Development of recombinase polymerase amplification-based colorimetric detection assay for rapid identification of invasive cassava mealybug, Phenacoccus manihoti Matile-Ferrero.

Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae), is an economically important invasive cassava pest responsible for the massive devastation of cassava in Asia and African continent. Initially, identifying this invasive pest posed challenges because it closely resembled native mealybug species. Additionally, the traditional morphological identification process is labor-intensive and time-consuming. Detecting invasive pests at an early stage is crucial, hence development of a rapid detection assay is essential. In the current study, we have developed a simple, rapid, sensitive, and efficient molecular detection assay for P. manihoti based on Recombinase Polymerase Amplification (RPA). The primers for the RPA assay were designed using unique nucleic acid sequences of P. manihoti, and the protocol was standardized. Specificity test demonstrated that the RPA assay could amplify DNA of P. manihoti only, and no amplification was observed in six other mealybug species. The specificity of assay was confirmed using SYBR green-based colorimetric detection and gel electrophoresis where positive samples showed 195 bp amplicon size in P. manihoti samples. The assay successfully amplified P. manihoti DNA in thirty minutes at an annealing temperature of 41° C in a water bath and displayed a sensitivity of 72.5 picograms per microliter. The assay's simplicity, rapidity, and high sensitivity make it a valuable tool for detecting and monitoring P. manihoti in quarantine stations and facilitating in development of a portable diagnostic kit.

Comparative effectiveness of biorational pesticides for management of Phenacoccus solenopsis Tinsley and Paracoccus marginatus Williams & Granara de Willink in Gymnema sylvestre (Retz.) R.Br. ex Sm.

The cotton mealybug, Phenacoccus solenopsis Tinsley and papaya mealybug, Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae) are becoming major threats to the production of Gymnema sylvestre R. Br. (Asclepiadaceae) in India. Management mainly depends on chemical insecticides which cause a serious problem of pesticide residue and insecticide resistance. The use of biorational insecticides such as biopesticides, botanicals, insect growth regulators, and microbial insecticides is important components of an Integrated Pest Management (IPM) program for successful management. We evaluated the bio-efficacy of twelve biorational insecticides, including entomopathogenic fungi (EPF), using the leaf spray method in laboratory conditions at 25 ± 1 °C, 70 % ± 5 % RH. The results revealed that the highest percent mortality was recorded by acetamiprid 20 % SP (100.00 %), followed by azadirachtin (98.27 %), Lecanicillium muscarium (2 × 109 spores/mL) (85.70 %) and Ocimum sanctum leaf extract (76.87 %) at 120 h after treatment (HAT) in P. solenopsis. In P. marginatus, 100.00 %, 96.39 % and 85.67 % and 74.90 % mortalities were achieved by acetamiprid 20 % SP, azadirachtin, L. muscarium (2 × 109 spores/mL) and O. sanctum leaf extract, respectively, at 120 HAT during the first spray. Various biorational insecticides showed a more or less similar trend of percent mortality in both species during the second spray. In both species, the lowest percent mortality was recorded by Andrographis paniculata leaf extract (46.29, 44.54) and (41.03, 46.39) at 120 Hours after treatment in the first and second spray, respectively. It was concluded that all the prescribed treatments are more effective than the control. Overall, azadirachtin recorded the highest percent mortality after acetamiprid and had the shortest LT50 (12.52 h) and (13.87 h) values in P. solenopsis and P. marginatus, respectively. Our study emphasizes that biopesticides like Azadirachtin 1 % EC (10000 ppm), L. muscarium (2 × 109 spores/mL) (5 mL/L) and O. sanctum leaf extract (5 %) may be recommended as alternatives to synthetic insecticides. Botanicals and EPF would be the most effective approach for sustainable integrated management of P. solenopsis and P. marginatus in the G. sylvestre ecosystem.

Transcriptome profiling and differential gene expression analysis provides insights into Lr24-based resistance in wheat against Puccinia triticina.

Leaf rust caused by Puccinia triticina is an important disease of wheat and Lr24 gene confers resistance to all known pathotypes of P. triticina in India. Transcripts associated with the Lr24 mediated resistance were identified through transcriptome sequencing and further expression analysis of differentially regulated genes was performed using qPCR technique. De novo transcriptome assembly showed 66,415 and 68,688 transcripts in resistant and susceptible genotypes, respectively. The study revealed that 5873 genes unique to resistant; 6782 genes unique to susceptible, while 10,841 genes were common to both. Gene Ontology distribution statistics showed 1030 and 1068 CDS in biological processes; 1234 and 1326 CDS in cellular processes; 1321 and 1352 CDS in molecular functions, respectively. A total of 659 genes were found to be differentially expressed, of which 349 were upregulated and 310 were downregulated in resistant genotype. Pathway analysis of transcripts appeared in resistant genotype revealed that 279 transcripts had homology with genes involved in signal transduction, 18 transcripts in membrane transport, one transcript in signaling molecules. Real-time PCR study showed that most of the up-regulated defense related genes expressed in early hours indicating that a cascade of defense starts early in Lr24 mediated resistance, which successfully inhibited pathogen establishment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02972-9.