Thermotolerance of Hemlock (Tsuga spp.) and Investigation of a Foliar Disorder in
Crapemyrtle (Lagerstroemia indica ?fauriei)
by
Matthew Stephen Wilson
A thesis submitted to the Graduate Faculty of
Auburn University
in partial fulfillment of the
requirements for the Degree of
Master of Science
Auburn, Alabama
August 09, 2010
Keywords: Heat stress, direct injury, electrolyte leakage, rabbit tracks
Copyright 2010 by Matthew Stephen Wilson
Approved by
Jeff L. Sibley, Chair, Professor of Horticulture
D. Joseph Eakes, Professor of Horticulture
Gary J. Keever, Professor of Horticulture
ii
Abstract
Foliar thermotolerance of six Tsuga species to direct heat injury was evaluated
using electrolyte leakage. Species evaluated included: T. canadensis, T. caroliniana, T.
chinensis, T. diversifolia, T. heterophylla, and T. dumosa (formerly T. yunnanensis).
Species were exposed to 12 temperatures (25-60?C) for 30 minutes and electrolyte
leakage from needle tissue was measured. T. canadensis and T. dumosa had similarly,
higher predicted temperature midpoints (Tm) and k-values than T. caroliniana suggesting
a higher resistance to direct heat injury when exposed to brief, supra-optimal
temperatures. While differences were found in Tm and k-values between T. canadensis
and T. dumosa when compared to T. caroliniana, no differences were found in Tm and k-
values for T. canadensis or T. dumosa when were compared to T. chinensis, T.
diversifolia, or T. heterophylla. Similarly, no differences were found for Tm and k-values
of T. caroliniana when compared to T. chinensis, T. diversifolia, or T. heterophylla.
An online survey was conducted in cooperation with seven nursery and landscape
associations in the southeastern U.S. to quantify the impact of a foliar disorder in
Lagerstroemia spp. on the ornamental horticulture industry. Industry members were
asked 16 questions to obtain demographic, disorder familiarity, and disorder significance
information. No significant correlations between gross business income of participants
and the ratings for the significance of ?rabbit tracks? to the industry were found.
Measures requiring a large amount of resources, expense, and costly solutions are not
iii
recommended for diagnosis work or treatment, as industry members did not indicate
significant losses or customer concern for effects of ?rabbit tracks? on plant material
quality.
A preliminary nutrition study was conducted on five Lagerstroemia indica
?fauriei cultivars using six modified nutrient solutions to evaluate foliar disorder
occurrence in relation to treatment. Nutrient solutions were modified to exclude one
macronutrient sulfur (S) or four micronutrients; copper (Cu), iron (Fe), manganese (Mn),
or zinc (Zn), as prescribed by treatment. No occurrence of ?rabbit tracks? for complete
nutrient solution-treated plants was seen. There was no significant difference between
any of the nutrient solutions for the occurrence of ?rabbit tracks? foliar disorder on the
five cultivars tested. Additionally, no significant relationship between modified nutrient
solutions and percent chlorosis coverage, chlorosis stage, chlorosis progression, spot
occurrence, spot pattern, or leaf distortion ratings were found. Chlorosis occurrence
ratings due to treatment were found to be significant across all cultivars with iron-
deficient nutrient solutions having higher incidence of chlorosis than zinc-deficient and
complete nutrient solution treatments.
iv
Acknowledgments
The author would like to express sincere gratitude and appreciation to Dr. Jeff
Sibley for providing the opportunity to pursue the Master of Science degree in
horticulture at Auburn. His guidance, support, and generosity will always serve as
reminders of the wonderful mentorship and friendship provided to the author. The author
would like to thank Drs. Gary Keever, Joe Eakes, John Ruter, and Brian Whipker for
their guidance and patience in the pursuit of this degree and to Drs. Joe Eakes and Jeff
Sibley for providing the opportunity and experience to instruct at the university level,
providing real-world experience and training for the road ahead. The author would like to
thank his parents Mark and Sandra Wilson, sister Kayla and brother-in-law Shane, and
grandparents Mary Wilson and Henry and June Richards for their love, support and
sacrifices to encourage the author to accomplish his goals and achievements. The author
would also like to thank B.J. Brymer, Chris Marble, Jeremy Pickens, and Daniel Wells
for the support and friendship provided through the partnership of Team MaterDirt,
?Strangers we met, Friends we became, and Brothers we remain.? In addition to
aforementioned acknowledgments, the author would like to express gratitude to fellow
Department of Horticulture faculty, staff, and graduate students for their friendship,
support, and assistance. The author would like to thank the Auburn University Campus
Club, Montgomery Federation of Garden Clubs, American Conifer Society, and Southern
Nursery Association for the provision of scholarships for the author?s academic pursuits.
v
Table of Contents
Abstract ............................................................................................................................ ii
Acknowledgments........................................................................................................... iv
List of Tables ................................................................................................................. vii
List of Figures ............................................................................................................... viii
Part I Thermotolerance of Hemlock (Tsuga)
Chapter I Introduction and Literature Review ..................................................................1
Hemlock Cultivation History and Species Descriptions .......................................1
Temperature Effects ..............................................................................................7
Electrolyte Leakage ............................................................................................14
Literature Cited ...................................................................................................18
Chapter II Foliar Thermotolerance of Tsuga spp ............................................................25
Abstract ...............................................................................................................25
Introduction .........................................................................................................26
Materials and Methods ........................................................................................29
Results and Discussion .......................................................................................31
Literature Cited ...................................................................................................34
Part II Investigation of a Foliar Disorder in Crapemyrtle (Lagerstroemia indica ?fauriei)
Chapter I Introduction and Literature Review ................................................................44
vi
The Genus Lagerstroemia...................................................................................44
Foliar Disorder in Crapemyrtle ...........................................................................46
Preliminary Work on ?Rabbit Tracks? Disorder ................................................47
Literature Cited ...................................................................................................52
Chapter II Survey of ?Rabbit Tracks? Foliar Disorder?s Extent, Effect, and
Significance to Ornamental Horticulture Industries ...........................................56
Abstract ...............................................................................................................56
Introduction .........................................................................................................57
Materials and Methods ........................................................................................58
Results and Discussion .......................................................................................60
Literature Cited ...................................................................................................65
Chapter III A Nutritional Approach to Diagnosing ?Rabbit Tracks? Foliar Disorder in
Five Cultivars of Lagerstroemia indica ?fauriei. ................................................75
Abstract ...............................................................................................................75
Introduction .........................................................................................................76
Materials and Methods ........................................................................................77
Results and Discussion .......................................................................................79
Literature Cited ...................................................................................................81
Chapter IV Final Discussion ...........................................................................................87
Appendix A Foliar Thermotolerance of Tsuga spp. in 2003 ..........................................91
Appendix B Survey of ?Rabbit Tracks? Foliar Disorder?s Extent, Effect, and
Significance to Ornamental Horticulture Industries .........................................102
Appendix C Preliminary Experiments Evaluating ?Rabbit Tracks? Occurrence to Various
Rates of Substrate and Foliar-Applied Nutrients ..............................................108
vii
List of Tables
Chapter I
Table 1. Accepted species for genus Tsuga .................................................24
Chapter II
Table 1. Response of Tsuga (hemlock) species to elevated foliar
temperature ....................................................................................37
Part II Chapter III
Table 1. Modified nutrient solution formulations with reagent stock
solution ml?L-1 of treatment solution for ?rabbit tracks?
crapemyrtle nutrition study in 2008. ..............................................82
Table 2. Lagerstroemia indica ?fauriei 'Yuma' leaf tissue nutrient
concentrations for six modified nutrient solutions.........................83
Table 3. Nutrient concentrations of irrigation water used in modified
nutrient solutions for "rabbit tracks" crapemyrtle hydroponic
study in 2008. .................................................................................83
Appendix A
Table 1A. Response of Tsuga (hemlock) species to elevated foliar
temperature ....................................................................................95
Appendix C
Table 1C. Effects of substrate-incorporated manganese rates on
occurrence of ?rabbit tracks? foliar disorder in
Lagerstroemia indica ?fauriei 'Natchez'. ....................................114
Table 2C. Effects of foliar-applied manganese rates on occurrence of
?rabbit tracks? foliar disorder in Lagerstroemia indica ?fauriei
'Natchez'. ......................................................................................114
viii
List of Figures
Chapter II
Figure 1. Foliar electrolyte leakage of Tsuga canadensis exposed to
25 to 60? C for 30 min. in 2008. ....................................................38
Figure 2. Foliar electrolyte leakage of Tsuga caroliniana exposed to
25 to 60? C for 30 min. in 2008. ....................................................39
Figure 3. Foliar electrolyte leakage of Tsuga chinensis exposed to
25 to 60? C for 30 min. in 2008. ....................................................40
Figure 4. Foliar electrolyte leakage of Tsuga diversifolia exposed to
25 to 60? C for 30 min. in 2008. ....................................................41
Figure 5. Foliar electrolyte leakage of Tsuga dumosa exposed to
25 to 55? C for 30 min. in 2008. ....................................................42
Figure 6. Foliar electrolyte leakage of Tsuga heterophylla exposed to
25 to 60? C for 30 min. in 2008. ...................................................43
Part II Chapter I
Figure 1. ?Rabbit tracks? foliar disorder in Lagerstroemia indica ?fauriei
?Tuskegee?......................................................................................55
Figure 2. Progressed ?rabbit tracks? foliar disorder in Lagerstroemia indica
?fauriei ?Tuscarora?. .....................................................................55
Chapter II
Figure 1. Participant response to ?rabbit tracks? crapemyrtle survey question:
Which state is your business located? ............................................66
Figure 2. Participant response to ?rabbit tracks? crapemyrtle survey question:
What form of communication did you receive knowledge of this
survey? Select all that apply. .........................................................66
ix
Figure 3. Participant response to ?rabbit tracks? crapemyrtle survey question:
What type of business do you manage/own? Select all that
apply. ..............................................................................................67
Figure 4. Participant response to ?rabbit tracks? crapemyrtle survey question:
What bracket best describes your gross sales? ..............................67
Figure 5. Participant response to ?rabbit tracks? crapemyrtle survey question:
If Grower/wholesaler/retailer, what term best describes your plant
selection? Select all that apply. ......................................................68
Figure 6. Participant response to ?rabbit tracks? crapemyrtle survey question:
What percentage of your crops/sales may be attributed to
crapemyrtle? ...................................................................................68
Figure 7. Participant response to ?rabbit tracks? crapemyrtle survey question:
Have you ever heard of a foliar disorder termed "Rabbit Tracking
or Rabbit Tracks" believed to affect crapemyrtle
(Lagerstroemia)?............................................................................69
Figure 8. Participant response to ?rabbit tracks? crapemyrtle survey question:
Have you ever seen the symptoms as shown in the image above
on crapemyrtles grown by you or another grower in late
spring-early summer?.....................................................................69
Figure 9. Participant response to ?rabbit tracks? crapemyrtle survey question:
Did you observe this disorder in field production, container
production, or the landscape? Select all that apply. .......................70
Figure 10. Participant response to ?rabbit tracks? crapemyrtle survey question:
What cultivars have you noticed to be affected greatest? ..............70
Figure 11. Participant response to ?rabbit tracks? crapemyrtle survey question:
Have you or another grower attempted to remedy this disorder
via chemical sprays, substrate amendments, or environmental
controls? .........................................................................................71
Figure 12. Participant response to ?rabbit tracks? crapemyrtle survey question:
If so, what remedies, if any, have you found to be beneficial in
remedying ?rabbit tracks?? ............................................................71
Figure 13. Participant response to ?rabbit tracks? crapemyrtle survey question:
Have you ever had customers/buyers comment on the "Rabbit
Tracks" symptoms? ........................................................................72
x
Figure 14. Participant response to ?rabbit tracks? crapemyrtle survey question:
Have customers/buyers refused to purchase affected plant
material? .........................................................................................72
Figure 15. Participant response to ?rabbit tracks? crapemyrtle survey question:
Rate the likelihood you, buyers, or customers would reject
purchasing affected material (0%- least likely, 100% most likely
to reject). ........................................................................................73
Figure 16. Average rating for rejection of ?rabbit tracks?-affected plant by
grower, retailer, and customer groups as rated by ?rabbit tracks?
crapemyrtle survey participants. ....................................................73
Figure 17. Participant response to ?rabbit tracks? crapemyrtle survey: Rate
your opinion of "Rabbit Tracks" significance to the industry. ......74
Chapter III
Figure 1. Top-view of hydroponic gutter system for ?rabbit tracks?
crapemyrtle nutrition study in 2008. ..............................................84
Figure 2. Top-view of polystyrene cover with gutter guard support and
subsample plot spacing and labels. ................................................84
Figure 3. Aeration system of hydroponic gutter system for rabbits tracks
crapemyrtle nutrition study in 2008. ..............................................85
Figure 4. Air tube submersion bracket for hydroponic gutter system in
?rabbit tracks? crapemyrtle nutrition study in 2008. .....................85
Figure 5. Rabbit tracks crapemyrtle nutrition study 81 days after planting
in 2008. ..........................................................................................86
Appendix A
Figure A1. Foliar electrolyte leakage of Tsuga canadensis exposed to
25 to 60? C for 30 min. in 2008. ....................................................96
Figure A2. Foliar electrolyte leakage of Tsuga caroliniana exposed to
25 to 60? C for 30 min. in 2003. ....................................................97
Figure A3. Foliar electrolyte leakage of Tsuga diversifolia exposed to
25 to 60? C for 30 min. in 2003. ....................................................98
Figure A4. Foliar electrolyte leakage of Tsuga heterophylla exposed to
25 to 60? C for 30 min. in 2003. ....................................................99
xi
Figure A5. Foliar electrolyte leakage of Tsuga mertensiana exposed to
25 to 60? C for 30 min. in 2003. ..................................................100
Figure A6. Foliar electrolyte leakage of Tsuga sieboldii exposed to
25 to 60? C for 30 min. in 2003. ..................................................101
Appendix B
Figure B1. Institutional review board letter of approval for survey. ............. 102
Figure B2. Flyer emailed to nursery and landscape association directors. .... 103
Figure B3. Preliminary on-line survey information and participant
approval page. .............................................................................. 104
Figure B4. First on-line survey question page. .............................................. 104
Figure B5. Survey of ??rabbit tracks?? foliar disorder?s extent, effect, and
significance to ornamental horticulture industries. ...................... 105
1
PART I THERMOTOLERANCE OF HEMLOCK (TSUGA)
Chapter I
Introduction and Literature Review
I. Hemlock cultivation history and species descriptions
The genus Tsuga currently consists of approximately 11 species (Table 1). Nine
of the 11 species are commonly used within the landscape/nursery industries (Bitner,
2007; Dirr, 1998; Swartley, 1984). A member of the Pinaceae family, Tsuga may be
differentiated from other family members such as Pinus, Picea, and Abies by its flat
evergreen leaves that are joined to the stem via woody, short decurrent bases and
pendulous female cones with imbricate scales (Radford et al., 1968). Tsuga, referred to
by the common name hemlock, is unlike its family members in that it has a soft, fine
texture in comparison to the coarse texture commonly associated with spruce (Picea) and
fir (Abies) (Dirr, 1998). This soft texture gives hemlocks a special place within the
landscape as specimen trees, unique hedges, in rock gardens, and other specialty uses
(Forrest, 2005).
Typically found in mountainous regions, hemlocks thrive in areas with abundant
rainfall and well-drained soil (Swartley, 1984). Hemlocks also may be found along
streambeds in deeply shaded forests (Bitner, 2007). Four native species and one hybrid of
Tsuga, as well as cultivated forms, can be found in North America. Along the eastern
sector of the continent two species can be found within forests, T. canadensis
2
(L.) Carr. (Canadian or eastern hemlock) and T. caroliniana Engelm. (Carolina hemlock),
and in the western sector the remaining three, T. heterophylla (Raf.) Sarg. (western
hemlock), T. mertensiana (Bong.) Carr. (mountain hemlock), and T. ?jeffreyi (Henry)
Henry [heterophylla x mertensiana]. In addition to native Tsuga species, four species of
Asian origin are found to be in cultivation in North America and around the world. These
species vary in origin, from T. chinensis (Franch.) Pritzel (Chinese hemlock), and Tsuga
dumosa (D. Don) Eichler (Himalayan hemlock) in Central-Southern China to T.
diversifolia (Maxim) Mast. (Northern Japanese hemlock) and T. sieboldii Carr. (Southern
Japanese hemlock) in Japan (Swartley, 1984).
Of the North American Tsuga, the Canadian hemlock (T. canadensis (L.) Carr.) is
the more prominently used specimen in the landscape. According to Dirr (1998), hemlock
was introduced into cultivation around 1736 with the number of named cultivars and
varieties currently exceeding 300. Canadian hemlock may be distinguished from other
native hemlocks by its flatly arranged leaves with stalked cones and broad cone scales
(Sargent, 1949). Noted for its conspicuous stomatal lines underneath leaves, relaxed
branch tips, and small pendulous cones, Canadian hemlock often adorns mixed borders
and plantings in the landscape as a prized specimen tree (Bitner, 2007). As a slow
growing conifer with large variability within seedlings, many varieties and cultivars of T.
canadensis have been developed such as ?Sargentii? with its pendulous branches and
spreading growth habit reaching 10 m (30 ft) in spread and ?Bennett? growing only 0.6 x
1.2 m (2 x 4 ft) within ten years (Bitner, 2007; Dirr 1998). With such extensive use in
cultivation, Canadian hemlock has encountered a major decline with the arrival of the
hemlock woolly adelgid (Adelges tsugae Annan.). Brought to North America in the
3
1920?s (Evans and Gregoire, 2007), from Asia and first recorded in eastern North
America in 1951, hemlock woolly adelgid (HWA) has spread to cover 21% of Canadian
hemlock?s native range consisting of the Appalachian Mountain range from Nova Scotia
to northern Alabama (Evans and Gregoire, 2007; Sargent, 1949). HWA is a member of
the order Homoptera, a category of insects with two pairs of wings and piercing-sucking
mouthparts that enable such insects to pierce plant tissues and extract sap,
photoassimilates, and water from the plant (Evans and Gregoire, 2007; Pedigo, 2002).
