Abstract

The influence of the grafting height (5, 10, 20 and 30 cm above the root collar) of P. edulis on P. gibertii was evaluated on the incidence of Fusarium wilt and horticultural performance. Plants of P. gibertii grafted on P. edulis and non-grafted plants of both species were also studied. In addition, histopathological studies were also performed on the roots of non-grafted P. edulis collected at three severity stages of Fusarium wilt. In greenhouse, the graft take was inversely related to the grafting height in general. In the field conditions, the plant growth of P. gibertii grafted on P. edulis was superior to its reciprocal grafting, even though the former combination was susceptible to Fop. Plants of P. edulis grafted on P. gibertii at all grafting heights did not present symptoms of Fop, and the number of fruit yield and quality were equivalent, but plant growth was decreased in relation to the non-grafted plants. Starch depletion in the root system of P. edulis was directly related to the severity of the Fusarium wilt. P. gibertii was confirmed as a Fusarium wilt resistant rootstock of P. edulis, with minimal influence of the grafting height for the control of the disease.

Key words
Passiflora edulis; Fusarium oxysporum f

INTRODUCTION

Soil-borne diseases are the main causes of economic losses in the yellow passion fruit (Passiflora edulis Sim) cultivation (Ortiz and Hoyos-Carvajal 2016ORTIZ E and HOYOS-CARVAJAL L. 2016. Standard methods for inoculations of F. oxysporum and F. solani in Passiflora. Afr J Agric Res 11: 1569-1575., Morgado et al. 2017MORGADO MADO, BRUCKNER CH, ROSADO LDS and SANTOS CEM. 2017. Growth dynamics and allometric relationships of Passiflora species rootstocks. Com Sci 8: 1-8.). Fusarium wilt, caused by Fusarium oxysporum f. sp. passiflorae (Fop), is considered the most important because it attacks the vascular system causing sudden wilting and early death of the plant (McGovern 2015MCGOVERN RJ. 2015. Management of tomato diseases caused by Fusarium oxysporum Crop Prot 73: 78-92.). Fop has been decreasing the productivity and lifespan of P. edulis crop worldwide (Fischer and Rezende 2008FISCHER IH and REZENDE JAM. 2008. Diseases of passion flower (Passiflora spp.). Pest Control Technol 2: 1-19.).

The grafting onto resistant rootstocks is used for the control of soil-borne diseases in several crops (Prunier et al. 1999PRUNIER JP, JULLIAN JP and AUDERGON JM. 1999. Influence of rootstock and height of grafting on the susceptibility of apricot cultivars to bacterial canker. Acta Hortic 488: 643-646., Pavlou et al. 2002PAVLOU GC, VAKALOUNAKIS DJ and LIGOXIGAKIS EK. 2002. Control of root and stem rot of cucumber, caused by Fusarium oxysporum f. sp. radicis-cucumerinum, by grafting onto resistant rootstocks. Plant Dis 86: 379-382., Sayler et al. 2002SAYLER RJ, SOUTHWICK SM, YEAGER JT, GLOZER K, LITTLE EL and KIRKPATRICK BC. 2002. Effects of rootstock and budding height on bacterial canker in French prune. Plant Disease 86: 543-546.) either anticipates the production (Castle 1995CASTLE W. 1995. Rootstock as a fruit quality factor in citrus and deciduous tree crops. New Zeal J Crop Hort Sci 23: 383-394., Karlidag et al. 2016KARLIDAG H, EREN G, KARAAT FE and KAN T. 2016. Grafting height effects on lateral branching shoot angles and growth of some fruit species saplings. Int J Agric Innov Res 5: 1-5.). The grafting of the yellow passion fruit on several resistant wild species was studied as an alternative for the production in areas with a history of Fop (Cavichioli et al. 2011aCAVICHIOLI JC, CORRÊA LS, GARCIA MJM and FISCHER IH. 2011a. Development, productivity and survival of yellow passion fruit trees grafted and cultivated in an area with a history of premature plant death. Rev Bras Frut 33: 567-574., Lima et al. 2017LIMA LKS, SOARES TL, SOUZA EH, JESUS ON and GIRARDI EA. 2017. Initial vegetative growth and graft region anatomy of yellow passion fruit on Passiflora spp. rootstocks. Sci Hortic 215: 134-141.). Passiflora gibertii N.E. Br. was highlighted with additional desirable traits, such as rapid plant growth, high seed germination and good anatomical compatibility for grafting with P. edulis (Cavichioli et al. 2011a, Yockteng et al. 2011YOCKTENG R, D’EECKENBRUGGE GC and SOUZA-CHIES TT. 2011. Passiflora In: Wilkins R (Ed), Wild Crop Relatives: Genomic and Breeding Resources Tropical and Subtropical Fruits. Germany: Springer, 256 p., Lima et al. 2017LIMA LKS, SOARES TL, SOUZA EH, JESUS ON and GIRARDI EA. 2017. Initial vegetative growth and graft region anatomy of yellow passion fruit on Passiflora spp. rootstocks. Sci Hortic 215: 134-141.).

