Shoot Development in Citrus Plants as Affected by Salicylates and Huanglongbing Infection

MISSING IMAGE

Material Information

Title:
Shoot Development in Citrus Plants as Affected by Salicylates and Huanglongbing Infection
Physical Description:
1 online resource (101 p.)
Language:
english
Creator:
Burani Arouca, Marina
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Horticultural Sciences
Committee Chair:
Spann, Timothy M
Committee Co-Chair:
Darnell, Rebecca L
Committee Members:
Schumann, Arnold W
Rogers, Michael E

Subjects

Subjects / Keywords:
citrus -- huanglongbing -- salicylates -- shoot
Horticultural Sciences -- Dissertations, Academic -- UF
Genre:
Horticultural Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
A debilitating disease called huanglongbing (HLB) seriously threatens the Florida citrus industry. All commercial citrus cultivars are susceptible to the presumed causal agent, a fastidious phloem-limited bacterium designated Candidatus Liberibacter asiaticus, which is vectored by the Asian citrus psyllid (Diaphorina citri).The symptoms are chlorotic blotchy mottling of leaves and tree decline. HLB-related phloem plugging interrupts the communication between source and sink organs, and nutrient transport. This study aimed to understand the effects of salicylates on the growth of citrus plants with and without HLB infection.In other species, salicylates are known to induce systemic acquired resistance;however, in HLB-affected citrus trees, salicylates appear to function as plant growth regulators. We hypothesized that salicylates induce development of new vegetative shoots in citrus, which may help HLB-affected plants cope with this disease by developing new vascular tissue free of the blockage that occurs in response to the infection. A series of growth chamber and field experiments were conducted to test this hypothesis, with the specific objectives to 1)determine if foliar applications of salicylates induce vegetative growth; and 2) quantify variation in budbreak and shoot development in citrus plants treated with salicylates. Healthy citrus plants treated with sodium and ammonium salicylate did not consistently respond to treatments.Results from our field study with HLB-affected citrus were also inconclusive,and generally did not support the hypothesis. This research contributes to our knowledge about salicylates acting as plant growth regulators in citrus, particularly with respect to the host-pathogen relationship of citrus and HLB.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Marina Burani Arouca.
Thesis:
Thesis (M.S.)--University of Florida, 2012.
Local:
Adviser: Spann, Timothy M.
Local:
Co-adviser: Darnell, Rebecca L.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2012
System ID:
UFE0045077:00001


This item is only available as the following downloads:


Full Text

PAGE 1

1 SHOOT DEVELOPMENT IN CITRUS PLANTS AS AFFECTED BY SALICYLATES AND HUANGLONGBING INFECTION By MARINA BURANI AROUCA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DE GREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

PAGE 2

2 2012 Marina Burani Arouca

PAGE 3

3 To my beloved parents, Eric and my best friends Rafa and Carol

PAGE 4

4 ACKNOWLEDGMENTS I would like to thank my precious family. Although distant, they have always been supportive by sending me their endless love, care and prayers. Special thanks to my parents who had always done the possible and the impossible for our family, always giving priceless guidance. To Rafa and Carol, fo r their understanding, friendship and f or always being there for me supporting my decisions. To Eric Gregory, for the constant motivation advice, patience and care To Sandy and Rob, Eric and Peggy thank you for the support and encouragement. Thank you a ll for being part of my journey; I am very blessed. Great thanks to my graduate committee members Dr. Spann, Dr. Darnell, Dr. degree program. I want to specially thank my ad visor, Dr. Spann who has supported me and offered me this invaluable opportunity. Thank you for your faith in me. Thank you for all my friends, for all the memories we built together and for your encouragement. I also want to thank Dennys Cornelio and La ura Waldo for their friendship and support in helping me get my tasks done. Appreciation goes to Curtis Smyder for all his assistance for answering all my questions every semester and for keeping up with all my paperwork. Thank you to Jennifer Dawson for a ll her support and making it easier and possible to participate in many on campus events and to enable my active membership with UF student clubs despite being off campus. I also want to thank Dr. Tim Ebert for all his help and patience with data analysis.

PAGE 5

5 Finally, I thank all the people who enriched my academic and personal experience during this last years and that somehow help ed me to conclude this project. Thank you for all I have learned from you.

PAGE 6

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 RATI ONALE AND SIGNIFICANCE ................................ ................................ ........ 13 2 LITERATURE REVIEW ................................ ................................ .......................... 16 Citrus ................................ ................................ ................................ ...................... 16 Huang longbing (HLB) ................................ ................................ ............................. 17 Current Management Practices ................................ ................................ ........ 21 The Plant Hormone Salicylic Acid and its Physiological Roles in Plants ................. 23 Plant Growth and Development Parameters in Salicylic Acid Studies .............. 26 Summary ................................ ................................ ................................ ................ 29 3 EFFECTS OF FOLIAR APPLICATIONS OF SALICYLATES ON VEGETATIVE SHOOT GROWTH AND OTHER PHYSIOLOGICAL PARAMETERS OF ................................ .................... 33 Introduction ................................ ................................ ................................ ............. 33 Materials and Methods ................................ ................................ ............................ 36 Plant Material and Experimental Conditions ................................ ..................... 36 Sali cylates Treatments ................................ ................................ ..................... 37 Data Collection ................................ ................................ ................................ 38 Leaf gas exchange measurements ................................ ............................ 38 Plant dry weights ................................ ................................ ........................ 38 Nutritional analysis of plant tissues ................................ ............................ 39 Plant growth ................................ ................................ ............................... 39 Electrolyte leakage and leaf sap pH measurements ................................ .. 39 Leaf chlorophyll content ................................ ................................ ............. 40 Experimental Design and Data Analysis ................................ ........................... 41 Results ................................ ................................ ................................ .................... 43 Discussion ................................ ................................ ................................ .............. 43 4 EFFECTS OF FOLIAR A PPLICATIONS OF SALICYLATES ON VEGETATIVE SHOOT GROWTH AND OTHER PHYSIOLOGICAL PARAMETERS OF ................................ ................................ 68

PAGE 7

7 Introduction ................................ ................................ ................................ ............. 68 Materials and Methods ................................ ................................ ............................ 72 Plant Material and Experimental Conditions ................................ ..................... 72 Determination of Healthy Versus HLB Affected Trees ................................ ..... 73 Study Trees Nutritional Status ................................ ................................ .......... 74 Salicylate Treatments ................................ ................................ ....................... 75 Data Collection ................................ ................................ ................................ 75 Bud growth ................................ ................................ ................................ 75 Vegetative growth ................................ ................................ ...................... 75 Elect rolyte leakage and leaf sap pH measurements ................................ .. 76 Experimental Design and Data Analysis ................................ ........................... 76 Results ................................ ................................ ................................ .................... 78 Discussion ................................ ................................ ................................ .............. 80 5 CONCLUSIONS ................................ ................................ ................................ ..... 91 LIST OF REFERENCES ................................ ................................ ............................... 93 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 101

PAGE 8

8 LIST OF TABLES Table page 3 1 Mean number of buds that grew, total shoot length, and total number of ammonium and sodium salicylat e 7 and 6 weeks after treatment (n = 8). .......... 52 3 2 Mean number of buds that grew (number of budbreaks), shoot length, and rates of ammonium and sodium salicylate 42 days after treatment (n = 8). ........ 53 3 3 Tissu applied ammonium and sodium salicylate 7 and 6 weeks after treatment, respectively (n = 8). ................................ ................................ ............................ 54 3 4 Tissue dry weights of applied ammonium and sodium salicylate 6 weeks after treatment (n = 8). ....... 55 3 5 foliar application with ammonium salicylate 7, 28 and 42 days after treatment (n = 8). ........................ 56 3 6 sodium salicylate 7 and 42 days after treatment (n = 8). ................................ .... 57 3 7 of ammonium salicylate at 1, 2, 3, 7 and 21 days after treatment (n=8). ............ 58 3 8 of sodium salicylate at 1, 2, 3, 7 and 21 days after treatment (n=8). .................. 59 3 9 different foliar applied rates of ammonium and sodium salicylate 7 and 6 weeks after treatment, respectively (n = 8). ................................ ........................ 60 3 10 ammonium salicylate 1, 2, 3 and 35 days (5 weeks) after treatment (n = 8). ...... 61 3 11 sodium salicylate 1, 2, 3 and 35 days (5 weeks) after treatment (n = 8). ............ 62 3 12 Mean leaf sap salicylate 1, 2, 3 and 35 days (5 weeks) after treatment (n = 8). ........................ 63 3 13 ar applied sodium salicylate 1, 2, 3 and 35 days (5 weeks) after treatment (n = 8). ........................ 64

PAGE 9

9 4 1 Leaf nutrient concentrations in leaves collected from healthy and HLB affected 'Valencia' trees 28 d after foliar application of sodium salicylate (n=5). ................................ ................................ ................................ .................. 84 4 2 Effect of foliar application of sodium salicylate on the average number of buds that grew on healthy trees and on HLB affected trees of 'Valencia' sweet orange over 21 days following treatment (n=5). ................................ ....... 85 4 3 The effect of foliar applied sodium salicylate on the number of nodes, length and dry weight of new shoots that grew from healthy trees and on HLB affected trees ................ 86 4 4 Effect of foliar application of sodium salicylate on the electrolyte leakage ratio of 'Valencia' trees after 1, 2, 7,14 and 21 days of the treatments (n=5). ............. 87 4 5 The effect of foliar applied sodium salicylate on the sap pH of leaves sampled from healthy trees and fr on HLB af fected trees orange 1 d after treatment (n=5). ................................ ................................ ........ 88

PAGE 10

10 LIST OF FIGURES Figure page 2 1 Salix species. ................................ ................................ ................................ ...... 31 2 2 The SAR process (Taiz and Zeiger, 2010). ................................ ....................... 32 3 1 Plant material.. ................................ ................................ ................................ .... 65 3 2 Cumulative nu applications of salicylate at five different rates.. ................................ .................. 66 3 3 ith foliar applications of salicylate at five different rates.. ................................ ......... 67 4 1 Examples of the study branches selected ................................ .......................... 89 4 2 Examples o f blotchy mottle symptoms of HLB collected from qPCR positive ................................ ............................. 90

PAGE 11

11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SHOOT DEVELOPMENT IN CITRUS PLANTS AS AFFECTED BY SALICYLATES AND HUA N GLONGBING INFECTION By Marina Burani Arouca December 2012 Chair: Timothy M. Spann Cochai r: Rebecca Darnell Major: Horticultural Science s A debilitating disease called huanglongbing (HLB) seriously threatens the Florida citrus industry. All commercial citrus cultivars are susceptible to the presumed causal agent, a fastidious phloem limited b acterium designated Candidatus Liberibacter asiaticus, which is vectored by the Asian citrus psyllid ( Diaphorina citri ). The symptoms are chlorotic blotchy mottling of leaves and tree decline. HLB related phloem plugging interrupts the communication betwee n source and sink organs, and photosynthate transport. This study aimed to understand the effects of salicylates on the growth of citrus plants with and without HLB infection. In other species, salicylates are known to induce systemic acquired resistance; however, in HLB affected citrus trees, salicylates appear to function as plant growth regulators. We hypothesized that salicylates induce development of new vegetative shoots in citrus, which may help HLB affected plants cope with this disease by develop ing new vascular tissue free of the blockage that occurs in response to the infection. A series of

PAGE 12

12 growth chamber and field experiments were conducted to test this hypothesis, with the specific objectives to 1) determine if foliar applications of salicylat es induce vegetative growth; and 2) quantify variation in budbreak and shoot development in citrus plants treated with salicylates. Healthy citrus plants treated with sodium and ammonium salicylate did not consistently respond to treatments. Results from o ur field study with HLB affected citrus were also inconclusive, and generally did not support the hypothesis. This research contributes to our knowledge about salicylates acting as plant growth regulators in citrus, particularly with respect to the host pa thogen relationship of citrus and HLB.

PAGE 13

13 CHAPTER 1 RATIONALE AND SIGNIFICANCE Citrus is one of the highest value crops in the world due to its nutritional and economic importance. The United States, specifically Florida, is the second largest producer of orange juice in the world. A severely debilitating and currently incurable industry. All commercial citrus cultivars are susceptible to the presumed causal agent, a fa stidious phloem limited bacterium designated Candidatus Liberibacter asiaticus, which, in Florida, is vectored by the Asian citrus psyllid, Diaphorina citri Typical symptoms on infected trees are pale yellow leaves and blotchy mottled leaves (variegated c harmful products of the bacteria. HLB infection affects the expression of hundreds of genes, as recent microar ray analysis studies have revealed. Some of these genes are specifically related to the mechanism of sieve pore plugging. The phloem protein 2 (PP2) proteins and callose, for example, are likely to be directly involved in this process. Therefore, phloem pl ugging appears to occur through production of compounds that block the sap flow, which may interrupt the communication between sources and sinks, as well as nutrient transport. Since no cures are available, current management strategies have focused on pre venting infection by reducing inoculum (infected trees), producing pathogen free nursery plants, vector control via chemical pesticide treatments, and nutritional programs to treat symptoms. These tools do not entirely prevent new infections, and

PAGE 14

14 have not eliminated concerns about the economic viability and the future of citrus production in Florida. Although HLB was first reported in Asia over 100 years ago, major research advances have only occurred in the last 5 years. However, the physiological and mole cular processes involved in the bacterium host plant interaction are still unclear. Recent studies focusing on the nutritional approach of managing disease symptoms are trying to determine the effects of the various components of what is commonly referred comprehensive combination of foliar applied nutrient sprays, foliar applied non nutrient products (e.g., phosphite, salicylates, Bacillus subtilis biofungicide, and hydrogen peroxide) intensive psyllid control, as well as the inclusion of often overlooked nutrients in ground applied fertilizer (e.g., calcium, boron, molybdenum). An interesting component of the MBC is the use of salicylates. In other plant species, salicylates are kn own to be inducers of systemic acquired resistance (SAR); however, in HLB affected citrus plants salicylates appear to be acting primarily as plant growth regulators (PGR). In light of this, we hypothesized that foliar applications of salicylates induce de velopment of new vegetative shoots in citrus, which may help HLB affected plants cope with this disease by developing new vascular tissue free of the blockage that occurs in response to the infection. Therefore, experiments were conducted to investigate th e effects of exogenously applied salicylates on healthy and HLB affected citrus plants with the objectives to 1) determine if foliar applications of salicylates induce vegetative growth; and 2) quantify variation in budbreak and shoot development in citrus plants treated with salicylates.

PAGE 15

15 The purpose of this research is to contribute to the current literature with new information about salicylates acting as PGRs in citrus in this host pathogen relationship, and also enhance our understanding of the plant re sponse when infected with Candidatus Liberibacter asiaticus. In addition, the aim of our study is to expand knowledge to improve current horticultural management strategies of this disease and potentially reduce economic damage.

