Reducing tree decline of Casuarina equisetifolia in Guam through replacement of bacterial wilt infected trees and research into the bacterial microbiomes of trees and associated termites

Obj. 2 Yr 1-2: UH Res & UOG Res:  Research into the bacterial wilt pathogen to determine the origin of Guam’s infection and its genomic biology.

Methods: Obj. 2, University of Guam: research was conducted to find a method suitable for isolation of R. solanacearum from Guam’s ironwood trees. Though R. solanacearum could be detected from wood chips, drill shavings, water from root drill shavings, and stems and branches of trees, attempts to isolate from these same samples failed.  The only means by which Rs could be isolated was by streaking ooze that formed on disks taken from stems, roots, or large branches of infected trees onto selective medium. To enhance the production of ooze, slices were placed on a saturated paper-towel in a moisture chamber for 24 hrs. Once formed, the ooze was streaked on Engelbrecht’s semi-selective medium (mSMSA) (Engelbrech 1994). Colonies were re-streaked onto SMSA, which was followed by streaking onto modified Kelman’s tetrazolium chloride medium (TZC) before grow-out on TZC (Norman and Alvarez 1989). Once grow-out plates had well-isolated fluidal growth, a transfer loop or spatula was used to transfer colonies to 3 ml of sterilized tap water in a 6 ml sterilized screw cap glass vials. The inoculation process is repeated as necessary to create a cloudy suspension. Duplicate vials were prepared, one for storage at room temperature in Guam and one which was shipped to the University of Hawaii for further evaluation.

Information recorded at each tree site included: tree tag number, date of collection, GPS location, visual decline rating (DS), site condition, height, DBH, altitude, geology characteristics, occurrence, root exposure, and presence of termites and Ganoderma wood rot. Tree drill shavings were collected from a single 3-inch deep hole using a ¼ inch drill bit. Shavings were assayed for R. solanacearum using R. solanacearum-specific immunostrip tests kits manufactured by Agdia Inc. of Elkhart, Indiana, USA (catalog number: STX 33900 and ISK 33900).  Ralstonia solanacearum immunostrip testing was conducted on samples: either 1.5-3.0 mg of wood shavings or 80 µL of water from soaked drill shavings.

Methods: Obj. 2: University of Hawaii: water cultures from Guam were streaked multiple times onto TZC to ensure the cultures purity. A single colony from modified SMSA media was picked and mixed in 50 microliters of nuclease-free water. The colony was denatured for 10 minutes at 95^oC and centrifuged for two minutes. The colony was used as a template to do endpoint PCR with Ralstonia solanacearum species complex specific primers. DNA extraction was performed with cultures found to be positive with PCR. The DNA extracted was used as a template to perform dnaA specific PCR for sending the samples for sequencing. Sequencing was done with both forward and reverse primers.

Engelbrecht MC (1994) Modification of a semi‐ selective medium for the isolation and quantification of Pseudomonas solanacearum. ACIAR Bacterial Wilt Newsletter Vol. 10: 3-5

Norman D, Alvarez AM (1989) A rapid method for presumptive identification of Xanthomonas campestris pv. diffenbachiae and other xanthomonads. Plant Dis. 73: 654-658

Obj. 3 Yr 1-2-3: UH Res., LSU Res., & UOG Res: Research into the flora of ironwood trees and the guts of termites.

   Obj. 3 Sub-obj. 1: UH Res. & UOG Res.: Research into the fungal flora of ironwood trees and their likely role in IWTD.

   Obj.3 Sub-obj. 2: UH Res. & UOG Res.: Research into the bacterial flora of ironwood trees and their likely role in IWTD.

Methods: Obj. 3: Sub-obj. 1 & Sub-obj. 2: University of Guam: In 2021, ironwood trees from four locations across Guam were surveyed for suitability for the bacterial and fungal microbiome study. The trees and their sites were evaluated for several characteristics. A tree was classified as damaged if it had been cut into, drilled into, or otherwise damaged by humans (microbiome possibly or likely compromised); and undamaged if it had never been cut into, drilled into, or otherwise damaged by humans (microbiome uncompromised). Damage caused by natural phenomena (storm damage, water damage, or any damage not caused by direct human contact with the tree) was not considered. Trees were also examined for the presence or absence of termites, and presence or absence of Ralstonia solanacearum. Effort was made to choose an equal number of R. solanacearum positive and R. solanacearum negative trees for inclusion into the study. The size and health status of the trees were not specific as the main objective was to identify 30 ironwood trees which were damaged by human activity and 10 ironwood trees which were undamaged by human activity.

