A new method for early detection of latent infection by ‘Candidatus Liberibacter asiaticus’ in citrus trees

CLas-infected and asymptomatic tree preparation

One-year-old seedlings of rough lemon (Citrus jambhiri Lush.) were prepared in 12 cm-diameter × 24 cm-deep pots. Graft-inoculation of 12 seedlings was carried out with a small amount of bark tissue of CLas-infected flat lemon trees (Citrus depressa Hayata.), which has been maintained in a quarantined greenhouse under a special permission (26Y1214) issued by the Ministry of Agriculture, Forestry and Fisheries (Fujikawa et al., 2013). CLas infection on seven rough lemon trees was confirmed by both the development of foliar symptoms and the presence of CLas by real-time PCR using HLBas/HLBr/HLBp primer–probe set (Li et al., 2006). Five rough lemon trees did not develop any foliar symptoms, and CLas was undetectable from leaves over two years in a monthly survey with both the conventional PCR (Fujikawa et al., 2012) and real-time PCR using HLBas/HLBr/HLBp primer–probe set (Li et al., 2006). These inoculated but asymptomatic rough lemon trees (five pots in all) were used as asymptomatic trees for this study.

Cutting preparation

Cuttings were prepared from the upper part of young green shoots and branches, which were taken from seven symptomatic and five asymptomatic rough lemon trees. Cuttings of approximately 4 cm in length containing one leaf with no disease symptoms were selected and treated with 1% “Menedael” Fe (II) aq. fertilizer (Menedael Co., Ltd., Osaka, Japan; Cat. No. 242461) and then dipped in “Rooton”, a synthetic auxin α-naphthyl acetamide, (Sumitomo chemical garden products inc., Tokyo, Japan; JAN code 4975292090625) for promotion of rooting. The cuttings that had been treated were planted in a box that contained autoclaved “Kanuma” pumice and kept at approximately 32°C for a month under natural light/dark conditions, covered with a plastic film. Care was taken to keep the Kanuma pumice gently wet but not saturated to ensure optimum rooting. Roots that regenerated a month after cutting were used for CLas detection in the real-time PCR (Li et al., 2006).

Cultivation test

For detecting CLas in cuttings with root-sprouts that tested negative in the first PCR test, subsequent cultivation of the cuttings in various soils was carried out in a quarantined greenhouse and monitored for two months. Five cuttings were individually transplanted into the following soils: two commercial soils which were “Kenbyo” nursery soil (Yaenougei Co., Ltd., Nagasaki, Japan) and “Sanyo composted bark” soil (Sanyo Chip Co., Ltd., Yamaguchi, Japan); one natural soil from an area where citrus greening disease has occurred, namely “Kunigami Maaji” clay soil collected from Kunigami-son village (around N26°44'44.8", E128°10'42.0"), Okinawa Prefecture; and two natural soils from areas where citrus greening disease has not occurred, which were Acrisols collected from Isen-cho (around N27°40'25.2", E128°56'16.2"), Tokunoshima Islands, Kagoshima Prefecture and Andosols collected from Tarumizu-city (around N31°28'38.1", E130°43'19.5"), Kagoshima Prefecture. The natural soils were collected in 2016. During soil sampling, the surface litter and soil at the sampling spot was removed using a spade. Using a plow at each sampling site, each natural soil was collected up to a depth of 10 cm and bulked. The precise locations of the sampling sites are not disclosed due to landowner privacy reasons. All soils were prepared with and without autoclaving. The autoclaving process was repeated twice. During the cultivation, 0.1g of “Hyponex” powder fertilizer (HYPONeX Japan Co., Ltd., Tokyo, Japan; JAN code 4977517003052) and one tablet of Aid-ball Ca (Sumitomo chemical garden products Inc., Tokyo, Japan; JAN code 4975292110118) were applied once a month. A single leaf and root were collected from each cutting on a monthly basis to determine the CLas infection status using real-time PCR (Li et al., 2006).

DNA preparation, PCR and real-time PCR

Leaf samples were cleaned and wiped with a kimwipe paper (Kimberly Clark Corp., Irving, TX) impregnated with 70% ethanol. Midribs were excised and chopped into 2–3 mm pieces, providing approximately 0.05 (±0.01 SD) g of the midribs for DNA extraction. For the root samples, a single root was vortexed vigorously to remove soils. Approximately 4 cm of the root segment was cut out from the bottom of the root and then wiped with a kimwipe impregnated with 70% ethanol. All sample materials were individually ground with a mortar and pestle, and then subjected to DNA extraction using the commercial kit DNeasy Plant Mini Kit (QIAGEN, Valencia, CA; Cat. No. 69106) according to the manufacturer’s instructions. DNA was eluted in 200 μL of a given AE buffer. CLas detection in cuttings was performed by TaqMan probe real-time PCR in a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific Inc., Waltham, MA) using the HLBas (5’-TCGAGCGCGTATGCAATACG-3’) / HLBr (5’-GCGTTATCCCGTAGAAAAAGGTAG-3’) / HLBp (6-FAM-AGACGGGTGAGTAACGCG-BHQ-1) primer–probe set (Li et al., 2006). Premix Ex Taq (Probe qPCR) (Takara Bio Inc., Shiga, Japan) was used according to the manufacturer’s protocol. The PCR reaction volume of 20 µL contained 0.4 µL of each primer, 10 µL of Premix Ex Taq, 0.8 µL of HLBp, 0.4 µL of ROX Reference Dye II, 2 µL DNA template, and 6 µL of sterile distilled water. The real-time PCR consisted of pre-denaturation at 95°C for 30 s, and 45 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. CLas concentration was calculated based on the relationship between quantity and molarity of CLas 16S rDNA (as outlined by Degen et al., 2006), and the standard curve analysis was determined using the amount of template DNA adjusted to the DNA content in 10⁸ cells/μL and a 10-fold dilution series. The CLas density in leaves was calculated from the total amount of DNA eluted from leaves according to the representative standard curve (y = −1.538x + 42.278, R² = 0.9999). The results of the CLas development were analyzed using Tukey-Kramer multiple comparison test in the R statistical software (v. 3.5.0)

Differences were considered significant when P < 0.01.

