Fusarium Species— a British Columbia Perspective in Forest Seedling Production

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Michael Peterson

MICHAEL PETERSON The Genus Fusarium: an Overview President and Principal Scientist Members of the genus Fusarium are among the Applied Forest Science Limited most important plant pathogens in the world. 4417 Bennett Road Fusarium spp. are a widespread cosmopolitan Victoria, British Columbia V9C 3Y3 Tel: 250.478.8358 group of fungi that commonly colonize aerial E-mail: [email protected] and subterranean plant parts, either as primary or secondary invaders. Fungi in this genus cause Peterson M. 2008. Fusarium species—a British Columbia perspective in a huge range of diseases on a wide range of host forest seedling production. In: Dumroese RK, Riley LE, technical coordinators. National Proceedings: Forest and Conservation Nursery Asso- plants. The fungus can be soil-, air-, and water- ciations—2007. Fort Collins (CO): USDA Forest Service, Rocky Moun- borne, or carried in or on plant residue or seeds, tain Research Station. Proceedings RMRS-P-57:109-125. Available at: http://www.fs.fed.us/rm/pubs/rmrs_p057.html and can be recovered from any part of a plant: roots, shoots, flowers, fruits, cones, and seeds ABSTRACT (Summerell and others 2003). This review provides a brief biological outline of some Summerell and others (2003) point out that species in the genus Fusarium and how these can be implicated as seedborne organisms leading to conifer Fusarium taxonomy has been plagued by chang- seed and seedling losses in British Columbia. Fusarium ing species concepts, with as few as 9 to over spp. are implicated with pre- and post-emergence 1,000 species being recognized by different taxon- damping-off, seedling wilt, late damping-off, root rot, omists during the past 100 years. Differing opin- and seedling mortality after outplanting. Current ions on species identification has stabilized since understanding of Fusarium spp., with regard to cone the 1980s following publications by Gerlach and and seed pest management in British Columbia, is outlined. Shortfalls that still exist and how these might be Nirenberg (1982) and Nelson and others (1983) addressed with the development of a vision for better who defined widely accepted morphological understanding this group of fungi and a mission state- species concepts. Since that time, however, the ment of how this might be achieved are presented. application of biological (Leslie 2001) and phylo- genetic (Nirenberg and O’Donnell 1998) species KEYWORDS concepts to new, as well as existing, strain collec- diseases, pest management, tions has resulted in further splitting of many of seedborne diseases, damping-off the previously described species. If changing

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these taxonomic designations were only rare, or duce sharply pointed macroconidia, while others of limited economic importance, they could be produce spores with rounder ends. The shapes of viewed as being merely pedantic. However, many these spores are used to differentiate morpholog- of these species can be important. For example, F. ically between species (Toussoun and Nelson andiyazi and F. thapsinum are major pathogens of 1968). Most Fusarium produce their macroconi- sorghum that differ from one another but had dia on sporodochia, which are cushion-shaped previously been grouped as F. moniliforme (Leslie fruiting structures covered with conidiophores 2001; Marasas and others 2001). Further descrip- (simple or branched hyphae bearing conidia) tion of taxonomy is well beyond the scope of this (Figures 1 and 2). Macro-conidia can also be note. However, its complexity and the recognized found, however, throughout the aerial mycelium. difficulty of rapidly identifying cultures to species Microconidia are 1- or 2-celled, ovoid or oblong, (Summerell and others 2003) has meant that and borne singly or in chains. These spores are research and development of cone and seed pest found scattered throughout the aerial mycelium. management associated with Fusarium spp. in The 1- or 2-celled microconidia are usually British Columbia (BC) has generally been limited smaller than the macroconidia. Macroconida and to genus. microconidia are produced from phialides (a A taxonomic treatment for Fusarium is pre- type of conidiogenous cell). Chlamydospores are sented for completeness: round, 1- or 2-celled, thick-walled spores pro- kingdom: Mycetae (fungi); division: Eumy- duced terminally or intercalary on older myceli- cota; subdivision: Deuteromycotina (the imper- um (Agrios 1988). Chlamydospores generally fect fungi); class: Hyphomycetes; order: function as resting spores, having the ability to Hyphales (Moniliales); genus: Fusarium. survive adverse conditions and enable the fungus Fusarium spp. are grouped in the subdivision to regenerate when favorable conditions for Deuteromycotina, which encompasses the imper- growth are reencountered. This is illustrated by a fect (asexual) fungi. Nelson and others (1983) disease triangle (Figure 3). In the presence of a point out that the perfect (sexual) states of Fusar- suitable host (for example, seedling) and ium are generally unfamiliar to many people pathogen (for example, Fusarium chlamy- working with these fungi. Plant pathologists most dospore), disease of the host will progress when often deal with the imperfect states, as the perfect the environment favors spore germination and states often have little to do with the disease prob- infection over time. lem under study. Some of the most successful Fusarium, for example, F. oxysporum and F. cul- Disease Cycle morum, appear to have lost their sexual ability Fusarium are soil inhabitants that overwinter and have adopted other methods of facilitating between crops in infected plant debris as mycelia genetic adaptations (Booth 1981). and in 3 spore forms. As chlamydospores, Fusari- um can remain in the soil for long time periods. General Characteristics Mycelium can infect healthy plant tissue in the Due to the great variability within this genus, it is same manner as spores do. Healthy plants can one of the most difficult of all fungal groups to become infected through their root tips; either distinguish taxonomically (Alexopoulus and directly, through wounds, or at the point of for- Mims 1979). Conidia (asexual spores) are hyaline mation of lateral roots (Agrios 1988). The fungus and can be divided into 3 groups: macroconidia, can grow as mycelium through the root cortex microconidia, and chlamydospores. Macroconi- intercellularly, ultimately advancing to the vascu- dia are several-celled, crescent or canoe-shaped lar tissue. As the mycelium continues to grow, spores. Their ends vary in that some species pro- usually up toward, and into the stem, it branches

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Figure 1. Structure of some fungi associated with disease (fungi imperfecti).

