Candidatus (Ca.) phytoplasma asteris subgroups display distinct disease progression dynamics during the carrot growing season

Aster Yellows phytoplasma (AYp; Candidatus (Ca.) Phytoplasma asteris) is an obligate bacterial pathogen that is the causative agent of multiple diseases in herbaceous plants. While this phytoplasma has been examined in depth for its disease characteristics, knowledge about the spatial and temporal dynamics of pathogen spread is lacking. The phytoplasma is found in plant’s phloem and is vectored by leafhoppers (Cicadellidae: Hemiptera), including the aster leafhopper, Macrosteles quadrilineatus Forbes. The aster leafhopper is a migratory insect pest that overwinters in the southern United States, and historical data suggest these insects migrate from southern overwintering locations to northern latitudes annually, transmitting and driving phytoplasma infection rates as they migrate. A more in-depth understanding of the spatial, temporal and genetic determinants of Aster Yellows disease progress will lead to better integrated pest management strategies for Aster Yellows disease control. Carrot, Daucus carota L., plots were established at two planting densities in central Wisconsin and monitored during the 2018 growing season for Aster Yellows disease progression. Symptomatic carrots were sampled and assayed for the presence of the Aster Yellows phytoplasma. Aster Yellows disease progression was determined to be significantly associated with calendar date, crop density, location within the field, and phytoplasma subgroup.

per hectare) or low density (556k per hectare) seeding rate. Each planting was further divided into five 118 rows of 18 beds with 4m bare alleys separating rows (90 beds per density). Each 3-row raised bed 119 measured 2m wide by 6m long. The first and last rows of beds were considered "edge" beds and were 120 bordered by other, non-carrot crops to the north (sorghum) and the south (green bean). Carrot beds 121 were subsequently managed with a standard fertilizer program, but no crop protection pesticides 122 (herbicides, fungicides, insecticides) were applied at any point in the season. Weeds were managed by 123 hand-pulling every two weeks.

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Using a random number generator, two beds from each row of the high-and low-density 131 for a total of 9 observations. The exact location of each phenotypically-described, diseased carrot was 132 recorded and marked within the field relative to the front stake of each row. Each symptomatic carrot 133 had a small sample of both petiole and stem removed for genetic confirmation of the presence of the analyze the genetic composition of subgroup and effector proportions. Subgroup identification was 174 determined using both nucleic acid sequencing and restriction fragment length polymorphism (RFLP).

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The identity and position of six unique, single nucleic acid polymorphisms (SNPs) were compared 176 between sequences to identify AYp subgroup. Furthermore, a RFLP assay using the restriction 177 endonuclease Hha1 (Promega Corporation), was conducted at 37°C for 90 min and run on 1.5% agarose 178 gel. RFLP was used as a supplementary assay to confirm the subgroup designation resulting from SNP 179 assessment of the sequencing data [11].  density planting, and an average of 11.0 ± 5.9% (edge plots) and 8.2 ± 2.2% (interior plots) in the low-216 density planting (Figure 1, Supplemental Table S3).
9 218 Figure 1. Mean Aster Yellows disease incidence in carrot plantings by planting density (high/low) and 219 plot location within the field (edge/interior).

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Aster Yellows disease incidence appeared to be influenced by planting density (high versus low September and October (Figure 2). The fraction of captured ALH carrying the AYp followed a similar 248 pattern, with a mean peak infectivity of 8.75% coincident with peak ALH populations (Figure 2). AYp averaging over both planting densities within the carrot field.

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Aster leafhopper abundance was greater in the high-density carrot plantings in July and August.

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ALH similarly peaked at 17.5% within the ALH populations in high density plantings on August 21 ( Figure   256 3). The combination of high ALH counts together with high phytoplasma detections within the ALH 257 coincide with high AYp disease incidence in carrot fields.  Table S2). Only SAP44 generated slightly more reads from 16SrI-B versus 16SrI-A within 295 infected samples.

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It has been previously established that AYp consists of multiple, genetically-distinct subgroups 334 that can cause AY disease in carrots [11]. In Wisconsin there are at least two AYp subgroups, 16SrI-A and proportion is important to highlight and suggests that the 16SrI-A subgroup is being selected for in 342 comparison to the 16SrI-B subgroup within our experiment. We did detect a small fraction of carrots 343 infected with both 16SrI-A and 16SrI-B, suggesting that the pathogen can co-occur within the same host.