Introduction
⌅“Huanglongbing” (HLB), also known as citrus greening, is the most serious disease threating the citrus industry worldwide (11.
Bové J-M. “Huanglongbing” or yellow shoot, a disease of Gondwanan
origin: will it destroy citrus worldwide? Phytoparasitica. 2014;
42:579-583. https://doi.org/10.1007/s12600-014-0415-4
). Three phloem-limited bacteria transmitted by
sap-sucking insects have been associated with its occurrence and named
according to their geographic origin as ‘Candidatus Liberibacter asiaticus’ (Asia and North America), ‘Ca. L. africanus’ (Africa), and ‘Ca. L. americanus’ (Brazil) (22.
Da Graça JV, Douhan GW, Halbert SE, Keremane ML, Lee RF, Vidalakis G,
Zhao, H. “Huanglongbing”: an overview of a complex pathosystem ravaging
the world’s citrus. Journal of Integrative P lant Biology.
2016;58(4):373-387. https://doi.org/10.1111/jipb.12437
). The most widespread pathogen is ‘Ca. L. asiaticus’, endemic in Asia, but detected as epidemic in the other geographic areas. In Cuba, recent studies reaffirmed ‘Ca. L. asiaticus’ as the only liberibacter associated with the occurrence of the disease (33. Luis M, Collazo C, Llauger R, Blanco E, Peña I, López D, et al. Occurrence of citrus “huanglongbing” in Cuba and association of the disease with ‘Candidatus Liberibacter asiaticus’. Journal Plant Pathology. 2009;91(3):709-712.
, 44. Luis-Pantoja M, Paredes-Tomás C, Uneau Y, Myrie W, Morillon R, Satta E, et al. Identification of ‘Candidatus Phytoplasma’ species in “huanglongbing” infected citrus orchards in the Caribbean. Eur. J. Plant Pathol. 2021; 160:185-198. https://doi.org/10.1007/s10658-021-02234-7
). The methods used to transmit these bacteria experimentally include tissue grafting and psyllid inoculation (55. Albrecht U, Hall DG, Bowman K.D. Transmission efficiency of ‘Candidatus Liberibacter asiaticus’ and progression of “huanglongbing” disease in
graft- and psyllid-inoculated citrus. HortScience. 2014; 49(3):367-377.
, 66.
Ammar ED, Hall DG, Hosseinzadeh S, Heck M. The quest for a non-vector
psyllid: Natural variation in acquisition and transmission of the
“huanglongbing” pathogen ‘Candidatus Liberibacter asiaticus’ by Asian citrus psyllid isofemale lines. PloS One. 2018;13(4): e0195804. https://doi.org/10.1371/journal.pone.0195804
, 77.
Ammar ED, George J, Sturgeon K, Stelinski LL, Shatters RG. Asian citrus
psyllid adults inoculate “huanglongbing” bacterium more efficiently
than nymphs when this bacterium is acquired by early instar nymphs.
Scientific Reports. 2020; 10:18244. https://doi.org/10.1038/s41598-020-75249-5
). The Asian citrus psyllid Diaphorina citri Kuwayama is reported as the main insect vector of ‘Ca. L. asiaticus’, as the transmission of the bacterium to healthy citrus plants using psyllids demonstrated (88. Capoor SP, Rao DG, Viswanath S. Diaphorina citri Kuway., a vector of the greening disease of citrus in India. Indian Journal Agricultural Sciences. 1967; 37:572-579.
). Low rates of successful inoculation of the bacterium with D. citri together with the pathogen acquisition from infected plant and the
latency period needed by the pathogen to multiply and circulate within
the psyllid, were reported by field observations (99. Canale MC, Tomaseto AF, Haddad ML, Coletta-Filho, HD, Lopes JRS. Latency and persistence of ‘Candidatus Liberibacter asiaticus’ in its psyllid vector, Diaphorina citri (Hemiptera: Lividae). Phytopathology. 2017; 107:264-272. https://doi.org/10.1094/PHYTO-02-16-0088-R
). Additionally, the transmission efficiency was
reported highly influenced by the environmental conditions under which
the plants were maintained (1010. Lopes SA, Frare GF, Bertolini E, Cambra M, Fernandes NG, Ayres AJ, et al. Liberibacters associated with citrus “huanglongbing” in Brazil: ‘Candidatus Liberibacter asiaticus’ is heat tolerant, ‘Ca. L. americanus’ is heat sensitive. Plant Disease. 2009; 93:257-262. https://doi.org/10.1094/PDIS-93-3-0257
, 1111. Lopes SA, Luiz FQBF, Martins EC, Fassini CG, Sousa MC, Barbosa JC, et al. ‘Candidatus Liberibacter asiaticus’ titers in citrus and acquisitions rates by Diaphorina citri are decreased by higher temperatures. Plant Disease. 2013; 97:1563-1570. https://doi.org/10.1094/PDIS-11-12-1031-RE
, 1212. Lopes SA, Luiz FQBF, Oliveira HT, Cifuentes-Arenas JC, Raiol-Junior LL. Seasonal variation of ‘Candidatus Liberibacter asiaticus’ titers in new shoots of citrus in distinct environments. Plant Disease. 2017; 101:583-590. https://doi.org/10.1094/PDIS-06-16-0859-RE
). Despite the lower variability of the results obtained by grafting-methods than those obtained by using D. citri, the insect transmission is a critical point as it provides the evidence needed to confirm the insect capability as vector (1313.
