The endophytic strain B. subtilis L1-21, an indigenous endophyte isolated from a healthy citrus host (Munir et al., 2020a) and deposited in the Chinese Culture Collection Bank, Beijing (Accession number: CGMCC15726), was selected for the greenhouse and citrus field experiments (Binchuan, Yunnan Province, China). The endophyte B. subtilis L1-21 and its RFP-tagged strain were stored in 40% glycerol (v/v) at −80°C; stock culture was renewed every 4 months on Luria Bertani (LB) agar and cultured for 24–48 h to check the stability of the potential endophyte. A pure culture of endophyte L1-21 was grown until the late log phase in LB broth for 24–48 h at 37°C in a shaking incubator (150 rpm). The colony forming units (cfu ml⁻¹) of the endophyte were checked using contrasting dilutions in sterile distilled water with 0.1% Tween 20 and plated on LB agar before foliar spraying on the citrus trees.

We designed a novel citrus half-leaf method to quantify CLas copies in leaves prior to and after treatment based on conventional and quantitative PCR (qPCR). Diseased citrus leaves [Citrus reticulata (C. reticulata)] were collected randomly from 16 years old citrus grove and brought to the laboratory in ice and immediately processed for experiment or stored in 4°C till further use. Firstly, DNA was extracted from one-half of a diseased leaf using the cetyltrimethylammonium bromide (CTAB) method (Araújo et al., 2002; Munir et al., 2019) and conventional PCR analysis, using the primer set Cal-R/Cal-F (Jagoueix et al., 1996), was followed by nested PCR analysis, using the primer set CG03F/CG05R (Zhou et al., 2007), for confirmation of “Ca. Liberibacter asiaticus.” Primers used in this study are given in Supplementary Table S1. Once confirmed as positive for CLas, the other half of the citrus leaf (4–5 cm) was suspended with 10⁴-10⁶ cfu ml⁻¹ of endophytes in a 5-ml Eppendorf tube and the different antibiotics, i.e., penicillin (100 μg μl⁻¹) dissolved in distilled water, spinosad (100 μg μl⁻¹) dissolved in hexane solution, and shenqimycin (100 μg μl⁻¹) dissolved in distilled water as chemical control (positive). LB broth and sterilized distilled water were used as uninfected control (negative). The Eppendorf tubes were kept for 96 h at room temperature, followed by extraction of DNA from the leaf midribs. The CLas 16S ribosomal RNA (16S rRNA) gene was amplified as discussed previously (Munir et al., 2019). The weight of each half leaf was recorded prior to the experiment. Each treatment comprised three replicate Eppendorf tubes that each contained six diseased citrus leaves midribs. The experiment was repeated five times. Treatment effects on CLas were tested by loading the amplified product on 1% agarose gel. In another experiment testing for CLas and endophytes abundance, diseased leaves were placed in Petri dishes (180 mm) and different treatments were performed. DNA was extracted from one-half of a diseased leaf using the CTAB method (Araújo et al., 2002) and amplified with the same primers as described previously. Once the leaf was confirmed as infected with CLas, the other half of the leaf was kept in a 180-mm Petri dish with 10⁴ and 10⁶ cfu ml⁻¹ or penicillin (100 μg μl⁻¹) dissolved in distilled water. LB and distilled water were used as controls. The Petri dishes were kept at room temperature for an interval of 1 day for collected leaves to be checked for pathogen concentration and endophytes for 4–5 days; then, DNA was extracted from the midribs and present endophytes were isolated (Araújo et al., 2002; Munir et al., 2018b). The weight of each half leaf was recorded prior to the experiment. Each treatment comprised three replicates of 12 diseased citrus leaves (six random top and bottom half leaves, each) and the experiment was repeated five times. The presence of the pathogen was confirmed using standard qPCR analysis and density (copies g⁻¹) was calculated based on a standard curve generated by cloning 382 bp of a specific DNA fragment located in the ribosomal protein (rplJ) (Munir et al., 2020a). The PCR reaction was performed in a 25-μl reaction mixture containing 1 × PCR buffer (SYBR Green Master Mix; Bio-Rad, United States), 0.8 μl of each primer (CQULA04R/CQULA04F), and the appropriate amount of DNA template in an RT-PCR system (StepOne Real-Time PCR System, Applied Biosystems, United States) as reported previously (Wang et al., 2006). Changes in CLas copies were tested from 0 to 4 days, based on CT values of pathogen copies g⁻¹ of the citrus leaf that were calculated using the standard curve generated previously; changes in endophyte density were recorded daily as log cfu g⁻¹ of citrus leaf midrib.

