Vol. 153, No. 1

JOURNAL OF BACTERIOLOGY, Jan. 1983, p. 452-457 0021-9193/83/010452-06$02.00/0 Copyright C 1983, American Society for Microbiology

Nutritional Requirements of Two Flower Spiroplasmas and Honeybee Spiroplasmat C. J.

CHANGt AND T.

A. CHEN*

Department of Plant Pathology, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903

Received 16 April 1982/Accepted 27 September 1982

A chemically defined medium (CC-494) was used to study the nutritional requirements of three spiroplasmas representing three distinct serogroups: flower spiroplasmas [Spiroplasma floricola and FS (SR-3)] and honeybee spiroplasma [HBS (AS-576)]. Glucose, fructose, and mannose were utilized by all three spiroplasmas. In addition, the honeybee spiroplasma could ferment trehalose, FS (SR-3) could ferment sucrose, and S. floricola could ferment trehalose, sucrose, and raffinose. The three spiroplasmas varied greatly in their requirements of amino acids for growth. S. floricola was the only strain that utilized arginine. HBS (AS-576) required at least one purine and one pyrimidine base (either free base or ribonucleoside) for growth, while both flower spiroplasmas grew with only one base in the medium. Oleic acid, cholesterol, and bovine serum albumin were essential to all three spiroplasmas. Palmitic acid, which was nonessential, promoted growth significantly.

Although more than 60 isolates of spiroplasmas, including strains pathogenic to plants, insects, and vertebrate animals and strains not yet shown to be associated with any disease, have been successfully cultivated (3, 4, 17, 18), the precise nutritional requirements for all of these isolates are still unclear. Previous studies (5-10, 12, 14, 16; C. J. Chang and T. A. Chen, Phytopathology 69:1024,1979; I. M. Lee, Ph.D. thesis, University of California, Riverside, 1977; I. M. Lee and R. E. Davis, Phytopathology News 12:215, 1978; K. M. Malloy and T. A. Chen, Phytopathology 70:465, 1980) with undefined media attempted to show the comparative capabilities of spiroplasmas to utilize various nutrients. The results were frequently different and conflicting. For example, Saglio et al. (14) showed that Spiroplasma citri was unable to metabolize arginine, whereas Townsend (16) reported that with a limited supply of a fermentable carbohydrate S. citri was able to metabolize arginine. Igwegbe et al. (9) showed that S. citri (Morocco isolate) could ferment mannose, whereas Davis' result (5) showed otherwise. Thus, data on substrate utilization and nutrient requirements of spiroplasmas based on cultivation in a complex medium are very difficult to interpret. t This is paper no. D-11160-1-82 of the journal series of the New Jersey Agricultural Experiment Station. i Present address: Department of Plant Pathology, University of Georgia, Georgia Experiment Station, Experiment, GA 30212.

We report herein the precise nutritional requirements for carbohydrates, amino acids, nitrogenous bases, ribonucleosides, fatty acids, and cholesterol of two flower spiroplasmas [Spiroplasmafloricola and FS (SR-3)] and honeybee spiroplasma [HBS (AS-576)] cultured in a chemically defined medium (2). Substrate utilization in undefined and defined media is discussed. MATERIALS AND METHODS

Spiroplasmas. S. floricola (ATCC 29989) (7), FS

(SR-3) (ATCC 33095), and HBS (AS-576) (ATCC 29416) were used throughout this study. They were maintained in CC-494 medium at 31 ± 1C and subcultured every 2, 4, and 3 days, respectively (2). Medium CC-494 contains nine major fractions: N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, 8 inorganic salts, 2 keto acids, 9 nucleosides and 1 nucleotide, 3 carbohydrates, 20 amino acids, 13 vitamins, lipids and bovine serum albumin (BSA), and 8 other components (e.g., NADP, flavin adenine dinucleotide, coenzyme A, etc.) (2). The basal medium was prepared by mixing the above 64 individually prepared stock solutions in HEPES (2). The lipid portion, a mixture of palmitic acid, oleic acid, cholesterol, Tween 40, and Tween 80, and the BSA were prepared separately (2) and added to the basal medium in the ratio of 1:4 (by volume). The pH of the basal medium and that of the lipid-BSA portion were adjusted to 7.5 before mixing. The completed medium was then filter sterilized (pore diameter, 0.45 ,um), and 2.5-ml portions were dispensed into test tubes. 452