and produces microconida. The proliferation of growing containers, or within attached extrane- fungal growth in a plant’s vascular tissue can ous root fragments. eventually cause the plant to wilt and die. Conifer Forest seedling nurseries represent artificial seedlings are especially susceptible to this patho- growing environments. In BC, seeds are sown in genic modality when subjected to drought stress soilless, peat-based growing media in Styro- and high transpirational demands. The fungus foam™ (Styroblock™) containers. Styro-block™ can continue to grow on the decaying tissue, containers generally reside on benches over con- where it can sporulate profusely, visibly present- crete or gravel. Seed germination and the early ing salmon to coral-pink colored sporo-dochia part of the growing cycle take place in polyethyl- on the lower portion of seedling stems. At this ene covered greenhouses where temperature, point, the spores can be spread to other plants or light, and moisture are closely controlled, and areas by wind, water, or through the movement of nutrients are applied through overhead irrigation. seedlings themselves (Agrios 1988). Fusarium are considered natural soil inhabitants and readily isolated from agricultural and Types of Disease forest soils. Understanding Fusarium-caused dis- In addition to vascular wilting, Fusarium can eases in forest seedling container nurseries, how- infect other plant parts close to the soil to induce ever, requires the recognition that in this growing root and stem rots. When seeds become contam- environment, Fusarium are introduced pests. inated or seedlings are infected with Fusarium, They are introduced to the container nursery via damping-off may occur. The Fusarium that cause seeds, water, and wind, or on old containers or vascular wilts can be spread in soil, dust, and irri- dirty equipment. Fusarium can lead to seed and gation water. Wind, rain, nursery equipment, and seedling losses in several ways: decaying plant tissue can also help to spread the fungus. Additionally, Fusarium can enter nurs- 1) seedborne contamination; eries as seed contaminants, be carried over from 2) pre- and post-emergence damping-off; previous years within surface cracks on dirty 3) seedling wilt;

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4) late damping-off; 5) seedling root rot; 6) seedling mortality after outplanting.

Seedborne Contamination Seedborne fungi are defined as those “that are dispersed in association with some kind of dispersal units of the host (that is, seeds)” (Ingold 1953). This definition includes all seed types and all associated microfungi, and is the one adopted with reference to conifer seedborne fungi in BC. Some authors classify fungi as being either seedborne or seed-transmitted (Thomsen and Figure 2. Fusarium culmorum showing sporodochia, macro- Schmidt 1999). They define seedborne fungi to conidia, and conidiophores. include all fungal types contaminating the surface of seeds or infecting seed tissues. Seed-transmitted fungi are those that cause no infection of a seed itself, but infect seedlings in the nursery or field (Neergaard 1979). It must be remembered that not all seedborne fungi are pathogenic, and they may include symbionts actually beneficial to the plant (Mallone and Muskett 1997). With regard to seed transmission of fusaria, we are more interested in it as a seedborne pathogen than a seedborne disease. Seedborne pathogens (as opposed to diseases) are defined here as organisms, whether on or in seeds, which may or Figure 3. Disease triangle indicates 3 conditions that must may not cause infections and symptoms on the be maintained over time for any disease to progress. seeds. Some seedborne pathogens may actively infect seeds, and may or may not cause symptoms by an organism followed by the establishment of on the seeds. Seedborne pathogens associated a relationship (that is, saprophytic or parasitic) with conifer seeds may inhabit the external or within the seeds. Once established, such a rela- internal tissues of seeds. Seedborne diseases tionship can give rise to outward hyphal growth occur on seedlings as a result of pathogens car- from within the seeds, which becomes apparent ried in or on the seeds. Evidence shows that upon penetrating the seed surface. While this Fusarium rarely exist within conifer seeds (Peter- hyphal growth can appear as a contaminant, it is son 2007). indicative of the presence of an infection deeper Seeds harbouring fungi can be described as within the seeds. In certain situations, it is possi- being either contaminated or infected. Contami- ble to disinfest seeds that are only superficially nation is used to denote the occurrence of a contaminated. Surface disinfestations of infected pathogen as either spores or mycelium on the seeds are of little value, as an internal relationship surface of seeds. Contamination may be entirely between the seeds and fungi will still exist. One of superficial, where spores or mycelium are usually the easiest ways to eliminate or reduce seedborne retained in small cracks or fissures in the seed- contamination is through the use of running coat. Infection refers to the penetration of seeds water during imbibition, followed by a post-strat-

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ification rinse with running water (Kolotelo and fined to individually contaminated seeds. Symp- others 2001). Disinfestation in this manner can toms of post-emergence damping-off include reduce the incidence of seedborne Fusarium by stem rotting at the groundline and subsequent reducing what has previously been observed as toppling of the seedling shoot. Post-emergence the tendency for contamination to actually damping-off results in damage and loss of infect- increase during stratification. ed germinants after the stems rot. However, the Seedborne contamination may occur through disease can also spread by spores produced on the indirect routes, such as via cone parts to the ovary infected stems, which can then infect adjacent and ovule tissues, or through direct routes when seedlings and cause further losses. seeds contact contaminated soil and water. Dirty equipment in a processing facility may also con- Seedling Wilt taminate seeds during interim storage, process- Conifer seedlings, especially Douglas-fir ing, or seed treatment for stratification. As spores (Pseudotsuga menziesii), are susceptible to seedling can be released throughout the year, at almost any wilt caused by Fusarium when fungal growth pro- time in the general lifecycle of major BC conifer liferates in the plant’s vascular tissue. This condi- seedlings, seeds are exposed to contamination tion is often encountered when cool and overcast over a wide range of conditions. Examination of weather in the late spring or early summer is fol- tree seed samples from over 2600 seedlots stored lowed by a sudden clear warming trend. Seedlings at the BC Ministry of Forests and Range (MoFR) that may otherwise have been tolerating a compro- Tree Seed Centre has indicated the frequency of mised vascular system are then subjected to seedborne Fusarium to be the same on seeds orig- drought stress induced by sudden high transpira- inating from seed orchards and those taken from tional demands. The avoidance or reversal of these natural stands (Peterson 2000). Spores freed from conditions (for example, increased irrigation) may soil or grasses within and around seed orchards either prevent or reverse the symptoms and mini- may be spread by irrigation sprinklers. This could mize any subsequent damage and loss. be exacerbated by the use of sprinklers to control pollination in the spring. Indirect contamination Late Damping-off through cone parts to the ovary and ovule tissues Late damping-off, also sometimes called Fusar- such as this could similarly occur in wild stands ium top blight, is often a progression from the via rainfall. Fungal inoculum (for example, intensification of seedling wilt. Symptoms include spores) reaching maturing cones on trees is needle chlorosis, browning, and desiccation with a thought to be one way that seeds can become hook or crooked-shaped leader tip. Fusarium top contaminated and Fusarium become seedborne. blight following wilt damage will not necessarily Seeds and cone parts harbouring the fungi can lead to seedling losses if the trees are promptly contaminate processing facility equipment, con- treated with a systemic fungicide. When seedling tributing to further contamination of otherwise death results, that is, late damping-off, the fungus clean seeds. Regardless of the initial source, seed- may continue to grow on the decaying tissue where borne Fusarium can intensify throughout a con- it can sporulate profusely, visibly presenting taminated seedlot during seed stratification. salmon to coral-pink colored sporodochia on the lower portion of seedling stems. Pre- and Post-Emergence Damping-off Pre-emergence damping-off is characterized Seedling Root Rot by seeds failing to germinate, or rotting of emerg- The symptoms of seedling wilt and late damping shoots or radicals with the associated seedling ing-off can also be indicative of Fusarium root losses. Damage and losses here are usually con- rot, which is further characterized by blackened,

