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

Progress report for SW19-906

Project Type: Research and Education
Funds awarded in 2019: $304,273.00
Projected End Date: 12/31/2022
Host Institution Award ID: G259-19-W7500
Grant Recipients: University of Guam; University of Hawaii; Louisiana State University
Region: Western
State: Guam
Principal Investigator:
Dr. Robert Schlub
University of Guam
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Project Information

Summary:

Ironwood (Casuarina equisetifolia (L) subsp. equisetifolia), recognized as an important tree species by the Pacific Islands area USDA Natural Resources Conservation Service, are dying in large part due to the disease bacterial wilt. The importance of ironwood is underscored by the fact that it has been on Guam for thousands of years and is tightly integrated into the island’s environment and its many ecosystems.  Ironwood is well suited to the shore lines of tropical islands, due to its ability to withstand salt spray, typhoon strength winds, and poor soil conditions. In a survey funded by WSARE in 2008, it was estimated that 51% of the trees were showing signs of a progressive dieback now referred to as ironwood tree decline (IWTD). The severity of the disease is believed to be in part due to the lack of genetic diversity of Guam’s tree population. The purpose of this project is to determine the origin of Guam’s ironwood tree bacterial wilt pathogen, Ralstonia solanacearum, to determine if the bacterium can be vectored by termites, to increase genetic diversity of Guam’s ironwood tree population, and to educate the public and scientific community about IWTD. From the analysis of 26 water cultures of the bacterial wilt pathogen by the University of Hawaii, it was determined that the bacterial wilt pathogen in Guam’s trees is best described as Ralstonia solanacearum species complex (RSSC). Their analysis revealed two strains and thus two potential origins of Guam’s pathogen. The most common was Phylotype I which is present in North and Eastern Asia, whereas less common was Phylotype II which occurs in Central America.  Based a collection of termites from 48 ironwood trees, the ‘higher’ termite, Nasutitermes takasagoensis was the most common accounting for 93% of the samples. The lack of Ralstonia spp. and the low presence and abundance of wetwood bacteria in the gut of these termites suggests that they are not a vector for these pathogens of IWTD. At taxonomic level two in SILVA (Phylum level), Spirochaetes (48.22 %) and Fibrobacteres (41.43 %) were found to be the most dominant phyla followed by Bacteroidetes (3.61%), Proteobacteria (3.35%), Margulisbacteria (0.84%), Acidobacteria (0.77%), Planctomycetes (0.65%), and others (1.61%).  It was determined that the presence or absence of Ralstonia in trees from which termites were collected showed no significant effect on the total bacteria community; however, the communities of termites collected from sick trees showed significantly higher bacterial richness compared to those from healthy trees. To increase genetic diversity of Guam’s ironwood tree population, off-island varieties were planted in agroforestry projects. Fifteen new windows were created consisting of 10 trees each and ten deteriorating windrows were refurbished with the addition of 6-15 trees each.  To educate the public and scientific community about IWTD, three workshops were presented. In February 2020, two half-day workshops were attended by 36 participants including farmers, property owners, home gardeners, professionals, and students at the University of Guam. In January 2022, an in-person and virtual ironwood tree decline (IWTD) conference was held for 3.5 days. In attendance were IWTD project researchers, IWTD research graduate students, and invited guests. Project researchers included Drs. Robert L. Schlub of the University of Guam, Claudia Husseneder of Louisiana State University, Zhong Chonglu of the Chinese Academy of Forestry, and from University of Hawaii Drs. Mohammad Arif and Shefali Dobhal. Graduate students reporting on IWTD research included Sujan Paudel of the University of Georgia, Garima Setia of Louisiana State University, and Dario Arizala of the University of Hawaii. The conference focused on exchange of knowledge and research discovery to ameliorate the impact of bacterial wilt in Casuarina equisetifolia, and unraveling the roles of Ralstonia solanacearum species complex, Ganoderma australe, wetwood bacteria, and termites in the decline of Guam’s ironwood.

Project Objectives:

Obj 1 Yr 1-2: UOG Ed: Through tree plantings, educate the public on the importance of planting new off-island cultivars (seedlots from international provenance trails).

   Obj. 1 Sub-obj. 1: UOG Ed: As part of the education effort, plant trees of mixed cultivars in 10 visually deteriorating windrows across Guam.

   Obj. 1 Sub-obj. 2: UOG Ed:  As part of the education effort, construct 15 new ironwood agroforestry projects using 150 trees consisting of a mixture cultivars.

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.

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.

   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.

Obj. 4 Yr 2-3: UOG Ed: Restoring ironwood as an agroforestry species in Guam through awareness and action of the local and scientific communities.

   Obj. 4 Sub 1: UOG Ed: Through two ½ day workshops, attendees will learn about the care of the ironwood trees and its uses in agroforestry. Ironwood trees will be given away.

   Obj. 4 Sub 2: UOG Ed, UH Ed & LSU Ed: PI and Extension/outreach representatives will conduct a four-day workshop/conference on bacterial wilt and other components of IWTD.

Timeline:

Cooperators

Click linked name(s) to expand/collapse or show everyone's info
  • Dr. Mohammad Arif (Researcher)
  • Jesse Bamba (Educator)
  • Claudia Husseneder (Researcher)
  • John Mesa - Producer
  • Bernard Watson - Producer

Research

Hypothesis:
  1. The fungal flora of healthy and declining trees are significantly different.
  2. The bacterial flora of healthy and declining trees are significantly different.
  3. The bacterial flora of the guts of termites from healthy and declining trees are significantly different.
  4. There is a higher change of detecting Ralstonia in the gut of termites from trees that test positive for RSSC than those that do not.
  5. Ironwood RSSC isolates are from a single origin.
Materials and methods:

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 95oC 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

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

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

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

 

Figure 4

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

Research results and discussion:

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

Results: Obj. 2, University of Guam: a total of 112 trees were sampled from 29 different locations in Guam. To determine the presence or absence of RSSC in the trees, drill shavings from each tree were tested with the RSSC specific Agdia immunostrips. Forty-five RSSC infected ironwood trees were identified. The map in Figure 5 shows the approximate locations of the sampled trees. Infected trees were widespread occurring in northern, central, and southeastern parts of the island. From visual inspection, trees in the southwestern area of the island appeared to less likely to test positive for RSSC.

 

map

Figure 5: Map of Guam with approximate locations of the ironwood trees sampled in the study. Trees that tested positives with Agdia immunostrip are in red, those that tested negative are in green.

 

Three types of ooze were found in root cross-sections (VO = viscous ooze, WO = clear watery ooze, and MO = mixture of viscous ooze and watery ooze). The white to off-white viscous ooze (VO) mostly occurring in the outer portion of a root slice, nearly always tested positive for RSSC. This ooze gave a higher percentage of successful RSSC isolations than other ooze types. Of the 95 trees that tested positive for Rs only 35 were found to be suitable for collection of root slices and the production of ooze (Figure 6).

 

Map of Rs samples sent to Hawaii

Figure 6: Guam Ironwood tree locations where tissue samples were collected and Ralstonia solanacearum successfully isolated and sent to Hawaii.

 

Thirty-five water cultures were prepared and sent to UH. The tree data from which the samples were collected is in Table 1.

Table 1:  Underlying data on RSSC isolates obtained from Guam

UOG isolatea

UOG

Clinic entryb

Ooze typec

Height

(m)d

DBH

(cm)e

DSf

Altitude (m)g

Geologyh

Sporocarpsi

Termitesj

                   

19-124

19-124

mo

19.2

26.51

3

71

0

0

1

19-127

19-127

mo

12.5

32.69

4

78

0

0

0

19-135

19-135

vo

128

25.79

3

176

0

1

2

19-147

19-147

mo

19.6

17.63

3

176

0

1

2

19-156

19-156

vo

15.5

9.39

4

26

0B

0

1

19-157

19-157

vo

9.8

23.56

3

19

0B

0

1?

