Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 215 - Biological Control of Pests Affecting Plants | 920 - Orange | 1100 - Bacteriology | 100% |
Plants host diverse endophytic bacteria that are an important component of the plant microbiome. Currently, endophytic bacteria remain poorly characterized. On the other hand, because endophytic bacteria have close contact with plants and overlap with many pathogens in colonizing the intercellular space, it has long been suggested that endophytic bacteria have great potential to control plant pathogens. Our long-term goal is to functionally characterize the endophytic bacteria of citrus and test their application in disease management. Our central hypothesis is that endophytic bacteria play important roles in suppressing infection by pathogenic microbes. In our previous study, we cultured a large number of endophytic bacteria from citrus roots and leaves. We will take advantage of our collection of endophytic bacteria to investigate their beneficial traits and test their application in disease control. The following objectives are proposed: investigation of the beneficial traits of the endophytic bacteria, genome sequencing of the endophytic bacteria, and engineering of synthetic endophytic bacterial microbiomes for disease management. We will use Xanthomonas citri subsp. citri (Xcc) as a model pathogen to investigate the control effect of the synthetic endophytic bacterial community. We will investigate the potential mechanisms responsible for the control effect against Xcc. Successful completion of the project will generate useful information regarding the function of endophytic bacteria. Engineering a synthetic root endophytic bacterial microbiome and a synthetic leaf endophytic bacterial microbiome has the potential to put the knowledge learned into action to benefit crop production.
Microbes can live externally on or internally in their host plants. Microbes that live outside their host plants are either epiphytic, i.e., living on the plant leaf surface, or rhizospheric, i.e., inhabiting plant roots within the soil. Conversely, microbes that live and thrive inside their host plant are called endophytic microbes. Endophytic microbes, especially endophytic bacteria, are often functional in that they may carry nutrients into plants, modulate plant development, increase the stress tolerance of plants, suppress the virulence of pathogens, increase disease resistance in plants, and suppress the development of competitor plant species (White et al., 2019). Endophytic bacteria have been suggested to significantly reduce the use of agrochemicals (fertilizers, fungicides, insecticides, and herbicides) in the cultivation of crop plants (White et al., 2019), contributing to sustainable agriculture and healthy food production. However, our understanding of the endophytic bacterial microbiome remains poor and the full potential of endophytic bacteria in disease management has not been realized. Our long-term goal is to functionally characterize the endophytic bacteria of citrus and test their application in disease management. Our central hypothesis is that endophytic bacteria play important roles in suppressing infection by pathogenic microbes. In our previous study, we cultured a large number of endophytic bacteria from the roots and leaves of healthy citrus. We will take advantage of our collection of endophytic bacteria to investigate their beneficial traits and test their application in disease control. The following specific objectives are proposed:
Objective 1. Investigation of beneficial traits of the endophytic bacteria Methods:1.1. Antimicrobial activity against XccThe antimicrobial activity of the endophytic bacterial isolates against Xcc strain 306 will be conducted as described previously (Wang et al., 2006b).1.2. Antimicrobial activity against C. acutatumHere, we will conduct antagonistic assays of endophytic bacterial isolates against C. acutatum as described in our previous study (Wang et al., 2006a; Riera et al., 2017). 1.3. Siderophore productionSiderophore production will be determined on chrome-azurol S (CAS) medium following the Universal Chemical Assay as described previously (Schwyn and Neilands, 1987; Trivedi et al., 2011).Objective 2. Genome sequencing of the endophytic bacteriaMethods:Genome sequencing. To isolate bacterial genomic DNA for genome sequencing, a modified E. Z. N. A Bacterial DNA Kit (OMEGA Bio-Tek, USA) protocol will be used. Bacterial genomes will be sequenced using both the Nanopore MinION (long reads) and Illumina (short reads) platforms to acquire complete genomes.Objective 3. Engineering synthetic endophytic bacterial microbiomes for disease managementMethods:3.1. Evaluation of the compatibility among bacterial isolatesWe will first test the compatibility among the entophytic bacterial isolates from the roots and leaves. A bacterial growth compatibility test will be performed in vitro on NA plates by cross-streaking fresh cultures of bacterial isolates. The plates will be incubated at 28°C for 24-48 h. Three biological replicates will be included for each test.3.2. Develop and test the SynComER in disease management3.2.1. Development of the SynComERTo develop and test the SynComER, we will use bacterial isolates that (i) are from the 28 isolates in Table 1, B. megaterium PT6, B. subtilis PT26A (Table 2), B. metallica A53, B. territorii A63, P. granadensis 100 and P. geniculata 95, (ii) are compatible with other members of the consortium (see Objective 3.1), and (iii) proliferate in nutrient-rich media. In our previous study, P. validus ATY16, B. megaterium PT6, and B. subtilis PT26A were among the top performers in term of beneficial traits (Fig. 2, Table 2), colonizing the endophytic compartment of citrus roots, and compatibility with each other (Fig. 5). We will further develop the SynComER based on these three members and add other isolates that are compatible with these three isolates.The bacterial isolates that meet the aforementioned criteria will be used together as the SynComER. 