Petri Dishes Research Articles

First Report of Xanthomonas euvesicatoria pv. sesami Causing Leaf Spot on Sesame (Sesamum indicum L.) in South Carolina, USA.

Sesame (Sesamum indicum L.) is an annual plant known as one of the first domesticated oilseed crops. It is cultivated worldwide, mostly in Asia, Africa, and the Americas (Singh, 2006). In August 2022 and September 2023, dark angular necrotic spots on leaves and stems (100% incidence), blights, and severe defoliation were observed in a 4-acre rainfed sesame field located in the Colleton County of South Carolina, USA (Fig. S1). Bacterial streaming from cut leaf lesions was observed from diseased plants in both years. Two plants were collected for pathogen isolation in 2023. Symptomatic leaves were surface sterilized with 70% ethanol for 1 min and dried in a laminar flow hood. For each isolate, four sterile toothpicks were used to poke lesion margins and stirred in 300 µl of sterile distilled water in a 2-ml sterile microcentrifuge tube and soaked at room temperature (c. 21 °C) for 10 min. Each bacterial suspension (10 µl) was streaked on nutrient agar (NA) in a Petri dish. Convex and mucoid yellow colonies formed after a 48-h incubation at 28°C in the dark. Two isolates (S813 and S814), one from each plant, were obtained by transferring single colonies to new NA plates. Both isolates were preliminarily identified as Xanthomonas [S813: X. campestris (P = 0.53); S814: X. campestris (P = 0.77)] using a Biolog Microbial Identification System (GEN III Microplate; Identification Database v.2.8.0.15G). PCR amplification of the atpD and dnaK genes was performed for both isolates using the conditions described in Félix-Gastélum et al. (2019). The sequences of both amplicons are 100% identical for each gene between the two isolates. PCR and sequencing of the gyrB gene was also done for S813 with the primers from Young et al. (2008). The atpD (S813/S814), dnaK (S813/S814), and gyrB (S813) sequences (GenBank accessions: PP507118 to PP507120) showed the best match with 100% identity to the corresponding gene sequences [GenBank accessions: KJ491167 (100% coverage), KJ491257 (99% coverage), EU285201 (100% coverage)] of the X. euvesicatoria pv. sesami (=X. campestris pv. sesami) type strain LMG865 (Constantin et al. 2015, Parkinson et al. 2009). A neighbor joining tree with the concatenated sequences of these three genes (2,210 nt) showed that S813 and LMG 865 had the closet relationship with X. euvesicatoria pv. alfalfae (CFBP3836, Fig. S2). To fulfill Koch's postulates, three healthy sesame plants (cultivar Shirogoma) were spray inoculated separately with each suspension of S813 and S814 in sterile tap water until runoff (approx. 5×108 CFU/ml). Two sesame plants were sprayed with sterile tap water and served as negative control. All plants were maintained in a greenhouse at approximately 28/20°C (day/night) with natural photoperiod. Dark leaf spots and leaf yellowing were observed on inoculated plants 7 to 14 days after inoculation. No disease symptom was observed on the control plants. Bacteria were reisolated from leaf spots of the inoculated plants and confirmed to be X. euvesicatoria pv. sesami based on atpD and dnaK sequences. The disease was first reported in Sudan (Sabet and Dowson, 1960), after which it was reported in USA (Isakeit et al., 2012) and Mexico (Félix-Gastélum et al. 2019). To the best of our knowledge, this is the first report of this disease in South Carolina, USA. Since the interest of sesame to the farmers is increasing in the southeastern USA, it is necessary to perform further research to examine the disease distribution and its economic impact.

First Report of Polygonatum kingianum Leaf Spot Caused by Alternaria alternata in Kunming, China.

Polygonatum kingianum Coll. et Hemsl., a Polygonatum species in the Asparagaceae family, plays an important role in Chinese herbal medicine (Zhao et al. 2018). P. kingianum is widely planted in the Southwestern China. In September 2023, we observed a leaf spot of P. kingianum with disease incidence of 100%, and disease index reached 60 in commercial plantings in Kunming, Yunnan province, China (24.3610°N, 102.3740°E). In the initial stage of infection, symptoms manifested as a small circular brown spot. As the spots gradually expanded, they formed oval to irregular shaped lesions with grayish-white or dark-brown borders. Progressively the entire leaf withered and died. For identification of the causal agent of the leaf spot, leaf sections (5×5 mm2) were cut from the margin of the lesion and soaked in 75% ethanol for 10 s, 1% sodium hypochlorite for 3 min, washed with sterile distilled water, dried on sterilized tissue paper and placed on potato dextrose agar (PDA). The Petri dishes were then incubated at 28℃ for 3 days with a 12-h photoperiod. A predominant fungus was isolated from 95% of the samples. Three monosporic isolates were screened using a single-spore isolation method. After 4 days of incubation the colonies were white, after 7 days turned yellow-white. Conidia were black-brown, oblong or fusiform, with 3-7 transverse septa and 0-3 longitudinal septa, with dimensions of 19.5 to 49.5 × 8.7 to 17.6 µm (n = 30). Total genomic DNA of these three isolates was extracted from mycelia by the cetyltrimethylammonium bromide (CTAB) protocol. The nucleotide sequences of the elongation factor 1-alpha (EF1α), nuclear ribosomal internal transcribed spacer (ITS), 28S nuclear ribosomal large subunit rRNA gene (LSU), 18S nuclear ribosomal small subunit rRNA gene (SSU), and the second largest subunit of nuclear DNA-directed RNA polymerase II (RPB2) gene regions were amplified using the primer pairs EF1-728F/EF1-986R (Carbone and Kohn 1999), ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Schoch et al. 2012), NS1/NS4 (Schoch et al. 2012), and fRPB2-5F/fRPB2-7Cr (Liu et al. 1999), respectively. Amplicons were cloned in a pMDTM19-T vector (code no. 6013, Takara, Kusatsu, Japan) and bidirectionally sequenced. All three isolates had identical nucleotide sequences. Sequences from one isolate (PkF03) were deposited in GenBank. BLASTn analyses showed that sequences of EF1α (GenBank accession no. PP695240), ITS (PP694046), LSU (PP683406), SSU (PP683407), and RPB2 (PP695241) of isolate PkF03 were 99.6 (KP125134), 100 (KP124358), 100 (KP124510), 99.9 (KP124980), and 100% (KP124826), respectively, identical with Alternaria alternata (Fr.) Keissl. strain CBS 118815. Based on the nucleotide sequences of EF1α, ITS, LSU, SSU, and RPB2, a maximum likelihood phylogenetic tree was constructed using MEGAX with Tamura-Nei model. Isolate PkF03 was grouped in the same clade as A. alternata. According to the morphology and sequence analyses isolate PkF03 was identified as A. alternata (Woudenberg et al. 2013). To determine pathogenicity of isolate PkF03, a spore suspension (106 spores/mL) was sprayed on 1-year-old healthy leaves of P. kingianum. The control leaves were sprayed with sterile water. All plants were incubated at 28℃, 70% relative humidity, and a 12-h photoperiod. The pathogenicity tests were repeated three times with six plants in each treatment. Fifteen days post-inoculation, the inoculated leaves showed brown-yellow lesions, whereas the control leaves remained symptomless. A. alternata was reisolated from infected leaves. To our knowledge, this is the first report of A. alternata causing leaf spot on P. kingianum in Kunming, China. The results provide a scientific basis for prevention and control of the disease.

