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Download Latest Progress Report PROGRESS REPORT PROJECT TITLE: Hyper-Thermostable Enzyme (Lactonases) for use as Microbial Biocontrol Agents for Plant Diseases PROJECT NUMBER: 4136-17SP REPORTING PERIOD: Oct. 1, 2019 – Jan. 31, 2020 PRINCIPAL INVESTIGATOR: Michael Sadowsky and Mikael Elias ORGANIZATION: University of Minnesota PHONE NUMBER: 612-624-2706 EMAIL: [email protected] Clavibacter michiganensis subsp. nebraskensis (Cmn) Dose Response Assay for Corn Leaves Maize growth Maize cv Viking seed (40-30UP) were grown in Euro pots (diameter 8 inch) with a sterilized soil mixture (50 standard soil/ 50 Germinating Mix) in a plant growth chamber under diurnal conditions with a 16 hour light at 22 ˚C and 8 hour dark cycle at 18˚C. Bacteria working solution Firstly, Clavibacter michiganensis subsp. nebraskensis (causing Goss's bacterial wilt & leaf blight on Maize) was grown on NBY agar at room temperature (for ~5 days). Then, a single colony of Cmn was transferred into the fresh NBY plate. After three days, Cmn was centrifuged 8 and suspended into 1X PBS to obtain working solution at OD540=0.1 ( about 10 cell/ml). Infection assays and results The corn leaves were scraped with sterilized sandpaper and inoculated serial diluted cells from 106, 105,104,103,102,10,0 cells on the scraped corn leaf, respectively. The effect of each concentration of Cmn on disease was evaluated using triplicate samples. After 18 days, Goss’s Wilt Symptoms were observed at two highest concentrations (106 and105) experiments. 106 105 104 103 102 10 0 Cmn + + - - - - - 1 To better quantify the effect on plant disease, we adopted the use of chlorosis assay that non- destructively measures plant chlorophyll in control and diseased corn leaf tissue. Chlorophyll Chlorophyll content in 6 replicates of corn with 106 cells Cmm Mean Day 0 37.7 44.8 46.9 49.4 47.6 45.5 45.3 Day 18 2.4 9.0 7.8 8.8 13.1 10.9 8.6 Day 18 Day 0 Results showed that dead/chlorotic leaves had 5-fold less chlorophyll than did controls. This assay will now be used on plants showing disease symptoms between these two extremes. We have now obtained a culture of Xanthomonas axonopodis pv. glycines causing bacterial pustule disease on soybean and we will test our enzymes against this pathogen. We also now have permission from APHIS to test Erwinia stewartii (Syn. Pantoea stewartii) causing Stewart's wilt (Stewart's disease, Stewart's bacterial wilt), Erwinia chrysanthemi pv zeae, and Dickeya zeae (bacterial stalk rot of maize) and we are trying to obtain these strain now to test on corn. To better assess the relationship between pathogen effects and quorum quenching lactonases we developed a system to measure this in a quantitative fashion. Here disease scale has 13 levels, representing 0-100% tissue necrosis/leaf. The test plant was maize, and this is seen below. Quantification of Disease Levels Scale designation Level Level Level Level Level Level Level Level Level Level Level Level Level 1 2 3 4 5 6 7 8 9 10 11 12 13 Percent of leaf with 0 2 5 10 20 30 40 50 60 70 80 90 100 necrosis 2 The assay appears to be useful for other pathogens as well. Below is the Corn Leaf Assay done using Clavibacter michiganensis subsp. nebraskensis (Cmn) Disease levels Treatment Rep. A Rep. B Rep. C Negative control 1 1 1 Buffer only 1 1 1 SsoPox only 1 1 1 Cmn only 9 9 9 Cmn+SsoPox 3 3 3 Bacterial strains and quorum quenching lactonases. Eleven pathogenic strains that we are using in this study include Clavibacter michiganensis subsp. nebraskensis, Erwinia chrysanthemi pv. Zeae, Pantoea stewartii Mergaert, Erwinia aroideae 8066, Erwinia atroseptica 8064, Xanthomonas campestris pv. campestris, Pseudomonas syringae pv syringae, Pseudomonas syringae pv phaseolicola, Erwinia carotovora, Xanthomonas campestris pv. phaseoli, and Pectobacterium carotovorum. The strains were grown in selected medium at 25-30˚C. There will be two kinds of the quorum quenching lactonases (SsoPox W2631, and GcL WT) used in this study. The characterizations of these lactonases were studied by Dr. Elias groups 1, 2. We previously reported successful protection of several plant species from bacterial diseases by using lactonase enzyme SsoPox. Here we described our initial assays on corn using Clavibacter michiganensis subsp. nebraskensis (Cmn). The goal was to determine if the presence of lactonase on the corn leaf surface (the phyllosphere) alters bacterial community structure, in addition to preventing disease. 