Beet Leafhopper Research Articles

Identification of regions of the Beet mild curly top virus (family Geminiviridae) capsid protein involved in systemic infection, virion formation and leafhopper transmission

Plant viruses in the genus Curtovirus (family Geminiviridae) are vectored by the beet leafhopper ( Circulifer tenellus) and cause curly top disease in a wide range of dicotyledonous plants. An infectious clone of an isolate of Beet mild curly top virus (BMCTV-[W4]), associated with an outbreak of curly top in pepper and tomato crops, was characterized and used to investigate the role of the capsid protein (CP) in viral biology and pathogenesis. Frameshift mutations were introduced into the overlapping CP and V2 genes, and a series of CP alanine scanning mutations were generated. All mutants replicated in tobacco protoplasts or systemically infected plants, consistent with these gene products not being required for viral DNA replication. The CP frameshift mutant and most C-terminal alanine scanning mutants did not systemically infect Nicotiana benthamiana plants or form detectable virions, and were not leafhopper-transmitted. In contrast, most N-terminal alanine scanning mutants systemically infected N. benthamiana and induced disease symptoms, formed virions and were leafhopper-transmissible; thus, these substitution mutations did not significantly alter the functional properties of this region. One N-terminal mutant (CP49-51) systemically infected N. benthamiana, but did not form detectable virions; whereas another (CP25-28) systemically infected N. benthamiana and formed virions, but was not insect-transmissible. These mutants may reveal regions involved in virus movement through the plant and/or leafhopper vector. Together, these results indicate an important role for virions in systemic infection (long-distance movement) and insect transmission, and strongly suggest that virions are the form in which BMCTV moves, long distance, in the phloem.

Association of Beet Leafhopper (Hemiptera: Cicadellidae) with a Clover Proliferation Group Phytoplasma in Columbia Basin of Washington and Oregon

At least 16 taxa of cicadellids and delphacids were tested by polymerase chain reaction (PCR) for the presence of a phytoplasma in the clover proliferation group, designated 16SrVI. Nucleic acid extracts from individual insects or groups of 5-10 were tested using PCR primers designed from the DNA sequence of 16S-23S rRNA or ribosomal protein genes of the pathogen. The beet leafhopper, Circulifer tenellus (Baker), was most often associated with the phytoplasma, with approximately 16% of the insects testing positive. The phytoplasma was occasionally found associated with Ceratagallia spp. Leafhopper species that were not associated with the phytoplasma included Macrosteles spp., Dikraneura spp., Colladonus montanus (Van Duzee), Circulifer geminatus (Van Duzee), Ballana spp., Amplysellus spp., Paraphlepsius spp., Texananus spp., Balclutha spp., Latalus spp., Erythroneura spp., Exitianus exitiosus (Uhler), and unidentified delphacids. The detected phytoplasma was similar to, or synonymous with, the beet leafhopper-transmitted virescence agent that is associated with the potato purple top disease in the Columbia Basin region of Washington and Oregon. This is in contrast to the phytoplasma associated with potato purple top disease in Mexico that is related to aster yellows (group 16SrI). The association of the group 16SrVI phytoplasma almost exclusively with the beet leafhopper suggests that this insect is the major vector of the phytoplasma in this region.

Association of Beet Leafhopper (Hemiptera: Cicadellidae) with a Clover Proliferation Group Phytoplasma in Columbia Basin of Washington and Oregon

Association of Beet Leafhopper (Hemiptera: Cicadellidae) with a Clover Proliferation Group Phytoplasma in Columbia Basin of Washington and Oregon

Moisture and temperature requirements for London rocket (Sisymbrium irio) emergence

Abstract London rocket is hypothesized to be the most important overwintering host of the beet leafhopper in southern New Mexico. Knowledge of the environmental factors affecting the emergence of this plant is essential to developing a predictive model for London rocket and leafhopper populations. The temperature and moisture optima for London rocket germination and emergence were assessed using growth chamber studies. Temperatures of 5 to 35 C and soil water tension treatments of − 0.01 to − 1.5 MPa were tested. Optimal seed burial depth also was assessed. Optimal London rocket germination occurred at approximately 15 C and maximum emergence was achieved at 15 to 20 C with soil moisture tensions of − 0.01 to − 1.2 MPa. London rocket emergence occurred best at a seed burial depth of 2 mm. Nomenclature: London rocket, Sisymbrium irio L.

