Serological classification of spiroplasmas: current status

Data concerning serological classification of spiroplasmas are in good agreement, but slightly different numerical designations have been given to existing groups. It is proposed that a standardized system be adopted based on information developed mainly by the IRPCM working team on spiroplasmas. The type species (Spiroplasma citri) should be redefined to include only the agent of citrus stubborn disease (subgroup I-1). Six other subgroups, including three proposed by Bové et al. in this volume (I-5, I-6, and I-7), are members of the Group I complex. Because subgroups I-1, I-2, and I-3 (1) show significant reciprocal differences in DNA-DNA homology and two-dimensional electrophoretic protein profiles, (2) occupy exclusive habitats, (3) are each associated with important diseases, and (4) consist of clusters of very similar or identical strains, it is suggested that Latin binomials could be assigned to subgroups I-2 and I-3. It is proposed that those criteria could serve as general guidelines for consideration of subgroups for species status in the class Mollicutes. The I-4 subgroup is assigned an uncertain status, pending comparisons with the LB-12 (I-5), M55 (I-6), and N525 (I-7) subgroups. To previously described serogroups we add the CN-5 Cotinus beetle spiroplasma (IX), the AES-1 mosquito strain (X), and the MQ-4 Monobia strain (XI).     

Spiroplasma Species, Groups, and Subgroups from North American Tabanidae

Twenty-one triply cloned spiroplasma strains from the United States east of the Rocky Mountains, all isolated from tabanid (Diptera:Tabanidae) flies or serologically related to strains from tabanids, were compared reciprocally by spiroplasma deformation (DF) and metabolism inhibition (MI) serological tests. Many of the strains were also tested against 28 antisera representing known spiroplasma groups, subgroups, and putative groups isolated from nontabanid hosts. Relationships among strains were indicated by reciprocal cross-reactivity in both DF and MI tests. The strains were found to represent 11 recognized spiroplasma groups or subgroups. On the basis of serological, biochemical, and genomic data, strain BARC 1901 from Tabanus lineola appeared to represent a previously unrecognized candidate group. Strain BARC 2649, also from T. lineola, also appeared to represent a new group, but its morphology, arginine utilization, and some one-way serological crossing patterns suggested that it may be distantly related to group VIII spiroplasmas. Morphological, serological, and genomic data were used to place tabanid spiroplasma strains into three informal clusters. These are (i) groups IV (strain B31) and XXXI (strain HYOS-1); (ii) the three existing subgroups and a new candidate subgroup of group VIII represented by strain BARC 1357 plus ungrouped strain BARC 2649; and (iii) 14 strains, including EC-1 and TATS-1 (group XIV); strains TN-1 and TAAS-2 (group XVIII); strains TG-1, TASS-1, and BARC 4689 (group XXIII), strains TALS-2 (group XXVII), strain TABS-2 (group XXXII), and strains TAUS-1 and TABS-1 (group XXXIII) and ungrouped but closely related strains BARC 1901, BARC 2264 and BARC 2555. Analysis of tabanids from other geographic regions probably will substantially increase the number of known spiroplasma groups from this insect family. Received: 23 April 1997 / Accepted: 31 May 1997     

Nutritional requirements of two flower spiroplasmas and honeybee spiroplasma.

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.     

The Effect of Nacl on Campylobacter Jejuni/Coli

The growth of 2 strains of Campylobacter jejuni/coli was investigated in 0–2.0 % NaCl in Brucella broth at 35° G and 30° C. Both strains tolerated more NaCl in the growth medium at 35° C than at 30° C. 2 % NaCl was bacteriocidic at both temperatures. The strains also grew in the medium without added NaCl. At 35° C, low concentrations of NaCl stimulated the growth of strain 5616, but not the growth of strain B33. At 30° C, strain 5616 grew in NaCl concentrations up to 1.0 % and strain B33 in 0 % and at the control concentration (0.5 % NaCl). The survival of 22 C. jejuni/coli strains in 2.0 % NaCl at 4° C and 35° C was also investigated. Human strains showed significantly greater tolerance to 2.0 % NaCl at both temperatures than did the strains isolated from animals. These findings suggest that the salting of food can be effective in preventing the growth or survival of C. jejuni/coli.     

