Inducing Resistance in Heliconia psittacorum cv . Golden Torch to Naturally Occurring Leaf Diseases

One of the bottlenecks to heliconia production is leaf diseases, which have the main characteristic of forming necrotic spots, that can reduce photosynthesis, damage the heliconia bracts and make the flowers unsuitable for commercialisation. The objective of the present study was to identify inducers capable of inducing resistance in H. psittacorum cv. Golden Torch, assessing by enzymatic action the reduction in the severity of the fungus complex associated to the cultivation of this species and verifying the action of the severity in relation to the net photosynthesis rate of the plants. The inducers were applied to the plants using a back spray and during application the plots were protected to prevent the products from drifting. The concentrations applied were Agro-Mos® (10 ml L), Bion® (0.5 g L), Quartz® (40 ml L), potassium phosphite (3 g L) and calcium phosphite (3 g L). During the experimental cultivation, the severity caused by the fungus complex and the photosynthetic rate of the plants were assessed and plant material was collected to analyse the enzymatic activity. The results indicated that the inducers Bion® and the phosphites applied reduced the fungus complex severity, increased the activity of peroxidase, polifenoloxidase and β-1.3 gluconase but did not cause alterations in the net photosynthesis rate of the plants. The value found for β > 1 suggested that the visual estimation of the fungus complex severity is a good visual indicator of the effect of the plant pathogens on the host photosynthetic rate. The results are concrete responses to producers in the sector on management alternatives for diseases associated to heliconia cultivation.


Introduction
Heliconia are tropical herbaceous plants, belonging to the family Heliconiaceae.They are popularly known and appreciated because the blossoms have a wide range of colours and shapes (Taniguchi, Castro, T. F. Silva, E. B. da Silva, & Martins, 2016).One of the bottlenecks to heliconia production is the occurrence of leaf diseases, which have the main characteristic of forming dark necrotic spots but can also damage the heliconia bracts and prevent their commercialisation.The leaf spots can also reduce photosynthesis because, according to Xavier et al. (2015), the main visual effect of leaf spot causing pathogens is reduced healthy, photosynthesising leaf area.been used to control post harvest diseases (Costa et al., 2010;Melo et al., 2016).Quartz® is a biological product based on Bacillus methylotrophicus, the genus Bacillus has been studied for plant nutritional quality, growth promotion and disease control (Harshavadhan, Kumar, Yathindra, Rajesh, & Hongal, 2016;Moreira & Araújo, 2013).Lastly the phosphites are a reduced form of phosphates and are suggested as biostimulators in horticulture that act on the plant metabolism (Gómez-Merino & Trejo-Téllez, 2015).These products may influence the activity of some enzymes that are in directly associated to plant defence processes, including the enzymes β-1.3-glucanases, peroxidases and polyphenol oxidases.
In this context the objective of the present study was to identify inducers capable of inducing resistance in H. psittacorum cv.Golden Torch, assessed by enzymatic action and reduced severity of the fungus complex associated to the cultivation of this species, and to verify the action of the severity on the net photosynthesis rate of the plant.

Experimental Field
The experiments were carried out at the Nucleus of Agronomic Biotechnology at the State University of Maranhão, Brazil (2°30′ S and 44°18′ W), where H. psittacorum cv.Golden Torch plants were cultivated in soil classified as dystrophic sandy red yellow Argissolo (EMBRAPA, 2013), corrected to pH 6 by applying limestone and fertilized monthly with cattle manure (0.2 kg/m 2 ).The cultivation was standardized by drastic pruning in all the area planted with H. psittacorum cv.Golden Torch and 30 days later the experiment was started by applying inducers to control the naturally occurring leaf plant pathogens associated to the culture.The inducer applications were repeated every 20 days, totaling five applications during the experiment.The inducers were applied using a back spray and during application the treatment plots were protected to prevent the products from drifting.The concentrations applied were: Agro-Mos® (10 ml L -1 ), Bion® (0.5 g L -1 ), Quartz® (40 ml L -1 ), potassium phosphite (3 g L -1 ) and calcium phosphite (3 g L -1 ).
During the experiment, the severity of the leaf spots nd the net photosynthetic rate of the plants were assessed and plant material was collected to analyse the enzymatic activity.

