Whole Plant Destructive Screening for Huanglongbing Susceptibility with Conetainer Seedlings Exposed to No-choice Asian Citrus Psyllid Inoculation

Huanglongbing (HLB) is associated with Candidatus Liberibacter asiaticus (Las) and is vectored by the Asian Citrus Psyllid (ACP, Diaphorina citri).  HLB is devastating the Florida citrus industry, with production reduced by 60% in the last 12 years, and HLB is considered the greatest threat to citrus production worldwide.  HLB-resistant plants are likely the best solution to this problem, but screening for resistance is slow, typically requiring 10-12 months following inoculation.  In this study we used no-choice ACP inoculation (two-week infestation of 15 ACP per plant from a Las+ colony) of small seedling trees in conetainers, and destructively sampled 10 trees of each genotype at 3, 6, and 12 weeks after inoculation.  At sampling, the trees were: 1) divided into leaves, stems and roots; 2) fresh weight of each tissue was determined; 3) tissues were ground in liquid nitrogen; 4) 200 mg of each tissue was sampled and DNA extracted; 5) qPCR was run to quantify Las using the LasLong probe/primer set; and 6) total Las load was calculated in each tissue type.  Kruskal-Wallis statistical analyses were used to assess the data in two ways, samples having non-detectable Las were designated as having 2 Las per sample (Ct=40) or these samples were excluded from analysis.  Only a few differences in interpretation resulted.  Percentage of samples (tissue in each genotype) with detectable Las ranged from 10-100% at week 3 (mean 52%), 40-83% in week 6 (mean 62%), and 40-90% in week 12 (mean 63%).  Across all samples: 1) Las per plant was 5 billion in week 3, 10 billion in week 6, and 45 billion in week 12; 2) both Las/plant and Las titer were significantly greater at week 12 compared to week 3; 3) there was no or little difference in Las/plant or Ct between tissue types; and 4) HLB-susceptible Citrus sinensis was clearly distinguished from HLB-resistant Poncirus trifoliata with higher titers and 35 X the Las/plant.  Comparing each genotype/tissue/timepoint: 1) only leaves showed a statistically significant increase in Las/plant and Ct over time, in all except Poncirus trifoliata which displayed a similar trend; and 2) by week 12 after inoculation, HLB-susceptible Citrus sinensis was clearly distinguished in all analyses from HLB-resistant Poncirus trifoliata and in most comparisons against their hybrid Carrizo, but only in roots.  Based on these results, genotype comparisons in susceptibility to Las can be expedited by sampling roots for Las at 12 weeks after conetainer tree inoculation.