PAGE 16

16 CHAPTER 2 LITERATURE REV IEW Citrus Citrus comprises a large group of plants in the Rutaceae, belonging to the closely related genera Citrus, Fortunella and Poncirus and their interspecific and intergeneric hybrids. Citrus is native to the subtropical and tropical regions of sout h Asia, primarily China and India, and today is grown around the world (Scora, 1975), and the main citrus product, orange juice, is an internationally traded commodity. So Paulo (Brazil) derived from sweet orange ( Citrus sinensis (USDA FAS, 2012). Schneider (1968) once described plants as living entit ies This is a proper term for this genus since leafy twigs, vascular tis sues, and the roots are periodically extended or replaced. These processes lead to the degeneration of some tissues in this perennial tree, after the newly formed ones reach maturity. The general branching system of orange trees produces dense growth, wit h innumerous small branches. In some climates, citrus enters a quiescent state during winter, but they do not shed their leaves. With warmer temperatures during January and February (South Hemisphere), axillary buds begin to break. Actively growing shoots, the (Schneider, 1968). The spring shoots will vary in composition from leafy (no flowers) to a mixture of vegetative and reproductive growth to entirely reproductive (leafless) Flower bearing shoots predominate on mature trees, and sterile shoots predominate on young trees (Sanz et al., 1987). The development of sho o ts in citrus is influenced by

PAGE 17

17 temperature and humidity. Shoot growth usually occurs in two annual well defined f lushes in cold climate s, while in warm tropical or subtropical climate s three to five growth flushes occur Root elongation like shoot growth occurs in flushes, and some researchers have suggested that shoot growth triggers root growth (Goldschmidt and S piegel Roy, 1996). Budbreak and shoot growth are affected by nutrition, hormones, soil temperature, photoperiod and light quality (Reed and MacDougal, 1937; Schneider, 1952, as cited in Schneider, 1968). Huanglongbing (HLB) All commercial citrus cultivars are susceptible to the presumed causal agent of HLB, Candidatus Liberibacter spp., a group of phloem limited bacteria (Folimonova et al., 2009). The natural vectors of this pathogen are two species of widely distributed psyllids, the Asian citrus psyllid ( Diaphorina citri ) and the African citrus psyllid ( Trioza erytreae ) (Achor et al., 2010; Bov, 2006; Brlansky and Rogers, 2007; Etxeberria et al., 2009; Gottwald, 2010; Kim et al, 2009). In Florida, HLB is associated with the Asian strain of the pathogen, C L. asiaticus (CLas), and is vectored by the Asian citrus psyllid (Gottwald et al., 2007) There are different responses among citrus species to HLB. Eureka lemon ( Citrus limononia Osbeck) and Persian lime ( C. aurantifolia Christm. Swingle) show less sever e symptoms and a much slower decline in health compared to Valencia sweet orange ( C. sinensis L. Osbeck) and Duncan and Ruby red grapefruit ( C. paradise MacFadyen) (Folimonova et al., 2009). Prior to symptom expression, HLB can have a variable incubation p eriod that is influenced by tree age and health. By the time symptoms become visible in an infected tree, there can be two to several asymptomatic infections already established in nearby

PAGE 18

18 trees (Gottwald, 2010). This situation poses a serious challenge for disease control. The bacterium and the vector are hosted by several ornamental plant species, further complicating efforts to eradicate and manage the disease (Halbert and Manjunath, 2004). Typical symptoms on infected trees include an overall pale yellow appearance, and a thick leathery texture (Bove, 2006). Typically symptomatic fruit are lopsided and small with aborted seeds, and there are fewer fruit per tree (Ba ssanezi et al., 2009; Bove, 2006). These symptoms may or may not occur together, making identification difficult (Achor et al., 2010; Bov, 2006; Etxeberria et al., 2009; Kim et al., 2009). Tatineni et al. (2008) found that CLas is unevenly distributed in the phloem of bark tissue and in the vascular tissue of leaf midribs, roots, and different parts of flowers and fruit. Recently, the genome sequence of CLas was determined. The genome showed no toxins, extracellular degrading enzymes or specialized secret ion systems (Duan et al., 2009). Thus, the symptoms of HLB appear to arise from plant responses to the bacteria. Unusually high levels of starch accumulation in leaves are considered to be among the causes of these symptoms since disintegration of the chlo roplast thylakoid system could produce the yellowing (Etxeberria et al., 2009; Fan et al., 2010; Schneider, 1968). Fan et al. (2010) compared sucrose, glucose and maltose in symptomatic leaves, and found the first two remained at high levels while maltose content decreased, when compared to levels from asymptomatic leaves.

PAGE 19

19 Etxeberria et al. (2009) suggested that the accumulation of starch denotes an imbalance in carbohydrate source sink relationships and investigated the magnitude of carbohydrate partitioni ng imbalances through the tree. They found that starch accumulated in photosynthetic cells, phloem elements, and vascular parenchyma cells of leaves and petioles. However, roots were depleted of starch, presumably resulting in decline of root function and a subsequent decline in tree health. The importance of sap movement was known since 1758, when Duhamel du Monceau conducted experiments showing that sap movement controlled plant growth and that phloem movement was responsible for root nutrition (Arteca, 1 994). Reduced levels of maltose are also suggested to contribute to starch accumulation in infected leaves, and to downregulate genes DPE2 and MEX1, both of which are starch breakdown related genes (Fan et al., 2010). In several areas of the world affected by HLB, this accumulation of starch has been used as a visual indicator of the disease (Takushi et al., 2007). Leaves are cut with a razor blade and the exposed surface is immersed in an iodine solution, after which the cut surface is observed for the cla ssical black staining (Etxeberria et al., 2009). Additionally, real time PCR (qPCR) is used for HLB detection in plant and insect tissue, although there is no distinction between live and dead bacteria (Li et al., 2006). Achor et al. (2010) described the s equence of events leading to the development elements and companion cells followed by some phloem cell collapse, presumed sugar backup in localized leaf blade areas leading to starch accumulation until chloroplast

PAGE 20

20 filamentous materials obstructed the sieve elements. Callose was probably the amorphous material and phloem protein 2 was ident ified as the filamentous material. The phloem blockage seems to result from plugging of sieve pores by callose and phloem protein and not from bacterial aggregation, since CLas does not occur in high enough quantities to form aggregates in citrus (Achor e t al., 2010). Callose plugs were extensive in sieve elements of leaf vascular tissue from sweet orange trees infected with CLas (Kim et al., 2009). Related genomic studies referred to P proteins 1 and 2, and callose as responsible for sieve tube pore plugg ing during wounding (Dinant et al., 2003; Knoblach and van Bel, 1998). Using microarray analysis, Kim et al. (2009) found hundreds of genes whose expression was affected by HLB infection. Examples include up regulation of ADP glucose pyrophosphorylase, sta rch synthase, granule bound starch synthase, starch debranching enzyme, and four starch biosynthesis genes. These genes appear to play a role in phloem disruption, starch accumulation, and sieve pore plugging. The PP2 gene was also up regulated in parallel with callose formation in phloem sieve tubes. It is possible that PP2 may interact with a variety of putative signaling RNAs, which in turn may interfere with communication between source and sink organs, as well as nutrient transport (Kim et al., 2009). Nutritional imbalances in CLas infected trees were found when leaves from symptomatic and asymptomatic branches, and non infected healthy trees were analyzed and compared by many researchers ( Aubert, 1979; Koen and Langenegger, 1970; Spann and Schumann, 20 09) In general, leaves on infected trees have increased K content while Ca, Mg, B, Mn and Zn content decrease.

PAGE 21

21 Current Management Practices No cure for HLB exists and current management strategies include chemical control to reduce psyllid populations, r emoval of infected trees to eliminate new sources of bacterial inoculum, production of pathogen free nursery plants and nutritional programs to treat visual disease symptoms (Albrecht and Bowman, 2008; Brlansky and Rogers, 2007; Brlansky et al., 2012; Gott wald, 2010). In Florida, there is no law or state enforcement that obliges citrus growers to remove infected trees, whether symptomatic or PCR positive for HLB. As a result, growers generally practice one of two management strategies, removing infected t rees or managing tree health with nutritional programs. Usually, but not always, the first situation applies to growers with low infection rate or below their perceived economic damage threshold, and the second is typically used by growers with high infect ion rates. Meanwhile, the chemical control of Asian Citrus Psyllid (ACP) is understood as key for any HLB management approach. Therefore, Citrus Health Management Areas (CHMAs) were created. CHMAs are groupings of commercial citrus groves in the same reg ion with the objective to work cooperatively to manage ACP at the population level. part of a coordinated ACP control program that will provide guidance on pesticide spr ay application timing and rotation of chemical modes of action, providing long lasting effective ACP control over a large area. Regardless of the effectiveness of insecticides, the movement behavior of ACP between groves makes the control of this insect di fficult without a coordinated program like CHMAs (Boina et al., 2009; Tiwari et al., 2010). Florida currently has 38 established CHMAs, and has observed a reduction of 68% in the incidence of ACP per block between 2011 and 2012 (Rogers, unpublished data).

PAGE 22

22 The nutritional program approach is intended to maintain the productivity of HLB infected trees. Foliar applications of plant nutrients are made using a mixture of nutrients and other various components (Rouse, unpublished data). It is well known that a well balanced nutrition program helps maintain plant health and productivity. At the same time, there is a lack of comprehension about the interaction between nutrition and vascular diseases, along with the fact that each plant disease complex is unique (D atnoff et al., 2007; Rouse, unpublished data; Spann and Schumann, 2009). The concept that leads growers to adhere to this management strategy is related to the plant response to the disease. As abovementioned, the plugging of the phloem that occurs after infection seems to be a response of the plant to the pathogen attack. This plugging leads to an interruption of sap flow, depriving the roots of carbohydrates. This starvation at least partially reduces root function, reducing water and nutrient uptake, ev entually leading to the appearance of leaf symptoms and root system decline. By supplying nutrients via foliar application, the plant seems to be able to temporarily avoid the decline. This supplemental supply could lead to new vascular system/phloem produ ction enabling the plant to maintain productivity despite infection. As mentioned, these foliar applications are often a mixture of nutrients and various other compounds, such as salicylates. The effect of each of the individual components, or the interact ion among them, is still uncertain (Rouse, unpublished data). Equally important are the questions that remain about long term maintenance of the nutrient management strategy and whether this will allow a continuous buildup and spread of inoculum, leading to concerns about future replanting and resetting and the ultimate survival of the Florida citrus industry.

PAGE 23

23 The Plant Hormone Salicylic Acid and its Physiological Roles in Plants In the early 1800s, Julius von Sachs proposed that plants produce substances that move in different directions controlling growth and development (Arteca, 1994; Davies, 2010). In 1951, a committee of scientists established the definition of plant whi ch in low concentrations regulate plant physiological processes Hormones usually move within the plant from a site of production to a site of action (Tukey et al., 1954). More recent studies show that transport is not an essential property of plant hormo nes, since some of them may bring changes within the same tissue or even cell where synthesized (Davies, 2010). Today, it is clear that specific substances have an important regulatory role on plant physiology. Agriculture has been using these substances f or different purposes such as seed germination, induction of rooting, control of fruit development, delaying or promoting ripening and abscission, weed control and many others (Arteca, 1994). Despite these advances, there are still many aspects of plant gr owth regulators to be learned in order to realize the full potential of integrating plant growth regulators into modern agro technology. Salicylic acid (ortho hydroxybenzoic acid) was identified in the 19 th century as the active component in the bark extr act of the willow tree and named for the willow genus, Salix (Figure 2 1). Chewing the bark of this tree was known as a means to relieve headaches and fevers (Arteca, 1994; Delaney, 2010; Pierpoint, 1997). In 1898, Bayer Co. introduced aspirin, the trade n ame for acetylsalicylic acid. Despite the fact that this compound is not naturally found in plants, it is readily converted into salicylic acid (SA) in aqueous systems (Raskin, 1992a). It was only recently found that this important signal molecule plays a role in many physiological processes such as flower induction,

PAGE 24

24 stomatal closure, heat production, defense against pathogen attack and growth induction (Raskin, 1992a, 1992b; Raskin et al., 1989). SA is found well distributed throughout the plant kingdom, a nd it has been identified in more than 34 plant species (Arteca, 1994). Differently from animals, plants are sessile organisms. This characteristic implies that these organisms have had to develop survival strategies other than movement to defend themselve s, compete and survive. Among these strategies are secondary metabolites, which help plants to defend themselves from herbivores and pathogens. Secondary metabolites are divided into three chemically distinct groups: phenolics, terpenes and nitrogen conta ining compounds. SA is an organic compound in the phenolic class. Phenolic compounds contain a phenol group, a hydroxyl functional group on an aromatic ring (Taiz and Zeiger, 2010). Salicylates are a class of compounds that have similar activity to SA (Art eca, 1994). In plants, SA biosynthesis was thought to be by the phenylpropanoid pathway (Verberne et al., 1999). However, more recent studies concluded that the biosynthesis of this compound is not fully defined and multiple biosynthetic pathways have been proposed, such as cinnamate and from isochorismate (Buchanan et al., 2000; Chen et al., 2009; Delaney, 2010; Shah, 2003). In a diverse phylogenetic range of plants, SA was identified in leaves and reproductive structures (Arteca, 1994). Levels typically f ound in leaves range from 0.05 to 5.0 g SA per gram of leaf tissue, and in thermogenic species it was observed at nearly 100 g SA per gram of reproductive tissue (Delaney, 2010).

PAGE 25

25 SA is mainly known by its involvement in plant defense strategies. This hormone is one of several compounds that is stimulated after a pathogen attack, as an important factor in systemic acquired resistance (SAR) (Figure 2 2), and also in local hypersensitive response (HR) (Gaffney et al., 1993; Verberne et al., 2000). Briefly, SAR is developed when a plant responds to an infection, increasing resistance to subsequent attacks. Resistance is extended to plant tissues distant from the initial infection site, can persist for weeks or months, and is effective against secondary infection by a broad spectrum of pathogens. O n the other hand, HR is the process in which cells surrounding the infection site die rapidly, in an attempt to block the spread of the pathogen and to deprive it of nutrients (Gaffney et al., 1993; Taiz and Zeiger, 2010). The application of exogenous SA can induce SAR, in accordance with the belief that SA is one of the endogenous signals to trigger SAR (Malamy el al., 1990; Metraux et al., 1990; White, 1979). SA can be transported over a long distance and via the vascular system to organs located far fro m the infection site (Shulaev et al., 1995); however, grafting and kinetic studies both showed evidence that SA is not the systemic signal in SAR, but is required in signal transduction. The signaling precedes the accumulation of SA in the plant (Delaney, 2010; Rasmussen et al., 1991; Vernooij et al., 1994). Furthermore, SA participates in the regulation of plant responses to abiotic stresses, such as low and high temperature, salt stress and oxidative conditions (Dat et al., 1998; Gunes et al., 2007; Janda et al., 1999; Larkindale and Knight, 2002; Larkindale et al., 2005; Mann et al., 2011; Shakirova, 2007; Yalpani et al, 1994).

