Water deficit increases the susceptibility of yellow passion fruit seedlings to Fusarium wilt in controlled conditions

Scientia Horticulturae 243 (2019) 609–621

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Water deficit increases the susceptibility of yellow passion fruit seedlings to Fusarium wilt in controlled conditions

T

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Lucas Kennedy Silva Limaa, Onildo Nunes de Jesusb, , Taliane Leila Soaresb, Saulo Alves Santos de Oliveirab, Fernando Haddadb, Eduardo Augusto Girardib a b

Centro de Ciências Agrárias, Ambientais e Biológicas, Universidade Federal do Recôncavo da Bahia, Cruz das Almas, BA, 44380-000, Brazil Embrapa Mandioca e Fruticultura, Cruz das Almas, BA, 44380-000, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Passiflora edulis Water deficit Fusarium wilt Inoculation Plant-pathogen interaction Anatomy

Fusarium wilt is considered the main fungal disease of yellow passion fruit plants in Brazil. There is ample anecdotal evidence of greater intensity of Fusarium wilt after water shortage in field conditions, but this association needs scientific confirmation. There is also a need to increase the efficiency of inoculation with Fop (Fusarium oxysporum f. sp. passiflorae) under controlled conditions for research purposes. Therefore, in this study we evaluated the effect of propagation method (from cuttings or seedlings) associated with controlled water deficit on the incidence of Fusarium wilt in yellow passion fruit plants. The artificial inoculation with Fop involved application of a spore suspension of 106 conidia mL−1 and infestation of the potting media with Fop grown in sand and cornmeal substrate. For anatomical analysis, root segments were used from inoculated and non-inoculated plants (control). Seedlings that were submitted to water deficit presented the highest incidence of Fusarium wilt, 75.0%, while in the irrigated control the incidence was below 40.0%. The mortality associated with Fop in cutting-propagated plants did not differ from non-inoculated plants. The plants subjected to water stress had greater presence of hyphae and chlamydospores and reduced starch concentration in the root cortex region. Propagation by seeds associated with controlled water stress can be used to screen accessions of P. edulis for resistance to Fusarium wilt.

1. Introduction The yellow passion fruit vine (Passiflora edulis Sims) is susceptible to various diseases that affect the aerial part and root system. Among these, Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. passiflorae (Fop), stands out as one of the main diseases (Ortiz and Hoyos-Carvajal, 2016), and can cause production losses greater than 80% (Fischer and Rezende, 2008; Freitas et al., 2016). In the absence of Fop, the plants can remain productive for at least three years, but the presence of this fungus usually reduces the productive lifespan to at most one year. The vascular system of infected plants presents a rusty coloration in the roots, stems and branches, due to the oxidation of phenolic compounds, necrosis of the root and collapse of the xylem. These alterations are caused by the physical structures of the pathogen, such as hyphae and spores, besides fungal toxins either the defense structures produced by the plant (Stangarlin and Leite, 2008; Fischer et al., 2010; Ortiz

et al., 2014). Control of this disease is very complex, because application of chemical pesticides alone does not result in significant suppression of the disease, and the pathogen can remain in the soil for several years in the form of chlamydospores, preventing planting in previously infected areas (Fischer and Rezende, 2008). The use of resistant varieties is considered the best strategy to minimize the damages caused by the disease, although truly resistant cultivars are not yet available (Freitas et al., 2016). The response of host plants to pathogens often depends on the developmental stage of the host when challenged by the pathogen (Whalen, 2005; Del Ponte et al., 2007). Fusarium wilt is favored by high air temperatures during the seedling stage, even though plants become more susceptible at flowering in field conditions (Ahmad et al., 2010). In tomato, the presence of root exudates stimulates the microconidia germination of F. oxysporum, and the specific stimulation level depends on physiological changes during the plant development (Steinkellner

Abbreviations: Fop, Fusarium oxysporum f. sp. passiflorae; DI, disease index; DAI, days after inoculation ⁎ Corresponding author. E-mail addresses: [email protected] (L.K.S. Lima), [email protected] (O.N.d. Jesus), [email protected] (T.L. Soares), [email protected] (S.A.S.d. Oliveira), [email protected] (F. Haddad), [email protected] (E.A. Girardi). https://doi.org/10.1016/j.scienta.2018.09.017 Received 9 October 2017; Received in revised form 18 July 2018; Accepted 6 September 2018 0304-4238/ © 2018 Published by Elsevier B.V.

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commercial nursery and the cuttings of the same variety were collected from two mother plants (10 months old) that had been kept in greenhouse. These source plants were near the start of flowering and were vigorous, well-nourished and free of pests and symptoms of diseases. The cuttings, presenting two buds without leaves with length of 17 ± 2 cm, were taken from mature and lignified branches with length of approximately 1.5 m. A segment of about 20 cm was removed from the apical region, and the resulting cuttings were placed for rooting in expanded vermiculite.

