Reduction of leaf tip burns of Ornithogalum dubium by controlling the temperature during bulb storage and greenhouse forcing to produce quality plants

: Production of quality potted Ornithogalum dubium Houtt. plants were investigated under multiple conditions: pre­planting treatment at 10, 16, and 22°C for 40 days from Sept. 21 (stage A; ST­A) during bulb storage and then bulbs were potted. After potting, post­planting treatment at 15/12, 18/15, and 21/18°C (day/night) during stage B for 35 days from Nov. 2 (stage B; ST­B), and at 15/12 and 21/18°C during stage C for 30 days from Dec. 7 (stage C; ST­C) dur­ ing greenhouse forcing was applied. Leaf tissue analyses for macro­ and micro­ nutrients were performed to investigate the cause of leaf tip burn symptom (LTB). Three criteria for quality of the plants at ﬂowering were established: (1) LTB occurs on less than 1.5 leaves per plant. (2) the number of days to ﬂower is less than 115 days, the length of the third leaf counted from the crown (the junction of the shoot and roots) is shorter than 11.5 cm, and the width is nar­ rower than 2.5 cm; the scape length is shorter than 15 cm, and there are more than 45 ﬂowers. (3) the leaf spread and morphology (leaf spread) and the pat­ tern of the scape


Introduction
Ornithogalum dubium Houtt. is a geophyte native to South Africa with yellow or orange flowers with a dark greenishbrown center (Du Plessis et al., 1989;Littlejohn and Blomerus, 1997). Temperature treat ments during the preplanting phase before potting the bulbs and the greenhouse forcing phase after planting the bulbs should be optimized to produce quality O. dubium potted plants. "Leaf tip scorch" was believed to be caused either by genetics or by high salt levels in the growing medium after treat ment with 5.5 grams of a slowrelease fertilizer. However, no information on the salt levels in the pot ting medium or leaf tissue analyses was presented (De Hertogh and Gallitano, 1997).
The boron (B) concentration of healthy and necrosed leaves of the Oriental hybrid lily 'Star Gazer' did not significantly differ, although those of Ca, magnesium (Mg), manganese (Mn), and copper (Cu) were higher in necrosed than in healthy leaves (Chang, 2002). In the older leaves of Curcuma 'Chiangmai University Pride' ('CMU Pride'), a high B concentration (122 ppm) at the margin of leaves may have caused the marginal leaf burn (Roh et al., 2006). It is unclear whether LTB in O. dubium is similar to marginal leaf burn observed in the older leaves of Curcuma 'CMU Pride'.
Leaf scorch symptoms were also reported in Lilium (Berghoef, 1986;Chang et al., 2008). In Lilium ×elegans, 'Red Carpet' and 'Sterling Star,' the scorch symptoms were caused by a low calcium (Ca) con centration resulting from an inefficient translocation of radioactive calcium ( 45 Ca) to the tip of the leaves (Roh, unpublished data). The critical Ca concentra tion associated with upper leaf necrosis of Lilium 'Star Gazer' was 0.3 to 0.4%, and upper leaf necrosis was caused by low Ca concentration (Chang, 2002) although "it is difficult to isolate a single characteris tic to explain the observed cultivar variation to upper leaf necrosis in Oriental hybrid lilies" (Chang et al., 2008).
Flowering of O. dubium was accelerated when the plants were forced at 27/22°C (Luria et al., 2002) or 19/13°C (Suh et al., 2000) compared with 17/12°C or 13/10°C, respectively. Flowering was accelerated in several new cultivars forcing at 22/18°C during a short day between the visible bud stage to flowering (Lee and Miller, 2015). The highest (visual) quality O. dubium cultivars were produced when forcing occurred at constant 17 to 19°C (Lee and Miller, 2015), which is comparable to 22/18°C for forcing seedraised bulbs (De Hertogh and Gallitano, 1997). However, the criteria used to assess the visual quality were not specified (Lee and Miller, 2015).
