Proceedings of the Florida State Horticultural Society

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.

Citrus huanglongbing (HLB), or citrus greening, is caused by a bacterial pathogen (Candidatus Liberibacter asiaticus, CLas) and is considered the most destructive citrus disease worldwide. The disease has affected millions of citrus trees both in Florida and around the world, resulting in billions of dollars in annual production losses. Infection by the CLas disrupts vascular movement, weakens the tree root system, and cause a rapid decline in tree health. Despite extensive research efforts, there are still no effective management tools to cure HLB-positive trees or prevent new infections. Current HLB management strategies include chemical application of insecticides to control the insect vector of the CLas bacterium (the Asian citrus psyllid) and traditional antibiotics that kill the CLas bacterium. Vector control has not been effective and antibiotic use has been complicated because the CLas bacterium resides inside the plant in the vascular system, and it is difficult to get enough antibiotics to penetrate at effective concentrations. We present a novel method of biological molecule delivery method referred to as Symbionts™. In Symbionts™, host cells are modified to divide autonomously using plant growth regulator genes from Agrobacterium tumefaciens and to produce biological molecules that can improve host-plant defenses against CLas. To produce the Symbiont, host cells expressing cargo of interest can either be induced on citrus trees by infection with A. tumefaciens, or cultured symbiont cells can be produced in the lab and transplanted (free of A. tumefaciens).

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an invasive citrus pest that vectors the phloem-dwelling bacterium, Candidatus Liberibacter asiaticus (CLas), which is a putative causal agent of citrus greening disease known as Huanglongbing (HLB). In Florida, the D. citri–CLas pathosystem complex is at present considered the greatest threat the citrus industry must manage. Brassinosteroids (Brs), a relatively new class of plant hormones, are approved for commercial use in Florida citrus and are believed to delay HLB progression. Individual protective covers (IPCs), a type of psyllid exclusion mesh bags, are increasingly being adopted to efficiently protect newly planted citrus trees from HLB infection. However, IPCs typically must be removed after 2–3 years due to tree growth. With the aim of prolonging tree health after IPC removal, we are investigating the efficacy Brs in protecting and/or preventing trees from ACP and CLas infection once they are left exposed. Upon IPC removal from trees covered for 3 years, monthly Br treatments started in trees from three mandarin cultivars (Early Pride, SugarBelle and Tango) on two different rootstocks, sour orange and US-942. The IPCs effectively excluded psyllids, prevented CLas infection, and clearly improved most of the horticultural traits measured. Although this is an on going experiment, we have already found that treatment with Brs reduced CLas bacterial titer by 10-fold on average two months after IPC removal. The long-term efficacy of the treatments still needs to be assessed and will be discussed.