First report of Triticum mosaic virus infecting oat crop in the United States.

Triticum mosaic virus (TriMV, genus Poacevirus, family Potyviridae) was first reported in 2006 (Seifers et al. 2008) to infect wheat, and since then, it has been established as a constraint for US wheat production (Byamukama et al. 2013). In the field, TriMV often exists as a coinfection with wheat streak mosaic virus (WSMV), and these two viruses interact synergistically to produce severe symptoms and greater yield loss (Byamukama et al. 2012; Tatineni et al. 2022). Both TriMV and WSMV are transmitted by wheat curl mites (Aceria tosichella Keifer) (McMechan et al. 2014). Wheat is the primary host reported for TriMV in the field, but Seifers et al. (2010) established oat, rye, barley, and several other cereals and grasses as hosts under controlled conditions. However, there are no documented cases of TriMV infecting oats in the field. Between 10-25 June, 2023, a total of 273 field oat plants showing foliar yellowing, yellow flecking, and streaking symptoms were collected from four different fields in Nebraska (Big Springs: 41.1029° N, 102.1451° W; Mead: 41.2292° N, 96.4938° W; Odell: 40.0459° N, 96.7984° W; Stumf: 40.5048° N, 101.4223° W). Total RNA was extracted using the MagMax Plant RNA Isolation kit (Thermo Fisher Scientific) and the KingFisher Flex Magnetic Particle Processor (Thermo Fisher Scientific) (Mondal et al. 2023). Sample RNA was assayed with a single-step multiplex reverse transcription polymerase chain reaction (RT-PCR) to determine presence of WSMV and TriMV. Out of 273 symptomatic oat plants, 254 (93.04%) tested positive for at least one virus. Out of total positive samples, 238 were positive for WSMV (93.70 %), 12 plants tested positive for both TriMV and WSMV (4.70%), and 4 plants were infected with TriMV alone (1.60%). As a secondary confirmation, amplified fragments from the TriMV single infection were gel purified using a gel extraction kit (QIAquick) and sequenced (Eurofins Genomics). The nucleotide sequences were analysed using the BlastN program, compiled, and edited in the BioEdit software (Hall 1999). Sequences were deposited in the NCBI GenBank database (accession number PP475806). Nucleotide BLAST searches of the target coat protein (CP) gene showed > 98% identity to the corresponding sequences in TriMV accession MK318274. For further validation, virus inoculum was prepared by grinding field-collected plant material from plants with only TriMV present in 20 mM sodium phosphate buffer, pH 7.0, and then mechanically inoculating two-week-old oats (cv. Shaw n=8) and wheat (cv. Sattler, n=8) plants. Three weeks post-inoculation, all the eight wheat plants exhibited mild yellowing and streaking symptoms, while oat plants did not show obvious foliar symptoms. All wheat and oat plants were further tested positive with DAC-ELISA (antibodies produced against TriMV CP at the USDA-ARS facility in Lincoln, NE) and with RT-PCR. The specific attribution of these symptoms to TriMV in oats is not possible as none produced prominent symptoms. Asymptomatic oat infection from symptomatic field-collected oat samples could be due to oat cultivar differences. Although the prevalence of TriMV in wheat has been established across the Great Plains of the United States, to our knowledge, this is the first report of TriMV infection in US oat fields. Our finding warrant further investigation into the incidence and impact of the virus in oat crop and its potential for serving as a asymptomatic virus reservoir.

Evidence of population expansion and insecticide resistance mechanism in invasive fall armyworm (Spodoptera frugiperda).