19-158

19-158

vo

8

19.74

2

21

0B

0

0

19-161

19-161

vo

12.8

31.52

1

117

1

0

0

19-170

19-170

mo

13.8

75.15

4

-3

0B

1

1

19-174

19-174

vo

9.1

32.48

4

19

0B

0

0(H)

19-194

19-194

vo

15.6

29.61

3

19

0B

0

1

19-200

19-200

vo

10.3

22.77

3

105

1

0

0

19-202

19-202

vo

10

37.89

3

96

1

0

1H

19-203

19-203

vo

11.9

31.52

4

93

1

0

1A

19-224

19-224

vo

15.6

69.74

4

17

0B

0

1H

19-227

19-227

vo

14.8

41.40

3

27

0B

0

1

19-228

19-228

mo

12.2

60.19

2

26

0B

0

1

19-229

19-229

vo

6.9

28.34

2

130

0

0

1H

20-016

20-016

vo

18

40.44

0

0

0

0

0

Table 1 Glossary: aUOG isolate, consisting of University of Guam clinic number with identifiers for multiple isolates; bUOG clinic number, identifies tree from with root samples were collected; cOoze type: vo = viscous ooze, mo = mixture of viscous ooze and watery ooze; dHeight, tree height; eDBH, tree diameter at breast height; fDS, decline severity, DS-0 (symptomless) to 4 (near dead); gAltitude, meters above sea level of tree site; hGeology, tree site parent material, 0= limestone upland, 0B= limestone beach, 1= volcanic upland, 1B=volcanic beach.

 

Results: Obj. 2, University of Hawaii: All the strains isolated from ironwood were able to grow on modified Kelman Tetrazolium Chloride (TZC) media and modified SMSA media. The growth was observed within 48 hours with light to dark pink pigmentation at 28oC. The identity of strains was first confirmed using colony PCR using Ralstonia solanacearum species complex (RSSC) specific end point PCR primers. The PCR products were electrophoresed at 100V for 40 minutes to visualize the amplicons. All the 25 strains isolated from ironwood were found to be positive with RSSC specific primers. Genomic DNA was extracted from all the strains using DNeasy Blood and Tissue kit (Qiagen, Valencia, CA). The extracted DNA was amplified using RSSC specific dnaA primers. All the strains were found to be positive. The BLASTn results showed all the strains from ironwood to be Ralstonia pseudosolanacearum.

A detailed report can be found here: WSARE_4.1.2020 - 3.30.21_Hawaii Report

 

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.

Results: Obj. 3, Sub-obj. 1 & Sub-obj. 2, University of Guam: UOG collected microbiome samples for Hawaii in four areas of the island (Figure 7). Tree information from the 40 ironwood trees from which microbiome samples were collected and sent to the University of Hawaii (Table 2).

 

IW microbiome collection sites for UH

Figure 7. Ironwood tree microbiome collection sites on Guam.

 

Table 2. University of Guam: Tree information from the 40 ironwood trees from which microbiome samples were collected and sent to the University of Hawaii.

