Blossom-end rot in tomatoes and peppers causes significant losses in the vegetable industry each year. This is followed by blackening and sometimes drying of the affected tissue. BER occurs during fruit development prior to physiological maturity. While calcium deficiency in fruit tissue and abiotic stress can increase BER, the biological mechanism of cell death remains unknown and current treatments are not fully effective. Many previous studies on the biological drivers of BER development in tomatoes and peppers have relied on comparing measurements from BER affected fruit to healthy fruit. This approach has identified several possible causes of cell death. Comparing BER affected tomatoes grown in a low calcium hydroponic solution to healthy fruit grown with a higher calcium hydroponic solution, Mestre et al. found a reduction in glutathione content, glutathione reductase activity, and catalase activity. Increased hydrogen peroxide and lipid peroxidation were also associated with BER affected fruit. Aloni et al. found depleted apoplastic and symplastic ascorbic acid contents and increased apoplastic ascorbate oxidase activities in BER affected peppers compared to healthy peppers. An investigation of intracellular wash fuids by Turhan et al. found increased hydrogen peroxide concentration and peroxidase activity in BER affected fruit compared to healthy fruit. While these results strongly support the trend of increased oxidative stress during blossom-end rot development, the conclusions thus far are mainly correlative in nature. Trough over expression of a vacuolar calcium importer, de Freitas et al. found that increasing vacuolar calcium and decreasing apoplastic calcium increased BER development in tomatoes. These results suggest that decreased apoplastic calcium can lead to BER development,raspberry plant container though the hypothesis that adequate apoplastic calcium incurs a protective effect has not yet been tested.
Additionally, confounding factors at the whole plant, fruit, and cellular level make it difficult to determine the sequence of events leading to cell death during BER development. To eliminate these confounding factors and establish causative evidence regarding the biological cause of BER, direct manipulation of the apoplastic calcium concentration and antioxidant capacity is needed. Pericarp discs have been used previously to study ripening in tomatoes. Te fruit used in these experiments were at the mature green stage and the discs studied during these experiments followed ripening trends regarding physiology, visual appearance, and original tissue location. This paper describes a new method for studying BER development in immature tomatoes using the pericarp disc system.Immature green tomatoes showing no symptoms of BER were observed to develop BER symptoms during storage after harvest, if harvested just prior to the usual timing of BER development on the plant . BER development of the plant occurred within the first 3 days after harvest and the symptoms resembled BER regarding location and visual development. These results were replicated with 21-day old fruit of the HM variety and in the Rutgers variety.Pericarp discs were made from the stem-end, blossom-end, and columella tissue of immature HM tomatoes 21 days after pollination . During the first 12 h of storage, disc color lightened slightly and developed a “frosted” appearance as described previously. In discs made from stem-end pericarp, this appearance remained the same for 4 days. In blossom-end pericarp discs, symptoms developed resembling the progression of BER development on the plant. Water soaking of the tissue was first observed on day 2 of storage, usually followed by tissue darkening. While water soaking often started on day 2, initiation of water soaking also occurred on day 3 and 4 in some blossom-end discs. Water soaking and tissue blackening was initially localized to one area of the disc, and subsequently spread to the rest of the disc, as observed in Additional file 2: Video S1. An example of these symptoms is shown in . These results were replicated in Rutgers and Ailsa Craig tomatoes . Darkening in top discs was observed only when the discs were excessively handled for color or weight loss analysis, and was visually similar to symptom development in blossom-end discs. In columella discs, symptoms developed similarly to that of pericarp discs.
Discs taken from closer to the stem-end often maintained a green-white appearance, while discs taken from near the blossomend darkened over the 4-day storage period . In blossom-end pericarp discs made from fruit already showing BER symptoms, the blackening spread from the BER affected area to the non-affected area. HM and Ailsa Craig tomatoes produced the most consistent results . Rutgers tomatoes were more susceptible to symptom development at sites of damage from handling in stem-end discs .Color was measured in HM discs and whole fruit harvested with BER symptoms . L* and a* values were used to quantify darkening and loss of green color, respectively. Visual evaluations of symptom development in discs were also completed for comparison . Discs exhibiting symptoms and BER tissue in whole fruit harvested with BER symptoms had lower L* values and higher a* values than stem-end discs or the stem-end of whole fruit harvested with BER symptoms. Blossom-end discs that did not develop symptoms had similar values to those of the stem-end discs.HM disc weight loss over a 4-day storage period was not significantly different between stem-end discs and asymptomatic blossom-end discs . Discs with a visual symptom rating of 2 had significantly higher weight loss than asymptomatic blossom-end discs and stem-end discs. Weight loss in Rutgers discs was higher than HM discs. Weight loss for stem-end, side, and blossom-end Rutgers discs was slightly higher from day 2 to day 3.5 than day 0 to day 2 . Weight loss in columella discs was similar from day 2 to day 3.5 and day 0 to day 2.Enzyme analysis and color measurements were taken after 4 days of storage on 24 top and 24 bottom discs. A Pearson correlation analysis between enzyme activities and color measurements from HM discs showed that both pyrogallol peroxidase and ascorbate oxidase were significantly negatively correlated with L* values and positively correlated with a* values . Te mean ascorbate oxidase activity was 193.8 nmol min−1 g−1 . Te mean pyrogallol peroxidase activity was 11.7 μmol min−1 g−1 . Both ascorbate oxidase and pyrogallol peroxidase activity means were significantly higher in blossom-end discs compared to stem-end discs.