Bosco D, Tedeschi R. Insect vector transmission assays. In: Dickinson
M, Hodgetts J, editors. Phytoplasma: Methods and protocols (Internet).
Totowa, N.Y.: Humana Press; 2012. https://doi.org/10.1007/978-1-62703-089-2
). D. citri was reported in Cuba in 1999,
but its ability to transmit HLB, assumed for years, has not yet been
confirmed experimentally. The objective of this study was to verify and
confirm the ‘Ca. L. asiaticus’ transmission to healthy plants of Citrus aurantium by D. citri under Cuban conditions.
Materials and methods
⌅Insect and plant material
⌅Specimens of D. citri were collected from citrus orchards with high incidence and severity of
HLB in Jaguey Grande, Matanzas province. The insects were caught on
symptomatic plants using a manual trap consisting of a 15 mL Falcon tube
coupled to another 50 mL tube (1414. Paredes-Tomás C, Luis-Pantoja M, Rodríguez-Tapia JL, Bertaccini A. Haplaxius crudus transmission of 'Candidatus Phytoplasma palmae' to coconut seedlings in Cuba. Phytopathogenic Mollicutes. 2023; 13(2): 177-182. https://doi.org/10.5958/2249-4677.2023.00078.6
). Two transmission trials were conducted (Fig. 1A, B and D). In the first transmission test, three seedlings of sour orange (Citrus aurantium) were used, and each plant was individually caged with 10-15 insects. In the second transmission test, 42 seedlings of C. aurantium were obtained from seeds germinated under insect-proof greenhouse and caged together with 200 insects. Before and after ‘Ca.
L. asiaticus’ inoculation in the seedlings, its presence was verified
by nested PCR in both experiments. The insects were left on the plants
until they died, at which point they were recovered and preserved in 95%
ethanol for subsequent molecular analysis. All the plants were
maintained under insect-proof greenhouse conditions and weekly inspected
for symptom appearance for ten months.
DNA extraction and nested PCR
⌅From
each citrus plant used for transmission trials, 0.4 - 0.6 g of midribs
and petioles were taken for DNA extraction. Pools of 10 insects were
used (three pools in the first assay and 20 pools in the second assay).
In the second trial, the pools were randomly selected at the time of
molecular analysis. In both cases (plants and insects), the total DNA
was extracted following the CTAB method described by Murray et al. (1515. Murray MG, Thompson WF. Rapid isolation of high molecular weight plant DNA. Nucleic Acid Research. 1980; 8(19):4321-4325.
).
The DNA pellets were suspended in 100 μl of sterile distilled water.
Amplification of ribosomal DNA was performed with the universal primer
pair fD1/rP1 (1616.
Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA
amplification for phylogenetic study. Journal of Bacteriology. 1991;
173:697-703.
) followed by nested PCR with the internal primers OI1/OI2c (1717. Jagoueix S, Bové J-M, Garnier M. PCR detection of the two ‘Candidatus’ liberobacter species associated with greening disease of citrus. Molecular and Cellular Probes 1996; 10:43-50.
), which targets the 16S rRNA gene from ‘Ca.
