Interaction Studies between Meloidogyne javanica and Fusarium oxysporum f. sp. lycopersici (Fol) Race 3 on Different Isolines of Tomato cv. Tasti Lee

The experiments were conducted between May 2019 and April 2020 at the University of Florida’s Gulf Coast Research and Education Center (GCREC), Wimauma, Florida. Both experiments evaluated the interactions between Meloidogyne javanica and Fusarium oxysporum f. sp. lycopersici (Fol) race 3 using isolines of the tomato cultivar Tasti Lee. The experiments are analyzed separately as the second experiment was slightly modified to reduce the high disease pressure that was noted in the first experiment. In both greenhouse experiments, the RKN inoculum was obtained from a pure culture of Meloidogyne javanica that was maintained on tomato (cv. Florida 47) in the greenhouse. Eggs were extracted from infected tomato roots using 10% commercial bleach and four minutes of hand shaking (Hussey and Barker, 1973).

Tomato seedlings were grown on bleach-sterilized Styrofoam trays (128 seedlings) with steam-pasteurized potting mix and a thin layer of vermiculite on the top. Tomato seedlings (6-week-old seedlings in experiment 1 and 4-week-old seedlings in experiment 2) were planted in 1,000 cc volume plastic pots with an outside diameter of 12.5 cm. Each pot was filled with steam-pasteurized soil at 70°C for 12 h using SST-15 1/8 cubic yard 120v Soil Sterilizer (Pro-Grow Supply Corp, Brooksville, WI, USA). The soil is classified as Myakka fine sand (Sandy, Siliceous Hyperthermic Oxyaquic Alorthod) with pH of 7.6 and 0.8% organic matter. Freshly extracted eggs were inoculated (~10,000 eggs/pot) by pipetting equal volumes (1 ml) in three 2.5 cm depressions surrounding tomato seedlings immediately after transplanting. A 20-20-20 fertilizer solution (10 g/ gallon) was provided to each plant at 100 ml/pot each week throughout the experiment. Greenhouse temperature was 26 ± 2°C with a relative humidity 60% throughout the experiments with natural light condition for both experiments.

Tomato cv. Tasti Lee (+I-3–Mi with Fol resistance but without RKN resistance) and its three novel isolines (−I-3, +Mi without Fol resistance but with RKN resistance, −I-3–Mi without both Fol and RKN resistance and +I-3+Mi = with both Fol and RKN resistance) were used for the experiments. The original Tasti Lee received I-3 gene from the parent line ‘Fla. 7907’ (Scott et al., 2008). Other three isolines were developed at Gulf Coast Research and Education Center (GCREC), Wimauma, Florida. Tasti Lee isoline without I-3 was developed by using non-I-3 version of ‘Fla. 7907’. The parent line ‘Fla. 8059’ received Mi gene from the tomato cultivar ‘Bush Early Girl’ and was used to develop Tasti Lee isoline with Mi gene. In all four isolines, both genes are in the heterozygous form (personal communication).

Fol 3 sand culture was used for the first experiment, and the liquid culture was used for the second experiment. The different inoculation method in the second experiment was done to reduce the disease pressure which was very high during the first experiment. For sand culture, A race 3 isolate of Fol, strain GEV 1400, collected in commercial field in Florida was used to produce the inoculum. The isolate was recovered from long-term storage at −80ºC by placing on a 1/2-strength potato dextrose agar (PDA) media. After 7 days at room temperature, plugs were removed from the edge of the media and placed into liquid potato dextrose broth (PDB). PDB cultures were incubated for a week at 30°C inside an orbital incubator shaker. A 200 ml suspension of Fol liquid culture with mycelium and spores was combined with a mixture of autoclaved cornmeal (500 g) and sand (2 kg) and placed in a 20 cm × 48 cm mushroom growing bag (Shroom Supply, Brooksville, Florida) with a 0.2 μ filter. Bags were sealed and incubated for two weeks at 27ºC and were cut open and allowed to dry for three more weeks. To prepare liquid culture, PDB (24 g/liter) was used to grow a pure culture of Fol race 3. Then, 1.5 ml of pure culture of Fol, stored in −20°C, was inoculated in a 1,000 ml bottle with autoclaved sterile water. The bottle was kept inside the orbital incubator shaker (Amerex Instruments, Inc, Lafayette, CA) for one week at 25°C. The final inoculum was diluted to have 1 × 10⁶ cfu/ml. The pathogen concentration was calculated using a hemacytometer (Hansen, 2000).