Wall-less prokaryotes from fall flowers in central United States and Maryland

Four spiroplasma strains and eleven isolates tentatively identified as acholeplasmas were obtained from fall flowers in Colorado, Nebraska, Illinois, and Maryland. Although the acholeplasma isolates were heterogeneous, all showed antigenic sharing with a group of unnamed organisms (L1 and related strains) isolated in othe studies from flowers in Florida. The W20 and W24 isolates from Nebraska were partially related to the L1 group by DNA-DNA homology and polyacrylamide gel electrophoresis (PAGE) analyses. A Colorado spiroplasma (W13) was identifed as a new strain of group IV complex. Three spiroplasma strains from flowers in Maryland old fields represent a new serovar with closest affinity to subgroup I-4 and to the LB12 and N525 serovars of group I. Widespread occurrence of acholeplasmas on flowers in this study, and on plant surfaces in general, suggests that, like spiroplasmas they probably will be found to reside in arthropods.     

Adaptation of red clover rhizobia to low temperatures

Summary Red clover Rhizobium strains, isolated from different locations between latitudes 60° and 63°30′ N in Finland, were tested for their adaptation to low temperatures. 31 strains were tested for growth at 5°C, 10°C, 15°C and 18°C in pure culture. No strain grew at 5°C. At the other temperatures there were differences between the strains, but the same strains grew fast at all temperatures. Ten strains were investigated for nodulation and acetylene reduction in phytotrons in two different climates, one simulating the growing season in southern and the other in northern Finland. There were differences between the strains in their ability to nodulate their host plant, and northern strains showed higher nitrogenase activity than southern strains in the cold climate.     

Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing

Aims: The potential effect of in‐premise plumbing temperatures (24, 32, 37 and 41°C) on the growth of five different Legionella pneumophila strains within free‐living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Methods and Results: Compared with controls that actively fed on Escherichia coli prey, when Leg. pneumophila was used as prey, strains Lp02 and Bloomington‐2 increased in growth at 30, 32 and 37°C while strains Philadelphia‐1 and Chicago 2 did not grow at any temperature within A. polyphaga. Strains Lp02, Bloomington‐2 and Dallas 1E did not proliferate in the presence of H. vermiformis nor did strain Philadelphia‐1 in the presence of N. fowleri. Yet, strain Bloomington‐2 grew at all temperatures examined within N. fowleri, while strain Lp02 proliferated at all temperatures except 41°C. More intriguing, strain Chicago 2 only grew at 32°C within H. vermiformis and N. fowleri suggesting a limited temperature growth range for this strain. Conclusions: Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. Significance and Impact of the Study: Temperature conditions and species of amoeba host supported in drinking water appear to be important for the selection of human‐pathogenic legionellae and point to future research required to better understand Legionella ecology.     

Studies on the color variation in larvae ofEphestia kühniella Zeller (Lepidoptera: Phycitidae) III. Fitness of different larval color strains

The population fitness in terms of the intrinsic rate of increase r_m was measured in eight pure (homogenic) strains of Ephestia kühniellaZeller with different larva color at an optimal temperature of 25°C, and in three strains at unfavorble temperatures of 15, 17, 28, and 30°C, to understand a mechanism of maintenance of a larval color variation found in wild populations. The survival rate, hatchability, and gross rate of reproduction were poorly correlated with the fitness but the mean generation time and net reproduction rate were correlated with the fitness significantly at 25°C. Intermediate color (pink) strain(s) grew faster, initiated reproduction earlier and had shorter longevities than other strain(s) under the range of 15 and 28°C; the fitness was highest in the intermediate larval color strains except at 30°C in which all strains had a negative rate of increase. The results were discussed with reference to the relationship between the larval color and fitness along with a maintenance mechanism of the variation.     