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thin and wispy roots with little sign of actively spruce (Picea sitchensis), yellow cedar (Chamae- growing root tips. The root cortex often easily cyparis nootkatensis), noble fir (Abies procera), strips away, leaving an exposed root stele. Fusarium amabilis fir (Abies amabilis), interior spruce root rot does not necessarily lead to seedling losses (Picea glauca and P. engelmannii, mountain hem- if the damage to the root system is limited. Root rot lock (Tsuga mertensiana), and interior lodgepole often occurs later in the growing season or can also pine (Pinus contorta var latifolia) (Kolotelo and occur if seedlings with infected roots are mishan- others 2001). dled after leaving cold storage. Fusaria are natural It was indicated earlier that, within the context rhizosphere inhabitants, and healthy, unstressed of the forest conifer container nursery system, seedlings can survive well in their presence. The Fusarium can be viewed as introduced pests. avoidance of stresses to the plants will limit damage Species of Fusarium that are part of this disease and losses caused by Fusarium root rot. complex can be introduced via air, water, on greenhouse equipment, on contaminated plant Seedling Mortality after Outplanting parts, as well as on seeds. Research and develop- Fusarium are commonly found on conifer ment of cone and seed pest management prac- seedling roots and in the root zone throughout the tices to reduce the negative effects of Fusarium on growing media plug. In BC, the presence of Fusar- conifer seedling production have focused on all ium on seedling roots in the absence of any disease of the previously discussed aspects of this disease symptoms is generally not sufficient grounds to complex. Each of these areas of investigation is reject seedlings scheduled for outplanting. Howev- summarized here. er, as seedlings commonly have Fusarium on or around their roots, it is important that proper Seedborne Contamination handling care is taken so that any fungi present do Initial Contamination not become aggressively pathogenic. Seedlings Several species of Fusarium, that is, F. sporotri- scheduled for outplanting must never be allowed choides, F. acuminatum, F. avenaceum and F. cul- to remain in boxes or in conditions where they can morum have been isolated from Douglas-fir seeds become overheated and the roots remain warm (Mallams 2004). Fusarium solani and F. oxyspo- and moist for prolonged periods. Under such con- rum are 2 other Fusarium species that Mallams ditions, Fusarium can rapidly spread from seedling (2004) notes have been isolated from diseased to seedling, as well as intensify within the roots of seedlings in fields at the J Herbert Stone Nursery, infected seedlings. When outplanted following Central Point, Oregon. However, as she did not these conditions, seedlings can quickly succumb to isolate these species from seeds, Mallams (2004) planting shock and, if exposed to a subsequent suggested that these infections occurred during heat or drought stress, will often die. or after sowing. Although F. acuminatum and F. avenaceum Cone and Seed Pest Management: Research to Date commonly colonize conifer seeds, James (2000) Tree species occurring in BC that are affected by found most F. acuminatum isolates he studied seedborne Fusarium, in decreasing order of fre- were not pathogenic to Douglas-fir. Other studies quency as indicated from fungal assays, include: by James (1985a, 1993) found F. acuminatum and Douglas-fir (Pseudotsuga menziesii var. menziesii), F. avenaceum both associated with pre- and post- western larch (Larix occidentalis), western white emergence damping-off of conifers, and he sug- pine (Pinus monticola), western redcedar (Thuja gests they were the result of seedborne inocula. plicata), ponderosa pine (Pinus ponderosa), grand Several different species of Fusarium can cause fir (Abies grandis), western hemlock (Tsuga het- root rot of container seedlings, with the major erophylla), subalpine fir (Abies lasiocarpa), sitka source of inocula being the seeds (Landis and

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others 1990). Seedborne Fusarium are usually mother plant (Baker 1948). Fusarium moniliforme, responsible for pre-emergence damping-off, but F. oxysporum, and F. scirpi have been isolated from can also lead to post-emergence damping-off as vascular bundles from all parts of cotton plants, well as Fusarium root rot and shoot blight, in this including bolls and seeds (Rudolph and Harris order of importance. 1945). Fusarium moniliforme has been shown to A sound understanding of 2 important seed- invade corn seeds through vascular tissues of the borne fungi, Caloscypha fulgens and Sirococcus stalk (Kingsland and Wernham 1962), while sys- conigenus, has led to management guidelines to temic infection in sweet corn plants by F. monili- reduce their incidence on seeds, thus lowering forme and F. oxysporum has been shown to occur outplant mortality attributable to their occur- with hyphae of each species growing in intercellu- rence. A similar understanding of infection lar spaces (Lawrence and others 1981). Mycelia of routes for seedborne Fusarium does not exist, and F. oxysporum f. sp. carthami have been observed in establishing this remains an essential first step to receptacles of safflower heads where hyphae tra- developing guidelines for reducing its incidence. versed through the abscission zone of the cypsil As a seedborne contaminant (that is, carried on and were associated with, but not limited to, seeds) or seed infection, Fusarium can attack the xylem (Klisiewicz 1963). Finally, mycelium roots and be implicated as a wilt following out- has been observed to be inter- and intracellular, planting. Observations during the winter of and also seen in the vascular elements of the 2003/04 by Applied Forest Science Limited (AFS), seedcoat and cotyledons in seeds of Fabaceae as part of their seedling diagnostic and adjudica- (Sharma 1992). tion services for the BC MoFR and private forest Littke (1996) speculated that seed association companies, indicate that Fusarium have the abili- with this pathogen originates from aerial deposi- ty to grow systemically within the vascular system tion on developing cones. He deduced that likely of 2-year-old seedlings (Peterson 2004a). As a routes of subsequent seed contamination exist as seedborne disease (that is, actively attacking a physical transfer from exterior cone parts seeds), Fusarium can be responsible for pre- (bracts and scales) to seedcoat surfaces during emergence damping-off where seeds fail to ger- seed development. AFS Limited routinely isolates minate. To function as a seedborne contaminant Fusarium from seed surfaces during fungal assay as well as a disease suggests more than one infec- testing for the BC MoFR Tree Seed Centre, con- tion route. Direct infection of angiosperm seeds firming that Fusarium inoculum exists on the occurs as systemic invasion via mother plant tis- seedcoats of many conifer species. Littke’s sues to the seed embryo, whereas indirect infec- hypothesis requires airborne Fusarium spore tion and contamination can occur via the stigma inoculum to be present in the vicinity of receptive to the seed embryo or via the flower/fruit to parts cones during pollination. Data collected by AFS of the ovary and ovule tissues (Maude 1996). Limited, as one of 24 locations across Canada over Direct infection via mother plant tissues is com- the past 10 years under the auspices of the Weath- mon for biotrophic fungi, which are parasitic in er Network/MetroMedia and calculated by Aero- nature and dependent upon the survival of their biology Research Laboratories, Ontario, showed host. Maude (1996) states, however, that this form airborne spore densities in the Victoria region to of seed infection is less likely to occur within nec- closely mimic those of conifer pollen density dur- totrophic fungi which degrade tissues as they ing the spring of 2003 (Peterson 2003). That air- advance, with the exception of wilt fungi, includ- borne spore inoculum can occur during times of ing Fusarium, which invade vascular tissue. As a pollination in the vicinity of Douglas-fir seed wilt fungus invading vascular tissues, F. oxysporum orchards supports Littke and could explain the has been shown to infect seeds via the xylem of the presence of inoculum on seed surfaces.

