Refereed journal articles

The development of upper leaf necrosis in Lilium 'Star Gazer'
-- J. Amer. Soc. Hort. Sci. 130(5): 759-766 . (2005)

A necrotic disorder occurs on upper leaves of many oriental hybrid lily (Lilium L.) cultivars, including the most-widely-grown 'Star Gazer'. We term this disorder “upper leaf necrosis” (ULN) and hypothesize that it is a calcium (Ca) deficiency. We demonstrated that Ca concentration in necrosed tissues was nearly six-fold below that of normal leaves (0.10% vs. 0.57% dry weight), and that Ca concentration was negatively associated with percentage necrosed leaf area. It was concluded that ULN is a Ca deficiency disorder. When the symptoms were slight, early ULN symptoms appeared as tiny depressed spots on the lower surface of the leaf, or as water-soaked areas when the disorder was severe. Most commonly, ULN began on the leaf margin. The injured areas turned brown, leading to leaf curling, distortion, or tip death. ULN occurred on leaves associated with flower buds and leaves immediately below the flower buds. For the plants grown from 16–18 cm circumference bulbs, the five leaves directly below the flower buds and larger leaves associated with the 1st and the 2nd flower buds were most susceptible. In general, flower buds were not affected by ULN, and continued to develop and flower normally, even though they were associated with subtending highly distorted leaves. Eighty-five percent of plants began to exhibit ULN symptoms 30–40 days after planting (i.e., 24–34 days after shoot emergence). This was the stage when the 6th or 7th leaf under the bottom flower bud was just unfolded. Light was not the main factor that initiated ULN, however, ULN severity was greatly increased by light reduction, as leaf transpiration was reduced.

The relationships between leaf enclosure, transpiration, and upper leaf necrosis on Lilium 'Star Gazer'
-- J. Amer. Soc. Hort. Sci. 129(1): 128-133. (2004)

Upper leaf necrosis (ULN) on Lilium 'Star Gazer' has been shown to be a calcium (Ca) deficiency disorder. Initial symptoms of ULN tend to appear on leaf margins. Before flower buds are visible, young expanding leaves are congested and overlap each other on the margin. In the current study, we examined the relationship between leaf enclosure, transpiration, and upper leaf necrosis. We demonstrated that low transpiration rate and enclosure of young leaves played an important role in the occurrence of ULN. Young expanding leaves are low transpiration organs. The younger the leaf, the lower the transpiration rate and Ca concentration. Leaf enclosure further reduced transpiration of these young leaves and promoted ULN. Upper leaf necrosis was suppressed by manually unfolding the leaves using a technique we refer to as artificial leaf unfolding (ALU). ALU minimized leaf congestion, exposing leaves that were previously enclosed. We demonstrated that the effect of ALU was not the consequence of thigmomorphogenesis, as ULN was not reduced by mechanical perturbation in lieu of ALU. With ALU, transpiration of upper leaves was significantly increased and Ca concentration of the “first leaf immediately below the flower buds” was increased from 0.05% to 0.20%. We concluded that leaf enclosure promoted ULN occurrence, and ALU suppressed ULN primarily by increasing transpiration. The use of overhead fans to increase airflow over the tops of the plants significantly reduced both ULN incidence and severity.

Efficacy of exogenous calcium applications for reducing upper leaf necrosis in Lilium 'Star Gazer'
-- HortScience 39(2): 272-275 (2004)
-- Kenneth Post Award, 2005

Upper leaf necrosis (ULN) on Lilium cv. Star Gazer is a calcium (Ca) deficiency disorder. In this study, we evaluated the efficacy of foliar Ca sprays and bulb Ca dipping on reducing ULN. Necrosis severity of a single leaf was determined by an index from 0 (healthy) to 5, based on symptoms progression and necrosed leaf area. Single leaf severity then was summed for all leaves to yield a whole plant severity rating. Single daily applications of 25 mM calcium chloride or calcium nitrate sprays for 14 days significantly suppressed the degree of symptom expression; whole plant severity was reduced from 18 (severely necrosed) to below 3 (essentially unnoticeable). Five single applications at 3.5-day intervals were not effective, even with at concentrations up to 150 mM. At concentrations of 100 mM and 150 mM, fourteen daily sprays of calcium chloride or calcium nitrate were toxic and caused leaf tip yellowing; calcium chloride caused more severe phytotoxicity than did calcium nitrate. For effectiveness of foliar Ca sprays, it was necessary to have the Ca solution reach the enclosed young expanding leaves. Pre-plant bulb immersion in calcium chloride was not effective even at concentrations as high as 400 mM for up to 16 hours.