et al., 2005). Another group also reported that seedlings exhibited greater susceptibility to disease compared to cuttings among the diff ;erent fungi evaluated including the Fusarium solani (Ahmad et al., 2013). In the yellow passion fruit, the highest percentage of dead plants by Fusarium wilt occurs in the reproductive phase of the crop after drought events followed by a period with high rainfall index associated with high temperatures, as these last two conditions favor the development of the fungus (Cavichioli et al., 2011). The water stress affects the susceptibility of various plant species to the pathogen (Choi et al., 2013; Ramegowda and Enthil-Kumar, 2015; Nejat and Mantri, 2017; Daranas et al., 2018), which can be associated with damage to the roots (Berta et al., 2005), alteration of the soil microbiota (Rolli et al., 2015), physiological changes (Ghaemi et al., 2010), quantity of available proteins (Ramegowda and Enthil-Kumar, 2015; Pandey et al., 2017), and alteration in the defense mechanisms (Pandey et al., 2017). In this way, the effects of biotic or abiotic stresses cannot be overlooked, since they both contribute to the manifestation of the symptoms of the pathogen, exerting a direct influence on the infection according to the susceptibility or resistance of the host plants (Ramegowda and SenthilKumar, 2015). The identification and the selection of more resistant genotypes are usually carried out in field conditions in areas infested by the pathogen. However, the long period required for evaluation and the high cost of cultivation make the selection process burdensome, and the influence of other diseases and environmental factors can hamper the diagnosis. Some studies have been conducted on passion fruit to establish efficient inoculation protocols to identify resistant individuals under controlled conditions (Fischer et al., 2010; Flores et al., 2012; Ortiz and Hoyos-Carvajal, 2016). The results obtained by these researchers are divergent, indicating the need for adjustments of the method for a more accurate identification of sources of resistance within Passiflora germplasm in a short time interval. Furthermore, this will enable assessment of various passion fruit genotypes simultaneously, facilitating the development of Fop resistant cultivars. In addition, an adequate inoculation protocol will support studies to better understand the genetic mechanisms of the plant-pathogen interaction, and histochemical and histopathological analyses for in loco evaluation of distinct patterns of infection between resistant and susceptible species, will allow the identification of chemical markers associated with resistance (Flores et al., 2012; Marques et al., 2013; Ortiz et al., 2014). Based on these considerations, the objective of this work was to evaluate an inoculation protocol of Fop using two methods of propagation (cuttings obtained in adult plants during the reproductive stage and seeds harvested in young plants) associated with controlled water deficit on the expression of Fop symptoms, as well as provide information about the anatomical alterations of the root of yellow passion fruit after the infection by Fop.

2.3. Formation of plantlets and inoculation with Fop The yellow passion fruit plantlets (BRS Gigante Amarelo) were obtained from seeds planted or cuttings rooted in small tubes with volume of 75 mL, containing vermiculite with medium granulometry. Thirty days after emergence of the seedlings (when they had six leaves) or rooting of the cuttings (with six leaves), the roots of all plants were injured with a scalpel to facilitate the penetration of the pathogen at the moment of inoculation (Supplementary Material, Fig. A). The monosporic isolate used for inoculation was Fop 05 (Fusarium oxysporum f. sp. passiflorae - Fop), obtained from the collection of the Phytopathology Laboratory of Embrapa Cassava and Fruits. The inoculation was made by the immersion of the roots for 10 min in a suspension adjusted to 106 conidia mL−1. The plantlets were then transplanted to polyethylene pots containing 1.2 L of washed and sterilized sand, previously infested with 50 g of substrate containing the same isolate (Fop 05) in the concentration of 106 CFU g−1. The substrate for inoculum production was prepared by mixing washed fine sand + cornmeal + water in a 9:1:2 proportion (m:m:m). Two hundred grams of this mixture was distributed in plastic bags with 1 kg capacity and autoclaved twice for 1.5 h. Then 20 disks (diameter of 5 mm) of potato dextrose medium (PDA) containing the Fop 05 isolate were added, and were cultured as proposed by Flores et al. (2012). The plastic bags were agitated every three days to obtain homogeneous colonization of the substrate by the fungi. 2.4. Water deficit after inoculation with Fop After the inoculation with Fop, the soil in all treatments was kept at field capacity for 15 days. This condition was maintained for the control treatments, while the water deficit was applied to the other treatments, with and without Fop. The moisture of the substrate was monitored on alternating days by the time domain reflectometry technique (TDR), utilizing probes with length of 10 cm (Coelho et al., 2006). Irrigation was suspended until the manifestation of partial wilting of the leaves, which occurred at relative moisture of around 0.12 ± 2 m3 m−3, and was resumed until full recovery of leaves turgidity, with moisture of 0.25 ± 2 m3 m−3, followed by another cycle of controlled water deficit until reaching the same levels mentioned. This process was repeated during the experiment period until the plants presented visual symptoms of the Fusarium wilt, which was set as the permanent wilting even after rehydration. In contrast, the plants in the control group (absence of Fop) submitted to water deficit regained turgidity of the leaves (Supplementary Material, Fig. B).

2. Materials and methods 2.1. Location of the experiment The study was conducted in a greenhouse at Embrapa Cassava and Fruits, located in the municipality of Cruz das Almas, Bahia state, Brazil (12°39′25′′S, 39°07′27′′W, 222m). ′25′′ S, 39°07′27′ the air temperature inside the greenhouse was maintained at 28 ± 2 °C and the relative humidity was 60%. The genotype evaluated was the cultivar ‘BRS Gigante Amarelo’ (P. edulis), which is considered susceptible to diseases caused by Fusarium species.

2.5. Experimental design and phytopathological variables studied The experimental design was completely randomized in a 2 × 2 × 2 triple factorial scheme (propagation method × water regime × pathogen inoculation), comprising eight treatments with four replications and 10 plants in each plot. For the anatomical analyses, at least four plants were collected per treatment.