The objectives of this research were to produce quality potted O. dubium plants by reducing the inci dence of LTB, accelerating flowering while ensuring the maximum number of flower buds, and desirable morphologies as influenced by temperature treat ment during the bulb handling and greenhouse forc ing stages to establish the optimum temperature regimes that reduce the incidence of LTB, and accel erate flowering with desirable morphologies. The number of leaves showing LTB, the time of flowering, and the leaf and floral morphology were used to develop criteria for quality O. dubium potted plants. The macro and microelements of the leaf tips show ing LTB/no LTB during growth and development were analyzed to determine the cause of LTB.

General cultural practices
Ornithogalum dubium bulbs (56 cm in circumfer ence) purchased from Agrexco (Jamaica Plain, NY, USA) were used in the experiment conducted between 1999 and 2002. The bulbs were stored dry or potted, with one bulb per 6.3 or 10 cm pot, filled with ProMix (ProMix BX mycorrhizae, Quakertown, PA, USA). Temperature treatments were performed in growth chambers as specified in each experiment with a 12 h day (29 W·m 2 )/night (day: 08:0018:00 hr; night: 18:0008:00 hr). At planting, 0.8 g of a slow release fertilizer (14 N 6.2 P 11.6 K, Osmocote, Scotts Co., Marysville, OH, USA) was applied to the surface of the growing medium. In addition, the plants received 200 ppm N from 15 N 16 P 18 K watersoluble fertilizer (JR Peter's Laboratory, Allentown, PA, USA) once a month during culture in the greenhouse.
The effect of the pre-plant bulb (ST-A) and post-plant forcing (ST-B and ST-C) temperatures on growth, flowering, and plant quality Orntihogalum dubium bulbs (78 cm in circumfer ence) from the previous experiment were harvested and stored dry at 20°C until potting on June 1 and then potted per 10 cm pot filled with ProMix and grown at 18/15°C. Leaf samples were collected for tissue analysis on July 12, 2000 from leaves without any LTB.
The tips and basal portions of the leaves were separated, and the width at midleaf was 2 cm. Samples were collected from the third and fourth leaves of each plant, counted from the tip of the bulbs, and were combined into a sample. The third leaf was collected from 15 plants, and the leaves were divided into three segments. The twocm long distal end (tip) did not show any incidence of LTB, the proximal end (base) of each leaf, and mid portion between the tip and base was used for macro and microelement tissue analysis.
In another experiment for macro and microele ment analysis, bulbs were harvested after forcing in 2000, and were potted in 2001. On Jan. 13, 2002, flowers began to develop and the flowers had fully developed color and were ready for anthesis. Leaves from the distal end (tip) with and without LTB ( Fig. 1) and the proximal end (base) were collected from 15 plants during forcing on Jan. 14 and 26, Feb. 3 and 10, and Mar. 4. The leaf samples were dried at 70°C for 4 days and were ground into fine particles using a mortar and pestle. Two replicates of the dried leaves were sent to the JR Peters Laboratory (Allentown, PA, USA).
Bulbs purchased on Sept. 15, 1999 were used in this experiment. Before potting, O. dubium bulbs were stored at constant temperatures of 10, 16, and 22 o C for 40 days from Sept. 21 (STA). Bulbs were planted 2 cm deep of the nose from the surface of the growing medium, and leaf emergence through the growing medium was recorded from potting when temperature treatment of STA started. Bulbs were potted (one bulb per 6.3 cm pot) filled with the ProMix medium, and the pots were placed in a greenhouse maintained at 15/12, 18/15, and 21/18 o C for 35 days from Nov. 2 (STB). On Nov. 2, a slow release fertilizer was applied to the surface of the growing medium, and plants received 200 ppm N watersoluble fertilizer once a month. The leaves emerged (about 0.5 cm or less above the nose of bulbs) before potting on Sept. 21. On Dec. 7, plants were divided into two groups and grown in a green house maintained at 15/12 and 21/18°C for 30 days (STC) ( Table 1). There were 16 plants per treatment. Starting from Jan. 6, all plants were grown in a green house maintained at 19/16°C until flowering, and data were collected at anthesis.