Growth and calcium partitioning in Lilium 'Star Gazer' in relation to leaf calcium deficiency
-- J. Amer. Soc. Hort. Sci. 128(6): 788-796 (2003)
-- Kenneth Post Award, 2004

Upper leaf necrosis (ULN) on Lilium cv. Star Gazer has been recently demonstrated to be a calcium (Ca) deficiency disorder. In the current studies, we confirmed this by using a Ca-free nutrient regime to reproduce ULN symptoms. The ability of a bulbous storage organ to supply calcium to a growing shoot is poorly understood. Therefore, we conducted experiments to determine Ca partitioning during early growth stages, and under sub-optimal Ca levels in order to determine how the bulb affects the symptomatology. The results indicated that ULN is originally caused by an insufficient Ca supply from the bulb. In the most susceptible period, bulb dry matter decreased dramatically and Ca concentrations in immature folded leaves dropped to very low levels. Consequently, necrosis began to appear on the upper, young leaves. The bulb was able to supply Ca to other organs, but only to a limited extent since Ca concentration in bulbs was low (0.04% w/w). In order to confirm this result, we cultivated lilies with low-Ca or Ca-free nutrient solution and obtained bulbs with extremely low internal Ca concentrations. Upon forcing these low-Ca bulbs, we found, as expected, prominent necrosis symptoms on the lower and middle leaves. Data suggested the lower and middle leaves relied more on Ca supplied from the bulb, while upper leaves and flowers relied more on Ca uptake from the roots. Different organs have different Ca requirements, and tissue sensitivity to Ca deficiency varies according to the growth stage.

Effects of solution calcium level and bulb calcium level on the
development of upper leaf necrosis on Lilium cv. Star Gazer
-- presented at ASHS 2003, Oct 2003

Upper leaf necrosis (ULN) on Lilium cv. Star Gazer is aresult of localized calcium (Ca) deficiency in the upper leaves. In the current study, we investigated the effects of solution Ca level and bulb Ca content on the development of ULN. Studying this way, we were able to examine the role of the bulb on ULN symptomatology. Bulbs (14-16 cm circum.) were planted in pure sand, and fertigated with nutrient solutions with 0, 0.2, or 4 mM Ca. The Ca concentrations of all organs were positively correlated, and ULN was negatively correlated, with solution Ca level. Regardless of nutrient Ca level (even Ca free), necrosis symptoms appeared only on the upper leaves. When leaves senesced, the bulbs (with various internal Ca concentrations) were harvested. Upon forcing these bulbs with extremely low internal Ca concentration (0.009% DW) in the next season, we found prominent necrosis symptoms on the lower and middle leaves. These results demonstrate ULN is primarily caused by an insufficient Ca supply from the bulb. The bulb is able to translocate limited Ca to the shoot; this is why ULN only shows on the upper leaves. Data suggest the lower and middle leaves rely more on Ca supply from the bulb, the upper leaves and flowers rely more on Ca uptake from the roots. 'Star Gazer' lily partitioned most Ca to leaves, little to bulb scales. However, the proportion of Ca partitioning to leaves decreased when solution Ca level was reduced, indicating that leaf Ca is very plastic, and depends on Ca availability to the roots.

Foliar Calcium Sprays Reduce the Severity of
Upper Leaf Necrosis on Lilium cv. Star Gazer
-- presented at IHC 2002/ASHS 2002, August 2002

Upper Leaf Necrosis (ULN) on Lilium cv. Star Gazer is a calcium (Ca) deficiency disorder. To determine if foliar Ca sprays are effective in reducing ULN, experiments were conducted to investigate the effect of Ca salts, Ca concentrations, and application frequency. Necrosis severity of a single leaf was determined by necrosed area using an index from 0 (healthy) to 5. Single leaf severity then was added up to give whole plant severity. Daily foliar Ca sprays significantly reduced the severity of ULN. They also tended to reduce the incidence of necrosis, although it was not statistically significant. Fourteen daily sprays of 25 mM calcium chloride or calcium nitrate were both effective. However, seven sprays at 3.5-day intervals were not effective, even with high concentrations up to 150 mM. Daily sprays of calcium chloride or calcium nitrate were toxic and caused leaf tip yellowing when the concentrations exceeded 50 mM. Calcium chloride seemed to be more toxic than calcium nitrate at the same concentration. For effectiveness, it is necessary to have the Ca solution reach the enclosed young expanding leaves. Since daily Ca sprays would be very labor intensive, they may not be a useful recommendation to growers.

