Hawaii has lost more than half of its endemic bird species, and one major factor is loss of habitat. This project examines inoculation of native tree species with an arbuscular mycorrhizal fungus as a means to improve growth in the nursery, as well as survival and growth in the field.

Contact Dr. Susan Miyasaka (miyasaka@hawaii.edu) Phone: 808-981-5180 Fax: 808-981-5190 FUNDING has been provided to CTAHR for this research from the USDA Cooperative State Research, Education, and Extension Service Project HAW00805-M.
	Koa seedlings grown at Lalamilo

OBJECTIVES

The overall objective of this project is to improve early growth and establishment of native Hawaiian forest tree species through use of beneficial arbuscular mycorrhizal fungi (AMF). Specifically, there are three objectives:

1) To determine the effect of pre-inoculation of koa with the Hawaiian strain of Glomus aggregatum in the nursery on subsequent growth and establishment in the field at Hakalau National Wildlife Refuge (NWR).

2) To isolate AMF that form effective associations with koa or other native tree species under acid soil conditions at the Hakalau NWR.

3) To determine the mycorrhizal dependence of Hawaiian tree species not previously studied, such as olapa (Cheirodendron trigynum), kawau (Ilex anomala), kolea (Myrsine lessertiana), pilo (Coprosma ochracea and C. rhynchocarpa), and naio (Myoporum sandwicense).

APPROACH

Objective 1: To determine beneficial effect of AMF on koa. A second field trial will be conducted with improved methods to ensure a greater root colonization by AMF in the greenhouse, more robust koa seedlings, a uniform N level in the nursery medium, and optimal P levels in the field for AMF activity. Koa will be grown in the nursery at Lalamilo station with two mycorrhizal treatments (control and inoculated with G. aggregatum). Seedlings will be grown for 3 to 4 months in the greenhouse at the Lalamilo station, then outplanted in a former pasture area at Hakalau NWR. Two P treatments will be applied at outplanting: 1) P will be applied at the same rate and method of application as the standard fertilization practice (13 g/tree applied in two dibble holes approximately 6 cm from tree); and 2) P will be applied at the rate determined to be optimal for mycorrhizal associations by means of a P sorption isotherm and mixed uniformly into the planting hole. Plots will consist of 40 tree seedlings spaced at approximately 3 m spacing, and the inner 16 trees will be measured. The experiment will follow a randomized complete block design with four treatments and four blocks. Measurements of basal diameter and height will be measured monthly for a year. Root samples will be collected monthly and observed for mycorrhizal colonization of roots. Pinnules will be collected monthly, and their P content will be determined. Analysis of variance will be conducted to determine significant treatment differences in growth, P content, and AMF colonization. Dr. Miyasaka will be responsible for multiplying AMF inoculum and for conducting this second field study.

Objective 2: Isolation of AMF adapted to acid soils. Native AMF from rhizosphere soils will be sampled from several healthy stands of koa and mamane at the Hakalau Forest NWR and used to inoculate koa or mamane seedlings in pot studies in the greenhouse at the UH-Manoa campus. Effectiveness of AMF will be determined by pinnule P content and growth over time of seedlings inoculated with various strains of AMF. Spores of effective AMF will be collected and multiplied using corn or sorghum as hosts. Preliminary identification of AMF species will be based on visual observations of spores. Dr. Habte will be responsible for the isolation of effective AMF strains from Hakalau Forest NWR.

Objective 3: Mycorrhizal dependence of other Hawaiian tree species. Mycorrhizal dependency will be determined on native Hawaiian tree species that have not been studied earlier, such as olapa, kawau, kolea, pilo, and naio. These tree species are planted as part of a continuing project at the Hakalau NWR. A volcanic ash soil will be sterilized and amended with P with the aid of a P sorption isotherm in order to obtain three levels of soil solution P (0, 0.02, and 0.2 mg P/L). Seedlings will be either inoculated with the Hawaiian strain of Glomus aggregatum or uninoculated (controls). Plants will be harvested and dry weights determined after approximately 3 to 6 months. Mycorrhizal dependency will be calculated. The graduate student, under the supervision of Dr. Habte, will be responsible for these studies.

5-month-old koa seedlings with frost protection devices installed (Hakalau NWR, November 2006)

Effect of Inoculation of Koa: Acacia koa seedlings were raised in the greenhouse at Lalamilo Experiment station during January to June 2006. Half the seedlings were inoculated with the effective AMF, Glomus aggregatum. In June 2006, percent colonization by AMF ranged from 50 to 80 percent in the inoculated seedlings and AMF were absent in the uninoculated seedlings. At Hakalau National Wildlife Refuge, soil in the planting hole was amended with dolomite to achieve a pH of 6.0 and phosphorus fertilizers were added for a total of 1.06 g per tree. Then koa seedlings were outplanted on 28 June 2006. Seedlings were watered manually on 29 June and 18 July 2006, due to droughty conditions. Fusilade was applied to control weedy grasses at each planting site on 14 September and 5 October 2006. At planting, the average height of uninoculated seedlings was significantly greater than that of inoculated seedlings; however, no differences between treatments were found for basal diameters. The height of inoculated seedlings increased over time to that similar to uninoculated seedlings, resulting in no significant height differences due to inoculation at 1, 2, and 3 months after planting. Similarly, there were no significant differences in basal diameters of koa due to pre-inoculation with AMF at 1, 2, and 3 months after planting. Mycorrhizal dependence of other Hawaiian tree species: Seeds have been collected of olapa, kawau, kolea, and pilo. A preliminary study is being conducted to determine whether acid scarification will improve uniformity of seed germination. Acid-scarification of kolea seed for 15 to 30 minutes resulted in greater uniformity of seed germination. We have started a pot study, examining the mycorrhizal dependence of kolea; however, this is a slow-growing seedling.

Mycorrhiza is the symbiotic association between a fungus and the roots of a host plant, in which the fungus obtains its supply of carbon compounds from the host and helps the host absorb mineral nutrients (particularly, phosphorus) via its hyphae. Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with many tropical and subtropical tree species. Inoculation of native Hawaiian plant species with AMF has been proposed as a conservation technique to improve growth and survival in the greenhouse and field. Tree seedlings raised in a nursery are particularly suitable to pre-transplant inoculation with effective AMF. This project will determine whether pre-inoculation of native Hawaiian tree seedlings with an effective AMF will increase growth in the greenhouse, as well as subsequent growth and establishment in the field, particularly under acid soil conditions.