3 Variation of maize phyllosphere microbiome due to interaction of Cmn and SsoPox Next-Generation Sequencing The V4 hypervariable region of 16S rRNA was amplified for bacteria and archaea by using primer set 515F (5’– GTG CCA GCM GCC GCG GTA A –3’) and 806R (5’– GGA CTA CHV GGG TWT CTA AT –3’). Sequencing was done by the University of Minnesota Genomics Center (Minneapolis, MN, USA) as previously described3. Briefly, samples were first amplified by using the following cycling conditions: 95˚C for 5 min, followed by 25 cycles of 98˚C for 20 s, 55˚C for 15 s, and 72˚C for 1 min. Adapters and barcodes were added by using an additional 10 cycles PCR. Amplicons were gel purified, pooled, and paired end sequenced at a read length of 300 nt on the Illumina MiSeq platform (Illumnia, Inc., San Diego, CA, USA). Bioinformatics Highly quality sequence data for analyses were obtrained by submitting the raw sequencing data through the SHI7 pipeline for quantity control (QC) 4, 5. In brief, sequencing adaptors were removed, the two paired-end reads were merged, and residual adaptors were further trimmed. Low quality reads were removed to keep QC values > 31 and highly quality reads were converted into the FASTA format and aligned on the basis of the Greengenes ver. 13.86. UCHIME software was used to identify and remove the probable Chimeric sequence7. All sequence data was rarefied to 63,000 sequence reads per sample before sustainable statistical analysis. Raw sequencing data will eventually be deposited in the Sequence Read Archive (SRA) of NCBI (https://www.ncbi.nlm.nih.gov/sra). Statistical analysis For statistical analysis, Alpha diversity (average species diversity) indices, as well as Good’s coverage, were calculated using the Shannon index and abundance-based coverage estimate through mother program. Visualization of the taxonomic distribution of microbial communities was performed by using the “ggplot2” package in R (Al-Masaudi et al., 2017). Differences in beta diversity among samples was evaluated by using analysis of similarity (ANOSIM), which employs Bray-Curtis dissimilarity matrices (BC)8, 9. Principal coordinates analysis (PCoA) was used to analyze the differences in microbial community structure between various DNA extraction methods. Linear discriminant analysis (LDA) of effect size (LEfSe) was used to identify OTUs that differed significantly influencing of SsoPox on phyllosphere. Moreover, indicator OTUs association with influencing of SsoPox on phyllosphere was determined based on multipatt function using the indcespecies package in R. The association was further considered significant using a false discovery rate. Attended conference Summarized the results of project and prepared the poster. Presented the poster in MN AG EXPO on January 24,2019. The results attracted a lot of attention. Lots of farmers were interested when the technology could be used in the field. We will continue to improve our technology and conduct the addition experiments in the field. 4 Corn field Study In order to further understand the capability of SsoPox on corn disease in the field, we conducted the corn field study in the past three months. We tested two diseases related to corns. The first disease is Bacterial Leaf Streak (BLS) causing by Xanthomonas vasicola pv. vasculorum (Xvv). We inoculated Xvv at 10^7 CFU/mL with the clapper, one upper leaf per plant. The second disease is Goss’s Wilt causing by Clavibacter michiganensis subsp. nebraskensis (Cmn). We inoculated Cmn at 10^8 CFU/mL with the clapper, one upper leaf per plant. In the treatment groups, we sprayed SsoPox on the surface of corn leaf, which had been inoculated Xvv or Cmn. In the control groups, we conducted the similar assays, but used BSA instead of SsoPox. We used the untreated corn plants as negative control groups. Each assay was performed using 3 replications, and each block was also performed using 3 plants replications. DNA extraction In order to determine the influence of leaf microbes in suppression of Goss’s wilt disease, we collected corn leaf samples from three plots at the Rosemount experiment station. Treatments used included a negative control group, Cmn-only group, Cmn plus SsoPox group, and Cmn plus BSA group. After enzyme treatment, 44 samples were collected and subjected to DNA extraction. We also extended this study to examine the shifts of the phyllosphere microbiome under the influence of SsoPox, by treating the leaves of filed grown soybean, black bean, and corn with enzyme under labs incubation conditions There were three treatments, including a negative control, a SsoPox group, and a BSA group. Each treatment had four replications. There were 48 and DNA was extracted by using PowerSoil DNA extraction kits. DNA was then subjected to DNA sequence and data analysis. Results 5 Composition of microbial communities in the maize phyllosphere Results in Table 1 show the mean coverage of all three treatment categories was greater than 99%, without significant difference between Cmn, Cmn+SsoPox, and control groups, respectively. Mean Shannon diversity indices for individual categories ranged from 1.04 to 1.64, and Shannon diversity differed significantly between Cmn groups to Cmn+SsoPox and control. The ranking of diversity was as follows: Cmn+SsoPox > Control > Cmn. However, there was no significant different between Cmn to Cmn+SsoPox in Chao1 and Sobs.
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