USDA Certification

Christian P. Giardina, USDA Forest Service, Hilo, Hawaii 96720

Summary


Treating Ohia with agents that confer resistance against Ceratocystis based diseases is poorly
understood, yet represents a centrally important piece of any long-term strategy for perpetuating Ohia
as a dominant component of Hawaii’s native forests. Questions about chemical treatments need to be
addressed for Ohia genotypes and species across the Hawaiian Islands. We will screen a new product,
BeGreen, with Ohia as a tool for reducing Ohia susceptibility to ROD.


Background


Because Ceratocystis wilt caused by C. lukuohia and Ohia canker caused by C. huliohia is infecting and
killing Ohia trees across Hawaii Island, and now on Kauai Island, with infection in some areas causing
high rates of mortality, identifying simple to apply treatments for various genotypes of Ohia that reduce
susceptibility to C. lukuohia and C. huliohia (known locally as rapid Ohia death – ROD) is critical to
managing the long-term consequences of ROD for forest health, and for informing conservation action
in Hawaii, including decisions about whether Ohia should be considered for restoration. Critical to
answering these questions is understanding how amendments, agents and treatments generally confer
resistance to ROD. To test BeGreen, we will screen Ohia with and without BeGreen treatments for
resistance to ROD using standard greenhouse methods. We will focus on Hawaii Island genotypes and
varieties. ROD has been found on at least one other Island, and so if results are promising, this work will
be foundational to expanding to other Ohia genotypes, other varieties of Metrosideros polymorpha, and
other Metrosideros species on other Hawaiian Islands. Selections on Hawaii Island will be made based
on morphological and geographic features. Ohia plant material is being generated through vegetative
cuttings and propagation. Studies show that this approach yields trees that can be tested in about 18
months. We have spent the past six months identifying mother trees, securing permits, and collecting
cuttings. These trees will be available for BeGreen effects on disease susceptibility in approximately 18
months, with reportable results on efficacy across genotypes in year three. From this work, positive
effects in the greenhouse will be evaluated and tests will move into a field trial on study gardens. ROD is
caused by a pair of closely related pathogens that represent perhaps the greatest threat to date to the
health of Hawaii’s native forest. By developing amendments that increase disease resistance of Ohia
trees to ROD, BeGreen will be able to provide an additional tool for restoring Ohia to ROD impacted
forests, but also urban open areas and private lands. Relying on modern greenhouse methods, we will
expose one set of Ohia genotypes to a BeGreen treatment (applied as a soil amendment, with repeat
monthly applications for 12 months), and these will be statistically paired to a second set Ohia
genotypes receiving a simple triple sixteen with micronutrients fertilizer application. A third set of plants
receiving no amendments will serve as a control. Preliminary findings could lead to innovative forest
restoration efforts at stand to landscape scales by providing one set of methods for enhancing Ohia’s
capacity to be resistant to ROD. Preliminary research shows that there is wide genetically driven
variability in the susceptibility of Ohia to ROD, and so this proposed work has a high likelihood of
identifying how BeGreen treatments can interact with level of susceptibility across Ohia genotypes.

Methods

Once seedlings are large enough to be tested, we will used assay based approaches of Ohia seedlings and saplings from Control, Fertilized (T16micros) and BeGreen treatments, relying on methods developed by Lisa Keith (ARS- PBARC) for testing the susceptibility of Metrosideros polymorpha to both C. lukuohia and C. huliohia. Preliminary screening has shown promising variation in the susceptibility of Ohia to ROD inoculations. This resistance testing has shown strong variation across a limited set of genotypes local to Hilo – from highly susceptible (mortality within 2 months) to apparent resistance (delayed mortality or in one case no mortality in inoculated seedlings after 8 months). We will expand screening of sampled diversity of genotypes and species for ROD resistance to include Ohia individuals treated for 12 months with fertilizer or BeGreen. We currently are working on refining nursery protocols for growing Ohia seedlings and rooted cuttings to accelerate time to inoculation. For screening across Hawaii Island, we are conducting a spatially extensive survey relying on seeds collected from diverse genotypes with representation from each district on Hawaii Island. This has involved: 1) locating, carefully tagging (without damaging), and collecting vegetative materials and/or seed from a minimum of 5 Ohia individuals per population; 2) sampling a wide diversity of populations (optimally 10) from each district; and 3) sampling all 6 districts of Hawaii Island (Kohala, Kona, Kau, Puna, Hilo and Hamakua). Collected material is being propagated at the IPIF greenhouse facilities, yielding some 6,000 plants. Seeds will be germinated and raised to seedling size sufficient for testing by Lisa Keith at the PBARC. Cuttings will be treated with rooting hormone, rooted into soil, placed into a mist chamber, established as rooted cuttings and grown until of sufficient size to be tested as above. Second, we will conduct a spatially targeted survey of survivor trees in otherwise heavily ROD impacted stands. This will involve locating and collecting both vegetative materials and seed from up to 10 Ohia individuals per ROD impact zone (>90% mortality), with the goal of sampling 20 ROD impact zones, yielding some 4,000 more plants. For this second set of targeted surveys, fertilizer and BeGreen trials will inform whether genetic basis for resistance can be enhanced with nutritional supplements, determined through inoculation based tests. To establish a control population for this process, we will rely on materials from the above randomly selected Ohia especially those adjacent to or physically as close as possible not yet ROD affected stands, anticipating not all of the above stands will represent an adjacent unaffected stand. The “survivor” trees in impacted areas are being located via aerial surveys and the exact location of sampled escape trees are being documented. For each parent tree, we are producing 10 or more rooted cuttings and 10 or more seedlings from seed to reach appropriate size (~40cm in height) for inoculation trials, organized into a randomized statistical design. We will inoculate with current protocols (e.g., C. lukuohia, C. huliohia, both Ceratocystis species, sterile inoculation) and assess accordingly. We will also consider different inoculation protocols, different ages or sizes of rooted cuttings, and different seedling sizes. This first round of inoculation testing work seeks to confirm that Ohia shows variation in resistance or susceptibility, and second whether BeGreen can confer additional resistance to seedlings exposed to ROD – above and beyond the nutritional beneftis of complete fertilization. Results will allow for follow up strategic planning on future work, with extra seed and cuttings collected for each sampled survivor tree or randomly selected control tree either stored (seed) or outplanted (cuttings). Based on the results from screening trials, BeGreen treated trees that show enhanced resistance to ROD will be included into field trials for further testing.

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