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Research by Peterson (2007) indicates that be a concern. Their observations indicated trends seedborne Fusarium on several conifer species only that were difficult to substantiate statistical- does not likely occur as internal infections, but is ly, indicating this to be an area in need of further limited to seedcoat contamination. These obser- investigation. vations appear to indicate that seedborne fungi in The potential for conifer seeds to become con- some conifer species do not occur systemically, taminated with Fusarium makes testing for its and more likely occurs following Littke’s (1996) presence a viable first step for managing it as a hypothesis. How seeds become initially contami- seedborne organism, thus allowing specific seed- nated remains uncertain, and more research is lots to be targeted for special treatment. The abil- needed to precisely define how and when this ity to spread or intensify within a seedlot, the fact occurs prior to developing pest management that some tree species are more susceptible to the guidelines to minimize this occurrence. effects of the fungi, as well as the fact that some species represent a higher potential monetary Spread and Intensification of Initial loss, are also reasons that seeds are tested for con- Seed Contamination tamination. A matrix established by the BC Regardless of the initial source, infested seed- MoFR Tree Seed Centre outlining the seed fungal lots can potentially cross contaminate those that testing priorities for the 3 important seedborne are uninfested, as well as intensify within them- fungi in BC has been developed. The priorities selves during imbibition and stratification. Cross- for Fusarium testing are such that subalpine fir, contamination between seedlots can occur wher- coastal Douglas-fir, western larch, western white ever mutual seedlot contact exists through shared pine, and ponderosa pine are all rated high; ama- seed handling equipment. This can occur when bilis fir, grand fir, western redcedar, interior Dou- unsanitized cone sacks are reused between cone glas-fir, western hemlock, Sitka spruce, interior harvests, or inoculum can potentially be trans- spruce, and Sitka x interior spruce hybrid are ferred from seed handling equipment during var- rated medium; and coastal and interior lodgepole ious stages of the seed extraction process. Kolo- pine, and yellow cedar are rated low priority telo and Peterson (2006) found some trends with (Kolotelo and others 2001). regard to the presence of Fusarium on cones, Past sampling has indicated average levels of debris, and seeds at various stages during cone and Fusarium on seeds to be typically less than 2.5%, seed processing. They found processing stages with a moderate degree of variation within seed- incorporating agitation to indicate increases in lots (Kolotelo and others 2001). Not all species of contamination. Kilning appeared to decrease con- Fusarium are pathogenic; those that are patho- tamination on seed and cone scales despite peak genic are often weakly so. In addition, past stud- kilning temperatures of 40 °C (104 °F) not totally ies to detect seedborne Fusarium in BC were eliminating the fungal contaminant. Their overall often limited to genus. Thus, when routine fungal finding was that initial seed contamination levels assays of seeds in BC were adopted, it was elected may not be indicative of final seedlot contamina- to detect levels within any seedlot at a relatively tion. This was emphasized by the fact that, conservative level of 5%. To detect levels of 5% despite BC interior Douglas-fir having a very low with a 95% degree of confidence requires a sam- incidence of seedborne Fusarium, some of the ple size of 500 seeds per seedlot for each seedlot associated debris had significant amounts of con- tested. Samples are not adjusted for seedlot size, tamination. Also, although initial levels on cone but sampling intensity is adjusted according to scales, seeds, and debris in some seedlots were the ISTA (1999) standards. The laboratory meth- high to moderate, the final contamination levels ods used to test seedborne Fusarium are outlined were very low and below what are considered to in Peterson (2007), and testing for its presence

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provides useful information for nursery growers. and do not readily infect the seed interior during The results of fungal assays are available for storage. Infection of an emerging shoot and/or each seedlot tested on the Seed Planning and Reg- radical may occur in the germination phase, but istry Information System (SPAR), in seedlot Kolotelo and others (2001) point out that early detail reports from SPAR, as well as on the sowing stages of seed colonization are primarily depend- request label sent to growers with each batch of ent on abiotic factors, such as environmental seeds. Knowing the percentage of contaminated water availability and temperature, rather than seeds within a seedlot provides growers, as well as seed moisture content. Strategies to reduce the others handling the seeds, with the option of tak- exposure of contaminated seeds to environmen- ing steps to minimize their impact on seedling tal conditions conducive to fungal growth can germination and growth. Historical records indi- help prevent any intensification of seedborne cate contamination levels of greater than 5% Fusarium within a seedlot. within any seedlot to be significant for disease Three strategies to minimize losses from seed- potential, and growers target seedlots with levels borne pathogens are: 1) eliminating or reducing higher than this. The main strategy for levels initial inoculum; 2) slowing the rate of pathogen above this are aimed at minimizing its ability to spread; and 3) shortening the time seeds are spread within a seedlot. exposed to the pathogen (Berger 1977). It is valu- Seed orchard seeds appear to be affected by able to view these strategies in the context of a Fusarium at the same rate as seeds collected from disease triangle with the seeds as host, Fusarium natural stands (Peterson 2000). Current knowl- the pathogen, and seed handling (from cone col- edge still does not provide a clear understanding lection, through extraction, storage, sowing, ger- of how cones become contaminated. More con- mination, and seedcoat loss) as the environment, trol is available, however, when collecting in seed representing each corner, respectively. Sanitation orchards compared to natural stands, and Kolote- encompasses cone collection procedures, seed lo and others (2001) point out 3 things that can orchard management and seed processing, falling be done when making these collections to pre- into Berger’s (1977) first category. Kolotelo and vent further spread of the fungus. First, cones others (2001) relate seed treatments and storage should be collected during dry weather whenever to the second, and stratification and germination possible. Second, cones should be stored in new, procedures to the third category. The first catego- or steam- or hot water-sterilized sacks to prevent ry is quite well understood, as presented by contamination from previous year’s collections. Eremko and others (1989), Leadem and others Informal investigations conducted by AFS Limit- (1990), and Portlock (1996). Some questions still ed for the BC MoFR have shown that cone sacks remain, however, with regard to when Fusarium can become contaminated, and the sacks them- become seedborne on seeds produced in orchards selves, especially when wet, will readily trap air- (Peterson 2007). Kolotelo and others (2001) pres- borne inoculum (Peterson 2004b). Third, filled ent a good summary of collection methods to sacks should be stored following the general rec- minimize seedborne disease, as well as discus- ommendations for all species described in Port- sions of seed processing. Initial research toward lock (1996). the potential for seedborne Fusarium to spread The ability of Fusarium-contaminated seeds to during seed processing has been started, but intensify within seedlots during imbibition and some questions still remain in this area (Kolotelo stratification, as well as the management prac- and Peterson 2006). Long term seed storage in BC tices to reduce this phenomenon, are extensively generally takes place at -18 °C (0 °F), and between reviewed in Kolotelo and others (2001). Seed- 4.9% and 9.9% moisture content, neither meas- borne Fusarium primarily exist as contaminants ure being conducive to fungal growth. Therefore

