Bacillus subtilis suppression of Clas in the laboratory
We used indigenous endophyte Bacillus subtilis L1-21 against Clas in the leaf midrib by means of citrus half-leaf method (Fig. 1a, b) maintained under room temperature in Eppendorf tube. Copy density of Clas in leaf material was reduced by 5 d after treatment with the endophyte at 104 and 106 cfu ml-1 (Fig. 1c); similarly, the antibiotics used in this experiment also reduced Clas copy density (Fig. 1d). Subsequent dilutions of the endophyte validated the results, confirming that Clas copy density inside the citrus leaf midrib was reduced by a single application of the endophyte.
To further confirm the efficacy of the endophyte against Clas, we used half-leaf method to test for pathogen copy density in one half of the leaf present in Petri dish treated with a single application of endophyte (104 and 106 cfu ml-1),or penicillin antibiotic (as a positive control) and LB broth and water (as negative controls). Presence of the pathogen was confirmed using standard qPCR analysis (see Methods), 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). Thus, the recombinant plasmid was designated as pMD18T-rplJ-HLB, and pathogen density was derived from treatment CT values of the standard curve [26]. qPCR amplification efficiency was 99% and melt curves confirmed specificity of the results. Initial copy densities of the pathogen in the leaf midribs treated with 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 (Fig. 1e; Table S3). Application of the endophyte reduced Clas copy density in the leaf midribs 1000-fold (1.12×107 to 3.72×104) and 100-fold (1.23×106 to 3.93×104) by 4 d after a single treatment of 104 cfu ml-1 and 106 cfu ml-1 (Fig. 1g; Table S3) of the endophyte, respectively. 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 d (Table S4).
Prior to application of the endophyte, microbe endophyte density in the citrus leaves was 105 cfu g-1; following a single treatment with endophytic strain L1-21, density increased to 109 cfu g-1 (Fig. 1f, h). Application of penicillin initially decreased leaf midrib microbe density, but this effect subsided with time as endophyte density returned to pre-treatment levels. Endophyte density was unaffected by the LB and water treatments. Application of penicillin reduced Clas copy density 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 d after treatment. These results clearly indicated that application of the endophytes reduced pathogen density in diseased citrus leaves.
Efficacy of short-term fieldapplications of Bacillus subtilisagainst Clas
In 2017-18, we tested the efficacy of B. subtilis L1-21 against Clas in two diseased citrus groves (100% HLB prevalence) containing 16 and 4-year old citrus trees, respectively, based on the assumption that it may play an important role in the prevention of colonization and development of the pathogen in citrus trees. We treated the Clas-infected mandarin groves with contrasting applications of the endophyte, antibiotics and bio-fertilizers to determine the most effective management strategy (Fig. 2). HLB affected citrus grove was completely recovered after 6-7 months of successful treatment with indigenous endophyte (Fig. 2b, c). Antibiotics and bio-fertilizers 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 citrus trees were tested for the total number of endophytes present inside each tree (Fig. 3a). B. subtilis L1-21 applied to the citrus trees spread to neighbouring, untreated trees (Fig. S1), so we tested microbe diversity of all citrus trees. Density of Clas and endophytes were measured monthly in leaf samples collected from each of the 162 citrus trees using conventional and nested PCR techniques (Fig. 3b-d). Health of leaves were improved following treatment with the endophyte for one year, where they changed from yellow to green, and we found negative effects of Clas on endophytic microbe community structure of citrus tree. Citrus trees were monitored monthly to assess visual disease symptoms, and following one year of monthly applications of B. subtilis L1-21, Clas density in the trees had been reduced. In the first quarter of experiment, the number of diseased trees had reduced to <100, where trees that had been characterised by yellowed and mottled leaves began to develop more robust shoots and leaves. The leaf density of citrus endophytes was initially reduced by the application of antibiotics, but later recovered to more than 107 cfu g-1. By the second quarter of the experiment, the number of diseased trees had reduced to 69, in which endophytes density was similarly increased; by the final quarter, the number of diseased citrus trees had been 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 and cheap option for citrus growers worldwide. Strengthening citrus microbe diversity using an endophyte with diverse antagonistic activities through trunk injection and foliar spray application may elevate microbe diversity in diseased citrus trees to levels where Clas pathogen may more easily be controlled (Fig. 3e). Previously, we found the number of endophyte types was lower in diseased citrus trees, indicating that greater diversity of indigenous citrus endophytes may contribute to the control of Clas in diseased trees[24].