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NUTRITIONAL REQUIREMENTS OF SPIROPLASMAS

Spiroplasma growth. Cell growth and yields were determined as previously reported (2). At least 10 passages (30 ,ul of inoculum was transferred into 2.5 ml of fresh medium at each passage) in a given test medium were made before results were considered positive. Growth yields were measured at 3, 2, and 4 days after inoculation for HBS (AS-576), S. floricola, and FS (SR-3), respectively, unless stated otherwise. Carbohydrte utilization. Each of the following carbohydrates was tested as the sole carbohydrate source in CC-494 at the concentrations shown in parentheses (2): D-glucose (44.4 mM), D-fructose (44.4 mM), D(+)mannose (44.4 mM), D-mannitol (43.9 mM), D-sorbitol (43.9 mM), a-lactose (23.4 mM), maltose (23.4 mM), sucrose (23.4 mM), D(+)-trehalose (23.4 mM), D(+)raffinose (15.9 mM), and starch (0.8%). All stock solutions were 20%, except for lactose and starch which were 10% and were filter sterilized (Millipore filter [Millipore Corp.], 0.45 ,um). Ribose and deoxyribose, which were the original constituents of medium CC-494, were deleted in this study. Amino acid requirements. The following 10 combinations were grouped from 20 amino acids (all L-form except glycine) according to their structural differences, their roles in the biosynthesis of sugars, and their essentiality for higher animals (see Table 2): glucogenic, 14 amino acids; ketogenic, 3; glucogenic and ketogenic, 3; essential, 10; nonessential, 10; uncharged nonpolar R group, 8; uncharged polar R group, 7; positively charged polar R group, 3; negatively charged polar R group, 2; and one group containing all 20 amino acids. The concentration of each amino acid was the same as that in medium CC-494 (2). Arginine metabolism. Arginine at a concentration of 20 mM (16) was used in medium CC-494 with the complete exclusion of glucose, ribose, and deoxyribose. Growth in this medium along with a change in pH towards basic is taken as an indication of the organism obtaining energy through the arginine dihydrolase pathway. Nitrogenous base utilization. The free bases adenine, guanine, cytosine, thymine, and uracil were used to replace nucleosides, deoxynucleosides (including 5methyldeoxycytidine), and UTP which were originally in medium CC-494. The following 10 combinations were grouped and tested (with millimolar concentrations given in parentheses): adenine (0.36)-guanine (0.20)-cytosine (0.27)-uracil (0.27)-thymine (0.24), guanine (0.20)-cytosine (0.27), adenine (0.36)-thymine (0.24)-uracil (0.27), adenine (0.36)-guanine (0.20), cytosine (0.27)-thymine (0.24)-uracil (0.27), adenine (0.72), guanine (0.32), cytosine (0.43), thymine (0.38), and uracil (0.43). Ribonucleoside requirement. The same 10 combinations as were used in the free nitrogenous base study were replaced with corresponding nucleosides. The concentration of each nucleoside was the same as that used in the original medium CC-494 (2). Deoxynucleosides (including 5-methyldeoxycytidine) and UTP, which were constituents of CC-494, were excluded. Fatty acids, cholesterol, and BSA. Based on the lipid composition in medium CC-494 (2), the following four groups were tested: (i) without cholesterol, (ii) without Tween 40 and palmitic acid, (iii) without Tween 80 and oleic acid, and (iv) without BSA. The BSA used was essentially fatty acid-free (less than 0.005%) and was purchased from Sigma Chemical Co., St. Louis, Mo.

453

(product no. A 6003), unless stated otherwise. The concentration of each ingredient remained the same as that in medium CC-494 (2).

RESULTS Carbohydrate utilization. The three spiroplasma strains showed differences in metabolizing various carbohydrates. All of them could ferment glucose, fructose, and mannose. In addition, HBS (AS-576) utilized trehalose, S. floricola utilized sucrose, trehalose, and raffinose, and FS (SR-3) utilized sucrose. None could utilize galactose, mannitol, sorbitol, lactose, maltose, and starch. The growth of each spiroplasma in different fermentable carbohydrates is shown in Table 1. Glucose was the best carbohydrate source for all of the spiroplasmas. There were significant differences in yield among the spiroplasmas growing in media supplemented with other carbohydrates.

