Molecular Surveillance, Prevalence, and Distribution of Cacao Infecting Badnavirus Species in Côte d'Ivoire and Ghana.

The cacao swollen shoot disease (CSSD) caused by a complex of badnavirus species presents a major challenge for cacao production in West Africa, especially Ghana and Côte d'Ivoire. In this study, CSSD species detection efficiency, diversity, and geographic distribution patterns in cacao plantations in Ghana and Côte d'Ivoire were investigated through field surveillance, PCR detection assays, sequencing of positive amplicons, and phylogeographic clustering. Cumulatively, the detection efficiency of the tested CSSD primer sets that were targeting the movement protein domain of the virus ranged from 0.15% (CSSD-3 primer) to 66.91% (CSSD-1 primer) on all the symptomatic cacao leaf samples assessed. The identified CSSD species differed phylogenetically and overlapped in distribution, with the cacao swollen shoot Togo B virus (CSSTBV) (n = 588 sequences) being the most prevalent and widely distributed compared to the other CSSD species that were encountered in both countries. Geographically, the cacao swollen shoot CE virus (CSSCEV) species (n = 124 sequences) that was identified was largely restricted to the bordering regions of Ghana and Côte d'Ivoire. These results provide updated knowledge of the geographic distribution of the key CSSD species and their diagnostic efficiency and, thus, provide guidance in identifying locations for structured testing of cacao germplasm and optimal diagnostics for the predominant CSSD species in Ghana and Côte d'Ivoire.

First report of sweepoviruses infecting Ipomeas batatas L. cultivars and landraces in Trinidad.

Root crops, referred to as ground provisions in the Caribbean, are traditional staples in Trinidad. One widely consumed example is sweet potato (Ipomeas batatas L.). The crop is mainly produced by subsistence farming which together with imports from neighboring Caribbean countries meet domestic demand (Singh et al. 2008). The Central Experiment Station, situated in the eastern part of Trinidad, maintains a sweet potato germplasm collection comprising both imported and locally-sourced landraces for cultivar development and distribution of propagules. In May 2017 chlorosis and leaf curling symptoms, typically associated with sweepoviruses, were observed on imported cultivars, Centennial, Jewel, 86 BM 31, TIB 313, TIB 8 21 1, and S128, and the landraces, Kick Up Jenny, John, and Carrot. Leaf samples from these nine symptomatic plants were collected for analysis, along with samples from the asymptomatic landrace, Chickenfoot. Total nucleic acids were extracted (Sharma et al. 2008) and the samples were assayed by PCR using degenerate primers SPG1 and SPG2 (Li et al. 2004) that target the replication associated protein gene (ORF C1), a highly conserved region of sweepoviruses. Amplicons of 912-bp were obtained from two of the nine symptomatic plants (TIB 8 21 1, Kick Up Jenny), but not from the asymptomatic Chickenfoot. The same samples were assayed by PCR amplification using primers SpvF and SpvR (Avelar 2015) which are specific to a highly conserved 632-bp region of the coat protein gene (ORF V1) of sweet potato leaf curl virus (SPLCV). All 10 samples tested positive for SPLCV, including the asymptomatic landrace, Chickenfoot. The ORF V1 PCR products from three of the 10 samples, namely Chickenfoot, TIB 8 21 1, and Kick Up Jenny, were cloned and sequenced (two clones per sample). Comparison of the sequences (GenBank accession nos. OR882007 [Chickenfoot], OR913125 [TIB 8 21 1] and OR913126 [Kick Up Jenny]) identified up to 4% nt sequence variability between samples. In BLASTn analysis, they were most closely related to the SPLCV isolate China:Sichuan (GenBank accession no. KJ013557), sharing 94 to 98% nt identity. Total nucleic extracts from one representative sample (TIB 8 21 1) was used as template for rolling circle amplification (RCA, TempliPhi Amplification Kit, GE Healthcare Life Sciences, Piscataway, NJ, USA). Digestion of the RCA product with StuI (Thermo Scientific, MA, USA) yielded ~2.8 kb DNA fragments indicative of monomeric full length genomes. Digested fragments were cloned, completely sequenced and deposited in GenBank under the accession nos. OR866202 (2,821 nts) and OR866203 (2,828 nts). Two species of sweepoviruses were detected. In BLASTn analysis, OR866202 showed 95% nt identity with sweet potato golden vein associated virus (SPGVaV) US:MS:1B-3 (GenBank accession no. HQ333143.1) which is a recombinant virus comprised of SPLCV and sweet potato leaf curl Georgia virus (SPLCGV) (Zhang and Ling 2011) and in BLASTx analysis OR866202 was most similar (92-99%) to SPLCV isolates from Brazil (GenBank accession nos. ACI23475.1, AGW16179.1, ACY79479.1), Peru (GenBank accession no. ACY79466.1) and China (GenBank accession nos. ACY79439.1). OR866203 shared 96% nt identity with SPLCV China:Henan25(8):2012 (GenBank accession no. KF040465.1) in BLASTn analysis and BLASTx analysis revealed ≥ 94% aa sequence identity with SPLCV from Brazil (GenBank accession nos. ACI23475.1, AGW16179.1, ADZ96559.1), Peru (GenBank accession no. ACY79479.1), China (GenBank accession no. ACY79466.1). and Spain (GenBank accession no. QWQ56365.1). Both Trinidad isolates also showed 90-96% nt identity with SPLCV from Korea (GenBank accession no.s KT992061.1, KT992064.1, unpublished). This is the first detection of sweepoviruses in Trinidad. SPGVaV has been reported in Brazil, the United States and Korea (Kil et al. 2014), while SPLCV has been described in other Caribbean islands, including Cuba, Jamaica, Puerto Rico, St. Vincent (Cuellar et al. 2015), and Barbados (Alleyne et al. 2019), as well as several countries in South America. Although Koch's postulates were not completed, our findings suggest that sweet potato crops in Trinidad harbor sweepoviruses, notwithstanding efforts to distribute pathogen-free materials and, in some instances, the apparent absence of visible symptoms on infected plants. Further studies on the management and impact of these viruses are necessary, including their prevalence in the sweet potato production regions of Trinidad.

