Highly Efficient Agroinoculation Method for Tomato Plants with <i>Tomato Yellow Leaf Curl Kanchanaburi Virus</i>

Abstract

Tomato yellow leaf curl disease caused by begomoviruses is a serious threat to tomato (Solanum lycopersicum L.) production. If begomoviruses, transmitted by whitefly (Bemisia tabaci), infect tomato plants during early growth, production can be almost entirely lost. Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), a bipartite Begomovirus, is emerging as an important threat to solanaceous crop production in Southeast Asia. The lack of mechanical transmission of some begomoviruses is a major experimental constraint. In this study, an agroinoculation method using TYLCKaV in tomato plants was established. Partial tandem repeats of TYLCKaV DNA A and DNA B were constructed and cloned to a binary pGreenII vector, and their infectivity was tested. Co-inoculation of TYLCKaV DNA A and DNA B to Nicotiana benthamiana L. produced typical begomoviral symptoms, and both of the viral DNA components accumulated in the upper uninoculated leaves, suggesting systemic infection of TYLCKaV. Two agroinoculation methods were conducted on tomatoes. First, excised sections of tomato shoots were agroinoculated with a soaking procedure. Although two Agrobacterium tumefaciens strains were tested, approximately 40% of inoculated plants only showed viral symptoms for EHA105. Second, agrobacterium from a cultured petri dish was directly inoculated with a colony inoculation procedure. When EHA105 was used, approximately 92% of inoculated plants showed viral symptoms. Sequencing the recovered viral DNA from the upper uninoculated leaf clarified that TYLCKaV had successfully infected the tomato plants. The colony inoculation procedure is labor-saving, and viral symptoms develop in susceptible tomato plants within approximately a month from sowing the seeds. This method could contribute to simple and speedy evaluation of TYLCKaV resistance of tomato plants.

Inoculation of Capsicums with <i>Pepper Yellow Leaf Curl Indonesia Virus</i> by Combining Agroinoculation and Grafting

“…These results showed that both components are necessary for symptom expression in N. benthamiana. Infection of N. benthamiana with bipartite begmovirus DNA A of TYLCKaV alone did not produce symptoms as also observed in our previous study (Koeda et al, 2017). Our study demonstrated that constructed clones had infectivity, and agroinoculation of PepYLCIV DNA A and DNA B can successfully induce virus symptoms.…”

“…In pepper-cultivating fields in Indonesia, mixed infection with PepYLCIV, TYLCKaV, and AYVV is frequently observed (Koeda et al, 2016). In our previous study, we successfully inoculated tomatoes with TYLCKaV by agroinoculation (Koeda et al, 2017). However, we did not succeed in the agroinoculation of peppers with TYLCKaV (data not shown).…”

“…In the present study, extracted DNA was used to amplify a full-length DNA B sequence using a rolling circle amplification (RCA)-based TempliPhi DNA amplification kit (GE Healthcare, Buckinghamshire, UK) according to the procedure described by Koeda et al (2017). Nucleotide sequencing was carried out by an ABI PRISM 3100 genetic analyzer with an ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA).…”

“…Agroinfiltration and a colony inoculation procedure were conducted according to the method reported by Koeda et al (2017). Agroinfiltration to N. benthamiana was conducted using pGreenII-p35S (empty), pGreenIIp35S-PepYLCIV-DNA-A, and pGreenII-p35S-PepYLCIV-DNA-B.…”

“…1a), PepYLCIV-[BA_D1-1] (Koeda et al, 2016), and DNA B sequence (Fig. 1a) of PepYLCIV-[BA_D1-1-B] were cloned into pGreenII-p35S according to Koeda et al (2017). For PepYLCIV DNA A, two fragments were amplified by PCR: fragment 1 (full-length copy of viral DNA A) with PepYLCIV fra1 F and PepYLCIV fra1 R primers, and fragment 2 (partial-length copy of viral DNA A) with PepYLCIV fra2 F and PepYLCIV fra2 R primers.…”