The copy density of Clas in leaf samples was reduced following 6 months of monthly treatments with B. subtilis L1-21 (Fig. 3f-g), confirming our hypothesis that indigenous citrus endophytes may reduce disease incidence in the field by >95% (Fig. 4a-f). After a year of monthly applications of the endophyte, levels of B. subtilis L1-21 in citrus leaf material 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. We have found similar effects of this endophyte on Clas in 2 other citrus groves in the study region and one each in the Maguan and Yongshan counties of Yunnan province (Fig. S2).
Effects of long-term field applications of Bacillus subtilis on Clas
To confirm the results from the one-year field experiments, we regularly treated diseased citrus groves, comprising 525 trees, with B. subtilis L1-21 in 2018-19; these regular applications reduced copy density of Clas in the citrus trees, so that the number of diseased trees was reduced. We selected 93 among the 525 trees, which represented three replicates of 31 trees, and found that after one year, regular applications of the endophyte had reduced the copy density of Clas from 109 to 104 g-1 of leaf midrib (Fig. 5a) (99% control; Table 5). Clas copy density was very high in April 2018, but decreased within 3 months (Fig. 5b). In April 2019, we found 16 of the 93 diseased trees contained 100 copies of the pathogen g-1 of leaf material, while 39 contained <100 copies, representing 91.39% reduction in Clas pathogen copy density (Fig. 5c), and there was a decrease in the number of yellow leaves and shoots and an increase in the growth of new shoots (Fig. 5d). Our findings indicated that Clas pathogen density remained constant in around 25% of the citrus trees during endophyte treatment and was undetected in 16 of the diseased citrus trees in April 2019. Thus, Clas was successfully eliminated from the diseased citrus trees by B. subtilis L1-21.
Analysing disease responsive genes
Further, molecular mechanisms in citrus plants after introduction of endophytes to diseased trees resulted in the up-regulation of important genes responsible for pathogen/disease resistance, chaperone family protein and respiratory burst oxidase, and differentially expressed pathogen resistance genes involved in different pathways may improve plant defences during biotic stress. We selected the top 20 abundant KEGG pathways that were triggered following application of the endophyte (Fig. 6) on diseased trees. The major upregulated KEGG pathways included biosynthesis of secondary metabolites; plant-pathogen interaction; and phenylpropanoid biosynthesis (padj<0.05) (Fig. 6a). In treatment of healthy citrus trees, major KEGG enrichment pathways comprised biosynthesis of secondary metabolites; metabolic pathways; biosynthesis of amino acids; and, phenylpropanoid biosynthesis (padj<0.05) (Fig. 6b). Upregulated pathogen resistance genes (in response to Clas) following endophyte application are pathogenesis-related 4 (PR4, Ciclev10029328m.g, Ciclev10029327m.g, Ciclev10029528m.g, Ciclev10029536m.g), disease resistance protein (CC-NBS-LRR class) family (Ciclev10030667m.g, Ciclev10024849m.g, Ciclev10024854m.g), chitin elicitor receptor kinase 1 (LYSM RLK1) (Ciclev10017678m.g), respiratory burst oxidase homologue D (RBOHD, Ciclev10027774m.g), mitogen-activated protein kinase 1 (MPK1, Ciclev10001531m.g), Chaperone protein htpG family protein (HSP90.5, Ciclev10030743m.g), CRINKLY4 related 3 (CCR3, Ciclev10030732m.g), and phloem protein 2-A12 (Ciclev10021489m.g) (Fig. 6c; Table S6) and downregulated genes involved in pathogen resistance were heat shock protein 70 (Ciclev10027981m.g) and 17.6 kDa class II heat shock protein (Ciclev10009756m.g), which are responsible for protein folding, indicating that the citrus trees treated with endophytes had previously contained Clas (Tables S7-S11). All read data are available in the SRA database (BioSample accession numbers SAMN15323747-SAMN15323758).
