Garima Setia, Junyan Chen, Robert Schlub and Claudia Husseneder

The decline of the ironwood trees in the U.S. territory of Guam has left scientists perplexed for years. The ironwood tree, Casuarina equisetifolia, has been a vital component of Guam's natural ecosystem for countless millennia. Ironwood trees play an important role in improving soil fertility by reducing erosion and fixing atmospheric nitrogen.

However, for the past few decades, ironwood trees began to mysteriously decline in dozens of locations. Ironwood tree decline (IWTD) in Guam is identified by symptoms, including yellowing and thinning foliage, dieback, and wetwood staining in cross-sections of tree stems. The affected ironwood trees don’t recover from IWTD and eventually perish over the span of several years.

Researchers from the University of Guam have been struggling to identify the culprits behind this decline. With the help of U.S. Department of Agriculture grants, LSU AgCenter entomologists joined the investigation to lend their expertise to solving the mystery.

The initial investigations were focused on plant pathogens, as they are known to cause diseases in various tree species. Ironwood trees showing signs of decline were found to be infected with a bacterial pathogen, Ralstonia solanacearum, which is known to cause wilt in trees and the formation of ooze. This pathogen was also shown to cause wilting symptoms in healthy ironwood seedlings. Additionally, two wetwood bacterial species from the genus Klebsiella were detected in the infected trees and were considered opportunistic pathogens for IWTD.

Insects are one of the main ways plant diseases caused by pathogens are transmitted. Some insects found on ironwood trees in Guam, like beetles and gall wasps, are not likely linked to IWTD. Through the collaboration of researchers from the University of Guam and the LSU AgCenter, termites have been shown as a significant factor impacting IWTD. The termites attacking ironwood trees in Guam were identified as Nasutitermes takasagoensis, Coptotermes gestroi and Microcerotermes crassus.

Could termites be the key to unlocking the mystery of IWTD? These tiny insects are known to harbor bacteria and feed on wood, making them the perfect suspects for transmitting IWTD-associated pathogens. To investigate the role of termite workers as potential vectors for IWTD, researchers analyzed the bacterial communities of termite workers collected from sick and healthy ironwood trees through 16S rRNA gene sequencing. This method uses massive parallel sequencing of marker genes in the bacterial genome to identify all the bacterial species present in the termites’ bodies. The taxonomic analysis revealed a diverse bacterial community in each of the three termite species with the spiral-shaped Spirochaetes as the most abundant phylum. Nasutitermes takasagoensis workers did not contain any bacteria belonging to Ralstonia species. A minor abundance of Klebsiella (unidentified species) was detected in only four out of 42 N. takasagoensis samples. In C. gestroi workers, Ralstonia pickettii was detected in one out of 27 samples, but this species is not a known wilt pathogen. Klebsiella was detected in only two out of 27 samples. Moreover, M. crassus workers did not show any evidence of Ralstonia or Klebsiella species. Further research was needed to explain the low abundance of IWTD-associated bacteria in termite samples despite feeding on diseased trees.

Feeding experiments were conducted by AgCenter researchers during a month-long trip to Guam. A four-choice test using natural ironwood pieces representing different levels of Ralstonia and wetwood bacteria of the genus Klebsiella, (Figure A) showed significant effects of food source (p = 0.01, One-Way ANOVA). Nasutitermes takasagoensis workers consumed significantly less wood categorized as "Ralstonia positive with high amounts of wetwood bacteria" compared to both "Ralstonia negative with low amounts of wetwood bacteria" wood (p = 0.01) and "Ralstonia negative with high amounts of wetwood bacteria" wood (p = 0.02, Tukey's Studentized Range Test). This feeding experiment indicates that N. takasagoensis workers prefer wood with lower pathogen loads over wood with higher pathogen loads. Moreover, when Ralstonia was force-fed to N. takasagoensis workers, the bacteria did not survive in the highly alkaline environment of their guts. Both findings could explain why the bacteria survey was not able to detect many tree-related pathogens in N. takasagoensis bodies. In contrast, C. gestroi workers didn't prefer wood with low Ralstonia concentrations in the tested range, but a pilot study using workers of a closely related species from Louisiana, Coptotermes formosanus, suggested reduced consumption by these workers with higher Ralstonia concentrations. This suggests a similar avoidance mechanism in C. gestroi workers as N. takasagoensis, but at a higher threshold for Ralstonia.

While termites are associated with IWTD, it was found that they are unlikely vectors of tree-related pathogens. However, termites might not be completely innocent in the case of IWTD. The mechanical tree damage due to feeding of termites might be serving as a point of entry for these soil-inhabiting IWTD-associated pathogens.

The mystery surrounding the definitive cause of IWTD on the island of Guam remains unsolved. Ongoing studies of the microbiome of healthy and diseased ironwood trees, as well as the soil surrounding them, can provide crucial insights into the factors behind IWTD. As this investigation continues, there is hope that a solution to this decline can be found, saving the ironwood trees of Guam and neighboring islands.

Garima Setia is an M.S. student, Junyan Chen is a Ph.D. candidate student, Claudia Husseneder is a professor in the LSU AgCenter Department of Entomology, and Robert Schlub is an emeritus professor in the Department of Plant Pathology, University of Guam.

This article appears in the summer 2023 edition of Louisiana Agriculture magazine.

Nasutitermes takasagoensis, the most prevalent termite species attacking ironwood trees in Guam. Photo by Claudia Husseneder

Figure A. Cross-sections of ironwood tree trunks being (a) R. solanacearum negative with low amounts of wetwood bacteria, (b) R. solanacearum positive with low amounts of wetwood bacteria, (c) R. solanacearum negative with high amounts of wetwood bacteria and (d) R. solanacearum positive with high amounts of wetwood bacteria. AgCenter file photos