The experiment was conducted from the period of May to June 2019. The original Tasti Lee and it’s three isolines as described earlier were evaluated in pots with and without RKN and with and without Fol 3 (~1,000 cfu/g soil). The experimental design was a three factorial one (4 × 2 × 2). Each treatment was replicated five times and completely randomized. Then, 10.2 g of Fol-inoculated sand (9.8 × 10⁴ cfu/g) was mixed with a kilogram of steam-pasteurized soil to obtain a Fol concentration of around 1,000 cfu/g. The Fol sand culture was mixed with the steam-pasteurized soil in a cement mixer for 10 min. The premixed soil was then divided to fill each experimental pot. Seedlings were transplanted immediately after filling the pots, and RKN eggs (~10,000 eggs/pot) were inoculated at the same time as described above.

The experiment was terminated after 30 days. Plant heights were measured every week for four consecutive weeks. Root gall ratings were conducted at the end of the experiment on a scale of 0–10 (0 = no galls and 10 = 100% of roots galled) (Zeck, 1971). Nematode eggs were extracted from each root system as described above and recorded, and reproduction factor (final population density/initial population density) was calculated. Shoot and root biomass was oven dried at 70°C for 5 days, and dry weight was recorded.

This experiment was conducted from the period March to April 2020. The experiment was conducted in the same manner as previously described with the following exceptions: only Tasti Lee isolines without Fusarium oxysporum f. sp. lycopersici (Fol) resistance (−I-3, +Mi, and −I-3Mi) were used, and two inoculation timings of Fol were included, at planting and 10 days after planting. The later FOL inoculation timing was included as this has previously shown to significantly impact the interaction between the two pathogens. Each treatment was replicated five times, and the entire experiment was established as a randomized block design. Ten extra pots were included for the isoline without Mi to evaluate the effect of Fol on early infection of RKN. All 10 pots were inoculated with RKN, of which five received Fol at planting, while the other five did not.

In this experiment, instead of Fol soil inoculum that was used in the first experiment, liquid culture of Fol was used for inoculation to reduce the disease pressure which was very high during the first experiment. A final concentration of 5 × 10⁷ cfu in 50 ml was applied into each pot immediately after seedling transplanting or 10 days after transplanting. RKN eggs (~10,000 eggs/pot) were inoculated at planting as described above.

The 10 extra pots were sampled at 10 days after transplanting (DAT), roots were gently washed free of soil, and RKN juveniles inside roots were stained (Thies et al., 2002). Roots of J2-inoculated plants were first cleaned in running tap water and then cleaned with 10% commercial bleach by immersing individual roots in a 50 ml beaker for 4 min. Roots were then thoroughly cleaned and further immersed in clean water for 15 min more in the beaker. The water was rinsed out, and the roots were dipped into 12.5% solution of McCormick red food color (McCormick & Co., Inc., Hunt Valley, MD). The root solution was brought to a boil and allowed to cool at room temperature. The stained roots were thoroughly rinsed with water and then destained by heating with acidified glycerol (10 drops 5N HCl + 100 ml glycerol) for 20 s. This destains the root tissues, but the J2 inside the root is still stained with red color. The root was then plated in a square-shaped petri dish (10 cm × 10 cm) to observe J2s inside the root under a stereomicroscope. The main experiment was terminated 42 DAT. Plant heights were measured every 2 weeks for six consecutive weeks. Root gall ratings were conducted at the end of the experiment as described above. Nematode eggs were extracted as described above and recorded. Shoot and root was oven dried at 70°C for 5 days, and dry weight was recorded.

Data were analyzed using SAS^® PROC GLIMMIX (SAS/STAT 15.1; SAS Institute Inc., Cary, NC), where tomato cultivars, RKN, and Fusarium inoculums were constants in both experiments, and block was used as a random effect in the second experiment. Student panel command was used to check the normality and homogeneity of the data before proceeding with analysis (Kozak and Piepho, 2018). Means of data were separated by using the Least squares means (LSMEANS) statement in SAS at P ≤ 0.05. Plant height data were analyzed using the repeated measures function. Egg count data were analyzed using negative binomial function in SAS. The ‘ilink’ option was used to get the real mean value of each dependent variable.

Fol and cultivar both showed a significant impact on RKN severity (Table 1). Root galling, egg production, and reproduction factor (RF) all were highly reduced in presence of Fol inoculum. As expected, Tasti Lee isolines with Mi gene significantly reduced root galling, egg production, and RF compared with the isolines without Mi gene. The interaction between Fol 3 and cultivars in terms of egg production was statistically significant (P-value = 0.003; Fig. 1) and so was RF (P-value = 0.004) as it depends on the final population density of the pest. RKN egg production in all Tasti Lee isolines was significantly higher when it was inoculated alone compared with when it was combined with Fol.