HWA?s feeding method, efficient reproduction mechanisms, and lack of predators have
allowed for mass infestations and continuous spread across North America, causing a
decline in native hemlock stands along the eastern portion of the continent (Evans and
Gregoire, 2007). Concern of HWA?s potential to endanger existing habitats and
landscapes of Tsuga is aiding research in finding and releasing predators with sufficient
control and adaptability to surrounding environment to control HWA populations
(McAvoy et al., 2007). In addition to developing predator controls for HWA, research
evaluating several Tsuga species for potential host plant resistance with regard to plant
nutrition and feeding preferences has been conducted to provide information useful for
breeding programs to introduce species/ hybrids of hemlock resistant to HWA related
death (Montgomery et al., 2009; Pontius et al., 2006; Tredici and Kitajima, 2004).
Another hemlock native along the eastern portion of North America is the
Carolina hemlock (T. caroliniana Engelm.). Carolina hemlock is more isolated in its
native range than Canadian hemlock as its range consists of southwestern Virginia to
Northern Georgia (Dirr, 1998), typically at higher elevations than T. canadensis.
Distinguishing characteristics from other native hemlocks are stalked cones with cone
4
scales longer than wide and obtusely pointed bracts and bristle-like leaves not appearing
to be flat, but two-ranked in arrangement off the stem (Bitner, 2007; Sargent, 1949;
Swartley, 1984). Similar to Canadian hemlock, Carolina hemlock has two white stomatal
lines underneath the leaf with a glabrous, dark green surface above. Reduced native range
and reported lack of drought tolerance have not encouraged Carolina hemlock?s use
within the landscape (Bitner, 2007; Dirr, 1998). Like Canadian hemlock, Carolina
hemlock is susceptible to HWA (Dirr, 1998; Montgomery et al., 2009).
North America also serves as the native range for two additional Tsuga species
and one hybrid along its West coast. Western hemlock?s (T. heterophylla (Raf.) Sarg.)
native range extends from southern Alaska to Idaho and northern California (Dirr, 1998).
Characterized by sessile cones with oval scales, T. heterophylla (Raf.) Sarg., also has
blackish green leaves arranged in upright ranks along the stem with broad white stomatal
lines underneath (Sargent, 1949; Swartley, 1984). Western hemlock is largely used by the
timber and forestry industry, along with limited use in landscapes of western North
America and western Europe (Bitner, 2007; Swartley, 1984). Western hemlock has
shown less mortality in response to HWA infestations than eastern counterparts which
was attributed to a greater presence of HWA-controlling predators in the West than host
plant resistance (Pontius et al., 2006).
Mountain hemlock (T. mertensiana (Bong.) Carr.), western hemlock?s companion
in western North America, shares a similar native range from southeastern Alaska
through British Columbia south to northern California (Sargent, 1949). More easily
distinguished from other species of the genus, mountain hemlock is characterized by its
convex leaves arranged along all sides of the stem with stomata on both lower and upper
5
leaf surfaces and long, cylindrical cones with oblong scales (Sargent, 1949). Mountain
hemlock also noticeably differs from other hemlocks by its blue-green leaves which have
a somewhat prickly appearance due to the leaf arrangement along all sides of the shoots
(Dirr, 1998). Mountain hemlock is found at high altitudes where it is tolerant of heavy
snow loads. Preferring cool moist soils, mountain hemlock is reported to have difficulty
adapting and growing in landscape and nursery situations because of excessive irrigation
and susceptibility to Phytophthora root rots (Dirr, 1998; Swartley, 1984). Mountain
hemlock serves the pulp industry mainly within the Pacific Northwest with a few
cultivars released for use in landscapes (Bitner, 2007). As with the western hemlock,
mountain hemlock has shown decreased mortality to HWA infestations in comparison to
eastern North American species (Pontius et al., 2006).
Along with the two western species of Tsuga, a hybrid of the mountain and
western hemlock (T.?jeffreyi (Henry) Henry) (USDA, 2007) exists. This hybrid is found
in parts of British Columbia and Washington and closely resembles mountain hemlock in
features and response to landscape situations but has been found to be highly susceptible
to Phytophthora root rots (Dirr, 1998; Swartley, 1984).
Of the four Asian species of Tsuga, Himalayan hemlock (T. dumosa (D. Don)
Eichler) and Chinese hemlock (T. chinensis (Franch.) Pritzel) are native to the mainland
of the continent and the other two native to the neighboring islands of Japan. Himalayan
hemlock?s native range is from north-central India?s Uttarakhand state along the
Himalayan Mountains to Bhutan just east of Nepal and south China (Swartley, 1984).
Himalayan hemlock may be distinguished among the hemlocks by its rigid leaves
pointing upwards along the upper side of the shoot with prominent white bands
6
underneath the leaves (Swartley, 1984). T. dumosa has the largest leaves of all the
hemlocks and has pendulous branching. Introduced into North America in 1838,
Himalayan hemlock is rarely found in landscapes. Recent observations (Wu and Raven,
1999) indicate that Yunnan hemlock (T. yunnanensis (Franch.) Pritzel), once considered a
separate species, may be a subspecies of T. dumosa due to morphological and
geographical distribution similarities (Table 1). Swartley (1984) describes Yunnan
hemlock as being similar to the Himalayan hemlock with its pendulous foliage and
creamy white bands underneath sparsely arranged large, shiny green leaves. Like
Himalayan, Yunnan hemlock is rarely found in North American landscapes, with little
available research concerning its HWA tolerance/resistance (Swartley, 1984).
Another mainland Asian hemlock includes T. chinensis (Franch.) Pritzel (Chinese
hemlock) which is native to China?s southwestern region of West Szechual in the
Sichuan Province (Dirr, 1998; Swartley, 1984). Chinese hemlock may be characterized
by a series of short leaves separating larger leaves evenly along the shoot and light green
surface with narrow light green bands underneath leaf (Swartley, 1984). Introduced in
1901, Chinese hemlock is growing in availability in North America due to its resistance
to the HWA (Dirr, 1998; Montgomery et al., 2009; Pontius et al., 2006; Tredici and
Kitajima, 2004).
Non-mainland Asian species of Tsuga include T. diversifolia (Maxim.) Mast.
(northern Japanese hemlock) and T. sieboldii Carr. (southern Japanese hemlock). The
northern Japanese hemlock, being the more popular of the two Japanese hemlocks, is
distinguished from other hemlocks by bristle-like foliage arranged tightly along the sides
of the shoots, ovoid, shiny brown cones, and dark orange-brown bark (Swartley, 1984).
7
Introduced in 1861 from Central and Southern Honshu regions of Japan, northern
Japanese hemlock has recently seen increased landscape use in Europe and North
America due to its tight leaf arrangement and broad pyramidal structure lending to its use
as specimen plants and bonsai plantings, as well as its noted resistance to the HWA
(Bitner, 2007; Dirr, 1998; Pontius et al., 2006; Swartley, 1984). Unlike its northern
counterpart, southern Japanese hemlock has not grown as quickly in demand for use in
bonsai or in the landscape. Introduced in 1850 from Japan?s South Honshu and southern
islands, southern Japanese hemlock is a broad, shrubby tree with dark shining green
foliage arranged along the bottom of shoots in irregular lengths with pendulous cones
having vertically ribbed and incurved scales (Dirr, 1998; Swartley, 1984). Just as the
northern Japanese hemlock, the southern Japanese hemlock has shown resistance to
HWA, furthering interests in its use in North American landscapes (Montgomery et al,
2009; Pontius et al., 2006).
II. Temperature Effects
No literature quantifying the performance and heat tolerance of Tsuga has been
published. Indigenous to numerous geographical locations, several species of hemlock
may perform satisfactory and be well suited to other regions of the world. Evaluations of
several species? resistance to HWA are prevalent and preventive measures to control
HWA infestations using predatory insects dominate the literature (Evans and Gregoire,
2007; Pontius et al., 2007). In addition to evaluating species? tolerances of insect
infestations for potential introduction into devastated areas, it is important to evaluate
performance of potential species within these new environments. Some Tsuga species
have been evaluated for their response to various environmental conditions, including
8
light gradients, acid concentrations in soil, carbon dioxide levels, and low temperatures
(Bond et al., 1999; Coleman et al., 1992; Khan et al., 2000; Murray et al., 1989; Ryan et
al., 1986).
In looking at the native range of a species, one may observe that temperature can
play a vital role in determining a species? ability to survive within a given environment
and climate. An example of this can be seen in the distribution of broadleaf evergreens
with broadleaves more commonly found in warm rather than cool climates. The disparate
distribution of broadleaf evergreens and conifers may be partially explained from the
standpoint of leaf area and its exposure to temperatures and conditions causing
desiccation, death of foliage, and plant death. Adaptations and acclimation to survive
these conditions is known as cold tolerance. As with cold tolerance, plants also have
limits as to the heat stress they can survive. This is often referred to as heat tolerance or
thermotolerance. Often used interchangeably with thermostability, thermotolerance and
heat tolerance are characterized by the ability of plant tissues to survive heat stress
whereas, thermostability is used to describe the ability of plant substances such as
proteins, lipids, or nucleic acids to remain viable upon exposure to heat stress (Levitt,
1980).
Influences that may affect thermotolerance include genetics, nutrition, soil
moisture, and maturity of the plant. Influences aside, temperature can affect the natural
distribution of a given species given its degree, variations during seasonal and diurnal
fluctuations, and the duration of exposure to high temperatures. Even though a species
may be tolerant of higher temperatures, the time of day and the duration of heat stress
with which it can withstand said temperatures without considerable damage also serve as
9
factors in heat tolerance. This degree versus duration dilemma is termed the heat dose
law as described by Belehradek (1957), which states that elevated temperatures held at
lower levels over extended periods of time may cause as much damage as higher
temperatures held over short periods of time. The relationship of high temperature and
duration may be further explained by the type of injury each combination may cause. The
two heat stress injury types are referred to as direct injury and indirect injury (Levitt,
1980).
A. Direct Injury
Direct injury may be described as the damage that occurs when tissues are
exposed to extreme temperatures for short periods of time. Direct injury is the damage
characterized under the degree portion of the heat dose law, where plants under supra-
optimal temperatures may suffer heat stress and death when exposed to extreme
temperatures for short periods of time (Levitt, 1980). Injury incurred under these brief,
intense temperatures is a mechanical means of damage as compartmentalization provided
by cell membranes fails and allows cell contents to flow out from the cell (Abass and
Rajashekar, 1991). Direct injury occurs when biological systems are damaged through
membrane damage via protein denaturation and lipid liquidfaction, nucleic acid damage,
photosynthesis inhibition, and ion imbalance (Levitt, 1980; Sage and Reid, 1994;
Schreiber and Berry, 1977; Sibley, 1997).
1. Membrane Damage
Direct injury is often associated and described via membrane damage where high
temperatures cross critical temperature thresholds causing cellular structures and
functions to be damaged, killing the protoplasm (Larcher, 1995; Levitt, 1980; Sibley;
10
1997). Membranes believed to experience stress are plasma, chloroplast, mitochondrial,
and nuclear membranes (Levitt, 1980). It is believed exposure overwhelms proteins and
lipids associated with membranes of cells and organelles within, causing leakage of
contents of these bodies into the protoplasm and space outside the cell. Bernstam and
Arndt (1937) observed the effects of 10 minute heat shocks on the slime mold Physarum
polycephalum where increasing temperatures caused discontinuation of cytoplasmic
streaming, leakage of protein metabolites, increased leakage of substances, and
respiration stoppage. With the leakage of cellular contents, disruption of cellular
processes accompanied. Aforementioned disruption of cellular processes of metabolic
nature led to the argument and investigation as to the actual cause for death, whether
direct injury to organ membranes or inhibition of metabolic pathways. Berry et al. (1975)
and Krause and Santarius (1975) observed that leakage of ions from plasma membranes
typically required as much as 11?C (19.8?F) higher temperatures than those necessary to
inhibit photosynthetic function via thylakoid membrane damage, suggesting inhibition of
metabolic pathways to be the contributing factor of death. Research conducted to further
investigate the nature of the injury suggested protein denaturation, lipid liquidfaction, and
nucleic acid damage to be potential contributors to direct heat injury and inhibition of
metabolic pathways (Levitt, 1980).
2. Protein Denaturation
With proteins and lipids serving as the main constituents of cellular membranes,
some previously and currently accepted theories of cellular death due to membrane
damage suggest that membrane damage occurs due to coagulation of proteins at increased
temperatures (Belehradek, 1935). However, early investigations concluded protein
11
coagulation was the cause of cell damage as many organisms are killed by temperatures
considered too low to denature proteins (Levitt, 1980). Contention and uncertainty
between the two theories led to Aleksandrov's (1964) theory that although not all proteins
making up cellular membranes are subject to coagulation at higher temperatures, one
protein sensitive to stress under high temperatures may provide a weak point for
membrane compromise. Cytoplasmic streaming is reduced at a lower temperature than
other cellular processes, suggesting that proteins associated with cytoplasmic streaming
are more susceptible to heat denaturation than others within the membrane (Aleksandrov,
1964). Fel'dman and Kamensteva (1967) further supported Aleksandrov by correlating
stability of urease with respiration and protoplasmic movement rates at different
temperatures. However, the correlation seemed to show a cause and effect relationship
between temperature and protein denaturation indicating that some other factors must be
considered such as reversible denaturation at higher temperatures (Levitt, 1980) and
water content of cell prior to stress (Lorenz, 1939). With protein denaturation not fully
accounting for cellular death, investigation into phospholipid and nucleic acid changes
have been looked at to explain direct heat injury (Levitt, 1980; Van Uden and Vidal
Leiria, 1976).
3. Lipid Liquidfaction
Theories supporting lipid liquefaction stated that membrane integrity loss
occurred due to the phase change of lipids within the membrane from a liquid crystalline
state to a solid gel form when exposed to high temperatures (Levitt, 1980). However,
recent work shows that this phase change occurs at lower temperatures than normal
growing temperatures and may not be the cause of cell death, but rather a result of death
12
(Lepeshlch, 1935; Levitt, 1980; Reinert and Steim, 1970). Although recent evidence
suggest it is not the cause for cell death, rising temperatures increase the mobility of
lipids encouraging the formation of lipid vesicles from the cell membrane (Levitt, 1980).
The formation of vesicles from the membrane is believed to cause weakening of the
membrane and increase susceptibility to cell death. It is this excessive fluidity paired with
denaturation and aggregation of proteins within the membranes that may serve as factors
for direct heat injury (Levitt, 1980).
4. Nucleic Acid Damage
In the same way proteins are subject to protein denaturation through temperature
increases, nucleic acids are sensitive to temperature increases as well. Since nucleic acids
do not serve as the primary constituents of cell membranes, they may serve as an indirect
factor in heat injury as they are involved in lipid and protein synthesis (Levitt, 1980;
Peacocke and Wallner, 1962). Investigations conducted on various bacteria and yeasts
revealed that although DNA bases were not affected by increased temperatures, various
types of RNA, mainly mRNA and tRNA, were found to be temperature-sensitive
(Malcolm, 1968; Nash et al., 1969) and restrictive to protein and lipid synthesis.
Inhibition of protein synthesis is important for membrane repair and shown to serve as a
factor for thermotolerance (Levitt, 1980).
Although research suggests indirect injury to be a leading cause of species death
and decline, direct injury to foliage and roots cannot be discounted as temperatures have
been shown to reach and exceed 50 ?C (122?F) in plant canopies (Pair and Still, 1982)
and 62.8?C (145?F) in container media (Martin and Ruter, 1996). Exposure to supra-
13
optimal temperatures for brief periods may contribute to cellular membrane and
metabolic pathway damage as the result of direct heat injury.
B. Indirect Injury
As current research suggests, direct injury alone may not fully account for the
death of various species exposed to elevated temperatures (Sibley et al., 1999).
Quantification of temperatures necessary to denature proteins, lipids, and nucleic acids
suggests that in addition to direct injury, indirect injury serves to limit and determine a
species? thermal tolerance. Referring to Belehradek?s heat dose law, indirect injury
characterizes the duration portion of the model. Indirect injury may be described as the
damage that occurs to an organism exposed to sub-lethal temperatures for extended
durations of time (Levitt, 1980). Species decline and death have often been shown to
occur at lower temperatures than those necessary to breakdown many biomolecules,
justifying indirect injury?s potential role in determining heat tolerance (Berry et al., 1975;
Krause and Santarius, 1975; Levitt, 1980; Malcolm, 1968; Nash et al., 1969).
Not characterized by the breakdown of biomolecules such as proteins and lipids,
indirect injury is commonly associated with the disruption of metabolic and cellular
processes. Prolonged disruption of metabolic processes along with the accumulation of
toxins, normally degraded during functioning metabolic pathways, are believed to cause
slow cellular decline and death (Larcher, 1995; Sibley et al., 1999). Measures to quantify
indirect injury include photosynthesis versus respiration rates, triphenyltetrazolium
chloride (TTC) tests, C14 assimilation, toxin accumulation, chlorophyll fluorescence, and
growth trials, while direct injury is measured through electrolyte leakage.
14
III. Electrolyte Leakage
Exposure to supra-optimal growth temperatures is one of the adverse conditions
container-grown ornamentals face in the intensive production cycle of nursery
production. Many ornamental plants are grown in colored containers set on pads often
lined with black weed barriers that reflect heat collected throughout the day. This can be
problematic for plants as the heat reflected off these surfaces collects in plant canopies
and container media, exposing them to supra-optimal temperatures that can cause direct
heat injury and reduced growth (Martin and Ruter, 1996; Pair and Still, 1982).
Temperatures have been shown to reach and exceed 50?C (122?F) in plant canopies (Pair
and Still, 1982) and 62.8?C (145?F) in container media (Martin and Ruter, 1996).
Exposure to supra-optimal temperatures for brief periods may contribute to direct damage
to cellular membranes and metabolic pathways as the result of direct heat injury.
Electrolyte leakage has been used as an accepted measure of cellular membrane
damage in foliage, fruit, and root tissues (Dexter et al., 1932; Ingram and Buchanan,
1981; Stergios and Howell, 1973; Sullivan, 1972; Yadava et al., 1978). Electrolyte
leakage measures thermal tolerance to direct injury (Levitt, 1980).
Direct injury occurs when tissues are exposed to extreme temperatures for short
periods of time, causing compartmentalization provided by cell membranes to fail and
allow cell contents to flow out from the cell (Abass and Rajashekar, 1991).
