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. 2019 Aug 14;14(10):e1652520. doi: 10.1080/15592324.2019.1652520

Healing of Gladioulus grandiflora corms and Fusarium oxysporum infection

Renata Ranielly Pedroza Cruz a, Wellington Souto Ribeiro c,✉,*, Silvanda de Melo Silva d, Fernando Luiz Finger a, José Cola Zanuncio b, Elida Barbosa Corrêa e, Riselane de Lucena Alcântara Bruno f, Karen Klotz Fugate g, Franciscleudo Bezerra da Costa c, Railene Herica Carlos Rocha Araújo b
PMCID: PMC6768183  PMID: 31409224

ABSTRACT

Gladiolus grandiflorus L. is highly susceptible to Fusarium and losses caused by this disease varies from 60% to 100%. Injuries caused during harvest, transport and inadequate storage, facilitate infection. The dynamics of wound healing can reduce infection by Fusarium. The objective was to characterize the wound healing in corms of G. grandiflora stored under refrigeration and how it affects the entry and establishment of F. oxysporum f. sp. gladioli infection. Corms were wounded and stored at 12 ± 4°C and relative humidity of 90 ± 5%. Cell damage, fresh weight loss, respiration, phenolic compounds, tissue darkening, suberization, lignification and resistance to infection were evaluated. Wounds on corms caused transepidermal damage with collapse and cell death. Physiological (increased loss of mass and respiration) and biochemical changes (deposition of lignin and suberin, enzymatic activity) occurred in the cells neighboring those death by the injury. The injury caused gradual darkening of the tissue, injured and neighbor. Fusarium oxysporum infection decreased with wound healing. The healing of injured G. grandiflora corms stored at 12ºC occurs from the 3rd day after injury by the accumulation of suberin, lignin, and melanin, inhibiting F. oxysporum f. sp. gladioli infection.

KEYWORDS: Corm storage diseases, flowers, Fusarium oxysporum, ornamental horticulture

Introduction

Gladiolus stands out among floral products for the variety of flower colors, high productivity, short production cycle, and high economic return.1 This species is seasonal and provides a stable microclimate for a large number of pests and diseases. Gladiolus plants are susceptible to Fusarium (F. moniliforme, F. oxysporum, F. solani, and F. roseum), the F. oxysporum f. sp. gladioli (L. Masey) W.C. Synder e H.N. Hans. is the most serious, affecting plants in the field2,3 and corms which deteriorate during storage.3,4 The disease can cause 60% to 100% of losses. Khan et al., 2017

Fusarium spp. is a common fungus in soils and persists as chlamydospores for years.5 Chlamydospores attach to the corms in the field and infect it during storage.6 Wounds caused during harvesting, handling, transport, and storage disrupt the integrity of tissues and cells7 and, when unhealed, facilitate Fusarium spp..8

Plant healing involves a series of molecular and cellular events that repair injured tissues9 and prevent further damage. Each plant cell is competent to induce defense responses.10 Activation of these responses may occur within minutes or several hours after injury10 and includes the generation/release, perception, and transduction of specific signals that activate a signaling cascade that induces defense responses in neighboring cells wounding11,12 also by activating specific genes.13 The proteins encoded by these genes produce substances capable of repairing injured tissue and toxins that inhibit the growth of insects, pests, and fungi.10,14-17

Tissue repair includes cell division, caliosis biosynthesis, lignin and suberin. Lignin and suberin are part of the polymer category.18 The biosynthetic pathway of the phenylpropanoids generates the monolignols (p-coumaric, coniferyl, and synaptic alcohols) and phenolic compounds that serve as substrates for the biosynthesis of lignin and suberin.18 The biosynthesis of both is induced after injury, contributing to the healing and formation of a layer resistant to water entry and pathogens.19 However, the order of which appears first and in what quantities, is relative.20 The production of substances or toxins by secondary metabolism includes terpenes (composed of five-carbon isoprene units), phenolic compounds (formed from an aromatic ring with one or more hydroxylic substituents) and nitrogenous compounds (formed primarily by amino acids)21,22 that inhibit the pathogenesis.