BACKGROUND: The invasive and calamitous polyphagous pest Spodoptera frugiperda or commonly known as fall armyworm (FAW) poses serious menace to the global agricultural production. Owing to the revamped invasion of FAW in 2018 in India, present study was undertaken for precise assessment of its genetic identity and pesticide resistance to aid in pest-management strategies. RESULTS: To evaluate the diversity in FAW population across Eastern India, mitochondrial COI sequences were used which revealed a low nucleotide diversity. Analysis of molecular variance indicated significant genetic variation between four global geographical FAW populations with lowest differentiation between India and Africa suggesting a present-day and shared origin of FAW. The study demonstrated existence of two different strains ('R' strain and 'C' strain) based on COI gene marker. However, discrepancies between COI marker and host plant association of FAW was observed. Characterization of Tpi gene revealed abundance of TpiCa1a followed by TpiCa2b and TpiR1a strains respectively. The FAW population showed higher susceptibility towards chlorantraniliprole and spinetoram than cypermethrin. Insecticide resistance genes depicted marked upregulation although with lot of variance. Chlorantraniliprole resistance ratio (RR) exhibited significant correlation with 1950 (Glutathione S-transferase, GST), 9131 (Cytochrome P450, CYP) and 9360 (CYP) genes, while spinetoram and cypermethrin RR was found to correlate with 1950 (GST) and 9360 (CYP) genes. CONCLUSION: This study manifests Indian subcontinent as the potential new hotspot for the growth and distribution of FAW population that can be effectively controlled using chlorantraniliprole and spinetoram. This study also adds novel significant information on FAW population across Eastern India for developing a comprehensive pest management approach for S. frugiperda.

Detection and in silico characterization of banana bunchy top virus in West Bengal, India: relevance to global genetic diversity and population structure.

Banana bunchy top disease is one of the major prevailing virus diseases associated with banana cultivation, spreading rapidly within a small scale of time. Till date there are only few extensive reports of completely sequenced isolates in India. A study was conducted to detect BBTV infection across 12 districts in West Bengal (WB) where extensive prevalence of the disease was ascertained. In silico characterization of the six genome components were accomplished which showed 84.90-99.86% similarity with other BBTV isolates reported worldwide. The phylogenetic analysis based upon DNA R and DNA S suggested formation of monophyletic cluster of majority of the WB isolates and its close association with Tripura, Manipur, Australia and Africa isolates indicating diversion from geographical differentiation. Dynamics of evolutionary pattern such as genetic diversity including Tajima's D test and Fu Li's Fs test, average number of nucleotide differences (K), Polymorphic sites (S); Fst distance; Mismatch distribution plot; Haplotype network, and selection pressure were performed based upon geographical distribution of the virus. Population genetics analysis of both Pacific Indian Ocean group and South East Asian group of the global BBTV population revealed low nucleotide diversity, high haplotype diversity, high gene flow within the group, and negative or purifying selection constraint indicating recent population expansion. Hence, this study portrays Indian subcontinent as the possible hotspot for rapid demographic expansion from a small virus population size, contributing valuable addition to the currently available information on BBTV worldwide. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00815-0.

First Record of Clonostachys rosea (Ascomycota: Hypocreales) Entomopathogenic Fungus in the Mango Hopper Amritodus atkinsoni (Hemiptera: Cicadellidae).

Mango hopper (Amritodus atkinsoni Lethierry) causes devastations in the early vegetative stage of the mango crop. The classical management of mango hopper is with systemic insecticides but their overuse has caused environmental pollution. Here, we have evaluated the entomopathogenic role of Clonostachys rosea through bioassay and optimized media for its large-scale culturing. The current study reveals the potentiality of C. rosea as entomopathogenic on A. atkinsoni. Initially, morphological and molecular characterization was used to validate local isolates' identity as C. rosea. Further, we have evaluated the entomopathogenic role of C. rosea through a bioassay, where the highest mean mortality in A. atkinsoni was observed at a treatment concentration of 3 × 108 conidia/mL, with 96.67% mortality after 168 h of infection. This work also provides insight into the laboratory-based media standardization for C. rosea, resulting in oatmeal agar media and broth as the most suitable artificial media, and 20 °C temperature for its mass culture. Thus, C. rosea is a novo-entomopathogenic fungus on A. atkinsoni and has a high potency to be included in the management of mango hopper pests.

Unraveling the Basis of Neonicotinoid Resistance in Whitefly Species Complex: Role of Endosymbiotic Bacteria and Insecticide Resistance Genes.

