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Baseline Sensitivity of Guignardia Citricarpa, the Causal Agent of Citrus Black Spot to Azoxystrobin, Pyraclostrobin and...

Permanent Link: http://ufdc.ufl.edu/UFE0045072/00001

Material Information

Title: Baseline Sensitivity of Guignardia Citricarpa, the Causal Agent of Citrus Black Spot to Azoxystrobin, Pyraclostrobin and Fenbuconazole
Physical Description: 1 online resource (94 p.)
Language: english
Creator: Hincapie Caputo, Martha A
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: azoxystrobin -- baseline -- black -- citricarpa -- citrus -- fenbuconazole -- g -- pyraclostrobin -- sensitivity -- spot
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Citrus Black Spot (CBS) caused by Guignardia citricarpa was first identified in Florida and the United States in March 2010. The economic impact of the disease is due to external blemishes on the fruit, making them unsuitable for the fresh market and to yield loss caused by premature fruit drop. Fungicide applications are the main control measures in other citrus production areas of the world. The purpose of this project was to evaluate the in vitro activity and baseline sensitivity of G. citricarpa isolates to azoxystrobin, pyraclostrobin and fenbuconazole. The effective concentration needed to reduce growth or germination by 50% (EC50) was determined for fifty isolates from the two Florida counties where CBS is found. The effect of salicylhydroxamic acid (SHAM) on the inhibition of mycelial growth and conidial germination by azoxystrobin and pyraclostrobin was also assessed. The EC50 mean for mycelial growth for azoxystrobin was 0.021 µg/ml and the means for fenbuconazole and pyraclostrobin were significantly lower at 0.009 and 0.008 µg/ml, respectively (P= . Similarly, the mean EC50 for azoxystrobin for conidial germination was 0.016 µg/ml and that for pyraclostrobin was significantly lower at 0.006 µg/ml (P= . There was no effect of SHAM on inhibition of mycelial growth and conidia germination by the QoI fungicides. However, SHAM slightly affected mycelium inhibition by pyraclostrobin. Results from this study will provide the baseline sensitivity to the fungicides tested and will help for future resistance monitoring of this newly introduced pathogen.
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 Martha A Hincapie Caputo.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Peres, Natalia.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0045072:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045072/00001

Material Information

Title: Baseline Sensitivity of Guignardia Citricarpa, the Causal Agent of Citrus Black Spot to Azoxystrobin, Pyraclostrobin and Fenbuconazole
Physical Description: 1 online resource (94 p.)
Language: english
Creator: Hincapie Caputo, Martha A
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: azoxystrobin -- baseline -- black -- citricarpa -- citrus -- fenbuconazole -- g -- pyraclostrobin -- sensitivity -- spot
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Citrus Black Spot (CBS) caused by Guignardia citricarpa was first identified in Florida and the United States in March 2010. The economic impact of the disease is due to external blemishes on the fruit, making them unsuitable for the fresh market and to yield loss caused by premature fruit drop. Fungicide applications are the main control measures in other citrus production areas of the world. The purpose of this project was to evaluate the in vitro activity and baseline sensitivity of G. citricarpa isolates to azoxystrobin, pyraclostrobin and fenbuconazole. The effective concentration needed to reduce growth or germination by 50% (EC50) was determined for fifty isolates from the two Florida counties where CBS is found. The effect of salicylhydroxamic acid (SHAM) on the inhibition of mycelial growth and conidial germination by azoxystrobin and pyraclostrobin was also assessed. The EC50 mean for mycelial growth for azoxystrobin was 0.021 µg/ml and the means for fenbuconazole and pyraclostrobin were significantly lower at 0.009 and 0.008 µg/ml, respectively (P= . Similarly, the mean EC50 for azoxystrobin for conidial germination was 0.016 µg/ml and that for pyraclostrobin was significantly lower at 0.006 µg/ml (P= . There was no effect of SHAM on inhibition of mycelial growth and conidia germination by the QoI fungicides. However, SHAM slightly affected mycelium inhibition by pyraclostrobin. Results from this study will provide the baseline sensitivity to the fungicides tested and will help for future resistance monitoring of this newly introduced pathogen.
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 Martha A Hincapie Caputo.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Peres, Natalia.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0045072:00001


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1 BASELINE SENSITIVITY OF Guignardia citricarpa THE CAUSAL AGENT OF CITRUS BLACK SPOT TO AZOXYSTROBIN, PYRACLOSTROBIN AND FENBUCONAZOLE By MARTHA HINCAPIE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Martha Hincapie

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3 To my family and my fianc Victor

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4 ACKNOWLEDGMENTS I first of all thank Dr. Natalia Peres for believing in me and for inspiring my career I also thank my fianc Victor Castillo for the encouragement and unconditional support through these years. I thank my family for their love and for supporting all my decisions. A special thank s to all members of the plant p athology laboratory at GCREC who contributed to this work. Thanks to Dr. Megan Dewdney and Dr. Gary Vallad for their contributions to this project. I also thank Dr. Sachindra Mondal and Nan Yi Wang for providing me with the isolates tested in this project. Thank you all.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 2 LITERATURE REVIEW ................................ ................................ .......................... 14 Citr us Production in Florida ................................ ................................ .................... 14 Citrus Black Spot ................................ ................................ ................................ .... 15 Citrus Black Spot, a Quarantine Disease in Florida ................................ ................ 16 Causal Organism ................................ ................................ ................................ .... 18 Disease Symptoms ................................ ................................ ................................ 21 Epidemiology and Life Cycle ................................ ................................ .................. 23 Disease Management ................................ ................................ ............................. 27 Cultural Control ................................ ................................ ................................ 27 Chemical Control ................................ ................................ ............................... 29 Strobilurin Fungicides ................................ ................................ ............................. 30 Azoxystrobin ................................ ................................ ................................ ...... 33 Pyraclostrobin ................................ ................................ ................................ ... 34 Triazole Fungi cides ................................ ................................ ................................ 35 Fenbuconazole ................................ ................................ ................................ ....... 36 Objective ................................ ................................ ................................ ................ 37 3 BASELINE SENSITIVITY OF CITRUS BLACK SPOT ISOLATES TO AZOXYSTROBIN, PYRACLOSTROBIN AND FENBUCONAZOLE. ...................... 38 Materials and Methods ................................ ................................ ........................... 41 Fungal Isolates and Culture ................................ ................................ .............. 41 Mycelium Growth Inhibition Assay ................................ ................................ .... 43 Spore Germination Inhibition Assay ................................ ................................ .. 44 Effect of Strobilurin Fungicides and SHAM on Mycelium Growth ...................... 45 Effect of Strobilurin Fungicides and SHAM on Spore Germination ................... 46 Effect of SHAM on M ycelium Inhibition ................................ ............................. 47 Azoxystrobin Technical vs. Commercial Grade and the Effect of the Different Grades and SHAM on Mycelium Inhibition. ................................ ................... 47 EC 50 Calculation and Statistical Analysis ................................ .......................... 48 Results ................................ ................................ ................................ ................... 48

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6 Mycelium Growth Inhibition Assay ................................ ................................ .... 48 Spore Germination Inhibition Assay ................................ ................................ .. 49 Effect of Strobilurin Fungicides and SHAM on Mycelium Growth ...................... 50 Effect of Strobilurin Fungicides and SHAM on Spore Germination ................... 51 Effect of SHAM on M ycelium Inhibition ................................ ............................. 52 Azoxystrobin Technical vs. Commercial Grade and the Effect of the Different Grades and SHAM on Mycelium Inhibition ................................ .................... 52 Discussion ................................ ................................ ................................ .............. 53 4 CONCLUSIONS ................................ ................................ ................................ ..... 81 LIST OF REFERENCES ................................ ................................ ............................... 83 BIOGRAPHIC AL SKETCH ................................ ................................ ............................ 94

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7 LIST OF TABLES Table page 3 1 List of isolates, location and source of isolates evaluated for baseline sensitivity.. ................................ ................................ ................................ .......... 42 3 2 Analysis of variance of the effective concentration of fungicides to inhibit mycelial growth by 50 % (EC 50 ) of fifty Guignardia citricarpa isolates. ................ 60 3 3 Mean effective concentration of fungicides to inhibit mycelial growth and spore germination by 50% (EC 50 ) of fifty Guignardia citricarpa isolates. ............ 60 3 4 Analysis of variance of the effective concentration of azoxystrobin and pyraclostrobin to inhibit spore germination by 50% (EC 50 ) of fifty Guignardia citricarpa isolates. ................................ ................................ ............................... 61 3 5 Analysis of variance of the effective concentration of azoxystrobin and pyraclostrobi n amended with 10 and 100 g/ml of SHAM on the inhibition of mycelial growth by 50% (EC 50 ) of fifteen Guignardia citricarpa isolates. ............ 62 3 6 Mean effective concentration of azoxystrobin and pyracl ost robin amended with 10 and 100 g/ml of SHAM to inhibit mycelial growth by 50% (EC 50 ) of fifteen Guignardia citricarpa isolates. ................................ ................................ .. 62 3 7 Analysis of variance of the effective concentration of azoxystrobin and pyraclostrobin with or without SHAM at 10 g/ml to inhibit spore germination by 50% (EC 50 ) of fifteen Guignardia citricarpa isolates. ................................ ...... 63 3 8 Mean effective concentration of azoxystrobin and pyraclostrobin with or without SHAM at 10 g/ml to inhibit spore germination by 50% (EC 50 ) of fifteen Guignardia citricarpa isolates. ................................ ................................ .. 63 3 9 Analysis of variance of the effective concentration to inhibit mycelial growth by 50% (EC 50 ) when using azoxystrobin technical grade vs. commercial grade with ten Guignardia citricarpa isolates. ................................ ..................... 64 3 10 Mean effective concentration to inhibit mycelial growth by 50% (EC 50 ) when using azoxystrobin technical grade vs. commercial grade with and without SHAM and ten Guignardia citricarpa isolates. ................................ .................... 64

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8 LIST OF FIGURES Figure page 2 1 Citrus black spot symptoms.. ................................ ................................ .............. 22 2 2 Life cycle of Guignardia citricarpa the causal agent of citrus black spot.. .......... 26 3 1 Frequency distribution of the effective concentration of azoxystrobin to reduce mycelial growth by 50% (EC 50 ) of Guignardia citricarpa isolates. ........... 65 3 2 Frequency distribution of the effective concentration of pyraclostrobin to reduce mycelial growth by 50% (EC 50 ) of Guignardia citricarpa isolates. ........... 66 3 3 Frequency distribution of the effective concentration of fenbuconazole to reduce mycelial growth by 50% (EC 50 ) of Guignardia citri carpa isolates. ........... 67 3 4 Inhibition of mycelial growth of Guignardia citricarpa by different concentrations of azoxystrobin.. ................................ ................................ ......... 68 3 5 Inhibition of mycelial growth of Guignardia citricarpa by different concentrations of pyraclostrobin.. ................................ ................................ ....... 69 3 6 Inhibition of mycelial growth of Guignardia citricarpa by different concentrations of fenbuconazole.. ................................ ................................ ...... 70 3 7 Frequency distribution of the effective concentration of azoxystrobin to reduce spore germination by 50% (EC 50 ) of Guignardia citricarpa isolates. ....... 71 3 8 Frequency distribution of the effective concentration of pyraclostrobin to reduce spore germination by 50% (EC 50 ) of Guign ardia citricarpa isolates. ....... 72 3 9 Inhibition of spore germination of Guignardia citricarpa isolates by different concentrations of azoxystrobin.. ................................ ................................ ......... 73 3 10 Inhibition of spore germination of Guignardia citricarpa isolates by different concentrations of pyraclostrobin.. ................................ ................................ ....... 73 3 11 Effect of salicylhydroxamic acid (SHAM) on the activity of azox ystrobin on mycelial growth of Guignardia citricarpa isolates.. ................................ .............. 74 3 12 Effect of salicylhydroxamic acid (SHAM) on the activity o f pyraclostrobin on mycelial growth of Guignardia citricarpa isolates.. ................................ .............. 75 3 13 Effect of salicylhydroxamic acid (SHAM) on the ac tivity of azoxystrobin on spore germination of Guignardia citricarpa isolates. ................................ ........... 76 3 14 Effect of salicylhydroxamic acid (SHAM) on the activity of pyraclostrobin on spore germination of Guignardia citricarpa isolates. ................................ ........... 77

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9 3 15 Inhibition of mycelial growth by different SHAM concentrations. ........................ 78 3 16 Inhibition of mycelium growth of Guignardia citricarpa by different concentrations of azoxystrobin technical grade and commercial grade.. ............ 79 3 17 Effect of salicylhydroxamic acid (SHAM) on the activity of technical and commercial grades of azoxystrobin on mycelial growth of Guignardia citricarpa isolates. ................................ ................................ ............................... 80

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science BASELINE SENSITIVITY OF Guignardia citricarpa THE CAUSAL AGENT OF CITRUS BLACK SPOT TO AZOXYSTROBIN, PYRACLOSTROBIN AND FENBUCONAZOLE By Martha Hincapie December 2012 Chair: Natalia Peres Major: Plant Pathology Citrus Black Spot (CBS) caused by Guignardia citricarpa was first identified in Florida and the United States in March 2010. The economic impact of the disease is due to external blemishes on the fruit, making them unsuitable for the fresh market and to yield loss caused by premature fruit drop. Fungicide appl ications are the main control measures in other citrus production areas of the world. The purpose of this project was to evaluate the in vitro activity and baseline sensitivity of G. citricarpa isolates to azoxystrobin, pyraclostrobin and fenbuconazole. Th e effective concentra tion needed to reduce growth or germination by 50% (EC 50 ) was determined for fifty isolates from the two Florida counties where CBS is found. The effect of salicylhydroxamic acid (SHAM) on the inhibition of mycelial growth and conidial germination by azoxystrobin and pyraclostrobin was also assessed. The EC 50 mean for mycelial growth for azoxystrobin was 0.021 g/ml and the means for fenbuconazole and pyraclostrobin were significantly lower at 0.009 and 0.008 g/ml, respectively (P= < 0 0001) Similarly, the mean EC 50 for azoxystrobin for conidial germination was 0.016 g/ml and that for pyraclostrobin was significantly lower at 0.006 g/ml (P= < 0 .0001) T here was no effect of SHAM on inhibition of mycelial growth and conidia germination by the Q o I fungicides. However,

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11 SHAM slightly affected mycelium inhibition by pyraclostrobin. Results from this study will provide the baseline sensitivity to the fungicides tested and will help for future resistance monitoring of this newly introduced pathogen.