Liberibacter’. DNA extracts from healthy plants maintained under
greenhouse conditions and from a field collected plant tested positive
for ‘Ca. L. asiaticus’ (44. Luis-Pantoja M, Paredes-Tomás C, Uneau Y, Myrie W, Morillon R, Satta E, et al. Identification of ‘Candidatus Phytoplasma’ species in “huanglongbing” infected citrus orchards in the Caribbean. Eur. J. Plant Pathol. 2021; 160:185-198. https://doi.org/10.1007/s10658-021-02234-7
) were used as negative and positive controls,
respectively. The PCR-reaction mixture contained 0.8 µM of each primer,
50 ng of DNA, 12.5 µL of 2X TopTaq Master Mix (Qiagen), and 1.5 U of Taq
DNA polymerase (Roche) for a final volume of 25 µL. PCR reactions were
carried out in a Biometra Thermal Cycler (Biometra, Germany) using an
initial denaturation stage at 94°C (only for the direct reaction) for 3
min; followed by 35 cycles (38 cycles for the nested reaction) of
denaturation at 94°C for 1 min (40 seconds for the nested reaction),
annealing at 52°C for 1 min (62°C for 40 seconds on the nested reaction)
and an extension at 72°C for 1 min; and a final extension at 72°C for 5
min (only for the direct reaction). The amplified DNAs (5 µL) were
separated by electrophoresis in 1% agarose gel (1X Tris-acetate/EDTA),
stained with ethidium bromide and visualized under ultraviolet light in a
transilluminator.
Sequences and analysis
⌅Four
amplicons (two from plants and two from insects in each experiment)
were purified with “High pure PCR product purification Kit” (Roche),
according to the manufacturer’s instructions and sequenced in both
directions by the Sanger Sequencing Service (Microsynth Seqlab,
Germany). The phylogenetic analysis was performed on the16S rRNA gene
partial sequences of ‘Ca. L. asiaticus’ from this work and of those from 54 of the 175 strains previously used (1818. Nelson WR. Reported single nucleotide polimorphisms on the 16S rRNA gene do not support haplotypes of ‘Candidatus Liberibacter asiaticus’. Citrus Research Technology. 2012;33(2):75-79. https://doi.org/10.5935/2236-3122.20120009
) selected from the GenBank database for their
geographical representativeness. After multiple alignment of the
sequences with ClustalW, a common segment of 852 bp was trimmed and used
in the subsequent analyses. The 16S ribosomal gene sequence of Escherichia coli (GenBank accession number V00348) was used as an outgroup to root the
phylogenetic trees produced. The evolutionary history was inferred by
using the Maximum Likelihood method based on the Kimura 2-parameter
model (1919
Kimura M. A simple method for estimating evolutionary rates of base
substitutions through comparative studies of nucleotide sequences. J.
Mol. Evol. 1980; 16:111-120.
). Initial tree(s) for the
heuristic search were obtained automatically by applying Neighbor-Join
and BioNJ algorithms to a matrix of pairwise distances estimated using
the Maximum Composite Likelihood (MCL) approach, and then selecting the
topology with superior log likelihood value. Bootstrap with 1,000
repetitions was used to estimate the stability of clades. Evolutionary
analyses were conducted in MEGA7 (2020.
Kumar S, Stecher G. Tamura K.. MEGA7: molecular evolutionary genetics
analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016;
33(7):1870-1874. https://doi.org/10.1093/molbev/msw054
).
To assess the presence of Single Nucleotide Polymorphisms (SNPs), 56 16S ribosomal gene sequences (including those from this study plus 54 sequences from GenBank) were aligned and trimmed to an 850 bp common fragment. Only the SNPs common to more than one strain were considered in the analysis, in order to identify the SNP-based lineages.
Results and discussion
⌅In both transmission trials, almost all the plants showed symptoms 6 months after inoculation. The symptoms included asymmetric blotching mottle and intense chlorosis of the leaves (Fig. 1C and E). Symptomatic plants as well as most of the pools of insect tested positive for ‘Ca. L. asiaticus’, only a few plants and some of the pools of insects (from the second trial) tested negative (Table 1).
| ‘Ca. L. asiaticus’ PCR detection | ||
|---|---|---|
| Number of positive plants/Total | Positive pools of insects /Total | |
| Trial 1 | 1/1 | 1/1 |
| 1/1 | 1/1 | |
| 1/1 | 1/1 | |
| Trial 2 | 30/42 | 17/20 |
As the partial sequences obtained from plants were identical to each other and nucleotide identity was observed between both pools of insects, one sequence per host was deposited in GenBank (accession numbers OQ892131 and OQ892130, respectively). These sequences shared 100% identity to each other in the 1,090 bp fragment overlapping and with the sequence of the strain Las_36 (GenBank accession number MK142763). In the phylogenetic tree, they clustered with strains from several regions in the American and Asian continents, including the Cuban strains previously reported (Fig. 2).
