携带不同原噬菌体的黄龙病菌在柑橘木虱体内的增殖及致病力

doi:10.3864/j.issn.0578-1752.2022.04.008

摘要/Abstract

摘要：

【背景】在中国,柑橘黄龙病（citrus Huanglongbing,HLB）是由候选韧皮部杆菌亚洲种（‘Candidatus Liberibacter asiaticus’,CLas）所引起的一种毁灭性病害,严重威胁着柑橘产业的可持续发展。前期研究表明CLas全基因组上鉴定出3种单独类型的原噬菌体基因序列,而广东黄龙病菌主要以Type-2原噬菌体类型和Type-1+3混合原噬菌体类型为主。这两种CLas菌株在传病媒介——柑橘木虱（Diaphorina citri）体内的增殖及其对寄主植物的致病力差异依然不清楚。【目的】比较Type-2-CLas菌株和Type-1+3-CLas菌株在柑橘木虱体内的增殖能力和对砂糖橘（Citrus reticulata Blanco cv. Shatangju）的致病力差异。【方法】嫁接准备含有不同CLas菌株的砂糖橘,分别将50头柑橘木虱若虫和50头成虫放置到含有不同CLas菌株的砂糖橘嫩梢上刺吸获菌 6、12、18 d,利用荧光定量PCR检测并统计柑橘木虱若虫期、成虫期对Type-2-CLas和Type-1+3-CLas的获菌率和获菌量差异。饲养分别携带Type-2-CLas和Type-1+3-CLas菌株的柑橘木虱种群,分别将携带Type-2-CLas和Type-1+3-CLas菌株的20头柑橘木虱成虫置于健康砂糖橘嫩梢上刺吸2周,移除木虱后统计柑橘木虱获菌率和获菌量,每月追踪砂糖橘症状变化和叶片病菌浓度,利用光学显微镜观察接种360 d时叶片韧皮部和薄壁细胞形态学变化情况。【结果】柑橘木虱以若虫形态对Type-2-CLas和Type-1+3-CLas菌株的获菌率和获菌量无显著差异,但柑橘木虱以成虫形态对Type-2-CLas的获菌率和获菌量显著高于Type-1+3-CLas菌株。在虫传后120 d时接种Type-2-CLas菌株的叶片附近新叶会呈现更严重的斑驳症状。Type-2-CLas菌株侵染砂糖橘一年后抽出的新叶转绿不正常,出现均匀黄化、小叶的明显症状,而Type-1+3-CLas菌株新叶症状则为典型斑驳和革质化。叶片解剖观察结果发现,在叶片病菌浓度相近的情况下,相比于接种了Type-1+3-CLas 的叶片,接种了Type-2-CLas菌株的叶片韧皮部细胞发生更严重的细胞塌缩,在叶脉和叶肉薄壁细胞内造成较多的淀粉粒积累。【结论】Type-2-CLas比Type-1+3-CLas更容易在柑橘木虱成虫体内增殖,且达到较高的病菌浓度,间接证明了Type-2-CLas菌株具有高传染性。感染Type-2-CLas的新叶呈现均匀黄化、叶小的症状,而感染Type-1+3-CLas的新叶呈现较为斑驳和革质化的症状,Type-2-CLas比Type-1+3-CLas造成更严重的叶脉韧皮部塌缩和淀粉粒积累情况,说明Type-2-CLas比Type-1+3-CLas对柑橘具有更强的致病力。

关键词: 柑橘黄龙病, 候选韧皮部杆菌亚洲种, 原噬菌体, 柑橘木虱, 致病力

Abstract:

