Introduction

Brazil is the global leader in the production and consumption of yellow passion fruit (Passiflora edulis Sims), with approximately 593,429 metric tons produced in an area of 41,584 hectares (IBGE 2020). However, the occurrence of phytosanitary problems has resulted reduced useful life and low productivity (14.3 t ha− 1) of orchards. Among these problems is passion fruit woodiness disease caused by cowpea aphid-borne mosaic virus (CABMV). It is considered the main viral disease affecting passion fruit in Brazil (Preisigke et al. 2020). As the name indicates, this virus is disseminated by aphid vectors in a non-circulatory and non-persistent manner (Garcêz et al. 2015; Spadotti et al. 2019). The high incidence of the disease has contributed to reduction of the crop cycle to a maximum of one year in several passion fruit production regions (Garcêz et al. 2015; Santos et al. 2015a, b).

The development of resistant cultivars is the most effective method for production in an area with CABMV. However, until now the available commercial yellow passion fruit (P. edulis) cultivars are susceptible to this virus. The identification of resistant species in germplasm banks is essential for the development of cultivars resistant to CABMV. The exploration of sources of resistance in wild species has been used with success in several crops (Rai et al. 2020; Cai et al. 2020; Sade et al. 2020). In the case of passion fruit, some wild species, such as P. setacea (Santos et al. 2019; Carvalho et al. 2019; Preisigke et al. 2020) and P. cincinnata (Jesus et al. 2016) have been used for introgression of resistant alleles of CABMV in P. edulis from interspecific hybridizations followed by backcrossing cycles.

Several mechanisms are responsible for genetic resistance to the virus, among which physical barriers stand out, by hindering the establishment of the pathogen, such as the presence of cutin, waxes and cell walls (Boutrot and Zipfel 2017; Gouveia et al. 2017; Andersen et al. 2018; Carvalho et al. 2019). Analysis of anatomical traits is also important for understanding the Passiflora spp. x CABMV interaction, by identifying cell structures that hinder the pathogen’s establishment. The use of fluorescence microscopy to identify callose, calcium oxalate crystals and the formation of tilose has been used in several species in interactions with different microorganisms (Hinch and Clarke 1982; Chen and Kim 2009; Ellinger and Voigt 2014; Palomares-Rius et al. 2017; Xiao et al. 2018; Lima et al. 2019).

Even in the main commercial species (P. edulis), the principal changes that occur with systemic spread of the virus within the plant resulting in leaf damage, such as mosaic symptoms, wrinkling and distortions, are still not fully understood. Therefore, it is necessary to better understand the Passiflora spp. x CABMV interaction to elucidate the morphoanatomical changes caused by CABMV and understand the mechanisms of action of this virus, which can shed light on this interaction as well as support control or coexistence strategies of this disease. Thus, the objectives of this study were: i) to evaluate the reaction to CABMV in eight species of Passiflora spp. under greenhouse conditions; and ii) to identify excethe possible leaf anatomical changes in these species at different levels of severity resulting from viral infection of P. edulis by means of light and fluorescence microscopy.

Materials and methods

Location of experiment and plant material

The experiment was carried out in the premises of Embrapa Mandioca e Fruticultura, located in the municipality of Cruz das Almas, Bahia, Brazil (12° 40’ 39” S, 39° 06’ 23” W, 226 m). Eight species of Passiflora spp. belonging to the Passiflora Germplasm Bank (PGB) of Embrapa were used: P. edulis Sims cv. BRS Gigante Amarelo (BRS-GA); P. alata Curtis (BGP393); P. foetida L. (BGP395); P. gibertii N.E.Br. (BGP414); P. malacophylla L. (BGP170); P. setacea DC. (BGP238); P. suberosa L. (BGP014) and P. subrotunda Mast. (BGP394).