Ironwood Tree Study WSARE # SW19-906                          
Woody tissue Collection Sites for the University of Hawaii October/November 2021                          
Tree Tag No.  Tree GPS Location Tree DS Tree Health Ranking Human impact Height (ft) DBH Single [S] or Multiple [M] (cm) Sporocarp Site condition Altitude (m) Altitude Classification Parent material Parent material Classification Termite activity Suspected termite species Rs (+/-) Date of Rs Testing  Conductivity value
µS/cm
Conductivity level  Date Conductivity tissue collected UOG Clinic no. assigned to shallow woody tissue sample UOG Clinic no. assigned to deep woody tissue sample Date of shallow and deep tissue sample collection
120 13.5669, 144.87727 Watson's Farm, Yigo 4 1 2 56.0 28.14 [S] 1 1 165 2 Limestone 1 1 N/A + 10/21/2021 750 2 11/3/21 21-267 21-268 11/3/21
121 13.56691, 144.87717 Watson's Farm, Yigo 4 1 2 51.0 29.11 [S] 0 1 160 2 Limestone 1 2   + 10/21/2021 820 2 11/3/21 21-265 21-266 11/3/21
276 13.56555, 144.87750 Watson's Farm, Yigo 4 1 0 36 26.27 [S] 0 1 164 2 Limestone 1 1 N/A + 10/21/2021 753 2 11/3/21 21-263 21-264 11/3/21
277 13.56715, 144.87656 Watson's Farm, Yigo 4 1 0 31 21.02 [S] 0 0 163 2 Limestone 1 2 Nt + 10/21/2021 478 1 11/3/21 21-277 21-278 11/3/21
279 13.56723, 144.87694 Watson's Farm, Yigo 2 1 0 59 24.66 [S] 0 1 161 2 Limestone 1 2 Nt + 10/22/2021 623 2 11/3/21 21-275 21-276 11/3/21
280 13.56694, 144.87735 Watson's Farm, Yigo 1 0 0 60 29.10 [S] 0 0 159 2 Limestone 1 1 N/A + 10/22/2021 858 3 11/3/21 21-269 21-270 11/3/21
282 13.56693, 144.87736 Watson's Farm, Yigo 1 0 0 45 31.53 [S] 0 0 159 2 Limestone 1 1 N/A + 10/22/2021 668 2 11/3/21 21-271 21-272 11/3/21
283 13.56708,144.87755 Watson's Farm, Yigo 2 1 0 61 47.70 [S] 0 0 161 2 Limestone 1 2 Nt + 10/22/2021 984 3 11/3/21 21-273 21-274 11/3/21
294 13.56715, 144.87604 Watson's Farm, Yigo 1 0 0 34 19.40 [S] 0 1 167 2 Limestone 1 1 N/A - 10/28/2021 243 0 11/3/21 21-279 21-280 11/3/21
275 13.565682, 144.877029 Watson's Farm, Yigo 4 1 1 34.0 25.87 [S] 0 0 169 2 Limestone 1 2 Nt + 10/21/2021 781 2 12/1/21 21-355 21-356 12/1/21
278 13.56717, 144.87664 Watson's Farm, Yigo 4 1 0 50 25.47 [S] 0 0 169 2 Limestone 1 2 Mc + 10/22/2021 582 2 12/1/21 21-343 21-344 12/1/21
281 13.5669, 144.87734 Watson's Farm, Yigo 1 0 0 38 18.60 [S] 0 0 169 2 Limestone 1 2 C + 10/22/2021 559 1 12/1/21 21-361 21-362 12/1/21
290 13.56695, 144.87644 Watson's Farm, Yigo 2 1 0 44 30.47 [M] 0 1 169 2 Limestone 1 2 Nt - 10/21/2021 266 0 12/1/21 21-337 21-338 12/1/21
291 13.56695, 144.87663 Watson's Farm, Yigo 2 1 0 49 21.34 [S] 0 1 169 2 Limestone 1 1 N/A - 10/21/2021 384 1 12/1/21 21-349 21-350 12/1/21
292 13.56715, 144.87598 Watson's Farm, Yigo 3 1 0 52 18.43 [S] 0 1 169 2 Limestone 1 1 N/A - 10/28/2021 412 1 12/1/21 21-331 21-332 12/1/21
285 13.53233, 144.87451 Yigo Experiment Station 3 1 0 36 50.13 [S] 0 1 116 2 Limestone 1 2 Nt + 10/22/2021 455 1 11/22/21 21-323 21-324 11/22/21
286 13.53233, 144.87451 Yigo Experiment Station 2 1 0 30 81.66 [S] 0 1 116 2 Limestone 1 1 N/A + 10/22/2021 970 3 11/22/21 21-325 21-326 11/22/21
287 13.53233, 144.87451 Yigo Experiment Station 0 0 0 34 43.25 [S] 1 1 116 2 Limestone 1 3 Nt + 10/22/2021 715 2 11/22/21 21-321 21-322 11/22/21
288 13.53205, 144.874420 Yigo Experiment Station 4 1 0 30 31.53 [S] 0 1 116 2 Limestone 1 1 N/A + 10/22/2021 475 1 11/22/21 21-327 21-328 11/22/21
289 13.5309, 144.87422 Yigo Experiment Station 4 1 0 43 28.30 [S] 0 0 116 2 Limestone 1 2 Mc + 10/22/2021 496 1 11/22/21 21-329 21-330 11/22/21
218 13.26318, 144.71814 Ija Experiment Station 3 1 1 50 68.66 [M] 1 0 74 1 Residium 2 2 Nt + 10/13/2021 851 3 11/17/21 21-303 21-304 11/17/21
219 13.26335, 144.71809 Ija Experiment Station 3 1 0 26 39.78 [S] 1 0 74 1 Residium 2 1 N/A + 12/26/2019 850 3 11/17/21 21-305 21-306 11/17/21
258 13.26563, 144.71668 Ija Experiment Station 4 1 1 21 32.99 [S] 1 1 85 1 Residium 2 1 N/A + 10/13/2021 1257 3 11/17/21 21-311 21-312 11/17/21
259 13.26561, 144.71660 Ija Experiment Station 3 1 1 30 47.78 [M] 1 1 85 1 Residium 2 1 N/A - 10/13/2021 800 2 11/17/21 21-307 21-308 11/17/21
260 13.26544, 144.71675 Ija Experiment Station 2 1 0 29 43.66 [S] 0 2 84 1 Residium 2 3 Nt - 10/13/2021 583 2 11/17/21 21-313 21-314 11/17/21
261 13.26526, 144.71742 Ija Experiment Station 0 0 0 26 19.40 [S] 0 1 96 1 Residium 2 0 N/A - 10/13/2021 338 1 11/17/21 21-315 21-316 11/17/21
262 13.26571, 144.71752 Ija Experiment Station 3 1 0 30 33.95 [S] 0 0 92 1 Residium 2 2 Ct - 10/13/2021 302 1 11/17/21 21-317 21-318 11/17/21
263 13.26629, 144.71758 Ija Experiment Station 1 0 1 62 48.51 [S] 1 1 90 1 Residium 2 3 Nt - 10/13/2021 538 1 11/17/21 21-319 21-320 11/17/21
264 13.26558, 144.71663 Ija Experiment Station 4 1 1 34 28.14 [S] 0 1 85 1 Residium 2 1 N/A - 10/13/2021 608 2 11/17/21 21-309 21-310 11/17/21
265 13.37512, 144.53713 Tarague Beach, AAFB 4 1 1 63.0 33.64 [M] 0 1 16 1 Coral Sand 3 1 N/A - 10/14/2021 762 2 10/14/21 21-293 21-294 11/10/2021
266 13.37515, 144.53708 Tarague Beach, AAFB 3 1 1 51.0 53.82 [M] 0 1 18 1 Coral Sand 3 1 N/A - 10/14/2021 530 1 10/14/21 21-287 21-288 11/10/2021
267 13.37521, 144.53709 Tarague Beach, AAFB 3 1 1 53.0 29.10 [S] 0 1 16 1 Coral Sand 3 1 N/A - 10/14/2021 285 0 10/14/21 21-289 21-290 11/10/2021
268 13.37520, 144.53710 Tarague Beach, AAFB 3 1 1 39.0 24.25 [S] 0 1 16 1 Coral Sand 3 1 N/A - 10/14/2021 377 1 10/14/21 21-291 21-292 11/10/2021
269 13.37522, 144.53709 Tarague Beach, AAFB 1 0 1 61.0 30.25 [M] 0 1 15 1 Coral Sand 3 1 N/A - 10/14/2021 475 1 10/14/21 21-285 21-286 11/10/2021
270 13.37526, 144. 53697 Tarague Beach, AAFB 1 0 1 62.0 75.67 [S] 0 1 17 1 Coral Sand 3 1 N/A - 10/14/2021 311 1 10/14/21 21-283 21-284 11/10/2021
271 13.37517, 144.53680 Tarague Beach, AAFB 0 0 1 67 80.04 [S] 0 1 17 1 Coral Sand 3 1 N/A - 10/14/2021 214 0 10/14/21 21-281 21-282 11/10/2021
272 13.37474, 144.53739 Tarague Beach, AAFB 0 0 1 36 74.70 [S] 0 1 30 1 Coral Sand 3 3 Nt - 10/14/2021 308 1 10/14/21 21-295 21-296 11/10/2021
273 13.37476, 144.53752 Tarague Beach, AAFB 0 0 1 35 93.96 [S] 0 1 25 1 Coral Sand 3 1 N/A - 10/14/2021 225 0 10/14/21 21-297 21-298 11/10/2021
274 13.37457, 144.53749 Tarague Beach, AAFB 1 0 1 55.0 61.05 [M] 0 1 32 1 Coral Sand 3 1 N/A - 10/15/2021 254 0 11/15/21 21-299 21-300 11/10/2021
231 13.56938, 144.93198 Andersen Golf Course 4 1 1 35 56.86[M] 1 2 162 2 Limestone 1 1 N/A  +  10/15/2021 500 1 11/10/21 21-301 21-302 11/10/2021

Glossary found here: FINAL trees for Hawaii microbiome 12-10-21

 

Results: Obj 3, Sub-obj. 2, University of Hawaii: Modified protocol for DNA isolation from greenhouse ironwood plants worked for greenhouse ironwood plants and was repeated 5 times to confirm the results. The obtained results were consistent (Figure 8). Left figure shows three band columns: standards, greenhouse ironwood, ironwood tree. Right figure shows five band columns: standards, ironwood tree, greenhouse ironwood, another ironwood tree and greenhouse ironwood.

 

Figure 8

Figure 8. Results of DNA isolation from greenhouse ironwood plants.

 

Hawaii has received approximately 101 samples from Guam. The samples’ 16S rRNA amplified gene region can be seen in Figure 9.

 

Figure 9

Figure 9. 16S rRNA amplified gene region of samples from Guam.

 

For the ITS sequencing results, 70 out of 101 samples were able to amplify for the ITS. However, we are trying to troubleshoot the problem and have all the samples amplified. The results for 70 samples amplification is seen in the gel image in Figure 10.

 

Figure 10

Figure 10. ITS sequencing results of 70 out of the 101 samples sent from Guam.  

 

Results: Obj. 3, Sub-obj. 3, University Of Guam: from 2019-2021, the University of Guam collected termites from infested ironwood trees across Guam. In 2019, a total of 64 termite samples from 32 ironwood trees were collected and sent to Louisiana State University for species identification and analysis of gut contents (Table 3 and Figure 11, blue dots). In 2021, an additional 37 termite samples from 34 ironwood trees were collected and sent to Louisiana State University for species identification and analysis of gut contents (Table 3 and Figure 11, red dots).

Table 3. University of Guam: Tree information from ironwood trees from which termites were collected and sent to Louisiana State University.

