Analysis of endophytic bacterial populations in the midvein of citrus leaves infected with Huanglongbing based on 16S rDNA amplicons

doi:10.16178/j.issn.0528-9017.20240461

Abstract

Abstract:

In this paper, the endophytic bacterial populations were analyzed in the midvein of the citrus leaves infected with Candidatus Liberibacter asiaticus which was the agent of citrus Huanglongbing. By conducting 16S rDNA V3-V4 region amplicon sequencing on a total of 24 infected samples (HLB) and 12 control samples (HC) and bioinformatics analysis, and filtering identified 60 annotated microbial OTUs (considered as 60 different microorganisms) were identified. It was found that the HC group had 54 OTUs, while the HLB group only had 38 OTUs. Among these, the HC group had 38 genera annotated at the genus level, while the HLB group had 30 genera. Candidatus Liberibacter was the dominant microrganism with an average percentage of 81.35% in the HLB group, Candidatus Liberibacter was not found in the HC group. Percentages of Candidatus Liberibacter and Roseburia in total were found to differ significantly between HC and HLB groups by t-test analysis. According to Spearman's rank correlation analysis, Candidatus Liberibacter was found to be positively correlated with three genera, Bacteroides, Fusobacterium and Roseburia, and negatively correlated with four genera, Sphingomonas, Amnibacterium, Methylobacterium and Beijerinckiaceae 1174-901-12.

Key words: citrus Huanglongbing, 16S rDNA, endophytic bacterial populations

CLC Number:

S436.66

ZHANG Liping, LIU Shunmin, PU Zhanxu, ZHU Li, LYU Jia. Analysis of endophytic bacterial populations in the midvein of citrus leaves infected with Huanglongbing based on 16S rDNA amplicons[J]. Journal of Zhejiang Agricultural Sciences, 2025, 66(2): 440-444.

Figures/Tables 4

References 9

[1]	JAGOUEIX S, BOVE J M, GARNIER M. The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria[J]. International Journal of Systematic Bacteriology, 1994, 44(3): 379-386.
[2]	JAGOUEIX S, BOVÉ J M, GARNIER M. PCR detection of the two Candidatus Liberobacter species associated with greening disease of citrus[J]. Molecular and Cellular Probes, 1996, 10(1): 43-50.
[3]	GARNIER M, JAGOUEIX-EVEILLARD S, CRONJE P R, et al. Genomic characterization of a Liberibacter present in an ornamental rutaceous tree, Calodendrum capense, in the Western Cape Province of South Africa: proposal of Candidatus Liberibacter africanus subsp. capensis[J]. International Journal of Systematic and Evolutionary Microbiology, 2000, 50 Pt 6: 2119-2125.
[4]	HALBERT S E, MANJUNATH K L. Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: a literature review and assessment of risk in Florida[J]. Florida Entomologist, 2004, 87(3): 330-353.
[5]	施雅曼, 曾吉兴, 薛进军, 等. 黄龙病对柑橘叶片同化物积累和矿质养分运输的影响[J]. 植物营养与肥料学报, 2023, 29(5): 949-960.
[6]	樊晶. 柑橘宿主对黄龙病病原菌侵染的应答机制[D]. 重庆: 重庆大学, 2010.
[7]	TATINENI S, SAGARAM U S, GOWDA S, et al. In planta distribution of Candidatus Liberibacter asiaticus as revealed by polymerase chain reaction (PCR) and real-time PCR[J]. Phytopathology, 2008, 98(5): 592-599.
[8]	郑雪芳, 刘波, 孙大光, 等. 柑橘黄龙病植株内生菌PLFAs多态性研究[J]. 中国生态农业学报, 2012, 20(7): 932-944.
[9]	吴柳, 白晓晶, 文庆利, 等. 柑橘黄龙病病原菌Las在叶圆片嫁接接种的锦橙中早期扩散研究[J]. 园艺学报, 2018, 45(11): 2121-2128.