PAGE 26

26 SA has also been identified as a signal initiator of the thermogenic process in the voodoo lily ( Sauromatum guttatum) During ant hesis, the temperature of the spadix can increase by 14 C. This process is initiated during floral development as a pathway of alternative oxidase, a nonphosphorylating mechanism. Therefore, at least in some Arum lilies, SA functions as an endogenous regulator of heat production (Buchanan et al., 2000; Delaney 2010; Raskin et al., 1987). The role of SA as a phytohormone in plant growth and development has also been studied, but most of these responses remain unknown. SA has been found to contribute to the regulation of processes such as seed germination, vege tative growth, photosynthesis, respiration, membrane permeability, enzyme activity, uptake of ions, thermogenesis, flower formation, seed production, senescence, and cell death (independent of HR) across a wide range of species (Hayat and Ahmad, 2007; Kutt imani and Velayutham, 2011; Raskin, 1992a; Rivas San Vicente and Plasencia, 2011; Singh et al., 2010). Plant Growth and Development Parameters in Salicylic Acid Studies Salicylic acid ef fects on plant growth have been tested by numerous authors. Shoot gro wth was promoted in a variety of plants; most notably in corn (maize) (El Khallal et al., 2009; Gautam and Singh, 2009; Khodary, 2004), cucumber (Singh et al., 2010), Clitoria (Martin Mex and Larqu Saavedra, 2001) onion (Amin et al., 2007), as well as ma ize under salt stress (El Khallal et al., 2009; Gautam and Singh, 2009; Khodary, 2004). HLB affected c itrus plants exposed to biotic and abiotic stresses such as heat, cold and disease stress were treated with sodium salicylate ranging from 0.10 to 0.18 mM resulting in young foliage growth when compared to the HLB affected control plants, three weeks after SA treatment. Flowering was also induced in the citrus

PAGE 27

27 plants treated with 0.10 mM of NaSA (Mann et al., 2011). Foliar applications of SA also resulted i n in crease of vegetative growth of 3 month old Pinus patula seedlings After nine monthly treatments with 10 10 and 10 8 M of SA plant height stem diameter and stem FW and DW increased when compared to untreated controls (San Miguel and Gutierrez, 2003 ). Leaf area and number increased in maize, wheat and onion treated with SA and corn and soybean treated with acetyl salicylic acid (Amin et al., 2007; El Khallal et al., 2009; Gautam and Singh, 2009; Hayat et al., 2005; Hussein et al., 2007; Khan et al., 2003) Dry mass increased in SA treated maize, Brassica juncea soybean, rosemary, barley, Pinus cucumber, wheat, onion, cowpea and groundnut compared to untreated controls (Amin et al., 2007; Chandra et al., 2007; El Khallal et al., 2009; Fariduddim et a l., 2003; Gautam and Singh, 2009; Hussein et al., 2007; Jayalakshmi et al., 2010; Khan et al., 2003; Khodary, 2004; Najafian et al., 2009; Pancheva et al, 1996; San Miguel and Gutierrez, 2003; Singh et al., 2010; Singh and Usha, 2003). Height increase wa s promoted by SA in maize, Pinus onion and ground nut (Amin et al., 2007; Hussein et al., 2007; Jayalakshmi et al., 2010; San Miguel and Gutierrez, 2003). Root length increase s with SA treatments in a variety of plants, such as Pinus and soybean (Gutierr ez Coronado et al., 1998; San Miguel and Gutierrez, 2003). San Miguel and Gutierrez (2003) found root length increased ~ 30% in pine when SA was applied They also found a 33% increase in root FW and a 45 to 54% increase in root DW Foliar applications of 10 8 10 4 and 10 2 M SA in soybean seedlings significantly

PAGE 28

28 increased the root after seven days of treatment in greenhouse and field conditions (Gutierrez Coronado et al., 1998). A decrease in net photosynthetic rate (Pn) and RuBisCO activity was observ ed in SA treated barley (Pancheva et al., 1996), but an increase in photosynthesis was found in Brassica juncea and B. napus sunflower, barley, and grapevine, and an increase in both Pn and RuBisCO activity was found in corn, wheat and cowpea (Arfan et a l., 2007; Chandra et al., 2007; Fariduddin et al., 2003; Ghai et al., 2002; Khan et al., 2003; Khodary, 2004; Noreens and Ashraf, 2008; Singh and Usha, 2003; Wang et al., 2010). Transpiration rate increase d when corn and groundnut were treated with SA (Kha n et al., 2003; Jayalakshmi et al., 2010). Applications of SA d ecrease d chlorophyll in Vigna mungo wheat, mung plants and barley (Anandhi and Ramanujam, 1997; Moharekar et al., 2003; Pancheva et al., 1996). On the other hand, there was increase in chloro phyll content in Brassica napus wheat, maize, Brassica juncea cucumber, onion and groundnut (Amin et al., 2007; El Khallal et al., 2009; Fariduddin et al., 2003; Gautam and Singh, 2009; Ghai et al., 2002; Hayat et al., 2005; Jayalakshmi et al., 2010; Kho dary, 2004; Purcarea and Cachita Cosma, 2007; Singh et al., 2010; Singh and Usha, 2003). Levels of starch and soluble sugars increased in SA treated maize, cucumber, onion, cowpea and mung bean (Amin et al., 2007; Chandra et al., 2007; El Khallal et al., 2 009; Kuttimani and Velayutham, 2011; Singh et al., 2010). Seed germination was enhanced in cucumber (Singh et al., 2010) however it was inhibited when a higher rate of SA was applied in cowpea (Chandra et al., 2007).

PAGE 29

29 An increase in yield was reported after SA treatments in Vigna mungo (Anandhi and Ramanujam, 1997), wheat (Arfan et al., 2007), onions (Amin et al., 2007), groundnut (Jayalakshmi et al., 2010), and mung bean (Kuttimani and Velayutham, 2011). Summary Candidatus Liberibacter asiaticus (C Las) is the presumed causual agent of a plant pathogen that threatens the Florida citrus industry. It is vectored by the Asian citrus psyllid ( Diaphorina citri ), and there are no known resistant commercial citrus cultivars to either the pathogen or the vector. The disease is a fastidious phloem limited bacterium that causes blotchy mottling or variegated chlorosis of foliage, and tree decline. According to studies using microarray analysis, the up regulation of genes involved with sieve pore plugging leads to these symptoms in HLB infected trees. The current management strategies focus on preventing new infections by reducing the inoculum (removing infected trees), production and planting of certified pathogen free nursery plants, vector control, and intensive nutri tional programs. Salicylates are common ingredients found in the foliar sprays of citrus nutritional progra ms. The purpose of this study was to better understand the effects of salicylates on important growth parameters of citrus trees, to contribute to th e current literature about salicylates use in citrus, and to improve current disease management strategies. Recently, salicylic acid has been the focus of research related to its function on systemic plant defenses to pathogens attacks, and its function a s an endogenous signaling molecule. Furthermore, efforts have been made to understand the role of SA during plant respon ses to abiotic stresses such as heat, chilling, drought, osmotic stress and heavy metal toxicity. In addition to stress responses, SA is a plant hormone that

PAGE 30

30 contributes to growth and development regulation, although most of the mechanisms that mediate these responses are remain mostly unknown. Physiological and biochemical responses to exogenously applied SA differ depending on the dose, plant species and developmental stage. The research presented here provide s basic information about physiological responses of young citrus trees to exogenously applied salicylic acid.

PAGE 31

31 Figure 2 1. Salix species. A) Salix b abylonica Photo by Living Afie ld B) S. alba by Prof. Dr. Otto Wilhelm Thom Flora von Deutschland, sterreich und der Schweiz 1885, Gera, Germany. A B

PAGE 32

32 Figure 2 2. SAR. From the infection site, SAR is transmitted through the phloem to other parts of the plant. SA and met hyl Salicylate (MeSA) increase significantly on this process and cause the production of pathogenesis related (PR) proteins. MeSA is often released and may serve as SAR inducing volatile signal in neighboring plants. ( Photo by Taiz and Zeiger, 2010).

PAGE 33

33 CHAPTER 3 E FFECTS OF FOLIAR APPLICATIONS OF SALICYLATES ON VEGETATIVE SHOOT Introduction Citrus production in Florida is an intensive activity due to the amount of pests, diseases and cultural practices that are necessary to keep productivity high. The disease of greatest threat to the Florida citrus industry today is Huanglongbing (HLB), a disease associated with a fastidious phloem limited bacterium, Candidatus Liberibacter asiaticus (CLas) (Folimonova et al., 2009). Typical symptoms on infected trees include an overall pale yellow appearance, with leaves displaying asymmetrical blotchy mottle 2006). Typically symptomatic fruit are lopsided and small with aborted seeds, and there are fewer fruit per tree (Bassanezi et al., 2006; Bove, 2006). These symptoms may or may not occur together, making identification difficult (Bov, 2006; Etxeberria et al., 20 09; Achor et al., 2010; Kim et al., 2009). The natural vector of this pathogen in Florida is the Asian citrus psyllid ( Diaphorina citri ) (Gottwald et al., 2007). Tatineni et al. (2008) found that CLas is unevenly distributed in the phloem of bark tissue a nd in the vascular tissue of leaf midribs, roots, and different parts of flowers and fruit. Recently, the genome sequence of CLas was determined. The genome showed no toxins, extracellular degrading enzymes or specialized secretion systems (Duan et al., 20 09). Thus, the symptoms of HLB appear to arise from plant responses to the bacteria. Achor et al. (2010) described the sequence of events leading to the of sieve elements and companion cells followed by some phloem cell collapse,

PAGE 34

34 presumed sugar backup in localized leaf blade areas leading to starch accumulation phloem blockage seems to res ult from plugging of sieve pores by callose and phloem protein and not from bacterial aggregation, since CLas does not occur in high enough quantities to form aggregates in citrus (Achor et al., 2010). No cure for HLB exists and current management strategi es include intensive pesticide use to reduce psyllid populations, removal of infected trees to eliminate bacterial inoculum, production of pathogen free nursery plants and nutritional programs to treat visual disease symptoms (Albrecht and Bowman, 2008; Br lansky and Rogers, 2007; Brlansky et al., 2012; Gottwald, 2010;). The nutritional program approach is intended to maintain the productivity of HLB infected trees. Foliar applications of plant nutrients are made using a mixture of nutrients and other vario us components (Rouse, unpublished data). It is well known that a well balanced nutrition program helps maintain plant health and productivity. At the same time, there is a lack of comprehension about the interaction between nutrition and vascular diseases, along with the fact that each plant disease complex is unique (Datnoff et al., 2007; Spann and Schumann, 2009). The concept that leads growers to adhere to this management strategy is related to the plant response to the disease. As abovementioned, the p lugging of the phloem system that occurs after infection seems to be a response of the plant to the pathogen attack. Presumably, this plugging leads to an interruption of sap flow, depriving the roots of carbohydrates. This starvation at least partially re duces root function, reducing water and nutrient uptake, eventually leading to the appearance of leaf symptoms and root

PAGE 35

35 system decline. By supplying nutrients via foliar application, the plant appears to be able to temporarily avoid the decline typically a ssociated with HLB. This supplemental supply could lead to new vascular system/phloem production enabling the plant to maintain productivity despite infection. As mentioned, these foliar applications are often a mixture of nutrients and various other compo unds, such as salicylate compounds, often referred to by the generic, although inaccurate, term salicylic acid (SA). The effect of each of the individual components, or the interaction among them, is still uncertain (Rouse, unpublished data). Various physi ological and biochemical effects of SA and salicylates have been documented in numerous crops (Raskin, 1992a) placing these compounds in the broad category of compounds commonly referred to as plant growth regulators (PGRs). The responses to SA and salicyl ates in citrus are generally unknown. HLB affected c itrus plants exposed to biotic and abiotic stresses such as heat, cold and disease (HLB) were exogenously treated with sodium salicylate ranging from 0.10 to 0.18 mM concentrations, resulting in new foli age growth on HLB affected citrus trees three weeks after SA treatment. Flowering was also induced in the citrus plants treated with 0.10 mM of sodium salicylate (Mann et al., 2011). We hypothesized that foliar applications of salicylates induce developmen t of new vegetative shoots in citrus, which may help HLB affected plants cope with this disease by developing new vascular tissue free of the blockage that occurs in response to the infection. Therefore, experiments were conducted to investigate the effect s of exogenously applied salicylates on citrus plants with the objectives to 1) determine if

PAGE 36

36 foliar applications of salicylates induce vegetative growth; and 2) quantify variation in budbreak and shoot development in citrus plants treated with salicylates. Materials and Methods Plant Material and Experimental Conditions Two experiments were conducted to test the effects of salicylates on budbreak and shoot growth of healthy non HLB affected sweet orange. The first experiment was conducted during autumn 2011 and used 1 year ( Citrus sinensis (L.) Osbeck ) 1A). The second experiment was conducted during spring 2012 and used 8 month 1B). Trees for both experiments were obtained from a commercial citrus nursery (Southern Citrus Nursery; Dundee, FL). All trees were grown in a standard commercial soilless potting medium consisting of peat, pine bark and pe rlite and were grown in 2.8 L citra pots (model controls lants and controls ). Plants were fertilized at the nursery using a slow release complete fertilizer. No additional fertilization was provided for the duration of the experiment. Plants were watered 3 times per week as needed. For both experiments, the pl ants were grown in custom built walk in growth chambers (Mechanical Refrigeration, Winter Haven, FL) at the University of Florida, Citrus Research and Education Center in Lake Alfred, Florida. Plants were placed on 70 cm high benches approximately 1.5 m fr om the light source and allowed to acclimate for about one week prior to treatment applications. Temperature and relative humidity were

PAGE 37

37 recorded at the top of the plant canopy every 15 min for the duration of the experiment (WatchDog 1000 series micro stat ion; Spectrum Technologies, Plainfield, IL). Average temperature was 31/25 C day/night with 58% average relative humidity (RH). Photosynthetically active radiation ( PAR m 2 s 1 with a photoperiod of 14 h. Salicylates Treatments For both experiments, plants were treated with either sodium salicylate (SIGMA ALDRICH, St. Louis, MO) or ammonium salicylate (MP Biomedicals, Solon, OH). For both c ompounds plants were treated foliarly with 0, 0.10, 0.15, 0.20, and 0.25 mM solutions. The salicylates and rates were selected based on previous experiments that showed efficacy on citrus within this range (Mann et al., 2011). Spray solutions of both salic ylates were made using laboratory grade chemicals mixed in deionized water (DI). Control (0 mM) plants were sprayed with DI water only. Treatments were applied as foliar sprays to runoff (~175 mL per plant), coating upper and lower leaf surfaces, using a 1 .5 L pump up hand sprayer. The soil surface was covered during spraying to avoid soil contamination and possible root uptake of the dripping solution. All plants were removed from the growth chamber during spray application and until spray solutions dried ( ca 1 hour) wee ks after treatment.

PAGE 38

38 Data Collection Leaf gas exchange measurements To assess the effect of the salicylate treatments on photosynthesis in both experiments, net CO 2 assimilation was measured on one fully expanded mature leaf on each plant using a portabl e photosynthesis system (LI 6400XT; LI COR, Lincoln, NE) fitted with a 6 cm 2 broadleaf chamber (model 6400 08; LI COR) with an LED light source (model 6400 02B; LI COR). The light source set to match the PAR levels (250 mol m 2 s ) of the growth chamber during measurements Net photosynthetic rate (Pn), stomatal conductance ( g s ) and stomatal CO 2 concentration (Ci) were recorded. made 1 and 6 weeks after treatment; measurements on ammonium salicylate treated measurements were made a t 24, 48 and 72 h after treatment to monitor short term responses and 1, 3 and 6 weeks after treatment, for both sodium and ammonium salicylate treated plants. Plant dry weights Plants were destructively harvested at the end of both experiments. Plant or gans (leaves, stems, and roots) were collected for dry weight determinations. The leaves were washed to remove surface contamination by rinsing in tap water, followed by a brief soak in deionized (DI) water with Liquinox soap (Alconox; White Plains, NY), followed by a rinse in clean DI water, followed by a 1 minute soak in a 5% hydrochloric acid solution, followed by a final rinse in DI water. Roots were separated from the rooting medium by carefully washing them in tap water. Tissue samples were oven

PAGE 39

39 dri ed at 65 C for 5 days to a constant weight, and leaf, stem, root and whole plant dry weights were determined. Nutritional analysis of leaf tissue Samples (2 g) of the washed, dried leaves were sent to a commercial laboratory for complete nutrient analysis (nitrogen, phosphorus, potassium, magnesium, calcium, sulfur, boron, zinc, manganese, iron, and copper; Waters Agricultural Laboratories, Inc. Camilla, GA). Plant growth For both experiments total shoot length (sum of the main stem and all lateral branche s), number of nodes and bud growth (number of bud break) were recorded. recorded prior to treatment and at the end of the experiment, and new bud growth (budbreak) was recorded after 3 days of treatment followed by weekly assessments thereafter. Electrolyte leakage and leaf sap pH measurements e collected for cell membrane permeability (assessed as electrolyte leakage) and leaf sap pH using the methodology described by Sairam et al. (1997), with the following adaptations. Leaves were rinsed in tap water, followed by a wash in deionized (DI) wate r with Liqui nox soap ( Alconox ), and a final rinse in DI water. Leaves were dried using paper towels and two punches of 6.35 mm diameter per leaf were collected (12 discs per plant total). Leaf discs were rinsed with DI water three times and surface dried with Kimwipes (Kimtech; Louisville, KY) before being placed into glass vials containing 15 mL of DI water. The

