First Report of Bean Leafroll Virus in Pea and Chickpea, in Canada.

Bean leafroll virus (BLRV; Bean leafroll virus), a single-stranded RNA virus in the genus Luteovirus, is phloem-limited and primarily transmitted by aphids in a non-propagative, persistent manner (Rashed et al., 2018; Kidanemariam and Abraham, 2023). BLRV infects various legumes and has been reported from major pulse-growing regions worldwide (Agindotan et al., 2019) but not in the Canadian Prairies. Its impact on crop yield varies with plant and virus genotypes and the timing of infection. Some pea fields have experienced disease rates of up to 80% (Clement et al., 2020; Hampton, 1983). Throughout the 2022 growing season (June and July), pulse fields from across Saskatchewan were randomly selected and surveyed, and symptomatic plants demonstrating leaf yellowing and chlorosis were collected and stored at -80°C before processing. Observed symptoms included necrotic spots, chlorosis, leaf mottling, leaf rolling in peas, severe bright yellowing, and leaf marginal necrosis in chickpeas. BLRV detection was performed on 35 leaves of the collected samples using both Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse transcription polymerase chain reaction (RT-PCR). ELISA testing followed the manufacturer's protocol using a commercial kit (Nano Diagnostics, San Jose, CA, USA). Total RNAs were extracted from the frozen samples using TRIzol (Invitrogen, Carlsbad, CA, USA). For the detection of the diverse BLRV isolates, sequences of various isolates were aligned and primers were specifically designed in-house, targeting the virus's highly conserved regions on the GP3 and 3' UTR (see Supplementary material). Additional primers were also designed targeting coat protein (CP) coding regions which were previously used for BLRV detection (Agindotan et al. 2019; Larsen & Webster 1999). PCR testing of 35 symptomatic samples including 12 pea plants and 23 chickpea plants, identified the presence of BLRV in two symptomatic samples, one each from a field pea (Pisum sativum L. var. CDC Inca) and a desi-type chickpea (Cicer arietinum L. var. CDC Leader). The infected pea and chickpea samples were found in Saskatoon, SK (Coordinates: 52°9'27''N,106°34'14"W), and the Leader area, southwest of Saskatchewan, SK (Coordinates: 50°52'14"N,109°23'11"W), respectively. PCR amplicons were purified and sent for Sanger sequencing. The reads were assembled to generate 1666 and 323 nucleotides from pea and chickpea, respectively, with a minimum of 2X coverage. Partial nucleotide sequences of the BLRV isolates obtained from pea (PsSK1) and chickpea (CaSK1) (GenBank accession numbers: PP240429, PP266588) showed (1521/1574 bp) 96.63% and (316/323 bp) 97.83% similarity with a BLRV reference isolate sequence (NC_003369) and to an isolate from Argentina (KR261610) which was reported on Medicago sativa L. with (1555/1574 bp) 98.79% and (319/323 bp) 98.76% similarity, correspondingly. Both infected samples were confirmed to be BLRV-infected through the ELISA and exhibited a high interaction ratio (PsSK1: 0.319 and CsSK1: 0.245) compared to a positive control (0.292) after 30 minutes as measured at 450 nm. This is the first report of BLRV in the pulse-growing region of the Canadian Prairies. In Saskatchewan, there is no history of BLRV despite the large amount of area growing susceptible crops. Therefore, the survey project that this study was part of was not intended to evaluate the severity of BLRV but rather to determine if there is any virus present that might have been overlooked. The samples were therefore taken randomly, with a focus on the number of fields and geographic coverage rather than focusing on multiple plants per field. Moreover, fields were not chosen based on symptoms but rather at random. Although, plants within fields were chosen because they displayed symptoms. Typically, a disease note includes estimates of severity and potential risk; however, that is not possible for this study. Rather, the fact that it was detected indicates a greater risk than previously perceived, since it was assumed that BLRV was not present. These findings highlight the need for further research on the virus's current status, its impact on crop production, and the resistance of pulse varieties grown in Saskatchewan.

Detection and Identification of a 'Candidatus Liberibacter solanacearum' Species from Ash Tree Infesting Psyllids.

'Candidatus Liberibacter' species are associated with severe, economically important diseases. Nearly all known species are putatively insect transmitted, specifically by psyllids. Detection of 'Ca. Liberibacter' in plants is complicated by their uneven distribution in host plants and largely fastidius nature. The death of black (Fraxinus nigra) and mancana (Fraxinus mandshurica) ash trees in Saskatchewan, Canada has been associated with infestation by the cottony ash psyllid (Psyllopsis discrepans). A combination of conventional PCR amplification and Sanger sequencing of the 16S recombinant DNA was used to detect and identify 'Ca. Liberibacter' in psyllids collected from ash trees in Saskatchewan. BLAST analysis of two 16S sequences that were 1,058 and 1,085 bp long (NTHA 5, GenBank accession number MK942379 and NTHA 6, GenBank accession number MK937570, respectively) revealed they were 99 to 100% similar to a 'Ca. Liberibacter solanacearum' sequence (GenBank accession number KX197200) isolated from the Nearctic psyllid (Bactericera maculipennis) of U.S. provenance. Sequencing the psyllid genes CO1 and Cyt-b confirmed that the psyllids from which the bacterial DNA was isolated were P. discrepans, based on comparisons with sequences in GenBank and BOLD and a reference sample from the United Kingdom. These results provide the first evidence that 'Ca. Liberibacter solanacearum' species are associated with psyllids collected from ash trees and specifically P. discrepans. The recent episodes of dieback of ash in Saskatchewan associated with psyllid feeding are consistent with disease symptoms caused by 'Ca. Liberibacter' pathogens, and this possibility warrants further study.

Development of economic thresholds for pea aphid (Hemiptera: Aphididae) management in lentil (Fabaceae) based on in-field insecticide efficacy trials.

Pea aphid (Acyrthosiphom pisum Harris, Hemiptera: Aphididae) presents a significant economic challenge to lentil (Lens culinaris Medik.) production in the major growing region of Saskatchewan, Canada. During 2019-2020, field experiments were conducted to optimize the management tools for pea aphid control on lentils. A randomized split-plot design was used with main plots consisting of different pea aphid pressures and subplots consisting of different insecticide treatments. The main plot design was aimed to assess the impact of A. pisum feeding on lentil yields during the late vegetative to early reproductive stages. Subplots of the study evaluated the efficacy of 3 insecticides in suppressing pea aphid populations on lentils. Lentil is susceptible to A. pisum feeding and requires management at low pest densities. The economic threshold for pea aphids on lentil crops varied depending on environmental conditions, ranging from 20 to 66 aphids per sweep, calculated using a discrete daily growth rate of 1.116. The estimated economic thresholds provided a 7-day lead time before aphid populations achieved the economic injury level (EIL). The EIL was defined as 78 ± 14 aphids per sweep net sample or 743 ± 137 cumulative aphid days from the first aphid present in the field. In addition, the results of the study found that, on average, foliar applications of insecticides containing the pyrethroid active ingredient lambda-cyhalothrin (IRAC group: 3A) reduced pea aphid populations by 83% compared with untreated control.