Bemisia tabaci (whitefly) is one of the most detrimental agricultural insect pests and vectors of many plant viruses distributed worldwide. Knowledge of the distribution patterns and insecticide resistance of this cryptic species is crucial for its management. In this study, genetic variation of mitochondrial cytochrome oxidase subunit 1 (MtCoI) gene of B. tabaci was analyzed followed by a study of the infection profile of various endosymbionts in 26 whitefly populations collected from West Bengal, India. Phylogenetic analysis revealed Asia I as the major cryptic species (65.38%), followed by Asia II 5, China 3, and Asia II 7, which were diversified into 20 different haplotypes. In addition to the primary endosymbiont (C. poriera), each of the four whitefly species showed a variable population of three secondary endosymbionts, majorly Arsenophonus with the highest infection rate (73.07%), followed by Wolbachia and Rickettsia. Further phylogenetic analyses revealed the presence of two subgroups of Arsenophonus, viz., A1 and A2, and one each in Wolbachia (W1) and Rickettsia (R3). Resistance to thiamethoxam, imidacloprid, and acetamiprid insecticides was analyzed for a clear picture of pesticide resistance status. The highest susceptibility was noted toward thiamethoxam (LC50 = 5.36 mg/L), followed by imidacloprid and acetamiprid. The whitefly population from Purulia and Hooghly districts bearing Asia II 7 and Asia II 5 cryptic species, respectively, shows maximum resistance. The differences in mean relative titer of four symbiotic bacteria among field populations varied considerably; however, a significant positive linear correlation was observed between the resistance level and relative titer of Arsenophonus and Wolbachia in the case of imidacloprid and thiamethoxam, while only Wolbachia was found in case of acetamiprid. Expression analysis demonstrated differential upregulation of insecticide resistance genes with Purulia and Hooghly populations showing maximally upregulated P450 genes. Moreover, thiamethoxam and imidacloprid resistance ratio (RR) showed a significant correlation with CYP6CM1, CYP6DZ7, and CYP4C64 genes, while acetamiprid RR correlated with CYP6CX1, CYP6DW2, CYP6DZ7, and CYP4C64 genes. Taken together, these findings suggested that P450 mono-oxygenase and symbiotic bacteria together affected whitefly resistance to neonicotinoids. Hence, a symbiont-oriented management programme could be a better alternative to control or delay resistance development in whitefly and can be used for pesticide clean-up in an agricultural field.

Copy number variation of two begomovirus acquired and inoculated by different cryptic species of whitefly, Bemisia tabaci in Okra.

The whitefly, B.tabaci is a major pest of agricultural crops which transmits begomovirus in a species-specific manner. Yellow vein mosaic disease (YVMD) and okra leaf curl disease (OLCD) caused by distinct begomovirus are a major limitation to production of okra in India. In this framework the present investigation reports, for the first time, comparative study of begomovirus species viz. yellow vein mosaic virus (YVMV) and okra enation leaf curl virus (OELCuV) ingested and egested by two cryptic species (Asia I and Asia II 5) of B.tabaci at different time interval using detached leaf assay. A gradual increase of both virus copies were observed with increased feeding exposure in Asia I and Asia II 5. Both the genetic groups of whitefly could acquire the viruses within just 5 minutes of active feeding however, a significant amount of variation was noted in virus uptake by the both. At 24 hours of active feeding Asia II 5 acquired more of YVMV whereas, Asia I ingested more OELCuV. Similarly, the genetic group acquiring higher titre of virus egested higher amount during inoculation period. On the whole, it can be presumed that Asia I is a more effective transmitter of OELCuV whereas, Asia II 5 of YVMV further suggesting increased risk of virus pandemics (both YVMV and OELCuV) in regions where Asia I and Asia II 5 is dominant.

Transcription dynamics of heat-shock proteins (Hsps) and endosymbiont titres in response to thermal stress in whitefly, Bemisia tabaci (Asia-I).