PAGE 40

40 vials were covered with plastic caps and placed in a forced air oven for 30 min at 40 C. Vials were removed and allowed to cool to room temperature. After cooling, vials were vortexed and electrical conductivity (EC1) (Corning pH ion meter 450; Corning, NY) and pH (Accumet pH, ion conductivity meter 50; Hampton, NH) were measured. The vial s were then heated in a temperature controlled water bath for 20 min at 95 C. After heating, vials were removed and allowed to stand at room temperature overnight. The vials were then vortexed and electric conductivity (EC2) was measured again. Electrolyt e leakage was calculated as the ratio of EC1 to EC2. mature leaves were selected on each replicate for cell membrane permeability and leaf sap pH assessments at 24 and 72 hours after treatment, followed by weekly assessments. Modifications on the methodology described by Sairam et al. (1997) led timings, two punches per leaf were collected resulting in three leaf discs of 6.35 mm diameter. Leaf discs were rinsed with DI water three times and surface dried with Kimwipes before being placed into glass vials containing 15 mL of DI water and covered with lids. After 24 hours, samples were stirred and ele ctrical conductivity (EC1) and pH were measured. Samples were then autoclaved for 20 minutes (121 C and 138 kPa) and allowed to cool and stand at room temperature for 24 hours. Samples were then vortexed and EC2 was measured. Electrolyte leakage was calculated as previously described. Leaf chlorophyll content ments, three fully expanded mature leaves were selected on each plant for the determination of chlorophyll content at 1, 2,

PAGE 41

41 and 3 days after treatment and also at 1, 2, 3, 4 and 5 weeks after the treatment. At each sampling time one punch per leaf ( 6.35 mm diameter) was collected (3 leaf discs per plant total). L eaf discs were placed into glass vials and 2 mL of N, N Dimethylformamide was added. The sample vials were covered with aluminum foil to exclude light and allowed to stand at room temperature for on e week. At the end of one week, samples were vortexed and a 1.5 mL aliquot was transferred to a quartz cuvette. Absorbance was read at 647 and 664 nm (GENESYS 10S UV Visible, Thermo Fisher Scientific, Waltham, MA). Chlorophyll concentration was calculated using the equations of Inskeep and Bloom (1985): Chlorophyll b =20.70 A 647 4.62 A 664 Chlorophyll a= 12.70 A 644 2.79 A 647 Total Chlorophyll = 17.90 A 647 + 8.08 A 664 Where A 647 = absorbance at 647 nm (maximum for chlorophyll b ), and A 664 absorbance at 66 4 nm (maximum for chlorophyll a ). Experimental Design and Data Analysis with one factor, salicylate treatment. There were eight single plant replicates for each concentration and source. Some responses were sampled over time and/or from different plant tissues. Therefore, other independent variables were added to the statistical model, as sampling design above described for each response. The complete model was response = salicylat e (5 levels) + time + plant tissue (3 levels), when time and plant tissue applies. Data from this study were analyzed as a one factor linear model with heterogeneous erro rs CRD and repeated measures The linear mixed model was

PAGE 42

42 analyzed using SAS (Version 9 .3; SAS Institute, Cary, NC) procedures: GLIMMIX, GLM and UNIVARIATE. Treatment factor was analyzed as a categorical factor. Significant differences were taken in account if the P value of the F test for the model effects was significant. Tukey Kramer mult performed in cases where models were significant by F test. For some variables, residuals did not accurately follow the normal distribution so the P values of the GOF (goodness of fit tests, e.g., the Shapiro Wilk test) were taken into consideration as criteria for data transformation (e.g. log, square root). Transformation was accepted in the cases that there was an improvement of the GOF test results, meaning that standardized errors are more normally distributed. experiments with only one factor, the salicylate treatment, and blocked for initial plant size. There were eight single plant replicates for each concentration and source. Data from this study were analyzed as a one factor randomized complete block design with fixed blocks (nuisance variable) and repeated measurements. The model was analyzed using the following SAS (Version 9.3; SAS Institute) procedures: GLIMMIX, GLM and UNIVARIATE. The treatme nt factor was analyzed as a categorical factor. Some responses were sampled over time and/or from different plant tissues (leaves, stem, roots). Therefore, other independent variables were added to the statistical model, as sampling design above described for each response. The complete model was response = salicylat e (5 levels) + time + plant tissue (3 levels), where time and plant tissue applied.

PAGE 43

43 Significant differences were taken in account if the P value of the F test for the model effects was significa nt. Tukey in cases where models were significant by F accurately follow the normal distribution so the P values of the GOF (goodness of fit tests, e.g., the Shapiro Wi lk test) were taken into consideration as criteria for data transformation (e.g. log, square root). Transformation was accepted in the cases that there was an improvement at the GOF tests results, meaning that standardized errors are more normally distribu ted. Results buds grow compared with control plants and those treated with 0.10 or 0.15 mM rates (Table 3 1). However, the total growth from the buds that grew following treatm ent with ammonium salicylate was similar for all treatment s when assessed as either length or number of nodes (Table 3 1). T he rate of bud break during the first two weeks following treatment appears greater in plants treated with 0.20 and 0.25 mM ammonium salicylate ( Figure 3 2A ) ; however this was not assessed statistically. No significant differences were found for budbreak shoot length or node sodium salicylate (Table 3 1). Likewise, there did not appear to be any affect of sodium 2B). N either salicylate treatment affected budbreak or final shoot length plants (Table 3 2). However, all treatments increased the final number of nodes per increase the rate of bud break, but may have actually delayed it; although treated plants did surpass control plants during the second week after treatm ent (Figure 3 3).

PAGE 44

44 (Table 3 4). Salicylate treatments did not affect leaf nutrient concentration in exception of manganese, which was significantly higher for 0.10 mM sodium salicylate treated plants compared with all other treatments. Similarly, there were no significant differences in the leaf nutrient concentration i with the exception of zin c, which was significantly lower for 0.15 mM compared with 0.25 mM sodium salicylate treated plants (data not shown). There was a significant interaction of treatment x time for photosynthetic rate of e 3 5 ). Photosynthetic rates generally declined over time regardless of treatment, but declined less at the highest ammonium salicylate rates. However, the highest salicylate rates also had the lowest photosynthetic rates at the first measurement 7 days a fter treatment (DAT). For time, but the main effects of both treatment and time were significant (Table 3 6 ). Photosynthetic rates were lower 42 DAT than at 7 DAT. At bo th measurements dates, 0.15 mM treated plants had the lowest photosynthetic rate, which was significantly lower compared with 0.25 mM treated plants. treated with ammonium sali cylate (Table 3 7 ). Photosynthetic rates increased slightly from 1 DAT to 2, 3 and 7 DAT, but then declined at 21 DAT. Plants treated with 0.10 mM ammonium salicylate had the highest photosynthetic rate at 1 DAT and generally maintained a higher rate compa red with the other treatments through 21 DAT. There

PAGE 45

45 was also sodium salicylate (Table 3 8 ). Similar to what was observed for ammonium salicylate, photosynthetic rates of sodiu m salicylate treated plants increased from 1 DAT to 2 DAT, but then slowly declined to 21 DAT. Plants treated with 0.15 and 0.25 mM sodium salicylate had the highest initial photosynthetic rates at 1 and 2 DAT; however, at 7 and 21 DAT 0.25 mM treated pla nts had the highest photosynthetic rates. concentrations among treatment s for either ammonium or sodium salicylate at any sampling time, with the exception of ammonium salicylate at 35 D AT, when control plants had significantly higher chlorophyll concentration than 0.15, 0.20 and 0.25 mM rates (data not shown). There was no effect of salicylate treatment on electrolyte leakage in plants (Table 3 h 0.25 mM sodium salicylate exhibited significantly higher leaf sap pH compared to all the other treatments. There was a significant interaction of treatment x time for electrolyte leakage in 10). Electrolyte leakage generally declined for all treatments from 1 DAT to 2 DAT, but plants treated with 0.10 and 0.15 mM increased again at 3 DAT. All treatments were similar at 35 DAT (Table 3 10). There was also a significant interaction of treatment x t ime for electrolyte leakage in 11). Plants treated with 0.10 and 0.15 mM sodium salicylate had higher electrolyte leakage than control s or 0.20 and 0.25 mM treated plants at 1 DAT, but declined to s imilar levels as the other treatments at 2 DAT. All treatments were similar at 35 DAT.

PAGE 46

46 There was a significant interaction of treatment x time for leaf sap pH for 12). Plants treated with 0.15 mM ammonium salicylate had the lowest pH 1 DAT and leaf sap pH declined significantly by 2 DAT. Leaf sap pH of p lants treated with 0.10, 0.15 and 0.20 mM ammonium salicylate generally declined from 1 DAT to 3 DAT and were significantly lower than control or 0.25 mM treatments at 3 DAT. There were no significant differences among treatments by 35 DAT. There was a significant interaction of (Table 3 13). Plants treated with 0 .10, 0.15 and 0.25 mM sodium salicylate had lower pH compared to the control and 0.20 mM treated plants at 1 DAT. Leaf sap pH in 0.10 and 0.15 mM treated plants increased and was similar to control plants at 2 and 3 DAT. Plants treated with 0.25 mM sodium salicylate declined further at 3 DAT. At 35 DAT all treatments had similar leaf sap pH. Discussion Foliar applications of salicylates increased budbreak in node number in compared with controls These responses oc curred primarily during the first two weeks after treatment. Similarly, Mann et al. (2011) showed that HLB affected citrus plants treated with sodium salicylate resulted in young foliage growth three weeks after treatment. Although no differences were obse rved i n final shoot length in our experiments, foliar applications of salicylic acid resulted in increased vegetative growth of 3 month old Pinus patula seedlings; however, treatments were applied monthly for 9 months (San Miguel and Gutierrez, 2003). An i ncrease in shoot length was also shown in 1 month old groundnut plants treated foliarly with salicylic acid; however, the rate that plants responded the most was 200 mg/L, much greater

PAGE 47

47 than the rates we worked with (Jayalakshmi, 2010). Shoot length also in creased in salicylic acid treated corn plants that were under salt stress (El Khallal et al., 2009; Gautam and Singh, 2009; Hussein et al., 2007; Khodary, 2004). Height of young cowpea plants responded negatively to treatments of 2.5 mM of salicylic acid ( Chandra et al., 2007), also a much greater rate than used in our study. Tissue dry weights did not increase in the current research as a result of ammonium or sodium salicylate treatments. However, when salicylic acid was applied to pine seedlings at 10 8 and 10 6 M stem dry weight increased by 50% compared to untreated controls (San Miguel and Gutierrez, 2003). Dry mass also increased with salicylic acid treatments compared to untreated controls in maize, Brassica juncea soybean, onion and groundnut (Ami n et al., 2007; Fariduddim et al., 2003; Jayalakshmi et al., 2010; Khan et al., 2003). However, all the plants in these studies were young seedlings when treated compared to the 1 year old plants used in our study. Although sodium salicylate affected the level of manganese at one rate in truly treatment related. However, there is evidence that salicylic acid can affect the uptake of nutrients. Maize plants treated wit h 0.01 to 1.0 mM of Ca(NO 3 ) 2 in association with salicylic acid had greater uptake of nitrogen and activity of nitrate reductase both in leaves and roots (Jain and Srivastava, 1981). Cucumber cotyledons treated with 0.05 mM of salicylic acid also showed a greater assimilation of nitrate, 14 days after treatment (Singh et al., 2010). soap for two spotted spider mite control at approximately 21 and 28 DAT. The mite

PAGE 48

48 infestation and the soap application may explain the decrease observed in photosynthetic rate beyond 7 DAT. Similarly, a decrease in net photosynthetic rate 6 and 24 hours after treatment was observed in salicylic acid treated (0.1 to 1 mM) 7 day old barley (Pancheva et al., 1996). Conversely, an increase in photosynthetic rate was found in Brassica juncea, corn and soybean when treated with salicylic acid. The 1 month old B. juncea plants were foliarly treated with 10 5 M of salicylic acid and the increase in photosynth etic rates was observed one month after treatment (Fariduddim et al., 2003). The study with corn and soybean plants record ed an increase in photosynthetic rates when foliarly treated with 10 5 M salicylic acid and also when treated with the same rate of ac etyl salicylic acid (Khan et al., 2003). Corn and sunflower under salt stress, wheat under water stress and grapevine under heat stress treated with salicylic acid also showed an increase in photosynthetic rate (Khodary, 2004; Noreens and Ashraf, 2008; Sin gh and Usha, 2003; Wang et al., 2010). A slight increase in photosynthetic rate was observed i ammonium salicylate over 1 to 7 DAT, but then declined at 21 DAT. This could be a short term effect of the salicylate treatment. Similar to what was observed for plants were higher 1 and 2 DAT compared to untreated plants, but then slowly declined to 21 DAT. Although there were some effects of salicyl ate treatments on photosynthetic rates, these effects were not due to changes in chlorophyll content as no biologically meaningful changes were detected. This is in contrast to onion seedlings, which had an increase in total chlorophyll when relatively hig h doses of salicylic acid (50 to 200 mg/L)

PAGE 49

49 were applied foliarly (Amin et al., 2007). Jayalakshmi et al. (2010) also measured an increase in chlorophyll content in leaves of one month old groundnut plants treated with 100 to 400 mg/L of salicylic acid. Con versely, 7 day old barley plants treated with 0.1 to 1 mM salicylic acid applied through the transpiration stream for two hours showed a decrease in total chlorophyll content (Pancheva et al., 1996). It is known that changes in the plasma membrane integrit y of the cell are one of the first responses observed after treatments with salicylic acid (Hayat and Ahmad, 2007). Electrolyte leakage was used as a measure of membrane permeability to study if salicylate treatment affected membrane integrity. Our study r evealed significant with ammonium salicylate had lower electrolyte leakage for all treatments at 2 DAT compared to untreated controls, but this affect was transient a nd largely gone by 3 DAT. higher electrolyte leakage than control or 0.20 and 0.25 mM plants at 1 DAT. But again, this affect was transient and gone at 2 DAT. There did n ot appear to be a consistent correlation between salicylate treatment and electrolyte leakage. Similarly, Mann et al. (2011) did not find significant differences among sodium salicylate treatments (0, 0.10, 0.14, 0.18 and 0.22 mM) on HLB affected 2 year ol measured over a 14 day period after treatment. Rosemary cuttings under salt stress were treated with a series of foliar applications of high rates of salicylic acid (150 to 450 mg/L) over a 150 day period and a decrease i n electrolyte leakage was measured (Najafian et al., 2009).

PAGE 50

50 Mann et al. (2011) studied the effects of sodium salicylate treatments on HLB affected 2 year time, with the largest incre ase 14 DAT on HLB affected trees treated with 0.14 mM of sodium salicylate had significantly higher leaf sap pH compared to all the other treatments 35 DAT. This is in co with ammonium and sodium salicylate, which generally had lower leaf sap pH compared with control trees from 1 to 3 DAT. At 35 DAT controls. These results indicate tha t there may be a varietal difference in the response to salicylates with respect to leaf sap pH. Plant sap pH can be used as an indicator of health and nutrient balance. The leaf sap pH may change in response to imbalances in ion uptake, since plant pH inc reases with the absorption of cations and decreases with the absorption of anions (Hiatt, 1967). The plant materials used i n this study were healthy plants and were not under any stress and had good nutritional status. This may explain why a greater effect on pH was not observed. In order words, salicylates may play a role in stress tolerance and results may be seen more strongly when plants are under some type of stress. Enforcing this hypothesis are the findings of Mann et al. (2011) who concluded that th e application of sodium salicylate to HLB affected plants (disease stress) increased the leaf sap pH, alleviating effects from HLB infection. The results of this study generally support the hypothesis that salicylates have a positive effect on vegetative growth of young healthy citrus plants. However, there is also evidence suggesting that the results may be greater when treated plants are under stress. The trends for both sodium and ammonium salicylate were similar, indicating

PAGE 51

51 that 0.20 mM and 0.25 mM rat es generally had a positive effect on citrus growth, particularly during the first 3 weeks after treatment. Further studies should be conducted to determine the optimum concentration and the effects of repeated applications.