Secondary metabolites expression
Secondary metabolites play an important role in defense mechanism of citrus trees and other plants. Significant regulation was noted in the diseased citrus leaves after treatment with endophytes indicating the positive affect of these agents against Clas pathogen. Important genes related to secondary metabolites are related to metabolism of terpenoids and polyketides. Geranylgeranyl pyrophosphate synthase 1 (Ciclev10012067m.g), isopentenyl diphosphate isomerase 1 (Ciclev10012312m.g), hydroxymethylglutaryl-CoA synthase/HMG-CoA synthase/3-hydroxy-3-methylglutaryl coenzyme A synthase (Ciclev10020042m.g), and squalene synthase 1 (Ciclev10028537m.g) are the important genes upregulated in the diseased citrus trees after treatment with endophytes (padj<0.05). Lipoxygenase 2 (Ciclev10014199m.g) involved in jasmonic acid mediated response in leaves, aldehyde dehydrogenase 3F1 (Ciclev10025492m.g), and GroES-like zinc-binding dehydrogenase family protein (Ciclev10020620m.g) are responsible for metabolism of lipids. Other important genes related to regulation of secondary metabolites are peroxidase superfamily protein (Ciclev10021010m.g), branched-chain amino acid transaminase 2 (Ciclev10001429m.g), S-adenosyl-L-methionine-dependent methyltransferases superfamily protein (Ciclev10026344m.g, Ciclev10012561m.g), and aldehyde dehydrogenase 2C4 (Ciclev10010514m.g) (Table S7). The oxidative stress created by Clas pathogen inside citrus trees are detoxified through genes such as glutathione S-transferases (Ciclev10008944m.g, Ciclev10032702m.g, Ciclev10033001m.g, Ciclev10005808m.g, Ciclev10005812m.g), thus helping the citrus trees to showed tolerance to HLB. Two genes for phenylalanine ammonia-lyase (Ciclev10027913m.g, Ciclev10027912m.g) were upregulated when endophytes were applied to healthy citrus trees.
Protein folding, chaperones, heat shock proteins
The most important mechanism involved in the disease symptoms of huanglongbing are the down regulation of heat shock proteins, and product of these genes protect the protein folding and function during pathogen attack. The correct function is maintained in the phloem and leaves in the presence of these genes. We found that the genes responsible for protein folding are up regulated in the citrus trees treated with endophyte such as Chaperone DnaJ-domain superfamily protein (Ciclev10002683m.g, Ciclev10016883m.g, Ciclev10009810m.g), Chaperone protein htpG family protein (Ciclev10030743m.g), and Co-chaperone GrpE family protein (Ciclev10008685m.g). The fold change for all of these genes were more than 1 and the FDR ratio was (padj<0.05). Ubiquitin mediated protein degradation plays an important role in plant-pathogen interactions. There are 10 ubiquitin related genes regulated after endophyte treatment inside the citrus trees, 9 of them were up-regulated and 1 was down-regulated. The up-expressed genes are ubiquitin-conjugating enzyme/RWD-like protein (Ciclev10021040m.g), Plant U-Box 15 (Ciclev10003715m.g), Transducin/WD40 repeat-like superfamily protein (Ciclev10007255m.g), senescence-associated E3 ubiquitin ligase 1 (Ciclev10030698m.g), Ubiquitin carboxyl-terminal hydrolase family protein (Ciclev10003568m.g), U-box domain-containing protein kinase family protein (Ciclev10018795m.g), plant U-box 29 (Ciclev10001380m.g), Rad23 UV excision repair protein family(Ciclev10011886m.g), and ubiquitin 4 (Ciclev10012291m.g). F-box/RNI-like superfamily protein (Ciclev10001527m.g) responsible for folding, sorting and degradation of proteins was downregulated (Table S8).
