Passifloraceae Rootstock Performance Against Soil Pathogens in Yellow Passion Fruit Crops (Passiflora edulis f. flavicarpa Degener)

Abstract

The response of five Passiflora species as rootstocks for yellow passion fruit was evaluated against the Meloidogyne incognita complex and Fusarium oxysporum f. sp. passiflorae. Individual, sequential, and simultaneous inoculations were applied, quantifying disease severity, nematode reproduction (RF), biomass, and plant vigour. In addition, integrated analysis was performed using the Combined Tolerance Index (CTI) to confirm the simultaneous interaction of the inoculation condition. The graft compatibility index (GCI) of the materials under study was also determined. The results showed critical functional differences; P. maliformis showed tolerance in terms of compensatory vigour but presented high susceptibility to the nematode and low graft affinity (GCI = 1.39). In contrast, P. platyloba emerged as the superior genotype, combining effective resistance to Meloidogyne (zero incidence at critical stages), excellent anatomical compatibility (deviation from the ideal of 0.04), and physiological stability superior to the control. Although P. nitida showed resilience in biomass under severe stress conditions, it is concluded that P. platyloba is the most promising alternative for use as rootstock. This is because its morphological affinity and health resistance ensure crop sustainability in field conditions and promote more sustainable agricultural practices.

1. Introduction

Tropical fruits belonging to the Passifloraceae family represent a viable alternative in sustainable agriculture. They vary in size, shape, and flavour depending on the species and are characterised by their juicy and aromatic pulp, which contains numerous seeds. Passion fruit (Passiflora edulis f. flavicarpa) is an agricultural crop of great importance in South America, especially in countries such as Brazil, Colombia, and Ecuador [1]. This fruit is valued for its high concentrations of vitamin C, polyphenols, and minerals, which furnish it with antioxidant properties and beneficial effects in terms of cardiovascular and metabolic health [2,3].

In addition, passion fruit pulp contains a wide range of bioactive compounds, including flavonoids, carotenoids and phenolic acids, which contribute significantly to antioxidant activity and nutritional value, increasing its importance in functional food markets. Recent studies have also highlighted that Passiflora fruits exhibit anti-inflammatory, antihypertensive and hypoglycaemic properties due to their phytochemical composition, reinforcing their potential role in promoting human health and dietary diversification [4].

This crop is of economic importance in Ecuador. In 2025, the harvested area of passion fruit in monoculture was 4287 ha, while in associated systems, it reached 1389 ha. In terms of production, the monoculture registered 33,397 metric tons, while the associated system produced 14,028 metric tons [5], the total production represents an estimated annual economic value of approximately USD 25,089,900, highlighting the relevance of this crop for rural livelihoods and agricultural development in the country [6].

However, despite its productive and economic importance, this crop faces limitations associated with soilborne pathogens, such as Fusarium oxysporum f. sp. passiflorae [7] and other Fusarium species reported in passion fruit production systems, including Fusarium solani and related species complexes identified through molecular characterization [8] and the nematode Meloidogyne incognita. These pathogens cause vascular wilt, root damage, and severe physiological stress, significantly affecting plant growth and productivity [9]. As a consequence, infections by these pathogens can reduce yield by up to 60% and shorten the productive lifespan of plantations from 36 to 18 month [10,11].

This situation has led producers to resort to the prolonged use of pesticides, such as propiconazole and difenoconazole [12], as well as nematicides like methyl bromide and carbamates (oxamyl and fenamiphos) [13,14]. The use of these pesticides cause environmental damage, such as soil and water contamination, since more than 95% of the applied agrochemicals can be lost outside the target organism [15]. The intensive use of agrochemicals in tropical fruit production has also been associated with biodiversity loss, soil degradation, and the emergence of resistant pathogen populations, which limits the long-term effectiveness of chemical control strategies [16].

Furthermore, these pesticides represent a risk to human health [17] and promote the development of microbial resistance. This same behaviour has been reported in passion fruit-producing areas under different production systems in Colombia [18]. In response to these challenges, integrated disease management strategies combining biological control agents, plant extracts, and resistant rootstocks have been proposed as sustainable alternatives for controlling soilborne pathogens in Passiflora crops [19].

Among these strategies, the use of resistant rootstocks has received increasing attention because it provides long-term protection against soilborne pathogens while reducing the dependence on chemical pesticides [10]. In addition to grafting strategies, other sustainable alternatives have been developed, including the application of bio-inputs, plant extracts, and nematicides aimed at reducing populations of phytopathogenic organisms in the soil. These, strategies have significantly contributed to reducing the populations of phytopathogenic organisms [20].

