commercial detergents are promising for the detergent industry. In addition, the kinetics analysis showed a low Km (1.10 mg/ml) and high Vmax (208.6 IU/mg) on the β-glucan substrate. The ability of EGaseBL for β-glucan and Avicel hydrolysis and broad pH/temperature stability makes it too a potential player in the biorefinery industry. Therefore, the recombinant EGaseBL has shown promise for varied industrial applications, and its future explorations could lead to astonishing bioprocess developments.A novel thermo-alkali stable and detergent compatible β-1,4-glucanase (EGaseThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The authors declare that this research did not involve human participants and/or animals. The authors declare that this work involved no unusual hazard, animal, or human subjects.VT: designed, performed experiments, data analysis, and manuscript writing. DS: conceived the study, research methodology and design, data interpretation, manuscript writing, and research supervision. All authors contributed to the article and approved the submitted version.VT duly received support from the Senior Research Fellowship (award no. 31/54(0144)/2019-EMR-1) from the Council of Scientific and Industrial Research (CSIR), New Delhi, India. DS received financial support from CSIR (grant MLP0143), New Delhi, under Niche Creating Project and Department of Biotechnology, New Delhi, in the form of GAP0130 under “Himalayan Bioresources Mission.”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.


Trichoglossum are described from south Mexico based on morphological and molecular evidence. Trichoglossumcaespitosum is characterized by the caespitose ascomata, rough and coiled paraphyses and the ascospores with 9–11 septa. Trichoglossumtropicale is characterized by the capitate ascomata, clavate and straight paraphyses and the ascospores with 10–12 septa. Both species grow in the tropical forests of the Yucatán peninsula. Here we provide descriptions and photographs for these species, together with a phylogenetic analyses based on the DNA sequences of nuc rDNA (ITS region and 28S gene) and a comparative table for the species known for America.Two new species of  Trichoglossum Boud. are characterized by club-like ascomata, usually with dark brown to black hues and acuminate setae covering both fertile and sterile parts of the ascomata, septate paraphyses, asci with 4 to 8 spores, and filiform, septate, brown ascospores. The genus is saprotrophic but is also present at the roots of plants as endophytic fungi  Boud., T.hirsutumvar.hirsutum (Pers.) Boud., T.hirsutumvar.heterosporum Mains, T.variabile (E.J. Durand) Nanff., T.velutipes (Peck) E.J. Durand, and T.walteri (Berk.) E.J. Durand ,.Universidad Autónoma de Yucatán (UADY), andInstituto Tecnológico de Ciudad Victoria (ITCV).Sampling of macrofungi was carried out in the Mexican state of Quintana Roo, in the Yucatán Peninsula Fig. . The repITCV and JF-526-ITCV), following a proteinase K protocol  according to 4)2SO4, 0.2% w/v Tween-20; Solis BioDyne, Tartu, Estonia] and 2.5 μl of proteinase K at 56 °C for 24 h, following by 15 min at 98 °C, and finally centrifuged for 2 min at 8000 rpm. Region nuc 28S rDNA (LSU) was amplified with the primers LR0R , Leucoglossumleucosporum (GB: KP272114/KP272115), and an uncultured Geoglossaceae (GB: D1273321/DQ273452). The molecular matrix was aligned using the MUSCLE algorithm (AIC) in MODELTEST 2.1.7  was performed in MrBayes 3.2.6  analysis was carried out using the GTR+I+G substitution model and bootstrap (BS) based on 100 replicates using MEGA 11  , and the specimen JF-526-ITCV with Trichoglossumhirsutum (GB: KC222146) (100% query cover and 95.65% identity). For ITS, the sequence retrieved for the specimen JF-208-ITS was 384 bp in length, spanning the 18S gene (SSU) only , so it could not be used for the phylogenetic analysis; however, the blastn analysis showed that the most similar sequences belong to the family Geoglossaceae: Gluginoglossum sp. (GB: OM672838), and two specimens of Trichoglossum sp. , all with 78% of query cover and 78.86% of identity. For the specimen JF-526-ITCV the sequence was 528 bp and contained ITS1, 5.8S and ITS2; the most similar sequences in the blastn analysis were several specimens of Trichoglossum sp. from New Zealand, showing a query cover of 100% and identity over 99.24% ; the most similar sequence of an identified specimen at the specific level was from T.hirsutum, also from New Zealand .For 28S, the blastn analysis of the collected specimens showed high levels of similarity (over 93%) with accessions of several genera within BI analysis . Within the clade of Geoglossomycetes, Sabuloglossumarenarium is sister to the rest of the species (with a low posterior probability of 0.73), and two clades are