Electrolyte leakage determines heat tolerance via electrical conductivity
measurements. As cells contain organelles, photoassimilates, nuclear bodies, and other
components necessary for cellular growth, maintenance, and reproduction, these
components can be exchanged between cells through protein channels within the
15
phospholipid bi-layer of cell membranes. Since these components are made of organic
and inorganic materials, they contain ions that contribute to electrical conductivity of the
solution in which they are suspended and can be measured using an electrical
conductivity meter. When plant tissue is placed into water containing no ions (de-ionized
water), some of these cellular components may flow from the cell into the water as
osmotic and solute potentials are equilibrated. When damage occurs to cellular
membranes through stress or physical rupture, cellular contents flow freely into the
surrounding solution. This ?leakage? of cellular components into the surrounding
solution can dramatically increase the electrical conductivity, depending on the severity
of the damage. Electrolyte leakage serves as a measure of membrane damage in relation
to stress (McKay, 1992; Sibley, 1997).
A model commonly used for electrolyte leakage procedures and data analyses for
ornamental plants was developed by Ingram (1985) and Ingram and Buchanan (1984) and
serves to provide the basis for direct heat stress determination of many ornamental plants.
As outlined by Ingram and Buchanan (1984), electrolyte leakage is conducted by
excising foliage, fruit, or roots from the plant and placing a known mass of each sample
into test tubes containing a known volume of de-ionized water. Samples are treated
within a thermostatically controlled water bath, cooler, or growth chamber at prescribed
temperatures. Upon completing prescribed temperature treatment, test tubes are placed on
ice and incubated in a cooler maintained at 4.4 ?C (40?F) for 24 hours. Following
incubation, samples are brought to room temperature and electrical conductivity
measured using a conductivity meter. Electrical conductivities are measured again
16
following autoclaving samples at 121?C and 1.4 kg?cm-2 (249.8?F and 20 lbs?in-2) for 20
minutes and incubated 24 hours at 4.4 ?C (40?F).
Once initial and final conductivity measurements are collected, electrolyte
leakage may be expressed as a ratio of the initial electrical conductivity post-treatment to
the conductivity post-autoclave. Electrolyte leakage response to temperature is expected
to fit the sigmoidal equation:
Le = [x - z) / (1 + exp [ - k (T - Tm] ) ] + z
where Le is the percent leakage, z = baseline level of electrolyte leakage, x = percent of
electrolyte leakage observed across all temperatures, Tm = predicted temperature
midpoint, also referred to as the ?inflection point? for the response curve, k = the slope of
the predicted response at Tm, and T = water bath temperature. The equation is used to
determine critical midpoint temperatures (Tm) by fitting electrolyte leakage data across
temperature treatments using Gauss-Newton method of non-linear regression approach.
The critical temperature midpoint (Tm) serves as the estimate where 50 percent of the
cells of a plant or one of its portions are damaged beyond repair and recovery/ survival is
unlikely.
Initial research conducted using electrolyte leakage determination were aimed at
determining cold hardiness for many plant species, especially for conifers (Burr et al.,
1993; Dexter et al., 1932; Murray et al., 1989; Stergios and Howell, 1973; Yadava et al.,
1978). Modified electrolyte procedures for agronomic crops such as sorghum and
soybeans followed (Martineau et al. 1979; Sullivan, 1972). With models for determining
heat tolerance via modified electrolyte leakage procedures for Pittosporum (pittosporum)
conducted by Ingram (1985) and Ingram and Buchanan (1984), evaluation of heat
17
tolerance of other ornamentals increased leading to electrolyte leakage?s acceptance for
direct heat injury determinations. Ornamentals evaluated for direct heat tolerance include:
Ilex (holly), Illicium (illicium), and Juniperus (juniper) (Ingram and Buchanan, 1981),
Pittosporum (pittosporum) (Ingram, 1985), Ilex (holly) (Ingram 1986; Ruter, 1993),
Nyssa (tupelo), Cephalanthus (button bush), and Taxodium (bald cypress) (Donovan, et
al., 1990), Magnolia (magnolia) (Martin et al., 1991), Acer (maple) (Sibley et al., 1999),
and Cornus (dogwood) (Hardin et al., 1999).
Based on the lack of information regarding thermotolerance of Tsuga species with
regard to direct heat injury, the objective of this project was to evaluate eight of the
recognized hemlock species (T. canadensis, T. caroliniana, T. chinensis, T. diversifolia,
T. dumosa / yunnanensis, T. heterophylla T. mertensiana, and T. sieboldii).
Thermotolerance of the leaf tissue cells was evaluated by assessing electrolyte leakage
following imposed direct-heat stress.
18
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T ab l e 1. A c c e p te d s p e c i e s f or ge n u s T s u ga
Z
S c i e n ti f i c N am e A u th or
Y
R e f e r e n c e
X
D ate
W
S ou r c e
V
T s u ga c an ad e n s i s (L .) Ca rri ? re T ra i t ? G ? n. Coni f. 189 1855 A CB
T s ug a a m e ri c a na (M i l l .) F a rw . Bul l . T orre y Bot . Cl ub 41: 629 1915 AC
T s ug a c a na de ns i s fo. pyra m i da l i s J e nki ns H e m l oc k A rbore t um Bul l . 6. 1934 B
U
T s ug a c a na de ns i s fo. a rg e nt e a N e l s on P i na c e a e : 32. 1866 B
T s ug a c a na de ns i s fo. a ure a (N e l s on) J ? g e r u Be i s s n. Z i e rg e h?l z e : 495. 1884 B
T s ug a c a na de ns i s fo. c a na de ns i s C
T
T s ug a c a na de ns i s fo. de ns i fol i a (J e nki ns ) S w a rt l e y H e m l . A rb. Bul l . 6. 1934 B
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T s ug a c a na de ns i s va r. m a c rophyl l a H ort A
S
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T s ug a dum os a s ubs p. l e pt ophyl l a (H a nd.-M a z z .) A .E . M urra y K a l m i a 12: 26 1982 AC
T s ug a dum os a va r. dum os a C
T s ug a dum os a va r. yunna ne ns i s (F ra nc h.) S i l ba P hyt ol og i a 68: 73 1990 AC
T s ug a dura D ow ni e N ot e s Roy. Bot . G a rd. E di nburg h 14(67): 16, pl . 194, f. 2 1923 C
T s ug a i nt e rm e di a H a nd.-M a z z . K a i s e rl . A ka d. W i s s . W i e n, M a t h.-N a t urw i s s . K l ., A nz . 61: 82 1924 C
T s ug a l e pt ophyl l a H a nd.-M a z z . K a i s e rl . A ka d. W i s s . W i e n, M a t h.-N a t urw i s s . K l ., A nz . 61: 83 1924 A BC
T s ug a w a rdi i D ow ni e N ot e s Roy. Bot . G a rd. E di nburg h 14(67): 17, pl . 17, f. 4 1923 C
T s ug a yunna ne ns i s (F ra nc h.) E . P ri t z . Bot . J a hrb. S ys t . 29(2): 217, i n obs . 1901 C
T s ug a yunna ne ns i s s ubs p. dura (D ow ni e ) A .E . M urra y K a l m i a 12: 26 1982 C
T s u ga h e te r op h yl l a (Ra f.) S a rg . S i l va 12: 73 1898 A BC
T s ug a a l be rt i a na (A . M urra y bi s ) S e ne c l . Coni f. 18 1868 B
T s u ga l on gi b r ac te ata W .C. Che ng Cont r. Bi ol . L a b. Chi n. A s s oc . A dva nc e m . S c i . 7(1): 1, f. 1 1932 A BC
T s u ga m e r te n s i an a (Bong .) Ca rri ? re T ra i t ? G ? n. Coni f. (e d. 2) 250 1867 A BC
T s ug a ba l fouri a na (Re hde r & E .H . W i l s on) W .R. M c N a b J . L i nn. S oc ., Bot . 19: 211 1882 C
T s ug a c ra s s i fol i a F l ous Bul l . S oc . H i s t . N a t . T oul ous e 69: 412 1936 AC
T s ug a hooke ri a na (A . M urra y bi s ) Ca rri ? re T ra i t ? G ? n. Coni f. (e d. 2) 252 1867 AB
T s ug a m e rt e ns i a na s ubs p. c ra s s i fol i a (Bong .) Ca rri ? re s ubs p. c ra s s i fol i a (F l ous ) S i l ba J . Int . Coni fe r P re s e rv. S oc . 15(2): 65. 2008 A
T s ug a m e rt e ns i a na s ubs p. g re ndi c one F a rj on P roc . K on. N e d. A ka d. W e t e ns c h., C 91(1): 39 1988 AC
T s ug a m e rt e ns i a na s ubs p. m e rt e ns i a na C
T s ug a m e rt e ns i a na va r. j e ffre yi (H e nry) C.K . S c hne i d. N E W 294 1913 AC
T s ug a m e rt e ns i a na va r. m a c rophyl l a (Bong .) Ca rri ? re va r. m a c rophyl l a Be i s s n. i n Be i s s n. H a ndb. N a de l hol z k. 404. 1891 A
T s ug a m e rt e ns i a na va r. m e rt e ns i a na C
T s ug a pa t t oni a na (J e ffre y e B A . M urra y bi s ) E ng e l m . G a rd. Chron., n.s ., 756 1879 BC
T s ug a w i l l i a m s oni i (N e w b.) de V os Be re d. W oorde nb. H e e s t . Coni f. 181 1867 BC
T s u ga ob l on gi s q u am ata (C.Y . Che ng & L .K . F u) L .K . F u & N a n L i N ovon 7(3): 263 1997 AC
T s u ga s i e b ol d i i Ca rri ? re T ra i t ? G ? n. Coni f. 186 1855 A BC
T s ug a a ra ra g i (S i e bol d) K oe hne D e ut . D e ndrol . 10 1893 AB
T s ug a ha nburya na B
T s ug a s i e bol di i va r. na na (E ndl .) Ca rri ? re C
T s ug a s i e bol di i va r. s i e bol di i C
T s ug a t s uj a A . M urra y bi s P roc . Roy. H ort . S oc . L ondon 1862(2): 508, f. 151-153 1862 AB
T s u ga x je f f r e yi (H e nry) A . H e nry P roc . Roy. Iri s h A c a d. 34: 55 1919 BC
Z
Da ta p r e s e n ted f r o m ta x o n o m ic s u r v e y o f p u b li s h e d n a m e s a n d d e s c r ip ti o n s f o r c u r r e n tl y a c c e p ted s p e c ies o f th e g e n u s T s u g a in 2 0 1 0 .
Y
A u th o r r e s p o n s ib le f o r r e c o r d e d s p e c ie e x is ten c e , d e s c r ip ti o n , a n d / o r a g r e e m e n t w it h p r e v io u s ly r e c o r d e d li ter a tu r e .
X
A c c e p ted li ter a tu r e c it in g c u r r e n t s p e c ie e x is ten c e a n d d e s c r ip ti o n b y r e f e r r e d a u th o r ( s ) .
W
P u b li s h in g d a te o f r e f e r e n c e d li ter a tu r e .
V
S o u r c e o f c u r r e n t ta x o n o m ic in f o r m a ti o n .
U
S w a r tl e y , J . C . , 1 9 8 4 . T h e C u lt iv a ted H e m lo c k s . T im b e r P r e s s , P o r tl a n d , O R . 1 8 6 .
T
T r o p ic o s . 1 0 M a y 2 0 1 0 . T r o p ic o s B o ta n ic a l I n f o r m a ti o n S y s tem a t th e M is s o u r i B o ta n ic a l G a r d e n , S t. L o u is . 1 0 M a y 2 0 1 0 . < h tt p :/ /w w w . tr o p ic o s . o r g /> .
S
I P NI . 6 M a y 2 0 1 0 . T h e I n ter n a ti o n a l P la n t Na m e s I n d e x . 1 0 M a y 2 0 1 0 . < h tt p :/ /w w w . ip n i. o r g /> .
25
Chapter II
Foliar Thermotolerance of Tsuga spp.
Abstract
Foliar thermotolerance of six Tsuga species to direct injury was evaluated using
electrolyte leakage. Species evaluated included: T. canadensis, T. caroliniana, T.
chinensis, T. diversifolia, T. heterophylla, and T. dumosa (formerly T. yunnanensis).
Bare-root liners of the six species were potted into containers and grown for nine months
prior to evaluation. Species were exposed to 25, 30, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5,
55, and 60?C (77, 86, 95, 99.5, 104, 108.5, 113, 117.5, 122, 126.5, 131, and 140?F) for 30
minutes in thermostatically controlled water baths, and electrical conductivity determined
subsequent each 24 hour incubation time following treatment and autoclaving.
Electrolyte leakage, expressed as a ratio of the electrical conductivity taken 24 hours
following treatment to the conductivity taken 24 hours following autoclaving, was
determined. Electrolyte leakage response to temperature was sigmoidal. T. canadensis
and T. dumosa had similarly, higher predicted temperature midpoints (Tm) and k-values
than T. caroliniana suggesting higher resistance to direct injury when exposed to brief,
supra-optimal temperatures. While differences were found for T. canadensis and T.
dumosa when compared to T. caroliniana, no differences were found in Tm and k-values
for T. canadensis or T. dumosa when each compared to T. chinensis, T. diversifolia, and
T. heterophylla. Similarly, no differences were found for Tm and k-values of T.
caroliniana when compared to T. chinensis, T. diversifolia, and T. heterophylla.
26
Introduction
Evaluation of plant response to environmental and biotic stress benefits industry
professionals by providing practical information useful in selecting species for production
and landscape use under various conditions. Research addressing heat tolerance of
species and cultivars provides valuable information for plant production in areas pre-
disposed to high temperatures. The genus Tsuga (hemlock) contains several species
indigenous to numerous geographical locations. No literature quantifying Tsuga?s
performance and heat tolerance has been published to date. With hemlock woolly adelgid
(Adelges tsugae Annan.), causing decline of North American hemlock stands (Evans and
Gregoire, 2007;), other species found to be more resistant to hemlock woolly adelgid
(HWA) infestations may perform satisfactory and be well suited to other regions of the
world (Montgomery et al., 2009; Tredici and Kitajima, 2004). Exposure to supra-optimal
temperatures is one of the adverse conditions container-grown ornamentals face in the
intensive production cycle of nursery production. Many ornamental plants are grown in
black containers set on pads lined with black weed barriers that transfer heat collected
throughout the day. This can be problematic for plants as the heat reflected off these
surfaces collects in plant canopies and container media exposing them to supra-optimal
temperatures that can cause direct heat injury and reduced growth (Martin and Ruter,
1996; Pair and Still, 1982). Temperatures have been shown to exceed 50?C (122?F) in
plant canopies (Pair and Still, 1982) and 62.8?C (145?F) in container media (Martin and
Ruter, 1996). Exposure to supra-optimal temperatures for brief periods may contribute to
direct damage to cellular membranes and metabolic pathways as the result of direct heat
injury.
27
Electrolyte leakage provides an indication of heat tolerance for cell membrane
stability in response to extreme temperatures due to direct injury (Levitt, 1980).
Electrolyte leakage can be used to predict heat tolerance via electrical conductivity
measurements. Cells contain organelles, photoassimilates, nuclear bodies, and other
components necessary for cellular growth, maintenance, and reproduction, with these
components often exchanged between cells through protein channels within the
phospholipid bi-layer of cell membranes. Since these components are made of organic
and inorganic materials, they contain ions that contribute to electrical conductivity of the
solution in which they are suspended, which can be measured using an electrical
conductivity meter. When plant tissue is placed into water containing no ions (de-ionized
water), these cellular components may flow from the cell into the water as osmotic and
solute potentials are equilibrated. When damage occurs to cellular membranes through
stress or physical rupture, cellular contents flow freely into the surrounding solution. This
?leakage? of cellular components into the surrounding solution can dramatically increase
the electrical conductivity, depending on the severity of the damage. Electrolyte leakage
serves as a measure of membrane damage in relation to stress (McKay, 1992; Sibley,
1997).
Electrolyte leakage has been used as an accepted measure of cellular membrane
damage in foliage, fruit, and root tissues (Dexter et al., 1932; Ingram and Buchanan,
1981; Stergios and Howell, 1973; Sullivan, 1972; Yadava et al., 1978). Electrolyte
leakage measures thermal tolerance with respect to direct injury (Levitt, 1980). Direct
injury occurs when tissues are exposed to extreme temperatures for short periods of time,
causing compartmentalization provided by cell membranes to fail and allow cell contents
28
to flow out from the cell (Abass and Rajashekar, 1991). Initial research conducted using
electrolyte leakage determination were aimed at determining cold hardiness for many
plant species, especially for conifers (Burr et al., 1993; Dexter et al., 1932; Murray et al.,
1989; Stergios and Howell, 1973; Yadava et al., 1978).
Modified electrolyte procedures for agronomic crops such as sorghum and
soybeans followed (Martineau et al., 1979; Sullivan, 1972). With models for determining
heat tolerance via modified electrolyte leakage procedures for Pittosporum (pittosporum)
conducted by Ingram (1985) and Ingram and Buchanan (1984), evaluation of heat
tolerance of other ornamentals increased leading to electrolyte leakage?s acceptance for
direct heat injury determinations. Ornamentals evaluated for direct heat tolerance with
predicted Tm values for foliage include: Ilex cornuta ?Burfordii? (46.5 ? 0.5?C), Illicium
anisatum (50.5 ? 0.5?C), and Juniperus chinensis ?Parsonii? (48.5 ? 0.5?C) (Ingram and
Buchanan, 1981), Pittosporum tobira (52.2 ? 0.2?C) (Ingram, 1985), Nyssa aquatica
(50.9 ? 0.2?C), Cephalanthus occidentalis (51.0 ? 0.2?C), and Taxodium distichum (46.6
? 0.6?C) (Donovan et al., 1990), Magnolia (52.5 ? 0.9-54.0 ? 0.4?C) (Martin et al.,
1991), Acer rubrum (52.0 ? 0.8-53.3 ? 0.5?C) (Sibley et al., 1999), and Cornus florida
(51.2 ? 0.5-52.4 ? 0.6?C) (Hardin et al., 1999).
However, electrolyte leakage may not serve as a complete indicator of a plant?s
performance under adverse conditions as photosynthetic and metabolic pathways are
often more sensitive to high temperatures than membranes (Berry and Bjorkman, 1980;
Larcher, 1995; Levitt, 1980; Sibley et al., 1999). Other means for evaluation of indirect
injury include chlorophyll fluorescence, photosynthesis versus respiration rates,
triphenyltetrazolium chloride (TTC) tests, and carbon (C14) partitioning/ assimilation
29
(Berry and Bjorkman, 1980; Ruter, 1993; Sibley, 1997). The purpose of the following
study was to examine the tolerance of six Tsuga species to direct foliar heat injury as
determined by electrolyte leakage.