Criteria establishment for quality evaluation
When 34 flowers reached anthesis, the date of flowering, the number of total leaves showing LTB ( Fig. 1) excluding the incipient LTB (Fig. 2) and the scape length, number of flowers, leaf spread, scape growth ( Fig. 2, 3) were recorded. The leaf spread was based on the angle of the leaf measured from the base to the tip using the following scores: 1: greater than 60° (pointing upward, erect); 2: between 30 and 60°; 3: old leaves prostrate and drooping downward at less than 15°. In addition, the scape growth was used as a quality criterion using the following scores: 1: no bending of the scape; 2: leaning sideways with out bending at the base of scape; 3: scape at the base starting to grow at about 40 o and continued sideways growth.

Data analysis
Data were analyzed using SAS ® System Version 9 for Microsoft® Windows® (SAS Institute Inc., 2002). The number of total leaves, number of leaves with LTB, and the scores of the leaf spread and scape ori entation were analyzed following a square root trans formation (x + 0.5) 1/2 . Means were compared using Duncan's Multiple Range test (DMRT) at the signifi cant level indicated in the tables.

Leaf tip burn symptoms and analysis of macro-and micro-elements in different leaf parts
The number of total leaves ranging from 4.8 to 5.6 was not affected significantly by the temperature treatments (data not presented). When bulbs were stored at 10°C/STA, followed by forcing at 15/12°C/STB and 21/18°C/STC, the number of leaves showing LTB was 1.1 out of 5.1 leaves. When the bulbs were stored and forced at 22°C/STA, 18/15°C/STB, and 15/12°C/STC, 4.3 out of 5.6 leaves showed LTB (Table 1). Fig. 2 Patterns of scape growth of O. dubium. A) straight growth and no bending of the scape (score 1). B) leaning to sideway without bending at the base of scape (score 2). C) scape at the base started to grow at about 30 degree angle, and continue to grow leaning to sideway (score 3). Incipient leaf tip burn (LTB) symptoms are indi cated with arrow sign. Plant A is considered the most ideal and highquality plant. Fig. 3 Leaf spread of Ornithogalum dubium. A) all leaves growing upward (erect) (score 1), B) Old leaves showing prostrate growth (spreading), while young leaves growing upward (score 2), and C) Old leaves drooping and curved inward toward the pot (prostrate) (score 3). Evident leaf tip burn (LTB) symptoms are indicated with arrow sign. The macro and microelement analysis of the leaves without apparent LTB revealed that the B con centration at the tip (distal end) of the leaves was 119 ppm, which is within the suggested acceptable range (30150 ppm) for general ornamentals ( Table 2). The B concentrations in the middle and base (proximal end) of the leaves were 34 and 21 ppm, respectively, which was close to the lower end of the range.
When the samples were collected in the early growth stages when no or incipient symptoms were observed on Jan. 14, Jan. 26, and Mar. 4, the B con centrations were 109, 96, and 108 ppm, respectively, which fell within the suggested acceptable range (Table 3). Macro and microelement analysis of the leaves without apparent LTB revealed that the B con centration in the tip of the leaves when collected on Jan. 14 was lower than 109 ppm (Table 3). However, B concentrations of leaves showing incipient symp toms and severe LTP when the leaves were collected on Feb. 3 and Feb. 8 were 218 and 230 ppm, respec tively, and these concentrations exceed the suggest ed acceptable range.
The Ca concentration was high in the leaf tips (0.76%), and the concentrations of the other macro and microelements fell within the acceptable ranges for each element, even when LTB was observed. The exception was zinc (Zn), whose concentration was slightly above the upper level (155 and 159 ppm) of the suggested range (150 ppm) ( Table 3). The nitro gen (N) concentration was above the lower level of the suggested range in all samples, regardless of leaf position, LTB symptoms, and leaf sampling time. The concentrations of Ca and Zn were lowest and highest, respectively, in the tip (distal end) and lowest in the middle and bottom (proximal end) of the leaves when LTB was not observed. At the tip of the leaves show  Level of significance y *** *** *** *** *** *** *** *** *** *** ** Suggested and acceptable range x 3.55.5 0.3501.0 2.08.8 0.83.0 0.21.5 30150 60200 50200 mag25 30150 0.55 ing evident and severe LPB Ca concentration was high er than in leaves showing incipient LTB symptom. The Mn concentration ranged from 25 to 40 ppm, which was lower than the lower level of the range (50 ppm). The concentrations of phosphorus (P), potassium (K), iron (Fe), Mn, Cu, and molybdenum (Mo) did not differ between the tips, middle, and bottom of the leaves.