The following materials were used to execute the collection of woody ironwood tissue for the University of Hawaii

• GPS (Garmin or Cell Phone)
• Flagging Tape
• Tree tags
• Copper Nails
• Insight LH Precision Laser Rangefinder with Hypsometer
• Measuring Tape
• Tree Log Data Sheet
• Machete
• Hammer
• Wide chisel (or scraper)
• Nitrile gloves
• 1/4 in and 5/16 in drill bits
• Cordless drill
• Agdia Rs Immunostrip test strips and buffer filled bags
• Paper funnels
• Glass vials
• Ziploc bags
• Cooler

At each site, healthy and unhealthy trees were tested for Rs (R. solanacearum) using Agdia immunostrip tests. Drill shavings were collected from beneath the bark layer with a ¼ inch drill bit approximately 1.5 inches into the trunk of the tree. Approximately 0.2 grams of drill shavings were placed in Agdia test buffer extraction pouch. Testing continued until approximately five Rs+ and five Rs- trees were identified at each site. In some situations, Rs+ trees were unable to be identified at one site, so additional Rs+ trees were identified at remaining sites to make up the difference. If the tree had been uncompromised before testing for Rs, the test drill hole was filled with a sterilized stainless-steel screw, thereby conserving the integrity of the microbiome of the tree. After trees for inclusion in the study were identified, the following tree information was collected: tree tag number, tree GPS, location, tree disease severity, level of human impact, height, diameter at breast height, presence or absence of sporocarps, site condition, altitude, termite activity, and suspected termite species (Nasutitermes sp., Cototermes sp., or Microcerotermes sp.)

Conditions in the field were kept as sterile as possible: nitrile gloves were worn when preparing the tree and collecting the samples to avoid human skin cells in samples, machete and chisel used to clear off the bark were cleaned with Clorox wipes, all drill bits were autoclaved or flamed in the field, and all vials were autoclaved and wrapped sterilely to avoid contamination in transport. All samples were stored in the refrigerator (4°C) until shipment. Samples were shipped out the same day they were collected and overnighted to the University of Hawaii.

To collect woody ironwood tissue samples for the microbiome analysis, a protocol was developed to collected both shallow (0-3 inches) and deep samples (4-7 inches).  At each depth, two combined samples were collected at 50 cm above the ground and on each side of the tree.  Prior to drilling, a clean Clorox-wiped scraper was used to scrape off outer bark. When the bark was nearly removed, the scraper was cleaned with Clorox wipe again. Scraper was then used to slice off additional layers until the bark layer was easily separated from sapwood (Figure 1). If the exposed tissue appeared to be living, samples of drill shaving were collected, if not, another location on the tree was chosen. 

Figure 1. An exposed area of sapwood used for microbiome sampling.

Sampling began with the shallow woody tissue sample: drill shavings from a 3-in deep hole were collected using a 12 in by 1/4 in drill bit. At least 2 g (20 mL) of shavings were collected into a sterile glass vial by using a sterilized paper funnel (Figure 2). Vial with shavings was immediately placed on ice in the field for transport back to the lab. After the shallow woody tissue sample was collected, a 5/16 in by 12 in drill bit was used to enlarge the diameter of the hole and to increase its depth to 4 in.  This would minimize the shavings contaminating the deep sample. For the deep sample, a sterile 12 in by 1/4 in bit was used to collect a total of 20 mL of drill shavings from the same drill holes, but at a depth of 4-7 inches into the tree. Again, shavings were collected into a sterile glass vial and vial with shavings was immediately placed on ice in the field for transport back to the lab. It should be noted that the depth of the deep sample was not allowed to exceed the radius of the tree. In these cases, depth was noted in the comments section.

Figure 2. Using a 12 in long by 1/4 in wide drill bit, drill shavings were collected into a sterile glass vial by using a sterilized paper funnel.

In the same area of the tree (50 cm above ground) where the bark was removed for microbiome samples, additional holes were drilled and shavings collected for conductivity measurements. Conductivity is a measure of water's capability to pass electrical flow. This ability is directly related to the concentration of ions in the water. It is a significant predictor of percent wetwood within a tree. Greater conductivity is associated with greater % wetwood. Drill shavings were collected to the depth of 4 inches on both sides of the tree. Multiple holes were drilled with a 5/16 drill bit until approximately 75 mL of shavings were collected into a clean ziplock bag. The bag was placed on ice and transported to the lab. Once at the lab, samples were grinded until small enough to pass through a window screen (square opening of 3 mm). Five grams of sieved sample was then placed in a clean glass vial and placed in the freezer for storage 1 to 90 days. Ground shavings, which were powder-like and allowed to thaw out if necessary, were poured into 50 mL of distilled water in a 125 mL flask. The mixture was placed on a hot plate, brought to a boil, boiled for 5 minutes, then removed. While hot, mixture was then immediately run through filter paper. Filtrate was allowed to cool to room temperature before measuring conductivity. Values were recorded as microSiemens per centimeter (μS/cm at 25 °C). Conductivity level was then classified using the value: 0= slight (<301); 1= low (301-574); 2= moderate (575-849); 3= high (>849).