3.2.2. Greenhouse tests of the SynComER effect on disease controlFor the greenhouse test of protection against Xcc infection, 20 plants of each treatment (SynComER1, SynComER2, and negative control) will be inoculated with Xcc in the greenhouse after the third treatment with the SynComER and when fully expanded immature leaves are available for each plant. To differentiate the effect of bacteria in the endophytic and rhizosphere compartments, the roots of 10 plants in each treatment will be washed with tap water and replanted in autoclaved soil. The remaining 10 plants in each treatment will be kept in the original pots. Two weeks after replanting, Xcc inoculation will be conducted by foliar spray as described in our previous study (Riera et al., 2018).3.2.3. Mechanisms of the SynComER effect on disease controlWe hypothesize that the application of the SynComER will activate plant immunity systemically. In our proof-of-concept tests, application of B. territorii A63, B. metallica A53 and P. geniculata 95 via soil drenching of the roots effectively induced plant defense and significantly reduced canker symptom development in leaves challenged with Xcc (Riera et al., 2018). To test whether application of the SynComER induces plant immunity systemically, we will collect Xcc-sprayed leaf samples of different treatments (SynComER1, SynComER2, and negative control) in the greenhouse test for gene expression, hormone and reactive oxygen species (ROS) analyses immediately after Xcc inoculation, 1 and 7 days post Xcc inoculation as described previously (Riera et al., 2018).3.2.4. Field tests of the SynComER effect on disease controlFor field trials, the remaining 80 plants of each treatment (SynComER1, SynComER2, and negative control) will be planted in a selected citrus grove approximately one week after the third treatment with the SynComER in April. The surrounding groves are naturally infected by Xcc with a canker incidence of >90%. To differentiate the effect of bacteria in the endophytic and rhizospheric compartments, the roots of 40 plants of each treatment will be washed with tap water before planting to remove bacteria on the root surface, and the remaining 40 plants/treatment will be planted directly. Treatments will be arranged in a randomized complete block design with six treatments replicated eight times in blocks of five contiguous trees. Assessments of the incidence of citrus canker on foliage will be conducted monthly on the three innermost trees of each plot in May to October. The field trial will be continued for three years.3.3. Develop and test the SynComEL effect on disease control3.3.1. Development of the SynComELTo develop and test the SynComEL, we will use bacterial isolates that (i) are among the approximately 30 selected leaf endophytic bacterial isolates belonging to Achromobacter, Bacillus, Bradyrhizobium, Brevundimonas, Burkholderia, Cellulosimicrobium, Chryseobacterium, Curtobacterium, Luteibacter, Methylobacterium, Paenibacillus, Pantoea, Pseudomonas, Serratia, Stenotrophomonas, and Streptomyces, (ii) are compatible with other members of the consortium (see Objective 3.1), and (iii) proliferate in nutrient-rich media. We will develop the SynComEL with a similar approach as that in Objective 3.2 by selecting several comparable top performers first and then adding other comparable bacterial isolates. The bacterial isolates that meet the aforementioned criteria will be used together as the SynComEL. Preparation of the SynComEL bacterial culture will be conducted as described in Objective 3.2. The final concentration of each bacterial isolate will be 1x107 CFU/mL (SynComEL1) and 1x108 CFU/mL (SynComEL2). Tap water will be used as a negative control.3.3.2. Greenhouse tests of the SynComEL effect on disease controlFor greenhouse assessments of protection against Xcc infection, we will conduct tests when fully expended young leaves are available on two-year-old Valencia sweet orange on Carrizo citrange rootstock.3.3.3. Mechanisms of the SynComEL effect on disease controlWe hypothesize that the application of SynComEL to leaves will exert a disease control effect by inducing plant immunity, antagonism, or siderophore production.3.3.4. Field tests of the SynComEL effect on disease controlFor the field trial, 3-year-old Hamlin sweet orange on Swingle citrumelo rootstock in a research grove owned by UF located in Lake Alfred, Florida will be used. The trees will be sprayed with SynComEL1, SynComEL1 followed by washing with tap water after 24 h, SynComEL2, SynComEL2 followed by washing with tap water after 24 h, copper hydroxide as a positive control, and tap water as a negative control.
