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.

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.

The comparison of species diversity and abundance of insect natural enemies in the domesticated species of cotton using the yellow pan trap method.

India is the world's largest cotton producer and the only country that grows all four cultivated cotton species. There have been very few studies on the diversity and abundance of natural enemies of cotton insect pests in these cultivated cotton species. Therefore, the current study (2016-2018) was conducted to assess the diversity and abundance of natural enemies that cultivated cotton species harbour. Phule Dhanwantari, Suraj, Suvin, RCH-2, and DCH-32 were the five genotypes used in the study, each with a distinct genetic background. Using the adiv 2.0.1 and vegan R packages, we identified significant differences in natural enemies in terms of species diversity, richness, evenness, and abundance. Analysis of Similarity (ANOSIM) and Non-metric Multidimensional Scaling (NMDS) indicated substantial differences in the natural enemy community structure among the examined genotypes. A total of 17,279 natural enemies were collected and identified across genotypes from seven predatory families and five parasitoid families. The percentage share of these natural enemy families across genotypes and years, in descending order, is Coccinellidae (28.23%) < Tachinidae (19.23%) < Braconidae (12.68%) < Chrysopidae (11.65%) < Chalcididae (9.41%) < Aphelinidae (6.33%) < Pentatomidae (3.29%) < Ichneumonidae (2.37%) < Syrphidae (2.33%) < Vespidae (1.81%) < Asilidae (1.79%) < Geocoridae (0.89%). Coccinellidae, Tachinidae, Braconidae, Chrysopidae, Chalcididae, and Aphelinidae are the six major families that account for more than 85% of all recorded natural enemies. These six families have a higher percentage share in Phule Dhanwantary (90%) compared to the other genotypes. The conservation and better utilization of these natural enemies are crucial for the ecological and safe management of insect pests in the cotton ecosystem.

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.

Development of loop mediated isothermal amplification (LAMP): A new tool for rapid diagnosis of cotton leaf curl viral disease.

Cotton leaf curl disease (CLCuD) ranks top among all endemic diseases transmitted by whitefly (Bemisia tabaci) affecting cotton (Gossypium hirsutum) causing severe economic losses to the cotton growers in the Indian subcontinent. For its effective management, robust tools for detection are a prerequisite and it is important to diagnose the virus titre in early stage of infection in plants as well as in the disease transmitting vector. Considering the limitations in current PCR-based techniques we have standardised rapid and sensitive Loop Mediated Isothermal Amplification (LAMP) protocol for the diagnosis of cotton leaf curl virus (CLCuV) in cotton leaves and in its transmitting vector whitefly. Perhaps, this is the first report of use of LAMP tool for rapid diagnosis of CLCuV in cotton and its transmitting vector the whitefly. Further, the colorimetric detection for diagnostic simplicity of amplified LAMP product by using different dyes lead to enhanced applicability of this technique in the field of disease diagnostics. The merit of present study is that the diagnostic failure of PCR and LAMP due to low virus titre in the infected leaf has been circumvented through the combination of rolling circle amplification (RCA) with LAMP. Thus RCA-LAMP can be an option for ultra-sensitive detection of samples with low virus titre. The potential applications of this advanced diagnostic tool in laboratory research on diagnosis of CLCuV, an important viral pathogen of cotton have been discussed.

Evidence for population expansion of Cotton pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in India.

Pink bollworm, Pectinophora gossypiella (Saunders) infestation on Bt cotton is a major concern to cotton production in India. The genetic diversity and phylogeographic structure of the insect in light of PBW resistance needs to be revisited. The objective of this study was to identify different haplotypes of pink bollworm and their distribution in India. To achieve this we studied the population structure in 44 cotton growing districts of India. The partial mitochondrial COI sequence analyses of 214 pink bollworm populations collected from 44 geographical locations representing 9 cotton growing states of India were analysed. Genetic diversity analysis exhibited presence of 27 haplotypes, among them Pg_H1 and Pg_H2 were the most common and were present in 143 and 32 populations, respectively. Distributions of pairwise differences obtained with partial COI gene data from the overall Indian populations are unimodal, suggesting population expansion in India. Significant neutrality test on the basis of Tajima' D and Fu's Fs presented a star-shaped haplotype network together with multiple haplotypes. The unimodal mismatch distribution, rejection of neutrality test with significant negative values supported the theory of demographic expansion in cotton pink bollworm populations in India. Genetic data not only provides us with a perspective of population genetics, but also that the two populations of pink bollworm, those occurring early in the season are genetically close to the late season populations with respect to their partial CO1 region. Resistance to Cry toxins does not seem to have had an impact on this region of the mt DNA in populations of pink bollworm.

