Development, Design, and Application of Efficient siRNAs Against Cotton Leaf Curl Virus-Betasatellite Complex to Mediate Resistance Against Cotton Leaf Curl Disease.

Cotton leaf curl disease (CLCuD), caused by the Cotton leaf curl virus, is one of the most irrepressible diseases in cotton due to high recombination in the virus. RNA interference (RNAi) is widely used as a biotechnological approach for sequence-specific gene silencing guided by small interfering RNAs (siRNAs) to generate resistance against viruses. The success of RNAi depends upon the fact that the target site of the designed siRNA must be conserved even if the genome undergoes recombination. Thus, the present study designs the most efficient siRNA against the conserved sites of the Cotton leaf curl Multan virus (CLCuMuV) and the Cotton leaf curl Multan betasatellite (CLCuMB). From an initial prediction of 9 and 7 siRNAs against CLCuMuV and CLCuMB, respectively, the final selection was made for 2 and 1 siRNA based on parameters such as no off-targets, good GC content, high validity score, and targeting coding region. The target sites of siRNA were observed to lie in the AC3 and an overlapping region of AC2-AC1 of CLCuMuV and ßC1 of CLCuMB; all target sites showed a highly conserved nature in recombination analysis. Docking the designed siRNAs with the Argonaute-2 protein of Gossypium hirsutum showed stable binding. Finally, BLASTn of siRNA-target positions in genomes of other BGVs indicated the suitability of designed siRNAs against a broad range of BGVs. The designed siRNAs of the present study could help gain complete control over the virus, though experimental validation is highly required to suggest predicted siRNAs for CLCuD resistance. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01191-z.

Investigating the second whitefly population outbreak within a decade in the cotton growing zone of North India.

The whitefly, Bemisia tabaci (Gennadius), is a polyphagous and major pest of cotton worldwide. Both adults and nymphs of B. tabaci affect the crop by causing direct and indirect damage. A severe whitefly outbreak was experienced during 2015 on cotton in North India and this was followed by a profound infestation during 2022. The present research rigorously examined whether the proliferation in the whitefly population was an outbreak or the result of a multi factor resurgence. During 2015, whitefly counts remained above the economic threshold level (ETL) between 28th and 35th Standard Meteorological Week (SMW). However, during 2022 above ETL population was observed in 27th SMW and it persisted until 36th SMW. The peak incidence of the whitefly was noticed during 31st and 29th SMW in 2015 and 2022, respectively. The early pest build up in 2022 and longer persistence (≥10 weeks) over the cotton season resulted in more damage to cotton crop. Additionally, pest survillence across the zone on the farmers' fields during 2022 revealed 44.4 per cent spots (585 out of 1,317 locations) above ETL while the corresponding locations in 2015 was 57% (620 out of 1,089). Thus, in 2022 infestation was not uniform in the entire zone wherein only few blocks of Punjab, Haryana and Rajasthan states of India experienced severe infestations of the whitefly. This study reports the complex of factors including weather, delayed sowing, use of tank mixtures/ subleathal doses of insecticides, pest resurgence etc. that might have possibly contributed to these upsurges in whitefly on cotton in north India.

Diversity study of Beauveria bassiana species for finding the most virulent strain to manage Bemisia tabaci in cotton.

Beauveria bassiana (Bal.-Criv.) is an important entomopathogenic fungus being used for the management of various agricultural pests worldwide. However, all strains of B. bassiana may not be effective against whitefly, Bemisia tabaci, or other pests, and strains show diversity in their growth, sporulation, virulence features, and overall bioefficacy. Thus, to select the most effective strain, a comprehensive way needs to be devised. We studied the diversity among the 102 strains of B. bassiana isolated from 19 insect species based on their physiological features, virulence, and molecular phylogeny, to identify promising ones for the management of B. tabaci. Strains showed diversity in mycelial growth, conidial production, and their virulence against B. tabaci nymphs. The highest nymphal mortality (2nd and 3rd instar) was recorded with MTCC-4511 (95.1%), MTCC-6289 (93.8%), and MTCC-4565 (89.9%) at a concentration of 1 × 106 conidia ml-1 under polyhouse conditions. The highest bioefficacy index (BI) was in MTCC-4511 (78.3%), MTCC-4565 (68.2%), and MTCC-4543 (62.1%). MTCC-4511, MTCC-4565, and MTCC-4543 clustered with positive loading of eigenvalues for the first two principal components and the cluster analysis also corresponded well with PCA (principal component analysis) (nymphal mortality and BI). The molecular phylogeny could not draw any distinct relationship between physiological features, the virulence of B. bassiana strains with the host and location. The BI, PCA, and square Euclidean distance cluster were found the most useful tools for selecting potential entomopathogenic strains. The selected strains could be utilized for the management of the B. tabaci nymphal population in the field through the development of effective formulations. KEY POINTS: • 102 B. bassiana strains showed diversity in growth and virulence against B. tabaci. • Bioefficacy index, PCA, and SED group are efficient tools for selecting potential strains. • MTCC-4511, 4565, and 4543 chosen as the most virulent strains to kill whitefly nymphs.