In this context, the use of resistant rootstocks has emerged as a sustainable and economically affordable management tactic to reduce the effect of soil pathogens [21]. Several types of wild Passiflora, such as P. edulis, P. maliformis, P. mucronata, and P. nitida, have shown varying levels of resistance or tolerance to F. oxysporum and M. incognita [22] In recent studies, it has been observed that grafts on P. nitida have mortality rates of less than 12.5% when faced with F. oxysporum f. sp. passiflorae, evidencing moderate to strong resistance (less than 12.5% mortality). Contrastingly, plants grafted on P. edulis as rootstock registered mortality rates above 56.25%, which confirms their high susceptibility [23]. It is important to consider the specificity of the special forms of F. oxysporum according to the agricultural species: for example, isolates from wild granadilla (P. nitida) are not pathogenic to passion fruit (P. edulis), which explains apparently opposite reports and justifies their comparative inclusion in this study [21]. In contrast, Meloidogyne spp. is markedly polyphagous and of low specificity: its host range encompasses most flowering plants and affects virtually all crops. In the presence of the pathogenic complex made up of nematode and fungus (M. incognita and F. solani), genotypes such as P. cincinnata, P. mucronata, and P. setacea have shown fewer symptoms compared to other rootstocks evaluated (P. giberti, P. nitida and P. morifolia), confirming that they are highly resistant (>80%) [24]. However, the comprehensive evaluation of their response to M. incognita complex and F. oxysporum f. sp. passiflorae, considering graft compatibility and temporal dynamics, has not yet been sufficiently explored to identify the most robust genotype.

The relationship between these pathogens is complicated and can alter the reaction of grafted crops. Research on cucurbits has shown that contamination with M. incognita can aggravate symptoms of F. oxysporum f. sp. niveum wilt and impair performance [25]. This highlights the importance of evaluating the combined reaction of rootstocks to both pathogens. Therefore, the purpose of this research was to evaluate, under greenhouse conditions, the resistance/tolerance of five species of Passiflora as yellow passion fruit rootstocks against infection by F. oxysporum and M. incognita, as well as the compatibility between the graft and rootstock, in order to identify Passiflora genotypes that contribute to decreasing the negative effect of soil pathogens on the plant and provide a sustainable alternative to reduce the use of chemicals.

2. Materials and Methods

2.1. Experimental Site

The research took place at the Central Experimental Station of the Amazon (EECA) belonging to the National Institute of Agricultural Research (INIAP), located in the parish of San Carlos, canton of Joya de los Sachas, province of Orellana, Ecuador, at an altitude of 250 m.a.s.l. It is located at coordinates 00°21′31.2′′ S and 76°52′40.1′′ W. Inside the greenhouse, an average temperature of 35 °C was recorded, maintaining a constant relative humidity of 70% [26].

2.2. Methodological Procedure

2.2.1. Plant Material

P. edulis f. flavicarpa Deg. (yellow passion fruit) was used as the scion for graft compatibility evaluation and as a non-grafted control. Five species of Passiflora were evaluated as potential rootstocks: P. platyloba (wild passion fruit), P. maliformis (conch apple or wild purple passion fruit), P. nitida (bell apple), P. edulis f. edulis (purple passion fruit or gulupa) and P. quadrangularis L. (giant granadilla or badea) (Figure 1A–F). Plants were grafted (cleft grafting) before the inoculation of the pathogens.

Seed Germination

After being extracted from ripe fruits from vigorous plants, the seeds were washed and then dried at 16 °C for 72 h. The seeds were subjected to an osmotic pre-germination treatment with PEG 6000 (polyethylene glycol 6000) at −1.2 MPa for 36 h in an incubator (Memmert model INCO2, Schwabach, Germany) at 25 °C, following the procedure adapted for passionflowers by [1].

The substrate used for sowing the seeds was sieved and then sterilised in a vertical stainless steel autoclave (Novatech model EV-24, Tlaquepaque, Mexico) at 120 °C for 20 min. The seeds were left to germinate in trays, then the resulting seedlings were transplanted into 3.5-pound nursery sleeves. They remained there for 60 days until they reached a minimum diameter of 1 mm in the stem, as at this point, the cambium is active and lignification is minimal, ensuring a comparable wound/inoculation and reducing variability, at which point inoculation was performed with the evaluated pathogens [26].

2.2.2. Experimental Design and Treatments

A randomised complete block design (RCBD) with three replications was used [17]. Factor A corresponded to the rootstock species P. platyloba, P. maliformis, P. nitida, P. edulis f. edulis and P. quadrangularis, and Factor B to five inoculation conditions: C1 = M. incognita (N); C2 = F. oxysporum f. sp. passiflorae (F); C3 = sequential inoculation with M. incognita followed by F. oxysporum at seven days (N→ F); C4 = sequential inoculation with F. oxysporum followed by M. incognita at seven days (F→ N); C5 = simultaneous inoculation with both pathogens (M. incognita and F. oxysporum) (N/F); and yellow passion fruit plants (P. edulis f. flavicarpa) without grafting [26]. These abbreviations were used consistently in the tables and in the Results and Discussion sections to refer to each infection condition.

2.2.3. Inoculum Extraction Process

Extraction Process of F. oxysporum f. sp. passiflorae

Fusarium oxysporum f. sp. passiflorae was isolated from yellow passion fruit stems with typical symptoms of vascular wilt. The fragments were disinfected with 1.5% sodium hypochlorite for 5 min. After being cleaned with sterile distilled water, these were put on Potato Dextrose Agar (PDA) medium and then incubated for 7 days at 27 °C. The whole time needed for isolation and purification, including initial plating and single-spore subculturing, is represented by the 18-day timeframe. Identification was made by observing their distinctive morphological features (microconidia in false heads, falcate macroconidia with 3–5 septa and intercalated and terminal chlamydospores), following the identification keys of Nelson et al. [27] and Leslie and Summerell [28]. In addition, pathogenicity tests were carried out on Passiflora leaves; these tests allowed the symptoms of necrosis to be replicated from 5 days after inoculation, which confirmed its pathogenicity [29].