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12 CHAPTER 1 INTRODUCTION The major citrus producing countries are Argentina, Australia, Brazil, China, Cuba, Egypt, India, Israel, Italy, Japan, Mexico, Morocco, South Africa, Spain, Turkey and the United States (Whiteside 2000). In the U.S., Florida is the largest citrus producer with 70% of the total citrus production during the last decade (USDA, 2011). In the 201 0 2011 season, Florida produc tion was valued at 165.9 million dollars on 219,068 ha (USDA, 2011) with the majority of the fruit going to processing. The majority of the commercial cultivars used as scion s or rootstock s belong to the genus Citrus or are trifoliate orange ( Poncirus trifoliata (L.) Raf. ), which is used only as a rootstock (Timmer et al., 2000). In Florida, commercially grown citrus includes oranges, grapefruit, tangelos and tangerines (USDA, 2012). Citrus black spot was exotic to Florida until April 2010 when it was found by the Florida Department of Agricultu re and Consumer Services, Division of Plant Industry (DPI) (Schubert et al., 2012 ). Black spot is caused by the fungus Guignardia citricarpa Kiely, anamorph stage Phyllosticta citricarpa (McAlpine) van der Aa. The disease causes blemishes on the fruit rind and premature fruit drop, although the internal fruit quality remains unaffected. Lesions on the leaves are usually rare and do not affect the tree, but infected leaves on the orchard floor are an important source of inoculum (Timmer et al., 2003). Most c ommercial citrus cultivars are susceptible to black spot including grapefruits, limes, mandarins, sweet oranges, and lemons; however sour oranges and Tahiti lime are asymptomatic (EPPO, 2009). The disease is favored in subtropical regions with high summer rainfall (Kotz, 2000), and the pathogen is widespread

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13 around the world (Sutton et al., 1998) although the disease has not been reported in regions with Mediterranean climates (Kotz, 2000). The pathogen produces two forms of reproductive structures. Ascos pores are formed in leaf litter, whereas conidia (pycnidiospores) are found in fruit lesions, peduncles and leaves ( Kotz, 1981, 2000 ). Both ascospores and pycnidiospores have an important role establishing epidemics, but when the disease becomes establish ed and depending on summer rainfall patterns, ascospores are a more important source of inoculum (Kotz, 1981). Citrus fruit remain susceptible to infection for four to five months after petal fall. After that period, fruit become resistant regardless of weather conditions or inoculum pressure (Kotz, 2000). Leaves remain susceptible from development up to 10 mo nths of age (Truter et al., 2007 ). In other countries where the disease is established, black spot is mainly controlled with application of fungic ides from different groups such as the benzimidazoles, strobilurins (Q o I), dithiocarbamates and copper products while fruit is actively growing (Agostini et al., 2006 ; Da Silva et al., 2009 ). On the other hand, cultural practices such as promotion of leaf litter decomposition (Bellotte et al., 2009) minimizing plant trash movement, avoidance of cultivars with off season blooms, increasing air flow in the canopy and using disease free nursery stock have been promot ed to reduce inoculum amount and spread ( Dewdney et al., 2012; FCHRP working group, 2010). Since only strobilurin fungicides and copper are registered for the control of citrus black spot in Florida, fungicide evaluation became necessary to determine minimally effective dosages and to monitor fut ure shifts in pathogen sensitivity.

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14 CHAPTER 2 LITERATURE REVIEW Citrus Production in Florida United States is one of the largest citrus producers in the world, and Florida has the greatest citrus production in the country (USDA, 2011). Since the introducti on of citrus into St. Augustine, Florida in the 1500s, citrus production has spread to other states such as Californ ia, Texas and Arizona (USDA and APHIS, 2006). Citrus varieties produced in Florida include oranges, grapefruit, tangelos and tangerines (US DA, 2012). Around 90% of the citrus production is destined for fruit processing and the remaining for the fresh market (Mossler, 2011). Most of the citrus growing areas are located on the ridge in central Florida and the flatwoods of the east coast and sou thwest Florida. Survey data of the 2008 2009 season indicated that the five Florida counties with the highest citrus production were in descending order: Polk, Highlands, Hendry, Desoto and Hardee (USDA and NASS, 2009). Freeze events during the 1980s prom oted the implementation of different production practices such as using s maller trees and closer spacing, which increase the tree number per acre, and the use of new varieties. Thus, although the acreage has been decreasing, the total number of trees per a cre has increased (Mossler, 2011). In 1988 the total commercial citrus acreage was 697,929 acres and it has decreased to 541,328 acres in 2011 (USDA, 2011). The hurricanes in 2004 and 2005 damaged many trees and reduced citrus production in Florida Also, these weather events contributed to the introduction and spread of citrus canker a nd huanglongbing which continue to threaten the citrus industry. Eradication attempts have been implemented to control the spread of citrus

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15 canker and huanglongbing, however they were considered to be unfeasible (USDA and APHIS, 2006). Nevertheless, preventive measures adopted by growers contributed to increase d citrus yield s (USDA and APHIS, 2006). For the 2003 2004 citrus season, Florida had a total production of 291.8 mill ion boxes H owever, by the following season, production declined to 169.3 million boxes; a 42% decrease (USDA, 2011). In the 2010 2011 season, Florida produced 166 million boxes of different citrus varieties, a 4% yield increase compared to the previous se ason (USDA, 2011), and it is estimated that for the 2011 2012 season there will be an other increase to 170 million boxes (USDA, 2012). Despite the relative increase in Florida production, the global production of oranges dropped by 7% in 2011 compared to the previous year, and juice production fell by 9% for the same year due to smaller crops in Brazil, Mexico and the EU (USDA, 2012). Citrus Black Spot Citrus black spot (CBS) is caused by the ascomycete Guignardia citricarpa Kiely, anamorph Phyllosticta ci tricarpa (McAlpine) Van der Aa. Among Citrus species, susceptible hosts include grapefruit ( C. paradisici ), lemon ( C. limon ), lime ( C. aurantifolia ), mandarin ( C. reticulata ) and orange ( C. sinensis ); however sour orange ( C. aurantium ) is not susceptible ( C. latifolia ) has been classified as an lime leaves, G. citricarpa does not elicit disease symptoms even at high inoculum pressure under field condi tions (Baldassari et al., 2007). Black spot occurs in subtropical areas with summer rainfall. It affects the fruit rind and when it first appears in a new region, it is usually found on highly susceptible lemons. Symptomatic fruit are unacceptable for fre sh fruit market, but they can be used

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16 for processing (Kotz, 1981). The disease reduces crop productivity due to premature fruit drop and it increase s the cost of production (Baldassari et al., 2007). The pathogen is found in many of the subtropical citrus production zones and is considered an economically important disease of citrus. The fungus was first described in New South Wales, Australi a in 1895, causing significant losses on Valencia oranges (Sutton and Waterston, 1998). Later, crop losses up to 80% due to black spot were reported in South Africa in 1929 (EPPO, 2009). The disease has spread to different countries such as Argentina, Brazil, China (Hong Kong), Indonesia, Japan, Kenya, Mozambique, Philippines, Swaziland, Taiwan, Uruguay, Venezuela, Zam bia, Zimbabwe (USDA and APHIS, 2010; Pa ul et al., 2005), and more recently the United States (Schubert et al., 2012) The disease, however, has not been reported i n Mediterranean countries ( Kotz 2000 ; Paul et al, 2005 ). The spread of G. citricarpa to loc ations previously free of the disease could occur through the movement of infected plant materials, such as contaminated nursery stock, rather than the movement of infected fruit (EPPO, 2009). Also, the spread of the disease will depend on the effectivenes s of quarantine measures and the actions taken after the first outbreak. Depending on the hosts and climatic conditions, the pathogen may take up to 5 to 30 years from the time the first symptoms are observed until it reaches epidemic rates (Kotz, 1981). Once the disease is established in a region it will not disappear; instead, epidemics will need to be managed usi ng effective control measure s (Kotz, 1981). Citrus Black Spot, a Quarantine Disease in Florida Environmental conditions in Florida are favor able for the development of CBS (Kotz, 1981). A predictive model used to determine the risk of CBS establishment after

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17 pathogen introduction to the United States found that Florida conditions are highly favorable for the disease (Magarey et al., 2011 ) Other areas, such as California, have a low risk for disease development due to the lack of rainfall and p rolonged leaf wetness periods that promote the disease ( Magarey and Borchert, 2003 ; Magarey et al., 2011 ). During a regular citrus grove sur vey on Mar ch 8 2010, a suspect sample of Valencia oranges from the Immokalee area was identified as citrus black spot. But, it Inspection Service (APHIS) confirmed the presence of G. citricarpa in Florida (APHIS, 2010). Citrus black spot was introduced into Florida through unknown means (Schubert et al., 2012 ) but a risk assessment analysis conducted by APHIS identified some factors that could have led to the introduction of G. citricarpa Since the disease is not present in neighboring countries such as Mexico, there is a low likelihood of introduction of the pathogen to the U.S by natural spread (Holtz, 2010). According to Kotz (1981), windborne spores of this pathogen, as a source of long distance spread, are not as dangerous as infected plant materials. In view of the number of interceptions of contaminated leaves and unregulated non commercial infected fruit over the last 23 year s by the Plant Protection and Quarantine (PP Q), there is a medium to high likelihood that the pathogen was introduced to the U.S through those pathways (Holtz, 2010). To prevent the spread of G. citricarpa a federal order from APHIS established restrictions on the interstate and intrastate movemen t of regulated articles (APHIS 2012). Regulated articles were identified as citrus fruit, plant

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18 parts such as leaves, budwood, and nursery stock or any other article that could possibly be a hazard for spread of CBS (APHIS, 2012). Currently in Florida, two counties, Collier and Hendry, are confirmed to have black spot and sections have been designated as quarantine areas by APHIS (APHIS, 2012). Several conditions to restrict the interstate movement of regulated articles (citrus fruit s and plant parts) were implemented. The restrictions include that the fruit must be washed, brushed, disinfested, treated and waxed at the packing house prior to shipment. Also, fruit should be free of leaves, stems or other regulated materials. For intrastate movement, vehicles transporting fruit should be covered at the cargo area with a tarpaulin. In addition, after the shipment all possible contaminated objects such as field boxes, bins, and tarpa ulins must be cleaned of debris; which must be heat treated, incinerated or bur ied in a landfill approved by APHIS (APHIS, 2012). Besides the implementation of regulatory measure s control str ategies must be undertaken. An e conomic assessment estimated that the c ost for the control of CBS will be at least $220 million annually in the United States ; however, due to preventive sprays of copper already use to control citrus canker the actual cost may be not as high as the predicted value (USDA, 2002). In addition to the cost of control measures, it is predicted that in Florida there could be up to $847 million in losses due to the disease (Holtz, 2010). Causal Organism Guignardia citricarpa was identified in New South Wales by Kiely in 1948 (Kiely, 1948). For years, the anamorph was known as Phoma citricarpa McAlpine, but it was chang ed to Phyllosticta citricarpa (McAlpine) van der Aa ( Kotz 2000) T he spermatial stage is Leptodothiorella ( Kotz 2000; Van der Aa 1973 ) which forms readily in culture

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19 (Wikee et al., 2011) and also appears on fallen leaves before the development of the pseudothecia (EPPO, 2009). Ps eu dothecia of G. citricarpa are the most important source of inoculum and occur in leaf litter but are never found on the fruit ( Kotz 2000). The 100 175 m dia. pseu dothecia are erumpent, globose, often irregularly shaped, dark brown, and unilocular with a central ostiole. Asci are 8 spored, bitunicate, clavate to broadly ellipsoid, with slightly square apex and well developed ocular chamber with dimensions of 40 65 x 12 15 m. Ascospores (4.5 6.5 x 12.5 16 m) are ellipsoid, aseptate, hyaline, sometimes slightly elongated, often guttulate, and their ends are obtuse with mucilaginous polar appendages ( Hanlin, 1990 ; Kotz 2000; Van der Aa, 1973 ). Spermatia (5 8 x 0.5 1 m) are hyaline, cylindrical to dumbbell shaped with guttules at the end, and straight or slightly curved (Van der Aa, 1973). Phyllosticta citricarpa pycnidia occur on leaves fruit lesions, and fruit peduncles and are formed in abundance on dead leaves. Pycnidia are globose, dark brown to black, and 115 190 m in diameter. Conidia are hyaline, one celled, obovate or ellipsoid, aseptate, multiguttulate bearing a single apical appendage, and 5.5 7 x 8 10.5 m ( Kotz 2000; Van der Aa, 1973). There has been confusion over the years about the id entity of the fungus that causes black spot. Guignardia citricarpa was reported to be in countries where black spot had never been observed, as well as in 21 plant families ( Everett and George, 2006 ; Kotz, 1981 ). How ever, the confusion about the pathogen identity was partially clarified in 1964 (McOnie, 1964). Two morphologically similar G. citricarpa strains that infect citrus were identified ( Kotz, 1981, 2000 ; McOnie, 1964 ). The pathogenic strain

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20 that caused black spot s ymptoms on citrus grew slowly i n culture and produced a yellow pigment at the edges of the colonies when grown on oatmeal agar (OA). The other strain did not cause black spot sy mptoms on citrus, grew faster i n culture and did not produce yellow pigm ent on OA ( Baldassari et al., 2006) With molecular techniques, the non pathogenic strain was shown to be a ubiquitous endophyte with a wide host range, G. mangiferae (anamorph P. capitalensis ) (Baayen et al., 2002; Baldassari et al., 2006). In order to accurately distinguish between G. citricarpa and G. mangiferae specific primers have been developed ( Bonants et al., 2003 ; Meyer et al., 2006; Peres et al., 2007; Van Gent Pelzer et al., 2006 ). The taxo nomy of this non pathogenic endophyte fungus is still in flux. A phylogenetic analysis carried out by Glienke et al. (2011) revealed that P. capitalensis is genetically distinct from G. mangiferae Likewise, Wang et al. (2011) found that P. capitalensis is olates were distinct from G. mangiferae concluding that G. mangiferae is not the teleomorph of P. capitalensis On the other hand, morphological analysis and sequences of the internal transcribed spacer (ITS) region conducted by Okane et al. (2003) identi fied G. endophyllicola as the teleomorphic stage of the endophytic P. capitalensis Recently, further Phyllosticta spp. have been found associated with citrus in Asia. Wang et al. (2011) and Wulandari et al. (2008) reported that P. citriasiana was isolated from tan spot symptoms on leaves and fruit peel of pomelos and P. citrichinaensis was isolated from pomelos, oranges, mandarins and lemons. The latter was considered a minor pathogen of citrus, with mild symptoms and minimal losses. Glienke et al. (2011) also described a new endophytic species, P. citribraziliensis occurring on citrus in Brazil.