【Background】In China, citrus Huanglongbing is a destructive disease associated with ‘Candidatus Liberibacter asiaticus’ (CLas), which currently threatening the sustainable development of citrus industry. Previous studies had identified three types of prophage sequences in the genome of CLas strains. Prophage group typing analysis revealed that the Type 2 prophage strains and Type 1+3 prophage strains dominated in Guangdong Province. However, the difference in propagation capacity of these two strains of CLas in Asian citri psyllid (Diaphorina citri) and pathogenicity of these two strains leading by insect transmission remains unknown. 【Objective】The objective of this study is to assess the difference in proliferation capacity between Type-2-CLas and Type-1+3-CLas in D. citri and the pathogenicity variations between them on Citrus reticulata Blanco cv. Shatangju. 【Method】The buds with different strains of CLas were grafted to healthy Shatangju. Fifty nymphal and fifty adult D. citri were caged on the young shoots of infected Shatangju to acquire different strains of CLas for 6, 12, and 18 days, respectively. Real-time quantitative PCR (qPCR) was used to detect and analyze the difference in the acquisition rate and quantity of Type-2-CLas and Type-1+3-CLas between the nymphal stage and the adult stage of D. citri. Further, twenty adult D. citri infected with Type-2-CLas or Type-1+3-CLas were placed on the young shoots of healthy Shatangju to inoculate for two weeks, respectively. The D. citri were collected and detected by qPCR. The general information of infection rate and quantity of CLas was collected. Symptom development of Shatangju and titers of CLas in leaves were monitored each month. Morphological change of leaves phloem and parenchyma cell was visualized by light microscopy 360 days after inoculation. 【Result】There was no significant difference between acquisition rate and quantity of Type-2-CLas and Type-1+3-CLas detected in D. citri that fed on the infected plant at the nymphal stage. In contrast to fed as nymphs, the acquisition rate and quantity of Type-2-CLas were significantly higher than Type-1+3-CLas when D. citri fed on infected plant at the adult stage. The leaves presented more severe mottled symptom after inoculation with Type-2-CLas at 120 d after D. citri removal. The immature leaves at young shoots had an obstacle on turning green, presenting leathery, small size, and yellowing which was infected with Type-2-CLas. The symptoms on Type-1+3-CLas infected plants were presented as typical mottled leaves and leathery. Anatomical analyses indicated that Type-2-CLas caused more severe damage to phloem and accumulated more starch in parenchyma cell even if the titer of Type-1+3-CLas was closed to Type-2-CLas. 【Conclusion】Compared to Type-1+3-CLas, Type-2-CLas was more capable of invading and proliferating in adult D. citri, accompanied by a higher titer of CLas which indirectly proved the high contagious capacity of Type-2-CLas. The young leaves infected with Type-2-CLas presented symptoms of uniform yellowing, small size, while infected with Type-1+3-CLas showed lightly mottled and leathery. Type-2-CLas destroyed more phloem cells and caused severe starch accumulation, indicating that Type-2-CLas had stronger pathogenicity to citrus than Type-1+3-CLas.

Key words: citrus Huanglongbing, ‘Candidatus Liberibacter asiaticus’ (CLas), prophage, Diaphorina citri, pathogenicity

黄家权,李莉,吴丰年,郑正,邓晓玲. 携带不同原噬菌体的黄龙病菌在柑橘木虱体内的增殖及致病力[J]. 中国农业科学, 2022, 55(4): 719-728.

HUANG JiaQuan,LI Li,WU FengNian,ZHENG Zheng,DENG XiaoLing. Proliferation of Two Types Prophage of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and their Pathogenicity[J]. Scientia Agricultura Sinica, 2022, 55(4): 719-728.

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引物名称 Primer name	引物序列 Primer sequence (5′→ 3′)	目的基因 Target gene	引物特异性 Specificity	来源 Source
SC1-gp060F	TCGCTCTCCTTCAAATTGCG	假设蛋白 Hypothetical protein	Type 1	本研究This study
SC1-gp060R	GTTTTAGTCCCGTCCGATGC	假设蛋白 Hypothetical protein	Type 1	本研究This study
SC2-gp045F	ACATCAGAGGCTACATCGGG	假设蛋白 Hypothetical protein	Type 2	本研究This study
SC2-gp045R	ACGTCTCGGTGGCTTAAAGA	假设蛋白 Hypothetical protein	Type 2	本研究This study
P-JXGC-8F	CGGCGCTGAACTCTTGTATT	R-M system	Type 3	文献[10] Reference [10]
P-JXGC-8R	AAGGGCGTTGTTCTTGTCAC	R-M system	Type 3	文献[10] Reference [10]
CLas-4G	AGTCGAGCGCGTATGCGAAT	16S rRNA gene	16S rRNA gene	文献[24] Reference [24]
HLBr	GCGTTATCCCGTAGAAAAAGGTAG
HLBp	FAM-AGACGGGTGAGTAACGCG-BHQ

