Progress report for SW19-906
Ironwood (Casuarina equisetifolia), recognized as an important tree species by the Natural Resources Conservation Service (NRCS) are under attack. Ironwood is one of the dominant, naturally occurring and propagated agroforestry species in the Pacific due to its ability to withstand salt spray, typhoon strength winds, and poor soil conditions. 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. Guam’s NRCS Environmental Quality Incentives Program rewards applications for planting ironwood as a riparian forest buffer or windbreak, and as a source of ironwood needle organic mulch. In a 2002 forestry inventory, it was reported that Guam’s tree population was healthy and numbered 116,000. 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 Guam Department of Agriculture ceased its yearly 25,000 seedling give-away program in large part due to IWTD. The seedling program provided trees to farmers, the public, and government agencies for windbreaks, riparian buffers, erosion control, land reclamation, and island beautification. In Guam’s 2010-2015 Statewide Forest Resource Assessment and Resource Strategy report it was stated that ironwood decline was impacting the health of Guam’s forests and that determining its cause and finding solutions should be a priority for the future. Bacterial wilt will be reduced as a result of tree plantings, increasing genetic diversity of tree population, advancing scientific discovery, and education of the public and scientific community. Seven hundred trees of four off-island cultivars will be out planted in 25 agroforestry projects on producer’s farms and given to the public and government agencies. Various biological components of Ralstonia solanacearum and wetwood bacteria will be examined to determine the origin of these bacteria. Since termites are significantly associated with IWTD, we will investigate whether they carry ironwood bacteria in their guts as a first step to elucidate their potential role as vectors. Over the course of the project, two ½ day workshops will be offered to the public, farmers and foresters, where they will receive instructions on tree care practices such as disinfecting pruning tools to reduce the spread of bacteria. They will also learn that the common termite species found in ironwood trees is Nasutitermes, which is not a threat to homes. Researchers will be brought to Guam for a 4-day workshop, and roundtable discussion about bacterial wilt and bacterial wetwood, and implications for Guam and the Pacific Islands if R. solanacearum were to move from ironwood into other tree species. Currently Guam is the only US location were R. solanacearum occurs in trees whereas in China bacterial wilt disease occurs in Casuarina, Olea, Morus, and Eucalyptus. Some rank bacterial wilt disease as the most important disease in China due to its wide distribution and cumulative losses on trees, ornamentals, medicinal plants and many crops.
Obj 1 Yr 1-2:UOG: Restoring ironwood as an agroforestry species in Guam by planting new agroforestry projects, restoring projects decimated by bacterial wilt, and establishing conditions for natural incorporation of new genes into Guam’s ironwood tree population.
Obj. 1 Sub-obj. 1: UOG: Test trees for bacterial wilt in 10 deteriorating agroforestry projects, those that test positive flag for replacement with
150 trees of mixed cultivars.
Obj. 1 Sub-obj. 2: UOG: Construct 15 new ironwood agroforestry projects using 150 trees consisting of a mixture of tree cultivars.
Obj. 2 Yr 1-2: UH: Restoring ironwood as an agroforestry species in Guam through 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 and LSU: Restoring ironwood as an agroforestry species in Guam through research into the bacterial flora of ironwood trees and the guts of termites.
Obj. 3 Sub-obj. 1: UH: Determine the role of vascular colonization of R. solanacearum and wetwood bacteria species Klebsiella sp. in IWTD.
Obj.3 Sub-obj. 2:UH: Determine bacterial flora of ironwood trees and their likely role in IWTD.
Obj.3 Sub-obj. 3: LSU: Determine if termites carry ironwood bacteria and thus, might be responsible for their movement.
Obj. 4 Yr 2-3:UOG: Restoring ironwood as an agroforestry species in Guam through awareness and action of the local and scientific communities.
Obj. 4 Sub 1:UOG: Through two ½ day workshops, attendees will learn about the care of the ironwood trees and its uses in agroforestry. A total of
400 trees will be given away.
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. Proceedings of the meeting will be added as an appendix to the Guam Ironwood Tree guide.
