Beet Curly Top Virus Research Articles

A robust SNP-haplotype assay for Bct gene region conferring resistance to beet curly top virus in common bean (Phaseolus vulgaris L.).

Beet curly top virus (BCTV), which is synonymous with curly top virus (CTV), causes significant yield loss in common bean (snap and dry beans) cultivars and several other important crops. Common bean cultivars have been found to be resistant to CTV, but screening for resistance is challenging due to the cyclical nature of epidemics and spotty feeding by the leafhopper that vectors the virus. We used an SNP dataset for the Snap Bean Association Panel (SnAP) agro-inoculated with CTV-Logan (CA/Logan) strain to locate the Bct gene region to a 1.7-Mb interval on chromosome Pv07 using genome-wide association study (GWAS) analysis. Recombinant lines from the SnAP were used to further narrow the Bct region to a 58.0-kb interval. A missense SNP (S07_2970381) in candidate gene Phvul.007G036300 Exonuclease V (EXO5) was identified as the most likely causal mutation, and it was the most significant SNP detected by GWAS in a dry bean population (DBP) naturally infected by the CTV-Worland (Wor) strain. Tm-shift assay markers developed for SNP S07_2970381 and two linked SNPs, S07_2970276 and S07_2966197, were useful for tracking different origins of the Bct EXO5 candidate gene resistance to CTV in common bean. The three SNPs identified four haplotypes, with haplotype 3-1 (Haplo3-1) of Middle American origin associated with the highest levels of CTV resistance. This SNP-haplotype assay will enable breeders to track resistance sources and to develop cultivars with better CTV resistance.

New Assays for Rapid Detection of Beet Leafhopper-Associated Plant Pathogens, 'Candidatus Phytoplasma trifolii', Beet Curly Top Virus, and Spiroplasma citri.

The beet leafhopper Circulifer tenellus is an important pest of agricultural crops in the United States, where it transmits beet curly top virus, beet leafhopper-transmitted virescence agent phytoplasma, and Spiroplasma citri to numerous crops, affecting yield and quality. Each of these pathogens have been linked to serious disease outbreaks within Washington State in the past century. To mitigate the risk of disease, growers target the beet leafhopper in their insect pest management programs. Knowledge of pathogen prevalence in beet leafhopper populations could help growers make better management decisions, but timely diagnostics is required. Four new assays were developed for the rapid detection of the beet leafhopper-associated pathogens. These include two assays that detect Beet leafhopper transmitted virescence agent (a PCR and a real-time PCR SYBR green assay), a duplex PCR assay that simultaneously detects beet curly top virus and Spiroplasma citri, and a multiplex real-time PCR assay for the simultaneous detection of all three pathogens. The screening of dilution series generated from plant total nucleic acid extracts with these new assays typically led to detection at levels 10- to 100-fold more sensitive than the conventional PCR assays currently used. These new tools will allow the rapid detection of beet leafhopper-associated pathogens in both plant and insect specimens and will have the potential to be used in diagnostic laboratories seeking to disseminate fast and accurate results to growers for implementation in their insect pest monitoring programs.

The Desert Whale: the boom and bust of hemp in Arizona

BackgroundThis paper examines the factors that led to the collapse of hemp grown for cannabidiol (CBD) in Arizona, the United States of America (USA), and particularly in Yuma County, which is a well-established agricultural area in the state.MethodsThis research uses a combination of mapping analysis along with a survey of hemp farmers to assess the reasons why the hemp industry collapsed as well as to foster solutions to these problems.ResultsIn 2019, 5430 acres were sown with hemp seed in Arizona with 3890 acres inspected by the state to determine if they could be harvested. By 2021, there were only 156 acres planted, and only 128 of those acres were inspected by the state for compliance. (Crop mortality accounts for the difference between acres sown and acres inspected.)ConclusionsA lack of knowledge about the hemp life cycle greatly contributed to the failure of high CBD hemp crops in Arizona. Other problems included noncompliance with tetrahydrocannabinol limits, poor sources for seeds and inconsistent genetics of the hemp varieties sold to farmers, and diseases that hemp plants were susceptible to such as Pythium crown and root rot and beet curly top virus. Addressing these factors will go far in making hemp a profitable and widespread crop in Arizona. Additionally, hemp grown for other traditional uses (e.g., fiber or seed oil) as well as new applications (e.g., microgreens, hempcrete, and phytoremediation) offers other pathways for successful hemp agriculture in this state.

Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System.

Beet Curly Top Iran Virus (BCTIV, Becurtovirus) is a dominant and widespread pathogen responsible for great damage and yield reduction in sugar beet production in the Mediterranean and Middle East. CRISPR-based gene editing is a versatile tool that has been successfully used in plants to improve resistance against many viral pathogens. In this study, the efficiency of gRNA/Cas9 constructs targeting the expressed genes of BCTIV was assessed in sugar beet leaves by their transient expression. Almost all positive control sugar beets revealed systemic infection and severe disease symptoms (90%), with a great biomass reduction (68%) after BCTIV agroinoculation. On the other hand, sugar beets co-agronioculated with BCTIV and gRNA/Cas9 indicated much lower systemic infection (10-55%), disease symptoms and biomass reduction (13-45%). Viral inactivation was also verified by RCA and qPCR assays for gRNA/Cas9 treated sugar beets. PCR-RE digestion and sequencing assays confirmed the gRNA/Cas9-mediated INDEL mutations at the target sites of the BCTIV genome and represented high efficiencies (53-88%), especially for those targeting BCTIV's movement gene and its overlapping region between capsid and ssDNA regulator genes. A multiplex CRISPR approach was also tested. The most effective four gRNAs targeting all the genes of BCTIV were cloned into a Cas9-containing vector and agroinoculated into virus-infected sugar beet leaves. The results of this multiplex CRISPR system revealed almost complete viral resistance with inhibition of systemic infection and mutant escape. This is the first report of CRSIPR-mediated broad-spectrum resistance against Becurtovirus in sugar beet.

Pest survey card on Beet curly top virus

This document provides the conclusions of the pest survey card that was prepared in the context of the EFSA mandate on plant pest surveillance (M-2020-0114) at the request of the European Commission. The full pest survey card for Beet curly top virus (BCTV) is published and available online in the EFSA Plant Pest Survey Cards Gallery at the following link and will be updated whenever new information becomes available: https://efsa.europa.eu/plants/planthealth/monitoring/surveillance/beet-curly-top-virus

Functional Analysis of V2 Protein of Beet Curly Top Iran Virus.

Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

Curly Top Disease of Hemp (Cannabis sativa) in California Is Caused by Mild-Type Strains of Beet curly top virus Often in Mixed Infection.

Interest in industrial hemp (Cannabis sativa) as a potential crop led to the establishment of commercial fields in a number of counties in California in 2019 and 2020. Plants in these fields developed different types of virus-like symptoms. The most prevalent type was stunted and bushy plants with distorted, upcurled, and yellowed leaves, which were similar to those associated with curly top disease (CTD) caused by the beet curly top virus (BCTV). This beet leafhopper-vectored virus is endemic in California and can cause economic losses to processing tomato production. Using a multiplex PCR test, BCTV infection was detected in 89% of hemp samples with CTD-like symptoms from Fresno, San Bernardino, and Ventura counties. Other symptom types had low incidence of BCTV infection and were associated with other factors. Hemp plants in California were infected only with the mild-type strains, BCTV-CO and BCTV-Wor, and often in mixed infection (43% of samples). Finally, using an infectious clone of a BCTV-CO isolate from hemp, we demonstrated that agroinoculated hemp plants developed these CTD-like symptoms, thereby fulfilling Koch's postulates for the disease.

Ty-5 Confers Broad-Spectrum Resistance to Geminiviruses.

The selection of resistant crops is an effective method for controlling geminivirus diseases. ty-5 encodes a messenger RNA surveillance factor Pelota with a single amino acid mutation (PelotaV16G), which confers effective resistance to tomato yellow leaf curl virus (TYLCV). No studies have investigated whether ty-5 confers resistance to other geminiviruses. Here, we demonstrate that the tomato ty-5 line exhibits effective resistance to various geminiviruses. It confers resistance to two representative begomoviruses, tomato yellow leaf curl China virus/tomato yellow leaf curl China betasatellite complex and tomato leaf curl Yunnan virus. The ty-5 line also exhibits partial resistance to a curtovirus beet curly top virus. Importantly, ty-5 confers resistance to TYLCV with a betasatellite. Southern blotting and quantitative polymerase chain reaction analyses showed that significantly less DNA of these geminiviruses accumulated in the ty-5 line than in the susceptible line. Moreover, knockdown of Pelota expression converted a Nicotiana benthamiana plant from a geminivirus-susceptible host to a geminivirus-resistant host. Overall, our findings suggest that ty-5 is an important resistance gene resource for crop breeding to control geminiviruses.

