Transmission of Xylella fastidiosa to Grapevine by the Meadow Spittlebug.

There is little information available on Xylella fastidiosa transmission by spittlebugs (Hemiptera, Cercopoidea). This group of insect vectors may be of epidemiological relevance in certain diseases, so it is important to better understand the basic parameters of X. fastidiosa transmission by spittlebugs. We used grapevines as a host plant and the aphrophorid Philaenus spumarius as a vector to estimate the effect of plant access time on X. fastidiosa transmission to plants; in addition, bacterial population estimates in the heads of vectors were determined and correlated with plant infection status. Results show that transmission efficiency of X. fastidiosa by P. spumarius increased with plant access time, similarly to insect vectors in another family (Hemiptera, Cicadellidae). Furthermore, a positive correlation between pathogen populations in P. spumarius and transmission to plants was observed. Bacterial populations in insects were one to two orders of magnitude lower than those observed in leafhopper vectors, and population size peaked within 3 days of plant access period. These results suggest that P. spumarius has either a limited number of sites in the foregut that may be colonized, or that fluid dynamics in the mouthparts of these insects is different from that in leafhoppers. Altogether our results indicate that X. fastidiosa transmission by spittlebugs is similar to that by leafhoppers. In addition, the relationship between cell numbers in vectors and plant infection may have under-appreciated consequences to pathogen spread.

Pierce's disease of grapevines: isolation of the causal bacterium.

A Gram-negative, rod-shaped bacterium has been consistently isolated from grapevines with Pierce's disease. Grapevines inoculated with the bacterium developed Pierce's disease, and the bacterium was reisolated from the plants. The bacterium was serologically and ultrastructurallv indistinguishable from the one in naturally infected plants, and also indistinguishable from a bacterium isolated from almonds with almond leaf scorch disease.

Pierce's Disease Bacterium: Mechanism of Transmission by Leafhopper Vectors.

The bacterium that causes Pierce's disease of grapevines is isolated most consistently from the foregut of its leafhopper vector Graphocephala atropunctata. As seen in light and scanning electron microscopy of infective leafhoppers, the bacteria are attached to the cibarial pump and the lining of the esophagus in the foregut where they appear to multiply. These findings suggest that the bacterium is transmitted from the foregut by egestion during feeding by infective leafloppers.

Cloning and Detection of DNA from a Nonculturable Plant Pathogenic Mycoplasma-like Organism.

The ability to detect, quantify, and differentiate nonculturable mycoplasma-like organisms (MLOs) would greatly facilitate epidemiological and taxonomical studies of this unique group of plant and insect pathogens. DNA isolated from extracts of insects infected with the Western X-disease MLO was cloned in Escherichia coli. X-disease-specific clones, when labeled and used as probes, readily detected X-disease MLOs in infected plants and insects but did not hybridize with DNA from healthy plants or insects, or from several other plant pathogenic MLOs or spiroplasmas. These methods provide both a sensitive diagnostic tool and a basis for genetically differentiating MLOs.

Fastidious xylem-limited bacterial plant pathogens.

Numerous bacteria have been isolated from within plants, and many reported from xylem, but only three species of xylem-limited bacteria (XLB) that are fastidious in cultural requirements, are plant pathogens, and exclusively occupy xylem, have been well characterized. Two XLB, Xylella fastidiosa and Pseudomonas syzygii, are transmitted by sucking insects that feed on xylem sap but are not transmitted mechanically from plant to plant. In contrast, Clavibacter xyli is mechanically transmitted to plants by cutting tools. All of these XLB occupy a highly specialized yet diverse ecological niche: the water-conducting systems of an extremely wide range of plant hosts. A variety of detection methods are available as diagnostic aids; each method has advantages and disadvantages; no single method is best for all uses. Molecular and genetic comparisons of strains of XLB lag behind progress being made for many other plant-pathogenic bacteria, but such studies are needed to answer important questions: (a) How do XLB move from cell to cell within plants? (b) What are the physiological and genetic bases of plant host specificity for XLB? (c) Why are only xylem-feeding specialists vectors of X. fastidiosa (and probably P. syzygii), when many leafhoppers feed regularly (but not continuously) on xylem?

The Fate of Xylella fastidiosa in Vineyard Weeds and Other Alternate Hosts in California.

