Characterization and control of “bottom box water spot”, a new citrus rind disorder in Spain
B. Orihuel-Iranzoa, I. Navarro, J. Breto and M.C. Mottura Department of Postharvest Technology, Productos Citrosol S.A., Partida Alameda parcela C, 46721 Potries, Valencia, Spain.Abstract
Citrus fruit rind disorders appear in many instances during postharvest handling and storage. In November of 2010 a rind disorder was observed in clementines and satsuma mandarins in Spain. The disorder symptomatically resembled the mandarin water spot that typically occurs in Spain associated with senescent fruit, but in this case it was affecting non-senescent and still greenish fruit. It appeared fundamentally in fruit to fruit contact rind areas or in fruit to harvest box contact rind areas. Incidence of the disorder was usually below 5% of the fruit. Typically, the disorder appeared only in fruit treated with a drencher fungicide treatment. We present a first characterization of this disorder called bottom box water spot. The disorder can be reduced or suppressed by using a treatment with a proprietary biostimulant formulation named Fortisol®Ca. In industrial scale trials, 1% biostimulant completely controlled the disorder when added to a imazalil+guazatine (IMZ+GZT) drench solution and also when added to a triple combination of both fungicides plus thiabendazole (TBZ) drench solution. Trials in the laboratory established the dose response for the biostimulant when counteracting the effect of the fungicidal suspensions in ‘Lanelate’, ‘Powell’ and ‘Valencia’ oranges; ‘Murcott’ and ‘Fortune’ mandarins; and ‘Primafiori’ and ‘Verna’ lemons. The disorder was caused by concentrations above the recommended dose of either IMZ or GZT fungicides. Except for IMZ damage on ‘Fortune’, we found a clear relationship between the dose of biostimulant added to the solution and the reduction of bottom box water spot; these relationships adjusted to polynomial equations with R2>0.90. In more practical terms, we found that concentrations higher than 0.8 or 1.0% were enough to control this disorder in most of the cultivars tested.
Keywords: rind disorders, fungicides, guazatine, imazalil, ortho-phenylphenol, prochloraz, pyrimethanil, thiabendazole, water spot, postharvest
INTRODUCTION
Citrus fruit rind disorders, although low in incidence, can cause important economic losses (Petrack et al., 2006). In many instances, these disorders appear during postharvest handling and storage (Lafuente and Zacarias, 2006). In mid-November 2010 a rind disorder was observed in ‘Clementine’ and ‘Satsuma’ mandarins from several areas of Spain (Figure 1). This disorder symptomatically resembled the mandarin water spot that typically occurs in Spain in senescent clementines and satsumas (Agustı et al., 1988; Almela and Agustı, 1992; Fornes et al., 2005), which has also been reported in California (Adaskaveg et al., 2010). While the mandarin water spot disorder is associated with senescent fruit and in many occasions appears following rainfall (Agustı et al., 1988; Almela and Agustı, 1992; Fornes et al., 2005; Adaskaveg et al., 2010), the observed disorder was affecting nonsenescent, still greenish, clementines and satsumas that still required approximately 2-3 days degreening to achieve commercial acceptable color. The disorder appeared fundamentally in fruit to fruit contact rind areas or in fruit to harvest box contact rind areas and very rarely was affecting more than 5% of the fruit. However, because of its unstable deteriorating nature, the affected peel area gets darker and enlarges in just a few days and
aE-mail: borihuel@citrosol.com
Acta Hortic. 1194. ISHS 2018. DOI 10.17660/ActaHortic.2018.1194.174
Proc. VIII International Postharvest Symposium:
Enhancing Supply Chain and Consumer Benefits – Ethical and Technological Issues
Eds.: F. Artés-Hernández et al.
consequently it could cause important economic losses. We realized that the disorder appeared only in fruit treated with a drencher fungicide treatment before degreening. These facts prompted our attempt to characterize this disorder that we called “bottom box water spot”.