Rapid detection of Tobacco streak virus (TSV) in cotton (Gossypium hirsutum) based on Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP).

Tobacco Streak Virus (TSV) belongs to the genus Ilarvirus of the family Bromoviridae an emerging pathogen posing threat to the crop species worldwide. Identification of symptoms due to TSV infection by visual observation of plants often results in misdiagnosis as symptoms produced by this virus can match with those reflecting physiological and nutritional disorders affecting cotton. Development of diagnostic tools with rapidity will have immense role to play in detection and management of the emerging virus. The protocol for rapid diagnosis of TSV infected samples by using Reverse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) was optimised and this is the first report of its use for diagnosis of TSV on cotton and Soybean. The colorimetric detection for diagnostic simplicity of amplified RT-LAMP product by using different dyes lead to enhanced applicability of this technique. The RT-LAMP diagnostic tool can be utilized not only for laboratory research but also for quarantine and field diagnosis of this important emerging pathogen affecting cotton.

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.

Field-evolved resistance of pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), to transgenic Bacillus thuringiensis (Bt) cotton expressing crystal 1Ac (Cry1Ac) and Cry2Ab in India.

BACKGROUND: Pink bollworm (PBW) adaptation to transgenic Bacillus thuringiensis (Bt) cotton expressing crystal 1Ac (Cry1Ac) (Bt-I) and Cry1Ac + Cry2Ab (Bt-II) was assessed in India during 2010-2017 in 38 districts of the 10 major cotton-growing states. RESULTS: PBW larval incidence on Bt cotton was nil in northern India, wherein the resistance ratios (RRs) to Cry1Ac were 26-262 and those to Cry2Ab were 1-108. In central and southern India, the annual average PBW larval recovery from Bt II cotton was high at 28.85-72.49% during 2014-2017. In central and southern India, the 50% lethal concentration (LC50 ) of Cry1Ac increased from a mean of 0.330 µg mL-1 (range 0.126-0.849 µg mL-1 ) in 2013 to a mean of 6.938 µg mL-1 (range 3.52 to 10.30 µg mL-1 ) in 2017 and the RR increased from a mean of 47.12 (range 18-121) in 2013 to a mean of 1387 (704-2060) in 2017, whereas the LC50 value for Cry2Ab increased from a mean of 0.014 µg mL-1 (range 0.004-0.094 µg mL-1 ) in 2013 to a mean of 12.51 µg mL-1 (range 3.92 to 28.10 µg mL-1 ) in 2017 and the RR increased from a mean of 5.4 (range 1-31) in 2013 to a mean of 4196 (1306-9366) in 2017. CONCLUSION: High PBW larval recovery on Bt-II in conjunction with high LC50 values for Cry1Ac and Cry2Ab in major cotton-growing districts of central and southern India provides evidence of field-evolved resistance in PBW to Bt-I and Bt-II cotton. © 2018 Society of Chemical Industry.

Mitochondria COI-based genetic diversity of the cotton leafhopper Amrasca biguttula biguttula (Ishida) populations from India.

Amrasca biguttula biguttula (Ishida), the cotton leafhopper, is a polyphagous insect pest of Asia and Southeast Asian countries. We sequenced a mitochondrial COI gene fragment from 67 individuals of cotton leafhopper collected from 7 major cotton growing states of North, Central, and South India. Genetic divergence analysis of leaf hopper population across India confirmed the presence of single species. Thirty haplotypes, in total, were determined across different regions of India. While population from North India was dominated by single haplotype, the south and central Indian populations show dispersion of different haplotypes across the region. The neutrality test rejection for the north Indian population also suggests population expansion. The genetic differentiation and gene flow analysis together confirmed the phylogeographic structure of the A. biguttula biguttula Ishida as isolated by distance.

Genetic Similarity between Cotton Leafroll Dwarf Virus and Chickpea Stunt Disease Associated Virus in India.

The cotton leafroll dwarf virus (CLRDV) is one of the most devastating pathogens of cotton. This malady, known as cotton blue disease, is widespread in South America where it causes huge crop losses. Recently the disease has been reported from India. We noticed occurrence of cotton blue disease and chickpea stunt disease in adjoining cotton and chickpea fields and got interested in knowing if these two viral diseases have some association. By genetic studies, we have shown here that CLRDV is very close to chickpea stunt disease associated virus (CpSDaV). We were successful in transmitting the CLRDV from cotton to chickpea. Our studies indicate that CpSDaV and CLRDV in India are possibly two different strains of the same virus. These findings would be helpful in managing these serious diseases by altering the cropping patterns.