Detection of CLas recovered from latent infection using cuttings

Most of the cuttings from asymptomatic trees survived and were well rooted (Figure 1a–c), but the regeneration of cuttings using symptomatic trees was evidently influenced by CLas infection. A total of 224 clones regenerated from 240 prepared cuttings using asymptomatic trees whereas a total of 24 clones were recovered from 45 prepared cuttings using symptomatic trees. One root sample was collected from each cutting and these were used for the primary PCR test. CLas was detected in a total of 115 cuttings in the real-time PCR (Fujiwara, 2021a), which corresponds to a 51.3% detection rate (Table 1), while none of the cuttings showed foliar symptoms of citrus greening. In addition to this test, nine leaf samples from cuttings showing CLas negative in roots were randomly selected and subjected to real-time PCR, the results of which showed no detection of CLas within the leaf samples. In regeneration of cuttings using symptomatic trees, CLas was detected in all root samples, which corresponds to a 100% detection rate (Table 1), as was detected in the symptomatic leaves.

Figure 1.

Preparation of cuttings using asymptomatic trees; a, cuttings grown in Kanuma soil a month after preparation; b–c, seedlings recovered from cuttings showing newly developed roots.

Effect of soil types on CLas recovery from latent infection

A subsequent cultivation test was performed (Fujiwara, 2021b) to assess the CLas recovery from latent infection using the cuttings which showed no CLas detection in the primary PCR test (Supplementary Figure 1, Extended data (Fujiwara, 2020)). To examine suitable soil types for CLas development in cultivation, we tested five different types of soils including two commercial soils (“Kenbyo” nursery soil and “Sanyo composted bark” soil) and three natural soils (Acrisols, Andosols, and “Kunigami Maaji” soil). Overall, a cultivation of the cuttings in prepared soils successfully provided CLas detection by real-time PCR from asymptomatic trees (Figure 2). In the nursery soil, CLas was detectable from both leaves and roots a month after transplanting (P < 0.01). The density of CLas in seedlings grown in the “Kenbyo” nursery soil with autoclaving (3.4 × 10⁵ cells/leaf and 2.1 × 10⁵ cells/root) was relatively higher than that in the same soil without autoclaving (3.8 × 10³ cells/leaf and 4.9 × 10³ cells/root), and the detection was stable throughout the cultivation for two months (Figure 2; Supplementary Figure 2, Extended data (Fujiwara, 2020)). In contrast, although CLas was likely to be detected in both leaves and roots grown in a composted soil (1.7 × 10³ cells/leaf and 4.5 × 10¹ cells/root with autoclaving), the CLas density detected in cultivations in the composted soil without autoclaving fluctuated highly (Figure 2; Supplementary Figure 3, Extended data (Fujiwara, 2020)). Seedlings grown in composted soil without autoclaving showed CLas development a month after cultivation, no CLas was detected in the subsequent cultivation. In the natural soils, which comprised Acrisols, Andosols, and “Kunigami Maaji” soils, it was also the case that CLas detection fluctuated in both the leaves and roots similar to that in the composted bark soil without autoclaving (Figure 2; Supplementary Figure 4–6, Extended data (Fujiwara, 2020)). Autoclaving of the Andosols and “Kunigami Maaji” soil had little effect on the propagation of CLas in the cuttings, showing 2.3 × 10³ cells/leaf and 1.1 × 10⁴ cells/root in Andosols without autoclaving; 5.2 × 10² cells/leaf and 9.5 × 10² cells/root in a Kunigami Maaji soil without autoclaving. In Acrisols, CLas development was relatively varied compared with other natural soils and appeared to be affected by the soil conditions with autoclaving (5.4 × 10³ cells/leaf and 2.2 × 10⁴ cells/root with autoclaving) (Figure 2; Supplementary Figure 4, Extended data (Fujiwara, 2020)). In short, the soil conditions of a nursery soil treated with autoclaving was suitable for early detections of CLas recovered from latent infection using cuttings.

Figure 2. Candidatus Liberibacter asiaticus (CLas) development in a subsequent cultivation using different types of soil.

Average of CLas density two months after cultivation was determined by real-time PCR and calculated using CLas density in five individual seedlings shown in Supplemental Figures 2–6 (see Extended data). CLas recovered from latent infection was likely to be detected from leaves and roots using nursery soil with autoclaving. Statistical significance was shown (*P < 0.01, Tukey-Kramer multiple comparison test). +: with autoclaved, -: without autoclaved.

Underlying data

Figshare: Raw data for “Detection of CLas from roots recovered from cuttings” shown in Table 1. https://doi.org/10.6084/m9.figshare.13985021.v1 (Fujiwara, 2021a).

This project contains the following underlying data:

Figshare: Raw data for “A subsequent cultivation in different soil types using CLas-negative cuttings” shown in Supplementary Figures 2–6. https://doi.org/10.6084/m9.figshare.13985198.v1 (Fujiwara, 2021b).

This project contains the following underlying data:

Extended data

Figshare: Supplementary Figures 1–7. https://doi.org/10.6084/m9.figshare.13466676.v1 (Fujiwara, 2020).

This project contains the following extended data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).