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21 Disease Symptoms Citrus black spot causes blemishes on the fruit rind and premature fruit drop although the internal quality remains unaffected. Lesions on the l eaves do not affect the tree, but t hose infected leaves on the ground are a n important source for inoculum (Timmer et al., 2003). CBS symptoms on fruit are variable and have been categorized with different names. Hard spot or shot hole spot is the most typ ical pre harvest symptom for diagnosis of the disease. The lesions appear when the fruit starts to mature, at color break on the side of the fruit that is most exposed to sunlight. The lesions are 3 10 mm diameter, circular, depressed, with brick red to b lack margins and gray white necrotic centers (Fig. 1A) On green fruit, the lesions are surrounded by a yellow halo ( Bonants et al., 2003; Kotz, 1981, 2000 ). Pycnidia may be found at the center of the lesion as slightly elevated black dots, but ascocarps are never formed i n fruit lesions ( Bonants et al., 2 003; Kotz, 1981 ). Even though hard spot is the mo st common symptom of black spot, the causal pathogen is isolated at low frequencies from these lesions ( Kotz 2000). Speckled blotch or false melanose is another symptom of black spot. The spots are numerous, small, 1 3 mm diameter, slightly depressed, tan, gray, reddish or dark brownish (Fig. 1B) Speckled blotch develops on maturing fruit, does not conta in pycnidia, but in some cases can turn into hard spot as the season progresses ( Bonants et al., 2003 ; Cooke et al., 2009 ; Kotz, 1981 ). Freckle spot or early virulent spot often appears when the fruit color has changed from green to orange. The lesions are irregularly shaped, slightly depressed, up to 7 mm long and pycnidia may be present. Freckle spot can coalesce to form one big

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22 lesion, which may turn into mature virulent spot during storage ( Cooke et al., 2009 ; Kotz, 1981 2000 ). Vi rulent spot lesions are small, reddish, irregularly shaped, and are expressed on heavily infected mature fruit at the end of the season (Fig. 1C) Large numbers of pycnidia can develop in the sunken lesions under high humidity conditions. Virulent lesions can cause important postharvest losses ( Cooke et al., 2009; Kotz, 1981 2000 ). Fi gure 2 1. C itrus black spot symptoms A. H ard spot; B. False melanose; C. V irulent spot; D. Cracked spot. Credit: University of Florida UF/IFAS citrus extension. Finally, c racked spot occurs on green and mature fruit. Lesions are large, flat, dark brown with raised cracks on the surface (Fig. 1D) It is suspected that is caused by an interaction between the pathogen and rust mites ( Phyllocoptruta oleivora ) (Dewdney et al., 2010). A. B. C. D.

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23 Leaf symptoms are rar e, and most often appear on lemons. Fruit peduncles can also show symptoms. When symptoms are present on leaves, they are small, round, sunken and necrotic with gray centers, surrounded by a dark brown margin and yellow halo ( Kotz 2000). Epidemiology and Life Cycle Inoculum availability, climatic conditions favorable for infection and susceptibility of the fruit are factors that influence in the epidemiology of CBS ( Kotz 2000). Both ascospores and pycnidiospores have a role in establishing epidemics. In South Africa, accordingly to summer rainfall patterns ascospores are an important source of inoculum (Kotz, 1981). However, i n Brazil, Spsito et al. ( 2008 ) found that conidia also have an important role in the disease epidemic. Ascospores are formed in pseudothecia in the leaf litter on the orchard floor, approximately 50 180 days after leaves drop. Development and maturation of ascospores seem to be influenced by the frequency of wetting and drying cycles of the leaf litter, as well as prevailing tempe ratures ( Kiely, 1948 ; Kotz, 1981, 2000 ). D ead leaves decompose before the development of pseudothecia in cooler production areas ( Kotz 2000). Lee and Huang (1973) found that moderate and evenly distributed rainfall favored pseudothecial development, whe reas prolonged rain resulted in leaf decay and elimination of the pathogen. In Ghana, Brentu et al. (2012) reported pseudothecial and ascospore formation in the leaf litter after 30 to 50 days of wetting and drying periods. Wind borne ascospores are releas ed during rainfall or irrigation events. The amount of rain has little effect on the number of ascospores released. The spores are discharged within the first hour of the rain and could continue for 12 hours or longer

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24 (Kotz, 1981). On the other hand, heav y showers could adversely affect the spore load in the air (Kotz, 1981). In a Brazilian study, ascospore production was not found to be related to total rainfall or temperature, but loosely related to leaf wetness duration (Reis et al., 2006). According t o Reis et al. (2006), a s long as the leaves continue to be moist, even a small amount of rain will tri gger the release of ascospores. Pycnidiospores are not wind borne, but those in leaf litte r could reach susceptible fruit by rain splash ( Kotz, 1981 20 00 ). Truter et al. (2007) reported that P. citricarpa pycnidiospores were not able to infect or colonize detached green leaves or leaf litter of Eureka lemons indicating that infected fruit lying on the ground do not represent inoculum source for detached leaves. However, the authors pointed out that the level of pycnidiospores on leaf litter may depend on the level of infection of the young leaves while attached to the tree. Pycnidiospores serve as a source of inoculum when dead twigs, out of season fru it or late hanging fruit remain on the trees, possibly being washed down to still susceptible young fruit (Kotz, 1981). In Florida, the cultivar Valencia is well known for producing two crops at the same time (Mossler, 2011), and those contaminated fruit cou ld potentially spread pycnidiospores to susceptible tissue. However, conidia do not survive for long periods (Kotz, 1981). Epidemiological studies conducted in Australia, established the importance of the ascospores as the main source of inoculum for path ogen spread and disease epidemics (Kiely, 1948). However, spatial pattern analysis in Brazilian groves determined that the spatial distribution of the disease was aggregated within trees with a maximum radius of 24.7 m, which indicates the limitation of th e pathogen to disperse over long distances (Spsito et al., 2007) This aggregation

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25 pattern observed in Brazil indirectly indicate d that conidia also have an important effect as a source of inoculum within trees in this region (Spsito et al., 2008). Citrus fruit remains susceptible to infection for four to five months after petal fall ( Kotz 2000). In Brazil, similar fruit susceptibility periods have been observed (Reis et al., 2005). After that period, fruit becomes resistant regardless weather cond itions or inoculum pressure ( Kotz 2000). Leaves remain susceptible from development for up to 10 months (Truter et al., 2007). For infection to occur, moisture is necessary for spore germination and appressorium formation ( Fig. 2 2). A wetting period of 24 48 hours is required for the fungus to infect (Kotz, 1981). From the appressorium, a thin penetration peg invades the cuticle and expands into a small mass of mycelium between the cuticle and the epidermal wall ( Kotz, 1981 ; McOnie, 1967 ). After infec tion is complete, the pathogen remains quiescent until the fruit become fully grown or mature when it grows further into the rind tissue producing black spot symptoms ( Kotz, 1981; McOnie, 1967 ). Kotz (1981) identified different factors that could affect symptom development: i. Temperature, rising temperatures stimulates symptom expression on mature fruit ; ii. Light, high light exposure of fruit induces lesion development; iii. Drought, fruit from wilted trees had more black spot lesions than the fruit fro m trees that were not wilted; iv. Maturity of the fruit, the more mature a fruit become (changing from green to yellow), the higher the chances for the symptoms to appear; v. Tree vigor, CBS sy mptoms are more severe on fruit from older trees than fruit fro m vigorous young trees. In Ghana, Brentu et al. (2012) also found th at disease incidence was lower in the young groves

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26 surveyed than i n older groves. Also disease severity within a tree was not uniform; fruit on the side exposed to more sunlight had highe r disease severity. Figure 2 2. Life cycle of Guignardia citric arpa the causal agent of citrus black spot. Figure by Hartzog, in: Holtz, 2010. In vitro studies with Phyllosticta species conducted by Hoch et al. (2006) indicated that conidial attachment, germination and appressorium formation proceeded under hydrophobic surfaces but not on nutrient agar which is hydrophilic. It is thought that Phyllosticta species evolved the re quirement to attach to hydrophobic surfaces because they are often found growing on hydrophobic plant tissues with waxy cuticle coatings (Hoch et al., 2006). Korf (1998) reported that conidial germination of P. citricarpa in vitro was increased by the addi tion of Valencia orange juice with a pH between 4.0 and 4.2. The extra stimulus required by conidia to germinate is thought to be provided by the juice nutrients. The same author also found that the optimal

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27 temperature for conidial germination was 22 o C, an d light was not important. Likewise, Mendes et al. (2005) found that the optimum temperature for conidial germination of P. citricarpa was between 18 and 28 o C, after 22 to 24 h of incubation. Meanwhile, for ascospores Timossi et al. (2003) found that 24 o C was the optimum temperature for germination after 16 hours of incubation. Disease Management Understanding different factors such as the life cycle of a pathogen, environm ental conditions conducive for disease and the host characteristics are essential f or the effective management of any disease (Maloy, 2005). Chemical and cultural practices currently use d to control black spot are based on information generated from regions where the disease has been present for a long time. Cultural Control From understanding the role of ascospores and pycnidia in the life cycle of G. citricarpa different cultural practices have been used in citrus orchards to reduce inoculum and to restrict pathogen spread. Sanitation practices in the orchard prior the harv esting time and after leaf drop may reduce or eradicate ascospores and pycnidiospores (Kotz, 1981). In Brazil, Spsito (2004) evaluated the effect of ascospore suppression by removing the leaves from the orchard floor, as well as pycnidiospores suppressio n by early harvesting late matured fruit. The author reported reduction in disease severity by tho se treatments. Moreover, Bellott e (2009 ) evaluated leaf litter decomposition by using urea, calcium nitrate, dolomite lime and two commercial products; all t reatments provided reduction in disease severity by reducing ascospore inoculum.

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28 Based on information on cultural practices in other areas, one recommended practice in Florida is to promote leaf litter decomposition. Leaf litter decomposition should start in mid March, and can be promoted through different methods. One method is to inc rease irrigation frequency by using microsprinklers at least 5 times a week for a half hour per irrigation period for 1.5 months. A second method is the application of urea (209.6 kg/ha) or ammonium sulfate (628.8 kg/ha) to the leaf litter. Nitrate fertili zers, however, did not reduce the spore numbers of the citrus pathogen Mycosphaerella citri the causal agent of greasy spot also found in the leaf litter. The third method is the application of dolomitic lime or calcium carbonate (2,495 kg/ha) to the leaf litter. All these methods have been shown to reduce the number of M. citri spores equally in the leaf litter, and it is expected that they can also reduce the ascospore numbers of G. citricarpa in the leaf litter ( Mondal and Timmer, 2003 ; Mondal et al., 2 007 ). Other recommended cultural p ractice to restrict the spread of the pathogen includes minimizing plant trash movement within grove or among groves. While most citrus leaves do not show black spot symptoms, they could carry the ascospores which are the main source of inoculum and inappropriate movement of asymptomatic leaves or other trash could transport the pathogen to other sites ( Dewdney et al., 2012; Florida CHRP working group, 2010). Avoidance of citrus cultivars with off season bloom and removal of declining trees is also recommended. Trees with diff erent ages of fruit allow fruit to fruit infection via conidia, amplifying the disease ( Dewdney et al., 2012; Florida CHRP working group, 2010 ). Furthermore, a good nutritional management program shou ld be implemented

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29 since stressed trees express more black spot symptoms (Florida CHRP working group, 2010 ; Kotz, 1981 ). To reduce the leaf wetness, it is recommended to increase the air flow in the canopy. Moreover, it is important to remove dead wood fro m the canopy because G. citricarpa can colonize and reproduce in dead twigs ( Dewdney et al., 2012; Florida CHRP working group, 2010 ; Kotz, 1981 ). To avoid introduction of black spot within a grove, it is important to use planting stock from disease free n urseries. In Florida, there is no nursery near known infected groves, but this may change as the distribution of the disease expands ( Dewdney et al., 2012; Florida CHRP working group, 2010 ). In Australia, where CBS has been present for many years, post har vest fruit exposure to temperatures above 20 o C is avoided, since high temperature s can trigger disease expression (Cooke et al., 2009). Chemical Control Fungicide applications are essential for the control of black spot where the disease has been establish ed. Protective and systemic products have been used to control CBS ( Goes 2002). From 1971 to 1982, single applications of benomyl to control the disease in South Africa were used until the pathogen became resistant to benomyl after 11 years of use (Herbert and Grech, 1985). Currently, in other areas, the disease is controlled with the application of fungicides from different groups such as benzimidazoles, strobilurins, dithiocarbamates and copper (Da Silva et al., 2009); however, only strobilurins and copper are registered for black spot control in Florida. Under Florida condit ions, monthly applications of copper or strobilurin fungicides (azo xystrobin, pyraclostrobin or tri floxystrobin) are recommended from early May to

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30 mid September to control black spot. Those products have shown to be effective against the disease in other r egions of the world (Dewdney et al., 2012 ; Fogliata et al., 2011; Goes 2002 ; Miles et al., 2004 ). Only four strobilurin applications are allowed per season. Thus, it is recommended to reserve strobilurin applications for periods when copper phytotoxicity may occur (temperatures exceeding 34 o C), especially when applied on fruit for the fresh market Two consecutive sprays of strobilurin fungicides should be avoided to manage the development of pathogen resistance. Application in nurseries should also be avo ided since these could lead to selection of resistant strains that could be distributed to groves (Dewdney, 2010; Dewdney et al., 2012; Florida CHRP working group, 2010). The best application method of those fungicides is throug h the use of air blast spra yers, using a volume of 2338 L /ha for application to ensure full coverage of fruit and leaves (Dewdney et al., 2010; Florida CHRP working group, 2010). As part of an integrated disease management, it has been considered important to establish spore trappi ng and to monitor environmental conditions (rainfall, dew periods and temperature) to determine the time and intensity of ascospore release to better time the application of protective fungicides ( Kotz 2000 ; USDA, 2002 ). Strobilurin Fungicides Quinone ou tside inhibitors (QoI), also known as strobilurins, are an important class of fungicides for agriculture. They were first marketed in 1996 and by 20 02, there were six commercially available strobilurin fungicides (Bartlett et al., 2002). By 1999, strobilur ins represented 10% of the global fungicide market with sales of $415 million and they are registered for use on 84 different crops in 72 countries (Bartlett et al., 2002).