刺吸时间 Access period	样本数量 Number of samples		获菌率 Acquisition rate (%)		单头木虱平均CLas数量 Mean CLas counts of individual D. citri
刺吸时间 Access period	Type 1+3	Type 2	Type 1+3	Type 2	Type 1+3	Type 2
若虫期刺吸 Access as nymph
6 d ^A	28	27	39.26±3.23a	70.37±3.70a	6.01×10⁴±1.62×10⁴a	6.98×10⁴±2.31×10⁴a
12 d	29	28	74.44±10.94b	80.83±10.83a	9.18×10⁵±2.73×10⁵b	3.58×10⁶±1.81×10⁶b
18 d	32	32	82.16±13.55b	93.33±6.67a	1.09×10⁶±4.04×10⁵b	7.99×10⁵±1.57×10⁵b
成虫期刺吸 Access as adult
6 d	28	30	10.00±0a	20.00±0a	3.65×10⁴±1.64×10³a	5.01×10⁴±4.24×10³a
12 d	30	28	20.00±0b	30.00±0b	2.46×10⁴±4.50×10³a	4.59×10⁶±1.27×10⁶b
18 d	30	30	23.33±3.33b	43.33±3.33c	2.38×10⁴±8.24×10³a	4.84×10⁴±1.34×10⁴a

菌株类型 Strains type	植株编号 Tree No.	柑橘木虱存活率 D. citri survival rate (%)	柑橘木虱获菌率 D. citri acquisition rate (%)	CLas-4G-qPCR的Ct值 Ct values of CLas-4G-qPCR
				0 d		60 d	120 d	360 d
				D. citri	FL	FL	FL/NFL	YFL
Type 1+3	CCT1-1	80.00	100.00	22.01±0.46	37.63±0.35	19.70±0.31	20.69±0.09	21.22±0.11
	CCT1-2	85.00	100.00	22.18±0.46	38.95±0.56	19.80±0.32	19.49±0.12	22.15±0.10
	CCT1-3	60.00	100.00	21.25±0.48	39.25±0.69	19.52±0.21	20.59±0.08	23.31±0.09
	CCT1-4	80.00	100.00	22.28±0.67	38.56±0.58	19.32±0.09	20.64±0.08	21.33±0.01
	CCT1-5	95.00	100.00	21.44±0.50	38.45±0.62	19.82±0.14	21.43±0.10	24.22±0.20
	CCT1-6	90.00	100.00	21.89±0.45	39.25±0.84	34.06±0.32	20.23±0.09	21.65±0.06
	CCT1-7	80.00	100.00	22.84±0.35	38.65±0.98	20.52±0.07	22.57±0.16	21.19±0.11
	CCT1-8	65.00	100.00	21.92±0.38	37.87±0.48	18.56±0.11	20.36±0.07	22.37±0.09
Type 2	CCT2-1	95.00	100.00	22.26±0.91	38.69±0.52	24.38±0.13	21.40±0.12	22.53±0.09
	CCT2-2	85.00	94.12	22.30±1.08	38.35±0.68	35.76±0.22	28.77±0.11	23.39±0.14
	CCT2-3	75.00	100.00	21.05±0.61	39.25±0.86	34.46±0.11	23.67±0.09	22.52±0.06
	CCT2-4	70.00	100.00	22.75±1.53	38.26±0.75	23.53±0.06	22.40±0.15	22.51±0.09
	CCT2-5	70.00	50.00	23.31±2.45	37.65±0.36	36.48±0.19	21.64±0.16	22.21±0.07
	CCT2-6	55.00	100.00	21.86±1.10	39.65±0.78	39.77±0.23	21.58±0.06	23.29±0.18
	CCT2-7	90.00	88.89	25.00±1.25	38.36±0.81	23.56±0.05	22.53±0.18	22.17±0.10
	CCT2-8	50.00	20.00	25.74±1.65	37.84±0.71	36.53±0.09	34.50±0.19	28.35±0.25