Planting Date Effects on Stand Establishment and Yield of Chile Pepper

Chile pepper (Capsicum annuum L.) yields are highly variable and are strongly influenced by disease and weather. The goal of two field experiments was to evaluate crop management factors, especially planting date, that could contribute to improved and more consistent crop production. Current practice in New Mexico is to direct seed the crop from 13 to 27 Mar. In the first experiment, chile pepper was direct seeded on three planting dates, 13, 20, and 27 Mar. 2000, without or with a fungicide treatment of pentachloronitrobenzene and mefenoxam for the control of damping off. The results indicate planting date had no effect on stand establishment or yield. Fungicide treatment, significantly reduced stand, but had no effect on yield. In the second experiment, chile pepper was direct seeded on six planting dates, 13, 20, 27 Mar. and 3, 10, 17, Apr. 2001, with or without an application of phosphorus fertilizer, P at 29.4 kg·ha-1, banded beneath the seed row. During the growing season, this experimental planting suffered, as did commercial plantings in New Mexico, from high mortality and stunting due to beet curly top virus, a disease transmitted by the beet leafhopper. The results indicate planting date had a significant effect on crop performance. The best stand establishment and highest yield were associated with the earliest planting date, 13 Mar. This date also resulted in the least viral disease damage. Phosphorus fertilizer had no effect on stand establishment or yield. Chemical names used: pentachloronitrobenzene (PCNB); (R)-2-[(2,6-dimethylphenyl)-methoxyacetylamino]-propionic acid methyl ester (mefenoxam).

Comparisons of Soil and Seed Applied Systemic Insecticides to Control Beet Curlv Top Virus in the San Joaquin Valley

Beet curly top virus (BCTV), a gemini virus, remains a problem for farmers in the San Joaquin Valley (SJV) of California. It is spread by the beet leaf hopper (Circulifer tene/lus Baker), which has become naturalized in the state. Recent dependence on sugarbeet cultivars without BCTV resistance has led to increased concern about the potential for a BCTV epidemic. Two trials were carried out in successive years in the western SJV to test the effects of alternative protective insecticides for control of BCTV on susceptible and resistant sugarbeet cultivars. Two rates ofimidicloprid applied as a seed treatment (45 g and 90 g a.i. per 100,000 seeds) were compared to the current standard treatment of phorate applied to soil at 83.8 g a. i. per 1000 m of row, and an untreated control. Natural BCTV infection occurred in both years, but the second trial took place during a major beet leafhopper population increase and infection occurred much earlier in crop development. Sugarbeet root and sugar yields declined linearly with increasing rates of infection (r2 = 0.856). Yields declined because roots were significantly smaller with the non-tolerant cultivar. Sugar percentage was unaffected by insecticide treatments, but differed by cultivar. Imidicloprid and phorate provided similar levels ofprotection to plants, but were not able to prevent large yield losses among susceptible cultivars. Plant resistance provided more protection than systemic

Adaptation from whitefly to leafhopper transmission of an autonomously replicating nanovirus-like DNA component associated with ageratum yellow vein disease.

Ageratum yellow vein disease is caused by the whitefly-transmitted monopartite begomovirus Ageratum yellow vein virus and a DNA beta satellite component. Naturally occurring symptomatic plants also contain an autonomously replicating nanovirus-like DNA 1 component that relies on the begomovirus and DNA beta for systemic spread and whitefly transmission but is not required for maintenance of the disease. Here, we show that systemic movement of DNA 1 occurs in Nicotiana benthamiana when co-inoculated with the bipartite begomovirus Tomato golden mosaic virus and the curtovirus Beet curly top virus (BCTV), but not with the mastrevirus Bean yellow dwarf virus. BCTV also mediates the systemic movement of DNA 1 in sugar beet, and the nanovirus-like component is transmitted between plants by the BCTV leafhopper vector Circulifer tenellus. We also describe a second nanovirus-like component, referred to as DNA 2, that has only 47% nucleotide sequence identity with DNA 1. The diversity and adaptation of nanovirus components are discussed.