Complete genome characterization of cacao leafroll virus, a newly described cacao-infecting polerovirus.

The complete genome sequence of cacao leafroll virus (CaLRV; family Solemoviridae, genus Polerovirus) was determined by high-throughput sequencing of total RNA isolated from symptomatic cacao Theobroma cacao L. plants (n = 4). The CaLRV genome sequences ranged from 5,976 to 5,997 nucleotides (nt) in length and contained seven open reading frames (ORFs). Nucleotide and amino acid (aa) sequence comparisons showed that, among selected well-characterized poleroviruses, the CaLRV genome shared the highest nt sequence identity of 62% with that of potato leafroll virus (PLRV, NC_076505). A comparison of the predicted aa sequence of the CaLRV coat protein indicated that cotton leafroll dwarf virus (CLRDV, NC_014545) and melon aphid-borne yellows virus (MABYV, NC_010809) were the closest relatives, sharing 57% aa sequence identity. Bayesian phylogenetic analysis based on complete genome sequences showed that CaLRV grouped with well-characterized poleroviruses that cause diseases of cereal and vegetable crops. During the course of publishing this work, the nearly complete genome sequence of a member of the same polerovirus species, referred to as "cacao polerovirus" (OR605721), with which CaLRV shares 99% nt sequence identity, was reported.

Optimal dsRNA Concentration for RNA Interference in Asian Citrus Psyllid.

The Asian citrus psyllid (ACP) is a citrus pest and insect vector of "Candidatus Liberibacter asiaticus", the causal agent of citrus greening disease. Double-stranded RNA (dsRNA) biopesticides that trigger RNA interference (RNAi) offer an alternative to traditional insecticides. Standardized laboratory screening of dsRNA requires establishing the minimal effective concentration(s) that result in effective RNAi "penetrance" and trigger RNAi, resulting in one or more measurable phenotypes, herein, significant gene knockdown and the potential for mortality. In this study, knockdown was evaluated for a range of dsRNA concentrations of three ACP candidate genes, clathrin heavy chain (CHC), vacuolar ATPase subunit A (vATPase-A), and sucrose non-fermenting protein 7 (Snf7). Gene knockdown was quantified for ACP teneral adults and 3rd instar nymphs allowed a 48 h ingestion-access period (IAP) on 10, 50,100, 200, and 500 ng/µL dsRNA dissolved in 20% sucrose followed by a 5-day post-IAP on orange jasmine shoots. Significant gene knockdown (p < 0.05) in ACP third instar nymphs and adults ranged from 12-34% and 18-39%, 5 days post-IAP on dsRNA at 10-500 and 100-500 ng/µL, respectively. The threshold concentration beyond which no significant gene knockdown and adult mortality was observed post-48 h IAP and 10-day IAP, respectively, was determined as 200 ng/µL, a concentration indicative of optimal RNAi penetrance.

Genetic Variability Among U.S.-Sentinel Cotton Plot Cotton Leafroll Dwarf Virus and Globally Available Reference Isolates Based on ORF0 Diversity.

The aphid-transmitted polerovirus, cotton leafroll dwarf virus (CLRDV), first characterized from symptomatic cotton plants in South America, has been identified in commercial cotton plantings in the United States. Here, the CLRDV intraspecific diversity was investigated by comparative sequence analysis of the most divergent CLRDV coding region, ORF0/P0. Bayesian analysis of ORF0 sequences for U.S. and reference populations resolved three well-supported sister clades comprising one U.S. and two South American lineages. Principal component analysis (PCA) identified seven statistically supported intraspecific populations. The Bayesian phylogeny and PCA dendrogram-inferred relationships were congruent. Population analysis of ORF0 sequences indicated most lineages have evolved under negative selection, albeit certain sites/isolates evolved under positive selection. Both U.S. and South American isolates exhibited extensive ORF0 diversity. At least two U.S. invasion foci were associated with their founder populations in Alabama-Georgia and eastern Texas. The Alabama-Georgia founder is implicated as the source of recent widespread expansion and establishment of secondary disease foci throughout the southeastern-central United States. Based on the geographically restricted distribution, spread of another extant Texas population appeared impeded by a population bottleneck. Extant CLRDV isolates represent several putative introductions potentially associated with catastrophic weather events dispersing viruliferous cotton aphids of unknown origin(s).