Materials and Methods
In January 2008, liners of six Tsuga species (T. canadensis (L.) Carr., T.
caroliniana Engelm., T. chinensis (Franch.) Pritzel, T. diversifolia (Maxim) Mast., T.
heterophylla (Raf.) Sarg., and T. dumosa (D. Don) Eichler (formerly T. yunnanensis)
were potted into 6.0 L (#2 trade gal.) containers in a substrate of 6 pine bark:1 builders?
sand (by volume) and amended with 6.6, 3.0, and 0.9 kg?m-3 (11.1, 5, and 1.5 lbs?yd-3) of
18N-2.6P-9.9K (18-6-12) 8-9 month Osmocote? (Scotts Co., Marysville, OH), dolomitic
limestone, and Micromax? (Scotts Co.), respectively. Plants were grown on a container
pad in Auburn, AL (32? 36?N?85? 29?W, USDA cold hardiness zone 8a) and irrigated
with approximately 1.3 cm (0.5 in) water daily for nine months prior to foliar membrane
thermostability determination in October 2008.
Electrolyte leakage procedures were similar to those described by Sullivan (1972)
and modified by Ahrens (1988), Ingram and Buchanan (1981), and Ruter (1993).
Recently matured needles from current-season growth were excised from petioles with a
scalpel, weighed to 0.75 g (0.03 oz) samples, placed in test tubes containing 3 ml (0.1 fl
oz) of de-ionized water and treated within a thermostatically controlled water bath at 12
temperatures [25, 30, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, and 60 ?C (77, 86, 95,
99.5, 104, 108.5, 113, 117.5, 122, 126.5, 131, and 140 ?F)] for 30 minutes. Each
temperature treatment contained replications of 5 test tubes for each species (temp N=30,
total N=360). Upon completing a prescribed temperature treatment, test tubes were filled
30
with 20 ml of de-ionized water, placed on ice, and incubated in a cooler maintained at 4.4
?C (40?F) for 24 hours. Following incubation, samples were brought to room temperature
and electrical conductivity measured (Accumet Excel XL50, Fisher Scientific, Pittsburgh,
PA). Samples were autoclaved at 121?C and 1.4 kg?cm-2 (249.8?F and 20 lbs?in-2) for 20
minutes and incubated for 24 hours at 4.4 ?C (40?F), after which electrical conductivities
were again determined. Electrolyte leakage was expressed as a ratio of the initial
electrical conductivity post-treatment to the conductivity post-autoclave. Electrolyte
leakage response to temperature has been reported to be sigmoidal with different species
(Ahrens and Ingram, 1988; Ingram and Buchanan, 1981; Martineau et al., 1979; Ruter,
1993; Sibley et al., 1999). The sigmoidal equation:
Le = [x - z) / (1 + exp [ - k (T - Tm] ) ] + z
where Le was the percent leakage, z = baseline level of electrolyte leakage, x = maximum
percent of electrolyte leakage observed across all temperatures, Tm = predicted
temperature midpoint, also referred to as the ?inflection point? for the response curve, k =
the slope of the predicted response at Tm, and T = water bath temperature used to
determine critical midpoint temperatures (Tm) by fitting electrolyte leakage data across
temperature treatments (Sibley et al., 1999). Gauss-Newton method of non-linear
regression was used to analyze electrolyte leakage data with correlations performed by
PROC CORR procedure using SAS Version 9.1.3 (SAS Institute, Cary, NC). Critical
midpoint temperatures and k-values of fitted response curves were determined for each
species (Table 1). Differences were determined by failures of species? Tm values, plus
and minus Tm variance, to overlap. K-values were used to distinguish slope differences
31
between Tm values and describe the range of temperatures around the Tm for which the
species is predicted to withstand before experiencing greater than 50% damage.
Results and Discussion
Gauss-Newton method of non-linear regression met convergence criteria for all
species and temperatures except for T. dumosa, where the 60?C (140?F) treatment failed
to converge due to variability. Analysis of T. dumosa was conducted using temperatures
25-55?C (77-131?F) in order to meet convergence criteria. Hereafter all further references
to T. dumosa are for 25-55?C (77-131?F). Electrolyte leakage and temperature were
correlated across all species (r= 0.77, p= <0.0001, N=355). T. canadensis and T. dumosa
had similarly, higher predicted temperature midpoints (Tm) and k-values than T.
caroliniana suggesting higher resistance to direct injury when exposed to brief, supra-
optimal temperatures. Predicted Tm for needles of T. dumosa 53.2 ? 0.2?C (127.7 ?
0.4?F) and T. canadensis 52.3 ? 0.2?C (126.2 ? 0.4?F) were ? 3.3 ?C (5.7?F) and ? 2.4?C
(4.2?F) greater than for needles of T. caroliniana 49.9 ? 0.7?C (122.0 ? 1.2?F) (Table 1)
(Figures 1-6). While differences were found for T. canadensis and T. dumosa when
compared to T. caroliniana, no differences were found in Tm and k-values for T.
canadensis or T. dumosa when each compared to T. chinensis, T. diversifolia, and T.
heterophylla. Similarly, no differences were found for Tm and k-values of T. caroliniana
when compared to T. chinensis, T. diversifolia, and T. heterophylla.
K-values indicated narrower response curves for needles of T. dumosa and T.
canadensis (0.97 ? 0.14 and 0.90 ? 0.16) compared to those of T. caroliniana (0.41 ?
0.10) (Table 1), indicating higher temperatures would be necessary to cause direct heat
injury to foliage of T. dumosa and T. canadensis. Narrow k-values and low variability
32
also indicated a narrower tolerance range for supra-optimal canopy temperatures around
the critical temperature midpoint, suggesting the Tm value predicted is an accurate
estimate of the beyond recovery temperature (Donovan et al., 1990; Martin et al., 1991;
Ruter, 1996). This narrow tolerance range is the result of the steep slope of the predicted
response curve and less variability found within the data for T. canadensis and T.
dumosa, indicating that the higher temperatures of the critical temperature midpoint range
for T. canadensis and T. dumosa are more accurate representations of the temperatures
necessary to induce direct injury to the foliage than those predicted for T. caroliniana.
In addition to data collected and analyzed in 2008, data were analyzed from a
similar study performed in 2003 using four of the same species, T. canadensis, T.
caroliniana, T. diversifolia, and T. heterophylla with two additional species (T.
mertensiana and T.sieboldii) (Appendix A). Predicted critical temperature midpoints (Tm)
in 2008 and 2003 were similar for T. canadensis and T. diversifolia, respectively (Tables
1 and A1). Predicted Tm values were lower for T. caroliniana in 2008 than 2003, where
T. caroliniana was shown to have one of the highest Tm values of the six species
evaluated (Table 1A). Data analyzed from 2003 also indicated that the k-value for T.
caroliniana was found to be highly variable in relation to T. caroliniana in 2008.
Comparison of the two data sets indicated that most Tm values in 2003 had more
variability than those in 2008. Variability between the two studies may have been due to
differences in tissue age, acclimation, collection procedures, and sample preparation.
While differences were observed for foliar thermotolerance as direct injury, the
occurrence of temperatures necessary to induce direct injury at the critical temperature
midpoint range is unlikely. While temperatures have been shown to reach and exceed
33
50?C (122?F) in plant canopies (Pair and Still, 1982), it is unlikely that said temperatures
are maintained long enough to significantly damage plants. Various factors such as
automated irrigation regimes and plant-to-plant shading may serve to alleviate high
temperatures within plant canopies, reducing the opportunity for foliage to be damaged
directly. The temperatures predicted from direct injury, while accurate indicators of
membrane thermotolerance, have little practical implication for species performance at
elevated temperatures for extended periods of time. Because photosynthetic and
metabolic pathways are often more sensitive to high temperatures than membranes, plants
may suffer damage and decline at lower temperatures occurring over longer periods of
time (Berry and Bjorkman, 1980; Larcher, 1995; Levitt, 1980; Sibley et al., 1999). This
damage and decline occurs as the result of indirect injury and may be a better indicator of
species performance and longevity in production and the landscape.
Further investigations into indirect injury and electrolyte leakage from hemlock
foliage and roots are necessary to verify aforementioned results as exposure duration,
tissue age, season, and stage of acclimation may contribute to varied responses for
thermotolerance (Donovan et al, 1990; Levitt, 1980; Martineau et al, 1979). With further
investigations into indirect heat injury, certain species of hemlock may prove more
suitable for production and use in heat-disposed areas than others.
34
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37
S pe c i e s Z on e
y
T
m
x
T
m
w
k - v a l u e s
v
T . c ana de ns i s 5 52.3 ? 0.2 126.2 ? 0.4 0.90 ? 0.1 6
T . c ar ol i ni ana 5 49.9 ? 0 .7 122.0 ? 1.2 0.41 ? 0.1 0
T . c hi ne ns i s 8 51.4 ? 0.9 124.6 ? 1.6 0.34 ? 0.0 8
T . di v e r s i f ol i a 8 52.1 ? 0.5 125 .7 ? 0.9 0.84 ? 0.3 1
T . du m os a
u
8 53.2 ? 0.2 127 .7 ? 0.4 0.97 ? 0.1 4
T . he t e r oph y l l a 5 51.2 ? 0.4 124 .1 ? 0.8 0.66 ? 0.1 5
v
K - v a l u e s f or T m
x
( C e l s i u s ) .
u
T s uga dum os a h a d 60 ? C ( 131 ? F ) t r e a t m e n t r e m ov e d du r i n g a n a l y s i s t o m e e t c on v e r g e n c e
c r i t e r i a f or n on - l i n e a r r e g r e s s i on a n a l y s i s .
w
M e a n s a n d s t a n da r d e r r or s f or pr e di c t e d c r i t i c a l t e m pe r a t u r e ( F a h r e n h e i t ) pa r a m e t e r s
de t e r m i n e d by l e a s t s qu a r e s a ppr oa c h G a u s s - N e w t on m e t h od o f n on - l i n e a r r e g r e s s i on .
T ab l e 1. R e s p on s e of T s u ga ( h e m l oc k ) s p e c i e s t o e l e vat e d f ol i ar t e m p e r at u r e
Z
.
x
M e a n s a n d s t a n da r d e r r or s f or pr e di c t e d c r i t i c a l t e m pe r a t u r e ( C e l s i u s ) pa r a m e t e r s de t e r m i n e d
by l e a s t s qu a r e s a ppr oa c h G a u s s - N e w t on m e t h od o f n on - l i n e a r r e g r e s s i on .
z
D a t a pr e s e n t e d f r om e l e c t r ol y t e l e a ka g e s t u dy i n 200 8.
y
U S D A H a r di n e s s Z on e s f or l oc a t i on of n u r s e r y r e s pon s i bl e f or t h e s u ppl y of s t oc ks or s e e dl i n g s .
38
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e 4 . F o l i a r E l e c t r o l y t e L e a k a g e o f T s u g a c a r o l i n i a n a e x p o se d t o t e m p e r a t u r e s f r o m 2 5 ? t o 6 0 ? C i n 2 0 0 8 .
O b s e r v e d
P r e d ic t e d
T
m
= 4 9 . 9 ? 0 . 7
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
e
ct
r
o
l
y
t
e
L
e
a
k
a
g
e
(
%
)
i r 6 . F o l i a r E l e c t r o l y t e L e a k a g e o f ga di v e r s i f o l i a e x p o se d t o t e m p e r a t u r e s f r o m 2 5 ? t o 6 0 ? C i n 2 0 0 8 .
T
m
= 5 2 . 0 ? 0 . 5
0
20
40
60
80
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
7 h e t e r o p h y l l a e x p o se d t o t e m p e r a t u r e s f r o m 2 5 ? t o 6 0 ? C i n 2 0 0 3 .
3 7 4
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
8 8
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
F i g u r e 9 . F o l i a r E l e c t r o l y t e L e a k a g e o f T s u g a m e r t e n s i a n a e x p o se d t o t e m p e r a t u r e s f r o m 2 5 ? t o 6 0 ? C i n 2 0 0 3 .
E
l
e
ct
r
o
l
y
t
e
L
e
a
k
a
g
e
(
%
)
i 1 0 . F o l i a r E l e c t r o l y t e L e a k a g e o f T s u g a c h i n e n s i s e x p o se d t o t e m p e r a t u r e s f r o m 2 5 ? t o 6 0 ? C i n 2 0 0 8 .
T
m
= 5 1 . 4 ? 0 . 9
E
l
e
ct
r
o
l
y
t
e
L
e
a
k
a
g
e
(
%
)
F i g u r e 1 1 . F o l i a r E l e c t r o l y t e L e a k a g e o f T s u ga s i e bo l di i e x p o se d t o t e m p e r a t u r e s f r o m 2 5 ? t o 6 0 ? C i n 2 0 0 3 .
T
m
= 5 6 . 3 ? 2 . 1
0
20
40
60
80
100
25 30 35 3 7 . 5 40 . 45 4 7 . 5 5 2 . 5 55 60
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e 1 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u ga c an ad e n s i s e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 8 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 2 . 3 ? 0 . 2
39
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e 1 2 . F o l i a r E l e c t r o l y t e L e a k a g e o f T s u g a y u n n a n e n s i s e x p o se d t o t e m p e r a t u r e s f r o m 2 5 ? t o 5 5 ? C i n 2 0 0 8 .
O b s e r v e d
P r e d ic t e d
T
m
= 5 3 . 1 ? 0 . 2
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e 2 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u ga c ar o l i n i an a e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 8 .
Ob s er v ed
P r ed i c t ed
T
m
= 4 9 . 9 ? 0 . 7
40
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e 3 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u g a c h i n e n s i s e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 8 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 1 . 4 ? 0 . 9
41
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e 4 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u g a d i ve r s i f o l i a e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 8 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 2 . 1 ? 0 . 5
42
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e 5 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u ga du m o s a ( T s u ga y u n n a n e n si s) e x p o se d t o 2 5 t o 5 5 ? C f o r 3 0 m i n . i n 2 0 0 8 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 3 . 2 ? 0 . 2
43
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
e
ct
r
o
l
y
t
e
L
e
a
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e 6 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u g a h e t e r o p h y l l a e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 8 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 1 . 2 ? 0 . 4
44
PART II INVESTIGATION OF A FOLIAR DISORDER IN
CRAPEMYRTLE (LAGERSTROEMIA INDICA ?FAURIEI)
Chapter I
Introduction and Literature Review
I. The Genus Lagerstroemia
The genus Lagerstroemia is comprised of approximately 50 species consisting
mainly of woody shrubs and small trees (Graham et al., 1987). Lagerstroemia is native to
Southeast Asia with origins in India, China, Korea, Japan, the Philippines, and Australia
(Egolf and Andrick, 1978; Furtado and Montien, 1969). As one of 31 genera currently
assigned to the Lythraceae family (Graham et al., 1993), Lagerstroemia is characterized
within the family by alternate to sub-opposite leaves sometimes whorled with three
leaves at each node (Dirr, 1998) and inflorescences of simple racemes, compound cymes,
and lateral/terminal panicles (Graham et al., 1987). Flowers are conspicuous ranging
from white to rose-purple in color. Flowers are perfect, with corolla segments 5-8-merous
and stamens ranging in count from 15 (L. subcostata Koehne) to 200. Stamens are often
dimorphic as characterized by L. indica L. (Graham et al., 1987).
Lagerstroemia speciosa (L.) Pers. and L. piriformis Koehne have been cultivated
in native habitats of Southeast Asia for centuries and prized for furniture making as
timber and wood quality from the two species share similar characteristics to teak
(Cabrera, 2004; Egolf and Andrick, 1978; Everett, 1969; Everett, 1981). L. speciosa is
45
also valued for medicinal use to treat diabetes, as chemical compounds within the leaves
used in a tea have had similar effects insulin (Cabrera, 2004; Kakuda et al., 1996).
Taxonomist Carl Linnaeus described and named Lagerstroemia after his friend Magnus
von Lagerstrom of the Swedish East Indies Company in 1759 (Byers, 1997). Byers
(1997), Dirr (1998) and Egolf and Andrick (1978) cite introduction of Lagerstroemia in
Europe and North America in the 1750s. Early use of Lagerstroemia or crapemyrtle,
?crape myrtle?, ?crepemyrtle, or ?crepe myrtle?, as commonly known and spelled,
consisted of a limited number species within the North American landscape. L. indica,
and L. reginae Roxb. are reported to be among the first seedlings planted in the gardens
of George Washington?s Mt. Vernon estate in 1799 (Byers, 1997; Egolf and Andrick,
1978). Crapemyrtle?s recognition within the American landscape developed slowly as
early introductions lacked cold hardiness, resistance to powdery mildew (Erysiphe
lagerstroemiae E. West), and variety of flower color (Egolf, 1981). Introductions of new
cultivars for the American landscape by Donald R. Egolf beginning in 1967, opened up a
greater use of the species with selections from the cross of Lagerstroemia indica and
Lagerstroemia fauriei Koehne (L. indica ?fauriei).
Early use of L. indica in the North American landscape can likely be attributed to
its long bloom period, variety of flower colors (red, pink, white, lavender, and purple),
and diversity of plant sizes and habits (Byers, 1997; Cabrera, 2004). However, increased
susceptibility to powdery mildew limited use in humid, southern landscapes where
powdery mildew could thrive and make plants unattractive by distorting growth (Knox,
1995). Until the 1960s, cultivars of L. indica were the predominant crapemyrtles in North
America. In 1956, John Creech of the USDA?s New Crops Research Branch, brought
46
seeds of L. fauriei back from Japan to evaluate. Exfoliating, cinnamon-colored bark,
greater cold hardiness, and reported resistance to powdery mildew of L. fauriei, led to
inter-specific breeding of L. indica and L. fauriei by Donald Egolf of the U.S. National
Arboretum (Byers, 1997; Cabrera, 2004; Knox, 1995). Egolf and Andrick (1978)
reported offspring of the inter-specific cross to exhibit powdery mildew resistance
previously known in L. fauriei parentage (Egolf, 1981). Further breeding from this single
ascension of L. fauriei served as the parent to subsequent cultivars (Cabrera, 2004;
Pooler, 2006). Resulting cultivars of the L. indica ?fauriei cross began to diversify the
flower color, bark color, size, and disease resistance for crapemyrtle selections for the
landscape, adding 22 hybrid cultivars to the over 200 registered cultivars of
Lagerstroemia (Cabrera, 2004; Pooler, 2006).