Ironwood Tree Study WSARE # SW19-906
LSU Termite Collection Sites 2019-2021
 
UOG Clinic no. assigned to termites Termite Collection Date Termite ID (morphological) Source of Termites Tree Tag No.  Tree GPS Location Tree Alive or Dead Tree DS Tree Health Ranking Tree Condition Height (m) DBH Single [S] or Multiple [M] (cm) Sporocarp Site condition Altitude (m) Altitude Classification Parent material Parent material Classification Rs Testing Date  Rs (+/-) Conductivity Value Conductivity Level
19-76 06-19-19 Nt tree 165 13.53216, 144.87418 UOG Yigo Station 0 3 1 1 9.0 18.57 [M] 0 1 148 2 limestone 1 02-05-21 (+) 367 Low
19-77 06-20-19 Nt tree 166 13.56583, 144.87688 Watson's Farm, Yigo 0 3 1 1 11.1 33.60 [S] 0 1 170 2 limestone 1 02-05-21 (+) 493 Low
19-78 06-20-19 Nt tree 167 13.56598, 144.87462 Watson's Farm, Yigo 0 4 1 1 10.0 9.16 [M] 0 1 164 2 limestone 1 02-05-21 (-) 185.4 Slight
19-79 06-20-19 Nt tree 168 13.56706, 144.87537 Watson's Farm, Yigo 0 2 1 2 13.0 24.20 [M] 1 1 171 2 limestone 1 02-18-21 (-) 415 Low
19-80 06-20-19 Nt tree 169 13.56660, 144.87416 Watson's Farm, Yigo 0 0 0 1 13.7 22.13 [S] 0 1 161 2 limestone 1 02-18-21 (-) 192.7 Slight
19-81 06-20-19 Nt tree 170 13.56456, 144.87746 Watson's Farm, Yigo 0 1 0 1 8.2 13.69 [S] 0 1 168 2 limestone 1 02-18-21 (-) 267 Slight
19-82 06-21-19 Nt tree 171 13.26583, 144.71691 UOG Ija Station 0 3 1 1 12.6 26.27 [S] 0 1 96 1 residum  2 03-16-21 (+) 482 Low
19-83 06-21-19 Nt tree 172 13.26529, 144.71629 UOG Ija Station 0 4 1 1 17.2 33.76 [S] 1 1 92 1 residum  2 03-16-21 (-) 378 Low
19-84 06-27-19 Nt tree 173 13.47111, 144.84528 Mangilao Golf Course 0 0 0 1 9.2 17.61 [M] 0 2 129 2 limestone 1 03-02-21 (-) 202 Slight
19-85 06-27-19 Nt tree 174 13.47082, 144.84503 Mangilao Golf Course 0 4 1 1 9.2 39.17 [S] 0 2 127 2 limestone 1 03-02-21 (+) 370 Low
19-86 07-01-19 Nt tree 176 13.37520, 144.73877 Windward Golf Course 0 4 1 2 12.0 33.28 [S] 1 2 117 2 residium 2 03-16-21 (-) 737 Moderate
19-87 07-01-19 Nt tree 175 13.37788, 144.74162 Windward Golf Course 0 1 0 2 10.7 18.47 [S] 1 2 126 2 residium 2 03-16-21 (+) 429 Low
19-88 07-09-19 Nt tree 177 13.64889, 144.85289 Ritidian 0 1 0 1 8.0 39.50 [S] 0 1 12 1 coral sand 3 02-19-21 (-) 361 Low
19-89 07-09-19 Nt tree 163 13.65222, 144.85764 Ritidian 0 0 0 1 10.5 46.70 [S] 0 1 21 1 coral sand 3 02-19-21 (-) 241 Slight
19-90 07-12-19 Nt tree 178 13.23476, 144.64574 Cocos Island 0 2 1 1 11.9 23.90 [S] 0 0 20 1 coral sand 3 04-08-21 (-) 227 Slight
19-91 07-12-19 Nt tree 179 13.23701, 144.65102 Cocos Island 0 1 0 1 10.3 17.80 [M] 0 0 12 1 coral sand 3 04-08-21 (-) 212 Slight
19-92 07-16-19 Nt tree 180 13.46753, 144.84236 Mangilao Golf Course 0 1 0 1 6.9 15.50 [M] 0 1 124 2 limestone 1 03-02-21 (+) 277 Slight
19-93 07-16-19 Nt tree 181 13.46464, 144.85080 Thousand Steps 0 0 0 1 5.9 28.18 [S] 0 0 17 1 limestone 1 03-24-21 (-) 247 Slight
19-94 07-17-19 Nt tree 182 13.24797, 144.72690 Ysrael Beach 0 4 1 1 9.4 24.20 [S] 0 0 5 1 coral sand 3 03-19-21 (-) 930 High
19-95 07-17-19 Nt tree 183 13.24741, 144.72708 Ysrael Beach 0 1 0 1 5.0 32.64 [S] 0 0 7 1 coral sand 3 03-19-21 (-) 230 Slight
19-96 07-18-19 Nt tree 229 13.50311, 144.78416 Sagan Kotturan Chamoru 0 1 0 1 11.9 33.92 [S] 0 0 45 1 limestone 1 03-26-21 (-) 190.1 Slight
19-98 07-30-19 Nt tree 185 13. 56830, 144.93179 AAFB 0 3 1 1 10.2 36.46 [M] 0 2 164 2 limestone 1 03-23-21 (-) 205 Slight
19-99 07-30-19 Nt tree 186 13.56314, 144.93079 AAFB 0 1 0 1 9.1 40.70 [M] 0 1 160 2 limestone 1 03-23-21 (-) 197 Slight
19-100 08-09-19 Nt tree 187 13.25978, 144.73688 Duenas Beach 0 0 0 2 12.4 63.70 [S] 0 1 11 1 coral sand 3 03-18-21 (-) 264 Slight
19-101 08-09-19 Nt tree 188 13.25963, 144.73735 Duenas Beach 0 1 0 1 11.4 41.20 [S] 0 1 12 1 coral sand 3 03-18-21 (-) 326 Low
19-102 08-20-19 Nt tree 191 13. 62340, 144.89664 Tarague Beach 0 0 0 1 11.6 39.00 [S] 0 2 21 1 coral sand 3 03-26-21 (-) 224 Slight
19-103 09-13-19 Nt tree 199 13.56594, 144.87816 Watson's Farm, Yigo 0 1 0 1 12.1 30.90 [S] 0 0 168 2 limestone 1 02-12-21 (-) 227 Slight
19-104 09-13-19 Nt tree 200 13.56674, 144.87776 Watson's Farm, Yigo 0 0 0 1 8.1 22.30 [S] 0 0 169 2 limestone 1 02-12-21 (-) 164 Slight
19-105 09-13-19 Nt tree 76 13.56864, 144.87701 Watson's Farm, Yigo 0 1 0 1 9.2 26.27 [S] 0 0 173 2 limestone 1 02-18-21 (-) 259 Slight
19-106 09-13-19 Nt tree 85 13.56741, 144.87413 Watson's Farm, Yigo 0 1 0 1 7.3 23.10 [S] 0 0 163 2 limestone 1 02-18-21 (-) 224 Low
19-107 09-13-19 Nt tree 86 13.56707, 144.87654 Watson's Farm, Yigo 0 0 0 1 12.4 37.70 [S] 0 0 171 2 limestone 1 02-12-21 (-) 225 Slight
19-108 09-13-19 Nt tree 87 13.53309, 144.87161  UOG Yigo Station  0 0 0 1 19.7 33.30 [S] 0 1 178 2 limestone 1 02-04-21 (-) 217 Slight
19-109 09-13-19 Nt tree 88 13.53297, 144.87364 UOG Yigo Station 0 1 0 0 8.9 25.20 [S] 0 1 142 2 limestone 1 02-04-21 (-) 185.1 Slight
20-122 07-15-20 Nt under bark 111 13.56711, 144.87700 Watson's Farm, Yigo 0 4 1 2 7 24.52 [S] 1 1 168 2 limestone 1 07-15-20 (+)    
20-123 07-15-20 Nt under bark 119 13.56692, 144.87740 Watson's Farm, Yigo 0 3 1 2 19.6 17.64 [M] 0 1 163 2 limestone 1 07-15-20 (+) 520 Low
