rRNA operons and genome size of 'Candidatus Liberibacter americanus', a bacterium associated with citrus huanglongbing in Brazil.

Huanglongbing is one of the most severe diseases of citrus worldwide and is associated with 'Candidatus (Ca.) Liberibacter africanus' in Africa, 'Ca. Liberibacter asiaticus' in Asia and the Americas (Brazil, USA and Cuba) and 'Ca. Liberibacter americanus' (Lam) in Brazil. In the absence of axenic cultures, genetic information on liberibacters is scarce. The sequences of the entire 23S rRNA and 5S rRNA genes from Lam have now been obtained, using a consensus primer designed on known tRNAMet sequences of rhizobia. The size of the Lam genome was determined by PFGE, using Lam-infected periwinkle plants for bacterial enrichment, and was found to be close to 1.31 Mbp. In order to determine the number of ribosomal operons on the Lam genome, probes designed to detect the 16S rRNA gene and the 3' end of the 23S rRNA gene were developed and used for Southern hybridization with I-CeuI-treated genomic DNA. Our results suggest that there are three ribosomal operons in a circular genome. Lam is the first liberibacter species for which such data are available.

Liberibacters Associated with Citrus Huanglongbing in Brazil: 'Candidatus Liberibacter asiaticus' Is Heat Tolerant, 'Ca. L. americanus' Is Heat Sensitive.

In São Paulo State, Brazil, 'Candidatus Liberibacter americanus' and 'Candidatus Liberibacter asiaticus' are associated with huanglongbing (HLB). Affected municipalities occur mainly in the central and southern regions, where the annual number of hours above 30°C is two to five times lower than that in the extreme northern and western regions. The influence of temperature on sweet orange trees infected with 'Ca. L. asiaticus' or 'Ca. L. americanus' was studied in temperature-controlled growth chambers. Symptom progression on new shoots of naturally infected and experimentally graft-inoculated symptomatic sweet orange trees was assessed. Mottled leaves developed on all infected trees at 22 to 24°C, but not on any 'Ca. L. americanus'-infected trees at 27 to 32°C. Quantitative, real time-PCR was used to determine the liberibacter titers in the trees. After 90 days, 'Ca. L. asiaticus'-infected trees had high titers at 32 and 35°C, but not at 38°C, while 'Ca. L. americanus'-infected trees had high titers at 24°C, but at 32°C the titers were very low or the liberibacters could not be detected. Thus, the multiplication of 'Ca. L. asiaticus' is not yet affected at 35°C, while a temperature of 32°C is detrimental to 'Ca. L. americanus'. Thus, 'Ca. L. americanus' is less heat tolerant than 'Ca. L. asiaticus'. The uneven distribution of these two liberibacters in São Paulo State might be in relation with these results.

A phytoplasma closely related to the pigeon pea witches'-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of São Paulo, Brazil.

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of São Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and 'Candidatus Liberibacter asiaticus'. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.

The tufB-secE-nusG-rplKAJL-rpoB gene cluster of the liberibacters: sequence comparisons, phylogeny and speciation.

The rplKAJL-rpoBC operon or beta operon is a classic bacterial gene cluster, which codes for proteins K, A, J and L of the large ribosomal subunit, as well as proteins B (beta subunit) and C (beta' subunit) of RNA polymerase. In the early 1990s, the operon was obtained as a 2.6 kbp DNA fragment (In-2.6) by random cloning of DNA from periwinkle plants infected with the Poona (India) strain of the huanglongbing agent, later named 'Candidatus (Ca.) Liberibacter asiaticus'. DNA from periwinkle plants infected with the Nelspruit strain (South Africa) of 'Ca. L. africanus' was amplified with a primer pair designed from In-2.6 and yielded, after cloning and sequencing, a 1.7 kbp DNA fragment (AS-1.7) of the beta operon of 'Ca. L. africanus'. The beta operon of the American liberibacter, as well as the three upstream genes (tufB, secE, nusG), have now also been obtained by the technique of chromosome walking and extend over 4673 bp, comprising the following genes: tufB, secE, nusG, rplK, rplA, rplJ, rplL and rpoB. The sequence of the beta operon was also determined for a Brazilian strain of 'Ca. L. asiaticus', from nusG to rpoB (3025 bp), and was found to share 99 % identity with the corresponding beta operon sequences of an Indian and a Japanese strain. Finally, the beta operon sequence of 'Ca. L. africanus' was extended from 1673 bp (rplA to rpoB) to 3013 bp (nusG to rpoB), making it possible to compare the beta operon sequences of the African, Asian and American liberibacters over a length of approximately 3000 bp, from nusG to rpoB. While 'Ca. L. africanus' and 'Ca. L. asiaticus' shared 81.2 % sequence identity, the percentage for 'Ca. L. americanus' and 'Ca. L. africanus' was only 72.2 %, and identity for 'Ca. L. americanus' and 'Ca. L. asiaticus' was only 71.4 %. The approximately 3000 bp nusG-rpoB sequence was also used to construct a phylogenetic tree, and this tree was found to be identical to the known 16S rRNA gene sequence-based tree. These results confirm earlier findings that 'Ca. L. americanus' is a distinct liberibacter, more distantly related to 'Ca. L. africanus' and 'Ca. L. asiaticus' than 'Ca. L. africanus' is to 'Ca. L. asiaticus'. The dates of speciation have also been estimated.

