Publications

4618

Abstract Whole‐genome sequencing of “ Candidatus Liberibacter asiaticus” (Las) indicated some polymorphic gene regions enabling the molecular characterization of this bacterium. Although the population diversity of Las in China has been previously studied, no reports have used a combination of a prophage region and short tandem repeat (STR) loci for phylogenetic relationship characterization. In this study, we investigated the genetic diversity and structure of 667 Las strains from nine provinces in southern China using multiple genetic loci including three type‐specific prophage loci, two STR loci, and a miniature inverted‐repeat transposable element (MITE) region. The results indicated that the genetic diversity varied according to the gene loci used. The prophage regions, including the MITEs, revealed significant genetic differences of Las populations in Yunnan and Guizhou from other provinces, while the STR loci only indicated a difference of Las population in Guizhou from those of other provinces. In particular, the Las population shown to be diverse in Jiangxi at the CLIBASIA_01215_STR locus was not diverse when measured using the other loci. Considering all loci, the Las populations from Yunnan and Guizhou were different, those from Guangdong and Guangxi were complex, while the Las population in Jiangxi was comparatively simple. Las populations from five neighbouring provinces, including Guangdong, Guangxi, Hunan, Fujian, and Jiangxi were grouped in a big cluster. The Las population structure changed with age according to the prophage type. This study evaluated the method used for studying the molecular diversity of Las populations and provided more detailed information on the geographical origin and epidemic trend of Chinese Las populations.

Abstract“Huanglongbing” (HLB) is one of the most devastating diseases of citrus orchards worldwide. Samples from 183 citrus plants of different cultivars and rootstock/cultivar combinations, showing HLB symptoms in three Caribbean countries (Cuba, Jamaica, and Guadeloupe-France), were collected to verify the possible co-infection of ‘Candidatus Phytoplasma’ and ‘Candidatus Liberibacter’ species. The 64% of the samples resulted positive to the ‘Ca. L. asiaticus’ and the 27% to diverse ‘Ca. Phytoplasma’-related species, moreover about the 14% of the samples infected with ‘Ca. Liberibacter’ were also found positive to phytoplasmas, indicating the presence of mixed infection especially in the orchards located in Cuba. Moreover, in one of the samples from Jamaica mixed phytoplasma infection was detected. Moreover the detection of only phytoplasmas in 11 symptomatic citrus samples collected from Cuba and Guadeloupe without ‘Ca. Liberibacter’ detection, confirmed that the symptomatology cannot be the sole criterium to discriminate between the presence of the two pathogens, and molecular detection is necessary to identify single or mixed infections. Diaphorina citri insects collected from Cuba and Guadeloupe resulted infected with ‘Ca. L. asiaticus’ confirming its active role in the dissemination of the pathogen. Only one insect of the Cicadidae family, collected in Guadeloupe, was found positive for phytoplasma presence. Considering that the phytoplasmas belonging to some ‘Candidatus species’ were detected in the three countries in different citrus varieties, a relevant role as phytoplasma reservoir can be attribute to citrus orchards.

In 2019, citrus production in Florida declined by more than 70%, mostly because of Huanglongbing (HLB), which is caused by the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas). Thermotherapy for HLB-affected trees was proposed as a short-term management solution to maintain field productivity. It was hypothesized that thermotherapy could eliminate HLB from affected branches; therefore, the study objectives were to show which time–temperature combinations eliminated CLas from woody tissues. Hardening, rounded Valencia twigs collected from HLB-affected field trees were treated in a steam chamber at different time–temperature combinations (50°C for 60 s; 55°C for 0, 30, 60, 90, and 120 s; 60°C for 30 s; and an untreated control). Three independent repetitions of 13 branches per treatment were grafted onto healthy rootstocks and tested to detect CLas after 6, 9, and 12 months. For the RNA-based CLas viability assay, three branches per treatment were treated and bark samples were peeled for RNA extraction and subsequent gene expression analyses. During the grafting study, at 12 months after grafting, a very low frequency of trees grafted with twigs treated at 55°C for 90 s and 55°C for 120 s had detectable CLas DNA. In the few individuals with CLas, titers were significantly lower (P ≤ 0.0001) and could have been remnants of degrading DNA. Additionally, there was a significant decrease (P ≤ 0.0001) in CLas 16S rRNA expression at 55°C for 90 s, 55°C for 120 s, and 60°C for 30 s (3.4-fold change, 3.4-fold change, and 2.3-fold change, respectively) in samples 5 days after treatment. Heat injury, not total CLas kill, could explain the limited changes in transcriptional activity; however, failed recovery and eventual death of CLas resulted in no CLas detection in most of the grafted trees treated with the highest temperatures or longest durations.