Drenching fungicidal treatments have to be applied before degreening to control decay (Smilanick at al., 2006a; Palou, 2014). In commercial fresh citrus shipments they are essential to control postharvest citrus diseases and maintain their quality and lengthen shelf life (Smilanick et al., 2006b). Postharvest treatment with synthetic fungicides such as imazalil (IMZ), pyrimethanil (PYR), guazatine (GZT) or ortho-phenylphenol (OPP) are the principal means used worldwide for effective control of citrus rot diseases (Naqvi, 2004; Smilanick et al., 2006b; Palou, 2014). However, if these treatments can cause burn or phytotoxic symptoms to the fruit rind, they should not be used regularly. In fact, this may be the reason why very few published studies can be found on the potential phytotoxic effects on citrus rind of these and other fungicides (Hopkins and Loucks, 1950; Orihuel Gasque and Meri Puig, 1957; Wardowski and Brown, 1991).
After studying the process to which many lots of fruit had been subjected, the overall picture that emerged was as follows: there was a huge variability among affected fruit lots, affected citrus growing areas and drencher treatments applied. While one fruit lot from one grove was affected, another lot of the same cultivar harvested the same day from another grove nearby and treated with the same drenching protocol was not affected. In addition, the disorder appeared only during a 2-3-week period during the months of November and December 2010, and mandarins harvested later from the same areas were not affected. In following years there were a few cases of the disorder appearing in other cultivars such as ‘Murcott’ mandarins and ‘Primafiori’ lemons. Since at that time we had observed that fruit from packing houses where the drencher treatment included our proprietary biostimulant Fortisol®Ca to stimulate citrus fruit defenses against fungal decay, had no bottom box water spot damage, we intended to determine its possible effect on the reduction of this disorder. Almost in parallel to the performance of this research, a peel disorder with similar symptoms named “Guazatine burn” was found by Citrus Research International (CRI) researchers in the Republic of South Africa (Erasmus et al., 2013). Findings of both research teams were very similar.
MATERIALS AND METHODS
Plant material
In industrial scale trials, mature green fruit from commercial orchards harvested in November 2010 in the Xeraco area (Valencia, Spain) were used. Clementines (Citrus reticulata Blanco ‘Clemenules’) and Satsumas (Citrus unshiu Marcovitch ‘Satsuma’) from two different groves each were used. Fruit from each grove was randomly divided in groups of 200 fruit (replicates) and set in plastic field boxes as those used for harvest. With ‘Clemenules’ and ‘Satsuma’, 4 and 2 replicates of 200 fruit each per treatment were included
in the trial, respectively.
For phytotoxicity experiments, commercial cultivars of oranges (Citrus sinensis L. Osbeck. ‘Lanelate’, ‘Powell’ and ‘Valencia’), mandarins (C. reticulata ‘Fortune’ and ‘Murcott’) and lemons (Citrus limon ‘Primafiori’ and ‘Verna’) were used. Fruit just harvested at commercial maturity from several Valencia and Murcia areas were selected and those with any rind disorder symptoms discarded.
Industrial scale trials
Treatments were applied by dipping each field box in a 100-L water bath during 30 s simulating a drenching bath. Commercial formulations at the recommended label doses, as stated in Figure 2 and Table 1, were used to prepare the treatment solutions (Citrosol LS 7.5: IMZ sulfate 7.5% w/v, Adama; Citropel: GZT acetate 20% w/v, Adama; Tiabendazol 60: thiabendazole (TBZ) 60% w/v, Syngenta; and Fortisol®Ca proprietary formulation, Productos Citrosol SA). A control treatment with water was included. After treatment, fruit were air dried at room temperature during 2 h. Then, citrus fruit were degreened at 22°C and 85% RH for 4 days before proceeding to estimate bottom box water spot damage as described below.
Table 1. Effect of two fungicidal treatments (IMZ+GZT: IMZ 450 mg L-1 + GZT 600 mg L-1; and IMZ+GZT+TBZ: IMZ 450 mg L-1 + GZT 600 mg L-1 + TBZ 1200 mg L-1) and the addition of 1% of Fortisol®Ca (FCa) to these treatments on the incidence of bottom box water spot damage expressed as blemish index (BI) on two satsuma groves. Control fruit were dipped in water. BI is the average of 2 replicates per treatment and was estimated on each fruit after 4 days degreening at 22°C and 85% RH.