The sweet potato whitefly, Bemisia tabaci (Gennadius), is one of the several species complexes of whitefly that are currently significant agricultural pests. Bemisia tabaci infests more than 600 plant species and thrives under a wide range of temperature conditions. In addition to the direct damage caused by sucking plant sap, it vectors several plant viruses. Heat-shock proteins play a pivotal role in enabling the insect to extend its geographical location, survival, and reproduction under different stress conditions. B. tabaci harbours several endosymbionts under the genera Portiera, Rickettsia, Hamiltonella, Wolbachia, Arsenophonus, Cardinium, and Fritschea that directly or indirectly affect its fitness. By accelerating cuticle biosynthesis and sclerotisation, symbiotic microbes can reduce or enhance tolerance to extreme temperatures and detoxify heavy metals. Thus, symbionts or microbial communities can expand or constrain the abiotic niche space of their host and affect its ability to adapt to changing conditions. The present study delineates the effect of thermal stress on the expression of heat-shock genes and endosymbionts in B. tabaci. Studies of the expression level of heat-shock proteins with the help of quantitative real-time polymerase chain reaction (qRT-PCR) showed that heat- and cold-shock treatment fuels the increased expression of heat-shock proteins (Hsp40 and Hsp70). However, Hsp90 was not induced by a heat- and cold-shock treatment. A significant decrease in the relative titre of secondary endosymbionts, such as Rickettsia, Arsenophonus, and Wolbachia, were recorded in B. tabaci upon heat treatment. However, the titre of the primary symbiont, C. Portiera, was relatively unaffected by both cold and heat treatments. These results are indicative of the fact that Hsp genes and endosymbionts in B. tabaci are modulated in response to thermal stress, and this might be responsible for the adaptation of whitefly under changing climatic scenario.

Effect of Neonicotinoids on Bacterial Symbionts and Insecticide-Resistant Gene in Whitefly, Bemisia tabaci.

The silverleaf whitefly, Bemisia tabaci (Gennadius, Hemiptera: Aleyrodidae), is a major threat to field and horticultural crops worldwide. Persistent use of insecticides for the management of this pest is a lingering problem. In the present study, the status of sensitivity of B. tabaci to two neonicotinoids, imidacloprid and thiamethoxam, was evaluated. The expression pattern of two cytochrome P450 (cyp) genes and changes in the relative amount of symbionts in insecticide-treated B. tabaci were also assessed. Quantitative PCR (qPCR) studies indicate that the CYP6CM1 and CYP6CX1 genes were always expressed higher in imidacloprid-treated whitefly, suggesting a correlation between gene expression and the insect's ability to detoxify toxic compounds such as insecticides. In addition, the thiamethoxam-treated population harbored higher Portiera and lower Rickettsia titers, whereas the imidacloprid-treated population harbored more Rickettsia at different time intervals. Interestingly, we also examined that an increase in exposure to both the insecticides resulted in a reduction in the mutualistic partners from their insect host. These differential responses of endosymbionts to insecticide exposure imply the complex interactions among the symbionts inside the host insect. The results also provide a deeper understanding of the molecular mechanism of resistance development that might be useful for formulating effective management strategies to control B. tabaci by manipulating symbionts and detoxifying genes.

Involvement of small heat shock proteins (sHsps) in developmental stages of fall armyworm, Spodoptera frugiperda and its expression pattern under abiotic stress condition.

Fall armyworm (FAW), Spodoptera frugiperda a recent invasive pest in India is reported to cause significant damage by feeding voraciously on maize and other economic crops from tropical to temperate provinces. It is becoming an arduous challenge to control the pest as it can survive in a wide range of temperature conditions and is already said to develop resistance towards certain insecticides. The small Heat shock proteins (hereafter, sHsps) are known to play an important role in adaptation of insects under such stress conditions. Our present study involved characterization of the three sHsps genes (sHsp19.74, sHsp20.7 and sHsp19.07) which encoded proteins of about 175, 176 and 165 amino acids with a conserved α-crystalline domain. Phylogenetic analysis of deduced amino acid sequences of the three genes showed strong similarity with the other lepidopteran sHsps. The effect of different growth stages on the expression profile of these stress proteins has also been studied and the Quantitative real time PCR (qRT-PCR) analysis revealed that the transcript level of sHsp19.07 and sHsp20.7 were significantly upregulated under extreme heat (44 °C) and cold (5 °C) stress. However, sHsp19.74 responded only to heat treatment but not to the cold treatment. In addition, the expression profile of all three sHsps was significantly lower in the larval stage (5th instar). Chlorantraniliprole treatment resulted in maximum expression of sHsp19.07 and sHsp20.7 after 12hr of exposure to the insecticide. Meanwhile, the same expression was observed after 8hr of exposure in case of sHsp19.74. These results proved that the sHsp genes of S. frugiperda were induced and modulated in response to abiotic stress, thus influencing the physiological function leading to survival of FAW in diversified climate in India.