Seeds (n = 30) of each genotype were soaked in 1.0 mL of the plant regulator GA4 + 7 + N-(phenylmethyl)-aminopurine (Promalin®) at a concentration of 400 mg L− 1 for 24 hours. After this period, the seeds were distributed in plastic pots with capacity of 1.0 L containing a mixture of coconut fiber and commercial substrate (Vivatto Plus®) in the proportion of 3:1 (v/v) plus 40 g of fertilizer (Osmocote®) for each 10 L of substrate. After seedling emergence (about 40 days after sowing), ten plants with greatest uniformity were selected to compose the experimental test. The plants were kept in a greenhouse with temperature around 28 ± 2 ºC and relative humidity of 75 ± 5%, and irrigation was performed manually daily using a watering can with capacity of 10 L.

Artificial inoculation and transmission electron microscopy (TEM)

The source of the CABMV inoculum was plants of P. edulis (susceptible) kept in a greenhouse. The treated plants received mechanical inoculation with CABMV while the plants in the control group did not, 45 days after the emergence of the plants, when they had at least four expanded leaves, following the method described by Gonçalves et al. (2018).

The virus used in the inoculations was obtained from apical leaf tissue of the plants kept in a greenhouse with severe symptoms of CABMV, and was identified by transmission electron microscopy (TEM) (Kitajima et al. 2008) and by RT-PCR in previous studies using the same plant as a source of inoculum (Gonçalves et al. 2018).

Assessment of symptoms and severity of CABMV

The reaction of the eight Passiflora species infected with CABMV was characterized based on the visual symptoms of the disease. The evaluations were carried out with the first fully expanded leaf from the apex, by observation of 10 leaves per plant. The first evaluation was performed 20 days after the first inoculation (DAI), and the others at intervals of seven days, ending with the sixth evaluation at 55 DAI. Symptom assessments used a diagrammatic grade scale based on severity levels that ranged from 1 to 4 asymptomatic (level = 1); leaf showing light mosaic (level = 2); leaf showing severe mosaic and blistering (level = 3); leaf showing severe mosaic, blistering and leaf deformations (level = 4), as established by Novaes and Resende (2003) (Fig. 1).

To quantify the severity of the symptoms of CABMV, the disease index (DI) was used (Mckinney 1923) at 55 DAI. This index is calculated based on the weighting of the scale of infection levels, applying the following formula: DI (%) = (GS x L)/ (TNL x HGS); where: GS = degree of scale determined for each leaf; L = number of leaves with each degree of symptoms (level); TNL = total number of leaves evaluated and HGS = maximum degree of infection.

Leaf anatomy

For anatomical analysis, segments of the internervural region (n = 6) were collected from leaves infected with CABMV and the respective controls of the eight Passiflora species, mature leaves are selected and fully expanded for all species (Fig. 1, 2, 3, and 4). For P. edulis, the leaf segments were collected according to the degree of severity of the symptoms caused by CABMV, according to the scale shown in Fig. 1. The process of fixing, blocking and cutting the samples was performed as described by Lima et al. (2019).

Fig. 1
figure 1

Light and fluorescence photomicrographs of P. edulis. a-d healthy leaf (control), the rectangle indicates the internervural region of the Passiflora leaf used for anatomical analysis; e-h leaf infected with the CABMV and asymptomatic (level = 1); i-l infected leaf showing light mosaic (level = 2); m-p) infected leaf showing severe mosaic and blistering (level = 3); q-t infected leaf showing severe mosaic, blistering and leaf deformations (level = 4). xy: xylem; ph: phloem; co: collenchyma; ue: upper epidermis; le: lower epidermis: pp: palisade parenchyma; sp: spongy parenchyma; vb: vascular bundle. Bars: 500 µm (b, f, j, n, r); 200 µm (c, d, g, h, k, l, o, p, s, t)

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Fig. 2
figure 2

Light and fluorescence photomicrographs of Passiflora leaves. Healthy P. alata (a-d); P. alata infected with CABMV (e-h), presenting intense mosaic, blistering and leaf deformation (e), with a slight change in the arrangement of the vascular bundles (f-h); healthy P. gibertii (i-l); P. gibertii infected with CABMV (m-p), presenting light mosaic (m) but without alteration in the infected cell structure (n-p); healthy P. malacophylla (q-t); P. malacophylla infected with CABMV (u-x); symptomies (u) and without anatomical modification (v-x). co: collenchyma; ue: upper epidermis; le: lower epidermis: pp: palisade parenchyma; sp: spongy parenchyma, tr: trichome, st: stoma. Bar: 500 µm (b, f, j, n, r, v); 200 µm (c, d, g, h, k, l, o, p, s, t, w, x)