The objective was to evaluate the healing events in G. grandiflora corms subjected to an injury that simulates those that occur under improper transport and handling conditions and to determine how these affect F. oxysporum infection and establishment under refrigerated conditions.

Material and methods

Raw material, healing induction, and storage conditions

Gladiolus grandiflora Hort. corms were planted on Bioplant® substrate (Bioplant Agrícola Ltda., Nova Ponte, Minas Gerais, Brazil) in 900 mL pots and plants were grown in a greenhouse. Plants were irrigated when necessary without fertilization. After 60 days, corms were harvested. The vegetative leaves and buds were removed with a knife and the corms were washed to remove substrate residues. Half of the corms were wounded superficially (2–3 times) by scraping with a steel brush (Sodomar 25 cm) 1 hour after harvest and the remaining corms were used as unwounded controls. Wounded and unwounded corms were stored at 12 ± 4°C and 90 ± 5% relative humidity for 21 d.23 The corms were evaluated after 0, 1, 2, 3, 4, 5, 6, 7, 14 and 21 d of storage.

Fresh weight loss

Losses in fresh mass were obtained on individual corms using a MARK 31000 semianalytical balance with an accuracy of ± 0.01 g. The results were expressed as percentage of fresh mass loss estimated by the equation: PMC = [(MFI – MFF) x 100]/MFI]; PMC = loss of corms mass (%); MFI = initial fresh mass loss (g) and MFF = final mass loss (g).

Respiratory rate

CO2 produced by G. grandiflora corms was measured by titration. Corms injured or not (control) were placed in hermetically sealed containers. A total of 10 mL of 0.5 N NaOH was placed in each vase and, after 24 h, titrated with 1N HCl. The results were expressed in mg of CO2 100 g−12 of fresh matter. The respiratory rate was estimated by the equation: mg CO2 100 g−1 fresh matter = (B-L) × C/MF; B = volume in mL spent for titration of the “control” (container without corm, only with NaOH); L = volume spent to neutralize NaOH; C = correction factor of NaOH (0.98); MF = corms fresh mass at the evaluation time. The hourly respiration rate was determined with the formula: mg CO2 Kg−12 d−12 = mg CO2 g−12 fresh matter × 1000/IT; IT = time interval between titrations (24 h).

Phenolic compounds

One milligram of fresh tissue was placed in 10 mL of methanol: acetic: water solution (50:3.7:46.3), sonicated for 15 min, and centrifuged at 16,000 g for 15 min. An aliquot of the extract (0.2 mL) was taken and a 1:10 (v/v) solution of Folin-Ciocalte: water was added. This solution was incubated for 10 min at room temperature,24 and after this period a total of 0.8 mL sodium carbonate (7.5%) was added to the solution which was mixed and incubated for 30 min at room temperature. Concentrations of soluble phenolic compounds were determined by absorbance at 473 nm with gallic acid as standard.

Tissue darkening

Tissue darkening was assessed by visual analysis.23 The images were captured with a Sony Cyber-Shot DSC-HX1 semiprofessional camera. The predominant color in the image was captured in the program Paint – Windows 10, using the color selection tool.

Lignification and suberization

Anatomical sections were manually removed from injured or unwounded (control) corm tissues with a razor blade. The sections were analyzed with UV epifluorescent light to detect polyphenols in suberin. Microscopy was performed using an HBO 50 W (L2), a short-arc mercury lamp equipped with a G-365 excitation filter, an FT-395 chromatic beam splitter, and an LP-429 barrier filter. The images were captured on a Zeiss Axioskop 50 microscope (Jena, Germany) equipped with a Zeiss AxioCam color camera. Tissue sections were stained with 5 N HCL-saturated phloroglucinol and examined under standard light microscopy for lignification detection.23,25 Lignification sites were identified by the specific reaction of the reagent with the coniferylaldehyde groups26 and cinnamaldehyde27 resulting in an orange-reddish coloration.