PAGE 52

52 Table 3 1. Mean number of bu ds that grew (number of budbreaks), total shoot length, applied rates of ammonium and sodium salicylate 7 and 6 weeks after treatment, respectively (n = 8). Application rate (mM) N o. of budbreaks Total shoot length (m) Total no. of nodes Ammonium salicylate 0 13 1c z 3.66 0.27 207 22 0.10 13 2bc 3.49 0.19 217 25 0.15 14 3bc 3.74 0.20 210 12 0.20 16 2ab 3.69 0.15 223 12 0.25 21 4a 4.09 0.30 225 13 Sodium salicylate 0 12 2 3.35 0.27 216 21 0.10 14 3 3.39 0.35 217 24 0.15 10 3 3.12 0.42 194 28 0.20 10 1 3.25 0.17 201 9 0.25 13 3 3.47 0.24 220 16 z Data were analyzed by one way ANOVA within a parameter for sal icylate treatment effects. Different letters indicate significant differences within a column within a salicylate type by Tukey's HSD test, = 0.05; no letters indicate no significant differences within a column.

PAGE 53

53 Table 3 2. Mean number of buds that grew (number of budbreaks), shoot length, and rates of ammonium and sodium salicylate 6 weeks after treatment (n = 8). Application rate (mM) Final no. budbreaks Final shoot length (cm) Final no. nodes Ammonium salicylate 0 5.0 1.4 z 98.4 9.9 54.0 7.1b 0.10 8.0 1.8 134.8 13.5 83.0 8.8a 0.15 6.0 1.1 115. 1 15.8 75.0 8.9a 0.20 5.0 1.2 93.4 16.1 59.0 8.0ab 0.25 7.0 1.6 107.1 17.7 77.0 9.3a Sodium salicylate 0 5.0 1.4 98.4 9.9 54.0 7.1b 0.10 7.0 1.3 106.5 9.5 65.0 4.8ab 0.15 7.0 1.2 119.5 7.2 74.0 6.0a 0.20 7.0 1 .1 121.3 10.4 81.0 8.0a 0.25 10.0 1.3 108.6 18.1 78.0 7.7a z Data were analyzed by one way ANOVA within a parameter for salicylate treatment effects. Different letters indicate significant differences within a column within a salicylate type by Tukey's HSD test, = 0.05; no letters indicate no significant differences within a column.

PAGE 54

54 Table 3 applied ammonium and sodium salicylate 7 and 6 weeks after treatment, respectively (n = 8) Application rate (mM) Leaves Stems Roots Dry weight (g) z Ammonium salicylate 0 y 24.51 2.13 x 34.42 2.54 20.86 2.20 0.10 24.38 1.65 32.81 2.39 20.29 1.86 0.15 24.29 1.42 31.81 1.40 18.20 1.11 0.20 25.02 1.01 35.86 2.92 21 .89 2.84 0.25 23.36 1.32 31.38 2.29 18.64 1.90 Sodium salicylate 0 18.61 2.13 29.22 1.99 20.74 1.95 0.10 15.99 2.04 23.68 2.29 16.96 1.61 0.15 16.35 2.26 24.61 1.95 19.08 1.29 0.20 17.94 1.70 24.17 1.35 1 8.16 1.05 0.25 17.20 1.50 25.53 2.15 17.35 1.64 z Values are means SE x Data were analyzed by one way ANOVA within a tissue type for salicylate treatment effects. No significant differences were detected.

PAGE 55

55 Table 3 4. Tissue dry w applied ammonium and sodium salicylate 6 weeks after treatment (n = 8). Application rate (mM) Leaves Stems Roots Dry weight (g) z Ammonium salicylate 0 y 9.94 0.61 y 11.49 0.90 9. 90 0.56 0.10 11.62 0.82 13.75 0.89 10.50 0.62 0.15 10.67 1.01 11.85 1.08 9.34 0.78 0.20 10.15 0.54 9.87 1.56 7.69 1.19 0.25 10.96 1.12 11.26 2.12 8.27 1.38 Sodium salicylate 0 9.94 0.61 11.49 0.90 9.90 0.56 0.10 10.9 3 0.96 11.86 1.37 9.58 0.98 0.15 11.33 0.76 12.75 1.10 10.55 0.76 0.20 11.27 0.41 11.66 0.68 10.14 0.40 0.25 11.36 0.75 10.85 1.75 8.24 1.26 z Values are means SE x Data were analyzed by one way ANOVA within a tissue t ype for salicylate treatment effects. No significant differences were detected.

PAGE 56

56 Table 3 ammonium salicylate 7, 28 and 42 days after treatment (n = 8). Application rate (mM) Net CO 2 2 1 ) 7 DAT 28 DAT 42 DAT 0 5.08 z 0.63 0.74 0.10 6.28 1.06 2.10 0.15 6.04 0.57 1.16 0.20 4.66 1.35 2.09 0.25 4.74 2.07 2.18 Statistical significance df F P Trt 4 2.03 0.0949 Time 2 120.10 <0.0001 Trt x time 8 2.09 0.0428 z Data were analyzed for salicylate treatment over time (repeated measures).

PAGE 57

57 Table 3 sodium salicylate 7 and 42 days after treatment (n = 8). Application rate (mM) Net CO 2 assimilation 2 1 ) 7 DAT 42 DAT 0 5.66ab z 2.81ab 0.10 7.16a 3.49ab 0.15 4.79b 1.78b 0.20 6.42ab 2.38ab 0.25 6.69a 3.66a Statistical significance df F P Trt 4 3.03 0.0229 Time 1 62.09 <0.0001 Trt x time 4 0.29 0.8831 z Data were analyzed for s alicylate treatment over time (repeated measures). Lowercase letters indicate mean separation within columns for treatment main effects = 0.05)

PAGE 58

58 Table 3 tions of ammonium salicylate at 1, 2, 3, 7 and 21 days after treatment (n=8). Application rate (mM) Net CO 2 2 1 ) 1 DAT 2 DAT 3 DAT 7 DAT 21 DAT 0 y 1.69 z 4.28 3.32 2.37 1.03 0.10 3.40 5.38 3.21 3.18 1.72 0.15 3.24 3.25 1. 81 3.39 1.28 0.20 1.26 2.38 3.65 2.96 1.46 0.25 1.23 3.10 2.80 2.41 1.38 Statistical significance df F P Trt 4 2.49 0.0639 Time 4 14.74 <0.0001 Trt x Time 16 2.08 0.0135 z Data were analyzed for salicylate treatment over time (repeated measures ).

PAGE 59

59 Table 3 of sodium salicylate at 1, 2, 3, 7 and 21 days after treatment (n=8). Application rate (mM) Net CO 2 m 2 s 1 ) 1 DAT 2 DAT 3 DAT 7 DAT 21 DAT 0 1.69 z 4.28 3.32 2.37 1.03 0.10 3.79 4.09 3.58 3.29 1.36 0.15 3.82 4.33 3.50 3.88 1.88 0.20 2.62 3.81 2.66 3.44 2.04 0.25 5.00 4.32 2.17 5.88 3.84 Statistical significance df F P Trt 4 3.67 0.0150 Time 4 8.17 <0.0001 Trt x time 16 1.73 0.0490 z Data were analyzed for salicylate treatment over time (repeated measures).

PAGE 60

60 Table 3 different foli ar applied rates of ammonium and sodium salicylate 7 and 6 weeks after treatment, respectively (n = 8). Application rate (mM) Electrolyte leakage ratio Leaf sap pH Ammonium salicylate 0 y 0.12 x 6.07 0.10 0.11 5.92 0.15 0.13 5.98 0.20 0.13 6.07 0. 25 0.13 6.08 Sodium salicylate 0 0.12 6.18b 0.10 0.13 5.82b 0.15 0.14 6.07b 0.20 0.14 6.12b 0.25 0.14 6.23a z Data were analyzed by one way ANOVA within a parameter and salicylate type for treatment effects. Different letters indicate significant differences within a column within a salicylate type by Tukey's HSD test, = 0.05; no letters indicate no significant differences within a column.

PAGE 61

61 Table 3 ammonium salicylate 1, 2, 3 and 35 days (5 weeks) after treatment (n = 8). Application r ate (mM) Electrolyte leakage ratio 1 DAT 2 DAT 3 DAT 35 DAT 0 0.15 z 0.13 0.15 0.15 y 0.10 0.16 0.11 0.15 0.13 0.15 0.15 0.11 0.21 0.14 0.20 0.16 0.11 0.09 0.16 0.25 0.15 0.10 0.08 0.16 Statistical significance df F P Trt 4 5.19 0.0027 Time 2 14.49 <0.0001 Trt x time 8 6.09 <0.0001 z Data were analyzed for salicylate treatment over time (repeated measures) for samples taken 1, 2 and 3 DAT. y Data were analyzed by one way ANOVA at 35 DAT for salicylate treatment effect s. No significant differences were detected ( P = 0.3683).

PAGE 62

62 Table 3 sodium salicylate 1, 2, 3 and 35 days (5 weeks) after treatment (n = 8). Application rate (mM) Electrolyte leakage ratio 1 DAT 2 DAT 3 DAT 35 DAT 0 0.15 z 0.13 0.15 0.15 y 0.10 0.19 0.10 0.13 0.13 0.15 0.22 0.10 0.10 0.13 0.20 0.16 0.10 0.13 0.14 0.25 0.15 0.16 0.14 0.15 Statistical significance df F P Trt 4 1.01 0.4158 Time 2 52.34 <0.0001 Trt x time 8 9.24 <0.0001 z Data were analyzed for salicylate treatment over time (repeated measures) for samples taken 1, 2 and 3 DAT. y Data were analyzed by one way ANOVA at 35 DAT for salicylate treatment effects. No significant dif ferences were detected ( P = 0.6841).

PAGE 63

63 Table 3 ammonium salicylate 1, 2, 3 and 35 days (5 weeks) after treatment (n = 8). Application rate (mM) Leaf sap pH 1 DAT 2 DAT 3 DAT 35 DAT 0 6.39 z 6.35 6.42 6.20 y 0.10 6.35 6.29 6.23 6.33 0.15 6.28 6.11 6.17 6.16 0.20 6.41 6.28 6.17 6.20 0.25 6.32 6.26 6.44 6.19 Statistical significance df F P Trt 4 9.19 <0.0001 Time 2 11.12 <0.0001 Trt x time 8 5.64 <0.0001 z Data were analyzed for salicylate treatment over time (repeated measures) for samples taken 1, 2 and 3 DAT. y Data were analyzed by one way ANOVA at 35 DAT for salicylate treatment effects. No significant differences were detected ( P = 0.9602).

PAGE 64

64 Table 3 13 salicylate 1, 2, 3 and 35 days (5 weeks) after treatment (n = 8). Application rate (mM) Leaf sap pH 1 DAT 2 DAT 3 DAT 35 DAT 0 6.39 z 6.35 6.42 6.20 y 0.10 6.31 6.37 6.30 6.15 0.15 6.32 6.39 6.37 6.17 0.20 6.39 6.30 6.33 6.20 0.25 6.32 6.31 6.18 6.21 Statistical significance df F P Trt 4 6.76 0.0005 Time 2 0.82 0.4470 Trt x time 8 3.03 0.0063 z Data were analyzed for salicylate treatment over time (r epeated measures) for samples taken 1, 2 and 3 DAT. y Data were analyzed by one way ANOVA at 35 DAT for salicylate treatment effects. No significant differences were detected ( P = 0.7890).

PAGE 65

65 Figure 3 1. Plant material. A) 1 year citrumelo rootstock. B) 8 month P hotos courtesy of Marina Burani Arouca. A A B A

PAGE 66

66 Figure 3 treated with foliar applications of salicylate at five different rates (expressed in mM concentration). A) Plants treated with ammonium salicylate. B) Plants treated with sodium salicylate.

PAGE 67

67 Figure 3 foliar applications of salicylate at five different rates (expressed in mM concentration). A) Plants treated with ammonium salicylate. B) Plants treated with sodium salicylate.

PAGE 68

68 CHAPTER 4 EFFECTS OF FOLIAR APPLICATIONS OF SALICYLAT ES ON VEGETATIVE SHOOT ORANGE TREES Introduction Citrus is one of the highest value crops in the world due to its nutritional and economic importance. The United States, specifically Florida, is the second largest producer of orange juice in the world (USDA FAS, 2012). In Florida, citrus production is an intensive activity due to the number of pests and diseases that must be managed. The disease of greatest threat to the Florida citrus industry t oday is Huanglongbing (HLB), a disease associated with a fastidious phloem limited bacterium, Candidatus Liberibacter asiaticus (CLas) (Folimonova et al., 2009). Typical symptoms on infected trees include an overall pale yellow appearance, with leaves disp thick leathery texture and tree decline (Bove, 2006). Typically symptomatic fruit are lopsided and small with aborted seeds, and there are fewer fruit per tree (Bassanezi et al., 200 6; Bove, 2006). These symptoms may or may not occur together, making identification difficult (Bov, 2006; Etxeberria et al., 2009; Achor et al., 2010; Kim et al., 2009). The natural vector of this pathogen in Florida is Asian citrus psyllid ( Diaphorina ci tri ) (Gottwald et al., 2007). Prior to symptom expression, HLB can have a variable incubation period that is influenced by tree age and health. By the time symptoms become visible in an infected tree, there can be two to several more asymptomatic infectio ns already established in nearby trees (Gottwald, 2010). This situation poses a serious challenge for disease control.

PAGE 69

69 Tatineni et al. (2008) found that CLas is unevenly distributed in the phloem of bark tissue and in the vascular tissue of leaf midribs, roots, and different parts of flowers and fruit. Recently, the genome sequence of CLas was determined. The genome showed no toxins, extracellular degrading enzymes or specialized secretion systems (Duan et al., 2009). Thus, the symptoms of HLB appear to ar ise from plant responses to the bacteria. Achor et al. (2010) described the sequence of events leading to the of sieve elements and companion cells followed by some phloem cell collapse, presumed sugar backup in localized leaf blade areas leading to starch accumulation phloem blockage seems to result from plugging of sieve pores by callose and phloem protein and not from bacterial aggregation, since CLas does not occur in high enough quantities to form aggregates in citrus (Achor et al., 2010). Communication between source and sink organs, as well as nutrient transport appears to be affecte d (Kim et al., 2009). This is supported by data indicating that when leaves from symptomatic and asymptomatic branches, and non infected healthy trees were analyzed and compared leaves from infected trees had increased K content while Ca, Mg, B, Mn and Zn content was lower compared with leaves from healthy trees ( Aubert, 1970; Koen and Langenegger, 1970; Spann and Schumann, 2009) No cure for HLB exists and current management strategies include intensive pesticide use to reduce psyllid populations, removal of infected trees to eliminate bacterial inoculum, production of pathogen free nursery plants and nutritional programs

PAGE 70

70 to treat visual disease symptoms (Albrecht and Bowman, 2008; Brlansky and Rogers, 2007; Brlansky et al., 2012; Gottwald, 2010;). The nu tritional program approach is intended to maintain the productivity of HLB infected trees. Foliar applications of plant nutrients are made using a mixture of nutrients and other various components (Rouse, unpublished data). It is well known that a well bal anced nutrition program helps maintain plant health and productivity. At the same time, there is a lack of comprehension about the interaction between nutrition and vascular diseases, along with the fact that each plant disease complex is unique (Datnoff e t al., 2007; Spann and Schumann, 2009). The concept that leads growers to adhere to this management strategy is related to the plant response to the disease. As abovementioned, the plugging of the vascular system that occurs after infection seems to be a response of the plant to the pathogen attack. Presumably, this plugging leads to an interruption of sap flow, depriving the roots of carbohydrates. This starvation at least partially reduces root function, reducing water and nutrient uptake, eventually lea ding to the appearance of leaf symptoms and root system decline. By supplying nutrients via foliar application, the plant appears to be able to temporarily avoid the decline typically associated with HLB. This supplemental supply could lead to new vascular system/phloem production enabling the plant to maintain productivity despite infection. As mentioned, these foliar applications are often a mixture of nutrients and various other compounds, such as salicylate compounds, often referred to by the generic, a lthough inaccurate, term salicylic acid (SA). The effect of each of the individual components, or the interaction among them, is still uncertain (Rouse,

PAGE 71

71 unpublished data). Various physiological and biochemical effects of SA and salicylates have been docume nted in numerous crops (Raskin, 1992a), placing these compounds in the broad category of compounds commonly referred to as plant growth regulators (PGRs). The responses to SA and salicylates in citrus are generally unknown. Citrus plants exposed to bioti c and abiotic stresses such as heat, cold and disease were exogenously treated with sodium salicylate ranging from 0.10 to 0.18 mM concentrations, resulting in new foliage growth on HLB affected citrus trees three weeks after SA treatment. Flowering was al so induced in the citrus plants treated with 0.10 mM of NaSA (Mann et al., 2011). However, work on young, healthy citrus plants using sodium and ammonium salicylate sources did not find any consistent effects on growth (the plants were too young to investi gate flowering) (Arouca chapter 3, 2012). This apparent discrepancy in results may indicate that citrus is only responsive to salicylates when under stress. We hypothesized that foliar applications of salicylates induce development of new vegetative shoo ts in citrus affected by HLB, which may help HLB affected plants cope with this disease by developing more vascular tissue free of the blockage that occurs in response to the infection. Therefore, an experiment was conducted to investigate the effects of e xogenously applied salicylates on citrus tree growth with the objectives to 1) determine if foliar application of salicylates induce vegetative growth; and 2) quantify variation in budbreak and shoot development in healthy and HLB affected citrus trees und er field conditions.