For confirmation of phloem colonization of the endophyte, the RFP-tagged strain of B. subtilis L1-21 was generated using genomic DNA by amplifying the mKate2 coding sequence with ribosome-binding site. Bam H1 and Hind III enzymes were used for product digestion and ligated with the plasmid pYC127 as discussed previously (Chai et al., 2008). The resulting plasmid designated as PY69 was used to make overexpression RFP strain of B. subtilis L1-21. A strain expressing red fluorescent reporter gene (mKate2) was grown until late log phase in LB medium with 10 μg μl⁻¹ chloramphenicol in shaking incubator at 37°C. Citrus plants (2 years old) in the greenhouse were foliar sprayed with RFP-tagged endophytic strain L1-21 and its colonization was visualized by confocal laser scanning microscope (CLSM) after 2 days. Citrus leaves were detached and gently washed for surface sterilization as performed previously to remove unattached cells from the citrus leaves (Munir et al., 2021). Leaves midribs were separated and put on the specimen holder by adding optimal cutting temperature (OCT) compound (Sakura, Europe). Specimen holder containing citrus leaves midribs were sliced briefly on the microtome-cryostat (Leica Biosystems, United States). Slice midribs were examined using CLSM (Olympus, FV10-ASW). The emission of RFP was measured at 405 to 635 nm after excitation at 559 nm, while citrus plant autofluorescence was measured after excitation at 405 and 635 nm for blue and green fluorescence, respectively. Images were captured using an automatic picture program FV1000 Viewer. The experiment was repeated three times, each time with three replicates.

The experimental field site was located at Binchuan, Dali City, Yunnan Province, China (100.5754° E, 25.8272° N), with an average annual temperature of 33–35°C and total annual rainfall of about 1,000 mm. Foliar symptoms in one citrus grove (C. reticulata), 16 years old, indicated > 90% CLas disease incidence, and the second citrus grove (C. reticulata), 4 years old, which was located 2 km away from the first citrus grove, exhibited 100% CLas infection as confirmed through conventional PCR and qPCR before the start of experiments. Two field trials were conducted from 2017 to 2019: in one citrus grove, 162 trees (Supplementary Figure S1) were treated with contrasting treatments and in the second citrus grove, 93 of 525 trees were selected (three replicates of 31 trees) for CLas titer test. Prior to the start of each trial, a field survey was carried out to visually determine individual tree HLB severity and detected the CLas pathogen using CLas-specific primers in PCR and qPCR analyses. The soil treatments at the citrus groves comprised fertilizer 1 (rapeseed cape with Bacillus amyloliquefaciens Y2, as a root growth promoting agent); fertilizer 2 (rapeseed cape without Bacillus amyloliquefaciens Y2); and no rapeseed cape as a control (F₀). Antibiotic penicillin (100 μg μl⁻¹) dissolved in distilled water was applied one time through trunk injection at the start of the experiment as shown in Supplementary Table S2. In addition, we applied 5 kg of fertilizer to each tree around a water drip of about 20 cm deep. The antibiotics and endophytes used in this study are given in Supplementary Table S2.

The endophyte application was carried in the field experiments. Two injection ports per tree were made by drilling 30 mm into the xylem tissue using a 7.14-cm drill bit, located at the opposite sides of the trunk at ~20 cm above the union bud. Antibiotic and endophyte (B. subtilis L1-21) treatments were injected from a pressurized bottle into each port using a micro drip infusioner at the recommended pressure (<50 psi). The first injection was done with penicillin (0.1%) for 4 days on 54 trees, with 27 trees as a control. The second injection was done 2 weeks after the antibiotic treatment with the endophyte (10⁶ cfu ml⁻¹) on 54 trees. We also applied 10⁶ cfu ml⁻¹ of the endophyte or penicillin as a monthly foliar spray on 27 trees for each treatment, until all leaves were wet, in the early morning or late evening. All the experiments were arranged as three replicates of three trees in a randomized complete block design.

Candidatus Liberibacter asiaticus causing HLB disease negatively affected the indigenous endophytic microbes. Therefore, we estimated the density of endophytic bacteria from three replicates of six citrus tree leaves that were sampled from the trees and transported to the laboratory in a cooler with ice. Endophytes were isolated from three replicates of treatment samples collected on the first week of each month in 2017–2018 as isolated previously (Araújo et al., 2002).