IDENTIFIKASI BAKTERI PADA PRODUK DARAH THROMBOCYTE CONCENTRATE

TThe Thrombocte Concentrate component is the blood component that is most easily contaminated with bacteria due to its processing and storage at room temperature. Blood or blood products that are ready to be transfused must be safe and free from the risk of infection by infectious diseases and contamination by microorganisms. Sources of bacterial contamination can come from donor skin that is less than aseptic, donor bacteremia and processing of blood products. This type of research is Pre-Experiment. The design of this research is One-Shot Case Study. In One-Shot Case Study design. The sample in this study used 1 Thrombocyte Concentrate blood product. Then it was replicated into 10 samples for Nutrient Agar media and 10 samples for Mac conkey Agar media. From the results of research conducted on 10 samples of Nutrient Agar (NA) media, it was found that there was bacterial growth in all petri dishes on Nutrient Agar (NA) media. ), while in petri dish number 10 it was found that 10% of gram-positive bacteria were in the form of bacilli (rods). From the results of research conducted on 10 samples of Mac Conkey Agar (MCA) media, no bacterial growth was found in all of the MCA media petri dishes. There was contamination of Gram-Positive bacteria in the form of Cocci (Rounds) and Bacillus (Sticks) in Thrombocyte Concentrate blood products using NA media in 10 samples out of the total samples examined were 10 samples of NA media. There was no Gram-Negative bacterial contamination in Thrombocyte Concentrate blood products using MCA media in 10 samples from the 10 samples examined.

First report of Colletotrichum plurivorum and Colletotrichum truncatum causing anthracnose on melon plants in Brazil.

Melon (Cucumis melo L.) is an economically important crop in Brazil, with an annual production of 699.281 tons (FAO 2024). Fungal diseases are one of the biggest problems in melon production, and melon growers in northeastern Brazil have reported over 80% of plants showing anthracnose symptoms in the fields during rainy seasons. Plants were wilted, displaying brown necrotic lesions and water-soaked spots with yellowish edges on the leaves and vines. Melon fruits displayed necrotic lesions on the outside. From June 2022 to June 2023, melon leaves (varieties Yellow, Galia, and Cantaloupe) from anthracnose-symptomatic plants were collected in four melon farms located in the municipalities of Afonso Bezerra, Mossoró, Tibau, and Upanema in the state of Rio Grande do Norte. Small fragments of symptomatic leaves were disinfected in 70% ethanol (30 sec) and 2.5 % sodium hypochlorite (1 min), rinsed in sterile distilled water, and plated on PDA Petri dishes with tetracycline (0.05g/liter). Plates were maintained in a bio-oxygen demand incubator (BOD) for 3 days at 28 ± 2 °C, under a 12 hr photoperiod. Eleven representative fungal colonies resembling Colletotrichum spp. were selected and monosporically grown on PDA for seven days for morphology, pathogenicity, and molecular analyses.ight colonies showed pinkish-dark brown with acervuli in the center and cottony mycelium, and showing black edges in some isolates, resembling C. plurivorum (Zhang et al. 2023). Conidia from those colonies were hyaline, cylindrical with obtuse ends, and 17.76 x 7.06 μm, n= 50. Three colonies developed pinkish-gray mycelia with numerous black microsclerotia, and the conidia were hyaline, falcate, and 27.38 x 4.10 μm, n= 50, resembling C. truncatum (Yu et al. 2023). The total DNA of the eleven isolates was extracted, and the internal transcribed space (ITS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin (TUB), and chitin synthase 1 (CHS-1) regions were partially amplified by PCR. Amplicons were sequenced and deposited to Genbank (Table eXtra1). A phylogenetic tree was built with the Maximum likelihood method with the concatenated sequences of the five partial gene sequences on Software MEGA (Version 11.0.10) (Tamura et al. 2021). The isolates CML5, CML8, CML9, CML10, CML11, CML14, CML15, and CML25 were grouped with Colletotrichum plurivorum CBS 125474 (orchidearum complex), and the isolates CML26, CML27 and CML28 with Colletotrichum truncatum CBS 15:35 (truncatum complex) with 87 % e 97 % of Bootstrap support, respectively. C. plurivorum was detected in four farms visited (we selected two representative isolates per farm), while C. truncatum isolates were all from the farm in Afonso Bezerra municipality. A pathogenicity test was performed following the method of Baishuan et al. (2023), micro-injuries were made in leaves of melon seedlings 'Goldex Yellow' and inoculated with a spore suspension of colonies with seven days of growth (106 spore/mL) of each isolate and sprayed to the point of dripping. Sterile water was used as mock. After nine days, anthracnose symptoms similar to those observed in the field were seen in all inoculated leaves, while no symptom was observed in the leaves of the mock plants. The pathogens were reisolated and their identification was confirmed by morphology and sequencing. Five seedlings were inoculated per isolate and mock, the assay was repeated, and the same results were observed. The species C. plurivorum has already been reported to cause disease in Cucumbers in Brazil (Silva et al. 2023) and C. plurivorum and C. truncatum in Citrullus lanatus in China (Guo et al. 2022). To the best of our knowledge, this is the first report of C. plurivorum and C. truncatum causing anthracnose in melon plants in Brazil.

First report of leaf spot disease caused by Cladosporium pseudocladosporioides on Lonicera caerulea L. in Heilongjiang Province, China.

