“Candidatus Liberibacter asiaticus” is associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease currently threatening world citrus production. HLB has a long history in China and was found in Brazil in 2004 and U.S.A. in 2005.  There is an urgent need to differentiate isolates of “Ca. L. asiaticus” from different geographical regions for effective control of HLB.  In this study, isolates of “Ca. L. asiaticus” collected from Brazil, China and the United States were evaluated based on two previously characterized genomic loci, one locus (trn1) with variable tandem repeat numbers (TRNs), and the other locus (snp1) is characteristic in single nucleotide polymorphisms (SNPs).  A total of 299 strains (84 Brazil, 132 China and 83 U.S.) were analyzed.  At the trn1 locus, “Ca. L. asiaticus” strains were divided into TRN-A and TRN-B groups.  TRN-A isolates dominated the China and U.S. populations but were not detected in the Brazil isolates.  In contrast, TRN-B dominated the Brazil isolates but occurred at low frequencies in China (3%) and U.S. (6%).  SNP Analyses at the snp1 locus established Term-A and Term-G groups.  Term-A group included all Brazil and China isolates, along with 6% U. S. isolates which were also TRN-B isolates.  The remaining (94%) U. S. isolates were in Term-G group.  By combining data from the analyses of the two genomic loci, it is shown that the TRN-A:Term A genotype was unique to China, TRN-A:Term-G genotype was unique to U.S., and the TRN-B:Term-A genotype dominated the Brazil isolates.  No TRN-B:Term-B isolates were found.

A study was conducted to assess (i) patterns of Las colonization in graft-inoculated 1-y-old seedlings of Cravo Rangpur lime, Sunki mandarin, and Swingle citrumelo and (ii) patterns of Las distribution in naturally infected 4 and 10-y-old trees of  sweet oranges ‘Pera’ on Sunki and ‘Folha murcha’ on 11 rootstocks. The seedlings were inoculated at the trunk, 40 cm above the substrate level. Samples of bark were collected at the inoculation site, 10, 20, and 30 cm below it, and from the root 45, 75, 105, and 135 days after inoculation (dai). Samples from trees included symptomatic leaves and bark from the trunk (10 cm above or below the grafting line) and roots. The samples were analyzed through qPCR to estimate log Las genome/gram tissue. In seedlings Las colonization pattern was similar for all rootstocks. Las was detected in the root 45 dai or 3 months before the symptoms developed on leaves. In Swingle maxima titers were lower but reached a plateau faster (45 to 75 dai) than in Sunki or Cravo (75 to 135 dai) for all sampled sites. In the field the distribution pattern of Las in the trees was also similar for all rootstocks, with higher titers detected in the leaves (5.29±0.22) than in trunks (4.38±0.49; 3.78±0.27) or roots (3.38±0.28). No correlation existed between the amount of symptom on the canopy and Las titer in the root. This lack of correlation plus the fast movement of Las from the inoculation site down to the root may explain the failure of pruning to control HLB.

A series of assays was conducted in an attempt to eliminate Ca. L. asiaticus (Las) from HLB affected plants using hot air (HA) or steam (ST). HA assays were conducted with 2-y-old greenhouse (GH) potted Valencia/Rangpur lime plants and 4-y-old Hamlin/Sunki field trees. GH plants were exposed to 38 to 44°C for 24 to 192 h in a growth chamber (GC). Field trees were exposed to sunlight under plastic cover sheets (≥40°C) for 24 to 96 h after the branches were pruned 1.5 m above ground. ST assays involved root-uncovered GH plants and 5-y-old pruned Hamlin/Swingle and Pera/Cravo field trees. GH plants were exposed to 45 to 60°C for 5 to 30 min and the trees to 55°C for 5 to 20 min and 60°C for 5 or 10 min. Plant and Las responses to heat were temperature/time dependent. All plants exposed to HA for ≥96 h at 44°C in GC and ca. 1/3 of the trees treated for ≥24 h at ≥40°C in the field died. Around 1/3 of the plants exposed to ST for 15 min at 50°C or 5 min at 55°C, and all plants at 60°C also died. In the field ST also damaged or killed the trees at 55°C for ≥30 min or 60°C for ≥20min. Most of the plants or trees showed typical HLB symptoms and were PCR+ 3 to 5 months after treatment. Under the trunk bark of field trees the temperature took over 10 min to reach maximum of 50°C, which was apparently not enough to kill Las. Las survival in the trunk and roots may explain the lack of success of the heat treatments against HLB.