Identification of Naturally Occurring Plant Pathogens in the Experimental Area
Leaves with disease symptoms were collected in the experimental area throughout assessment period.The collected material was isolated and identified according to Sardinha et al. (2012).Due to the similar symptomology of the various diseases associated to H. psittacorum cv.Golden Torch cultivation the term fungus complex was used for the group of plant pathogens identified in experimental area.

Determining Leaf Spot Severity and Net Photosynthesis
Leaf spot severity and net photosynthesis were assessed at 30, 60 and 120 days after the start of the experiment.All treatments were compared to each other to demonstrate the results.To quantify the leaf spot, four leaves with similar physiological age were randomly collected with various infection levels.All the material was scanned and the lesions quantified using the software WinDias-Image Analysis System.
The gas exchanges of the H. pisitacorum cv.Golden Torch leaves were measured using the Li-6400 XT infrared gas analyser (LI-Cor, Lincoln, NE, USA), in response to irradiance of 2000 μmol photons m -2 s -1 and 400 μmol mol -1 CO 2 .Measurements were only taken between 8 a.m. and 10:30 a.m. and on uniformly sunny days to minimize the sources of diurnal heterogeneity.

Severity X Leaf Photosynthesis
The relation between the fungus complex severity and the relative net photosynthetic rates (ratio between the net photosynthesis of diseased leaves and the mean net photosynthetic rate of healthy leaves, P x /P o ) was determined by the model by Bastiaans (1991).The β values were estimated by the equation P x /P o = (1 -x) β .The T test was carried out to verify whether the β values differed from the unit.

Enzymatic Analyses
Plant material for the enzymatic analysis was collected 0, 24, 48, 96 and 120 hours after applying the products, 50 days after the drastic pruning.Immediately after collection, the samples were frozen using liquid nitrogen, identified and kept in an ultrafreezer at -80 °C until the assessments.

Obtaining the Extract for Enzyme Quantification
The extraction was made according to the methodology adapted by Simões et al. (2015).Liquid nitrogen was used to homogenize 1 g fresh leaf tissue in 13 mL potassium phosphate buffer 0.2 mol (pH 6.0) previously kept at 4 ºC.The extract was centrifuged at 13.000 × g for 21 minutes at 4 ºC.The extract was stored in an ultrafreezer at -80 °C until the analyses were made.2.5.2Peroxidase (POD, EC: 1.11.1.7)and Polyphenol Oxidase (PPO, EC: 1.10.3.1)Activity The POD trial was determined by adding 300 μL of the supernatant to the reaction medium containing 1000 μL phosphate buffer 0.2 mol (pH 6.0), 100 μL guaiacol (0.5%) and 100 μL hydrogen peroxide (0.08%).The readings were made on a spectrophotometer at 470 nm and 25 ºC, for three minutes.The peroxidase activity was calculated based on the molar extinction coefficient of 26.6 mM cm -1 for guaicol, and expressed in µmol g -1 min -1 .(MF) The PPO trial was determined by adding 50 μL of the supernatant to reaction medium containing 1650 μL phosphate buffer 0.2 mol (pH 6.0) and 1300 μL catechol (0.2 mol).The readings were made in a spectrophotometer at 425 nm and 25 ºC, for two minutes.The PPO activity was calculated based on the molecular extinction coefficient of 34 mmol cm -1 for catechol and expressed in µmol g -1 min -1 (MF).
2.5.3 β-1.3-Gluconase (EC: 3.2.1.39)Activity β-1.3-gluconase was determined by the dosage of glucose released with lamarine hydrolysis (Tuzun, Rao, Vogeli, Schardl, & Kuc, n.d.).The following were transferred to two test tubes: 25 µL enzymatic extract, 200 µL potassium phosphate buffer (0.2 mol and pH 6.0) and 200 µL laminarine (5 mg mL -1 ).This material was incubated at 37 ºC for 30 minutes and then 1 mL Somogyi reagent (Smogyi, 1952) and 5 ml deionised distilled water were added and shaken for 10 minutes.After shaking, the material was heated to 100 ºC for 15 minutes and chilled in a ice bath.Then 1 mL Nelson reagent (Smogyi, 1952) and 15 ml deionised distilled water were added and shaken for 15 minutes.The spectrophotometric readings at 760 nm of the samples were compared with glucose standards.The standard glucose curve was prepared by adding the standard, in the same way as the samples, substituting the laminarine with glucose solutions ranging from 0 to 800 mg L -1 .

