Search

Background: Asian citrus psyllid (ACP), also known as Diaphorina citri, is the natural vector of Candidatus Liberibacter asiaticus (CLas), which is responsible for Huanglongbing (HLB), a devastating citrus disease. Previously, the pathogen was successfully excluded from diseased citrus plants by using the indigenous endophyte Bacillus subtilis L1-21. However, the pathogen elimination and colonization potential of B. subtilis L1-21 in the carrier vector ACP, as well as the recruitment of native microbial communities of psyllid in the presence of endophytes, are still unknown., Results: Initially, we suggested that endophyte L1-21 reduced the CLas copies in ACP from 6.58 × 10 6 to 5.04 × 10 4 per insect after 48 h, however, the pathogen copies remained stable in the negative control. The endophyte was stable for 48 h after application. Among the bacterial genera those highlighted in ACP were Candidatus Liberibacter, Pseudomonas, Candidatus Profftella, Methylobacterium-Methylorubrum, Pantoea, Curtobacterium, Wolbachia, Actinomycetospora, and Bacillus. Interestingly, B. subtilis L1-21 easily colonizes the midgut of ACP but cannot be detected in eggs. When ACP with endophyte L1-21 was allowed to feed on new citrus leaves, the highest colonization was observed. We also found that psyllids carrying endophyte L1-21 after feeding on citrus leaves reduced the CLas copies in leaves on the 0, 3rd and 5th day from 8.18 × 10, 4 2.6 × 10, 3 and 0 pathogen copies/g fresh midvein, respectively., Conclusions: We propose that B. subtilis L1-21 is a native endophyte in citrus and psyllid, which efficiently reduces the CLas pathogen in both citrus and psyllids, provides a more protective effect by increasing the number of cultivable endophytes, and successfully colonizes the midgut of ACP. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Huanglongbing (HLB), a deadly citrus disease which has significantly downsized the entire industry worldwide. The intractable and incurable disease has brought the citriculture an enormous loss of productivity. With no resistant varieties available, failure of chemical treatments despite repeated applications, and hazardous consequences to environmental health, have led to large-scale research to find a sustainable cure. Inside plants, the key determinants of health and safety, live the endophytic microbes. Endophytes possess unrivaled plant benefiting properties. The progression of HLB is known to cause disturbance in endophytic bacterial communities. Given the importance of the plant endophytic microbiome in disease progression, the notion of engineering microbiomes through indigenous endophytes is attracting scientific attention which is considered revolutionary as it precludes the incompatibility concerns associated with the use of alien (microbes from other plant species) endophytes. In this review, we briefly discuss the transformation of the plant-pathogen-environment to the plant-pathogen-microbial system in a disease triangle. We also argue the employment of indigenous endophytes isolated from a healthy state to engineer the diseased citrus endophytic microbiomes that can provide sustainable solution for vascular pathogens. We evaluated the plethora of microbiomes responses to the re-introduction of endophytes which leads to disease resistance in the citrus host. The idea is not merely confined to citrus-HLB, but it is globally applicable for tailoring a customized cure for general plant-pathogen systems particularly for the diseases caused by the vascular system-restricted pathogens.

The native microbiomes of citrus trees play important roles in plant health, with good communication between the native microbiome and the host plant. Here, we report on the native endophytes in 24 citrus varieties in nine citrus growing regions in China; some of the trees were healthy and others had asymptomatic or symptomatic huanglongbing, which is caused by the pathogen Candidatus Liberibacter asiaticus (CLas). We used culture-dependent analysis and characterized the isolates by partial 16S rRNA gene sequencing. The endophytes were compared between different citrus varieties, regions, and disease states (healthy, asymptomatic, and symptomatic). The total number of endophytes isolated from most of the citrus varieties was 10 4 -10 6 CFU/g of leaves, but it differed significantly by disease state, with the highest numbers in the healthy leaves and the lowest in the symptomatic leaves (p < 0.05). Among the citrus varieties, the Valencia variety had the maximum number of endophyte species (22). The most dominant endophytes were Bacillus subtilis, B. velezensis, Curtobacterium luteum, and Microbacterium testaceum. The higher frequency of B. subtilis in the healthy/asymptomatic plants compared to the symptomatic plants suggests that it has a role in huanglongbing resistance. Native endophyte communities in various citrus varieties could be used to improve citrus growth and combat CLas.

Huanglongbing (HLB) is one of the most destructive citrus plant diseases worldwide. It is associated with the fastidious phloem-limited α-proteobacteria 'Candidatus Liberibacter asiaticus', 'Ca. Liberibacter africanus' and 'Ca. Liberibacter americanus'. In recent years, HLB-associated Liberibacters have extended to North and South America. The causal agents of HLB have been putatively identified, and their transmission pathways and worldwide population structure have been extensively studied. However, very little is known about the epidemiologic relationships of Ca. L. asiaticus, which has limited the scope of HLB research and especially the development of control strategies. HLB-affected plants produce damaged fruits and die within several years. To control the disease, scientists have developed new compounds and screened existing compounds for their antibiotic and antimicrobial activities against the disease. These compounds, however, have very little or even no effect on the disease. The aim of the present review was to compile and compare different methods of HLB disease control with newly developed integrative strategies. In light of recent studies, we also describe how to control the vectors of this disease and the biological control of other citrus plant pathogens. This work could steer the attention of scientists towards integrative control strategies.