- - Producer
- - Producer
- Termites will be collected from Guam’s Casuarina equisetifolia trees and the University of Louisiana will identify them.
- Casuarina equisetifolia trees in which termites were collected will be tested for Ralstonia solanacearumon using Agdia® Inc. Ralstonia solanacearum (Rs) immunodiagnostic strips.
- The University of Hawaii will begin to analyze Ralstonia solanacearum samples sent from Guam.
- The University of Hawaii will identified Guam’s Ralstonia solanacearum origin.
Restoring ironwood as an agroforestry species in Guam through research into the bacterial flora of ironwood trees and the guts of termites.
The following materials were used to execute the collection:
- Glass Vials
- 10 mL of 95% UN1170 Ethyl Alcohol 3, PGII 190 Proot
- 10 mL of 70% UN 1170 Ethyl Alcohol 3, PGII 190 Proot
- Aspirator ( X2)
- GPS (Garmin)
- Tree tags
- Copper Nails
- Insight LH Precision Laser Rangefinder with Hypsometer
- Measuring Tape
- Tree Log Data Sheet
- Termite Data Sheet
- Petri Dish
- Flagging Tape
Ironwood or Casuarina equisetifolia L. trees were examined for signs of active termite infestation at various locations (Figure 1). Trees were examined for the presence of termites and evidence of an active infestation. The size and health status of the trees were not specific as the main objective was to identify five ironwood trees with active worker and soldier termites in each given location.
For each ironwood tree with 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, and collection area of termite ( i.e mud trail, above or below ground colonies).
Subsequently, 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 with 10 mL of 95% and 70% ethyl alcohol. 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. Sampling has been completed and 64 samples were sent to Louisiana State University for species identification and analysis of gut contents.
Figure 1: Ironwood tree termite collection sites on Guam.
Louisiana State University:
Morphological Identification of Termites
Diagnostic characters of soldier termites were examined under a stereo microscope (Leica MZ16) for the morphological identification of termite species. The soldiers of Nasutitermes takasagoensis have a pyriform head which is modified to form a nasute, antennae have 12-14 segments, pronotum is saddle shaped having a length twice the width with round lateral margin and rostrum is conical with a basal width nearly as long as half of length with four setae at the base and two to four setae at the tip of the rostrum (Chhotani 1997, Liang and Li 2016). The two Coptotermes species, that have been previously reported from Guam, are Coptotermes gestroi and Coptotermes formosanus,. Soldiers of both species have a long tongue-shaped labrum extending beyond the middle of the mandibles and a large opening on the forehead called the fontanelle (Su and Scheffrahn 1998). Coptotermes gestroi soldiers have one pair of setae near the rim of the fontanelle, while in Coptotermes formosanus, two pairs originate around the fontanelle. The lateral profile of the top of the head just behind the fontanelle shows a weak bulge in Coptotermes gestroi that is absent in Coptotermes formosanus.
Materials and Methods: Restoring ironwood as an agroforestry species in Guam through research into the bacterial wilt pathogen to determine the origin of Guam’s infection and its genomic biology.
Isolates of Ralstonia solanacearum were collected from ironwood trees and water cultures prepared. Water cultures were sent to the University of Hawaii for PCR confirmation and for Genome sequencing.
University of Guam: During this study 112 trees from 29 locations across Guam were visually inspected and sampled. 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 woodrot. 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.
Though R. solanacearum could be detected from wood chips, drill shavings, water from root drill shavings, 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 on to 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 6 ml sterilized screw cap glass vials. This 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 (Figure 2).
Figure 2: Guam Ironwood tree locations where tissue samples were collected and Ralstonia solanacearum successfully isolated and sent to Hawaii.
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.
University of Hawaii:
The water cultures were streaked into Tetrazolium chloride media and semi-selective modified SMSA media. The cultures were streaked multiple times in TZC to ensure the cultures are pure. 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 950C 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. Analyses are on-going.
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
64 termite samples from 32 ironwood trees were sent to Louisiana State University for species identification and analysis of gut contents.