II. Foliar Disorder in Crapemyrtle
Growers began to notice a perplexing leaf disorder occurring on cultivars of the
Lagerstroemia indica ?fauriei cross in the 1980s. Most prevalent on cultivars of
?Muskogee?, ?Tuskegee?, ?Miami?, and ?Natchez? hybrid crapemyrtles (Cabrera and
Lopez, 2004), the foliar disorder has been routinely identified in container production and
landscapes, with a few reports in field production. The disorder is casually referred to as
?rabbit tracks? or ?rabbit tracking? and typically occurs on the second flush of foliage in
the spring. Symptoms of the disorder include bronze elliptical spots on both sides of the
mid-vein leaving a series of translucent spots visible from upper and lower leaf surfaces
and distortion of new growth when severe (Figure 1). Disorder progression suggests the
effect of the disorder is aesthetic and not detrimental to affected plant health and survival
as the disorder?s symptoms appear to diminish with continued shoot growth (Figure 2).
47
III. Preliminary Work on Rabbit Tracks Foliar Disorder
Preliminary experiments conducted by Sibley and Ruter (Wilson et al., 2007)
during the 1990s and early 2000s sought to determine the cause of ?rabbit tracks? foliar
disorder through a nutritional approach. Initial work began in 1993 and 1994 with tissue
samples collected from affected and unaffected ?Natchez? crapemyrtles in the landscape.
Eight plants were included in this analysis with four affected and four unaffected plants.
Fifty leaves and one soil sample from each specimen were analyzed for interactions
between specific nutrient concentrations and occurrence of disorder. Data suggested
investigation of several nutrients as potential causes for the disorder for which the
following experiments were conducted.
In 1995, Sibley evaluated the effects of three manganese and three zinc
concentrations (0, 1x, and 2x) within modified Hoagland?s nutrient solutions on
Lagerstroemia indica ?fauriei ?Tuskegee? and ?Natchez?. Plants were grown in 100%
coarse sand in # 1 trade-gallon containers. Plants were grown in a controlled environment
greenhouse and examined for presence of ?rabbit tracks? disorder. Conclusions of both
the manganese and zinc studies suggest further investigation of other nutrients is
warranted because both cultivars exemplified the disorder in all treatments (Wilson et al.,
2007).
In 1997, an experiment was conducted using 54 ?Natchez? crapemyrtle 10 cm (4
in) potted liners, rooted in unamended 6 pine bark: 1 sand substrate. Liners were potted
up to #3 trade-gallon containers with four different rates of dolomitic limestone [0, 3, 6,
and 9 kg?m-3 (0, 5, 10, and 15 lbs?yd-3)]. Differing lime rates were used to evaluate the
48
effect of limited nutrient availability caused by increased substrate pH on the presence of
?rabbit tracks?. Results of the experiment did not indicate correlation between substrate
pH and the presence of the ?rabbit tracks? foliar disorder (Wilson et al., 2007).
Sibley continued nutritionally based experiments in 1999 evaluating four
concentrations of phosphorus (0x, 1x, 2x, and 3x) based on recommended phosphorus
rates using phosphoric acid, H3PO4 in a modified Hoagland?s solution. Bare root liners of
?Natchez? crapemyrtles were grown using 100% sand in #1 trade-gallon containers inside
a controlled environment greenhouse. All of the plants in the phosphorus experiment
displayed the disorder in all treatments suggesting further investigation (Wilson et al.,
2007).
In 2003 and 2004, Sibley and Ruter (Wilson et al., 2007) conducted a study to
examined the role of nickel in the occurrence of ?rabbit tracks? using a foliar spray and
substrate drench. This experiment was conducted using the hybrid crapemyrtle cultivars
?Miami?, ?Muskogee?, and ?Natchez?. One year old plants in #3 trade-gallon containers
were potted up to #7 trade-gallon containers in 6 pine bark : 1 sand substrate. Treatments
consisted of a foliar spray of urea at 2 lbs?100 gal-1 with a surfactant at 0.14 fl oz?gal-1,
along with 0.30 oz?gal-1 nickel sulfate foliar spray applied at 100 gal?acre-1 and a drench
treatment with 0.16 oz?gal-1 nickel sulfate drench applied at 16.9 fl oz per container.
Treatments were applied once in the fall and again in the spring during leaf expansion.
Plants were grown outdoors on a container pad under overhead irrigation. Results of the
nickel study did not explain the cause of the leaf disorder in the three hybrid crapemyrtle
cultivars using nickel foliar spray and drench as all the plants displayed the disorder
(Wilson et al., 2007). Additional preliminary experiments evaluating various rates of
49
substrate and foliar-applied nutrients on the occurrence of ?rabbit tracks? were conducted
in 2007 and 2008 (Appendix C).
In addition to nutrition based experiments, an enzyme-linked immunosorbent
assay (ELISA test) was conducted in 1997 and 2008 to see if any viruses or bacteria were
present in samples of affected leaf tissue that could be transferred to young tobacco plants
or evaluated in a plate reader. In reviewing the ELISA test for the presence of plant
viruses, no transferable virus was found. Not all plant viruses are easily transferable to
other plants and not all crops are susceptible to other plant species? viruses. Tobacco was
used due to its susceptibility to many known plant viruses. ELISA tests conducted in
2008 for the bacterium Xylella fastidiosa for which crapemyrtle is a host (Huang, 2007)
exhibited positive readings for all samples. The peroxidase reagent kit used for most
ELISA test of Xylella fastidiosa was believed to react with phytochemicals associated
with crapemyrtle causing all samples to exhibit bacteria. According to personal
communication with John Murphy, an Auburn University plant pathology professor,
further work using polymerase chain reaction (PCR) method to determine Xylella
presence and ?rabbit tracks? foliar disorder interaction and development of ELISA
reagent kits compatible with Lagerstroemia are needed (Murphy, personal
communication).
Cabrera and Lopez of Texas A&M University conducted a cultivar and foliar
nutrient survey of 13 crapemyrtle cultivars of three species and one hybrid (5 L. indica, 3
L. fauriei, 4 L. indica ?fauriei, and 1 L. speciosa) as a part of a salinity tolerance
evaluation in 2003. Cabrera and Lopez (2004) reported zero percent of L. speciosa
cultivars exhibited symptoms of ?rabbit tracks? disorder whereas, 94.4 and 97.5% of the
50
cultivars for L. fauriei and L. indica ?fauriei exhibited symptoms of the disorder (N=390,
at 30 plants per cultivar). Increased incidence for disorder occurrence in cultivars of L.
fauriei and L. indica ?fauriei suggests that abnormalities associated with gene expression
for cultivars with L. fauriei parentage may be a factor for ?rabbit tracks? presence as seen
in flea beetle and powdery mildew resistance between L. indica and L. fauriei (Cabrera et
al., 2003; Hagan et al., 1998). In addition to potential genetic influences, Cabrera and
Lopez (2004) observed that some of the nutrient content of foliage samples were strongly
associated with ?rabbit tracks? occurrence. Manganese (Mn) and zinc (Zn) were observed
to have a potential relationship with disorder presence as the percentage of plants
exhibiting ?rabbit tracks? was highest for plants with lower Mn levels (L. fauriei and L.
indica ?fauriei). In addition to Mn, Zn was found strongly associated with the foliar
disorder. Observations of the raw data indicated foliar Zn concentrations less than 90 to
100 ppm were found to be associated with increased percentage of affected plants
(Cabrera and Lopez, 2004). Consistent with visual symptoms, the foliar nutrient survey
suggests micronutrient (Fe, Mn, Cu, and Zn) deficiency or toxicity may be involved as
the disorder appears on growth subsequent to the first foliage flush. The occurrence of
symptoms on secondary growth indicates a potential immobile nutrient
deficiency/toxicity as most micronutrient elements are unable to translocate from older
foliage to newer foliage (Mengel and Kirkby, 2001). In addition to genetic and nutrient
content, studies examining interactions between genetic and nutritional influences are
warranted. Conversation with Fenny Dane, an Auburn University horticulture professor,
nutrient status and other environmental factors may initiate or terminate gene expression
for protein biosynthesis leading to disorders of photosynthetic and metabolic enzyme
51
production, carbon partitioning, and plant growth and development (Dane, personal
communication; Galangau et al., 1988; Migge et al., 2000), potentially inducing the
?rabbit tracks? foliar disorder.
Cabrera and Lopez (2004) and Wilson et al. (2007) are the only literature
describing work to diagnose ?rabbit tracks? foliar disorder in Lagerstroemia. The purpose
of the following work was to explore the significance of ?rabbit tracks? disorder to the
nursery and landscape industry through an industry sponsored survey and to examine the
disorder?s occurrence through a hydroponic nutrition study.
52
Literature Cited
Byers, D. 1997. Crapemyrtle: a grower?s thoughts. Owl Bay, Auburn, AL.
Cabrera, R.I. 2004. Evaluating and promoting the cosmopolitan and multipurpose
Lagerstroemia. Acta Hort. 630:177-184.
Cabrera, R.I., J.A. Reinert, and C. McKenney. 2003. Resistance among crape myrtle
(Lagerstroemia spp.) cultivars to the flea beetle (Altica litigate). South. Nurs.
Assoc. Res. Conf. 48:164-167.
Cabrera, R I. and R.E. Lopez. 2004. A leaf interveinal chlorosis-necrosis disorder in
crape myrtle. South. Nurs. Assoc. Res. Conf. 49:90-93.
Dane, F. 2010. Personal communication. 18 June 2010.
Dirr, M.A. 1998. Manual of woody landscape plants: their identification, ornamental
characteristics, culture, propagation, and Uses. 5th ed. Stipes, Champaign, IL.
Egolf, D.R. 1981. ?Muskogee? and ?Natchez? Lagerstroemia. HortScience 16:576.
Egolf, D.R. and A.O. Andrick. 1978. The Lagerstroemia handbook/checklist. Amer. Soc.
Botanical Gardens and Arboreta, Inc. Los Angeles.
Everett, T.H. 1969. Living trees of the world. Doubleday, N.Y.
Everett, T.H. 1981. The New York botanical garden illustrated encyclopedia of
horticulture. Vol. 6. Garland, N.Y.
Furtado, C.X. and S. Montien. 1969. A revision of Lagerstroemia L. (Lythraceae). Gard.
Bul. Straits Settlem. 24:185-335.
53
Galangau, F., F. Daniel-Vedele, T. Moureaux, M.F. Dorbe, M.T. Leydecker, and M.
Caboche. 1998. Expression of leaf nitrate reductase genes from tomato and
tobacco in relation to light-dark regimes and nitrate supply. Plant Physiol. 88:383-
388.
Graham, A., J.W. Nowicke, J.J. Skvarla, Graham, S.A., V. Patel, and S. Lee. 1987.
Palynology and systematics of the Lythraceae. II. Genera Haitia through Peplis.
Amer. J. Bot. 74:829-850.
Graham, S.A., J.V. Crisci, and P.C. Hoch. 1993. Cladistic analysis of the Lythraceae
sensu lato based on morphological characters. Bot. J. Linn. Soc. 113:1-33.
Hagan, A.K., G.J. Keever, C.H. Gilliam, J.D. Williams, and G. Creech. 1998.
Susceptibility of crapemyrtle cultivars to powdery mildew and Cercospora leaf
spot in Alabama. J. Environ. Hort. 16:143-147.
Huang, Q. 2007. Natural occurrence of Xylella fastidiosa in a commercial nursery in
Maryland. Can. J. Plant Path. 29:299-303.
Kakuda, T., I. Sakane, T. Takihara, Y. Ozaki, H. Takeuchi, and M. Kuroyanagi. 1996.
Hypoglycemic effect of extracts from Lagerstroemia speciosa L. leaves in
genetically diabetic KK-A(y) mice. Biosci. Biotechnol. Biochem. 60:204-208.
Knox, G.W. 1995. Crape myrtles for the deep south. Amer. Nurseryman 6 (11):70-77.
Mengel, K. and E.A. Kirkby. 2001. Principles of plant nutrition. 5th ed. Kluwer Academic
Publishers, Dordrecht, the Netherlands.
Migge, A., C. Bork, R. Hell, and T.W. Becker. 2000. Negative regulation of nitrate
reductase gene expression by glutamine or asparagines accumulating in leaves of
sulfur-deprived tobacco. Planta. 211:587-595.
54
Murphy, J.F. 2008. Personal communication. 22 July 2008.
Pooler, M.R. 2006. Crapemyrtle ? Lagerstroemia indica. P. 428-449. In: N.O. Anderson
(editor). Flower breeding and genetics: Issues, challenges, and opportunities for
the 21st century. Vol. 2. Springer, N.Y.
Wilson, M.W., J.L. Sibley, D.J. Eakes, and J.M. Ruter. 2007. Investigation of rabbit
tracks: a foliar disorder of crapemyrtle. South. Nurs. Assoc. Res. Conf. 52:590-
593.
55
Figure 1. ?Rabbit tracks? foliar disorder in Lagerstroemia indica ?fauriei
?Tuskegee?.
Figure 2. Progressed ?rabbit tracks? foliar disorder in Lagerstroemia indica ?fauriei
?Tuscarora?.
56
Chapter II
Survey of ?Rabbit Tracks? Foliar Disorder?s Extent, Effect, and
Significance to Ornamental Horticulture Industries
Abstract
With rapid growth and use of hybrid crapemyrtles, growers began to notice a
perplexing leaf disorder occurring on cultivars of the Lagerstroemia indica L. ?
Lagerstroemia fauriei Koehne crosses in the 1980s. The disorder is casually referred to as
?rabbit tracks? or ?rabbit tracking? and typically occurs on the second flush of foliage in
the spring. No work quantifying the impact of the foliar disorder to ornamental
horticulture industries has been published to date. In August 2008, an online survey was
conducted in cooperation with seven nursery and landscape associations in Alabama,
Florida, Georgia, Louisiana, South Carolina, Tennessee, and Texas. Industry members
within the seven associations were asked 16 questions to obtain demographic, disorder
familiarity, and disorder significance information. No significant correlations between
gross income of participants and the ratings for significance to the industry for ?rabbit
tracks? foliar disorder were found. Percentages of participants? responses were reported.
Thirty percent of industry members surveyed reported never hearing the term ?rabbit
tracks? and 40% reported experience with the disorder. Data collected from the survey
served to estimate the need for significant measures to diagnose and treat the disorder.
57
Measures requiring large amount of resources, expense, and costly solutions are not
recommended for diagnosis work or treatment, as industry members did not indicate
significant losses or customer concern for ?rabbit tracks? effect to plant material quality.
Introduction
With rapid growth and use of hybrid crapemyrtles, growers began to notice a
perplexing leaf disorder occurring on cultivars of the Lagerstroemia indica L.,
Lagerstroemia fauriei Koehne crosses in the 1980s. Most prevalent on cultivars of
?Muskogee?, ?Tuskegee?, ?Miami?, and ?Natchez? hybrid crapemyrtles (Cabrera and
Lopez, 2004), the foliar disorder has been routinely identified in container production and
landscapes, with a few reports in field production. The disorder is casually referred to as
?rabbit tracks? or ?rabbit tracking? and typically occurs on the second flush of foliage in
the spring. Symptoms of the disorder include bronze elliptical spots on both sides of the
mid-vein leaving a series of translucent spots visible from upper and lower leaf surfaces
and distortion of new growth when severe. Disorder progression suggests the effect of the
disorder is aesthetic and not detrimental to affected plants? health and survival as the
disorder?s symptoms appear to diminish with continued growth of subsequent foliage.
Preliminary work including a cultivar survey and nutrition experiments have attempted to
diagnose the cause of ?rabbit tracks? with no work quantifying the impact of the foliar
disorder to ornamental horticulture industries.
Cabrera and Lopez (2004) and Wilson et al. (2007) are the only literature
describing work to diagnose ?rabbit tracks? foliar disorder in Lagerstroemia. From casual
observation and correspondence with growers, Cabrera and Lopez (2004) stated ?rabbit
tracks? did not seem to have apparent effects on the popularity or sales of crapemyrtle. In
58
Alabama, the nursery, landscape, and horticulture retail industries contributed $2.7 billion
dollars and 41,800 jobs to Alabama?s economy in 2007 (ALNLA, 2009). Previous work
performed by Adrian et al. (1998) evaluated the fixed and variable costs associated with
producing crapemyrtle over three years using three production systems. Average
production cost for producers using the three production systems ranged from $21.52 to
$23.73 per plant with total costs ranging from approximately $450,000 to $508,000 for a
6 ha (15A) nursery with a 4 ha (10A) production area (Adrian et al., 1998) plus 2.52 %
annual inflation rate for years 1996 to 2007 (USBLS, 2010). While most growers
produce additional plant material besides crapemyrtle, work conducted by Adrian et al.
(1998) showed the production price associated with growing each plant. Crop failure due
to disease or improper management can cause growers to incur larger costs as the
production costs can account for most of the wholesale/retail value of the crop, leaving
small profit margins. The purpose of the following work was to explore the ?rabbit
tracks? disorder?s significance to the nursery and landscape industry through an industry
sponsored survey in eight southeastern states.
Materials and Methods
In July 2008, the internal review board (IRB) of Auburn University approved an
online survey to nursery and landscape grower associations to evaluate the significance of
?rabbit tracks? to their industry (Appendix B). Nursery and landscape grower
associations were identified to participate in an online survey through the American
Nursery and Landscape Association website (ANLA, 2009). Association directors of ten
southeastern U.S. states (Alabama, Arkansas, Florida, Georgia, Louisiana, Mississippi,
North Carolina, South Carolina, Tennessee, and Texas) were contacted and asked to
59
participate in the on-line survey. Association directors were given an opportunity to
preview the online survey and suggest changes. All associations with the exception of
Arkansas, Mississippi, and North Carolina agreed to participate. In August 2008,
approval from association directors was obtained and a letter to industry professionals
was given to associations to include on association websites, emails, newsletters, and
magazines (Appendix B). Associations were responsible for disseminating information to
its members to avoid collection of personal information as requested by association
directors for protection of member information and for IRB approval.