Extraction of Meloidogyne incognita in the Root

Nematode extraction was performed following the methodology of [30]. The nematodes were collected from infected roots of naranjilla (Solanum quitoense). The roots were washed under running water to remove soil debris, then cut into segments of about 1 cm under sterile conditions and 10 g of root tissue was weighed. Subsequently, it was divided into portions of 100 g to extract the inoculum. For extraction, the root tissue was processed in a household blender (Oster, FL, USA) with 100 mL of distilled water at low speed for 20 s in two 10-s intervals with a 5-s pause between each cycle. The homogenate was filtered through a system of 60, 100, and 500 mesh sieves in descending order. The first two sieves were washed with distilled water to drag the juveniles towards the final sieve. The extract was collected, measured at 100 mL, and homogenised by agitation. A 4 mL aliquot was used to identify and count second-instar juveniles (J2) under a trinocular light microscope (IM-5, OPTIKA, Bergamo, Italy), equipped with LWD IOS objectives, X-LED illumination for optimal visibility, and EWF10X/22 mm eyepieces. J2 were identified by their typical morphological characteristics (filiform body, well-defined stylet and oesophagus typical of M. incognita), following the keys of Hunt and Handoo [31] and the pictorial guide of Ángel-García et al. [32].

The same procedure was repeated for the extraction of nematodes in the roots of Passiflora evaluated as rootstocks.

Extraction of Meloidogyne incognita in Soil

The extraction process of M. incognita in soil was carried out via the incubation method, using subsamples of 100 cm³ of previously homogenised soil. Each subsample was placed on a system of aluminium plates with plastic mesh and tissue paper, forming a thin layer of moistened soil that was incubated for three days. Subsequently, the water suspension with nematodes was recovered by washing it through a 25 μm sieve; it was then measured at 100 mL and homogenised. From this suspension, a 4 mL aliquot was taken for identification and counting under a microscope (expressing the results as population density of nematodes per 100 cm³ of soil) in order to verify the homogeneity of the inoculum between experimental units [26].

2.2.4. Inoculation of Seedlings

Seedlings of 60-day-old passionflowers were inoculated with M. incognita at a dose of 1200 J2 (applied 5 cm deep into the rhizosphere) and subsequently covered with 2 cm of sterilised soil. For inoculation with F. oxysporum f. sp. passiflorae, the stem incision method was used: 20 mL of a suspension of 5 × 10⁶ mL⁻¹ conidia was applied to the wound, administered by directed irrigation at the base of the stem in order to facilitate the entry of the pathogen into the vascular tissue and ensure its establishment. The mechanical wound was performed with a previously disinfected stylet, reaching a depth equivalent to 50% of the stem diameter, which had a root neck diameter of approximately 6 mm [33] The control plants were not inoculated [32].

2.2.5. Experiment Handling Conditions

The inoculated and grafted plants were kept in the greenhouse at 25–28 °C and 70–80% relative humidity with manually controlled irrigation to maintain the field capacity of the substrate. Next, 300 mL of water per plant was applied, adjusted according to the humidity of the substrate. The inoculated plants were irrigated with distilled water to avoid contamination [26]. In the grafted plants, 5 g of NPK 15–15–15 was applied to the substrate after 30 days. According to phytosanitary monitoring, the presence of chopper insects was detected. For the control, an application of abamectin at 0.5 mL·L⁻¹ was performed. The trial was conducted in high-capacity germination trays to maintain homogeneous conditions of humidity and ventilation and optimise the experimental space.

2.2.6. Response Variables and Measurements

Assessments were performed at 15 and 30 days after inoculation [34,35] with the purpose of recording both the initial phase of symptom expression and the advanced progression of diseases. The following phytopathological variables were considered: wilt severity, yellowing, incidence, lesion size, and reproduction index. These variables, recognised in the phytopathological literature, allowed us to comprehensively characterise the progression, magnitude, and temporal dynamics of the damage caused by pathogens in susceptible hosts [26].

Fusarium-Associated Variables

Wilt severity: This was determined using a visual scale from 0 to 6, in which each category corresponds to the percentage of foliage affected (

Table 1. Taylor and Sasser Wilt Severity Scale [36].

Class	Intensity *
0	No symptoms (0%)
1	Mild wilting (1–15%)
2	Moderate wilting (16–25%)
3	Severe wilting (26–35%)
4	Very severe wilting (36–45%)
5	Severe wilting and stunting of the plant (>50%)
6	Death of the plant

* Percentage of visible impairment of spots on leaves and shoots.

). Leaf yellowing was determined using an ordinal visual scale from 0 to 4, where: 0 = no yellowing; 1 = mild yellowing; 2 = moderate yellowing; 3 = severe yellowing; and 4 = very severe or generalised yellowing, visually assessed in the foliage of each plant.

