Pathogenicity of Fusarium species on Acacia koa seedlings

Fusarium isolates obtained from diseased Acacia koa Gray (Fabaceae) plants, adjacent soil, and seeds and seedpods may or may not be pathogenic on young seedlings under greenhouse conditions. This includes isolates of Fusarium oxysporum, the putative cause of koa wilt and dieback disease (“koa wilt”) in Hawai‘i. We tested 10 Fusarium isolates, made up of 4 different species (F. solani, F. subglutinans, F. oxysporum, F. semitectum), for their pathogenic potential on koa seedlings under greenhouse conditions. All tested Fusarium isolates completely colonized seedling root systems and became systemic, spreading to aboveground plant tissues (stems, branches, and leaves). Virulence was quantified on the basis of disease symptoms (mortality, wilting, foliar chlorosis, or necrosis) and effects on seedling height, stem diameter, and root volume. Results varied, ranging from nonpathogenic to high levels of virulence. Pathogenic screening of many more isolates will be necessary to identify pathogens that can be effectively used to screen families of koa for potential resistance to the koa wilt and dieback disease that is seriously affecting this important Hawaiian tree species. Dudley NS, James RL, Sniezko RA, Yeh A. 2007. Investigating koa wilt and dieback in Hawai‘i: pathogenicity of Fusarium species on Acacia koa seedlings. Native Plants Journal 8(3):259–266. K E Y W O R D S reforestation, dieback disease, Fabaceae, Fusarium oxysporum, Fusarium semitectum, Fusarium solani, Fusarium sterilihyphosum, Fusarium subglutinans N O M E N C L AT U R E Fungi: Nelson and others (1983) Insects: ITIS (2007) Plants: USDA NRCS (2007) ative koa (Acacia koa Gray [Fabaceae]) is a dominant canopy tree in many Hawaiian forest ecosystems. Koa is present on the main Hawaiian Islands, growing in moist habitats at elevations from 90 to 2100 m (295 to 6890 ft). Koa has always been a vital part of Hawaiian culture; koa wood was used for building many original Hawaiian structures, as well as being prized for seafaring canoes. This important tree species is currently playing a primary role in an expanding Hawaiian wood industry, being used for producing furniture, musical instruments, bowls, surfboards, and handicrafts. The major factor limiting establishment and maintenance of koa is a wilt disease that causes dieback that adversely affects tree survival and growth. Significant tree mortality following koa plantings due to this disease has restricted koa expansion, particularly in certain areas where disease severity is high. Koa wilt and dieback disease (“koa wilt”) is putatively caused by the fungus Fusarium oxysporum f.sp. koae, which was first described in Hawai‘i by Gardner (1980). Disease impacts and biology were subsequently investigated (Anderson and others 2002). Limited genetic analysis of several Hawaiian isolates of F. oxysporum f.sp. koae indicated potentially low genetic diversity, possibly due to recent introductions of pathogenic strains of this fungus into the state (Anderson and others 2004). If pathogenic strains were recently introduced from a source nonnative to Hawai‘i, the severe disease impacts currently occurring may be a reflection of an invasive, well-adapted pathogen. Improved techniques for detection and management of this disease are urgently needed to limit pathogen spread and to reduce disease consequences. Several other Fusarium species, particularly F. solani, are routinely isolated from diseased koa seedlings and trees (Daehler and Dudley 2002; James 2004). In addition, 13 different Fusarium spp. were recently isolated from koa seeds and seedpods (James and others 2006). Some of these organisms may be involved in disease etiology and epidemiology and require testing for their ability to induce disease. Family variation in koa field trials strongly suggests the presence of resistance to F. oxysporum f. sp. koae (Dudley 2002). Although koa wilt has significantly affected koa plantings on certain sites, some seed sources appear mostly unaffected. Therefore, genetic disease resistance likely occurs and may be widespread within some families. Frequency of resistance in natural koa populations is unknown but is expected to be low. Inoculation methods for screening potential disease resistance in seedlings are available (James and others 1989; James 1996; Anderson and others 2002). Rapid screening of many koa families would provide valuable information on resistance potential. Following laboratory and greenhouse experiments, outplanting field validation of resistance screening results will be needed (Sniezko 2003). The present work was designed to evaluate pathogenic potential of selected Fusarium isolates associated with diseased koa plants and nearby soil to help clarify their potential roles in disease etiology. Tests were conducted under controlled conditions on koa seedlings within a greenhouse. MATERIALS AND METHODS Many isolates of Fusarium were obtained during routine isolations from koa seedlings, large trees exhibiting koa wilt symptoms, and seeds and seedpods. Isolates comprised several different Fusarium species (James 2004; James and others 2006), which were determined on the basis of morphological characterization (Nelson and others 1983). Some of these species have not previously been described on koa and their potential role in disease etiology is unknown. Therefore, seedling inoculation tests were initiated to help elucidate disease potential of selected representative isolates. Ten isolates encompassing 4 different Fusarium species were evaluated for their pathogenic potential (Table 1). Fungal inoculum was prepared using the procedures of Miles and Wilcoxin (1984). Perlite, an inert, inorganic, siliceous rock of volcanic origin that is commonly used in potting mixtures, was the matrix for fungal growth. Yellow cornmeal (150 g [5.3 oz]) was moistened with 300 ml (10 fl oz) warm 1% potato dextrose agar (PDA), to which 75 g (2.6 oz) of perlite were added. The perlitecornmeal-PDA matrix was autoclaved at 121 °C (250 °F) for 60 min, cooled, inoculated with spore suspensions of test fungi, and incubated at about 24 °C (75 °F) in the dark for at least 15 d. After incubation, inoculum was dried in open Petri plates within a cabinet. Inoculum dried within 5 to 7 d and did not become contaminated with other microorganisms because the food base was completely colonized by inoculated fungal isolates. Once dry, inoculum was refrigerated until needed. Inoculum was ground to a fine powder and thoroughly mixed with a commercial peat moss and perlite growing medium (Sunshine Mix 4, Aggregate Plus, Sungro Horticulture, Bellevue, Washington) at a concentration of 1:50 (w:w). The inoculum-growing medium mixture was placed into plastic containers (“dibble tubes” 115 mm3), which had previously been sterilized by immersion in hot water (71 °C [160 °F] for 5 min). Seeds of koa from a single family were nicked at their distal end with nail clippers to break dormancy, soaked in water for about 12 h, and sown into flats containing a 50:50 (v:v) mixture of vermiculite (StaGreen Horticultural Vermiculite, St Louis, Missouri) and perlite (Redco II, North Hollywood, California). Flats were periodically watered and monitored for germination. Following germination, when radicles were approximately the same length as cotyledons, seedlings were carefully extracted from the flat and transplanted into the plastic containers containing the inoculum-growing medium I N V E S T I G AT I N G K O A W I LT A N D D I E B A C K I N H AWA I ‘ I NATIVEPLANTS | FALL 2007 260 N