UV-LASER adjuvant-surfactant-facilitated delivery of mobile dsRNA to tomato plant vasculature and evidence of biological activity by gene knockdown in the potato psyllid.

BACKGROUND: Double-stranded RNA (dsRNA) biopesticides are of interest for the abatement of insect vectors of pathogenic bacteria such as 'Candidatus Liberibacter', which infects both its psyllid and plant hosts. Silencing of genes essential for psyllids, or for Liberibacter, is anticipated to lead to mortality or impeded bacterial multiplication. Foliar delivery is preferred for biopesticide application; however, the cuticle impedes dsRNA penetration into the vasculature. Here, conditions were established for wounding tomato leaves using ultraviolet light amplification by stimulated emissions of radiation (UV-LASER) to promote dsRNA penetration into leaves and vasculature. RESULTS: UV-LASER treatment with application of select adjuvants/surfactants resulted in vascular delivery of 100-, 300- and 600-bp dsRNAs that, in general, were correlated with size. The 100-bp dsRNA required no pretreatment, whereas 300- and 600-bp dsRNAs entered the vasculature after UV-LASER treatment only and UV-LASER adjuvant/surfactant treatment, respectively. Of six adjuvant/surfactants evaluated, plant-derived oil combined with an anionic organosilicon compound performed most optimally. Localization of dsRNAs in the tomato vasculature was documented using fluorometry and fluorescence confocal microscopy. The biological activity of in planta-delivered dsRNA (200-250 bp) was determined by feeding third-instar psyllids on tomato leaves post UV-LASER adjuvant/surfactant treatment, with or without psyllid cdc42- and gelsolin dsRNAs. Gene knockdown was quantified by quantitative, real-time polymerase chain reaction with reverse transcription (RT-qPCR) amplification. At 10 days post the ingestion-access period, knockdown of cdc42 and gelsolin expression was 61% and 56%, respectively, indicating that the dsRNAs delivered to the tomato vasculature were mobile and biologically active. CONCLUSION: Results indicated that UV-LASER adjuvant/surfactant treatments facilitated the delivery of mobile, biologically active dsRNA molecules to the plant vasculature. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification and distribution of the "Candidatus Liberibacter asiaticus"-Asian citrus psyllid pathosystem in Saudi Arabia.

Citrus greening disease was first reported in Saudi Arabia during the 1970's when characteristic foliar and fruit symptoms were observed in commercial citrus groves, however, "Candidatus Liberibacter asiaticus" (CLas) was not detected in symptomatic trees until 1981-1984 when CLas-like cells were observed by transmission electron microscopy in leaves collected from symptomatic citrus groves in southwestern Saudi Arabia. Despite the anticipated establishment of the CLas-Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) pathosystem, CLas presence has not been verified in suspect trees nor have ACP infestations been documented. Given the recent expansion of citrus production in Saudi Arabia, a systematic country-wide survey was carried out to determine the potential CLas distribution in the thirteen citrus-growing regions of the country. Citrus trees were surveyed for presence of CLas-psyllid vector(s) and characteristic disease symptoms in commercial and urban citrus trees. Adult psyllids collected from infested citrus trees were identified as ACP based on morphological characteristics. Real-time, quantitative PCR amplification (qPCR) of the CLas ß-subunit of the ribonucleotide reductase (RNR) gene from citrus leaf and fruit samples and/or ACP adults, revealed trees were positive for CLas detection in ten of the 13 survey regions, however, CLas was undetectable in ACP adults. Phylogenetic and SNPs analyses of a PCR-amplified, cloned fragment of the CLas 16S rRNA gene (~1.1 kbp) indicated Saudi Arabian isolates were most closely related to Florida, USA isolates. Analysis of climate variables indicated that the distribution of the ACP-CLas pathosystem observed in Saudi Arabia was consistent with published predictions of terrains most likely to support establishment.

Differential gene expression of Asian citrus psyllids infected with 'Ca. Liberibacter asiaticus' reveals hyper-susceptibility to invasion by instar fourth-fifth and teneral adult stages.