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Fig. 3
figure 3

Light and fluorescence photomicrographs of Passiflora leaves. Healthy P. suberosa (a-d); P. suberosa infected with the CABMV (e-h) and with normal distribution of infected tissues (f-h); healthy P. foetida (i-l); P. foetida infected with CABMV showing symptoms of light mosaic (m) and no change in the structures of infected tissues (n-p). co: collenchyma; ue: upper epidermis; le: lower epidermis: pp: palisade parenchyma; sp: spongy parenchyma, tr: trichome, st: stoma, vb: vascular bundle. Region of vascular tissue present in the parenchymatic tissue of the leaf (circle). Bar: 500 µm (b, f, j, n); 200 µm (c, d, g, h, k, l, o, p)

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Fig. 4
figure 4

Light and fluorescence photomicrographs of Passiflora leaves. Healthy P. setacea (a-d); P. setacea infected with CABMV (e-h) without changes in infected cell tissues (f-h); healthy P. subrotunda (i-l); P. subrotunda infected with CABMV (m-p), with symptoms of light mosaic (m), mild cell disorganization and prominent palisade parenchyma in the leaf mesophyll region (n-p). co: collenchyma, ph: phloem, xy: xylem, ue: upper epidermis, le: lower epidermis, pp: palisade parenchyma, sp: spongy parenchyma, tr: trichome, vb: vascular bundle, st: stoma. Bar: 500 µm (b, f, j, n), 200 µm (c, d, g, h, k, l, o, p)

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For fluorescence microscopy, the leaf segments were stained with Lugol’s iodine for 3 min, then washed with tap water and stained with 1% aniline for 8 min and finally in Lugol again for 30 s. The slides were assembled with tap water (Kraus and Arduin 1997). The blue aniline dye produces a blue color in callose tissue and Lugol acts on the cell walls, giving a gray and yellowish coloration to lignified areas. The slides containing the histological sections were analyzed and photo documented with an Olympus BX51 light microscope coupled to an Olympus DP175 digital camera (Olympus, Tokyo, Japan) with an ultraviolet filter that emits fluorescence to check for the presence of callose.

Quantitative analyses of anatomical traits (LBT = leaf blade thickness; UET = upper epidermis thickness; LET = lower epidermis thickness; PPT = palisade parenchyma thickness; SPT = spongy parenchyma thickness) were performed based on the measurements of the six leaf histological sections (n = 6), measured five times (6 cuts x 5 measurements) along each photomicrograph using the ImageJ 1.46r program (Rasband 2016).

Data analysis

The experimental design was completely randomized, distributed in eight treatments (species), with eight replications, in addition to two plants of each species as a control. To determine the disease severity index, control plants of all species and asymptomatic plants of P. edulis were not considered in the statistical analyses.

For grouping the species, the DI values (%) were used at 55 DAI according to the Scott-Knott test (p ≤ 0.05). This grouping was used to categorize the reaction of species in a group based on the DI scale: 0.00 to 10.99% - resistant (R); 11.00 to 25.99% - moderately resistant (MR); 26.00 to 60.99% - susceptible (S); and ≥ 61.00% - highly susceptible (HS). The analysis of the anatomical measurements of P. edulis was enerated according to the different levels of leaf severity (levels 1 to 4, Fig. 1) and box plots were used to visualize the data dispersion. In addition, the control and level 4 (highest level of severity of CABMV) for this species were analyzed as positive controls. The anatomical trait data of the other Passiflora species were subjected to analysis of variance and compared by the F-test (p ≤ 0.05). All statistical analyses were performed using the ‘agricolae’ package in the R program (R Development Core Team 2020).