Characterization of Spiroplasma citri adhesion related protein SARP1, which contains a domain of a novel family designated sarpin

Transmission of the plant pathogen Spiroplasma citri by its leafhopper vector, Circulifer tenellus, involves adherence to and invasion of insect host cells. The S. citri adhesion related protein P89 (SARP1) was purified by immunoprecipitation using anti-SARP1 monoclonal antibodies. The protein's N-terminal amino acid sequence was determined and used to design a degenerate oligonucleotide. The labeled oligonucleotide hybridized to a 3.5 kb MboI fragment from S. citri DNA, which was then cloned and sequenced. Additionally, a 1.9 kb RsaI fragment of S. citri DNA, partially overlapping the MboI fragment, was isolated and characterized. Sequence analysis of the two clones revealed four open reading frames. ORF1 (675 bp) encodes the C-terminal part of a Soj-like protein. ORFs 1 and 2 were separated from ORFs 3 and 4 by a putative transcription termination site, indicated by a hairpin structure. ORF3 encodes an amphiphilic 798 amino acid long protein with a cleavable signal peptide and a predicted transmembrane helix near the C-terminus. The mature protein of 85.96 kDa has a calculated p I value of 5.5 and has an N-terminal amino acid sequence consistent with that determined from the purified SARP1. At the N-terminus of this protein is a region consisting of six repeats, each 39–42 amino acids, a motif belonging to a previously unrecognized family of repeats found in a variety of bacterial proteins. The taxonomically spotty presence of this ‘sarpin’ domain and the relationship of the repeats to each other suggests a convergent evolution in multiple lineages.

A century of plant virus management in the Salinas Valley of California, ‘East of Eden’

The mild climate of the Salinas Valley, CA lends itself well to a diverse agricultural industry. However, the diversity of weeds, crops and insect and fungal vectors also provide favorable conditions for plant virus disease development. This paper considers the incidence and management of several plant viruses that have caused serious epidemics and been significant in the agricultural development of the Salinas Valley during the 20th century. Beet curly top virus (BCTV) almost destroyed the newly established sugarbeet industry soon after its establishment in the 1870s. A combination of resistant varieties, cultural management of beet crops to provide early plant emergence and development, and a highly coordinated beet leafhopper vector scouting and spray programme have achieved adequate control of BCTV. These programmes were first developed by the USDA and still operate. Lettuce mosaic virus was first recognized as causing a serious disease of lettuce crops in the 1930s. The virus is still a threat but it is controlled by a lettuce-free period in December and a seed certification programme that allows only seed lots with less than one infected seed in 30 000 to be grown. ‘Virus Yellows’ is a term used to describe a complex of yellows inducing viruses which affect mainly sugarbeet and lettuce. These viruses include Beet yellows virus and Beet western yellows virus. During the 1950s, the complex caused significant yield losses to susceptible crops in the Salinas Valley. A beet-free period was introduced and is still used for control. The fungus-borne rhizomania disease of sugarbeet caused by Beet necrotic yellow vein virus was first detected in Salinas Valley in 1983. Assumed to have been introduced from Europe, this virus has now become widespread in California wherever beets are grown and crop losses can be as high as 100%. Movement of infested soil and beets accounts for its spread throughout the beet-growing regions of the United States. Control of rhizomania involves several cultural practices, but the use of resistant varieties is the most effective and is necessary where soils are infested. Rhizomania-resistant varieties are now available that perform almost as well as the non-resistant varieties under non-rhizomania conditions. Another soil-borne disease termed lettuce dieback, caused by a tomato bushy stunt-like tombusvirus, has become economically limiting to romaine and leaf lettuce varieties. The virus has no known vector and it seems to be moved through infested soil and water. Heavy rains in the past 4 years have caused flooding of the Salinas River and lettuce fields along the river have been affected severely by dieback. Studies are now in progress to characterize this new virus and identify sources of resistance. Agriculture in the Salinas Valley continues to grow and diversify, driven by demands for ‘clean’, high quality food by the American public and for export. The major aspects of plant virus control, including crop-free periods, breeding for resistance, elimination of inoculum sources, and vector control will continue to be vital to this expansion. Undoubtedly, the advances in crop production through genetic manipulation and advances in pest management through biological control will eventually become an important part of agricultural improvement.