Lesion size (mm): This was quantified 30 days after inoculation by measuring the necrotic area visible on the stem at the neck of the plant, at the point where the wound was made for the entry of F. oxysporum f. sp. passiflorae. The lesion was delimited with the help of a millimetre pattern and measured with a calliper (CD-6, Mitutoyo, Kawasaki, Japan), expressing the result as an affected surface in square millimetres at the inoculation site.

Variables Associated with Meloidogyne incognita

Severity by galls numbered 0–5: At the end of the bioassay, each root system was washed and the total number of galls produced by Meloidogyne spp. in each plant was counted. This count was classified on the root-galling severity scale proposed by Ballén-Taborda et al. [37] (

Table 2. Root-galling severity scale by Ballén-Taborda et al. [37].

Clase	Intensity
0	0 galls
1	1–2 galls
2	3–10 galls
3	11–30 galls
4	31–100 galls
5	>100 galls

).

Reproduction index (RI = FP/IP): This was calculated from the number of nematodes recovered by extraction from the roots and soil. The final population (FP) was obtained by adding the juvenile J2 and eggs of M. incognita present in the root system and in 100 cm³ of soil per plant, while the initial population (IP) corresponded to the 1200 J2 applied in the inoculation. The RI was expressed as a continuous numerical value. Soil counts were included to verify inoculum homogeneity between experimental units and to complement the characterisation of pathogen interaction with the rootstock root system [26] (

Table 3. Host categorisation according to the reproduction index (FP/IP) of M. incognita and the relative performance of the control.

Nematode Reproduction Index	Host Performance Compared to the Control
	Minor	Equal or Greater
FP/IP < 1	2. Resistant–Not tolerant	1. Resistant–Tolerant
FP/IP > 1	4. Susceptible–Not tolerant	3. Susceptible–Tolerant

FP/IP reproduction rate of M. incognita. Plants are categorised as resistant if FP/IP is less than 1 and as tolerant if their yield is equivalent to or greater than 1 of the control [26,38].

).

Growth Variables and Aboveground Biomass of Seedlings

The growth and biomass of the seedlings were evaluated using three groups of physiological variables: (i) height increase, calculated as the difference between the final and initial height measured from the neck to the apex, which enables one to estimate the vigour and elongation capacity of the plant; (ii) stem diameter, measured with a digital calliper (CD-6, Mitutoyo, Kawasaki, Japan); and (iii) fresh weight and dry weight of foliage and root (g). Fresh weight was obtained by weighing aboveground and belowground biomass on a precision digital balance: Citizen SCALE CG 4102C analytical balance (Citizen Group, Gardena, CA, USA). For the dry biomass, the foliage and roots were placed in a forced-air stove (BINDER, ED 260, Tuttlingen, Germany) at 70 °C until a constant weight was reached and then weighed on the same digital scale.

Compatibility Variables

To obtain this parameter, nine plants were taken from the grafted treatments and divided into three replications, giving a total of 45 experimental units. The plants used in this study were not inoculated with nematodes or Fusarium.

Compatibility Index (GCI): This was determined as the ratio between the stem’s diameter above the junction and its diameter below the junction. Values close to 1 indicate high compatibility, while a constriction (GCI < 1) or a marked bulge (GCI > 1) indicates possible incompatibility or growth imbalance. The measurement was performed at 30 days [39,40].

$$\mathrm{GCI} ={\displaystyle \frac{{\mathrm{Diameter}}_{\mathrm{above}}}{{\mathrm{Diameter}}_{\mathrm{below}}}}$$

(1)

2.3. Statistical Analysis

The data were analysed with the Infostat software version 2017 (29 September 2020, Córdoba, Argentina). A parametric analysis of variance (ANOVA) (p < 0.05) with Tukey’s test at 5% for mean separation was applied.

To assess the overall response of rootstocks to combined biotic stress and mitigate the variability inherent in complex interactions, physiological (benefit) and pathological (damage) variables were integrated by calculating a Combined Tolerance Index (CTI) [41]. The CTI was constructed by normalising the variables in a range of 0 to 1.

The benefit variables (B) correspond to the fresh weight (B_FW) and dry weight (B_DW) of the biomass. In this context, xᵢ is the variable’s observed value for the experimental unit, xₘᵢₙ is its lowest recorded value, and xₘₐₓ is its greatest value, where the normalised (scaled) benefit score for plant i is obtained by Bᵢ_esc. Better physiological performance is indicated by higher readings [42].

