Huanglongbing and citrus variegated chlorosis integrated management based on favorable periods for vector population increase and symptom expression

Huanglongbing (HLB, associated with ‘Candidatus Liberibacter asiaticus’ and transmitted by the Asian citrus psyllid Diaphorina citri) and citrus variegated chlorosis (CVC, caused by Xylella fastidiosa subsp. pauca and transmitted by sharpshooter species) have been managed by vector control and removal of symptomatic trees. Although vectors and new symptomatic trees can be detected year-round, peaks of vector populations are higher in spring and summer and the most symptomatic trees are found in autumn and winter. This work aimed to compare the management of both diseases during these favorable periods. The experiment was conducted over five years in a commercial orchard and had a 3 by 2 factorial design. The factor “vector control” had 3 levels: monthly vector control year-round (VCYR); monthly vector control in spring and summer (VCSS); and vector control when a threshold level of 10% occupancy was detected (VCOT). The factor “inoculum removal” had 2 levels: monthly eradication year-round (TEYR); and monthly eradication in autumn and winter (TEAW). Host flush, both HLB and CVC vector populations, and the number of symptomatic citrus plants were visually assessed. The level of vectors over the seasons, as measured using the average area under the curve (AUC), was similar for all treatments with the exception of psyllid abundance, which was around 4.5 times higher for VCSS than other treatments. For both diseases, no difference in the average AUC of disease progress and disease final incidence was observed. VCOT or adjusted VCSS associated to TEAW could be integrated for sustainable citrus production.

Differential Responses of Tobacco to the Citrus Variegated Chlorosis and Coffee Stem Atrophy Strains of Xylella fastidiosa

Xylella fastidiosa comprises a diverse group of xylem-limited, insect-transmitted bacterial pathogens. In Brazil, the citrus variegated chlorosis (CVC) and coffee stem atrophy (CSA) diseases are caused by X. fastidiosa subspecies pauca transmitted by common insect vectors. No simple protocol allowing strain discrimination exists, making epidemiological studies, which are important for devising control measures, difficult to undertake. Here, we show that both strains can easily be distinguished based on the pattern of leaf symptoms that they induce on pin prick-inoculated tobacco seedlings, namely small orange lesions and large necrotic lesions induced by the CVC and CSA strains, respectively. These differential responses allowed us to investigate whether mixed strain infections would occur in citrus or coffee trees in the field. Seedlings were individually inoculated with X. fastidiosa colonies recovered from citrus or coffee plants from various locations at three different times. No mixed infections were detected. In two experiments, the citrus and coffee strains infected only their original hosts as well as tobacco. The usefulness of this tobacco bioassay as a tool to study X. fastidiosa spread was demonstrated. It provided evidence that, over the years, the CVC and CSA pathogens have remained limited to their original hosts, despite crop proximity and the presence of sharpshooter vectors that favor transmission of the bacteria to and between both host species.

Scion Substitution: A New Strategy to Control Citrus Variegated Chlorosis Disease

Citrus variegated chlorosis (CVC) disease, caused by the xylem-limited and insect-transmitted bacterium Xylella fastidiosa, has caused severe losses in orange production in Brazil. Disease control requires insecticide applications, tree removal, and pruning of symptomatic branches. Pruning success has been erratic, especially in areas of high disease incidence. In this work, in planta X. fastidiosa distribution and the effectiveness of severe pruning procedures for curing diseased adult trees were investigated. Most sampled upper parts of the trees contained X. fastidiosa, but at higher frequencies in symptomatic branches. Removal of all main branches (decapitation) was not effective and revealed a 20 to 30% incidence of latent infections. Trunk decapitation resulted in a higher number of healthy scions but killed 10 to 30% of the remaining trunks. Removal of all scion and grafting the newly sprouted shoots of ‘Rangpur’ lime (Citrus limonia Osbeck) or ‘Cleopatra’ (Citrus reshni Hort. ex Tan.) rootstocks with healthy buds allowed production of fast-growing and productive new scions that remained free from CVC for at least 2 years in four locations. With this method, highly affected trees do not need to be fully removed and the costs involved in this practice and in young tree acquisition and plantings are circumvented; therefore, it is a feasible option for less technically inclined small growers in Brazil.

Proteomic and Metabolomic Analyses of Xylella fastidiosa OMV-Enriched Fractions Reveal Association with Virulence Factors and Signaling Molecules of the DSF Family

Xylella fastidiosa releases outer membrane vesicles (OMVs) known to play a role in the systemic dissemination of this pathogen. OMVs inhibit bacterial attachment to xylem wall and traffic lipases/esterases that act on the degradation of plant cell wall. Here, we extended the characterization of X. fastidiosa OMVs by identifying proteins and metabolites potentially associated with OMVs produced by Temecula1, a Pierce’s disease strain, and by 9a5c and Fb7, two citrus variegated chlorosis strains. These results strengthen that one of the OMVs multiple functions is to carry determinants of virulence, such as lipases/esterases, adhesins, proteases, porins, and a pectin lyase-like protein. For the first time, we show that the two citrus variegated chlorosis strains produce X. fastidiosa diffusible signaling factor 2 (DSF2) and citrus variegated chlorosis-DSF (likewise, Temecula1) and most importantly, that these compounds of the DSF (X. fastidiosa DSF) family are associated with OMV-enriched fractions. Altogether, our findings widen the potential functions of X. fastidiosa OMVs in intercellular signaling and host–pathogen interactions.

Comparison of protocols for the extraction of genomic DNA from Sharpshooters (Hemiptera: Cicadellidae) for the detection of Xylella fastidiosa

ABSTRACT: The bacterium Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC), is dependent on vector insects for its spread and infection of citrus hosts. The insects, leafhoppers (Hemiptera: Cicadellidae) transmit the bacteria to healthy plants after feeding on infected plants. The objective of this study was to develop and compare methods for extracting genomic DNA from sharpshooters to detect X. fastidiosa. The DNA extraction from insects was performed according to a phenol-chloroform based DNA extraction in conjunction with two commercial kits, Dneasy® Plant Mini Kit and blood & tissue Dneasy® Handbook (Qiagen Inc., Valencia, CA, USA). The heads of the following species of sharpshooter were used: Dilobopterus costalimai, Acrogonia citrina, Oncometopia facialis, Bucephalogonia xanthophis, Macugonalia leucomelas and Homalodisca ignorata. Based on the numeric differences between independent samples, the results showed the use of the phenol:chloroform extraction method (36/50 positives) and the DNeasy® Plant Mini Kit (33/50) resulting in the most detections of X. fastidiosa from leafhopper samples. As these two methods detected Xylella in the greatest number of infected samples, they may be more efficient to use for detection purposes in leafhoppers.