Searching and oviposition behavior of a mymarid egg parasitoid, Anagrus nigriventris, on five host plant species of its leafhopper host, Circulifer tenellus

Searching and oviposition behavior and parasitization ability of Anagrus nigriventris Girault (Hymenoptera: Mymaridae), an egg parasitoid of beet leafhopper, Circulifer tenellus (Baker) (Homoptera: Cicadellidae), were examined on five host plant species of beet leafhopper: sugar beet ( Beta vulgaris L.), red stem filaree ( Erodium cicutarium [L.]), peppergrass (Lepidium nitidumNuttall), desert plantain ( Plantago ovata Forsskal), and London rocket (Sisymbrium irio L.). Beet leafhopper embeds its eggs in the tissues of these plant species. For each plant species, A. nigriventris behavior was examined on plants with and without beet leafhopper eggs. Experimental design was a 5 (plant species) by 2 (host eggs present/absent) factorial. Additionally within each treatment, parasitoid behavior was observed over a 22-h period at five different observation periods: t D 0, 3, 6, 9, and 22 h wheretD 0 h represents initial exposure of the insect with the plant. The behavioral events observed were: ‘fast walking’ (general searching), ‘slow walking’ (intensive searching), ovipositor probing, grooming, feeding, and resting. Significant differences ( D 0:05) among plant species in time spent on the plant, percentage of host eggs parasitized, and behavioral variables associated with intensive searching and oviposition all indicated that the plant species fell into two groups: ‘preferred’ plants (sugar beet, London rocket, and peppergrass), and ‘unpreferred’ plants (filaree and plantago). These variables also indicated that the parasitoids spent more time on, searched more, probed more, and oviposited more in plants with host eggs than plants without host eggs. Consistent effects of time (over the observation periods from t D 0t ot D 22 h) generally were detected only in the preferred plant species that had host eggs present. In these cases, intensive searching and probing decreased as time advanced, while variables related to general searching (‘fast walking’) and abandoning host egg patches (leaving the plant) tended to increase over time.

Spiroplasma citriSurface Protein P89 Implicated in Adhesion to Cells of the VectorCirculifer tenellus

ABSTRACT Two microtiter plate assays were developed to study the adherence of the plant-pathogenic mollicute Spiroplasma citri to a monolayer of cultured cells of its leafhopper vector, Circulifer tenellus. Adherence was significantly reduced by prior treatment of the spiroplasmas with proteinase K or pronase. Electrophoresis and western blotting of spiroplasma membrane proteins, before and after exposure of intact spiroplasmas to proteases, revealed the concomitant reduction in intensity of a major membrane protein (P89) and a new polypeptide of approximately 46 kDa in protease-treated preparations (P46). Triton X-114 phase partitioning demonstrated that P89 and P46 are amphiphilic, and labeling of the new polypeptide P46 with anti-P89 serum suggested that this molecule may be a breakdown product of P89. Regeneration of P89 after proteinase K treatment of spiroplasmas was directly associated with restoration of the pathogen's attachment capability. Treatment of spiroplasmas with any of several carbohydrates and glycoconjugates or with tetramethyl-urea, a compound that interferes with hydrophobic associations, had a negligible effect on attachment. These results suggest that a spiroplasma surface protein, P89, has a role in S. citri adherence to C. tenellus cells.

Oviposition behavior of Anagrus nigriventris,an egg parasitoid of beet leafhopper, Circulifer tenellus

Oviposition behavior of Anagrus nigriventris,a parasitoid of the eggs of the beetleafhopper,Circulifer tenellus, wasdescribed on sugar beet. Ovipositor probes ofrelatively long duration were accompanied by a periodof abdominal vibrations toward the end of the probe. These periods began as high frequency, low amplitudevibrations in the abdomen, and climaxed as a tremblingof the whole body in an up and down direction.Oviposition by the parasitoid was associated with theoccurrence of abdominal vibrations during a probe andwith probe durations ≥50 s; however thepresence/absence of abdominal vibrations during aprobe was considered a more reliable indicator ofoviposition than was a threshold probe duration of 50s. The majority of probes (63% of all probes) were inempty plant tissue, lasted 15 s, and were notassociated with abdominal vibrations. While probeswithout abdominal vibrations at host egg sites (14%of all probes) lasted an average of 48 s, probes withabdominal vibrations at host egg sites (23% of allprobes) lasted an average of 102 s, and all were ≥50s. It is hypothesized that the relative frequency ofthe 3 types of probes could be affected by the degreeof wasp experience, by the level of host cues on thesubstrate, and by the relative importance of ovipositor probing (vs. antennation) in the hostdetection process. Data from the study suggest thatA. nigriventris is able to discriminate againstpreviously attacked host eggs.