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storage itself does not present a significant threat ontagne and Wang 1976; Wenny and Dumroese to spread or intensification. 1987). Research in BC with regard to cone and seed The use of running water to imbibe seeds fol- pest management and seedborne Fusarium with- lowed by a post-stratification running water rinse in Berger’s (1977) third category to minimize loss is the simplest strategy to reduce the intensifica- to seedborne disease, that is, stratification and tion of seedborne contamination (Kolotelo and germination, has concentrated on treating tested others 2001). Running water treatments appear to seedlots having significant contamination by reduce the incidence of post-stratification seed- cleaning seed surfaces. Most research in this area borne Fusarium (James 1985b; Dumroese and has aimed at reducing seedcoat infestations others 1988; Axelrood and others 1995). The (James 1985b; Axelrood and others 1995). And method used at the BC MoFR Tree Seed Centre is the importance of this research is emphasized by to imbibe seeds in mesh bags in a tank of running the findings that infestation levels of can increase water for 24 to 48 hours. However, this requires significantly during seed imbibition and stratifi- significant water resources. Kolotelo and others cation (Axelrood and others 1995; Hoefnagels (2001) suggest complete water changes every 4 to and Linderman 1999). Likewise, dry seed levels 8 hours will have similar effects. This is likely due (< 1%) of Fusarium, below what is considered a to the response noted by Neumann (1996), that potential disease threat (> 5%), can substantially the first 6 hours of imbibition is critical for what increase during stratification to as high as 10% she termed “bulking up” of seedborne Fusarium. (Neumann 1996). Neumann investigated poten- Seed imbibition at the BC MoFR Tree Seed Centre tial external sources that may have contributed to generally involves soaking several sowing requests these increases, for example, airborne inoculum of differing seedlots and conifer species in the in the drying room and the soaking mesh and/or same tanks for a running water soak. However, tanks. It was ultimately deemed, however, that as Neumann (1995) identified a potential for cross- the observed “bulking up” of seedborne Fusarium contamination between low- and highly-infested occurred during the first 6 hours of imbibition, a Douglas-fir and western larch seedlots when faster water flow over the seeds during this time soaked together, and these are now soaked in indi- might be a simple cultural control to prevent this vidual tanks at the BC MoFR Tree Seed Centre. escalation. Further studies by Neumann (1997) Very effective chemical seed sanitation can be concluded that simple cleaning of soaking tanks achieved using hydrogen peroxide. Differences in between seedlots could reduce inoculum and the concentration and treatment duration, stratifica- potential for cross-contamination. tion timing, and conifer species tolerance exist, Applying fungicides directly to seedcoats to and an excellent summary of hydrogen peroxide control seedborne Fusarium has been investigat- seed treatment is presented in tabular form in ed. However, it is difficult to find fungicides that Kolotelo and others (2001). It is worth noting meet the many requirements necessary for their that for 12 conifer species, 4 hydrogen peroxide safe and effective application (Bennett and others concentrations, up to 10 exposure durations, and 1991). These range from being suitably effica- for both pre- and post-stratification treatments, cious under different climatic conditions, being no reductions in germination are indicated and non-phytotoxic, being residue-free, as well as neither were any increases in fungal contamina- non-toxic to humans and wildlife. Earlier tion. In BC, the recommended hydrogen peroxide research findings of the negative effects of fungi- technique is to treat post-stratification seeds by cides on seed germination, as well as variable effi- immersing them in a 3% hydrogen peroxide solu- cacy and handling difficulties have all led to their tion, at 3:1 solution to seed volume ratio, for 30 reduced usage (Lock and Sutherland 1975; Lam- minutes to 4 hours followed by a running water

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rinse. The potential for reducing fungal levels on Greenhouse sanitation, including floors, seeds with hydrogen peroxide clearly exists. How- benching, pallets, and Styroblock™ containers, ever, some Abies species do not respond consis- will all reduce levels of inoculum in the immedi- tently, and for this reason Kolotelo and others ate vicinity of germinating seeds. However, these (2001) suggest more research and operational strategies are targeted more to reduce risk to studies are needed to address this. seedlings in the post-emergence environment. Aside from employing the strategies outlined in Pre- and Post-emergence Damping-off the previous section to reduce the ability for Pre-emergence Damping-off Fusarium to intensify on contaminated seeds Seedborne Fusarium are most often responsible during imbibition and stratification, some other for pre-emergence damping-off, that is, seeds that methods can be employed to reduce pre-emer- become infected and fail to germinate. Technically, gence damping-off. For seedlots with a greater the seed contents do not become infected prior to than 5% incidence of contamination, it is recom- their being exposed to the environmental condi- mended that greenhouse temperatures be opti- tions of moisture and temperature that allow seed- mized to encourage rapid germination. This will coat inoculum to germinate. If these conditions often reduce the incidence of pre-emergence are present while actual seed germination is slow damping-off and promote rapid shedding of the to initiate, the seed contents may become infected seedcoat. and rot. However, what commonly happens below ground is that the beginnings of a radical and Post-emergence Damping-off shoot will emerge and can become infected by any Young germinant or seedling root infections, seedcoat inoculum present, with the result that no resulting from roots growing in close proximity sign of a germinating seedling will appear above to germinating chlamydospores, can lead to stem the soil line. Pre-emergence damping-off refers to rotting at the groundline, which typifies post- both of the above situations. Given the appropriate emergence damping-off. Seedborne Fusarium conditions, pre-emergence damping-off might still can also be responsible for post-emergence occur in the absence of seedborne Fusarium. This damping-off when the seeds germinate. However, can happen if sufficient inoculum is present in the for reasons such as a slow-to-shed seedcoat, for growing media, most often encountered when example, inoculum contacting and infecting the dirty growing containers carry over inoculum emergent tissues will often cause the new shoot to from the previous year. Neumann (1993) did not rot at the goundline. Young germinants rotting at find planting mix or water to be a source of inocu- the goundline and breaking or falling over at this lum in a 2-year study of seedborne Fusarium and point typify symptoms of post-emergence damp- root colonization of container-grown Douglas-fir, ing-off. The strategy of encouraging rapid loss of but she did suggest that other sources of inoculum the seedcoat will reduce the time any contamina- likely came from wooden pallets. Axelrood and tion on the seedcoat surface is likely to be in con- Peters (1993) found 50% of the cavities in opera- tact with the germinating needles and stem, and tionally sanitized Styro-block™ containers to con- can reduce losses here. It is also important during tain infested root fragments. They also found 60% this growth phase to irrigate early in the day to of the growing cavities to be contaminated with encourage rapid drying of seedling foliage, which Fusarium on their surfaces. Fusarium have also will also help reduce the spread of spore inoculum. been found on the wooden pallets used to support Sanitation of older Styroblock™ containers can growing containers, as well as on plant debris significantly extend the useful life of these growing beneath these pallets (Neumann and Axelrood containers by reducing pathogen inoculum associ- 1992). ated with old rough surfaces and associated extra-

