ABSTRACT
Fusarium wilt caused by Fusarium oxysporum f. sp. pisi (Fop) is an important disease and major obstacle to pea production, causing huge losses to growers. The focus of this study was on isolation followed by morphological, molecular characterization and analyzing the growth of the casual agent under variable temperature, pH and Nitrogen levels. The morphological features of radial growth, sporulation, pigmentation and mycelial characterization were examined and the variability of all isolates was presented. Molecular characterization of the fungus by ITS rDNA sequencing revealed that all 13 isolates belong to Fusarium oxysporum species. Six isolates were tested for temperature, pH and nitrogen dosage optimization studies. Seven different temperatures, viz., 21, 23, 25, 27, 29, 31, 33°C and pH values, having 3, 4, 5, 6, 7, 8, and 9 pH, as well as nitrogen dosage levels of 0 g, 3 g, 5 g, 7 g, 9 g, 11 g, and 13 g were tested against all six isolates, respectively. The results showed that all isolates exhibited the highest growth at a temperature of 25°C and the optimal temperature range for growth of Fusarium oxysporum was 23-27°C. All isolates showed the highest growth at pH5. Change in the nitrogen doses of the base ended in formation of thick, dense, fluffy mycelium of the casual agent. Six isolates were used for combination studies with seven different levels of temperatures, pH levels and nitrogen dosages. The density plots revealed the variations in the growth of the isolates with changes in temperature, pH and nitrogen levels, which can lead to mutations or genetic changes in the pathogens that could potentially introduce new threats to pea cultivation.
ABSTRACT
This study reports a simple template-based reverse transcription-polymerase amplification assay (ST-RT-RPA) for detection of citrus tristeza virus (CTV) from crude plant extract lysed in NaOH:EDTA (1:1) without the need of tedious RNA isolation. The developed assay showed versatility in its usage as amplification can be performed at wide temperature range (14°C to 42°C) and incubation time (4 to 32 min), although the best conditions were 38°C for 30 min. The developed ST-RT-RPA assay could detect the CTV up to 10-8 dilution of crude plant extract of NaOH:EDTA and up to 0.01 fg µl-1 of RNA of CTV-infected plant tissues and 0.001 ag µl-1 of plasmid DNA containing viral insert, thus exhibiting sufficient sensitivity. ST-RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus yellow vein clearing virus, and Candidatus Liberibacter asiaticus) and was more sensitive in detection of CTV infection in field samples as compared to standard reverse transcription-polymerase chain reaction (RT-PCR) with later showing false negative in 7.92% of samples tested after 1 week of sampling. The developed ST-RT-RPA assay used minimally processed crude plant extract as template, tolerant to sample degradation in transit and storage, while it can be easily performed at wide temperatures and could be adopted in resource-poor setup.
Subject(s)
Citrus , Reverse Transcription , Recombinases/metabolism , Edetic Acid , Sodium Hydroxide , RNA , Citrus/metabolism , Sensitivity and Specificity , Nucleic Acid Amplification TechniquesABSTRACT
Tea is an important beverage consumed worldwide. Of the different types of tea available, herbal tea is an important beverage consumed owing to its popularity as a drink and stress relieving factors, several different herbal concoctions made from seeds, leaves, or roots are currently consumed and sold as herbal teas. The herbal teas are not the usual tea but "tisanes." They are caffeine free and popular for their medicinal property or immune boosters. Herbal tea formulations are popularly sold and consumed by millions owing to their health benefits as they are rich in antioxidants and minerals. However, plants are also known to contain toxic and anti-nutritional factors. Anti-nutritional factors are known to interfere with the metabolic process and hamper the absorption of important nutrients in the body. These anti-nutritional factors include saponins, tannins, alkaloids, oxalates, lectins, goitrogens, cyanogens, and lethogens. These chemicals are known to have deleterious effects on human health. Therefore, it is important to understand and assess the merits and demerits before consumption. Also, several techniques are currently used to process and reduce the anti-nutrients in foods. This review is focused on comparing the contents of various anti-nutritional factors in some underutilized plants of North-East India used as herbal tea along with processing methods that can be used to reduce the level of these anti-nutrients.
ABSTRACT
Chilli is infected by at least 65 viruses globally, with a mixed infection of multiple viruses leading to severe losses being a common occurrence. A simple diagnostic procedure that can identify multiple viruses at once is required to track their spread, initiate management measures and manage them using virus-free planting supplies. The present study, for the first time, reports a simplified and robust multiplex PCR (mPCR) assay for the simultaneous detection of five RNA viruses, capsicum chlorosis orthotospovirus (CaCV), chilli veinal mottle virus (ChiVMV), large cardamom chirke virus (LCCV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV), and a DNA virus, chilli leaf curl virus (ChiLCV) infecting chilli. The developed mPCR employed six pairs of primer from the conserved coat protein (CP) region of the respective viruses. Different parameters viz., primer concentration (150-450 nM) and annealing temperature (50 °C), were optimized in order to achieve specific and sensitive amplification of the target viruses in a single reaction tube. The detection limit of the mPCR assay was 5.00 pg/µL to simultaneously detect all the target viruses in a single reaction, indicating a sufficient sensitivity of the developed assay. The developed assay showed high specificity and showed no cross-amplification. The multiplex PCR assay was validated using field samples collected across Northeast India. Interestingly, out of 61 samples collected across the northeastern states, only 22 samples (36%) were positive for single virus infection while 33 samples (54%) were positive for three or more viruses tested in mPCR, showing the widespread occurrence of mixed infection under field conditions. To the best of our knowledge, this is the first report on the development and field validation of the mPCR assay for six chilli viruses and will have application in routine virus indexing and virus management.