The effect of the pre-planting and post-planting forcing temperatures on growth, flowering, and LTB expression Leaf emergence was affected significantly by tem perature treatments during STA and STB and the interaction of STA and STB (Table 4). Leaves emerged late when bulbs were stored at 10°C before potting during STA (10°C/STA) compared to when bulbs were stored at 16 and 22°C/STA, and the forc ing temperatures were 15/12 o C or 18/15 o C/STB and 15/12°C or 18/15°C/STB (Table 4). The number of days to flower was affected significantly by tempera ture treatment during STA, STB, and STC and the interaction of STA and STB. The earliest flowering (87 days) occurred when bulbs were treated at 10°C/STA and 21/18°C/STB and STC. Bulbs stored at 22°C/STA took longer than 112 days to flower, regardless of the temperature during STB and STC. The average days to flowering was 116 days, whereas the days to flow ering was <100105 days at 21/18°C/STC.
The number of flowers (<36 flowers) was only affected by bulb storage temperature during STA  (Table 4). More than 44 flowers were produced when bulbs were treated at 16 and 22°C/STA, regardless of the temperature during STB and STC. The scape length was >20.4 cm when bulbs were stored at 10°C/STA, regardless of the tempera ture during STB and STC. When the temperature during STA was increased from 10 to 22°C, a signifi cantly higher number of flowers was produced on short scapes when bulbs were stored at 22°C. For plants that required an average of 115 days to flower, the scape length was 12.4 cm, and 52 flowers were produced in the treatment of 16°C/STA, 21/18°C/STB, and 15/12°C/STC (Table 4). However, a significantly higher number of leaves (3.31) show ing LTB was produced compared to 0.50 leaves in treatment (10°C/STA, 21/18°C/STA, and 21/ 28°C/STC) and 1.13 leaves in treatment (10°C/STA, 15/12°C/STA, and 21/28°C/STC). As the bulb stor age temperature was increased to 16 or 22°C, the number of leaves showing LTB increased to 2.19 leaves (16°C/STA, 15/12°C/STA, and 15/12°C/STC) to 4.31 leaves (22°C/STA, 18/15°C/STA, and 15/12°C/STC).
The scape growth was primarily influenced by the temperature during STA, resulting in scores of <1.5 when bulbs were stored at 10°C/STA, regardless of the temperature during STB and STC (Table 4). The scape growth when bulbs were stored at 16 and 22°C/STA had scores of >2.3, regardless of the tem perature during STB and STC. The leaf spread showed a complex response; STA, STB, and STC had a significant effect, as did the interaction of STA and STB and STB × STC. However, the leaf spread had scores <1.9 when the plants were forced at 15/12°C/STC, followed by storing the bulbs at 10 or 16 o C/STA. In contrast, the scores were >1.9 regard less of the temperature treatments during STA, STB, and STC. The length of the leaves was<10.4 cm when bulbs were stored at 10°C/STA, regardless of the forcing temperature during STB and STC, and was >12 cm when bulbs were stored at 22 o C/STA. Bulbs stored at 16°C/STA had leaf lengths between those of the 10°C and 22 o C/STA treatments. The leaf width was <2.3 cm when bulbs were stored at 10°C/STA.