26 vials containing R. solanacearum in water from 26 different ironwood trees across Guam were prepared, one for storage at room temperature in Guam and one which was shipped to the University of Hawaii for further evaluation.
32 ironwood trees were tested for R. solanacearum with Agdia® Inc. Ralstonia solanacearum (Rs) immunodiagnostic strips. Six were positive. The data will be used by LSU when they perform termite gut content analysis.
University of Loiuisiana:
The ‘higher’ termite, Nasutitermes takasagoensis was found in 45 out of 48 (93%) samples received from Guam (Table1). The dominant presence of N. takasagoensis in ironwood trees was consistent with previous findings by Park et al. (2019), who identified termites based on morphological characters and confirmed their identification via DNA barcoding. The morphological characters of the Coptotermes sp. found in three samples were not clearly diagnostic for either species. Since the small sample size did not warrant inclusion into the gut microbiota study, we did not employ additional DNA barcoding methods to discriminate among C. gestroi or C. formosanus. Based upon Park et al. (2019) study, the Coptotermes species attacking iron wood trees is likely to be C. gestroi.
Progress till Date:
1. DNA extraction and quantification:
The termite samples were crushed and the DNA was extracted using Qiagen Dneasy Blood and Tissue kit based on manufacturer’s instructions. The quantity of DNA was confirmed using a Qubit Fluorometer.
2. Primer selection:
16s full- length (1.4k) sequences of different Ralstonia solanacearum strains from NCBI GenBank were aligned using Geneious software and examined for variability among different V- regions ( V1-V3 and V4 regions). V1-V3 region showed more variability and was selected for performing sequencing operations.
The extracted DNA will be sequenced on the Illumina platform and analyzed for bacterial diversity, and the presence of plant pathogens, such as Ralstonia solanacearum bacteria in termites.
Chhotani, O. B. 1997. The fauna of India and the adjacent countries. Isoptera (Termites):(family Termitidae), Vol. 2. Zoological Survey of India, Calcutta, India.
Liang, W. R., and H. F. Li. 2016. Redescriptions of three Nasutitermesspecies (Isoptera: Termitidae: Nasutitermitinae) occurring in Taiwan. Ann. Entomol. Soc. Am. 109: 779–795.
Park, J.-S., Husseneder, C., & Schlub, R. L. 2019. Morphological and Molecular Species Identification of Termites Attacking Ironwood Trees, Casuarina equisetifolia (Fagales: Casuarinaceae), in Guam. Journal of Economic Entomology, 112(4), 1902–1911.
Su, N.-Y., and R. H. Scheffrahn. 1998. Coptotermesvastator Light (Isoptera: Rhinotermitidae) in Guam. Pro. Hawaii. Entomol. Soc. 33: 13–18.
University of Hawaii: See materials and methods.
We are only 6 months into the project.
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 3). 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 maintain 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.
Figure 3: Principle Investigator Dr. Robert Schlub and Pest Management students at an ironwood tree workshop held at the University of Guam.
Educational & Outreach Activities
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 4). 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 regard to ironwood tree 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).
Figure 4: 15 of the 25 participants who attended the WSARE funded ironwood tree workshop.
Restoring ironwood as an agroforestry species in Guam and increasing genetic diversity.
Ironwood (Casuarina equisetifolia) seeds sourced from different geographical locations (Table 1) were planted into seed cones. Saplings were raised for 4-6 months then transplanted to different locations across Guam (Figure 5). Fifteen new windrows were created consisting of 10 trees each and four deteriorated windrows were refurbished with 15 trees each, which completes these objectives. (Figure 6). Property owners were educated on tree care best management practices during planting.
Figure 5: Locations of windrows planted across Guam (3 locations in south central Guam not shown).
Figure 6: 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 1: Locations of Casuarina equisetifolia seeds used for the purpose of planting new windrows and increasing genetic diversity.
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Ironwood tree genetic diversity and bacterial disease
Planting genetically diverse ironwood trees in windrows on farms will protect crops from damage and help prevent destruction of windrows from diseases in the future.