4.8. Plant Treatments

Bioassays were performed using 4-day-old Bactericera cockerelli adults. Individual tomato plants, covered with an antiaphid mesh, were infested with 30 unsexed adults, and the insect–plant interaction was allowed for two days. After that, B. cockerelli adults were removed from the plants by gentle aspiration. Next, plants were tested through polymerase chain reaction (PCR) to verify infection. The bioassays were carried out only for CaLso-positive plants and their respective controls.

A first bioassay was performed in November–December 2018 in Saltillo, Mexico, to evaluate the effect of using different doses of PNB in infected tomato plants (nine plants per dosage were utilized). These investigations included infected plants treated with 70, 40, or 20 ppm of PNB and its corresponding controls. Additionally, uninfected plants were treated with 70 ppm of PNB as a second control.

Each plant was initially sprayed with 3 mL of the corresponding PNB concentration, followed by a respray one week later. After that, all plants were kept in separate tunnels with antiaphid mesh to avoid B. cockerelli reinfestation. The plants were grown under greenhouse conditions and were monitored every week for symptoms. Finally, 25 days post-treatment, leaves were collected, flash-frozen in liquid nitrogen, ground in a mortar and pestle, and stored at –80 °C until needed for deoxyribonucleic acid (DNA) isolation.

A second bioassay was performed in March–April 2019. Infected tomato plants were separately treated with two different bactericides. For PNB, plants were initially sprayed and resprayed at day 7 with a solution containing 70 ppm of PNB. After this treatment, plants were allowed to grow and appraised every week for three weeks to estimate an adequate treatment interval (7, 14, or 21 days). This experiment was performed with a series of 18 plants, including 18 (untreated) control plants. For comparison, 18 plants were treated with a solution containing 600 ppm of oxytetracycline (second bactericide) and evaluated 21 days after treatment. For both bioassays, plants were allowed to grow under similar conditions after their respective treatment and were monitored for symptoms. Finally, leaves were collected, flash-frozen in liquid nitrogen, and stored at –80 °C until needed for DNA isolation.

Composite DNA samples were obtained from the combined and isolated DNA from leaves from each treatment in both bioassays. DNA isolation was performed using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions, treated with DNAase-free RNAase.

The samples were separated on 1.2% agarose gels (Certified Molecular Biology Agarose, Bio-Rad Laboratories, Hercules, CA, USA) to verify DNA isolation quality. All DNA samples were quantified using the ND-1000 Spectrophotometer (NanoDrop Products, Wilmington, DE, USA).

Endpoint PCR was used to verify the presence of CaLso in DNA isolated from infested tomato plants and B. cockerelli adults. Candidatus Liberibacter Zebra Chip (CLZC) specific primers were used by following a protocol described elsewhere [8]: CLZC-F 5′-ACCCTGAACCTCAATTTTACTGAC-3′ and CLZC-R 5′-TCGGATTTAGGAGTGGGTAAGTGG-3′. All PCR amplifications were performed using Taq PCR Master Mix Kit (QIAGEN, Hilden, Germany) under the following conditions: 1 cycle at 94 °C for 3 min; 35 cycles at 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 45 s; followed by a final process at 72 °C for 5 min. Two amplicons were obtained from infested plants and insects (~185 bp), separated on 2% agarose gels, and purified using QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). Next, these fragments were quantified and sequenced (ELIM Biopharmaceuticals, Inc., Hayward, CA, USA). The obtained sequences were compared with the aid of the database from the National Center for Biotechnology Information, USA. Genomic DNA was isolated from leaves of 5-week-old healthy plants (control), PNB-treated plants, and infected tomato plants (CaLso and CaLso PNB-treated plants). Quantitative real-time PCR (qPCR) was employed to detect CaLso differences in all treatments during the bioassays. CLZC specific primers were used for CaLso detection, and RPL2 (Tomato Ribosomal Protein L2) was used as an endogenous reference gene [9], employing the following primers: RPL2-F 5′GAGGGCGTACTGAGAAACCA-3′ and RPL2-R 5′-CTTTTGTCCAGGAGGTGCAT-3′. The analysis was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), CLZC and RPL2 primers at 300 nM, and 100 ng of genomic DNA using Hard-Shell PCR Plates (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer’s protocol. All qPCR analyses were achieved on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For the first bioassay, six treatments with nine biological samples with three technical replicates per sample were used for qPCR investigations. The second bioassay was carried out with five treatments and three control treatments with six biological samples with three technical replicates per sample. The qPCR was performed under the following conditions: 1 cycle at 95 °C for 10 min, 35 two-step cycles each at 95 °C for 15 s and 55 °C for 60 s, and melting curve of 65–95 °C for 5 s. Data were obtained with CFX Manager Version 3.1 Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Quantification of CaLso in the infected plants vs. control plants was obtained relative to the endogenous RPL2 gene according to the comparative C_t method (2^−∆∆Ct)[32].