An overview of pathogens associated with biotic stresses in hemp crops in Oregon, 2019-2020

Hemp (Cannabis sativa) acreage in Oregon has increased by approximately 240 times in the last five 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 polymerase chain reaction (PCR), or reverse transcription (RT)-PCR. Diseases with fungal or oomycete, and virus causes accounted for 26.5%, and 42.9% of submissions, respectively; co-infection 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.

Genome analysis of Spiroplasma citri strains from different host plants and its leafhopper vectors

Background Spiroplasma citri comprises a bacterial complex that cause diseases in citrus, horseradish, carrot, sesame, and also infects a wide array of ornamental and weed species. S. citri is transmitted in a persistent propagative manner by the beet leafhopper, Neoaliturus tenellus in North America and Circulifer haematoceps in the Mediterranean region. Leafhopper transmission and the pathogen’s wide host range serve as drivers of genetic diversity. This diversity was examined in silico by comparing the genome sequences of seven S. citri strains from the United States (BR12, CC-2, C5, C189, LB 319, BLH-13, and BLH-MB) collected from different hosts and times with other publicly available spiroplasmas. Results Phylogenetic analysis using 16S rRNA sequences from 39 spiroplasmas obtained from NCBI database showed that S. citri strains, along with S. kunkelii and S. phoeniceum, two other plant pathogenic spiroplasmas, formed a monophyletic group. To refine genetic relationships among S. citri strains, phylogenetic analyses with 863 core orthologous sequences were performed. Strains that clustered together were: CC-2 and C5; C189 and R8-A2; BR12, BLH-MB, BLH-13 and LB 319. Strain GII3–3X remained in a separate branch. Sequence rearrangements were observed among S. citri strains, predominantly in the center of the chromosome. One to nine plasmids were identified in the seven S. citri strains analyzed in this study. Plasmids were most abundant in strains isolated from the beet leafhopper, followed by strains from carrot, Chinese cabbage, horseradish, and citrus, respectively. All these S. citri strains contained one plasmid with high similarity to plasmid pSci6 from S. citri strain GII3–3X which is known to confer insect transmissibility. Additionally, 17 to 25 prophage-like elements were identified in these genomes, which may promote rearrangements and contribute to repetitive regions. Conclusions The genome of seven S. citri strains were found to contain a single circularized chromosome, ranging from 1.58 Mbp to 1.74 Mbp and 1597–2232 protein-coding genes. These strains possessed a plasmid similar to pSci6 from the GII3–3X strain associated with leafhopper transmission. Prophage sequences found in the S. citri genomes may contribute to the extension of its host range. These findings increase our understanding of S. citri genetic diversity.

First Report of Curly Top of Coriandrum sativum Caused by Beet curly top virus in the Columbia Basin of Washington State.