Results

Severity classification of passion fruit species to CABMV

The transmission electron microscopy (TEM) analysis performed on the parenchyma of the apical leaves of the P. edulis plants without infection, as expected, did not show viral structures in the transmission electron photomicrographs (Supplementary Fig. 1a). Plants with typical symptoms of CABMV (Supplementary Fig. 1b), used for mechanical inoculation of species (Table 1), showed the presence of cylindrical inclusions typical of viruses of the genus Potyvirus (Supplementary Fig. 1b).

Table 1 Incidence, minimum, maximum and average value of severity and classification of passion fruit species artificially infected with cowpea aphid-borne mosaic virus (CABMV)
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The CABMV severity in the species evaluated ranged from 0.0–58.2%, according to the disease index (DI) at 55 DAI. Among the species evaluated, P. suberosa (BGP014), P. setacea (BGP238), P. malacophylla (BGP170) and P. gibertii (BGP414) were classified as resistant, with DI ranging from 0.0–2.4%. In contrast, the species P. alata (BGP393) and P. edulis (BRS Gigante Amarelo), were classified as susceptible, with DI values of 58.2% and 51.9%, respectively (Table 1).

Anatomical alterations of passion fruit species infected with CABMV

In the controls, normal tissue distribution was observed in the eight species of Passiflora. Four species infected with CABMV (P. foetida, P. subrotunda, P. alata and P. edulis) presented different variations of leaf symptoms (from light mosaic to leaf deformations) and changes in the distribution of the internal leaf tissues P. gibertii, had mild symptoms of CABMV and did not present any alteration in cell structures. Similarly, infected P. malacophylla, P. setacea and P. suberosa also showed no changes in cell structures, without symptoms of the disease (Figs. 1, 2, 3 and 4).

The anatomical analyses in P. edulis (Fig. 1a-t) allowed the identification of major anatomical changes in this species as the symptoms of passion fruit woodiness disease progressed, including reduction of parenchymal tissues and alteration in the distribution of vascular bundles in the central vein of the leaves. The control plants did not have morphological (Fig. 1a) or anatomical (Fig. 1b-d) alterations. Greater thickness of the collenchyma in the central vein (Fig. 1f) was observed in P. edulis plants without (level 1) infection severity (Fig. 1e-h). Similar behavior was also observed at the other levels of disease severity (Fig. 1j, n, r).

Changes in the arrangement of the vascular bundles were observed only after level 3 (Fig. 1n-o). The xylem and phloem vessels were disassociated from their natural conformation forming three vascular bundles (Fig. 1n), more visibly with fluorescence microscopy (Fig. 1o). This observation was more evident in the greatest degree of severity (level 4), in which the leaves had intense mosaic formation, blistering and deformation. In addition, the presence of four vascular bundles was observed at this severity level, in addition to reduction in the diameter of the xylem vessels (Fig. 1r, s).

Palisade and spongy parenchyma tissues of P. edulis were also reduced when infected with CABMV. This also occurred in the leaves that did not show visual symptoms of the disease (Fig. 1h). Additionally, the cells of the spongy parenchyma showed deformations and hypertrophy in the shape of the cells, hypertrophy and depressions in certain regions of the leaf blade with the progression of the symptoms of the disease (Fig. 1p, t) compared to the control (Fig. 1d).

In the control plants of P. alata, as well as all six wild Passiflora species evaluated, the presence of leaf mosaic was not observed (Figs. 2a, i and q, 3a and i and 4a and i). Plants infected with CABMV, on the other hand, showed some degree of viral infection (Fig. 2e, m, 3m, 4m) except for P. malacophylla (Fig. 2u), P. suberosa (Fig. 3e) and P. setacea (Fig. 4e), which did not show symptoms of CABMV.

In P. alata, no significant anatomical changes were seen after inoculation, instead only the start of dissociation in the arrangement of the vascular bundles (Fig. 2f-g) when compared to the control treatment (Fig. 2b-c). The palisade parenchyma was not altered due to the manifestation of symptoms, whereas the spongy parenchyma showed slight hypertrophy when infected (Fig. 2h).