Following multiple alignment of 56 16S ribosomal gene sequences (including those from this study plus 54 sequences from GenBank), an 850 bp common fragment was used to assess SNPs numbers and their distribution across the selected accessions. The comparison of this portion of the 16S rRNA gene sequences of ‘Ca. L. asiaticus’ strains showed that 29 accessions were identical (including all Cuban sequences and others from American and Asian areas). A total of 50 SNPs was observed in the remaining 27 sequences, mostly occurring in a single accession (35 SNPs, 70%). However, 15 SNPs (30%) were observed common to more than one strain, suggesting the presence of ‘Ca. L. asiaticus’ populations with 16S rRNA gene variability; in particular nine SNP-based lineages were identified (Table 2).
| Position of the nucleotide in the trimmed sequence | SNP | GenBank accession number of sequences (Country/State) | Lineage number |
|---|---|---|---|
| 40 | A/T | FJ750456; FJ750457 (USA: Florida) | 1 |
| 163 | T/C | DQ432005; DQ432002; DQ431999 (China: Guangdong) | 2 |
| 186 | A/T | DQ432005; DQ432002; DQ431999 (China: Guangdong) | 2 |
| 210 | A/G | DQ432004; DQ431997 (China: Guangdong) | 3 |
| 234 | C/T | FJ750456; FJ750457 (USA: Florida) | 1 |
| 508 | C/T | FJ750456; FJ750457 (USA: Florida) | 1 |
| 596 | C/T | DQ778016 (China: Jianxi); FJ263702 (USA: Florida) | 4 |
| 597 | T/G | DQ778016 (China: Jianxi); FJ263702 (USA: Florida) | 4 |
| 632 | A/G | FJ263701; FJ263700 (USA: Florida) | 5 |
| 669 | T/A | DQ432003 (China: Jianxi); GQ369792 (India: Saktara-Tripura) | 6 |
| 675 | T/C | DQ303210; DQ157275 (China: Guangdong) | 7 |
| 682 | A/C | FJ750459; FJ750458 (USA: Louisiana) | 8 |
| A/- | FJ263704; FJ263699 (USA: Florida) | 9 | |
| 705 | -/C | FJ750459; FJ750458 (USA: Louisiana) | 8 |
| 809 | A/T | DQ432005; DQ432002; DQ431999 (China: Guangdong) | 2 |
The epidemiology of vector-borne plant diseases directly depends on the insect vector (2121.
Johannesen J, Foissac X, Kehrli P, Maixner M. Impact of vector
dispersal and host-plant fidelity on the dissemination of an emerging
plant pathogen. PLoS ONE. 2012; 7(12): e51809. https://doi.org/10.1371/journal.pone.0051809
), and the precise identification of the insect
vector species is a critical step. Despite the accepted hypothesis of
the role of D. citri in the spread of ‘Ca. L. asiaticus’ in the Cuban national context (2222. González C, Hernández D, Cabrera RI, Tapia JR. Diaphorina citri Kuw., inventario y comportamiento de los enemigos naturales en la
citricultura cubana. FAO Plant Protection Bulletin. 2007: 3-9.
),
there is not experimental confirmation to support this assumption. The
experimental trials carried out with naturally infected D. citri adults collected from citrus orchards with high incidence of HLB confirmed the ability of this insect to transmit ‘Ca.
L. asiaticus’ to citrus seedlings in Cuban environments too. In all
cases in which field-collected infected insects were used on
experimental plants, they were able to transmit the bacterium, and the
plants showed HLB symptoms after six to eight months, a time span that
corresponds with the incubation period reported for ‘Ca. L. asiaticus’ (2323.
Bové J-M. Huanglongbing: a destructive, newly-emerging, century-old
disease of citrus. Journal Plant Pathology. 2006; 88(1):7−37.
, 2424.
De-Souza IP, Galdeano, DM, Lopes JRS, Machado MA. Development on
infected citrus over generations increases vector infection by 'Candidatus Liberibacter asiaticus’ in Diaphorina citri. Insects. 2020; 11(8):469. https://doi.org/10.3390/insects11080469
, 2525. Lopes SA, Cifuentes-Arenas JC. Protocol for successful transmission of ‘Candidatus Liberibacter asiaticus’ from citrus to citrus using Diaphorina citri. Phytopathology. 2021;111(12):2367-2374. https://doi.org/10.1094/PHYTO-02-210076-R
). These results were confirmed by PCR detection
and sequence identification of the bacterium. In the second trial,
despite the collective manner of being inoculated, almost all the plants
showed symptoms. The positive tests for most of the insect pools are
consistent with the infectivity ability reported for D. citri (88. Capoor SP, Rao DG, Viswanath S. Diaphorina citri Kuway., a vector of the greening disease of citrus in India. Indian Journal Agricultural Sciences. 1967; 37:572-579.