Figure 2. Average of bottom box water spot incidence expressed as blemish index (BI). Data from 4 replicates of fruit per treatment from two clementine groves subjected to two treatments each (IMZ 450 mg L-1 and GZT 600 mg L-1) and a control (WATER). BI was estimated on each fruit after 4 days degreening at 22°C and 85% RH. Bars for each grove with unlike letters are significantly different by Fisher’s Protected LSD test (p≤0.05).
Laboratory phytotoxicity experiments
Commercial fruit free of any rind disorder symptoms were used in these experiments. Cotton wool disks, 25 mm in diameter, were soaked with 1 mL treatment solution. Cotton wool disks were covered by a cardboard (30×30 mm wide) and fixed to the fruit with a 30 mm wide parafilm band. Fruit were treated at 22°C and 85% RH for 24 h. Afterwards, the cotton circles were removed from the fruit and the still wet area was air dried at room temperature during 2 h before proceeding to estimate the damage. As an additional control, the rest of the fruit peel was also checked to make sure no damage had appeared in untreated areas. For phytotoxicity experiments, 15 to 20 fruit for each combination of cultivar and fungicide were used. To reduce variability, different treatments were applied to a single fruit. The commercial formulations mentioned above were used to prepare the solutions for each treatment. In order to be able to reproduce, under laboratory conditions and with presumably not sensitive fruit, the bottom box water spot damage and study the effect of different concentrations of Fortisol®Ca counteracting the damage, concentrations of 4000 ppm GZT and 900 ppm IMZ were used. Furthermore, the effect of adding 1% Fortisol®Ca to some standard fungicide treatments was studied with ‘Valencia’ oranges. Treatments selected for this study were the drencher treatments used in Spain and/or in South Africa at the concentrations stated in the caption of Figure 3 (Mirage 40 ECNA: Prochloraz (PCL) 40% w/v, Adama; Citrosol LS 7.5; Philabuster®: IMZ 20% w/v + PYR 20% w/v, Janssen Pharmaceutica; Citrocil: IMZ 7.5% w/v + OPP 10% w/v, Productos Citrosol S.A.; Citropel; and Citrocide®PLUS: 15% w/w peracetic acid + 23% H2O2 w/w, Productos Citrosol S.A).
Figure 3. Effect of the addition of 1% Fortisol®Ca (1% FCa) to different fungicide treatments on the reduction of bottom box water spot damage (blemish index, BI) in ‘Valencia’ oranges (fungicide treatments: Citrocil: IMZ 525 mg L-1 + OPP 700 mg L-1; Citropel: GZT 600 mg L-1 + Citrocide Plus 0.4%; Citropel + Philabuster: GZT 600 mg L-1 + IMZ 400 mg L-1 + PYR 400 mg L-1 + Citrocide Plus 0.4%; Citrosol LS: IMZ 450 mg L-1 + Citrocide Plus 0.4%; Mirage: PCL 600 mg L-1 + Citrocide Plus 0.4%; Philabuster: IMZ 400 mg L-1 + PYR 400 mg L-1 + Citrocide Plus 0.4%). Bars within each treatment with unlike letters mean significantly different BI by Fisher’s Protected LSD test (p≤0.05).
Estimation of bottom box water spot damage
A visual rating scale was used to estimate the incidence of bottom box water spot damage on fruit rind. A rating scale from 0 to 3 based on rind surface affected by bottom box water spot symptoms was used, where: 0 = no damage; 1 = total damaged area <8 mm in diameter; 2 = total damaged area ≥8<15 mm in diameter; 3 = total damaged area ≥15 mm in diameter. Bottom box water spot damage was expressed as Blemish Index (BI). BI was calculated according to the formula: BI = Σ (peel damage rating × number of fruit within each class) / total number of fruit.