2.2. Plant material and growing conditions Two propagation methods of the passion fruit plants were evaluated, seedlings and cuttings, regarding the manifestation of symptoms of Fusarium wilt. BRS Gigante Amarelo seeds were obtained from a

2.6. Evaluation of incidence and severity of Fusarium wilt To assess the Fusarium wilt in the stems and roots, visual 610

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for 72 h each, and lastly in pure historesin, where they remained for one week. The samples were imbedded for polymerization at room temperature for 48 h. Histological sections (5 μm) were obtained with a Leica RM 2155 rotary microtome (Leica, Nussloch, Germany) and were mounted on slides and stained (Marques et al., 2013). The sections were analyzed and photographed with an Olympus BX51 photomicroscope coupled to an Olympus DP175 digital camera (Olympus, Tokyo, Japan). Others sections with thickness of 5 μm were stained with Lugol’s iodine for 5 min, as is recommended to identify starch, detected by blue-black or very dark brown color. Aiming to evaluate the starch density and distribution within root tissues, a digital analysis of the microscopy images was conducted. For the assessment, three to five images from the treatments subjected to the water deficit (with and without Fop inoculation) were scaled to an interest area of 1000 x 1000 μm. The interest area was divided in 64 quadrants with 125 x 125 μm each, followed by the evaluation of the percentage of area occupied by starch, using the Image Analysis Software for plant disease quantification ‘Assess v1.0’. For the statistical analysis, each quadrant were evaluated for the type of contained tissue (epidermis, cortex, phloem or xylem), followed by a pairwise comparison of inoculated and non-inoculated by means of ANOVA and F-test. An additional analysis was conducted based on the comparison of isopach maps generated for both treatments, by transforming the position of the quadrat in a “x/y” grid vector, being the y-axis the vertical distance from the root epidermis to the center (pith), and the x-axis the distance from the center (pith) to the epidermis in a perpendicular direction. A contingence table was constructed using both x and y axis, and the % of area occupied with starch for each position; followed by the generation of the contour surface maps using the software SigmaPlot version 11.0. The presence of callose in vascular tissue and the tissue lignification of infected plants were identified by fluorescence microscopy. Sections from the roots of P. edulis plants submitted to water deficit and inoculation with Fop were stained with Lugol’s iodine for 3 min, washed with tap water and stained with 1% aniline blue for 8 min and then with Lugol’s iodine again for 30 s, and mounted on slides with tap water. The aniline blue stain produces a blue coloring of callose while Lugol’s iodine acts on the cell walls, giving a gray and yellowish color to lignified tissues. The possible presence of callose in the root system of the passion fruit plants infected by Fop was investigated by examining the slides with a fluorescence microscope with ultraviolet filter (Axioskop 2, Carl Zeiss, Jena, Germany).

symptomatology criteria were used, both external (dead regions, stems cracks and callus formation) and internal (darkened vascular color and callus formation). The evaluation of the disease’s symptoms was based on a scale from 0 to 3, where 0 = healthy plant; 1 = plant without external symptoms but with internal symptoms (internal darkening of stem and roots); 2 = plant with external and internal symptoms (callus formation, necrosis, darkening of tissues, cracks and vascular darkening); 3 = wilted and dead. The disease index (DI) was calculated according to McKinney (1923). The calculated data (percentage of incidence of symptomatic plants and DI) were subjected to variance analysis and the treatment means were pairwise compared by F test, using the agricolae package of the R program (R Development Core Team, 2016). For confirmation of the Koch’s postulates, excisions of the stem and roots of the symptomatic and asymptomatic plants were utilized to re-isolate the pathogen. 2.7. Kaplan–Meier estimates of the time-to-death of plants The survival analysis was conducted using nonparametric Kaplan–Meier (KM) curve (Kaplan and Maier, 1958). In this study, the event of interest was the death of the plant caused by Fop which was verified every two days. Differences of the survival curves from the methods of propagation with and without water deficit, and the interaction between these factors were tested using the nonparametric F-test of Cox (P ≤ 0.05). The KM estimator, Ŝ (t), is the nonparametric maximum likelihood estimate of the probability that a plant will survive up to time tj and is defined as: k

S¨ (t ) =

∏ ⎜⎛ j=1

nj−dj ⎞ ⎟

⎝ nj ⎠

where nj is the number of individuals alive just before time tj, and dj is the number of deaths at time tj, where tƙ ≤ t ≤ t(ƙ + 1) for ƙ = 1, 2,… r (Kaplan and Meier, 1958). In this study, the KM estimator defines the proportion of plant surviving to a specific time, t. 2.8. Histopathological analysis of the roots After the visual detection of disease symptoms on inoculated plants (wilting and internal darkening of vessels), 10 root fragments of five plants with lengths between 2 and 4 cm were collected to evaluate the colonization of the host plant’s tissues by Fop, using the root clearing and staining technique (Phillips and Haymann, 1970). The same procedure (clearing and staining of the roots) was also carried out for the non-inoculated plants as control. Quantification of the number of chlamydospores was performed in five photographed segments of the root. In each image, five squares of 200 × 200 μm were randomly distributed in the image and the number of chlamydospores was counted with the ImageJ software (Rasband, 1997-2016). The obtained data were subjected to normality tests and analysis of variance (ANOVA), using the agricolae package of the R program (R Development Core Team, 2016).