Target Audience
Scientific community, citrus growers, graduate students, agricultural industry
Changes / Problems
Nothing Reported
Training & Professional Development
We trained one graduate student, one undergraduate student in citrus microbiome and canker research.
Dissemination Streams
The results have been published in two manuscripts, presented in meetings and invited seminars.
Next Reporting Steps
Objective 1. Investigation of beneficial traits of the endophytic bacteria. We will test more endophytic bacterial isolates from citrus leaves for the following beneficial traits: antagonism against selected citrus pathogens and siderophore production. Objective 2. Genome sequencing of the endophytic bacteria. We will conduct genome sequencing of our endophytic bacterial isolates with beneficial traits from citrus roots and leaves. We have already sequenced multiple beneficial isolates. We will conduct genomic analyses of those isolates. Objective 3. Engineering synthetic endophytic bacterial microbiomes for disease management. Continue the field trials. We will test whether genome editing of certain host genes can improve microbiome for disease control.
Target Audience
Scientific community, citrus growers, graduate students, agricultural industry
Changes / Problems
Nothing Reported
Training & Professional Development
We trained one graduate student, one undergraduate student in citrus microbiome and canker research.
Dissemination Streams
The results have been published in two manuscripts, presented in meetings and invited seminars.
Next Reporting Steps
Objective 1. Investigation of beneficial traits of the endophytic bacteria. We will test more endophytic bacterial isolates from citrus leaves for the following beneficial traits: antagonism against selected citrus pathogens and siderophore production. Objective 2. Genome sequencing of the endophytic bacteria. We will conduct genome sequencing of our endophytic bacterial isolates with beneficial traits from citrus roots and leaves. We have already sequenced multiple beneficial isolates. We will conduct genomic analyses of those isolates. Objective 3. Engineering synthetic endophytic bacterial microbiomes for disease management. Continue the field trials. We will test whether genome editing of certain host genes can improve microbiome for disease control. <br><br>
<br>What was accomplished under these goals? Objective 1. A total 303 citrus leaf and root endophytic bacterial isolates were tested for the antimicrobial activity against Xcc on nutrient agar (NA) plates. 19 bacterial isolates were identified as Bacillus, Brevibacillus and Pseudomonas respectively showed inhibitory activity against Xcc growth. In addition, we have isolated over 100 bacterial isolates from another site in Florida citrus grove. Among them, 14, 10, and 6 bacterial isolates showed antagonistic activity against X. citri subsp. citri, citrus melanose pathogen Diaporthe citri, and citrus root rot pathogen Phytophthora nicotianae, respectively. A total 125 endophytic bacteria were screened for siderophore production. 16 bacteria from Bacillus, Brevibacillus and Pseudomonas respectively produced siderophore. Objective 2. The endophytic bacteria B. megaterium PT6, B. subtilis PT26A and Paenibacillus sp. ATY16 WERE whole-genome sequenced. Genome mining for secondary metabolites biosynthesis genes revealed that B. subtilis PT26A contains six gene clusters coding for non-ribosomal peptide synthetase (NRPS) and three gene clusters coding for polyketide synthetase (PKS), based on antiSMASH analysis. B. megaterium PT6 contains five gene clusters for NRPS and two gene clusters for PKS. Paenibacillus sp. ATY16 has eight gene clusters for NRPS and two gene clusters for PKS. In addition, the whole-genomes of the leaf entophytic bacteria Brevibacillus sp. EBL1, Pseudomonas sp. EPL5, and Pseudomonas sp. EPL8 were sequenced. Genome mining for secondary metabolites biosynthesis genes revealed that Brevibacillus sp. EBL1 contains three gene clusters coding for non-ribosomal peptide synthetase (NRPS) and two gene clusters coding for polyketide synthetase (PKS). Pseudomonas sp. EPL5 contains four gene clusters for NRPS and two gene clusters for PKS. Pseudomonas sp. EPL8 has five gene clusters for NRPS and two gene clusters for PKS.). Pseudomonas sp. EPL5 and Pseudomonas sp. EPL8 also contain genes putatively responsible for siderophore biosynthesis, including the genes entD and pchB. Objective 3. 