Amino acid metabolism. Both S. floricola and FS (SR-3) grew in any of the 10 amino acid combinations, whereas HBS (AS-576) grew only in four: (i) 20 amino acids, (ii) glucogenic amino acids, (iii) nonessential amino acids, and (iv) uncharged polar R group amino acids. The growth of each spiroplasma under different combinations of amino acids is shown in Table 2. The medium supplemented with 20 amino acids supported the highest growth for all three spiroplasmas. Generally, as more amino acids were deleted from the medium, a lower yield resulted. For example, HBS (AS-576) reached 2.46 x 109 cells per ml with 20 amino acids compared with 1.03 x 108 cells per ml when only seven uncharged polar R group amino acids were supplied. Arginine metabolism. Based on cell growth and pH change of the culture medium it was

determined that S. floricola, among the three

TABLE 1. Growth comparison of HBS (AS-576), S.

floricola, and FS (SR-3) in various carbohydratesa Growth (cells/ml)

Carbohy-

drate

Glucose Fructose Mannose Sucrose Trehalose

S

576)

2.38 2.00 3.62 1 1.51

x x

109

109

S. floricola

FS (SR-3)

2.71 x 109 2.33 x 109

2.23 x 109 1.93 x 109

1.20 x 109 1.60 x 109 7.84 x 108

x 1

xc

1.51 x 109 1.21 x 109

x 10P 7.99 x 107 Raffinose a Other tested, nonutilized carbohydrates for all of these spiroplasmas are galactose, mannitol, sorbitol, lactose, maltose, and starch.

b Growth reached 1.53 second transfer. c , No growth.

x

109 cells per ml in the

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CHANG AND CHEN

J. BACTERIOL.

TABLE 2. Growth of HBS (AS-576), S. floricola, and FS (SR-3) with different amino acid combinationsa Amino acid

combinationb

Growth (cells/ml)

HBS (AS576) 2.46 x 109 1.45 x 109

S. floricola

FS (SR-3)

3.59 x 108 1.83 x 108 20 amino acids 1.12 x 107 1.98 x 108 Glucogenic c 1.94 x 107 6.00 x 106 Ketogenic 1.23 x 107 3.70 x 107 Glucogenic and ketogenic 2.86 x 5.72 x 10' Essential 1.62 x 108 1.80 x 107 1.05 x 108 Nonessential 2.25 X 107 6.86 x 107 Uncharged nonpolar R group 2.45 x 10' 1.38 x 108 1.03 x 108 Uncharged polar R group 1.91 x 107 7.53 x i07 Positively charged polar R group 2.69 x 10' 8.00 x 107 Negatively charged polar R group a BSA fraction V (Sigma Chemical Co., product no. A 4503) was used in this study for S. floricola and FS (SR-3). b Glucogenic amino acids: alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, methionine, proline, serine, threonine, and valine. Ketogenic amino acids: leucine, lysine, and tryptophan. Glucogenic and ketogenic amino acids: isoleucine, phenylalanine, and tyrosine. Essential amino acids: arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Nonessential amino acids: alanine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine. Uncharged nonpolar R group amino acids: alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Uncharged polar R group amino acids: asparagine, cysteine, glutamine, glycine, serine, threonine, and tyrosine. Positively charged polar R group amino acids: arginine, histidine, and lysine. Negatively charged polar R group amino acids: aspartic acid and glutamic acid. c -,No growth.

strains tested, could obtain energy through an arginine dihydrolase pathway. Growth of S. floricola in media containing either glucose (44.4 mM) or arginine (20 mM) reached its peak in 2 and 5 days, respectively. S. floricola grew much slower in the arginine medium; its yield was reduced almost fivefold. Nitrogenous base requirement. The requirement for nitrogenous bases differed between the two flower spiroplasmas and the honeybee spiroplasma. At least one free pyrimidine base and one free purine base must be supplied in the medium to support the growth of HBS (AS-576). On the other hand, any pyrimidine or purine base could support the growth of S. floricola and FS (SR-3). Growth of the three spiroplasmas varied greatly with different combinations of nitrogenous bases (Table 3). The highest yield was obtained when all five nitrogenous bases were added. Growth in the medium containing guanine and cytosine was significantly higher than in the medium containing adenine, thymine, and uracil. Ribonucleoside requirement. Similar results were obtained when five ribonucleosides were used to replace the corresponding five nitrogenous bases. HBS (AS-576) required at least one

ribonucleoside from a pyrimidine base and one from a purine base, whereas S. floricola and FS (SR-3) grew in a medium supplemented with any single pyrimidine or purine ribonucleoside. Growth of the three spiroplasmas with different ribonucleoside combinations is shown in Table 4. The medium supplemented with all five ribonucleosides supported the highest growth for the three spiroplasmas. The addition of guanosine and cytidine resulted in higher yields than with adenosine, thymidine, and uridine. Other combinations resulted in significantly different and reduced yields for the two flower spiroplasmas.