OTU num	HC组占比/%	HLB组占比/%	Taxonomy
OTU 1	0.848 701	0.786 940	Unassigned
OTU 2	0.149 473	0.211 752	Unassigned
OTU 3	0	0.001 052	Candidatus Liberibacter asiaticus
OTU 4	0.000 307	0.000 045	Methylobacterium fujisawaense
OTU 5	0.000 305	0.000 001	Pantoea brenneri
OTU 6	0.000 095	0	Flavobacteriales endosymbiont
OTU 7	0.000 114	0.000 007	Amnibacterium
OTU 8	0.000 074	0.000 013	Beijerinckiaceae 1174-901-12
OTU 9	0.000 029	0.000 050	Fusobacterium
OTU 10	0.000 029	0.000 004	Methylobacterium sp.
OTU 11	0.000 159	0.000 005	Massilia sp. 9096
OTU 12	0.000 077	0	Hymenobacter
OTU 13	0.000 005	0.000 007	Myxococcales
OTU 14	0.000 029	0	Marmoricola
OTU 15	0.000 095	0.000 003	Hymenobacter
OTU 16	0.000 011	0.000 007	Acinetobacter
OTU 17	0.000 130	0.000 008	Sphingomonas
OTU 18	0.000 019	0.000 004	Bacteria
OTU 19	0.000 013	0.000 007	Methylobacterium
OTU 20	0.000 045	0.000 001	Hymenobacter
OTU 21	0.000 021	0.000 003	Methylobacterium
OTU 22	0.000 011	0.000 020	Bacteroides vulgatus
OTU 23	0.000 011	0.000 003	Rhizobiales bacterium
OTU 24	0.000 027	0.000 001	Myxococcales
OTU 25	0.000 080	0.000 001	Saccharimonadales
OTU 26	0.000 003	0.000 003	Clostridium pasteurianum
OTU 27	0.000 013	0	Spirosoma sp.
OTU 28	0.000 003	0.000 016	Roseburia
OTU 29	0.000 005	0.000 001	Friedmanniella
OTU 30	0.000 008	0.000 001	Unassigned
OTU 31	0.000 005	0.000 005	Mesorhizobium
OTU 32	0.000 008	0	Acetobacteraceae
OTU 33	0.000 005	0	Edaphobacter sp.
OTU 34	0.000 003	0.000 004	Ruminococcus gnavus group
OTU 35	0.000 005	0	Caulobacteraceae
OTU 36	0.000 005	0	Pseudomonas sp.
OTU 37	0.000 013	0.000 004	Lactobacillus
OTU 38	0.000 005	0.000 001	Thermoanaerobacterium
OTU 39	0.000 011	0.000 001	Hymenobacter
OTU 40	0	0.000 001	Clostridium sensu stricto
OTU 41	0.000 011	0	Acidiphilium
OTU 42	0	0.000 004	Larkinella
OTU 43	0.000 013	0	Variovorax
OTU 44	0.000 005	0	Roseomonas sp.
OTU 45	0.000 005	0	Terriglobus
OTU 46	0	0.000 007	Bacteroides
OTU 47	0	0.000 003	Sediminibacterium
OTU 48	0.000 008	0	Cardinium endosymbiont of Marietta sp.
OTU-49	0.000 011	0.000 004	Pseudomonas
OTU 50	0.000 008	0	Actinoplanes sp.
OTU 51	0.000 005	0	Myxococcales
OTU 52	0.000 005	0.000 001	Belnapia soli
OTU 53	0.000 008	0	Bdellovibrio
OTU 54	0.000 008	0	Hymenobacter
OTU 55	0.000 003	0	Acidiphilium
OTU 56	0.000 003	0.000 004	Parasutterella
OTU 57	0.000 021	0.000 004	Comamonas sp. JL25
OTU 58	0.000 005	0	Burkholderia sp.
OTU 59	0.000 016	0	Hymenobacter
OTU 60	0.000 005	0	Myxococcales
OTU 61	0.000 003	0	Escherichia coli TA464
OTU 62	0	0.000 001	Spirosoma sp. TX0653

