Thermotolerance

For aging assays, synchronous populations were obtained by allowing 5–10 hermaphrodites to lay eggs for 4 h to overnight. For ease of analysis, we used the adult sterile strain, fem-1(hc17), as the wild-type strain to avoid progeny overgrowth in lifespan assays. Therefore, eggs were shifted to 25 °C, the nonpermissive temperature for fertility of fem-1(hc17). Lifespan scoring was initiated after hermaphrodites completed the final larval molt, on the first day of adulthood. For aging assays with BB extracts, treatments were added to NGM agar plates on the first day of the lifespan assay. For lifespan assays with fertile strains, hermaphrodites were transferred daily for the first 4 days of adulthood to avoid progeny overgrowth. In these cases, all treatment plates were prepared on day 0 of adulthood. Statistical analyses and survival plots of lifespan data were performed with JMP analysis software (SAS Institute Inc, Cary, NC, USA). Pharynx pumping rates were scored on adults at room temperature (24 °C) under a Nikon SMZ1500 stereomicroscope (Nikon, Melville, NY, USA).
Thermotolerance assays were performed with hermaphrodites on adult day 5, after the majority of egg-laying had ceased. Animals were transferred onto 3-cm NGM agar plates supplemented as indicated and then incubated at 35 °C for 16 h. Survival was scored as the number of animals responsive to gentle touch as a fraction of the original number of animals on the plate. Animals that had died from dessication on the sides of the plate were censored. Paraquat-induced oxidative stress assays were performed with fem-1(hc17) hermaphrodites at 25 °C as for aging assays, except paraquat was added to NGM medium to 10 mm final concentration (ChemService, West Chester, PA, USA). For hydrogen peroxide, we scored 5-h survival of adult day 5 fem-1(hc17) animals in S-basal medium with indicated concentrations of hydrogen peroxide.
To determine lipofuscin levels, adult hermaphrodites were anesthetized in 0.2% sodium azide and mounted on 2% agarose pads for visualization of intestinal fluorescence on a Nikon E800 microscope using an Endow GFP filter with a mercury UV source (Nikon). Images were captured using a constant exposure time with a Hamamatsu ORCA digital CCD camera (Hamamatsu, Bridgewater, NJ, USA) using OpenLab software (Improvision, Lexingon, MA, USA). Lipofuscin levels were measured using ImageJ software (NIH Image) by determining average pixel intensity in each animal's intestine.
To measure levels of 4-HNE, animals were collected, fixed with 4% formaldehyde and permeabilized by digestion with type IV collagenase (Sigma Chemical Company), as described (Loer & Kenyon, 1993 (link)). Fixed and permeabilized specimens were incubated with anti4-HNE antisera (1 : 100 dilution, Genox Corp, Baltimore, MD, USA) for 2 h at 24 °C, washed and incubated overnight at 4 °C with Alexafluor-546-conjugated goat anti-mouse secondary antibody (1 : 100) (#A-11003, Invitrogen, Carlsbad, CA, USA). Stained animals were mounted on 2% agarose pads and fluorescence visualized as for lipofuscin with appropriate filter sets. 4-HNE immunofluorescence was measured in the pharynx terminal bulb and somatic gonad using ImageJ software.

All biochemically verified isolates were subjected to PCR analysis for the detection of the 23S rRNA gene, which revealed the presence of thermotolerant Campylobacter spp., and then for the identification of the species, two differentiation genes (mapA for C. jejuni and ceuE gene for C. coli) were used. The QIAamp DNA Mini kit (Qiagen, Germany, GmbH) was used to extract DNA in accordance with the manufacturer's instructions. Briefly, 200 µl of the sample suspension was treated at 56 °C for 10 min with 10 µl of proteinase K and 200 µl of lysis solution. Then, 200 µl of 100% ethanol was added to the lysate after incubation. After that, the sample was washed and centrifuged in accordance with the manufacturer's instructions with the help of 100 µl of elution buffer, and DNA was extracted.
The oligonucleotide primers used in this study were provided by Metabion (Germany) (supplementary table 1). A 25-µl reaction containing 12.5 µl of Emerald-Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer at a 20 pmol concentration, 5.5 µl of water, and 5 µl of DNA template was used. Thermal cycler 2720 from Applied Biosystems was used to perform the reaction.
The PCR products were separated using 5 V/cm gradient electrophoresis on a 1.5% agarose gel (Applichem, Germany, GmbH) in 1 × TBE buffer at room temperature. Each gel slot had 20 µl of the product for gel analysis. The fragment sizes were calculated using the Generuler 100 bp ladder (Fermentas, Germany) and the Gelpilot 100 bp ladder (Qiagen, Gmbh, Germany). A gel documentation system (Alpha Innotech, Biometra) took pictures of the gel, and computer software was used to analyze the data.