The fate of Xylella fastidiosa, the bacterium that causes Pierce's disease of grape, was assessed in 29 species of plants associated with vineyards in California's San Joaquin Valley. Bacterial populations and movement in greenhouse-grown plants were measured 1, 3, and 9 weeks after mechanical and insect inoculation. X. fastidiosa was recovered in 27 of 29 species in greenhouse tests, with common sunflower (Helianthus annuus), cocklebur (Xanthium strumarium), annual bur-sage (Ambrosia acanthicarpa), morning glory (Ipomoea purpurea), horseweed (Conyza canadensis), sacred datura (Datura wrightii), poison hemlock (Conium maculatum), and fava bean (Vicia faba cv. Aquadulce) being infected in more than 50% of inoculation attempts. Twenty-three species supported bacterial populations in excess of 104 CFU/g of plant tissue. X. fastidiosa populations increased for 9 weeks after inoculation in six species, and were static or declined in 16 species. Although the blue-green sharpshooter (Graphocephala atropunctata) was more efficient than mechanical inoculation for infecting plants, median populations and systemic movement for the two methods did not differ significantly. X. fastidiosa colonization of greenhouse and field-grown plants was compared in five alternate hosts. X. fastidiosa was recovered from 23 and 34% of field-grown plants, compared with 49 and 67% of greenhouse-grown plants, in the winter and summer, respectively, with at least 10 times fewer bacteria in field plants in the winter. Although X. fastidiosa has a wide host plant range, its fate in most species is variable and heavily influenced by field conditions.

Vector Transmission of Xylella fastidiosa to Dormant Grape.

Homalodisca coagulata (Say) is a sharpshooter leafhopper vector of the bacterial plant pathogen Xylella fastidiosa. Introduced into California about 15 years ago, this insect triggered recent outbreaks of Pierce's disease of grapevine in the state. H. coagulata has been observed feeding on dormant grapevines during the winter, raising the possibility of X. fastidiosa transmission during that season. We tested whether H. coagulata can acquire X. fastidiosa from and inoculate the bacterium to dormant grape (Vitis vinifera) in the laboratory and in the field. Usually, >90% of H. coagulata survived on dormant plants in the laboratory and field. Field experiments showed that H. coagulata can inoculate X. fastidiosa into dormant plants, yet field acquisition experiments did not result in transmission. Transmission to dormant plants during the winter is a potential problem in California vineyards adjacent to citrus groves or other habitats with large overwintering populations of H. coagulata. Because dormant plants have positive root pressure, our findings provide evidence that X. fastidiosa transmission does not require negative pressure in plant xylem to be inoculated into plants.

Populations of Xylella fastidiosa in Plants Required for Transmission by an Efficient Vector.

ABSTRACT Xylella fastidiosa, a xylem-limited bacterium that causes Pierce's disease (PD) of grapevine and other diseases, is transmitted efficiently by xylem-feeding leafhoppers. Acquisition of a PD strain of X. fastidiosa by the blue-green sharpshooter (BGSS) from five plant host species-grapevine (Vitis vinifera), Himalayan blackberry (Rubus discolor), California mugwort (Artemisia douglasiana), watergrass (Echinochloa crus-galli), and Bermuda grass (Cynodon dactylon)-was tested at various time intervals after vector inoculation. The minimum incubation periods in plant hosts before BGSS acquired X. fastidiosa were 4, 22, 29, and 25 days for grapevine, blackberry, mugwort, and watergrass, respectively. There were no transmissions by vectors or recoveries of X. fastidiosa by culturing from Bermuda grass in 133 attempts, including 80 attempts with the green sharpshooter, Draeculacephala minerva. The first acquisitions and subsequent transmissions by BGSS occurred after X. fastidiosa multiplied to a population of about 10(4) CFU/g of stem tissue. Higher populations of bacteria in plants resulted in higher rates of transmission. In grapevine, the rate of transmission increased over time (4.5% in the first 10 days to 55% after day 25) as the maximum number of viable CFU of X. fas-tidiosa recovered by culturing also increased (from 5 x 10(5) CFU/g during the first 10 days to 5 x 10(8) after day 25).

Causal Role of Xylella fastidiosa in Oleander Leaf Scorch Disease.

ABSTRACT A lethal leaf scorch disease of oleander (Nerium oleander) appeared in southern California in 1993. A bacterium, Xylella fastidiosa, was detected by culturing, enzyme-linked immunoassay, and polymerase chain reaction in most symptomatic plants but not in symptomless plants or negative controls. Inoculating oleanders mechanically with X. fastidiosa cultures from diseased oleanders caused oleander leaf scorch (OLS) disease. The bacterium was reisolated from inoculated plants that became diseased. Three species of xylem sap-feeding leafhoppers transmitted the bacterium from oleander to oleander. The bacterium multiplied, moved systemically, and caused wilting in Madagascar periwinkle (Catharanthus rosea) and leaf scorch in periwinkle (Vinca major) in a greenhouse after inoculation with needle puncture. No bacterium was reisolated from grapevine (Vitis vinifera), peach (Prunus persica), olive (Olea europaea), California blackberry (Rubus ursinus), or valley oak (Quercus lobata) mechanically inoculated with OLS strains of X. fastidiosa. A 500-bp sequence of the 16S-23S ribosomal intergenic region of oleander strains showed 99.2% identity with Pierce's disease strains, 98.4% identity with oak leaf scorch strains, and 98.6% identity with phony peach, plum leaf scald, and almond leaf scorch strains.