To increase association and participant response, pre-contact emails, short web
address, and easy-to-answer questions were incorporated into the survey?s design and
implementation (Dillman, 2007). The survey was designed and hosted on
SurveyMonkey.com? (SurveyMonkey, 2008). The survey used a short web uniform
resource locator (URL) to redirect participants to the online survey hosted on
SurveyMonkey.com? server (www.auburn.edu/rtsurvey). Incorporating a short web
address ensured that the survey could be easily passed along to industry members through
word-of-mouth communication and email (Dillman, 2007). Upon following the link to
the survey, respondents were directed to a survey introduction page (Appendix B), which
explained the purpose of the survey and assured participants that no personally
identifying information was collected from the survey. Survey respondents were asked to
click on a button to agree to participate in the survey. Upon agreement to the terms and
conditions of the survey, participants were directed to the first page of the survey for a
series of 14 multiple-choice questions with two optional short answer questions (16 total)
(Appendix B). Questions related to the nature, size, location (state), and income of their
60
business, whether they had previous ?rabbit tracks? foliar disorder knowledge, what
cultivars experienced the symptoms, and experience/opinion of the disorder?s
significance to the industry (Appendix B). Response collection for the survey began on
August 20, 2008 with an initial deadline for October 31, 2008 (72 days). Due to late
dissemination of association media and to collect more responses, the deadline was
extended to December 31, 2008 (133 days).
Upon closure of the survey, survey data were collected and entered into an Excel?
spreadsheet (Microsoft, 2007). Data were imported into statistical software (SPSS, 2007)
where variable and response data were given values to perform correlations using the
Pearson correlation procedure (SPSS, 2007).
Results and Discussion
Data revealed that 61 horticulture industry members participated in the survey
(N=61). Since no collection of email addresses or personal information was conducted
through the survey, there was no definite number as to how many were ultimately
contacted about the survey. Correspondence with association directors estimates that a
total of approximately 5,500 potential contacts were made through association emails,
websites, newsletters, and magazines. States with the greatest participation in the survey
with 41% and 31% of the total surveyed were Florida and Texas (Figure 1), which are
among the largest crapemyrtle producers in the U.S. Association emails (68.8%) and
website postings (15.6%) were reported to be the most effective in gaining participation
in comparison to association newsletters (7.8%) and magazine (1.5%) articles, personal
communication (6.2%), or general internet searches (0.0%) (Figure 2). Problems
associated with online surveys are that online surveys are not favored among all groups
61
of respondents as elderly or access-challenged participants may find electronic surveys
challenging (Dillman, 2007). Mail-in surveys with included return-postage may increase
response, but require additional labor and expense to enter data and pay for postage and
survey labor. The survey was conducted electronically to reduce data entry time, expense,
and error.
In order to determine the significance of ?rabbit tracks? to the horticulture
industry, correlations were performed using the Pearson correlation procedure (SPSS,
2007) for the gross income of participants versus the ratings for ?rabbit tracks?
significance to the industry. Correlations did not indicate a significant relationship
between significance ratings for ?rabbit tracks? and gross business income (p=0.265,
N=55). No correlations between gross income and other ratings were significant,
therefore only frequencies in relation to total responses (percentages) were further
reported.
The survey was designed to gather demographic information, industry experience
with ?rabbit tracks?, and industry ratings for significance of ?rabbit tracks? to the
ornamental horticulture industry. Of the participants that responded to the survey, 62.3%
were growers, 11.5% landscapers, 3.3% retailers, 3.3% re-wholesalers (landscape
distribution center), 1.6% grower/retailer, 4.9% grower/landscaper, 3.3% grower/re-
wholesaler, 3.3% grower/retailer/landscaper, and 6.6% of others were industry
professionals such as consultants and extension specialists (Figure 3). Participants were
allowed to select all applicable categories to describe their business. Gross sales of
industry professionals? business averaged over $500,000 with 20% earning less than
$200k, 20% between $200-$500k, 18.3% between $500k-$1million, and 41.7% earning
62
over 1 million dollars (Figure 4). Participants described their plant selection
produced/offered as consisting largely of landscape staples (i.e. common shrubs, trees,
etc.) (43.9%), 28.1% specialty crops (i.e. only trees, azaleas, etc.), 15.8% niche market
crops (herbs, natives, etc.), and 12.2% indoor/foliage plants (Figure 5). Of the
respondents, 91.4% attributed 0 to 25% of their total sales to crapemyrtle, 6.9%: 26-50%,
0.0%: 51-75%, and 1.7%: 76-100% (Figure 6).
Of those who responded, 31.0, 67.2 and 1.7% of those surveyed had, had not, or
possibly had heard the term ?rabbit tracks?, referring to crapemyrtle foliar disorder
(Figure 7). However, 41.4, 48.3, and 10.3% reported had, had not, or possibly had seen
the ?rabbit tracks? foliar disorder in crapemyrtle when presented with a photograph
(Appendix B) of the disorder (Figure 8). Participants acknowledging yes to noticing the
disorder, revealed location of experience with ?rabbit tracks? disorder. Twenty-three
percent saw the disorder in container production, 4.9% in the landscape, 1.6% in field
production, and 8.2% in all three locations (Figure 9). Along with location of disorder
occurrence, respondents were asked to name the cultivars of crapemyrtle on which they
observed ?rabbit tracks?. ?Natchez? was observed to have the highest occurrence (38.9%),
followed by ?Muskogee? (25.9%), ?Tuscarora? (11.1%), ?Tuskegee? (3.7%), and
?Firebird? (3.7%) (Figure 10). With ?Natchez? as the most commonly commercially
grown cultivar, it is possible that cultivars with greater frequency of ?rabbit tracks?
occurrence were not grown by all growers surveyed. Thus, other cultivars may prove to
show greater propensity to exhibit the disorder than ?Natchez?. Respondents with
disorder experience reported 83.3% had not tried to remedy the disorder, while 16.7%
had tried to remedy the disorder (Figure 11). Participants reported attempting nothing
63
(33.3%), fungicide (22.2%), micronutrient application (22.2%), or others had
discontinued crapemyrtle production as a means to cure the disorder and believed the
solutions to be beneficial in alleviating or remedying the disorder (Figure 12).
To determine the significance to the industry of the ?rabbit tracks? disorder, sales
estimates and ratings were collected. It was found that 77.4% of industry members never
had customers comment on the presence of ?rabbit tracks? on affected products, while
22.6% experienced customer comments (Figure 13). In addition, only 6.7% of
participants ever had buyers/customers refuse to purchase disorder-affected plants
(Figure 14). When asked to rate the likelihood (1=lowest, 4=highest) that a grower,
retailer, or customer would refuse to purchase affected plant material, respondents
estimated that retailers are the most likely to reject plants affected by the disorder (2.6)
followed by growers (2.5 and customers (2.2) (Figures 15 and 16). When asked to rate
the significance of ?rabbit tracks? foliar disorder to the ornamental horticulture industry,
industry members reported medium (38.1%) to low (36.4%) significance ratings
compared to high (18.2%) and no significance (7.2) (Figure 17).
Conclusions from the ?rabbit tracks? survey reveal that industry members do not
view the ?rabbit tracks? foliar disorder of crapemyrtle as a significant detriment to the
ornamental horticulture industry. Thirty percent of industry members surveyed reported
never hearing the term ?rabbit tracks? and 40% reported experience with the disorder.
Data collected from the survey served to estimate the need for measures to diagnose and
treat the disorder. Measures requiring large amount of resources, expense, and costly
solutions are not recommended for diagnosis work or treatment, as industry members did
64
not indicate significant losses or customer concern for ?rabbit tracks? effect to plant
material quality.
65
Literature Cited
Adrian, J.L., C.C. Montgomery, B.K. Behe, P.A. Duffy, and K.M. Tilt. 1998. Cost
comparisons for infield, above ground container and pot-in-pot production
systems. J. Environ. Hort. 16:65-68.
ALNLA. 2009. Economic impact of Alabama?s green industry: green industry growing.
Alabama Nursery and Landscape Assn. Spec. Rpt. 7.
Cabrera, R I. and R.E. Lopez. 2004. A leaf interveinal chlorosis-necrosis disorder in
crape myrtle. South. Nurs. Assoc. Res. Conf. 49:90-93.
Dillman, D.A. 2007. Mail and internet surveys: the tailed design method. 2nd ed. John
Wiley and Sons, Inc. N.J.
Microsoft. 2007. Microsoft excel? 2007 for windows?. Microsoft Corp., Inc. Redmond,
Wash.
SPSS. 2007. IBM SPSS statistics 15. SPSS Inc. Chicago.
SurveyMonkey. 2008. SurveyMonkey.com? . SurveyMonkey.com, LLC. Palo Alto,
Calif. .
USBLS. 2010. Inflation calculator. U.S. Bureau of Labor Statistics, Washington, D.C. 20
Jun 2010. < http://www.bls.gov/data/inflation_calculator.htm/>.
Wilson, M.W., J.L. Sibley, D.J. Eakes, and J.M. Ruter. 2007. Investigation of rabbit
tracks: a foliar disorder of crapemyrtle. South. Nurs. Assoc. Res. Conf. 52:590-
593.
66
67
68
69
70
71
72
73
74
75
Chapter III
A Nutritional Approach to Diagnosing ?Rabbit Tracks? Foliar Disorder
in Five Cultivars of Lagerstroemia indica ?fauriei
Abstract
In September 2008, five Lagerstroemia indica ?fauriei cultivars ?Miami?,
?Muskogee?, ?Natchez?, ?Tuscarora?, and ?Yuma? were positioned with roots suspended
by polystyrene disc wedges into gutters of six nutrient solutions. Treatments were used to
test for ?rabbit tracks? foliar crapemyrtle disorder and included one complete nutrient
solution and five modified nutrient solutions to exclude sulfur (S), copper (Cu), iron (Fe),
manganese (Mn), or zinc (Zn). Disorder and chlorosis occurrence, chlorosis coverage,
chlorosis stage, chlorosis progression, spot occurrence, spot pattern, and leaf distortion
ratings were taken 81 days later in December 2008. Data analyses revealed no evidence
to suggest any of the modified nutrient solution treatments had an effect on the
occurrence of ?rabbit tracks? foliar disorder for the five cultivars tested. No relationship
occurred between modified nutrient solutions and chlorosis coverage, chlorosis stage,
chlorosis progression, spot occurrence, spot pattern, or leaf distortion ratings. However,
chlorosis occurrence ratings due to treatment were found to be significant across all
cultivars. Death of nearly 11% of the plants studied resulted in less ability of the
generalized mixed linear models procedure (Proc Glimmix) to determine any significant
relationship between disorder or symptom occurrences.
76
Introduction
With rapid growth and use of hybrid crapemyrtles, growers began to notice a
perplexing leaf disorder occurring on cultivars of Lagerstroemia indica ?fauriei crosses
in the 1980s. Most prevalent on cultivars of ?Muskogee?, ?Tuskegee?, ?Miami?, and
?Natchez? hybrid crapemyrtles (Cabrera and Lopez, 2004), the foliar disorder has been
routinely identified in container production and landscapes, with a few reports in field
production. The disorder is casually referred to as ?rabbit tracks? or ?rabbit tracking? and
typically occurs on the second flush of foliage in the spring. Symptoms of the disorder
include bronze elliptical spots on both sides of the mid-vein leaving a series of
translucent spots visible from upper and lower leaf surfaces and distortion of new growth
when severe. Disorder progression suggests the effect of the disorder is aesthetic and not
detrimental to affected plants? health or survival as the disorder?s symptoms appear to
minimize with continued growth of subsequent foliage.
Consistent with visual symptoms, foliar nutrient surveys conducted by Sibley
(1993 and 1994), as well as Cabrera and Lopez (2003) suggest micronutrient (Fe, Mn,
Cu, and Zn) deficiency or toxicity may be involved as disorder appears on subsequent
growth to first foliage flush (Cabrera and Lopez, 2004; Wilson et al., 2007). Symptoms
occurring on secondary growth indicate immobile nutrient deficiency/toxicity as most
micronutrient elements are unable to translocate from older foliage to newer foliage
(Mengel and Kirkby, 2001).
Cabrera and Lopez (2004) and Wilson et al. (2007) are the only literature
describing work to diagnose ?rabbit tracks? foliar disorder in Lagerstroemia. The purpose
77
of the following work was to diagnose a causal agent for ?rabbit tracks? foliar disorder by
inducing disorder in a hydroponic nutrition study using six nutrient solutions.
Materials and Methods
In September 2008, 24, 6.4?10.2?154.9 cm (2.5?4?61 in) polyvinyl chloride
(PVC) residential drainage gutters with PVC end-caps [10.0 L (2.6 gal)] spaced 10.2 cm
(4 in) apart were constructed to contain six hydroponic nutrient solutions. Polystyrene
foam sheets were cut to fit on top of the gutters and were supported with PVC gutter
guards used to keep residential gutters clear of debris (Figure 1). Holes were cut 10.2 cm
(4 in) apart in the polystyrene sheets and gutter guards for plant spacing and root
clearance. Polystyrene sheets were labeled according to nutrient solution for gutter and
labeled for cultivar of Lagerstroemia indica ?fauriei used in the study (Figure 2). Main
effects nutrient solution and cultivar subplot designs were randomly assigned in SAS 9.1
(SAS, 2008). Gutters were aerated with two 80 watt air pumps (EcoPlus? Commercial
Air 5) using 0.64 cm (0.25 in) Raindrip? black, polyethylene tubing (NDS, Inc.,
Woodland Hills, Calif.) (Figure 3). Air pumps provided approximately 1.2?10-5m3?s-1
(0.03 gal?s-1) to obtain a 70% air to solution exchange rate for each gutter. Air tubing was
submersed within each gutter using a poly-coated shelving bracket to maintain complete
aeration of solution (Figure 4). A bench top warming coil was placed under gutters to
maintain water temperature of 23.9?C (75?F). A datalogger (Spectrum Technologies,
Inc.) with ambient and water temperature sensors was placed on the bench with gutters
readings were recorded every 30 minutes. Supplemental lighting initiated on October 15
to maintain and extend day length to represent photoperiod from May 18 to July 16, 2008
(NOAA, 2008) was provided using high-pressure sodium lamps at approximately 96.2
78
?mols?m-2. Lamps were set to turn on prior to sunrise and extend after sunset for
corresponding sunrise and sunset times (i.e. Sunrise May 18, 2008: 4:44; Sunset: 18:43
CST) (NOAA, 2008).
On September 18, 2008, five Lagerstroemia indica ?fauriei cultivars ?Miami?,
?Muskogee?, ?Natchez?, ?Tuscarora?, and ?Yuma? were positioned with roots suspended
by polystyrene disc wedges into gutters of six nutrient solution treatments (Figure 5).
Three 15.2 cm (6 in) tall bare-root liners for each cultivar were planted in each gutter at
10.2 cm (4 in) spacing (N= 15 plants/gutter, N=360). Plants were rooted in fall of
previous year and had one season of growth. Liners were trimmed to 15.2 cm (6 in) in
height and all foliage removed to stimulate new growth. Plants were arranged using a
completely randomized split-plot design with four treatment replications containing three
subsamples for each cultivar. Gutters were filled with 10 L of one of six #1 modified
Hoagland nutrient solutions (Table 1), one containing a complete formulation of all
prescribed elements and five modified to exclude one macronutrient sulfur (S) or four
micronutrients; copper (Cu), iron (Fe), manganese (Mn), or zinc (Zn) (Whipker, 2008).
All nutrient solutions were mixed with municipal water inside the greenhouses. Nutrient
content of irrigation water was analyzed at the conclusion of the study (Table 3). Nutrient
solutions were completely exchanged on a weekly basis with additional nutrient solutions
added as necessary to maintain volume.
The experiment was terminated on December 8, 2008 (81 days after planting), as
solution temperatures began to drop to 16?C (60.8?F) and cause dormancy of plants to
initiate. Plants did not exhibit the amount of growth typical for a growing season for
crapemyrtle. Growth in nutrient solutions induced more root development and growth
79
than foliage. Plant foliage increased approximately 20.4 cm (8 in). Foliar nutrient
analyses of two representative plants from each treatment were conducted using L. indica
?fauriei ?Yuma?. All leaves of plants analyzed were dried for 48 h at 68.3?C (155?F) and
sent to the Auburn University Soil Testing Laboratory for complete nutrient analyses
(Table 2). Data for ?rabbit tracks? occurrence and nutrient disorder symptoms were
collected using a binomial rating scale (0=no disorder, 1=disorder present) and
multinomial rating scale (i.e. chlorosis coverage ratings; 0=no chlorosis, 1=entire
chlorosis, 2=interveinal chlorosis, and 3=intraveinal chlorosis). Other disorder symptoms
collected included chlorosis occurrence, stage, and coverage ratings, chlorosis/necrosis
spot occurrence and spot pattern ratings, and leaf distortion occurrence. Occurrence and
rating data were analyzed using generalized linear mixed models with Proc Glimmix
procedure in SAS (Version 9.2; SAS Institute, Cary, NC). Treatment was included in the
model as the fixed factor with cultivar and cultivar/treatment interactions included as
random factors.
Results and Discussion
Data analyses revealed no evidence to suggest any of the modified nutrient
solution treatments had an effect on the occurrence of ?rabbit tracks? foliar disorder for
the five cultivars tested (p=1.000). No significant relationship between modified nutrient
solutions and percent chlorosis coverage, chlorosis stage, chlorosis progression, spot
occurrence, spot pattern, or leaf distortion ratings was observed. However, chlorosis
occurrence ratings due to treatment were found across all cultivars with iron-deficient
nutrient solutions having higher incidence of chlorosis than zinc-deficient and complete
nutrient solution treatments (p=0.473). Additionally, the lack of significant control or
80
induction of the rabbit tracks disorder in all the treatments and the presence of the
disorder in the study may serve to refute the hypothesis that drought could be a causal
agent for the disorder as all plants were immersed in the nutrient solution without drought
occurrence.
Death of nearly 11% of the plants studied, resulted in less ability of the
generalized mixed linear models procedure (Proc Glimmix) to determine any significant
relationship between disorder or symptom occurrences. Death in combination with the
onset of dormancy and irrigation water nutrient content (Table 3), were likely
contributors to analyses weaknesses. Replication of the experiment during the optimal
growing season for crapemyrtle (May-July) may provide more conclusive results as to the
role of nutrients in the occurrence of ?rabbit tracks?. Results from such work may
confirm or refute prior hypotheses proposed by investigations from Cabrera, Lopez and
Sibley (Cabrera and Lopez, 2004; Wilson et al., 2007). Further studies investigating
?rabbit tracks? occurrence in relation to modified nutrient solutions are needed to
diagnose the causal agent for rabbit track foliar disorder in crapemyrtle.