Studies with passion fruit, in general, do not make inferences of the effect of grafting height on the horticultural performance and incidence of Fop, even though this influence is reported for other grafted crops and diseases (Prunier et al. 1999PRUNIER JP, JULLIAN JP and AUDERGON JM. 1999. Influence of rootstock and height of grafting on the susceptibility of apricot cultivars to bacterial canker. Acta Hortic 488: 643-646., Sayler et al. 2002SAYLER RJ, SOUTHWICK SM, YEAGER JT, GLOZER K, LITTLE EL and KIRKPATRICK BC. 2002. Effects of rootstock and budding height on bacterial canker in French prune. Plant Disease 86: 543-546., Chalise et al. 2013CHALISE B, PAUDYAL KP and SRIVASTAVA SP. 2013. Effect of grafting height on success and subsequent growth of acid Lime (Citrus aurantifolia Swingle) saplings. Nepal J Sci Technol 14: 25-32., Yazdani et al. 2016YAZDANI Z, JAFARPOUR M and SHAMS M. 2016. Effect of scion source budding method and graft union height on sweet cherry budding compatibility on Mahaleb rootstock. Trends Appl Sci Res 6: 1-4., Karlidag et al. 2016KARLIDAG H, EREN G, KARAAT FE and KAN T. 2016. Grafting height effects on lateral branching shoot angles and growth of some fruit species saplings. Int J Agric Innov Res 5: 1-5.). ,Nakasone and Paull (1998)NAKASONE HY and PAULL RE. 1998. Tropical and Subtropical Fruits. Oxon, UK: CAB International, p. 443. report that higher grafting height of the purple passion fruit on the yellow one is associated to a decreased incidence of Fusarium wilt. Cleft grafting of P. edulis on P. gibertii at 6 to 8 cm above the soil level resulted in high plant survival to Fusarium wilt in São Paulo State, Brazil (Cavichioli et al. 2011a). However, infected plants of this combination were observed in Bahia State, Brazil, which was attributed to the low height in hypocotyledonar grafting (Santos et al. 2016SANTOS CHB, OLIVEIRA EJ, LARANJEIRA FF, JESUS ON and GIRARDI EA. 2016. Growth fruit set and fusariosis reaction of yellow passion fruit grafted onto Passiflora spp. Rev Bras Frut 38: 1-5.).

In this work, we evaluated the influence of the grafting height of P. edulis on P. gibertii on the incidence of Fusarium wilt and horticultural performance. In addition, the grafting of P. gibertii on P. edulis and histopathological aspects of the latter’s root system were investigated to understand this pathogen-host relationship.

MATERIALS AND METHODS

LOCATION OF EXPERIMENT AND PLANT MATERIAL

Experimental works were carried out in the municipality of Cruz das Almas, Bahia State, Brazil (12° 39’ 25’’ S, 39° 07’ 27’’ W, 222 m. a. s. l.). Plantlets were grown in greenhouse from January to April 2016, at 28±2 °C and relative humidity of 60%. Later, the plantlets were transplanted into a field with a high incidence of Fusarium wilt (Santos et al. 2016SANTOS CHB, OLIVEIRA EJ, LARANJEIRA FF, JESUS ON and GIRARDI EA. 2016. Growth fruit set and fusariosis reaction of yellow passion fruit grafted onto Passiflora spp. Rev Bras Frut 38: 1-5.) and cultivated from April 2016 to August 2017. During the field evaluation period, the average air temperature was 25.4 °C, with rainfall of 1047 mm and relative humidity of 77%.

The scion variety was BRS Rubi do Cerrado, a commercial yellow passion fruit hybrid (P. edulis), and the wild species P. gibertii (BGP008 accession) was used as the rootstock. Non-grafted seedlings of both species were controls. Reciprocal grafting was also performed to evaluate if the P. gibertii scion would induce some resistance to Fop in the rootstock of P. edulis (Figure 1).

Figure 1
(a) Non-grafted P. edulis and P. gibertii plants used as controls (b) grafting of P. edulis/P. gibertii at four grafting heights and (c) their reciprocal grafting of P. gibertii/P. edulis.

EVALUATION IN GREENHOUSE

The experimental design in the greenhouse was completely randomized blocks in a factorial scheme (2 x 4 + 2), with two scion-rootstock combinations (P. edulis/P. gibertii and P. gibertii/P. edulis) and four grafting heights (5, 10, 20 and 30 cm above the root collar, Figure 1), plus two additional treatments (non-grafted seedlings of each species), distributed in four replications of 10 plants per plot.

The rootstock seedlings grafted at 20 and 30 cm height were sown 30 days before those grafted at 5 and 10 cm height. This procedure was performed so that the grafts were performed at the same time at all grafting heights studied. The seeds were planted in trays using vermiculite potting mix. Fifteen days after emergence, the rootstock seedlings were transplanted into polyethylene bags (22 x 12 cm) containing a mixture of humus, vermiculite, coconut fiber and commercial potting mix Vivato® (2:1:1:1, v:v).