Two fields of coriander (Coriandrum sativum L.) seed crops of proprietary cultivars were observed in the Columbia Basin of Washington in July 2020 with 40 and 90% incidence of plants showing stunting and leaf and stem discoloration, sometimes with mild leaf curl. Foliar discoloration ranged from yellow to red and purple. Sweep-netting along the field edges collected one beet leafhopper (Circulifer tenellus Baker; BLH), the known vector of Beet curly top virus (BCTV), Beet leafhopper transmitted virescence agent (BLTVA) phytoplasma, and Spiroplasma citri, all of which affect Solanaceae and Apiaceae crops in Washington (Crosslin et al. 2006; Johnson and Martin 1998; Lee et al. 2006). Nucleic acids extracted from leaves and petioles of 12 coriander plants (8 from Field 1 and 4 from Field 2) using the Dellaporta method, and from the BLH using the CTAB method (Crosslin et al. 2006) were subjected to PCR assays to detect the BLH-transmitted pathogens which cause yellow and purple discoloration in potato (Solanum tuberosum L.) and carrot (Daucus carota subsp. sativus (Hoffm.) Arc.) in this region. BLTVA was targeted using a species-specific nested PCR assay with primers P1 and P7, followed by primers FU5 and BLTVA-int (Crosslin et al. 2006); S. citri was targeted using primers P89-F and P89-R (Yokomi et al. 2008); and BCTV was targeted using curtovirus primers BCTV2-F and BCTV2-R (Strausbaugh et al. 2008). BLTVA and S. citri were not detected in the plants, but curtovirus was detected in 10 of the 12 plants. All three pathogens were detected from the single BLH. A 519 bp region of the curtovirus capsid protein gene was amplified from seven plants (5 from Field 1 and 2 from Field 2) and the BLH, and cloned into TOP10 Escherichia coli cells using the pCR-2.1 TOPO vector (Invitrogen, Carlsbad, CA). Three clones were sequenced from each sample. For each of six plant samples and the BLH, the three clones were identical and consensus sequences were generated (GenBank Accessions MW234419 to MW234425). For the seventh plant, two clones were identical in sequence (MW234426) and the third contained 12 single nucleotide polymorphisms (MW234427). All sequences were subjected to an NCBI BLASTn analysis and showed 98.3 to 99.8% identity with BCTV sequences. Additional PCR assays with primers BMCTV-C1 2213F and BMCTV-C1 2609R (Strausbaugh et al. 2008), targeting the C1 gene of the Worland strain of BCTV, detected BCTV-Worland-like strains in all plants and the BLH, confirming that BCTV was present and indicating that the strain-specific primer pair was more sensitive than the universal curtovirus primers. Yield losses in the two fields were approximately 60%, with reduced seed size but not seed quality. BCTV infections in coriander crops have been observed in the Columbia Basin in 2002, 2005, 2008, and 2013, with yield losses ranging from 10 to 100% per field, though official reports were not made following the diagnoses (Crosslin, du Toit, and Frost, unpublished data). BCTV has caused millions of dollars of losses in the U.S. in crops such as sugar beet (Beta vulgaris subsp. vulgaris L.), tomato (S. lycopersicum L.), and pepper (S. annuum L.) (Johnson and Martin 1998). This is the first publication of BCTV affecting seed production of the specialty crop C. sativum. The observation of 90% incidence of symptoms in one field suggests that resistant cultivars and/or insect pest management practices are needed to prevent significant impacts of BCTV on coriander seed production in this semi-arid region.

Genome analysis of Spiroplasma citri strains from different host plants and its leafhopper vectors

Background Spiroplasma citri comprises a complex of bacteria that cause diseases in citrus, horseradish, carrot, sesame, and also infect a wide array of ornamental and weed species. S. citri is transmitted in a persistent propagative manner by the beet leafhopper, Neoaliturus tenellus in North America and Circulifer haematoceps in the Mediterranean region. Leafhopper transmission and the pathogen’s wide host range serve as drivers of genetic diversity. This diversity was examined by comparing the genome sequences of seven S. citri strains from the United States (BR12, CC2, C5, C189, LB319, BLH-13, and BLH-MB) collected from different hosts and times with other publicly available spiroplasmas.Results Phylogenetic analysis using 16S rRNA sequences from 39 spiroplasmas obtained from NCBI database showed that S. citri strains, along with S. kunkelii and S. phoeniceum, two other plant pathogenic spiroplasmas, formed a monophyletic group. To refine genetic relationships among S. citri strains, phylogenetic analyses with 863 core orthologous sequences were performed. Strains that clustered together were: CC-12 and C5; C189 and R8-A2; BR12, BLH-MB, BLH13 and LB 319. Strain GII3-3 remained in a separate branch. Sequence rearrangements were observed among S. citri strains, predominantly in the center of the chromosome. One to nine plasmids were identified in the seven S. citri strains analyzed in this study. Plasmids were most abundant in strains isolated from the beet leafhopper, followed by strains from carrot, Chinese cabbage, horseradish and citrus, respectively. All these S. citri strains contained one plasmid with high similarity to plasmid pSci6 from S. citri strain GII3-3X which is known to confer insect transmissibility. Additionally, 17 to 25 prophage-like elements were identified in these genomes, which may promote rearrangements and contribute to repetitive regions. Conclusions The genome of seven S. citri strains were found to contain a single circularized chromosome, ranging from 1.58 Mbp to 1.74 Mbp and 1,597-2,232 protein-coding genes. These strains possessed a plasmid similar to pSci6 from the GII3-3X strain associated with leafhopper transmission. Prophage sequences found in the S. citri genomes may contribute to the extension of its host range. These findings increase our understanding of S. citri genetic diversity.