3. Results 3.1. Incidence and severity of Fusarium wilt At the end of the experiment period (210 days), all non-inoculated plants propagated by seeds and cuttings, with and without water deficit, had normal plant growth with healthy roots, were free of dead regions and had internal stem coloration typical of the species (Fig. 1A–C). Seedlings that were inoculated and submitted to water deficit started to show visual symptoms of Fusarium wilt 66 days after inoculation (DAI) (Fig. 1D–E). The presence of Fop was confirmed by reisolation of the pathogen, while the irrigated and inoculated seedlings started showing symptoms at 82 DAI. Among the symptomatic inoculated plants, some were observed with advanced deterioration of the roots (Fig. 1F-H), some with emission of new roots (Fig. 1G), and callus formation for emission of new roots (Fig. 1H). On the 150th day after inoculation, 30.8% of the irrigated seedlings presented symptoms of Fusarium wilt. In counterpart, in the plants submitted to water deficit, this value was more than two times higher, at 66.6% (P = 0.01). Another aspect found in the control plants (P. edulis seedlings) was the greater apparent volume of the root system (Fig. 2B–G) in

2.9. Anatomical analysis of the root system using the double staining method To differentiate the cell wall of the plant, structures of the pathogen and substances produced by the infected plants, a double staining method was used (Marques et al., 2013). Segments of secondary roots of five plants with lengths between 2 and 4 cm from the non-inoculated and inoculated plants propagated by seeds and kept under controlled water deficit were fixed in a solution of formaldehyde, acetic acid and 70% ethanol (FAA 70) for 48 h and then were conserved in 70% ethanol for one week (Johansen, 1940). After this period, the root segments were dehydrated in an increasing ethanol series (85–100%) for 9 h, slowly infiltrated in historesin: ethanol in proportions of 1: 2 and 1: 1 611

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Fig. 1. Passiflora edulis seedlings, submitted to water deficit with Fusarium oxysporum f. sp. passiflorae (Fop). A–C) Control plant, with turgid leaves, healthy roots and stem; D–H). Inoculated plant submitted to water deficit with symptoms of Fusarium wilt, presenting root necrosis and external and internal darkening of the stem. Some plants also presented growth of new roots (G), Callus formation (H) and External aspect of plant stem with symptoms of Fusarium wilt (I and J), indicating responses of the species to infection by Fusarium oxysporum f. sp. passiflorae 75 days after inoculation under controlled conditions. The arrow indicates the callus formation and new root growth.

3.2. Survival analysis

comparison to the plants submitted to controlled water deficit (Fig. 1F–J). Points of infection by the pathogen, consisting of necrotic areas, were observed in the root insertion-axillary region (Fig. 2B–C), as well as external darkening (Fig. 2E–F; I–K) and internal darkening of the stem (Fig. 2G). Swelling in the collar region was also detected, caused by multiplication of cortical parenchyma cells (Fig. 2D), in both stressed and unstressed plants (Figs. 1H and 2D). In cuttings subjected to water deficit, typical symptoms of Fusarium wilt were also observed in the plant (Fig. 2H), and stem and roots (Fig. 2I–K). On the other hand, the plants propagated by cuttings showed high mortality at 10 DAI independently of inoculation and water regime (42.2% on average). This result was probably a reflection of the low adaptation of the evaluated cultivar material to this propagation method, not having a direct relation with the inoculation process. Based on the box plot analysis (Fig. 3) for the disease severity of Fusarium wilt, the disease index (DI) showed that the use of water deficit did not influence the severity of the disease in both propagation methods (Fig. 3A-B). The inoculated seedlings presented the highest median severity of Fusarium with DI of 73.7% when compared to the inoculated cuttings (DI = 54.7%, P = 0.05) (Fig. 3C). However, the water regime condition did not influence on the severity of the Fusarium wilt (Fig. 3D).

The survival analysis results were significant according to the F-test of Cox between seedlings and cuttings (P < 0.0001), between plants subjected or not to the water deficit (P = 0.012), and between treatments within the plants propagated by seeds (P = 0.0001). The mortality associated with Fop in cutting-propagated plants did not differ between inoculated and non-inoculated plants (P = 0.3273) (Fig. 4A–D). Approximately 60% of the seedlings showed symptoms of the disease, while only 10% (P < 0.001) of the cuttings showed the typical wilting (Fig. 4A). Water deficit contributed to increase the susceptibility to Fop to an incidence of 50%, compared to the irrigated and inoculated plants, where only 30% of the plants exhibited symptoms (P = 0.012) (Fig. 4B). Of the seedlings that were subjected to controlled water deficit (Group 1), 75% showed symptoms, while this was 40% for the irrigated seedlings (Group 3). As expected, the control treatments with water deficit and without Fop (Group 2) and without water deficit and without Fop (Group 4) remained asymptomatic (P = 0.0001) (Fig. 4C). The survival curves among plants from cuttings were similar by the Ftest of Cox (Fig. 4D), with approximately 10% of the plants being 612

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Fig. 2. Typical symptoms of Fusarium wilt in Passiflora edulis plants inoculated and propagated by seeds, without controlled water deficit (A–G), and by cuttings with controlled water deficit (H–K). A) Plants inoculated, B–C) Root insertion-axillary region, D) Callus, E–F) External darkening and G) Internal darkening of the stem, (H) Plant propagated by cuttings with controlled water deficit and I-K) External darkening of the stem and roots. pi: points of infection; ca: callus.

M–N). Among the inoculated plants, there was generalized presence of chlamydospores (intracellular) in plants propagated by seeds (Fig. 5D) and cuttings (Fig. 5L) submitted to water deficit. Seedlings subjected to controlled water deficit (Fig. 5D) had a larger number of chlamydospores than those not deprived of water (Fig. 5H). The water deficit did not cause substantial alterations in the shape and structure of the root cells (Fig. 5), because the plants were only collected after rehydration. Besides this, no histopathological

symptomatic in both the water-restricted (Group 5) and irrigated (Group 7) plants. 3.3. Histopathological analysis The roots of the non-inoculated yellow passion fruit plants were healthy, with total absence of structures of the pathogen, and with characteristic growth in the root elongation region (Fig. 5A–B, E–F, I–J, 613

