*2.2. Experimental Design*

In an upland rice field of 6 ha severely infected with *M. graminicola* in 2018 (root gall index 8 in a scale range of 0–10 [16]), the experimental layout was a randomized complete block design with 5 blocks. Each block was divided into three plots (5 × 5 m) where 3 managemen<sup>t</sup> treatments were assigned randomly: Uncultivated (U); Treated (T), where three separate cycles of trap crop were carried out; Control (C), where the rice was sown and left to grow until the end of the three cycles in T plots.

The experimental area was located 15 m from the north edge of the selected field and plots were separated by 2-m-wide untreated buffers to avoid effects from the migration of nematodes, and to facilitate operations within the different plots.

At the end of April 2019 (T0), the experimental area was ploughed, the plots were delimited, and soil sampling was carried out as described below. In plots C and T, 0.60 kg/plot of long-grain rice cv. S. Andrea was sown, and only in T at the second leaf stage (BBCH-scale 12 [17]), after 15 days, rice plants were destroyed with a registered herbicide. This cycle was repeated three times, as illustrated in Figure 1. In particular, between the destruction of the rice plants of the previous cycle and the sowing of the next ones, a week was always allowed to pass.

**Figure 1.** Illustration of the experimental design used to evaluate the trap crop technique in the managemen<sup>t</sup> of *Meloidogyne graminicola*. Uncultivated (U), Control (C), and Treated (T). (Drawings by Giuseppe Mazza).

#### *2.3. Evaluation of Nematode Density in the Soil*

To evaluate the number of eggs and juveniles of *M. graminicola* and compare the population density before (T0) and after (T1) the trap crop technique experiment, in each plot, three soil samples (approximately 0.5 kilo/sample) were randomly collected using a hand shovel. All samples were individually placed in a plastic bag, labeled, and then brought to the laboratory of the Minoprio Foundation (Como, Italy). These materials were stored in a climatic chamber at about +4 ◦C until they were processed for analysis. For each sample, 200 cc of soil were placed in a plastic bucket, and 6 L of water were added. The resulting slurry was vigorously swirled for about 30 s, and after 45 s of sedimentation time, the supernatant suspension was decanted through a 40 μm sieve. Water was again added to the soil in the bucket and the process was repeated twice.

To dissolve the gelatinous matrices of the egg masses and obtain the suspension with nematodes, the sodium hypochlorite (5% NaOCI) technique described in Byrd et al. [18] and the centrifugal flotation method [19] were carried out. Nematodes were collected in a glass dish for examination and counted under an optical microscope LEICA MZ12 (Leica Microsystems, Heerbrugg, Switzerland).

#### *2.4. Evaluation of the Plant Population Density*

After the third cycle of trap crop, all experimental areas were mechanically worked to destroy rice plants and weeds, taking care not to transport soil from one plot to another with the machines. Subsequently, each plot was sown with the same amount of rice (see above) at the same time. At the second leaf stage of the plants, in order to record the number of rice plants per unit area, a circle frame (0.3 m2) quadrant was used. It was randomly launched five times in each plot and all plants rooted inside the circle were counted.

#### *2.5. Evaluation of Root-Gall Index and Plant Growth*

To evaluate the damages on plants, the gall index was assessed on the rooting system and the plant growth was measured on the aerial part of the same plants.

At the same time of the evaluation of the plant population density, a representative sample (20–23 rice plants/plot) was collected with the whole root system. Plants of each plot were placed in a labeled plastic bag and analyzed in the lab within 24 h of collection.

The roots were rinsed with tap water, placed on paper towels to eliminate excess water, and observed to assess the severity of root damage caused by the amount of galling. The evaluation of root gall indices was studied visually using the root evaluation chart developed by Bridge and Page [16].

The same plants were individually photographed (Canon PowerShot G3—Ota, Tokyo, ¯ Japan) with a bare scale, and the plant length (distance from the coleoptilar node to the tallest leaf) was recorded using ImageJ program (Image Processing and Analysis in Java) Version 1.53a (Wayne Rasband, National Institute of Health, Washington, DC, USA).