Blue honeysuckle (Lonicera caerulea L.) has contributed to maintaining the forest's ecological balance and remarkable frost-resistant abilities, helping it withstand extremely cold conditions (-46 °C) and a wide pH range (5 to 8) (Sharma and Lee 2021). Between September 2022 and September 2023, leaf spots were observed on approximately 30% of blue honeysuckle plants of the 'Lanjingling' cultivar grown in a 1.13 ha field in Da Hinggan Ling Prefecture (50.32° N, 124.13° E) in Heilongjiang Province, China. The leaves of the affected plants displayed black-colored spots. To identify the causal agents, 10 healthy and symptomatic leaves were randomly collected from ten healthy and infected individual plants, respectively. Small (3 to 4 mm) segments of the symptomatic tissues were immersed in 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried in a paper towel, and plated on 9-cm Petri dishes containing potato dextrose agar (PDA). Ten fungal colonies developed on the PDA plates with an isolation frequency of 100% from 10 symptomatic leaves, and all colonies displayed a morphology consistent with Cladosporium spp. (Bensch et al. 2018). Cladosporium-like fungi were not isolated from healthy leaves. Dark olive-colored mycelia were observed, with straight unbranched conidiophores bearing terminal light brown-colored limoniform conidia (1.80 to 4.50 × 2.10 to 12.60 μm) and surrounded by a thin line of white mycelium (Delisle-Houde et al. 2024). To confirm this identification, PCR amplification of two representative strains LD-299 and LD-300 genomic DNA was performed with ITS1/ITS4 (White et al. 1990) and ACT512F/ACT783R (Carbone and Kohn 1999) primers. Basic local alignment search tool (BLAST) analyses of the National Center for Biotechnology Information database showed that sequences of the ITS (PP600316, PP600317) and ACT (PP624334, PP624335) all revealed 100% (493/493 nt, 493/493 nt; 181/181 nt, 181/181 nt) shared identity with Cladosporium pseudocladosporioides strain ex-type MF473195 and HM148674 (Bensch et al. 2010), respectively. Using a neighbor-joining phylogenetic analysis based on the ITS and ACT sequences, isolates LD-299 and LD-300 clustered in the same clade of C. pseudocladosporioides. Therefore, based on its morphological characteristics and molecular phylogeny, the two isolates were identified as C. pseudocladosporioides (Cosseboom and Hu 2023). A pathogenicity test was performed using nine healthy two-year-old blue honeysuckle Lanjingling plants. Three plants were inoculated with either the LD-299 or the LD-300 conidial suspension (1 × 106 spores/ml) or with clean water as an experimental control (Aydoğdu et al. 2023). All plants were cultured in a greenhouse (28℃, 75% relative humidity, 12 h light and dark cycle), and each experiment was replicated three times. Typical leaf spot symptoms were first observed on the inoculated leaves after 10 days. Morphological and molecular characterization of re-isolated pathogens from the artificially infected leaves indicated that the two isolates were identical, thereby confirming Koch's postulates. Cladosporium pseudocladosporioides previously caused leaf spot disease on artichoke (Cynara scolymus) in Türkiye (Aydoğdu et al. 2023). To the best of our knowledge, this is the first report of C. pseudocladosporioides causing leaf spots on blue honeysuckle in China. Blue honeysuckle production losses due to the leaf spots are critical for growers. Therefore, further focus should be given to investigate the host range and geographic distribution of C. pseudocladosporioides.

First report of leaf spot disease caused by Cladosporium pseudocladosporioides on Lonicera caerulea L. in Heilongjiang Province, China.

Blue honeysuckle (Lonicera caerulea L.) has contributed to maintaining the forest's ecological balance and remarkable frost-resistant abilities, helping it withstand extremely cold conditions (-46 °C) and a wide pH range (5 to 8) (Sharma and Lee 2021). Between September 2022 and September 2023, leaf spots were observed on approximately 30% of blue honeysuckle plants of the 'Lanjingling' cultivar grown in a 1.13 ha field in Da Hinggan Ling Prefecture (50.32° N, 124.13° E) in Heilongjiang Province, China. The leaves of the affected plants displayed black-colored spots. To identify the causal agents, 10 healthy and symptomatic leaves were randomly collected from ten healthy and infected individual plants, respectively. Small (3 to 4 mm) segments of the symptomatic tissues were immersed in 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried in a paper towel, and plated on 9-cm Petri dishes containing potato dextrose agar (PDA). Ten fungal colonies developed on the PDA plates with an isolation frequency of 100% from 10 symptomatic leaves, and all colonies displayed a morphology consistent with Cladosporium spp. (Bensch et al. 2018). Cladosporium-like fungi were not isolated from healthy leaves. Dark olive-colored mycelia were observed, with straight unbranched conidiophores bearing terminal light brown-colored limoniform conidia (1.80 to 4.50 × 2.10 to 12.60 μm) and surrounded by a thin line of white mycelium (Delisle-Houde et al. 2024). To confirm this identification, PCR amplification of two representative strains LD-299 and LD-300 genomic DNA was performed with ITS1/ITS4 (White et al. 1990) and ACT512F/ACT783R (Carbone and Kohn 1999) primers. Basic local alignment search tool (BLAST) analyses of the National Center for Biotechnology Information database showed that sequences of the ITS (PP600316, PP600317) and ACT (PP624334, PP624335) all revealed 100% (493/493 nt, 493/493 nt; 181/181 nt, 181/181 nt) shared identity with Cladosporium pseudocladosporioides strain ex-type MF473195 and HM148674 (Bensch et al. 2010), respectively. Using a neighbor-joining phylogenetic analysis based on the ITS and ACT sequences, isolates LD-299 and LD-300 clustered in the same clade of C. pseudocladosporioides. Therefore, based on its morphological characteristics and molecular phylogeny, the two isolates were identified as C. pseudocladosporioides (Cosseboom and Hu 2023). A pathogenicity test was performed using nine healthy two-year-old blue honeysuckle Lanjingling plants. Three plants were inoculated with either the LD-299 or the LD-300 conidial suspension (1 × 106 spores/ml) or with clean water as an experimental control (Aydoğdu et al. 2023). All plants were cultured in a greenhouse (28℃, 75% relative humidity, 12 h light and dark cycle), and each experiment was replicated three times. Typical leaf spot symptoms were first observed on the inoculated leaves after 10 days. Morphological and molecular characterization of re-isolated pathogens from the artificially infected leaves indicated that the two isolates were identical, thereby confirming Koch's postulates. Cladosporium pseudocladosporioides previously caused leaf spot disease on artichoke (Cynara scolymus) in Türkiye (Aydoğdu et al. 2023). To the best of our knowledge, this is the first report of C. pseudocladosporioides causing leaf spots on blue honeysuckle in China. Blue honeysuckle production losses due to the leaf spots are critical for growers. Therefore, further focus should be given to investigate the host range and geographic distribution of C. pseudocladosporioides.