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Fig. 3. Blox plot of the disease index (DI) of Fusarium wilt of P. edulis plants propagated by seed and cuttings and submitted to controlled water deficit. A) Comparison of plants propagated by seeds with and without water deficit and inoculated with Fusarium oxysporum f. sp. passiflorae (Fop); B) Comparison of plants propagated by cuttings and without water deficit and inoculated with Fop; C) Comparison of plants propagated by seed and cuttings, and D) Comparison of the plants submitted to controlled water deficit. The line shown in the box is the median value. The box boundaries upper and lower quartiles, whiskers caps represent the minimum and maximum values. Error bar = standard error of the mean. * Indicates significant difference by the F-test at 5% of probability.

extend to the metaxylem (Fig. 7E-G). Since the plants were only collected after wilting, which is considered the last stage of the Fop x Passiflora spp. interaction, part of the roots was markedly deteriorated. In the non-inoculated seedlings, it was noticed that more starch was distributed along the cortical parenchyma compared to the vascular tissue (Fig. 7I–L). In contrast, the inoculated seedlings apparently presented greater deposition of starch in the vascular region, and very low concentrations in the cortical parenchyma (Fig. 7M–P). Furthermore, just below the first layer of cells (epidermis), there was scattered distribution of starch along the root cortex of the noninoculated seedlings (Fig. 7L), and absence of carbohydrates in the metaxylem (Fig. 7K). In turn, in the inoculated plants, the distribution occurred along the vascular rays in parallel direction (Fig. 7N), with low concentration in the cortex (Fig. 7M,O). In the fluorescence microscopy analysis, the secondary roots of plants grown from seeds and non-inoculated presented higher concentration and wider distribution of starch along the cortex (Fig. 7Q, ST) and near the vascular rays (Fig. 7R), while in the inoculated plants there was lower availability along the cortical parenchyma and vascular rays (Fig. 7U–Y). No deposition of callose plates was observed in inoculated plant tissue stained with aniline blue, but lignification was

differences were observed between the plants obtained from seeds and cuttings that can be associated with the colonization method of Fusarium f. sp. passiflorae (Fop). In the longitudinal sections, it was possible to identify hyphae colonizing the intercellular and intracellular spaces (Fig. 5C, G, K, O) and the presence of chlamydospores in the root cap (Fig. 5P). Besides this, spores with germinative tubes were identified in the intracellular space (Fig. 5H) and penetration point (Fig. 5C) in the root epidermis. However, no physical barriers were observed, such as increased cell wall thickening that could indicate some response to the infection. As the highest incidence of Fop was observed in seedlings, the quantification of the number of chlamydospores in the roots was only carried out in this method of propagation (Fig. 6). The secondary roots of the control plants had normal distribution of tissues of the cortex and vascular rays for the species, with absence of fungal structures (Fig. 7A–C) and presence of fibers in the cortical parenchyma (Fig. 7D). The roots of the plants propagated by seeds and inoculated contained a larger number of intracellular hyphae (Fig. 7E–H) and presence of gel along with starch in the vascular rays (Fig. 7G). In the infected roots, it was possible to observe disorganization of cells of the cortex in comparison with the control plants, but this did not

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Fig. 4. Survival analysis of P. edulis plants inoculated with Fusarium oxysporum f. sp. passiflorae (Fop) and propagated by seeds and cuttings (A), and submitted to water deficit (B). Comparison between treatments within the group of plants propagated by seeds (C) and cuttings (D). Group 1: propagated by seeds with controlled water deficit with Fop; Group 2: propagated by seeds with controlled water deficit without Fop; Group 3: propagated by seeds without controlled water deficit with Fop; Group 4 propagated by seeds without controlled water deficit without Fop (Fig. 6C); Group 5: propagated by cuttings with controlled water deficit with Fop; Group 6: propagated by cuttings with controlled water deficit without Fop; Group 7: propagated by cuttings without controlled water deficit with Fop; Group 8: propagated by cuttings without controlled water deficit without Fop (Fig. 6D).

4. Discussion

observed in the vascular region of inoculated plants (Fig. 7V–X). Since the starch accumulation in the vessel was an uncommon behavior for the interaction between Fop x Passiflora spp., a more deep investigation were conducted, based on the analysis of the digital images (as described in the methodology, Fig. 8A). The comparison of the area occupied by starch within each different tissue was in agreement with the first observation of the microscopic images (Fig. 8B), since the non-inoculated plants normally present the higher amount of starch in the cortex region (6.2% of area occupied by starch), being significantly different (P = 0.01) from those inoculated (2.58% of area occupied by starch). A significant difference was also noticed to the accumulation of starch in the xylem vessels, being the inoculated plants with 11.6% of area occupied by starch, and the non-inoculated with only 0.51% of area occupied by starch (P = 0.01). There were no significant differences among the other tissues of the roots (phloem and epidermis) (Fig. 8B). The contour surface analyses revealed the spatial distribution and density of the starch granules on the different tissues. The normal characteristic of the starch accumulation patterns was the presence of starch on the cortical parenchyma in low densities (Fig. 8C–D), in comparison with the diseased plants that showed increased starch accumulation in the xylem vessels, and lower starch density in the cortex (Fig. 8E–F).

