Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (16): 3182-3191.doi: 10.3864/j.issn.0578-1752.2024.16.007

• PLANT PROTECTION • Previous Articles     Next Articles

Optimization and Application of Rapid Evaluation System for Citrus Huanglongbing Resistance Mediated by Agrobacterium rhizogenes

CAO Peng1(), ZHOU JinHuan1, WANG XinLiang1, LI ChuXin1, LI JiaXIN1, JIANG Pei2, LIU JinXiang1, SONG Zhen1()   

  1. 1 Citrus Research Institute, Southwest University, Chongqing 400712
    2 National Agricultural Technology Extension and Service Center, Beijing 100125
  • Received:2024-04-20 Accepted:2024-06-13 Online:2024-08-16 Published:2024-08-27
  • Contact: SONG Zhen

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Abstract:

【Objective】Citrus Huanglongbing (HLB) is a destructive citrus disease primarily caused by Candidatus Liberibacter asiaticus (CLas). The objective of this research is to investigate the growth pattern of CLas in citrus hairy roots at the initial stage of infection, optimize the rapid evaluation system of Agrobacterium rhizogenes-mediated HLB resistance and apply it to the resistance evaluation of antimicrobial peptides.【Method】A plant expression vector, pGNGM1300, was employed to transform different citrus varieties via the hairy root transformation system, which was mediated by A. rhizogenes K599. Subsequently, varieties with rapid induction and high transformation rate were then selected. Following inoculation with CLas, the presence of CLas in hairy roots was consistently demonstrated through the use of real-time quantitative PCR (qPCR), which allowed for the monitoring of its growth pattern. Further, the optimization of the HLB resistance evaluation system permitted the HLB resistance evaluation of antimicrobial peptides to be conducted via (RT-) qPCR analysis of CLas and related genes, quantification of callose deposition, microstructure observation, and symptom assessment.【Result】Among the 11 tested citrus varieties, citron (Citrus medica) exhibited the fastest induction of hairy roots (15 d), accompanied by the highest induction rate (73.75%) and a substantial transgene positivity rate (53.54%) in hairy roots. The results of regular qPCR monitoring indicated that CLas began to colonize roots on 20-30 days post-inoculation (dpi). At 30-50 dpi, there was a notable increase in the CLas content. The content of the CLas exhibited a slight fluctuation at a range of 60 to 120 dpi, though the discrepancy with 50 dpi was not statistically significant. The results of the antimicrobial peptide resistance evaluation, conducted on MaSAMP (stable antimicrobial peptide), HBD-4 (homo sapiens defensin beta 4), and CB (cecropin B), revealed that at 50-120 dpi, the content of CLas in both MaSAMP- and CB-expressing plants was significantly lower than that of the control. However, except that at 90 dpi, the CLas content was also significantly lower in HBD-4-expressing plants than the control. The callose content in all three plants expressing antimicrobial peptides was significantly lower than that of the control (60 dpi). There was no visible cell wall thickening in the phloem, and no significant deposition of starch grains or callus was observed. Furthermore, none of the plants exhibited signs of root death at 90 dpi.【Conclusion】This study elucidates the growth dynamics of CLas in citrus hairy roots during the early stages of infection and optimizes a rapid scoring system for A. rhizogenes-mediated HLB resistance. In addition, the results show that overexpression of MaSAMP, HBD-4 and CB effectively suppresses CLas proliferation, reduces callose deposition and alleviates HLB symptoms. These findings have the potential to be used in the prevention and control of HLB.

Key words: citrus Huanglongbing, antimicrobial peptide, Agrobacterium rhizogenes, hairy root transformation, resistance evaluation

Table 1

Information on the main primers used in this study"

引物名称Primer name 引物序列Primer sequence (5′-3′) 用途Usage
CLas16S-F TGAGTGCTAGCTGTTGGGTG CLas16S基因的qPCR分析
qPCR analysis for CLas16S gene
CLas16S-R CTGCGCGTTGCATCGAATTA
Cs18S-F AATTGTTGGTCTTCAACGAGGAA 柑橘内参基因
Citrus reference gene
Cs18S-R AAAGGGCAGGGACGTAGTCAA
CsCalS7-qF GACGCCTAACCGAGTACCTGC CsCalS7的qPCR分析
qPCR analysis for CsCalS7
CsCalS7-qR GTGCAGCTGGTGATCCATCA
CsPP2B15-qF GCTCTGAGATCTAGGGTTTCCG CsPP2B15的qPCR分析[24]
qPCR analysis for CsPP2B15
CsPP2B15-qR CCCCTTTCAGATGCTGACCC
2OG oxygenase-qF CGAAAGCACATGATGGAAAG 2OG oxygenase的qPCR分析[24]
qPCR analysis for 2OG oxygenase
2OG oxygenase-qR GCCACGAGTGAAGAAATGGT

Fig. 1

Induction hairy roots of citrus varieties mediated by A. rhizogenes K599"

Table 2

Hairy root induction and transformation of citrus varieties mediated by A. rhizogenes"

品种
Variety		 枝条总数
Total number of branches		 生根枝数
Number of branches with hairy roots		 生根时间
Hairy root
induction time (d)		 生根总数
Total number
of hairy roots		 阳性根数
Total number of
positive hairy
roots		 生根率
Hairy root induction rate (%)		 阳性根率
Hairy root
positive
rate (%)
锦橙C. sinensis 60 13 30 58 2 21.67 3.44
尤力克柠檬C. limon 262 179 20 722 313 68.32 43.35
香橼C. medica 160 118 15 480 257 73.75 53.54
枳壳P. trifoliata 40 6 60 19 9 15.00 47.36
卡里佐枳橙
C. sinensis×P. trifoliata		 40 14 30 53 8 35.00 15.09
粗柠檬C. limonia 40 29 20 116 46 72.50 39.65
墨西哥莱檬C. aurantiifolia 40 25 20 267 76 62.50 28.46
摩洛哥酸橙C. aurantium 40 20 25 88 48 50.00 54.54
甜橙C. sinensis 40 6 60 27 14 15.00 51.85
沙田柚* C. grandis 345 5 180 11 4 1.45 36.37
纽荷尔脐橙* C. sinensis 385 24 120 40 15 6.23 37.50
锦橙* C. sinensis 50 10 60 13 10 20.00 76.92

Table 3

Detection of CLas in hairy roots of C. medica by qPCR"

处理
Treatment		 植株编号
Plant number		 20 dpi 30 dpi 40 dpi
CLas Ct18S CLas Ct18S CLas Ct18S
pGNGM1300 cp2333-3 37.87 18.43 35.43 19.63 28.01 17.08
cp2333-5 38.87 17.15 36.61 19.06 31.72 18.87
cp2333-9 35.51 19.48 33.79 18.04 29.4 16.31
cp2333-12 36.96 18.77 31.68 17.64 29.41 17.62
cp2333-16 37.49 17.72 35.84 18.67 28.53 18.79
cp2333-17 37.71 18.57 33.31 17.86 27.21 18.94
CLas阳性对照
Positive control		 +CK 22.89 16.11 22.56 15.63 21.81 15.05
CLas阴性对照
Negative control		 -CK — 17.61 — 17.47 — 18.06

Fig. 2

(RT-) qPCR detection of CLas and related genes in transgenic hairy roots of C. medica"

Fig. 3

The effects of the antimicrobial peptide on CLas and related genes in transgenic hairy roots of C. medica"

Fig. 4

Callose content detection in transgenic hairy roots of C. medica"

Fig. 5

Observation of microscopic structure and phenotype of transgenic hairy roots of C. medica"

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