At 45 days after transplanting, the cleft grafting method was performed and the rootstocks were cut with scissors at heights of 5, 10, 20 and 30 cm. At the cutting site, a longitudinal cut of 1.0 cm was opened with a scalpel blade. In the open cut, the scion with about 4.0 cm long, obtained from the meristematic apex of seedlings from passion fruit seeds ‘BRS Rubi do Cerrado’, was inserted. The base of the scion was cut into wedge-shaped double bevel and its leaves were cut in 1/3 of its area. Subsequently, the graft was inserted in the rootstock carefully, in order to coincide with the cambium tissues; the grafting region was protected by clip grafted (Figure 1).

The variables studied were graft length (GL, in cm) measured from the graft region to the tip of the plant, diameter of the scion stem (DC, in mm) measured with a pachymeter at 2 cm above the grafting region, and graft take (%) at 60 days after grafting (DAG).

EVALUATION IN FIELD CONDITIONS

At 60 DAG, all the grafted plantlets and seedlings were transplanted into the field to evaluate the incidence of Fusarium wilt and the horticultural performance. The experimental design was the same of the greenhouse, except that six plants were used in the plot. The soil of the experimental area is classified as allic distrocohesive yellow latosol, with the following chemical characteristics (0-20 cm): pH (water) 6.5, phosphorus (P) 60.85 mg dm^-3, potassium (K) 0.20, calcium (Ca) 2.26, magnesium (Mg) 0.99, aluminum (Al) 0.0, sodium (Na) 0.09, hydrogen + aluminum (H + Al) 1.25, base saturation (SB) 3.24, cation exchange capacity (CTC) 4.79 cmol_c dm^-3, base saturation percentage (V%) 73.65%, and organic matter (OM) 13.2 g kg^-1.

The plants were trained by single-wire trellises with 2.0 m height and spacing between plants was 1.8 m x 2.0 m. Drip irrigation was carried out three times a week with a duration of 30 min each, with an average water volume of 10 L per plant per irrigation. The plants were fertilized every 20 days for the first six months with 30 g of ammonium sulphate per plant (afterward increased to 80 g) plus 30 g of potassium chloride and 20 g of simple superphosphate. The other cultural practices were carried out following the recommendations for cultivation of passion fruit of Lima et al. (2011)LIMA AA, BORGES AL, FANCELI M and CARDOSO CEL. 2011. Passion flower: conventional production system. In: Pires MM, São José AR and Conceição AO (Eds), Passion fruit: technological advances and sustainability. Ilhéus: Edit, 238 p..

The plant growth index (GRI) was estimated up to 220 days after transplanting as established by Jesus et al. (2016)JESUS ON, SOARES TL, GIRARDI EA, ROSA RCC, OLIVEIRA EJ, CRUZ NETO AJ and OLIVEIRA JRP. 2016. Evaluation of intraspecific hybrids of yellow passion fruit in organic farming. Afr J Agric Res 11: 2129-2138.. The number of fruits per plant and crop yield (kg ha^-1) were also measured in the first production cycle. To evaluate fruit quality, five ripe fruits were randomly collected per plot at peak plant production during the months of November and December 2016. Fruit quality was studied only for the plants of the P. edulis/P. gibertii combination and the non-grafted seedlings of P. edulis. The physical and chemical characteristics measured were: fruit mass (g), length and diameter (cm), skin thickness (mm) and mass (g), pulp mass (g), soluble solids content (SS, in °Brix), titratable acidity (TA) expressed in mg citric acid per 100 mL juice, as determined through titration with NaOH at 0.1 mol L^-1, and the maturity index (SS/TA) (Jesus et al. 2017JESUS ON, OLIVEIRA EJ, FALEIRO FG, SOARES TL and GIRARDI EA. 2017. Iustrated morpho-agronomic descriptors for Passiflora spp. Brasília: Embrapa Mandioca e Fruticultura 1: 122.).

HISTOPATHOLOGICAL ANALYSIS OF ROOTS

To support the understanding of the interaction P. edulis x Fop, histopathological analysis was performed on non-grafted seedlings of BRS Rubi do Cerrado (n = 3) at different Fusarium wilt severity stages: (i) visually healthy plants or controls (Figures 2a, c), (ii) plants with slight leaf wilt (onset of symptoms) (Figures 2b, d, stage 1), (iii) generalized presence of wilted leaves adhered to the plant (Figures 2e, g, stage 2), and (iv) necrosis of the roots with most of the leaves already fallen (Figures 2f, h, stage 3). Secondary root fragments (n = 6, with a mean diameter of 1.0 mm and 2-4 cm length) were collected throughout the evaluation period from plants at all wilt stages, and were analyzed to confirm the presence of the pathogen as the causal agent (Figures 2d, g, h).

Figure 2
Morphological aspects of non-grafted plants of yellow passion fruit (Passiflora edulis Sims.) at different stages of Fusarium wilt. a-c: healthy plant. b-d: first stage of wilt. e- g: second stage of wilt. f-h: third stage of wilt. st: stem, vd: vascular discoloration, sb: secondary branch, ro: root, Fop: Fusarium oxysporum f. sp. passiflorae.