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31 methoxyacrylic acid, a secondary metabolite are strobilurin A, oudemansin A and myxothiazol A, and they are produced by Basidiomycete wood rotting fungi such as Strobilurus tenacellus (Bartlett et al., 2002). The mode of action of strobilurin fungicides is based on the inhibition of mitochondria l respiration by binding at the Q o site of the cytochrome b Cytochrome b is located in the bc 1 complex (complex III) in the inner mitochondrial membrane of fungi and other eukaryotes. When binding to the Q o site takes place, electron transfer between cytochrome b and cytochrome c 1 is blocked, disrupting production of pathogen ATP (Bartlett et al., 2002 ; Gisi and Sierotzki, 2008 ). For true fungi, s pore germination and for fungi like organism s zoospore motil ity, are stages that are particularly sensitive to strobilurins since these stages are highly energy demanding and this mode of action disrupts energy production (Bartlett et al., 2002). Several plant pathogens can avoid the toxic effects of Q o I fungicide s by the expression of alternative oxidase pathway, to sustain ATP synthesis (Jin et al., 2009). The alternative oxidase pathway takes place in the inner mitochondrial membrane (Vanlerberghe and McIntosh, 1997) and it can be inhibited by salicylhydroxamic acid (SHAM) and n propyl gallate ( Schonbaum et al., 1971 ; Siedow and Bickett, 1981 ). For this reason, SHAM is usually added to Q o I fungicides when tested in vitro (Duan et al., 2012). The add ition of SHAM to azoxystrobin to in vitro tests with Sclerotinia sclerotiorum allowed pathogen inhibition, whereas there was none when azoxystrobin was used alone (Duan et al., 2012). On the other hand, the mixture of SHAM and

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32 azoxystrobin in in vitro tests with Colletotrichum capsici Botrytis cinerea Rhizoctonia sola ni and Magnaporte grisea showed a synergistic effect on my celium inhibition; however, as time passed, mycelium respiration did rise and SHAM did not reduce the oxygen consumption (Jin et al., 2009). It is thought that alternative respiration does not have an important role during infections in planta, possibly due to host flavones that interfe re with the activation of this pathway (Vincelli and Dixon 2002), but it maintains viability of the fungus in vitro thus, it should be blocked during in vitro studies (Duan et al., 2012). All commercial formulations of strobilurin fungicides have broad sp ectrum activity against the four major groups of plant pathogenic fungi, but the level of control varies according to the type of strobilu rin used. Studies of toxicity in this group of fungicides indicates that th ey represent minimal risk to human health, as well as to the environment since they are readily degraded through adsorption, microbial degradation and photolysis (Bartlett et al., 2002). The first report of resistance to Q o I fungicides was in 1998 in wheat powdery mildew ( Blumeria graminis f.sp. t ritici ). In 1999, barley powdery mildew ( B. graminis f.sp. hordei ) also developed resistance to these fungicides (Heaney et al., 2000). In those cases, resistance was associated with a single point mutation in the cytochrome b gene which leads to a change from glycine (G) to alanine (A) at amino acid residue 143 (G143A) (Gisi et al., 2000; Heaney et al., 2000). It was later confirmed with other fungal plant patho gens that this point mutation was responsible for the loss of disease control when Q o I fungicide s were used as the sole product (Gisi and Sierotzki, 2008). A second amino acid substitution that has been shown to reduce sensitivity to Q o I fungicides is

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33 the replacement of phenylalanine with leucine at position 129 (F129L) (Pasche et al., 2004). A third amino acid substitution from glycine to arginine at the position 137 (G137R) was detected recently in Pyrenophora tritici repentis conferring to the pathogen a reduced sensitivity to the Q o I fungicide s (Siertozki, 2007) Managing the build up of pathogen resistance t o Q o I fungicides is an important matter, especially for the citrus ind ustry in Florida, since the availability of effective alternatives is limited for rotational purposes and inappropriate use would reduce the number of products for black sp ot. For this reason, it is important to follow the guidelines established by the Fungicide Resistance Action Committee (FRAC) for the use of Q o I fungicides (FRAC, 2010). Azoxystrobin Azoxystrobin is one of the strobilurin fungicides labeled for use on citr us ; it was first marketed in 1996 by Syngenta Crop Protection. It has a broad spectrum of activity, can be used in a wide range of crops and could increase yield. Azoxystrobin can be taken up into the leaf cells and also can move to new growing parts of th e plant through systemic xylem movement (Bartlett et al., 2002). It can be considered as a protective and curative fungicide and can be used as a foliar, seed or soil treatment s (Schutte et al., 2003). Field evaluations of azoxystrob in carried out by Miles et al. (2004) in Queensland, Australia, demonstrated that azoxystrobin was as effective as or more so than the industry standard copper/mancozeb for co ntrolling citrus black spot and reduced fruit rind damage compared to the standard products. Schutte et al. ( 2003 ) also reported up to 100% control of black spot in South Africa when azoxystrobin was used in tank mixtures with mancozeb and mineral oil.

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34 However, in vitro studies on mycelium inhibition of G. citricarpa indicated that even at high concentratio ns of azoxystrobin, the pathogen could not be completely inhibited, although sporulation rate was reduced up to 100% (Possiede et al., 2009). Pyraclostrobin Pyraclostrobin is one of the newest strobilurin fungicides on the market. It was first marketed i n 2002 by BASF Corporation. It has broad spectrum activity and can be used on a wide range of crops (Bartlett et al., 2002). Pyraclostrobin is not a xylem systemic fungicide. In vitro studies conducted by Karadimos et al (2005) using pyraclostrobin to contr ol Cercospora beticola on sugar beet suggested that this strobilurin fungicide has some translaminar activity which helps it to penetrate the leaf tissue and be deposited on the cuticle of the opposite leaf surface, inhibiting spore germination, spore production and mycelium gro wth. Similar results were obtained by Ammermann et al (2000), which strongly suggest that pyraclostrobin has protectant, curative and translaminar activity affecting different developmental stages of the fungus (Stierl et al., 2000). Pyraclostrobin has s hown good black spot control in the field. Rodriguez et al (2010) demonstrated up to 88% disease control when pyraclostrobin was applied twice in a season. Almeida (2009) also reported good protective activity of pyraclostrobin even at a high inoculum pre ssure in Brazil. Fogliata et al (2011) compared the efficacy of three different strobilurins with copper and mancozeb in the control of CBS on lemons in Argentina. Two applications of azoxystrobin, pyraclostrobin and trifloxystrobin in a season provided u p to 96% control of the disease and there was no difference in the efficacy of the strobilurins. These results suggest that the strobilurin fungicides provide good control of black spot even under environmental conditions favorable to the

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35 disease, but the high risk of pathogen resistance to these fungicides should limit their usage (Fishel, 2012). Triazole Fungicides The fungicides belonging to the triazole group are also known as the demethylation inhibitors (DMI). Among other fungicides in this group are the imidazoles, piperazines, pyrid ines and pyrimidines (FRAC, 2012 ). DMI fungicides inhibit demethylation at the 14 methylene dihydrolanosterol which are the substrates for the cytochrome P450 demethylase in the b iosynthesis of fungal sterols, for example, ergosterol ( Gisi et al., 2000). Most fungi are able to synthesize ergosterol as their main sterol (Mercer, 1991). The absence of ergosterol and the increase of other compounds promote fungal cell wall disorganiza tion inducing disruption of the membrane (Zambolim et al., 2007). Sterol biosynthesis inhibiting fungicides were developed and registered in the 1970s for many crops (Mercer, 1991). These fungicides have local systemic activity in the apoplast and have pr otective and curative activity against a wide spectrum of foliar, root and seedling diseases, and for instance they can be applied as foliar, seed or soil treatments ( Agrios, 2005 ; Bushong and Timmer, 2000 ) There is a medium risk for the development of pa thogen resistance t o the DMI fungicides (FRAC, 2012 ). A single point mutation on the CYP51 gene causing an amino acid change from tyrosine (Y) to phenylalanine (F) in the 136 position (Y136F) was responsible for conferring resistance to Erysiphe graminis a nd Uncinula necator to the DMIs (Gisi et al., 2000). Moreover, five different mutations ( G129A, Y132H, S405F, G464S, and R467K) at the CYP51 gene of Candida albicans conferred resistance to the azole fungicide group (Sanglard et al., 1998). Although there are differences in the

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36 spectrum of activity of the DMIs, cross resistance is expressed among all DMI compounds that are active again st the same pathogen ( FRAC, 2012; Gisi et al., 1997). For this reason, the FRAC guidelines should be taken in to considerat ion for resistance management of these fungicides ( FRAC, 2012; Zambolim et a l., 2007 ). In vitro studies on the contro l of CBS in South Africa using fungicides from the DMI group showed that difenoconazole considerably reduced mycelial growth of the pathogen (Korf, 1998); however, in another study using imazalil, mycelial growth could not be completely inhibited even at high doses of this fungicide (Deising et al., 2007). Overall, fungicides of this group are considered as non toxic for birds and bees, but appropriate disposal of the product must be followed to not ha rm the environment (Fishel, 2011). Fenbuconazole Fenbuconazole is a triazole fungicide that was first introduced in 1988 (Russell, 2005). The fungicide has protective activity against a broad spectrum of pathogens of many crops ( Bushong and Timmer, 2000 ; Russell, 2005 ). Although fenbuconazole is registered for citrus in Florida, it is not labele d for the control of blac k spot. Fenbuconazole has been reported to be effective for the control of different fungal pathogens in citrus. Field experiments showed that fenbuconazole provided good control of Elsino fawcettii the causal agent of citrus scab (Timmer and Zitko, 1997 ) as well as controlling M citri (Mondal and Timmer, 2006). Nevertheless, the effectiveness of the fungicide in controlling greasy spot was reduced when the applications were conducted after inoculation indicating that fenbuconazole had better activity w hen applied preventive ly (Mondal and Timmer, 2006). Holb and Schnabel (2006) found that protective applications of fenbuconazole were significantly more

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37 effective in controlling mycelial growth and disease development of Monilinia fructicola than curative applications. Regardless of the good control of some citrus pathogens, fenbuconazole has been ineffective against melanose caused by Diaporthe citri and Alternaria brown spot caused by Alternaria alternata ( Bushong and Timmer, 2000; Timmer and Zitko, 1997 ) Objective The objective of this project is to evaluate strobilurin and triazole fungicides for in vitro activity and to determine the baseline sensitivity of Guignardia citricarpa isolates from Florida

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38 CHAPTER 3 BASELINE SENSITIVITY OF CITRU S BLACK SPOT ISOLATE S TO AZOXYSTROBIN, PYRACLOSTROBIN AND F ENBUCONAZOLE Citrus black spot (CBS), caused by Guignardia citricarpa Kiely, anamorph stage Phyllosticta citricarpa (McAlpine) Van der Aa, was an exotic disease to Florida. In April 2010, the presence of the disease in Florida was confirmed by the U.S Department of APHIS, 2010 ; Schubert et al., 2012 ). Most commercially grown citrus species in cluding grapefruit, tangerines, sw eet oranges and lemons are susceptible to CBS. However, sour orange has been shown to be resistant (EPPO, 2009). Citrus black spot causes extensive blemishes on the fruit rind affecting the fruit appeal for the fresh market, although the internal quality remains unaffected. On the other hand, premature fruit drop may occur, reducing crop yield ( Baldassari et al., 2006; Timmer et al., 2003 ). If not contro lled in Florida, an estimated $ 847 million dollars in losses could occur due to the disease (Holtz, 2010 ). Control of black spot is mainly based on preventive applications of fungicides during the period of fruit susceptibility (Schutte et al., 2003); nevertheless, cultural practices have been implemented in Florida to reduce inoculum and pathogen spread (De wdney et al., 2012; Florida CHRP working group, 2010). In other areas where CBS is present, the disease is controlled with fungicides from different groups such as the benzimidazoles, strobilurins (Q o I), dithiocarbamates, and copper products ( Da Silva et a l., 2009 ; Schutte et al., 2003 ). Currently in Florida, only strobilurin and copper products are registered for the control of black spot. Monthly applications of copper and strobilurins ( azoxystrobin, pyraclostrobin or trifloxystrobin) are recommended from early May to mid September; but there is a label limit of four strobilurin applications in a season. For this reason, it is suggested to reserve strobilurin fungicides for times when

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39 there is concern about copper phytotoxicity (temperatures exceeding 34 o C ), especially when applied for fresh fruit (Dewdney, 2010; Dewdney et al., 2012; Florida CHRP working group, 2010). Strobilurin fungicides block electron transport at the Quinol oxidizing site of the cytochrome b complex (complex III) disrupting ATP produ ction ( Bartlett et al., 2002 ; Gisi and Sierotzki, 2007 ). Spore germination is the fungal stage that is particularly sensitive to strobilurins (Bartlett et al., 2002). The mode of action of this group of fungicides is highly specific and many different path ogens have lost sensitivity to Q o I fungicides due to a single point of mutation that leads to a change from glycine (G) to alanine (A) at amino acid residue 143 in the cytochrome b gene (G143A) ( Gisi and Sierotzki, 2007; Gisi et al., 2000; Heaney et al., 2000). A second amino acid substitution that has been shown to reduce sensitivity to Q o I fungicides is the replacement of phenylalanine with leucine at position 129 (F129L) (Pasche et al., 2004). A third amino acid substitutio n from glycine to arginine at the position 137 (G137R) was detected recently in Pyrenophora tritici repentis conferring to the pathogen a reduced sensitivity to Q o I fungicide s (Siertozki, 2007) Fenbuconazole, which belongs to the triazole group of fung icides, has been used in Florida since 1999 for the control of greasy spot ( caused by Mycosphaerella citri ) on grapefruit (Mossler, 2011) and is also effective against citrus scab ( caused by Elsino fawcettii ) (Timmer and Zitko, 1997). Triazoles are known as demethylation inhibitors (DMI) and act by inhibiting the biosynthesis of fungal sterols, such as ergosterol (Gisi et al., 2000). Resistance to DMIs fungicides has been reported. Five different mutations ( G129A, Y132H, S405F, G464S, and R467K) at the CYP 51 gene of Candida albicans