Statistical analysis
Mean values were compared using analyses of variance (ANOVA) and separated by Fisher’s Protected Least Significant Difference test (p≤0.05) on the basis of statistically significant differences. StatGraphics 5.0 Plus software was used. In industrial scale trials, due to the very low level of incidence, the statistical analysis was performed as described above but multiplying the BI per 100.
RESULTS AND DISCUSSION
Industrial scale trials
The results obtained in the first industrial scale trial when treating ‘Clemenules’ clementines with 450 mg L-1 IMZ or 600 mg L-1 GZT are presented in Figure 2. While damage in fruit from Grove 1 was extremely low, BI in fruit from Grove 2 was double after GZT treatment. For both groves, GZT-treated fruit showed more peel damage than IMZ-treated clementines; in fact, only for Grove 2 a very low level of damage was seen in IMZ-treated fruit. It should be emphasized that when only water was applied no peel damage was produced.
Similar results were obtained in the second industrial trial with satsumas (Table 1). The peel was not damaged by the water control, but the treatment with IMZ+GZT produced a very high level of damage, especially in fruit from Grove 2. The damage was eliminated by the addition of Fortisol®Ca at 1% to the treatment mixture. Similar results were obtained with the triple combination of fungicides IMZ+GZT+TBZ, and the damage was also completely eliminated by the addition of Fortisol®Ca at 1% to the treatment mixture. The addition of 1% of Fortisol®Ca to the fungicidal treatments had a protective effect against bottom box water spot damage, suppressing completely the disorder symptoms. This protective effect of Fortisol®Ca appears to be quite exceptional, since we have only found in the scientific literature a precedent about the effect of hexamine minimizing the chemical peel burn caused by dip treatments with OPP (Hopkins and Loucks, 1950; Orihuel Gasque and Meri Puig, 1957). This was a serious limitation for citrus decay control in the 1940s and 1950s (Hopkins and Loucks, 1950) until these researchers associated OPP with hexamine in the industrial formulations. Its introduction allowed the effective use of OPP minimizing phytotoxicity risk.
Dose response curves for Fortisol®Ca reducing and eliminating bottom box water spot
Since only a few fruit lots are affected by the disorder and fruit sensitivity is not previously known, to be able to study the dose response for the Fortisol®Ca effect counteracting the damage provoked by the fungicidal suspensions, the chemical concentrations had to be increased and the rind maintained wet for 24 h. In this way we were able to obtain evident and repeatable symptoms of the peel damage. Since the two fungicides more widely used in Spain at that time were GZT and IMZ, they were used to induce the damage.
In the case of GZT, all citrus varieties studied showed medium or high levels of damage with a GZT dose of 4000 mg L-1 (Figure 4), between 4 to more than 6 times the recommended dose. The only exception was ‘Verna’ lemons that showed rather low levels of damage. When the above mentioned GZT dose was applied without Fortisol®Ca in the solution, bottom box water spot damage reached a BI of 1.68; 1.88 and 2.24 in ‘Lanelate’, ‘Valencia’ and ‘Powell’ oranges, respectively. When the same treatment was applied to mandarins and lemons, BI were 2.60 and 2.95 for ‘Murcott’ and ‘Fortune’, and 1.05 and 2.74 for ‘Verna’ and ‘Primafiori’, respectively (Figure 4). For IMZ, the dose had to be increased twice up to 900 mg L-1 to cause damage (Figure 4) and the highest BIs were also observed in ‘Fortune’ and ‘Murcott’ mandarins, and ‘Primafiori’ lemons, 1.70; 2.40 and 2.03, respectively (Figure 4). It may be that for whatever reason(s) the particular fruit lots tested from these cultivars were particularly sensitive but, according to our experience serving Spain’s fresh citrus industry, ‘Fortune’ and ‘Murcott’ clementines, and ‘Primafiori’ lemons are more sensitive cultivars to bottom box water spot than the other cultivars tested in this study. In
all cases, when Fortisol®Ca was added to the fungicide solution at doses between 0.20 and 1.50%, bottom box water spot damage was reduced dramatically in comparison to the treatments without the biostimulant (Figure 4). For both fungicide treatments and for all cultivars, except for IMZ in ‘Fortune’ mandarins, there was a clear relationship between the doses of Fortisol®Ca added to the solution and the reduction in bottom box water spot damage expressed as BI (Figure 4). In all these cases, the increase of Fortisol®Ca dose from 0.20 to 1.5% drastically reduced bottom box water spot damage to levels far below any commercial incidence (BI<1). In fact, in several cases bottom box water spot damage was completely eliminated. Furthermore, the effect of Fortisol®Ca counteracting the bottom box water spot peel damage followed a very clear dose-response pattern, which in most cases adjusted to polynomial equations with R2>0.90, as shown in Figure 4.