The benefit variables were rescaled using the following formula:

$${\mathrm{B}}_{\mathrm{i}\text{\_}\mathrm{esc}} = {\displaystyle \frac{x_i{-x}_{min}}{x_{max}{-x}_{min}}}$$

The damage variables (D) included three components: number of root nematodes (D_nem), reproduction index (D_RI), and lesion size (D_les). Where the scaled damage score for plant i is represented by Dᵢ_esc; higher values denote less harm (more tolerance). Each variable was individually rescaled inversely, where 1 represents the absence of harm (maximum tolerance) and 0 represents the maximum damage observed in the trial:

$${\mathrm{D}}_{\mathrm{i}\text{\_}\mathrm{esc}} = 1 - {\displaystyle \frac{x_i{-x}_{min}}{x_{max}{-x}_{min}}}$$

These components were then integrated into a single average damage value ($\overline{D_{esc}}$) for each experimental unit:

$$\overline{{\mathrm{D}}_{\mathrm{esc}}} = {\displaystyle \frac{{\mathrm{D}}_{\mathrm{nem}\text{\_}\mathrm{esc}}+{\mathrm{D}}_{\mathrm{R}\mathrm{I}\text{\_}\mathrm{esc}}+{\mathrm{D}}_{\mathrm{les}\text{\_}\mathrm{esc}}}{3}}$$

The same procedure was applied to the benefit variables (B) to obtain $\overline{B_{esc}}$. Finally, the crude CTI was calculated as the difference between the averages of the benefit and injury components, normalising the final result in a range of 0 to 1 for statistical comparison, using the formula:

$${\mathrm{CTI}}_{\mathrm{raw}} = \overline{{\mathrm{B}}_{\mathrm{esc}}} - \overline{{\mathrm{D}}_{\mathrm{esc}}}$$

Due to the sample size (n = 3) and the non-parametric distribution of the integrated variables, the Mann–Whitney U test was used to determine differences between each rootstock and the control (P. edulis f. flavicarpa). Each treatment consisted of three independent biological replicates; in total, 180 experimental units were evaluated, considering six plant materials, five inoculation conditions, and two evaluation times. A significance level of p ≤ 0.05 was considered. In addition, the magnitude of the effect was quantified by calculating the mean differential (∆), defined as the arithmetic difference between the average of the rootstock evaluated and the average of the control (${\overline{x}}_{material}-{\overline{x}}_{control}$). This indicator allowed us to determine the direction of the biological response, where positive values indicate a performance that lies above the commercial reference and negative values indicate a detriment in the variable analysed.

Additionally, differences with values of 0.05 < p ≤ 0.10 were discussed as biologically relevant trends since, in complex pathosystems, consistent response patterns usually precede strict statistical significance.

3. Results

3.1. Response of Passiflora Rootstocks to Different Inoculation Conditions

Under exclusive inoculation with M. incognita (N), significant differences were detected between the genotypes evaluated (p < 0.05; Tukey’s test), with responses that varied according to the time of exposure. At 15 days after inoculation, P. maliformis stood out for its early vigour in fresh biomass and weight, although it showed primary susceptibility with high nodulation. This growth trend was consolidated at 30 days, where P. maliformis reached a total weight of 50.97 g and a final height of 223.67 cm, statistically sharing the latter metric with P. platyloba. However, from a phytosanitary point of view, P. platyloba was the most outstanding rootstock (letter b in Tukey’s test) in contrast to P. edulis (66.67%) and the other genotypes, which reached 100% levels of involvement (Table S1).

The CTI analysis showed responses that depended on genotype and time of exposure. At 15 days after inoculation, a superior early defensive response was observed in three genotypes: P. maliformis, P. platyloba, and P. quadrangularis had higher CTI values than the control (P. edulis f. flavicarpa), with mean differences (ΔCTI) of +0.33, +0.30 and +0.19, respectively. These differences were not statistically significant according to Tukey’s test, but they indicate a biological trend of greater initial tolerance to the nematode (

Table 4. Summary of the differential performance (ΔCTI) of five Passiflora rootstocks with respect to the control (P. edulis f. flavicarpa) under different inoculation conditions.

Inoculation Condition	Time (Days)	ΔCTI of P. maliformis	ΔCTI of P. platyloba	ΔCTI of P. quadrangularis	ΔCTI of P. nitida	ΔCTI of P. edulis f. edulis
M	15	+0.33 * ± 0.08	+0.30 ± 0.10 *	+0.19 ± 0.11 *	+0.10 ± 0.10	+0.03 ± 0.17
M	30	+0.20 ± 0.09	+0.25 ± 0.05 *	+0.11 ± 0.12 *	+0.04 ± 0.06	−0.06 ± 0.11 *
F	15	+0.32 ± 0.05 *	+0.32 ± 0.01	+0.05 ± 0.22	−0.08 ± 0.09	+0.05 ± 0.12
F	30	−0.16 ± 0.25	−0.18 ± 0.03	−0.12 ± 0.21	−0.33 ± 0.19 *	−0.26 ± 0.13 *
N→F	15	−0.10 ± 0.18	−0.13 ± 0.22	−0.26 ± 0.11 *	−0.23 ± 0.18 *	−0.17 ± 0.12 *
N→F	30	−0.21 ± 0.03 *	−0.18 ± 0.19	−0.16 ± 0.06 *	−0.18 ± 0.06 *	−0.01 ± 0.08
F→N	15	+0.22 ± 0.61	+0.32 ± 0.27 *	−0.10 ± 0.22	−0.10 ± 0.16	0.00 ± 0.03
F→N	30	+0.11 ± 0.23	0.00 ± 0.19	+0.13 ± 0.26	+0.14 ± 0.17	−0.02 ± 0.01
N/F	15	−0.23 ± 0.10 *	+0.07 ± 0.32	−0.06 ± 0.12	0.00 ± 0.05	−0.05 ± 0.08
N/F	30	+0.09 ± 0.19	+0.17 ± 0.23 *	+0.26 ± 0.13 *	+0.36 ± 0.02 *	+0.21 ± 0.29 *

Inoculation conditions are described in Section 2.2.2. Positive value (+): Greater tolerance than the control. Negative value (−): Lower performance/tolerance than the control. Values are expressed as mean ± standard error (SE). The asterisk (*) indicates a significant difference compared to the control according to the Mann–Whitney test (p ≤ 0.05).