identified, one formed by species of Glutinoglossum, Hemileucoglossum, Leucoglossum, Geoglossum, an uncultured Geoglossaceae, and the specimen JF-526-ITCV sister to Trichoglossumwalteri, with a high support . The second clade is well supported , and it is composed exclusively with the rest of the species of Trichoglossum in which we find the specimen JF-208-ITCV, which is sister to T.rasum with a high posterior probability (PP = 1). Our phylogenetic analysis of T.hirsutum specimens suggests that the species is polyphyletic in its current circumscription. The topology of the ML tree (not shown) shows some incongruences with the BI majority rule tree, such as the position of Hemileucoglossumpusillum and of the specimen JF-208-ITCV, which is here sister to the clade of T.hirsutum-T.octopartitum-T.rasum but with low support (BS = 68). The remaining relationships were congruent with those obtained with the Bayesian Inference analysis.The majority rule consensus tree produced by the sis Fig.  shows a Taxon classificationFungiGeoglossalesGeoglossaceaede la Fuente, J. García & Raymundosp. nov.04656CAC-FCEE-5A36-BE95-10207C85F9E2 84300818°31'N, 88°18'W, 01 December 2015, de la Fuente JF-208-ITCV; Isotype UADY 04867. GenBank: OM727118.Mexico. Quintana Roo: Othón P. Blanco Municipality, Chetumal, alt. 8 m, Trichoglossumcaespitosum is characterized by the unique combination of characters: Caespitose habit, paraphyses with rugose and coiled tips, and the ascospores of 119–127 × 5–7 µm with 9–11 septa.caespitosum in reference to the caespitose habit.Named Ascomata black, 18–30 × 4–10 mm, clavate to spathulate, stipitate, erect, caespitose, with compressed ascogenous portion of 3–7 mm long, 0.5–1 mm thick, glossoid, ellipsoidal, flattened, sometimes curved, black, hirsute with brownish to black setae projecting from the hymenium; stipe 1–33 mm long, up to 6 mm thick, cylindrical, solid, black to dark brown, hirsute.Setae 250–300 × 5–9 µm, septate, smooth, straight, dark brown to black. Paraphyses filamentous, septate, with rugose, with wide, coiled to clavate terminal cells of 16–28 × 3–8 µm. Asci 183–221 × 16–20 µm, cylindrical to clavate, rounded at apex, short-pedicellate at the base, hyaline, thin walled, octosporic, inamyloid. Ascospores 119–127 × 5–7 µm, filiform, mostly 9–11 septate, slightly curved, hyaline when young, brown to olivaceous when mature, narrowed and rounded at both ends, thin walled, smooth.Manilkarazapota in urban vegetation.Known from the Mexican state of Quintana Roo, growing on soil under Trichoglossumrasum Pat. is morphologically similar by the octosporic asci, clavate spathulate ascoma with dark brown hues, and the tropical distribution but it differs in the bigger ascomata (10–60 mm), smaller setae (200–250 × 5–12 µm), larger ascospores (100–140 × 5–8 µm) with 3–7 septa  with 3–7 septa , straight paraphyses with bulbose tips, and the ascospores of 122–132 × 5–5.5 µm with 10–12 septa. Trichoglossumtropicale is phylogenetically close to T.walteri but that species has ascospores of 60–125 × 5–6 μm with 7 septa and paraphyses curved to circinate ; differs in the thicker setae and larger ascospores (90–150 × 5–7 µm) with 15 septa  with 7–11 septa , and bigger ascospores (80–150 × 6 µm) with 9–14 septa , as well as the Geoglossum, Glutinoglossum, and Trichoglossum clades. The inclusion of Trichoglossumwalteri and T.tropicale in this clade make the genus non-monophyletic. Both species, T.walteri and T.tropicale, form a small clade close to Glutinoglossum, nevertheless both species have setae, an absent feature within Glutinoglossum. Based on morphology and phylogenetic data (data set nuc rDNA), we describe and propose Trichoglossumcaespitosum and T.tropicale as new species that inhabit tropical vegetations and associate with Manilkarazapota and Birsonymacrassifolia respectively in the Yucatán Peninsula.The results of the phylogenetic analyses are concordant with those of Trichoglossumcaespitosum is very close morphologically and phylogenetically to T.rasum , but differs because of its ascospores of 100–140 × 5–8 μm, 3–9 septa, bulbous and curved tips paraphyses; this species was described from New Caledonia  from North Carolina but differs in that it has spores with a size of 90–100 × 4–5.5 μm and paraphyses curved to circinate, besides, the type specimen of T.walteri is from Australia, but it was cited from Brazil, Jamaica, and the United States of America (aledonia  and latealedonia  and Indialedonia . Trichogcospores . Accordied study . The mai America .T.tropicale. Nevertheless, those species are not described yet. T.cf.octopartitum. ITS sequences provided by Trichoglossum are presented in Table According to the blastn analysis of the ITS region, several sequences from New Zealand are close to the one obtained from