Virus-mediated change in clumping properties ofDrosophila SR spiroplasmas

Transovarially transmitted SR spiroplasmas inDrosophila cause an abnormal sex ratio (SR condition: male-specific killing) in the host fly progenies. A reaction known as clumping takes place between different SR spiroplasma strains in which spiroplasmas instantly form aggregates upon mixing of the two strains. Each strain of SR spiroplasma carries an associated virus that is lytic to certain other strains. When the virus, HIV, from the recently discovered non-male-killingDrosophila hydei spiroplasma (HIS) is injected into host flies carrying the SR spiroplasma ofD. nebulosa (NSR), the latter spiroplasmas either undergo complete lysis and disappear, or survive with decreased numbers and with an abnormal morphology, and are transmissible from generation to generation in host flies. The surviving spiroplasmas possess two viruses, the endogenous virus of thenebulosa spiroplasma, spv-1, and the newly introduced superinfecting virus, HIV. This combination leads to a change in the surface properties of the superinfected spiroplasmas that is manifested in their ability to form clumps with normalnebulosa spiroplasmas, but does not interfere with male killing. This change in spiroplasma phenotype is discussed in terms of host-phenotype modification by infecting viruses.     

Growth rates of Eucheuma denticulatum (Burman) Collins et Harvey and Kappaphycus striatum (Schmitz) Doty under different conditions in warm waters of Southern Japan

Green and brown strains of Eucheuma denticulatum and new cultivated strain Kappaphycus striatum were collected from commercial cultivation ground in Philippines. Daily growth rates (DGR) of the materials were measured under different temperatures and photon fluence in laboratory controlled conditions and at different depths in the sea using the floating raft culture method in Uranochi Inlet, Tosa Bay, Southern Japan. The highest DGR of E. denticulatum (brown strain) (2.76% ±0.64) and K. striatum (green strain) (4.5% ±1.51) were recorded at 25°C. K. striatum (both strains) had the highest DGR and grew over a wide range of temperatures. Observation on the effect of photon fluence showed that K. striatum (brown strain) grew well (DGR = 5.16%±1.3) at 145 μmol photon m^-2 s^-1.In the floating raft cultivation, K. striatum (green strain) had a DGR of3.1% ± 0.93 higher than E. denticulatum (brown strain)(2.73% ± 0.43). This revised version was published online in August 2006 with corrections to the Cover Date.     

The Minimum Growth Temperature of Obligately Thermophilic Bacteria as Influenced by Inhibitors in Complex Growth Media

The inability of several obligately thermophilic bacteria to grow at or below 37°C in common laboratory media was shown to be caused by inhibitors present in these media. The inhibitors could be removed by extraction with chloroform and by adsorption on ion exchange resins or starch. It was concluded that they were of organic nature, probably bile acids. Four laboratory strains of Bacillus stearothcrtno-philus grew readily at 37°C in detoxified media. With one of them (NCA 1503) growth was obtained at 34–35°C. A fresh soil isolate having a minimum growth temperature of 40°C in glucose nutrient broth grew at 30°C in this medium after the inhibitors had been removed.     

Metarhizium spp. isolates from madagascar: Morphology and effect of high temperature on growth and infectivity to the migratory locust,Locusta migratoria