In P. gibertii, anatomical changes in the distribution of vascular bundles were not seen. On the other hand, the xylem vessels had altered disposition, mainly when analyzed by fluorescence microscopy (Fig. 2). Similarly, in P. malacophylla there was also a slight change in the arrangement of the vascular bundle of the central vein observed both by light and fluorescence microscopy (Fig. 2v, x). Despite the leaf anatomical changes observed in this species, no visual symptoms were observed in the leaves, so they were classified as resistant to the virus.

In relation the anatomical analyses in P. suberosa, there were no changes in the central vein and leaf mesophyll between the infected and control plants (Fig. 3b, f). This species presented the largest palisade and spongy parenchyma, which may be associated with resistance to passion fruit woodiness disease (Fig. 3d, h). P. foetida also showed no anatomical changes in plants infected with CABMV (Fig. 3m-p), although mild symptoms of the disease were observed in this species, classified as moderately resistant (Table 1). In addition, the presence of trichomes in the abaxial and adaxial surfaces was observed in the leaves of the control and infected plants (Fig. 3l, p).

In P. setacea (classified as resistant, DI = 0.0%), no anatomical changes associated with the virus were identified, with the presence of phenolic compounds in the region of the central vein of the control and infected leaves (Fig. 4a-h). The species P. subrotunda, classified as moderately resistant, (DI = 18.8%), showed similarity between the control and infected treatments in the distribution of the tissues of the central vein (Fig. 4j, n), but a reduction in the spongy parenchyma of the infected plants was observed with greater agglutination of cells in this region in relation to the control plants, in which the cells were well spaced, facilitating gas exchange (Fig. 4l, p).

Considering the morphoanatomical analyses of P. edulis in relation to viral infection, significant hypertrophy (p ≤ 0.05) was observed in the leaf blade of plants infected with and without CABMV symptoms in relation to the control treatment (Fig. 5a).

Fig. 5
figure 5

Box plots of the anatomical traits of Passiflora edulis plants infected with CABMV. The line shown in the box is the median value and the x indicates the average of the data. Different letters on the top whiskers indicate statistically different means (Scott-Knott test, p ≤ 0.05)

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The upper epidermis thickness was not affected by the severity of passion fruit woodiness disease (Fig. 5b). The dispersion of the morphoanatomical data indicates that leaves without (level 1) and with lower severity level (level 2) showed greater amplitude in the distribution of data in relation to the control plants (Fig. 5).

Based on the morphoanatomical analyses of P. edulis control plants and the highest severity of CABMV (level = 4), significant reductions (p ≤ 0.05) were observed in the leaf blade thickness (Fig. 6a), lower epidermis (Fig. 6c) palisade parenchyma (Fig. 6d) and spongy (Fig. 6e), with reductions of 24.5%, 13.9%, 18.6% and 34.8% compared to the control treatments, respectively. The upper epidermis thickness of P. edulis remained unchanged (Fig. 6b).

Fig. 6
figure 6

Quantitative anatomical traits of Passiflora species with the passion fruit woodiness disease: P. edu: P. edulis, P. ala: P. alata, P. foe: P. foetida, P. gib: P. gibertii, P. mal: P. malacophylla, P. set: P. setacea, P. sub: P. suberosa and P. subr: P. subrotunda. (**), (*) The top of the error bars indicates statistically different means according to the F-test at 1 and 5% probability. LBT: leaf blade thickness; UET: upper epidermis thickness; LET: lower epidermis thickness; PPT: palisade parenchyma thickness; SPT: spongy parenchyma thickness

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In P. alata, significant anatomical changes were observed due to inoculation with CABMV (p ≤ 0.05) for all the traits except LET and PPT. This species, together with P. edulis, had the highest incidence and severity of CABMV according to the disease index (Table 1). The leaf blade thickness (Fig. 6f), upper epidermis (Fig. 6g) and spongy parenchyma (Fig. 6j) of P. alata plants subjected to infection with CABMV was significantly reduced by 23.3%, 24.7% and 34.7%, compared to the control treatment, respectively.