The bacterial pathogen Candidatus Liberibacter asiaticus (CLas) is the causal agent of citrus greening disease. This unusual plant pathogenic bacterium also infects its psyllid host, the Asian citrus psyllid (ACP). To investigate gene expression profiles with a focus on genes involved in infection and circulation within the psyllid host of CLas, RNA-seq libraries were constructed from CLas-infected and CLas-free ACP representing the five different developmental stages, namely, nymphal instars 1-2, 3, and 4-5, and teneral and mature adults. The Gbp paired-end reads (296) representing the transcriptional landscape of ACP across all life stages and the official gene set (OGSv3) were annotated based on the chromosomal-length v3 reference genome and used for de novo transcript discovery resulting in 25,410 genes with 124,177 isoforms. Differential expression analysis across all ACP developmental stages revealed instar-specific responses to CLas infection, with greater overall responses by nymphal instars, compared to mature adults. More genes were over-or under-expressed in the 4-5th nymphal instars and young (teneral) adults than in instars 1-3, or mature adults, indicating that late immature instars and young maturing adults were highly responsive to CLas infection. Genes identified with potential for direct or indirect involvement in the ACP-CLas circulative, propagative transmission pathway were predominantly responsive during early invasion and infection processes and included canonical cytoskeletal remodeling and endo-exocytosis pathway genes. Genes with predicted functions in defense, development, and immunity exhibited the greatest responsiveness to CLas infection. These results shed new light on ACP-CLas interactions essential for pathogenesis of the psyllid host, some that share striking similarities with effector protein-animal host mechanisms reported for other culturable and/or fastidious bacterial- or viral- host pathosystems.

LbCas12a mediated suppression of Cotton leaf curl Multan virus.

Begomoviruses are contagious and severely affect commercially important fiber and food crops. Cotton leaf curl Multan virus (CLCuMuV) is one of the most dominant specie of Begomovirus and a major constraint on cotton yield in Pakistan. Currently, the field of plant genome editing is being revolutionized by the CRISPR/Cas system applications such as base editing, prime editing and CRISPR based gene drives. CRISPR/Cas9 system has successfully been used against biotic and abiotic plant stresses with proof-of-concept studies in both model and crop plants. CRISPR/Cas12 and CRISPR/Cas13 have recently been applied in plant sciences for basic and applied research. In this study, we used a novel approach, multiplexed crRNA-based Cas12a toolbox to target the different ORFs of the CLCuMuV genome at multiple sites simultaneously. This method successfully eliminated the symptoms of CLCuMuV in Nicotiana benthamiana and Nicotiana tabacum. Three individual crRNAs were designed from the CLCuMuV genome, targeting the specific sites of four different ORFs (C1, V1 and overlapping region of C2 and C3). The Cas12a-based construct Cas12a-MV was designed through Golden Gate three-way cloning for precise editing of CLCuMuV genome. Cas12a-MV construct was confirmed through whole genome sequencing using the primers Ubi-intron-F1 and M13-R1. Transient assays were performed in 4 weeks old Nicotiana benthamiana plants, through the agroinfiltration method. Sanger sequencing indicated that the Cas12a-MV constructs made a considerable mutations at the target sites of the viral genome. In addition, TIDE analysis of Sanger sequencing results showed the editing efficiency of crRNA1 (21.7%), crRNA2 (24.9%) and crRNA3 (55.6%). Furthermore, the Cas12a-MV construct was stably transformed into Nicotiana tabacum through the leaf disc method to evaluate the potential of transgenic plants against CLCuMuV. For transgene analysis, the DNA of transgenic plants of Nicotiana tabacum was subjected to PCR to amplify Cas12a genes with specific primers. Infectious clones were agro-inoculated in transgenic and non-transgenic plants (control) for the infectivity assay. The transgenic plants containing Cas12a-MV showed rare symptoms and remained healthy compared to control plants with severe symptoms. The transgenic plants containing Cas12a-MV showed a significant reduction in virus accumulation (0.05) as compared to control plants (1.0). The results demonstrated the potential use of the multiplex LbCas12a system to develop virus resistance in model and crop plants against begomoviruses.

Sentinel plot surveillance of cotton leaf curl disease in Pakistan- a case study at the cultivated cotton-wild host plant interface.

A sentinel plot case study was carried out to identify and map the distribution of begomovirus-betasatellite complexes in sentinel plots and commercial cotton fields over a four-year period using molecular and high-throughput DNA 'discovery' sequencing approaches. Samples were collected from 15 study sites in the two major cotton-producing areas of Pakistan. Whitefly- and leafhopper-transmitted geminiviruses were detected in previously unreported host plant species and locations. The most prevalent begomovirus was cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu). Unexpectedly, a recently recognized recombinant, cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Ra) was prevalent in five of 15 sites. cotton leaf curl Alabad virus (CLCuAlV) and cotton leaf curl Kokhran virus-Kokhran, 'core' members of CLCuD-begomoviruses that co-occurred with CLCuMuV in the 'Multan' epidemic were detected in one of 15 sentinel plots. Also identified were chickpea chlorotic dwarf virus and 'non-core' CLCuD-begomoviruses, okra enation leaf curl virus, squash leaf curl virus, and tomato leaf curl New Delhi virus. Cotton leaf curl Multan betasatellite (CLCuMuB) was the most prevalent CLCuD-betasatellite, and less commonly, two 'non-core' betasatellites. Recombination analysis revealed previously uncharacterized recombinants among helper virus-betasatellite complexes consisting of CLCuKoV, CLCuMuV, CLCuAlV and CLCuMuB. Population analyses provided early evidence for CLCuMuV-Ra expansion and displacement of CLCuKoV-Bu in India and Pakistan from 2012-2017. Identification of 'core' and non-core CLCuD-species/strains in cotton and other potential reservoirs, and presence of the now predominant CLCuMuV-Ra strain are indicative of ongoing diversification. Investigating the phylodynamics of geminivirus emergence in cotton-vegetable cropping systems offers an opportunity to understand the driving forces underlying disease outbreaks and reconcile viral evolution with epidemiological relationships that also capture pathogen population shifts.