3.1. The compatibility among 17 root entophytic bacterial isolates including B. megaterium PT6, B. subtilis PT26A, B. metallica A53, B. territorii A63, Paenibacillus sp. ATY16, P. granadensis 100 and P. geniculata 95 were tested on NA plates. B. megaterium PT6, B. subtilis PT26A, B. metallica A53, Paenibacillus sp. ATY16 and B. territorii A63 are compatible with each other and no antagonistic activity was observed after co-culturing the bacteria at 28°C for 48 h. The compatibility among root entophytic isolates of Paenibacillus sp. ATY16, B. megaterium PT6, B. subtilis PT26A and leaf entophytic isolates of Brevibacillus sp. EBL1 and Pseudomonas sp. EPL5, Bacillus sp. LAL3 were tested on NA plates. These six bacteria are compatible.. 3.2. Leaf entophytic isolates of Brevibacillus sp. EBL1 and Pseudomonas sp. EPL5 were determined to inhibit the citrus canker pathogen X.. citri growth both in laboratory and greenhouse tests. The bacterial mixture applied by foliar spray conferred better efficacy of control of citrus canker than single strains in greenhouse assays. In addition, the expression of citrus defense-related genes including PR1, PR2, and PR5 were significantly induced by the bacterial mixture spray treatment, as determined by quantitative reverse-transcription PCR (qRT-PCR). To investigate the contribution of antagonism and siderophores production to the control of citrus canker, deletion mutants for genes encoding the LuxR-type transcriptional regulator PcoR and RfiA in Pseudomonas sp. EPL5 were constructed. Phenotype assays of those mutants are underway to confirm the gene's function in antimicrobial and/or siderophore production. Those mutants are also to be tested for inhibition of the citrus canker pathogen X. citri growth in laboratory and greenhouse settings. Root entophytic bacteria Paenibacillus sp. ATY16, B. megaterium PT6, and B. subtilis PT26A were tested for prevention of citrus canker in greenhouse. The bacteria were applied alone and in combination, respectively, through root inoculation by soil drench 3 days prior to Xcc inoculation via leaf spray of Valencia sweet orange plants. The results show that plants treated with the bacterial consortium developed less canker lesions on the leaves than those treated with water. Additionally, the expression of the genes encoding allene oxide cyclase (AOC), allene oxide synthase (AOS), basic helix-loop-helix (bHLH) transcription factor MYC2, and the plant defensin gene PDF1.2 were significantly induced by the bacterial mixture root inoculation. 3.3. In March 2022, a field trial for control of citrus canker was initiated with 4-year-old "OLL-8" orange trees planted in 2018 at Lake Alfred, Polk County, FL. This has been reported last year. In March 2023, a field trial for control of citrus canker was initiated in the same citrus grove as 2022. The experiment is a randomized complete block design (RCBD) with six treatments replicated five times in blocks of five contiguous trees. The six foliar treatments include: 1) copper hydroxide at 2.0 g a.i. per tree per application (Kocide 3000; 30% metallic Cu a.i.; DuPont); 2) SynComEL3 (B. megaterium PT6, B. subtilis PT26A, Bacillus sp. LAL3, and Brevibacillus sp. EBL1) at 108 cfu/ml. per tree per application; 3) SynComEL4 (B. megaterium PT6, B. subtilis PT26A, Bacillus sp. LAL3, Brevibacillus sp. EBL1 and Pseudomonas sp. EPL5) at 108 cfu/ml; 4) copper hydroxide rotated with SynComEL3; 5) copper hydroxide rotated with SynComEL4; and 6) water only as untreated control. The treatments were applied as foliar spray every 3 weeks, starting on March 23, 2023, during the spring flush, until October 12, 2023 during the fall flush. The incidence of citrus canker on foliage was assessed as the percentage of canker-infected leaves on sets of leaf flushes: spring-first summer, spring-summer, and