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40 conferred resistance to the azole fungicide group (Sanglard et al., 1998). Moreover, a single point of mutation in the CYP51 gene, leading to an amino acid change from tyrosine to phenylalanine at position 136 (Y136F) conferred resistance to Erysiphe graminis and Uncinula necator (Gisi et al., 2000). Since black spot control mainly relies on fungicide applications, it is necessary to determine the baseline sensitivity of the pathogen to monitor for future shifts in population sen sitivity. Azoxystrobin and pyraclostrobin have been reported to provide good control of black spot in the field as well as in in vitro studies ( Almeida, 2009 ; Fogliata et al., 2011 ; Miles et al., 2004; Rodriguez et al., 2010; Schutte et al., 2003 ). Fenbuco nazole however, has not been tested f o r control of black spot. Due to the site specific mode of action of strobilurin and DMI fungicides, the potential of resistance development should not be ignored. Although G. citricarpa resistance to strobilurins or DMIs has not been reported, in vitro studies determined that mycelium growth was not completely inhibited even at high concentrations of azoxystrobin (Possiede et al., 2009). On the other hand, the same author pointed out tha t the variation in sensitivity to the fungicide is possibly related to genetic variability of G. citricarpa isolates. The baseline sensitivity of other Florida citrus pathogens such as Colletotrichum acutatum Alternaria alternata Elsino fawcettii Diapo rthe citri and Mycosphaerella citri to azoxystrobin, pyraclostrobin and fenbuconazole has been determined (Mondal et al., 2005). Most isolates from the different pathogens were sensitive or tolerant to the fungicides tested with the exception of A. alterna ta to azoxystrobin ( Mondal et al., 2005). More recently, resistance to this fungicide has be en reported (Vega et al., 2012)

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41 The evaluation of azoxystrobin, pyraclostrobin and fenbuconazole for in vitro activity will help to determine the baseline sensitivi ty of G. citricarpa isolates from Florida and to monitor future shifts in sensitivity of this recently introduced pathogen. Materials and Methods Fungal Isolates and Culture Fifty isolates of G. citricarpa from the two Florida counties where CBS is present (Table 3 1), were evaluated for their in vitro sensitivity to the fungicides azoxystrobin, pyraclostrobin and fenbuconazole. Briefly, symptomatic fruit were washed, surface disinfested in a 5% NaOCl solut ion, rinsed in sterile deionized water (SDW) and air dried for 2 3 h. The lesions were excised, placed in 50% ethanol for 30 sec then 5 % NaOCl solution and then rinsed with SDW for 7 min The lesions were thoroughly rinsed and dried in a laminar air flow hood. Sections of the lesions were placed on carrot agar (CA; Peres et al. 2007) and incubated at room temperature with 12 h of light. If pycnidia were selected for isolation, instead of laying individual pycnidia on CA, they were placed on moistened steri le filter paper and incubated overnight. Pycnidia were selected under the stereomicroscope and individually placed on CA. All isolations were incubated for 5 6 days. Isolates with typical morphology were placed on the indicator media oatmeal agar and obser ved for yellow halo production (Baayen et al. 2002). Each isolate was also subjected to PCR identification with the primer sets NP Br ITS Gc and NP Br ITS Gm as described by Peres et al. ( 2007). The isolates were single spored before the commenc ement of the assays. To obtain single spore isolates, a 10 5 conidia/ml suspension was made and a 20 l aliquot was spread onto potato dextrose agar (PDA). Plates were incubated for two days and germinated conidia were picked from the media

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42 surface with a f lame sterilized needle under a stereomicroscope. The conidia were place d onto fresh PDA plates and incubated for a week. For long term storage, all isolates were kept on sterile filter paper in sealed plastic containers containing CaSO 4 desiccant at 20 o C. For mycelium and conidium production, G. citricarpa isolates were tran sferred to half strength potato dextrose agar ( PDA) and grown for 14 days at 25 o C Table 3 1. List of isolates, location and source of isolates evaluated for baseline sensitivity. Is olations were made from Valencia fruit in 2010 and 2011. Isolate no. Location County Symptom type or structure 11 27 b Collier Pycnidia 11 28 a c Collier Pycnidia 11 29 a c Collier Pycnidia 11 30 Collier Pycnidia 11 31 Collier Pycnidia 11 32 a b c Collier Pycnidia 11 33 b Collier Pycnidia 11 34 a b c Collier Pycnidia 11 35 Collier Pycnidia 11 36 a c Collier Pycnidia 11 120 b Collier Freckle spot 11 121 Collier Freckle spot 11 122 b Collier Freckle spot 11 123 a b c Collier Freckle spot 11 124 Collier Freckle spot 11 125 Collier Freckle spot 11 126 a b c Collier Freckle spot 11 127 a b c Collier Freckle spot 11 128 a c Collier Freckle spot 11 129 a c Collier Freckle spot 11 133 Collier Pycnidia 11 134 Collier Pycnidia 11 135 Collier Pycnidia 11 136 Collier Pycnidia 11 137 b Collier Pycnidia 11 138 b Collier Pycnidia

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43 Table 3 1 Continued. a Isolates used to determine the effe ct of SHAM at 10 and 100 g/ml on mycelial growth inhibition and spore germination inhibition b Isolates used to test the effect of different SHAM concentrations. c Isolates used to test azoxystrobin technical grade vs. commercial grade Mycelium Growth Inhibition Assay Commercial formulations of the following fungicides were used: azoxystrobin (Abound, Syngenta Crop Protection), pyraclostrobin (Headline SC, BASF C orporation) and fenbuconazole (Enable 2F, Dow AgroSciences). Thes e fungicides were diluted in SDW to prepare stock solutions of 100 and 1 mg of active ingredient/ml. From 1 mg/ml stock, 0, 3.5, 35 and 350 l and from 100 mg/ml stock, 35 and 350 l Isolate no. Location County Symptom type or structure 11 139 Collier Pycnidia 11 140 Collier Pycnidia 11 141 Collier Pycnidia 11 142 Collier Pycnidia 11 150 a Hendry Hard spot 11 151 b Hendry Hard spot 11 152 a Hendry Hard spot 11 153 Hendry Hard spot 11 154 Hendry Hard spot 11 155 Hendry Hard spot 11 156 b Hendry Hard spot 11 157 Hendry Hard spot 11 158 Hendry Hard spot 11 159 b Hendry Hard spot 11 160 a b Hendry Hard spot 11 161 b Hendry Hard spot 11 162 b Hendry Hard spot 11 163 a b Hendry Hard spot 11 164 a Hendry Hard spot 11 165 Hendry Hard spot 11 166 b Hendry Hard spot 11 167 Hendry Hard spot 11 168 b Hendry Hard spot 11 169 Hendry Hard spot

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44 were added to molten half strength PDA (3500 ml) after cooling to 55 o C to obtain final concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 g of active ingredient per ml. Twenty ml of amended PDA was poured into 100 mm diameter petri dishes using a sterile bott le top dispenser (Fisherbrand). Three mm diameter mycelium plugs from the actively growing area of the fungal colony were placed at the center of each plate. Three replicates were used for each fungicide concentration. Plates were incubated for 14 days at 25 o C under continuous light, and colony diameter was determined for each of the 50 isolate s as the average of two perpendicular measurements. The diameter of the mycelium plug was subtracted from the average colony diameter for each replicate. The perce nt inhibition of the fungicide amended plates was calculated relative to the growth of the non amended control for each isolate. Each experiment was conducted twice. Spore Germination Inhibition Assay Pycnidiospore production was done as described by Ku o and Hoch (1996); however, some modifications were implemented. Isolates of G. citricarpa were cultured on PDA for 14 days. Then, conidia were washed off using 4 ml of sterile water with 0.02% Tween 20. Th e suspension was transferred to four micro centrifuge tubes each with 1 ml of the suspension and centrifuged at 5000 rpm for 5 min. The supernatant was discarded and the pellet resuspended with sterile water. Once the pellets from the four tubes were combined for each isolate, they were cent rifuged again at 5000 rpm for 5 min. This step was repeated twice. Finally, the conidial concentration was adjusted at 10 6 spores/ml using a hemacytomete r.

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45 A germination medium consisting of 2% of Valencia orange juice (pH 4.0) was prepared to stimulate c onidial germination (Korf, 1998). Germination was assessed in hydrophobic slides (Fisher Scientific) and each well contained a volume of 10 l. Ten fold serial dilutions of strobilurin fungicides ranging from 0.001 to 1 g/ml were prepared prior their addi tion to the wells. Each well received 7.5 l of 2% Valencia juice, 1.25 l of the corresponding fungicide concentration and 1.25 l of the adjusted conidial suspension. Control wells contained 1.25 l of SDW instead of the fungicide. Hence, the suspension in each well had a 3:1 ratio of Valencia orange juice to medium. Hydrophobic slides were placed into a humidified petri dish to prevent the medium from desiccation (Kuo and Hoch, 1996; Noronha, 2002 ). All slides were kept in a humid chamber for 20 h. Afte r 20 h of incubation, a cover slip was placed on each slide and 100 conidia were observed under the microscope at 400x to determine the percent germinated conidia. A conidium was considered germin ated if the germ tube was equal or longer than the length of the conidium. There were three replicates for each of the 50 isolate s and the experiment was conducted twice. The percent spore inhibition was calculated for each isolate strobilurin fungicide experiment combination. Effect of Strobilurin Fungicides and S HAM on Mycelium Growth Fifteen G. citricarpa isolates were tested to determine whether salicylhydroxamic acid (SHAM) affected the response of fungal growth to azoxystrobin and pyraclostrobin. SHAM was dissolved in methanol at 0.1 mg/ml. The amount of methanol in the media was 0.1% (vol/vol). Half strength PDA (900 ml) was amended with 9 and 90 mg of SHAM to obtain final concentrations of 10 and 100 g/ml.

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46 Th e effect of SHAM at 10 and 100 g/ml on fungal growth was evaluated in combination with final fungicide concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 g/ml. Percent growth inhibition was based on comparison with the SHAM plates with no fungicide. Inocu lation method and measurements were done as described for mycelium inhibition assay, with three replications per concentration, and the experiment was conducted twice. Effect of Strobilurin Fungicides and SHAM on Spore Germination The same isolates used i n the previous experiment were used to evaluate the effect of SHAM on spore germination inhibition. Different SHAM concentrations were tested prior to the addition of strobilurin fungicides. A stock suspension of SHAM at 0.1 mg/ml was serially diluted to obtain final concentrations in each well of 10, 25, 50 and 100 g/ml of SHAM. The content of methanol in each well was 0.1% vol/vol. Each well received 7.5 l of 2% Valencia orange juice, 1.25 l of the adjusted spore suspension and 1.25 l of the corresp onding SHAM concentration. Control wells received 1.25 l of SDW instead of SHAM. Thus, the suspension in each well had a 3:1 ratio of Valencia orange juice to medium. The effect of SHAM at 10 g/ml on spore germination was evaluated in combination with f inal strobilurin fungicides concentrations of 0, 0.001, 0.01, 0.1, and 1 g/ml. In order to maintain a 3:1 ratio of Valencia Juice to medium and SHAM, there were modifications in the amount of medium added to each well. Therefore, each well received 7.5 l of 2% orange juice, 0.83 l of each fungicide concentration, 0.83 l of SHAM and 0.83 l of the adjusted conidial suspension. Control wells received 0.83 l of SD W instead of fungicide.

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47 After the slides were loaded with the suspension, the same procedur e as described in the spore inhibition assay was followed There were three replications per isolate concentration and the experiment was conducted twice. Percent inhibition was determined for each isolate fungicide experiment combination. Effect of SHAM on M ycelium Inhibition The effect of different concentrations of SHAM was tested on mycelium growth of twenty isolates of G. citricarpa without the addition of strobiliurin fungicides. SHAM was diluted at 0.1 mg/ml in methanol at 0.1% vol/vol of media. H alf strength PDA (1200 ml) was amended with 12, 30, 60 and 120 mg of SHAM to obtain final concentrations of 10, 25, 50 and 100 g/ml. There were three replications per concentration and the experiment was done twice. Inoculations and measur ements were done as described for the mycelium inhibition assay. Percent inhibition was calculated relative to the non amended SHAM plates and was subjected to Analysis of Variance and mean separation using the Least Significant Difference (LSD). Azoxystrobin Technical vs Commercial Grade and the Effect of the Different Grades and SHAM on Mycelium Inhibition To test whether or not there was a difference between commercial formulation of azoxystrobin (Abound, Syngenta Crop Protection) or te chnical grade active ingredient, ten isolates of G. citricarpa were tested against the two forms of the chemical. Azoxystrobin commercial and technical grade stock solutions of 100 g of active ingredient per ml were diluted in water or acetone respectively and added to molt en PDA at 0, 0.001, 0.01, 0.1, 1 and 10 g/ml.

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48 In addition, the effect of SHAM at 100 g/ml added to commercial and technical grade azoxystrobin was also tested on mycelium growth inhibition. The dilution method was done as described for the effect of SHAM on mycelium growth assay. For both test s there were three replications per isolate fungicide concentration and each experiment was repeated twice. Inoculation method, measurements and calculation of percent inhibition was done as described for myc elium inhibition assay. EC 50 Calculation and Statistical Analysis For mycelium inhibition assays, the effective concentration to reduce growth by 50% (EC 50 ) was determined by fitting a four parameter logistic (sigmoidal) function when azoxystrobin was used and a three parameter function when pyraclostrobin and fenbuconazole were used. On the other hand, for spore inhibition assays, EC 50 was determined by fitting a three parameter sigmoidal f unction for both strobilurins. The effect of fungicide, isolate, experiment and all the two way interactions were investigated by an analysis of variance. Treatment means were separated using the Least Significant Difference (LSD) with PROC GLM (SAS 9.3, SAS Institute, Carry, NC). Results Mycelium Growth Inhibition Assay To test for the homogeneity of variance, the standardized residuals were plotted against predicted values. In addition, the normality of the data distribution was tested with a univariate analysis. Since variances were equal, data from different experiments were pooled to calculate the mean EC 50 for each fungicide isolate combination. The EC 50 of G. citricarpa isolates to azoxystrobin, pyraclostrobin and fenbuconazole was determined and t he analysis of variance showed that the fungicide

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49 effect was highly significant (P < 0 .0001). No interactions between experiment, fungicide and isolate were found (Table 3 2). The mean EC 50 value of the 50 isolates for azoxystrobin was significantly higher (P < 0 .0001) than the EC 50 values for fenbuconazole and pyraclostrobin ( Table 3 3). T here was no statistical difference between the mean EC 50 values for fenbu conazole and pyraclostrobin (Table 3 3). For azoxystrobin, 86% of the isolates had an EC 50 between 0.02 to 0.03 g/ml (Fig. 3 1). For pyraclostrobin, however, the EC 50 values were more evenly distributed over the range, and 48% of the isolates were between 0.003 to 0.006 g/ml ( Fig. 3 2). For fenbuconazole, 98 % of the isolates had EC 50 values b etween 0.007 and 0.01 g/ml (Fig. 3 3). Azoxystrobin inhibited mycelial growth up to 75% at 1 g/ml, but only 66% at 10 g/ml (Fig. 3 4). This reduction in growth inhibition at the highest concentration was not observed either with pyraclostrobin or fenbuc onazole. At 10 g/ml, growth inhibition was 90% and 97% for pyraclostrobin (Fig. 3 5) and fenbuconazole (Fig. 3 6), respectively. Spore Germination Inhibition Assay Preliminary results from the spore germination test using 0, 0.001, 0.01, 0.1, 1 and 10 g of azoxystrobin and pyraclostrobin per ml indicated that 10 g/ml completely inhibited spore germination. Thus, the highest fungicide concentration, 10 g/ml, was eliminated from all spore inhibition assays. A significant effect for fungicide, isolate, an d fungicide by isolate interaction were shown by ANOVA (Table 3 4). For azoxystrobin, 64% of the isolates had an EC 50 between 0.02 and 0.03 g/ml (Fig. 3 7); wh ereas 66% of the isolates had an EC 50 between 0.003 and 0.006 g/ml for pyraclostrobin (Fig. 3 8).