Five strains ofMetarhizium anisopliae (Metsch.) Sorokin and one strain ofMetarhizium flavoviride Gams &; Rozsypal originally isolated in Madagascar were studied. Measurements of conidia and, for the first time, also of blastospores produced in a liquid medium were used for species and variety determination. Blastospores ofM. flavoviride were more homogenous in their size than those ofM. anisopliae. Growth at high temperatures between 25° and 40°C showed that 4 isolates ofM. anisopliae grew at 36°C andM. flavoviride grew at 38°C. Using alternating day/night temperatures (8/16 h) the three strains tested could also tolerate 40°/25°C. In bioassays, fiveMetarhizium spp. isolates were tested against third and fourth instar larvae ofLocusta migratoria (L.) at two alternating day/night temperatures of 30°/25°C and 36°/25°C. In the cooler regime, all strains caused a mortality of 50% within 5.9 to 8.5 days (median lethal time), while in the 36°/25°C treatment only the thermophilicM. flavoviride and oneM. anisopliae strain isolated from a soil sample gave comparable results with median lethal times of 6.8 and 7.3 days, respectively.     

Obligately and facultatively autotrophic,sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts

A variety of autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria were isolated from thermogenic composts at temperatures of 60–80° C. All were penicillin G sensitive, which proves that they belong to the Bacteria domain. The obligately autotrophic, non-spore-forming strains were gram-negative rods growing at 60–80°C, with an optimum at 70–75°C, but only under microaerophilic conditions (5 kPa oxygen). These strains had similar DNA G+C content (34.7–37.6 mol%) and showed a high DNA:DNA homology (70–87%) with Hydrogenobacter strains isolated from geothermal areas. The facultatively autotrophic strains isolated from hot composts were gram-variable rods that formed spherical and terminal endospores, except for one strain. The strains grew at 55–75° C, with an optimum at 65–70° C. These bacteria were able to grow heterotrophically, or autotrophically with hydrogen; however, they oxidized thiosulfate under mixotrophic growth conditions (e.g. pyruvate or hydrogen plus thiosulfate). These strains had similar DNA G+C content (60–64 mol%) to and high DNA:DNA homology (> 75%) with the reference strain of Bacillus schlegelii. This is the first report of thermogenic composts as habitats of thermophilic sulfur- and hydrogen-oxidizing bacteria, which to date have been known only from geothermal manifestations. This contrasts with the generally held belief that thermogenic composts at temperatures above 60° C support only a very low diversity of obligatory heterotrophic thermophiles related to Bacillus stearothermophilus. Received: 20 July 1995 / Accepted: 25 September 1995     

Powder puff spiroplasma: A new epiphytic mycoplasma

A spiroplasma (strain PPS1) isolated from healthy flowers ofCalliandra haematocephala in Florida has been found to be a member of a serogroup of the Spiroplasmataceae. It is distinct fromSpiroplasma citri and from other described spiroplasmas as determined by growth inhibition, fluorescent antibody, and ELISA serological tests. PPS1 was also distinguished fromS. citri and several other spiroplasmas by the guanine + cytosine content of its DNA. PPS1 requires sterol for growth, is inhibited by digitonin, grows at 20–30°C, and does not hydrolyze arginine or urea. The ready isolation of this and similar organisms from surfaces of healthy plants emphasizes that caution should be exercised in attempts to isolate cell wall-less prokaryotes from the interior of diseased plants. Although some strains of spiroplasmas are known as insect pathogens in nature, the ecological role(s) of the flower-inhabiting spiroplasmas has yet to be fully determined.     

Antibiotic sensitivities in vitro of diverse spiroplasma strains associated with plants and insects

Fifteen spiroplasma strains, representing five serological subgroups classified in three distinct serogroups, and four strains unassigned to serogroups were examined for sensitivity to antibiotics. The data contribute to the characterization of spiroplasmas and enlarge comparisons between plant pathogenic strains and strains that are evidently a part of the normal epiphytic microflora.     

