EVALUATION OF CITRUS GERMPLASM FOR RESISTANCE OR TOLERANCE TO HUANGLONGBING DISEASE IN PUERTO RICO

Huanglongbing (HLB) caused by the bacteria 'Candidatus Liberibacter asiaticus' (CLa) is a devastating disease in citrus production regions worldwide. The bacterium can attack all parts of the trees canopy and death of trees is observed in susceptible genotypes. The levels of resistance/tolerance in Citrus spp. from the "Rico" collection in Adjuntas are unknown. These genotypes are of interest for Puerto Rico because although some selections were positive to CLa, they did not show severe symptoms and are still productive. Thereby, the levels of resistance/tolerance to HLB in this collection need to be assessed. The objectives of this study are: (1) evaluate the response of the "Rico" citrus genotypes to HLB in the greenhouse, (2) quantify the concentration of the bacteria in the plant tissue (3) identify genotypes with higher levels of resistance/tolerance. Fourteen scions of the "Rico" collection and lime 'Tahití' will be grafted in 'Swingle' and 'HRS 812' rootstocks. Buds from infected HLB tissue will also be grafted into the rootstocks. The plants will be maintained under greenhouse and disease severity will be evaluated periodically. The identification of highly resistant genotypes in local germplasm would be essential for the management of HLB. Likewise, recommendations of better scions and rootstocks will be provided at the end of this project for future field studies.

Grafting Citrus spp. and inoculation with CLa in the rootstocks:Scions of fourteen Citrus spp. of different origins belong to the "Rico" collection and lime (cv. Tahití) as susceptible control, will be grafted on 'Swingle citrumelo' (Hutchison, 1974) and HRS 812 (Bowman et al., 1997) rootstocks by the T-budding methodology (Bir and Ranney, 2014). Stem buds of approximately 6 cm-length from HLB infected tissue will be grafted into healthy tissue of the rootstocks using the methodology described above (Bir and Ranney, 2014). The healthy phloem will have contact with the infected one allowing the spread of CLa into the scions.Greenhouse evaluation of HLB Severity:At least four scions/varieties grafted in HRS 812 and Swingle citrumelo rootstocks will be placed in a randomized block design with four replications in a greenhouse in the Isabela AES-UPR. Plants will be kept with a temperature of 24-28 C and 12 h light. HLB severity will be evaluated monthly using a scale from 1-7 described by Rouse et al. (2010); where 1= vigorous tree without HLB symptoms, 2= vigorous tree with mild greening symptoms , 3= trees with mild decline and 10% of greening symptoms in the tree's canopy, 4= trees with moderate decline and 25% of greening symptoms in the tree's canopy, 5= trees with severe decline and 50% of greening symptoms in the tree's canopy, 6=trees with severe decline and 75% of greening symptoms in the tree's canopy, and 7= death of the tree. HLB scores of 1-2 will be considered resistant, 3-4 intermediate, and 5-7 susceptible. Evaluations will be conducted for one and half year.DNA extraction, qPCR, and DNable procedures:Total DNA will be extracted from 200 mg of fresh leaf midribs in a bulk sample of each genotype using the DNeasy Plant Mini Kit (Quiagen, Valencia, CA). DNA will be diluted to 10 μg/ml and 2 μl will be used for molecular analysis.The qPCR will be performed in a 25 μl reaction volume including 250 nM of each primer, 150 nM of target probes, 300 nM (each) internal control primers, 150 nM internal control probes, 6.0 mM Mg Cl2, 250 μM dNTPS, and 1-unit Platinum of Tag DNA polymerase (Invitrogen, Carlsbad, Ca). Li et al. (2006) describe in detail the procedure and the amplification conditions of the qPCR mentioned above. Data will be analyzed using the Smart Cycler software version 2.0 D.Furthermore, DNable procedure will be conducted according to the protocol described by Quantifier-Envirologix in the user Manual DNable to corroborate the presence or absence of CLa in the samples identified by qPCR, previously.Data analysis:Data will be analyzed using SAS 9.4 PROC GLM (SAS, 2012). Mean HLB disease score and area under disease progress curve (AUDPC; Cooke, 2006) will also be calculated. Fisher's least significant difference at P ≤ 0.05 will be calculated for HLB severity and AUDPC values. Moreover, correlations between HLB severity and ct values from qPCR will be conducted.