In P. foetida, only the spongy parenchyma thickness (p ≤ 0.05) changed after infection with the virus, with an increase of 11.9% compared to the control treatment (Fig. 6). The morphological and anatomical evaluation of P. gibertii showed the opposite behavior to P. alata and P. edulis, with hyperplasia of the leaf blade (Fig. 6p), palisade parenchyma (Fig. 6s) and spongy parenchyma (Fig. 6t), in thickness increases of 16.6%, 13.2% and 21.9% in the infected plants compared to the controls, respectively. A change in the lower epidermis thickness was also observed, with a significant reduction (p ≤ 0.05) of 13.7% in the infected plants (Fig. 6r).

In P. malacophylla, there was morphometric alteration of the anatomical traits LBT (Fig. 6u) and PPT (Fig. 6x) when the plants were infected with CABMV, with increases of 16.6% and 22.1%, in relation to their respective controls. In relation to P. setacea, plants infected by the virus showed a significant increase (p ≤ 0.01) of 16% in LET compared to the control plants (Fig. 6ab). For the other morphometric traits evaluated, there were no differences (p > 0.05) between the infected and non-infected plants of P. setacea (Fig. 6z, aa, ac, ad).

The leaves of P. suberosa did not show CABMV symptoms, but a significant reduction (p ≤ 0.01) of PPT was observed in the presence of the virus, with 20.9% less than the control plants (Fig. 6ah). There was no change for the other traits between infected and non-infected plants of P. suberosa (Fig. 6ae, af, ag, ai). The leaves of P. subrotunda showed hypertrophy in the spongy parenchyma (p ≤ 0.05), 16.5% lower in the infected plants compared to the controls (Fig. 6an). The other traits did not differ significantly (p ≥ 0.05) (Fig. 6aj, ak, al, am). The presence of light mosaic on the leaves of P. subrotunda was not enough to significantly alter the anatomical structures of the infected plants in relation to the controls.

Discussion

The wild species of passion fruit that showed complete resistance (ID = 0.0%) to CABMV in our study are natural sources of resistance and/or tolerance genes, both to biotic (Gonçalves et al. 2018) and abiotic factors (Souza et al. 2018; Lima et al. 2020). Studies carried out by several authors have also reported that the wild species P. setacea, P. suberosa and P. malacophylla (Oliveira et al. 2013; Freitas et al. 2015, 2016; Santos et al. 2015a; Gonçalves et al. 2018; Sacoman et al. 2018) present complete resistance to CABMV, so they can be used in interspecific hybridizations to obtain new resistant cultivars. Santos et al. (2015a) demonstrated success in obtaining 156 interspecific hybrids from the cross between P. edulis x P. setacea, of which 25 were considered resistant to CABMV in field conditions. Other authors have also reported resistance of P. setacea to CABMV (Oliveira et al. 2013; Gonçalves et al. 2018).

Despite the immunity observed in P. suberosa to CABMV, this species belongs to the subgenus Decaloba (2n = 24; 36) (Melo et al. 2016), while P. edulis belongs to the subgenus Passiflora (2n = 18) (Melo et al. 2016; Pamponét et al. 2019). The lack of chromosomal homology between these two subgenera makes it difficult to obtain interspecific hybrids resistant to CABMV under natural conditions. Although, studies demonstrate success in obtaining interspecific hybrids of Passiflora even in the absence of chromosomal homology (Soares et al. 2015; Soares et al. 2018). This probably occurred because interspecific barriers of incompatibility in Passiflora are relatively fragile (Meletti et al. 2005).

It is interesting to note that the species P. malacophylla considered resistant to CABMV can be used in hybridization programs to transfer resistance alleles to commercial species (P. edulis), since both have the same number of chromosomes (2n = 18) (Souza et al. 2003; Pamponét et al. 2019), increasing the chances of obtaining commercial hybrids tolerant to CABMV.

Several authors have reported anatomical changes in different species of plants infected by viruses (Takimoto et al. 2009; Gomes et al. 2010), including Passiflora species (Gonçalves et al. 2018). Viral infection is a controlled and multifaceted process, in which the virus appropriates the host’s cell functions to replicate itself (Netherton and Wileman 2011; Niehl and Heinlein 2011). We found that P. edulis showed the greatest morphological and anatomical changes in the leaves, with a potential impact on plant metabolism by reducing leaf expansion and production of photoassimilates (Bolton 2009; Fernández-Calvino et al. 2014; Murray et al. 2016). As symptoms progressed, changes in cell conformation and vascular bundles became more pronounced in symptomatic plants of P. edulis (Figs. 1 and 5).

