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Mycelial Compatibility and Pathogenic Diversity among Sclerotium Rolfsii Isolates in Southeastern United States

Permanent Link: http://ufdc.ufl.edu/UFE0044734/00001

Material Information

Title: Mycelial Compatibility and Pathogenic Diversity among Sclerotium Rolfsii Isolates in Southeastern United States
Physical Description: 1 online resource (71 p.)
Language: english
Creator: Xie, Chenzhao
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: diversity -- pathogenicity -- rolfsii -- sclerotium
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: Sclerotium rolfsii is a soil-borne fungus that causes southern blight to a wide range of plants in the tropical and sub-tropical regions of the world. This study aimed to characterize the phenotypic and pathogenic diversity among isolates collected from the southern United States. Eighty-four isolates collected from Virginia, South Carolina, Georgia, Florida, Louisiana, and Texas were paired and assigned to twenty-three mycelial compatibility groups (MCGs), of which 11 MCGs consisted of a single isolate. Isolates within a MCG typically originated from different hosts and different geographical areas with the exception of MCG 11. Fourteen out of 16 isolates in MCG 11 originated from peanuts in Georgia, while the other two isolates originated from snap bean and potato in Virginia. Significant differences in the size and number of sclerotia produced in vitro existed between isolates from peanut and other hosts. Nineteen isolates representative of the most common MCGs were tested for pathogenicity on tomato, pepper, and peanut plants.  All isolates were pathogenic to all hosts, but virulence differed significantly among isolates. Isolates originally collected from peanut were the most virulent on all tested plants compared to isolates collected from tomato and pepper. The peanut cultivar Georgia Green was more susceptible to peanut isolates from Georgia than to other isolates collected from other hosts or other geographical regions. Of the two tomato entries, the commercial tomato cv. Tygress exhibited more tolerance than the previously reported resistant breeding line 5635M to many of the S. rolfsii isolates tested, with the exception of the peanut isolates collected from Georgia.
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 Chenzhao Xie.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Vallad, Gary Edward.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044734:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044734/00001

Material Information

Title: Mycelial Compatibility and Pathogenic Diversity among Sclerotium Rolfsii Isolates in Southeastern United States
Physical Description: 1 online resource (71 p.)
Language: english
Creator: Xie, Chenzhao
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: diversity -- pathogenicity -- rolfsii -- sclerotium
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: Sclerotium rolfsii is a soil-borne fungus that causes southern blight to a wide range of plants in the tropical and sub-tropical regions of the world. This study aimed to characterize the phenotypic and pathogenic diversity among isolates collected from the southern United States. Eighty-four isolates collected from Virginia, South Carolina, Georgia, Florida, Louisiana, and Texas were paired and assigned to twenty-three mycelial compatibility groups (MCGs), of which 11 MCGs consisted of a single isolate. Isolates within a MCG typically originated from different hosts and different geographical areas with the exception of MCG 11. Fourteen out of 16 isolates in MCG 11 originated from peanuts in Georgia, while the other two isolates originated from snap bean and potato in Virginia. Significant differences in the size and number of sclerotia produced in vitro existed between isolates from peanut and other hosts. Nineteen isolates representative of the most common MCGs were tested for pathogenicity on tomato, pepper, and peanut plants.  All isolates were pathogenic to all hosts, but virulence differed significantly among isolates. Isolates originally collected from peanut were the most virulent on all tested plants compared to isolates collected from tomato and pepper. The peanut cultivar Georgia Green was more susceptible to peanut isolates from Georgia than to other isolates collected from other hosts or other geographical regions. Of the two tomato entries, the commercial tomato cv. Tygress exhibited more tolerance than the previously reported resistant breeding line 5635M to many of the S. rolfsii isolates tested, with the exception of the peanut isolates collected from Georgia.
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 Chenzhao Xie.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Vallad, Gary Edward.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044734:00001


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1 MYCELIAL COMPATIBILITY AND PATHOGENIC DIVERSITY AMONG Sclerotium rolfsii ISOLATES IN SOUTHEASTERN UNITED STATES By CHENZHAO XIE 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 Chenzhao Xie

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3 To my family and all my friends

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4 ACKNOWLEDGMENTS I would like to thank my family for all the time and money you invested in me. I would l ike to e specially thank my sister and her husband for their support and valuable advice. I would also like to thank my advisor, Dr. Vallad, for his persistent encouragement, support, and scientific guidance during my studies with him. I really appreciate a ll the opportunities you created for me, especially the opportunities to practice my oral English. I would like to thank my graduate committee members, Dr. Rollins, Dr. Robert s and Dr. Scot t for all the ir encouragement and support as well I would like to extend my deep est gratitude to my lab members for their support and help in the lab and greenhouse. I would like to pay special thanks to Dr. Cheng Hua Huang for his help with statistics and other scientific advice; and to his wife Yachiao Wang, for kind care in my everyday life. I would like to thank my fellow graduate student Mehul Bhakta for his support with my greenhouse work. Finally I would like to thank my husband and all my friends who helped me achieve this degree and who have filled my life with joy and happiness.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURE S ................................ ................................ ................................ .......... 8 CHAPTER 1 LITERATURE REVIEW ................................ ................................ ........................... 11 Tomato Production in Florida ................................ ................................ .................. 11 Common Diseases of Tomato Caused by Soilborne Pathogens ............................ 11 Fumigation for the Control of Soilborne Pathogens ................................ ................ 12 Sclerotium rolfsii Sacc ................................ ................................ ............................. 14 Biology ................................ ................................ ................................ .............. 15 Life Cycle ................................ ................................ ................................ .......... 16 Pathogenicity and Virulence Factors ................................ ................................ 17 Population Diversity ................................ ................................ .......................... 19 Integrated Management for the Disease Caused b y Sclerotium rolfsii ............. 25 Host resistance ................................ ................................ .......................... 25 Chemical control ................................ ................................ ........................ 25 B iological control ................................ ................................ ........................ 26 Research Objectives ................................ ................................ ........................ 27 2 MORPHOLOGICAL CHARACTERization OF SCLEROTIUM ROLFSII ISOLATES AND PATHOGENICIT Y /VIRULENCE TESTS UNDER GREENHOUSE CONDITIONS ................................ ................................ ............... 28 Introduction ................................ ................................ ................................ ............. 28 Materials and Methods ................................ ................................ ............................ 30 Fungal Materials ................................ ................................ ............................... 30 Mycelial Compatibility Groups (MCG) ................................ .............................. 30 Morphological Characterization ................................ ................................ ........ 31 Plant Materials for Pathogenicity Tests ................................ ............................ 31 Pathogenicity Tests ................................ ................................ .......................... 32 Statist ical Analysis ................................ ................................ ............................ 33 Results ................................ ................................ ................................ .................... 33 Mycelial Compatibility Groups ................................ ................................ .......... 33 Oth er Morphological Characters ................................ ................................ ....... 34 Pathogenicity/Virulence Tests ................................ ................................ .......... 35 Discussion ................................ ................................ ................................ .............. 37

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6 3 SUMMARY AND CONCLUSIONS ................................ ................................ .......... 61 LIST OF REFERENCES ................................ ................................ ............................... 65 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 71

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7 LI ST OF TABLES Table page 2 1 Sclerotial characteristics and mycelia compatibility group (MCG) designation for Sclerotium rolfsii isolates sorted by geographic origin, host of origin, sclerotia color and size, year of isolation, and sclerotia number in medium ...... 43 2 2 Sclerotium rolfsii isolates sorted by mycelial compatibility group (MCG). ............ 46 2 3 Statistical contrasts for sclerotia size and number of sclerotia per isolate produced in vitro by Sclerotium rolfsii isolates collected from tomato, pepper, and peanut. ................................ ................................ ................................ ......... 47 2 4 Statistical contrasts for southern blight severity caused by Sclerotium rolfsii isolates collected from tomato, pepper, and peanut on tomato cultivar Tygress and breedling line 5635M using 10 sclerotia per plant. ......................... 48 2 5 Analysis of variance of the effect of inoculation load on the severity of southern blight caused by Sclerotium rolfsii isolates SR 1, 29, and 41 on tomato cultivars Tygress and breeding line 5635M plants 28 days after inoculating. ................................ ................................ ................................ ......... 49 2 6 Effect of inoculation level on the severity of southern blight on tomato plants 28 days after inoculating. ................................ ................................ .................... 50 2 7 Analysis of variance of the effect of Sclerotium rolfsii isolates on southern blig ht severity on tomato cultivar Tygress and b reeding line 5635M using 4 sclerotia per a plant. ................................ ................................ ........................... 51 2 8 Statistical contrasts of Sclerotium rolfsii isolates collected from tomato, pepper, and peanut on south ern b light severity of tomato cultivar Tygress and breeding line 5635M using 4 sclerotia per a plant. ................................ ...... 52 2 9 Statistical contrasts of Sclerotium rolfsii isolates collected from tomato, pepper, and peanut on south ern blig ht severity of peanut cultivar Georgia Green and pepper cultivar Tom Cat. ................................ ................................ .. 53

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8 LIST OF FIGURES Figure page 2 1 An A) incompatible and a B) compatible reaction between paired isolates of Sclerotium rolfsii for designating mycelial compatibility grou ps. ......................... 54 2 2 Respective overhead and side profiles of a Sclerotium rolfsii isolate from tomato, SR 1 A) and C), and a isolate from peanut, SR 754 B) and D) showing the difference in colony morphology. ................................ .................... 55 2 3 Symptoms of southern blight caused by Sclerotium rolfsii using the A) stem inoculation method with foliar wilt and blight symptoms observed on B) pepper, C) tomato, an d D) peanut. ................................ ................................ ..... 56 2 4 Symptoms of A) stem rot and wilt, and B) stem scab lesions on pepper compared to symptoms of C) stem rot and wilt, and D) stem scab lesions on tomato caused by Sclerot ium rolfsii ................................ ................................ ... 57 2 5 An example of a pepper plant A) not exhibiting any wilt symptoms following inoculation with Sclerotium rolfsii but still showing an apparent B) scab lesion at the ino culation site and an example of a C) stem scab near the soil line of another inoculated pepper plant. ................................ .............................. 58 2 6 Typical symptoms of wilt observed A) on a pepper plant 28 days after inoculati ng with Sclerotium rolfsii compared to a B) pepper plant that recovered after the same inoculation period, with a C) close up view of the upper foliage. ................................ ................................ ................................ ...... 59 2 7 Mean wilt severity caused by 19 isolates of Sclerotium rolfsii 28 days after S. rolfsii included: SR1, 5, 8, 12, 14, 19, and SR 47 originati ng from tomato; SR 21, 41, 105, and 920 from pepper; SR 29, 30, 32, and SR 40 from peanut; SR 23 from pumpkin; SR 25 from sweet potato, SR 38 from peanut leaf; and SR 44 from snap bean. Vertical bars on each column represent the 95% confidence intervals. A n asterisk near the isolate name in (C) denotes a significant difference in wilt severity between the two tomato cultivars ( = 0.05). An asterisk near the isolate name means there were significant difference between two tomato cultivars. ................................ ............................ 60

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MYCELIAL COMPATIBILITY AND PATHOGENIC DIVERSITY AMONG Sclerotium rolfsi i ISOLATES IN SOUTHEASTERN UNITED STATES By Chenzhao Xie August 2012 Chair: Gary E. Vallad Major: Plant Pathology Sclerotium rolfsii is a soil borne fungus that causes southern blight to a wide range of plants in the tropical and sub tropical regions of the world. This study aimed to characterize the phenotypic and pathogenic diversity among isolates collected from the southern United States. Eighty four isolates collected from Virginia, South Carolina, Georgia, Florida, Louisiana, and Texas were paire d and assigned to twenty three mycelial compatibility groups (MCGs ) of which 11 MCGs consisted of a single isolate. Isolates within a MCG typically originated from different hosts and different geographical areas with the exception of MCG 11. Fourteen out of 16 isolates in MCG 11 originated from peanuts in Georgia, while the other two isolates originated from snap bean and potato in Virginia. Significant differences in the size and number of sclerotia produced in vitro existed between isolates from peanut and other hosts. Nineteen isolates representative of the most common MCGs were tested for pathogenicity on tomato, pepper, and peanut plants. All isolates were pathogenic to all hosts, but virulence differed significantly among isolates Isolates original ly collected from peanut were the most virulent on all tested plants compared to isolates collected from tomato and

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10 pepper. The peanut cultivar Georgia Green was more susceptible to peanut isolates from Georgia than to other isolates collected from other h osts or other geographical regions Of the two tomato entries, the commercial t omato cv. Tygress exhibited more tolerance than the previously reported resistant breeding line 5635M to many of the S. rolfsii isolates tested with the except ion of the peanut isolates collected from Georgia.