Enzyme activity assays

Peroxidase (POD), polyphenol oxidase (PPO), and catalase (CAT) activities were determined using the protein extraction and activity assay methods of Aebi,28 Campos et al.,29 Wuyts et al.,30 and Ferrareze et al.31 Protein extracts were prepared by adding five volumes (w/v) of extraction buffer to corm fresh tissue. Extraction buffers were for POD: 0.1 M potassium phosphate buffer, pH 7.0, 10 mM sodium bisulfite, and 0.5 M NaCl; for PPO: 0.1 M potassium phosphate buffer, pH 6.5, and 1% polyvinylpyrrolidone-40 (PVP-40); for CAT: 0.1 M potassium phosphate, pH 7.0, 1 mM EDTA, 1 mM PMSF, and 1% (w/v) PVP. Tissue suspensions and extraction buffer were sonicated for 15 min at 4°C, filtered over Miracloth (EMD Millipore, Billerica, MA, USA), and centrifuged at 16,000 g for 15 min at 4°C. Supernatants were used for enzyme activity and total soluble protein assays. Enzyme activities were measured spectrophotometrically using a Shimadzu model UV-1601 dual-beam spectrophotometer (Kyoto, Japan). POD activity assays contained protein extract, 0.1 M potassium phosphate buffer, pH 6.5, 10 mM guaiacol, and 4 mM hydrogen peroxide. The activity was determined at 25°C using the maximum change in absorbance at 470 nm during the first 3 min of the reaction and an extinction coefficient of 26.6 mM−12 cm−12. PPO activity assays contained protein extract, 0.1 M phosphate buffer, pH 6.5, and 50 mM catechol. The activity was determined at 25°C using the maximum change in absorbance at 420 nm. CAT activity assays contained protein extract, 50 mM phosphate buffer, pH 7.0, and 10 mM H2O2. The activity was determined at 25°C using the change in absorbance at 240 nm and an extinction coefficient of 39.4 mM−12 cm−12 (Koduri & Tien, 1995). Total soluble protein concentrations were determined using Bio-Rad Protein Assay Reagent (Hercules, CA, USA) with bovine serum albumin as a standard.

Obtaining the Fusarium oxysporum f. sp. gladioli (L. Masey) W.C. Synder e H.N. Hans. isolates and infection

Fusarium oxysporum f. sp. gladioli isolates were obtained from commercial corms in the Alagoa Nova municipality, Paraíba state, Brazil. Cultures were started with 100 mm x 15 mm mycelial fragment isolates placed in the geometric center of Petri dishes containing potato dextrose agar medium (PDA, Difco, Sparks, MD) and incubated at 25°C for 5 days until agar was completely covered by fungi. After 0, 1, 2, 3, 4, 5, 6, 7, 14 and 21 days, the gladiolus corms were inoculated with a 10 mm diameter circular fragment of the fungal covered PDA plate. After inoculation, the corms were stored at 12 ± 4°C and 90 ± 5% relative humidity for 30 days.23 Disease severity in internal tissues was assessed at 30 days by excision and weighing of the infected tissue from each corm. The infection percentage was estimated by the total weight of the root and that of the infected portion.

Data analysis

The experiment was set up in a completely randomized design (CRD) with treatments composed of wounded or unwounded G. grandiflora corms with three replications. The data were tabulated in Excel 2016 (Microsoft, Redmond, Washington, EUA) and submitted to analysis of variance with SAS® (Software Business Analytics and Business Intelligence, Cary, North Caroline). Means were compared using the Tukey test at 1% of probability. Means and standard errors were plotted using SigmaPlot™ 10.0 program (Systat Software Inc. San Jose, California).

Results

Injuries resulted in transepidermal damage with collapse and death of cells in G. grandiflora corms (Figure 1a). Physiological changes (increased mass losses and respiration) and biochemical changes (lignin, melanin and suberin deposition) occurred in the cells neighboring those killed by the wound.

Figure 1.

Figure 1.

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Cross-sectional view of Gladiolus grandiflora corm injury. Arrows indicate cell wall rupture and opening and plasma membrane (Objective 20x) (a). Simplified scheme of darkening reactions and melanin formation after wounding (b).

