Characterizing the Interaction Between Root-knot Nematodes and Soil-borne Fungi which are Pathogenic to Passion Fruit (Passiflora edulis)

Seeds of passion fruit cultivar UENF Rio Dourado were purchased from Rio Norte Sementes (Campos, Brazil) and sown in 300-cm³ plant growth tubes (Supplementary Fig. 1A) filled with a substrate composed of washed riverbed sand and soil (2:1). The substrate was subjected to solarization in tubular metal containers (12-cm diameter) for 3 d before use. The temperatures inside the containers (that is, 60–80^oC) are known to eradicate both fungi and nematodes (Martins et al., 2003). The plants were cultivated in a growth chamber under a 12-hr photoperiod, with illumination of 106 m × mol^-1 × m^-2 provided by 40-watt fluorescent light tubes. The day and night temperatures were set to 27 and 26^oC, respectively.

The plants were assigned to one of the following treatments: (i) inoculation with the nematode 60 d after seeding (DAS); (ii) inoculation with the nematode at 60 DAS and fungus at 120 DAS; or (iii) inoculation with the fungus at 120 DAS. Noninoculated plants served as controls. The treatments were randomly arranged in trays, with 10 replicates/treatment, for a total of 40 plants. This experimental design was employed to investigate the interactions of M. incognita-Neocosmospora sp. (isolates 311 and 511); M. incognita-F. nirenbergiae (isolate 022); M. javanica-Neocosmospora sp. (311 and 511); and M. javanica-F. nirenbergiae (022). For each interaction, two identical and independent assays (1 and 2) were conducted.

M. incognita and M. javanica were obtained from cultures maintained in tomato (Solanum lycopersicum L.) plants in a greenhouse at Universidade Federal de Viçosa (Brazil). Their identity was ascertained by morphology and esterase phenotyping (Esbenshade and Triantaphyllou_, 1985). These cultures were transferred to and maintained in passion fruit plants in a greenhouse. To obtain nematode inocula, infected roots were cleaned with tap water and submitted to nematode extraction through the method of Coolen and D’Herde (1972) but without adding kaolin in the blender. The resulting suspension was passed through 65- and 500-mesh sieves (250 and 25 mm openings, respectively). The nematode eggs and second-stage juveniles (J²) retained on the 500-mesh sieve were suspended in water, and three 1 mL aliquots were counted on Peter’s slides under a stereomicroscope. The inoculum was calibrated to 350 eggs + J²/mL of water. For inoculation, two 5-cm deep holes were made in the soil around the plant, into which 10 ml of the inoculum (3,500 eggs + J²) was applied. Ten tomato seedlings “Santa Cruz”, known to be susceptible to root-knot nematodes, were inoculated using the same procedures to attest to the viability of the inocula. These plants were cultivated under the same condition as the passion fruit, except in 5 L plastic pots.

Neocosmospora sp. 311, 511, and F. nirenbergiae 022 originated from passion fruit orchards which were affected by collar rot and fusarium wilt in the municipalities of Campos dos Goytacazes, Viçosa, and Cáceres (Brazil), respectively. Stock cultures maintained in anhydrous silica gel were routinely revived, inoculated in passion fruit plants, cultivated in a growth chamber, and re-isolated to avoid losing pathogenicity. For inoculum preparation, the fungi were cultivated in potato dextrose agar (PDA) medium in Petri dishes for 7 d, at 24^oC, and in a 12-hr light/ dark cycle. Inoculation was performed according to the method of Fischer et al. (2010): a cleft (10-mm long, 5-mm wide, 1-mm deep) was made in the plant stem with a sterile blade, at 1 cm above the soil. A 6-mm wide agar disk collected from the edge of the fungus colony was placed on the exposed stem region and sealed with a sterile plastic film.