The Bemisia tabaci cryptic (sibling) species group - imperative for a taxonomic reassessment.

The taxonomy of the Bemisia tabaci cryptic species group remains a challenge due to the lack of morphological differentiation and porous species boundaries among its members. Additionally, it is unclear whether B. tabaci consists of several species in evolutionary stasis with limited morphological change or is the result of a recent adaptive radiation characterized by great ecological diversity but little morphological divergence. Here, a historical overview of the development of the nomenclature used to classify B. tabaci is provided covering changes after synonymizing several species in 1957 until recent insights gained from whole-genome sequencing data. The article discusses the limitations of using a 3.5% mtCOI threshold and argues that a 1% nuclear divergence cutoff better reflects ecological and biogeographic species boundaries. Finally, a plan of action is outlined for naming B. tabaci species using a Latin binomial system in accordance with the International Comission on Zoological Nomenclature (ICZN) regulations.

Native and Non-Native Bemisia tabaci NAFME Haplotypes Can Be Implicated in Dispersal of Endemic and Introduced Begomoviruses in Oman.

Irrigated agriculture and global trade expansion have facilitated diversification and spread of begomoviruses (Geminiviridae), transmitted by the Bemisia tabaci (Gennadius) cryptic species. Oman is situated on major crossroads between Africa and South Asia, where endemic/native and introduced/exotic begomoviruses occur in agroecosystems. The B. tabaci 'B mitotype' belongs to the North Africa-Middle East (NAFME) cryptic species, comprising at least eight endemic haplotypes, of which haplotypes 6 and/or 8 are recognized invasives. Prevalence and associations among native and exotic begomoviruses and NAFME haplotypes in Oman were investigated. Nine begomoviral species were identified from B. tabaci infesting crop or wild plant species, with 67% and 33% representing native and exotic species, respectively. Haplotypes 2, 3, and 5 represented 31%, 3%, and 66% of the B. tabaci population, respectively. Logistic regression and correspondence analyses predicted 'strong'- and 'close' virus-vector associations involving haplotypes 5 and 2 and the exotic chili leaf curl virus (ChiLCV) and endemic tomato yellow leaf curl virus-OM, respectively. Patterns favor a hypothesis of relaxed virus-vector specificity between an endemic haplotype and the introduced ChiLCV, whereas the endemic co-evolved TYLCV-OM and haplotype 2 virus-vector relationship was reinforced. Thus, in Oman, at least one native haplotype can facilitate the spread of endemic and introduced begomoviruses.

In Silico Identification of Cassava Genome-Encoded MicroRNAs with Predicted Potential for Targeting the ICMV-Kerala Begomoviral Pathogen of Cassava.

Cassava mosaic disease (CMD) is caused by several divergent species belonging to the genus Begomovirus (Geminiviridae) transmitted by the whitefly Bemisia tabaci cryptic species group. In India and other parts of Asia, the Indian cassava mosaic virus-Kerala (ICMV-Ker) is an emergent begomovirus of cassava causing damage that results in reduced yield loss and tuber quality. Double-stranded RNA-mediated interference (RNAi) is an evolutionary conserved mechanism in eukaryotes and highly effective, innate defense system to inhibit plant viral replication and/or translation. The objective of this study was to identify and characterize cassava genome-encoded microRNAs (mes-miRNA) that are predicted to target ICMV-Ker ssDNA-encoded mRNAs, based on four in silico algorithms: miRanda, RNA22, Tapirhybrid, and psRNA. The goal is to deploy the predicted miRNAs to trigger RNAi and develop cassava plants with resistance to ICMV-Ker. Experimentally validated mature cassava miRNA sequences (n = 175) were downloaded from the miRBase biological database and aligned with the ICMV-Ker genome. The miRNAs were evaluated for base-pairing with the cassava miRNA seed regions and to complementary binding sites within target viral mRNAs. Among the 175 locus-derived mes-miRNAs evaluated, one cassava miRNA homolog, mes-miR1446a, was identified to have a predicted miRNA target binding site, at position 2053 of the ICMV-Ker genome. To predict whether the cassava miRNA might bind predicted ICMV-Ker mRNA target(s) that could disrupt viral infection of cassava plants, a cassava locus-derived miRNA-mRNA regulatory network was constructed using Circos software. The in silico-predicted cassava locus-derived mes-miRNA-mRNA network corroborated interactions between cassava mature miRNAs and the ICMV-Ker genome that warrant in vivo analysis, which could lead to the development of ICMV-Ker resistant cassava plants.