Root samples were fixed, embedded, sliced in sections and visualized according to the method described by Lima et al. (2017). Three slides containing 10 sections were prepared for each treatment, totaling 30 units. To determine the presence of starch, sections containing 5 μm thick root segments were stained with Lugol’s iodine for 5 min for observation of cell structure with dark blue or brown color (Berlyn and Miksche 1976BERLYN GP and MIKSCHE JP. 1976. Botanical microtechnique and cytochemistry. Ames: Iowa State University, 326 p.).

For fluorescence microscopy, the root 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 and assembled with tap water (Kraus and Arduin 1997KRAUS JR and ARDUIN M. 1997. Basic manual of methods in plant morphology. Rio de Janeiro: Eduar, Seropédica, 198 p.). The blue aniline dye produces a blue color in callose tissue and Lugol acts on the cell walls, giving gray and yellowish coloration to lignified ones. To verify the presence of callose in the Fop-infected yellow passion fruit root system, a fluorescence microscope with ultraviolet filter was used (Axioskop2, Carl Zeiss, Jena, Germany). The slides were also analyzed under a light microscope.

Hyphae of the pathogen were visualized using fresh root fragments 2-4 cm long to evaluate the colonization of host tissues by Fop, using the clearing and staining technique (Phillips and Haymann 1970PHILLIPS JM and HAYMANN AS. 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for assessment of infection. Trans Br Mycol Soc 55: 158-161.).

STATISTICAL ANALYSES

Data were submitted to analysis of variance and the means were compared by the Tukey test at 5% probability. When necessary, data were transformed by to satisfy the premises of normal distribution. Survival analysis was performed using the nonparametric Kaplan-Meier (KM) curve (Kaplan and Maier 1958KAPLAN EL and MEIER P. 1958. Nonparametric estimation from incomplete observations J Am Stat Assoc 53: 457-481.). In this study, the event of interest was the death of the plant caused by the Fusarium wilt, which was recorded every two days based on the typical visual symptoms (Figure 2). The differences between survival curves in the different combinations of scion-rootstock and grafting heights were tested using the nonparametric log-rank, Peto and Peto’s Wilcoxon, and Cox tests (p ≤ 0.05). In addition to the survival analysis, the average life expectancy at 122 days (coinciding with the initial reproductive stage of the plants) was estimated using the lifetime table and the number of plants affected by Fusarium wilt using the boxplot statistical algorithm.

RESULTS

EVALUATION IN GREENHOUSE

Biometric variables and percentage of graft take presented significant differences (p ≤ 0.05%) in relation to the scion-rootstock combinations and grafting heights (Figure 3). There was interaction between the scion-rootstock vs. grafting height factors for the variables graft length and stem diameter (Figures 3a, d).

Figure 3
Biometric variables of Passiflora edulis grafted on P. gibertii their reciprocal grafting (P. gibertii/P. edulis) non-grafted P. edulis and P. gibertii plants and the grafted plants and non-grafted plants. (a-c): graft length. (a) scion-rootstock combination x grafting height. (b) comparison between non-grafted plants and (c) comparison between grafted and non-grafted plants. (d-f): stem diameter. (d) scion-rootstock combination x grafting height. (e) comparison between non-grafted plants and (f) comparison between grafted and non-grafted in general. (g) graft take as a function of grafting height of P. edulis/P. gibertii. (h) plant survival (%) of the non-grafted plants and (i) survival between grafted and non-grafted plants. Error bar = standard error of the mean different letters indicate significant differences by the Tukey test (p ≤ 005). * Indicates significant difference by the F-test (p ≤ 005).

P. gibertii/P. edulis presented higher graft length than the P. edulis/P. gibertii combination at all grafting heights except for 30 cm (Figure 3a). Plants with grafts at 5 cm showed the least growth, especially when P. edulis was grafted on P. gibertii (Figure 3a). In relation to non-grafted plants, P. gibertii presented greater plant height with 76.0 cm, superior to P. edulis with 41.0 cm (Figure 3b). Mean plant height of non-grafted plants (58.4 cm), was higher than average graft length of all scion-rootstock combinations with 45.8 cm (Figure 3c).

For the stem diameter, there were different responses between the combinations and grafting heights (Figure 3d). P. gibertii/P. edulis presented the largest stem diameters at all grafting heights except 5 cm, at which the reciprocal combination (P. edulis/P. gibertii) had wider stem diameter (Figure 3d). The stem diameter of non-grafted P. edulis was 3.66 mm, 27% thicker than P. gibertii with 2.68 mm (Figure 3e). The grafted and non-grafted plants did not differ significantly for stem diameter (Figure 3f).

Regarding the graft take of P. edulis/P. gibertii, the grafting height at 10 cm provided the highest percentage (92%), although it did not differ significantly from plants grafted at 5 cm, with 87% (Figure 3g). In contrast, the lowest percentages were observed at 20 cm and 30 cm, both with 62% (Figure 3g). In general, the non-grafted plants showed higher plant survival (93%) than the grafted plants with 75% (Figure 3i). This behavior was not maintained among non-graft plants (Figure 3h).