Figure 1

Plant height and dry biomass were negatively impacted by Fol, and RKN also slightly reduced plant height and biomass (Table 2). Interaction among RKN, Fol and cultivar for final height was statistically significant (P-value = 0.045; Fig. 2). RKN alone did not affect plant height much in any of the isolines, but Fol alone or in combination with RKN significantly reduced plant height in all Tasti Lee isolines relative to height given by non-treated control and RKN, with differing effect depending on presence or absence of I-3. Isolines with I-3 gene had better plant height under Fol treatment alone compared to the isolines without I-3, but height was further reduced when Fol and RKN were inoculated concomitantly at planting; Fol alone and in combination with RKN equally reduced plant height in isolines that lacked I-3. Interactions between Fol and time and RKN and time for plant height were statistically significant (P-value <0.0001 for both interactions, Fig. 3). Both pests reduced plant height by time, and the reduction due to Fol was greater. Plant biomass, at the end of the experiment, was also significantly reduced by Fol alone and the combination of Fol and RKN (Figs. 4 and 5). Similarly, shoot dry weight was better in the isolines with I-3 compared with the isolines without I-3 under Fol treatment alone, but biomass was further reduced when Fol and RKN were concomitantly inoculated; likewise, Fol alone and in combination with RKN equally reduced biomass in isolines that lacked I-3 (Fig. 4). Root dry weight was also significantly reduced by Fol and Fol+RKN treatments in all isolines (Fig. 5). Contrast analysis showed that, overall, the reduction in biomass due to Fol alone and to Fol+RKN was similar and much higher (>4 times) compared with the damage caused by RKN alone (Table 5).

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Figure 5

At the end of the experiment (42 DAT), Fol inoculated at planting (0DAT) negatively impacted RKN reproduction and the isoline without Mi gene had higher root gall ratings, egg counts, and RF (Table 3). Gall rating tended toward being slightly reduced by Fol inoculated at 0 DAT, but differences were not statistically significant (P-value = 0.278). Fol inoculated at 10 DAT did not show any negative impact on RKN galling or reproduction. The interaction between Fol and cultivars for RKN egg production was statistically significant (P-value = 0.001; Fig. 6). In the isoline without Mi, egg production was significantly reduced (by 90%) when Fol and RKN were inoculated together as compared with RKN alone. But in the isoline with Mi, there was no significant effect of Fol on RKN egg production. Early nematode infection at 10 DAT in the isoline without Mi was significantly less in the plants inoculated with Fol at planting compared with plants without Fol (Fig. 7). Fol applied 10 days after transplanting did not decrease the RKN egg production in either isoline (Fig. 6). The Tasti-Lee isolines were significantly different for gall rating, egg count, and RF of M. javanica (Table 3). The Tasti-Lee isoline with Mi reduced root galls by 23% and nematode egg production and RF approximately twofold compared to the isoline without Mi gene.

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Figure 7

RKN and Fol inoculated at 0 DAT both reduced plant height and plant biomass until the end of the experiment (Table 4). Fol inoculated at 10 DAT did not negatively impact plant height but reduced shoot dry weight. There was significant interaction between RKN and Fol for plant height at 28 and 42 DAT (P-value: <0.0001 and 0.0001, respectively; Fig. 8). At both time points, plant height was significantly reduced by Fol inoculated at planting in presence of RKN compared with absence of RKN. Fol when inoculated 10 DAT did not have negative impact on plant height either with or without RKN. As in the first experiment, the interaction between Fol and time and between RKN and time for plant height was statistically significant (P-values 0.008 and <0.0001, respectively, Fig. 9). Both pests reduced plant height over time, but the reduction due to RKN was more prominent. Different than in the first experiment, Fol inoculated at planting slightly decreased the plant height, and the Fol inoculated at 10 DAT did not have any impact on plant height reduction. The interaction between RKN and Fol for plant biomass was also statistically significant (P-value = 0.0004 and 0.008, respectively; Fig. 10). Root and shoot dry weights were significantly reduced by Fol inoculated at planting in presence of RKN (Fig. 10 and Fig. 11). Fol when applied at 10 DAT also significantly reduced shoot dry weight in presence of RKN compared to its absence. Fol alone, inoculated at 0 DAT and 10 DAT, also reduced shoot biomass compared to non-treated control. The highest shoot dry weight was observed in the plants with control treatment (no RKN or Fol inoculated). Contrast analysis showed that, overall, the reduction in biomass due to Fol inoculated at 0 DAT and to RKN was similar and much lower compared with the reduction made by combination of RKN and Fol inoculated at planting (Table 5). The shoot and root biomasses were reduced by more than four and five times, respectively, by the concomitant inoculation of RKN and Fol at planting compared with the reduction caused by Fol (0 DAT) alone or by RKN alone. Reduction to shoot biomass due to the combination of RKN and Fol when inoculated at 10 DAT was also doubled when compared with Fol (10 DAT) alone.

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