).
The adults used in the transmission study were collected from
high-infected orchards; thus, it is very likely that they emerged from
nymphs under infected conditions, a fact that could explain the high
transmission ability observed (2626.
Inoue H, Ohnishi I, Ito T, Tomimura K, Miyata S, Iwanami T, Ashihara,
W. Enhanced proliferation and efficient transmission of ‘Candidatus Liberibacter asiaticus’ by adult Diaphorina citri after adquisition feeding in the nymphal stage. Annals of Applied Biology. 2009; 155:29-36. https://doi.org/10.1111/j.1744-7348.2009.00317.x
).
The four 16S rRNA amplicons obtained from
two plants and two pools of insects sharing 100% nucleotide identity
each other and clustering together with ‘Ca. L. asiaticus’
strains in a phylogenetic subclade, and the identity detected between
the Cuban strains and those previously reported (33. Luis M, Collazo C, Llauger R, Blanco E, Peña I, López D, et al. Occurrence of citrus “huanglongbing” in Cuba and association of the disease with ‘Candidatus Liberibacter asiaticus’. Journal Plant Pathology. 2009;91(3):709-712.
, 44. Luis-Pantoja M, Paredes-Tomás C, Uneau Y, Myrie W, Morillon R, Satta E, et al. Identification of ‘Candidatus Phytoplasma’ species in “huanglongbing” infected citrus orchards in the Caribbean. Eur. J. Plant Pathol. 2021; 160:185-198. https://doi.org/10.1007/s10658-021-02234-7
), suggest the presence of low genetic variability
in the Cuban strains. This was further confirmed by the alignment of
the 16S ribosomal gene partial sequences of a number of ‘Ca. L.
asiaticus’ strains, including those obtained in this work. Moreover, the
identification of nine 16Sr SNP lineages is consistent with what
Adkar-Purushothama et al. reported (2727. Adkar-Purushothama CR, Quaglino F, Casati P, Gottravalli Ramanayaka J, Bianco, PA. Genetic diversity among ‘Candidatus Liberibacter asiaticus’ isolates based on single nucleotide
polymorphisms in 16S rRNA and ribosomal protein genes. Annals of
Microbiology. 2009; 59(4):681-688. https://doi.org/10.1007/bf03179208
). Our analysis suggested the presence of genetic
variability in the strains from some areas of China and USA. However,
the Cuban sequences (together with other 27 sequences) belonged to a
lineage without SNPs, which supports the presence of low genetic
variability for bacteria infecting citrus in this country. This could
imply the presence of a stable bacterial population and epidemic in
severe stable conditions. Despite the presence of molecular variability
in the 16S ribosomal gene sequence, this region has a high degree of
conserved information confirmed also by other authors (1818. Nelson WR. Reported single nucleotide polimorphisms on the 16S rRNA gene do not support haplotypes of ‘Candidatus Liberibacter asiaticus’. Citrus Research Technology. 2012;33(2):75-79. https://doi.org/10.5935/2236-3122.20120009
, 2727. Adkar-Purushothama CR, Quaglino F, Casati P, Gottravalli Ramanayaka J, Bianco, PA. Genetic diversity among ‘Candidatus Liberibacter asiaticus’ isolates based on single nucleotide
polymorphisms in 16S rRNA and ribosomal protein genes. Annals of
Microbiology. 2009; 59(4):681-688. https://doi.org/10.1007/bf03179208
); therefore, additional molecular markers, such
as prophage regions or numbers of simple tandem repeats, should be
developed to conduct more focused epidemiological studies (2828.
Paredes-Tomás C, Luis-Pantoja M, Ramos-Leal M and Bertaccini A. 2024.
Obtaining positive controls for the detection of two polymorphic regions
in Cuban strains of 'Candidatus Liberibacter asiaticus'. Rev. Protección Veg. 2024: 39: 1-5. Cu-ID: 2247/v39e09.
). This is the first experimental confirmation of the ability of D. citri to transmit ‘Ca. L. asiaticus’ to sour orange (Citrus aurantium)
seedlings in Cuba. More epidemiological data should be developed to
provide information that allows a better understanding of the HLB
pathosystem in this and other neighboring countries.
Conclusions
⌅The ability of D. citri to transmit strains of ‘Ca. L. asiaticus’ to sour orange (Citrus aurantium) seedlings under our conditions was successfully confirmed for the first time. This is the first report of experimental evidence supporting the role of D. citri as a vector of HLB in Cuba.