The Rep and C1 of Beet curly top Iran virus represent pathogenicity factors and induce hypersensitive response in Nicotiana benthamiana plants

Beet curly top Iran virus (BCTIV) is a member of the genus Becurtovirus (Family Geminiviridae) with a circular single-strand DNA genome. BCTIV causes leaf curling and vein swelling symptoms in plants. However, the potential pathogenicity factor/s in BCTIV is/are not known. This study presents characterization of complementary-sense transcripts of BCTIV and the viral factors in directing the pathogenicity and hypersensitive response (HR) in Nicotiana benthamiana plants. In both local and systemic infection, splicing of the complementary transcripts of BCTIV was observed. Notably, a small number (8.3%) of transcripts were spliced to produce Rep (C1:C2) transcripts after deletion of 155 nt (position 1892-2046 from BCTIV). Expression of BCTIV genes in N. benthamiana using tobacco rattle virus (TRV)-based vector showed that Rep together with C1 are the main pathogenicity factors which cause typical viral leaf curling symptoms. In addition, the V2 caused a mild leaf curling, thickening, and asymmetric leaves, while the V1, V3, and C2 had no clear effect on the plant phenotype. Transient expression of individual viral genes showed that both the C1 and Rep trigger a HR response in N. benthamiana. The higher expression of HR marker genes, harpin-induced 1 (Hin1) and hypersensitivity-related (Hsr203JI), supported the role of C1 and Rep in HR response in plants. It is concluded that Rep and C1 are the main pathogenicity factors that also trigger HR response in plants.

Leaf Bacteriome in Sugar Beet Shows Differential Response against Beet curly top virus during Resistant and Susceptible Interactions

Beet curly top virus (BCTV) significantly reduces sugar beet yield in semi-arid production areas. Genetic resistance to BCTV is limited; therefore, identification of additional resistance-associated factors is highly desired. Using 16S rRNA sequencing and BCTV resistant (R) genotypes (KDH13, KDH4-9) along with a susceptible (S) genotype (KDH19-17), we investigated leaf bacteriome changes during BCTV post inoculation (pi). At day 6 (~6-week-old plants), Cyanobacteria were predominant (~90%); whereas, at week 4 (~10-week-old plants) Firmicutes (11–66%), Bacteroidetes (17–26%), and Verrucomicrobia (12–29%) were predominant phyla and genotype dependent. Both Bacteroidetes and Verrucomicrobia, increased post infection only in the R lines. The bacterial genera Brevibacillus increased at 6 dpi, and Akkermansia and Bacteroides at 4 wkpi in the R lines. Linear discriminant analysis effect size (LEfSe) identified potential biomarkers in the R vs. S lines. Functional profiling revealed bacterial enrichment associated with the TCA cycle, polyisoprenoid, and L-methionine biosynthesis pathways only in KDH4-9 at 6 dpi. At 4 wkpi, bacteria associated with tryptophan and palmitate biosynthesis in the R lines, and uridine monophosphate, phosphatidyl glycerol, and phospholipid biosynthesis in the S line, were enriched. Future characterization of bacterial genera with antiviral properties will help establish their use as biocontrol agents/biomarkers against BCTV.

Insect vector manipulation by a plant virus and simulation modeling of its potential impact on crop infection

There is widespread evidence of plant viruses manipulating behavior of their insect vectors as a strategy to maximize infection of plants. Often, plant viruses and their insect vectors have multiple potential host plant species, and these may not overlap entirely. Moreover, insect vectors may not prefer plant species to which plant viruses are well-adapted. In such cases, can plant viruses manipulate their insect vectors to preferentially feed and oviposit on plant species, which are suitable for viral propagation but less suitable for themselves? To address this question, we conducted dual- and no-choice feeding studies (number and duration of probing events) and oviposition studies with non-viruliferous and viruliferous [carrying beet curly top virus (BCTV)] beet leafhoppers [Circulifer tenellus (Baker)] on three plant species: barley (Hordeum vulgare L.), ribwort plantain (Plantago lanceolata L.), and tomato (Solanum lycopersicum L.). Barley is not a host of BCTV, whereas ribwort plantain and tomato are susceptible to BCTV infection and develop a symptomless infection and severe curly top symptoms, respectively. Ribwort plantain plants can be used to maintain beet leafhopper colonies for multiple generations (suitable), whereas tomato plants cannot be used to maintain beet leafhopper colonies (unsuitable). Based on dual- and no-choice experiments, we demonstrated that BCTV appears to manipulate probing preference and behavior by beet leafhoppers, whereas there was no significant difference in oviposition preference. Simulation modeling predicted that BCTV infection rates would to be higher in tomato fields with barley compared with ribwort plantain as a trap crop. Simulation model results supported the hypothesis that manipulation of probing preference and behavior may increase BCTV infection in tomato fields. Results presented were based on the BCTV-beet leafhopper pathosystem, but the approach taken (combination of experimental studies with complementary simulation modeling) is widely applicable and relevant to other insect-vectored plant pathogen systems involving multiple plant species.