). At 30 days, P. maliformis lost its comparative advantage and did not differ statistically from the control (p = 0.70), while P. platyloba and P. quadrangularis maintained higher CTI values, forming a statistical group different from the control in Tukey’s test (p < 0.05). In contrast, P. edulis f. edulis showed a lower CTI than the control, indicating greater sensitivity to the progression of damage caused by M. incognita. Meanwhile, P. nitida did not differ from the control (Table S1). The combined performance of P. platyloba reduced nematode reproduction, stabilised biomass accumulation, and had excellent graft compatibility. This makes it the most balanced rootstock under simultaneous or sequential pathogen pressure, which better reflects field conditions, even though it displayed higher wilt severity in a single Fusarium inoculation.

In exclusive inoculation with F. oxysporum f. sp. passiflorae (F), rootstocks showed a differentiated tolerance dynamics over time. At 15 days, P. maliformis stood out for its early vigour with the largest stem diameter (4.89 mm), while P. platyloba obtained the highest fresh root biomass (16.33 g). This vegetative superiority was consolidated after 30 days, when both genotypes formed a higher statistical group (letter a in Tukey’s test, p < 0.05) in plant height, with 186.00 cm in P. maliformis and 181.00 cm in P. platyloba, significantly surpassing susceptible genotypes such as P. edulis f. edulis (59.33 cm). P. platyloba showed the highest root-gall severity (2.33); even so, it maintained greater biomass, which corresponds to a mechanism of tolerance rather than resistance (Table S1).

The response of the genotypes was predominantly homogeneous. At 15 days, P. maliformis and P. platyloba tended to have greater initial tolerance (ΔCTI of +0.32) without significant differences with respect to the control (Table 4). At 30 days under Fusarium inoculation, all materials, including the control, presented negative ΔCTI values; the magnitude of this reduction was different between materials. P. quadrangularis (−0.12) showed a smaller decrease compared to the control in Tukey’s test, suggesting better relative tolerance, while P. nitida (−0.33 *) and P. edulis f. edulis (−0.26 *) showed significantly greater reductions, indicating greater susceptibility to the pathogen (Table 4).

Sequential inoculation was carried out with Meloidogyne incognita followed by F. oxysporum f. sp. passiflorae (N→F). A clear divergence between genotypes was observed at 30 days. Passiflora edulis f. edulis showed a tolerance mechanism focused on vigour, reaching the highest total plant weight (51.82 g) and having a smaller-sized vascular lesion in the stem (11.88 mm) compared to the control (P. edulis f. flavicarpa at 18.42 mm), despite allowing for a high reproduction of nematodes. P. platyloba and P. maliformis restricted the root population (<30 individuals versus >175 in the control) but had a lower biomass accumulation compared to P. edulis f. edulis and the control, placing them at an intermediate level of growth that did not differ from the lower group in Tukey’s test (Table S1).

The CTI evidenced clear physiological data for wild genotypes compared to the commercial control (P. edulis f. flavicarpa). At 15 days, rootstocks P. quadrangularis, P. nitida, and P. edulis f. edulis showed lower CTI values than the control, with ΔCTI values of −0.17 to −0.26, indicating a greater early sensitivity to combined nematode and fungal stress. In contrast, P. platyloba presented a smaller reduction (ΔCTI −0.13), with no significant difference with respect to the control. At 30 days, this physiological disadvantage was consolidated in P. maliformis (ΔCTI −0.21), as well as in P. quadrangularis and P. nitida, while P. platyloba remained the closest wild rootstock in terms of its CTI in comparison to the control (Table 4).

In sequential inoculation with F. oxysporum f. sp. passiflorae followed by M. incognita (F→N), at 30 days, P. platyloba emerged as the most promising rootstock, combining superior vegetative vigour (having a maximum final height of 219.33 cm and the highest total plant weight of 46.23 g) with complete resistance to secondary infection by nematodes (0% incidence) despite presenting a considerable vascular lesion in the stem (19.42 mm), comparable to that observed in P. nitida. Meanwhile, P. maliformis showed a pattern of tolerance, maintaining high growth (147.33 cm) and limiting fungal lesion to the lowest value of the group (14.88 mm), although it allowed for a 100% incidence of nematodes, suggesting that its defence focuses on restricting the damage caused by Fusarium rather than on avoiding infection by M. incognita. In contrast, P. nitida was apparently resistant to nematode penetration (0% incidence), registering lower heights (42.33 cm) and the most extensive vascular lesion (20.42 mm). This indicates a high physiological sensitivity to the primary attack of F. oxysporum f. sp. passiflorae and a low tolerance to combined stress (Table S1).