The fresh mass losses of wounded and unwounded G. grandiflora corms were similar in the first 3 days of storage and higher in the injured ones, from the fifth (8.2%) to the twenty-first day (28.4%) (Figure 2a). The respiration rate of the injured G. grandiflora corms was higher between the first and fifth day, lower on the twenty-first day and similar to the control on the sixth to fourteenth days of storage. The respiration rate in the control was relatively constant during storage (Figure 2b). The concentration of phenolic compounds in injured G. grandiflora corms increased during the 3 days after the injury, with greatest concentrations occurring on the third day in storage. Phenolic compounds were not detected during storage in uninjured corms (Figure 2c).

Figure 2.

Figure 2.

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Fresh weight losses (%) (a); respiratory rate (mg CO2 Kg−12 h−12) (b) and phenolic compounds (mg gallic acid g−12) (c) of Gladiolus grandiflora corms injured or not and storage at 12°C for 21 days.

The enzymatic activity increased in injured G. grandiflora corms and has not been altered in those not injured (control). Peroxidase (POD) activity increased on the first day after injury and decreased until it remained constant from the seventh day. Polyphenoloxidase (PPO) activity increased on the first day after the injury, remained high until the fourth day, and then decreased and remained constant. Catalase (CAT) activity increased on the first day after the injury and decreased from the fourth day (Figure 3).

Figure 3.

Figure 3.

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Peroxidase (POD) (a), polyphenol oxidase (PPO) (b) and catalase (CAT) (c) activities of Gladiolus grandiflora corms injured or not and storage at 12°C for 21 days.

The wound caused gradual tissue darkening during storage, from brown to light brown (Figure 4).

Figure 4.

Figure 4.

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Suberization (100x) in wounded and unwounded corms is indicated by autofluorescence (blue images) from polyphenols in suberin. Lignification sites in wounded and unwounded corms (200x) were identified by gold–brown pigmentation from specific reaction of phloroglucinol with the coniferylaldehyde groups and cinnamaldehyde. The surface discoloration is indicated by predominant color of the surface corm wounded in the program paint – windows 10. Bar = 100 µm.

Suberization preceded lignification. The formation of a thin, continuous suberin layer was observed, on the third day, in the cells at the wound site. Fluorescent parallel cells layers were observed below the wound surface, from the seventh to the twenty-first day after the injury, forming an extensive continuous suberin layer. Isolated, irregular and discontinuous lignification sites were observed along the outer (intercellular) cell wall and adjacent to the inner (intracellular) wall on the third day after injury. Over subsequent days, lignin became thicker and continuous on the cell walls of some wounded cells, but without forming a continuous layer which could close off the wound site (Figure 4).

Wounding of G. grandiflora corms was associated with a 12% increase in corm infection by F. oxysporum f. sp. gladioli relative to unwounded controls on the first day after injury and improved infection resistance in corms, 3 days after injury. The infection, however, increased from the fourth to the seventh days of storage in injured corms, remaining high until the fourteenth day and declining on the twenty-first day of storage. Infection in uninjured corms was restricted to the corm outer layers and did not penetrate the internal tissues (Figure 5).

Figure 5.

Figure 5.

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Infected tissue (%) of Gladiolus grandiflora corms stored at 12°C and 90% relative humidity after 30 days. On the third day, it is observed that there was no internal infection of the tissues by Fusarium oxysporum.

Discussion

Cell collapse and death in injured corms is due to the rupture and opening of the cell wall and plasma membrane, resulting in the decompartmentalization and cellular content leaching similar to that reported for Arabidopsis,32 whose cell collapse caused by injuries resulted in its death. Cell collapse and death occur due to an integrity loss of the wall and plasma membrane, flooding the cytosol with ions that unbalance cellular functions.33,34 Cell death may impair plant tissue health.35 However, cell wall fragments, such as cellobiosis or oligogalacturonides can activate immune responses36 in neighboring living cells that undergo physiological (increased loss of mass and respiration) and biochemical changes (lignin, melanin and suberin deposition) to heal injured tissue.37 This was reported for Arabidopsis, whose cells near to those killed by the injury had rapid protein aggregation to aid in tissue healing32 (Engelsdorf et al., 2018). These physiological and biochemical changes were induced in Beta vulgaris altissima L.,23 Solanum tuberosum L.,38 B. vulgaris esculenta L.39, and Daucus carota L.40 rendering injured tissue relatively impermeable, thereby limiting water loss and microbial invasion.41 The gradual darkening of damaged tissue is due to melanin formation resulting from decompartmentalization and the cellular content leaching that exposed enzymes to substrates and O2, as reported for sugar beet, which also underwent tissue darkening during healing.23,41

