Project Overview
Information Products
Commodities
- Additional Plants: trees
Practices
- Crop Production: agroforestry
Abstract:
Ironwood (Casuarina equisetifolia), recognized as an important tree species by the Natural Resources Conservation Service (NRCS), are in decline. In a survey funded by WSARE in 2008, it was estimated that 51% of the trees on Guam were showing signs of a progressive dieback now referred to as ironwood tree decline (IWTD). Between 2010-2019, data analyses identified 4 predictors of IWTD: the bacterial wilt pathogen Ralstonia solanacearum (RSSC) species complex, the butt and root rot fungus Ganoderma australe, termites, and bacterial wetwood. 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 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. Due to the similarities between IWTD in Guam and bacterial wilt caused by RSSC in China, the focus of this project was to examine the various biological components of RSSC and wetwood bacteria. Currently Guam is the only US location were RSSC has been confirmed 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. The purpose of this project is to reduce the impact of bacterial wilt in Guam by (1) determining the origin of Guam’s ironwood tree bacterial wilt pathogen, (2) determining if the bacterium can be vectored by termites, (3) strengthen Guam’s ironwood tree population through increasing the genetic diversity of Guam’s ironwood tree population, and (4) educating the public and scientific community about bacterial wilt and IWTD.
Twenty-six water cultures of the bacterial wilt pathogen were collected on Guam and used in Sujan Paudel's 2020 thesis research at the University of Hawaii. In this study, he helped develop an efficient protocol for the isolation and characterization of RSSC strains from the declining trees. Both R. pseudosolanacearum (Rps) and R. solanacearum (Rs; strains were isolated from 2008 survey) were found to be associated with decline, although the latter species was found to be much less numerous (3) compared to the former species (35). The phenotypic characterization assays (Biolog) showed a similar utilization pattern for the Rps and Rs strains. The ironwood Rps population in Guam was found to be highly clonal, with the least nucleotide diversity and a contracting population structure. The MLST analysis identified North and Eastern Asia, Indonesia, and Northern Australia as potential origins of ironwood Phylotype I strains, whereas Central America, South-Eastern USA, Northern Latin America, and the Caribbean may be the potential origins of ironwood Phylotype II strains. The genomes of seven strains representing different phylogenetic groups were sequenced and annotated using long- and short-read sequencers. Ten more genomes representing different phylotypes were retrieved from the NCBI GenBank database. The reconstructed genealogy of the 17 Ralstonia strains using ClonalFrameML revealed a ratio of recombination and mutation rates equal to R/θ = 0.09592, an average length of recombined fragments δ = 22.3743, a divergence of DNA imported by recombination ν = 0.160891 and a relative effect of recombination and mutation r/m = (R/θ) x δ x ν = 0.345. The results indicated that homologous recombination had played a significant role in diversification/evolution of these strains. The most recombined strains were those from phylotype I within the lineage 3. The fastGEAR was used to identify ancient and recent recombination events across the core-genome of the 17 Ralstonia strains. Intriguingly, only one ancestral recombination event of 290-bp donated by the lineage 3 to lineage 2 seems to have occurred during the evolution time. Conversely, 572 recent recombination events were found across the population, which indicates that Ralstonia strains are still evolving; possibly to adapt to a constraining environment. A multiplex assay for specific detection and discrimination of reclassified Ralstonia species was also developed. The assay was used to identify Ralstonia from infected samples.
Termite samples collected on Guam for inclusion in Garima Setia's 2023 Louisiana State University thesis research were as follows: 42 Nasutitermes takasagoensis, 27 Coptotermes gestroi, and 6 Microcerotermes crassus. Garima’s thesis showed that none of the termite species (N. takasagonesis, C. gestroi and M. crassus) that attack ironwood trees in Guam were vectors for IWTD pathogens. The IWTD-associated pathogens were either absent or were scarcely detected in the termite worker samples collected from sick and healthy ironwood trees in Guam. Only Klebsiella sp. was detected in the worker samples of N. takasagonesis. Putative pathogens from genera Ralstonia, Klebsiella, Enterobacter, Pantoea, and Citrobacter were detected in low amounts (0.02% relative abundance) in the worker samples of C. gestroi. No pathogenic bacterial species associated with IWTD were detected in the worker samples of M. crassus. Bacterial communities of N. takasagonesis workers were found to be impacted by the presence of Ralstonia, tree health, plot average DS, plot average decline, proportion of dead trees in the plot, proportion of trees with termites in the plot, altitude, parent material, and site management, while those of C. gestroi workers were impacted by tree health and site management. The number of M. crassus samples was too limited to investigate the effects of those factors on the bacterial community. Feeding experiments were performed to investigate if wood consumption by termite workers was influenced by the bacterial load of ironwood. Ralstonia spp. were not detected in any of the N. takasagoensis samples, regardless if they were collected from trees with confirmed Ralstonia infection or healthy trees. One C. gestroi sample and none of the M. crassus samples showed Ralstonia. Four N. takasagoensis samples and one C. gestroi sample showed Klebsiella species, albeit in low amounts.
A total of 101 samples collected from 40 ironwood trees distributed across five geologically distinct locations on the island of Guam were sent to the University of Hawaii and used in Diksha Klair’s 2022 thesis research. Klair’s study focused on phenotypic metadata and 16S rRNA (V3) and ITS (ITS) amplicon-based microbiome analyses. The dominant bacterial and fungal phyla identified were Proteobacteria (75.6%) and Basidiomycota (61.44%), respectively. However, the average relative abundance of 24.04% of reads remained unclassified for fungal classification owing to a poorly annotated fungal database. At the genus level, Ralstonia was identified with higher richness from infected samples and, interestingly, greater abundance in shallow than in deep woody sample types. Irrespective of the other associated factors, the bacterial microbiota showed a close microbial association based on the relative abundance (disease severity) of Ralstonia, whereas the fungal microbiota formed an association based on identified Ganodermataceae. The study found significantly lower bacterial and fungal diversity and richness in Ralstonia-infected trees compared to healthy trees, putting plants' ability to host non-pathogenic endophytic microbiota at risk. In addition, conductivity, tree height, human impact, and the abundance of Ganodermataceae all had a significant impact on fungal diversity. Results suggested that deciphering the endophytic microbiota of ironwood trees and its association with Ralstonia will help to understand this complex pathosystem and can also act as a potential source for the formulation of biocontrol agents and the development of efficient disease management strategies. To increase the genetic diversity of Guam’s ironwood tree population, off-island varieties were planted in agroforestry projects. Fifteen new windrows consisting of 10 trees each were created, 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 the 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 the exchange of knowledge and research discoveries 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.
In conclusion, this study provides research findings that are applicable for management of ironwood trees under the threat of bacterial wilt on Guam and other islands. The formation of ooze in cross-sections of large roots or branches is a strange indicator that a tree is infected with R. solanacearum or harmful levels of wetwood bacteria. Now knowing that high conductivity of water extracted drill shavings is a predictor of poor tree health, silviculturists have a quick, easy method to evaluate tree stress. This project provides evidence that Guam’s bacterial wilt pathogen most likely originated in China; therefore, trees that show resistance to this disease in China hold promise for Guam as well. From termite gut analyses, it was determined that termite workers are not vectors for the bacterial wilt pathogen or wetwood bacteria associated with IWTD; therefore, there is no evidence that controlling termite infestations will reduce the spread of Ralstonia. The findings suggest that Ralstonia infection reduces the plant’s ability to promote or possibly select beneficial endophytic microbiota. Linking population of beneficial endophytic microbiota with tree health will provide a means by which various tree management practices and environment conditions can be evaluated for their impact on overall tree health. Professionals and the general public are now being advised to reduce lawnmower and weed-trimmer damage to roots and the base of trees as a means to reduce infection and spread of pathogens. To reduce transmission of RSSC and wetwood bacteria when pruning, individuals are instructed to disinfect all tools. The public is also being advised to remove severely declined trees as a means to protect nearby healthy trees. We now know that most of the infected trees on Guam occur in cohorts of less than 12 trees but single infected trees are not uncommon. Even within tree cohorts, IWTD is slow to spread. It usually takes years from symptom onset to tree death. IWTD seems to be unique to Guam and has no single cause, though the majority of the tree loss can be contributed to R. solanacearum.
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.