Introduction

Zebra chip disease of potato (ZC) has been the subject of recent comprehensive reviews (Wenninger and Rashed 2024; Prager et al. 2022). The first reports of potato Zebra chip disease in the United States occurred at the turn of the 21st century (Munyaneza et al. 2007) and has since become an increasingly serious economic disease in commercial potato production globally. The phloem-limited bacterium, Candidatus Liberibacter solanacearum (Lso), causes ZC disease (Munyaneza 2012). Lso is persistently transmitted to plants by adult-stage tomato-potato psyllid Bactericera cockerelli. Harmful symptoms manifest in many ways, so control through tolerance is complex and therefore difficult. The logical preference would be to have true resistance—finding potato plants that are unsuitable hosts for both the psyllid and the bacterium. Some populations of the wild potato species S. verrucosum (Cooper and Bamberg 2016) and S. bulbocastanum (Cooper and Bamberg 2014) have been reported to have strong resistance to the psyllid vector.

For the approach of seeking resistance to the bacterium, Wallis and Rashed (2015) cite wild potato species S. chacoense, tarijense, raphanifolium, etuberosum, berthaultii as being in the pedigrees of breeding lines showing tolerance. When a spectrum of wild species from USPG was screened by Levy et al. (2018), plants from populations of S. microdontum (mcd) appeared to show no symptoms compared to uninfected controls, leading to the current experiment with a focus examining the multiplication of Lso in infected plants of that species. S. microdontum is a remarkable species by virtue of its many useful traits (Bamberg and del Rio 2014), and while it has had little documented use in breeding, recent genetic surveys show it has a significant presence in the cultivated potato genome (Hoopes et al. 2022).

With nearly 100 populations in USPG, mcd was a good candidate species from which to select a core subset of about 50 populations that capture nearly all the detected genetic variation, and also captures nearly all of the most favorable state of documented trait variation. The original AFLP-based core subset selected is described in Bamberg and Del Rio (2014), and the SNP-based core of two random clones in vitro from each core population that followed is described in Ma et al. (2022).

Materials and Methods

Detailed information on source populations for the mcd germplasm used in this study can be obtained from the United States Potato Genebank (USPG) using the USDA-ARS Germplasm Resources Information Network (GRIN) (Available at: https://npgsweb.ars-grin.gov/gringlobal/search.aspx).

The tested plants listed in Table 1. were 84 individual seedling clones from 50 mcd populations maintained in vitro at USPG (Bamberg et al. 2016), and grown and tested for Lso at Wapato. S. tuberosum cultivar ‘Russet Burbank’, obtained as commercial seed tubers was used as a control. Plants were grown in 900 ml plastic pots using standard methods of potting medium, fertilization, and lighting at 16 h days to produce uniform plants.

Table 1 Ranked relative lso resistance of S. microdontum clones
Full size table

Psyllids were obtained from a laboratory colony maintained on potato cultivar ‘Ranger Russet’ and the tomato cultivar ‘Moneymaker’. The psyllid colony was established initially from the western haplotype collected from potato fields near Prosser, WA in the spring of 2012, and new psyllids were periodically introduced to maintain genetic diversity. Subsets of colony insects were confirmed by PCR to harbor Lso.

Assays were conducted in two separate trials started from tissue culture plantlets in a completely randomized design with two replicates. Inoculation with Lso was done by confining three infected reproductively mature (> 7 day old) female psyllids to separate terminal leaves of plants in a cage of fine mesh in a greenhouse. The psyllids were removed from plants after one week and leaves were collected after four weeks. DNA was extracted from psyllids and plants using commercial kits (Qiagen). Real-time quantitative PCR (qPCR) was used as an indirect measure of Lso titers in leaves. Each reaction consisted of 10 µL of 2X SYBR Green Master Mix (Roche), 8 µl PCR grade water, 0.5 µl of each primer (final concentration of 125 nM), and 1 µL of template cDNA. The forward primer used was LsoF (CGA GCG CTT ATT TTT AAT AGG AGC) and the reverse primer used was HLBr (GCG TTA TCC CGT AGA AAA AGG TAG) (Li et al. 2009). Samples were run on a Roche Lightcycler 480 with an initial denaturation step of 10 min at 95 °C, 45 cycles of 95 °C for 10 s, 60 °C for 10 s, 72 °C for 30 s, and a final melt curve. The melt curves were checked at the end of each run to ensure there was no off-target amplification. Ct values were compared to those of serial dilutions of plasmid standards to quantify the number of copies of Liberibacter present in each sample. Clone susceptibility was expressed as mean log titer (MLT) with a conservative Ct of 45 set as the standard for zero Lso. Five of most resistant and five of the most susceptible clones were retested in a second trial in by the same method except in triplicate, assessed at both 2 and 4 weeks, and including an average symptom score (1 to 5, where a lower score is more healthy).

Results and Discussion

All mcd are not resistant to Lso. In fact, most (74 of 84) were less resistant to Lso than the susceptible cultivar control (up to 177% MLT of Russet Burbank control). However, 12 were more resistant than control, six having 61–93% MLT of Russet Burbank control, and 6 having a zero MLT value. When 5 clones with zero MLT were retested in a second trial in triplicate, all were confirmed more resistant than the control. Two clones were confirmed as 0% of control MLT, mcd14B1 and mcd62B1. However, considering plant vigor and symptoms, the two we recommend as having the most robust resistance are clones mcd15B2 and mcd62B1 (as shaded in Table 1.).

The resistance of an individual mcd clone is also not very consistent within its source population. When the average MLT difference of individuals from within the same population was calculated (1.32) it was only slightly less than the difference in random pairs of individuals (1.42). This is not unexpected considering that DNA marker data on this set of materials (unpublished data not shown) fails to most closely pair 8 of 50 possible matches of siblings from the same population as being most genetically similar. Thus, fine screening and population development may need to be done to purify and genetically fix the strongest expression of resistance.

Conclusions

Strong Lso resistance in the standard cultivar breeding pool has been difficult to find, so identifying it here in two S. microdontum clones should provide exciting opportunities for mapping the trait, other research, and breeding.