Progress report for SW19-906

Restoring ironwood as an agroforestry species in Guam through research into the bacterial flora of ironwood trees and the guts of termites.

Guam:

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
• Cooler

Methods:

     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.

Guam:

Method

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

WSARE_4.1.2020 – 3.30.21_Hawaii Report