A New Biological Product Shows Promising Control of the Northern Root-Knot Nematode, Meloidogyne hapla, in Greenhouse Tomatoes

M. hapla colonies were maintained in the MSU Plant Science Greenhouses on Michigan State University’s main campus. Root-knot eggs were collected from roots from an infested parsnip field in September 2019, and colonies were reared in gallon-sized polystyrene pots on tomatoes (cv. Rutgers). Inoculum (M. hapla eggs) for these trials were collected from tomato colonies using the NaClO root extraction process described in Hussey and Barker (1973).

A greenhouse trial was established in 2021, to evaluate the potential efficacies of four product solutions against the plant-parasitic nematode, M. hapla. Steam sterilized soil was mixed with play sand at a 30:70 ratio, and approximately 500 cm³ of the soil mixture was placed into each of the 46 fresh polystyrene pots. Four seeds of a susceptible variety of tomato (cv. Rutgers) were seeded into pots. Four days later, the plants were thinned to two seedlings per pot. Any pots containing plants that contained less than two seedlings, or which showed signs of unthrifty growth, were removed prior to inoculation. Remaining pots were each inoculated with approximately 2000 M. hapla eggs each (N=36). One week following plant emergence, treatments were applied as a soil drench. Fifty milliliters of distilled water was applied to the noninoculated control pots. Velum Prime was applied to respective pots at a reduced rate equivalent to 6.5 oz/A. Next, 50 ml of respective solutions (MN11.1, MN11.2, MN21.1, MN21.2) were applied to the respective remaining pots, each with six replicates. Pots were labelled correspondingly and arranged in a randomized complete block design (RCBD) on the greenhouse bench and monitored weekly. Each week, plants were evenly fertilized with a 20-20-20 fertilizer solution to promote normal plant growth. Additionally, two weeks following product applications, plants were visually evaluated for signs of phytotoxicity. Following trial conclusion, above ground plant heights and weights were measured, and roots were separated from soil. Soil and roots were collected into plastic bags for further processing.

The experiment was repeated as previously described with slight modifications. The optimal formulations of each product were selected to repeat (MN11.2 and MN21.2). Inoculated, nontreated controls (positive controls) and noninoculated controls (negative controls) treatments were included for comparison. Each of the five treatments (positive control, MN11.2, MN21.2, Velum Prime, and the negative control) had twelve replicates. Six of the replicates were deconstructed at five weeks post-application (5 WPA), and the other six were concluded at ten weeks post-application (10 WPA). At both takedowns, the plant parameters (above ground height and root mass) and nematode parameters (juvenile nematodes/100 cm³ soil, mature female galls/1 g roots) described above were collected from each pot.

Soil within each bag was homogenized and a 100 cm³ volume subsample was collected for processing. Immediately following the trial conclusion, we used the sugar floatation centrifugal method to elucidate vermiform nematodes (Jenkins, 1964). Root-knot nematode juveniles were enumerated using a counting dish and an inverted Nikon TMS microscope at x200 and x1000 magnification, within five days of processing.

Roots were gently cleared of soil, rated on the Gall Indices Rating developed by Hussey and Janssen (2002): 0 indicating no galls and up to 5 indicating over 75% of the root system contains galls. Root systems were then weighed and recorded. Following evaluation, 1 g of lateral roots was collected from each plant. Each root sample was individually cut into 1 cm pieces and stained using the NaOCl acid fuchsin-glycerin procedure (Bybd et al., 1983). Samples were then temporarily stored in plastic petri dishes with glycerol until reading. From there, we used a dissecting scope at 35X to quantify galls within three days of staining (Fig. 1).

Figure 1:

Data analysis was completed via RStudio version 1.3.1093 (R Core Team, 2020). For each variable, a one-way ANOVA followed by Tukey’s HSD post-hoc analysis was conducted to determine significance between treatments for each plant and nematode parameter collected. Unless otherwise stated, p-values were obtained via a Tukey post-hoc analysis (a=0.05).

During both trials, we did not observe any notable phytotoxic defects to plant coloration or growth for any of the treated plants. In the first trial, the positive control (inoculated, nontreated) possessed the lowest root mass of the five treatments. All of the positive control plants were heavily galled with lower masses (Figs. 2A,B). Root masses of plants treated with Velum Prime and MN21.2 were 5.76x and 5.72x heavier than the positive (inoculated, nontreated) control plants, respectively (P < 0.05). In the second experiment at 5 WPA, the positive control again possessed the lowest root mass. However, at 10 WPA, the root mass of the positive control was not significantly lower than any of the four treatments (Fig. 2B). The pots treated with MN21.2 were the only treatments to show significant increases on root mass in both trials. There were no significant trends observed from plant height from either trial (P > 0.05).

Figure 2:

In the first greenhouse trial, plants with a higher Gall Index Rating (GIR) indicated more severe infestations and presence of galling on roots (Fig. 3A). Untreated (positive control) plants were significantly more galled than treatments M11.2, MN21.1, MN21.2, and Velum-applied plants (P < 0.01). In the repeated trial, plants treated with MN21.2 had a lower average GIR than both the inoculated positive control and plants treated with MN11.2 (Fig. 3B; P < 0.001). Plants treated with Velum Prime or MN21.2 possessed a significantly lower GIR than the positive control plants during both the first and second trials.

Figure 3:

In the first greenhouse trial, all four treatments (MN11.1, MN11.2, MN21.1, and MN21.2) were lower than concentrations in the positive control plant roots. Both versions of MN21 were lower than MN11.1 (P < 0.05). In the second trial, MN21.2 reduced final (10 WPA) juvenile soil populations by 89.13% and mature female galls by 78.55% (P < 0.01). Velum Prime decreased final (10 WPA) juvenile soil populations by 90.08% and mature female galls by 79.51% (P < 0.01). MN21.2 significantly reduced female galls per 1 g roots for both trials (P < 0.01).