Publications

4509

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.

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.

‘Candidatus Liberibacter asiaticus’ (CLas) is a pathogen causing Huanglongbing (HLB, yellow shoot disease), which is highly destructive to citrus production. The CLas strains harbor prophages. We identified two unique prophages, designated as P-PA19-1 and P-PA19-2, in CLas strain PA19 from Pakistan using next-generation sequencing analysis. P-PA19-1 prophage has high sequence similarity (identity: 78.23%) at the early-gene region of prophage SC1 (Type 1), but it is significantly divergent in the late-gene region (identity: 62.03%). P-PA19-2 was highly similar to SC2 (Type 2) in the late gene region (identity: 97.96%), and also in the early gene region except for a deletion of a 7,179-bp nucleotide sequence that contains a CRISPR/cas system in SC2. Both P-PA19-1 and P-PA19-2 had circular plasmid forms, and only P-PA19-2 was found integrated in the PA19 chromosome. The two new prophages were only found in Pakistani samples. Identification of prophages enhances our understanding of CLas genomic diversity and also the biology and evolution of CLas prophages.

Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of ‘Candidatus Liberibacter asiaticus’ (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of ‘Ca. L. asiaticus’ was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The ‘Ca. L. asiaticus’ isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of ‘Ca. L. asiaticus’ and CTV isolates and their association with citrus decline in Bhutan.