Regulatory Roles of Small Non-coding RNAs in Sugar Beet Resistance Against Beet curly top virus

Beet curly top virus (BCTV) mediated yield loss in sugar beets is a major problem worldwide. The circular single-stranded DNA virus is transmitted by the beet leafhopper. Genetic sources of BCTV resistance in sugar beet are limited and commercial cultivars rely on chemical treatments versus durable genetic resistance. Phenotypic selection and double haploid production have resulted in sugar beet germplasm (KDH13; 13 and KDH4-9; 4) that are highly resistant to BCTV. The molecular mechanism of resistance to the virus is unknown, especially the role of small non-coding RNAs (sncRNAs) during early plant–viral interaction. Using the resistant lines along with a susceptible line (KDH19-17; 19), we demonstrate the role of sugar beet microRNAs (miRNAs) in BCTV resistance during early infection stages when symptoms are not yet visible. The differentially expressed miRNAs altered the expression of their corresponding target genes such as pyruvate dehydrogenase (EL10Ac1g02046), carboxylesterase (EL10Ac1g01087), serine/threonine protein phosphatase (EL10Ac1g01374), and leucine-rich repeats (LRR) receptor-like (EL10Ac7g17778), that were highly expressed in the resistant lines versus susceptible lines. Pathway enrichment analysis of the miRNA target genes showed an enrichment of genes involved in glycolysis/gluconeogenesis, galactose metabolism, starch, and sucrose metabolism to name a few. Carbohydrate analysis revealed altered glucose, galactose, fructose, and sucrose concentrations in the infected leaves of resistant versus susceptible lines. We also demonstrate differential regulation of BCTV derived sncRNAs in the resistant versus susceptible lines that target sugar beet genes such as LRR (EL10Ac1g01206), 7-deoxyloganetic acid glucosyltransferase (EL10Ac5g12605), and transmembrane emp24 domain containing (EL10Ac6g14074) and altered their expression. In response to viral infection, we found that plant derived miRNAs targeted BCTV capsid protein/replication related genes and showed differences in expression among resistant and susceptible lines. The data presented here demonstrate the contribution of miRNA mediated regulation of metabolic pathways and cross-kingdom RNA interference (RNAi) in sugar beet BCTV resistance.

Beet curly top virus (curly top)

Beet curly top virus (curly top)

Beet curly top Iran virus

This datasheet on Beet curly top Iran virus covers Identity, Hosts/Species Affected, Further Information.

Screening of Resistant Genotypes of Sugar Beet to Sugar Beet Cyst Nematode and Beet Curly Top Virus and Underlying Biochemical Mechanisms Involved in Their Resistance

Screening of Resistant Genotypes of Sugar Beet to Sugar Beet Cyst Nematode and Beet Curly Top Virus and Underlying Biochemical Mechanisms Involved in Their Resistance

First Insights Into the Virus and Viroid Communities in Hemp (Cannabis sativa)

Hemp (Cannabis sativa L.) production has increased significantly in recent years; however, the crop has been understudied in the U.S. since its production declined in the late 1950s. Disease identification and management is an increasing challenge for hemp growers across the country. In 2019, beet curly top virus (BCTV) was first reported in hemp in Colorado. Hence, we were motivated to understand the diversity and prevalence of BCTV strains infecting hemp in Colorado. We detected BCTV at high incidence rate (81%) in leaf samples from 12 counties. Two different strains of BCTV, Worland (Wor) and Colorado (CO) were present as a single or mixed infection in hemp leaf samples. Phylogenetic analysis revealed BCTV sequences from hemp formed a distinct group along with BCTV strains CO and Wor. To determine other potential viral and viroid pathogens in hemp, we performed next generation sequencing (NGS). Virome analysis revealed the presence of both virus and viroid sequences that had high nucleotide sequence identity with GenBank accessions for cannabis cryptic virus, cannabis sativa mitovirus, citrus yellow vein associated virus, opuntia-like virus and hop latent viroid. In contrast, tobacco streak virus sequences were highly variable compared to sequences in GenBank suggesting a possible new genotype of this virus. The data presented here has important implications for the epidemiology and management of the various diseases of hemp and will lead to the development of integrated pest management strategies designed to interrupt transmission cycles and facilitate efficient crop production.