Genome Analysis of Spiroplasma Citri Strains From Different Host Plants and Its Leafhopper Vectors

Background Spiroplasma citri comprises a complex of bacteria that cause diseases in citrus, horseradish, carrot, sesame, and also infect a wide array of ornamental and weed species. S. citri is transmitted in a persistent propagative manner by the beet leafhopper, Neoaliturus tenellus in North America and Circulifer haematoceps in the Mediterranean region. Leafhopper transmission and the pathogen’s wide host range serve as drivers of genetic diversity. This diversity was examined by comparing the genome sequences of seven S. citri strains from the United States (BR12, CC2, C5, C189, LB319, BLH-13, and BLH-MB) collected from different hosts and times with other publicly available spiroplasmas.Results Phylogenetic analysis using 16S rRNA sequences from 39 spiroplasmas obtained from NCBI database showed that S. citri strains, along with S. kunkelii and S. phoeniceum, two other plant pathogenic spiroplasmas, formed a monophyletic group. To refine genetic relationships among S. citri strains, phylogenetic analyses with 863 core orthologous sequences were performed. Strains that clustered together were: CC-12 and C5; C189 and R8-A2; BR12, BLH-MB, BLH13 and LB 319. Strain GII3-3 remained in a separate branch. Sequence rearrangements were observed among S. citri strains, predominantly in the center of the chromosome. One to nine plasmids were identified in the seven S. citri strains analyzed in this study. Plasmids were most abundant in strains isolated from the beet leafhopper, followed by strains from carrot, Chinese cabbage, horseradish and citrus, respectively. All these S. citri strains contained one plasmid with high similarity to plasmid pSci6 from S. citri strain GII3-3X which is known to confer insect transmissibility. Additionally, 17 to 25 prophage-like elements were identified in these genomes, which may promote rearrangements and contribute to repetitive regions. Conclusions The genome of seven S. citri strains were found to contain a single circularized chromosome, ranging from 1.58 Mbp to 1.74 Mbp and 1,597-2,232 protein-coding genes. These strains possessed a plasmid similar to pSci6 from the GII3-3X strain associated with leafhopper transmission. Prophage sequences found in the S. citri genomes may contribute to the extension of its host range. These findings increase our understanding of S. citri genetic diversity.

Prediction of Early Season Beet Leafhopper Populations in Southern New Mexico

Curly top is an important widespread disease in semiarid regions that can be caused by several Curtovirus and Becurtovirus species. The strains of beet curly top virus (BCTV) have been some of the most widely reported to be associated with curly top. The viruses causing curly top are phloem limited and transmitted by the beet leafhopper (BLH), Circulifer tenellus Baker (Hemiptera: Cicadellidae). The BLH can also transmit other important pathogens such as phytoplasmas. Both the virus and insect vector have a broad host range of crops and weeds, including the winter annual weed London rocket (Sisymbrium irio L.). Prior prediction of disease would allow growers a window of opportunity to make informed management choices. A prediction model of BLH abundance was developed for southern New Mexico based on fall precipitation, which corresponds with London rocket emergence, and BLH sticky trap catch data for 2001 to 2018. Regression analyses showed positive associations between BLH numbers and October + November rainfall (P < 0.001) for two areas within southern New Mexico. A third area, where good weed management was used, had lower BLH numbers, and the relationship with precipitation was not significant (P = 0.190). Cumulative-season BLH abundance was correlated with BLH abundance in late April (r = 0.43) and late May (r = 0.56), indicating that early season knowledge of BLH abundance is useful for planning later season management. Although models based on October + November precipitation are good predictors of BLH abundance through June, they may not predict year-long BLH abundance because other environmental and biological factors contribute to subsequent BLH success and movement.