Treating discs with a 15 min soak in either 10 g/L calcium chloride or 500 mM ascorbic acid was effective in inhibiting the development of visual symptoms. Both visual evaluation and color data indicates that symptoms only developed in deionized water pH 5.58 and DI water pH 2.00 treated discs. Ascorbic acid treatment caused a mild loss of green color as seen in the slight increase in a* value compared to calcium treatments. Aside from this color change, ascorbic acid and calcium treated discs appeared healthy with no water soaking or darkening. Discs treated with DI water pH 2.00 showed water soaking and tissue degradation, and exhibited a gray color. Gray color development became apparent 3 days after harvest and disc formation, and became more noticeable on day 3.5, similar to the timing of tissue darkening observed in DI water-treated discs. In addition to 15 min soaking treatments, vacuum infiltration was tested as a method for inducing the uptake of treatment solutions. However, vacuum infiltration was found to induce a translucent and water-soaked appearance in all treatments, making water-soaked symptoms associated with blossom-end rot hard to identify . Te vacuum infiltration method was not pursued further.Symptom development in harvested whole fruit and disc systems closely resembled symptom development in tomato fruit on the plant. Harvested whole fruit developed symptoms only at the blossom-end of the fruit. Similarly,container raspberries only discs from the blossom-end of fruit developed symptoms in the pericarp disc system. Similar to trends in whole fruit, columella tissue taken from near the blossom-end of the fruit developed BER like symptoms while top columella tissue often did not develop symptoms. Visual BER symptom development in whole fruit on the plant begins with water soaking of the tissue at the blossom-end, usually in one or a small number of spots. Water-soaked tissue begins to darken until it has a blackened appearance. In discs, symptoms developed in a similar manner, starting with the development of a water soaked and somewhat translucent appearance. Water soaked areas then darkened. Drying of the affected tissue in discs was limited, likely due to the high relative humidity environment, though weight loss evidence suggests that increased water loss can occur during symptom development.
Drying of the BER affected area is also common in fruit that develop BER on the plant.Ascorbate oxidase activity was used as a measure of the overall trend in antioxidant depletion in BER affected fruit. Maintaining ascorbic acid concentrations in fruit has been suggested as key to BER resistance and increasing ascorbic acid concentrations in vulnerable fruit tissues is a possible treatment to inhibit BER incidence. However, conflicting ascorbate concentrations have been reported in BER fruit compared to healthy fruit. Tus, direct measurement of ascorbic acid is an unreliable indicator for the state of the antioxidant system and was deemed inappropriate for the purpose of comparing BER processes in whole fruit to symptom development in discs. Alternatively, ascorbate oxidase activity has been shown to be increased in BER affected peppers compared to healthy peppers. In tomato, ascorbate oxidase was found to be transcriptionally upregulated in fruit treated with the BER-inducing phytohormone gibberellin. Additional file 3: Figure S2 shows ascorbate oxidase activity in healthy top, healthy bottom, and BER affected pericarp tissue from BER affected fruit, and healthy top and healthy bottom pericarp tissue from BER unaffected fruit. This data indicates that the ascorbate oxidase activity from stem-end pericarp tissue of a BER-affected fruit is similar to the stem-end and blossom-end of healthy fruit. In our pericarp disc system, ascorbate oxidase activity was significantly correlated with objective measures of tissue darkening , suggesting that similar antioxidant depletion processes occur during symptom development in discs as in whole fruit.Peroxidase activity was chosen as a second indicator of enzymatic similarities between symptom development in discs and BER development on the plant. Peroxidase activity measured using an aromatic electron donor was shown to be greatly increased in BER fruit compared to healthy fruit. This trend has been linked to increased lignification, and peroxidase trends presented by Reitz and Mitcham are similar to those found for ascorbate oxidase activity in Additional file 3: Figure S2. In our disc system, peroxidase activity was significantly correlated to measures of tissue darkening , suggesting a similar trend to that of increased peroxidase activity in BER affected whole fruit on the plant as compared to healthy fruit.Regulation of respiration is vital to plant health, with increased respiration indicating an increase in energy consumption and possibly stress. Results presented here indicate symptom development is associated with an increase in respiration rate. Furthermore, the correlation between respiration on day 1 and color measurement on day 2 indicates this increase in respiration rate occurs prior to visual symptoms. Blossom-end disc respiration decreased to near the level of the stem-end discs on day 3, possibly due to cell death or exhaustion of the available chemical energy pool. This also indicates that processes associated with blossom-end rot are likely active prior to visible symptom development.Calcium deficiency in soil and fruit tissue has long been associated with BER incidence. However, abiotic stress can also increase BER incidence, bringing into question the direct relationship between BER occurrence and calcium deficiency. We applied calcium chloride and ascorbic acid to disc tissues to test the direct effect of increased calcium and antioxidant potential onBER development. Both treatments eliminated symptom development as evaluated visually and by colorimeter. Calcium localization within the cell is a key factor in BER incidence. De Freitas et al. directly studied the effect of calcium localization at the cellular level by increasing vacuolar calcium through increased expression of the sCAX1 gene. Increased vacuolar calcium was associated with decreased apoplastic calcium and increased BER incidence. Calcium applied to the pericarp disc systems would likely diffuse into the apoplast, testing the opposite localization state compared to that of De Freitas et al.. As expected, the opposite effect was observed when calcium was applied. Correlation of BER symptom development with increased ROS accumulation and depleted antioxidant activity has been well established in the literature.