PAGE 72

72 Materials and Methods Plant Material and Experimental Conditions A field experiment was conducted for 21 days during August 2012 at the University of Florida, Citrus Research and Education Center in Lake Alfred, Florida year Citrus sinensis uncoated Lamellic Quartzipsamments), on spacing 2.1 m by 9.1 m, was used for the experiment. The block was managed according to standard commercial pract ices with respect to irrigation fertilization, pruning and pest control. For this trial selected branches of approximately 1 cm diameter with 8 to 12 fully expanded shoots from the first summer flush, avera ging 36 to 42 nodes per branch, were used. Treatments were applied to the entire tree, but the experimenta l unit was the selected branch Three different types of branches selected from two different types of trees were studied: HLB symptomatic branches fr om HLB symptomatic (qPCR+) trees (symptomatic), HLB asymptomatic branches from HLB symptomatic (qPCR+) trees (asymptomatic), and healthy branches from apparently healthy (qPCR undetected, symptomless) trees (healthy) (Figures 4 1, 4 2). Each treated tree h ad three selected branches for study; therefore, healthy trees had three healthy study branches and HLB affected trees had six study branches, three symptomatic and three asymptomatic. Weather conditions at 2 m height were recorded every 15 minutes by the IFAS FAWN weather station at the CREC located approximately 200 m from the study site. Over the study period the weather conditions averaged 26.5 C air temperature, 82.3% RH and total rainfall was 206 mm.

PAGE 73

73 Determination of Healthy Versus HLB Affected Tre es Healthy and HLB affected trees were determined by both visual symptom presence (Figure 4 2) and polymerase chain reaction (PCR). For the purposes of this study, healthy trees refer to those that did not display symptoms of HLB and had undetected result s for Candidatus Liberibacter through Real Time PCR (qPCR) analysis at the time of this study. HLB affected trees refer to those that presented classical HLB symptoms, such as blotchy mottle leaf patterns (Figure 4 2), and tested positive for Candidatus Li beribacter through qPCR analysis. Professional scouts performed visual inspections of all trees on 20 July 2012 and determined HLB symptomatic trees based on the IFAS Extension recommended procedures (Burrow, Futch and Spann, 2011). The infection rate of the studied block was 52% at that time. The experiment was designed based on these results and the distribution of the HLB affected trees within the block. In order to determine the presence of the bacteria associated with HLB, C. Liberibacter asiaticus, q PCR was performed. Five mature leaves from each tree (not necessarily from selected study branches) were collected and immediately stor ed in a cooler. Collection of the leaves was based on HLB symptom s e specially the typical blotchy mottle. L eaves wi th da mage from pest attacks, other diseases and herbicide phytotoxicity were avoided in this collection. About 100 mg of petiole and mid vein material from each sample of 5 leaves was used to extract total DNA with DNeasy Plant Mini Kit (Qiagen, Valencia, CA) f ollowing according to the method of Li et al. (2006), which included two primer probe sets of HLBasrp (designed based on 16S rDNA of Las genome) and COXfpr (designed based

PAGE 74

74 o n citrus mitochondrial cytochrome oxidase gene and used as positive internal control to assess the quality of the DNA extracts and reaction mixtures). The qPCR reactions were performed on an Applied Biosystems 7500 Fast qPCR System (Life Technologies, Car lsbad, CA), and the protocol was as follows: 10 min incubation at 95 C, then 40 cycles of 15 s at 95 C and 1 min at 60 C, and fluorescent signals were collected at the 1 min at 60 C stage of each cycle. The 20 L qPCR reaction mixture contained 10 L o f 2x ABI TaqMan Universal PCR Master Mix (Life Technologies), 2 L of DNA template, appropriate amount of primer/probe stock (to reach the optimized final concentrations) and Nuclease Free Water (Qiagen). All reactions were performed in duplicates, and ea ch run contained one negative, one positive and one healthy control. qPCR data were analyzed with the Applied Biosystems software (Version 1.4.0.). Study Trees Nutritional Status To assess the nutritional status of the trees used in the study samples wer e taken from each tree consisting of 20 mature leaves per tree, 10 from each side of the canopy 28 days after treatment. Fully expanded leaves were collected from non fruiting spring flushes as described by Obreza and Morgan ( 2011). Diseased and insect da maged leaves were avoided. Immediately upon collection, leaves were placed in a cooler for transport to the laboratory. The day of collection, the leaves were washed to remove surface contamination by rinsing in tap water, followed by a brief soak in deion ized (DI) water with Liquinox soap, followed by a rinse in clean DI water, followed by a 1 minute soak in a 5% hydrochloric acid solution, followed by a final rinse in DI water. After washing, paper towel dried samples were refrigerated and sent to a

PAGE 75

75 comm ercial lab (Waters Agricultural Laboratories, Camilla, GA) for analysis the following morning. Salicylate Treatments Pairs of trees [an HLB affected (PCR +) and healthy tree (PCR undetected) adjacent to one another] were treated with foliar sprays of sodi um salicylate at five different rates based upon previous work (Mann et al., 2011): 0, 0.10, 0.15, 0.20 and 0.25 mM. Five replicate pairs of trees were used for each treatment (50 trees total). Treatments were applied between 0800 and 1000 HR All treatmen ts were prepared using tap water and laboratory grade chemicals (Sigma Aldrich, St Louis, MO). The control trees were sprayed with water and the remaining treatments were applied in order of increasing concentration using a 40 L 12 V garden sprayer. All tr eatments were applied to run off (~4 L of solution per tree). After application there was no rain for at least 48 hours. Data Collection Bud growth Prior to treatment, the total number of nodes per branch was recorded to determine the maximum number of b uds that could potentially grow per branch, and ranged from 36 to 42 nodes per branch. After treatment, the number of buds beginning to grow (bud break) was recorded weekly for each branch. The orientation of each branch (east or west facing) was recorded to determine if the microclimate on the different sides of the canopy was a significant factor. Vegetative growth New vegetative shoots that grew during the study on the selected branches were collected at the end of the study and individual shoot length a nd number of nodes was

PAGE 76

76 recorded. The shoots were then dried at 65 C to a constant weight and dry weights were recorded. Electrolyte leakage and leaf sap pH measurements At the beginning of the study three fully expanded mature leaves were selected on eac h study branch (nine leaves per branch type per tree) for cell membrane permeability (assessed as electrolyte leakage) and leaf sap pH assessments at 24 and 48 hours after treatment, followed by weekly assessments thereafter. The methodology of Sairam et a l. (1997) as modified and described by Burani Arouca (Ch 3, 2012) as diameter) was punched from each of the selected leaves per branch resulting in nine leaf discs collected fro m each branch type per tree: healthy, symptomatic, asymptomatic. The leaf discs were placed into 50 mL conical centrifuge tubes (Model 12 565 270; Fisher Scientific ) containing 15 mL of DI water, and covered with lids. After 24 hours at room temperature th e samples were stirred and electrical conductivity (EC1) and pH of the solution were measured. Samples were then autoclaved for 20 minutes (121 C and 138 k Pa) and allowed to cool and stand at room temperature for 24 hours. After 24 hours the samples were stirred and the EC was measured again (EC2). Electrolyte leakage was calculated as the ratio of EC1 to EC2. Experimental Design and Data Analysis There was some consideration given that this was a split plot experimental design. If one considers healthy versus infected trees to be treatments, then a split plot to one tree, and then apply salicylate to this pair of trees. The key here is the random

PAGE 77

77 scouted to find infected trees. Infected trees next to healthy ones were chosen as a pair, and the salicylate treatment was applied to the pair. In the split plot design the experimental unit i s a tree where block consists of two trees and the infection is a treatment. In our design, we consider infection as a condition and the experimental unit is the pair of trees. The three samples from the trees for each condition (healthy, asymptomatic, and symptomatic branches) are therefore subsamples and not replicates. Another consideration is that there is an expectation that neighboring trees are more highly correlated than more distant neighbors. Soil conditions, hydrology, microclimate, and the suit e of weeds and insect pests will all be more similar for neighboring trees compared to trees separated by several rows. So if one considers the tree as the experimental unit, then one must also argue that the trees are independent. Yet we have just shown t hat they are not. While the end result in the grove might look the same for either approach, we feel that for both statistical and biological reasons it makes more sense that the pair of trees is the experimental unit. Data from this study were analyzed as linear mixed model using SAS (Version 9.3; SAS Institute) procedures: GLIMMIX, GLM and UNIVARIATE in order to identify the effects of the different doses of the treatment, the effects of plant health status and finally the effects of the shoots visual sta tus healthy, asymptomatic and symptomatic, at a certain time point and over time. Variables that were studied over time were a nalyzed as repeated measures since samples were taken from the same leaves over time.

PAGE 78

78 Number of nodes, length and dry weight of the new shoots at the end of the third week were analyzed as averages per type of branch per tree, considering that each tree had more than one study branch. Significant differences were taken in account if the P value of the F test for the model effects w as significant. Tukey performed in cases where models were significant. For some variables, residuals did not follow a normal distribution so the P values of the GOF (goodness of fit tests, e.g., the Shapiro Wilk t est) were taken into consideration as criteria for data transformation (e.g., log, squared root). Transformation was accepted in the cases that there was an improvement in GOF test results, meaning that standardized errors are more normally distributed. Th e statistical model was composed by treatment as a categorical variable, by pair (taking into consideration that trees were paired), by east west facing side of tree (study branches were in both sides of the trees) and, where it applied, day (as the variab le for sampling timings), health status of the branches (healthy, asymptomatic and symptomatic) and health status of the tree (healthy or HLB affected). Results No significant differences were found for any nutrient concentration based on salicylate treat ment, tree health status or the interaction of treatment x health status (Table 4 1). All nutrient levels were within the optimum range for Florida citrus except for copper, which was in the high range and zinc. P lants treated with 0 and 0.1 mM showed opti mum levels of zinc, however plants treated with higher concentrations of sodium salicylate (0.15, 0.20 and 0.25 mM) were zinc deficient (Obreza and Morgan, 2008).

PAGE 79

79 There were no statistical differences ( P = 0.69 ) in the number of buds that broke on the ob served branches during the 21 d observation period following treatment based on salicylate treatment, branch health status or the interaction of treatment x branch health (Table 4 2). The total number of buds that broke averaged 22, 24, 24, 16 and 33 for t he 0, 0.10, 0.15, 0.20 and 0.25 mM sodium salicylate rates, respectively, and across branch health those branches treated with 0.25 mM sodium salicylate had the greatest bud break, although not significantly so Additionally, across salicylate treatments, more buds (~3) grew on symptomatic branches compared to healthy and asymptomatic branches, but again this was not statistically significant. Branches on the east facing side of the trees tended to have greater bud break compared to those on the west facing side, but again this was not statistically significant (data not shown). The new shoots that grew from the buds that broke on the observed branches generally produced less than three nodes during the 21 day observation period (Table 4 3). There were no s ignificant differences found for the number of nodes on the new shoots, but there appeared to be a differential response among the healthy, asymptomatic and symptomatic branches. New shoots produced on healthy branches appeared to be most responsive (produ ced more nodes) at the highest and lowest salicylate rates (0.10 and 0.25 mM), asymptomatic branches appeared to be most responsive at the lowest rate (0.10 mM) and symptomatic branches appeared to be most responsive at the highest rate (0.25 mM), but agai n, there was no statistical significance (Table 4 3). The length and dry weight of these new shoots general ly followed the same pattern as the number of nodes, and similarly there were no significant differences (Table 4 3).

PAGE 80

80 Electrolyte leakage was used a s a measure of membrane integrity and to see if salicylate treatment could affect any potential changes in membrane integrity due to HLB. Assessment of the electrolyte leakage ratio at 1, 2, 7, 14, and 21 days after salicylate application did not reveal an y significant differences among treatments, branch health, sampling time or interact ions of these factors (Table 4 4 ). Averaged across branch health and time, electrolyte leakage ratios for the five treatments were 0.13, 0.12, 0.14, 0.14 and 0.14 for 0.0, 0.10, 0.15, 0.20 and 0.25 mM rates of sodium salicylate, respectively. Leaf sap pH was measured to assess whether it was significantly altered by HLB and if salicylate treatment could affect the possible HLB induced changes. pH values were significantly d ifferent at the five different sampling times (1, 2, 7, 14 and 21 days after treatment), but there was no interaction with treatment or branch health (data not shown). In general, there were no significant effects of treatment on leaf sap pH; however, bran ch health was a significant source of variability, symptomatic branches had lower leaf sap pH compared with healthy and asymptomatic branches. As an example of these differences, the leaf sap pH data 1 day after salicylate treatment application are shown i n Table 4 5. Discussion For the trees used in this stud y, nutrients tested for were in general, in the optimum range based on the citrus nutritional guidelines of Obreza and Morgan (2008), regardless of salicylate treatment. This indicates that the fert ilization practices applied in the study block have been able to ameliorate the commonly found nutritional deficiencies associated with HLB (Aubert, 1970; Koen and Langenegger, 1970; Spann and Schumann, 2009). In studying the effects of salicylates on youn g healthy citrus

PAGE 81

81 plants, Burani Arou ca (Ch 3, 2012) did not find effects of ammonium or sodium salicylate treatments on leaf nutrient concentrations. Maize plants treated with 0.01 to 1.0 mM of Ca(NO3)2 in association with salicylic acid had greater uptake of nitrogen and activity of nitrate reductase in both leaves and roots (Jain and Srivastava, 1981). Similarly, cucumber cotyledons treated with 0.05 mM salicylic acid had greater nitrate assimilation 14 days after treatment compared to untreated controls (Singh et al., 2010). It is possible that the salicylate treatments applied to citrus affected nutrient uptake and assimilation, but that the large standing biomass of a citrus tree relative to a maize plant or cucumber cotyledon coupled with the relativel y short duration of the study masked these effects. More detailed work in this area is warranted. Burani Arouca (Ch 3, 2012) found that foliar applications of salicylates affected citrus plants treated with foliar applied sodium salicylate ranging from 0.10 to 0.18 mM resulted in bud break and new shoot growth three weeks after salicylate treatment compared to control plants These effects were not generally supported by the current study, although there was a trend toward increased budbreak on HLB affected branches treated with 0.25 mM ammonium or sodium salicylate. The trees used in the study by Mann et al. (2011) were significantly smaller than those used in the present study. It is possible that the volume of solution applied to wet the trees in the two studies effectively resulted in different rates being applied even though the soluti on concentrations were the same, and may account for the somewhat varying results.