OTU num	HC组占比/%	HLB组占比/%	Taxonomy
OTU 1	0.848 701	0.786 940	Unassigned
OTU 2	0.149 473	0.211 752	Unassigned
OTU 3	0	0.001 052	Candidatus Liberibacter asiaticus
OTU 4	0.000 307	0.000 045	Methylobacterium fujisawaense
OTU 5	0.000 305	0.000 001	Pantoea brenneri
OTU 6	0.000 095	0	Flavobacteriales endosymbiont
OTU 7	0.000 114	0.000 007	Amnibacterium
OTU 8	0.000 074	0.000 013	Beijerinckiaceae 1174-901-12
OTU 9	0.000 029	0.000 050	Fusobacterium
OTU 10	0.000 029	0.000 004	Methylobacterium sp.
OTU 11	0.000 159	0.000 005	Massilia sp. 9096
OTU 12	0.000 077	0	Hymenobacter
OTU 13	0.000 005	0.000 007	Myxococcales
OTU 14	0.000 029	0	Marmoricola
OTU 15	0.000 095	0.000 003	Hymenobacter
OTU 16	0.000 011	0.000 007	Acinetobacter
OTU 17	0.000 130	0.000 008	Sphingomonas
OTU 18	0.000 019	0.000 004	Bacteria
OTU 19	0.000 013	0.000 007	Methylobacterium
OTU 20	0.000 045	0.000 001	Hymenobacter
OTU 21	0.000 021	0.000 003	Methylobacterium
OTU 22	0.000 011	0.000 020	Bacteroides vulgatus
OTU 23	0.000 011	0.000 003	Rhizobiales bacterium
OTU 24	0.000 027	0.000 001	Myxococcales
OTU 25	0.000 080	0.000 001	Saccharimonadales
OTU 26	0.000 003	0.000 003	Clostridium pasteurianum
OTU 27	0.000 013	0	Spirosoma sp.
OTU 28	0.000 003	0.000 016	Roseburia
OTU 29	0.000 005	0.000 001	Friedmanniella
OTU 30	0.000 008	0.000 001	Unassigned
OTU 31	0.000 005	0.000 005	Mesorhizobium
OTU 32	0.000 008	0	Acetobacteraceae
OTU 33	0.000 005	0	Edaphobacter sp.
OTU 34	0.000 003	0.000 004	Ruminococcus gnavus group
OTU 35	0.000 005	0	Caulobacteraceae
OTU 36	0.000 005	0	Pseudomonas sp.
OTU 37	0.000 013	0.000 004	Lactobacillus
OTU 38	0.000 005	0.000 001	Thermoanaerobacterium
OTU 39	0.000 011	0.000 001	Hymenobacter
OTU 40	0	0.000 001	Clostridium sensu stricto
OTU 41	0.000 011	0	Acidiphilium
OTU 42	0	0.000 004	Larkinella
OTU 43	0.000 013	0	Variovorax
OTU 44	0.000 005	0	Roseomonas sp.
OTU 45	0.000 005	0	Terriglobus
OTU 46	0	0.000 007	Bacteroides
OTU 47	0	0.000 003	Sediminibacterium
OTU 48	0.000 008	0	Cardinium endosymbiont of Marietta sp.
OTU-49	0.000 011	0.000 004	Pseudomonas
OTU 50	0.000 008	0	Actinoplanes sp.
OTU 51	0.000 005	0	Myxococcales
OTU 52	0.000 005	0.000 001	Belnapia soli
OTU 53	0.000 008	0	Bdellovibrio
OTU 54	0.000 008	0	Hymenobacter
OTU 55	0.000 003	0	Acidiphilium
OTU 56	0.000 003	0.000 004	Parasutterella
OTU 57	0.000 021	0.000 004	Comamonas sp. JL25
OTU 58	0.000 005	0	Burkholderia sp.
OTU 59	0.000 016	0	Hymenobacter
OTU 60	0.000 005	0	Myxococcales
OTU 61	0.000 003	0	Escherichia coli TA464
OTU 62	0	0.000 001	Spirosoma sp. TX0653