spring-summer-fall in June, September, and November, 2023, respectively. The results show that all treatments significantly reduced incidence of canker on the three sets of flushes and fruit compared with the untreated control and, copper spray had the best performance in terms of control of foliar and fruit canker. The untreated spring-summer-fall flushes evaluated in November had 49.4% canker-diseased leaves, while the copper hydroxide, SynComEL3, and SynComEL4 sprayed had 14.7, 25.3, and 26.4% canker-diseased leaves, respectively, and the treatments copper hydroxide rotated with SynComEL3 and copper hydroxide rotated with SynComEL4 had 21.1 and 20.6% canker-diseased leaves. In the untreated trees evaluated in November, 42.6% fruit were infected by citrus canker, while in the copper hydroxide, SynComEL3, and SynComEL4 sprayed trees, 12.2, 20.1, and 19.5% fruit were infected by citrus canker, respectively, and the treatments copper hydroxide rotated with SynComEL3 and copper hydroxide rotated with SynComEL4 had 15.6 and 15.8% fruit infected by canker. The harvest fruit yield was not statistically significant among the treatments including untreated control. Additionally, a field trial for testing the SynComE for preventing citrus canker and HLB diseases by root application in a UF-CREC research block was initiated in May 2023. The experiment included two treatments: water as untreated control and SynComE (including the root entophytic bacteria Paenibacillus sp. ATY16, B. megaterium PT6, and B. subtilis PT26A) and both had 40 replicate trees. The treatments were applied by root soaking prior to planting and by soil drench monthly after planting, respectively. The incidence of citrus canker in untreated control and SynComE treatment was 37.5% and 22.5% at 8 months post the first treatment application. The incidence of citrus HLB in untreated control and SynComE treatment was 57.5% and 37.5% at 8 months post the first treatment application. <br><br><b>Publications</b><br>
Target Audience
Scientific community, citrus growers, graduate students, agricultural industry
Changes / Problems
Nothing Reported
Training & Professional Development
We trained one graduate student, one undergraduate student in citrus microbiome and canker research.
Dissemination Streams
The results have been published in one manuscript, presented in meetings and invited seminars.
Next Reporting Steps
Objective 1. Investigation of beneficial traits of the endophytic bacteria. We will test more endophytic bacterial isolates from citrus leaves for the following beneficial traits: antagonism against selected citrus pathogens and siderophore production. Objective 2. Genome sequencing of the endophytic bacteria. We will conduct genome sequencing of our endophytic bacterial isolates with beneficial traits from citrus roots and leaves. We have already sequenced multiple beneficial isolates. We will conduct genomic analyses of those isolates. Objective 3. Engineering synthetic endophytic bacterial microbiomes for disease management. Continue the field trials. <br><br>
<br>What was accomplished under these goals? Objective 1. A total 192 citrus leaf and root endophytic bacterial isolates were tested for the antimicrobial activity against Xcc on nutrient agar (NA) plates. Nine bacteria preliminarily identified as Bacillus, Brevibacillus and Pseudomonas respectively showed inhibitory activity against Xcc growth. In addition, we have isolated over 100 bacterial isolates from another site in Florida citrus grove, tested their inhibitory abilities against the three phytopathogens in vitro. Among them, 14, 10, and 6 bacterial isolates showed antagonistic activity against X. citri subsp. citri, citrus melanose pathogen Diaporthe citri, and citrus root rot pathogen Phytophthora nicotianae, respectively. A total 51 endophytic bacteria were screened for siderophore production. Seven bacteria from Bacillus, Brevibacillus and Pseudomonas respectively produced siderophore in chrome-azurol S (CAS) medium. Objective 2. The endophytic bacteria B. megaterium PT6, B. subtilis PT26A and Paenibacillus sp. ATY16 WERE whole-genome sequenced. Genome mining for secondary metabolites biosynthesis genes revealed that B. subtilis PT26A contains six gene clusters coding for non-ribosomal peptide synthetase (NRPS) and three gene clusters coding for polyketide synthetase (PKS), based on antiSMASH analysis. B. megaterium PT6 contains five gene clusters for NRPS and two gene clusters for PKS. Paenibacillus sp. ATY16 has eight gene clusters for NRPS and two gene clusters for PKS. In addition, the whole-genome sequencing for the leaf entophytic bacteria Brevibacillus sp. EBL1, Pseudomonas sp. EPL5, and Pseudomonas sp. EPL8 is ongoing. Objective 3. 