Fatty acid, cholesterol, and BSA requirement. All three spiroplasmas have the same requirements for fatty acids, cholesterol, and BSA. Oleic acid, cholesterol, and BSA were found to be essential because no growth was observed when they were individually deleted from the medium. Although palmitic acid was not required, it promoted significant growth of the three spiroplasmas. For example, growth yields of HBS (AS-576), S. floricola, and FS (SR-3) without 'the supplementation of palmitic acid reached 1.73 x 108, 4.37 x 108, and 5.37 x 108, respectively, as compared with the growth yields with the supplementation of palmitic acid

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VOL. 153, 1983

TABLE 3. Growth comparison of HBS (AS-576), S. floricola, and FS (SR-3) with different nitrogenous bases Growth yield (cells/ml) FS (SR-3) Nitrogenous base(s) HBS (ASS. floricola 576) 7.88 x 108 3.23 X 108 1.80 x 109 Adenine, guanine, cytosine, thymine,

uracil Guanine, cytosine Adenine, thymine, uracil Adenine, guanine Cytosine, thymine, uracil Adenine Guanine Cytosine Thymine Uracil

5.57 x 107 7.06 x 105

a

Control'

5.44 x 108 1.31 x 107

2.26 x 108 4.31 X 106

7.76 x 106 1.87 X 107

2.37 x 108 3.52 x 107

9.80 2.35 4.58 2.76 2.05

7.06 2.44 5.91 2.51 6.58

x 106 x 108 x 107

x 107 x 107

x 106 x 108 x 107 x 107 x 107

a , No growth. b No nitrogenous bases added.

which were 2.69 x 109, 2.00 109, respectively.

x

109, and 1.96

x

DISCUSSION Carbohydrate utilization. Our study indicates that the spiroplasmas of distinct serogroups show differences in the utilization of carbohydrates. Of the 12 carbohydrates tested, S. floricola could utilize 6, whereas HBS (AS-576) and FS (SR-3) each could utilize 4. Apparently S. floricola has more enzymes for carbohydrate utilization than the other two spiroplasmas. Glucose, fructose, and mannose were fermented by all three spiroplasmas. In addition, trehalose could be used by HBS (AS-576) and S.

floricola, sucrose by S. floricola and FS (SR-3), and raffinose by S. floricola. Considering that trehalose is the major disaccharide found in the hemolymph of most of the insect habitats of honeybee spiroplasma, it is not surprising that this spiroplasma could cleave trehalose into two glucose residues. Therefore, it is quite possible that S. floricola, at some time in its life cycle, may also reside in the hemolymph of an insect. Both S. floricola and FS (SR-3) were originally isolated from the surface of flowers. It has been suspected that the two spiroplasmas multiply in the nectaries of flowers, but no direct evidence of this has ever been reported. The ability of S. floricola and FS (SR-3) to ferment sucrose

is consistent with this.

TABLE 4. Growth comparison of HBS (AS-576), S. floricola, and FS (SR-3) with different ribonucleosides Growth (cells/ml) S. floricola

FS (SR-3)

2.02 x 109

1.57 x 109

1.93 x 109

1.86 x 109 1.63 x 108

1.07 x 109 5.88 x 108

1.48 x 109 1.42 x 109

Ribonucleoside(s)

HBS (AS576)

Adenosine, guanosine, cytidine, thymidine, uridine Guanosine, cytidine Adenosine, thymidine, uridine Adenosine, guanosine Cytidine, thymidine Adenosine Guanosine Cytidine Thymidine Uridine Controlb a _, No growth. b

No ribonucleosides added.

a

4.42 4.79 3.56 2.77 1.94 9.51 1.12

x x x x x x

108 108 108 108 108 107

x 10"