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neous root material from past years use. Peterson The potential for beneficial soil or growing (1990) achieved significant reductions in levels on media amendment should be mentioned here, as old Styroblock™ containers using a variety of san- the use of artificial media is particularly suited to itation techniques, and developed these into a set this. Suppressive growing media can be created by of practical guidelines for the sanitation of nursery introducing beneficial organisms, or by using seedling containers using either heat or chemical media components that suppress disease organ- methods (Peterson 1991). The adoption of many isms. An excellent review of this technology is of these guidelines is commonly used to extend presented by Linderman (1986). One soil-inhab- container life while reducing the presence of iting fungus, that is, Trichoderma harzianum,is inoculum in the container seedling root zone. actively antagonistic to Fusarium, as it competes with the pathogen for substrate. T. harzianum is Seedling Wilt the active ingredient in the biological fungicide Following germination and subsequent RootShield®, and it is best applied as a green- seedling growth, it is important to reduce stress on house and nursery soil amendment early in the the plants. Seedlings can tolerate low levels of growing season. Fusarium on their roots. However, heat or drought Seedling wilt caused by Fusarium usually only stress can impair the seedling’s ability to transport affects young germinants. Resistance of conifers water and nutrients, especially if fungi have to wilt diseases develops with ageing, and plant entered the roots and xylem tissues. Seedlings that resistance to wilt pathogens is known to depend continue to grow and become infected by either on the synthesis rate of phenolics, with free and chlamydospores, introduced air- or water-borne bound phenolics preventing or retarding disease spore inoculum, or from infected root fragments development. Shein and others (2003) treated or dirty container surfaces, can be influenced by Scots pine (Pinus sylvestris) seedlings with viru- heat or drought stress leading to top blight or wilt- lent spore suspensions of F. sporotrichiella and ing. Top blight or wilt, also sometimes called late deduced conifer seedling resistance to wilt dis- damping-off, often shows symptoms of needle eases to be correlated with the synthesis rate and chlorosis, browning, and desiccation with a hook accumulation of insoluble phenolic polymers. or crooked-shaped leader tip. The ability to resist wilt was higher in plants Top blight and wilt damage will not necessari- unable to synthesize these polymers as they accu- ly lead to seedling losses if the trees are promptly mulated with age. treated with a systemic fungicide. In Canada, Sen- ator® 70WP is registered for use on container Late Damping-off greenhouse conifers for controlling Fusarium, If left unchecked, seedling wilt can progress as and can be applied at 14-day intervals to provide foliage becomes chlorotic to brown, severe needle systemic control. Also, as this damage is often ini- necrosis occurs, needles drop, and the seedling tiated by a sudden heat or drought stress in the dies. Seedlings at this point usually become presence of the pathogen, the avoidance or rever- crooked at the leader and appear to die from the sal of these conditions can either prevent or tip down. Little can be done to reverse the disease reverse the symptoms and minimize any subse- at this point. It is important, however, to remove quent damage and loss. Sutherland and others affected seedlings, as they can lead to increased (1989) point out that Fusarium can enter seedling spore inoculum in the greenhouse. Left in con- roots early in a growing season, with disease tainers, infected seedlings often develop salmon- development being delayed until the seedlings pink sporodocia that produce and release conidia become stressed for moisture and nutrients. that can be splashed from irrigation water to infect adjacent seedlings. Not only is it important

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to remove any infected seedlings at this stage, but rot often occurs later in the growing season, or can attention must be paid to seedling growth. also occur if seedlings with infected roots are mis- Seedlings usually present the above described handled after leaving cold storage. Fusarium root symptoms during their rapid growth phase. This rot does not necessarily lead to seedling losses if is characterized by accelerated tissue growth and the damage to the root system is limited. expansion, and is generally considered the time when seedlings are most succulent and suscepti- Seedling Mortality after Outplanting ble to infection. Dennis and Trotter (1995) point Seedlings with minor amounts of Fusarium on out that environmental and cultural manipula- their root surfaces, or low levels of root infection, tion during the rapid growth phase must concen- often readily survive being outplanted when han- trate on providing the seedling with select grow- dled properly and not exposed to severe planting ing conditions in order to accentuate its growth shock. However, when infested seedlings remain in potential. They also emphasize that seedling envi- storage or shipping containers under warm, moist ronment and culture have a significant impact on conditions for extended periods prior to planting, whether disease develops or not, pointing out the disease can rapidly develop into root rot, that disease-causing fungi can infect seedlings at severely jeopardizing seedling survival. In British an early stage of development, and then remain Columbia, the presence of Fusarium on seedling latent and cause disease later in the growing sea- roots in the absence of any disease symptoms is son when plants become stressed. generally not sufficient grounds to reject seedlings scheduled for outplanting. In fact, Axelrood and Seedling Root Rot others (1998) concluded that Fusarium are proba- Fusarium root rot, characterized by blackened, bly of little consequence with regard to the mortal- thin and wispy roots with little sign of actively ity of seedlings on reforestation sites after they growing root tips, is often the final stage of a dis- were unable to find a significant difference ease continuum that may have begun with the between seedling infections and root colonization. seeds, or at least at the time of sowing. Not all However, the mean age of the outplanted and nat- species of Fusarium are pathogenic (James and urally regenerated seedlings examined was 5.6 and others 1989), and many of those that are patho- 4.7 years, respectively, and they did not take into genic are weakly so. However, Neumann (1993) account seedling mortality that may have arisen points out a very important adaptive characteristic immediately after outplanting. Thus, their results that contributes to its persistent ability as a perhaps speak more for the long-term than for pathogen, and that is that Fusarium are often fac- what might happen in the short-term, when plant- ultative parasites well adapted for survival in either ing shock may have a role in initial survival. dormant (chlamydospores) or saprophytic states Fusarium can be isolated from visually healthy (Bruehl 1987). Saprophytic survival in container nursery-grown conifer seedlings (Bloomberg nursery settings occurs when dead root fragments, 1966; James 1986; Kope and others 1996). carried over on old containers, have been colo- Because of this, Axelrood and others (1998) point nized by saprophytic Fusarium following parasitic out that the recovery of Fusarium from the roots colonization during the previous year. Thus, of nursery-grown conifers does not necessarily although often a weak pathogen that is tolerated in indicate a disease situation. Instead, they state a stress-free environment, as a facultative parasite that this can be indicative of a potential for dis- it persists in seedling containers, alternating ease to develop following outplanting if con- between saprophytic and more aggressively patho- ducive environmental (refer to environment cor- genic phases while the environment corner of the ner of disease triangle) conditions are present. disease triangle changes as seedlings develop. Root Container seedlings commonly have Fusarium on