All assays were conducted until 165 DAS, except for those involving Neocosmospora sp. 311. This isolate always induced rapid decline of the plants, forcing evaluations at 135 DAS due to plant death. At the end of the assays, the plants were removed from the growth tubes, and the root system was gently cleaned with tap water. Nematode and fungal pathogenicity was expressed according to (i) the severity of shoot symptoms: leaf chlorosis, wilting, and leaf chlorosis, wilting and necrosis; and whole plant wilting and death (these symptoms were photographed and ranked on the modified scale of Santos (1997) – 1: no symptoms; 2: chlorosis restricted to one or two leaves; 3: leaf chlorosis in the whole plant and some leaf wilting; 4: whole plant wilting and necrosis of leaves; and 5: plant death); (ii) variables related to plant growth: plant length (PL) (from apical meristem to the most distal rootlet, in mm); shoot length (SL) (in mm); shoot fresh mass (SM) (in g); root system fresh mass (RSM) (in g) and root system volume (RSV) (calculated through water displacement in a graduated glass tube (in mm³); and (iii) the volume of lesioned tissue (VLT) in the collar-stem region (in mm³). Because fungi-induced lesions in the stem typically ascended uniformly in a sagittal plane, the length and width of the lesions were measured with a digital pachymeter (Supplementary Fig. 1B,C) and applied in the formula to calculate the volume of a cylinder – π × r² × h – in which r is the radius and h is the height. The fungal damage was also expressed by the length of the lesion (LL).

Nematode reproduction was expressed as the final population (Pf), used in turn to compute the reproduction factor (RF = Pf/Pi). To obtain Pf, eggs and J₂ were extracted from roots through trituration in a commercial blender (method of Boneti and Ferraz, 1981). The resulting suspension were sieved and counted as described before. This procedure was also employed in the tomato plants which were used to attest to the viability of the inoculum.

To confirm re-isolation of the fungi, fragments from inner tissues were collected in the boundary between lesioned and healthy tissues in the collar-stem region. These tissues were then disinfested in a 70% aqueous ethanol solution for 1 min and in a 0.5% aqueous sodium hypochlorite solution for 1 min. After double cleansing in sterile distilled water, the fragments were transferred to PDA medium and cultivated as described before. Seven days later, the colonies (Supplementary Fig. 1D) were identified to confirm the re-isolation of Neocosmospora sp. and F. nirenbergiae.

The pathogens significantly affected variables expressing growth of passion fruit plants – SM, RSM, PL, and SL (Supplementary Table 3).

In the assays involving Neocosmospora sp. 311, the fungus negatively affected (P < 0.05) PL, SL, SM and RSM in most assays (Figs. 1,2). M. incognita and M. javanica did not affect plant growth (P > 0.05). Synergy between pathogens occurred only in the reduction of SM in plants co-parasitized by Neocosmospora 311 and M. incognita, in one assay.

In the assays involving Neocosmospora sp. 511, the fungus negatively affected (P < 0.05) plant growth in most assays (Figs. 3,4), while M. incognita and M. javanica did so occasionally. Synergy occurred only in the reduction of RSM in plants co-parasitized by Neocosmospora sp. 511 and M. javanica, in one assay.

In the assays involving F. nirenbergiae, the fungus negatively affected SM and RSM only, in one assay (Figs. 5,6). The nematodes had no effect on plant growth, and no synergy between pathogens occurred.

The nematodes and the fungi, acting alone or combined, had only occasional negative effect (P < 0.05) on the plants' RSV (Table 1).

Neocosmospora sp. 311, Neocosmospora sp. 511, and F. nirenbergiae 022 lesioned the collar-stem region of all plants in all assays. The lesions ascended on the stem up to 86 mm, 38 mm, and 56 mm from soil, respectively. No lesions developed in the nematode-parasitized plants or in the noninoculated control plants. The co-parasitism by M. incognita or M. javanica had no effect (P > 0.05) on the VLT in the collar-stem region (Table 2).

According to the modified scale of Santos (1997), there were no differences (P > 0.05) between the severity of symptoms induced by the nematodes and fungi, alone or combined (Table 3). Nonetheless, visually fungal symptoms were more severe than those caused by nematodes, sometimes leading to plant death (Supplementary Figs. 2,3).

M. javanica and M. incognita induced root galls on all inoculated plants, but the maximum RF observed was 0.44 for M. incognita and 0.61 for M. javanica (Table 4). In the tomato plants used to attest to the viability of the inocula, the mean M. incognita RF values were 26 (standard deviation [SD] = ± 14.5) and 9.9 (SD = ± 1.52) in assays 1 and 2, respectively. For M. javanica, the values were 48 (SD = ± 27.38) and 8.1 (SD = ± 1.32), respectively. Fungal co-parasitism affected (P < 0.05) nematode reproduction positively or negatively in at least one of the assays, but the plants remained resistant to the nematodes (RF < 1).

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6