Spiroplasma citri Movement into the Intestines and Salivary Glands of Its Leafhopper Vector, Circulifer tenellus

ABSTRACT Spiroplasma citri, a helical, wall-less prokaryote in the class Molli-cutes, is transmitted by the beet leafhopper, Circulifer tenellus. Invasion of leafhopper tissues and cytopathological effects by S. citri were investigated by transmission electron microscopy. All eight cell types of the principle salivary glands, as well as the adjacent muscle cells and the cells of the accessory salivary glands, were colonized by the spiroplas-mas. In both midgut epithelia and salivary gland cells, spiroplasmas usually occurred in membrane-bound cytoplasmic vesicles that often were located near the cell periphery. In several salivary gland cells, spiroplas-mas were also observed within membranous pockets apparently formed by invagination of the plasmalemma beneath intact basal lamina. These observations are consistent with spiroplasma entry into the insect cells by receptor-mediated endocytosis. Cytopathological effects of spiroplasma infection in salivary cells included loss of membrane and basal lamina integrity, presence in some cells of irregular inclusion-like structures containing dense matrices of filamentous material that labeled with anti S. citri antibodies, and apparent disorganization of the endoplasmic reticulum. Compared to the tightly aligned fiber bundles in healthy muscle cells, bundles in spiroplasma-containing muscle cells appeared fragmented and loosely arranged. Such symptoms could contribute to the reduction in longevity and fecundity that has been previously reported for S. citri-infected C. tenellus.

Systemic Insecticides and Plant Age Affect Beet Curly Top Virus Transmission to Selected Host Plants.

Greenhouse and field studies were conducted to assess the effects of systemic insecticides and plant age on beet curly top virus (BCTV) transmission to sugar beet, tomato, pepper, melon, and cowpea. Sugar beets were the most susceptible to BCTV infection. For all BCTV-susceptible plants tested, younger plants showed greater susceptibility to infection than did older plants, even when inoculations were delayed by only 1 week. Systemic insecticides applied to test plants resulted in increased beet leafhopper (Circulifer tenellus) mortality and decreased the percent BCTV transmission. Soil treatment with imidacloprid (250 g a.i./ha) gave significantly better reductions in BCTV transmission than did dimethoate foliar sprays (280 g a.i./ha). These data suggest that the use of specific systemic insecticides only when crop plants are most susceptible to BCTV infection could be an effective alternative component of the curly top disease control program.

Curly top virus found in perennial shrubs in foothills

Beet curly top geminivirus damages numerous crops, including beans, beets, spinach, peppers, melons and tomatoes. Both the virus and its vector, the beet leafhopper, overwinter in the foothills surrounding the Central Valley. The known host range of the virus is now recognized to include many native and introduced perennial shrubs in the foothills. This is the first reported detection of the virus in plants in the families Ephedraceae, Rhamnaceae and Salicaceae.

Comparative ultrastructure of the salivary glands of two phytopathogen vectors, the beet leafhopper, Circulifer tenellus (Baker), and the corn leafhopper, Dalbulus maidis DeLong and Wolcott (Homoptera : Cicadellidae)

The salivary glands of 2 leafhoppers, Circulifer tenellus and Dalbulus maidis (Homoptera : Cicadellidae) were examined by light and electron microscopy. Centrally located and occupying both the head and thorax, the salivary glands consist of 2 paired parts, the accessory glands and the principal glands. In C. tenellus and D. maidis, the accessory glands are large, multicelled lobes that lie anterior to the principal gland. They join the principal glands near the common salivary duct-gland junction via a thinner tubular duct. The principal glands of both species consist of large binucleate cells that differ in cytology and arrangement. These cells are easily distinguished by unique staining characteristics. Circulifer tenellus salivary gland cells are arranged in 2 lobes, the anterior lobe, made up of 3 concentric rings around the salivary duct and the posterior lobe, arranged in a loose pyramid extending above the foregut. Dalbulus maidis glands are similarly organized around the salivary duct.