Six of the forty trees were chosen to be cut down (felled) for collection of additional microbiome samples: root sample, 150 cm felled sample, 300 cm felled sample, and 450 cm felled sample. Criteria included trees being undamaged (never having been drilled into, cut, or otherwise damaged by human activity), and half Rs positive/ half Rs negative. Two roots were sampled for each tree. When possible, each root had to be at least 2 inches in diameter. For each root, the spot where the root starts to go underground was marked (this might be directly at the trunk base or a foot or more away). Starting from the mark, the root was uncovered for a distance of 15 inches. Soil was removed from the exposed root using water and a soft scrub brush. At the 12 in distance, a drill sampling area was created by removing the bark with a disinfected machete. Using a 1/4 in drill bit, drill shavings were collected to a depth of 2 inches into each root by using a sterilized weigh boat to catch the shavings. The combined shavings were transferred into a glass vial until a volume of 20 ml was reached. Vial was immediately placed on ice in the field. Additional shavings were collected in a ziploc bag and tested for Rs using an Agdia immunostrip test.

After collecting the root sample, the trees were cut down (felled). The outer bark was removed from one side of the tree at distances of 150 cm, 300 cm, and 450 cm from the tree’s base using a disinfected machete (Figure 3, Figure 4). At each distance, drill shavings were collected to a depth of 2 inches using a sterilized weigh boat (Figure 4). The shavings were transferred into a glass vial until a volume of 20 ml was reached and then placed on ice to transport to the lab.

Figure 3. Felled tree showing 150 cm, 300 cm, and 450 cm collection sites cleaned and bark removed.

Figure 4. The 150 cm felled sample is collected using a sterilized weight boat (two people in front), while the 300 cm felled sample collection area has bark removed to sapwood in preparation for sample collection (one person in back). 

Methods: Obj. 3, Sub-obj. 1 & Sub-obj. 2: University of Hawaii:

Genome sequencing of Ralstonia strains for evolutionary analyses: selecting ironwood strains from current and past Guam collections, their DNA was extracted from a half loopful of pure bacterial colonies, grown overnight on DPA (dextrose 5 g/L, peptone 10 g/L and agar 17 g/L) at 26 ± 2°C, by using the Genomic-tips 500/G kit (Qiagen) according to the manufacturer’s instruction. DNA concentration of the seven samples was measured using a Qubit 4 fluorometer (Thermo Fisher Scientific, Life Technologies, Carlsbad, CA); additionally, the DNA quality was assessed in a 1 % agarose gel electrophoresis.

The isolated DNA were sent for illumine (Novaseq) sequencing. Short-read sequencing of the seven Ralstonia strains was performed using Illumina NovaSeq system at the UC Davis Genome Center. DNA libraries were prepared using Seqwell plexWell LP384 Library Preparation Kit with 10 ng gDNA required for Illumina sequencing platform (seqWell, Beverly, MA). The prepared library was amplified with 8 PCR cycles, analyzed using Bioanalyzer 2100 (Agilent, Santa Clara, CA), and quantified by Qubit 4 Fluorometer Instrument (LifeTechnologies, Carlsbad, CA), and equimolar library pool was quantified and sequenced using qPCR with a Kapa Library- Quant kit (Kapa Biosystems/Roche, Basel Switzerland). Sequencing was run with paired-end 150 bp reads, using an and Illumina NovaSeq system. The sequences have been received.

We also plan to run Nanopore MinIon to get long reads. Long-reads of high quality whole genomic DNA will be obtained using a MinION Sequencer (Oxford Nanopore Technologies, Oxford, UK). DNA libraries will be prepared using the Rapid Barcoding Kit (SQK-RBK004) (Oxford Nanopore Technologies Inc) with 400 ng of high-quality genomic DNA. The libraries will be sequenced in a flow-cell version R10 and run for 72 hours. Sequencing will be monitored in real time using the MinKNOWN software version 4.0.20 (Oxford Nanopore Technology). The generated FAST5 sequences files from Nanopore sequencing will be base called using MinKNOWN software version 4.0.20 (Oxford Nanopore Technology). Hybrid assemblies will be generated using both short and long reads, and evolutionary analyses will be conducted. The evolutionary pipelines (ClonalFrameML and fastGEAR) have been established and running on our lab server.