Quality criteria of the finished potted plants
Criteria for the quality of finished potted O. dubi-um plants have not been established even in 2019. The quality can be evaluated in reducing the inci dence leaf tip burn (LTB) at the tip of the leaves (Fig.  1) and by the rate of flowering without sacrificing the number of flower buds. Areas of LTB are indicated by the arrows in O. dubium seedlings in figures 2 and 3. However, the criteria used to assess the visual quality were not specified (Lee and Miller, 2015). Therefore, the following three quantitative crite ria to assess the plant quality were established in this study. Criterion I: LTB occurs on <1.5 leaves per plant. The lower the score of the scape growth and leaf spread, the higher the plant quality is. Criterion II: the number of days to flowering is <115 days, the length <11.5 cm, the width of the third leaf from the crown is <2.5 cm, the scape length is <15 cm with >45 flowers. Criterion III: the leaf spread and scape growth have a score <1.5.

Analysis of the macro-and micro-elements in the third and/or fourth leaves showing various symptoms and suggested concentration for quality plant production
The cause of LTB was investigated based on the macro and microelement concentrations in the tis sue. Macro and microelements analysis of O. dubium leaves without apparent LTB revealed that the B concentration in the leaf tips (distal end) was 119 ppm, which was within the suggested acceptable range for general ornamentals (30150 ppm) ( Table 2 and 3). The concentrations in the middle and bottom (proximal end) of the leaves were slightly higher or lower, respectively, than the lower level of the range of B concentration. When the samples were collected in the early growth stages when no or incipient LTB symptoms were observed on Jan. 14, Jan. 26, and Mar. 4, the B concentrations were 109, 96, and 108 ppm, respectively, which fell within the suggested acceptable range.
Leaf tip burn in O. dubium in young and old leaves is considered to be different from marginal leaf burn observed in old leaves of Curcuma 'CMU Price'. The latter is related to high concentrations of B (119 ppm), Fe (189 ppm), and Mn (273 ppm) and low N concentration (1.7%) (Roh et al., 2006). Boron may accumulate at the tip or margin of leaves (Jones, 1970) and may cause toxic effects (Oertli, 1962). Therefore, LTB in Ornithogalum is a unique physio logical expression resulting from high B concentra tion at the leaf tips and developing regardless of the leaf age; these symptoms were observed in O. thyr-soides and O. arabicum (Roh, personal observation). The incidence of LTB in Watsonia laccata (Jacquin) Ker Aawler is considered a similar disorder as in O. dubium and adversely affects potted plant produc tion in W. laccata (Suh et al., 2011). Leaf tip scorch observed in O. dubium (De Hertogh and Gallitano, 1997), upper leaf necrosis observed in Oriental hybrid lilies (Chang et al., 2008), and burn appearing at certain developmental stages before the visible bud stages in Lilium 'Pirate' (Berghoef, 1986) The LTB is, therefore, caused by a high B concen tration (218230 ppm) and possibly a high Zn concen tration (155159 ppm), which was higher in this study than the high level of the suggested range, even when grown in medium low in B and Zn. Boron con centration in the growing medium was <6 ppm (Tech Data PROMIX_HP_Mycorrhizae_4253; accessed on Nov. 1, 2020). The source of B accumulated at the tip of leaves showing LTB in Ornithogalum is considered resulting from B accumulation at the margin of old leaves after translocation from rhizome and leaves as discussed in Curcuma (Roh et al., 2006;Roh and Lawson, 2009). Since Zn has intermediate mobility and occurs in the middle leaves (Shelp et al., 1995;McCauley et al., 2009), it may not be associated with LTB observed in the young and old leaves of O. dubium.

The effect of the temperatures in the pre-planting bulb (ST-A) and post-planting forcing periods (ST-B and ST-C) on growth, flowering, and plant quality
Regardless of the temperature treatments during the preplanting bulb (STA) and postplanting forcing (STB and STC) periods, the LTB symptoms were not entirely eliminated in O. dubium, which was in agree ment with the results for W. laccata (Suh et al., 2011). In our experiment, the number of leaves showing LTB was 1.1 out of 5.1 leaves which was the fewest when received 10°C/STA, followed by forcing at 15/12°C/STB and 21/18 o C/STC (Table 1). Therefore, the flowering responses and leaf and scape morphologies, as evaluated in criteria II and III, respectively, were further considered. The overall average number of days to flower was 116 days (Table 4), which was close to the 115 days estab lished for criterion I. Therefore, it is suggested to store bulbs at lower than 22°C, which is lower than the 25°C suggested by De Hertogh and Gallitano (1997). Our data suggested that storing bulbs at 10 o C ensures that plants will flower in <115 days.