An Experimental Inoculation System to Study Citrus-Xylella fastidiosa Interactions.

Difficulties in reproducing the citrus variegated chlorosis (CVC) disease symptoms in experimental plants have delayed implementation of studies to better understand the essential aspects of this important disease. In an extensive study, cultivars of sweet orange (Citrus sinensis) were inoculated with Xylella fastidiosa using procedures that included root immersion, and stem absorption, pricking, or infiltration of the inoculum into plants of different ages. Inoculum consisted of 5-day-old cultures or cell suspensions of CVC strain 9a5c diluted in phosphate-buffered saline. Inoculated plants and controls were grown, or transferred just after inoculation, to 5-liter pots or 72-cell foam trays. Approximately 4, 5, 9, and 12 months after inoculation, leaves were collected and processed for polymerase chain reaction analysis or X. fastidiosa isolation on BCYE agar medium. Root immersion and stem inoculation of 4- and 6-month-old plants resulted in low percentages of symptomatic (0 to 7%) and plants positive by isolation (0 to 9%). Pinpricked or injected stems of 1-month-old seedlings resulted in high percentages of plants symptomatic (29 and 90% in Pera Rio, 75, 59, and 83% in Valencia, and 77% in Natal) or positive by isolation (26 and 93% in Pera Rio, 98, 96, and 83% in Valencia, and 77% in Natal). In foam trays, the seedlings grew less, the incubation period was shorter, and disease severity was higher than in pots. This system allows testing of higher numbers of plants in a reduced space with a more precise reproduction of the experimental conditions.

Sudden Death of Citrus in Brazil: A Graft-Transmissible Bud Union Disease.

Citrus Sudden Death (CSD), a new, graft-transmissible disease of sweet orange and mandarin trees grafted on Rangpur lime rootstock, was first seen in 1999 in Brazil, where it is present in the southern Triângulo Mineiro and northwestern São Paulo State. The disease is a serious threat to the citrus industry, as 85% of 200 million sweet orange trees in the State of São Paulo are grafted on Rangpur lime. After showing general decline symptoms, affected trees suddenly collapse and die, in a manner similar to trees grafted on sour orange rootstock when affected by tristeza decline caused by infection with Citrus tristeza virus (CTV). In tristeza-affected trees, the sour orange bark near the bud union undergoes profound anatomical changes. Light and electron microscopic studies showed very similar changes in the Rangpur lime bark below the bud union of CSD-affected trees: size reduction of phloem cells, collapse and necrosis of sieve tubes, overproduction and degradation of phloem, accumulation of nonfunctioning phloem (NFP), and invasion of the cortex by old NFP. In both diseases, the sweet orange bark near the bud union was also affected by necrosis of sieve tubes, and the phloem parenchyma contained characteristic "chromatic" cells. In CSD-affected trees, these cells were seen not only in the sweet orange phloem, but also in the Rangpur lime phloem. Recent observations indicated that CSD affected not only citrus trees grafted on Rangpur lime but also those on Volkamer lemon, with anatomical symptoms similar to those seen in Rangpur lime bark. Trees on alternative rootstocks, such as Cleopatra mandarin and Swingle citrumelo, showed no symptoms of CSD. CSD-affected trees did recover when they were inarched with seedlings of these rootstocks, but not when inarched with Rangpur lime seedlings. These results indicate that CSD is a bud union disease. In addition, the bark of inarched Rangpur lime and Volkamer lemon seedlings showed, near the approach-graft union, the same anatomical alterations as the bud union bark from the Rangpur lime rootstock in CSD-affected trees. The dsRNA patterns from CSD-affected trees and unaffected trees were similar and indicative of CTV. CSD-affected trees did not react by immunoprinting-ELISA using monoclonal antibodies against 11 viruses. No evidence supported the involvement of viroids in CSD. The potential involvement of CTV and other viruses in CSD is discussed.