Plants are constantly subjected to biotic either abiotic stresses, and the responses to these factors vary depending on the plant’s resistance or susceptibility (Ramegowda and Enthil-Kumar, 2015; Pandey et al., 2017). The results reported here indicate that the controlled water deficit had a direct influence on the manifestation of symptoms in the yellow passion fruit plants inoculated with Fop. Some researchers have observed that greater incidence of Fusarium wilt in yellow passion fruit occurs under high air temperatures and when transpiration demand is intense (Cavichioli et al., 2011), and water availability in the soil is limited (Ramegowda and Enthil-Kumar, 2015; Pandey et al., 2017). The water deficit caused by increased temperature can substantially alter the plant’s physiology, to the point of suppressing resistance to various pathogens (Wang et al., 2009). In yellow passion fruit, high susceptibility to Fop has been observed in field conditions after drought events (Cavichioli et al., 2011). This result was corroborated in the present study in greenhouse conditions when the plants were submitted to controlled water deficit. The majority of studies evaluating the inoculation of the yellow passion fruit has disregarded the joint effect of biotic and abiotic factors on the symptoms of Fusarium wilt (Fischer et al., 2010; Flores et al., 2012; Ortiz and Hoyos-Carvajal, 2016), thus revealing distinct responses regarding intensity of the disease. However, the results obtained in this study open the possibility of using water deficit associated 615

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Fig. 5. Secondary roots of Passiflora edulis plants propagated by seeds and cuttings, with and without controlled water deficit, inoculated or not with Fusarium oxysporum. Roots of seedlings with controlled water deficit without Fop (A–B) and with Fop (C–D), without controlled water deficit, noninoculated (E–F) and inoculated (G–H). Roots of cuttings with water deficit, noninoculated (I–J) and inoculated (K–L), without controlled water deficit, without Fop (M–N) and with Fop (O–P). chl: chlamydospores; hy: hyphae; sp: spores, gt: germinative tube; pp: penetration point. Bars: 250 μm (F), 100 μm (a, c, d, e, g, h, i, j, k, l, m, o) and 50 μm (b, n, p).

Flores et al. (2012), utilizing fusaric acid and a spore suspension, obtained Fusarium wilt incidences of 59.4% and 56.0%, respectively, after inoculation in P. edulis. Faithful reproduction of the natural exposure conditions of the plant

with Fop inoculation to select Passiflora genotypes for resistance to this pathogen, since 75% of the inoculated Passiflora edulis plants (susceptible) propagated by seeds and subjected to water deficit showed typical internal symptom of the disease, that is, necrosis of the vascular system.

Fig. 6. Number of chlamydospores in roots of P. edulis seedlings inoculated with Fop with water deficit and without water deficit. Analysis of the values through the Box-Plot box diagram for the propagation by seeds with and without water deficit. *Indicates significant difference by F-test at 1% of probability. ch: chlamydospores, sp: spores, gt: germinative tube. Bars: 100 μm. 616

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Fig. 7. Secondary roots of Passiflora edulis propagated by seeds and submitted to controlled water deficit. A–D) secondary roots stained with cotton blue and safranin, showing normal arrangement of the tissues. E–H) roots inoculated with Fusarium oxysporum f. sp. passiflorae with presence of hyphae in the cortical parenchyma and gel in the metaxylem (G). I–K) non-inoculated roots stained with Lugol’s iodine to detect starch with abundant concentration in the cortical parenchyma and absence in the metaxylem (K). M–P) inoculated roots with larger concentration of starch in the vascular rays. Q–T) healthy roots visualized by fluorescence microscopy with normal arrangement of tissues and abundant starch, identified by the brown color. U–Y) inoculated roots with absence or low concentration of starch along the cortical parenchyma. ep: epidermis, mx: metaxylem, ge: gel: co: cortex; st: starch; fb: fiber; hy: hyphae, arrows indicate the Fop hyphae. Bars: 250 μm (A, I, M, R, Q, R, U, S, V, X); 100 μm (B, C, J, K, N, O, Y); 50 μm (D, E, F, L, P, T); and 25 μm (G, H). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

inoculation, using plants propagated by seeds submitted to controlled water deficit followed by rehydration. This allows a substantial reduction in time needed to select resistant genotypes in comparison to field studies with Passiflora, which last an average of 400 days and are substantially expensive. Regarding the propagation method, Fop incidence and severity were higher in P. edulis seedlings that were submitted to the water deficit. On the other hand, cuttings obtained in adult plants at reproductive stage were less affected, suggesting that Fop incidence is not directly related to the advanced phenological stage of the plant. Similarly, Junqueira

to the pathogen is fundamental in artificial inoculation to obtain responses similar to those observed in the field. According to Flores et al. (2012), the correlation of the response between genotypes selected in vitro using Fop filtrate (fusaric acid) and entire plants inoculated with the pathogen is essential to confirm the validity of in vitro selection of genotypes, since the reaction of plants to inoculation with the pathogen is more reliable. Besides this, validation in fields with a history of the disease is necessary to assure the effectiveness of the method. The assessment of the resistance to Fop under the controlled conditions evaluated in this study can be concluded in 150 days after 617

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Fig. 8. A) Quadrant sampling method estimative of starch accumulation on different root tissues; B) comparison of area occupied by starch in each root tissue from inoculated and noninoculated plants with water deficit treatment; C–D) microscopic image from root cut of noninoculated plants and comparison with the contour surface map of the starch accumulation on the cortex region; E–F) microscopic image from root cut of inoculated plants and comparison with the contour surface map of the starch accumulation on the xylem vessel region. Different colors, color gradient and isolines (lines of constant value) indicates the percentage of the area occupied by starch in relationship to the tissue location. ep: epidermis; co: cortex; and vascular tissue (xy: xylem and ph: phloem) Bars: 200 μm (C and E). * Indicates significant difference by F-test at 1% of probability.