Figure 4. Effect of increasing doses of Fortisol®Ca (from 0.0% to 1.5%) on the incidence of bottom box water spot damage on orange, clementine and lemon varieties caused by 4000 mg L-1 GZT and 900 mg L-1 IMZ. Each point represents the blemish index (BI) mean estimated over 15 to 20 rind areas. Vertical lines represent ±SE of the mean.
To further study the effect of Fortisol®Ca on the elimination of any peel damage provoked by any other fungicide formulation or drencher mixture, and since in the above experiments a concentration of 1% Fortisol®Ca in most cases eliminated almost all damage, similar experimental work was performed with ‘Valencia’ oranges applying commercial fungicides and drencher mixtures at label concentrations. Only for the Mirage treatment and the Citropel + Philabuster treatment, bottom box water spot damage was detected above commercial incidence, BI≥1 (Figure 3). For the rest of the treatments, bottom box water spot damage was BI≤1, a level of damage of low commercial concern in most cases. As it was observed in industrial scale trials, the addition of 1% Fortisol®Ca to the treatment
suspension significantly reduced bottom box water spot damage, except for Citrocil and Philabuster treatments (Figure 3). Nevertheless, these two fungicide mixtures produced the lowest peel damage (Figure 3).
Furthermore, these results agree with those shown by Erasmus et al. (2014) at the CRI Postharvest Technical Forum “Smartie session”. Erasmus et al. found that the addition of 1% of Fortisol®Ca to GZT treatments at doses between 500 and 2000 ppm notably reduced guazatine burn in clementines and navel oranges, with the efficacy of the biostimulant counteracting the effect of the chemical increasing with the GZT concentration and the corresponding levels of peel damage.
Overall, data by Erasmus et al. (2013, 2014) and our findings suggest that the main cause of this rind disorder is a chemical peel burn induced by the application of a postharvest fungicide treatment (Figure 2; Table 1), the level of damage increasing with the concentration of the chemical(s) applied (Erasmus et al., 2013, 2014). However, there are other factors that may affect the occurrence of this rind disorder, such as the climatic conditions (low RH and/or wind) before or during harvest, which may increase the sensitivity of the peel to chemical burn. This may explain why the sensitivity varies between seasons, harvesting dates, and fruit lots, although the same fungicide treatments are applied (Figure 2; Table 1).
The frequency of occurrence of bottom box water spot is generally very low (Erasmus et al., 2013). In addition, when standard fungicide treatments are applied under industrial conditions by drenching or dipping, the level of damage may be generally so low that it is even not detected in the packing lines (Figure 2; Table 1). However, in particular cases, bottom box water spot damage may become of great commercial relevance due to its evolving nature.
CONCLUSION
According to the results presented here, Fortisol®Ca appears as an extremely valuable tool to prevent the bottom box water spot rind damage reported in this study. The postharvest application of Fortisol®Ca at a minimum dose of 0.8% to the fungicide suspensions applied in drencher or water tanks prevents or significantly reduces the incidence of this peel disorder. Fortisol®Ca may be routinely applied in drencher treatment, particularly during periods of fruit sensitivity, as a preventive treatment against this rind damage. Additionally, since the water treatment (control) applied to sensitive fruit does not induce any damage, and the damage increases with the concentration of fungicides applied, this peel disorder could be also called “postharvest chemical burn”, although because of its evolving nature it should be considered a rind disorder.
