Janice Uchida, Department of Plant & Environmental Protection Sciences, UH-CTAHR

J. B. Friday, Department of Natural Resources and Environmental Management, UH-CTAHR

Shaobin Zhong, Department of Plant & Environmental Protection Sciences, UH-CTAHR

The causes of koa wilt and decline needs to be determined. The project attempts to improve koa silvaculture and reforestation efforts by addressing the role of pathogens and their distribution.

Contact
Dr. Janice Uchida (juchida@hawaii.edu)
Phone: 808-956-2827
Fax: 808-956-2832

Click here to link to Koa Wilt Webpage.
Click here to link to Koa Pest and Disease Image Gallery.

FUNDING has been provided to CTAHR for this research from the USDA Cooperative State Research, Education, and Extension Service Project HAW00951-M.

OBJECTIVES

The soil borne pathogen, Fusarium oxysporum, has been identified as a pathogen from wilting koa by other researchers. Because it has been associated with root rots and has caused damping-off of young seedlings, the foliar phase of many trees were not extensively sampled. Recent isolations from the top of diseased trees reveals that F. oxysorum is not present in trunk and branch infections. Thus, our major goal is to conduct a disease analysis of koa from multiple sites on the diseased tree. There are also some sites that have not been evaluated and trees from those sites require evaluation. A second goal will be to evaluate the root system of healthy trees to determine if F. oxysporum is present. A third goal will be to test the pathogenicity of all isolates collected to determine which are pathogens and which are saprobes. The susceptibility of other Acacia species to F. oxysporum will be determined and if any resistant root stock is found these will be used for grafting with A. koa. A fourth goal will be to begin a molecular analysis of F. oxysporum to find specific nucleic acid fragments that can be used to develop a rapid assay of F. oxysporum in the field. We will communicate with growers and forestry leaders during the project. Dr. Friday is the extension agent for forestry and we will hold at least one workshop per year to share our finding with members of the community interested in Koa silvaculture and restoration of forests.

APPROACH

Dr. Friday will collect the samples from the Big Island. He is very familiar with previous studies and locations that have been sampled in the past. Specimens will be sent to the Agricultural Diagnostic Service Center on Oahu and then to Dr. Uchida's lab. Dr. Uchida will do all isolations and pathogen identification. Isolations will be from roots, collar, stem/trunk, branches, twigs, leaves and seeds. Water agar which allows the growth of all pathogens will be used as opposed to selective media which focus on the isolation of only certain fungi. Seeds will be collected and planted in Dr. Uchida's clean greenhouse section. These will be inoculated with the various fungi collected to establish pathogenicity. A smaller group of plants will be kept for 1-3 years and these will be inoculated to determine if larger plants are also susceptible. This has not been done for Fusarium oxysporum. All inoculated fungi will be reisolated to complete Koch's postulates. Similarly, seeds of other Acacia species will be planted and inoculated with F. oxysporum to determine susceptibility. Isolates of F. oxysporum will be given to Dr. Zhong, a molecular biologist who will begin the bioassay of this fungus. If good biomolecular markers can be found, he will use this preliminary information to get a larger grant to proceed with this study. Dr. Friday will use the information we gather to develop recommendations for the growers. We will have a hands-on workshop with disease specimens, fungal cultures and other experimental results.

A statewide survey of Koa plants was begun with diseased samples collected by Dr. J.B. Friday and sent to UH Manoa for pathogen collection. Over 200 isolation plates and over 300 pure cultures of fungi were established and 65 isolates of Fusarium, 15 Calonectria colhounii, 2 of Cylindrocladium, Geotrichum, Leptosphaerulina, Verticillium, 19 Phoma-like, 5 Coniothyrium, 14 Gliocladium, 4 ascomycetes, 4 basidiomycetes, 1-2 Topospora, Stemphylium, Lasiodiplodia-like, Urocladium, Phlyctaena, Sclerobasidium-like, Colletotrichum, and 10 dark mycelium unidentified cultures. Many Trichoderma, Aspergillus, Penicillium, yeast, Cylindrocarpon, Cladosporium, Candida, Pestalotia, Rhizopus, and Curvularia were saprophytic and discarded. Fusarium oxysporum has been associated with Koa decline. At a koa group meeting, on Dec 13, 2005, it was decided that with the limited funding available, the Fusarium isolates should be tested on koa seedlings first. Site/Summary of Fusarium and Calonectria isolations: Site 1: Oahu. Tent. ident of F. solani fr shoots; Fusarium fr roots/seed pod; Site 2: GBR farm in Laupahoehoe. 3 Fusarium. Site 3: Keauhou, Volcano area: 2 Fusarium. Site 4: Anahola and Puu Opea Rd Kokee, Kauai: F. oxysporum fr roots. Site 5: Flume Rd. Calonectria colhounii from leaves/twigs, pathogenic. Fusarium fr roots. Site 6A: Kapapala, Big Isle. Site 6B: Keanakolu, Big Island. 2 Fusarium fr roots. Site 7: Waikii, Kamuela. F. oxysporum fr roots. Site 8: Kokee, Kauai. 29 inner trunk/bark/roots with F. oxysporum. Pathogenicity: In 12, 2005, over 30 isolates of Fusarium were available for testing but severely limited by funding. Jonathan Ho, a student with Dr. Uchida did part of the work. Plants were drenched or sprayed with a spore suspension of separate Fusarium. Inoculated plants were non-wounded, kept in a glasshouse and monitored weekly. After 4-14 days, leaf wilts, stem infection, and death occurred. 4 pathogenicity tests were completed and tests are continuing. K69, K70, and K72 were pathogenic and tentatively id as F. oxysporum. Confounding factors: All seeds were collected from a single tree but genetic differences in susceptibility occurred. Every effort is made to provide a uniform environment for the plants. However, since a long time is needed per test, water was occasional insufficient and a few roots could have been damaged. Rapid growth made testing of exact aged plants difficult. Identification and Pathogenicity of Calonectria. 15 C. colhounii fr Flume Rd were isolated. This species is characterized by: cylindrical conidia, 63 to 72 micrometers X 5.7 to 5.9 micrometers in width w/3 septations, yellow-brown perithecia, w/ 4 ascospores/asci. Plants were sprayed or drenched with a spore suspension, as described above, kept in a glasshouse and monitored weekly. 24 hrs after inoculation, numerous water soaked flecks were formed on leaves, expanded to gray to light brown spots, causing defoliation by the fifth day . On phyllodes, abundant dark brown flecks developed by the second day and leaves defoliated in a 1-2 weeks. Stem developed and over 70% of plants were dead in 3 months. For larger seedlings, 38% were dead in 3 months.

Two new pathogens have been identified and confirmed on diseased koa (Acacia koa), Calonectria colhounii and Fusarium oxysporum. Koch's postulates were completed and no wounded plants were used. This is the firest report of C. colhounii on koa. Pathogenicity tests for many for potential fungal pathogens collected from diseased koa will be continued.

PUBLICATIONS (not previously reported): 2005/10 TO 2006/09

1. Uchida, J.Y., Kadooka, C, Aragaki, M and Ho, J. 2006. Calonectria colhounii causing disease of koa trees in Hawaii. American Phytopathological Society Annual Conference in Quebec, Canada. July 28, 2006. Phytopathology 96:S116 Abstract.

2. Friday, J. B. and Nelson, S. 2006. Koa disease image gallery. http://www.ctahr.hawaii.edu/nelsons/koa/koa.html