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50 Conidial germination was inhibited up to 94% and 91% at 1 g/ml by azoxystrobin and pyraclostrobin, respectively (Figs. 3 9 and 3 10). No shift in the spore inhibition was observed at the highest concentration of azoxystrobin as occurred in the myceli um inhibition assay. A separate analysis of variance was conducted to look at the differences in the fungicide and isolate interactions. The mean azoxystrobin EC 50 for the isolate 11 155 (EC 50 = 0.030 g/ml) was significantly higher than for isolates 11 15 9 (EC 50 = 0.002 g/ml) and 11 156 (EC 50 = 0.001 g/ml), but were not different to the other 47 isolates (range EC 50 = 0.003 to 0.03 g/ml) (P <0.0001). The mean pyraclostrobin EC 50 value for the isolate 11 162 (EC 50 = 0.024 g/ml) was significantly higher than for the other 49 isolates evaluated (range EC 50 = 0.001 to 0.01 g/ml) (P= 0.0072) (data not shown) Effect of Strobilurin Fungicides and SHAM on Mycelium Growth The addition of SHAM at 10 and 100 g/ml to media amended with azoxystrobin had no effect on the mycelial growth EC 50 values of the 15 G. citricarpa isolates. However, the addition of SHAM at the same concentrations to pyraclostrobin had a significant effect (P= 0.017 and 0.006 ) respectively, on the EC 50 for mycelial growth (Table 3 5 and 3 6). N o other factors or interactions between factors were significant. At the highest concentration of azoxystrobin, the growth inhibition was 67%, but the addition of SHAM at 10 and 100 g/ml increased growth inhibition to 83%. But, when SHAM was added at 100 g/ml with the lowest rate of azoxystrobin, the growth inhibition was reversed since more mycelial growth was observed on those plates than on the SHAM plates with no fungicide ( Fig. 3 11). Although SHAM increased growth inhibition at 10 g/ml for azoxyst robin, pairwise comparison indicated that there was no

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51 difference in the EC 50 values and for this reason SHAM was not used to determine the baseline sensitivity of the 50 G. citricarpa isolates. With the addition of SHAM to pyraclostrobin, a similar patte rn of growth inhibition was observed as with pyraclostrobin alone ( Fig. 3 12). The addition of SHAM at 10 g/ml to the lowest concentration of pyraclostrobin inhibited growth more than SHAM at 100 g/ml or pyraclostrobin alone. However, t he EC 50 values fro m both SHAM concentrations were the same, and pairwise comparison indicated that the addition of SHAM to pyraclostrobin had an effect in the EC 50 values (Table 3 6) Effect of Strobilurin Fungicides and SHAM on Spore Germination Different SHAM concentratio ns were tested prior to the addition of the strobilurin fungicides. Complete spore germination inhibition was observed using only SHAM at 25, 50 and 100 g/ml. C rystals formed on the hydrophobic slides when SHAM was added at 100 g/ml. Apparently, there is a reaction between the acid in the orange juice and the high concentr ation of SHAM which induces crystal formation. SHAM at 10 g/ml was tested for activity on spore germination inhibition when added to azoxystrobin and pyraclostrobin. An ANOVA indicated that SHAM had no effect on the sensitivity of conidial germination when added to strobilurins (Table 3 7). The isolate had a significant effect (P= 0.0007) in the experiment with azoxystrobin; however interactions between factors were not significant for either fungicide. The mean EC 50 value for the isolates tested with azoxystrobin only was 0.015 g/ml and with SHAM was 0.016 g/ml. When pyraclostrobin was used alone, the mean EC 50 value was 0.006 g/ml and the EC 50 mean with SHAM was 0.005 g/ml (Table 3 8). The percent spore inhibition when azoxystrobin or pyraclostrobin was used alone was very similar to the inhibition when SHAM was added to the fungicides (Figs. 3 13 and 3 14).

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52 Without SHAM, the maximum spore germ ination inhibition was 93% for azoxystrobin and 92% for pyraclostrobin. With SHAM at 10 g/ml, the maximum spore germination inhibition was 95% and 97% for azoxystrobin and pyraclost robin, respectively (Figs. 3 13 and 3 14). Effect of SHAM on M ycelium Inhi bition Analysis of variance of growth inhibition by SHAM at 10, 25, 50, and 100 g/ml indicated that there was a significant difference among SHAM concentrations (P <0.0001). The main effects of experiment and isolate were not significant and no significan t interactions between factors were detected (data not shown). The maximum mycelial growth inhibition was obtained with SHAM at 25 g/ml, inhibiting growth by 18.4% (Fig. 3 15). Azoxystrobin Technical vs. Commercial Grade and the Effect of the Different Grades and SHAM on Mycelium Inhibition No significant effect between the two azoxystrobin grades was demonstrated by ANOVA. Also, no significant effect o f the experiment or isolate was found and interactions between factors were not significa nt (Tables 3 9 and 3 10). The percent mycelium inhibition by both azoxystrobin grades was similar at the different concentrations. At 1 g/ml of commercial grade, there was a 74% inhibition, but when the concentration increased to 10 g/ml, inhibition was reduced to 67%. The same trend was also observed when using technical grade; at 1 g /ml, there was 76% inhibition of mycelial growth, but at 10 g/ml the inhibition was reduced to 69% (Fig. 3 16). The effect of SHAM at 100 g/ml added to the different azo xystrobin grades was also tested. There was no effect of the addition of SHAM to technical or commercial

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53 grade as determined by ANOVA (P= 0.2006) (Table 3 10). However, pairwise comparison of EC 50 values from technical grade with or without SHAM were signi ficant (P= 0.0001), indicating that although there was no difference in the addition of SHAM when comparing the two azoxystrobin grades, the addition of SHAM did affect the EC 50 value of azoxystrobin technical grade. The mycelial growth inhibition by azox ystrobin commercial grade at 1 g/ml with SHAM was 88%, but at 10 g/ml, the inhibition was reduced slightly to 81%. The growth inhibition by technical grade at 1 g/ml wi th SHAM was 83% and at 10 g/ml, contrary to the commercial grade, the inhibition was higher, 91% (Fig. 3 17). Despite of the effect of SHAM on the growth inhibition when the maximum rate of azoxystrobin technical grade was used, it had no significant effect on the EC 50 values of either grade of azoxystrobin. Discussion Citrus black spot, an emerging disease in Florida, is mainly controlled with applications of strobilurin and copper fungicides. Establishment of the baseline sensitivity of G. citricarpa isolates never exposed to strobilurins or DMIs in FL which are currently used and pote ntially effective fungicides will help to monitor future shifts in pathogen sensitivity. Azoxystrobin, pyraclostrobin and fenbuconazole are currently used on Florida citrus; however, fenbuconazole use is limited to diseases other than black spot. Pathogen s are prone to the development of resistance to strobilurins and DMI fu ngicides; hence the importance of this project In our study, we established the baseline sensitivity of G. citricarpa isolates to azoxystrobin, pyraclostrobin and fenbuconazole. Baseline sensitivity derived from mycelium inhibition of other citrus pathogens to azoxystrobin were higher than G.

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54 citricarpa when compared to our results; the mean EC 50 of E. fawcettii was 0.06 g/ml, the mean EC 50 for C. acutatum was 0.40 g/ml, the D. citri EC 50 mean was 0.08 g/ml, and the EC 50 mean for M. citri was 1.62 g/ml (Mondal et al., 2005). Vega et al. (2012) found for Q o I sensitive conidia of A. alternata a mean EC 50 of 0.44 g/ml f or azoxystrobin Although Mondal et al. (2005) reported EC 50 values higher than 100 g/ml from A. alternata mycelium, whereas the values from sensitive isolates conidia were much lower, indicating that mycelium of this pathogen is insensitive to azoxystrob in ( Mondal et al., 2005 ; Vega et al., 2012 ). In addition, the baseline from mycelium of isolates of Plasmopara viticola showed a large range of EC 50 values from 0.04 to 0.78 g/ml (Wong and Wilcox, 2000). The most similar baseline EC 50 as from sensitive isolates of C. graminicola which had a range of EC 50 values from 0.05 to 0.1 g/ml (Avila Adame et al., 2003). In vitro activity of G. citricarpa to azoxystrobin was also evaluated in Brazil; even at higher doses of azoxystrobin (10 g/m l), mycelium growth could not be totally inhibited and some isolates had a decrease in mycelium inhibition when compared to the lowest concentr ation of the fungicide (1 g/ml ) (Possiede et al., 2009). Similar results were obtained in this study, where a re duction in inhibition was observed at 10 g/ml with all the isolates tested. On the other hand, the same authors reported complete sporulation inhibition by azoxystrobin, suggesting that spore production may be more sensitive to the effect of strobilurins. Few reports on in vitro activity of pyraclostrobin are available. In this study, isolates of G. citricarpa were highly sensitive to pyraclostrobin. Mondal et al. (2005) established the baseline sensitivity of five fungal citrus pathogens and their EC 50 values to pyraclostrobin were higher when compared to our results. Similarly, G. citricarpa

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55 isolates were highly sensitive to fenbuconazole. Results, similar to ours, from the sensitivity of Monilinia oxycocci isolates to fenbuconazole found that the path ogen EC 50 values ranged from 0.0001 to 0.01 g/ml (McManus et al., 1999). Baseline from other DMI fungicide such as cyproconazole, showed that sensitive isolates of M. graminicola had EC 50 values between 0.01 to 0.05 g/ml (Gisi et al., 2000) which are hi gh er than to our baseline values. Likewise, never exposed isolates of Uncinula necator to other DMI fungicides had higher EC 50 values; ranging from 0.03 to 0.09 g/ml (Erickson and Wilcox, 1997). Development of resistance to this group of fungicides had be en reported, and it is considered to be the result of a slow, stepwise loss of control (FRAC, 2005). Although resistance to DMI is associated with multiple mutations at the target gene, demethylase, high levels of resistance are observed after a stepwi se adaptation (polygenic resistance) ( FRAC, 2005 ; Gisi et al., 2000 ). Spore germination is a particularly sensitive fungal stage to strobilurins (Bartlett et al., 2002). In this study the EC 50 values from conidia of G. citricarpa to azoxystrobin were lowe r than EC 50 values reported from conidia germination of Erysiphe graminis f.sp. tritici on cereals, where the EC 50 ranged from 0.022 to 0.235 g/ml (Chin et al., 2000). Our results showed that t he baseline from mycelial growth was higher than for spore ger mination, implying that G. citricarpa conidia are more sensitive to Q o I fungicides. In other studies, spore germination has also been shown to be more sensitive to pyraclostrobin. EC 50 values of pyraclostrobin for Uncinula necator were similar to our resul ts, ranging from 0.0016 to 0.010 g/ml with a mean of 0.0044 g/ml (Won g and Wilcox, 2002). Despite belonging to the same group of fungicides, G. citricarpa was more sensitive to pyraclostrobin than to azoxystrobin for mycelium and

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56 spore inhibition. In another study with Alternaria solani pyraclostrobin had greater spore germination inhibition than azoxystrobin (Pasche et al., 2004). Vega et al. (2012) also reported that conidia of A. alternata were more sensitive to pyraclostrobin than to azoxystrobin. Some fungal plant pathogens evade the toxic effects of Q o I fungicides by the alternative respiration pathway, which allows the generation of ATP without electron transport through complex III and thus, allows the fungus to survive ev en at high doses of fungicide ( Olaya and Kller, 1999; Ziogas et al., 1997). It has been reported that residual growth of M. grisea sustained by alter native respiration was involved in the spontaneous emergence of Q o I resistant cytochrome b target site mut ants (Avila Adame and Kller, 2003 b ). Nevertheless, for several fungal species this resc ue mechanism had little effect i n the field when Q o I fungicides were used to control the disease ( Olaya and Kller, 1999; Ziogas et al., 1997). It has been proposed tha t for those fungal species, plant antioxidants such as flavone s present in the host can silence this mechanism during infection by quenching the reactive oxygen (Avila Adame and Kller, 2003 a ). Therefore, differences between in vitro and in vivo for the sa me studied pathogen are likely to be found (Olaya and Kller, 1999). For this reason, the alternative respiration should be blocked for some pathogens, by using salicylhydroxamic acid (SHAM) during in vitro experiments. The response of G. citricarpa isola tes to different concentrations of SHAM was variable between mycelium growth and spore germination. SHAM at 25 g/ml show ed the highest mycelium inhibition; meanwhile, higher concentrations completely inhibited spore germination. No other studies where SHA M at low doses inhibited pathogen