The similarity in the fresh mass losses between injured or uninjured corms after the first 3 days of storage was due to the relative humidity gradient between air and wounded tissues after leaf and root hair removal from all corms. The high fresh mass losses in the first days after root and tuber harvesting are common and it was observed in Beta vulgaris altissima L.23,42 and Solanum tuberosum L.,37 being attributed to common wounds during collection, stacking and handling. The greater fresh mass losses in injured corms from the fifth to the twenty-first day of storage indicate an inability of the tissue to avoid water losses due to incomplete wound healing. The fresh mass losses in plant organs are parameter used to evaluate wound healing37,43 and it was used to determine S. tuberosum37,44,45 and Ipomoea batatas (L.) Lam46 and B. vulgaris altissima,23 which were considered completely healed when water loss was reduced by the hydrophobic barriers accumulation (suberin and lignin).37

The higher respiration rate in injured corms in the first 5 days of storage is related to the metabolic energy and substrate needed for biopolymer biosynthesis to aid in defense and regeneration. These are synthesized from carbon intermediates from glycolysis, the oxidative pathway of the pentoses phosphate and the mitochondrial tricarboxylic acid cycle. These pathways intensify in response to injury and other biotic and abiotic stresses47,48 to avoid oxidative damage and regenerate tissues. The respiration rate increase is possibly due to a demand for metabolic energy and substrates for the healing processes as found in B. vulgaris altissima23 and other higher plants.49 Compounds and biopolymers involved in tissue defense and regeneration include phenolics, phenylpropanoids, specific fatty acids,50,51 and suberin polyphenols,52 the principal compounds accumulated in potato tubers, and its oxidation implies suberization.53 Similar respiration of the injured corms between the sixth (330 mg CO2 kg−12) and the fourteenth (297 mg CO2 kg−12) days indicates the stabilization of the production of substrate for the biosynthesis of defense and tissue regeneration compounds and biopolymers as reported for B. vulgaris altissima.54,55 Respiration rate stabilization or reduction is used to determine wound healing in plant tissues, since increased respiration is directly related to cellular mechanisms to maintain metabolic homeostasis during stress.47 The respiration rate decrease in B. vulgaris altissima was directly related to healing.23,49,56 The lower respiration rate in injured corms on the 21st day of storage may be due to the decrease in live-cell density caused by cell collapse and death, which is necessary for tissue respiration.37

The increase in the phenolic compound concentration in injured corms, with a peak on the third day, may be due to the increase in respiration providing additional carbon intermediates for their biosynthesis. An increase in respiration rate and increased concentration of phenolic compounds were reported for potatoes during wound healing.53 The increase of these compounds resulted in incremental lignin and suberin biosynthesis that coincided with higher peroxidase (POD) activity.53 Suberin biosynthesis depends on phenolic compound accumulation,57 whose concentration is associated with injuries.58 An increase in phenolic compound concentration has also been reported after injury to sugar beet and carrot roots.23,59,60

The darkening, during healing, due to rupture of membrane integrity and, presumably, the complete oxidation of phenolic substrates by PPO, POD, and CAT.41,61 The enzymatic darkening is associated with the availability/content of polyphenols62 PPO and POD activity,63 Coseteng & Lee, and presence of O2. PPO, present in almost all plant tissues, is composed of four copper atoms and a binding site for two aromatic compounds and O2, catalyzing the O-hydroxylation of O-monophenols in O-diphenols and yielding O-quinones64 These o-quinones are polymerized with other quinones or phenolics, giving brown, black, brown or red pigments, called melanins,65,66 as reported for beet, which also underwent darkening of the tissue during healing23,41 due to the availability of phenolic compounds and increased PPO activity. In potato, the injury caused in the peeling and cutting stages of the sticks breaks the integrity of the membranes and causes the complete oxidation of the substrates by the PPO in the first 10 min after the cellular decompartmentalization.67 POD induction in B. vulgaris altissima wounds53 and B. vulgaris23 coincided with the formation of melanin, indicating that these enzymes are probably involved in tissue darkening during healing of gladiolus.

