Grafting Compatibility, Scion Growth, and Fusarium Wilt Disease Incidence of Intraspecific Grafted Tomato

The experiment was conducted in a greenhouse condition. Four tomato accessions, LE314, LE472, LE482, and LE501, selected previously from tomato germplasm in the Tropical Vegetable Research Center (TVRC), Kasetsart University, Kamphaeng Saen Campus for their resistance to Fol race 1, were used as rootstocks (RS). A susceptible tomato cultivar, ‘Sidathip 3’ (SDT3), was used as a scion (SC). This cultivar is a table tomato fruit type with a small fruit size of 3 × 4 cm. The fruit weight is more than 20 g. The seeds of all Fol-resistant tomato accessions were sown directly into 50-cell plug trays filled with a ready-to-use mixture of the substrate (Kekkilä Professional, Finland). Eight days later, the seeds of SDT3 were sown similarly. The 21-day-old seedlings at the three- to four-true-leaves stage were transferred to a 6 × 8-inch plastic pot with a soil mixture (with topsoil and ready-to-use soil mixture 2 : 1 by volume). All seedlings were grown in a greenhouse for 30 days under the natural sunlight and temperature of 30–33 °C/25–28 °C (day/night). Seedlings were watered once a day.

Thirty days after germination, the susceptible SDT3 cultivar (SC) was grafted onto four Fol-resistant accessions, LE314, LE472, LE482, and LE501 (RS), using the cleft grafting method. The grafted plants were covered with transparent plastic to avoid water loss and grown in the greenhouse for two weeks. The grafted plants were watered twice daily and supplemented with 20 g of dry pelleted fertilizer with N-P-K of 15-15-15 weekly until the 30-day. The experiment included intraspecific heterograft (SDT3/LE314, SDT3/LE472, SDT3/LE482, and SDT3/LE501) and self-grafted tomato plants SDT3/SDT3 and LE472/LE472). The plant survival rate (SR) evaluation and Fol inoculation detailed below were performed at 20 days after grafting (DAG). Evaluation of grafting compatibility was taken at 21, 28, 35, and 42 DAG. The samples of grafting points were collected at 4 and 15 DAG for gene expression analysis.

Grafting compatibility was evaluated by measuring the diameter of the grafting point (GP), the scion (SC), and the rootstock (RS) at 1 cm above and below the grafting point using the formula (Zeist et al. 2018): GI = {[(GP − RS) + (GP − SC)/2] + (RS − SC)}/2.

The plant survival rate was calculated at 20 DAG as a percentage of surviving plants relative to the total number of grafted plants. The plant height (PH), measured from the base of the scion stem to the apex (cm), and the number of branches (NB) were evaluated at 21, 28, 35, and 42 DAG. The size of the fruit and fruit yield were evaluated at 45 DAG.

1 cm-long stem samples were collected at the grafting point of LE472/LE472 and SDT3/LE472 at 4 and 15 DAGs. Total RNA was extracted using TRIzol reagent (Invitrogen). The synthesis of complementary DNA (cDNA) was carried out using a 2-step RT-PCR Kit (Vivantis, Malaysia). Expression of the peroxidase gene was performed by qPCR. The primer sequences for the peroxidase gene (Solyc02g084800.2) were 5′-TTTGTTGAGGCTCCATTTCC-3′ and 5′-TTTGTGGGCATTCTTTCTCC-3′ (Wang et al. 2019). The selected primers of the housekeeping baseline gene, 18S rRNA (X51576), were 5′-GGGCATTCGTATTTCATAGTCAGA-3′ and 5′-GTTCTTGATTAATGAAAACATCCT-3′ (Mascia et al. 2010).