Upper Leaf Necrosis on Oriental Hybrid Lilies
-- presented at ASHS 2001, July 2001

A necrotic disorder happened to upper leaves of many popular Oriental hybrid lilies, we termed it "upper leaf necrosis" (ULN). On 'Stargazer,' ULN happened to leaves associated with flower buds and the few leaves right under flower buds when these leaves were expanding. The two leaves right under the first flower bud and the larger leaf associated with the first flower bud were most susceptible. ULN most commonly began to develop on the leaf margins and was initially characterized by water-soaked tissues, ranging in size from 1 mm to 2 cm, depending on the severity. These necrosed areas turned brown soon, and led to leaf curling, distortion, or tip death. We demonstrated the calcium (Ca) concentration in necrosed leaves was much less than normal leaves and it seemed to be a localized Ca deficiency disorder. Young expanding leaves had low transpiration rate, and low transpiration was correlated with low Ca concentration. Bulb size had a significant impact on ULN. The plants grown from 16/18 cm bulbs were much more susceptible compared to those from 14/16 cm and 12/14 cm. Plants grown from 16/18 cm bulbs had more leaves, and thus made the shoot apex more enclosed when the leaves were unfolding. The enclosure might further reduce the transpiration of the young leaves, and increase ULN incidence. There was a critical period that Oriental hybrid lily 'Stargazer' was more susceptible to ULN, so that most plants showed the symptoms 35-40 days after planting.

Dissertation summary of Ph.D. degree:

Upper Leaf Necrosis on Lilium cv. Star Gazer
- A Calcium Deficiency Disorder

A necrotic disorder that we termed "upper leaf necrosis" (ULN) occurs on the upper leaves of Lilium cv. Star Gazer. The disorder resembles calcium (Ca) deficiency of other crops, so we hypothesized it was a Ca deficiency disorder. We acquired the following data to support this hypothesis: 1) The Ca concentration in necrosed tissue was only 0.10% (w/w), much less than that in healthy tissue (0.57%). Leaf Ca concentration was negatively correlated with necrosed area. 2) By using Ca-free nutrient solution in sand cultures with smaller bulbs that are not normally susceptible, we were able to reproduce the symptoms of ULN. 3) Supplying supplemental Ca directly to young leaves significantly reduced ULN severity.

ULN occurred only on upper leaves. It was initially characterized by depressed regions on lower leaf surface or water-soaked tissues, that often led to leaf curling, distortion, or tip death. For plants grown from bulbs 16/18 cm circumference, most began to show symptoms 30-40 days after planting. At this stage, plants were in the rapid growth phase, with a height of 32 cm, and 6 leaves immediately below flower buds remaining to unfold.

ULN was primarily caused by insufficient Ca supply from the bulb. While the bulb did export Ca to the shoot, the amount was limited. Accordingly, symptoms appeared on the upper, young leaves. Leaf overlap (enclosure), which reduced the transpiration of young leaves, was an important factor that encouraged the occurrence of ULN. By manually unfolding leaves, transpiration of young upper leaves was increased, and ULN was prevented. Plants grown from large bulbs (> 16 cm) were much more susceptible, a more enclosed shoot apex might be the major cause. Low light intensity reduced transpiration, and led to a more severe ULN. Mist also increased the risk of ULN.

Foliar Ca sprays significantly reduced the severity of ULN; fourteen daily sprays of 25 mM calcium chloride or calcium nitrate were both effective. Using overhead fans to increase air flow on the tops of plants significantly reduced both incidence and severity, offering another promising method to control this disorder.

Thesis summary of MS degree:
   

The most suitable tissue of Pleione formosana Hayata for tissue culture was the shoot tip from flower bud of large matured corm, which was harvested in winter and then stored at 5 C for one to two months. Somatic protocorm formation and proliferation of shoot tip were promoted by 1/3 MS basal salts. However, adventitious roots induced easily and few somatic protocorms were formed from shoot tip using Knudson's C medium. After cold storage, the shoot tips had little response to plant growth regulators for somatic protocorm proliferation. The proliferation of shoot tip seemed to perform better in solid media than in liquid ones. The growth of protocorm and bulbing through tissue culture were faster than seedlings germinated from seeds.

Rhizomes of Cymbidium sinense Willd differentiated into buds directly with BA ( higher than 1 ppm ). The organogenesis of rhizome was affected appartently by BA/NAA ratio. Shoots also formed from rhizomes without plant growth regulators after a long period of growth. Neither light nor cultural methods (solid or liquid culture) seemed to affect the shoot formation. Activated charcoal inhibited the differentiation of bud, probably because it absorbed some metabolites which secreted by explant might be beneficial to shoot formation when they coexisted with BA.