Fate of Pierce's Disease Strains of Xylella fastidiosa in Common Riparian Plants in California.

The fate of strains of the bacterium Xylella fastidiosa that cause Pierce's disease of grapevines was investigated in 33 species of mostly perennial plants common in riparian habitats in northern coastal California grape-growing regions. Plants were inoculated in the field with needle puncture using cultured cells of X. fastidiosa as inoculum or inoculated in the laboratory with infective insect vectors (Graphocephala atropunctata). Populations of X. fastidiosa were highest in most plant species within 3 to 6 weeks of inoculation, followed by declines in populations of viable bacteria over the next 3 to 4 months. Homogenates of petioles of California black walnut (Juglans hindsii) and coffeeberry (Rhamnus californica) inhibited in vitro growth of X. fas-tidiosa, precluding culture of the bacterium from these plants. Big leaf maple (Acer macrophyl-lum), California buckeye (Aesculus californica), California blackberry (Rubus ursinus), coast live oak (Quercus agrifolia), elderberry (Sambucus mexicana), French broom (Genista monspessulanus), periwinkle (Vinca major), valley oak (Quercus lobata), and the grape root-stock Vitis rupestris supported systemic populations of X. fastidiosa that survived throughout the year outdoors in Napa Valley, California.

Homalodisca coagulata (Hemiptera, Cicadellidae) Transmission of Xylella fastidiosa to Almond.

Almond leaf scorch (ALS) is caused by the bacterium Xylella fastidiosa, transmitted by sharpshooter leafhoppers and spittlebugs. The recent invasion of a X. fastidiosa vector, Homalodisca coagulata (Hemiptera, Cicadellidae), into California may have major consequences to the spread of ALS because this insect feeds readily on trees, including stone fruit species. We found that, under laboratory conditions, H. coagulata acquired X. fastidiosa from symptomatic almond plants with low efficiency relative to grape (3.3 to 10% per individual per day). Inoculation efficiency also was low, approximately 4% per insect per day. H. coagulata inoculated 1-year-old woody tissues of almond plants at similar rates as green shoots. H. coagulata transmitted two X. fastidiosa grape strains from grape source plants to grape and almond. We also observed X. fastidiosa transmission to dormant almond plants. X. fastidiosa populations in the petioles of field-collected symptomatic almond leaves were not higher than 107 CFU/g of tissue, suggesting that low bacterial populations within almond are partially responsible for the lower acquisition rates observed from diseased almond compared with diseased grape, which are usually within the range of 108 to 109 CFU/g. The relevance of our findings to ALS epidemiology, considering H. coagulata as a vector, is discussed.

Multiplication and Movement of a Citrus Strain of Xylella fastidiosa Within Sweet Orange.

Populations of cultivable cells of a citrus variegated chlorosis (CVC) disease strain of Xylella fastidiosa in stems and leaf veins of sweet orange (Citrus sinensis (L.) Osbeck) seedlings were estimated by dilution plating at 1, 2, 4, 8, and 16 weeks after needle inoculation. Cell populations ranged from log 4 to log 5 CFU/g of tissue after 1 week and increased to log 5 to log 7 CFU/g (median log 6) after 8 to 16 weeks. Recovery of greater than log 5 CFU/g from stem nodes distal to the inoculation site indicated systemic movement of the bacteria. Foliar symptoms in inoculated seedlings first appeared after 8 weeks. Population estimates from leaf veins of CVC-affected trees in citrus groves were in the same range but slightly lower (average log 5.8 CFU/g). X. fastidiosa was isolated from citrus more efficiently in periwinkle wilt-GelRite (PWG) and periwinkle wilt (PW) media than in charcoal-yeast extract with ACES buffer (BCYE) medium The relatively lower populations of cultivable cells of X. fastidiosa in citrus with CVC symptoms, compared with those reported in grapevines with Pierce's disease, suggest that most cells of X. fastidiosa within symptomatic citrus may be dead, explaining in part the low rates of vector transmission from citrus to citrus.

Effects of temperature on the flight activity of Graphocephala atropunctata (Hemiptera: Cicadellidae).