3.1. The compatibility among 17 root entophytic bacterial isolates including B. megaterium PT6, B. subtilis PT26A, B. metallica A53, B. territorii A63, Paenibacillus sp. ATY16, P. granadensis 100 and P. geniculata 95 were tested on NA plates. B. megaterium PT6, B. subtilis PT26A, B. metallica A53, Paenibacillus sp. ATY16 and B. territorii A63 are compatible with each other and no antagonistic activity was observed after co-culturing the bacteria at 28°C for 48 h. The compatibility among root entophytic isolates of Paenibacillus sp. ATY16, B. megaterium PT6, B. subtilis PT26A and leaf entophytic isolates of Brevibacillus sp. EBL1 and Pseudomonas sp. EPL5, Bacillus sp. LAL3 were tested on NA plates. These six bacteria are compatible.. 3.2. Leaf entophytic isolates of Brevibacillus sp. EBL1 and Pseudomonas sp. EPL5 were determined to inhibit the citrus canker pathogen Xanthomonas citri ssp. citri growth both in laboratory and greenhouse tests. The bacterial mixture applied by foliar spray conferred better efficacy of control of citrus canker than single strains in greenhouse assays. In addition, the expression of citrus defense-related genes including PR1, PR2, and PR5 were significantly induced by the bacterial mixture spray treatment, as determined by quantitative reverse-transcription PCR (qRT-PCR). To investigate the contribution of antagonism and siderophores production to the control of citrus canker, constructions of deletion mutants for antimicrobial or siderophore gene for Pseudomonas sp. EPL5 are underway. Root entophytic bacteria Paenibacillus sp. ATY16, B. megaterium PT6, B. subtilis PT26A were tested for prevention of citrus canker in greenhouse. The bacteria were applied alone and in combination, respectively, through root inoculation by soil drench 3 days prior to Xcc inoculation via leaf spray of Valencia sweet orange plants. The results show that plants treated with the bacterial consortium developed less canker lesions on the leaves than those treated with water. Additionally, the expression of the genes encoding allene oxide cyclase (AOC), allene oxide synthase (AOS), basic helix-loop-helix (bHLH) transcription factor MYC2, and the plant defensin gene PDF1.2 were significantly induced by the bacterial mixture root inoculation. 3.3. In March 2022, a field trial for control of citrus canker was initiated with 4-year-old "OLL-8" orange trees planted in 2018 at Lake Alfred, Polk County, FL. This grove is naturally infected by citrus canker. Treatments were arranged in a randomized complete block design (RCBD) with four treatments replicated five times in blocks of five contiguous trees. The four foliar treatments were copper hydroxide at 2.0 g a.i. per tree per application (Kocide 3000; 30% metallic Cu a.i.; DuPont), SynComEL1 (B. megaterium PT6, B. subtilis PT26A, Paenibacillu sp. ATY16 and Brevibacillus sp. EBL1) at 108 cfu/ml. per tree per application, SynComEL2 (B. megaterium PT6, B. subtilis PT26A, Brevibacillus sp. EBL1 and Pseudomonas sp. EPL5) at 108 cfu/ml, and water only as untreated control. The treatments were applied as foliar spray every 3 weeks, starting on March 14, 2022, during the spring flush, until October 6, 2022 during the fall flush. The incidence of citrus canker on foliage were assessed as the percentage of canker-infected leaves on sets of leaf flushes: spring-first summer, spring-summer, and spring-summer-fall in June, August, and November, 2022, respectively. The results show that all treatments significantly reduced incidence of canker on the three sets of flushes