The rising threat of geminiviruses: molecular insights into the disease mechanism and mitigation strategies.

BACKGROUND: Geminiviruses are among the most threatening emerging plant viruses, accountable for a huge loss to agricultural production worldwide. These viruses have been responsible for some serious outbreaks during the last few decades across different parts of the world. Sincere efforts have been made to regulate the disease incidence by incorporating a multi-dimensional approach, and this process has been facilitated greatly by the advent of molecular techniques. But, the mixed infection due to the polyphagous nature of vectors results in viral recombination followed by the emergence of novel viral strains which thus renders the existing mitigation strategies ineffective. Hence, a multifaceted insight into the molecular mechanism of the disease is really needed to understand the regulatory points; much has been done in this direction during the last few years. The present review aims to explore all the latest developments made so far and to organize the information in a comprehensive manner so that some novel hypotheses for controlling the disease may be generated. METHODS AND RESULTS: Starting with the background information, diverse genera of geminiviruses are listed along with their pathological and economic impacts. A comprehensive and detailed mechanism of infection is elaborated to study the interactions between vector, host, and virus at different stages in the life cycle of geminiviruses. Finally, an effort isalso made to analyze the progress made at the molecular level for the development of various mitigation strategies and suggest more effective and better approaches for controlling the disease. CONCLUSION: The study has provided a thorough understanding of molecular mechanism of geminivirus infection.

Evaluation of the Bioefficacy and Insecticide Compatibility of Entomopathogens for Management of Whitefly (Hemiptera: Aleyrodidae) on Upland Cotton Under Laboratory and Polyhouse Conditions.

Entomopathogens (EPFs) are potential alternatives to chemical insecticides for managing Bemisia tabaci (Genn.), an invasive pest of the cotton crop. EPFs alone may not always provide enough insect pest control, but combining EPFs with pesticides, provided both components are compatible, can make an integrated pest management program considerably more effective. Hence, the bioefficacy of EPFs against whitefly, their compatibility with pesticides, and the factors responsible for determining compatibility were studied. The highest nymphal mortality was recorded with the Beauveria bassiana strains Bb-4511 (95.1%) and Bb-4565 (89.9%), and Metarhizium anisopliae Ma-1299 (86.7%) at 1 × 106 conidia ml-1. Lower LC50 values were observed for Cordyceps javanica Cj-089 and Bb-4511, 0.2 × 104 and 0.5 × 104 conidia ml-1, respectively. The toxicity index values in insecticide sensitivity assays ranged from 19.4 to 119.6% among all the EPFs. Comparatively, all the EPFs except Bb-4543 and Bb-4565 showed compatible to moderately toxic reactions to neonicotinoids and spinosyns. Organophosphates (ethion) and pyrethrins (bifenthrin) were toxic to very toxic to all the EPFs except Bb-4511, Fv-083, and Ma-1299. Cj-102 and Cj-089 were compatible with 50% of the average recommended dose of bifenthrin and ethion, and the average recommended dose for the field application of neonicotinoids and spinosyns. Principal component analysis showed that spore production and toxicity index values correlate with each other and are responsible for determining the EPF compatibility with insecticides. The EPF spore production and toxicity index are important factors for determining chemical compatibility. Compatible EPFs can be used individually or in combination as promising and compatible biological alternatives to insecticides in the management of whitefly in cotton.

Evaluation of bioefficacy potential of entomopathogenic fungi against the whitefly (Bemisia tabaci Genn.) on cotton under polyhouse and field conditions.

The whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), is becoming a serious problem on Bt cotton. It causes enormous crop loss through its direct feeding and as a vector of cotton leaf curl virus. Chemical-dependent management is harming the environment and increased insecticide resistance is often observed in the fields. Identification of most virulent strains of entomopathogenic fungi (EPF) is essential to serve as an important component of an IPM program for management of B. tabaci. Compared to B. tabaci adults, the nymphal stage is reported to be more susceptible to entomopathogens, and targeting nymphs also helps vector management. We evaluated the bioefficacy of EPF and chemical pesticides against nymphs of B. tabaci on Bt cotton under polyhouse and field conditions. The bioefficacy index (BI) was considered as a mechanism to select the most effective EPF strains for field evaluation. The highest nymphal mortality under polyhouse conditions was recorded for Metarhizium anisopliae NA-01299 (86.7%), Beauveria bassiana MT-4511 (85.1%), Cordyceps javanica IT-10498 (81.1%), IT-10499 (81%), and B. bassiana NA-0409 (78.2%) relative to other EPF strains, spiromesifen (69.6%), buprofezin (62.2%) and pyriproxyfen (52.7%) at 7-days-post-spray treatment (DAS). However, among all the EPF, the highest BI was recorded in C. javanica IT-10499 (77%), IT-10495 (75.4%), Fusarium verticillioides IT-10493 (74.6%), and B. bassiana MT-4511 (73.1%). The pooled data of two-year field trials (2017-18 & 2018-19) revealed that the highest nymphal mortality was recorded for MT-4511 (85%), IT-10499 (83.2%), and pyriproxyfen 10% EC (78.6%) at 7-DAS. The BI-based selection of EPF proved to be a useful predictor of field efficacy. A sequential spray of the selected EPF would be a vital approach for resilient and sustainable integrated management of the B. tabaci nymphal population under field conditions.

Dominance of recombinant cotton leaf curl Multan-Rajasthan virus associated with cotton leaf curl disease outbreak in northwest India.

Cotton leaf curl disease (CLCuD), caused by whitefly (Bemisiatabaci) transmitted single-stranded DNA viruses belonging to the Genus, Begomovirus (family, Geminiviridae) in association with satellite molecules; is responsible for major economic losses in cotton in three northwest (NW) Indian states Haryana, Punjab, and Rajasthan. Annual CLCuD incidences during 2012 to 2014 were estimated to be 37.5%, 63.6%, and 38.8% respectively. Cotton leaves were collected from symptomatic plants annually for three years and subjected to DNA isolation, followed by rolling circle amplification (RCA), cloning, and DNA sequencing of apparently full-length begomoviral genomes and associated betasatellites and alphasatellites. Among the thirteen CLCuD-begomoviral genomes recovered, eight were identified as Cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Ra), one as -Pakistan (PK) and another as -Faisalabad (Fai), whereas, three were as Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu), indicating that CLCuMuV-Ra was the most prevalent begomovirus species. Five of the eight CLCuMuV-Ra sequences were found to be recombinants. The CLCuMuV-Ra- associated satellites consisted of Cotton leaf curl Multan betasatellite (CLCuMB), and Gossypium darwinii symptomless alphasatellite (GDarSLA), and Croton yellow vein mosaic alphasatellite (CrYVMoA). The second most abundant helper virus species, CLCuKoV-Bu, was associated with CLCuMB and GDarSLA.

Compatibility of entomopathogenic fungi with insecticides and their efficacy for IPM of Bemisia tabaci in cotton.

Bemisia tabaci, a vector of cotton leaf curl virus disease, is among the most devastating pests causing huge economic losses due to reduced cotton yield and quality. The excessive use of chemical pesticides causes insecticide resistance. Entomopathogenic fungi (EPFs) have a role as mycoinsecticides. The combined use of these insecticides is a promising pest-control option to minimize adverse chemical effects. Thus, we have evaluated 10 EPFs under polyhouse conditions for their virulence against whitefly nymphs and their compatibility with chemical and botanical insecticides. The highest overall biological efficacy index was recorded with Ij-102, followed by Bb-4511, and Ij-089. An in vitro compatibility study was conducted to evaluate the effect of botanical and chemical pesticides on mycelial growth and spore production using the poisoned food technique. The effect of pesticides on the reduction of mycelial growth and conidial production ranged from -169 to 94.1% and -25.6 to 87.6%, respectively. However, Ij-089, Ij-102, Ma-1299, and Bb-4511 were found to be the most compatible with the chemical and botanicals evaluated. Comparatively, spiromesifen, diafenthiuron, buprofezin, pyriproxyfen, and flonicamid were more compatible with EPFs at half doses, as compared to the other chemical pesticides, namely imidacloprid, fipronil, profenophos, and triazophos. These results might provide the basis for future work and indicate that applications of EPFs showing the best virulence and compatibility have the maximum likelihood for the management of B. tabaci in the field in an integrated pest management system.