Low preplanting temperature treatments in the range of 9 to 27°C for 3 weeks were shown to accel erate flowering (Lee and Miller, 2015). However, this depends on the cultivar and bulbhandling methods since leaves did not emerge and plants failed to flower when bulbs were treated at 10°C. The initia tion of the inflorescence can be inhibited by more than 6 weeks because breaking dormancy may require temperatures higher than 10 o C (Roh and Joung, 2004). The optimum preplanting temperature was reported as 22 to 28°C immediately after har vesting the bulbs to promote the early development of the first inflorescence and initiation of the second inflorescence (Roh and Joung, 2004). In O. arabicum, flower initiation and development were accelerated by storing initially at 30°C for 12 weeks, followed by 20°C for 4 weeks, and finally at 13°C for 8 weeks before planting .
Forcing at 21/18°C/STB and STC after potting the bulbs caused the plants to flower in 100 to 105 days, and the response was faster when bulbs were treat ed at 16°C/STA. Therefore, the recommended tem peratures for storing bulbs to ensure flowering in <115 days are 10 or 16°C/STA followed by forcing at 21/18 o C/STB or preferably 21/18°C/STC (10°C/STA or at 16°C/STA, 21/18°C/STB or 21/18°C/STC). De Hertogh and Gallitano (1997) suggested to force bulbs in a greenhouse at a minimum of 22/18°C to flower in 96 days (this is similar to the period in STB and STC in this study). Temperatures below 15 o C during an unspecified growth period did not increase the growth rate (Littlejohn and Blomerus, 2000), although a constant temperature of 17 to 19°C was most suitable for forcing, considering the forcing time, plant height, and visual quality of Ornithogalum cultivars. However, the criterion for visual quality was not specified (Lee and Miller, 2015).
The number of flowers was significantly influ enced by bulb storage temperature during STA, and plants produced <36 flowers (10 o C /STA, 18/15 o C /STB, and 15/12 o C/STC). However, more than 44 flowers were produced when bulbs were treated at 16 and 22 o C during STA, regardless of the tempera ture during STB and STC. The number of flowers was reduced when forcing occurred at 32/27 o C; how ever, the flowering rate was not mentioned (Luria et al., 2002). A significantly higher number of flowers (52 flowers) produced on the short scapes (12.4 cm) when bulbs were stored at 22 o C/STA and forced at 21/18 o C/STB) and 15/12 o C/STC. Depending on the bulb size, 20 flowers each were produced on two flower stalks, and the bulbs stored at 9 o C produced floral stalks (31.3 cm) (De Hertogh and Gallitano, 1997).
Three criteria were established to determine the quality of O. dubium plants. Criterion I focus on the occurrence of LTB that are observed in less than 1.5 leaves per plant. Leaf tip burn cannot be avoided, but can be controlled by maintaining the forcing temper ature at a minimum of 10 o C or potentially 16 o C dur ing STA. This treatment does not delay flowering. Leaf tip burn was observed in O. dubium in both young and old leaves and was caused by a high B concentration (218230 ppm) and possibly by a high Zn concentration (155159 ppm). Calcium was not the primary cause for LTB. Criterion II focuses on growth and flowering, leaf and scape morphologies; flowering occurs less than 115 days, the length and width of the third leaf is shorter than 11.5 cm and narrower than 2.5 cm, respectively, the scape length is shorter than15 cm, and there are more than 45 flowers. Criterion III focuses on the leaf morphology and the scape growth have a score of less than 1.5. The recommended treatment to meet the three cri teria and to produce high quality plants at flowering (plant A, Fig. 2) is to treat bulbs at 10 or 16 o C before planting (STA), and at 15/12 o C in STB and at 21/18 o C during STC during forcing. However, it was not possi ble to produce finished plants without severe LTB symptoms. Although LTB can not be prevented in O. dubium, it can be minimized by temperature manipu lation.