Genes responsible for photosynthesis and carbohydrate metabolism
The HLB affected citrus leaves results in downregulation of important genes involved in photosynthesis processes. In the present study, we also showed that when diseased citrus leaves were treated with indigenous endophytes, only one of the gene ferrodoxin 3 (Ciclev10029499m.g) related to photosynthesis was up-expressed while two genes, APE1 (Ciclev10012434m.g) and PSB28 (Ciclev10022322m.g) related to acclimation of photosynthesis to environment and photosystem II reaction center, respectively were down-expressed indicating the pathogen is present inside the diseased leaves. Significant genes responsible for ethylene (Ciclev10000608m.g, Ciclev10014617m.g, Ciclev10031204m.g) were up-regulated. HLB causes accumulation of starch inside the phloem and other photosynthetic cells, resulting in blockage of important nutrients inside leaves. We found that our endophytes treatment resulted in downregulation of beta glucosidase 46 (Ciclev10014887m.g) and beta glucosidase 11 (Ciclev10019719m.g), which are responsible for starch accumulation. It has been reported that Clas pathogen negatively affected the metabolism of carbohydrate inside citrus trees. The present study found that some of the genes were upregulated related to carbohydrate metabolism when endophytes application was employed. Significant genes are aldehyde dehydrogenase 3F1 (Ciclev10025492m.g), Galactose mutarotase (Ciclev10012004m.g),GroES-like zinc-binding dehydrogenase (Ciclev10020620m.g), hydroxymethylglutaryl-CoA synthase / HMG-CoA synthase / 3-hydroxy-3-methylglutaryl coenzyme A synthase (Ciclev10020042m.g), Alpha amylase(Ciclev10007401m.g), arginosuccinate synthase (Ciclev10019860m.g), and phosphoglucose isomerase 1 (Ciclev10000603m.g) with log fold change (LFC) of 1.77, 1.69, 1.57, 1.47, 1.45, 1.14, and 1.01, respectively, with FDR (padj<0.05) (Table S9).
Related to cell wall breakdown
The genes involve in cell wall breakdown are mostly expressed in the citrus leaves affected with huanglongbing, indicating the symptoms development are associated with these genes in HLB progression. The cellulose/transferases are all associated with the cell wall breakdown. Our study indicated that cellulose synthase/transferases (Ciclev10007586m.g, Ciclev10023570m.g, Ciclev10014586m.g) genes are down regulated after applying endophytes, which is the main genes in breakdown of cell wall (Table S10).
Transcription factors
A total of 23 important transcription factors were identified when citrus trees were treated with endophytes (Table S11). Among them 18 TFs are upregulated and 5 are down-expressed. These transcription factors has important role in plant defense response, biotic and abiotic stress, plant immunity, leaf senescence, stomatal movement, and jasmonate metabolism. Several families of transcription factors, such as WRKY (11, 28, 33, 40, 50, 55), MYB (1, 15, 116), and EIN3 (Ciclev10000608m.g) are associated with the plant immunity and defense up-expressed in the citrus trees after endophyte treatments. The WRKYs transcription factors are also involved in the tolerance to Clas pathogen in citrus. Heat shock transcription factor A4A (Ciclev10015413m.g) and heat shock transcription factor A8 (Ciclev10015541m.g) are up-expressed in the citrus leaves indicating the important regulator in several environmental stress. The other important TFs responsible for plant defense mechanism are respiratory burst oxidase homologue D (Ciclev10027774m.g) and Leucine-rich repeat protein kinase family protein (Ciclev10019897m.g), expressed to a log fold change of 4.02 and 2.29, respectively (padj<0.05) (Table S11).