After 15 days, only P. platyloba tended to display better physiological performance than the control P. edulis f. flavicarpa (ΔCTI +0.32), suggesting a transient ability to maintain vigour or limit initial damage under this F→N sequence (

Table 5. Compatibility Index (GCI) at 30 days post-graft per material.

Species	GCI	Compatibility ‖1 − GCI‖ 30 dpi (↓)	Quality
P. platyloba	0.96 ± 0.09 bc	0.04 ± 0.09 bc	Excellent
P. quadrangularis	0.94 ± 0.08 abc	0.06 ± 0.08 abc	Very good
P. edulis f. edulis	0.91 ± 0.06 c	0.09 ± 0.06 c	Very good
P. maliformis	1.39 ± 0.07 a	0.39 ± 0.07 a	Low
P. nitida	1.28 ± 0.07 ab	0.28 ± 0.07 ab	Medium

Different letters in each parameter indicate significant statistical differences (p = 0.05). The tests assume equal variances. Values are expressed as mean ± standard error (SE). GCI (30 dpi): Values closer to 1 are interpreted as better. This is summarised with the distance |1 − GCI| (lower = better compatibility). Lower values mean a GCI closer to 1. Tests were adjusted for all pair comparisons within a row of each innermost subtable using the Bonferroni correction.

). At 30 days, the rootstocks P. maliformis (ΔCTI = +0.11), P. quadrangularis (+0.13) and P. nitida (+0.14) presented positive ΔCTI values, indicating a superior performance to the control. P. platyloba showed an equivalent behaviour (ΔCTI = 0.00), while P. edulis f. edulis presented a slightly negative value (−0.02), without showing significant differences compared to the control in this condition (Table 4).

Regarding simultaneous inoculation with M. incognita and F. oxysporum f. sp. passiflorae (N/F), at 15 days, P. quadrangularis presented the highest susceptibility to nematode reproduction, with the highest root load of individuals and the highest reproduction factor (RF = 2.10 a), while P. platyloba maintained lower values (1.09 b). In parallel, P. maliformis recorded the highest visual severity and nodulation, indicating a marked sensitivity to early damage. At 30 days, P. nitida emerged as the most tolerant material in terms of total biomass (14.45 g), statistically differentiating itself from the control (P. edulis f. flavicarpa at 7.16 g) and maintained good visual health with a yellowing index of 1.00. In contrast, the control showed the worst overall health performance, allowing the greatest multiplication of the nematode (total RF = 2.10). The case of P. platyloba is unusual because, although it restricted the reproduction of the nematode (RF = 1.09), it presented the highest yellowing index (3.33), which suggests that its defence mechanism under simultaneous attack compromises its photosynthetic or nutritional status (Table S1).

At 15 days, P. maliformis tended to show lower physiological performance than the control (P. edulis f. flavicarpa with a ΔCTI of −0.23), while the rest of the genotypes did not differ statistically from the control (Table 4). At 30 days, the CTI of the control decreased to −0.42, while the genotypes showed a superior resilience capacity. P. nitida, P. quadrangularis, P. edulis f. edulis, and P. platyloba maintained CTI values that were higher than the control, with ΔCTI values of +0.17 to +0.36. Thus, the data indicate that under advanced co-infection, several wild genotypes have a relatively higher CTI than the control (Table 4).

3.2. Compatibility

The evaluation of the compatibility index (GCI) at 30 days post-graft allowed us to classify the genotypes according to the diametrical affinity between graft and rootstock (Table 5). P. platyloba presented the most outstanding behaviour, with a GCI of 0.96, which indicates an excellent bond quality and suggests homogeneous vascular healing without visible deformations. Similarly, P. quadrangularis and P. edulis f. edulis showed very good compatibility, with GCI values of 0.94 and 0.91, respectively. These results reflect balanced isodiametric growth and high mechanical stability of the graft–rootstock junction.

In contrast, P. nitida registered a GCI of 1.28 and P. maliformis a GCI of 1.39, evidencing less morphological compatibility. This mismatch is associated with a marked overgrowth or “bulging” in the junction area, a condition that is usually related to incomplete vascular continuity and a restricted flow of photoassimilates between the graft and rootstock.

3.3. Selecting the Best Rootstock

The choice of the ideal genotype was based on a multi-criteria analysis that weighed graft compatibility, sanitary resistance (restriction of nematodes and fungal lesions), and maintenance of vigour under stress. P. platyloba (wild passion fruit) emerged as the rootstock with the most balanced and promising performance. This material stood out for presenting a graft compatibility classified as “excellent” at 30 days, with a minimum deviation from the ideal (1-GCI = 0.04), which guarantees an optimal vascular connection. In terms of health, it demonstrated effective resistance against M. incognita, maintaining an incidence of 0% both in exclusive inoculation and in the F→N sequence at 30 days. Within this sequence, it combined sanitary resistance with the highest vegetative vigour, achieving the best plant height (219.33 cm) and weight (46.23 g). Although it showed physiological stress (yellowing) under simultaneous co-infection, its ability to block nematode reproduction and maintain graft integrity positions it as the best option.