First Report of Nigrospora oryzae Causing Leaf Spot Disease on Amorphophallus albus in Shaanxi Province of China.

Amorphophallus albus P. Y. Liu & J. F. Chen is a typical cash crop widely planted in southwest China (Gao et al., 2022). In early August of 2021, a peculiar leaf spot disease was first detected on A. albus in Ankang Academy of Agricultural Sciences manufacturing base (32°69'N, 109°02'E), Shaanxi, China. Small irregular yellow-brown spots (1 to 2 mm) were observed on the surface of A. albus leaf. Following infection of the leaf, it expanded (3 to 5 mm) and became necrotic. Nine planting bases were investigated, and approximately 75% of plants were symptomatic during the rapid expansion period of bulb growth in Hanyin, Langao and Hanbin counties, Ankang City, Shaanxi, China. Higher disease incidence was observed at temperatures above 30℃ and humidity above 80%. Twenty-seven symptomatic tissues of infected leaves were first surface sterilized by immersion in 75% ethanol for 1 minute, followed by rinsing three times in sterile distilled water. The tissues were then cut into 4-5 mm pieces, plated on 1.5% potato dextrose agar (PDA), and incubated at 28±2°C. The hyphal tip from the growing edge of colonies cultured for three days at 28±2℃ was transferred to PDA to obtain pure cultures. Fungal colonies were white, then grey to black with an unevenly distributed, fast-growing aerial mycelium covering the petri dish within five days at 28±2℃. The colony turned dark brown when maintained in the dark at 28±2℃ after seven days, then grayish brown upon sporulation after 15 days (Fig.1f-g). Conidia were brown or black, smooth, spherical to sub-spherical, single-celled (8-12 µm × 10-13µm, average 9-11.5 µm in diameter, n=5µm). The nutritional hyphae exhibited septa, and a portion of the aerial hyphae formed a long, rough conidium, giving rise to a nearly spherical apical sac (Fig.1h). The surface gave rise to several small peduncles bearing clusters of surfaced spherical conidia (Fig.1i). Surfaced spherical conidia were generated on the surface of the small peduncle (Fig.1j). These morphological features were consistent with Nigrospora oryzae (Li et al., 2017). Genomic DNA was extracted from mycelia of the pathogen using an Ezup column fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS), LSU (28S) and BenA gene of the isolate were amplified by PCR (Wang et al., 2017) and sent for sequencing. The resultant sequence (GeneBank ID of gene ITS, LSU, BenA are OR723825, OR775345, OR277316, respectively) were compared with the voucher specimens. BLAST results showed >99% identity with those of N.oryzae (GeneBank ID of N.oryzae strain LC2707 ITS, LSU, BenA are KX985954, KY806242, KY019481, respectively). A neighbor joining phylogenetic tree with the concatenated sequences of these genes showed that A-pb169 had the closest match with N. oryzae (Fig. 2). For pathogenicity testing, fifty plants in a period of rapid expansion of bulb growth were selected. Four leaves per plant were inoculated by sprayed till runoff with a conidial suspension of the pathogen (50 µL, 1×106 conidia/ml sterile water), and incubated at 30±2℃ and 80 ± 5% humidity. Control plants received sterile water. On the third day after inoculation, a yellow-brown spot appeared on leave surfaces, the spot gradually expanded; the infection rate was 90 to 95%. Fifteen days after inoculation, infected leaves showed symptoms like those observed in the field, whereas 100 control leaves sprayed with sterile water remained symptomless (Fig.1 a-e). The pathogen was reisolated from infected leaves and confirmed as N. oryzae by morphology and molecular identification. To our knowledge, this is the first report of leaf spot disease of A. albus caused by N. oryzae in China. Since its one of the major cash crops of the southeastern China, further work is necessary to determine its spread and economic impact as well as developing sustainable disease management options.

A cell-based fluorescent system and statistical framework to detect meiosis-like induction in plants.

Genetic gains made by plant breeders are limited by generational cycling rates and flowering time. Several efforts have been made to reduce the time to switch from vegetative to reproductive stages in plants, but these solutions are usually species-specific and require flowering. The concept of in vitro nurseries is that somatic plant cells can be induced to form haploid cells that have undergone recombination (creating artificial gametes), which can then be used for cell fusion to enable breeding in a Petri dish. The induction of in vitro meiosis, however, is the largest current bottleneck to in vitro nurseries. To help overcome this, we previously described a high-throughput, bi-fluorescent, single cell system in Arabidopsis thaliana, which can be used to test the meiosis-like induction capabilities of candidate factors. In this present work, we validated the system using robust datasets (>4M datapoints) from extensive simulated meiosis induction tests. Additionally, we determined false-detection rates of the fluorescent cells used in this system as well as the ideal tissue source for factor testing.

Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage.

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) from the umbilical cord (UC) has gained increasing interest in the biomedical field as an outstanding source of MSCs. However, challenges such as limited supply and lack of standardization in existing methods have arisen. This article presents a novel method for enhancing MSC yield by dissecting intact WJ from the umbilical cord. The method employs blunt dissection to remove the epithelial layer, maintaining the integrity of the entire WJ and resulting in an increased quantity and viability of harvested MSCs. This approach significantly reduces WJ waste compared to conventional sharp dissection methods. To ensure the purity of WJ-MSCs and minimize external cellular influence, a procedure utilizing internal tension to peel off the endothelium after flipping the UC was conducted. Additionally, the Petri dish was inverted for a short time during explant culture to improve attachment and cell outgrowth. Comparative analysis demonstrated the superiority of the proposed method, showing a higher yield of WJ and WJ-MSCs with better viability than traditional methods. The similar morphology and expression pattern of cell surface markers in both methods confirm their characterization and purity for various applications. This method provides a high-yield and high-viability approach for WJ-MSC isolation, demonstrating great potential for the clinical application of MSCs.