RNA interference-mediated knockdown of genes involved in sugar transport and metabolism disrupts psyllid Bactericera cockerelli (Order: Hemiptera) gut physiology and results in high mortality.

Introduction: The causal agent of zebra chip of potato and vein-greening diseases of tomato is "Candidatus Liberibacter solanacearum" (CLso), a fastidious bacterium transmitted by the potato psyllid. In the absence of disease-resistant cultivars, disease management has relied on minimizing vector population size to reduce CLso transmission, which requires frequent insecticide applications. There is growing interest in the use of RNA interference (RNAi) technology to supplant traditional insecticides with biopesticides. This requires knowledge of genes essential for insect livelihood whose knockdown leads to significant mortality or other phenotypes. Such candidate genes can be evaluated by reverse genetics approaches to further corroborate predicted gene function. Methods: Here, five potato psyllid genes involved in sugar homeostasis in the potato psyllid gut, α-glucosidase1 (AGLU1), aquaporin2 (AQP2), facilitated trehalose transporter1 (TRET1), Trehalase1 (TRE1), and Trehalase2 (TRE2), were investigated as candidates for effective gene silencing. Potato psyllid dsRNAs were designed to optimize knockdown of gene targets. Third instar PoP nymphs were given a 48-hr ingestion-access period (IAP) on individual or groups of dsRNA in 20% sucrose. Mortality was recorded 0, 3, 5, 7, and 9 days post-IAP. Gene knockdown was analyzed 9 days post-IAP by quantitative real-time reverse-transcriptase polymerase chain reaction amplification. Results: The individual or stacked dsRNA combinations resulted in 20-60% and 20-40% knockdown, respectively, while subsequent psyllid mortality ranged from 20-40% to >60% for single and stacked dsRNA combinations, respectively. Reverse genetics analysis showed that simultaneous knockdown of the five selected candidate genes with predicted functions in pathways involved in sugar-homeostasis, metabolism, and -transport yielded the highest mortality, when compared with single or combinations of targets. Discussion: Results confirmed the functions afforded by psyllid gut genes responsible for osmotic homeostasis and sugar metabolism/transport are essential for livelihood, identifying them as potentially lucrative RNAi biopesticide targets and highlighted the translational relevance of targeting multiple nodes in a physiological pathway simultaneously.

Sub-optimal temperatures lead to altered expression of stress-related genes and increased 'Candidatus Liberibacter solanacearum' accumulation in potato psyllid.

Introduction: The potato psyllid Bactericera cockerelli is the insect vector of the fastidious bacterium 'Candidatus Liberibacter solanacearum'. The bacterium infects both B. cockerelli and plant species, causing zebra chip (ZC) disease of potato and vein-greening disease of tomato. Temperatures are known to influence the initiation and progression of disease symptom in the host plant, and seasonal transitions from moderate to high temperatures trigger psyllid dispersal migration to facilitate survival. Methods: 'Ca. L. solanacearum' -infected and uninfected psyllids were reared at previously established 'permissible', optimal, and 'non-permissible' and temperatures of 18°C, 24°C, and 30°C, respectively. Gene expression profiles for 'Ca. L. solanacearum'-infected and -uninfected adult psyllids reared at different temperatures were characterized by Illumina RNA-Seq analysis. Bacterial genome copy number was quantified by real-time quantitative-PCR (qPCR) amplification. Results: Relative gene expression profiles varied in psyllids reared at the three experimental temperatures. Psyllids reared at 18°C and 30°C exhibited greater fold-change increased expression of stress- and 'Ca. L. solanacearum' invasion-related proteins. Quantification by qPCR of bacterial genome copy number revealed that 'Ca. L. solanacearum' accumulation was significantly lower in psyllids reared at 18°C and 30°C, compared to 24°C. Discussion: Temperature is a key factor in the life history of potato psyllid and multiplication/accumulation of 'Ca. L. solanacearum' in both the plant and psyllid host, influences the expression of genes associated with thermal stress tolerance, among others, and may have been instrumental in driving the co-evolution of the pathosystem.

Characteristics of environmental RNAi in potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Psylloidea: Triozidae).

