Bisi, Rayane Barcelos

Candidatus Liberibacter asiaticus (CLas) is an unculturable, phloem-limited gram-negative bacterium associated with citrus Huanglongbing (HLB) disease in Florida and most other citrus-producing countries around the world. HLB is considered the most destructive disease of citrus and is severely affecting the fruit production worldwide. Natural transmission of CLas occurs by the Asian citrus psyllid (ACP) vector Diaphorina citri. Under greenhouse conditions, the experimental transmission of CLas to non-infected trees usually is accomplished using infected ACP or by grafting infected citrus tissue. CLas was confirmed infecting citrus in Florida in 2005 and many characteristics of the disease have been described. However, the transmission of CLas for controlled greenhouse studies is often prob lematic and poorly understood. Due the complexity in maintenance of ACP colonies for controlled vector transmission, the experimental infection of new plants is accomplished primarily by grafting, but several factors may influence the reliability of this method. This study evaluates the propagation and transmission efficiency of CLas using graft inoculation methods under greenhouse conditions. Three experiments were conducted for with graft inoculation: 1) seedlings of 12 cultivars; 2) ‘Tango’ mandarin on 13 rootstock cultivars; and 3) ‘Navel’ orange on 14 rootstock cultivars. Plants were inoculated with buds from infected greenhouse-grown ‘Valencia’ orange plants that were poly merase chain reaction (PCR) positive for CLas and symptomatic for HLB. Three bud pieces were used to inoculate each plant using the inverted-T method.

Huanglongbing (HLB) is a major disease of citrus associated with phloem-limited bacteria in the genus Candidatus Liberibacter that affects all known citrus species and relatives, with many commercial cultivars being greatly damaged. Testing cultivar tolerance to HLB in field conditions is difficult because of the erratic spread of the bacteria, scion and rootstock interactions, and influence of many biotic and abiotic factors on the tree response to the disease. This study aimed to validate the effect of CLas infection on different citrus species and hybrids thought to have different levels of tolerance to the disease using CLas graft inoculation under controlled greenhouse conditions. Young potted seedlings from 12 different citrus germplasm selections were graft-inoculated with CLas or mock-inoculated. Plants were monitored periodically during 18 months for canopy growth, HLB and nutritional leaf symptoms, and leaf CLas titers. The leaf nutrient content was measured at the end of the experiment. Roots were also assessed at 18 months after inoculation (mai) for CLas titers and biomass distribution. There were significant differences in most analyzed variables of healthy and infected plants. Some plants of all cultivars were successfully infected; however, overall, the CLas transmission rate was low and inconsistent. Ct values of roots were generally higher than those in leaves at 18 mai. HLB symptoms were not observed on seedlings until 1 year after inoculation; at 18 mai, infected trees of all cultivars were HLB symptomatic. Significant shoot and root biomass reductions (44%–75%) in infected ‘Cleopatra’, ‘Duncan’, ‘Olinda Valencia’, ‘Sunburst’, and ‘Valencia 1-14-19’, considered susceptible to HLB, were measured. These cultivars also showed more severe HLB symptoms than the presumed tolerant cultivars such as Microcitrus inodora, Rich 16-6 trifoliate orange, and US-897. This study provides new knowledge of the efficacy and value of greenhouse screening of citrus germplasm for response to HLB to support the development of new cultivars with improved HLB tolerance or resistance.