Leaf samples from all the treatments were collected monthly from the citrus groves to quantify CLas titer. Leaves were treated as above before DNA extraction. Methods for DNA extraction, PCR, and qPCR were those described previously.

Endophytic bacteria populations were calculated, based on average logarithm (base 10) cfu values, and analyzed using the GraphPad Prism version 8 (San Diego, California, USA). Pathogen copies g⁻¹ of diseased citrus leaf material were calculated using a standard curve value (Munir et al., 2020a). Treatment effects were tested using the ANOVA in the SPSS Statistics version 22.0 (IBM Corporation, Armonk, New York, USA) and treatment means were compared using Duncan's multiple range test at p < 0.05.

We used the indigenous endophyte B. subtilis L1-21 against CLas in the leaf midrib by means of citrus half-leaf method (Figures 1A,B) maintained under room temperature in Eppendorf tube. CLas copies in leaf material were reduced by 5 days after treatment with the endophyte at 10⁴ and 10⁶ cfu ml⁻¹ (Figure 1C); similarly, the antibiotics used in this experiment also reduced CLas copies (Figure 1D). Subsequent dilutions of the endophyte validated the results, confirming that CLas copies inside the citrus leaf midrib were reduced by a single application of the endophyte. Since the leaves midribs were cut and put on shaking in water, we suggested that pathogen ooze out of the midribs and no positive band was observed.

To further confirm the efficacy of the endophyte against CLas, we used the half-leaf method to test pathogen copies in one half of the leaf present in Petri dish treated with a single application of the B. subtilis L1-21 (10⁴ and 10⁶ cfu ml⁻¹) or penicillin antibiotic (as a positive/chemical control) and LB broth and water (as negative/uninfected controls). The presence of the pathogen was confirmed using standard qPCR analysis and density (copies g⁻¹) was calculated based on a standard curve (Munir et al., 2020a). CLas pathogen copies in the leaf midribs treated with the endophyte L1-21 (10⁴ and 10⁶ cfu ml⁻¹), penicillin, LB, and water were 1.12 × 10⁷, 1.23 × 10⁶, 4.71 × 10⁵, 2.38 × 10⁵, and 1.51 × 10⁶, respectively (Figure 1E; Supplementary Table S3). Application of the endophyte reduced CLas copies in the leaf midribs 1,000-fold (1.12 × 10⁷ to 3.72 × 10⁴) and 100-fold (1.23 × 10⁶ to 3.93 × 10⁴) by 4 days after a single treatment of 10⁶cfu ml⁻¹ and 10⁴ cfu ml⁻¹ (Figure 1F; Supplementary Table S3) of the endophyte, respectively. Application of penicillin reduced CLas in diseased citrus leaves 10-fold (4.71 × 10⁵ to 1.64 × 10⁴) and there was no effect of LB or water controls by 4 days after treatment. These results clearly indicated that application of the endophytes reduced CLas pathogen in diseased citrus leaves. The half-leaf method was also employed to check the pathogen reduction in individual leaves using top and bottom parts to confirm validation of results and we showed that CLas could be reduced to more than 90% after 3–4 days (Supplementary Table S4).

Prior to application of the endophyte, indigenous citrus endophyte density in the citrus leaves was 10⁵ cfu g⁻¹; following a single treatment with endophytic strain L1-21, the density of other endophytes present inside leaves increased to 10⁹ cfu g⁻¹ (Figure 2A). Treated endophyte L1-21, which displayed pinkish color on LB media, was recovered from the diseased citrus leaves during different time intervals with different treatments mentioned (Figure 2B). The application of penicillin initially decreased leaf midrib microbe density, but this effect subsided with time as the endophyte density returned to pretreatment levels. The endophyte density was unaffected by the LB and water treatments.

To prove the hypothesis that the indigenous endophyte B. subtilis L1-21 colonization inside citrus midribs phloem helps in excluding the CLas, which resides in citrus phloem, an RFP-tagged L1-21 strain was constructed that can express mKate2 genes. After observation with CLSM, we found that RFP-tagged L1-21 successfully colonizes the citrus midribs (Figure 3). Two days after inoculation, we observed that a lot of cells are present inside the phloem. These results indicated that the endophyte not only colonizes the citrus phloem completely but also reduces the pathogen load present in the citrus phloem as proved above.