Effect of Silybum marianum (L.) Gaertn. Leaf and Seed Extracts Prepared Using Different Solvents on Root-Knot Nematode

Objective: The nematicidal effect of milk thistle leaves and seeds prepared with different solvents on Meloidogyne incognita was investigated. Materials and Methods: Acetone, ethanol and distilled water were used. The in vitro was carried out in 6 cm petri dishes. The extractions were studied with 500 and 1000 μg/ml (ppm). The in vitro and pot experiments designed random plots with 5 replications for each extraction, solvent and concentration. Four hundred second stage juvenile larvae (J2) were used as inoculum and dead individuals were counted after 48 hours. Five days after transplantation, nematode inoculation was carried out with 500 J2 per pot. After 24 hours, 30 ml of the solution was applied to the soil at 1000 ppm concentration. The experiment was terminated for 50 days. Then, gall and egg mass counts were made. Results: In vitro, the mortality rate at 1000 ppm was found to be similar in acetone (78.0%) and ethanol (80.8%) solvents in leaf extraction, while the highest was detected in ethanol (94.0%) in the seed extract. In distilled water solvent, 68.0% mortality was determined in the leaf extract and 62.2% mortality in the seed extract. There was no statistically significant difference between the leaf and seed extracts in number of galls and egg masses. No statistical difference could be determined between the solvents in the number of egg masses in seed extraction. While the number of galls in the leaf extract was found to be higher than in acetone (8.8 unit/root) and ethanol (8.0 unit/root) in distilled water (18.0 unit/root) and the difference between them was found to be significant, no statistically significant difference in the number of egg mass between the solvents. Conclusion: It was observed that all solvents of the leaf and seed extract suppressed galls and egg masses by more than 80% compared to the control.

P-216 Whole genome amplification (WGA) of spent culture media may not be a reliable marker of blastocyst prioritization for transfer

Abstract Study question Does the success or failure of DNA amplification from spent culture media of good-quality blastocysts correlate with their ploidy status and clinical pregnancy outcomes? Summary answer Samples with successful DNA amplification exhibited similar rates of euploidy and clinical pregnancy rates compared to spent culture samples with failed DNA amplification. What is known already Studies have detected DNA in spent culture media/blastocoelic fluid (BF) from fully developed blastocysts. After amplification, this DNA is sequenced to provide information on their ploidy status. Some authors reported simply detection of amplified DNA as an indicator of blastocyst viability, so that spent culture media/BF with DNA amplification derive from abnormal embryos, and failed DNA amplification from euploid blastocysts. Along with this line of rationale, some authors associated DNA amplification failure with higher clinical pregnancy rates. Reproducibility of such findings is warranted which may stem from various factors, such as culture conditions, amplification methods and statuses of blastocysts analysed. Study design, size, duration This preliminary set of results is part of a cohort (nonselective) ongoing study, from which 62 patients participated so far from two tertiary IVF centers during the period of January 2022 until October 2023. Only ICSI patients aged <43-year-old with >6 mature oocytes were included. Single frozen blastocyst transfers were performed blinded to the results of the whole genome amplification (WGA) and/or DNA sequencing and pregnancy outcomes recorded. Participants/materials, setting, methods Embryos were singly cultured into 20µl GTL-media in Embryoscope+ incubator (Vitrolife). On day 3, embryos were washed repeated times and placed into new culture dish. Spent media was collected from ≥BL4BB blastocysts (Gardner) on days 5/6 after laser collapse prior vitrification. DNA from culture medium were amplified using MALBAC (Yikon-Genomics). After WGA, DNA was quantified using Qubit 3.0 fluorometer (cut-off of 0.6 ng/µl) and then subjected to next-generation sequencing (NGS) with Illumina MiSeq platform. Main results and the role of chance A total of 104 spent culture media samples were analysed from individual blastocysts. About 70% of samples of spent media had amplified DNA and were sequenced further. From those sequenced samples, 55% resulted in euploidy and 45% in abnormal ploidy status. Embryo transfers took place from blastocysts cultured in 82/104 samples. A total of 36 (44%) clinical pregnancies were obtained from 82 transfers/samples from which, 78% derived from samples with DNA amplification and 22% from samples with failed DNA amplification. Outcomes of clinical pregnancies were verified for both conditions, samples with DNA amplification or failed DNA amplification. A total of 28/55 (51%) samples with DNA amplification resulted in clinical pregnancies, while only 30% (8/27) of failed DNA amplification samples did so (P = 0.06). Positive predictive values (PPV) and negative predictive values (NPV) of ploidy status of sequenced samples in relation to ongoing pregnancy and live birth rates are ongoing. Limitations, reasons for caution The limited sample size. Wider implications of the findings The presence of DNA in the spent culture media may offer an attractive alternative as a non-invasive test for aneuploidy. Research is still ongoing and challenging. Nevertheless, truncating the test at DNA amplification phase seems not a reproducible methodology to serve as a criterion to prioritize embryos for transfer. Trial registration number This research project is financially supported by Theramex Birth Grant program, a competitive grant supporting IVF research in Europe.

P-219 Does a dry or humid incubation environment affect continuous and undisturbed culture media protocols?