Taxonomy	HC组总序列数	HLB组总序列数	HC组占比/%	HLB组占比/%
Candidatus Liberibacter	0	794	0	81.35
Methylobacterium	140	44	21.08	4.51
Fusobacterium	11	38	1.66	3.89
Bacteroides	4	20	0.60	2.05
Beijerinckiaceae 1174-901-12	32	12	4.82	1.23
Roseburia	1	12	0.15	1.23
Sphingomonas	49	6	7.38	0.61
Amnibacterium	43	5	6.48	0.51
Acinetobacter	4	5	0.60	0.51
Hymenobacter	95	4	14.31	0.41
Massilia	60	4	9.04	0.41
Mesorhizobium	2	4	0.30	0.41
Comamonas	8	3	1.20	0.31
Pseudomonas	6	3	0.90	0.31
Lactobacillus	5	3	0.75	0.31
Parasutterella	1	3	0.15	0.31
Ruminococcus gnavus group	1	3	0.15	0.31
Larkinella	0	3	0	0.31
Clostridium sensu stricto 12	1	2	0.15	0.20
Sediminibacterium	0	2	0	0.20
Pantoea	115	1	17.32	0.10
Spirosoma	5	1	0.75	0.10
Thermoanaero bacterium	2	1	0.30	0.10
Friedmanniella	2	1	0.30	0.10
Belnapia	2	1	0.30	0.10
Spirosoma	5	1	0.75	0.10
Thermoanaero bacterium	2	1	0.30	0.10
Friedmanniella	2	1	0.30	0.10
Belnapia	2	1	0.30	0.10
Clostridium sensu stricto 1	0	1	0	0.10
Candidatus Uzinura	36	0	5.42	0
Marmoricola	11	0	1.66	0
Variovorax	5	0	0.75	0
Acidiphilium	5	0	0.75	0
Bdellovibrio	3	0	0.45	0
Candidatus Cardinium	3	0	0.45	0
Actinoplanes	3	0	0.45	0
Burkholderia-Caballeronia-Paraburkholderia	2	0	0.30	0
Edaphobacter	2	0	0.30	0
Terriglobus	2	0	0.30	0
Roseomonas	2	0	0.30	0
Escherichia-Shigella	1	0	0.15	0

Taxonomy	HC组总序列数	HLB组总序列数	HC组占比/%	HLB组占比/%
Candidatus Liberibacter	0	794	0	81.35
Methylobacterium	140	44	21.08	4.51
Fusobacterium	11	38	1.66	3.89
Bacteroides	4	20	0.60	2.05
Beijerinckiaceae 1174-901-12	32	12	4.82	1.23
Roseburia	1	12	0.15	1.23
Sphingomonas	49	6	7.38	0.61
Amnibacterium	43	5	6.48	0.51
Acinetobacter	4	5	0.60	0.51
Hymenobacter	95	4	14.31	0.41
Massilia	60	4	9.04	0.41
Mesorhizobium	2	4	0.30	0.41
Comamonas	8	3	1.20	0.31
Pseudomonas	6	3	0.90	0.31
Lactobacillus	5	3	0.75	0.31
Parasutterella	1	3	0.15	0.31
Ruminococcus gnavus group	1	3	0.15	0.31
Larkinella	0	3	0	0.31
Clostridium sensu stricto 12	1	2	0.15	0.20
Sediminibacterium	0	2	0	0.20
Pantoea	115	1	17.32	0.10
Spirosoma	5	1	0.75	0.10
Thermoanaero bacterium	2	1	0.30	0.10
Friedmanniella	2	1	0.30	0.10
Belnapia	2	1	0.30	0.10
Spirosoma	5	1	0.75	0.10
Thermoanaero bacterium	2	1	0.30	0.10
Friedmanniella	2	1	0.30	0.10
Belnapia	2	1	0.30	0.10
Clostridium sensu stricto 1	0	1	0	0.10
Candidatus Uzinura	36	0	5.42	0
Marmoricola	11	0	1.66	0
Variovorax	5	0	0.75	0
Acidiphilium	5	0	0.75	0
Bdellovibrio	3	0	0.45	0
Candidatus Cardinium	3	0	0.45	0
Actinoplanes	3	0	0.45	0
Burkholderia-Caballeronia-Paraburkholderia	2	0	0.30	0
Edaphobacter	2	0	0.30	0
Terriglobus	2	0	0.30	0
Roseomonas	2	0	0.30	0
Escherichia-Shigella	1	0	0.15	0