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or around their roots (Landis 1976; Graham and 1) Seedborne contamination: Linderman 1983; James 1985a), and it is therefore a) How seeds become contaminated is important that care is taken so any fungi present still not clear; does not become aggressively pathogenic. b) It remains unclear how seed Seedlings scheduled for outplanting must never be contamination may be exacerbated allowed to remain in boxes, or in conditions where during cone and seed processing. they can become overheated and the roots remain warm and moist for prolonged periods. Under 2) Pre- and post-emergence damping-off: such conditions, Fusarium can rapidly spread from a) Develop better Standard Operating seedling to seedling, as well as intensify within the Procedures (SOP) to eliminate Fusarium roots of infected seedlings. When outplanted fol- as introduced greenhouse pests, that is, lowing these conditions, seedlings can quickly suc- benching, pallets, and container cumb to planting shock and, if exposed to a subse- sanitation; quent heat or drought stress, will often die. b) Improve seedcoat disinfestations procedures. Cone and Seed Pest Management: Vision for the Future 3) Seedling wilt: Understanding the disease biology of the major a) Improve SOP to reduce risks to fungal pathogens of forest nursery conifer sudden heat or drought stress induced seedlings in BC has been an important step transpirational demands. toward developing pest management plans to eliminate or minimize their impact on cone pro- 4) Late damping-off: duction and seed handling, as well as forest nurs- a) Improved understanding of 2 and 3 ery seedling production and increased seedling will help resolve this. survival after outplanting on reforestation sites. Fungi in the genus Fusarium can negatively 5) Seedling root rot: affect the reforestation value chain from the time a) Improved understanding of all steps of cone and seed production, through seed han- 1, 2, 3 and 4 will reduce losses to root rot; dling and processing, as well as during the course b) Improved handling practices during of nursery operations, to successful survival of storage and especially post-storage will outplanted seedlings. Increased understanding of reduce losses to root rot. Fusarium host-pathogen interactions throughout many aspects of conifer seedling production in 6) Seedling mortality after outplanting: BC is desirable. a) Plantation failure usually occurs when A vision of increased seed and seedling sur- Fusarium have survived through all the vival from cone and seed production to outplant- components of the reforestation value ing at reforestation sites is attainable through bet- chain described above, and a satisfactory ter understanding of the disease mechanisms environmental component of the disease associated with these fungi, and will lead to the triangle is met at the reforestation site. development of more effective cone and seed pest Improved understanding of value chain management plans. steps 1 through 5 could lower the incidence Increased understanding is needed of the fol- at the pathogen corner of the disease lowing Fusarium host-pathogen interactions of triangle to below what is necessary to cause conifer seedling production in BC: significant losses at the reforestation site.

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Cone and Seed Pest Management: Axelrood PE, Neumann M, Trotter D, Radley R, Shrimpton G, Mission Statement Dennis J. 1995. Seedborne Fusarium on Douglas-fir: path- For the vision of increased seed and seedling sur- ogenicity and seed stratification method to decrease vival from cone and seed production to outplanting Fusarium contamination. New Forests 9:35-51. at reforestation sites to be attainable, better under- Axelrood PE, Chapman WK, Seifert KA, Trotter DB, Shrimpton standing of the disease mechanisms associated with G. 1998. Cylindrocarpon and Fusarium root colonization of Fusarium and seedling production are needed. Douglas-fir seedlings from British Columbia reforestation Many of these mechanisms are understood individ- sites. Canadian Journal of Forest Research 28:1198-1206. ually, perhaps the most important being the fact Baker KF. 1948. Fusarium wilt of garden stock (Matthiola that the fungi are facultative parasites. As such, it has incana). Phytopathology 38:399-403. the ability to move in and out of a pathogenic or Bennett MA, Callan NW, Fritz VA. 1991. Seed treatments for saprophytic relationship with its host, depending, in disease control. HortTechnology 1:84-87. part, on the conditions at the environment corner Berger RD. 1977. Application of epidemiological principles to of the disease triangle. The ability to survive as a achieve plant disease control. Annual Review of Phy- saprophyte on tissues it has previously colonized as topathology 15:165-183. a parasite allows some fusaria to enter the reforesta- Bloomberg WJ. 1966. The occurrence of endophytic fungi in tion value chain as a seedborne contaminant and Douglas-fir seedlings and seed. Canadian Journal of still pose a threat to seedling survival many months Botany 44:413-420. later at the reforestation site. Better understanding Booth C. 1981. Perfect states (teleomorphs) of Fusarium of the key components of this value chain and the species. In: Nelson PE, Toussoun TA, Cook RJ, editors. interactions between host, pathogen, and environ- Fusarium: diseases, biology and taxonomy. University Park ment will allow the development of cone and seed (PA): Pennsylvania State University Press. p 446-452. pest management plans so that interventions can be Bruehl GW. 1987. Soilborne plant pathogens. New York (NY): made to break these disease triangle connections MacMillan Publishing Company. 368 pp. where possible. Dennis J, Trotter D. 1995. Life on the edge of the curve or the To achieve the vision of increased seed and current status of root rots in coastal Douglas-fir seedlings. seedling survival from cone and seed production In: Kooistra CM, editor. Proceedings of the 1995, 1996, to outplanting at reforestation sites requires 1997 Forest Nursery Association of British Columbia Annu- building on the current state of knowledge with al Meetings. Victoria (BC): Forest Nursery Association of regard to Fusarium as cone and seed pests. Specif- British Columbia. ically, better understanding is needed as to how Dumroese RK, James RL, Wenny DL, Gilligan CJ. 1988. Douglas- seeds become contaminated; how seed contami- fir seed treatments: effects on seed germination and seed- nation may be exacerbated during cone and seed borne organisms. In: Landis TD, technical coordinator. Pro- processing needs to be examined; and what ceedings, combined meeting of the Western Forest Nursery improvements if any, can be made to seedcoat Associations; 1988 Aug 8-11; Vernon, British Columbia. Fort disinfestation procedures. Collins (CO): USDA Forest Service, Rocky Mountain Research Station. General Technical Report RM-167. 6 p. References Eremko RD, Edwards DGW, Wallinger D. 1989. A guide to collect- Agrios GN. 1988. Plant pathology. 3rd ed. San Diego (CA): ing cones of British Columbia conifers. Victoria (BC): Forestry Academic Press, Inc. 803 pp. Canada and BC Ministry of Forests. FRDA Report Number 055. Alexopoulos CJ, Mims CW. 1979. Introductory mycology. Graham JH, Linderman RG. 1983. Pathogenic seedborne Fusar- Toronto (ON): John Wiley and Sons. ium oxysporum from Douglas-fir. Plant Disease 67:323-325. Axelrood PE, Peters R. 1993. Influence of nursery cultural Gerlach W, Nirenberg HI. 1982. The genus Fusarium — a pictori- practices on Cylindrocarpon and Fusarium root rot infection al atlas. Mitteilungen aus der Biologischen Bundesanstalt of Douglas-fir. Victoria (BC): British Columbia Ministry of für Land-und Forstwirtschaft, Berlin-Dahlem 209:1-406. Forests, Major Service Contract # 08384.