Citrus and Coffee Strains of Xylella fastidiosa Induce Pierce's Disease in Grapevine.

Xylella fastidiosa causes citrus variegated chlorosis (CVC) disease in Brazil and Pierce's disease of grapevines in the United States. Both of these diseases cause significant production problems in the respective industries. The recent establishment of the glassy-winged sharpshooter in California has radically increased the threat posed by Pierce's disease to California viticulture. Populations of this insect reach very high levels in citrus groves in California and move from the orchards into the vineyards, where they acquire inoculum and spread Pierce's disease in the vineyards. Here we show that strains of X. fastidiosa isolated from diseased citrus and coffee in Brazil can incite symptoms of Pierce's disease after mechanical inoculation into seven commercial Vitis vinifera varieties grown in Brazil and California. Thus, any future introduction of the CVC strains of X. fastidiosa into the United States would pose a threat to both the sweet orange and grapevine industries. Previous work has clearly shown that the strains of X. fastidiosa isolated from Pierce's disease- and CVC-affected plants are the most distantly related of all strains in the diverse taxon X. fastidiosa. The ability of citrus strains of X. fastidiosa to incite disease in grapevine is therefore surprising and creates an experimental system with which to dissect mechanisms used by X. fastidiosa in plant colonization and disease development using the full genome sequence data that has recently become available for both the citrus and grapevine strains of this pathogen.

Coffee Leaf Scorch Caused by a Strain of Xylella fastidiosa from Citrus.

Citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS) are two economically important diseases in Brazil caused by the bacterium Xylella fastidiosa. Strains of the bacterium isolated from the two plant hosts are very closely related, and the two diseases share sharpshooter insect vectors. In order to determine if citrus strains of X. fastidiosa could infect coffee and induce CLS disease, plant inoculations were performed. Plants of coffee, Coffea arabica 'Mundo Novo', grafted on Coffea canephora var. robusta 'Apuatão 2258' were mechanically inoculated with triply cloned strains of X. fastidiosa isolated from diseased coffee and citrus. Three months postinoculation, 5 of the 10 plants inoculated with CLS-X. fastidiosa and 1 of the 10 plants inoculated with CVC-X. fastidiosa gave positive enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR). Eight months postinoculation, another six plants inoculated with CVC-X. fastidiosa gave positive PCR results. The two X. fastidiosa strains were isolated from the inoculated plants and showed the same characteristics as the original clones by microscopy, ELISA, and PCR. None of the plants inoculated with sterile periwinkle wilt (PW) medium as controls gave positive reactions in diagnostic tests, and none developed disease symptoms. Six months postinoculation, seven plants inoculated with CLS-X. fastidiosa and eight inoculated with CVC-X. fastidiosa began to develop characteristic CLS symptoms, including apical and marginal leaf scorch, defoliation, and reductions of internode length, leaf size, and plant height, terminal clusters of small chlorotic and deformed leaves, and lateral shoot dieback. We have demonstrated that X. fastidiosa from citrus plants is pathogenic for coffee plants. This has important consequences for the management of CLS disease and has implications for the origin of citrus variegated chlorosis disease.

Catharanthus roseus, an Experimental Host Plant for the Citrus Strain of Xylella fastidiosa.

