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

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.     

Detection and Molecular Characterization of Phytoplasma associated with Lethal Yellowing Disease of Coconut Palms in Cuba

Lethal Yellowing (LY) disease of coconut palm (Cocos nucifera L.) in Cuba has been reported since the end of the 19th century. In order to ascertain the presence of phytoplasmas associated with this disease, leaf samples were taken from plants showing typical disease symptoms and assayed for the LY agent by the polymerase chain reaction (PCR) using LY‐specific primers. Selected PCR amplification products were cloned, sequenced and compared to that of a Mexican LY isolate from the Yucatán region. The results obtained confirm the presence of LY phytoplasma in Cuba. Cuban and Mexican isolates show an overall high degree of sequence similarity with occasional point mutations and small deletions or insertions. Based on these identified genetic differences, LY isolates from the Havana and the Yucatán region cluster together and apart from isolates originating at Maisí in eastern Cuba.     

Comparative genomics identifies genes shared by distantly related insect-transmitted plant pathogenic mollicutes

Phytoplasmas and spiroplasmas are distantly related insect-transmitted plant pathogens within the class Mollicutes. Genome sequencing projects of phytoplasma strain Aster Yellows-Witches' Broom (AY-WB) and Spiroplasma kunkelii are near completion. Complete genome sequences of seven obligate animal and human pathogenic mollicutes (Mycoplasma and Ureaplasma spp.), and OY phytoplasma have been reported. Putative ORFs predicted from the genome sequences of AY-WB and S. kunkelii were compared to those of the completed genomes. This resulted in identification of at least three ORFs present in AY-WB, OY and S. kunkelii but not in the obligate animal and human pathogenic mollicutes. Moreover, we identified ORFs that seemed more closely related between AY-WB and S. kunkelii than to their mycoplasma counterparts. Phylogenetic analyses using parsimony were employed to study the origin of these genes, resulting in identification of one gene that may have undergone horizontal gene transfer. The possible involvement of these genes in plant pathogenicity is discussed.     

Independent origins of resistance or susceptibility of parasitic wasps to a defensive symbiont

Insect microbe associations are diverse, widespread, and influential. Among the fitness effects of microbes on their hosts, defense against natural enemies is increasingly recognized as ubiquitous, particularly among those associations involving heritable, yet facultative, bacteria. Protective mutualisms generate complex ecological and coevolutionary dynamics that are only beginning to be elucidated. These depend in part on the degree to which symbiont‐mediated protection exhibits specificity to one or more members of the natural enemy community. Recent findings in a well‐studied defensive mutualism system (i.e., aphids, bacteria, parasitoid wasps) reveal repeated instances of evolution of susceptibility or resistance to defensive bacteria by parasitoids. This study searched for similar patterns in an emerging model system for defensive mutualisms: the interaction of Drosophila, bacteria in the genus Spiroplasma, and wasps that parasitize larval stages of Drosophila. Previous work indicated that three divergent species of parasitic wasps are strongly inhibited by the presence of Spiroplasma in three divergent species of Drosophila, including D. melanogaster. The results of this study uncovered two additional wasp species that are susceptible to Spiroplasma and two that are unaffected by Spiroplasma, implying at least two instances of loss or gain of susceptibility to Spiroplasma among larval parasitoids of Drosophila.     

Shoot Proliferation and Leaf Malformation of Celosia argentea and Celosia spicata Caused by a Phytoplasma of the 16SrIII‐J Group

Ornamental plants of Celosia argentea L. and Celosia spicata L. displaying typical phytoplasma‐induced symptoms were observed in Piracicaba, State of São Paulo, south‐eastern Brazil. Our aim was to identify the possible phytoplasma involved. PCR revealed the association of phytoplasma with diseased plants of both species. Based on actual and virtual RFLP analysis and phylogenetic analysis, the phytoplasma was characterized as a member of the 16SrIII‐J subgroup. Transmission of the pathogen by dodder supported the evidence that the symptoms observed in naturally diseased plants were induced by a phytoplasma. Our results show that C. spicata is a new host for phytoplasma and that this is the first report of a 16SrXIII‐J phytoplasma infecting plants of C. argentea and C. spicata in Brazil.     

