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Banana bunchy top virus (BBTV) causing bunchy top disease, is one of the most devastating diseases of banana and plantain. All the six genomic components of isolates from different parts of the world have been well characterised, with most of the studies focusing on replicase gene and coat protein gene. Overexpression of coat protein (CP) in Escherichia coli system can contribute significantly in structural as well as immunological studies. In the present investigation, the full length BBTV CP was cloned to pGEX-4T-2 expression vector and overexpressed in various Escherichia coli strains to obtain high quality and quantity of the CP. An augmented overexpression and stability of recombinant coat protein was achieved by molecular manipulation of the clone by restriction-free (RF) cloning platform. The RF cloning was employed to replace the thrombin cleavage site in the vector backbone, which was also present in the protein of interest, and to incorporate TEV protease site to cleave fusion protein at this specific site, and separate the affinity tag. The RF method allows direct transformation of the PCR product to undergo ligation in vivo and obtain the transformants thereby avoiding the restriction digestion and ligation of the product to the linearized plasmid. From a litre culture, 1.084 mg/ml of fusion protein with GST tag was obtained after GSH sepharose affinity column chromatography. The fluorescence spectra indicated partial disordered tertiary structure of the fusion protein. Cleavage of tag was attempted using TEV protease overexpressed and purified in the laboratory. [ABSTRACT FROM AUTHOR]

Banana bunchy top virus (BBTV) causing bunchy top disease, is one of the most devastating diseases of banana and plantain. All the six genomic components of isolates from different parts of the world have been well characterised, with most of the studies focusing on replicase gene and coat protein gene. Overexpression of coat protein (CP) in Escherichia coli system can contribute significantly in structural as well as immunological studies. In the present investigation, the full length BBTV CP was cloned to pGEX-4T-2 expression vector and overexpressed in various Escherichia coli strains to obtain high quality and quantity of the CP. An augmented overexpression and stability of recombinant coat protein was achieved by molecular manipulation of the clone by restriction-free (RF) cloning platform. The RF cloning was employed to replace the thrombin cleavage site in the vector backbone, which was also present in the protein of interest, and to incorporate TEV protease site to cleave fusion protein at this specific site, and separate the affinity tag. The RF method allows direct transformation of the PCR product to undergo ligation in vivo and obtain the transformants thereby avoiding the restriction digestion and ligation of the product to the linearized plasmid. From a litre culture, 1.084 mg/ml of fusion protein with GST tag was obtained after GSH sepharose affinity column chromatography. The fluorescence spectra indicated partial disordered tertiary structure of the fusion protein. Cleavage of tag was attempted using TEV protease overexpressed and purified in the laboratory. [ABSTRACT FROM AUTHOR]

Cucumber mosaic virus (CMV) infected banana leaf samples were collected based on characteristic symptoms and screened by direct antigen coating immunosorbent assay and reverse transcriptase polymerase chain reaction. The expression clone of CMV coat protein was prepared using the expression vector pRSETC and the host Escherichia coli BL21 (DE3) pLysS. Then, E. coli cells were induced by Isopropyl â-D-1- thiogalacto pyranoside (IPTG) for expression of 25 kDa recombinant CMV coat protein. After induction, the E. coli cells were sonicated and the coat protein was ultra-pelleted. Sucrose density gradient (10-40%) was prepared and the coat protein ultra- pellet which was dissolved in Tris-NaCl buffer of pH 8.0 was subjected to sucrose density gradient ultra-centrifugation at 26,000 rpm for 3 h at 4°C for purification. The SDS-PAGE gel profile of sucrose fractions indicated more recombinant CP in 10- 20 per cent sucrose fractions. Along with the recombinant CP, contaminant (host derived) proteins were also observed. When the coat protein ultra-pellet was dissolved in SAT buffer of pH 5.5 and subjected to sucrose gradient ultracentrifugation as above, the 25 kDa coat protein was absent in sucrose fractions and was observed in cell pellet which indicated the insolubility of the protein in the buffer. [ABSTRACT FROM AUTHOR]