Suberization, before lignification, in injured G. grandiflora corms can be explained by the hydroxycinnamic acid predominance in cell walls, as reported for Clivia miniata,68,69 Quercus suber,70 and Solanum tuberosum.71,72 Hydroxycinnamic acid is the only polyphenolic component found in suberized cells that are not found in lignified cells.73 The formation of a thin and continuous suberin layer in the injured corms shows that suberization was more important than the lignification for closing off wound sites in G. grandiflora corms since continuity is more important than thickness.46 The induction kinetics of healing events in Arabidopsis roots peaked 2 days after injury, related to increased suberization.74 Lignification occurred in isolated, irregular and discontinuous sites in the walls of those cells bordering the wound, indicating low efficiency as a hydrophobic barrier and protection of the injured tissues similar to our results. This suggests that lignin is not one of the main contributors to wound healing in gladiolus corms. Differences in lignin and suberin biosynthesis are still unknown,23,53 but include the induction of phenylalanine ammonia (PAL) by injury, associated with phenylpropanoid biosynthesis18 and biosynthesis of specific fatty acids.50,51 Inhibition of PAL activity reduced the accumulation of suberin in injured potato tissues, suggesting that this enzyme is directly related to the suberin accumulation in tubers. The sweet potato scarification differs between varieties, temperature, and relative humidity during storage46,75 with the formation of a thick lignin layer (17 cell layers) at 60% RH and another thin (4–6 cell layers) in roots maintained at 95% RH.76 In sugarbeet roots, suberization preceded lignification at 12°C23 and lignification preceded and exceeded suberization in the healing process of stored beetroots.41 Correlations between lignin or suberin formation and peroxidase activities have been found in other plant species and organs, suggesting that these enzymes participate in the regulation of lignin and suberin biosynthesis.23 CAT activity, which has been implicated in suberin formation in potato,43 increased in response to wounding and may be related to suberin and lignin biosynthesis.

The greater infection of corms injured by F. oxysporum sp. gladioli on the first day after injury was likely due to loss of the outer protective layers of the corm by the collapse and death of directly wounded cells. Improved resistance of injured corms after the third day after injury is likely due to deposition of suberin/lignin and accumulation of melanin or other antifungal substances, as reported during root healing of D. carota77 and S. tuberosum 10,78,79. However, increases in infection in injured corms from the fourth to the seventh days of storage are difficult to explain but may indicate that suberization and lignification layers were insufficient to provide strong resistance against this pathogen during storage. Another possibility is that after 3 d, injured corms dehydrating more rapidly than uninjured corms. It is possible that dehydration makes them more susceptible to disease (this occurs in beets). The durability of E. carotovora subsp. carotovora resistance in injured potato tubers of the ‘BelRus’ variety was explained by the segmented deposition of suberin within each cell layer, first on the external tangential cell walls followed by the radial walls and then on the inner tangentials. This was not observed for tubers of the ‘Superior’ variety, indicating that the absence of segmented deposition was responsible for the development of resistance to E. carotovora subsp. carotovora.79

Conclusion

Wounding leads to increased water loss, increased phenolic compound accumulation and transient increases in respiration rate. The main healing mechanisms of G. grandiflora injured corms were the deposition and accumulation of lignin, melanin, and suberin in the damaged tissues. Wound healing was evident by the 3rd day after injury as suberized cell layers formed at the wound site and signs of lignification were found at this time. These wound healing processes provided some resistance against infection, although overall, injured corms were more susceptible to infection than unwounded corms.

Acknowledgments

To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support.

Conflicts of Interest: The authors declare no competing financial and non-financial interests.

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