Two-fold serial dilutions were performed using cDNA from a representative sample to generate standard curves and threshold values (C_T) for a housekeeping gene (18S rRNA) and the gene of interest. A standard curve-based calculation method was used to estimate the efficiency of the primer with the following equation: Efficiency (%) = [10^(−1/slope) − 1] × 100. For gene expression analysis, the cDNA template was diluted to a 1 : 2 concentration. The real-time PCR reaction used in both the efficiency test and the analysis of gene expression was performed with the PCR mixture containing 1 μl of the diluted cDNA, 5 μl of 2X SensiFast SYBR NO-ROX mix (Meridian Bioscience), and 0.4 μl each of 10 μM Primer-F and Primer-R. The reaction of PCR was performed in triplicate for each cDNA sample, along with no template control in parallel to each gene. All reactions were processed in PCRmax ECO 48 REAL TIME PCR PLATE (PCRmax). The PCR reaction was performed in PCRmax Eco 48 real-time PCR (PCRmax). The 2-step cycle of the PCR condition consisted of enzyme activation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 20 s. The dissociation curve analysis was performed after 40 cycles to identify the presence of primer dimers and analyze the specificity of the reaction. The 18S rRNA gene was used as an internal control. The C_T data of homo- and hetero-grafted tomatoes at each time point obtained from peroxidase gene amplification were normalized with those from 18S rRNA to give the relative gene expression. The fold changes in the target gene and the normalized to 18S rRNA samples, relative to homograft samples (LE472/LE472) at 4 or 15 DAG, were calculated for each sample using the 2^−ΔΔCT (Livak & Schmittgen 2001) method described previously (Mascia et al. 2010).

The grafted and nongrafted plants at 20 DAG were inoculated with a suspension of spores from the Fol isolate TFPK401 race 1 in the concentration of 1×10⁶ spore per ml using the root dip method. The mock-inoculated plants were treated with autoclaved distilled water to serve as controls. The inoculated plants were transferred to 6 × 8 inch plastic pots with autoclaved and watered before transplantation. SDT3 (nongrafted) was used as a susceptibility control. The tested plants were grown in a greenhouse condition with natural sunlight. The disease severity score (DSS) and disease index (DI) were adopted from Marlatt et al. (1996) with some modifications. Symptoms were scored at the vegetative stage using a five-grade severity scale, with (1) denoting symptomless, (2) chlorotic plants, (3) chlorotic plants and wilting, (4) wilting plants, and (5) plant death. Disease scoring was recorded at 60 days post-inoculation (DPI) with SDT3 (nongrafted) as a susceptibility control, and disease symptoms were shown on a scale of 5. The DI was calculated using the following formula: DI = [(ni × si)/(N × S)] × 100, where ni – number of wilting plants, si – disease severity score, N – total number of tested tomatoes, and S – highest disease severity score.

The experiment was carried out using a completely randomized design (CRD). 10 biological replicates were used for measuring and evaluating the plant survival rate (SR), grafting compatibility, and Fol resistance. All data collected were subjected to analysis of variance (ANOVA) using Statistix 8.0. Means were compared using Tukey's mean comparison test (p = 0.05).

The percentage of successful grafting was observed after 20 DAG in intraspecific self-grafted tomatoes (SDT3/SDT3) and heterografted tomatoes (SDT3/LE314, SDT3/LE472, SDT3/LE482, and SDT3/LE501). In both graftings, 100% plant survival rate was observed. The rootstocks were compatible with the SDT3 scion genotype and demonstrated that the grafting condition was performed favored healing without interfering in the formation of the graft union (Fig. 1). There was an increase in GI in all grafted tomatoes ranging from 0.06–0.08 to 0.11–0.14 in the course of 21–42 DAG, but these differences did not differ significantly until 42 DAG, although self-grafted plants showed a slightly lower GI than hetero-grafted ones (Table 1).

Figure 1

The height of the scions of the self-grafted and heterografted genotypes grew consequently with time but did not differ significantly within term points (Fig. 2A). A similar pattern was observed in the number of branches (Fig. 2B). The average fruit size ranged from 2.75–3.27 cm × 3.17–3.66 cm and fruit yield ranged from 141.90 to 160.48 g per plant, showing no significant differences between grafting components (Table 2).

Figure 2

The relative expression of the peroxidase gene was analyzed in self-grafted LE472/LE472 and heterografted SDT3/LE472 at 4 and 15 DAG. The expression of the SDT3/LE472 sample increased threefold and was significantly higher than that of LE472/LE472 at 15 DAG (Fig. 3).

Figure 3

The results showed that all intraspecific root-stocks significantly reduced symptoms of fusarium wilt disease at 60 days post-inoculation. The heterografted plants showed a DSS of 1 and 0% of disease index (DI), compared to a self-grafted and nongrafted SDT3 showing 5 DSS and 100% DI (Fig. 4).

Figure 4