81
Literature Cited
Cabrera, R I. and R.E. Lopez. 2004. A leaf interveinal chlorosis-necrosis disorder in
crape myrtle. South. Nurs. Assoc. Res. Conf. 49:90-93.
Mengel, K. and E.A. Kirkby. 2001. Principles of plant nutrition. 5th ed. Kluwer Academic
Publishers, Dordrecht, the Netherlands.
Mills, H.A. and J.B. Jones. 1996. Plant analysis handbook ii. MicroMacro Publishing,
Inc. Athens, Ga.
NOAA, 2008. National oceanic & atmospheric administration earth system research
laboratory sunrise/sunset calculator. Washington, D.C. 20 August 2008.
< http://www.srrb.noaa.gov/highlights/sunrise/sunrise.html>.
SAS Institute Inc. 2009. SAS OnlineDoc? 9.2 Cary, NC: SAS Institute Inc.
Whipker, B.E. 2008. Personal communication. 15 August 2008.
Wilson, M.W., J.L. Sibley, D.J. Eakes, and J.M. Ruter. 2007. Investigation of rabbit
tracks: a foliar disorder of crapemyrtle. South. Nurs. Assoc. Res. Conf. 52:590-
593.
82
T ab l e 1. M od i f i e d n u t r i e n t s ol u t i on f or m u l at i on s w i t h r e age n t s t oc k s ol u t i on m l ? L
- 1
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F or m u l a t i on a m ou n t s w e r e pr e s c r i be d u s i n g w or k a n d pr ot oc ol f r om W h i pke r , B .E . 20 08.
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83
N P K Ca Mg Fe B Mn Zn Cu
S ol u t i on
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Fe 3.25 0.25 1.80 0.92 1.15 47.45 45.47 157.01 19.07 7.09
Mn 4.63 0.48 2.04 1.27 1.17 54.75 44.18 139.61 14.18 6.72
Zn 4.23 0.52 2.18 1.40 1.19 112.68 48.31 187.70 18.45 17.37
C om pl e t e 3.94 0.50 1.88 1.32 1.17 62.83 47.02 209.26 16.66 10.33
S u f f i c i e n c y R a n g e
W
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Z
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Y
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X
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T ab l e 2. L age r s t r oe m i a i n di c a ? f au r i e i ' Y u m a' l e af t i s s u e n u t r i e n t c on c e n t r at i on s f or s i x m od i f i e d n u t r i e n t s ol u t i on s .
Z
W
S u f f i c i e n c y r a n g e r e por t e d f or s u m m e r l e a v e s of 9 h y br i d c r a pe m y r t l e s i n bot a n i c a l g a r de n / a r bor e t u m .
M i l l s , H .A . a n d J .B . J on e s . 19 96. P l a n t a n a l y s i s h a n dbo ok i i . M i c r oM a c r o P u bl i s h i n g , I n c . A t h e n s , G a .
T a b l e 3 . N u t r i e n t c o n c e n t r a t i o n s o f i r r i g a t i o n w a t e r u s e d i n m o d i f i e d n u t r i e n t s o l u t i o n s f o r "r a b b i t t r a c k s " c r a p e m y r t l e h y d r o p o n i c
NO 3 -N P K Ca Mg Fe B Mn Zn Cu
S a m p l e ppm ppm
I r r i g a t i o n W a t e r 0 .2 8 0 .3 4 3 .0 7 2 8 .8 1 1 0 .5 4 < 0 .1 < 0 .1 < 0 .1 0 .2 4 < 0 .1
Z M e a n n u t r i e n t c o n c e n t r a t i o n v a l u e s a r e t h e a v e r a g e s o f 4 s a m p l e s .
s t u d y i n 20 08 . Z
84
85
86
87
Chapter IV
Final Discussion
Thermotolerance of Hemlock (Tsuga spp.)
Results from work conducted in 2003 and 2008 indicate that T. canadensis and T.
dumosa had similarly, higher predicted temperature midpoints (Tm) and k-values than T.
caroliniana suggesting higher resistance to direct injury when exposed to brief, supra-
optimal temperatures with no differences found in Tm and k-values for T. canadensis or
T. dumosa when each was compared to T. chinensis, T. diversifolia, or T. heterophylla.
Similarly, no differences were found for Tm and k-values of T. caroliniana when
compared to T. chinensis, T. diversifolia, or T. heterophylla.
Nature of work conducted and results from 2003 and 2008 indicate a need for
further investigations into the thermotolerance or heat tolerance of Tsuga species. Though
direct foliar injury may reveal a facet of hemlock tissues? ability to withstand intense
temperatures for brief periods of time, direct injury lacks the ability to quantify the
capacity of a species to sustain necessary growth to survive in adverse conditions. While
conclusions derived from direct injury evaluations may serve as an indicator to predict
the ability/inability of a species to survive these conditions, investigations into species
performance, growth, and adaptation are necessary to validate claims from direct injury
observations. Evaluations into response of Tsuga species to elevated temperatures with
other diagnostic means such as net photosynthesis, night respiration rates,
triphenyltetrazolium chloride (TTC) tests, C14 assimilation, toxin accumulation,
88
chlorophyll fluorescence, and growth trials are warranted to fully understand both direct
and indirect injury.
Plants with tissues observed to survive intense temperatures have also displayed
poor suitability to elevated supra-optimal temperatures because of reduced photosynthetic
capacity and increased respiration rates, damage to photosystem pathways, and decreased
carbon assimilation, all areas accepted as necessary for plant growth and survival. More
work investigating these factors is warranted in validating direct injury prediction claims
for the ability of various species of hemlock to grow and survive suitably in conditions
and locations predisposed to elevated temperatures. Investigations into indirect injury are
better suited to predict the survival of a species in heat-disposed areas as the temperatures
used in indirect injury work are more representative of the natural environmental
conditions a species would be exposed to in heat-disposed climates.
Survey of ?Rabbit Tracks? Foliar Disorder?s Extent, Effect, and Significance to
Ornamental Horticulture Industries
Resulting analysis of survey data revealed important information for approaching
?rabbit tracks? foliar disorder of crapemyrtle diagnosis. While the survey was conducted
after initial experiments to determine the disorder?s causal agent, information gathered
served to quantify the disorder?s significance and effect to various ornamental
horticulture industries. Less than half of the industry members surveyed had heard or
seen ?rabbit tracks? foliar disorder in crapemyrtle, revealing limited awareness of the
disorder among industry members. There was no significant correlation between business
gross income and disorder significance to industry ratings, indicating low economic
89
significance for larger industry members. Overall significance to industry ratings ascribed
low to medium industry significance ratings for the disorder.
Data collected from the survey served to estimate the need for significant
measures to diagnose and treat the disorder. Measures requiring large amount of
resources, expense, and costly solutions are not recommended for diagnosis work or
treatment, as industry members did not indicate significant losses or customer concern for
?rabbit tracks? effect to plant material quality.
A Nutritional Approach to Diagnosing ?Rabbit Tracks? Foliar Disorder in Five
Cultivars of Lagerstroemia indica ?fauriei
Data analysis revealed no evidence to suggest any of the modified nutrient
solution treatments had a significant effect on the occurrence of ?rabbit tracks? foliar
disorder for the five Lagerstroemia indica ?fauriei cultivars tested ?Miami?, ?Muskogee?,
?Natchez?, ?Tuscarora?, and ?Yuma?. No significant relationship between modified
nutrient solutions and chlorosis coverage, chlorosis stage, chlorosis progression, spot
occurrence, spot pattern, or leaf distortion ratings. However, chlorosis occurrence ratings
due to treatment were found to be significant across all cultivars. Additionally, the lack of
significant control or induction of the rabbit tracks disorder in all the treatments and the
presence of the disorder in the study may serve to refute the hypothesis that drought
could be a causal agent for the disorder as all plants were immersed in the nutrient
solution without drought occurrence.
Death of nearly 11% of the plants studied, resulted in less ability of the
generalized mixed linear models procedure to determine any significant relationship
90
between disorder or symptom occurrences. Death in combination with the onset of
dormancy and irrigation water nutrient content, were likely contributors to analyses
weaknesses. Replication of the experiment during the optimal growing season for
crapemyrtle (May-July) may provide more conclusive results as to the role of nutrients in
the occurrence of ?rabbit tracks?. Results from such work may confirm or refute prior
hypotheses proposed by investigations from Cabrera, Lopez and Sibley. Further studies
investigating ?rabbit tracks? occurrence in relation to modified nutrient solutions are
needed to diagnose the causal agent for rabbit track foliar disorder in crapemyrtle.
91
Appendix A
Foliar Thermotolerance of Tsuga spp. in 2003.
Abstract
Initial measures for foliar thermotolerance of six Tsuga species were evaluated
using electrolyte leakage in 2003. Species evaluated included: T. canadensis, T.
caroliniana, T. diversifolia, T. heterophylla, T. mertensiana, and T.sieboldii. Liners of the
six Tsuga species were potted into containers and grown for twelve months prior to
evaluation. Species were exposed to 25, 30, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, and
60?C (77, 86, 95, 99.5, 104, 108.5, 113, 117.5, 122, 126.5, 131, and 140?F) for 30
minutes in thermostatically controlled water baths, and electrical conductivity determined
subsequent each 24 hour incubation time following treatment and autoclaving.
Electrolyte leakage, expressed as a ratio of the electrical conductivity taken 24 hours
following treatment to the conductivity taken 24 hours following autoclaving, was
determined. Electrolyte leakage response to temperature was sigmoidal. Predicted
temperature midpoints (Tm) of T. canadensis, T. caroliniana, T. heterophylla, T.
mertensiana, and T.sieboldii were ? 1.6?C (2.9?F) higher than that of T. diversifolia.
Highly variable Tm and k-values void significant extrapolations and conclusions
concerning heat tolerance.
92
Materials and Methods
In September 2002, liners of six Tsuga species (T. canadensis (L.) Carr., T.
caroliniana Engelm., T. diversifolia (Maxim) Mast., T. heterophylla (Raf.) Sarg, T.
mertensiana (Bong.) Carr., and T. sieboldii Carr.) were potted into 6.0 L (#2 trade gal.)
containers in a substrate of 6 pine bark:1 builders? sand (by volume) and amended with
6.6, 3.0, and 0.9 kg?m-3 (11.1, 5, and 1.5 lbs?yd-3) of 18N-2.6P-9.9K (18-6-12) 8-9 month
Osmocote? (Scotts Co., Marysville, OH), dolomitic limestone, and Micromax? (Scotts
Co.), respectively. Plants were grown on a container pad in Auburn, AL (32? 36?N?85?
29?W, USDA cold hardiness zone 8a) and irrigated with approximately 1.3 cm (0.5 in)
water daily for twelve months prior to foliar membrane thermostability determination in
September 2003.
Recently matured needles from current-season growth were excised from petioles
with a scalpel, weighed to 0.5 g (0.02 oz) samples, placed in test tubes containing 1 ml
(0.03 fl oz) of de-ionized water and treated within a thermostatically controlled water
bath at 12 temperatures [25, 30, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, and 60 ?C (77,
86, 95, 99.5, 104, 108.5, 113, 117.5, 122, 126.5, 131, and 140 ?F)] for 30 minutes. Each
temperature treatment contained replications of 6 test tubes for each species (temp N=36,
total N=432). Upon completing a prescribed temperature treatment, test tubes were filled
with 20 ml of de-ionized water, placed on ice, and incubated in a cooler maintained at 4.4
?C (40?F) for 24 hours. Following incubation, samples were brought to room temperature
and electrical conductivity measured (Accumet Excel XL50, Fisher Scientific, Pittsburgh,
PA). Samples were autoclaved at 121?C and 1.4 kg?cm-2 (249.8?F and 20 lbs?in-2) for 20
minutes and incubated for 24 hours at 4.4 ?C (40?F), after which electrical conductivities
93
were again determined. Electrolyte leakage was expressed as a ratio of the initial
electrical conductivity post-treatment to the conductivity post-autoclave.
Electrolyte leakage response to temperature was sigmoidal as reported with other
species. Therefore, a sigmoidal equation was used to understand the data:
Le = [x - z) / (1 + exp [ - k (T - Tm] ) ] + z
where Le was the percent leakage, z = baseline level of electrolyte leakage, x = percent of
electrolyte leakage observed across all temperatures, Tm = predicted temperature
midpoint, also referred to as the ?inflection point? for the response curve, k = the slope of
the predicted response at Tm, and T = water bath temperature used to determine critical
midpoint temperatures (Tm) by fitting electrolyte leakage data across temperature
treatments. Gauss-Newton method of non-linear regression was used to analyze
electrolyte leakage data with correlations performed by PROC CORR procedure using
SAS Version 9.1.3. Critical midpoint temperatures and k-values of fitted response curves
were determined for each species (Table A1). Differences were determined by failures of
species? Tm values, plus and minus Tm variance, to overlap. K-values were used to
distinguish slope differences between Tm values and describe the range of temperatures
around the Tm for which the species is predicted to withstand before experiencing greater
than 50% damage.
Results and Discussion
Gauss-Newton method of non-linear regression met convergence criteria for all
species and temperatures. Electrolyte leakage and temperature were correlated across all
species (r= 0.74, p= <0.0001, N=432). Predicted temperature midpoints (Tm) revealed
species T. canadensis 53.7 ? 0.4?C (128.7 ? 0.7?F), T. caroliniana 54.2 ? 0.6?C (129.6 ?
94
1.1?F), T. heterophylla 53.7 ? 0.4?C (128.7 ? 0.6?F), T. mertensiana 55.6 ? 0.6?C (132.2
? 1.1?F), and T.sieboldii 56.3 ? 2.1?C (133.3 ? 3.8?F) had higher Tm values ? 1.6?C
(2.9?F) than T. diversifolia 52.1 ? 0.5?C (125.8 ? 0.8?F) (Table A1) (Figures A1-A6).
Highly variable Tm and k-values void significant extrapolations and conclusions
concerning heat tolerance.
T. canadensis and T. heterophylla had similar Tm values to T. caroliniana and T.sieboldii
(? 1.6?C / 2.8?F) and narrower k-values (0.075 ? 0.05) than T. caroliniana and
T.sieboldii, indicating higher temperatures would be necessary to cause direct heat injury
to foliage of T. canadensis and T. heterophylla. Narrow k-values and low variability also
indicated a narrower tolerance range for supra-optimal canopy temperatures around the
critical temperature midpoint, suggesting the Tm value predicted is an accurate estimate
of the beyond recovery temperature. High variability for Tm and k-values for T.
caroliniana, T. mertensiana, and T. sieboldii, warrants further replications in determining
their foliar thermotolerance with other Tsuga species.
Following the work in 2003, it was determined that further investigations of
electrolyte leakage from hemlock foliage were necessary to verify aforementioned results
as tissue age and stage of acclimation may contribute to varied responses for
thermotolerance. Replications of the aforementioned experiment and procedures along
with experiments examining different tissue age and acclimation may better predict the
heat tolerance of hemlock species populations. With further investigations into direct heat
injury, certain species of hemlock may prove more suitable for production and use in
heat-disposed areas than others. These investigations began in 2008.
95
S pe c i e s T
m
y
T
m
x
k - v a l u e s
w
T . c ana de ns i s 53.7 ? 0.4 128.7 ? 0.7 0.50 ? 0.0 9
T . c ar ol i ni ana 54.2 ? 0.6 129.6 ? 1.1 0.58 ? 0.1 8
T . di v e r s i f ol i a 52.1 ? 0.5 125 .8 ? 0.8 0.50 ? 0.0 9
T . he t e r oph y l l a 53.7 ? 0.4 128 .7 ? 0.6 0.54 ? 0.0 9
T . m e r t e ns i ana 55.6 ? 0.6 132 .2 ? 1.1 0.47 ? 0.0 9
T . s i e bol di i 56.3 ? 2.1 133 .3 ? 3.8 0.31 ? 0.1 0
w
K - v a l u e s f or T m
x
( C e l s i u s ) .
T ab l e A 1. R e s p on s e of T s u ga ( h e m l oc k ) s p e c i e s t o e l e vat e d f ol i ar t e m p e r at u r e .
z
z
D a t a pr e s e n t e d f r om e l e c t r ol y t e l e a ka g e s t u dy i n 200 3.
y
M e a n s a n d s t a n da r d e r r or s f or pr e di c t e d c r i t i c a l t e m pe r a t u r e ( C e l s i u s ) pa r a m e t e r s de t e r m i n e d
by l e a s t s qu a r e s a ppr oa c h G a u s s - N e w t on m e t h od o f n on - l i n e a r r e g r e s s i on .
x
M e a n s a n d s t a n da r d e r r or s f or pr e di c t e d c r i t i c a l t e m pe r a t u r e ( F a h r e n h e i t ) pa r a m e t e r s
de t e r m i n e d by l e a s t s qu a r e s a ppr oa c h G a u s s - N e w t on m e t h od o f n on - l i n e a r r e g r e s s i on .
96
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e A 1 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u g a c a n a d e n s i s e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 3 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 3 . 7 ? 0 . 4
97
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e A 2 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u g a c a r o l i n i a n a e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 3 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 4 . 2 ? 0 . 6
98
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e A 3 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u g a d i ve r s i f o l i a e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 3 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 2 . 1 ? 0 . 5
99
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
e
ct
r
o
l
y
t
e
L
e
a
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e A 4 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u g a h e t e r o p h y l l a e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 3 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 3 . 7 ? 0 . 4
100
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
e
ct
r
o
l
y
t
e
L
e
a
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e A 5 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u g a m e r t e n s i a n a e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 3 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 5 . 6 ? 0 . 6
101
0
20
40
60
80
100
25 30 35 3 7 . 5 40 4 2 . 5 45 4 7 . 5 50 5 2 . 5 55 60
E
l
ect
r
o
l
y
t
e
L
ea
k
a
g
e
(
%
)
T e m p e r a t u r e ( C )
F i g u r e A 6 . F o l i a r e l e c t r o l y t e l e a k a g e o f T s u g a s i e b o l d i i e x p o se d t o 2 5 t o 6 0 ? C f o r 3 0 m i n . i n 2 0 0 3 .