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57 mycelium or germination were found. Nevertheless, in an other study, sub lethal doses of the fungicide mefenoxam enhanced Pythium damping off disease; which was related 2011). Hormesis is a physiological process where factors that trigger homeostatic disruptions at high doses produce adaptive responses at low doses and can result in metabolic stimulation (Calabrese and Baldwin, 2002). Although in our experiment low doses of SHAM increased the inhibition of mycelium, it may be possible that adaptive responses of G. citricarpa to high doses of SHAM stimulate pathogen growth, but i nhi bited growth at low doses More research would be needed to confirm these results as well as the hypothesis. In the current study, SHAM did not affect the activity of azoxystrobin. Neither mycelium growth nor spore germination was significantly affected by the addition of SHAM to the media. Similar results were obtained with the citrus pathogen A. alternata where the addition of SHAM did not increase the activity to azoxystrobi n (Mondal et al., 2005). Nevertheless Vega et al. (2012) found for th e conidia of the same pathogen t hat SHAM did affect growth in an isolate dependent manner. The sensitivity of C. graminicola and Penicillium digitatum also showed no significant synergistic effect of SHAM on their sensitivity to azoxystrobin ( Avila Adame et al., 2003 ; Kanetis et al., 200 8 ). Our results also indicated that the addition of SHAM to pyraclostrobin did affect the activity of mycelium growth although the EC 50 values were similar. But, spore germination was not affected by SHAM. Comparable to our results, the addition of SHAM to pyraclostrobin significantly reduced the colony diameter of the citrus pathogen D. citri ; even though EC 50 values could not be calculated due to reversed inhibition at low doses of the fungicide (Mondal et al., 2005). From our results, there was no effect of

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58 SHAM on the EC 50 values, indirectly indicating that G. citricarpa is not using the alternative respiration pathway. Interestingly, in our study, 10 g/ml azoxystrobin showed lower mycelium growth inhibition than at 1 or 0.1 g/ml. Thus, to evaluate wh ether or not the grade of the active ingredient was influencing these results, technical grade was compared to commercial grade. The results indicated that there were no differences between the two grades; therefor e, the same pattern was exhibited for both The addition of SHAM slightly increased the inhibition at 10 g/ml; however it did not affect their EC 50 values. Although there is no evidence that G. citricarpa is using alternative respiration, t he pathogen may be activating this pathway specifically a t 10 g/ml of azoxystrobin and SHAM is blocking this pathway or else the mycelium is not taking up the fungicide well at higher concentrations and SHAM increases the permeability. Overall, G. citricarpa isolates were very sensitive to azoxystrobin, pyracl ostrobin and fenbuconazole. In other regions with citrus black spot, azoxystrobin and pyraclostrobin were tested for field efficacy with positive results ( Almeida, 2009; Fogliata et al., 2011; Miles et al., 2004; Rodriguez et al., 2010; Schutte et al., 2003 ). In Florida citrus, fenbuconazole is already used to control citrus scab, greasy spot and sooty mold ( Mondal and Timmer, 2003, 2005; Timmer and Zitko, 1997 ). In this study, we present evidence that fenbuconazole is highly effective inhibiting G. cit ricarpa growth in vitro ; hence the potential use of this fungicide in th e field must be evaluated When chemical control measures fail, it may be due to selection pressure for pathogen resistance. The high specificity of the mode of action of fungicides, together with sexual recombination that occurs in some fungal pathogens promote the selection

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59 of resistant isolates after prolonged periods of use (Brent and Hollomon, 1998; Jutsum et al., 1998). Through the years, several pathogens have become insensitive to strobilurin and DMI fungicides ( FRAC, 2012; Gisi et al., 2000; Heaney et al., 2000 ; Rosenzweig et al., 2007; Vega et al., 2012; Zambolim et al., 2007 ) and there is a high risk of G. citricarpa developing resistance. Consequently, resistance management practices are important for the continued use of strobilurins and DMI fungicides. The baseline sensitivity established in this study will help to monitor future shifts in Florida populations of G. citricarpa to assure the continued effectiveness of the spr ay programs that are currently recommended. The efficacy of fenbuconazole to inhibit G. citricarpa in vitro was shown. The next step will be to demonstrate through field trials that it is a good option for black spot management and a viable rotational prod uct.

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60 Tab le 3 2. Analysis of variance of the effective concentration of fungicides to inhibit mycelial growth by 50% (EC 50 ) of fifty Guignardia citricarpa isolates. a Azoxystrobin, pyraclostrobin and fenbuconazole. b Significant effects are underlined. Table 3 3. Mean effective concentration of fungicides to inhibit mycelial growth and spore germination by 50% (EC 50 ) of fifty Guignardia citricarpa isolates. a Average of two independent experiments. b Minimum and maximum mean EC 50 value of mycelium and spore inhibition for each fungicide. c Mean separation within column followed by the same lower case letter are not significantly different according to t d Mean separation within rows followed by the same capital letter are not significantly different according to t e Fenbuconazole was not tested in the spore inhibition assay. Source of variation df SS MS F value P value Experiment (E) 1 0.0001032 0.0001032 1.03 0.3115 Fungicide (F) a 2 0.011019 0.005510 55.22 <.0001 b Isolate (I) 49 0.006079 0.000124 1.24 0.1800 E x F 2 0.000233 0.000117 1.17 0.3149 E x I 49 0.004195 0.000086 0.86 0.7203 F x I 98 0.013431 0.000137 1.37 0.0589 Fungicide Mycelium inhibition Spore inhibition EC 50 g/ml a Azoxystrobin (0.01 0.08) b 0.021 a c A d (0.001 0.03) 0.016 aB Fenbuconazole (0.006 0.01) 0.009 b n/a e Pyraclostrobin (0.002 0.03) 0.008 bA (0.001 0.02) 0.006 bB

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61 Tab le 3 4. Analysis of variance of the effec tive concentration of azoxystrobin and pyraclostrobin to inhibit spore germination by 50% (EC 50 ) of fifty Guignardia citricarpa isolates. a Azoxystrobin and pyraclostrobin. b Significant effects are underlined. Source of variation df SS MS F value P value Experiment (E) 1 0.000070 0.000070 2.62 0.1117 Fungicide (F) a 1 0.005375 0.005375 202.70 <.0001 b Isolate (I) 49 0.003955 0.000081 3.04 <.0001 E x F 1 0.000089 0.000089 3.34 0.0738 E x I 49 0.001063 0.000022 0.82 0.7581 F x I 49 0.003198 0.000065 2.46 0.0010

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62 Table 3 5. Analysis of variance of the effective concentration of azoxystrobin and pyraclostrobi n amended with 10 and 100 g/ml of SHAM on the inhibition of mycelial growth by 50% (EC 50 ) of fifteen Guignardia citric arpa isolates. a Fungicides azoxystrobin (Azo) and pyraclostrobin (Pyra) were amended with 10 and 100 g/ml of SHAM. b P values for comparison of the EC 50 values of SHAM amended and nonamended was determined from F test. c Significant effects are underlined. Table 3 6. Mean effective concentration of azoxystrobin and pyraclostrobin amended with 10 and 100 g/ml of SHAM to inhibit mycelial growth by 50% (EC 50 ) of fifteen Guignardia citricarpa isolates. No SHAM SHAM 10 SHAM 100 EC 50 g/ml Azoxystrobin 0.017 a a 0.016 a 0.013 a Pyraclostrobin 0.008 a 0.005 b 0.005 b a Mean separation within rows followed by the same letter are not significantly different according to t Azo SHAM 10 a Azo SHAM 100 Pyra SHAM 10 Pyra SHAM 100 Source of variation P values b Experiment (E) 0.9990 0.7159 0.2747 0.1750 SHAM (S) 0.7136 0.0726 0.0171 c 0.0063 Isolate (I) 0.2596 0.7842 0.0824 0.1355 E x S 0.9771 0.7383 0.6400 0.8935 E x I 0.1320 0.2526 0.2018 0.5521 S x I 0.3197 0.7236 0.6543 0.1103

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63 Table 3 7. Analysis of variance of the effective concentration of azoxystrobin and pyraclostrobin with or without SHAM at 10 g/ml to inhibit spore germination by 50% (EC 50 ) of fifteen Guignardia citricarpa isolates. Azo SHAM 10 a Pyra SHAM 10 Source of variation P values b Experiment (E) 0.1759 0.2455 SHAM (S) 0.3849 0.5887 Isolate (I) 0.0007 c 0.1239 E x S 0.1885 0.5132 E x I 0.1667 0.7403 S x I 0.5737 0.8411 a Fungicides azoxystrobin (Azo) and pyraclostrobin (Pyra) were amended with 10 g/ml of SHAM. b P values for comparison of the EC 50 values of SHAM amended and un amended was determined from F test. c Significant effects are underlined. Table 3 8. Mean effective concentration of azoxystrobin and pyraclostrobin with or without SHAM at 10 g/ml to inhibit spore germination by 50% (EC 50 ) of fifteen Guignardia citricarpa isolates. No SHAM SHAM 10 EC 50 g/ml Azoxystrobin 0.015 a a 0.016 a Pyraclostrobin 0.006 a 0.005 a a Mean separation within rows followed by the same letter are not significantly different according to t

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64 Table 3 9. Analysis of variance of the effective concentration to inhibit mycelial growth by 50% (EC 50 ) when using azoxystrobin technical grade vs. commercial grade with ten Guignardia citricarpa isolates. a Fungicides azoxystrobin technical grade and azoxystrobin commercial grade (Abound) were tested for their activity on mycelium inhibition. Table 3 10. Mean effective concentration to inhibit mycelial growth by 50% (EC 50 ) when using azoxystrobin technical grade vs. commercial grade with and without SHAM and ten Guignardia citricarpa isolates. Azoxystrobin technical Azoxystrobin commercial EC 50 g/ml No SHAM 0.017 a a A b 0.016 aA SHAM 100 0.013 b A 0.014 aA a Mean separation within column s followed by the same lower case letter are not significantly different according to t b Mean separation within row s followed by the same capital letter are not significantly different according to t Source of variation df SS MS F value P value Experiment (E) 1 0.000062 0.000062 3.25 0.1049 Fungicide (F) a 1 0.000000 0.000000 0.00 0.9939 Isolate (I) 9 0.000513 0.000057 2.97 0.0603 E x F 1 0.000075 0.000075 3.92 0.0791 E x I 9 0.000428 0.000048 2.48 0.0961 F x I 9 0.000138 0.000015 0.80 0.6274

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65 Figure 3 1. Frequency distribution of the effective concentration of azoxystrobin to reduce mycelial growth by 50% (EC 50 ) of Guignardia citricarpa isolates. n= 50

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66 Figure 3 2. Frequency distribution of the effective concentration of pyraclostrobin to reduce mycelial growth by 50% (EC 50 ) of Guignardia citricarpa isolates. n= 50

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67 Figure 3 3.Frequency distribution of the effective concentration of fenbuconazole to reduce mycelial growth by 50% (EC 50 ) of Guignardia citricarpa isolates. n= 50

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68 Figure 3 4. Inhibition of mycelial growth of Guignardia citricarpa by different concentrations of azoxystrobin. Points represent the average of two experiments and three replicates for each concentration. Y= 13.84+56.96/(1+exp( (x ( 1.87))/0.26)) R 2 = 0.98 n= 50

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69 Figure 3 5 Inhibition of mycelial growth of Guignardia citricarpa by different concentrations of pyraclostrobin. Points represent the average of two experiments and three replicates for each concentration. Y= 91.51/(1+exp( (x ( 2.30))/0.79)) R 2 = 0.99 n= 50

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70 Figure 3 6. Inhibition of mycelial growth of Guignardia citricarpa by different concentrations of fenbuconazole. Points represent the average of two experiments and three replicates for each concentration. Y= 94.39/(1+exp( (x ( 2.12))/0.42)) R 2 = 0.99 n= 50

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71 Figure 3 7. Frequency distribution of the effective concentration of azoxystrobin to reduce spore germination by 50% (EC 50 ) of Guignardia citricarpa isolates. n= 50

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72 Figure 3 8. Frequency distribution of the effective concentration of pyraclostrobin to reduce spore germination by 50% (EC 50 ) of Guignardia citricarpa isolates. n= 50

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73 Figure 3 9. Inhibition of spore germination of Guignardia citricarpa isolates by different concentrations of azoxystrobin. Points represent the average of two experiments and three re plicates for each concentration Figure 3 10 Inhibition of spore germination of Guignardia citricarpa isolates by different concentrations of pyraclostrobin. Points represent the average of two experiments and three replicates for each concentration. Y= 217.79/(1+exp( (x 0.57)/1.98)) R 2 = 0.99 n= 50 Y= 145.35/(1+exp( (x ( 1.02))/1.99)) R 2 = 0.99 n= 50

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74 Figure 3 11. Effect of salicylhydroxamic acid (SHAM) on the activity of azoxystrobin on mycelial growth of Guignardia citricarpa isolates. Points represent the average of two experiments and three replicates for each concentration. Y= 14.73+56.38/(1+exp( (x ( 1.91))/0.20)) R 2 = 0.99 Y= 16.44+66.48/(1+exp( (x ( 1.95))/0.36)) R 2 = 0.99 Y= 17.22+102.41/(1+exp( (x ( 2.03))/0.28)) R 2 = 0.99 n= 15

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75 Figure 3 12. Effect of salicylhydroxamic acid (SHAM) on the activity of pyraclostrobin on mycelial growth of Guignardia citricarpa isolates. Points represent the average of two experiments and three replicates for each concentration. Y=90.54/(1+exp( (x ( 2.31))/0.73)) R 2 = 0.99 Y=92.56/(1+exp( (x ( 2.98))/0.75)) R 2 = 0.99 Y= 96.0/(1+exp( (x ( 2.50))/0.77)) R 2 = 0.99 n= 15

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76 Figure 3 13. Effect of salicylhydroxamic acid (SHAM) on the activity of azoxystrobin on spore germination of Guignardia citricarpa isolates. Points represent the average of two experiments and three replicates for each concentration. Y= 203.19/(1+exp( (x 0.31)/1.92)) R 2 = 0.99 Y= 2066/(1+exp( (x 8.13)/2.68)) R 2 = 0.99 n= 15

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77 Figure 3 14. Effect of salicylhydroxamic acid (SHAM) on the activity of pyraclostrobin on spore germination of Guignardia citricarpa isolates. Points represent the average of two experiments and three replicates for each concentration. Y= 120.5/(1+exp( (x ( 1.77))/1.51)) R 2 = 0.99 Y= 202.97/(1+exp( (x 0.17))/2.26)) R 2 = 0.99 n= 15

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78 Figure 3 15. Inhibition of mycelial growth by different SHAM concentrations. Bars with the same letter are not significantly different according to t The error bars represent the standard error. n= 2 0 a b c d

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79 Figure 3 16. Inhibition of mycelium growth of Guignardia citricarpa by different concentrations of azoxystrobin technical grade and commercial grade. Points represent the average of two experiments and three replicates for each concentration. Y= 2.92+69.88/(1+exp( (x ( 1.98))/0.32)) R 2 = 0.99 Y= 13.34+57.62/(1+exp( (x ( 1.91))/0.19)) R 2 = 0.99 n= 10