Method for bacterial flora Standardized DNA isolation method from ironwood plant:

Ironwood seeds sent by Dr. Robert Schlub in 2020 were sown for standardizing experiments and protocols. The critical and most important factor while sowing the ironwood seed was to place the seed horizontally 2-3 cm deep in the conical pots. The seeds germinate in 2-3 weeks depending upon the temperature condition. Ironwood seed germinated in 2 weeks under greenhouse conditions. The germination rate was lower comparatively to seeds sown in the month of January.

The modified protocol for DNA isolation from ironwood wood tissues was standardized.

DNA extraction from wood (Sapwood and heartwood) was quite difficult due to presence of higher quantity of secondary metabolites phenolic and lignin compounds. Therefore, after experimenting 8-10 different protocols we were able to finalize the protocol as follows:

• Preparation of CTAB buffer (250ml)

1. NaCl (2.5M) – Weigh 14.625 gm and dissolve in 100 ml of autoclaved distilled water
   2. Add Tris (1M) – 25 ml
   3. Add EDTA (0.5M) – 10 ml
2. Add 5 gm of CTAB and stir until dissolved
   5. Make the volume up to 250 ml with autoclaved distilled water 6. Adjust pH- 8.0 with conc. HCl
3. Preparation of 10% PVP
   Add 10 gm of PVP and dissolve in 100 ml of distilled water in a beaker. Filter using 0.22             μM filter membrane. Take a syringe of 10 ml, intake 8-9 ml of prepared solution at a         time, fit the tip of syringe to 0.22 μM filter membrane without touching the surface of      membrane, adjust the nozzle of filter to 50 ml centrifuge tube and gently press the          syringe. Stored at room temperature.

Modified protocol for DNA isolation from greenhouse ironwood plants

1. Pre-heat 5 ml of CTAB buffer in water bath (65°C) in 50 ml centrifuge tube. Once it is heated, add 500 μl of 10% PVP and 10 μl of β-mercaptoethanol.
2. Weigh 500 gm of plant tissue and grind in an autoclaved mortar pestle using Liquid Nitrogen. Grind well and make it in a powder form.
3. Mix well the grinded tissue in the 5 ml of CTAB buffer prepared in step1.
4. Use a wide borer tip (make a cut of around 1 cm on 1 ml tip using blade) to pour 500 μl solution from step 3 into bead beater tubes.
5. Grind in the bead beater for 2 minutes at maximum speed.
6. Incubate at 65°C for 1 hour and 30 minutes in a water bath. Vortex the tubes for 5 seconds after a 20-minute interval.
7. Take out the tubes from the water bath and let it cool down for 2-3 minutes under.
8. Add 5 μl of RNase (100mg/ml) and mix well. Incubate the tube at 37°C and 140 rpm for

one hour in room 312 incubator with shaker.
*Following steps to be done on DNA isolation workbench. Take aliquots of buffer solutions required for DNA isolation.
*Buffers used in the following steps are taken from Qiagen DNeasy Plant Mini Kit (Catalog no. 69104)

1. Add 130 μl of AP1, mix well with pipette and incubate in ice for 5 minutes.
2. Centrifuge the tubes at 20 X1000 g for 5 minutes.
3. Pipet the lysate into a QIAshredder spin column placed in a 2 ml collection tube.
4. Centrifuge for 2 min at 20 X1000 g.
5. Transfer the flow-through into a new tube without disturbing the pellet if present. Add 1.5 volumes of Buffer AW1 and mix by pipetting.
6. Transfer 650 μl of the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge for 1 min at ≥6000 x g (≥8000 rpm). Discard the flowthrough. Repeat this step with the remaining sample
7. Place the spin column into a new 2 ml collection tube. Add 500 μl Buffer AW2, and centrifuge for 1 min at ≥6000 x g. Discard the flow-through.
8. Add another 500 μl Buffer AW2. Centrifuge for 2 min at 20,000 x g. Note: Remove the spin column from the collection tube carefully so that the column does not come into contact with the flow-through.
9. Transfer the spin column to a new 1.5 ml or 2 ml microcentrifuge tube.
10. Add 25 μl Buffer AE for elution. Incubate for 5 min at room temperature (15–25°C).
11. Centrifuge for 1 min at 8000 g.
12. Repeat step 17
13. Store DNA in aliquots at -20°C.

The samples from Guam were processed immediately for DNA isolation using the modified method described above. The first run was with an in house 16S primer set to check the quality. The DNA isolated from 101 samples sent to Microbial Genomics and Analytical Laboratory core for 16S and ITS library preparation. All the samples were amplified for 16S rRNA gene region.