compared with the untreated control and, copper sprays had the best performance in terms of control of foliar canker. The untreated spring-summer-fall flushes evaluated in November had 43.5% canker-diseased leaves, while the copper hydroxide, SynComEL1, and SynComEL2 sprayed had 16.6, 20.9, and 23.1% canker-diseased leaves, respectively. In addition, the copper hydroxide, SynComEL1, and SynComEL2 treatments had a higher average fruit yield per plot than untreated control, although the differences were not statistically significant. In March 2023, a field trial for control of citrus canker was initiated in the same citrus grove as 2022. The experiment is a randomized complete block design (RCBD) with four treatments replicated five times in blocks of five contiguous trees. The four foliar treatments include copper hydroxide at 2.0 g a.i. per tree per application (Kocide 3000; 30% metallic Cu a.i.; DuPont), SynComEL3 (B. megaterium PT6, B. subtilis PT26A, Bacillus sp. LAL3, and Brevibacillus sp. EBL1) at 108 cfu/ml. per tree per application, SynComEL4 (B. megaterium PT6, B. subtilis PT26A, Bacillus sp. LAL3, Brevibacillus sp. EBL1 and Pseudomonas sp. EPL5) at 108 cfu/ml, and water only as untreated control. The treatments were applied as foliar spray every 3 weeks, starting on March 23, 2023, during the spring flush and, will continue till the fall flush in October 2023. <br><br><b>Publications</b><br>
Target Audience
Scientific community, citrus growers, graduate students, agricultural industry
Changes / Problems
No.
Training & Professional Development
We trained one graduatestudent, one undergraduate student in citrus microbiomeand canker research.
Dissemination Streams
The results have been published in 2 manuscripts, presented in meetings and invited seminars.
Next Reporting Steps
Objective 1. Investigation of beneficial traits of the endophytic bacteria. We will test more endophytic bacterial isolates from citrus leaves for the following beneficial traits: antagonism against selected citrus pathogens and siderophore production. Objective 2. Genome sequencing of the endophytic bacteria. We will conduct genome sequencing of our endophytic bacterial isolates with beneficial traits from citrus roots and leaves. We have already sequenced multiple beneficial isolates. We will conduct genomic analyses of those isolates. Objective 3.Engineering synthetic endophytic bacterial microbiomes for disease management. We will test thesyntheticcommunity ofendophytic bacteria (SynComE) in disease management of citrus canker. We have already developed a SynComE consisting of four bacterial isolates. We have set up the field trial. <br><br>
<br>What was accomplished under these goals? Objective 1. Investigation of beneficial traits of the endophytic bacteria. We have tested more than 100 beneficial bacteria isolates on antagonistic activities against Xanthomonas citri subsp. citri and other traits such as siderophore production. Two isolates belonging to Pseudomonas sp. were determined to have antimicrobial activity against Xcc. Objective 2. Genome sequencing of the endophytic bacteria. The citrus root endophytic bacterial isolates B. megaterium PT6 and B. subtilis PT26A that are antagonistic to Xcc and contain multiple beneficial traits have been whole-genome sequenced and data analyses are ongoing. Genome mining for secondary metabolites biosynthesis genes in B. subtilis PT26A revealed there are six gene clusters coding for non-ribosomal peptide synthetase (NRPS) and three gene clusters coding for polyketide synthetase (PKS). Objective 3.Engineering synthetic endophytic bacterial microbiomes for disease management. The compatibility among 16 root entophytic bacterial isolates including B. megaterium PT6, B. subtilis PT26A, B. metallica A53, B. territorii A63, P. granadensis 100 and P. geniculata 95 were tested in vitro on NA plates. B. megaterium PT6, B. subtilis PT26A, B. metallica A53, and B. territorii A63 are compatible with each other, as no antagonistic activity was observed after cross-streaking fresh cultures of bacterial isolates and incubation at 28°C for 48 h. <br><br><b>Publications</b><br>