In 2017–2018, the efficacy of potential endophyte B. subtilis L1-21 was tested against CLas in two diseased citrus groves (>90% and 100% HLB prevalence, respectively) containing 16- and 4-year-old citrus trees, respectively. We treated the CLas-infected mandarin groves with contrasting applications of the endophyte, antibiotic, and biofertilizers to determine the most effective management strategy (Supplementary Figure S1). HLB-affected citrus grove was completely recovered after 6–7 months of successful treatment with the indigenous endophyte (Supplementary Figures S1B,C). Antibiotic and biofertilizers were applied once at the start of the experiment and endophytes were applied monthly, following leaf sampling to check densities of CLas and the endophyte. Prior to the start of the experiment, all the citrus trees were tested for the total number of endophytes present inside each tree (Figure 4A). Bacillus subtilis L1-21 applied to the citrus trees spreads to neighboring, untreated trees (Supplementary Figure S2); therefore, the endophytic density of all the citrus trees were presented on monthly basis. The density of CLas and endophytes was measured monthly in leaf samples collected from each of the 162 citrus trees using conventional and nested PCR techniques (Figures 4B–D). The health of leaves was improved following treatment with the endophyte for 1 year, where they changed from yellow to green, and we found negative effects of CLas on the endophytic microbiomes of the citrus tree. Citrus trees were monitored monthly to assess visual disease symptoms and following 1 year of monthly applications of B. subtilis L1-21, CLas density in the trees reduced significantly. In the first quarter of the experiment, the number of diseased trees reduced to <100, where trees that had been characterized by yellow and mottled leaves began to develop more robust shoots and leaves. The leaf density of citrus endophytes was initially reduced by the application of antibiotic but later recovered to more than 10⁷ cfu g⁻¹. By the second quarter of the experiment, the number of diseased trees reduced to 69, in which the endophyte density was similarly increased; by the final quarter, the number of diseased citrus trees reduced to three, and trees in the grove finally yielded fruit suitable for the commercial market. Thus, we confirmed that the endophyte B. subtilis L1-21 was the most effective control agent for the management of HLB and a reliable yet cheap option for citrus growers worldwide. Strengthening citrus microbe density using an endophyte with diverse antagonistic activities through trunk injection and foliar spray application may elevate microbe density in diseased citrus trees to levels where CLas may more easily be controlled (Figure 4E). Previously, we found the number of the endophyte types was lower in diseased citrus trees, indicating that greater density of indigenous citrus endophytes may contribute to the control of CLas in diseased trees (Munir et al., 2019).

The copies of CLas in leaf samples were reduced following 6 months of monthly treatments with B. subtilis L1-21 (Figures 4F,G), confirming our hypothesis that indigenous citrus endophytes may reduce disease incidence in the field by >95%. After a year of monthly applications of the endophyte, population of B. subtilis L1-21 in citrus leaf midribs increased from 10³ to around 10⁹ cfu g⁻¹ in most trees, showing that indigenous endophytes may represent a novel management strategy for the control of CLas in citrus plants (Figure 5). We have found similar effects of this endophyte on CLas in 2 other citrus groves in the study region and one each in the Genma county of Yunnan Province (Figure 6).

To confirm the results from the 1-year field experiments, we regularly treated diseased citrus groves, comprising 525 trees, with B. subtilis L1-21 in 2018–2019; these regular applications reduced copies of CLas in the citrus trees so that the number of diseased trees was reduced. Finally, we selected 93 among the 525 trees, which represented three replicates of 31 trees and found that after 1 year, regular applications of the endophyte had reduced the CLas from 10⁹ to 10⁴ g⁻¹ of leaf midrib (Figure 7A) (99% control; Table 1). CLas copies were very high in April 2018 but decreased within 3 months (Figure 7B). In April 2019, we found 16 of the 93 diseased trees contained 100 copies of the pathogen g⁻¹ of leaf material, while 39 diseased trees contained < 100 copies, representing a 91.39% reduction in CLas (Figure 7C) and there was a decrease in the number of yellow leaves and shoots and an increase in the growth of new shoots (Figure 7D). Our findings indicated that CLas density remained constant in around 25% of the citrus trees during the endophyte treatment and was undetected in 16 of the diseased citrus trees in April 2019. Thus, we suggested that CLas was successfully eliminated from diseased citrus trees by B. subtilis L1-21.