Obj. 3: UH Res., LSU Res., & UOG Res: Research into the flora of ironwood trees and the guts of termites.

   Obj.3 Sub-obj. 3: LSU Res. & UOG Res.: Research to determine if termites carry ironwood bacteria and thus, might be responsible for their movement.

Methods: Obj. 3, Sub-obj. 3, University of Guam: from 2019-2020 termites were collected on the island of Guam. For each ironwood tree with an active infestation, general data was recorded, including: Tree tag number, date of collection, GPS location, visual rating, site condition, height, DBH, altitude, geology characteristics, occurrence, root exposure, collection area of termite ( i.e mud trail, above or below ground colonies), and presence or absence of Ralstonia solanacearum (tested using Agdia Immunostrip buffer test pouch). A minimum of 21 active termites (15 workers/6 soldiers) per tree were collected directly from the infestation site with a general aspirator and immediately transferred into vials. For each tree, the samples including soldier and worker termites which were partitioned into 10 mL of 70% ethanol (for morphological identification) and 10 mL of 95% ethanol (for Illumina sequencing). Each individual vial was labeled by tree number in the field, placed in a vial box, then placed on ice in a cooler. The vials were further processed through labeling by clinic number, then in a cardboard vial box with the lid closed and placed in the laboratory freezer.

     Termite sampled ironwood trees were tested for Ralstonia solanacearum by selecting two locations on the stem breast height above the soil line. An electric drill with a 1.5 in. sterilized drill bit was used to create the initial break in the periderm, then a 5/32 in. sterilized drill bit was used to collect drill shavings by drilling a 4 cm deep hole in the tree. The shavings were collected in plastic bags and subsequently labeled. To reduce the chances of secondary infection by other insects or pathogens, the two holes were plugged with a sterilized Flat Head #12 (7.32) stainless steel screw. The shavings were then brought back to the University of Guam Cooperative Extension Plant Pathology Lab and tested for Rs using the Agdia® Inc. Ralstonia solanacearum (Rs) immunodiagnostic strips. The test was performed on the same day the samples were collected. Following the manufacturer’s instructions, 0.15 g of the drill shavings were placed in BEB1 sample extraction bags. The sample was allowed to set three minutes before inserting the immunostrip.

Drill shavings were collected for conductivity determinations. In the case of LSU, the bark was removed just to the point that the live tissue layer appeared (phloem) before being drilled and shavings collected. In the lab, tissue samples were grinded and sieved through 1/8 in galvanized hardware cloth. Five grams of sieved tissue was then placed in a clean glass vial and placed in the freezer for storage. Processing method included the following: the entire 5 g of sieved tissue was transferred into a 125 mL flask containing 50 mL of steam-distilled water, shaken, and placed on a hot plate. At first sign of boiling (approximately 2 minutes later), mixture was boil rapidly for an additional 5 minutes. While hot, using a Vortex-genie 2, the sample was vortexed vigorously for one minute at the medium setting. Afterwards, it was placed in an ice bath and cooled to 60°C, then filtered through Whatman #3 paper by pulling a vacuum. After the filtrate cooled to room temperature, its conductivity was measured using a Hach 51800-10 sensION 5 Waterproof Conductivity Meter and recorded as microSiemens per centimeter (μS/cm at 25 °C). Conductivity level was then classified using the value: 0= slight (<301); 1= low (301-574); 2= moderate (575-849); 3= high (>849).

Methods: Obj.3, Sub-obj. 3, Louisiana State University: forty-five termite samples were collected in 2019-20 by the University of Guam from healthy as well as sick ironwood trees present on 14 distinct locations on the island of Guam. Geology related (location, parent material classification, site management), tree-related (tree DS, tree health, presence or absence of Ralstonia, altitude classification) and plot-related (plot average DS, plot average health, stand maturity estimate, percentage of trees with termites in the plot, percentage of dead trees in the plot) parameters were recorded. The samples including soldiers and worker termites were partitioned into 70% ethanol (for morphological identification) and 95% ethanol (for Illumina sequencing) and were shipped to Louisiana State University. In the attached report by LSU, a full description of the materials and methods for the following topics can be found: (1) description of the factors collected from ironwood tree plots by University of Guam; (2) Morphological species identification of termites; (3) DNA extraction; (4) Primer selection; (5) DNA amplification and sequencing; (6) Bioinformatics analysis; (7) termite feeding experiments.

Full Report: LSU WSARE R&E Progress Report 4.1.21 - 3.30.22