Ob s er v ed
P r ed i c t ed
T
m
= 5 6 . 3 ? 2 . 1
102
Appendix B
Survey of ?Rabbit Tracks? Foliar Disorder?s Extent, Effect, and
Significance to Ornamental Horticulture Industries
Figure B1. Institutional review board letter of approval for survey.
103
Figure B2. Flyer emailed to nursery and landscape association directors.
Have You Seen This?
The picture above depicts symptoms of a foliar disorder on crapemyrtle termed ?Rabbit Tracks?. Since the mid-1980?s,
growers have noticed this disorder on cultivars of the L. indica ?L. fauriei cross such as ?Natchez?, ?Miami?,
?Muskogee?, ?Tuskegee?, and ?Tuscarora?. Symptoms of this disorder typically are characterized by bronze, elliptical
spots surrounded by chlorosis along both sides of the mid-vein. As symptoms become more severe, distortion of the
leaf margins (margin curl) may occur. This foliar disorder occurs on the second flush of growth in the spring (May-
June) with subsequent growth of second flush often minimizing symptoms during growing season causing the affected
plants to appear to ?grow out? of the disorder.
Currently, Dr. Jeff Sibley, a horticulture professor at Auburn University, is researching potential causes of the disorder.
Along with finding its cause, part of the research includes gathering information from growers, landscapers, and
retailers/wholesalers about their professional experience with the disorder and their opinion of the disorder?s
significance to the industry. The (insert state grower/landscape association name) would like to encourage members to
participate in an online survey to aid in the research of this disorder. The survey consists of 14 simple, multiple choice
and two optional, short answer questions. The survey is open to growers, landscapers, retail/wholesale businesses, and
researchers, irregardless of disorder knowledge and may be accessed at the following web address:
www.auburn.edu/rtsurvey
Your participation is essential and invaluable in providing researchers with information concerning this disorder and
improvement of the horticulture industry.
Surveys are to be completed and submitted by XX/XX/XX. Questions or concerns may be addressed to:
Matt Wilson
Graduate Research Assistant
Department of Horticulture
101 Funchess Hall
Auburn University, AL 36849-5408
wilsoms@auburn.edu
104
Figure B3. Preliminary on-line survey information and participant approval page.
Figure B4. First on-line survey question page.
105
Figure B5. Survey of ?rabbit tracks? foliar disorder?s extent, effect, and significance
to ornamental horticulture industries.
1. What type of business do you manage/own? Select all that apply.
Grower
Landscape Distribution Center (Re-wholesaler)
Retail
Landscape
Other (please specify)
2. What bracket best describes your gross sales?
<$200,000
$200,001-$500,000
$500,001-$1 Million
$1 Million+
3. If Grower/wholesaler/retailer, what term best describes your plant selection? Select all
that apply.
Foliage (indoor plants)
Unique-Unusual (i.e. herbs, niche market items)
Staples (common landscape plants)
Specialty (i.e. only trees, azaleas, etc.)
4. What percentage of your crops/sales may be attributed to crapemyrtle?
0-25%
26-50%
51-75%
76-100%
5. Have you ever heard of a foliar disorder termed "Rabbit Tracking or Rabbit Tracks"
believed to affect crapemyrtle (Lagerstroemia)?
Yes
No
Maybe
6. Have you ever seen the symptoms as shown in the image below on crapemyrtles grown
by you or another grower in late spring-early summer?
Yes
No
Maybe
106
7. Answers No to Question 6, go to Question 13. Answers Yes or Maybe. Did you
observe this disorder in field production, container production, or the landscape? Select
all that apply.
Field
Container
Landscape
8. What cultivars have you noticed to be affected greatest?
9. Have you or another grower attempted to remedy this disorder via chemical sprays,
substrate amendments, or environmental controls?
Yes
No
10. If so, what remedies, if any, have you found to be beneficial?
107
11. Have you ever had customers/buyers comment on the "Rabbit Tracks" symptoms?
Yes
No
Maybe
12. Have customers/buyers refused to purchase affected plant material?
Yes
No
13. Rate the likelihood you, buyers, or customers would reject purchasing affected
material (0%- least likely, 100% most likely to reject).
Buyer 0-25% 26-50% 51-75% 76-100%
Grower
Retailer
Customer
14. Rate your opinion of "Rabbit Tracks" significance to the industry.
None Low Medium High
Significance
to industry
15. What form of communication did you receive knowledge of this survey? Select all
that apply.
Email
Newsletter
Magazine
Personal Communication
Website
Web Search
Other (please specify)
16. Which state is your business located?
Alabama
Arkansas
Florida
Georgia
Louisiana
Mississippi
North Carolina
South Carolina
Tennessee
Texas
108
Appendix C
Preliminary Experiments Evaluating ?Rabbit Tracks? Occurrence to
Various Rates of Substrate and Foliar-Applied Nutrients
Experiment 1 Occurrence of ?Rabbit Tracks? Foliar Disorder in ?Natchez?
Crapemyrtle using Various Substrate-Incorporated Manganese Rates
In May 2007, 2.7 L (2.9 qt) containers of Lagerstroemia indica ?fauriei
?Natchez? were potted into 10.20 L (10.78 qt) containers in a substrate of 6 pine bark:1
builders? sand (by volume) and amended with 9.9, 3.0, and 0.9 kg?m-3 (16.6, 5, and 1.5
lbs?yd-3) of 18N-2.6P-9.9K (18-6-12) 8-9 month Polyon? (Agrium Inc., Loveland, CO),
dolomitic limestone, and Micromax? (Scotts Co., Marysville, OH), respectively.
Treatments were based on the standard rate of Mn supplied by Micromax? (Scotts Co.) at
0.9 kg?m-3 (1.5 lbs?yd-3) (0.02 kg?m-3/0.04 lbs?yd-3 Mn) serving as the 1x rate. Additional
amounts of manganese sulfate (MnSO4) were withheld or incorporated in each substrate
as treatments to obtain 0x, 1x, 2x, 3x, and 4x Mn rates. At planting, each plant was cut to
45 cm (18 in) in height to stimulate new growth. Plants were grown on a container pad in
Auburn, AL (32? 36?N?85? 29?W, USDA cold hardiness zone 8a) with overhead
irrigation at approximately 1.3 cm (0.5 in) water daily for two months. Plants were
arranged using a complete randomized design with 5 treatments and 10 repetitions
(N=50). In July 2007, disorder occurrence ratings were conducted from 0 to 5 (0=no
109
disorder symptoms and 5=showing most). Analysis of the data revealed significant
treatment effects from the addition of MnSO4 (p=0.0333, ?=0.05) with a linear decrease
in disorder symptoms as MnSO4 was increased (p=0.0023, ?=0.05) (Table C1). Further
investigation of substrate-applied manganese effects on ?rabbit tracks? occurrence for
crapemyrtle is warranted to confirm results.
Experiment 2- Occurrence of ?Rabbit Tracks? Foliar Disorder in ?Natchez?
Crapemyrtle Using Various Manganese Concentrations Applied as a Foliar Spray.
In June 2007, 2.7 L (2.9 qt) containers of Lagerstroemia indica ?fauriei
?Natchez? were potted into 10.2 L (10.8 qt) containers in a substrate of 6 pine bark:1
builders? sand (by volume) and amended with 9.9, 3.0, and 0.9 kg?m-3 (16.6, 5, and 1.5
lbs?yd-3) of 18N-2.6P-9.9K (18-6-12) 8-9 month Polyon? (Agrium Inc., Loveland, CO),
dolomitic limestone, and Micromax? (Scotts Co., Marysville, OH), respectively. At
potting, each plant was cut to 63 cm (25 in) in height to stimulate new growth.
Treatments consisted of seven rates of manganese (Mn) based on the standard rate
supplied by Micromax? (Scotts Co.) when incorporated at 0.9 kg?m-3 (1.5 lbs?yd-3) (0.02
kg?m-3/0.04 lbs?yd-3 Mn) which served as the 1x rate. Additional amounts of manganese
sulfate (MnSO4) were withheld or added to 700 ml (23.7 fl oz) water and sprayed to the
foliage of prescribed treatment plants to obtain 2x, 3x, 4x, 5x, and 6x Mn rates. Plants
under 0x and 1x treatments contained 0.0 and 0.9 kg?m-3 (0.0 and 1.5 lbs?yd-3) Micromax?
applied to substrate only (no foliar application). Plants were grown on a container pad in
Auburn, AL (32? 36?N?85? 29?W, USDA cold hardiness zone 8a) with overhead
irrigation at approximately 1.3 cm (0.5 in) water daily for two months. Plants were
110
arranged using a complete randomized design with 7 treatments and 5 repetitions of each
treatment (N=35). In July 2007, disorder occurrence ratings were conducted from 0 to 5
(0=no disorder symptoms and 5=showing most). Analysis of the data revealed significant
treatment effects with the addition of MnSO4 at 1x, 2x, 3x, 4x, and 5x rates (p=0.0195,
?=0.05) with a linear decrease in disorder symptoms as MnSO4 was increased (p=0.0019,
?=0.05) (Table C2). Analysis of data also revealed a slight, but insignificant quadratic
relationship between treatment and disorder ratings due the limited control of the disorder
provided by the highest manganese rate (6x). This could be due to antagonistic effects
that high concentrations of manganese could have on the availability of other nutrients
with potential roles in the occurrence of the foliar disorder, inducing foliar disorder
symptoms due to competition with manganese to be absorbed and utilized by the plants.
Experiment 3- Occurrence of ?Rabbit Tracks? Foliar Disorder in ?Natchez?
Crapemyrtle Using Copper Hydroxide Foliar Spray Applications at Various Stages
of Development.
In August 2007, 2.7 L (2.9 qt) containers of Lagerstroemia indica ?fauriei
?Natchez? were potted into 10.2 L (10.8 qt) containers in a substrate of 6 pine bark:1
builders? sand (by volume) and amended with 9.9, 3.0, and 0.9 kg?m-3 (16.6, 5, and 1.5
lbs?yd-3) of 18N-2.6P-9.9K (18-6-12) 8-9 month Polyon? (Agrium Inc., Loveland, CO),
dolomitic limestone, and Micromax? (Scotts Co., Marysville, OH), respectively. At
potting, plants were cut to 63 cm (25 in) in height, as prescribed by treatment, to
stimulate new growth. Treatments consisted of no Kocide? or one foliar spray
application of copper hydroxide (Kocide? 2000, DuPont, Wilmington, DE) at the rate of
111
3.4 kg?ha-1 (3 lbs?a-1) during three stages of growth (before pruning, first stage of growth-
after pruning, and second stage of growth-after pruning). Stages of growth were
determined in relation of time and pruning application. All plants were pruned on same
day with Kocide? applied immediately prior to pruning, 1 month after pruning (first
growth stage), or 2 months after pruning (second growth stage). Plants were grown on a
container pad in Auburn, AL (32? 36?N?85? 29?W, USDA cold hardiness zone 8a) with
overhead irrigation at approximately 1.3 cm (0.5 in) water daily for two months. Plants
were arranged using a complete randomized design with 4 treatments and 3 repetitions of
each treatment (N=12). Visual observations conducted on September 29 and October 6
reveal that no foliar disorder appeared on any treatments. Plants were moved into the
greenhouse in October and plants were grown for two additional months for observations.
On December 7, no disorder symptoms were observed for any treatments. The
experiment was terminated with no data collected. Further studies evaluating the impact
of copper on ?rabbit tracks? foliar disorder are warranted (Chapter 3).
Experiment 4- Occurrence of ?Rabbit Tracks? Foliar Disorder in ?Muskogee? and
?Tuskegee? Crapemyrtles Using Various Rates of Substrate-Incorporated Sulfur
and Gypsum.
In September 2007, 6.4 cm (2.5 in) rooted cuttings of Lagerstroemia indica
?fauriei ?Muskogee? and ?Tuskegee? were potted into 2.7 L (2.9 qt) containers in a
substrate of 6 pine bark:1 builders? sand (by volume) and amended with 9.9 and
0.9 kg?m-3 (16.6 and 1.5 lbs?yd-3) of 18N-2.6P-9.9K (18-6-12) 8-9 month Polyon?
(Agrium Inc., Loveland, CO) and Micromax? (Scotts Co., Marysville, OH), respectively.
112
Each plant was cut to 5 cm and 10 cm (5 cm for ?Tuskegee? and 10 cm for ?Muskogee?)
in height 24 days after planting to stimulate new growth. Treatments consisted of various
rates and combinations of elemental sulfur (Hi-Yield? Chemical Co., Bonham, TX) and
pelletized gypsum (CaSO4) as follows:
1. 0.0 sulfur, 0.0 gypsum, 5 dolomitic lime kg?m-3 (Control)
2. 0.0 sulfur, 0.0 gypsum, 0 dolomitic lime kg?m-3
3. 0.6 sulfur, 0.0 gypsum, 0 dolomitic lime kg?m-3
4. 1.2 sulfur, 0.0 gypsum, 0 dolomitic lime kg?m-3
5. 0.0 sulfur, 1.2 gypsum, 0 dolomitic lime kg?m-3
6. 0.0 sulfur, 2.4 gypsum, 0 dolomitic lime kg?m-3
7. 0.0 sulfur, 3.6 gypsum, 0 dolomitic lime kg?m-3
8. 0.6 sulfur, 1.2 gypsum, 0 dolomitic lime kg?m-3
9. 0.6 sulfur, 2.4 gypsum, 0 dolomitic lime kg?m-3
10. 0.6 sulfur, 3.6 gypsum, 0 dolomitic lime kg?m-3
11. 1.2 sulfur, 1.2 gypsum, 0 dolomitic lime kg?m-3
12. 1.2 sulfur, 2.4 gypsum, 0 dolomitic lime kg?m-3
13. 1.2 sulfur, 3.6 gypsum, 0 dolomitic lime kg?m-3
Plants were grown on a container pad in Auburn, AL (32? 36?N?85? 29?W, USDA cold
hardiness zone 8a) with overhead irrigation at approximately 1.3 cm (0.5 in) water daily
for one month before being moved to a greenhouse to prevent dormancy after which, they
were hand watered daily with approximately 0.6 cm (0.3 in) of water. Plants were
arranged using a complete randomized design with 13 treatments and 5 repetitions of
each treatment (N=65). On December 7, no disorder symptoms were observed for any
treatment, therefore the experiment was terminated with no data collected. Further studies
evaluating the potential impact of sulfur and calcium on ?rabbit tracks? foliar disorder are
warranted (Chapter 3).
113
Experiment 5- Occurrence of ?Rabbit Tracks? Foliar Disorder in ?Natchez?
Crapemyrtle Using Various Rates of Substrate-Incorporated Copper Sulfate.
In June 2008, 2.7 L (2.9 qt) containers of Lagerstroemia indica ?fauriei
?Natchez? were potted up into 10.2 L (10.8 qt) containers in a substrate of 6 pine bark:1
builders? sand (by volume) and amended with 10.5, 3.0, and 0.9 kg?m-3 (17.6, 5, and 1.5
lbs?yd-3) of 17N-2.1P-9.1K (17-5-11) 8-9 month Polyon? (Agrium Inc., Loveland, CO),
dolomitic limestone, and Micromax? (Scotts Co., Marysville, OH), respectively.
Treatments consisted of various rates of substrate-incorporated copper sulfate (CuSO4)
[0, 3.0, 4.5, 5.9, 7.4, 8.9, 10.1, and 11.9 kg?m-3 (0, 5, 7.5, 10, 12.5, 15, 17, and 20 lbs?yd-
3)]. At potting, each plant was cut to 25.4 cm (10 inches) in height and were grown on a
container pad in Auburn, AL (32? 36?N?85? 29?W, USDA cold hardiness zone 8a) and
overhead irrigated with approximately 1.3 cm (0.5 in) water daily for two months. Plants
were arranged using a randomized complete block design with 8 treatments and 10
repetitions of each treatment (N=80). In July of 2008, no disorder symptoms were
observed for any treatment, therefore the experiment was terminated with no data
collected. Further studies evaluating the impact of copper on ?rabbit tracks? foliar
disorder are warranted (Chapter 3).
114
M e d i an R at i n g
z
M n R at e k g? m
-3
M i c r om ax
?
m g? l
-1
M n S O 4 S c al e 0 - 5
0x 0. 0 0. 0 3. 0
1x 0. 9 0. 0 2. 0
2x 0. 9 69 .5 1. 0
3x 0. 9 13 8. 9 1. 0
4x 0. 9 20 8. 4 1. 0
5x 0. 9 27 7. 9 1. 0
6x 0. 9 34 7. 4 2. 0
z
M e di a n i n c i de n c e r a t i n g s f or r a bb i t t r a c ks c a l c u l a t e d u s i n g P R O C G L I M M I X i n
S A S 9 .2 w ith ? = 0 .0 5. R a tin g w a s f rom 0 ( n o oc c u rre n c e ) t o 5 (di s or de r
pr e v a l e n t ) .
T r e at m e n t
T ab l e C 2. E f f e c t s o f f ol i ar - ap p l i e d m an ga n e s e r at e s o n o c c u r r e n c e o f r ab b i t
t r ac k s f ol i ar d i s or d e r i n L ag e r s t r oe m i a i n di c a ? f au r i e i ' N at c h e z ' .
M e d i an R at i n g
z
M n R at e k g? m - 3 M i c r om ax
?
k g? m
-3
M n S O 4 S c al e 0 - 5
0x 0. 0 0. 0 3. 0
1x 0. 9 0. 0 2. 0
2x 0. 9 0. 06 95 3. 0
3x 0. 9 0. 13 89 2. 0
4x 0. 9 0. 20 84 1. 0
T ab l e C 1. E f f e c t s o f s u b s t r at e - i n c or p or at e d m an ga n e s e r at e s o n o c c u r r e n c e
of r ab b i t t r ac k s f ol i ar d i s or d e r i n L ag e r s t r oe m i a i n di c a ? f au r i e i ' N at c h e z ' .
T r e at m e n t
z
M e di a n i n c i de n c e r a t i n g s f or r a bb i t t r a c ks c a l c u l a t e d u s i n g P R O C G L I M M I X i n
S A S 9 .2 w i t h ? = 0.05. R a t i n g w a s f r om 0 ( n o oc c u r r e n c e ) t o 5 ( di s or de r
pr e v a l e n t ) .