Ten potato cultivars (Table 1) were planted, grown, and harvested in greenhouses at the Horticulture Teaching Research and Extension Center at Texas A&M University, located near Somerville (Latitude: 30.5223 and Longitude: -96.4307), Texas, USA. Atlantic, Russet Burbank, Russet Norkotah and Yukon Gold are commercial cultivars used as references for different market classes. Atlantic is a chipping variety; Russet Burbank is a French fry processing potato; Russet Norkotah is a popular russet skin potato for the fresh market; Yukon Gold is a fresh market yellow cultivar. The Texas A&M University Potato Breeding Program released the other six potato clones used in the study. The Texas A&M potato breeding program has been selecting potato clones for the last 30-40 years under high-temperature conditions where several days with temperatures beyond 35°C are common (Vales et al., 2019 ). Thus, the clones from Texas A&M can be considered to possess heat tolerance characteristics. COTX09022-3RuRe/Y was released under the experimental code; it has russet skin, red eyes and yellow flesh. TX1523-1Ru/Y (Sierra Gold™) has russet skin and yellow flesh. Reveille Russet, Russet Norkotah 278, Russet Norkotah 296, and Vanguard Russet are fresh market potatoes with russet skin and white flesh.

To compensate for innate differences in trait components, heat tolerance was quantified using the percentage of the control for each genotype. Pots were considered to be the experimental units. For each pot in the heat stress treatment, the chlorophyll retention at 4, 8, 12 or 16 days was expressed as a percentage of Day 0 (control). The rationale for using chlorophyll content at Day 0 as the control was previously described in Ristic et al. (2008) (link), and this method was later on used by Sharma et al. (2017) (link) and Fu et al. (2022) (link). Briefly, the rationale stood on the observations that during the first 26 days after flowering the chlorophyll contents were not significantly changed under the control condition for hexaploid common wheat, tetraploid wheat and maize (Ristic et al., 2008 (link)). The rationale for using percentage of control rather than heat susceptibility index (HSI, Fischer and Maurer, 1978 (link)) was given in Fu et al. (2015) (link), and this method was later on used by Fu et al. (2022) (link); Fu et al. (2017 (link)). Briefly, the rationale stood on the facts including (1) that percent of control is always non-negative and a HSI can be negative in some cases and a negative number can cause problems in some statistical analyses such as PROC GLIMMIX which lacks convergence for a negative number, and (2) percent of control for each accession is more intuitive and is unaffected by the group of accessions under evaluation and, in contrast, HSI is the opposite (Fu et al., 2015 (link)). For the yield traits, percentage of control was calculated as follows: Percent of Control = 100 x (Y_h/Y_c).
where Y_h is the value for each experimental unit in the heat stress treatment and Y_c is the average value for the corresponding experimental units in the non-stress control condition. Higher mean percentage of control indicates greater heat tolerance. The experiment was analyzed as a completely randomized design. Data were obtained for two runs of the experiment (Exp. 1 and Exp. 2). Each experiment was analyzed separately. Genotype was analyzed as a fixed effect. Analysis of variance was performed with SAS 9.4 (SAS Institute, 2010 ) PROC GLIMMIX. The Gaussian (normal), Poisson, lognormal, and negative binomial distributions were tested for best fit on the basis of standard fit statistics and examination of the residuals. For each variable, the negative binomial distribution was used because it had the best fit. Least squares means for genotypes (lines) were back-transformed for presentation using the ilink function of GLIMMIX. Least squares means of genotypes were compared with an F test, and then separated using the Tukey-Kramer multiple comparison test. Correlations were calculated using SAS PROC CORR.