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11 CHAPTER 1 LITERATURE REVIEW Tomato Production in Florida Tomato ( Solanum lycopersicum L.) is an important vegetable grown in the United States and much of the rest of the world. Fresh market tomato production occurs throug hout the southeastern United States (Alabama, Florida, Georgia, North Carolina, South Carolina, Tennessee, and Virginia) and in California ( 70 ) California and Florida are the leading producers of fresh market tomatoes in the United States. However, Florida and Mexico are the main sources of many fresh vegetable crops to the United States market from November through May. Fresh market tomato production in Florida was valued at $631 million, firs t in the United States accounting for 45% of the total value (USDA NASS, 2010 ). Commercial tomato es are produced in five production regions in Florida : Miami Dade County (Homestead), Palm Beach/St. Lucie counties, southwest Florida (Immokalee/Naples), Mana tee/Hillsborough counties (Ruskin), and northwest Florida (Quincy) ( 44 ) Various kinds of tomatoes are grown in Florida, and are classified into horticultural types according to the fruit type, which include round, plum, cherry and grape ( 67 ) In Florida, the large fruited round tomatoes are the most common type produced commercially, where they are usually harvested at the mature green stage and treated with ethylene in ripening roo ms prior to shipmen t (14 ). Common Diseases of Tomato Caused by Soilborne Pathogens While tomato production in Florida is challenged by many diseases during the growing season, some of the most serious are those caused by soilborne pathogens. These diseases include, Bacteri al wilt (caused by Ralstonia solanacearum ), Fusarium

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12 wilt (caused by Fusarium oxysporum f. sp. lycopersici ), Fusarium crown and root rot (caused by F. oxysporum f. sp. radicis lycopersici ), southern blight (caused by Sclerotium rolfsii ), timber rot (caused by Sclerotinia sclerotiorum ), and Verticillium wilt (caused by Verticillium albo atrum and V. dahlia ). Control of these diseases has re lied heavily on the use of soil applied chemical fumigants prior to planting to limit the levels of these pathogens in t he soil. Nearly all commercial tomatoes grown in Florida are produced on raised, plastic mulched beds that are fumigated for the control of soilborne pathogens, in addition to weeds and other pests ( 60 ) Fumigation for the Control of Soilborne Pathogens Commercial tomato production in Florida requires intense management of soil moisture and fertility in order to produce an optimal quantity of high quality fruit, along with the use of broad spectrum soil fu migants and additional pesticides to manage other weeds and pests. Pre plant fumigants are typically used for the production of high value vegetables, tree fruits, and nursery crops as a means to suppress the level of insects, nematodes, diverse plant path fumigant combines the properties of a herbicide, fungicide, and nematicide along with the ability to dissipate from the soil rapidly, while not posing any serious long term impact to the environment ( 60 ) The most commonly used soil fumigants are methyl bromide, chloropicrin, 1, 3 dichloropropene, metam potassium, metam sodium, and methyl iodide. Methyl bromide is an ideal broad spectrum fumigant that h as been used for synergistic activity that can broaden the pest control spectrum ( 59 ) Effectiveness was primarily due to the high vapor pressure of methyl bromide that enabled it to distribute

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13 evenly through the soil profile and dissipate rapidly impa rting short plant back interval compared to other fumigants ( 36 ) This property also made methyl bromide ideal for quarantine purposes, for the control of fungi, nematodes, insect s, and vermin in internationally traded nuts, grains, fresh fruit, vegetables, and other commodities; and for the fumigat ion of structures ( 69 ) California and Florida were the largest users of ( 59 ) In Florida, methyl bromide was widely used fo r the production of many vegetables, including tomatoes, peppers, eggplants, cucumbers, squash, and watermelons. In September of 1987, the Montreal Protocol on Substances that Deplete the Ozone Layer was signed by 24 countries leading to the phase out of a ll substances believed to deplete atmospheric ozone. This was followed by the Clean Air Act in the United States mandating that the United States Environmental Protection Agency (EPA) list any substance with an ozone depletion potential (ODP) 0.2 a s a Class I ozone depleter ( 42 ) Methyl bromide with an estimated ODP of 0.65 wa s categorizes as an ozone depleting chemical in 1992. Due to this reason methyl bromide was scheduled for phase out by 2005, with the exception of pre shipment and quarantine uses, critical agricultural uses, and mandated emergency uses. With the phase ou t process of methyl bromide, Florida vegetable growers have observed a resurgence of several diseases caused by soilborne pathogens ( 65 ) Early reports estimated that of the loss of methyl bromide would seriously affect Florida fresh vegetable and fruit industry, including tomatoes, strawberries, watermelon, peppers, eggplant, cucumbers, and squash; with economic losses exceeding $500 million ( 59 )

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14 Therefore, a number of alternative fumigants to methyl bromide have been researched and labeled for agricultural use such as the methyl isothiocyanate generators (metam potassium and metam sodium), 1,3 dichloropropene, chloropicrin, dimethyl disulfide, and methyl iodide. However, the performance of these alternatives are more sensitive to environmental factors such as soil moisture and soil temperature, and the permeability of the plastic mulch ( 41 ) Sclerotium rolfsii Sacc Southern blight caused by the fungus Sclerotium rolfsii Sacc. (teleomorph Athelia rolfsii (Curzi) C.C. Tu & Kimbr.) is a serious disease affecting diverse crops grown around the world especially in tropical and subtropical regio ns. Based on host range, S. rolfsii is also one of the most destructive fungal pathogens able to cause disease on about 500 plant species across 100 plant families, including several crops of economic importance in Florida like tomato, potato, pepper, cant aloupe, celery, carrot, cabb age, bean, eggplant, and peanut ( 4 19 ) T he disease has long been a major problem in the production of row crops like peanut in the southeast United States. However, the disease has become more problematic in vegetable production coinciding with the adoption of alternative fumigant s to methyl bro mide and the adoption of organic and other low input production strategies. Losses attributed to this disease can be profound. For example, yield losses in peanut production to S. rolfsii typically do not exceed 25%, and are around 7 10% annually on avera ge, but losses may reach to 80% in serious disease years in southeast United States. Annual economic loss of peanut associated to S. rolfsii alone was estimated at $36.8 million in Georgia from 1988 through 1994 ( 23 )

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15 Biology S. rolfsii is an aerobic basidiomycete fungus. Primary growth consists of vegetative mycelia that are white and fluffy over an extensive temperature range of 8 40 C; with the optimal temperature range for mycelium growth and sclerotia production of between 27 30 C. Sclerotia are the sole asexual reproductive propagules produced by S. rolfsii yet sclerotia are not truly spores, but are composed of highly compacted, melanized hyphae. These hard, round, sclerotia are produced from the main hyphal strands about seven days after infection, and range from 0.3 mm to 2.0 3.0 mm in diameter. Sclerotia are initially white, and then become yellow, tan, or brown as they mature. During sclerotium development, drops of clear exudates are produced at the surface, which contain protei ns, cations, amino acids, carbo hydrates, enzymes, and oxalic acid. However, droplet s do not appear on the sclerotia surface in soil or in cultures exposed to ambient air ( 14 52 ) An outer melanized rind, an underlying cortical layer, and an innermost medulla comprise the mature scleotia. Nutrient composition of mature sclerotia consists of amino acids, fatty acids, sugars and lipids ( 34 ) Sclerotia can germinate in two different ways, eruptive or hyphal, depending on the environmental conditions. Eruptive germination is characteriz ed by mycelium aggregrating and then bursting through the sclerotial rind, le aving an empty sclerotial rind. Hyphal germination differs from eruptive germination by the growth of individual strands from the sclerotium surface that originate from inner medu lla. Exposing dry sclerotia to volatile compounds, such as primary alcohols, can induce eruptive germination ( 50 ) The optimal germination temperature f or sclerotia is between 25 30 C when th e soil moisture holding capacities are between 25 75% ( 53 )

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16 The frequency of eruptive germination is highest for sclerotia at the surface of field soil, and decreases at soil depths of 5 25 mm. Germination frequency continues to decrease gradually with increasing soil depths below 25 mm, to near zero at soil depths greater than 70 mm. Low oxygen levels, the buildup of carbon dioxide, and the physical pressure exerted by the overlying soil are all cited as reasonable explanations for the low germination of sclerotia at increasing soil depths ( 53 ) Life Cycle Sclerotium rolfsii overwinters as sclerotia and mycelium in or o n infected plant tissues and debris. As sclerotia have a high demand for oxygen, they commonly germinate when they are in the upper regions of the soil ( less than 5cm depth), explaining why the disease is more damaging in sandy soils ( 51 ) Sclerotia can be easily disseminated through transplant seedlings, water, wind or any cultural practice that moves infested soil or plant debris. Sclerotia germinate and hyphal growth resumes once favorable environmental conditions are present. Susceptible plant tissues, lower stems, roots, and fruit can be directly penetrated by contacting hyphae under ideal conditions. Wounds can facilitate infection as well. During the infection proc ess, the fungus secretes oxalic acid and endopolygalacturonase, which degrade plant tissues and cells, and eventu ally lead to decay ( 51 ) Diseases caused by S. rolfsii are favored by warm, wet weather and keep spreading when hyphae and sclerotia reach new susceptible plant tissues while these favorable conditions persist These conditions not only favor rapid disease development but also plant to plant spread of the pat hogen. This explains why plants infected with S. rolfsii are typically clustered or clumped in the field D isease develops better in loose

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17 sandy soil than in fine textured like silt or clay soil, and better when the soil moisture holding capacity is 50 to 75% than at saturation ( 56 ) The exact role of the teleomorphic stage (sexual or perfect stage) in the d isease epidemiology of S. rolfsii remains unclear. Although this perfect stage and basidiospore production can be induced under special laboratory conditions, it has r arely been observed in nature. Greenhouse trials have clearly demonstrated that basidiosp ores can infect host tissues ( 51 ) Usually the disease is a single cycle within the growing season, with limited spread to neighboring plant s under conductive condition s. Any potential of a secondary cycle caused by basidiospores is not clear. However, aerial leaf spots caused by S. rolfsii were reported in India ( 55 ) and may support basidiospores as a source of inoculum in the field. Genetic analys is or conventional spore trapping techniques will need to be employed to determine if basidiospores are airborne and their relative contribution to disease epidemiology ( 5 1 ) Pathogenicity and Virulence Factors Punja et al ( 51 ) reported that oxalic acid and endopolygalacturonase are produced in concert with rapid mycelial growth, and a re the factors critical to pathogenicity. Oxalic acid plays an essential role during disease progress in many pathogenic fungi, and was shown to be essential for pathogenicity of Sclerotinia sclerotiorum ( 37 ) Differences in virulen ce among isolates of S. rolfsii were related to the level of oxalic acid produced as well as the level of cell wall degrading enzymes and mycelium growth rate ( 49 ) Oxalic acid has been reported to weaken or degrade the plant cell wall by acidifying the infected tissue and chelating Ca 2+ within the cell wall. The lowered pH at

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18 the infection site also improves the activity of many fungal enzymes secreted during the infection process ( 6 ) Cessna et al ( 13 ) found that oxalic acid, as a pathogenicity factor for S. sclerotiorum can suppress the oxidative burst of the host plant, which is required for several defense re sponses in most plant species. Kim et al ( 28 ) found that oxalic acid elicits programmed cell death in infected tissues during the development of disease caused by S. sclerotiorum. Similar to S. sclerotiorum Punja ( 51 ) reported that virulence among S. rolfsii isolates was related to oxalic acid production, rapid growth rate and the level of activity of endo polygalacturonase. Endo polygalacturonase is excreted at the early phase of infection, deplet ing pectic substances from cell walls, which aids to macerate host tissue. Cellulase and other enzymes like polygalacturonase and pectinmethylgalacturonase, help destroy cell walls completely. High levels of oxalic acid, endo polygalacturonase, endo pectin methylgalacturonase, were detected in both liquid S. rolfsii culture media and in infected carrot root tissues ( 51 ) The high virulence associated with some S. rolfsii i solates was attributed to the rapid hyphal growth rate, extensive production of oxalic acid, and high activities of endo polygalacturonase and endo pectinmethylgalacturonase. Large quantites of oxalic acid are produced at the initial stage of infection low ering the tissue pH at the infection site. This enhances conditions for optimum activity of endo polygalacturonase and cellulose to degrade cell walls. Endo polygalacturonase is a critical enzyme produced during the early phase of pathogenisis process that depletes pectic substances from middle lamella of cell walls ( 51 ) Pectic enzymes also play a significant role in S. rolfsii path o genesis by macerating pectin substance in the invaded tissues and depleting the galacturonic acid content of

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19 cell walls in diseased tissues ( 7 ) In additio n to these hydrolytic enzymes, x ylanase and mannanase are reportedly produced to degrade xylans and mannans respectively. Xylans are the main non cellulosic polysaccharides in hardwood and annual plants; and mannans are the most abundant hemicelluloses in softwoods ( 61 ) Population Diversity Heterokaryon formation between two fungal individuals is a common phenomenon in Ascomycetes and Basidiomy cetes which comprise two different forms, sexual heterokaryon and vegetative heterokaryon ( 33 ) Strains that can form stable patible However, the two forms of compatibility are quite different from each other, vegetative compatibility is the ability of two strains to fuse and fo rm a stable heterokaryon ( 29 ) governed by specific vic (vegetative incompatibility) or het (heterokaryon incompatibility) loci ( 63 ) Commonly, one or more mating type loci which may have two or more alleles govern sexual compatibility ( 33 ) Anastomosis of vegetatively incompatible strains leads to hyphal fusion, and then rapidly results in compartmentation and death of the fused and adjacent cells, lysis of anastomosing and adjacent c ells, and hyphal vacuolization On media plate s, a macroscopic barrage zone caused by lysis appears between two vegetatively incompatible strains. The biological significance of this phenomenon to fungi remains an open debate. Vegetative incompatibility prevents the formation of a stable heterokaryon between unlike individuals and then further limits the migration of nuclei and the exchange of genomic information; it also reduces the risk of horizontal transmission of infectious cytoplasmic elements, such as mycoviruses, transposons, double stranded R NAs,

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20 senescence plasmids, and other debilitated organelles ( 24 63 ) These functions of het genes are assumed to pre serve genetic individuality ( 63 ) Two genetic systems, allelic and nonallelic, controlling vegetative incompatibility have been identified. Both of these two s ystems function effectively when a specific genetic difference happens between the two strains. In allelic systems, incompatibility is triggered when two strains carrying different alleles at one or more het loci interact. Generally, allelic incompatibilit y does not affect sexual compatibility. In nonallelic systems, genetic differen ce at two distinct loci leads to incompatibility, and this incompatibility cannot be overcome during the sexual stage of the life cycle ( 24 ) In Ascomycetes, most incompatibility studies have focused on Podospora anserina Neurospora crassa Sclerotinia sclerotiorum Cochliobolus heterostrophus and Fusarium oxysporum ( 24 63 ) In N. crassa at least 11 het loci exist and all of them are allelic, each with two alleles ( 63 ) Among these 11 het loci, het c causes a relatively mild incompatibility reaction compared to other het loci interactions in N. crassa An incompatible reaction at this locus is characterized by slow hyphal growth, and curly mycelium ( 63 ) w here about 15% of the cells died in partial diploid situations. In contrast to the het c locus heteroallelism at the het 6 locus, which contains two tightly linked het genes, leads to a severe inhibition of hyphal growth in heterokaryons or partial diploids ( 63 ) For P. anserina both allelic and non allelic genetic systems exist. Thirteen nonallelic loci control vegetative incompatibility, of which het c/het d, het c/het e and het r/het v have been best described. Loci c and e have more than one allele while the v locus operates in both allelic and nonallelic systems ( 24 ) These three nonallelic

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21 systems, het c/het d, het c/het e and het r/het v can also be suppressed by mutations at other loci, identified as mod A and mod B ( 24 ) Vegetative compatibility has been used as a means to measur e population diversity in a number of plant pathogenic fungi by the existence of barrage or heterokaryon tests. Placing isolates into vegetative compatibility groups (VCG) was utilized as an indicator of genetic diversity in the population analysis of Aspe rgillus flavus isolates from a single cotton field ( 8 ) Thirteen VCGs were identified within a total of 61 isolates in a single field and the population was unique in contrast to other local field populations. The frequency and number of VCGs detected changed from 1987 through 1989. In 1989 the population consisted of more VCGs than in 1987 and 1988; and VCG A, which was the dominant VCG in the field in 1988, had been displaced from the field population. Isolates within the same VCG a re assumed have the ability to exchange genetic information during the parasexual cycle ( 33 ) Mycelia compatibility refers to the ability of two strains of the same fungal species to fuse together and form a single colony, whereas incompatible strains cannot. Mycelial compatibility has been widely used as an effective means to study potential genetic diversity within field populations of various fungi, and is considered as one of several ev ents associated with vegetative incompatibility ( 29 ) Mycelia compatibility group (MCG ) assignment is a popular method to describe S. rolfsii diversity, which is based on the hyphal interaction between diff erent isolates. Isolates from the same MCG/VCG are assumed to have similar genetic b ackground relative to isolates from different groups ( 29 33 )

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22 Some studies have suggested that MCG/VCG facilitate genetic information exchange in fungi in which the sexual stage is not available or has minimal impact on the disease cycle, and is important in analyzing field populatio ns ( 29 33 ) The barrage pheno menon is commonly used to identify incompatible isolates on agar medium. Kohn et al ( 29 ) paired 35 isolates of Sclerotinia sclerotiorum from different hosts, and found that compatibility between pairs consisted of four forms, i) one strain simply overgrew the other one without obvious hyp hal fusion; ii) the direct fusion of hyphae; iii) one hyphae wound around the other one or the formation of a simple appressorium; or iv) anastomosis followed by the formation of a cluster of hyphal initials at the fusion site. No matter the type of compat ibility, there was no deterioration of hyphae after hyphal contact in compatible pairings. In contrast, anastomosis was rarely observed in incompatible pairings and was usually followed by hyphal deterioration within the interaction zone. Based on their re sults, Kohn et al. ( 29 ) concluded that mycelial compatibility/incompatibility reactions are an effective means to categorize intraspecific heterogeneity; the high level of mycelial incompatibility among S. sclerotiorum strains may indicate the high level of vegetative incompatibility in as comycetes. Various DNA based methods have also been used to as a means to measure population diversity in a number of plant pathogenic fungi ( 38 ) In addition to the various ribosomal subunits, the internal transcr ibed spacer (ITS) regions between these subunits, and the intergenic spacer (IGS) regions that lie between ribosomal operons are common genomic targets used to measure fungal diversity at the molecular level. Within the genome of all eukaryotes, including fungi, ribosomal genes lie within a single operon composed of several open reading frames that encode the 5S rDNA, 5.8S

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23 rDNA, 1 8S rDNA, and 28S rDNA subunits. There are two ITS sequences within each ribosomal subunit gene that are removed during post trans criptional processing; ITS1, Non coding IGS sequences While the coded ribosomal DNA sequences are highly conserved, even between closely related species, small differences within the non coding ITS and IGS sequences have been used to measure di versity within a species and evolutionary relationships between closely related species. Therefore, molecular analyses based on differences within ITS and IGS sequences are commonly used for the identification and characterization of population diversity f or many microorganisms, especially fungi ( 12 ) Cilliers et al. ( 15 ) used an AFLP fingerprinting method to measure diversity within 73 isolates o f S. rolfsii collected from white lupin, peanut, sunflower, wild carrot, and soybean throughout South Africa. Overall, isolates from the same MCG were genetically diverse, even though the isolates were morphologically identical and usually came from the sa me host plant. Clear differentiation among isolates within an MCG could be found based on AFLP analysis, although isolates tended to cluster together based on MCG. Therefore, Cilliers et al. ( 15 ) concluded that a smaller degree of genetic variation existed among S. rolfsii isolates from a single MCG compared to isolates from different MCGs whe re a larger degree of variation existed. However, Nalim et al ( 40 ) found a different situation upon examining 3 66 peanut isolates from Texas. Isolates were assigned into 25 MCGs; all isolates within an MCG shared the same restriction patterns

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24 based on restriction analysis of the ITS region. Therefore, it was concl uded that isolates within a MCG are clonal. Based on these results, different levels of genetic diversity may exist among populations of S. rolfsii in different geographical areas. Almeida et al ( 1 ) identified 13 MCGs among 30 isolates of S. rolfsii suggesting considerable genetic variability exists among Bra zilian S. rolfsii isolates. However, they did not find a clear correlation between MCG and host. They further investigated genetic variability using a RAPD analysis and found a high correlation between genetic distances and mycelial compatibility. They fou nd compatible reactions were restricted between isolates with an aver age genetic distance of 10.1%. Incompatible reactions, however, occurred between isolates with an average genetic distance of 54.9%. The authors thought that heterokaryotic isolates, inte rspecific hybridization, gene duplications, and the sexual state, which may occur in Brazil, as possible mechanisms accounting for the high level of genetic variation observed. Yet, no correlation was found between the host or geographic origin of the isol ates and genetic distance as measured by RAPD analysis. Therefore, populations of S. rolfsii in different geographic areas may have different levels of genetic variability. To date, no studies have assessed S. rolfsii diversity with in the southern United S tates. Knowledge of genetic diversity among S. rolfsii isolates from outbreaks occurring on important economic crops like peanuts and vegetables such as tomato in Florida and other southern states is essential for regional resistance breeding efforts, and important to determine whether any level of host specialization exists among isolates from different areas and different hosts.

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25 Integrated Management for the Disease Caused by Sclerotium rolfsii Management of S. rolfsii broad host range that includes over 500 plant species and the ability of sclerotia to survive for 3 4 years in the soil ( 39 ) Integration of cultural and chemical management strategies may help reduce the impact of southern blight during vegetable production. Host resistance The use of resistant cultivars is always a preferred method of disease manage ment. Unfortunately, resistance to S. rolfsii has either not been identified or is limited for many host plant species. Resistance has been identified for some hosts. Three peanut cultivars, Georgia 03L, Georgia 02C and AP 3, were described to have a moderate level of resistance, and could help reduce grower reliance on fungicides for the management of S. rolfsii on peanut ( 71 ) Two other peanut cultivars, DP 1 and Georgia 07W were registered resistant to S. rolfsii in 2008, but DP 1 is not avai lable due to its limited commercial success ( 25 ) In tomato, six breeding lines, 5635M, 5707M, 5719M, 5737M, 5876M, and 5913M were released jointly from the Texas A&M Univer sity Research Center and the Coastal Plain Experiment Station, University of Georgia ( 32 ) However, Bhakta ( 5 ) observed little difference between 5913M and the susceptible breeding line Fla.7776 against the tomato isolate GCT 1, but found ac cession PI126932 with resistance to three S. rolfsii isolates from tomato, peanut and sweet potato. Chemical control The use of soil fumigants, such as methyl bromide, methyl iodide, chloropicrin, and metam sodium or metam potassium, are the most practic al means to treat seed beds and fields for a number of soilborne plant pathogens, including S. rolfsii ( 39 ) These

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26 fumigants must be applied days to weeks before planting. However, the availability of methyl bromide is limited due to its status as an ozone depleting material, and in 2012 the manufacturer of methyl iodide voluntarily suspended sales in the United States (http://current.com/http://www.montereycountyweekly.com/weblogs/news blog/2012/mar/20/breaking arysta to pull methyl iodide from us/). Certain fungicides, such as chlorpyrifos and pentachloronitrobenzene (PCNB), can effectively limit dis ease incidence when applied prior to planting ( 23 ) Both are registered for use on a limi ted number of vegetable crops. Some commercially available strobilurin fungicides (azoxystrobin, pyraclostrobin, and fluoxastrobin) are also labeled for the control of southern blight on certain vegetables, but labeling is based on efficacy on peanut ( 71 ) Azoxystrobin could significantly reduce peanut stem rot disease at 0.34 kg/ha and at 0.47 kg/ha it performed superior to chlorothalonil alone ( 26 ) Biological control A number of studies have shown that some antagonistic microbial agents can suppress S. rolfsii such as Trichoderma harzianum and T. viride Gliocladium virens Bacillus sub tilis and Penicillium spp. Soils amended with G. virens provided significant control of southern blight by reducing disease in pepper fields from 87 to 39% and fr om 42 to 18% in 1991 and 1992, respectively ( 58 ) Gliocladium virens also significantly reduced the number of viable sclerot ia in the soil from 47.9 sclerotia per 100cm 3 soil to below detectable levels when combined with a solarization treatment ( 57 ) which greatly limited the incidence of southern blight in subsequent tomato production. Trichoderma koningii was reported to effectively prevent S. rolfsii spread in potted outdoor and field grown tomatoes, reducing the sclerotia concentration in field soil from1.3 per cm 2 soil to 0.4 0.3 per cm 2 ( 30 ) Papavizas ( 45 ) found that G. virens suppressed dam ping off by

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27 30 50% and suppressed southern blight by 36 74%. All of the sclerotia of one strain were colonized and completely destroyed by G. virens after 28 days in the soil. However, many of these experiments were conducted under cont rolled conditions. T he effectiveness of currently labeled biological pesticides under field conditions is not clear, and the exact mechanisms by which these agents suppress S. rolfsii are largely unknown. Research Objectives Information regarding the diversity of among S. ro lfsii in the southern United States is critical for regional row crop and vegetable breeding programs. The objectives of this s tudy were to 1) characterize S. rolfsii isolates collected from southern United States for differences in cultural morphology and mycelial compatibility; 2) to test S. rolfs ii isolates for differences in virulence and host adaptation against tomato, pepper, and peanut; and 3) to test the specificity of previously reported resistant tomato lines against diverse isolates of S. rolfsii A specific emphasis was placed on isolates from peanut and vegetable production areas to identify any possible relationship between MCG and the isolates host of origin, geographical origin, morphological features, and pathogenicity towards peanut, tomato and pepper.

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28 CHAPTER 2 MORPHOLOGICAL CHARAC TERIZATION OF SCLERO TIUM ROLFSII ISOL ATES AND PATHOGENICITY /VIRULENCE TESTS UNDER GREENHOU SE CONDITIONS Introduction Sclerotium rolfsii is an aerobic fungus that produces a fluffy or compact colony on artificia l media. Mycelia growth rate in vitro is high, with the diameter of a single colony spreading to 85mm after 8 days on potato dextrose agar (PDA) medium ( 48 ) ,and a rate of growth as fast as 0.79 mm h 1 during the fi rst 48 hours ( 31 ) Othe r characteristics like the color, size, and number of sclerotia on media are also commonly described morphological characteristics. Another useful character istic to describe isolates and their relationship to isolates from other hosts and geographic areas is the mycelial compatibility grouping (MCG) ( 31 43 54 ) Based on mycelial compatibility, a number of genetic diversity studies have been performed on S. rolfsii to characterize the population structure or to monitor the distribution and spread of isolates over time. In one study, Harlton et al ( 27 ) identified 49 MCGs among 119 S. rolfsii isolates collected from different hosts and areas. They observed that isolates from one host and geographic area frequently belonged to the same MCG. For example, of the 21 annual bluegr ass isolates collected from California, 16 isolates belonged to the same MCG. However, the 16 isolates from 12 different crops collected from North Carolina were distributed in 12 different MCGs. Therefore, Harlton et al ( 27 ) believed that there was some correlation between MCG and geographical area or host of origin among isolates of S. rolfsii Another study by Nalim et al ( 40 ) examined 366 isolates collected from Texas peanut fields that were assigned to 25 MCGs. They concluded that isolates from the same MCG were clona l based on restriction analysis of the ITS and rDNA regions, identical genomic amplification patterns, and c olony. Okabe and Matsumoto ( 43 )

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29 reached a similar conclusion after they compared MCGs and corresponding restriction analysis of the ITS of 185 isolates collected from peanut fields in Jap an. However, Punja and Sun ( 54 ) found that most isolates within an MCG could be distinguished based on small differences in RAPD PCR banding patterns. Clonality was only observed in certain S. rolfs ii MCGs with isolates collected from the same host and geographic region. Cilliers et al ( 15 ) collected 73 S. rolfsii isolates from different hosts and locations throughout South Africa and identified nine MCGs; isolates within the same MCG could be clearly differentiated based on AFLP, but isolates within an MCG clustered closer together tha n isolates from different MCGs. This suggested that genetic variability within an MCG is smaller than variability between MCGs. Over all, these studies are in agreement with published studies of other fungal systems that used MCG to infer genetic relatedne ss among isolates. However, the degree of genetic variation between and within a MCG may vary based on the geographic origin of the isolates or the specific host. Pathogenic variability among S. rolfsii isolates has been previously reported. Difference in pathogenicity among S. rolfsii isolates from Piper betle and rice was observed by Thompson ( 68 ) on various plants; Cooper ( 16 ) reported that the aggressiveness of peanut isolates collected from North Carolina ranged from weak to strong. The objective of this study was to characterize S. rolfsii isolates collected from the southern Unite d States for MCG an d other morphological features. A specific emphasis was placed on isolates from peanut and vegetable production areas to identify any

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30 possible relationship between MCG and the isolates host of origin, geographical origin, morphological f eatures, and pathogenicity towards peanut, tomato, and pepper. Materials and Methods Fungal Materials Eighty four isolates of S. rolfsii from Virginia, South Carolina, Georgia, Florida, Louisiana, and Texas were isolated from infected tissues of several ho sts to include tomato, pepper, peanut, and snap bean (Table 2 1). All isolates were purified from initial cultures by picking single scl erotia to increase the isolate. All isolates were stored as dry sclerotia at room temperature. Individual colonies fo r subsequent experiments were initiated from single sclerotia, disinfested with 75% ethanol prior to placing on the center of PDA plates and maintained at room temperature. Mycelial Compatibility Groups (MCG) For grouping isolates based on mycelial compati bility, fresh mycelial discs (5 mm diameter) were cut off from the edge of an active colony (4 5 days old), transferred to new 85 15 mm petri dishes with PDA, and incubated at room temperature (24 28 C) Three isolates per a plate were spaced 2 to 2.5 c m apart and visually examined after 5 to 8 days for the presence of an aversion or a barrage zone. Pairings were marked either as incompatible when an antagonistic barrage zone was observed between two paired isolates and were put into different groups; or compatible when mycelia from two isolates intermingled without a barrage zone between them, in which case they w ere placed into the same group Eventually, a representative tester isolate was designated for each MCG to streamline testing of new isolates. Each test was repeated two separate times.

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31 Morphological Characterization A single sclerotium was placed in the center of a petri dish with PDA medium (25 ml) and incubated at room temperature. During incubation, the colony was characterized as either rai sed or flat and sclerotia characteristics, such as the size, color, and number of sclerotia produced, were also recorded. After 4 weeks, fifteen mature sclerotia were randomly picked from each petri dish and their diameter was measured using the right ang le method ( 48 ) with an optical microscope at 40 X magnification. Each isolate had three replicate s per petri dish Plant Materials for Pathogenicity Tests inis Vegetable Seeds, Inc., St. Louis, MO, USA), a commercial determinate fresh market round tomato produced in Florida; the advanced breeding line 5635M, previously characterized as resistant to S. rolfsii ( 32 ) (obtained from Dr. J. Scott, University of Florid a); the pepper pepper produced in Florida; and the peanut cultivar ( 9 ) a commercial peanut with moderate resistance to S. rolfsii and commonly pr oduced in Florida an d Georgia. Plants were grown in a sterilized peat potting mix ture (Speedling Inc., Sun City, FL, USA), fertilized as needed with Miracle Gro water soluble plant food ( The Scotts Company LLC, Marysville, OH, USA) every 7 days, and maintained in a temperat u re controlled greenhouse at 30 C 5 C and natural daylight. Tomato and pepper seeds were germinated in Speedling trays. Four weeks after sowing, seedlin gs were transplanted into 10 cm d iameter plastic pots, one plant per a pot. Peanut seeds were germinat ed directly in 6 inch diameter plastic pots, two seedlings each pot. All

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32 plants were watered by drip tape. The experiment was conducted three separate times, from May to November, 2010, June to November 2011, and April to May 2012. Pathogenicity Tests A si ngle isolate representative of each MCG group was used for pathogenicity tests. Isolates were initially cultured on PDA plates at room tempe rature for three to four weeks. Mature sclerotia were then collected and air dried for at least seven days before be ginning te sts. Plants were inoculated at eight weeks after germination by wrapping 10 sclerotia around the stem of each plant just above the soil line with tape (Figure 2 3 A). The experiment was arranged as a completely randomized design with 16 replicate plants per isolate ; a set of non inoculated plants served as a control. Plants were maintaine d in greenhouse conditions for four weeks. Diseas e symptoms were recorded every seven days after inoculation using a visual scale of 0 to 6 for the severity of wi lting: 0 = no symptoms, 1 = less than 12.5% wilted foliage, 2 = 12.5% 25% wilted foliage, 3 = 25% 50% wilted foliage, 4 = 50% 75% wilted foliage, 5 = 75% 100% wilted foliage, 6 = Dead plant. The pathogenicity test was conducted twice. The pepper di sease severity was also scored using a stem lesion scale in the first experiment: 1= no stem lesion, 2 = small stem lesion ( 25% of the stem circumference), 3 = moderate stem lesion (26 50% of the stem circumference), 4 = large stem lesion (> 50% of the s tem circumference), and 5 = dead plant (stem completely girdled) ( 24 ). After the first series of experiments were completed, the response of tomato plants (both varieties) to the various isolates could not be ascertained, as all plants quickly succumbed to all S. rolfsii isolates when10 sclerotia per a plant was used. To better ascertain the effect of inoculum load on disease severity, a separate series of tomato pathogenicity tests were conducted. Different inoculation levels of 2, 4, 6, or 8 sclerotia

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33 per tomato plant were tested on both Tygress and 5635M using isolates SR 1, 29 and 41 from tomato, pepper, and peanut respectively, that varied in aggressiveness from previous studies. Each treatment had 10 individual plants, the experiment was repeated twi ce. Based on the results of these trials, the tomato pathogenic ity trials were repeated using four sclerotia per plant with 15 plants per isolate ; this experiment was also repeated twice Statistical Analysis Experiments were arranged as a completely rand omized design for statistical analyses ; data obtained from isolate studies were pooled across repeated trials. Disease severity values were converted to mid percentages and then normalized using an arcsine square root transformation for the final statistic al analyses. Generalized linear models employed in PROC GLIMMIX of SAS (version 9.2; SAS Institute, Gary, NC) were used to determine the effect of isolate and host on disease severity and their interaction. Experiments and replicates were considered random but cultivars and isolates were considered fixed. test or 95% confidence intervals was used to compare treatment means, and the in comparisons among treatments. Results Mycelial Compatibility Groups An aversion zone was observed between incompatible isolates after 7 to 10 days of growth. As initial contact between isolates occurred, hyphae lysed and died leaving a zone of c learin g (Figure 2 1 A). Colony boundaries of compatible isolates could not be easily discerned over the same period of time (Figu re 2 1 B). In total, 84 isolates of S.

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34 rolfsii were assigned into 23 MCGs based on pairing isolates with testers Eleven MCGs (MCG 4, 7, 9, 14, 15, 16, 18, 19, 20, 22, and 23) consisted of a single isolate, while MCG 11 was the largest group consisting of 16 isolates. Ten out of 14 peanut isolates from Georgia belonged to MCG 11; in comparison, the other isolates from vegetables and orn amentals were distributed into 13 different MCGs (Table 2 2). Other Morphological Characters The isolates of S. rolfsii varied in colony morphology, the average diameter of sclerotia, and the average number of sclerot ia produced on PDA. Most isolates pro duced sclerotia that were tan in color, but a few isolates (SR 63, 64, and 978 etc.) formed light brown, brown, or dark brown sclerotia (Table 2 1). All strains consistently formed sclerotia except for SR 876, which sometimes produ ced no sclerotia or as f ew as ten sclerotia per PDA plate. Average sclerotia size ranged from 0.56 mm to 1.91 mm in diameter, with the the smallest sclerotia produced by tomato strain SR 1 and the largest produced by peanut isolate SR 55 Significant difference existed among isol ates in the average sclerotia size and average number of sclerotia produced in vitro ( P < 0.0001 and P < 0.0001 respectively). In general, isolates originating from peanut produced larger sclerotia on PDA plates than those isolates originating from other h osts. Of the 22 peanut isolates, 19 were in the top one third of all isolates based on sclerotia diameter. As a group, sclerotia produced by peanut isolates were significantly larger than those produced by isolates from tomato ( P < 0.0001) and pepper ( P = 0.0282) (Table 2 3), while the difference in sclerotia size between tomato and pepper isolates was not significant ( P = 0.1193). Sclerotia size was different among d ifferent MCGs as well (Table 2 3 ). The group with the largest number of isolates MCG 11 t hat included 14 peanut isolates and two other isolates from potato and snap bean produced

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35 significantly bigger sclerotia compared to MCG 1, 8 and 21 ( P < 0.0001) with an average diameter 1.35mm. Isolate SR38 in MCG 16 was collected from a peanut leaf, wa s inc ompatible with other peanut isolates and produced sclerotia that were distinctly smaller than other peanut isolates, but similar to other non peanut isolates. Sclerotia number only differed significantly between MCG 11 and 21, but not between MCG 11 a nd MCG 1 or between MCG 11 and MCG 8 (Table 2 3 ) On average, isolate SR 980 produced the fewest number of sclerotia at 49 sclerotia per plate, while isolate SR 70 produced the greatest number of sclerotia at 1427 per plate. Fifty nine out of 84 isolates p roduced a large number of sclerotia, with an average of >300 sclerotia per a PDA plate; seven isolates formed less than 100 sclerotia/plate, of which five (SR 34, 40, 754, 877, and 880) were from peanut (Table 2 2). In addition, when compared to other isol ates grown on PDA the mycelia of peanut isolate colonies remained flat along the media surface giving the colony a thin, compact morphology (Figure 2 2 B and D). In contrast, colonies of most other isolates produced abundant aerial hyphae that extended u p in the air and gave the colony a fluffy appearance (Figure 2 2 A and C). C olonies from all isolates consisted of white mycelial growth. Pathogenicity/Virulence Tests Isolates of S. rolfsii collected from tomato (n = 7), peanut (n = 5), pepper (n = 4), sw eet potato (n=1), pumpkin (n=1), and snap bean (n=1) were pathogenic on tomato (Tygress and 5635M), pepper, and peanut (Figure 2 3 B, C, and D), causing stem lesions and some level of wilt. The time required for initial wilt symptoms to appear across hosts ranged from 4 to 10 days after inoculation, or 7 days on average. Peanut plants required the longest time of 10 days after inoculation for initial wilt symptoms to

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36 appear. Overall, the temporal development of symptoms was very uniform across isolates on each host. No symptoms of wilt were obs erved on non inoculated plants. Symptoms on tomato (Tygress and 5635M) were more severe than those exhibited on pepper or peanut. Although severity among isolates on tomato differ ed statistically in the first trial ( Tygress, P < 0.0157; 5635M, P < 0.0002 ) significant difference s among tomato, pepper, and peanut isolates groups were not detected presumably because the tomato plants were inoculated with ten sclerotia per a plant. Additional experiments found that toma to cultivar ( P = 0.0024) and varying inoculum load ( P < 0.0001) from two, four, six, or eight sclerotia per a tomato plant had a significant effect on wilt severity with no significant interaction between isolate a nd inoculum load ( P = 0.8694 ; Table 2 5 ) An inoculum load of six or eight sclerotia per a plant produced higher disease compared to two and four sclerotia per a plant (Table 2 6). Based on these findings, four sclerotia per a plant was chosen for subsequent pathogenicity tests on tomato, which detected significant differences in disease severity among isolates for Tygress ( P < 0.0001) and 5635M ( P < 0.0001). Severity among isolates 28 days following inoculation ranged from 18.1 to 71.8 % on 5635M, which was significantly higher than on Tygress ( P < 0.0001) which ranged from 14.5 to 64.2 % (Figure 2 7 ) As a group, isolates collected from peanut were more aggressive than those collected from pepper or tomato when used to inoculate tomato plants ( P < 0.0001 and P < 0.0001 respectively ) ; difference s b etween tomato isolates and pepper isolates for disease severity 28 days after inoculati on were not significant ( P = 0.2321). The i nteraction between tomato cultivar and isolate o n disease severity was significant

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37 ( P = 0.0008; Table 2 7 ); t en out of 19 isol ates produced different levels of disease severity on the two tomato varieties (Figure 2 7 C ). For peanut the four isolates collected from peanut produced the highest levels of disease severity compared to those isolates collected from tomato ( P < 0.0001 ) or pepper ( P < 0.0053); no significant difference was observed between tomato and pepper isolates ( P = 0.2845). The same trend was observed on pepper plants with isolates collected from peanut causing statistically higher levels of disease than those iso lates collected from tomato or pepper ( P = 0.0001 and P = 0.0053 respectively ) (Table 2 9) Discussion Based on m orphological character ization substantial morphological variability exist s among S. rolfsii isolates from the southe aste rn United States. All isolates produced typical silky white mycelia, with either a fluffy or compact colony type when grown o n PDA medium, consistent with other studies ( 18 31 51 ) Both f luffy and compa ct colony types were represented among isolates characterized from India and Viet n am peanut fields ( 18 31 ) ; however in this study, only fl at and compact colony were observed among peanut isolate s collected in Georgia Isolates from Georgia peanut, produced larger sclerotia than those produced by isolates from tomato and pepper. Okabe et al ( 43 ) also described a group of S. rolfsii isolates that produce d larger, irregularly shaped sclerotia with stipes compared to other groups of isol ates, and Punja ( 43 ) suggested that these isolates be reclassified as S. delphinii instead of S. rolfsii Punja and Damiani ( 48 ) demonstrated that S. delphinii could be distinguished from S. rolfsii based on larger sclerotia size (0.75 1.80 mm) reddish brown sclerotia color, and generally lower levels of oxalic acid and pectinase

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38 enzymes than S. rolfsii Ansari and Agnihotri ( 3 ) reported a positive correlation between oxalic acid production and the virulence of S. rolfsii isolates. In this study, peanut isolates produced larger sclerotia than other isolates, but were not irregular ly shape d and were not reddish brown in color as previously reported ( 48 ) but rather were spherical in shape and pro duced dark color ed sclerotia. I solates collected from peanut also were the most aggressive across the three different host plants tested. The mechanisms behind these phenomena are not clear, but could be due to genetic differen ces or differences in nutriti onal requirements ( 18 ) ; further genetic studies need be conducted to elucidate this. In this study, peanut isolates collected in Georgia produced larger sclerotia on PDA media than isolates collected from other hosts similar to resul t s reported for peanut isolates in Viet n am ( 31 ) Le et al ( 31 ) reported that three peanut isolates in one MCG produced approximately two times larger sclerotia than other two groups which included both peanut isolat es and tomato or other isolates All of the 84 collected isolates produced sclerotia except for SR 876, which was originally collected in 2003 from peanut. This may be a special character of this isolate as the result of a specific mutation affecting sclerotia development or caused by a nutri tional deficiency that is limiting its ability t o produc e sclerotia Three peanut isolates from Viet n am were also described to produce fewer sclerotia than other peanut isolates ( 31 ) The composition and distribution of S. rolfsii MCGs within a population can vary greatly depending on the host plant and geographic region. S ome studies documented a divers e collection of fungal compatibility groups occurring on a limited range of host s

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39 or within a specific geographic region E xamples of a single fungal compatibility group being limited to a specific host or region have also be en documented ( 27 33 62 ) Results of this study are in line with other published studies on S. rolfsii with examples of different levels of MCG diversity depending on the host or geographic region Isolates collected from tomato were very diverse, with 31 isolates distributed into 12 MCG groups in this s tudy, from which 29 of the isolates were collected from different tomato production areas of Florida. Previous studies reported similar results. For example, Nalim et al. ( 40 ) described 25 MCGs among 366 peanut isolates in Texas where the number of MCGs found in sever al surveyed fields ranged from one to five Cill iers et al. ( 15 ) found that isolates from the same host plant appeared to group into diff erent MCGs as well. Tomato isolates in this study, were mostly collected from different fields; however, in one case seven isolates representing four MCGs were collected from a single field in Florida. In contrast, isolates from peanut were less diverse ba sed on MCG in this study Nine out of the 12 peanut isolates collected over a 10 year period from Georgia belonged to MCG 11, while the other three were in MCG 13 and MCG 17. Isolates in the same MCG were shown to be genetically more similar than isolates from different MCGs ( 15 27 31 ) According ly the po pulation of S. rolfsii isolate s affecting Florida tomatoes is more diverse than the population affecting Georgia peanuts. Selection pressure resulting from the introduction of resistant host varieties, c hanges in environment could contribute to the rise of new strains ; while recombination and mutations within the het genes could also lead to new MCGs. Finally, the i ntroduction of new isolates through human activi ti es, contaminated plant materials, and transportation could also introduce new isolates

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40 Myceli al compatibility is a useful way to survey the distribution and spread of isolates over time and geographic areas within a fungal species, especially among pathogenic fungi ( 2 ) In this study, several MCGs were found in several broad geographic areas of the south. For example, MCG 10 consisted mostly of tomato isolates from Florida. However, two tomato isolates from Texas and Georgia, as well as two snap bean isolates from South Carolina also belonged to MCG 10. Similarly, the largest represented MCG in this study, MCG 11, consisted of 16 isolates of which 13 were peanut isolates from Georgia, and included another peanut isolate from north Florid a, and two Virginia isolates collected from snap bean and potato. This distribution suggests that some MCGs are more predominant in certain areas, but wi th some movement between areas. Movement may be related to human activities, such as the movement of in fected plant materials, contaminated soil, or equipment that help to spread the pathogen over long distances. However, these apparent differences in distribution could be due to sampling bias. The pathogenicity test results showed that all the 19 tested isolates of S. rolfsii were pathogenic to the four different host s H owever significant differences in virulence were observed depending on the host of origin, similar to other reports ( 22 64 ) Georgia peanut isolates SR29, SR30, SR32, and SR40 were consistently the most virulent isolates tested on tomato, pepper, and peanut. In general, isolates originating from tomat o and pepper were equally virulent on tomato, pepper, and peanut plants based on the final disease severity. Results in this study showed that peanut isolates produced sclerotia with a significantly larger diameter than those produced by other isolates Th ere may be a positive correlation between sclerotia size and virulence ; since larger

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41 sclerotia could secrete a larger dose of oxalic acid and other cell wall degrading enzymes necessary for disease development ( 51 ) T he increased virulence associated with the Georgia peanut isolates may be due to increased selection pressure from the release of the peanut cultivar Georgia Green which was a tolerant cultivar planted to 85% of the peanut production acreage in Georgia ( 71 ) The two tomato varieties used in this study were highly susceptible to S. rolfsii when directly inoculated with ten sclerotia per a plant. Tomato breeding line 5635M was released from Texas in 19 92 as a source of resistance to southern blight, which was attributed to the development of a reinforced periderm at the infection site ( 32 ) Even after reducing the inoculum to four sclerotia per a plant, the two tomato lines were still susceptible to peanut i solates with the average disease severity of 59.0% and 58.6 % respectively. Bhakta ( 5 ) considered tomato plants with a disease severity rating based on wilting foliage of 37.5% or less as resistant Based on this criterion both Tygress and 5635M were resistant to 15 out of 19 tested S. rolfsii isolates. Bhakta ( 5 ) also found that another Texas breeding line 5913M was resistant to a peanut isolate WM609, (named SR 32 in this study), but suscepti ble to another tomato i solate. However, in this study 5635M was susceptible to strain SR 32 (and the other peanut isolates), but resistant to the isolates from tomato and other hosts The resistant breeding lines released from Texas may have resistance to different isolates of S rolfsii Unfortunately, the average disease severity of tomato isolates on Tygress plants was 6.67% compared to 20.38% on 5635M tomato plants suggesting that the commercial hybrid cultivar Tygress has better resistance to many S. rolfsii isolates present in southeastern United States than the breeding line from Texas

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42 On pepper plants, the average stem lesion scores were higher than average wilt scores because some plants with severe stem lesions did not develop any wilt symptoms. Similar observations wer e made for the southern blight resistant pepper cultivar Golden California Wonder which was reported to contain resistance conferred by a single recessive gene ( 20 ) Interestingly, the re s ponse of pepper cultivar varied across isolates from high ly resi stan t (SR 1, 5, and 105) to high ly susceptib le (SR 30 and 40); subsequent studies should investigate these potential differences across a larger group of pepper cultivars and breeding lines.

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43 Table 2 1. Sclerotial characteristics and mycelia compatibility group (MCG) designation f or Sclerotium rolfsii isolates sorted by geographic origin, host of origin, sclerotia color and size, y ear of isolation and sclerotia number in medium Geographic origin Original host Isolate number Sclerotia color Year of isolation MCG number Sclerotia size (mm) Number of sclerotia (per plate) Florida Tomato SR 1 Tan 2009 1 0.56 0.03 1273 146 SR 2 Brown 2009 1 0.73 0.02 648 128 SR 3 Tan 2009 2 0.93 0.09 341 105 SR 4 Tan 2009 2 0.96 0.05 219 60 SR 5 Tan 2009 1 0.72 0.03 839 73 SR 6 Tan 2009 3 0.82 0.08 576 192 SR 7 Brown 2009 1 0.94 0.13 434 104 SR 8 Brown 2009 4 0.88 0.08 492 57 SR 9 Brown 2009 5 0.89 0.08 500 134 SR 10 Tan 2009 6 1.02 0.16 526 316 SR 11 Brown 2009 6 0.94 0.17 577 309 SR 12 Brown 2009 7 1.02 0.06 367 17 SR 13 Dark 2009 6 1.17 0.05 163 24 SR 14 Tan 2009 6 0.85 0.05 432 57 SR 15 Tan 2009 8 1.04 0.04 357 84 SR 16 Tan 2009 6 0.90 0.09 380 27 SR 17 Tan 2009 8 0.99 0.15 311 68 SR 18 Brown 2009 3 0.96 0.12 461 107 SR 19 Brown 2009 9 0.82 0.12 731 42 SR 20 Tan 2009 8 0.82 0.20 424 65 SR 22 Tan 2010 10 0 .86 0.16 466 50 SR 47 Tan 2010 6 0.99 0.10 1201 154 SR 48 Tan 2010 8 0.96 0.06 752 77 SR 49 Tan 2010 6 1.02 0.06 935 104 SR 50 Tan 2010 1 0.95 0.03 605 85 SR 51 Tan 2010 20 0.85 0.04 802 51 SR 52 Tan 2010 10 0.96 0.04 598 21 SR 69 Tan 2010 22 0.86 0.03 1258 33 SR 72 Tan 2010 8 0.90 0.13 869 63

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44 Table 2 1. Continued Geographic origin Original host Isolate number Sclerotia color Year of isolation MCG n umber Sclerotia size (mm) Number of sclerotia (per plate) Florida Pepper SR 21 Tan 2009 2 1.02 0.09 528 46 SR 73 Tan 2010 21 0.93 0.08 291 25 SR 105 Tan -8 0.60 0.03 1009 129 SR 920 Tan -14 0.64 0.01 914 34 Peanut SR 754 Dark brown 19 98 11 1.27 0.09 91 6 SR 876 Tan -12 --SR 877 Tan 2003 12 1.45 0.15 87 4 SR 948 Tan 2004 11 0.93 0.04 228 18 SR 68 Brown 2010 11 1.46 0.24 379 48 Watermelon SR 978 Light brown 1905 2 0.57 0.08 797 27 SR 70 Tan 2010 8 0.91 0.05 1427 128 Cantaloupe SR 71 Tan 2010 8 0.97 0.13 1159 45 Pumpkin SR 23 Brown 1998 5 0.90 0.04 516 233 Ruellia simplex SR 24 Tan 2003 5 0.94 0.03 502 249 Iris sp., pod SR 979 Tan 2008 12 1. 60 0.25 77 16 Unknown SR 980 Tan 2008 12 1.49 0.15 49 11 SR 113 Dark brown -11 1.21 0.13 151 28 SR 74 Brown -17 1.06 0.04 777 137 Georgia Peanut SR 27 Brown 2009 11 1.28 0.04 226 94 SR 28 Tan 2009 11 1.28 0.24 239 32 SR 29 Dark tan 2009 11 1.45 0.05 146 15 SR 30 Tan 2009 13 1.41 0.04 142 6 SR 31 Tan 2009 11 1.31 0.03 152 19 SR 32 Tan 2009 11 1.22 0.02 294 16 SR 33 Tan 2009 11 1.44 0.05 170 31 SR 34 Tan 20 09 11 1.58 0.06 97 17 SR 35 Brown 2009 8 0.75 0.04 1130 90 SR 36 Brown 2009 13 1.34 0.05 170 12 SR 37 Tan 2004 11 1.45 0. 2 0 228 28

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45 Table 2 1. Continued Geographic origin Original host Isolate number Sclerotia color Year of isolation MCG n umber Sclerotia size (mm) Number of sclerotia (per plate) Georgia Peanut SR 38 Tan 1991 16 0.99 0.04 759 25 SR 39 Dark 2000 11 1.20 0.02 267 74 SR 40 Tan 1993 17 1.80 0.05 66 16 SR 66 Brown 2010 11 1.62 0.13 509 70 Tomato SR 58 Tan 2010 10 0.90 0.11 1152 222 Snap bean SR 67 Tan 2010 17 1.15 0.24 427 115 Louisiana Sweet potato SR 25 Tan 2009 15 1.00 0.06 529 242 SR 26 Tan 2009 5 0.92 0.09 531 45 South Carolina Pepper SR 41 Tan 2010 18 0.76 0.20 794 78 SR 42 Tan 2010 1 0.85 0.05 381 61 Eggplant SR 43 Brown 2010 1 0.82 0.08 463 40 Snap bean SR 44 Brown 2010 10 1.00 0.02 348 279 SR 45 Tan 2010 10 1.01 0.09 371 42 Virginia Snap bean SR 59 Tan 2009 17 1.59 0.13 313 59 SR 60 Tan 2009 17 1.49 0.11 370 21 Pepper SR 61 Tan 2010 13 1.07 0.05 966 44 Potato SR 62 Tan 2010 11 1.44 0.08 313 51 Texas Peanut SR 53 Tan 2010 21 1.45 0.15 -SR 54 Tan 2010 23 1.11 0.02 949 73 SR 55 Tan 2010 21 1.91 0.12 168 2 Tomato SR 56 Tan 2010 22 1.06 0.07 649 210 Unknown SR 46 Tan 2010 19 0.95 0.12 638 62 Pothos SR 63 Light brown 2010 21 1.19 0.03 241 13 Vanda SR 64 Light br own 2010 21 1.24 0.06 153 12 SR 65 Brown 2010 21 1.26 0.13 133 22 Note: Thirty six isolates were kindly provided by Dr. T. Brenneman (SR 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40), Dr. A. Gevens (978), Dr. M. Elliott (979 a nd 980), Dr. H. Dankers (SR 25 and SR 26), Dr. T. Kucharek ( SR 876, 877, 920, and 948), Dr. A. P. Keinath (SR 41, 42, 43, 44, and 45), A., Dr. J. Woodward (SR 53, 54, 55, and 56), and Dr. M. Paret (SR 71, 72, 73, and 74). The value -is used for when th e pertinent information is unknown. ** means standard deviations

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46 Table 2 2. Sclerotium rolfsii isolates sorted by mycelial compatibility group (MCG). Note: I solates followed by an asterisk were used in pathogenicity studies MCG number Isolates 1 SR 1 SR 2, SR 5, SR 7, SR 42, SR 43, SR 50 2 SR 3, SR 4, SR 18, SR 21 SR 978 3 SR 6, SR 47, SR 49 4 SR 8 5 SR 9, SR 23 SR 24, SR 26 6 SR 10, SR 11, SR 13, SR 14 SR 16 7 SR 12 8 SR 15, SR 17, SR 20, SR 35 SR 48, SR 70, SR 71, SR 72 SR 105* 9 SR 19 10 SR 22, SR 44 SR 45, SR 52, SR 56, SR 58 11 SR 27, SR 28, SR 29 SR 31, SR 32 SR 33, SR 34, SR 37, SR 39, SR 60, SR 62, SR 66, SR 68 SR 113, SR 754, SR 948, 12 SR 876, SR 877, SR 979, SR 980 13 SR 30 SR 36, SR 61 SR 883, 14 SR 920 15 SR 25 16 SR 38 17 SR 40 SR 59, SR 60, SR 67, SR 74 18 SR 41 19 SR 46 20 SR 51 21 SR 53, SR 55, SR 63, SR 64, SR 65, SR 73 22 SR 69 23 SR 54

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47 Table 2 3. Statistical contrast s for sclerotia size and number o f sclerotia per isolate produced in vitro by Sc l ero t ium rolfsii isolates collected from tomato, pepper, and peanut. Note: MCG 11 contains 14 peanut isolates, one snap bean isolate, and one potato isolate; MCG 1 contains five tomato isolates, one pepper isolate, and one eggplant isolate; MCG 8 contains f ive tomato isolates, one pepper isolate, one peanut isolate, one watermelon isolate, and one cantaloupe isolate; MCG 21 contains three peanut isolates, one pepper isolate, one Pothos isolate, and one Vanda isolate. Sclerotia size Sclerotia number F Value Pr > F F Value Pr > F Tomato isolates vs. peanut isolates 56.02 < 0.0001 66.16 < 0.0001 Tomato isolates vs. pepper isolates 2.45 0.1193 156.11 < 0.0001 Pepper isolates vs. peanut isolates 4.90 0.0282 50.26 < 0.0001 MCG 11 vs. MCG 1 29.59 < 0 .0001 0.00 0.9561 MCG 11 vs. MCG 8 41.88 < 0 .0001 0.00 0. 9486 MCG 11 vs. MCG 21 37.02 < 0 .0001 75.00 < 0.0001

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48 Table 2 4 Statistical contrasts for so uthern blight severity caused by Sc lerotium rolfsii isolates collected from tomato, pepper, and peanut on tomato cultivar Tygress and breedling line 5635M using 10 sclerotia per plant Tygress 5635M F Value P > F F Value P > F Tomato isolates vs peanu t isolates 0.35 0.5558 1.13 0.2876 Tomato isolates vs pepper isolates 0.01 0.9201 3.84 0.0501 Pepper isolates vs peanut isolates 0.43 0.5131 0.67 0.4127

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49 Table 2 5 Analysis of variance of the effect of inoculation load on the severity of sou thern blight caused by Sclerotium rolfsii isolates SR 1, 29, and 41 on tomato cultivars Tygress and breeding line 5635M plants 28 days after inoculating Effect F Value Pr > F Isolate 5.29 0.1551 Inocul um load 17.29 < 0 .0001 Cultivar 9.47 0.0024 Isolate inoculum load 0.41 0.8694 Iso late Cul tivar 0.11 0.8982 Ino culate Cul tivar 1.16 0.3255 Iso late Ino culate Cul tivar 0.50 0.8081

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50 Table 2 6 Effect of inoculation level on the severity of southern blig ht on tomato plants 28 days after inoculating Note: Estimate s followed by the same letter are not significantly different ( = 0.05). N umber of sclerotia/plant W ilt severity (%) 8 58.5 A 6 58.2 A 4 40.6 B 2 27.4 C

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51 Table 2 7 Analysis of variance of the effect of Sclerotium rolfsii isolates on southern blight severity on tomato cultivar Tygress and breeding line 5635M using 4 sclerotia per a plant Effect F Value Pr >F Cultivar 57.95 < 0.0001 Isolate 36.73 < 0.0001 Cu ltivar Isolate 2.42 0.0008

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52 Table 2 8 Statistical contrasts of Sclerotium rolfsii isolates collected from tomato, pepper, and peanut on southern b light severity of tomato cultivar Tygress and breeding line 5635M using 4 s clerotia per a plant F Value Pr > F Tomato isolates vs. pepper isolates 1.43 0.2321 Peanut isolates vs. Tomato isolates 117.94 <.0001 Peanut isolates vs. Pepper isolates 69.92 <.0001

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53 Table 2 9 Statistical contrasts of Sclerotium rol fsii isolates collected from tomato, pepper, and peanut on southern blight severity of peanut cultivar Georgia Green and pepper cultivar Tom Cat Peanut Pepper F Value P > F F Value Pr > F T omato isolates vs. pepper isolates 0.39 0.5412 1.21 0.2845 Peanut isolates vs. Tomato isolates 61.60 <.0001 22.86 0.0001 Peanut isolates vs. Pepper isolates 55.25 <.0001 9.88 0.0053

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54 A B Figure 2 1. An A) incompatible and a B) compatible reaction between pai red isolates of Sclerotium rolfsii for designating mycelial compatibility groups

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55 A B C D Figure 2 2. Respective overhead and side profiles of a Sclerotium rolfsii isolate from tomato, SR 1 A) and C) and a isolate from peanu t, SR 754 B) and D) showing the difference in colony morphology

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56 A B C D Figure 2 3. S ymptoms of southern blight caused by Sclerotium rolfsii using the A) stem inoculation method with foli ar wilt and blight symptoms observed on B) pepper, C ) tomato, and D) peanut.

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57 A B C D Figure 2 4. Symptoms of A) s tem rot and wilt and B) s tem scab lesion s on pepper compared to symptoms of C) s tem rot and wilt and D) s tem scab lesion s on tomato caused by Sclerotium rolfsii

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58 A B C Figure 2 5. An example of a p epper plant A) not exhibiting any wilt symptoms following inoculation with Sclerotium rolfsii but still showing an apparent B) scab lesion at the inoculation site and an example of a C) s tem scab near the soil line of anot her inoculated pepper plant

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59 A B C Figure 2 6. Typical symptoms of w ilt observed A) on a pepper plant 28 days after inoculati n g with Sclerotium rolfsii compared to a B) pepper plant that recovered a ft er the same inoculation period, w ith a C) close up view of the upper foliage

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60 Figure 2 7. Mean wilt severity caused by 19 isolates of Sclerotium rolfsii 28 days after inoculati n g of A ) pepper cv. B) peanut cv. and C) tom ato breeding line 5635M and Isolates of S. rolfsii included: SR1, 5, 8, 12, 14, 19, and SR 47 originating from tomato; SR 21, 41, 105, and 920 from pepper; SR 29, 30, 32, and SR 40 from pe anut; SR 23 from pumpkin; SR 25 from sweet potato, SR 38 from peanut leaf; and SR 44 from snap bean Vertical bar s on each column represent the 95% confiden ce interval s An a sterisk near the isolate name in ( C ) denotes a significant difference in wilt seve rity between the two tomato cultivar s ( = 0.05) An asterisk near the isolate name means there were significant difference between two tomato cultivars. A B C * *

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61 CHAPTER 3 SUMMARY AND CONCLUSIONS Southern blight of tomato caused by Sclerotium rolfsii Sacc. is a difficult disease to manage through tradi tional cultural practices due to its wide host range and the ability of sclerotia to survive for long periods of time in soil. Although it is a minor soilborne pathogen of vegetables in Florida, the economic losses resulting from this disease has increased with the gradual phase out of methyl bromide for soil fumigation ( 59 65 ) Additional characterization of S. rolfsii such as genetic diversity, geographical distribution, pathogenicity and host specialization, is essential for the development of effective management strategies. Previous studies found that MCG diversity can vary greatly within a specific host or within a limited geographical area ( 11 66 ) This stud y confirmed this conclusion, for example, seven isolates of S. rolfsii collected from a single tomato field at the Gulf Coast Research and Education Center, Wimauma, FL were differentiated into three MCGs. Isolates in MCG 5 originated from four different h ost plants. Several studies have demonstrated that isolates from the same MCG are genetically more similar than isolates from different MCGs ( 29 33 ) Based on these observations, 31 tomato isolates from Florida distributed into 13 different MCGs indicate that a high level of genetic d iversity exists among Florida tomato isolates. In contrast, 10 out of 14 peanut stem isolates collected from Georgia belonged to the same MCG, although they were collected from different peanut fields in Georgia over a 10 year period. This result suggests that the level of diversity of S. rolfsii among Georgia peanut fields is relatively low. This difference in population structure between tomato and peanut isolates may be the result of differences in cropping history or even due to differences in gen etic

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62 d iversity within the crop. In Georgia, the peanut cultivar Georgia Green was the commercial standard for a number of years, accounting for approximately 85% of the peanut acreage in Georgia ( 71 ) However in Florida, tomato production utilizes a divers e array of tomato types and varieties with varying levels of genetic diversity, which may hinder any selective pressure on the S. rolfsii population. However, it is also possible that some of these differences may r epresent regional differences. Molecular markers are a useful tool for directly evaluating genetic diversity and phylogenetic relationships within and between species ( 35 ) Further studies using molecular markers are required to improve our understanding of population structure and dispersal of S. rolfsii isolates throughout the southern United States. Earlier work demonstrated that differences in virulence exist among isolates of S. rolfsii ( 62 64 ) similar to the findings of this study. Georgia peanut isolates always exhibited the highest level of disease severity compared to the other isolates, regardless of the host plant. In contrast, tomato isolates and pepper isolates diff ered in virulence among the four different host plants. Peanut cultivar Georgia Green is predominant in Georgia, which was released by Georgia Agricultural Experiment Stations in 1995 with excellent yielding ability ( 9 ) and was considered as a resistant culti var to stem rot caused by S. rolfsii in 1999 ( 10 ) In 2008, Georgia Green covered about 85% of Georgia peanut acreage but then was described as a stem rot susceptible cultivar. A fter almost ten years of repeat planting of the same cultivar in the same areas, it is quite possible that new S. rolfsii emerged that were better adapted to G eorgia Green with enhanc ed virulence. The larger sclerotia size of Georgia peanut isolates may be another result of this process that help to enhance the virulence by secreting a

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63 larger volume of oxalic acid and other cell wall degradin g enzymes. This resu lt could also suggest that a new, more virulent isolate was introduced in this area and became the predominate one. Host resistance to S. rolfsii has been reported in peanut, cowpea, pepper, alfalfa, and tomato ( 5 10 17 21 46 47 ) ; however, the actual mechanisms conferring resistan ce or partial resistan ce among these hosts is unclear. Documented sources of resistance to southern blight in tomato are limited. To date, wild Peruvian accessions PI 126932 and PI 126432 of S.pimpinellifolium are the only source Six breeding lines (5635M, 5707M, 5719M, 5737M, 5876M and 5913M) were released from Texas A&M University, each one containing S. pimpinellifolium in their genetic background ( 32 ) Incorporating resistance against S. rolfsii into cultivated tomato cultivars would provide a cost effective and an environmentally safe method for man aging this disease. Bhakta ( 5 ) found that one of the six Texas breeding lines 5913M was resistant to a peanut isolate of S. rolfsii from Georgia but susceptible to a Florida tomato isolate, while another breeding line 5635M tested in this study did not show the same result. On the c ontrary, in this study 5635M w as susceptible to peanut isolates, including the one used severity than the other isolates on Tygress and 5635M, including tomato isolates from Florida. Interestingly, Tygr ess was more resistant towards the non peanut isolates than 5635M, suggesting that resistance in tomato may be isolate specific. However, additional stud ies will need to be conducted S ince tomato isolates collected in Florida were less virulent on Tygress a comme rcial cultivar grown in Florida future studies

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64 should assess the susceptibility of other commercial tomato varieties so growers could potentially choose more tolerant varieties in problematic areas. The phase out of methyl b romide has brought m ore attention to soilborne disease s such as southern blight to growers and researchers alike. Although southern blight has traditionally been considered a minor disease of tomato production in Florida, it now has the potential to become more problematic. T his study evaluated population structure of the pathogenic fungus S. rolfii and observed that high levels of genetic diversity existed among tomato isolates in Florida, with evidence of either host or geographic specialization among peanut isolates in Geor gia. Tomato pathogenicity tests detected evidence of resistance, but suggest that it may be specific to certain groups of isolates; which makes bre eding efforts more challenging. However, some commercial tomato cultivars, like Tygress, may already have som e level of resistance to isolates common to Florida and should be investigated further.

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65 LIST OF REFERENCES 1 Almeida, A., Abdelnoor, R., Calvo, E., Tessnman, D., and Yorinori, J. 2001. Genotypic diversity among Brazilian isolates of Sclerotiu m rolfsii Phytopathology 149 :493 502. 2. Anderson, J. B., and Kohn, L. M. 1995. Clonality in soilborne, plant pathogenic fu ngi. Annu Rev Phytopathol 33 :369 391. 3 Ansari, M., and Agnihotri, S. 2000. Morphological, physiological and pa thological variations among Sclerotium rolfsii isolates of soyabean. Indian Phytopathology 53:65 67. 4 Aycock, R. 1966. Stem rot and other diseases caused by Sclerotium rolfsii or the status of Rolfs' fungus after 70 years. Tech. Bull 174 N. Carol, agric Exp. Stn. 5. Bahakta, M. S. 2010. Genetics of Southern Blight Resistance in Tomato ( Solanum lycopersicum L.). Master Thesis, University of Florida, U.S.A. 6 Bateman, D., and Beer, S. 1965. Simultaneous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis by Sclerotium rolfsii Phytopathology 55:204. 7 Bateman, D. F. 1970. Depletion of the galacturonic acid content in bean hypocotyl cell walls during pathogenesis by Rhizoctonia solani and Sclerotium rolfsii Phyto pathology 60:1846 1847. 8 Bayman, P., and Cotty, P. J. 1991. Vegetative compatibility and genetic diversity in the Aspergillus flaws population of a single field. Can. J. Bot 69. 9 :8 06. 1 0 Branch, W., and Brenneman, T. 1999. Stem rot disease evaluation of mass selected peanut populations. Crop Protection an International Journal of Pes t Disease and Weed Control 18 :127 130. 11 Brayford, D. 1990. Vegetative incompatibility in Phom op sis from elm. Mycol Res 94 :745 752. 12 Buchan, A., Newell, S., Moreta, J., and Moran, M. 2002. Analysis of internal transcribed spacer (ITS) regions of rRNA genes in fungal communities in a southeastern U S salt marsh. Microb Ecol 43 :329 340. 13 Cessna, S. G., Sears, V. E., Dickman, M. B., and Low, P. S. 2000. Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the ho st plant. Science's STKE 12 :2191.

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66 14. Christias, C. 1980. Nature of the sclerotial exudate of Sclerotium rolfsii Sacc. Soil Biology and Bi ochemistry 12 :199 201. 15. Cilliers, A., Herselman, L., and Pretorius, Z. 2000. Genetic variability within and among mycelial compatibility groups of Sclerotium rolfsii in Sou th Africa. Phytopathology 90 :102 6 1031. 16 Cooper, W. E. 1953. Variation in pathogenicity of Sclerotium rolfsii Sacc. isolates on peanuts. Phytopathology 43:289 290. 17 Dukes, P. D., Ferry, R.L., Jones, Al. 1983. Evaluating peppers, cowpeas, sweet potato, and tomatoes for resistanc es to southern blight incited by Sclerotium rolfsii Sacc. Phytopathology 73:785 786. 18 Durgaprasad, S., Reddy, N. P. E., Kishore, C., Reddy, B. V. B., Sud hakar, P., and Rao, S. Variability among the isolates of Sclerotium rolfsii (Sacc.) causing stem rot of peanut (Arachis hypogaea L.). Page 72. 19 Farr, D. F., Bills, G. F., Chamuris, G. P., and Rossman, A. Y. 1989. Fungi on plants and plant products in the United States. APS press. 20. Fery, R. L., and Dukes, P. D. 2005. Potential for utilization of pepper germplasm with a variable reaction to Sclerotium rolfsii Sacc. to develop southern blight resistant pepper ( Capsicum annuum L.) cultivars. Plant Genetic Resources: Charact erization and Utilization 3 :326 330. 21 Fery, R. L., and Dukes, P. D. 200 2. Southern blight ( Sclerotium rolfsii Sacc.) of cowpea: yield loss estimates and sources of resistance. Crop Prot 21 :403 408. 22 Flores Moctezuma, H., Montes Belmont, R., Jimnez Prez, A., and Nava Jurez, R. 2 006. Pathogenic diversity of Sclerotium ro lfsii isolates from Mexico, and potential control of southern blight through solarization and orga nic amendments. Crop Prot 25 :195 201. 23 Franke, M., Brenneman, T., Stevenson, K., and Padgett, G. 1998. Sensitivity of isolates of Sclerotium rolfsii from peanut in Georgia to selec ted fungicides. Plant Dis 82 :578 583. 24 Glass, N. L., and Kaneko, I. 2003. Fatal attraction: nonself recognition and heterokaryon incompatibility in filame ntous fungi. Eukaryot Cell 2 :1 8. 25 Gorbet, D., and Tillman, B. 2008. P :200 204.

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67 26 Hagan, A., Rivas Davila, M., Bowen, K., and Wells, L. 2004. Comparison of Fungicide Programs for the Control of Early Leaf Spot and Southern Stem Rot on Selected Peanut Cultivars 1. Peanut Scien ce 31 :22 27. 27 Harlton, C. E., Levesque, C. A., and Punja, Z. K. 1995. Genetic Diversity in Sclerotium ( Athelia ) r olfsii and Related Species. Phytopathology 85 :1269 1281. 28. Kim, K. S., Min, J. Y., and Dickman, M. B. 2008. Oxalic acid is an elicitor o f plant programmed cell death during Sclerotinia sclerotiorum disease development. Molecular p lant microbe interactions 21 :605 612. 29 Kohn, L., Stasovski, E., Carbone, I., Royer, J., and Anderson, J. 1991. Mycelial incompatibility and molecular markers identify genetic variability in field populations of Sclerotinia sclerotiorum Phytopathology 81 :480. 30 LATUNDE DADA, A. 1993. Biological control of southern blight disease of tomato caused by Sclerotium rolfsii with simplified mycelial formulations of T richoderma koningii Plan t Pathol 42 :522 529. 31 Le, C. N., Mendes, R., Kruijt, M., and Raaijmakers, J. M. 2012. Genetic and Phenotypic Diversity of Sclerotium rolfsii i n Groundnut Fields in C entral Vietnam. Plant Dis 96 :389 397. 32 Leeper, P. W., Phatak, S. C., Bell, D. K., George, B. F., Cox, E. L., Oerther, G. E., and Scully, B. T. 1992. Southern bligh t resistant tomato breeding lines: 5635M, 5707M, 5719M, 5737M, 5876 M, and 5913M. Hortscience 27 :475 478. 33. Leslie, J. F. 1993. Fungal vegetative compatibil ity. Annu Rev Phytopathol 31 :127 150. 34 Liu, M. F., Wu, L.C. 1971. The effect of amino acids on the growth and morphogenesis of Sclerotium rolfsii Sacc. Plant Prot. Bull. (Taiwan) 13:87 96. 35 Majer, D., Mithen, R., Lewis, B. G., Vos, P., and Oliver, R. P. 1996. The use of AFLP fingerprinting for the detection of genetic varia tion in fungi. My col Res 100 :1107 1111. 36. Martin, F. N. 2003. Development of Alternative Strategies for Management of Soilborne Pathogens Currently Controlled with Methyl Bromid e 1. Annu Rev Phytopathol 41 :325 350. 37 Maxwell, D. P. a. L., R. D. 1970. Oxalic Acid Pr oduction by Scleotinia sclerotiorum in Infected Bean and in Culture. Phytopathology 60:1395 1398. 38 McDonald, B. A., and McDermott, J. M. 1993. Population genetics of plant pat hogenic fungi. Bioscience 43 :311 319.

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71 BIOGRAPHICAL SKETCH Chenzhao Xie grew up in Yutian, Hebei province, China. She graduated from Hebei Agric ultural University with a Bachelor of Science degree in 2001. In the summer of 2008, she received a Master of Science degree in plant p athology from China University o f Florida as a master s student.