We verified by pathogenicity tests that the herbaceous plant Catharanthus roseus (Madagascar periwinkle) can be used as an experimental host for the strain of Xylella fastidiosa that causes citrus variegated chlorosis (CVC). Plants were mechanically inoculated with CVC strain 9a5c, the genome of which was recently sequenced. Plants were inoculated with the virulent 8th passage (9a5c-8) and the 51st passage (9a5c-51). Leaf deformation and stunting were seen 2 months after inoculation on 18 of 21 plants with 9a5c-8 and 8 of 21 plants with 9a5c-51. The plants were infected with X. fastidiosa as shown by polymerase chain reaction. The bacterium could be reisolated from all plants tested, showing that CVC-X. fastidiosa multiplied and moved systemically in C. roseus plants causing dysfunction in plant growth. The disease symptoms evolved within 4 months post-inoculation to a severe leaf chlorosis in all inoculated plants. The localization of X. fastidiosa in the xylem was verified by immunofluorescence. Genes coding for proteins with homologies to plant sterol-C-methyltransferase, a transketolase-like protein, subunit III of photosystem I, and a desiccation protectant protein were found to be differentially expressed in symptomatic C. roseus plants as a response to infection with X. fastidiosa in comparison to healthy plants. A tentative correlation between the pattern of expression of these C. roseus genes with the mechanism of pathogenicity of X. fastidiosa is discussed.

Distribution of Xylella fastidiosa in Citrus Rootstocks and Transmission of Citrus Variegated Chlorosis Between Sweet Orange Plants Through Natural Root Grafts.

To study translocation of Xylella fastidiosa to citrus rootstocks, budsticks from citrus variegated chlorosis (CVC)-affected cv. Pera sweet orange (Citrus sinenesis (L.) Osb.) were top grafted on 15 citrus rootstocks. Disease symptoms were conspicuous 3 months later on all 15 rootstocks tested. The presence of X. fastidiosa was confirmed by light microscopy, double-antibody sandwich enzyme-linked immunosorbent assays, and polymerase chain reaction in rootlets and main roots of CVC-symptomatic Pera sweet orange in 11 of the 15 rootstocks tested. These results suggest that bacterial translocation from the aerial plant parts to the root system occurs but is not essential for X. fastidiosa to induce symptoms in the aerial parts. Bacterial translocation to the roots was not correlated with CVC leaf-symptom severity in the Pera scion. To determine if CVC disease could be transmitted by natural root grafts, two matched seedlings of each of four sweet orange cultivars (Pera, Natal, Valencia, and Caipira) were transplanted into single pots. One seedling rootstock of each pair was inoculated by top grafting with a CVC-contaminated budstick while the other seedling rootstock was cut but not graft inoculated. Transmission of X. fastidiosa from an inoculated plant to a noninoculated plant sharing the same pot was observed in all four sweet orange cultivars tested. Transmission was confirmed by observation of natural roots grafts between the two plants, presence of X. fastidiosa in the root grafts, and disease development in the uninoculated plants. This is the first report of transmission of CVC disease through natural root grafts.

Vestibular stimulation and eye contact in autistic children.

Five autistic boys ages 5-1 to 5-10 were studied to determine whether stimulation of the macular receptors of the inner ear through linear motion influences the boys' eye contact with the investigators. The duration of eye contact was measured during linear motion on a motor-driven oscillator and on two hand-operated swings and compared to the duration of eye contact when the macular receptors were not stimulated. Because the boys varied in cooperation, the tests could not be performed using the same apparatus with each child; therefore results were analyzed separately for each child. Four of the five boys showed longer eye contact while on the motor driven oscillator (p less than .0005), and two of these also showed longer contact when on a manually operated swing (p less than .025). The fifth child resisted the use of the oscillator and did not show longer eye contact while on it (p greater than .05), but did so when on two different swings (p less than .005).

Crossing the body midline in learning-disabled and normal children.

The SVCU scores of 179 learning-disabled children, ages 5 to 8, were compared with the scores of 120 normal children, using both the actual Space Visualization Contralateral Use (SVCU) score, and the SVCU category of "normal, suspect, or possible deficit," in order to evaluate whether using guidelines to interpret the SVCU as a function of age enabled differentiation between normal children and children with learning disabilities. The SVCU measure clearly differentiated between groups at the younger ages, although the difference was not as clear for the older age groups. It was suggested that the SVCU score be used in conjunction with other observations of midline crossing. Alternative interpretations of the SVCU score are discussed.

Possible pubertal effect on therapeutic gains in an autistic girl.