EVALUATION IN FIELD CONDITIONS

In field conditions there was no effect of grafting height on the plant growth index (GRI 7.57) (Figure 4a). When the P. gibertii scion was grafted on the P. edulis rootstock, a higher GRI 6.72 (p = 0.01) occurred, 20% higher than the inverse combination (Figure 4b). Regarding the non-grafted plants, opposite behavior was observed, with higher GRI for P. edulis (9.53) than P. gibertii (5.89) (Figure 4c). There were no differences between the grafted and non-grafted GRI plants of 7.57 and 7.71, respectively (Figure 4d).

Figure 4
Growth rate index (GRI) number of fruits per plant and productivity. (a) GRI at different grafting heights. (b) comparison of P. edulis grafted on P. gibertii and their reciprocal grafting. (c) comparison between non-grafted P. edulis and P. gibertii plants. (d) comparison between grafted and non-grafted plants in general under field conditions. (e) Number of fruits and (f) Productivity at different grafting heights. Error bar = standard error of the mean different letters indicate difference of the means by the Tukey test (p ≤ 005). **Indicates significant difference by the F-test (p ≤ 001).

The number of fruit per plant and the productivity were not influenced by the evaluated grafting heights, and there was no difference in these variables between the grafted plants and non-grafted P. edulis plants (Figures 4e-f).

The Peto and Peto’s Wilcoxon, log-rank and Cox tests indicated that the survival curves generated by the Kaplan-Meier nonparametric method differed significantly (p < 0.0001) between the non-grafted P. edulis and P. gibertii seedlings and the combinations P. gibertii/P. edulis and P. edulis/P. gibertii (Figure 5a).

Figure 5
(a) Cumulative survival rate of P. edulis (non-grafted) P. gibertii P. gibertii/P. edulis and P. edulis/P. gibertii to Fusarium wilt (Fop). (b) boxplot of Fop survival (days) of P. edulis (non-grafted) and P. gibertii/P. edulis. (c) average survival time to Fop of the non-grafted P. edulis and P. gibertii and combination P. gibertii/P. edulis and P. edulis/P. gibertii. (d) cumulative survival ratio to Fop of P. gibertii/P. edulis at grafting heights of 5 cm, 10 cm, 20 cm and 30 cm. (e) boxplot of Fop survival (days) of P. gibertii/P. edulis at different grafting heights. (f) mean survival time to Fop (days) of P. gibertii/P. edulis with grafting heights of 5 cm, 10 cm, 20 cm and 30 cm.

The use of P. gibertii/P. edulis grafting affected the susceptibility to Fusarium wilt of P. gibertii, which is resistant to the disease. The manifestation of symptoms by P. gibertii/P. edulis plants started at 150 days after planting (DAP), and, at the end of the study, only 32% of plants were still alive (Figure 5a).

The non-grafted seedlings of P. edulis were more susceptible to Fusarium wilt, with the onset of symptoms at 450 DAP and only 20% of survival at the end of the study (Figure 5a). Based on the box plot analysis, the manifestation of Fusarium wilt symptoms occurred earlier in non-grafted P. edulis, with the first quartile between 110 and 200 DAP, while for the combination of P. gibertii/P. edulis the first quartile was between 175 and 248 DAP (Figure 5b). The average life expectancy also followed the same trend, with 168 and 245 days for P. edulis and P. gibertii/P. edulis, respectively (Figure 5c). P. edulis plants grafted on P. gibertii and P. gibertii seedlings did not present typical symptoms of Fusarium wilt (Figure 5a), and had mean life expectancy of 327 days (Figure 5c).

Survival to Fop of the P. gibertii/P. edulis combination was significantly lower independent of the grafting height used, and no variation was observed between the survival curves by the three tests (Peto and Peto’s Wilcoxon, p = 0.31964, Cox, p = 0.45898, and log-rank, p = 0.16047) (Figure 5d). Lower dispersions attributed to Fop were obtained at 5 and 20 cm grafting heights, with the first quartile between 220-260 and 180-210 days at these grafting heights, respectively, when compared to grafts at 10 and 30 cm (Figure 5e). The mean survival time was lower (175 days) for the grafting performed at 5 cm (Figure 5f) and the intensification of mortality occurred from 250 days after planting (Figure 5d).

Based on the physicochemical characteristics of the fruits of P. edulis grafted on P. gibertii, there were no differences (p ≤ 0.05) between the grafting heights for all the evaluated characteristics (Table I). The grafting height did not influence the average number of fruit per plant when compared to that of non-grafted P. edulis (Figure 4e).

HISTOPATHOLOGICAL ANALYSIS OF ROOTS

The fluorescence microscopy images of the P. edulis seedling (non-grafted) plants without visual symptoms of Fusarium wilt revealed the presence of fibers in the cortical parenchyma of the root with light staining and deposition of starch in the same region extending to the xylem vessels (Figure 6a, e). In the first wilting stage, there was a marked reduction in the presence of starch in comparison to the control, in addition to a smaller volume of fibers (Figures 6b, f). In the second wilt stage, complete absence of starch was observed in both the cortex and xylem (Figures 6c, g), and in the third wilt stage, this behavior was maintained, with further deterioration of vascular tissues (Figures 6d, h). No callus deposition was observed in the xylem vessels in any of the wilt stages, indicating that the species P. edulis does not have this metabolite compound, which constitutes a physical barrier that prevents the colonization of the pathogen (Figures 6f-h).