Graphocephala atropunctata (Signoret) is the principal vector of Xylella fastidiosa (Wells, Raju, Hung, Weisberg, Mandelco-Paul and Brenner), the bacterium that causes Pierce's disease of grapevine in coastal California. Monitoring the activity of C. atropunctata in the early spring is important for timing insecticide sprays and assessing the potential for disease spread to adjacent vineyards. Trapping studies with yellow sticky traps over 3 yr in Napa Valley, CA, established a significant correlation between early spring trap catch and temperature. Sticky trap catches of G. atropunctata occurred in the springs of 1996-1998 only when temperature was greater than or equal to 14.5 degrees C. In 1997 and 1998, the degree-hours (> 14.5 degrees C) per day from sunrise to sunset during March and April, but not in May, correlated significantly with trap catches. The temperature threshold of 14.5 degrees C in the early spring can be used to improve the timing of insecticidal applications aimed at reducing C. atropunctata populations in vineyards affected by Pierce's disease.

Occurrence and transmission of facultative endosymbionts in aphids.

The occurrence of a secondary bacterial symbiont (PASS) of pea aphid, Acyrthosiphon pisum (Harris), was detected by polymerase chain reaction (PCR) with specific nucleotide primers based on PASS 16S rDNA nucleotide sequences from over 80% (50/57) of clones of pea aphid collected from widely separated locations in California. PASS was also detected by PCR in both red and green phenotypes of rose aphid,Macrosiphum rosae (L.), but not in six other species of aphids examined, including blue alfalfa aphid (A. kondoi Shinji). The nucleotide sequences of the PCR-amplified, partial 16S rDNAs (1060 bp) from pea aphid and rose aphid were identical and 99.9% similar to the published 16S rDNA of PASS. PASS and a recently described new rickettsia of pea aphid (PAR) were transmitted by needle injection of hemolymph from positive pea aphid clones into negative clones and into blue alfalfa aphids. Both PASS and PAR were maintained in the offspring of some of the injected mother aphids via high rate of maternal transmission.

Phylogenetic affiliation of BEV, a bacterial parasite of the leafhopper Euscelidius variegatus, on the basis of 16S rDNA sequences.

The phylogenetic relationship of a nonflagellated, Gram-negative, rod-shaped intracellular bacterial parasite (BEV) of the leafhopper Euscelidius variegatus to other bacteria within the class Proteobacteria was determined by sequence analysis of 16S rDNAs. The presence of specific signature nucleotides showed this bacterium to be a member of the gamma-3 subdivision of the Proteobacteria. Phylogenetic analysis based on maximum parsimony placed BEV within a clade in the Enterobacteriaceae, which includes a number of bacteria that are facultative symbiotes of insects and have a common ancestor with Proteus vulgaris. Within this clade, BEV is most closely related to a bacterium identified as the secondary endosymbiote of another homopteran, the pea aphid, Acyrthosiphon pisum.

Transmission via plants of an insect pathogenic bacterium that does not multiply or move in plants.

A bacterial parasite (designated as BEV) of the leafhopper Euscelidius variegatus, which is passed transovarially to offspring, was transmitted from insect to insect via feeding of the insects in plants. The rate of bacterial infection of leafhoppers fed upon plants that had previously been exposed to BEV-infected leafhoppers declined with an increase in the time that infected leafhoppers had been off rye grass. Transmission of BEV also occurred on sugar beet and barley but not celery. The bacterium was also transmitted to and acquired from membrane-encased artificial diets. There was no evidence that the bacterium was transmitted via plant surfaces, but transmission and direct culture assays from plants indicated that the bacterium did not multiply or move within plants. This parasite-host relationship may represent a primitive stage in either the evolution of intracellular symbiosis with its insect host or to alternative parasitization of plant and insect hosts via insect transmission, as is the case for insect-vectored plant pathogens.

A new rickettsia from a herbivorous insect, the pea aphid Acyrthosiphon pisum (Harris).

An undescribed, maternally heritable, rod-shaped bacterium (or "tertiary symbiont") was detected by microscopy in hemolymph of about half (59/122) of pea aphid [Acyrthosiphon pisum (Harris)] clones collected from widely separated locations in California. On the basis of molecular phylogenetic analysis of 16S rDNA sequences, the bacterium was clearly placed among other Rickettsia in the alpha-subgroup of Proteobacteria, close to Rickettsia bellii-a rickettsia found in ticks. A PCR assay was developed to detect this bacterium in pea aphid clones with specific 16S rDNA PCR primers. Results of PCR-based assays completely correlated with detection by microscopy. This is the first confirmed detection of a Rickettsia in a herbivorous insect.