Publications

3534

In China, citrus Huanglongbing (HLB) disease is caused by the Candidatus Liberibacter asiaticus bacterium, which is carried by the Asian citrus psyllid Diaphorina citri Kuwayama. It was hypothesized that the epidemic of the HLB may related with the rate of bacterium presence in the insect vector and bacterium content in plant tissues, as well as the phyllosphere microbe communities changes. This study systematically analyzed the presence or absence of Ca. L. asiaticus in citrus tree leaves and in the insect vector D. citri over a 6-year period using real-time PCR. In addition, changes in the number of bacteria carried by D. citri over 12 months were quantified, as well as the relationship between the proportion of D. citri carrying Ca. L. asiaticus and the proportion of plants infected with Ca. L. asiaticus were analyzed. Results showed that the proportion of D. citri carrying bacteria was stable and relatively low from January to September. The bacteria in citrus leaves relatively low in spring and summer, then peaked in December. The proportion of D. citri carrying bacteria gradually declined from 2014 to 2019. The proportion of D. citri carrying Ca. L. asiaticus showed a significant positive correlation with the proportion of diseased citrus. The phyllosphere bacterial and fungal communities on the healthy citrus leaf were significantly different with the disease leaf in April and December. Pathogenic invasions change the citrus phyllosphere microbial community structure. It could be summarized that citrus Huanglongbing correlated with incidence of Diaphorina citri carrying Candidatus Liberibacter asiaticus and citrus phyllosphere microbiome.

Background: ‘Candidatus Liberibacter asiaticus’ (CLas) is a major causal agent of citrus greening disease. The disease primarily involves an asymptomatic, often latent infection of CLas. However, there is no effective technique to distinguish latent-infected trees from healthy ones. This study describes the development of a new detection method for latent CLas infection using cuttings. Methods: Root tissues regenerated from cuttings using symptomatic and asymptomatic citrus trees were prepared for real-time a polymerase chain reaction (PCR) test which was used to investigate latent CLas. When some of the regenerated roots were negative for CLas in the first real-time PCR assay, a subsequent cultivation in soils was performed using the CLas-negative cuttings. CLas development during cultivation was evaluated by a second real-time PCR assay using soil-grown roots from seedlings. Results: Previously, CLas had not been detected from leaves of the latent-infected trees in our greenhouse by real-time PCR. In this study, however, CLas was detected at a moderate frequency from the root tissues of cuttings derived from the latent-infected trees, by the same PCR test. For cuttings with regenerated roots that tested negative for CLas by real-time PCR, CLas was frequently detected from roots grown in nursery soil with autoclaving, after cultivation for a month or more. Conclusions: Latent infection with CLas was detectable by real-time PCR using root tissues regenerated by cuttings and roots grown in nursery soil with autoclaving. These results suggest that the new method of investigation would provide great opportunities for early detection of CLas in asymptomatic citrus trees from field surveys, and would accelerate the eradication practice of citrus greening.

The bacterium ‘Candidatus Liberibacter solanacearum’ is a recent plant pathogen of several crops in Solanaceae and Apiaceae and is associated with economically important diseases. The bacterium is a carrot seed borne pathogen that can also be transmitted from potato mother tubers and by psyllid vectors. The psyllid Bactericera trigonica Hodkinson was described carrying CaLso associated with vegetative disorders in carrot and celery crops in Spain and its competence to transmit this phloem-limited bacterium among vegetables is currently being investigated. Here electrical penetration graphs showed that B. trigonica fed in the phloem of carrot and celery and probed the phloem in potato, but not in tomato plants. The bacterium was efficiently transmitted to carrot and celery plants when either single B. trigonica or groups of ten fed on these species. An inoculation access period of 24 hours was sufficient for a single B. trigonica to transmit the bacterium to carrot (67.8%), celery (21.1%) and eventually to potato and tomato (6.0%). Higher transmission rates were obtained with 10 individuals on celery (100%), carrot (80%), potato (10%) and tomato (10%). Bactericera trigonica laid eggs, and the hatched nymphs develop into adult on carrot and celery, but not on potato and tomato. CaLso was detected in 20% of the eggs laid by females carrying the bacterium. The results confirmed that B. trigonica is a vector of the bacterium to carrot and celery, and it is discussed the potential role of this psyllid in the transmission of the pathogen to potato and tomato plants.

Plants inoculated with the huanglongbing (HLB)-associated bacterium, Candidatus Liberibacter asiaticus (CLas) typically must be monitored for 8–10 months to identify differences in susceptibility between genotypes. Continuous light is reported to accelerate development of HLB symptoms and field observations suggest that trees girdled by tags or tree ties showed greater symptoms. Therefore, an experiment was conducted assessing HLB susceptibility as influenced by light/dark periods of 12 hours: 12 hours and 24 hours: 0 hours, in combination with scoring tree trunks to disrupt phloem. Sixty trees of each of three citrus genotypes (‘Kuharske’, previously shown to be HLB resistant; rough lemon, previously shown to be HLB tolerant; and ‘Valencia’, highly HLB susceptible) were bud grafted using two CLas-infected buds (rough lemon and citron) per tree on 26 Mar. 2012, and were placed in controlled growth rooms (one 12 hour light: 12 hour dark and one constant light) on 4 June 2012. Ten trees of each genotype in each growth room were scored 10 cm above the soil (cutting through the bark but not the wood) with a knife on 18 July 2012 and the scoring was repeated at the same scoring wounds on 30 Aug. 2012. Trees were removed from growth rooms on 12 Dec. 2012 and subsequently maintained in a greenhouse. At two to three month intervals between June 2012 and May 2013, HLB symptoms and stem diameter at 5 cm above the soil were assessed, and three leaves per tree were collected for quantitative polymerase chain reaction (qPCR) determination of CLas titer. Six months after inoculation and 3 months following imposition of treatments, the ‘Valencia’ scored in the 12 hour light: 12 hour dark regime, the ‘Valencia’ non scored trees in 24 hours of light and the ‘Kuharske’ scored trees in 24 hours of light displayed higher CLas titers than most other trees. After an additional two months, both scored and non-scored trees of all three genotypes in 24 hours of light had significantly elevated CLas titers compared with trees in 12 hour light: 12 hour dark regime, but within most treatments all three genotypes had titers which were not statistically different from each other. Growth of ‘Kuharske’ and rough lemon was enhanced; whereas ‘Valencia’ growth was reduced when graft-inoculated plants were maintained in continuous light. Scoring enhanced early CLas development in ‘Kuharske’ when combined with continuous light, had no effect in rough lemon, and showed inconsistent effects in ‘Valencia’. Although continuous lighting enhanced disease progression, it did not reveal differences in HLB susceptibility.