Figure 6
Anatomical aspects of non-grafted P. edulis in three stages of Fusarium wilt. (a-e): fluorescence microscopy of the root of control plants not inoculated showing the presence of fibers and starch. (b-f): first wilt stage with marked reduction in the presence of starch and absence of callus. (c-g): absence of starch and callus and lower fluorescence of the fibers. (d-h): absence of starch with disintegration of tissues. (i-m): root control with presence of starch in the cortical parenchyma. (j-n): low starch concentration in the first wilt stage (k-l, o-p): absence of starch in the parenchyma of the cortex and vascular system. (q): control plant root with absence of pathogen. (r-t): presence of intracellular hyphae and chlamydospores. hy: hyphae, ch: chlamydospores, ro: root, ep: epidermis, stc: starch, mx: metaxylem, fb: fibers. Bars: (a, c, d) = 250 μm (k) = 125 μm (b, e-i, l, o, p, s) = 100 μm (j, m, n, q, r, t) = 50 μm.

The presence of starch in the cortical parenchyma (Figures 6i, m) was identified in the transversal sections of the root of healthy plants stained with Lugol’s iodine, while in the fungus-infected plants, low starch concentration was observed in the first wilt stage (Figures 6j, n) in comparison to the control, and complete absence of this polysaccharide in the other wilt stages (Figures 6k, l, o, p).

With the root clearing technique, it was possible to identify the presence of hyphae inside cells in secondary roots in the first wilt stage (Figure 6r) and the presence of chlamydospores in the last wilt stage (Figure 6t). No pathogen structures were observed in control plants, which had healthy roots with well-delimited cells (Figure 6q).

DISCUSSION

The definition of grafting techniques for yellow passion fruit is important to obtain vigorous plants associated with resistance to the main soil-borne diseases that constrain cultivation in several regions of Brazil. In the present study, results indicated that as the grafting height increases, there is a reduction in the graft take in the nursery.

In field conditions, no differences in the plant growth were observed regarding grafting heights, indicating that the effect is probably temporary in the greenhouse, due to the healing time for the graft between the scion and the rootstock, and the reestablishment of the vascular connections (Lima et al. 2017LIMA LKS, SOARES TL, SOUZA EH, JESUS ON and GIRARDI EA. 2017. Initial vegetative growth and graft region anatomy of yellow passion fruit on Passiflora spp. rootstocks. Sci Hortic 215: 134-141.). However, the grafting height influenced on the number of leaves and shoots of sweet cherry (Yazdani et al. 2016YAZDANI Z, JAFARPOUR M and SHAMS M. 2016. Effect of scion source budding method and graft union height on sweet cherry budding compatibility on Mahaleb rootstock. Trends Appl Sci Res 6: 1-4.), and on the plant height and branching of apple (Karlidag et al. 2016KARLIDAG H, EREN G, KARAAT FE and KAN T. 2016. Grafting height effects on lateral branching shoot angles and growth of some fruit species saplings. Int J Agric Innov Res 5: 1-5.). Four grafting heights were evaluated on different cultivars of apple, pear, quince and sweet cherry, and the results showed significant effects of the parameters evaluated in all species and cultivars (Karlidag et al. 2016KARLIDAG H, EREN G, KARAAT FE and KAN T. 2016. Grafting height effects on lateral branching shoot angles and growth of some fruit species saplings. Int J Agric Innov Res 5: 1-5.).

On the other hand, the growth rate of P. gibertii when grafted on P. edulis was higher than the inverse combination. These results are very interesting, since they indicate that the reduction in the vegetative vigor of P. edulis when grafted on P. gibertii is associated with the developmental limitation of the root system of the rootstock and not with the vascular connection process. This assumption is supported by the larger GRI of P. gibertii/P. edulis combination in relation to the non-grafted P. gibertii plants. P. edulis grafted on P. gibertii showed lower growth speed compared to P. edulis. A lower root dry mass in P. edulis/P. gibertii was observed in potted plants in greenhouse in relation to the self-graft of P. edulis (Morgado et al. 2015MORGADO MADO, BRUCKNER CH, ROSADO LDS and SANTOS CEM. 2015. Development of passion fruit seedlings grafted on wild Passiflora species. Rev Bras Frut 37: 471-479.). These results corroborate the size of the root system of P. gibertii as the limiting factor for the development of P. edulis scion. The root system of the rootstock is of fundamental importance, since the water flow to the scion controls many plant processes, such as growth, mineral nutrition, photosynthesis, transpiration and, consequently, productivity (Martínez-Ballesta et al. 2010MARTÍNEZ-BALLESTA MC, ALCARAZ-LÓPEZ C, MURIES B, MOTA-CADENAS C and CARVAJAL M. 2010. Physiological aspects of rootstock–scion interactions. Sci Hort 127: 112-118., Gambetta et al. 2012GAMBETTA GA, MANUCK CM, DRUCKER ST, SHAGHASI T, FORT K, MATTHEWS MA and MCELRONE AJ. 2012. The relationship between root hydraulics and scion vigour across Vitis rootstocks: what role do root aquaporins play? J Exp Bot 63: 6445-6455.).