The French plantain cultivar, Nendran (Musa AAB) is the most widely and commercially cultivated variety of banana in Kerala. But this cultivar is highly susceptible to foliar fungal diseases especially leaf spot diseases. Among the leaf spot diseases, Sigatoka leaf spot caused by Mycosphaerella spp. poses a serious threat to banana cultivation. The studies on characterization of the pathogen associated with Sigatoka leaf spot disease complex in Kerala revealed that the predominant leaf spot disease is Eumusae leaf spot disease caused by Mycosphaerella eumusae. With an objective to develop a fungicidal recommendation for the sustainable management of this disease, a field experiment was conducted during 2016-17 at Banana Research Station, Kannara by evaluating the efficacy of six chemical fungicides viz., trifloxystrobin (25%) + tebuconazole (50%) (Nativo® - 0.4g/L), propiconazole (Tilt® - 1ml/L), copper hydroxide (Kocide® - 2g/L), pyraclostrobin (Headline® - 1g/L), hexaconazole (5%) + captan (70%) (Taqat® - 2g/L) and Bordeaux mixture (1%). All the chemical fungicides used in the study were found to be superior compared to unsprayed control. However, there was a variation in the efficacy between the treatments. The foliar application of the systemic fungicide trifloxystrobin + tebuconazole (0.4g/L) recorded the lowest per cent disease severity (PDS) of 15.43 per cent with the eighth leaf of the plant as the youngest leaf spotted (YLS) and the maximum disease development time (DDT) of 50.66 days. Among the contact fungicides evaluated, the lowest per cent disease severity of 16.65 per cent was recorded in plants sprayed with copper hydroxide which recorded DDT of 49.33 days and YLS of 7.29. The analysis of fungicide residue in the fruits revealed that there was no residue of the chemicals left in the fruit. The study revealed that four times foliar spraying of systemic fungicide, trifloxystrobin + tebuconazole (0.4g/L) or contact fungicide, copper hydroxide (2g/L) starting from the initial appearance of the disease on the lowest leaves of 75 per cent plants was safe and effective for the management of Eumusae leaf spot disease of banana. [ABSTRACT FROM AUTHOR]

Tomato leaf curl virus disease (ToLCD), caused by whitefly transmitted begomo virus is a devastating disease affecting tomatoes worldwide. Twenty seven genotypes (Five breeding lines from World Vegetable Centre, Taiwan, nine commercial hybrids, eight germplasm accessions from NBPGR, one local collection from Idukki and four KAU varieties) were screened for ToLCV resistance under natural field conditions at Thrissur, Kerala during summer 2021. Among the screened genotypes, Ansal was found to be highly resistant, while, Kaustubh, EC 519806, Arka Rakshak and local collection (Idukki) were identified as resistant genotypes. Among the genotypes evaluated, four genotypes were moderately susceptible (AVTO 1726, AVTO 1727, Anagha and Akshaya), twelve were susceptible (AVTO 1707, AVTO 1706, AVTO 0922, Virang, Durg, Abhiraj, EC 528360, EC 620428, EC 521067 B, Manuprabha, Manulekshmi and EC 620486) and six were highly susceptible (Aryaman, Raymond, Pranay, EC 538153, EC 315489 and EC 567305) to ToLCV. The genotypes found to be highly resistant and resistant were found to be resistant to ToLCV even after whitefly mediated artificial inoculation. The resistant and highly resistant genotypes remained asymptomatic, whereas, the susceptible genotypes (Anagha and Manuprabha) exhibited ToLCV symptoms. The genotypes varied significantly with respect to plant height, number of primary branches/plant and days to flowering. Correlation between trichome density, and disease severity showed that glandular trichome density on abaxial and adaxial surfaces of leaf was significantly and negatively correlated with per cent disease incidence and disease severity index. However, the negative correlation between the abaxial glandular trichome density and ToLCV incidence was stronger. The glandular trichome density of the resistant genotypes identified in the field screening was found to be high. Non-glandular trichome density on abaxial and adaxial side of the leaf was significantly and positively correlated with per cent disease incidence and disease severity index. The resistant genotypes identified in the present study can be used for resistance breeding in tomato against ToLCV disease. [ABSTRACT FROM AUTHOR]