Early Events of Citrus Greening (Huanglongbing) Disease Development at the Ultrastructural Level

Abstract

Citrus greening (Huanglongbing [HLB]) is one of the most destructive diseases of citrus worldwide. The causal agent of HLB in Florida is thought to be 'Candidatus Liberibacter asiaticus'. Understanding of the early events in HLB infection is critical for the development of effective measures to control the disease. In this work, we conducted cytopathological studies by following the development of the disease in citrus trees graft inoculated with 'Ca. L. asiaticus'-containing material under greenhouse conditions to examine the correlation between ultrastructural changes and symptom production, with the main objective of characterizing the early events of infection. Based on our observations, one of the first degenerative changes induced upon invasion of the pathogen appears to be swelling of middle lamella between cell walls surrounding sieve elements. This anatomical aberration was often observed in samples from newly growing flushes in inoculated sweet orange and grapefruit trees at the early "presymptomatic" stage of HLB infection. Development of symptoms and their progression correlated with an increasing degree of microscopic aberrations. Remarkably, the ability to observe the bacterium in the infected tissue also correlated with the degree of the disease progression. Large numbers of bacterial cells were found in phloem sieve tubes in tissue samples from presymptomatic young flushes. In contrast, we did not observe the bacteria in highly symptomatic leaf samples, suggesting a possibility that, at more advanced stages of the disease, a major proportion of 'Ca. L. asiaticus' is present in a nonviable state. We trust that observations reported here advance our understanding of how 'Ca. L. asiaticus' causes disease. Furthermore, they may be an important aid in answering a question: when and where within an infected tree the tissue serves as a better inoculum source for acquisition and transmission of the bacterium by its psyllid vector.

Modeling huanglongbing transmission within a citrus tree

“…The asymptomatic flush become symptomatic due to an increase in concentration of the pathogen, making the symptomatic flush more infectious to vectors than the asymptomatic flush (22). In one study more active bacterial cells were observed in presymptomatic than in highly symptomatic leaf samples (23), suggesting that the bacteria are dormant or dead in highly symptomatic tissue. However, a positive correlation between concentration of CLas and HLB symptom expression has also been shown (22).…”

“…(iv) Symptomatic flush is assumed to be more infectious than asymptomatic flush on the basis of higher CLas DNA densities in symptomatic flush (22). Recently, large numbers of active CLas cells were found in tissue samples from presymptomatic young flushes whereas more inactive bacteria were observed in highly symptomatic samples (23). If we assume that the symptomatic flush is less infectious than the asymptomatic one, then the number of subsequent infections will be reduced.…”

“…The time taken for an infected tree to become unproductive and die depends on the tree age, because multiplication of CLas is fastest in young citrus plants (22). In graft-inoculated young trees, production of additional new growth decreased until about 9 mo after inoculation, when severely affected branches stopped producing new growth (23). Most of the infected plants eventually died over the following year.…”

“…This might be attributed to the fact that as much of the tree becomes symptomatic, growth of new flush decreases (23), so that the vectors will search for other trees with flush to lay their eggs (31). Indeed, the parameter reflecting the flush maturation rate and thus the availability of flush ( ξ) has the most effect on the dynamics of the HLB infection.…”

“…In practice, such a situation may be present when nutritional products and systemic acquired resistance inducers are used to ameliorate the effect of symptoms produced by HLB in citrus groves (3) or when a resistant rootstock or interstock is used (32). The actual movement of CLas through a tree is evident from systemic spread of the pathogen in plants that are inoculated through grafting in greenhouses where vectors are absent (23). However, the rates of movement and multiplication of the pathogen inside trees are not well understood (3).…”