Development of a reverse transcription loop-mediated isothermal amplification assay with novel quantitative pH biosensor readout method for SARS-CoV-2 detection.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a molecular amplification method that can detect SARS-CoV-2 in a shorter time than the current gold-standard molecular diagnostic reverse transcription-polymerase chain reaction (RT-PCR). However, previously developed RT-LAMP assays have mostly relied on highly subjective visual colorimetric interpretation. In this study, an RT-LAMP assay was developed with quantitative measurement of reaction pH using a novel portable pH biosensor compared to qualitative colorimetric interpretation and gel electrophoresis, with 57 clinical COVID-19 samples used for validation of the test. The LoD of the assay is 103 copies/µL. The highest sensitivity was found in the qualitative methods (93.75%), while the highest specificity and likelihood ratio was found in the pH sensor (87.5% and 6.72). On the sensor measurement, a significant difference (p < 0.0001) was observed between the average pH of the RT-PCR (+) COVID-19 (6.15 ± 0.27), while the average pH of the RT-PCR (-) samples (6.72 ± 0.22). Correlation analysis revealed a strong correlation (r = 0.78, p < 0.0001) between the Ct values obtained from RT-PCR with the biosensor pH readout. RT-LAMP with the quantitative pH sensor readout method has the potential to be further developed as an objective molecular assay for rapid and simple detection of SARS-CoV-2.

Effects of multiple halotolerant rhizobacteria on the tolerance, growth, and yield of rice plants under salt stress.

Halotolerant bacteria get adapted to a saline environment through modified physiological/structural characteristics and may provide stress tolerance along with enhanced growth to the host plants by different direct and indirect mechanisms. This study reports on multiple halotolerant plant growth-promoting rhizobacteria isolated from the coastal soils in Bangladesh, in fields where the halophytic wild rice Oryza coarctata is endemic. The aim was to find halotolerant bacteria for potential use as biofertilizer under normal/salt-stressed conditions. In this study, eight different strains were selected from a total of 20 rhizobacterial isolates from the saline-prone regions of Debhata and Satkhira based on their higher salt tolerance. 16S rRNA gene sequencing results of the rhizobacterial strains revealed that they belonged to Halobacillus, Bacillus, Acinetobactor, and Enterobactor genera. A total of ten halotolerant rhizobacteria (the other 2 bacteria were previously isolated and already reported as beneficial for rice growth) were used as both single inoculants and in combinations and applied to rice growing in pots. To investigate their capability to improve rice growth, physiological parameters such as shoot and root length and weight, chlorophyll content at the seedling stage as well as survival and yield at the reproductive stage were measured in the absence or presence (in concentration 40 or 80 mmol/L) of NaCl and in the absence or presence of the rhizobacteria. At the reproductive stage, only 50% of the uninoculated plants survived without setting any grains in 80 mmol/L NaCl in contrast to 100% survival of the rice plants inoculated with a combination of the rhizobacteria. The combined halotolerant rhizobacterial inoculations showed significantly higher chlorophyll retention as well as yield under the maximum NaCl concentration applied compared to application of single species. Thus, the use of a combination of halotolerant rhizobacteria as bioinoculants for rice plants under moderate salinity can synergistically alleviate the effects of stress and promote rice growth and yield.

Interactive effects of nitric oxide and glutathione in mitigating copper toxicity of rice (Oryza sativa L.) seedlings.

Nitric oxide (NO) and glutathione (GSH) are 2 vital components of the antioxidant system that play diverse roles in plant responses to abiotic stresses. Recently, we have reported that exogenous supply of both these molecules reduced copper (Cu) toxicity in rice seedlings. Individual as well as co-treatment of sodium nitroprusside (SNP: a NO donor) and GSH with Cu significantly mitigated the adverse effects of Cu, evident in the reduced level of oxidative markers such as H2O2, superoxide (O2(·-)), malondialdehyde (MDA), and proline (Pro). GSH content and most of the antioxidative and glyoxalase enzymes were up-regulated upon Cu stress, indicating their responses were co-related with the level of stress. Our results indicated that direct ROS scavenging, reduced Cu uptake, and the balanced antioxidative and glyoxalase systems, at least in part, successfully executed NO- and GSH-mediated alleviation of Cu toxicity in rice seedlings. In addition, the combined effect of adding SNP and GSH together was more efficient than the effect of adding them individually. Here, we are speculating that 1) GSH and Pro could be used as potential markers for copper stress, and 2) adding SNP and GSH might produce S-nitrosoglutathione (GSNO) which could be a source of bioactive NO and may affect many regulatory processes involved in Cu-stress tolerance. We further note that the combined effect of adding SNP and GSH was pronounced in inhibiting the uptake and translocation of Cu in rice seedlings.

Exogenous sodium nitroprusside and glutathione alleviate copper toxicity by reducing copper uptake and oxidative damage in rice (Oryza sativa L.) seedlings.

Nitric oxide (NO) and glutathione (GSH) regulate a variety of physiological processes and stress responses; however, their involvement in mitigating Cu toxicity in plants has not been extensively studied. This study investigated the interactive effect of exogenous sodium nitroprusside (SNP) and GSH on Cu homeostasis and Cu-induced oxidative damage in rice seedlings. Hydroponically grown 12-day-old seedlings were subjected to 100 µM CuSO4 alone and in combination with 200 µM SNP (an NO donor) and 200 µM GSH. Cu exposure for 48 h resulted in toxicity symptoms such as stunted growth, chlorosis, and rolling in leaves. Cu toxicity was also manifested by a sharp increase in lipoxygenase (LOX) activity, lipid peroxidation (MDA), hydrogen peroxide (H2O2), proline (Pro) content, and rapid reductions in biomass, chlorophyll (Chl), and relative water content (RWC). Cu-caused oxidative stress was evident by overaccumulation of reactive oxygen species (ROS; superoxide (O2 (•-)) and H2O2). Ascorbate (AsA) content decreased while GSH and phytochelatin (PC) content increased significantly in Cu-stressed seedlings. Exogenous SNP, GSH, or SNP + GSH decreased toxicity symptoms and diminished a Cu-induced increase in LOX activity, O2 (•-), H2O2, MDA, and Pro content. They also counteracted a Cu-induced increase in superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and glyoxalase I and glyoxalase II activities, which paralleled changes in ROS and MDA levels. These seedlings also showed a significant increase in catalase (CAT), glutathione peroxidase (GPX), dehydroascorbate reductase (DHAR), glutathione S-transferase (GST) activities, and AsA and PC content compared with the seedlings stressed with Cu alone. Cu analysis revealed that SNP and GSH restricted the accumulation of Cu in the roots and leaves of Cu-stressed seedlings. Our results suggest that Cu exposure provoked an oxidative burden while reduced Cu uptake and modulating the antioxidant defense and glyoxalase systems by adding SNP and GSH play an important role in alleviating Cu toxicity. Furthermore, the protective action of GSH and SNP + GSH was more efficient than SNP alone.