et al. (2006), evaluating cuttings and seedlings of P. edulis under field conditions, observed that cuttings were less affected by soil-borne pathogens (20.6%) than seedlings (64.6%). Ahmad et al. (2013), to compare the disease incidence in the seedlings (sexual propagation)

and shoot cuttings (asexual propagation) of shisham (Dalbergia sissoo Roxb. ex DC.) after artificial inoculation with diff ;erent fungal pathogens, observed that plants of propagated by cuttings showed lower incidence soil-borne diseases. However, these results do not corroborate

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Fusarium wilt incidence in commercial fields as well. Investigations on this control measure in areas affected by the disease are in progress, and additional care must be dedicated to other aspects of the vegetative propagation which include the use of disease-free mother plants, nursery infrastructure for cutting, genetic variability to avoid self-incompatibility problems, and faster commercial-scale propagation protocols. Analyzing the structure of the secondary root of P. edulis inoculated withFop and submitted to water deficit, the presence of pectin gels in the metaxylems was observed. Some authors mention that the formation of pectin gels in the xylem vessels of the inoculated plants submitted to controlled water deficit is associated with the responses induced by the pathogen (Ortiz et al., 2014). However, this post-infection barrier was not sufficient to limit the advance of the pathogen and prevent colonization of the tissues. Similar responses to those observed in this study were reported by Ortiz et al. (2014), with formation of gels in the xylem vessels of P. edulis inoculated with Fop. Probably the signaling for inducement of synthesis of the compound occurred tardily, when the Fop spores had already colonized the plant, and the deposition of the gels associated with polysaccharides of fungal origin might have contributed by obstructing the vessels and promoting wilting of the plant (Stangarlin and Leite, 2008). Large amounts of starch polysaccharides were also observed in the vascular rays of the roots. The few studies in this respect have reported that plants submitted to inoculation show reduced deposition of this polysaccharide in the roots (Keunen et al., 2013; Manila and Nelson, 2014), since it is utilized as a substrate for the colonization of the plant. Similar results were observed in olive (Olea europaea L.), which showed reduction of starch content in the xylem vessels with increased severity of Verticillium dahlia wilt symptoms (Trapero et al., 2018) . Another study showed that plants submitted to water deficit have a higher concentration of carbohydrates in the roots (Muller et al., 2011). The abnormal accumulation of starch in plant tissue was also noticed in literature, and one of the possible explanations is that the plant could enhance the starch accumulation as a way to supply substrate for the synthesis of molecules associated with response to stress (O’Brien et al., 2014; Higuchi et al., 2015). The lower starch content found in olive plants subjected to drought stress compared to well-watered plants, as well as its recovery after resumption of irrigation, supports this role of starch (Trapero et al., 2018). According to Nyitrai et al. (2004), some stress-inducing compounds (such as heavy metals) could lead to non-specific signals, which involve changes in the hormonal balance (probably cytokinins), followed by an increase on CO2 fixation and starch accumulation in bean leaves. However, the role of the starch accumulation on the xylem vessels due to the infection by Fop remains uncertain and needs further investigation. The distribution of fibers and lignification of the cortical walls in the secondary roots were low, both in the control and inoculated plants. This might be associated with the susceptibility of yellow passion fruit (P. edulis) to Fop, because the cell wall is considered to be the first barrier against the penetration or colonization of the pathogen. Besides this, alterations can occur in this membrane that prevent colonization by the pathogen (Miedes et al., 2014). Another defense strategy of plants against attack by pathogens is the rapid deposition of callose in the vascular tissue to strengthen the xylem walls and impede the pathogen’s advance, thus constituting a physical barrier to colonization (Nishimura et al., 2003). However, in the present study we did not visualize callose plates in the roots of the inoculated P. edulis plants, suggesting that this species cannot form this physical barrier to impede colonization of Fop in the vascular tissue, or that callose plates are only formed in the stem. However, the emission of new roots in inoculated plants was not observed. These results indicate that physiological and structural mechanisms exist in response to infection, but these are triggered tardily by the host. This behavior was also observed in tomato plants infected by F. oxysporum f. sp. radicislycopersici (McGovern, 2015).