BACKGROUND: Asian citrus psyllid (ACP), also known as Diaphorina citri, is the natural vector of Candidatus Liberibacter asiaticus (CLas), which is responsible for Huanglongbing (HLB), a devastating citrus disease. Previously, the pathogen was successfully excluded from diseased citrus plants by using the indigenous endophyte Bacillus subtilis L1‐21. However, the pathogen elimination and colonization potential of B. subtilis L1‐21 in the carrier vector ACP, as well as the recruitment of native microbial communities of psyllid in the presence of endophytes, are still unknown. RESULTS: Initially, we suggested that endophyte L1‐21 reduced the CLas copies in ACP from 6.58 × 106 to 5.04 × 104 per insect after 48 h, however, the pathogen copies remained stable in the negative control. The endophyte was stable for 48 h after application. Among the bacterial genera those highlighted in ACP were Candidatus Liberibacter, Pseudomonas, Candidatus Profftella, Methylobacterium‐Methylorubrum, Pantoea, Curtobacterium, Wolbachia, Actinomycetospora, and Bacillus. Interestingly, B. subtilis L1‐21 easily colonizes the midgut of ACP but cannot be detected in eggs. When ACP with endophyte L1‐21 was allowed to feed on new citrus leaves, the highest colonization was observed. We also found that psyllids carrying endophyte L1‐21 after feeding on citrus leaves reduced the CLas copies in leaves on the 0, 3rd and 5th day from 8.18 × 10,4 2.6 × 10,3 and 0 pathogen copies/g fresh midvein, respectively. CONCLUSIONS: We propose that B. subtilis L1‐21 is a native endophyte in citrus and psyllid, which efficiently reduces the CLas pathogen in both citrus and psyllids, provides a more protective effect by increasing the number of cultivable endophytes, and successfully colonizes the midgut of ACP. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) has turned into a devastating botanical pandemic of citrus crops, caused by Candidatus Liberibacter asiaticus (C Las). However, until now the disease has remained incurable with very limited control strategies available. Restoration of the affected microbiomes in the diseased host through the introduction of an indigenous endophyte Bacillus subtilis L1-21 isolated from healthy citrus may provide an innovative approach for disease management. A novel half-leaf method was developed in vitro to test the efficacy of the endophyte L1-21 against C Las. Application of B. subtilis L1-21 at 104 colony forming unit (cfu ml−1) resulted in a 1,000-fold reduction in the C Las copies per gram of leaf midrib (107 to 104) in 4 days. In HLB-affected citrus orchards over a period of 2 years, the C Las incidence was reduced to < 3%, and C Las copies declined from 109 to 104 g−1 of diseased leaf midribs in the endophyte L1-21 treated trees. Reduction in disease incidence may corroborate a direct or an indirect biocontrol effect of the endophytes as red fluorescent protein-labeled B. subtilis L1-21 colonized and shared niche (phloem) with C Las. This is the first large-scale study for establishing a sustainable HLB control strategy through citrus endophytic microbiome restructuring using an indigenous endophyte. [ABSTRACT FROM AUTHOR]

The endophytic bacteria Bacillus velezensis L1-21 was isolated from citrus huanglongbing (HLB) field diseased by Candidatus Liberibacter asiaticus, a fastidious phloem limited bacteria. We hypothesized that endophytes from citrus may be transfer to other plant hosts and could retain potential biocontrol. The bacteria was evaluated from citrus leaves by plating and assessed by 16S ribosomal RNA (16S rRNA), RNA polymerase B subunit (rpoB), and a DNA gyrase B subunit (gyrB) genes. Isolation was carried out more frequently from the healthy and asymptomatic citrus leaves. The possible biocontrol strain was tested by dual culture test to evaluate antagonistic effects against the bacterial plant pathogens Erwinia carotovora (B2), Xanthomonas citri (G), Erwinia sp. (HBYL), Pseudomonas syringe (JB), Xanthomonas oryzae (LKF), and Ralstonia solanacearum (WL); and soil borne fungi, F. oxysporum, and F. verticillium. Bacillus velezensis exhibited remarkable antibacterial activity against all the tested bacterial pathogens. While during colonization experiment of various hosts, a green fluorescent protein (GFP) labeled Bacillus velezensis L1-21-gfp, obtained by natural transformation of GFP-labeled vector of pHT01-P43GFPmut3a in L1-21 cytoplasm, was used. Fluorescence microscopy analysis revealed that endophytic strain can colonize outside and inside the citrus, green soy bean, tomato, green bristle grass, grape, chilli, Sonchus oleraceus, Malvastrum, eggplant, and Malva verticillata. The colonization range was 104-106 CFU/gram per leaf sample indicating its multiple colonization abilities. The higher frequency of Bacillus velezensis in healthy and asymptomatic citrus suggested a possible role of this endophyte to strengthen the citrus microbiome against huanglongbing. [ABSTRACT FROM AUTHOR]