5.19 1.32 3.14 6.37 2.47 7.84 1.46

x 107 x

108

x x x x

107 107

108 107 x 108

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CHANG AND CHEN

Our results contradict earlier reports on carbohydrate utilization studies. For example, HBS (AS-576) was reported to use glucose, fructose, maltose, trehalose, and starch (K. M. Malloy and T. A. Chen, Phytopathology 70:465, 1980) but not mannose (5). Since in our defined medium the presence or absence of a particular sugar can be precisely controlled, different results of carbohydrate utilization obtained from undefined and chemically defined conditions should be expected. Recently, Malloy and Chen (Phytopathology 71:892, 1981) showed that di-, oligo-, and polysaccharides are converted to glucose when incubated with horse serum or serum fraction in the absence of spiroplasma. Therefore, the validity of using carbohydrate utilization as one of the criteria for mycoplasma taxonomy when using undefined media becomes questionable. Amino acid and arginine metabolism. It is possible that free amino acids are present in the complex components of undefined culture media because the three spiroplasmas required all 20 amino acids for maximum growth in our defined medium. Cell yields decreased with reduced amino acid supplements. This is not surprising since the amino acids are important metabolic building blocks for spiroplasmas which carry a minimum genome size. However, it was unexpected that both S. floricola and FS (SR-3) grew in a medium supplemented with only two negatively charged polar R group amino acids (aspartic and glutamic acids) or with any of the 10 amino acid combinations used in this study. This suggests that they are able to transaminate amino acids readily. On the other hand, HBS (AS576) required at least seven uncharged polar R group amino acids in the medium, indicating a lesser transamination ability. Positive results have been reported for HBS (AS-576) and S. floricola (5) for arginine metabolism. In our defined medium, however, only S. floricola utilized arginine, but growth was much lower compared with that when glucose was used as the energy source. Since ATP production from arginine catabolism is less than that through glycolysis, the arginine dihydrolase pathway may function as a minor energy-generating system for S.

floricola.

Like carbohydrate utilization studies, studies on arginine metabolism obtained from defined and undefined conditions produced conflicting results. Previous studies (5, 9, 16) indicated that the addition of minute amounts of glucose was required for arginine metabolism of S. citri. In

our defined medium, however, S. floricola uti-

lized arginine without the addition of any fer-

mentable carbohydrate. However, S. floricola

required a period of adjustment before adapting to the arginine medium, while HBS (AS-576) and

J. BACTERIOL.

FS (SR-3) did not survive through the first passage. Nitrogenous base and ribonucleoside requirement. Our studies indicated that spiroplasmas, like other mycoplasmas, lack the orotic acid pathway for pyrimidine synthesis and the enzymatic pathway for de novo synthesis of purine bases (13). HBS (AS-576) could only interconvert bases within the same group (either purine or pyrimidine), whereas S. floricola and FS (SR-3) could convert not only within groups but also between groups. HBS (AS-576) required the same base precursors as Mycoplasma mycoides subsp. Mycoides (11) and Acholeplasma laidlawii strain B (15). 5. floricola and FS (SR-3) require fewer base precursors for nucleic acid synthesis than any of the above-mentioned organisms. The nitrogenous bases and ribonucleosides used in this study were Sigma-grade chemicals from Sigma Chemical Co. Their purity was not tested in this laboratory. Therefore, further study is needed to find out if the growth of both flower spiroplasmas in a medium supplemented with only one base or ribonucleoside was due to trace amounts of contamination in the chemicals. Deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxycytidine, and 5-methyldeoxycytidine), ribose, and deoxyribose are constituents of medium CC-494 (2). In both nitrogenous base and ribonucleoside utilization studies they were therefore deleted from the medium. Thus, our results have clearly shown that spiroplasmas are able to synthesize ribose and deoxyribose from other fermentable carbohydrates and to make deoxynucleotides from either nitrogenous bases or ribonucleosides. Fatty acids, cholesterol, and BSA. The requirement for sterols by spiroplasmas and mycoplasmas has been recognized universally. Cholesterol is supplied from the serum in the culture media. Besides sterols, a number of glycerides, free fatty acids, phospholipids, and proteins also play important roles in spiroplasma nutrition. Only in the development of chemically defined media (2) have we realized the precise importance of these active chemical components from the serum. In our medium, CC-494, horse serum was replaced by a combination of palmitic acid, oleic acid, cholesterol, and BSA. CC-494 supported excellent growth of S. floricola, FS (SR-3), and HBS (AS-576). We found that oleic acid, cholesterol, and BSA were required by the three spiroplasmas. Palmitic acid was not essential but promoted significantly better growth. This has confirmed the previous suggestion that spiroplasmas are incapable of de novo biosynthesis of long-chain fatty acids and cholesterol for their cell membranes from acetyl coenzyme A (13).