Abstract Study question Can continuous embryo culture medium maintain a safe and effective osmolality in dry and humidified incubators under both light viscosity and heavy viscosity oil overlay? Summary answer Undisturbed embryo culture is possible in 25uL and 100uL media volumes with both light and heavy oil and in both dry and humidified environments. What is known already Ideal osmolality for pre-implantation embryos is 255-295 mOsm/kg. Much debate surrounds continuous embryo culture, without refreshing of the medium, providing a suitable concentration of osmoles during embryo culture in both humidified and dry incubators. Meanwhile, ideal pH or measure of free hydrogen ions available in the embryo culture medium is 7.20-7.50. Contemporary knowledge that undisturbed embryos are able to regulate their own needs during culture has replaced belief that embryos require specific and varying environmental pH induced at different developmental stages. Thus, environmental pH needs to be maintained and stable in order to optimize health and outcomes of resulting blastocysts. Study design, size, duration Osmolality, pH, and mass were measured in humidified and dry incubators over 8 days of continuous embryo culture conditions. Four dishes each of 25uL and 100uL drops of embryo culture medium (CSCM-NXC) were analyzed per incubator type (Non-humidified – Miri, Humidified – Astec). Additionally, each micro-volume was analyzed under both Light Oil for Embryo Culture and Heavy Oil for Embryo Culture (Irvine Scientific). In total, 2352 data points for osmolality, pH, and mass were examined. Participants/materials, setting, methods A private IVF laboratory conducted the testing with no patient samples introduced to the embryo culture media. To mitigate early evaporative loss, dish preparation was strictly time and temperature controlled. Osmolality was measured in triplicate on days 0-7 using Advanced Micro-Osmometer Model 330 (Advanced Instruments). Mass measures were taken in triplicate on days 0-7 using the Torbal AGZN120 Analytical Balance. pH was measured in triplicate using the i-STAT 300 (Abbott) on days 0,3,5,7. Main results and the role of chance The pH in 25uL and 100uL drops under light oil and heavy oil remained well within suitable ranges and stable when compared day to day, from day 0 (M = 7.276, σ = 0.011) through day 7 (M = 7.303, σ = 0.011). The average aggregate pH overall was 7.290 (σ = 0.012). Aggregate osmolality, measured as milliosmoles per kilogram (mOSM/kg), over the course of incubation in all conditions also remained well within acceptable safety range (M = 277mOsm/kg, σ = 5). Dry incubator incubation exhibits a 7% increase from day 0 to day 6 (M = 270mOsm/kg, σ = 4 and M = 289mOsm/kg, σ = 5) compared to a 2% increase in humidified incubation (M = 269mOsm/kg, σ = 5 and M = 274mOsm/kg, σ = 5) regardless of oil type or microdrop size, both remaining within accepted limits. Additionally, when monitoring mass in grams (g) in 25uL as a function of evaporative loss, humidified incubation reduces from 12.6346g to 12.5566g (day 0 to day 7). In dry incubation, there is negligible change day 0 to day 7 (12.6543g and 12.6538g). Similarly, in 100uL, the change in humidified incubators is 13.37222g to 13.25025g (day 0 to day 7) and dry incubation reduces from 13.3236g to 13.2963g. The changes in mass of prepared culture dishes maintains consistency with the study findings for osmolality. Limitations, reasons for caution As CSCM-NXC is for use through day 6 of embryo culture, the extended simulated embryo culture length is for data collection purposes only. There is no developmental data to include in this study. Additional studies must be conducted in order to explore any differences when embryos are present in culture. Wider implications of the findings The ability to culture embryos continuously in dry incubator environments with micro-volumes of culture medium is widely debated. These many data point study has shown that safe and efficacious embryo culture can be undertaken undisturbed, therefore relaxing some of the burdensome and labor-intensive processes in laboratories employing this equipment. Trial registration number not applicable

P-274 Humid or dry culture with medium renewal: impact on A-Grade Day 5 blastocysts. Randomized prospective cohort study

Abstract Study question Could the humid embryo culture impact the development of top-graded blastocysts? Summary answer While no differences were observed in the overall blastocyst development rate, humidity conditions emerged as a significant factor influencing the achievement of A-Grade D + 5 blastocysts. What is known already In recent years, several publications have highlighted the advantages of humid culture (HC) over dry culture (DC) (<45% relative humidity, RH). HC, with its lower risk of media evaporation and consequent osmolality changes. Nevertheless, to the best of our knowledge, this is the first prospective study addressing this aspect through oocytes from the same cohort. Study design, size, duration We have been conducting a randomized control trial (RCT) consisting on 379 MII oocytes from 30 patients from August 2022 until January 2024 (and ongoing). After ICSI procedure, sibling oocytes were divided following 1:1 ratio into both humid (>70% HR) and dry (0% HR) chambers of a Geri incubator (Genea Biomedx, Australia). Ejaculated sperm ICSI cycles with >5 fresh MII were included in the analysis. Time-lapse cleavage timings were manually gathered. Participants/materials, setting, methods Extended culture to the blastocyst stage (D + 5, D + 6 or D + 7) was performed in 80μL of single step G-TL medium (Vitrolife, Sweden). HC remained undisturbed while medium was renewed on D + 3 in DC by changing the culture dish. Fertilization, overall blastocyst development and A-Grade Day 5 blastocysts rates (ASEBIR’s morphology criteria) were compared. Statistical analysis consisted on Chi-squared test, t-student test and logistic regression for computation of Odds Ratio (OR, 95%CI). Main results and the role of chance While no statistically significant differences were observed in the fertilization rate (DC: 76.4%; HC: 69.1%, NS) or the overall usable blastocyst development rate (DC: 48.6%; HC: 53.1%, NS), noteworthy distinctions emerged in the proportion of top-graded embryos based on ASEBIR’s morphology criteria. Specifically, the A-Grade Day 5 blastocyst development rate displayed significant disparities when comparing all fertilized embryos in each culture type (DC: 19.9%; HC: 32.3%; p < 0.05) and also among all usable blastocysts (DC: 40.8%; HC: 60.9%, p < 0,05). The Odds Ratio for achieving A-Grade Day blastocysts depending on type of humidity conditions was 1.87 (1,099 – 3,235) for the humid culture. Moreover, time-lapse imaging facilitated the detection of direct cleavage presence, which was similarly distributed between both incubation types (DC: 27/146; HC: 23/130, NS). However, only 3 embryos showing direct cleavage and cultured in HC reached the blastocyst stage at Day 6, while in dry incubator, 10 embryos with direct cleavage were able to develop to usable blastocysts, three of them being A-Grade on Day 5. Limitations, reasons for caution This study is currently ongoing, aiming to increase the sample size. Future analyses will explore clinical outcomes, especially for underrated embryos, day 6 blastocysts, and those with morphodynamic phenomena. While A-grade blastocyst rate variations may not impact pregnancy outcomes directly, they could influence cumulative success rates. Wider implications of the findings These data highlight that day+3 medium renewal doesn’t equalize conditions to humid culture. Ongoing efforts involve validating these insights through an expanded sample size and extensive outcome analysis. Investigating the long-term impact on clinical outcomes and refining culture protocols can amplify the relevance of these findings in assisted reproductive technologies. Trial registration number pending_protocolOK

P-195 Euploid embryo transfer in a hyaluronic acid-enriched media results in a higher ongoing pregnancy rate than euploid embryo transfer in media not containing hyaluronic acid