Infection and replication sites of Spiroplasma kunkelii (Class: Mollicutes) in midgut and Malpighian tubules of the leafhopper Dalbulus maidis

Spiroplasma kunkelii distribution and infection mechanisms in the intestines and Malpighian tubules of Dalbulus maidis were investigated by transmission electron microscopy. Spiroplasmas were found between microvilli and in endocytic vesicles of the midgut epithelium. At the basal part, cytoplasmic vesicles contained multiple spiroplasmas with tube-like extensions and spiroplasmas accumulated between the laminae rara and densa of the basal lamina. Tip structures of flask-shaped spiroplasmas pierced the lamina densa that was discontinuous in close proximity to spiroplasmas. Spiroplasmas were found in hemolymph, crossed the basal lamina of Malpighian tubule epithelium and accumulated at high numbers in muscle cells that had cytopathogenic changes. S. kunkelii had perithrochous approximately 8nm diameter structures determined to be fimbriae protruding from the cell surface, and similar structures were adhering to the basal lamina of midgut epithelium and to external lamina of muscle cells. Further, spiroplasmas had pili-like appendages at one or both cell poles and appeared to conjugate. This is the first time that fimbriae and pili have been observed in a mollicutes.     

Detection of contaminants in cell cultures,sera and trypsin

The aim of this study was standardization and application of polymerase chain reaction (PCR) for the detection of contaminants in cell cultures, sera and trypsin. Five PCR protocols were standardized to assess the presence of genetic material from mycoplasma, porcine circovirus 1 (PCV1), bovine leukemia virus (BLV) or bovine viral diarrhea virus (BVDV) in cell culture samples. PCR reactions for the genes GAPDH and beta-actin were used to evaluate the efficiency of nucleic acid extraction. The PCR protocols were applied to 88 cell culture samples from eight laboratories. The tests were also used to assess potential contamination in 10 trypsin samples and 13 fetal calf serum samples from different lots from five of the laboratories. The results showed the occurrence of the following as DNA cell culture contaminants: 34.1% for mycoplasma, 35.2% for PCV1, 23.9% for BVDV RNA and 2.3% for BLV. In fetal calf sera and trypsin samples BVDV RNA and PCV1 DNA was detected. The results demonstrated that cell culture, sera and trypsin used by different laboratories show a high rate of contaminants. The results highlight the need for monitoring cell cultures and controlling for biological contaminants in laboratories and cell banks working with these materials.     

I-SceI-mediated plasmid deletion and intra-molecular recombination in Spiroplasma citri

S. citri wild-type strain GII3 carries six plasmids (pSci1 to -6) that are thought to encode determinants involved in the transmission of the spiroplasma by its leafhopper vector. In this study we report the use of meganuclease I-SceI for plasmid deletion in S. citri. Plasmids pSci1NT-I and pSci6PT-I, pSci1 and pSci6 derivatives that contain the tetM selection marker and a unique I-SceI recognition site were first introduced into S. citri strains 44 (having no plasmid) and GII3 (carrying pSci1-6), respectively. Due to incompatibility of homologous replication regions, propagation of the S. citri GII3 transformant in selective medium resulted in the replacement of the natural pSci6 by pSci6PT-I. The spiroplasmal transformants were further transformed by an oriC plasmid carrying the I-SceI gene under the control of the spiralin gene promoter. In the S. citri 44 transformant, expression of I-SceI resulted in rapid loss of pSciNT-I showing that expression of I-SceI can be used as a counter-selection tool in spiroplasmas. In the case of the S. citri GII3 transformant carrying pSci6PT-I, expression of I-SceI resulted in the deletion of plasmid fragments comprising the I-SceI site and the tetM marker. Delineating the I-SceI generated deletions proved they had occurred though recombination between homologous sequences. To our knowledge this is the first report of I-SceI mediated intra-molecular recombination in mollicutes.     

Genomic variations of Mycoplasma capricolum subsp. capripneumoniae detected by amplified fragment length polymorphism (AFLP) analysis

The genetic diversity of Mycoplasma capricolum subsp. capripneumoniae strains based on determination of amplified fragment length polymorphisms (AFLP) is described. AFLP fingerprints of 38 strains derived from different countries in Africa and the Middle East consisted of over 100 bands in the size range of 40-500 bp. The similarity between individual AFLP profiles, calculated by Jaccard's coefficient, ranged from 0.92 to 1.0. On the basis of the polymorphisms detected, the analysed strains can explicitly be grouped into two major clusters, equivalent to two evolutionary lines of the organism found by 16S rDNA analysis. The present data support previous observations regarding genetic homogeneity of M. capricolum subsp. capripneumoniae, and confirm the two evolutionary lines of descent found by analysis of 16S rRNA genes.