RNA interference (RNAi) has potential to become a major tool for integrated management of insect pests of agricultural crops based on sequence-specificity and low doses of rapidly biodegradable dsRNA. Deploying 'environmental RNAi' for control of insect vectors of plant pathogens is of increasing interest for combatting emerging plant diseases. Hemipteran insect vectors, including psyllids, are vascular feeders, making their development difficult to control specifically by targeting with pesticidal chemistries. Psyllids transmit "Candidatus Liberibacter solanacearum" the causal organism of potato zebra chip and tomato vein greening diseases, transmitted, respectively, by the potato or tomato psyllid (PoP). Until now, the optimal effective concentration(s) of double-stranded RNA (dsRNA) required for significant gene knockdown and RNAi persistence in PoP have not been determined. The objective of this study was to optimize RNAi in young PoP adults and 3rd instars for screening by oral delivery of dsRNAs. The minimal effective dsRNA concentrations required for robust knockdown and persistence were evaluated by delivering seven concentrations spanning 0.1 ng/µL to 500 ng/µL over post ingestion-access periods (IAP) ranging from 48 h to 12 days. The PoP gene candidates evaluated as targets were vacuolar ATPase subunit A, clathrin heavy chain, and non-fermenting protein 7, which were evaluated for knockdown by qPCR amplification. The minimum and/or the second most effective dsRNA concentration resulting in effective levels of gene knockdown was 100 ng/µL for all three targets. Higher concentrations did not yield further knockdown, indicating potential RISC saturation at the higher doses. Gene silencing post-IAP of 100 ng/µL dsRNA persisted for 3-5 days in adults and nymphs, with the PoP 3rd instar, followed by teneral and mature adults, respectively, exhibiting the most robust RNAi-response.

Characterization of the Asian Citrus Psyllid-'Candidatus Liberibacter Asiaticus' Pathosystem in Saudi Arabia Reveals Two Predominant CLas Lineages and One Asian Citrus Psyllid Vector Haplotype.

In Saudi Arabia (SA), the citrus greening disease is caused by 'Candidatus Liberibacter asiaticus' (CLas) transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. The origin and route(s) of the ACP-CLas pathosystem invasion in SA have not been studied. Adult ACP were collected from citrus trees in SA and differentiated by analysis of the mitochondrial cytochrome oxidase I (mtCOI) and nuclear copper transporting protein (atox1) genes. A phylogenetic analysis of the Wolbachia spp. surface protein (wsp) gene was used to identify the ACP-associated Wolbachia spp. A phylogenetic analysis of the atox1 and mtCOI gene sequences revealed one predominant ACP haplotype most closely related to the Indian subcontinent founder populations. The detection and identification of CLas in citrus trees were carried out by polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA gene. The CLas-integrated prophage genomes were sequenced, annotated, and used to differentiate CLas populations. The ML and ASTRAL trees reconstructed with prophages type 1 and 2 genome sequences, separately and concatenated, resolved two major lineages, CLas-1 and -2. The CLas-1 clade, reported here for the first time, consisted of isolates from SA isolates and Pakistan. The CLas-2 sequences formed two groups, CLas-2-1 and -2-2, previously the 'Asiatic' and 'Floridian' strains, respectively. Members of CLas-2-1 originated from Southeast Asia, the USA, and other worldwide locations, while CLas-2-2 was identified only in Florida. This study provides the first snapshot into the status of the ACP-CLas pathosystem in SA. In addition, the results provide new insights into the pathosystem coevolution and global invasion histories of two ACP-CLas lineages with a predicted center of origin in South and Southeast Asia, respectively.

A Previously Undescribed Polerovirus (Solemoviridae) Infecting Theobroma cacao Germplasm.

Cacao Theobroma cacao L. (Malvaceae) is an economically important crop cultivated in tropical climates for the bean from which chocolate and other products are made (Zarrillo et al., 2018). Virus-like symptoms consisting of discoloration, leaf distortion with downward rolling of leaves, and yellow speckling or mottling (Fig. S1), were observed in imported cacao germplasm at the USDA-ARS-SHRS cacao quarantine facilities in the fall of 2020. Total RNA was isolated from leaves collected from four symptomatic plants using silica RNA extraction method (Rott and Jelkmann, 2001). Ribosomal RNA (rRNA)-depleted RNA samples were used for cDNA library construction, followed by high throughput sequencing on Illumina® NovaSeq 6000 platform (Novogene Corp., Sacramento, CA). Quality-filtered, 150-bp paired-ended reads (2,601,293-3,104,474) were assembled de novo using SPAdes v.3.14.1 (Nurk et al., 2013). The contigs (200,799 to 276,851) were queried against the NCBI virus reference sequence (RefSeq) database using the discontiguous megablast algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi?). The resultant contigs (n=1,344) were 150-nt to 1463-nt in length (k-mer coverage from 6.3x to 26,721.7x) and shared their highest nucleotide (nt) identity with potato leafroll virus (PLRV; NC_001747; genus Polerovirus; family Solemoviridae), at 69.1%-72.8%. The contigs pooled from the four samples were assembled into 15 scaffolds. BLASTn analyses of the 15 scaffolds against the RefSeq database indicated the best matches were to thirteen other polerovirus species, with top hits to cereal yellow dwarf virus-RPV (D10206) and pepper vein yellows virus (LC528383), having similarity scores of 66.2% and 100% respectively. The 15 scaffolds matched to the 5' terminal end, ORF1-2, ORF3, ORF4 and ORF3-5 of the different polerovirus genomes. For confirmatory sequencing, total RNA was subjected to reverse transcription using SuperScript IV (Invitrogen, Carlsbad, CA), followed by RT-PCR amplification with general polerovirus primers PoconF/PoconcpR (Xiang et al., 2008) expected to yield an amplicon of ~1,400-bp located at the 3' end of the RNA-dependent, RNA polymerase (RdRp), including the complete coat protein (CP) and movement protein (MP) genes. Amplicons were ligated to pGEM-T Easy vector (Promega, Madison, WI) and sequenced bi-directionally by Sanger sequencing (Eton Bio, Research Triangle Park, NC). BLASTn analysis of the polerovirus-like nt sequences (GenBank accession nos. (ON745771-ON745774) indicated the closest relatives were potato leafroll virus (OK058524) and cucumber aphid-borne yellows virus (FJ460218), at 71% and 73%, respectively. The CP amino acid (aa) sequence shared the greatest similarity to cereal yellow dwarf virus RPV (NP_840023), at 53%, and the MP aa sequence shared the greatest aa similarity to wheat yellow leaf dwarf virus-GPV (YP_003029842), at 38%. These results provide robust support for the association of a previously undescribed polerovirus with symptomatic cacao trees, herein named, cacao leafroll virus (Solemoviridae; Polerovirus). Although Koch's postulates have not been completed to confirm causality, the presence of this virus in cacao germplasm undermine efforts to distribute pathogen-free germplasm and may pose a risk to cacao production in trees established from virus-infected plant material. To our knowledge, this is the first report of a polerovirus infecting cacao trees. All trees of these accessions at the quarantined facility in Miami, FL have been destroyed.