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80 Figure 3 17. Effect of salicylhydroxamic acid (SHAM) on the activity of technical and commercial grade s of azoxystrobin on mycelial growth of Guignardia citricarpa isolates. Points represent the average of two experiments and three replicates for each concentration. Y= 84.73+176.02/(1+exp( (x ( 2.85))/0.80)) R 2 = 0.99 Y= 24.86+109.46/(1+exp( (x ( 2.03))/0.24)) R 2 = 0.99 n= 10

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81 CHAPTER 4 CONCLUSIONS Since the discovery of citrus black spot in Florid a, quara nt ine measures were established to prevent the spread of the disease In addition, cultural and chemical control practices were recommended based on information from other areas where CBS is found. The Q o I fungicides azoxystrobin and pyrac lostrobin are registered in Florida for the control of black spot. The DMI fungicide fenbuconazole is also registered for use on Florida citrus, but only for greasy spot and scab Since pathogens have a history of resistance to these fungicides the establ ishment of baseline sensitivity to currently used and potentially effective fungicides becomes an important mechanism to detect population resistance. In this study, we evaluated the sensitivity of G. citricarpa isolates to azoxystrobin, pyraclostrobin and fenbuconazole. These fungicides provided good intrinsic activity against black spot. From the baseline values, pyraclostrobin and fenbuconazole inhibited mycelial growth by 50% at a lower concentration than did azoxystrobin. Moreover, spore germination wa s more sensitive than mycelium growth to the Q o I fungicides, which corresponds to the mode of action of this group of fungicides. Also, pyraclostrobin was more effective than azoxystrobin on inhibiting spore germination. Alternative respiration, a pathway used by many fungi to avoid disruption of the mitochondrial electron transport chain, such as activated b y Q o I fungicides, can be blocked by using SHAM in vitro No evidence was found that G. citricarpa isolates activated this pathway since the EC 50 values where SHAM was not used did not differ significantly from those where SHAM was used, for both azoxystrobin and

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82 pyraclostrobin. At the same time, low doses of SHAM directly inhibited mycelial growth and spore germination. Interestingly, mycelial growth was increased at 10 g/ ml compared to 1 and 0.1 g/ ml of azoxystrobin. For this reason, commercial and technical azoxystrobin grades were eva luated, but the growth was not different at the higher concentration by either gra de SHAM was tested in combination with the fungicides as well and no significant differences in the EC 50 values were observed One possible reason of the increased mycelium growth at the maximum rate of azoxystrobin is that the fungus activates a low lev el of alternative respiration at 10 g/ ml and SHAM is blocking it, or SHAM is modifying the medium permeability enhancing the fungicide take up by the mycelium. More research will be necessary to determine if this hypothesis is correct. Since black spot is an emerging disease, detection of resistant isolates to the fungicides tested was not expected. However, if precautionary measures are not taken, after prolonged used, the development of G. citricarpa resistance to strobilurins is likely. Results from t his study will help to monitor population shifts in sensitivity to these fungicides which would contribute to the long term effectiveness of currently used spray programs in Florida. At the same time, this study confirmed the potential use of fenbuconazole to control black spot; hence, field experiments should be conducted to establish its efficacy.

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83 LIST OF REFERENCES Agrios G. N. 2005. Plant p athology. Academic Press San Diego, CA. Agostini, J. P., Peres, N.A., Mackenzie, S.J., Adaskaveg, J.E., Timmer, L.W. 2006. Effect of fungicides and storage conditions on postharvest development of citrus black spot and survival of in fruit tissues. Plant Dis. 90:1419 1424. Almeida, T. 2009. Mancha preta dos citros: express o dos sintomas em frutos pela in oculao com conidios e controle do agente causal ( Guignardia citricarpa ). Faculdade de Ciencias Agrarias e Veterinarias. Universidade Estadual Paulista. So Paulo, Brazil. Ammermann, E., Lorenz, G., Schelberger, K., Mueller, B., Kirstgen, R. 2000. BAS 50 0 F the new broad spectrum strobilurin fungicide. Pest s and D is eases 2:541 548. Avila Adame, C. and Kller, W. 2003 a Characterization of spontaneous mutants of Magnaporthe grisea expressing stable resistance to the Qo inhibiting fungicide azoxystrobin. Curr. Genet. 42:332 338. Avila Adame, C. and Kller, W. 2003 b Impact of alternative respiration and target site mutations on responses of germinating conidia of Magnaporte grisea to Q o inhibiting fung icides. Pest Manag e. Sci. 59:303 309. Avila Adame, C.,Olaya, G., Kller, W. 2003. Characterization of Colletotrichum graminicola isolates resistant to strobilurin related QoI fungicides. Plant Dis. 87:1426 1432. Baayen, R.P., Bonants, P.J.M., Verkley, G., Carroll, G.C., van der Aa, H. A., de We erdt, M., van Brouwershaven, I.R., Schutte, G. C., Maccheroni, W., de Blanco, C. Glienke, Azevedo, J. L. 2002. Nonpathogenic i solates of the citrus black spot fungus, Guignardia citricarpa identified as a cosmopolitan endophyte of woody plants, G. mangife rae ( Phyllosticta capitalensis ). Phytopathology 92:464 477. Baldassari, R., Wickert, E., de Goes, A. 2007. Pathogenicity, colony morphology and diversity of isolates of Guignardia citricarpa and G. mangiferae isolated from Citrus spp. Eur. J. Plant Pathol. 120:103 110. Bartlett, D.W., Clough, J.M., Godwin, J.R., Hall, A.A., Hamer, M., Parr Dobrzanski, B. 2002. The strobilurin fung icides. Pest Manage. Sci. 58:649 662. Bellott e, J. A. Kupper, K., Rinaldo, D., Souza, A., Pereira, F., Goes, A. 20 09. Acceleration of the decomposition of Sicilian lemon leaves an auxiliary measure in the control of citrus black spot. Tropical Plant Pathol. 34:71 76. Bonants, P.J.M., Carroll, G.C., de Weerdt, M., van Brouwershaven, I.R., Baayen, R.P 2003. Development and validation of a fast PCR based detection method for

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84 pathogenic isolates of the citrus black spot fungus, Guignardia citricarpa Eur. J. Plant Pathol. 109:503 513. Brent, K., and Hollomon, D. 1998. Fungicide resistance: the assessme nt of risk. Fungicide Resistance Action Committee, UK. Brentu, F., Oduro, K., Offei, S., Odamtten, G., Vicent, A., Peres, N. A. Timmer, L. W 2012. Crop loss, aetiology, and epidemiology of citrus black spot in Gh ana. Eur. J. Plant Pathol. 133:657 670. Bushong, P. M. and Timmer, L.W. 2000. Evaluation of postinfection control of citrus scab and melanose with benomyl, fenbuconazole, and azoxystrobin. Plant Dis 84:1246 1249. Ca labrese, E. and Baldwin, L. 2002 Defining hormesis. H uman & Experimental Toxic ology 21:91 97. Chin, K. M., Chavaillaz, D., Kaesbohrer, M., Staub, T., Felsenstein, F. G. 2000. Characterizing resistance risk of Erysiphe graminis f.sp. tritici to strobilurins. Crop P rotection 20:87 96. Cooke, T., Persley, D ., House, S. 2009. Diseases of fruit crops in A ustralia CSIRO, Australia. Da Silva, A., De Goes, A., Wickert, E., Almeida, T., Machado, M.A. 2009. Mancha preta dos citros: epidemiologia e manejo. Revista Laranja 30:45 64. Deising, H.B., Reimann, S., Pa scholati, S. F. 2008. Mechanisms and significance of fungicide resistance. Braz. J. Microbiol. 39:286 295. Dewdney, M. M. 2010. Fungicide resistance: why it happens and how it may affect you. Citrus Industry 91:3. Dewdney, M. M. Peres, N. A. Ritenour M., Roberts, P. 2010. Citrus black spot. UF/IFAS Extension. Available at : http://www.crec.ifas.ufl.edu/extension/black_spot/citrus_black_spot.shtml Accessed Augus t 13, 2012. Dewdney, M. M Estes, M.R., Peres, N.A. 2012. 2012 Florida C itrus P est M anagement G uide: citrus black spot. In: Rogers, M.E., Dewdney, M.M. and Spann, T.M. (eds.) University of Florida IFAS Extension. Publ.No. PP279. Duan, Y., Liu, S., Ge, C. Feng, X., Chen, C., Zhou, M. 2012. In vitro inhibition of Sclerotinia sclerotiorum by mixtures of azoxystro bin, SHAM, and thiram. Pestic. Biochem. Physiol. 103:101 107.

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85 Erickson, E. and Wilcox, W. 1997. Distribution of sensitivities to three sterol de methylation inhibitor fungicides among populations of Uncinula necator sensitive and resistance to triadimefon. Phytopathology 87:784 791. European and Mediterranean Plant Protection Organization (EPPO). 2009. Guignardia citricarpa Bulletin 39:318 327. European and Mediterranean Plant Protection Organization (EPPO). 2009. Guignardia citricarpa data sheet on quarantine pests. Available at: http://www.eppo.int/ QUA RANTINE/fungi/Guignardia_citricarpa/GUIGCI_ds.pdf Accessed August 3, 2012. Everett, K. R. and Rees George, J. 2006. Reclassification of an isolate of Guignardia citricarpa from New Zealand as Guignardia mangiferae by sequence analysis. Plant Pathol. 55 :194 199. Fishel, F.M. 2011. Pesticide toxicity profile: triazole pesticides. Florida Cooperative Extension Service, IFAS.Publ. No. PI105. Fishel, F.M. 2012. Fungicide Resistance Action Committee's (FRAC) classification scheme of fungicides according t o mode of action. University of Florida IFAS Extension. Publ. No. PI94. Florida Citrus Health Response Program Working Group. 2010. Strategies for effective eradication of citrus black spot in Collier and Hendry Counties. Available at: http://www.crec.ifas.ufl.edu/extension/greening/PDF/Citrusblackspotcontrolrecomme ndations20100527.pdf Accessed August 13, 2012. Fogliata, G.M, Muoz, M.L., Rojas, A.A. Ploper, D. 2011. Eficacia de tres estrobilurinas para el control de mancha rojiza ( Guignardia mangiferae ) y mancha negra ( Guignardia citricarpa ) en frutos de limn, en Tucumn, Repblica Argentina. Revista In dustrial y Agr cola de Tucumn 88:37 45. Fungicide Resistanc e Action Committee (FRAC). 2005. Introduction and general information about sterol biosynthesis i nhibitors (SBI's) Available at: http://www.frac.inf o/frac/work/work_sbif.htm Accessed October 15, 2012. Fungicide Resistance Action Committee (FRAC). 2010. QoI working group of FRAC. Available at: http://www.frac.info/frac/work/FRAC_QoI_Minutes_2010_final_RG.pdf Accessed August 15, 2012. Fungicide Resistance Action Committe e (FRAC). 2012. FRAC code list 2012 : Fungicides sorted by mode of action (including FRAC code numbering) Available at: http:/ /www.frac.info/frac/index.htm Accessed August 10, 2012.

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86 Garzn C.D., Molineros J.E., Ynez J. M., Flores, F., Jimenez Gasco M., Moorman, G. 2011. Sublethal doses of mefenoxam enhance Pythium damping off of geranium. Plant Dis. 95:1233 1238. Gisi, U.,Chi n, K. M., Knapova, G., Kng Frber, R., Mohr, U., Parisi, S., Sierotzki, H., Steinfeld, U. 2000. Recent developments in elucidating modes of resistance to phenylamide, DMI and strobilurin fungicides. Crop Protection 19:863 872. Gisi, U., Hermann, D., Ohl, L., Steden, C. 1997. Sensitivity profiles of Mycosphaerella graminicola and Phytophthora infestans populations to different classes o f fungicides. Pesti c. Sci. 51:290 298. Gisi, U. and Sierotzki, H. 2008. Fungicide modes of action and resistance in downy mildews. Eur. J. Plant Pathol. 122:157 167. Glienke, C., Pereira, O.L., Stringari, D., Fabris, J., Kava Cordeiro, V., Galli Terasawa, L., Cunnington, J., Shivas, R.G., Groenewald, J.Z., Crous, P.W. 2011. Endophytic and pathogenic Phyllosticta species, with reference to those associated wi th citrus black s pot. Persoonia 26:47 56. Goes, A. 2002. Efeito da combinao de fungicidas sistmicos e protetores no controle da mancha preta dos frutos ctricos causada por Guignardia citricarpa Summa Ph ytopathologica 28:9 13 Hanlin, R. T.1990. Illustrated g enera of Ascomycetes. American Phytopathological Society, St. Paul, Minn Heaney, S. P., Hall, A.A., Davies, S.A., Olaya, G. 2000. Resistance to fungicides in the Qol STAR cross resistance group: current perspectives. The BCPC Conference: Pests and D iseases 2:755 762 Herbert, J.A., Grech, N.M. 1985. A strain of Guignardia citricarpa the citrus black spot pathogen, resistant to benomyl in South Africa. Plant Dis. 69: 1007. Hoch, H.C., Shaw, B.D., Carroll, G. C. 2006. Generality of the prerequisite of conidium attachment to a hydrophobic substratum as a signal for germination among Phyllosticta species. Mycologia 98:186 194. Holb, I.J. and Schnabel, G. 2007. Differenti al effect of triazoles on mycelial growth and disease measurements of Monilinia fructicola isolates with reduced sensitivity to DMI fungicides. Crop Protection 26:753 759. Holtz, T. 2010. Risk assessment of Citrus spp. fresh and dried fruit as a pathway of the introduction of Guignardia citricarpa Kiely, the organism that causes citrus black spot disease. Available at:

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94 BIOGRAPHICAL SKETCH Martha Hincapie was born in Santa Marta, Colombia She graduated from with an accounting specializat ion in 2003. In 2004, she won a scholarship from Dole Fru it Company to conduct agronomic studies at EARTH University in Costa Rica. During her bachelo r studies she did an intern ship at the Fundao Mokiti Okada in Brazil where she became interested in the field of plant pathology After her graduation in 2007, strawberry pathology lab for two years. Then she began a s a plant pathology graduate student. of baseline sensitivity of Guignardia citricarpa the causal agent of citrus black spot, to azoxystrobin, pyraclostrobin, and fenbuconazole.