Detection of Pathogens Associated with Psyllids and Leafhoppers in Capsicum annuum L. in the Mexican States of Durango, Zacatecas, and Michoacán

In fall 2014, 5 to 75% percent of chili and bell pepper (Capsicum annuum L.) in commercial fields located in the Mexican states of Durango, Zacatecas, and Michoacán had symptoms of deformed, small, mosaic, curled, and chlorotic leaves; shortened internodes; plant dwarfing; or phyllody and rosetting leaf tips. At the same time, leafhoppers and psyllids were observed in the fields, and more than 50 beet leafhoppers (Circulifer tenellus) and nearly 300 potato psyllids (Bactericera cockerelli) were collected from the pepper plants and adjacent weeds. Based on the insect pressure and observed symptoms, nearly 400 pepper samples were collected across this region of Mexico and tested for the presence of leafhopper- and psyllid-associated pathogens. In all, 76% of the pepper samples were found to be infected with ‘Candidatus Liberibacter solanacearum’, beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma, a strain of a curtovirus, or a combination of any two or three of these pathogens. Additionally, 77% of the collected leafhoppers and 40% of the psyllids were infected with one or more of these pathogens, in addition to Spiroplasma citri. Specifically, the leafhoppers were infected with BLTVA phytoplasma, S. citri, or a strain of curtovirus. Of particular interest, potato psyllids were not only infected with ‘Ca. L. solanacearum’ but also with phytoplasmas that belong to the groups 16SrVI subgroup A and 16SrI subgroup A. The presence of mixed infections in pepper plants and the insect vectors highlights the need for growers to effectively control both leafhoppers and potato psyllids from solanaceous crops in this region of Mexico in order to prevent the spread of these bacterial and viral pathogens.

Beet curly top virus Strains Associated with Sugar Beet in Idaho, Oregon, and a Western U.S. Collection

Curly top of sugar beet is a serious, yield-limiting disease in semiarid production areas caused by Beet curly top virus (BCTV) and transmitted by the beet leafhopper. One of the primary means of control for BCTV in sugar beet is host resistance but effectiveness of resistance can vary among BCTV strains. Strain prevalence among BCTV populations was last investigated in Idaho and Oregon during a 2006-to-2007 collection but changes in disease severity suggested a need for reevaluation. Therefore, 406 leaf samples symptomatic for curly top were collected from sugar beet plants in commercial sugar beet fields in Idaho and Oregon from 2012 to 2015. DNA was isolated and BCTV strain composition was investigated based on polymerase chain reaction assays with strain-specific primers for the Severe (Svr) and California/Logan (CA/Logan) strains and primers that amplified a group of Worland (Wor)-like strains. The BCTV strain distribution averaged 2% Svr, 30% CA/Logan, and 87% Wor-like (16% had mixed infections), which differed from the previously published 2006-to-2007 collection (87% Svr, 7% CA/Logan, and 60% Wor-like; 59% mixed infections) based on a contingency test (P < 0.0001). Whole-genome sequencing (GenBank accessions KT276895 to KT276920 and KX867015 to KX867057) with overlapping primers found that the Wor-like strains included Wor, Colorado and a previously undescribed strain designated Kimberly1. Results confirm a shift from Svr being one of the dominant BCTV strains in commercial sugar beet fields in 2006 to 2007 to becoming undetectable at times during recent years.