All plants of P. edulis/P. gibertii and seedlings of P. gibertii survived throughout the evaluation period in an area with a Fop history, regardless the grafting height, indicating that the resistance of P. gibertii occurs at the root level. Therefore, the lower survival of P. edulis/P. gibertii reported by Santos et al. (2016) may be associated with the crop management, climatic conditions or genetic variation within the rootstock seedlings, among other factors. There are few studies on the effect of the grafting height on the incidence of soil-borne diseases of fruit crops. The incidence of Pseudomonas sp. was reduced in apricot and Prune due to grafting height of 180 cm in relation to 30 cm above the root collar (Prunier et al. 1999PRUNIER JP, JULLIAN JP and AUDERGON JM. 1999. Influence of rootstock and height of grafting on the susceptibility of apricot cultivars to bacterial canker. Acta Hortic 488: 643-646., Sayler et al. 2002SAYLER RJ, SOUTHWICK SM, YEAGER JT, GLOZER K, LITTLE EL and KIRKPATRICK BC. 2002. Effects of rootstock and budding height on bacterial canker in French prune. Plant Disease 86: 543-546.). This range of grafting height is obviously of no practical use in vine crops such as passion fruit.

On the other hand, the use of P. gibertii as a scion grafted on P. edulis resulted in high mortality of plants associated with Fop throughout the same period. These results confirm the existence of inhibitory compounds of infection and/or colonization of the pathogen in the roots of P. gibertii, and that there is no flow of these substances from the scion to the roots of the susceptible species, P. edulis. Recent studies indicate the movement of miRNA via phloem from shoot to root in vines, which, however, is strongly dependent on the environment (Yang et al. 2015YANG Y, MAO L, JITTAYASOTHORN Y, KANG Y, JIAO C, FEI Z and ZHONG GY. 2015. Messenger RNA exchange between scions and rootstocks in grafted grapevines. BMC Plant Biol 15: 251-258.).

The maintenance of the yield and quality of the yellow passion fruit in grafted plants is also fundamental for the acceptance of rootstocks by growers. The results obtained showed that there were no changes in the physical and chemical attributes as a function of the grafting heights tested in relation to the non-grafted P. edulis plants. The number of fruit and productivity were also not influenced by the grafting height or in comparison with non-grafted seedlings of P. edulis. The productivity in the first production cycle in the grafted plants observed in this study was below (39%) the national average which is of 14.1 t ha^-1 (IBGE 2017IBGE - INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA. 2017. Survey of national passion fruit production in the year 2017. Available in: <https://sidraibgegovbr/Tabela/1613> Access in: November 15, 2017.
https://sidraibgegovbr/Tabela/1613>... ), which is calculated on the basis of at least two production cycles over the year. Nevertheless, the average life expectancy of non-grafted P. edulis was only 168 days, indicating that the number of live plants in the second production cycle will be less than 40%. Cavichioli et al. (2011a) found a production of 3,517 kg ha^-1 in P. edulis/P. gibertii from December 2006 to February 2007, which did not differ from self-grafted P. edulis.

Grafting is an important alternative for living with Fusarium wilt until obtaining a variety of P. edulis with resistance to the pathogen. The grafting should only be used in areas infested with Fop, because although there is no compromise of the physical and chemical characteristics of the fruits, the vigor and productivity of grafted plants, when compared with those of seedling plants, might be reduced in areas without the disease. Cavichioli et al. (2011b)CAVICHIOLI JC, CORRÊA LDS, BOLIANI AC and SANTOS PCD. 2011b. Physical and chemical characteristics of yellow passion fruit fruits grafted on three rootstocks. Rev Bras Frut 33: 906-914., when evaluating the quality of passion fruit fruits from plants of P. edulis grafted on three rootstocks (P. edulis, P. alata and P. gibertii), verified that grafted plants produced fruits satisfying marketing standards, despite being shorter in length than the non-grafted plants. No significant rootstock effects on antioxidants contents or fruit quality of P. edulis were observed in the graft combinations of P. edulis/P. mucronata and P. edulis/P. gibertii (Salazar et al. 2016SALAZAR AH, SILVA DFP and BRUCKNER DH. 2016. Effect of two wild rootstocks of genus Passiflora L. on the content of antioxidants and fruit quality of yellow passion fruit. Bragantia 75: 164-172.). Grafting did not affect the fruit quality of peach (Picolotto et al. 2010PICOLOTTO L, FACHINELLO JC, BIANCHI VJ, MANICA-BERTO R, PASA MDS and SCHMITZ JD. 2010. Yield and fruit quality of peach scion by using rootstocks propagated by air layering and seed. Sci Agric 67: 646-650.), whereas rootstocks are major factors for the fruit yield and/or quality of pear (Ikinci et al. 2014IKINCI A, BOLAT I, ERCISLI S and KODAD O. 2014. Influence of rootstocks on growth yield fruit quality and leaf mineral element contents of pear cv ‘Santa Maria’ in semi-arid conditions. Biol Res 47: 1-8.), watermelon (Turhan et al. 2012TURHAN A, OZMEN N, KUSCU H, SERBECI MS and SENIZ V. 2012. Influence of rootstocks on yield and fruit characteristics and quality of watermelon. Hortic Environ Biotechnol 53: 336-341.), sweet cherry (López-Ortega et al. 2016LÓPEZ-ORTEGA G, GARCÍA-MONTIEL F, BAYO-CANHA A, FRUTOS-RUIZ C and FRUTOS-TOMÁS D. 2016. Rootstock effects on the growth yield and fruit quality of sweet cherry cv. ‘Newstar’ in the growing conditions of the region of Murcia. Sci Hortic 198: 326-335.) and citrus (Castle 1995CASTLE W. 1995. Rootstock as a fruit quality factor in citrus and deciduous tree crops. New Zeal J Crop Hort Sci 23: 383-394.). Such diferences are also associated to the interaction between the scion/rootstock combination x environmental and management conditions (Al-Jaleel et al. 2005AL-JALEEL A, ZEKRI M and HAMMAM Y. 2005. Yield, fruit quality, and tree health of ‘Allen Eureka’ lemon on seven rootstocks in Saudi Arabia. Sci Hortic 105: 457-465.).