An Overview of Pathogens Associated with Biotic Stresses in Hemp Crops in Oregon, 2019 to 2020.

Hemp (Cannabis sativa) acreage in Oregon has increased by approximately 240 times in the last 5 years, and a greater number of hemp diseases have been observed. This special report documents pathogens, particularly those causing virus and virus-like diseases, that have been detected from field and greenhouse-grown hemp crops in Oregon, based on plant samples submitted to the Hermiston Agricultural Research and Extension Center Plant Clinic of Oregon State University in 2019 and 2020. Symptoms and signs were used to evaluate disease types and determine diagnostic assays used on each submission. Plants with signs or symptoms of fungal or oomycete infection were cultured to isolate pathogenic organisms and plants with symptoms suspected to be caused by virus infection were assayed for the presence of Beet curly top virus (BCTV), viroids, and phytoplasmas using PCR, or reverse transcription (RT)-PCR. Diseases with fungal or oomycete, and virus causes accounted for 26.5 and 42.9% of submissions, respectively; coinfection of viral and fungal or oomycete pathogens were detected from 6.1% of submissions between 2019 and 2020. BCTV, a curtovirus, and hop latent viroid (HLVd) were the predominant pathogens detected from field and indoor grown hemp. Worland-like strains of BCTV represented 93% of all curtovirus detections. Eighty percent of HLVd detections occurred from plants that originated from indoor growing facilities. Based on BCTV vector, beet leafhopper, prevalence, field-grown hemp in western production regions may be affected by curly top and increasing hemp acreage in the landscape may have potential implications on other crops affected by curtoviruses. Virus and virus-like diseases could be a limiting factor for hemp production in some regions of the United States.

Targeted mutagenesis in plants using Beet curly top virus for efficient delivery of CRISPR/Cas12a components

Genome editing using CRISPR/Cas is rapidly being developed for gene targeting in eukaryotes including plants. However, gene targeting by homology-directed DNA recombination (HDR) is an infrequent event compared to the dominant DNA repair by non-homologous end-joining. Another bottleneck is the ineffective delivery of CRISPR/Cas components into plant cells. To overcome these constraints, here a geminiviral replicon from Beet curly top virus (BCTV) has been produced with a wide host range and high DNA accumulation capacity for efficient delivery of CRISPR/Cas12a components into plant cells. Initially, a BCTV replicon was prepared after removing the virion sense genes from an infectious full-length clone for agrobacterium mediated infection. This replicon expressed a green fluorescent protein (GFP) marker gene at a high level compared to T-DNA binary vector. In transient assay, the BCTV replicon produced a higher rate of mutagenesis and HDR in the GFP transgene in Nicotiana benthamiana through efficient delivery of CRISPR/Cas12a components compared to the cognate T-DNA control. This was through a range of complete or partial HDR for conversion of GFP into YFP after exchange of a single amino acid (Thr224Tyr) in the target gene. In addition, induced mutagenesis and HDR in the target gene were heritable. Thus, the BCTV replicon provides a new tool for efficient delivery of CRISPR/Cas12a components that could be used in a wide range of dicotyledonous plants. The established GFP to YFP system and the GFP mutant line produced also enable further optimization and understanding of HDR in plants via CRISPR/Cas12a system using geminiviral replicons.