Differential Transcriptional Responses in Two Old World Bemisia tabaci Cryptic Species Post Acquisition of Old and New World Begomoviruses.

Begomoviruses are transmitted by several cryptic species of the sweetpotato whitefly, Bemisia tabaci (Gennadius), in a persistent and circulative manner. Upon virus acquisition and circulative translocation within the whitefly, a multitude of molecular interactions occur. This study investigated the differentially expressed transcript profiles associated with the acquisition of the Old World monopartite begomovirus, tomato yellow leaf curl virus (TYLCV), and two New World bipartite begomoviruses, sida golden mosaic virus (SiGMV) and cucurbit leaf crumple virus (CuLCrV), in two invasive B. tabaci cryptic species, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED). A total of 881 and 559 genes were differentially expressed in viruliferous MEAM1 and MED whiteflies, respectively, compared with their non-viruliferous counterparts, of which 146 genes were common between the two cryptic species. For both cryptic species, the number of differentially expressed genes (DEGs) associated with TYLCV and SiGMV acquisition were higher compared with DEGs associated with CuLCrV acquisition. Pathway analysis indicated that the acquisition of begomoviruses induced differential changes in pathways associated with metabolism and organismal systems. Contrasting expression patterns of major genes associated with virus infection and immune systems were observed. These genes were generally overexpressed and underexpressed in B. tabaci MEAM1 and MED adults, respectively. Further, no specific expression pattern was observed among genes associated with fitness (egg production, spermatogenesis, and aging) in viruliferous whiteflies. The weighted gene correlation network analysis of viruliferous B. tabaci MEAM1 and MED adults identified different hub genes potentially implicated in the vector competence and circulative tropism of viruses. Taken together, the results indicate that both vector cryptic species and the acquired virus species could differentially affect gene expression.

Comparison of Auxenochlorella protothecoides and Chlorella spp. Chloroplast Genomes: Evidence for Endosymbiosis and Horizontal Virus-like Gene Transfer.

Resequencing of the chloroplast genome (cpDNA) of Auxenochlorella protothecoides UTEX 25 was completed (GenBank Accession no. KC631634.1), revealing a genome size of 84,576 base pairs and 30.8% GC content, consistent with features reported for the previously sequenced A. protothecoides 0710, (GenBank Accession no. KC843975). The A. protothecoides UTEX 25 cpDNA encoded 78 predicted open reading frames, 32 tRNAs, and 4 rRNAs, making it smaller and more compact than the cpDNA genome of C. variabilis (124,579 bp) and C. vulgaris (150,613 bp). By comparison, the compact genome size of A. protothecoides was attributable primarily to a lower intergenic sequence content. The cpDNA coding regions of all known Chlorella species were found to be organized in conserved colinear blocks, with some rearrangements. The Auxenochlorella and Chlorella species genome structure and composition were similar, and of particular interest were genes influencing photosynthetic efficiency, i.e., chlorophyll synthesis and photosystem subunit I and II genes, consistent with other biofuel species of interest. Phylogenetic analysis revealed that Prototheca cutis is the closest known A. protothecoides relative, followed by members of the genus Chlorella. The cpDNA of A. protothecoides encodes 37 genes that are highly homologous to representative cyanobacteria species, including rrn16, rrn23, and psbA, corroborating a well-recognized symbiosis. Several putative coding regions were identified that shared high nucleotide sequence identity with virus-like sequences, suggestive of horizontal gene transfer. Despite these predictions, no corresponding transcripts were obtained by RT-PCR amplification, indicating they are unlikely to be expressed in the extant lineage.