Citrus is among the most important plants in the fruit industry severely infected with pathogens. Citrus green mold caused by Penicillium digitatum is one of the most devastating diseases during post-harvest stages of citrus fruit. In this study, a potential endophyte Bacillus subtilis L1-21, isolated from healthy citrus plants, was assessed for its biocontrol activity against the pathogen P. digitatum. Based on an in vitro crosstalk assay, we suggested that B. subtilis L1-21 inhibits the pathogen with an inhibition zone of 3.51 ± 0.08 cm. Biocontrol efficacy was highest for the fermented culture filtrate of B. subtilis L1-21. Additionally, using GC-MS analysis, 13 compounds were detected in the extract of this endophyte. The culture filtrate in Landy medium could enlarge and deform pathogen spores and prevent them from developing into normal mycelium. Accordingly, the Landy culture filtrate of B. subtilis L1-21 was stable in the temperature range of 4–90 °C and pH of 3–11. Further, MALDI-TOF-MS for B. subtilis L1-21 detected surfactin, fengycin, bacillaene and bacilysin as potential antifungal compounds. GFP-tagged B. subtilis L1-21 easily colonized in citrus fruit peel and pulp, suggesting its role in eliminating the fungal pathogen. Altogether, it is highly expected that the production of antifungal compounds, and the colonization potential of B. subtilis L1-21 are required against the post-harvest P. digitatum pathogen on citrus fruit. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas) brings a great concern about the phloem nutrient transport in diseased plants. There is an urgent need to find the best management strategies to reduce the losses in the citrus industry worldwide. Endophytic bacteria are negatively affected by CLas pathogen, and these endophytes are associated with improved availability of nutrients and pathogen resistance. This study underpins the relationship between CLas pathogen, endophyte population and nutrients availability in citrus plants. The citrus plants were treated with Bacillus subtilis L1-21 and Hoagland solution to find out synergism efficacy to mitigate citrus HLB. We showed that citrus shoots in the presence of 50% Hoagland solution displayed maximum number of endophytes with 6.28 × 103 to 3.04 × 105 CFU/g. Among 50 candidate strains, B. subtilis L1-21 emerged as potential antagonist against surrogate strain Xanthomonas citri subsp. citri. The citrus half-leaf method identified that application of endophyte L1-21 with 50% Hoagland solution successfully reduces the CLas abundance. We point out that this combination results in a higher number of endophytes population with 2.52 × 104 to 9.11 × 106 CFU/g after 60 days, and reduces CLas pathogen abundance in asymptomatic HLB plants. In HLB symptomatic citrus plants, B. subtilis L1-21 potentially increases the endophyte population from 1.11 × 104 to 5.26 × 107 CFU/g in the presence of Hoagland solution, and pathogen abundance was reduced from 9.51 × 105 to 1.06 × 104 copies/g. Altogether, we suggested that the presence of endophyte L1-21 with Hoagland solution is more effective in HLB asymptomatic citrus plants, but a slight reduction of pathogen was observed in symptomatic plants. The findings revealed the role of indigenous citrus endophyte B. subtilis L1-21 along with other nutrients in the reduction of CLas pathogen abundance inside symptomatic and asymptomatic plants in citrus endophyte–nutrient–pathogen interplay. [ABSTRACT FROM AUTHOR]

• Metabolomics approaches were employed to gain insight into mechanisms involved in citrus defense. • Potential changes in diseased plants were observed with Bacillus subtilis L1-21. • Metabolites up-regulated were xanthine, leucic acid, and α-Linolenic acid • Changes in diseased citrus physiology after endophyte L1-21 could give us future direction. Huanglongbing (HLB) is one of the most serious citrus diseases, caused by phloem limited endophytic bacteria Candidatus Liberibacter asiaticus (C las), affecting worldwide citrus production. Metabolomics approaches were employed to gain insight into mechanisms involved in defense against C las in endophyte Bacillus subtilis L1-21 treated diseased and healthy citrus plants. Using LC-ESI-MS/MS, we compared the metabolic profile of citrus plants before and after treatment with endophyte L1-21. Our analysis indicated large differences in citrus metabolites after endophyte L1-21 application. In total, seven hundred and fourty two metabolites were detected with highest percentage recorded for organic acids, flavone, amino acid derivatives, flavone C-glycosides, nucleotide derivatives, and flavonol. Interestingly, differentially expressed metabolites (DEMs) analysis revealed the amino acids, such as lysine and tyrosine which are involved in plant defense agianst pathogen attack were regulated in diseased citrus plants after endophyte application (padj<0.05). In addition, other important metabolites up-regulated were xanthine, leucic acid, and α-Linolenic acid implicated in different plant defense pathways against C las. Furhter, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed important pathways related to purine metabolism, biotin metabolism, and betalain biosynthesis, terpenoid-quinone biosynthesis, phenylalanine, tyrosine and lysine biosynthesis, isoflavonoid biosynthesis (padj<0.05). Taken together, this is the first study using native endophytes in diseased and healthy state of citrus which has proven to be useful in disease management by strengthening the defense of citrus to C las pathogen. [ABSTRACT FROM AUTHOR]