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NUTRITIONAL REQUIREMENTS OF SPIROPLASMAS

Using defined medium we are convinced that spiroplasmas do require cholesterol, which is a taxonomic criterion for the family Spiroplasmataceae. Apparently spiroplasmas need BSA in the medium to act as a carrier and detoxifier for the free fatty acids (1). The failure of the 6 out of the 10 amino acid combination media to support the growth of HBS (AS-576) and the fact that no lag phase growth was observed when subculturing all three spiroplasma strains lead to the assumption of no proteolytic activity for the three spiroplasmas. However, a further investigation of BSA functioning as a nitrogen source is needed. ACKNOWLEDGMENTS This research was supported by state, U.S. Hatch Act, and National Science Foundation grant no. PCM-80-23948 funds. LITERATURE CITED 1. Brown, J. R. 1978. Albumin, structure, biosynthesis, function. Proceedings of the 11th FEBS Meeting. Pergamon Press, New York. 2. Chang, C. J., and T. A. Chen. 1982. Spiroplasmas: cultivation in chemically defined medium. Science 215:1121-1122. 3. Davis, R. E. 1978. Spiroplasma associated with flower of the tulip tree (Liriodendron tulipifera L.). Can. J. Microbiol. 24:954-959. 4. Davis, R. E. 1979. Spiroplasmas: newly recognized arthropod-borne pathogens, p. 451-484. In K. Maramorosch and K. F. Harris (ed.), Leafhopper vectors and plant disease agents. Academic Press, Inc., New York. 5. Davis, R. E. 1979. Spiroplasmas: helical cell wall-free prokaryotes in diverse habitates, p. 59-65. In H. S. J. Su and R. E. McCoy (ed.), Proceedings of the Republic of China-United States Cooperative Scientific Seminar on

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Mycoplasma Diseases of Plants. National Science Council Symposium, Series I, Taipai, Taiwan. 6. Davis, R. E., I.-M. Lee, and L. K. Basciano. 1979. Spiroplasmas: serological grouping of strains associated with plants and insects. Can. J. Microbiol. 25:861-866. 7. Davis, R. E., I.-M. Lee, and J. F. Worley. 1981. Spiroplasma floricola, a new species isolated from surfaces of flowers of the tulip tree, Liriodendron tulipifera L. Int. J. Syst. Bacteriol. 31:456-464. 8. Freeman, B. A., R. Sissenstein, T. T. McManus, J. E. Woodward, I. M. Lee, and J. B. Mudd. Lipid composition and lipid metabolism of Spiroplasma citri. J. Bacteriol. 125:946-954. 9. Igwegbe, E. C. K., C. Stevens, and J. J. Hollis, Jr. 1979. An in vitro comparison of some biochemical and biological properties of California and Morocco isolates of Spiroplasma citri. Can. J. Microbiol. 25:1125-1132. 10. Jones, A. L., R. F. Whitcomb, D. L. Williamson, and M. E. Coan. 1977. Comparative growth and primary isolation of spiroplasmas in media based on insect tissue culture formulations. Phytopathology 67:738-746. 11. Mitchell, A., and L. R. Finch. 1977. Pathways of nucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides. J. Bacteriol. 130:1047-1054. 12. Mudd, J. B., M. Ittig, B. Roy, J. Latrille, and J. M. Bove. 1977. Composition and enzyme activities of Spiroplasma citri membranes. J. Bacteriol. 129:1250-1256. 13. Razin, S. 1978. The mycoplasmas. Microbiol. Rev. 42:414-470. 14. Saglio, P., M. L'Hospital, D. Lafleche, G. Dupont, J. M. Bove, J. G. Tully, and E. A. Freundt. 1973. Spiroplasma citri gen. and sp. n.: a mycoplasma-like organism associated with "stubborn" disease of citrus. Int. J. Syst. Bacteriol. 23:191-204. 15. Touretellotte, M. E., H. J. Morowitz, and P. Kasimer. 1964. Defined medium for Mycoplasma laidlawii. J. Bacteriol. 88:11-15. 16. Townsend, R. 1976. Arginine metabolism by Spiroplasma citri. J. Gen. Microbiol. 94:417-420. 17. Whitcomb, R. F. 1980. The genus Spiroplasma. Annu. Rev. Microbiol. 34:677-709. 18. Whitcomb, R. F. 1981. the biology of Spiroplasmas. Annu. Rev. Entomol. 26:397-425.