Statistical Analysis
The experimental field measured 22 × 14 meters and the experimental plots 2 × 2 m, totaling 4 m² and 1 m² central useful area per plot.A completely randomised design was used, placed in random blocks, with four replications.The data found for severity and relative net photosynthesis were correlated and submitted individually to analysis of variance in a factorial scheme (6 products × 3 periods), while the enzymatic activity was also submitted to analysis of variance, but analyzed separately in each period.The means of the parameters, when significant, were compared by the Tukey test (p < 0.05), using the software STATISTICA (Stat-Soft, Tulsa, EUA).

Identification of Naturally Occurring Plant Pathogens in the Experimental Area
The following naturally occurring plant pathogens were identified in the experiment: Curvularia eragrostides (Henn.)J. A. Mey, Pestalotiopsis sp., Colletotrichum gloeosporioides (Penz), Curvularia lunata (Wakker) Boedijn and Alternaria sp.The plant pathogens cited formed the fungus complex associated with H. psittacorum cv.Golden Torch cultivation, present in the lesions, that tended to coalesce causing complete drying of the leaves and served as inoculum source for the bract infection.

Severity
The statistical analysis was significant for the measured factors.Generally the fungus complex severity, associated with H. psittacorum cv.Golden Torch cultivation, increased during the three assessment periods.However, the fungus complex was shown to expand its colonisation differentially when plants treated with inducers were compared with the control treatment (Figure 1).

Figure
The maxim observed i The PPO calcium ph the contro were diffe phosphite, with Heliconia ium phosphite ng the period on® presented n (Figure 6).
None of the inducers assessed interfered directly in the net photosynthesis rate of H. psittacorum cv.Golden Torch, but the severity and net photosynthesis rate were strongly and negatively correlated, suggesting that fungus infection, if it is not controlled, may reduce net photosynthesis in the plant.
The β value estimated at 8.5 (±0.78) indicated that the plants infected by the fungus complex had photosynthesis damage not only in the lesion area, but also in the apparently healthy region of the leaf.Consequently, the calculated severity is a good indicative of the effect of the fungus complex on photosynthesis in H. psittacorum cv.Golden Torch plants.Although the net photosynthetic rate measurements demonstrated that the fungus complex reduced photosynthesis in the remaining green tissue, these measurements did not elucidate the mechanism responsible for the reduction or indicate the localization of this effect.Johnson (1987) considered that pathogen presence in diseased tissue may influence the crop development by reducing the solar radiation interception (RI) by the green matter or by interference in the radiation use efficiency (RUE).This experiment demonstrated that the spots caused by the fungus complex is an example where both effects occur.References were not found for this parameter in this pathogen system.However, this effect has been observed in pathogen systems such as Phaeosphaeria maydis in corn (Godoy, Amorim, & Bergamin Filho, 2001) and Corynespora cassiicola in soybean (Xavier et al., 2015).
These results suggested that when promoting reduction in the fungus complex severity associated to H. psittacorum cv.Golden Torch the phosphites, indirectly, permitted the plant to maintain its net photosynthesis rate close to that found in healthy plants, that would probably permit better quality flower production.These results have been confirmed in research already carried out by the authors (data in publication) when the effects were observed of the inducers, potassium phosphite and calcium phosphite, applied in the field, on the quality of H. psittacorum cv.Golden Torch flowers post harvest.

Considerations
The inducer Bion® and the phosphites applied in the field were efficacious in reducing the fungus complex severity associated to H. psittacorum cv.Golden Torch The net photosynthesis rate was not affected by the inducers assessed, but the fungus complex can reduce the photosynthesis rate as severity increases.
The value found for β bigger than 1, suggests that the visual estimate of the fungus complex severity is a good in visual indicator of the effect of the plant pathogens on the photosynthetic rate of the host.
The results presented here contribute to the understanding of a little studied pathogen system and give concrete responses to tropical flower producers on management alternatives for the diseases associated to heliconia cultivation.

Table 1 .
O associated
-gluconase, increased their activity and the plants treated with this inducer characterized the process of resistance induction.