Chemically Defined Medium for Cultivation of Several Epiphytic and Phytopathogenic Spiroplasmas

A chemically defined medium, LD82, was formulated for in vitro cultivation of spiroplasmas. Medium LD82 supported good growth for four epiphytic and insect-pathogenic spiroplasmas, Spiroplasma floricola 23-6^T, Spiroplasma sp. strain SR3, Spiroplasma sp. strain brevi, and Spiroplasma sp. strain AS576, and of the phytopathogenic spiroplasmas Spiroplasma citri Maroc R8A2^T and PC1. Titers of all six strains grown in defined medium LD82 reached 2.0 × 10⁹ to 6.0 × 10⁹ CFU/ml of culture. All spiroplasma strains tested formed colonies readily on agar medium LD82. None of the spiroplasmas formed typical fried-egg colonies. All formed diffuse colonies, but the forms of colonies differed somewhat among the spiroplasma strains. In preliminary studies of nutritional requirements, phospholipids slightly enhanced the growth of the epiphytic and insect-pathogenic strains in medium LD82 and were found essential for good growth of S. citri.     

Growth promotion of wheat seedlings by <Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis> 1P (MTCC 8707) a cold tolerant bacterial strain from the Uttarakhand Himalayas

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a gram-positive, rod-shaped, yellow pigmented bacterium isolated from soil on nutrient agar plates at 4°C. The identity of the bacterium was arrived on the basis of the biochemical characterization, BIOLOG sugar utilization pattern and sequencing of the 16S rRNA gene. It grew at temperatures ranging from 4 to 42°C, with temperature optima at 30°C. It expressed multiple plant growth promotion attributes such as phosphate solubilization, indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN) production, differentially at suboptimal growth temperatures (15 and 4°C). At 15°C it solubilized phosphate (21.1 μg of P ml⁻¹ day⁻¹), and produced IAA (14.9 μg ml⁻¹ day⁻¹) in tryptophan amended media. Qualitative detection of siderophore production and HCN were possible at 15°C. At 4°C it retained all the plant growth promotion attributes. Seed bacterization with the isolate, positively influenced the growth and nutrient uptake parameters of wheat seedlings in glass house studies at suboptimal cold growing temperatures.     

Influences of Culture Temperature on the Growth, Lipid Content and Fatty Acid Composition of Aurantiochytrium sp. Strain mh0186

The growth, lipid content, and fatty acid composition of Aurantiochytrium sp. strain mh0186 at different temperatures were investigated. Strain mh0186 grew well at 15–30°C, but weakly at 10°C. The biomass at 15–30°C was significantly higher than at 10 and 35°C, and the total lipid at 15–35°C was significantly higher than that at 10°C. The amount of DHA in the total fatty acid was highest at 10°C and decreased in response to temperature increase. The content of DHA (mg/g-dry cell weight) at 15–30°C were significantly higher than those at 35°C and those at 15–25°C were significantly higher than those at 10 and 35°C. The DHA yield at 15–35°C was significantly higher than those at 10 and 35°C. Unsaturation of fatty acid was regulated by temperature and was enhanced in response to temperature decrease. The ratio of DHA to DPA varied at different temperatures.     

Spiralin: Major membrane protein specific for subgroup I-1 Spiroplasmas

Preparations of spiralin from membranes ofSpiroplasma citri, strain C189, purified by sequential solubilization with detergents followed by agarose-suspension electrophoresis induced rabbit antibodies that were largely specific forSpiroplasma citri Group I-1 spiroplasmas, as demonstrated by metabolic inhibition (MI), growth inhibition (GI), and deformation (DF) tests. By contrast, antibodies againstS. citri whole-membrane protein preparations reacted broadly with representative type cultures of seven subgroups of theS. citri complex. Neither antimembrane nor antispiralin sera reacted withS. floricola, S. mirum, or Group IV, (VI), (VII), or (VIII) spiroplasmas. Minor cross-reactions in MI and DF tests between antispiralin serum and Subgroup I-2 and I-3 antigens may have represented shared epitopes in a set of homologous membrane proteins of the three spiroplasmas, or antibodies against highly antigenic traces of other common membrane proteins in the purified spiralin preparations. The unique antigenic properties of spiralin, the most abundant protein in theS. citri membrane, explain in part the unique profiles shown by this spiroplasma species in comparative taxonomic serological tests.