A deaf, partially sighted, severely retarded autistic girl, 11 years, 6 months of age, received approximately 2 years of occupational therapy, where sensory integration procedures were employed to reduce self-stimulation. Videotaped time samples of the amount of stereotypies showed a consistent reduction from the time of starting therapy to an interruption for vacation and surgery for scoliosis 46 weeks later. On returning to therapy after a 9-week break, self-stimulation had greatly increased and did not return to the presurgery level during an additional 55 weeks of therapy, 30 of which followed the removal of a cast. Menarche occurred 1 week after removing the cast. Increased self-stimulation is linked to reduced inclination toward environmental interaction as well as to an interruption of intervention and possible pubertal effect. Brief reports on four other autistic adolescents who received similar therapy are consistent with the conjecture of frequent pubertal regression.

Influence of sensory integration procedures on language development.

The relationship between language development and sensory integration was explored through single case experimental studies of one female and three male aphasic children ranging in age from 4 years, 0 months to 5 years, 3 months. Other agencies had assessed all the children in the area of language development at least 6 months before the start of occupational therapy. Three of the four children had received either speech therapy, special education specific to aphasia, or both, before starting occupational therapy. Additional baseline data on language expression and comprehension, as well as on sensory integrative functioning, were gathered before beginning a year of occupational therapy that involved sensory integration procedures. Inspection of rate of language growth before and after starting occupational therapy showed a consistent increase in rate of growth in language comprehensive concomitant with occupational therapy compared to previous growth rate. The two children with depressed postrotary nystagmus demonstrated notable gains on expressive language measures.

Hyper-responsivity to touch and vestibular stimuli as a predictor of positive response to sensory integration procedures by autistic children.

Sensory processing disturbance in autistic children as a predictor of response to sensory integrative procedures was investigated. Ten autistic children, ages 3-1/2 to 13 years (mean, 7.4 years), were initially evaluated in regard to their hypo-, hyper-, or normal responsivity to visual, auditory, tactile, vestibular, proprioceptive, olfactory, and gustatory stimuli. After evaluation, each child received therapy that provided somatosensory and vestibular stimulation and elicited adaptive responses to these stimuli. At the end of one year of therapy, each child's progress was judged in relationship to that of the others, and the group was divided into the six best and the four poorest respondents. Stepwise discriminant analysis identified which initial test variables predicted good or poor responses to therapy. The good respondents showed tactile defensiveness, avoidance of movement, gravitational insecurity, and an orienting response to an air puff. Results suggest that children who registered sensory input but failed to modulate it responded better to therapy than those who were hypo-responsive or failed to orient to sensory input.

Dichotic listening performance in learning-disabled children.

A dichotic listening test was administered to 114 learning-disabled children aged 6 to 10 years and the ratio of correct right ear responses to correct left ear responses was computed. Three subgroups were formed: those with a low right-left ear ratio, those with an average ratio, and those with a high ratio. The groups were given a battery of sensory integrative, postural-ocular, auditory-language, and academic tests. Mean scores were computed for each group. All three groups were considerably lower in their overall accuracy of response to the dichotic listening test than was a normative sample. The groups with low right-left ear ratios were less likely to have a somatosensory disorder than the other two groups, but they were more apt to have a language problem. There was a much larger percentage of children with very high right-left ratios (right ear very "superior" to left ear) than would be expected in a random population.

Cluster analyses of measures of sensory integration.

Cluster analyses of intercorrelation of scores on tests of sensory integrative, language, auditory, intellectual, and academic tests administered to young learning-disabled school children demonstrated a close relationship between academic achievement, the right ear score on dichotic listening, language expression, and auditory memory. Visual perception tests and the Auditory Association sub-test of the Illinois Test of Psycholinguistic Abilities showed a close association with IQ. Others associations were consistent with the view that when the two cerebral hemispheres do not corroborate, both hemispheres tend to develop similar motor and language functions. Analyses of the intercorrelation of scores on the sensory integration, auditory, and language tests resulted in four major factors: auditory-language functions, postural-ocular reactions, eye-hand coordination, and somatosensory and motor planning or praxis. A fifth factor, visual perception, contributed less to the final factor structure.

Effect of sensory integrative therapy on the coordination of children with choreoathetoid movements.

Learning-disabled children with deficits in sensory integration as well as choreoathetosis and who received sensory integrative therapy gained more on an eye-hand coordination test than did control children. Using a one-tailed test, levels of significance difference between the mean gain scores of the experimental and control groups were .058 for the more accurate hand and .061 for the less accurate hand. It is not possible to determine whether the therapeutic effect was on the motor or on the sensory integrative aspect of coordination or on both.