20-124 07-15-20 Nt mud tunnel 112 13.56736, 144.87631 Watson's Farm, Yigo 0 3 1 2 7.4 13.38 [S] 0 1 158 2 limestone 1 07-15-20 (+) 412 Low
20-125 08-01-20 Nt arboreal nest 122 13.43109, 144.80084 UOG, Mangilao 0 4 1 0 15.1 17.52 [M] 0 0 67 1 limestone 1 08-01-20 (+)    
20-126 08-01-20 Nt under bark 153 13.43128, 144.80281 UOG, Mangilao 0 3 1 2 11.9 41.40 [S] 0 2 81 1 limestone 1 08-01-20 (+) 598 Moderate
20-129 08-01-20 Nt under bark 226 13.46781, 144.84233 Mangilao Golf Course 0 1 1 2 7.8 27.71 [S] 0 2 129 2 limestone 1 08-01-20 (+) 581 Moderate
20-130 08-01-20 Nt mud tunnel 217 13.53231, 144.87267 UOG Yigo Station 0 4 1 2 13.9 31.21 [S] 0 1 173 2 limestone 1 08-01-20 (+) 713 Moderate
20-131 08-01-20 Nt mud tunnel 217 13.53231, 144.87267 UOG Yigo Station 0 4 1 2 13.9 31.21 [S] 0 1 173 2 limestone 1 08-01-20 (+)    
20-132 08-02-20 Nt mud tunnel 215** 13.26533, 144.71634 UOG Ija Station 0 4 1 1 10 37.90 [S] 0 1 96 1 residum  2 08-02-20 (+) 344 Low
20-133 08-02-20 Nt under bark 216** 13.26578, 144.71692 UOG Ija Station 0 4 1 2 11.9 31.53 [S] 0 1 93 1 residum  2 08-02-20 (+) 403 Low
20-134 08-02-20 Nt under bark 208 13.26590, 144.71707 UOG Ija Station 0 4 1 2 15.8 35.67 [S] 1 1 82 2 residum  2 08-02-20 (+) 639 Moderate
20-135 08-02-20 Nt arboreal nest 209 13.26595, 144.71707 UOG Ija Station 0 4 1 2 8.9 29.78 [M] 0 1 110 2 residum  2 08-02-20 (+) 449 Low
21-150 9/17/2021 C tree 306 13.28276, 144.75675 UOG Inarajan Station 1 n/a n/a n/a n/a n/a n/a n/a 16 1 limestone  1 n/a n/a n/a n/a
21-151 9/17/2021 C tree 306 13.28276, 144.75675 UOG Inarajan Station 1 n/a n/a n/a n/a n/a n/a n/a 16 1 limestone  1 n/a n/a n/a n/a
21-153 9/17/2021 C stake 302 13.26530, 144.71624 UOG Ija Station 1 n/a n/a 1 7.6 29.77 [M] 0 1 89 1 residum  2 n/a n/a n/a n/a
21-154 9/17/2021 C tree 302 13.26530, 144.71624 UOG Ija Station 1 n/a n/a 1 7.6 29.77 [M] 0 1 89 1 residum  2 n/a n/a 351 Low
21-155 9/17/2021 C tree 216 13.26578, 144.71692 UOG Ija Station 0 4 1 2 11.9 31.53 [S] 0 1 93 1 residum  2 08-02-20 (+)    
21-156 9/17/2021 C tree 301 13.36490, 144.64920 Nimitz Park 0 0 0 0 13.1 75.99 [S] 0 2 4 1 Coral sand 3 3/4/2022 (-) 300 Slight
21-157 9/17/2021 C tree 378 13.47956, 144.75429 Paseo Park, Hagatna 0 0 0 0 9.4 60.70 [M] 0 2 6 1 Limestone 1 3/4/2022 (-) 232 Slight
21-159 9/17/2021 C tree 250 13.47040, 144.84463 Mangilao Golf Course 1 n/a n/a 2 6.7 17.35 [S] 1 1 128 2 Limestone 1 n/a n/a 802 Moderate
21-160 9/22/2021 C tree 32 13.40239, 144.66307 Apaca Point, Agat 0 0 0 1 13.1 103.13 [M] 1 1 6 1 residum  2 3/4/2022 (-) 237 Slight
21-161 9/22/2021 C tree 32 13.40239, 144.66307 Apaca Point, Agat 0 0 0 1 13.1 103.13 [M]  1 1 6 1 residum  2 3/4/2022 (-)    
21-162 9/22/2021 C tree 379 13.47870, 144.73200 Governor's Complex, Aniquia 0 0 0 2 10.9 50.13 [M] 0 2 6 1 residum  2 3/4/2022 (-) 172.8 Slight
21-164 9/22/2021 C tree 154 13.53327, 144.87158 UOG Yigo Station 0 4 1 1 38.4 33.96 [S] 1 2 144 2 Limestone 1 2/23/2022 (+) 384 Low
21-165 9/22/2021 C tree 297 13.56553, 144.87749 Watson's Farm, Yigo 0 4 1 1 33.0 93.98 [S] 1 1 159 2 Limestone 1 3/17/2022 (+) 1,113 High
21-166 9/22/2021 C tree 296 13.56528, 144.87704 Watson's Farm, Yigo 0 3 1 1 12.8 41.2 [S] 0 1 159 2 Limestone 1 3/17/2022 (+) 444 Low
21-168 9/27/2021 C tree 240 13.55782, 144.93009 AAFB 0 4 1 2 12.5 55.36 [M] 0 2 155 2 Limestone 1 3/3/2022 (-) 308 Low
21-170 9/27/2021 C tree 242 13.56132, 144.93056 AAFB 0 1 0 0 13.1 83.17 [M] 0 1 155 2 Limestone 1 3/3/2022 (-) 395 Low
21-171 9/27/2021 C tree 243 13.62518, 144.89525 AAFB, tarague beach 0 4 1 2 13.1 47.7 [S] 0 2 9 1 Coral sand 3 3/11/2022 (+) 703 Moderate
21-172 9/27/2021 C tree 244 13.62629, 144.89449 AAFB, tarague beach 0 3 1 0 16.2 50.94 [S] 0 1 9 1 Coral sand 3 3/11/2022 (-) 521 Low
21-174 9/27/2021 C tree 246 13.37650, 144.73789 Windward hills golf course 0 4 1 2 11.3 46.42 [M] 0 2 115 2 residum  2 3/10/2022 (+) 264 Slight
21-176 9/29/2021 C tree 250 13.47040, 144.84463 Mangilao Golf Course 1 n/a n/a 2 6.7 17.35 [S] 1 1 128 2 Limestone 1 n/a n/a n/a n/a
21-180 9/29/2021 C tree 251 13.37163, 144.76817 Country Club of the Pacific 0 3 1 2 9.4 54.17 [S] 0 2 26 1 residum  2 3/10/2022 (-) 298 Slight
21-181 9/29/2021 C tree 253 13.26586, 144.71689 UOG Ija Station 1 n/a n/a n/a n/a n/a n/a n/a 75 1 residum  2 n/a n/a n/a n/a
21-182 9/29/2021 C tree 254 13.26566, 144.71705 UOG Ija Station 0 3 1 0 10.4 30.72 [S] 0 1 86 1 residum  2 3/2/2022 (+) 584 Moderate
21-183 9/29/2021 C tree 303 13.26587, 144.71694 UOG Ija Station 1 n/a 1 1 28.0 45.9[M] 0 2 86 1 residum  2 n/a n/a n/a n/a
21-184 9/29/2021 C tree 304 13.26592, 144.71698 UOG Ija Station 1 n/a 1 1 23.0 39.24[M] 1 2 86 1 residum  2 n/a n/a n/a n/a
21-185 9/29/2021 C tree 305 13.26597, 144.71705 UOG Ija Station 1 n/a 1 1 28.0 40.42[S] 0 2 86 1 residum  2 n/a n/a n/a n/a
21-186 9/29/2021 C tree 255 13.27161, 144.66414 Merizo Cemetary 0 3 1 0 40.0 29.10[S] 0 0 25 1 residum  2 3/18/22 (+) 540 Low
21-188 9/29/2021 C tree 257 13.29509, 144.66092 Fort Soledad 0 3 1 2 35.0 47.7[S] 0 2 42 1 residum  2 3/18/22 (-) 442 Low
                                             
**Tree 215 was the source tree for two Ralstonia solanacearum species complex isolates used by Sujan Paudel in this thesis research at the University of Hawaii in August 2020. https://scholarspace.manoa.hawaii.edu/handle/10125/70339; Paudel hawii 0085O 10777.pdf
The corresponding UOG isolate numbers are 192021 and 19202 and the associated UOG clinic number is 19-202.
   
**Tree 216 was the source tree for one Ralstonia solanacearum species complex isolate used by Sujan Paudel in this thesis research at the University of Hawaii in August 2020. https://scholarspace.manoa.hawaii.edu/handle/10125/70339; Paudel hawii 0085O 10777.pdf
The corresponding UOG isolate number is 19203 and the associated UOG clinic number is 19-203. 
   

Glossary found here: LSU Ironwood termite metadata 4-4-22

 

IW Termite collection sites

Figure 11. Ironwood tree termite collection sites on Guam. Blue dot = 2019 collection site; Red dot = 2021 collection site.

 

Results: Obj.3, Sub-obj. 3, LSU: Based a collection of termites from 48 ironwood trees, the ‘higher’ termite, Nasutitermes takasagoensis was the most common accounting for 93% of the samples. The lack of Ralstonia spp. and the low presence and abundance of wetwood bacteria in the gut of these termites suggest that they are not a vector for these pathogens of IWTD. At taxonomic level two in SILVA (Phylum level), Spirochaetes (48.22 %) and Fibrobacteres (41.43 %) were found to be the most dominant phyla followed by Bacteroidetes (3.61%), Proteobacteria (3.35%), Margulisbacteria (0.84%), Acidobacteria (0.77%), Planctomycetes (0.65%), and others (1.61%).  It was determined that the presence or absence of Ralstonia in trees from which termites were collected showed no significant effect on the total bacteria community; however, the communities of termites collected from sick trees showed significantly higher bacterial richness compared to those from healthy trees. In the attached report by LSU, a full description of the results can be found.

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

Research conclusions:

A great deal of research has been accomplished in the past 2.5 years; however, there are still too many results pending for a comprehensive conclusion at this time. I am comfortable with reporting the following. From the analysis of 26 water cultures of the bacterial wilt pathogen by the University of Hawaii, it was determined that the bacterial wilt pathogen in Guam’s trees is best described as Ralstonia solanacearum species complex (RSSC). Their analysis revealed two strains and thus two potential origins of Guam’s pathogen. The most common was Phylotype I which is present in North and Eastern Asia, whereas less common was Phylotype II which occurs in Central America. Based a collection of termites from 48 ironwood trees, the ‘higher’ termite, Nasutitermes takasagoensis was the most common accounting for 93% of the samples. The lack of Ralstonia spp. and the low presence and abundance of wetwood bacteria in the gut of these termites suggest that they are not a vector for these pathogens of IWTD. At taxonomic level two in SILVA (Phylum level), Spirochaetes (48.22 %) and Fibrobacteres (41.43 %) were found to be the most dominant phyla followed by Bacteroidetes (3.61%), Proteobacteria (3.35%), Margulisbacteria (0.84%), Acidobacteria (0.77%), Planctomycetes (0.65%), and others (1.61%).  It was determined that the presence or absence of Ralstonia in trees from which termites were collected showed no significant effect on the total bacteria community; however, the communities of termites collected from sick trees showed significantly higher bacterial richness compared to those from healthy trees. 

Participation Summary
3 Producers participating in research

Research Outcomes

5 New working collaborations

Education and Outreach

30 Consultations
3 Curricula, factsheets or educational tools
6 On-farm demonstrations
1 Published press articles, newsletters
17 Webinars / talks / presentations
4 Workshop field days
11 Other educational activities: Educated property owners and managers on planting and maintaining ironwood tree windrows. Ironwood tree seedlings were provided free of charge, and in some cases, property owners were assisted with planting.

Participation Summary:

4 Farmers participated
24 Ag professionals participated
Education and outreach methods and analyses:

Obj 1 Yr 1-2: UOG Ed: Through tree plantings, educate the public on the importance of planting new off-island cultivars (seedlots from an international provenance trails)

Ironwood (Casuarina equisetifolia) seeds sourced from different geographical locations (Table 4) were planted into seed cones. Saplings were raised for 4-6 months then transplanted to different locations across Guam in February 2020 (Figure 12). Saplings were used for both new and replacement windrows.

    Obj. 1, Sub-obj. 1, UOG: Plant trees of mixed cultivars in 10 visually deteriorating windrows across Guam.

Ten deteriorating windrows were identified by visual surveillance of sites for IWTD. These windrows were refurbished with 6-15 trees each, for a total of 138 trees planted (Table 5). Property owners were educated on the best tree care and management practices during planting. 

   Obj. 1, Sub-obj. 2, UOG:  Construct 15 new ironwood agroforestry projects using 150 trees consisting of a mixture of tree cultivars.

Fifteen new windrows were created consisting of 10 trees each, for a total of 150 trees (Figure 13). Property owners were educated on the best tree care and management practices during planting.

Locations of windrows planted across Guam

Figure 12: Locations of windrows planted across Guam.

 

Planting new windrows

Figure 13: Extension Associate Joseph Afaisen planting a new windrow at Cooperator Vincent Velasquez’s farm. The rebar and fencing is used to protect the sapling from wild pigs.

 

Table 4: Locations of Casuarina equisetifolia seeds used for the purpose of planting new windrows and increasing genetic diversity.

          LATITUDE: LONGITUDE: ALT M:
Location: STATE: DEG MIN DEG MIN  
(R)BAKO NATIONAL PARK MALAYSIA 1 44 110 30 30
(R)KUANTAN PENANG MALAYSIA 3 48 103 20 0
(R)DESARU JOHOR MALAYSIA 1 30 104 16 0
HORNO IS. MANUS PAPUA NEW GUINEA  2 19 147 49 1
(R)ELA BEACH PAPUA NEW GUINEA  9 5 147 17 10
(R)KOLOMBANGARA SOLOMON ISLANDS 8 7 57 8 2
(R)BAN KAM PHUAM RANO THAILAND 9 21 98 27 10
(R)HADSAMIRA SONGKHLA THAILAND 7 9 100 37 2
MELVILLE ISLAND AUSTRALIA 11 5 130 40 10
(R)AGAMY EGYPT 31 13 29 45 9
(R)CHANDIPUR BALASORE INDIA 21 30 86 54 2
HAMBANTOTA SRIL 6 8 81 7 16
VILLACLARA PLANTED CUBA 23 0 80 30 60
(R)BAOBAB KENYA  4 0 39 6 25
(R)EFATE VANUATU 17 45 168 18 30
(R)YANJING CHINA 23 0 113 3 4
(R)DAODONG SF CHINA 19 58 110 59 10
BEIHAI GUANGXI CHINA 21 35 109 0 2
(R)NINH TUAN VIETNAM 11 33 108 59

2

 

Table 5. Deteriorating windrows refurbished across Guam.

Location

Date planted

Number of trees planted

Tree origins used

GPS- beginning of windrow

GPS- end of windrow

Palm Tree Golf Course, AAFB

June 2019

12

Guam (9), Vanuatu (3)

13.57476, 144.93803

13.56912, 144.93175

Palm Tree Golf Course, AAFB

June 2019

6

Guam (2), Vanuatu (2), China (2)

13.56127, 144.93317

13.56187, 144.93282

Bernard Watson Farm, Yigo

June 2019

15

Vanuatu (3), Malaysia (3), Egypt (2), PNG (1), China (3), Vietnam (1), Sri Lanka (2)

13.56637, 144.87721

 

13.56702, 144.87700

Bernard Watson Farm, Yigo

June 2019

15

Vanuatu (3), Malaysia (3), Egypt (2), PNG (1), China (3), Vietnam (1), Sri Lanka (2)

13.56587 144.87730

13.56583, 144.87688

 

UOG Yigo Experiment Station

August 2020

15

PNG (2), Thailand (4), Egypt (4), India (4), Malaysia (1)

13.5335  144.8716

13.5335, 144.8710

UOG Yigo Experiment Station

August 2020

15

Thailand (4), Egypt (4), India (5), China (2)

13.5324   144.8745

13.5320, 144.8745

Mangilao Golf Course

August 2020

15

Egypt (2), Thailand (5), Guam (4), India (2), Kenya (1), China (1)

13.4684, 144.8423

13.4680, 144.8423

Mangilao Golf Course

August 2020

15

Thailand (9), Guam (4), Australia (2)

13.4680, 144.8423

13.4676, 144.8424

Windward Hills Golf Course

August 2020

15

Egypt (2), Thailand (4), Guam (4), India (4), PNG (1)

13.3754, 144.7480

13.3759, 144.7380

Windward Hills Golf Course

August 2020

15

Thailand (4), Guam (4), India (4), PNG (1), Australia (2)

13.3759, 144.7379

13.3765, 144.7379

 

Obj. 4 Yr 2-3: UOG Ed: Restoring ironwood as an agroforestry species in Guam through awareness and action of the local and scientific communities.

   Obj. 4, Sub-obj. 1, UOG: Through two ½ day workshops, attendees will learn about the care of the ironwood trees and its uses in agroforestry. Ironwood trees will be given away.

½ Day Workshop February 28, 2020:

A hands-on half-day workshop/training was held for 11 University of Guam Pest Management students in the afternoon on Saturday February 28, 2020 (Figure 14). In an effort to bring attention to the current studies and familiarize the students with ironwood tree decline, participants were exposed to a variety of practices and techniques used in studying these phenomena.

Activity 1: Windbreak planting and genetic diversity: Participants were provided different ironwood trees sapling varieties from around the world and were tasked to refill the missing ironwood trees in a windbreak located at the University of Guam Agriculture and Life Science Building.  Participants were also instructed on the importance of increasing the genetic diversity of Guam’s ironwood trees. All students successfully planted one tree.

Activity 2: Ironwood tree seed germination: Ironwood trees seeds from different areas of the world were given to the participants. Each participant was tasked to plant the seeds in cone planters filled with damp #4 Sunshine mix. Dr. Schlub demonstrated the proper depth the seeds should be buried. Each participant had seeds from different geographical locations and 20 cones and successfully planted the seeds. The saplings from the seeds will be maintained and used in planting windbreaks during the next rainy season.

Activity 3: Evaluating ironwood trees and testing for Ralstonia  solanacearum (Rs): Participants were divided into three groups and taught how to visually determine ironwood tree decline severity. They logged the geographical location and altitude of a tree using a GPS device, determined the height of a tree using a Precision Laser Rangefinder with Hypsometer, and measured the circumference of the tree at breast height to calculate the diameter. Also, through visual inspection of the tree they determined if the tree had present or past evidence of termite activity as well as if there were Ganoderma sporocarps. Finally, they used a drill to collect wood shavings from the tree and tested for the presence of Rs in the laboratory using Agdia immunostrip test kits.

Activity 4. Inoculation of tomato seedlings with Ralstonia  solanacearum (Rs) previously isolated from ironwood trees: Each participant inoculated tomato seedlings with Rs isolated from ironwood trees and grown on media. Some plants were lightly scraped on the stem near the crown and Rs was applied using a cotton swab. Some plants were lightly scraped with nothing applied. Half of the plants inoculated with Rs developed severe bacterial wilt symptoms, while no symptoms developed on the plants that weren’t inoculated. Photos of the plants were taken and sent to the participants.

Workshop on February 28, 2020

Figure 14: Principle Investigator Dr. Robert Schlub and Pest Management students at an ironwood tree workshop held at the University of Guam on February 28, 2020.

 

 

½ Day Workshop February 29, 2020

     A half-day workshop was attended by 25 participants including farmers, property owners, home gardeners, professionals, and students at the University of Guam’s College of Natural and Applied Sciences’ Agriculture and Life Science building on Saturday, February 29, 2020 from 8:30 to 11:30 am (Figure 15). The focus of the workshop was to educate the community about the ironwood tree, the ironwood tree decline (IWTD) and the current response to address the decline.

        PowerPoint was used for all workshop presentations/discussions. Extension Associate DonaMila Taitano opened the workshop with a discussion of this grant’s objectives. Principle Investigator Dr. Robert Schlub then gave a presentation in regards to ironwood trees on the world stage and on Guam including: distribution, uses, economic value, and ecological value. Then Dr. Schlub discussed threats to the ironwood trees around the world and on Guam including: economic losses and ecological losses, uniqueness of ironwood trees on Guam, ironwood tree diseases, and the importance of increasing the genetic diversity of Guam’s ironwood trees. Finally, DonaMila Taitano discussed planting and care of the ironwood tree and gave out a total 30 ironwood sapling varieties from different worldwide locations for those requesting the trees.

     A pre-test and a post-test were given to the participants to determine the knowledge gained. The results were significant, with an average score on the pre-test of 33% correct and an average score on the post test of 88%.

     Workshop participants were asked to evaluate the workshop on a scale of 1 to 5 in different categories, with one being poor and five being excellent: 100% rated information provided at 4 or 5 (82% at 5); 100% percent rated quality of speakers at 4 or 5 (90% at 5); 82% rated overall organization at 4 or 5 (55% at 5); 100% rated overall benefit from attending the workshop at 4 0r 5 (77% at 5). 

Workshop on February 29, 2020

Figure 15: 15 of the 25 participants who attended the WSARE funded ironwood tree workshop on February 29, 2020.

 

   Obj. 4 Sub 2: UOG/UH/LSU: PI and Extension/outreach representative in the third year will conduct a four-day workshop/conference on bacterial wilt and other components of IWTD.

From January 3-7, 2022, a four- and a half-day workshop/conference was held at the University of Guam.