The histopathological evaluations of roots of non-grafted P. edulis plants at different Fusarium wilt stages demonstrated that as disease progresses, there is a significant reduction of starch in the roots, and the presence of hyphae and chlamydospores, especially in the last wilt stage. The fiber distribution and lignification of the cortical walls in the secondary roots was low, both in control plants and in the different wilt stages, with reduction in the fluorescence of the fibers in the infected plants. These physical structures represent an important barrier to pathogen infection and the their fragility may be associated with the susceptibility of yellow passion fruit to Fop, since the cell wall is considered the first barrier to penetration or colonization of the pathogen. Furthermore, changes in this membrane can occur that make it impossible for the pathogen to colonize the plant (Underwood 2012UNDERWOOD W. 2012. The plant cell wall: a dynamic barrier against pathogen invasion. Front Plant Sci 3: 1-6., Miedes et al. 2014MIEDES E, VANHOLME R, BOERJAN W and MOLINA A. 2014. The role of the secondary cell wall in plant resistance to pathogens. Front Plant Sci 5: 1-13.).

The available information in the literature on starch dynamics in the plant-pathogen interaction is scarce, and the few studies that have been carried out show that pathogen-affected plants present a reduction in the deposition of this polysaccharide in roots (Keunen et al. 2013KEUNEN E, PESHEV D, VANGRONSVELD J, ENDE WVD and CUYPERS A. 2013. Plant sugars are crucial players in the oxidative challenge during abiotic stress: extending the traditional concept. Plant Cell Environ 36: 1242-1255., Manila and Nelson 2014MANILA S and NELSON R. 2014. Biochemical changes induced in tomato as a result of arbuscular mycorrhizal fungal colonization and tomato wilt pathogen infection. Asian J Plant Sci 4: 62-68.). In the healthy plants, greater starch accumulation was observed in the root cortex, and as the wilt stage intensified, the starch concentration declined or was too low to detect. This reduction may also be associated with the stage of wilt observed in plants regardless of the pathogen, since studies show that starch degradation is associated with osmotic adjustment to water deficit (Ponce et al. 2008PONCE G, RASGADO FA and CASSAB GI. 2008. Roles of amyloplasts and water deficit in root tropisms. Plant Cell Environ 31: 205-217.). Nevertheless, the most important anatomical and histopathological changes due to Fop infection were the concentration of starch, hyphae and chlamydospores as a function of the wilt stage.

CONCLUSION

Graft take of P. edulis on P. gibertii in greenhouse was higher at 5 and 10 cm height. In field conditions, the grafted P. gibertii/P. edulis plants presented higher growth than the reciprocal grafting and non-grafted P. gibertii. However, they were susceptible to Fop, indicating that resistance occurs at the root level. Grafting height from 5 cm up to 30 cm above the root collar did not influence the Fusarium wilt incidence of P. edulis grafted on P. gibertii, thus this combination is considered resistant to Fop. Fruit yield and quality were also not influenced by the grafting height or in relation to the P. edulis seedlings. Based on the fluorescence and histopathological analyses of the roots, it was possible to identify changes in starch concentration and presence of hyphae and chlamydospores as a function of the Fusarium wilt stage in non-grafted P. edulis plants. These phenomena are possibly related to the infection and colonization processes in this pathosystem.

ACKNOWLEGMENTS

To the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship grant of the first author (L.K.S.L.), to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support (process 473643/2013-8) and also for the Regional Scientific and Technological scholarship (DCR0013/2015) granted to the fourth author (T.L.S), to Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) for financial support (DCR0013/2015), and to Embrapa Mandioca e Fruticultura and Embrapa Cerrados for technical support and supply of plant materials.

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