Transmission characteristics of Spiroplasma citri and its effect on leafhopper vectors from the Circulifer tenellus complex

Summary.Several leafhopper variants of the Circulifer tenellus complex were collected in “citrus stubborn” affected areas in Israel. Two of these variants transmitted the Spiroplasma citri to Matthiola incana after being injected with the disease agent. The variant from Atriplex halimus was designated Circulifer tenellus‐A (CTA) and the variant from Portulaca oleracea was designated Circulifer tenellus‐? (CTP). Transmission characteristics were determined for both leafhoppers. A high rate of transmission (43.3%) was obtained by single CTA leafhoppers that were injected with the Amiad S. citri isolate from the Upper Galilee, compared with 7% transmission obtained with the CTP leafhoppers. The Gilgal S. citri isolate from the Jordan Valley, was not transmitted by either. Injection was more effective than acquisition access feeding to render the leafhopper infective for both CTA and CTP. The minimum acquisition access period needed for the CTA variant to transmit the Amiad isolate was 1 h. Longer AAPs did not necessarily result in a higher rate of transmission. The minimum incubation period was 6 days and the maximum was 32 days. The LP50 calculated from the logarithmic curve y = 45.74Ln(x)–53.68 was 9.64 days. The minimum inoculation access period (IAP) was lh. The same transmission parameters for the CTP variant could not be determined, as no transmission was obtained even when groups of five‐six insects were placed on a single plant.

Extensive chromosome aberrations in Spiroplasma citri Strain BR3.

Genetic variations in the plant pathogen, Spiroplasma citri strain BR3, were characterized through physical genome mapping of the original isolate, BR3-3X, and two derivatives, BR3-T and BR3-G, obtained after several years of different maintenance conditions. BR3-T was transmitted from plant to plant via its natural insect vector, the leafhopper Circulifer tenellus, while BR3-G was maintained only in plants by periodic grafting and has lost its ability to be insect transmitted. By pulsed field gel electrophoresis (PFGE) analysis and DNA hybridization, extensive changes in chromosomal DNA restriction patterns relative to the parent, BR3-3X, were observed in both BR3-T and BR3-G, each of which also had a larger genome size than the parent line. Genetic organization was relatively conserved between BR3-T and BR3-3X. In contrast, a large chromosomal inversion and deletions of approximately 10 kb near each of the inversion borders were observed in BR3-G. One of the deletions, which included several possibly functional genes, was closely linked to a SpV1-related transposase gene. The locations of the deletion borders were also determined. The results of this study demonstrated remarkable genome instability of spiroplasmas.

Olfactory response of Anagrus, nigriventrls (HYM.: Mymaridae): Effects of host plant chemical cues mediated by rearing and oviposition experience

The egg parasitoid Anagrus nigriventris Girault is an important natural enemy of the beet leafhopper, Circulifer tenellus (Baker) which has a broad host range that includes cultivated sugar beets and the weed, Russian thistle. When parasitoids were reared on leafhopper eggs deposited in sugar beets, females were attracted to sugar beet volatiles and preferred them over a blank control and over Russian thistle volatiles in Y- tube olfactometer tests. No preference was detected for either plant by female wasps that were reared on Russian thistle. Wasps reared on Russian thistle and allowed an oviposition experience on sugar beet showed a strong attraction to sugar beet volatiles; however, wasps reared on sugar beets and allowed oviposition experience on Russian thistle demonstrated no preference between the two plants. The implications of parasitoid response towards plant volatiles and their importance in biological control are discussed.

Molecular characterization of Spiroplasma citri BR3 lines that differ in transmissibility by the leafhopper Circulifer tenellus

Four lines of Spiroplasma citri strain BR3, derived from different maintenance conditions over several years and differing in their ability to be transmitted by the beet leafhopper Circulifer tenellus were characterized. The lines included BR3-T (transmissible), maintained in turnip by leafhopper transmission; BR3-G (now nontransmissible), maintained in plants by periodic graft transmission; BR3-P (now rarely transmissible), subcultured in artificial medium over 130 times; and BR3-M (transmissible), subcultured 43 times. Although all four lines had similar overall protein profiles, the two transmissible lines each contained two proteins missing in the non- or rarely transmitted lines. In addition, one protein was unique to BR3-M and another was unique to BR3-P. Spiralin, a major S. citri membrane protein, had dual mobility in line BR3-G only. Patterns of extrachromosomal DNA and restricted total DNA also were similar, although differences occurred among the four lines. The genome of line BR3-G was larger than those of the other lines and unique restriction bands occurred in this line. Protein and DNA profiles of six to eight individual clones of each line also were compared. Protein patterns within each clone were indistinguishable except for a difference in the migration rate of spiralin in clones of BR3-G. Restricted total DNA showed differential patterns among clones of each line, possibly reflecting differences in extrachromosomal DNA. Molecular differences among the spiroplasma lines may reflect the selection pressures of the different environments in which they were maintained and suggest genes and proteins that may be involved in the biological phenotypes of these lines.Key words: spiroplasma, Mollicutes, insect transmission.