Abstract Study question Will using a hyaluronan-rich transfer medium increase the ongoing pregnancy rate and live birth rate when transferring euploid embryos cultivated in a dry incubator? Summary answer Transferring euploid embryos in a media with high concentrations of hyaluronic acid increases the ongoing pregnancy rate compared with media with no hyaluronic acid. What is known already Cochrane Review found that solutions with high concentrations of hyaluronic acid increase the chance of live birth by adding probably 1 additional live birth for every 14 embryos transferred in a high-concentration hyaluronic acid solution for patients aged 27 to 35. A study involving 85 frozen embryo transfer cycles showed a positive effect of high concentrations of hyaluronic acid on the results of transfer of morphologically poor euploid blastocysts. Previous studies were conducted in humidified incubators where hyaluronic acid-rich media was equilibrated without oil overlay. Our clinic uses dry incubators, necessitating oil overlay with all media, including hyaluronic acid-rich media. Study design, size, duration A randomized prospective study involving 255 patients aged 19-44 undergoing a transfer of a single euploid embryo between June 2023 and November 2023. Patients were randomly assigned to the study and control groups. Blastocysts were incubated after thawing for 3 hours either in a hyaluronic acid-enriched medium or in a medium without hyaluronic acid. Participants/materials, setting, methods Embryos underwent PGT-A at the blastocyst stage on either day 5 or day 6, using the EmbryoMap method. Trophectoderm biopsy was performed using the Flicking technique without a laser. 5-7 cells were removed for analysis. Blastocysts were vitrified following biopsy. Hyaluronic acid-enriched media was equilibrated for 16-18 hours in a 5%O2 6%CO2 atmosphere in a well of 4-well culture dish under oil overlay (600 microliters of media and 500 microliters of oil). Main results and the role of chance In this ongoing study, 127 transfers of euploid blastocysts in a hyaluronic acid-rich media resulted in 70 ongoing pregnancies, with a 55.1% ongoing pregnancy rate. In comparison, 128 transfers of euploid blastocysts in a media with no hyaluronic acid resulted in 65 ongoing pregnancies, with a 50.8% ongoing pregnancy rate. The relative improvement in the ongoing pregnancy rate is 8.5%. Our data suggests that using a hyaluronic acid-rich medium for the transfer of euploid blastocysts of good morphology can improve clinical pregnancy rates, although the effect is less dramatic than was shown in a 2023 study by Nakagawa et al. for morphologically poor euploid blastocysts, where more than twofold increase in clinical pregnancy rates was detected. Limitations, reasons for caution Specific media equilibration protocol is required for the incubation and transfer of embryos in a hyaluronan-rich transfer medium for IVF clinics utilizing dry incubators. Further research is required to confirm results and to establish whether transferring embryos in hyaluronic acid-enriched media increases the live birth rate after euploid embryo transfer. Wider implications of the findings Our results show that it is feasible to use a hyaluronic acid-enriched medium in a dry incubator with an oil overlay and that using it according to described protocol could increase the ongoing pregnancy rate after a single euploid blastocyst transfer. Trial registration number not applicable

The effect of exogenous melatonin administration before superovulation on embryo yield in Assaf ewes.

The aim of this study was to determine the effect of exogenous melatonin administration on transferable embryos by increasing total antioxidant status before superovulation in Assaf ewes. Selected ewes were randomly divided into two equal groups: melatonin (n = 9) and control (n = 9). In the melatonin group, a melatonin implant (18 mg melatonin, Regulin®, Ceva, Turkey) was placed under the skin of the ear 7 days prior to insertion of the progesterone-containing sponge. In the control group, a physiological saline solution was injected under the skin of the ear on the same day. The same superovulation protocol was used in both groups. In addition, blood samples for determination of Glutathione peroxidase, superoxide dismutase, total antioxidant status and total oxidant status concentrations were collected on five different days, including the day of melatonin implant placement (Day-7), vaginal sponge insertion (Day 0), vaginal sponge removal (Day 11), mating (Day 12-13) and uterine flushing (Day 19). Embryos were collected by laparotomy on the 7th day after mating. Uterine flushing taken into petri dishes were scanned under a stereomicroscope, and the quality and developmental stages of the embryos were recorded. In the study, total corpus luteum count and total cell count were found to be higher in the control group than in the melatonin group (p < .05). When the results were evaluated in terms of oxidative stress index, a negative correlation was found between the total number of corpus luteum, number of cells obtained, count of transferable embryos and number of Grade 1 embryos on Day 0. There was also a positive correlation oxidative stress index and the number of unfertilized oocytes on Day-7. As a result, exogenous melatonin administration prior to superovulation during the breeding season is thought to have a negative effect on embryo yield and quality. Therefore, the use of exogenous melatonin in MOET studies during the breeding season is recommended to be investigated in new studies.

Antimicrobial efficacy of calcium silicate-based bioceramic sealers against Enterococcus faecalis and Staphylococcus aureus – An in vitro study

Context: The main objective of endodontic therapy is to achieve a sterile environment and three-dimensional filling of the root canals. Endodontic sealers play an important role in the obturation of the root canal system as they may provide a seal, which prevents the penetration of bacteria. Sealers are meant to entomb residual bacteria, prevent leakage of nutrients, and ideally possess antibacterial properties. Aim: The aim of the study was to evaluate and compare the antimicrobial effectiveness of calcium silicate-based bioceramic root canal sealers against Enterococcus faecalis and Staphylococcus aureus using the agar diffusion method. Materials and Methods: The materials were divided into four groups of nine samples each for both E. faecalis and S. aureus. Group I (control group) – MTA-Fillapex (Angelus), Group II – BioRoot RCS (Septodont), Group III – Bio-C Sealer (Angelus), and Group IV – Dia-Root Bio Sealer (Diadent). To evaluate the antibacterial activity of the samples, the agar diffusion method was utilized. To begin, a bacterial suspension was evenly spread onto sterile Petri dishes using sterile swabs. Four wells of 4 mm diameter were then punched, and the sealers were mixed following the manufacturer’s instructions and placed onto the prepared wells. The Petri dishes were then incubated at 37°C for 72 h. After incubation, the zones of inhibition around each well were measured at 24 h, 48 h, and 72 h, and the resulting values were statistically analyzed. Statistical Analysis Used: The data were analyzed using the ANOVA and post hoc Tukey’s test. Results: Calcium silicate-based root canal sealers showed significantly better efficacy than the control group. BioRoot RCS (3.44 ± 1.13 mm) showed maximum antibacterial effect against E. faecalis at 24 h, 48 h, and 72 h time intervals. Bio-C Sealer (16.44 ± 1.42 mm) showed maximum antibacterial effect against S. aureus at 24 h, 48 h, and 72 h time intervals. The effectiveness of root canal sealants eventually decreased over a period. Based on analysis using the ANOVA test, a statistically significant difference (P ≤ 0.05) was observed between the mean values of antimicrobial efficacy of sealers against both E. faeca lis and S. aureus. Conclusions: The study’s findings lead to the conclusion that BioRoot RCS showed a maximum antibacterial effect against E . faecal is and Bio-C Sealer showed maximum antibacterial effect against S. aureus at 24 h, 48 h, and 72 h.