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Hoefnagels MH, Linderman RG. 1999. Biological suppression Kolotelo D, Peterson M. 2006. Fusarium spp. Trends in conifer of seedborne Fusarium spp. during cold stratification of cone and seed processing (CSP) [PowerPoint presenta- Douglas-fir seeds. Plant Disease 83(9):845-852. tion]. Presentation at the IUFRO Tree Seed Symposium; Ingold CT. 1953. Dispersal in fungi. London (United King- 2006 July 18-21; Fredericton, New Brunswick. dom): Oxford University Press. Kolotelo D, Van Steenis E, Peterson M, Bennett R, Trotter D, Den- [ISTA] International Seed Testing Association. 1999. Interna- nis J. 2001. Seed handling guidebook. Victoria (BC): British tional rules for seed testing. Seed Science and Technology. Columbia Ministry of Forests, Tree Improvement Branch. Supplement 27. Kope HH, Axelrood PE, Sutherland JR, Reddy MS. 1996. Preva- James RL. 1985a. Studies of Fusarium associated with con- lence and incidence of the root-inhabiting fungi, Fusari- tainerized conifer seedling diseases: (2). Diseases of west- um, Cylindrocarpon and Pythium on container-grown Dou- ern larch, Douglas-fir, grand fir, subalpine fir, and pon- glas-fir and spruce seedlings in British Columbia. New derosa pine seedlings at the USDA Forest Service Nursery, Forests 12:55-67. Coeur dAlene, Idaho. Missoula (MT): USDA Forest Service, Lamontange Y, Wang BSE. 1976. Germination of polyram Northern Region. Forest Health Protection Report 85-12. 7 p. treated white spruce seeds from various provenances. Tree James RL. 1985b. Diseases of conifer seedlings caused by Planters’ Notes 27(1):5-6, 22. seedborne Fusarium species. In: Shearer RC, technical Landis TD. 1976. Fusarium root disease of container-grown coordinator. Proceedings of a symposium on conifer tree tree seedlings. Lakewood (CO): USDA Forest Service, Rocky seed in the Inland Mountain West. Missoula (MT): USDA Mountain Region. Forest Insect and Disease Management, Forest Service, Intermountain Research Station. General Biological Evaluation R2-76-16. 7 p. Technical Report INT-203. p 267-217. Landis TD, Tinus RW, MacDonald SE, Barnett JP. 1990. The James RL. 1986. Mortality of containerized western larch container tree nursery manual. Vol. 5. The biological com- seedlings at the Champion Timberlands Nursery, Plains, ponent: nursery pests and mycorrhizae. Washington (DC): Montana. Missoula (MT): USDA Forest Service, Northern USDA Forest Service. Agriculture Handbook 674. Region. Forest Pest Management Report Number 86-16. Lawrence EB, Nelson PE, Ayers JE. 1981. Histopathology of James RL. 1993. Fusarium species associated with post-emer- sweet corn seed and plants infected with Fusarium monili- gence damping-off and root disease of young container- forme and F. oxysporum. Phytopathology 67:1461-1468. grown Douglas-fir seedlings USDA Forest Service Nursery, Leadem CL, Eremko RD. Davis IH. 1990. Seed biology, collec- Coeur d Alene, Idaho. Missoula (MT): USDA Forest Service, tions and post-harvest handling. In: Lavender DP, Parish R, Northern Region. Nursery Disease Notes #129. 5 p. Johnson CM, Montgomery G, Vyse A, Willis RA, Winston D, James RL. 2000. Pathogenic characteristics of Fusarium editors. Regenerating British Columbias forests. Vancouver acuminatum isolated from inland Pacific Northwest nurs- (BC): University of British Columbia Press. p 193-205. eries. Missoula (MT): USDA Forest Service, Northern Leslie JF. 2001. Population genetics level problems in the Gib- Region. Forest Health Protection Report 00-16. 8 p. berella fujikuroi species complex. In: Summerell BA, Leslie James RL, Dumroese RK, Gilligan CJ, Wenny DL. 1989. Patho- JF, Backhouse D, Bryden WL, Burgess LW, editors. Fusari- genicity of Fusarium isolates grown from Douglas-fir seed um: Paul E Nelson Memorial Symposium. St Paul (MN): and container-grown seedlings. Moscow (ID): University of American Phytopathological Society. p 113-121. Idaho College of Forestry, Wildlife and Range Sciences. Linderman RG. 1986. Managing rhizosphere microorganisms Bulletin Number 52. 10 p. in the production of horticultural crops. HortScience Kingsland GC, Wernham CC. 1962. Etiology of stalk rot of corn 21(6):1299-1302. in Pennsylvania. Phytopathology 52:519-523. Littke W. 1996. Seed pathogens and seed treatments. In: Lan- Klisiewicz JM. 1963. Wilt-incitant F. oxysporum f. sp. carthami dis TD, South DB, technical coordinators. National proceed- present in seed from infected safflower. Phytopathology ings, forest and conservation nursery associations. Portland 53:1046-1049. (OR): USDA Forest Service, Pacific Northwest Research Sta- tion. General Technical Report PNW-GTR-389. p 187-191.

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