Part one: Building Resiliency of Trees in the Northern Mariana Islands Workshop

A full-day workshop was held for 19 agricultural professionals on January 3, 2022. Participants included agricultural professionals from Guam: 4 from Guam Department of Agriculture, 6 from Guam Plant Extinction Prevention Program (GPEPP), 4 from Colorado State University Center for Environmental Management of Military Lands (CEMML), 4 from University of Guam, and 1 from Andersen Air Force Base, Guam (Figure 16). Four agricultural professionals from the Northern Mariana Islands (including forestry and extension agents from Saipan, Rota, and Tinian) were invited to the workshop, but were unable to travel due to COVID restrictions. The workshop was held at the University of Guam’s College of Natural and Applied Sciences’ Agriculture and Life Science building on Monday, January 3, 2022 from 8:00 am to 4:30 pm. The workshop was presented by Dr. Robert Schlub of the University of Guam and guest presenter Dr. Anand Persad of ACRT Services. The focus of the workshop was to update the citizens of Guam and the Northern Mariana Islands on the health of its Ironwood tree, and to provide them with tree care practices that can be used to reduce the impact of pests and diseases in Ironwood as well as other trees of the region.

The following was the agenda for the workshop: 2022 Building Resiliency Workshop

Activities included morning classroom instruction, which covered topics such as Guam’s tree species, plant health care, and soil-borne bacteria and fungi impacting the health of Guam’s trees. In the afternoon, participants were taken on a short walking tour of campus where they examined ironwood trees for symptoms of ironwood tree decline and the presence of other decline predictors/contributors (Figure 17). After the tour, participants spent the rest of the day in the lab learning various activities, including how to test samples for Ralstonia solanacearum, how to identify major termites that attack ironwood trees, examining fruiting structures of wood-rotting fungi under a microscope, examining tree disks for bacterial ooze and wetwood, and how to propagate ironwood trees.

Workshop in January 2022

Figure 16. Participants, presenters, and staff who attended the WSARE-funded Tree Resiliency workshop at UOG in January 2022.

 

IW Demonstration Garden

Figure 17. Dr. Robert Schlub talks to participants about symptoms of ironwood tree decline as they tour the UOG Ironwood Tree Demonstration Garden.

 

Part two: Ironwood (Casuarina equisetifolia) decline conference

            A 3.5 day in-person and virtual ironwood tree decline (IWTD) conference was held from Tuesday, January 4 to Friday, January 7, 2022 at the University of Guam Agriculture and Life Sciences building. Known ironwood tree researchers from across the globe were invited to attend in-person or virtually via zoom. The International Union of Forest Research Organizations (IUFRO) also posted an advertisement for the University of Guam IWTD conference on their twitter and Facebook pages, where an additional 300-350 people were notified of the conference and how to contact UOG if they were interested in attending virtually. Four researchers were able to attend the conference in-person: Dr. Anand Persad of ACRT Services, Dr. Sujan Paudel of the University of Hawaii, Dr. Claudia Husseneder of Louisiana State University, and graduate student Garima Setia of Louisiana State University. Many of the invited international researchers were unable to travel due to COVID restrictions. Virtual participants included researchers from the University of Hawaii, University of Florida, Louisiana State University, University of Pretoria (South Africa), the Indian Council of Forestry, the Chinese Academy of Forestry, the Commonwealth Scientific and Industrial Research Organization (Australia), and the U.S. Forest Service. During the two days of lectures, 31 people participated virtually via zoom and 4 people participated in-person, for a total of 35 participants. 17 formal presentations were given over the 3.5 day conference, including presentations by in-person and virtual attendees, and national and international researchers. The conference focused on exchange of knowledge and research discovery to ameliorate the impact of bacterial wilt in Casuarina equisetifolia, and unraveling the roles of Ralstonia solanacearum species complex, Ganoderma australe, wetwood bacteria, and termites in the decline of Guam’s ironwood.

The following was the agenda for the ironwood tree decline conference: 2022 IWTD Conference

The first day of the conference (Tuesday, January 4th) was spent giving the in-person researchers a tour of Guam’s ironwood decline sites across the island. Sites included the Guam National Wildlife Refuge (north) and the UOG Ija Experiment Station (south) (Figure 18). Participants were able to see the effects of IWTD in-person, ask questions, and take photos.

Conference attendees at Ritidian beach

Figure 18. In-person conference attendees pose for a picture with Dr. Robert Schlub on Ritidian beach, Guam National Wildlife Refuge.

 

The second day of the conference consisted of in-person and virtual classroom lectures. In-person participants were able to connect with virtual participants via zoom (Figure 19, Figure 20). Lecture topics and presenters can be seen within the agenda above.  

Sujan Paudel presentation

Figure 19. Sujan Paudel of the University of Hawaii gives an in-person presentation on Genetic diversity and genealogy of the Ralstonia solanacearum species complex associated with Ironwood decline in Guam. Virtual participants could view his presentation via zoom meetings.

 

Indian Council of Forestry presentation

Figure 20. Indian Council of Forestry researcher Dr. Abel Nicodemus gives a virtual presentation via zoom. Dr. Nicodemus and his colleague Dr. Arumugam Karthikeyan are visible in the upper right-hand corner of the screen.

 

The third day of the conference consisted of in-person and virtual lectures in the morning (Figure 21). In the afternoon, in-person participants departed for a field trip to Bernard Watson’s farm. At the farm, participants were able to see first-hand the effects of IWTD on farm windrows (Figure 22). Participants also saw new windrow plantings (Figure 23). Lastly, an ironwood tree which was a known positive of R. solanacearum was cut down at the trunk and participants were able to see evidence of wetwood, and bacterial ooze (R. solanacearum) contributing to IWTD (Figure 24).

Anand Persad presentation

Figure 21. Dr. Anand Persad gives a presentation on “Climate and Island States.”

 

Windrow at Watson farm

Figure 22. An ironwood tree windrow seen at Bernard Watson’s farm. This windrow has not yet been fully decimated by IWTD.

 

3 yr old windrow at Watson farm

Figure 23. Researchers examine planted ironwood tree windrows at Bernard Watson’s farm. These trees are approximately 3 years old.

 

Conference attendees examine wetwood

Figure 24. Extension agents show researchers the wetwood and signs of bacterial ooze on a freshly cut-down ironwood tree stump. This tree was known positive for R. solanacearum.

 

The fourth (and final) day, WSARE Research Project team members who were able to attend in-person held an informal discussion of project goals and accomplishments.

 

6 Farmers intend/plan to change their practice(s)
6 Farmers changed or adopted a practice

Education and Outreach Outcomes

6 Producers reported gaining knowledge, attitude, skills and/or awareness as a result of the project
Key areas taught:
  • Agroforestry
Key changes:
  • Ironwood tree genetic diversity and bacterial disease

Success Stories

    Success stories:

    The Western IPM Center posted an online article:

    “Protecting the trees that protect Guam”: http://westernipm.org/index.cfm/ipm-in-the-west/natural-areas/protecting-the-trees-that-protect-guam/

     

    The Western IPM Center made a short video documentary on Dr. Robert Schlub and his research of Ironwood trees on Guam:

    “Ironwood trees on Guam- an important species that has many benefits for the island- are dying. See how scientists are working to understand and reverse the decline”: Ironwood Tree Decline on Guam - YouTube

     

    A forester from the Guam Department of Agriculture had the following comments on the University of Guam’s Tree Resiliency Workshop held in January 2022:

    “On behalf of my staff I'd like to thank you for inviting us to the workshop. It was very informative and well organized. I would like to request that if you have any extra binders with the presentation handouts and lecture materials please send them my way as I would distribute them to my other staff members.”

    By distributing workshop materials to staff members who could not attend, objectives of the workshop can better reach the Guam forestry community, and therefore, have a positive influence on the farmers that these forestry agents interact with on a daily basis.

Information Products

    Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.