field observations that report higher incidence of the Fusarium wilt during the initial reproductive stage of the yellow passion fruit (Cavichioli et al., 2011). This dissemination pattern in the field is probably associated with the period of time necessary for the release of exudates by the roots, and further perception by the pathogen for infection and colonization of the plant (McGovern, 2015). Besides that, the higher transpiration in adult plants, due to larger leaf area, flowering and fruit set, predisposes the plants to water stress that may contribute to a higher incidence of Fusarium wilt. It is important to notice that the plants used in this work had the same number of leaves (n = 6), even though cuttings had noticeably larger leaf area of fully expanded trilobated leaves if compared to the seedlings (Supplementary Material, Fig. A). Several authors have mentioned that the biometric and physiological responses of Passiflora plants propagated by seeds and cuttings may vary according to the species studied (Almeida et al., 1991; Santos et al., 2016). The mechanisms responsible for the distinct susceptibility to Fop between cuttings and seedlings are still unknown. Possible explanations may be associated with plant vigor, osmotic regulation capacity, level of suberization and/or size of the xylem vessels, since these characteristics may be associated with plant resistance to wilt-causing pathogens either tolerance to water stress (Pouzoulet et al., 2014; Barrios-Masias et al., 2015). In addition, some genes expressed in seedlings are not always expressed in adult plants (Burdon et al., 2014), for example for some crops adult plant resistance (APR) genes confer a durable partial resistance and are often polygenic in nature (Hickey et al., 2011; Riaz et al., 2017;). These APR genes confer susceptibility of plants at the seedling stage and resistace in later stages (Yuan et al., 2018), although in passion fruit several genotypes die in the field durant adult phase due to Fop (Junqueira et al., 2006; Cavichioli et al., 2011). Some authors have collaborated with information on the APR in relation to plants infected by Fusarium (Wang et al., 2018). Therefore, new studies focusing on the morphological, biometric, physiological and anatomical aspects and comparison of Fop incidence in field and greenhouse conditions are mandatory to elucidate the mechanisms associated with the differences in mortality of passion fruit plants propagated by cuttings and seeds and under water stress condition. The control of diseases caused by soil-borne pathogens is very complex, as is the case of F. oxysporum, which is among the 10 most soil-borne pathogens in the world in terms of economic importance (Kang et al., 2014). The micelles of pathogens can survive in association with plant tissues in saprophytic form as well as in alternative hosts, and their thick-walled chlamydospores can survive for long periods (McGovern, 2015). The higher concentration of chlamydospores in the plants submitted to water deficit can be explained by the greater vulnerability of these plants under this abiotic condition. The formation of chlamydospores in F. oxysporum is related to stress factors such as the absence of hosts and unfavorable environmental conditions (DaamiRemadi et al., 2009). In other species of plants, as for example in tomato, a higher incidence of Fusarium wilt was caused by the greater sporulation of the fungi under salt stress conditions (Daami-Remadi et al., 2009). Besides this, the alternation of dry with moist periods stimulates the multiplication of hyphae and chlamydospores in the soil (Couteaudier and Alabouvette, 1990) and inside the roots (Lin and Heitman, 2005). In this study, it might have caused micro-fissures in the roots that allowed the pathogen to enter (McGovern, 2015), or altered the plant’s metabolism, raising susceptibility and culminating in obstruction of the vessels and wilting of the plant. Commercially, the yellow passion fruit is propagated in Brazil by sexual method using seeds, because this is a cheaper and more available material. Cuttings were used in experiments to multiply selected genotypes resulting in superior traits, such as early-bearing, yield and fruit uniformity (Koch et al., 2001; Santos et al., 2012). In this study, plants propagated by cuttings had four times less mortality by Fop than seedlings in controlled conditions. This is very promising in terms of the disease management, since cutting propagation could decrease the 619

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The results obtained in this study open the possibility of using water deficit cycles in the selection of P. edulis genotypes for resistance to Fop in controlled conditions, because the stressed plants were more susceptible to Fop, since they showed lower accumulation of starch in the cortex than the non-inoculated plants. This finding will contribute to elucidate the mechanisms of the plant-pathogen interaction and will shed more light on the effect of water restriction on the susceptibility of yellow passion fruit to this pathogen. However, this study needs to be expanded to other species of Passiflora to identify individuals that are resistant to Fop for subsequent use in interspecific hybridization programs or as rootstock. Also, additional investigations at the molecular level can confirm the anatomical observations related to the deposition of starch and the concentration of hyphae and chlamydospores, as well as elucidate and identify the pathways related to resistance or susceptibility of Passiflora genotypes to Fusarium wilt.

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5. Conclusion The results obtained in this study indicate that the seed propagation associated with controlled water deficit influences the incidence of Fusarium wilt in Passiflora edulis. Controlled water deficit cycles favor the earlier expression of symptoms, noted by the greater development of hyphae and presence of chlamydospores. The yellow passion fruit plants inoculated with Fop presented anatomical alterations in response to the infection by the pathogen, but these were not sufficient to prevent colonization. Conflicts of interest The authors declare no financial or other competing conflicts of interest. Acknowledgements To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support and for thepost-doctoral scholarship –PDJ (151086/2018-4) given to the first author, the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) and CNPq for the scholarship for regional scientific and technological development (DCR0013/2015) granted to the third author, and Embrapa Mandioca e Fruticultura and Embrapa Cerrados for financial and technical support (MP 02.12.02.006.00.00) and supply of plant materials. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2018.09.017. References Ahmad, M.A., Iqbal, S.M., Ayub, N., Ahmad, Y., Akram, A., 2010. Identification of resistant sources in chickpea against Fusarium wilt. Pak. J. Bot. 42, 417–426. Ahmad, B.I., Khan, R.A., Siddiqui, M.T., 2013. Incidence of dieback disease following fungal inoculations of sexually and asexually propagated shisham (Dalbergia sissoo). Eur. J. Forest Pathol. 43, 77–82. Almeida, L.P., Boaretto, M.A.C., Santana, R.G., 1991. Estaquia e comportamento de maracujazeiros (Passiflora edulis Sims f. flavicarpa Degener) propagados por via sexual e vegetativa. Rev. Bras. Frutic. 13, 157–159. Barrios-Masias, F.H., Knipfer, T., McElrone, A.J., 2015. Differential responses of grapevine rootstocks to water stress are associated with adjustments in fine root hydraulic physiology and suberization. J. Exp. Bot. 66, 6069–6078. Berta, G., Sampo, S., Gamalero, E., Massa, N., Lemanceau, P., 2005. Suppression of Rhizoctonia root-rot of tomato by Glomus mossae BEG12 and Pseudomonas fluorescens A6RI is associated with their effect on the pathogen growth and on the root morphogenesis. Eur. J. Plant Pathol. 111, 279–288. Burdon, J.J., Barrett, L.G., Rebetzke, G., Thrall, P.H., 2014. Guiding deployment of resistance in cereals using evolutionary principles. Evol. Appl. 7, 609–624. Cavichioli, J.C., Corrêa, L., Garcia, M.J., Fischer, I.H., 2011. Desenvolvimento, produtividade e sobrevivência de maracujazeiro-amarelo enxertado e cultivado em área com histórico de morte prematura de plantas. Rev. Bras. Frut. 33, 567–574.

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