Genome-based identification of beet curly top Iran virus infecting sugar beet in Turkey and investigation of its pathogenicity by agroinfection

Beet curly top disease (BCTD) is a yield-limiting viral infection of sugar beet (Beta vulgaris) throughout the arid and semi-arid regions of the world. Two virus species, belonging to two different genera of the family Geminiviridae (Curtovirus and Becurtovirus) had been described as the disease’s causative agents on sugar beet. Despite the detection of the BCTD in some sugar beet fields of Turkey sixty years ago, the genome based characterization of BCTD-associated viruses have not been studied previously. In this study, 628 sugar beet plants exhibiting BCTD symptoms were collected from fourteen cities in central Anatolia, the major sugar beet production areas in Turkey. PCR assays of these samples using the respective Curtovirus and Becurtovirus genus-specific primers indicated that the Turkish sugar beet samples' viral sequences belong only to the genus Becurtovirus. The results of sequencing and phylogenetic analysis of the partial genome of the virus obtained from fourteen cities confirmed that BCTD-associated virus in Turkish sugar beet fields is beet curly top Iran virus (BCTIV-Becurtovirus) species. The whole genome of the collected viruses from fourteen cities were amplified by the rolling circle amplification (RCA) and the five most phylogenetically diverse viruses obtained from Afyon, Ankara, Adapazarı, Yozgat and Aksaray were sequenced. The results of whole genome sequence analysis indicated >98 % sequence identities with that of a BCTIV variants reported from Urmia province (bordering Turkey) of Iran. A virus genome from Yozgat city had a genomic sequence identity of >97 % with those of BCTIV isolated from cowpea, tomato, pepper and sugar beet in the northern part of Iran. These results suggested that the spread of BCTIV through the region could create a significant threat to the production of sugar beet as well as other agricultural crops. A tandem dimer of a BCTIV-Turkish variant isolated from Ankara city was cloned into Agrobacterium plasmid to be used for agro-infection studies. Agroinoculation of this construct on sugar beet leaves generated severe BCTD symptoms (84 %) which were also confirmed by RCA and qPCR analysis. These results constituted the first genome based characterization of BCTIV Turkish variants and the first report of BCTIV spreading out of Iran.

First Report of 'Candidatus Phytoplasma trifolii' Related Strain Associated with Yellowing and Witches'-Broom of Industrial Hemp (Cannabis sativa) in Arizona.

In Arizona, industrial hemp (Cannabis sativa) is a newly cultivated crop for fiber, oil, cosmetic products, and health food. During July to September 2020, two fields of industrial hemp crops were identified in southern Arizona with 10 to 30% incidence of plants showing witches' broom. Disease incidence was assessed by counting symptomatic plants in 4 randomly selected rows of 25 plants in each field. Symptoms ranged from leaf mottling and yellowing on mildly affected plants to leaf curling and shortened internode length of stem on severely affected plants (Fig. 1). Shoots were randomly collected from eight symptomatic plants and three asymptomatic plants in the same area. Genomic DNA was extracted from 200 mg of each sample using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. Phytoplasma was tested by a real-time PCR assay and TaqMan probe targeting the 23S ribosomal RNA gene that detects a wide range of known Phytoplasmas (Hodgetts et al., 2009). Beet curly top virus (BCTV) was targeted using BCTV-specific primers BCTV1 and BCTV2 following a method by Rondon (Rondon et al., 2016). BCTV was not detected in the plants, but Phytoplasmas were detected in all eight symptomatic plants, but not in the three control plants. The positive DNA samples were used to identify the phytoplasma by nested PCR using universal phytoplasma-specific primer pairs P1/P6 (Deng, S. et al. 1991) and R16F2n/R16R2 (Gundersen et al., 1996) targeting the 16S rRNA gene and the resulting 1.25 kb fragment in 4 positive samples was subjected to Sanger sequencing (Eton Bioscience, San Diego). All 4 sequences were identical and deposited in GenBank under accession MW981356. BLASTn results indicated 100% identity with that of several 'Candidatus Phytoplasma trifolii' strains on potato (KR072666, KF178706) in Washington and chile peppers (HQ436488) in New Mexico. It also shared 99.84% identity with the sequence of the reference strain of Candidatus Phytoplasma trifolii' (AY390261) that caused clover proliferation. The phytoplasma AZH1 was classified as a member of subgroup A within group16SrVI using iPhyClassifier, an interactive online tool for phytoplasma classification and taxonomic assignment (Zhao et al., 2013). Phylogenetic analysis revealed that the phytoplasma AZH1 clustered with other isolates of 'Candidatus Phytoplasma trifolii' (Fig. 2), including the strain NV1 associated with witches' broom on C. sativa in Nevada (Feng et al. 2019). This is the first report of 'Candidatus Phytoplasma trifolii' related strain associated with yellowing and witches' broom on hemp in Arizona. This finding is significant as the observation of symptoms at 30% incidence in one field suggested that the identified pathogen may pose a significant threat to the production of industrial hemp production in Arizona.