PAGE 82

82 Mann et al. (2011) also observed a flowering response with 0.10 mM of sodium salicylate treatment. No such response was observed in the current study. In the current stud y, salicylate treatment did not significantly affect dry weight of the new shoots. However, there is evidence in the literature that salicylate treatment can affect shoot dry weight in woody plants. San Miguel and Gutierrez (2003) found a 50% increase in s tem dry weight on Pinus seedlings treated for 9 months with 10 8 and 10 6 M salicylic acid. The variability in the current study was large, suggesting that future work should use a larger sample size and some responses may best be observed in longer term s tudies with multiple salicylate applications as in Pinus. Biotic and abiotic stresses are known to have an effect on plant antioxidant systems and this effect can be investigated by the analysis of membrane stability (Sairam et al., 1997). Changes in plas ma membrane integrity have been reported to be one of the first responses observed after treatment with salicylic acid (Hayat and Ahmad, 2007). Our study did not reveal any significant differences in electrolyte leakage among salicylate treatments for both healthy and HLB affected samples. Likewise, Burani Arouca (Ch 3, 2012) did not find a consistent correlation between salicylate Similarly, Mann et al. (2011) did not find sig nificant differences among sodium salicylate treatments (0, 0.10, 0.14, 0.18 and 0.22 mM) applied to HLB affected 2 year old Besides electrolyte leakage, plant sap pH can also provide insights as to plant health. Plant sap pH relates to the acid/base balance and it has been noted that diseased plants tend to have more acidic sap than healthy plants (Hayat and Ahmad,

PAGE 83

83 2007; Mann et al., 2011). There were no significant differences in sap pH among salicylate treatments in the curr ent study; however, there was a significant difference due to health status, with samples from healthy and asymptomatic branches having higher sap pH compared with samples from symptomatic branches. Mann et al. (2011) found that sodium salicylate treatment s to HLB affected 2 year field tended to increase sap pH over time (14 days). Although the low sap pH of symptomatic tissue was detected in the current study, no change over time associated with salicylate treatment was found. Aga in, it may be that the size differential of the trees in the two studies resulted in dose differences that could account for the variable results. Results of this study generally do not support the hypothesis that sodium salicylate induces shoot growth an d, thus, may help citru s plants cope with HLB. However there were nonsignificant trends in the data that indicate that further research would be warranted. Further studies are also necessary to account for possible interactions of salicylates with compoun ds commonly applied foliarly in commercial citrus production (e.g., pesticides, nutrients, fungicides), the residues of which may have confounded the treatment effects in the current study. Future experiments should also study the effects of repeated appli cations over a longer period of time under field conditions.

PAGE 84

84 Table 4 1. Leaf nutrient concentrations in leaves collected from healthy and HLB affected 'Valencia' trees 28 d after foliar application of sodium salicylate (n=5). Salicylate rate (mM) Dis ease status Nutrient conc. N P K Mg Ca S B Zn Mn Fe Cu % mg/kg 0 Healthy 3.27 z 0.14 1.11 0.33 4.14 0.38 105.0 32.0 40.2 86.8 26.8 HLB 3.23 0.14 1.07 0.33 4.22 0.39 112.8 21.6 28.6 88.0 30.4 0.10 Healthy 3.24 0.14 1.02 0.34 4.26 0.39 113.6 1 9.0 25.2 77.2 22.4 HLB 3.24 0.14 1.10 0.33 4.11 0.38 111.4 26.8 31.4 82.6 21.6 0.15 Healthy 3.22 0.14 1.09 0.33 4.17 0.38 119.4 17.0 24.4 78.8 19.2 HLB 3.16 0.14 1.02 0.35 4.37 0.40 119.2 17.4 25.8 75.8 19.4 0.20 Healthy 3.21 0.13 1.04 0.34 4.29 0 .38 114.0 16.6 22.6 79.6 18.8 HLB 3.16 0.14 1.12 0.33 4.15 0.37 115.2 17.0 21.2 80.0 19.4 0.25 Healthy 3.18 0.13 1.02 0.34 4.40 0.39 113.2 16.8 21.8 75.4 17.8 HLB 3.15 0.13 1.15 0.33 4.14 0.37 109.2 16.0 19.8 75.8 18.6 z There were no significant differences found for any nutrient for salicylate rate, disease status or their interaction.

PAGE 85

85 Table 4 2. Effect of foliar application of sodium salicylate on the average number of buds that grew on healthy branches from healthy trees and HLB asymptomati c and symptomatic branches on HLB affected trees of 'Valencia' sweet orange over 21 days following treatment (n=5). Application rate (mM) Avg. no. of growing buds per branch z Branch type Healthy Asymptomatic Symptomatic 0 0.4 0.2 y 1.8 1.4 1.4 0.5 0.10 1.6 0.8 1.6 0.7 1.0 0.3 0.15 1.6 1.0 1.0 0.8 1.6 1.4 0.20 0.4 0.2 0.8 0.6 0.8 0.5 0.25 2.0 1.5 2.4 2.2 1.8 1.1 z Values are means SE y There were no significant differences found for branch type, application rate or their interaction.

PAGE 86

86 Table 4 3. The effect of foliar applied sodium salicylate on the number of nodes, length and dry weight of new shoots that grew from healthy branches from healthy trees and HLB asymptomatic and symptomatic branches on HLB affecte d trees Application rate (mM) Branch type Healthy Asymptomatic Symptomatic No. of nodes z 0 1.1 0.7 y 2.6 1.7 2.1 1.0 0.10 3.2 1.5 3.0 1.2 1.7 1.0 0.15 2.0 1.3 2.1 1.3 1.3 1.2 0.20 0.7 0.4 1.2 0.9 1.4 0.9 0.25 2.5 1.5 1.9 1.3 2.7 0.9 Shoot length (mm) z 0 4.3 3.9 y 18.2 15.1 8.3 4.9 0.10 21.6 9.9 18.1 8.2 3.4 1.8 0.15 19.7 14.6 20.3 13.1 4.6 4.4 0.20 1.2 1.0 6.1 5.7 11.7 7.6 0.25 21.4 15.2 11.7 8.4 15.7 12.8 Shoot dry weight (mg) z 0 3.7 3.3 y 83.0 79.6 29.7 20.0 0.10 167.3 96.0 126.0 51.4 16.6 12.7 0.15 114.2 100.1 246.6 193.6 13.7 13.6 0.20 0.7 0.6 13.3 13.1 39.6 26.5 0.25 251.8 243. 1 44.2 33.1 58.0 53.6 z Values are means SE y There were no significant differences found for the number of nodes, shoot length or shoot dry weight.

PAGE 87

87 Table 4 4. Effect of foliar application of sodium salicylate on the electrolyte leakage ratio o f 'Valencia' trees after 1, 2, 7,14 and 21 days of the treatments (n=5). Application rate (mM) Branch type Electrolyte leakage ratio Days after treatment 1 2 7 14 21 0 Healthy 0.12 0.14 0.12 0.12 0.14 Asymptomatic 0.10 0.13 0.13 0.10 0 .12 Symptomatic 0.11 0.12 0.13 0.11 0.13 0.10 Healthy 0.13 0.13 0.13 0.15 0.13 Asymptomatic 0.12 0.13 0.13 0.15 0.13 Symptomatic 0.10 0.11 0.11 0.10 0.11 0.15 Healthy 0.13 0.14 0.13 0.14 0.12 Asymptomatic 0.14 0.15 0.13 0.16 0.13 Symptomatic 0. 12 0.13 0.11 0.13 0.13 0.20 Healthy 0.12 0.14 0.13 0.18 0.13 Asymptomatic 0.15 0.12 0.14 0.22 0.13 Symptomatic 0.12 0.12 0.14 0.17 0.13 0.25 Healthy 0.16 0.13 0.13 0.15 0.14 Asymptomatic 0.12 0.12 0.17 0.26 0.12 Symptomatic 0.12 0.12 0.14 0.11 0.12 df F P Treatment 4 1.37 0.2437 Day 4 1.78 0.1326 Branch 2 1.22 0.2970 Treatment x Day 16 1.22 0.2509 Treatment x Branch 8 0.95 0.4784 Day x Branch 8 1.23 0.2839

PAGE 88

88 Table 4 5. The effect of foliar applied sodium salicylate on the sap pH of leaves sampled from healthy branches from healthy trees and HLB asymptomatic and symptomatic branches on HLB affected trees 1 d after treatment (n=5). Application rate (mM) Leaf sap pH z Branch type Healthy Asymptomatic Symptomatic 0 5.98 y 6.04 5.91 0.10 6.01 6.03 5.84 0.15 5.97 6.02 5.88 0.20 5.98 6.00 5.82 0.25 6.01 5.97 5.83 df F P Trt 4 0.4587 0.7657 Branch 2 18.66 <0.0001 Trt x Branch 8 0.4454 0.8888 z Leaf sap pH = pH recorded for nine leaf discs (6. 35 mm diameter) per branch after standing at room temperature in 15 mL of deionized water for 24 h. y Values are means.

PAGE 89

89 Figure 4 1. Examples of the study branches selected: A) healthy branch, B) HLB affected asymptomatic bran ch, C) and D) HLB affected symptomatic branches. P hotos courtesy of Marina Burani Arouca. A B D C C D

PAGE 90

90 Figure 4 2. Examples of blotchy mottle symptoms of HLB collected from qPCR positive P hoto courtesy of Marina Burani Arouca.

PAGE 91

91 CHAPTER 5 CONCLUSIONS We studied growth and physiological parame ters on three different sweet orange application of salicylates. We concluded that salicylates may have a positive effect on vegetative growth of young healthy citrus evidence suggesting that the results may be greater when treated plants are under stress (Mann et al., 2011) The trends for both sodium and ammonium salicylate were similar, indicating that 0. 20 mM and 0.25 mM rates generally had a positive effect on citrus growth, particularly during the first 3 weeks after treatment. However, results from this study with respect to treatment of HLB affected citrus were inconclusive, and generally did not supp ort the hypothesis that salicylate treatment induces shoot growth of HLB affected trees, which may in turn help citrus plants cope with HLB infection. Further studies should be conducted to determine the optimum concentration and the effects of repeated a pplications of salicylates on both healthy and HLB affected citrus trees. Future projects should also take into account the possible interactions of salicylates with other chemicals used in commercial citrus production that may interact with salicylates on the leaf surface, and the possible benefits from the use of surfactants for the foliar application of salicylates. The purpose of this research was to contribute to the current literature with new information about salicylates acting as plant growth regul ators in citrus, particularly with respect to the host pathogen relationship of citrus and HLB, and also to enhance our understanding of the plant growth responses when infected with Candidatus

PAGE 92

92 Liberibacter asiaticus. In addition, the aim of the study was to expand knowledge to improve current horticultural management strategies of HLB and potentially reduce economic damage.

PAGE 93

93 LIST OF REFERENCES Achor D.S., E. Etxeberria, N. Wang, S.Y. Folimonova, K.R. Chung, and L.G. Albrigo. 2010. Sequ ence of anatomical symptom observations in citrus affected with Huanglongbing disease. Plant Pathol. J ournal 9:56 64. Albrecht, U. and K.D. Bowman. 2008. Gene expression in Citrus sinensis (L.) Osbeck following infection with the bacterial pathogen Candida tus Liberibacter asiaticus causing Huanglongbing in Florida. Plant Science 175:291 306. Amin A., A. EL Sh, M. Rashad, and H.M.H. EL Abagy. 2007. Physiological Effect of Indole 3 Butyric Acid and Salicylic Acid on Growth, Yield and Chemical Constituents of Onion Plants. J. Appl. Sci. Res. 3:1554 1563. Anandhi, S. and M.P. Ramanujam. 1997. Effect of salicylic acid on black gram ( Vigna mungo ) cultivars. Ind. J. Plant Physiol 2 :138 141. Arfan, M., H.R. Athar, and M. Ashraf. 2007. Does exogenous application of salicylic acid through the rooting medium modulate growth and photosynthetic capacity in two differently adapted spring wheat cultivars under salt stress? Plant Physiol. 164:685 694. Arteca, R. N. 1994. Plant Growth Substances: Principles and Applications. Chapman & Hall, New York, NY. Aubert, B. 1979. Progrs accompli dans la lutte contre le greening des citrus la Runion. Revue Agricole et Sucrire 58:53 56. Bassanezi, R.B., L.H. Montesino, and E.S. Stuchi. 2009. Effects of huanglongbing on fruit qualit y of sweet orange. Eur. J. Plant Pathol. 125:565 572. Boina, D. R., W.L. Meyer, E.O. Onagbola, L.L. Stelinski. 2009. Quantifying dispersal of Diaphorina citri (Hemiptera: Psyllidae) by immunomarking and potential impact of unmanaged groves on commercial ci trus management. Environ. Entomol. 38:1250 1258. Bov, J.M. 2006. Huanglongbing: A destructive, newly emerging, century old disease of citrus J. Plant Pathol. 88:7 37. Brlansky, R.H., and M.E. Rogers. 2007. Citrus Huanglongbing: Understanding the Vector P athogen Interaction for Disease Management. Accessed on 6 July 2012. < http://www.apsnet.org/publications/apsnetfeatures/Pages/Huanglongbing.aspx > Brlansky, R.H., M .M., Dewdney, and M.E., Rogers. 2012. Florida Citrus Pest Management Guide: Huanglongbing (Citrus Greening). Plant Pathology Department, Florida Cooperative Extension Service, IFAS, UF. < http://edis.ifas.ufl.edu/cg086 >.

PAGE 94

94 Buchanan, B. B., W. Gruissem, and R.L. Jones. (eds.). 2000. Biochemistry & Molecular Biology of Plants. ASPB, Rockville, MD. Burrow, J.D., S.H. Futch, and T.M. Spann. 2008. Revised December 2011. Scouting for citrus gr eening. (HS1147). Gainesville: University of Florida Institute of Food and Agricultural Sciences. Accessed on 6 July 2012. < https://edis.ifas.ufl. edu/ch200 >. Chandra, A., A. Anand, and A. Dubey. 2007. Effect of salicylic acid on morphological and biochemical attributes in cowpea. Environ. Biol. 28:193 196. Chen, X., Z. Zheng, J. Huang, Z. Lai, and B. Fan. 2009. Biosynthesis of salicylic acid in pl ants. Plant Signaling Behavior 4:493 496. Citrus Health Management Areas (CHMAs). 2008. Revised 17 October 2012. Gainesville: University of Florida Institute of Food and Agricultural Sciences. Accessed on 6 July 2012. < http://www.crec.ifas.ufl.edu/extension/chmas/chma_overview.shtml >. Dat, J., H. Lopez Delgado, C. Foyer, and I. Scott. 1998. Parallel changes in H 2 O 2 and catalase during thermotolerance induced by salicylic acid or heat acclimation in mustard seedlings. Plant Physiol. 116:1351 1357. Datnoff, L. E., W. H. Elmer, and D.M. Hubber. (eds). 2007. Mineral Nutrition and Plant Disease. APS Press, Saint Paul, MN. Davies, P. J. 2010. Plant Hormones: Biosynthesis, Signal Tra nsduction, Action! Springer, New York, NY. Delaney, T. P. 2010. Salicylic acid, pp. 681 699 In: Davies, P. J (ed). Plant Hormones: Biosynthesis, Signal Transduction, Action! Springer, New York, NY. Dinant S., A.M. Clark, Y. Zhu, F. Vilaine, J.C. Palauqui, C. Kusiak, and G.A. Thompson. 2003. Diversity of the superfamily of phloem lectins (phloem protein 2) in angiosperms. Plant Physiol. 131:114 128. Duan, Y., L. Zhou, D.G. Hall, W. Li, H. Doddapaneni, H. Lin, L. Liu, C.M. Vahling, D.W. Gabriel, K.P. Williams A. Dickerman, Y. Sun, and T. Gottwald. 2009. Complete Candidatus Liberibacter Molecular Plant Microbe Interactions 22:1011 1020. El Khallal, S. M., T.A. Hathout A. El Raheim, A. Ashour, and A.A. Kerrit. 2009. Brassinolide and salicylic acid induced growth, biochemical activities and productivity of maize plants grown under salt stress. Res. J. Agric. Biol. Sci. 5:380 390.