Bacillus thuringiensis volatile organic compounds (VOCs) inhibit the growth of the pathogenic amphibian fungus, Batrachochytrium dendrobatidis while autoclaved secondary metabolites promote fungal growth

Environmental microorganisms have a complex, dynamic relationship with flora and fauna, with consequences for the host organism that range from immunogenic to immunoprotective. The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), can infect the skin of amphibians and may lead to the deadly disease, Chytridiomycosis. Some skin-associated microorganisms of amphibians have the potential to prevent or mitigate Bd infection by producing anti-fungal metabolites. Yet, the majority of environmentally derived bacteria that may inhabit amphibian skin have not been investigated for their interaction with Bd. The soil-dwelling bacteria, Bacillus thuringiensis (Bt), is a ubiquitous species that is widely applied as a natural insecticide and produces anti-fungal metabolites as a byproduct of metabolism. With the increased use of Bt-based insecticides and the potential benefit of antifungal properties, we investigated the effects of Bt against Bd. This study investigates (1) the impact of Bt volatile metabolites on the growth of Bd and (2) the impact of autoclaved Bt products on the growth of Bd in vitro. Our findings showed growth inhibition of Bd (p < 0.001) by Bt volatile organic compounds (VOC) when cultured in split petri plates, relative to the control. Yet, in a liquid culture of Bd, autoclaved secondary metabolites from Bt demonstrated a significant increase in Bd growth (p < 2.2e-16). Together these results suggest that Bt may produce metabolites that beneficially inhibit Bd in some conditions while negatively enhancing the growth of the Bd in others.

SPECIATION OF HEAVY METALS AND MICROBIAL CONTAMINANTS IN HARMATTAN DUST WITHIN THE FEDERAL POLYTECHNIC KAURA NAMODA MAIN CAMPUS, ZAMFARA STATE

Harmattan dust is a natural phenomenon contributing to air pollution due to the presence of heavy metals and microorganisms. This research aimed to determine the speciation of heavy metal and microbial contaminants in Harmattan dust samples within the polytechnic Kaura Namoda. The composite sample of each month, namely, December 2023, January 2024, and February 2024, was collected with the aid of a sterilized petri dish. Five extraction phases were carried out, in order of mobility of the metals: exchangeable ion &gt; metal bound with carbonate &gt; metal bound to iron and manganese oxide &gt; metals bound to organic matter and sulfide &gt; residual phase. This was achieved using Tessie’s method and AAS for extraction and analysis. The result for the heavy metals shows Cr (13.6%) and Zn (25.5%) are more abundant in the exchangeable phases in December and January. Metals bound to Fe and MnO extraction phases have Zn (25.50%) abundance in December and January. The residual phase of extraction shows the highest percentage of pb (95.3%) and nickel (85.40%) for December and January. The bacteria and fungi identified are Bacillus cereus, Staphylococcus, Aspergillum, and Clodosporium, respectively. There is no significant difference in physicochemical parameters between the Harmattan dust samples collected in December, January, and February. The percentages of heavy metals in the Harmattan dust are high in the immobility phase of extraction, and bacterial and fungal levels are high in December and January. There is a need for preventive and protective measures to mitigate the health effects associated with...

Automated Imaging to Evaluate the Exogenous Gibberellin (Ga3) Impact on Seedlings from Salt-Stressed Lettuce Seeds.

Salinity stress is a common challenge in plant growth, impacting seed quality, germination, and general plant health. Sodium chloride (NaCl) ions disrupt membranes, causing ion leakage and reducing seed viability. Gibberellic acid (GA3) treatments have been found to promote germination and mitigate salinity stress on germination and plant growth. 'Bauer' and 'Muir' lettuce (Lactuca sativa) seeds were soaked in distilled water (control), 100 mM NaCl, 100 mM NaCl + 50 mg/L GA3, and 100 mM NaCl + 150 mg/L GA3 in Petri dishes and kept in a dark growth chamber at 25 °C for 24 h. After germination, seedlings were monitored using embedded cameras, capturing red, green, and blue (RGB) images from seeding to final harvest. Despite consistent germination rates, 'Bauer' seeds treated with NaCl showed reduced germination. Surprisingly, the 'Muir' cultivar's final dry weight differed across treatments, with the NaCl and high GA3 concentration combination yielding the poorest results (p < 0.05). This study highlights the efficacy of GA3 applications in improving germination rates. However, at elevated concentrations, it induced excessive hypocotyl elongation and pale seedlings, posing challenges for two-dimensional imaging. Nonetheless, a sigmoidal regression model using projected canopy size accurately predicted dry weight across growth stages and cultivars, emphasizing its reliability despite treatment variations (R2 = 0.96, RMSE = 0.11, p < 0.001).

Photoperiod Regulates Aerobic Methane Emissions by Altering Plant Growth and Physiological Processes

Previous studies have shown that light quality and quantity affect methane emissions from plants. However, the role of photoperiod in plant-derived methane has not been addressed. We studied the effects of two photoperiods—long-day (16 h light/8 h dark), and short-day (8 h light/16 h dark)—on growth and methane emissions of lettuce (a long-day plant), mung bean (a short-day plant), and tomato (a day-neutral plant) under a temperature regime of 22/18 °C. All species were grown under both light durations. First, seeds were germinated in Petri dishes for one week, then plants were transferred to pots and randomly assigned to one of the two experimental conditions. Under each condition, twelve plants were grown for 21 days; at that time, plant growth and physiological traits, including plant dry mass, growth index, photosynthesis, chlorophyll fluorescence, total chlorophyll, nitrogen balance index, flavonoids, and anthocyanin, were measured. Lettuce plants under the short-day photoperiod had the highest methane emissions. Long-day plants that were exposed to short-day conditions and short-day plants that were exposed to long-day conditions were stressed; day-neutral plants were also stressed under short days (p&lt; 0.05). All three species had decreased total dry mass under short-day conditions, most likely because of decreased photosynthesis and increased transpiration and stomatal conductance. Methane emission was positively correlated with shoot/root mass ratio, nonphotochemical quenching and anthocyanin; but was negatively correlated with stem height, dry mass, photosynthesis, water-use efficiency, total chlorophyll, and flavonoids (p &lt; 0.05). This study revealed that, besides light intensity and quality, light duration can also affect methane emissions from plants.