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4870

Psyllids are vectors of fastidious plant pathogenic ‘Candidatus Liberibacter’ species that infect both the psyllid vector and plant host. Understanding the molecular and cellular basis of ‘Ca. Liberibacter’ interactions with the psyllid host will aid in identification of effectors involved in invasion and multiplication and facilitate transmission to the host plant. The differential expression of previously identified genes/loci with predicted involvement in tomato host–plant– ‘Ca. L. solanacearum’–prophage–Wolbachia endosymbiont dynamics was quantified by RT-qPCR amplification. Fifteen ‘Ca. Liberibacter solanacearum genes and/or prophage loci and four predicted Wolbachia spp. loci were analyzed in potato psyllids in a 14-day time-course study, post-48-h acquisition-access period by potato psyllids on ‘Ca. L. solanacearum’-infected tomato plants. The ‘Ca. L. solanacearum’-infected tomato host plants were used as an infected host ‘calibrator’ species lacking involvement of psyllid effectors. ‘Ca. L. solanacearum’ genes with predicted functions in adhesion, motility, transport, and virulence that are associated with the prophage lysogenic lifestyle were differentially expressed. In contrast, the prophage-loci expression was synchronous with early or late phase of psyllid-‘Ca. L. solanacearum’ infection, respectively. The observations are consistent with the previously in silico-predicted ‘Ca. L. solanacearum’ gene and prophage/Wolbachia loci functions and time-course global expression patterns. Knockdown of ‘Ca. L. solanacearum’ genes involved in invasion, biofilm formation, and colonization would be expected to impair the vertical and horizontal transmission of ‘Ca. L. solanacearum’ to psyllid offspring and host plants, respectively.

‘Candidatus Phytoplasma solani’ is the causal agent of the Bois noir (BN), affecting grapevine worldwide. The complex epidemiology of BN, which involves multiple ‘Ca. P. solani’ host plants and insect vectors, as well as the occurrence of recovery (loss of symptoms on grapevine canopy), makes disease investigations and containment in vineyards difficult. To achieve early detection of ‘Ca. P. solani’, a droplet digital PCR (ddPCR)-based approach and quantitative (q)PCR assay were compared, testing specific primers based on the elongation factor Tu (tuf) gene using SYBR Green chemistry. The regression curve analysis of the ddPCR assay showed good linearity. Compared with the qPCR method, the sensitivity of ddPCR improved about 10-fold. The analysis of grapevine roots spiked with serial dilutions of ‘Ca P. solani’. PCR tuf fragments showed that qPCR was inhibited, while ddPCR was not affected. Testing 66 grapevine samples from 50 grapevine plants, the ddPCR provided superior diagnostic performance compared to qPCR in roots of symptomatic plants (75% detected by ddPCR, 41.6% by qPCR), roots of recovered plants (58.8% detected by ddPCR, 25% by qPCR), and asymptomatic leaf tissues from recovered plants (75% detected by ddPCR, 25% by qPCR). The ddPCR analysis allowed us to detect ‘Ca. P. solani’ on 40% of leaf samples from recovered plants and 20% of roots from asymptomatic plants. No differences among ddPCR and qPCR were found in detecting phytoplasma on symptomatic leaf samples. The ddPCR assay allowed the absolute quantification of ‘Ca. P. solani’ in complex matrices, such as roots, and when low titer of phytoplasma is present.

This is a general protocol for the vegetative propagation of citrus material infected with the bacterium Candidatus Liberibacter asiaticus (CLas), the presumed causal agent of citrus greening disease (HLB), using an aeroponic cloning apparatus. The following procedure details a workflow using an EZ-Clone Pro Low Cloning System from Hydrobuilder.com. This system comes in varying cell-sizes (number of clones). We have found the most versatile to be the 16- or 32-cell size based on ease of preparation, keeping algae growth to a minimum, and maintenance. Cloning versus seed sowing has been useful in producing infected material for experimental assays requiring cloned, mature citrus trees as opposed to genetically variable plants from seeds. Where seed germination can take up to 12+ months to achieve plants with stem diameters ≥ 10 mm and 5+ years for flowering, infected branches from flowering trees can be directly propagated using this protocol within a few months and retain their maturity. Clone viability rate differs by citrus cultivar and general health of the cutting used, with citron and CLas negative material having the highest success rate. Infected citron cuttings have had a 95% survival rate, regardless of the diameter of the cutting. The cutting diameter ranged from 2mm-12mm and was limited to 12mm maximum, which is the size of the collar on the EZ-Clone Pro Low cloner.

ABSTRACT The phyllody severity in sesame crop ranged from 9% to 20% under field conditions, and the maximum was recorded in Thiruvannamalai district of Tamil Nadu, India. The observed morphological characters of the transmitting insect vector Orosius albicinctus were light ochraceous colour with irregular striations and dark brown with black mottling. The infected sesame samples were subjected to PCR amplification by using universal primer pair P1/P6, followed by nested PCR using R16F2n/R16R2 primers, which amplified ~1.25 kb, which was also occurred in the Parthenium weed plant. Further, BLAST analysis showed that the sequences were aligned with the 16SrII‐B group phytoplasma consisting of Candidatus Phytoplasma aurantifolia (94.87%), sesame phyllody (97.97%), Blainvillea acmella phytoplasma (97.70%) and Mollugo disticha phyllody phytoplasma (98.28%). Since there is no adorus impact despite the vast host range, polyphagous insect vectors, lack of environmentally acceptable effective insecticide, and unavailability of resistant sources, management of phyllody disease has been attempted with biotic inducers (salicylic acid [SA], methyl jasmonate [MeJA] and beta amino butyric acid [BABA]) individually (50, 100 and 150 ppm) and in combination. Among the inducers tested in vitro, SA 50 ppm showed a higher vigour index (1401) in the seed infiltration method of seed treatment, further confirmed in blotter paper technique. The same treatment showed a high germination percentage (86%) with more shoot length on the 60th day (67.0 cm) and five branches/plant under glasshouse experiment. In addition, SA 50/100 ppm increased the phenolic activity and plant defence enzymes; hence it has been further test verified at two different field locations, and the results revealed that seed infiltration treatment with SA 50/100 ppm, followed by foliar application of SA (50/100 ppm) at 30, 45 and 60 DAS, was shown to be effective by recording lesser disease severity compared to control (42.2%). Also, salicylic acid treatment showed higher germination (87.6%), more capsules/plant, thereby incorporation of inducers like salicylic acid in the integrated disease management will serve as a viable and sustainable management option.