P. maliformis was identified as a high-vigour alternative, with the highest growth in biomass and height under conditions of exclusive infection (N or F). However, its use has limitations: it showed a “low” graft quality due to a significant anatomical mismatch (GCI: 1.39) and allowed a 100% incidence of nematodes in most conditions, indicating that its tolerance is based on compensatory growth rather than resistance to the pathogen. On the other hand, P. nitida and P. edulis f. edulis showed resistance under specific conditions. P. nitida was the most resistant material under the condition of simultaneous co-infection of pathogens, retaining the highest dry weight and visual health. P. edulis f. edulis (gulupa or purple passion fruit) exhibited a tolerance mechanism in the N→F sequence, significantly limiting the progression of the Fusarium lesion and maximising plant weight despite the presence of nematodes.

4. Discussion

In the present study, the dynamics of the Fusarium–Meloidogyne interaction were governed by the order of inoculation, confirming that biotic predisposition is a key factor in the severity of damage. Simultaneous inoculation (N/F) and the N→F sequence increased the size of the lesion caused by Fusarium in most genotypes. This pattern is consistent with evidence in other crops, such as: in okra (Abelmoschus esculentus), where M. incognita predisposes the plant to wilt and governs severity [43]; in cotton (Gossypium hirsutum), where co-occurrence increases the aggressiveness of the Fusarium race; and in tomato (Solanum lycopersicum), where co-infection aggravates root deterioration [41]. Mechanically, nematode-induced wounds and physiological alterations facilitate fungal vascular colonisation; however, notable exceptions were observed, particularly in the N + F sequence, where Passiflora edulis f. edulis significantly restricted lesion expansion relative to the control, indicating a specific tissue tolerance mechanism that remains effective even under nematode pressure. Given that the aggressiveness of the isolates is associated with their region of origin, it is necessary to conduct studies aimed at characterising the genetic resistance of the isolates in order to identify and understand the possible genetic differences that exist between them [19].

Regarding the response to M. incognita, contrary to previous reports of susceptibility, P. platyloba showed remarkable resistance, with 0% incidence and very low populations of nematodes, behaving as a non-host genotype or with resistance by antibiosis, by limiting the survival and reproduction of the pathogen in the root [44]. This pattern contrasts with the high susceptibility observed in the control and in P. quadrangularis, which recorded the highest reproduction rates in co-infection. The findings qualify what has been described for other wild passionflowers, where M. incognita is usually a severe limiting factor [10,11] and suggest that P. platyloba has pre-infectious barriers or hypersensitive response mechanisms not yet described for this pathosystem. The reproduction index (RF) confirmed the dependence on the host [44] and P. nitida and P. platyloba were the genotypes that most restricted the multiplication of the nematode in sequential infection scenarios.

In terms of biomass, the evidence suggests that the effect of co-infection is not uniform but strongly depends on the context of infection and genotype. In sequential scenarios, P. maliformis and P. platyloba tended to sustain greater dry matter accumulation, indicating that they can offset part of the physiological cost of attack through adjustments in growth and resource use. On the other hand, under simultaneous inoculation, the control and susceptible genotypes, such as P. quadrangularis, showed a strong drop in biomass, a pattern consistent with what has been recorded for tomatoes and other tropical crops [41,45]. This variability, observed between inoculation conditions, reinforces the interaction of genotype, time, environment, and pathogen, since the aggressiveness of F. oxysporum and the pressure of Meloidogyne differentially modulate the expression of damage [7,46,47].

Finally, the compatibility assessment (GCI) at 30 days post-graft was key to the final selection of rootstocks. P. platyloba and P. quadrangularis showed excellent compatibility, ensuring a vascular connection and adequate mechanical stability of the graft [47]. In sum, P. platyloba offers the most consistent balance between health, vigour, and compatibility [48,49]. These results make it necessary to qualify the use of P. maliformis, which, although it has been used in the field due to its tolerance to Fusarium [50], presents compatibility mismatches that can compromise the translocation of photoassimilates and reduce the longevity of the plantation. Overall, GCI information supports the priority of P. platyloba. Finally, despite its limitations in initial growth, P. nitida could be considered a niche resource for simultaneous high-pressure situations of both pathogens, given its unique resilience in biomass under that specific condition, which is essential for the long-term sustainability of commercial Passiflora systems.

5. Conclusions

This study confirmed that the Meloidogyne–Fusarium interaction generates a negative synergism dependent on the inoculation condition. Simultaneous co-inoculation and nematode-predisposed sequencing maximised vascular damage, increased wilt severity, reduced biomass, and the CTI of several genotypes. Under these conditions, the root system was highly compromised, and the crop showed a general deterioration of its physiological performance, which shows that the order and overlap of infections not only add effects but also amplify the impact of the pathogenic complex on the plant.

P. platyloba was considered the most suitable rootstock. It combined effective resistance against M. incognita (zero incidence in critical stages) with excellent graft compatibility. On the other hand, P. maliformis showed high susceptibility to nematode reproduction and low graft-binding quality, limiting its use in long-term plantations. Based on these findings, it is advisable to incorporate P. platyloba as rootstock in Passiflora-integrated management programmes.