More work needs to be done to better understand the stress factors that may increase fruit sensitivity, and a histological study should be undertaken to better distinguish the disorder from a symptomatological point of view.
Literature cited
Adaskaveg, J.E., Forster, H., and Connell, J.H. (2010). Etiology and management of a mandarin rind disorder in California. Plant Dis. 94 (12), 1485–1490 https://doi.org/10.1094/PDIS-07-10-0484. Agustı, M., Almela, V., and Guardiola, J.L. (1988). Aplicacion de acido giberelico para el control de alteraciones de la corteza de las mandarinas asociadas a la maduracion. Invest. Agr. Prod. Prot. Veg. 3, 125–127. Almela, V., and Agustı, M. (1992). Principales Alteraciones de los Frutos Cıtricos y Su Control (Valencia, Spain: Ed. Edipublic).
Erasmus, A., Njombolwana, N., Cronje, P., and Fourie, P. (2013). Sour rot, guazatine and burn. Paper presented at: CRI Postharvest Technical Forum, Citrus Research International Extension Department (Pty) Ltd., Postharvest Workshops (South Africa).
Erasmus, A., Christie, C., Savage, C., and Fourie, P. (2014). The smartie session. Paper presented at: CRI Postharvest Technical Forum, Citrus Research International Extension Department (Pty) Ltd., Postharvest Workshops (South Africa).
Fornes, F., Almela, V., Abad, M., and Agustı, M. (2005). Low concentration of chitosan coating reduce water spot
incidence and delay peel pigmentation of Clementine mandarin fruit. J. Sci. Food Agric. 85 (7), 1105–1112 https://doi.org/10.1002/jsfa.2071.
Hopkins, E.F. and Loucks, K.W. (1950). Prevention of the phytotoxic action of sodium orthophenylphenate on citrus fruits by hexamine. Science 112 (1920), 720–721.
Lafuente, M.T., and Zacarias, L. (2006). Postharvest physiological disorders in citrus fruit. Stewart Postharvest Rev. 2 (1), 1–9 https://doi.org/10.2212/spr.2006.1.2.
Naqvi, S.A.M.H. (2004). Diagnosis and management of pre and post-harvest diseases of citrus fruit. In Diseases of Fruits and Vegetables. Diseases and Management, S.A.M.H. Naqvi, ed. (Dordrecht, The Netherlands: Kluwer Academic Publishers), p.339–359 http://dx.doi.org/10.1007/1-4020-2606-4_8.
Orihuel Gasque, B., and Meri Puig, J. (1957). Influencia de algunos factores sobre la aparicion de cierto grado de fitotoxicidad en la naranja “W. Navel” frente al uso del ortofenilfenato sodico-hexamina. Anales de Edafologıa y Fisiologıa Vegetal XVI (6), 751–783.
Palou, L. (2014). Chapter 2: Penicillium digitatum, Penicillium italicum (Green Mold, Blue Mold). In Postharvest Decay Control Strategies, S. Bautista-Banos, ed. (Elsevier Inc.), pp.383.
Petrack, P.D., Kelsey, D.F., and Grierson, W. (2006). Physiological disorders. In Fresh Citrus Fruits, 2nd edn, Wardowski, Miller, Hall and Grierson, eds. (Longboat Key, Florida: Florida Science Source, Inc.), pp.602.
Smilanick, J.L., Brown, G.E., and Eckert, J.W. (2006a). Postharvest citrus diseases and their control. In Fresh Citrus Fruits, 2nd edn, Wardowski, Miller, Hall, and Grierson, eds. (Longboat Key, Florida: Florida Science Source, Inc.), pp.602.
Smilanick, J.L., Mansour, M.F., and Sorenson, D. (2006b). Pre- and postharvest treatments to control green mold of citrus fruit during ethylene degreening. Plant Dis. 90 (1), 89–96 https://doi.org/10.1094/PD-90-0089.
Wardowski, W.F., and Brown, G.E. (1991). Postharvest decay control recommendations for Florida citrus fruit. Circular 952 (Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida).