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The etiology of false broomrape of tobacco

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Title:
The etiology of false broomrape of tobacco
Creator:
Mixson, Wayne Clark, 1944-
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English
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47 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Burley tobacco ( jstor )
Cauliflower ( jstor )
Diseases ( jstor )
Inoculation ( jstor )
Peas ( jstor )
Plant gall ( jstor )
Plant roots ( jstor )
Plants ( jstor )
Species ( jstor )
Symptomatology ( jstor )
Dissertations, Academic -- Plant Pathology -- UF
False broomrape ( lcsh )
Plant Pathology thesis Ph. D
Tobacco -- Diseases and pests ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1972.
Bibliography:
Includes bibliographical references (leaves 44-46).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Wayne Clark Mixson.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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The Etiology of False Broomrape of Tobacco By WAYNE C. MIXSON A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE U-NIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSO.^HY UNIVERSITY OF FLORIDA 1972

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ACKNOWLEDGMSXTS The author wishes to express his thanks to Dr. Carol R, Killer, former Supervistory Couumittee Chairman and assistant professor of plant pathology at the University of Florida, for his assistance during this study. Special thanks go to Dr. R. E. Stall, dairnian of the Supervisory Committee, for his guidance and advice as well as critical assistance during the preparation of this manuscript. Thanks also go to the other members of the S'upervisory Committee, Dr. V, G. Perry, Dr. L. H. Purdy, and Fred Clark, Acknowledgments are also extended to Forrest Parrott of Tabacalera Nicaraguence, Managua, Nicaragua, and Paul Gardner, Tabacalera Hondurena, San Pedro Sula, Honduras, for their assistance during the author's trips to Central America. Special consideration is also extended to the British American Tobacco Company for making the author's trips to Central America possible. 11

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS j_i LIST 0? FIGURES ^ LIST OF TABLES ^^^ ABSTRACT ^^^^ INTRODUCTION MATERIALS AND METHODS 5 Inoculation and Transmission Methods 5 Sources and Types of FBR Tissue 6 Effect of Temperature and Size of Plant on Fasciation 12 Host Range and Resistance Within Nicotiana species and Cultivars of N. tabacum 17 Effect of Fertilization Levels on FBR Development 17 Determination and Isolation of Causal Agent j^g RESULTS 21 Inoculation and Transmission Methods „Sources and Types of FBR Tissue 23 Effect of Temperature, Time From Inoculation, and Size of Plants on Fasciation 27 111

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Page Host Range and Resistance Within Nicotiana Species and Cultivarc of N. tabacurn 30 Effect of Fertilization Levels on FBR Development ....,,..., 32 Isolation of the Causal Agent 32 Identification of FBR-72-65. ....... 38 DISCUSSION 40 SUlvfl-IARY 42 LITERATURE CITED 44 BIOGRAPHICAL SKETCH 47 IV

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Figure 10 LIST OF FIGURES Burley tobacco plants in a Kentucky field stunted by FBR Fasciated tissue from burley tobacco plants in Kentucky infected with FBR Fasciated tissue from fluecured in Honduras infected with FBR Hardened FBR galls on roots of tobacco Page Fasciated tissue from burley tobacco plants in Nicaragua infected with FBR q Fasciated tissue from cigar wrapper tobacco in Nicaragua infected with FBR 9 Above ground plants originating from below ground fasciated tissue caused by FBR , -^q 11 Cauliflower plant found naturally infected with FBR in Nicaragua 23 14 Succulent FBR galls on roots of tobacco ^5 Succulent leafy FBR tissue and small plants originating from underground tissue on roots of tobacco ^g

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Figure page 11 Severely FBR infected root systems resulting in stunting of burley tobacco cor.rj-.red to uninfected plant in Nicaragua ....... 24 12 Small plants forming from fasciated tissue near the soil surface on burley tobacco in Nicaragua 25 13 Root systems of FBR inoculated tobacco plants grown at 25 C (right), 30 C (center), and 35 C (left) 29 14 FBR symptoms produced by inoculation of tobacco roots with bacterial isolate, FBR-70-33 35 15 FBR symptoms produced by inoculation of tobacco roots with bacterial isolate, FBR-72-65 36 VI

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Table LIST OF TABLES Page Effect of dilution of crude extract of false broomrape tissue on percent infection of tobacco roots ...,.,,, 22 Reaction of the tobacco cultivar Hicks to inoculation with false broomrape at three temperature regimes and three time periods 28 Reaction of several genera of plants including Nicotiana species and N, tabacum cultivars. ,~ 31 Effect of fertilization levels on false broomrape development of burley and flue-cured tobacco. ..... 33 Results of physiological. Gram stain, and optimum temperature tests conducted on known C. fascians and bacterial isolate, FBR72-65 39 VI 1

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Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ETIOLOGY OF FALSE BROOMRAPE OF TOBACCO By Wayne C. Mixson December, 1972 Chairraanj Dr. R. E. Stall Major Departments Plant Pathology The false broomrape (FBR) pathogen was easily transmitted from tobacco to tobacco using any type of fasciated tissue other than the small plants formed above ground from the fasciated tissue near the soil surface. The best method for inoculation was to 1) grind FBR tissue in a sterile mortar, with additions of small amounts of 0.1 M potassium phosphate buffer at pH 7.0 until the horaogenate has a consistency of a thick paste; 2) carelessly remove 20 cm tall plants from soil; 3) dust clean roots with 600 mesh carborundum and rub the roots gently with a cotton swab; 4) immerse the roots of the plants immediately in the fresh FBR homogenate and rub the roots gently with a cotton swab; and 5) transplant the inoculated plants immediately. This method gave 100% transmission. No differences were found between FBR from the United States and Central America. FBR development Vlll

PAGE 9

is associated with the temperature most favorable for plant growth. At least 4 weeks were required for gall development. All species of Nicotiana tested were susceptible to FBR as were all 14 cultivars of N. tabacum tested. Cauliflower with FBR was observed growing in Nicaragua. Sweet potato, cabbage, and sweet pea were susceptible to FBR in greenhouse inoculations. A bacterium was isolated from FBR tissue and upon subsequent inoculations caused FBR symptoms on tobacco. This isolate, FBR-72-65, was identified as a species of Corynebacterium and is probably C. fascians . A known isolate of C. fascians (ICPP CF-19) caused similar symptoms on tobacco. Two colony types were observed (smooth and rough) for FBR-72-65 and ICPP CF-19 and only the smooth types were pathogenic. Results of all physiological tests with the FBR bacterium agreed with the Sergey's Manual description of C. fascians except for nitrate reduction. The known C. fascians isolates also were anomolous with the nitrate reduction test. The best medium for isolation of the causal bacterium was found to be one containing 40 g Difco PDA, 2 g Na2HP0^, 2 g NaCl, 1 g sodium citrate, 1 g asparagine, and 300 mg potassium dichromate per 1000 ml of water. IX

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INTRODUCTION False broomrape (FBR) is a disease of the root system of tobacco (Nicotiana tabacum L.)Symptoms of FBR include a fasciation of the root tissue in which masses of white, succulent, and irregular outgrowths form. Under certain conditions differentiation of chlorophyll containing leaves (15) and/or a complete plant may occur from the outgrowths. Thompson (19) placed FBR tumors into the following three groups: 1) heterotopic teratomas, 2) leafy teratomas, and 3) secondary root teratomas. All the galls, except those on the main root (heterotopic teratomas), had tumor gaps and traces connecting the tumors to the vascular system of the tobacco plant. Valleau (22) named the disease false broomrape because he first identified the disease as broomrape which is caused by Orobanchi spp . , a parasitic seed plant. Thompson (19) stated that the tumors are somewhat similar to crown gall caused by Aqrobacterium tumefaciens (Smith and Town) Conn and tumors that are hereditary in nature. FBR appears to have different etiology from either of these two tumor problems, however. FBR was first reported in Kentucky on burley tobacco in 1951 (22) and hc.^ since been reported in Florida (24),

PAGE 11

North Carolina (8), Georgia (6), and Maryland (18). It has caused little economic loss to the total tocacco crop in the United States (12), however. Jenkins (6) reported that the disease was sporadic and never caused any serious losses in Georgia and Massey (U) found the same to be true in Kentucky. Economic losses do occur in individual fields, however. Plants in some fields in Kentucky were observed in 1969 by the author to be severely diseased (Fig. 1). It was estimated tnat the yield in these fields would be reduced at least 20%. Tne Diseased plants were 1 to 3 ft in height while the healthy plants were 5 to 6 ft in height. When the root systems on the stunted plants were examined, all of them had galls ranging in diameter from 2 to 8 cm on the tap root. The most severe losses apparently occur in Central America where FBR has been observed in Honduras, Nicaragua, El Salvador, and Panama (12, 15). In the Central American countries FBR is more severe on burley than flue-cured tobacco. The author also observed FBR on shade tobacco in the burley area of Nicaragua in 1970 and 1971, A severe outbreak in the Jalapa Valley resulted in a 28% reduction in yield (15). Several researchers have tried and failed to isolate a causal agent of FBR (3, 8, 19). Some proposed that FBR is caused by the bacterium, C. fascians Tilford (Dowson) (8, 12, 17, 19, 22). Valleau noted the similarity of this disease to leafy gall of tobacco described by Tilford (20)

PAGE 12

Figure 1 . Burley tobacco plants in a Kentucky field stunted by FBR. .-=^/./i

PAGE 13

and Lacey (iO). Thompson (19) and Newman (17) inoculated tobacco with C. fascians and obtained symptoms almost identical to FBR but were unable to complete Koch's postulates. Kelman (8) did the same but did not believe that C. fascians was the causal organism because of differences in symptoms and the fact that FBR developed only after wounding, whereas C, fascians did not. C. fascians was first identified as a pathogen of sweet pea by Tilford (20) and was found to cause root fasciations, leafy galls, and several other disorders on many plants particularly on ornamental plants (1, 16). Jacobs and Mohanty (5) found that C. fascians has a very wide host range >and confirmed the existence of host specificity among strains of the pathogen. Lacey (10) isolated a bacterium associated with leafy gall of sweet peas and found that these strains were pathogenic on Nicotiana qlutinosa and N. tabacum . vicia faba L. and Phaseolu s vulgaris L. were susceptible to the tobacco strain (10). FBR has the potential of becoming a serious disease on tobacco in the United States as well as on other economic crops. As a result of a review of the literature which revealed that seve-al researchers have conflicting views as to the causal agent of the disease and subsequent personal observations of the disease in fields of tobacco in Kentucky and Central America, the author conducted research to assist in identifying the causal agent which, in turn, might aid in its control.

PAGE 14

hiATERIALS AND METHODS Iioculation and Transmission Methods In initial experiments inoculation techniques for transmission of FBR to tobacco were with macerated, fasciated tissue because no causal agent had been identified. Transmission of the causal agent of FBR by method described by Dukes was tried (3). The Dukes* method is 1) grind FBR tissue in a sterile mortar, with additions of small amounts of 0.1 M potassivun phosphate buffer at pH 7.0; 2) grind until the homogenate has the consistency of a thick paste j 3) dust clean roots of healthy tobacco plants in the 4-5 leaf stage of growth that were carefully removed from the soil with 600 mesh carborundum and rub the roots gently with cotton swabs; 4) immerse the roots of the plants immediately in the fresh FBR homogenate and rub the roots gently with a cotton swab; and 5) transplant the inoculated plants iiTimediately. Kelman's method (8) of inoculation was also tried. This method was simpler in that the plants' roots were wounded and immersed in a suspension of fragments of FBR tissue. Several variations of Dukes' method were also tested. These were 1) identical procedure but the plants were pulled carelessly from the soil; 2) the roots were deliberately rubbed for injury; 3) the same 5

PAGE 15

as number 1 without carborundum; 4, 5, 6) repeat the above three metnods but the homogenata had been filtered througn Whatman no. 1 filter paper; 7) identical to number 1 but with homogenate diluted lilO, lilO^, lilO^, lilO , and lilO^j 8) inject homogenate into roots with a hyperdermic syringe and 27 gauge needle; and 9) transplant in naturally and artifically infested soil (plants were pulled from soil, soil carefully washed from roots, and then rubbed with carborundtun) . Method 1 was used to inoculate plants in subsequent tests unless otherwise indicated, and the cultivar Kicks was also used unless otherwise indicated. Sources and Types of FBR Tissue Fasciated tissue was obtained from burley tobacco in Kentucky and Nicaragua (Fig. 2 and 3), cigar wrapper tobacco in Nicaragua (Fig. 4 and 5), and flue-cured tobacco in Honduras (Fig. 6). FBR was observed under natural conditions at these locations for differences in symptoms. Tissue was obtained from many fields in Nicaragua and Honduras and kept separate to screen for possible differences that might exist. Tobacco plants were inoculated with each of the above sources of diseased materials, grown in a greenhouse for several weeks, and observed for symptom expression. Tissue from the fasciated roots of a cauliflower ( Brassica plearacea var. botrytis L.) plant found in Nicaragua

PAGE 16

Figure 2. Fasciated tissue from bur ley tobacco plants in Kentucky mtected with FBR,

PAGE 17

Figure 3. Fasciated tissue from burley tobacco plants in Nicaragua infected with FBR.

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Figure 4. Fascia ted tissue from cigar wrapper tobacco in Nicaragua infected with FBR.

PAGE 19

10 Figure 5, Above ground plants originating from below ground fasciated tissue caused by FBR.

PAGE 20

il Figure 6. Fasciated tissue from flue-cured tobacco in Honduras infected with FBR.

PAGE 21

12 was also used to test for transmission to tobacco (Fig. 7), Tobacco plants were inoculated with various types of fasciated tobacco tissue. This tissue was either dried or living. The living tissue was further subdivided into hardened galls (Fig. 8), succulent galls (Fig, 9), succulent leafy tissue (Fig. lO), and small plants that had developed from galls when they occurred near the soil surface (Fig. 10). The galls were also sectioned to obtain inner and outer tissue. Effect of Temperature and Size of Plant on B'asciation Fifteen tobacco plants of the Hicks cultivar were inoculated and placed in each of three growth chambers set at 25, 30, and 35 C. Five plants were removed at each of three time intervals of 3, 4, and 6 weeks. The plants were weighed and examined for fasciation. Any galls were then removed and weighed. Inoculated plants were compared with uninoculated checks. This experiment was repeated. Tobacco plants of the same cultivar at heights of 10, 20, 40, 60, 80, and 100 cm were inoculated to determine the optimum size for FBR development. Inoculated plants were held in a greenhouse, and the root systems were observed at intervals of 3, 4, 5, and 6 weeks.

PAGE 22

Figure 7, Cauliflower plant found naturallyinfected with FBR in Nicaragua.

PAGE 23

Figure 8. Hardened FBR galls on roots of tobacco.

PAGE 24

15 Figure 9. Succulent FBR galls on roots of tobacco.

PAGE 25

16 Figure 10, Succulent leafy FBR tissue and small plants originating from underground tissue on roots of tobacco.

PAGE 26

17 Host Range and Resistance VJithin Nicotiana Species and Cultivars of N« tabacum Two species, N. glutinosa L. and N. rustica L. , were inoculated along with the following cultivars of N. tabacum i Coker 80-F, F-17, N. C. 95, Hicks, Va 115, N. C. 2326, Coker 254, Coker 319, Coker 298, Speight G-7, McNair 14, Maryland 64, Bur ley 21, and Pennbel 69. Peanut (Archis hypogea L.), bean ( Phaseolus vulgaris L.), cauliflower, sweet potato ( Ipomoea batatus L.), cabbage ( Brassica olearacea var. capita ta L.), sweet pea ( Lathyrus odoratus L.), and garden pea ( Pi sum sativium L. ) were also inoculated. Plants of the above species were root inoculated and the sweet and garden pea plants were also inoculated as described by Jacobs and Mohanty (5)» In this method the seeds were soaked in a slurry of FBR tissue ^ and planted in sterile soil in the greenhouse. Effect of Fertilization Levels on FBR Development Twentyfive tobacco plants approximately 20 cm in height were inoculated with a slurry of FBR tissue and planted in 4-inch clay po^s containing sterile soil in the greenhouse. Five different fertilization treatments were established within each group of 25 plants (5 plants/treatment). The treatments were no fertilizer, 1.5, 3, 4.5, and 6 g/pot of 3-6-9 analysis. Plants were maintained for 5 weeks following inoculation and the plants' roots, shoots, and resulting galls were weighed. Twenty-five

PAGE 27

18 uninoculated plants were also planted in the soil with the five fertilizer treatments, « Determination and Isolation of Causal Agent Since several researchers have speculated that FBR is caused by a bacterium (3, 4, 8, 12, 17, and 19), experiments were designed to eliminate other causal agents and to isolate a causal agent. A slurry of FBR tissue was made and filtered through a Whatman no. 1 filter paper several times to remove plant material. The resulting filtrate was then filtered through a 0.45/a Millipore filter to remove any bacteria that were present. Presumably a virus would pass through into the filtrate. Roots of tobacco plants were inoculated with 1) the slurry before the first filtration, 2) the filtrate obtained through the Whatman filter, and 3) the filtrate obtained through the Millipore filter. A suspension containing tobacco mosaic virus (TMV) was also passed through a 0.45^ Millipore filter and then inoculated on tobacco to determine, if indeed, a virus particle did pass through the filter. As a check the FBR filtrate obtained after Millipore filtration was plated on nutrient agar to determine if any bacteria were allowed through. Streptomycin was added to a FBR filtrate through Whatman no. 1 paper to obtain the following concentrations! ppm, 50 ppm, 100 ppm, 200 ppm, 300 ppm, and 400 ppm. Each of these was inoculated on roots of tobacco plants.

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19 The plants were allowed to grow for 5 weeks and were observed for gall development. Attempts were made to isolate the causal organism, and most of these attempts were directed at the isolation of a bacterium. The procedure in the first attempt was similar to that used by Tilford (20) in 1936 in his original isolation of C. fascians from sweet peas. A large piece of tissue was taken from the base of the fasciated growth, dipped in alcohol, flamed, and then crushed in 5 ml of sterile water. After 2 to 4 hr one drop of the liquid was transferred with a sterile pipette to a sterile dish; two drops were placed in a second dish} and three drops were placed in a third. Potato dextrose agar (PDA) at 48 C was added to the plates and held at 25 C. This same procedure was used in other isolations except the drops were placed on solidified PDA and smeared with a sterile glass rod. This was also done without the 2 to 4 hr waiting period and repeated on nutrient agar (NA). In another attempt the medium described by Burkholder (2) for the isolation of C. sepidonicum , the causal agent of tuber ring rot and wilt of potato, was used. To make Burkholder's medium approximately 300 g of sliced potatoes were boiled and the broth made up to a liter. To this was added 5 g peptone, 2 g Na2HP04, 2 g NaCl, 1 g sodium citrate, 1 g asparagine, 6 g dextrose, 12 g agar.

PAGE 29

20 Burkholcer's medium was also tried using potassium cichrornate to retard the growth of saprophytes. This was used at 100 ypa, 2C0 ppm, 300 ppm, and 400 ppm, Kado a;id Hes>cetc*s laediLuu D2 (9;^ which was proposed for isolation of Corynebacteriurg spp . was tried. This mediun contained 10 g glucose, 4 g casein hydrolysate, 2 g yeast extract, 1 g NH^^Cl, 0.3 g MgS0^.7H20, 5 g LiCl, 1.2 g Tris, 40 mg polymyxin sulfate, 2 nig sodium azide, and 15 g agar por liter of water. This was adjusted to pH 7.8 with KCl before autoclaving. Polymyxin sulfate and sodium azide were added after the medium had cooled to 50 C. Each of the types of gall tissue listed previously was used in isolation attempts with each media. The most widely used method of isolation was to macerate a young, succulent gall in sterile water, filter through Whatman no. 1 filter paper, and then dilute to 1:10, lilO^, and 1:10-'. Tnese dilutions were added to the plates with sterile pipettes in quantities of 0.1. 0.5, and 1 ml and smeared with a sterile glass rod. The plates were incubated at 25 or 30 C. At the beginning of the isolation experiments, different bacterial colonies were removed from the plate, purified, and inoculated to tobacco. These were also stored in sterile water and held at 10 C. Eventually, colonies visible within 24 hr -.-re no longer isolated. Only the slower growing colonies were used, especially the ones that became visible in 3 or more days.

PAGE 30

RESULTS Inoculation and Transmission Methods Dukes* method of inoculation gave 100% transmission of FBR if the slurry was used within 1 hr after preparation. When the plants used in inoculation studies were carefully removed from the soil and not injured before inoculation, 80% of the inoculated plants had FBR, whereas 100% were diseased when the plants were carelessly pulled from the soil or if they were injured by rubbing. This means that excessive injury to the plant was not required for transmission of FBR. Injury to the tap root produced a larger gall and more stunting than when the tap root was not injured. After filtration through Whatman no. 1 filter paper to remove coarse plant parts, the filtrate was diluted 3 it 10 before transmission dropped below 50%. The 1:1, IslO, and l:lo2 dilutions gave 100% transmission (Table 1). Inoculation of a specific root with FBR filtrate using a hypodermic needle was successful, and a dilution of 1:102 gave 100% transmission. . This is a very good method to use when a small amount of inoculum, is available. It can also be used without disturbing the plant because soil can be carefully removed from only a small portion 21

PAGE 31

22 Table 1 . Effect of dilution of crude extract of false broonirape tissue on percent infection of tobacco roots. Dilution % infection ItO 100 111 100 lilO .100 ltl02 100 lil03 48 1«104 15 1»105

PAGE 32

23 of the root system before inoculation. FBR was easily transmitted using either naturally or artifically infested soil. When plants were carefully removed from the soil and transplanted in naturally or artifically infested soil, the transmission was 65%. When the plants were carelessly pulled from the soil and then planted in infested soil, the transmission percentage rose to 100%. This methoc produced galls on the fine lateral roots more often than did dipping the root system in the FBR homogenate. Based on the above experiments the most efficient inoculation method was pulling the plants carelessly from the soil, washing the soil away, and then dipping the root system in a Is 10 dilution (filtered or unfiltered) of FBR homogenate. This method consistently gave 100% transmission. Sources and Types of FBR Tissue Field observations of FBR on burley and flue-cured tobacco disclosed no differences in symptoms. No differences were noted in symptoms in the United States or in Central America. The disease was more severe in Central America (Fig. 11) and has occurred every year for the past 10 years, whereas in the United States the disease may not appear every year. Also FBR affected plants tended to form small plants above the ground (Fig. 12) in Central America more often than in the United States.

PAGE 33

, -'I •,. •' . 24 Figure 11. Severely FBR infected root systems resulting in stunting of burley tobacco compared to uninfected plant in Nicaragua.

PAGE 34

25 Figure 12. Small plants forming from fasciated tissue near the soil surface on burley tobacco in Nicaragua.

PAGE 35

26 The soils of the two areas were very similar. The main difference seemed to be the lack of cold in Central America, although other reasons may exist for more severe disease in Central America. SeverEil fields were observed in Honduras and Nicaragua, both with and without FBR. Analysis of soil from these fields was made before the crop was transplanted. There was nothing significant about fields with or without FBR as far as elements, organic matter, or pH. Diseased roots were brought to the University of Florida from Kentucky and Central America. Tobacco plants inoculated with FBR homogenate from various fields and maintained in the greenhouse were no different in symptomotology. Each source of fasciated tissue produced small tobacco plants from the fasciated tissue that occurs near the soil surface. The cauliflower plants found in Nicaragua with FBR symptoms evidently were affected by the FBR causal agent. It was readily transmitted to tobacco and back to cauliflower. The symptoms on tobacco inoculated with the cauliflower homogenate were very similar to the symptoms produced with FBR tissue from tobacco. FBR was successfully transmitted from all types of fasciated tissues but not from the shoots that developed from such tissues. Old galls were dried at room temperature and stored at room temperature for 18

PAGE 36

27 months, and 50% transmissibility was still attained. FBR could be transmitted, with equal frequency, from hardened galls, succulent galls, and leafy underground tissue. Effect of Temperature, Time From Inoculation, and Size of Plants on Fasciation The growth of fasciated tissue was correlated with optimum conditions for growth of the tobacco plant. The results of the growth chamber experiments are given in Table 2 and Fig. 13. The optimum temperature for gall development was 25-30 C. The growth of the plants after inoculation was correlated with temperature more than with FBR, although the plants with the larger galls were slightly smaller at a given temperature than were uninoculated plants at the same temperature. No galls were observed to develop at any of the three temperatures during the first 3 weeKs following inoculation. At the end of 4 weeks galls were foiind at ju C and 35 C but not at 25 C. Plants at all three temperatures had FBR at the end of 6 weeks with plants at 35 C having a smaller number ana weight. Tobacco plants 20 cm in height proved optimal for inoculation purposes. Galls developed faster on plants of this height than on plants of any of the other heights. The larger plants, 40-60 cm tall, were better for point inoculation, but for screening purposes the 20 cm plants were superior. The plants required at least 4 weeks for symptom expression regardless of size.

PAGE 37

28 *i l-J > c • o (0 •H TJ 4^ o RS •H f-H V) D (U O a c G) •H a H 4J p (U CQ OJ JSd l^ x: •H -p T-l fr^ Ti u C (0 (0 > •iH CO 4J 0) H 6 3 H O 01 dJ M o 0) ra u X5 3 4J 4J nJ s^ 0) G x: a 4J B o in 'T CM F-l CM i I CO rH -H CM I I o ^r CM m o o iH (M + I CM -Hr« + 1 + 1 + 1 + + + + + + a\ + I CM ^ iH CM .H fH I I CO 0) «u O 0) H (0 at 0) CJ •o •o •0 c: CD (U 0) 0) TJ -P V -P •O 4-) OQ fO 0) rtJ Q) (3 0) 4-» rH 4J rH +> -( M (C 3 rt 3 (TJ 3 a H O rH U -< 3 3 3 CO O C o c U C (1) O-H O-H rH A-> C C c c C C (0 •H 3 •H 3 •H 3 a rH in O in c CM cn n H

PAGE 38

jL-a Figurs 13. Rcot systems of FER inoculated tobacco plants grovn at 25 C (right), 30 C (center), and 35 C (left).

PAGE 39

30 Host Range and Resistance V/ithin Nicotiana STDecies and Cultivars of N. tabacum Three species of Nicotiana were inoculated along with several other cultivars of N. tabacum and other genera of plants. These are listed in Table 3 along with their reaction to FBR, All three species of Nicotiana were equally susceptible to inoculation with FBR tissue. Of the 20 plants of each of the different cultivars of N. tabacum inoculated with FBR and grown in the greenhouse, five were removed from the soil, observed for FBR development, weighed, and measured at four different time intervals. No differences were noted in susceptibility among the 14 cultivars (Table 3). All plants developed fasciated tissue at the same rate and none were more retarded in overall growth than the others. Three of the other genera of plants evaluated proved susceptible to FBR (Table 3). Cauliflower, which was observed naturally diseased with FBR in Nicaragua, was susceptible in greenhouse inoculations along with cabbage. After several attempts FBR was transmitted to sweet potato. Sweet pea plants appeared to be susceptible, although the fascia tion did not develop to the extent reported by Tilford (20), Mohanty (16), Jacobs (5), and Lacey (11), Baker (1) reported Phaseolus vulgaris to be susceptible to C, fascians, but the cultivar of bean used in this work was not susceptible to FBR transmission from tobacco. Neither garden pea nor peanut was diseased.

PAGE 40

31 Table 3. Reaction of several genera of plants including Nicotiana species and N, tabacum c ultivars. Genera Reaction* Cauliflower Brassica olearacea var. botr ytis + Peanut Arachis hypoqea Bean Phaseolus vulgaris Sweet potato xpomoea batatus var. m capi tata + Cabbage Brassica olearacea + Sweet pea Lathyrus odoratus ^ Garden pea Pisurn sativium mtum Tomato Lycopersicon escute ^^ Tobacco N. rustica •f N. CTlutinosa + N. tabacuiTi Coker 80-F + F-17 + N. C. 95 + Hicks + Va 115 + N. C. 2326 + Coker 254 ^ Coker 319 -iCoker 298 + Speight G-7 + .. McNair 14 4Maryland 64 + Bur ley 21 + * + i nrsi na^J-oc Pennbel 69 +

PAGE 41

32 Effoct of Fertilization Levels on FBR Develop;;-,ant Fertilization had no effect on the initiation of fasciation (Table 4). Increasing the fertilization rate of the tobacco plants did not result in FBR being initiated sooner. Higher rates of fertilization did result in a faster development of the fasciated tissue once it was initiated. Isolation of the Causal Agent FBR was rot transmitted to tobacco by using bacterial free filtrates. Neither was it transmitted using a Whatman filtrate treated with streptomycin at 100 ppm and above. When the filtrate and treated filtrate were plated on nutrient agar, few bacterial colonies grew and at 300 ppm no bacterial colonies grew. TMV particles passed through the Millipore filter since plants inoculated with TMV filtrate developed TMV, These two experiments indicate that a bacterium but not a virus is the causal agent. Ease of transmission would tend to rule out a mycoplasma as the causal agent. The mediura chosen for most of the isolation attempts contained the following per 1000 ml: 40 g Difco PDA, 2 g Na2KP04, 2 g NaGl, 1 g sodium citrate, 1 g asparagine, and 100 mg potassium dichromate. Kado's medium, PDA, and NA were not successful because many saprophytic bacteria grew on them.

PAGE 42

33 Table 4. Effect of fertilization levels on false broomrape development of burley an4 flue-cured tobacco. Fertility level* g/pot Tumor Weight (g) Burley Flue-cured 5 5 1.5 6 7 3.0 10 11 4.5 11 13 6.0 13 16

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34 Seventythree bacterial isolations were made from the different types of FBR tissue. Most were slow growing and creiae, beige, yellow, or ye Howorange in color. These isolates .ere stored in sterile water at 10 C until the inoculated plants reacted postively or negatively after a 6 weoc period. The 33rd isolate obtained (FBR-70-33) was from a gall on a plant in the greenhouse induced by tissue from burley tobacco in Kentucky. After 4 weeks four out of five tobapcb plants inoculated with this isolate expressed symptoms of FBR (Fig. 14). Five more tobacco plants were then inoculated with FBR-70-33. No FBR symptoms were expressed after 7 weeks. This was repeated with the same results. Attempts to isolate the bacterium from the galls of FBR-70-33 inoculated plants were not successful. Isolation attempts were continued, and isolate FBR-72-65 was obtained from a hardened gall of burley tobacco brought from Nicaragua. This isolate appeared on the potassium dichromate medivun after 3 days as a pinpoint yellow-orange colony. This was the only colony of this type observed on any of the 20 plates that were smeared with 0.5 ml of the 1:10 dilution of the FBR homogenate previously described. This isolate stained Gram positive. All five plants inoculated with the isolate expressed FBR symptoms (Fig. 15). After a suspect bacterium was isolated, a culture of C. fascians was requested from M. P. Starr, Curator of the

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35 Figure 14. FBR symptoms produced by inoculation of tobacco roots with bacterial isolate, FBR-70-33.

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36 Figure 15. FBR symptoms produced by inoculation of tobacco roots with bacterial isolate, FBR-72.65.

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37 International Collection of Plant Pathogenic Bacteria, Davis, California, for comparison. This isolate of C. fascians (ICPP CF-19) also produced FBR symptoms on tobacco plants. When the two bacteria, ICPP CF-19 and FBR-72-65, vGre cultured on Difco PDA, both turned deep orange after 5 days. When FBR-72-65 was observed with the aid of a dissecting microscope, two colony types were evident, one rough and one smooth. The majority of the colonies were of the rough type. When the C. fascians isolate obtained from Starr was observed under the microscope, the same two colony types were observed. But in this instance the majority were of the smooth type. The smooth isolate was then designated FBR-72-65-1S and the rough isolate, FBR-72-65-iR, Tobacco plants were inoculated with each colony type from both ICPP CF-19 and FBR-72-65. In both instances the smooth isolates proved pathogenic and the rough ones did not. Selective development of the rough isolate in culture probably explains why the original FBR-72-65 and FBR-70-33 seemed to lose pathogenicity on subsequent inoculations. The smooth colony type was reisolated from galls caused by inoculation with FBR-72-65, and subsequent inoculations produced FBR symptoms. C. fascians grew on Burkholder 's medium with additions of up to JOO ppm potassium dichromate which severely retarded growth of saprophytic bacteria and fungi. With this medium reisolation of FBR-72-65 from

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38 a gall resulting from inoculation by FBR-72-65 was accomplished . Identification of FBR-72-.65 Physiological tests were undertaken using the known C. fascians (rough and smooth) and FBR-72-65-1S and -IR. These and the results are listed in Table 5 along with the cnaracterization from Bergey's Manual . All test results of the FBR-72-65-iS and -iR agreed with the results of the known C. fascians (rough and smooth). The nitrate reduction tests did not agree with Bergey's Manual . Based on these tests and comparisons with the known C. fascians, FBR-72-65 is a species of Corynebacterium and in all likelihood is C, fascians.

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39 Table 5. Results of physiological. Gram stain, and optimum temperature tests conducted on icnown C. fascians and bacterial isolate, FBR-72-65, "" C. fascians C. fascians FBR-72-65 Test Berqey's Manual "~ICPP CF-19 Gelatin lignification Nitrate reduction + « Starch hydroylis Hydrogen sulfide + + Litmus milk blued + +. + + Acid formation Dextrose Maltose Xylose + Lactose + + + + + + Gram stain + + + + Optimum temperature 25-28 C 25-30 C 25-30 C

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DISCUSSION Tobacco plants may be severely stunted and losses in yield may result if the tobacco plants become infected with FBR soon after transplanting. The size plant used for transplanting is easily inoculated with the causal agent of FBR. If the seedbed is contaminated, this may be the source of inoculum for previously uninfested fields. In greenhouse experiments FBR was easily transmitted when tobacco plants were planted in soil from a FBR infested field. Old, dried, fasciated tissue is a good source of FBR inoculum. No differences were found between any of the sources of FBR. When they were inoculated on tobacco plants in a greenhouse, the symptoms expressed were identical. All would form small tobacco plants from fasciated tissue near the soil surface. Thompson (19) did not find this to be true in earlier experiments. Tobacco plants could be inoculated with macerated tissue from any type of fasciated tissue and FBR was transmitted. This inoculum could be filtered and diluted i:iO and still be viable. All bacteria could be removed from this filtrate, and FBR symptoms could still be reproduced on tobacco plants. This disease h^.3 been reported in Florida (24), 40

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41 North Carolina (8), Georgia (6), Maryland (l«), and Central Ar.arica (i:,i, lb;. Many researchers have tried and failed to determine the causal agent (3, 8, 17). It was proposed that FBR is caused by the bacterium C. fascians (8, 12, 17, 19, 22;. Worlcing on this premise, organisms other than a bacterium were ruled out, and isolation techniques for the isolation of a bacterium were employed using a medium similar to one suggested by Burkholder (2). The medium was the same except Difco PDA and 300 mg potassium dichromate were used. On this medium C. fascians is allowed to grow without being overgrown by faster growing saprophytic organisms. After several attempts at isolation a bacterium was isolated that reproduced FBR symptoms when inoculated on tobacco. This same bacterivim was reisolated. Several physiological tests were conducted on the isolate, FBR-72-65, from fasciated tobacco tissue as well as a C. fascians isolate from M. P. Starr. They all agreed with the results in Bergey's Manual except the nitrate reduction test. Starr's C. fascians isolate did not agree with Sergey's Manual in this respect. Based on these tests and growth characteristics the isolate was identified as C. fascians.

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SUMMARY FBR is a very easily transmitted disease and no extra or excessive injury to the root systems of tobacco plants is necessary. Ample injury is inflicted to a tobacco plant during normal transplanting procedures. Any type of gall tissue can be used to transmit the causal agent of FBR. Dried tissue is also effective. This means that FBR can easily be carried over from one growing season to another. The temperature optimum for plant growth, 25-30 C, is optimum for FBR development, and almost any size or age of plant can be infected. At least 4 weeks are required for expression of symptoms. All Nicotiana species tested were susceptible to FBR as well as some 14 cultivars of N. tabacum. Cauliflower was found to be naturally infected with FBR in Central America. This FBR was easily transmitted to tobacco and back to cauliflower. Sweet potato, cabbage, and sweet pea were also suscepts to the disease. Increasing fertilization levels did not cause FBR to be initiated sooner but did result in a faster development of the fasciated tissue. This could explain why FBR is more severe on burley tobacco than flue-cured since burley received extremely high rates of fertilizer. 42

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43 A bacterium was isolated from fasciated tissue from Central America, and this bacterium produced FBR symptoms on tobacco plants in the greenhouse. The same bacterium was reisolated from these plants using a medium containing 40 g Difco PDA, 2 g Na2HP04, 2 g NaCl, 1 g sodium citrate, 1 g asparagine, and 300 mg potassium dichromate per liter of water. This bacterium was identified as Corynebacterium fascians. Two colony types were present, a rough colony which was not pathogenic and a smooth type which was pathogenic. More work is need with this disease and the causal agent. Since the causal agent was found to be a bacterixim, a control method can probably be developed. In as much as the disease is not at present a wide spread economic problem, the control would probably be with chemicals and/or crop rotation. The possibility of a wild host should also be investigated as well as cultural and environmental conditions that may favor the disease and influence its severity.

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LITERATURE CITED 1. Baker, Kenneth F. 1950. Bacterial fasciation disease of ornamental plants in California. Plant Dis. Rsptr. 34i 121-125. 2. Burkholder, Walter I-I. 1938. The occurrence in the United Szates of the tuber ring rot and wilt of the potato. Aarier. Potato J. 15:243-245. 3. Dukes, P. D., S. ?. Jenkins, Jr., and R. W. Toler. 1963. La improved inoculation technique for transmission of false broomrape to flue-cured tobacco. Plant Dis. Reptr. 47:895-897. 4. Dukes, P. D,, S. F. Jenkins, Jr., and R. W. Toler. 1964. Influences of soil temperature on the development of false brocvirape on the roots of flue-cured tobacco. Phytopathology 54:622. (Abstr . ) 5. Jacobs, S. E. and U. Mohanty. 1951. Studies in bacteriosis XXVII. Factors influencing infection '^y C o " y n a ba c t e r i ur a fas cians (Tilford) Dowson. •Ann. Appi. Biol. S'SsJjS. 6. Jenkins, S« F., Jr., P. D. Dukes, and S. S. Thompson. 1963. Flue-cured tobacco diseases in Georgia, 1959-62. Plant Dis. Reptr. 47:9:^98. 7. Johnson, J. T. and L. W. Nielsen. 1970. Sweet potato a suscepa of false broomrape. Plant Dis. Reptr. 54; 97^-980. 8. Kel.-.an, A., G. B. Lucas, and K. R, Garriss. 1955. F.-lse broor.-irapa of tobacco in North Carolina. E:.ij:.^ Mitchell Sci. See. J. 71:173-174. (Abstr.) 9. Kado, C. I. and M. G. Heskett. 1970. Selective media for _3olation of Agrobacterium , Corynebacteriu.-.. , Erwinia , Pseudomonas , and Xanthoracnas . Phytopathology 60:969-976. 44

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45 10. Lacey, M. S. 1936. Studies in bacteriosis XXII. I. The isolation of a bacterium associated with the fasciation of sweet peas, "cauliflower" strawberry plants and leafy gall of various plants. Ann. Appl. Biol. 23:302-310. 11. Lacey, M. S. 1939. Studies in bacteriosis XXIV. Studies on a bacterium associated with leafy galls, fasciations and "cauliflower" disease of various plants. Part III. Further inoculation experiments and cultural studies. Ann. Appl. Biol. 26«262-278. 12. Lucas, G. B. 1958. Diseases of tobacco. Scarecrow Press, New York, 498 p. 13. Massey, Ira. 1970. (Personal communication) 14. Marcelli, E. 1950. Carattere endemico dei "tumori radicali" presumibilimente da. Bact. tumefasciens, in campo di tabacco. (Nota preliminare) Tabacco 54i 199-201. 15. Miller, C. R, and G. B. Lucas. 1970. False broomrape, a destructive tobacco disease in Nicaragua. Phytopathology 60t 1541 . (Abstr.) 16. Mohanty, U. 1951. Coryne bacterium fascians (Tilford) Dowsonj Its morphology, physiology, nutrition and taxonomic position. Trans. Brit. Mycol. Soc. 34» 23-24. 17. Newman, M. A. and R. W. Toler. 1970. Characteristics of false broomrape disease and properties of the etiological agent. Phytopathology 50t 1305-13U6. (Abstr. ) 18. SJcoog, H. A. 1963. Diseases of Maryland tobacco. Maryland Agr. Exp. Sta. Bull. 419. 12 p. 19. Thompson, Samuel Stanley, Jr. 1965. False broomrape of tobacco: anatomy and morphology of tumors) influence of environment; reaction of some Nicotiana species to the disease. Master's Thesis, Purdue University. 81 p. 20. Tilford, P. E. 1936. Fasciation of sweet pea caused by Phytomonas fascians n. sp. J. Agr. Res. 53:383-4107"^ 21. Trotter, A. 1946, Sulla presenza di tumori radicali nelle coltivanzioni di tabacco di pieno campo. Ric. Ossvz. Divulg. fitopat. Campania ed Mezzogiorno (Portici) 10:65-80. (Abstr. Rev. App. Mycol. 26:34. 1947)

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46 22. Vallenu, W. D. 1953. False broomrape and leaf curl I Two new tobacco diseases in Kentucky, Plant Dis. Reptr. 37i 538-539. 23. Valleau, W. D,, E, M. Johnson, and S. Diachun. 1954. Tobacco diseases. Kentucky Agr. Exp. Sta. Cir. 522. 55 p. 24. Weber, G. F. 1954. False broomrape of tobacco in Florida. Plant Dis. Reptr. 38»121.

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BIOGRAPHICAL SKETCH Wayne Clark Mixson was born on a farm in Marion County, Florida, on November 29, 1944. He completed twelve years of school at Reddick, Florida, and was graduated from North Marion High School in 1962. He attended the University of Florida at Gainesville. In 1965 he received the degree of Bachelor of Science in Agriculture with a major in agronomy and in 1969 the degree of Master of Science in Agriculture with a major in agronomy. In September, 1969, he began studies toward the degree of Doctor of Philosophy. He is a member of the American Phytopathological Society, the American Society of Agronomy, Crop Science Society of America, and the Florida Turf-Grass Association. He is employed as a research agronomist by 0. M. Scott and Sons, Apopka, Florida. Wayne C. Mixson is married to the former Sharon Kay Freimuth of Lowell, Florida, a librarian with a Master of Science degree in Library Science. 47

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t"HM I cortify thc^t I have read this study and that in my opinion it conforrus to accepta'oie standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Joert E. Chairman Professor o Pathology Stall, Plant I certify -chat I have read this study and that in L\y opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. \Aa^
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I cortify thi.t I have read this study z-.-id that in r.y opinion it cc.aforns u-^ acceptable standards of scholarly presentation and is fully adequate, in scope and qur.lity, as a dissertation for tha dc^-ree of Doctor of Philosophy. -"rca Clark \ Professor of AgronoiTiy This dicwv-rtation vas subr;.itted to the Dean of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfill^r.ent of the requirements for t..3 degree of Doctor of Philosophy, DeceiuL'Dr, 1^72 « .gri.cui.wur Daan, Graduate School