PAGE 95

95 Etxeberria, E., D. Achor, P. Gonzalez, a nd G. Albrigo. 2009. Anatomical distribution of abnormally high levels of starch in HLB affected Valencia orange trees Physiol. Molecular Plant Pathol 74:76 83. Fan, J., C. Chen, and R.H. Brlansky. 2010. Changes in carbohydrate metabolism in Citrus sinen Plant Pathol. 59:1037 1043. Fariduddin, Q., S. Hayat, and A. Ahmad. 2003. Salicylic acid influences net photosynthetic rate, carboxylation efficiency, nitrate reductase activity and seed yield in Brass ica juncea Photosynthetica 41 :281 284. Florida Automated Weather Network. 2012. Lake Alfred report for 2012. University of Florida Institute of Food and Agriculture Science. Folimonova, S.Y., and D.S. Achor. 2010. Early events of citrus greening (Huanglon bing) disease development at the ultrastructural level. Phytopathol. 100:949 958. Folimonova, S .Y., C.J. Robertson, S.M. Garnsey, S. Gowda, and W.O. Dawson. 2009. Examination of the responses of different genotypes of citrus to huanglongbing (citrus greeni ng) under different conditions. Phytopathol. 99:1346 54. Gaffney, T.F., L. Friedrich, L. Vernooij, D. Negrotto, G. Nye, S. Uknes, E. Ward, H. Kessmann, and J. Ryals. 1993. Requirement of salicylic acid for the induction of systemic acquired resistance. Sc ience 261:754 6. Gautam, S., and P.K. Singh. 2009. Salicylic acid induced salinity tolerance in corn grown under NaCl stress. Acta Physiol. Plant. 31:1185 1190. Ghai, N., R.C. Setia, and N. Setia. 2002. Effects of paclobutrazol and salicylic acid on chloro phyll content, hill activity and yield components in Brassica napus L. (cv. GSL 1). Phytomorphol 52 : 83 87. Goldschmidt, E.E. and P. Spiegel Roy. 1996.Biology of citrus. 1 st ed. Cambridge University Press, New York, NY. Gottwald, T.R. 2010. Current epidem iological understanding of citrus huanglongbing. Annu. Rev. Phytopathol 48:119 139. Gottwald, T. R., J.V. da Graa, and R.B. Bassanezi. 2007. Citrus Huanglongbing: The pathogen and its impact. Online. Plant Health Progress. Accessed on 6 July 2012. < http://www.plantmanagementnetwork.org/pub/php/review/2007/huanglongbing/ > Gunes, A., A. Inal, M. Alpaslan, F. Eraslan, E.G. Bagci, and N. Cicek. 2007. Salicylic acid indu ced changes on some physiological parameters symptomatic for

PAGE 96

96 oxidative stress and mineral nutrition in maize ( Zea mays L.) grown under salinity. J. Plant Physiol. 164:728 36. Gutirrez Coronado, M.A., Trejo Lpez, C., and Larqu Saavedra. 1998. Effects of salicylic acid on the growth of roots and shoots in soybean. Plant Physiol. Biochem. 36:563 565. Halbert, S.E., and K.L. Manjunath. 2004. Asian citrus psyllids (Sternorrhyncha: psyllidae) and greening disease of citrus: a literature review and assessment o f risk in Florida. Florida Entomol. 87:330 353. Hayat, S., Q. Fariduddin, B. Ali, and A. Ahmad. 2005. Effect of salicylic acid on growth and enzyme activities of wheat seedlings. Acta. Agron. Hung 53 :433 437. Hayat, S., B. Ali, and A. Ahmad. 2007. Salicyl ic Acid: Biosynthesis, Metabolism and Physiological Role in Plants, pp. 1 14 In: Hayat, S., and A. Ahmad (eds). Salicylic Acid: A Plant Hormone, Springer, Dordrecht, The Netherlands. Hiatt, A.J. 1967. Relationship of cell sap pH to organic acid change duri ng ion uptake. Plant Physiol. 42:294 298. Hussein, M. M., L.K. Balbaa, and M.S. Gaballah. 2007. Salicylic acid and salinity effects on growth of maize plants. Res. J. Agric. Biol. Sci. 3:321 328. Inskeep, W.P., and P.R. Bloom. 1985. Extinction coefficients of chlorophyll a and b in N,N dimethylformamide and 80% acetone. Plant Physiol. 77:483 485. Jain, A. and H.S. Srivastava. 1981. Effect of salicylic acis on nitrate reductase activity in maize seedlings. Physiol. Plant. 51:339 342. Janda, T., G. Szalaij, I Tari, and E. Paldi. 1999. Hydroponic treatment with salicylic acid decreases the effects of chilling injury in maize (Zea mays) plants. Planta. 208:175 180. Jayalakshmi, P., P.S. Devi, N.D. Prasanna, G. Revathi, and S.K. Shaheen. 2010. Morphological and physiological changes of groundnut plants by foliar application with salicylic acid. Bioscan 5:193 195. Khan, W., B. Prithviraj, and D. L. Smith. 2003. Photosynthetic responses of corn and soybean to foliar application of salicylates. J. Plant Physiol 160 :485 492. Khodary, S. F. A. 2004. Effect of salicylic acid on the growth, photosynthesis and carbohydrate metabolism in salt stressed maize plants. Intl. J. Agric. Biol 6 :5 8. Kim, J., U.S. Sagaram, J.K. Burns, J. Li, and N. Wang. 2009. Response of sweet orange ( Citrus sinensis microscopy and microarray analyses. Phytopathol 99:50 57.

PAGE 97

97 Knoblauch, M., A.J.E., van Bel. 1998. Sieve tubes in action. Plant Cell 10:35 50. Koen, T.J., and W. Langenegger. 1970. Ef fect of greening virus on the macro nutrient content of citrus leaves. Farming South Africa 45:65. Kuttimani, R., A. Velayutham. 2011. Foliar Application of Nutrients Enhances the Yield Attributes and Nutrient Uptake of Greengram. Agric. Sci. Digest 31:202 205. Larkindale, J., and M.R. Knight. 2002. Protection against heat stress induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene and salicylic acid. Plant Physiol. 128:682 95. Larkindale, J., J.D. Hall, M.R. Knight, and E. Vier ling. 2005. Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiol. 138:882 97. Li, W., J.S. Hartung, and L. Levy. 2006. Quantitative real time PCR for detection and identifi cation of Candidatus Liberibacter species associated with citrus huanglongbing. J. Microbiol. Methods 66:104 115. Malamy, J., J.P. Carr, D.F. Klessig, and I. Raskin. 1990. Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection. Science 250:1002 1004. Mann, K. K., A.W. Schumann, and T.M. Spann. 2011. Response of citrus to exogenously applied salicylate compounds during abiotic and biotic stress. Proc. Fla. State Hort. Soc. 124:1 10. Martin Mex, R., and A. Larqu Saavedra. 2001. Effect of salicylic acid in clitoria (Clitoria ternatea L.) bioproductivity in Yucatan, Mxico. J. Plant Growth Regul. 28:97 99. Metraux J.P., H. Signer, J.A. Ryals, E. Ward, and M. Wyss Benz, J. Gaudin, K. Raschdorf, E. Schmid, W. Blum, B. Inverardi. 1990. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250:1004 6. Moharekar, S. T., S.D. Lokhande, T. Hara, R. Tanaka, A. Tanaka, and P.D. Chavan. 2003. Effect of salicylic acid on chlorophyll and ca rotenoid contents of wheat and moong seedlings. Photosynthetica 41 :315 317. Monselise, S. P. 1979. The use of growth regulators in citriculture: A Review. Scientia Hort. 11:151 162. Najafian, S., M. Khoshkhui, and V. Tavallali. 2009. Effect of salicylic ac id and salinity in rosemary ( Rosmarinus officinalis L.): Investigation on changes in gas exchange, water relations, and membrane stabilization. Adv. Environ. Biol. 3:322 328.

PAGE 98

98 Noreen, S., and M. Ashraf. 2008. Alleviation of adverse effects of salt stress on sunflower ( Helianthus Annuus L.) by exogenous application of salicylic acid: growth and photosynthesis. Pak. J. Bot 40:1657 1663. Obreza, T.A., and K.T. Morgan. 2008. Reviewed January 2011. Nutrition of Florida citrus trees. 2 nd edition. Accessed on 6 Ju ly 2012. < http://edis.ifas.ufl.edu/pdffiles/SS/SS47800.pdf >. Pancheva, T. V., L.P. Popova, and A.M. Uzunova. 1996. Effect of salicylic acid on growth and photosynthesis in barley plants. J. Plant Physiol 149 :57 63. Pierpoint, W.S. 1997. The natural history of salicylic acid; plant product and mammalian medicine. Interdisciplinary Sci. Rev 22 :45 52. Purcarea, C., D. Cachita Cosma. 2007. Comparative studies about the influence of salicylic an d salicylic acid on content of assimilatory pigments in the primary leaves of wheat ( Triticum aestivum ) plantlets. Fascicula Biologie 14:77 80. Raskin, I. A. Ehmann, W.R. Melander, B.J. Meeuse. 1987. Salicylic Acid: A natural inducer of heat production in Arum lilies Science 237:1601 1602. Raskin, I. 1992a. Role of salicylic acid in plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:439 463. Raskin, I. 1992b. Salicylate, a new plant hormone. Plant Physiol. 99 :799 803. Raskin, I., I.M. Turner, and W.R. Melander. 1989. Regulation of heat production in the inflorescences of an Arum lily by endogenous salicylic acid. Proc. Natl. Acad. Sci. USA. 86: 2214 2218. Rasmussen, J.B., R. Hammerschmidt, and M.N. Zook. 1991. Systemic induc tion of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syringae pv syringae. Plant Physiol. 97:1342 1347. Rivas San Vicente, M., and J. Plasencia. 2011. Salicylic acid beyond defence: its role in plant growth and development. J Exp. Bot. 62:3321 3338. Sairam, K., P.S. Deshmukh, and D.S. Shukla. 1997. Tolerance of drought and temperature stress in relation to increased antioxidant enzyme activity in wheat. J. Agron. Crop Sci. 178:171 178. San Miguel, R., and M. Gutirrez. 2003. Salicylic Acid Increases the Biomass Accumulation of Pinus patula. South. J. Appl. For. 27:52 54. Sanz, A., C. Monerri, J. Gonzlez Ferrer, and J.L. Guardiola. 1987. Changes in carbohydrates and mineral elements in Citrus leaves during flowering and fruit set. Physiol. Plant. 69:93 98.

PAGE 99

99 Schneider, H. 1968. Anatomy of greening diseased sweet orange shoots. Phytopathol. 58:1155 60. Schneider, H. 1968. The anatomy of citrus. p. 1 85. In: W. Reuther, L. D. Batchelor, and H. J. Webber (eds.). The citrus industry. Vol. 2. University of California Division of Agricultural Science, Berkeley. Scora, R.W. 1975. On the history and origin of citrus. Bull. Torrey Bot. Club 102:369 375. Shah, J. 2003. The salicylic acid loop in plant defense. Current Opinion in Plant Biolo gy 6:365 371. Shakirova, F. M. 2007. Role of hormonal system in the manisfestation of growth promoting and antistress action of salicylic acid. p. 69 90. In: Hayat, S. and A. Ahmad (eds.). Salicylic acid: A plant hormone. Springer, Dordrecht, The Netherlan ds. Shulaev, V., J. Lon, and I. Raskin. 1995. Is salicylic acid a translocated signal of systemic acquired resistance in tobacco? Plant Cell. 7: 1691 1701. Singh, P. K., V.K. Chaturvedi, and B. Bose. 2010. Effects of salicylic acid on seedling growth and n itrogen metabolism in cucumber ( Cucumis sativus L.). J. Stress Physiol. Biochem. 6:102 113. Singh, B., and K. Usha. 2003. Salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress. Plant Growth Regulation 39: 137 141 Spann, T. M., and A.W. Schumann. 2009. The role of plant nutrients in disease development with emphasis on citrus and huanglongbing. Proc. Fla. State Hort. Soc. 122:169 171. Syvertsen, J.P., and L.G. Albrigo.1980. Some effects of grapefruit tree canopy p osition on microclimate, water relations, fruit yield, and juice quality. J. Amer. Soc. Hort. Sci. 105:454 459. Takushi, T., T. Toyozato, S. Kawano, S. Taba, A. Ooshiro, and M. Numazawa. 2007. Starch method for simple, rapid diagnosis of citrus huanglungbi ng using iodine to detect high accumulatuion of starch in citrus leaves. Ann. Phytopathol. Soc. Jpn 73:3 8. Taiz, L., and E. Zeiger. 2010. Plant Physiology. Sinauer Associates, Sunderland, MA. Tatineni, S., U.S. Sagaram, S. Gowda, C.J. Robertson, W.O. Daw son, T. Iwanami, N. Candidatus revealed by polymerase chain reaction (PCR) and Real Time PCR. Phytopathol. 98:592 599.

PAGE 100

100 Thom, O. W. 1885. Flora von Deutschland, sterreich und der Schweiz. G era, Germany. Tiwari, S., H. Lewis Rosenblum, K. Pelz Stelinski, and L.L. Stelinski. 2010. Incidence of Candidatus Liberibacter asiaticus infection in abandoned citrus occurring in proximity to commercially managed groves. J. Econ. Entomol. 103:1972 1978. Tukey, H.B., F.W. Went, R.M. Muir, J. van Overbeek. 1954. Nomenclature of chemical plant regulators: Report by a Committee of the American Society of Plant Physiologists. Plant Physiol. 29:307 308. United States Department of Agriculture Foreign Agricultur al Service. 2012. Oranges, Fresh: Production, Supply and Distribution in Select Countries. 26 July 2012. < http://www.fas.usda.gov/psdonline/psdHome.aspx >. Accessed on 22 October 2012. Verberne, M.C., R. Verpoorte, J.F. Bol, J. Mercado Blanco, and H. Linthorst. 2000. Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance. Nature Biotechnol. 18:779 783. Verberne, M.C., R.A. Budi Muljono, and R. Verpoorte. 1999. Salicylic acid biosynthesis. p.295 314. In: Hooykaas, P.P.J., M.A. Hall, and K.R. Libbenga (eds.). Biochemistry and molecular biology of plant hormones. Elsevier Science B.V., Amsterdam. Vernooij B., L. Friedrich, A. Morse, R. Reist, R. Kolditz Jawh ar, E. Ward, S. Uknes, H. Kessmann, and J. Ryals. 1994. Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal transduction. The Plant Cell 6:959 96. Wang, L., L. Fan, W. Loescher, W. D uan, G. Liu, J. Cheng, H. Luo, and S. Li. 2010. Salicylic acid alleviates decreases in photosynthesis under heat stress and accelerates recovery in grapevine leaves. BMC Plant Biology 10:1 10. White, R.F. 1979. Acetylsalicylic acid (aspirin) induces resist ance to tobacco mosaic virus in tobacco. Virology 99 :410 412. Yalpani, N., A. Enyedi, J. Leon, and I. Raskin. 1994. Ultraviolet light and ozone stimulate accumulation of salicylic acid, pathogens is related proteins and virus resistance in tobacco. Planta. 193:372 376.

PAGE 101

101 BIOGRAPHICAL SKETCH Marina Burani Arouca was born in So Paulo, Brazil in 1987. After graduating ic Engineering. During the first year as an undergraduate, Marina already demonstrated her interest in fruit crops. She worked as a student assistant researching propagation methods for almost two years. During the final two years she researched, under an assistantship program, plant nutrition, evaluating growth methods in fruit crops. She was involved in research clubs and frequently joined meetings related to her research. Prior to graduation, she spent one semester working as an intern at the University of Florida, Citrus Research and Education Center, Lake Alfred under the supervision of Dr. Gene Albrigo. After graduating at the beginning of 2010, Marina assisted in technical and production management tasks in the family farming company during spring of that same year. In August 2010, she started her Master of Science degree in the Horticultural Sciences Department at the University of Florida under the mentorship of Dr. Timothy Spann. Her M.S. thesis focused on the vegetative growth responses of young sw eet orange plants to salicylate foliar sprays.