Crimean‑Congo hemorrhagic fever (CCHF) is a vector‑borne viral disease, widely distributed in different regions of the world. The fever is caused by the CCHF virus (CCHFV), which belongs to the Nairovirus genus and Bunyaviridae family. The virus is clustered in seven genotypes, which are Africa‑1, Africa‑2, Africa‑3, Europe‑1, Europe‑2, Asia‑1 and Asia‑2. The virus is highly pathogenic in nature, easily transmissible and has a high case fatality rate of 10‑40%. The reservoir and vector of CCHFV are the ticks of the Hyalomma genus. Therefore, the circulation of this virus depends upon the distribution of the ticks. The virus can be transmitted from tick to animal, animal to human and human to human. The major symptoms include headache, high fever, abdominal pain, myalgia, hypotension and flushed face. As the disease progresses, severe symptoms start appearing, which include petechiae, ecchymosis, epistaxis, bleeding gums and emesis. Enzyme‑linked immunosorbent assay, quantitative polymerase chain reaction, antigen detection, serum neutralization and isolation of the virus by cell culture are the diagnostic techniques used for this viral infection. There is no specific antiviral therapy available thus far. However, ribavirin has been approved by the World Health Organization for the treatment of CCHFV infection. Awareness campaigns regarding the risk factors and control measures can aid in reducing the spread of this disease to a greater extent, particularly in developing countries.

We evaluated transgenic lines of sugarcane modified with the barley chitinase class-II gene to create resistance against the red rot causative agent Colletotrichum falcatum Went. Local sugarcane cultivar SP93 was transformed with a 690-bp coding sequence of the chitinase-II gene under the influence of a polyubiquitin promoter. Transgenic sugarcane lines (T 0) overexpressing the chitinase gene were obtained through a particle bombardment method with 13.3% transformation efficiency. Four transgenic sugarcane lines, SCT-03, SCT-05, SCT-15, and SCT-20, were tested for resistance against red rot by in vitro antifungal assays. Crude protein extracts from transgenic sugarcane plants SCT-03, SCT-05, SCT-15, and SCT-20 inhibited the mycelial growth of C. falcatum by 49%, 40%, 56%, and 52%, respectively, in a quantitative in vitro assay. Our findings revealed that two transgenic lines, SCT-15 and SCT-20, exhibited the highest endochitinase activity of 0.72 and 0.58 U/mL, respectively. Furthermore, transgenic lines SCT-15 and SCT-20 exhibited strong resistance against inoculated C. falcatum in an in vitro bioassay, as they remained healthy and green in comparison with the control sugarcane plants, which turned yellow and eventually died 3 weeks after infection. The mRNA expression of the transgene in the C. falcatum-inoculated transgenic sugarcane lines increased gradually compared to the control plant. The mRNA expression was the highest at 72 h in both transgenic lines and remained almost stable in the subsequent hours.

Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35kDa exhibited highest expression at 0.5mM concentration of IPTG. Expressed recombinant protein of 35kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80μg and 200μg.

Chilo partellus is an invasive polyphagous pest that has not been effectively managed with chemical pesticides. To select potential dsRNAs for use in an alternate control strategy, it is crucial to identify and evaluate stable reference genes for knockdown expression studies. This study evaluates the expression stability of seven candidate reference genes in C. partellus larvae fed on crude bacterially-expressed dsRNAs and purified dsRNAs at different time intervals, as well as the developmental stages and sexes. The expression stabilities of the reference genes were evaluated with different software programmes, such as BestKeeper, NormFinder, deltaCt, geNorm, and RefFinder. The overall results rank ELF as the most stably expressed reference gene when larvae were fed with crude bacteria-induced dsRNAs and purified dsRNA. However, Tubulin and HSP70 were more stable under different developmental stages and sexes. The expression levels of larvae that were fed crude bacteria-induced dsRNAs of Chitinase and Acetylcholinesterase were normalized with the four most stable reference genes (ELF, HSP70, V-ATPase and Tubulin) and the least stable reference gene (18S and HSP70) based on the geNorm algorithm. The least stable reference gene showed inconsistent knockdown expression, thereby confirming that the validation of a suitable reference gene is crucial to improve assay accuracy for dsRNA-targeted gene selection in C. partellus.