Morphoanatomical measurements were also affected by CABMV in asymptomatic plants (Fig. 5), indicating that the virus promotes changes in the anatomical structure of the leaves even in the absence of visible symptoms. Although there was no mosaic or deformation, there was apparent chlorosis in asymptomatic plants (Fig. 1e), indicating some physiological disorder caused by the presence of the virus. In this respect, Vitis vinifera L. plants infected with tomato ringspot virus (ToRSV) showed chlorotic leaves, without leaf deformation, but with changes in the cytoplasm, chloroplasts, nucleus, cell wall, xylem and phloem. ToRSV mainly affected chloroplasts, with deformation in thylakoids, accumulation of starch granules and vacuolar deformation (El-Banna et al. 2014). Further studies are essential to associate these anatomical changes (Fig. 5) with molecular and or enzymatic aspects of Passiflora edulis.

The fluorescence microscope technique to identify callose compounds from the plant-virus interaction was not effective, since there was no difference between the control and the infected plants. Callose is deposited on the plasma membrane and cell wall after mechanical, chemical, physiological or biotic damage (Chen and Kim 2009). In addition, no induction of defense associated with the formation of calcium oxalate crystals, tylosis and high deposition of callose in the xylem and phloem vessels was observed, which would prevent the virus from colonizing vascular tissue. It is important to mention that this response induction is more common in plant-fungus interactions (Ellinger and Voigt 2014; Lima et al. 2019).

Anatomical changes directly interfere with the cell wall structure and/or the conformation and distribution of organelles, thereby altering the biochemical processes of signaling and synthesis and impacting productivity (Grove and Marsh 2011; El-Banna et al. 2014; Xiao et al. 2016).

In this study, viral infection caused greater anatomical change at the level of the spongy parenchyma, reducing the intercellular spaces in P. edulis, P. alata (classified as susceptible) and in P. subrotunda (classified as moderately resistant). This region of the mesophyll is fundamental for physiological functioning, because it is where the gas exchange takes place between the stomatal chamber and the external environment (Xiao et al. 2016). The probable consequence is reduction in the photosynthetic rate of the plant (Su et al. 2018).

The anatomical changes observed in our study were directly associated with CABMV severity, considering that greater changes in tissue conformation and dimensions were observed in P. edulis and P. alata, while the other species, such as P. suberosa, P. gibertii and P. malacophylla, showed only slight changes in the dimensions of the infected tissues, confirming the results obtained by Gonçalves et al. (2018). Virus resistance involves a series of physiological, biochemical, anatomical, molecular and genetic processes (Alexander and Cilia 2016). Therefore, knowing the mechanisms associated with viral infection is essential to better understand the plant-pathogen interaction. Furthermore, it is important to understand the molecular processes that affect resistance to CABMV in Passiflora spp. through further investigation of Passiflora x CABMV interaction.

Based on the severity of the disease, it was possible to identify resistance to CABMV in the wild species P. malacophylla, P. setacea and P. suberosa. These species did not show leaf symptoms and changes in the arrangement of cell structures when infected by CABMV, showing potential for use in interspecific hybridizations with susceptible commercial species (P. edulis and P. alata) or use of other unconventional breeding strategies. The species P. subrotunda and P. foetida were classified as moderately resistant to CABMV, with slight anatomical disorganization. Viral infection directly affected the distribution and organization of vascular bundles and spongy parenchyma hypertrophy in P. edulis and P. alata. Considering the different levels (1 to 4) of severity of CABMV in P. edulis, viral infection promoted changes in leaf anatomy even in asymptomatic plants (level 1) and major anatomical changes occurred as the symptoms of disease progressed, including parenchyma tissue reduction (level 2) and complete change in the arrangement of vascular bundles (phloem and xylem) in the central leaf vein (levels 3 and 4).