Dana L. Richter

Genetic transformation of the ectomycorrhizal fungus Paxillus involutus by particle bombardment

Paxillus involutus was transformed using gold particle mediated gene transfer. Transformation was determined using the hygromycin B phosphotransferase gene (hph) as the selectable marker and the β-glucuronidase gene (GUS) as a reporter gene. Southern blot analysis confirmed that the vector DNA for both hph and GUS genes was integrated into the fungal genome. Variations in the number of multiple gene copies and insertions were found in the transformants. The hygromycin resistant transformants were mitotically stable maintaining both the hph and GUS genes in the fungal genome six months following transformation. Western blot analysis determined that the GUS gene was capable of transcribing and translating its protein product in the transformed fungus. Enzyme assays of GUS extracts determined that β-glucuronidase was active in the transformed fungi. Pure culture synthesis experiments showed that the ability of P. involvutus to form ectomychorrhizae with Pinus resinosa was not altered by transformation. These results provide the first report of a successful transformation of an ectomycorrhizal fungus using particle bombardment.

Mycorrhizal fungus colonization of Pinus resinosa Ait. Transplanted on northern hardwood clearcuts

Abstract An ordered succession in mycorrhizal fungus colonization occurred on red pine ( Pinus resinosa Ait.) root systems transplanted on three recently-cleared northern hardwood sites in northern Michigan, U.S.A. Ordered changes were related to tree age from transplanting, and were demonstrated experimentally by quantification of mycorrhizal morpho-types, laboratory isolation of fungi from mycorrhizae, physiological categorization of fungi recovered from mycorrhizae and surveys of fruiting bodies associated with seedlings and transplants. With increasing plantation age, (1) the number of non-mycorrhizal root tips encountered declined to zero, (2) the common nursery mycorrhizae decreased in abundance, (3) new mycorrhizal types (especially Cenococcum and Suillus ) became more abundant, (4) cellulolytic fungi and ectendomycorrhizal fungi were isolated from mycorrhizae less frequently, and (5) ectomycorrhizal fungi were isolated from mycorrhizae more frequently. The number of apparently different mycorrhizal fungus species isolated from mycorrhizae increased from five in the first year after transplanting to 22 in the fourth year.

Field survival of containerized red and jack pine seedlings inoculated with mycelial slurries of ectomycorrhizal fungi

Red pine and jack pine seedlings growing in styroblocks were inoculated 8 wk after sowing with mycelium/agar slurries of 3 mycorrhizal fungi (Laccaria bicolor, Scleroderma citrinum, and an unidentified basidiomycete), and one suspected mycorrhizal fungus (Cantharellula umbonata). Seedlings inoculated with L. bicolor developed mycorrhizae earlier and in greater numbers than the other inoculation treatments, with red pine out-performing jack pine in both respects. At 34 wk following sowing, seedlings were outplanted on a cleared xeric site in Baraga Co., in Michigans Upper Peninsula. Seedlings inoculated with C. umbonata failed to form mycorrhizae and were not outplanted. Inoculation treatments did not affect shoot or root weight at outplanting. Red pine inoculated with L. bicolor averaged 21% and 19% greater survival compared with control seedlings after one and two years in the field, respectively. Other inoculation treatments failed to increase seedling survival for either tree species. Jack pine demonstrated higher overall survival than did red pine for both years in all corresponding inoculation treatments.

Effects of substrate on laboratory spalting of sugar maple

Abstract Spalting is the coloration of wood caused by fungal colonization. Woodturners, craftspeople, and artists appreciate spalted wood for its aesthetic appeal and uniqueness. Laboratory-induced spalting aims at a repeatable procedure in which wood is inoculated with selected fungi to obtain natural color with high aesthetic appeal, low weight loss and good machinability. Vermiculite (a natural clay with a high capacity for water holding and cation exchange) has been the primary incubation substrate for spalting research despite soil being the standard substrate for soil block decay testing. In this research, we explored the differences between these two substrates and their effects on the growth of spalting fungi on sugar maple (Acer saccharum) wood. Five fungi, Trametes versicolor, Xylaria polymorpha, Arthrographis cuboidea, Ceratocystis pilifera, and Ceratocystis virescens, were tested for their weight loss and spalting abilities on 14-mm sugar maple cubes incubated in both soil and vermiculite. Weight losses from all fungi were either unaffected or reduced by incubation in vermiculite compared to soil. In vermiculite, X. polymorpha produced more zone lines and A. cuboidea produced more pigment than blocks incubated in soil. Growth in vermiculite decreased weight loss of blocks inoculated with T. versicolor and X. polymorpha, while bleaching was unaffected regardless of substrate. External blue stain was higher on blocks inoculated with either Ceratocystis species and incubated in soil. These results indicate that vermiculite is a better substrate for spalting regardless of fungus due to the higher external pigmentation, lower weight loss, and better color contrast on the sugar maple blocks incubated in this substrate.

Ectomycorrhizal colonization of Quercus rubra seedlings in response to vegetation removals in oak and pine stands

Abstract Ectomycorrhizal (ECM) colonization of northern red oak (Quercus rubra L., NRO) seedlings in response to different degrees of overstory and understory removal was investigated in NRO and red pine (Pinus resinosa Ait.) stands in northern Lower Michigan. The experimental design consisted of two stand types (oak and pine), three blocks nested within stand type, four levels of canopy cover (clearcut, 25% first year), 75%, and uncut), and two understory treatments (shrub removal and untreated control). NRO acorns from a common seed source were sown in the spring of 1991 and the emerged seedlings were sampled to quantify their ECM during the first two growing seasons. Photosynthetically acitive radiation (PAR) transmittance was recorded during the second growing season. Soil moisture and temperature were also measured at two- to three-week intervals for the first two years. ECM colonization (%) was significantly greater in the 50% canopy cover treatment (37.5) than in the clearcut (22.3) and uncut (20.8) treatments during the first growing season. In contrast, during the second growing season, percent ECM in the 25% canopy cover treatment (45.8) was significantly greater than in the clearcut treatment (20.4), but did not differ from the 75% cover (40.4) and uncut (37.6) treatments. ECM number per gram dry root was also significantly larger in the 25% canopy cover treatment (4595) than in the clearcut treatment (2588). Significantly more ECM (number per three lateral roots) were found in the untreated understory of the pine stand type (121) when compared to the shrub-removal treatments (103 and 107 for oak and pine stands, respectively) and untreated understory of the oak stand type (104) during the second growing season. Our results indicated that intermediate canopy levels stimulated the development of ECM, whereas complete removal of overstory and understory reduced such development. These results may aid forest managers in manipulating the field mycorrhizal condition of oak seedlings through silvicultural practices.

Effects of red pine (Pinus resinosa Ait.) mycorrhizoplane-associated actinomycetes onin vitro growth of ectomycorrhizal fungi

Mycorrhizoplane-associated actinomycetes were isolated using an enrichment technique from red pine (Pinus resinosa Ait.) roots of seedlings recently outplanted onto cleared northern hardwood sites in the Upper Peninsula of Michigan, USA. Interactions were assessedin vitro between actinomycete isolates and three commonly occurring ectomycorrhizal fungi (Laccaria bicolor (Maire) Orton,L. laccata (Scop.: Fr.) Berk. and Br., andThelephora terrestris Fr.). Most actinomycete isolates exerted a range of effects on the growth of the three fungus isolates during the four week test period, inhibiting some while stimulating others; several inhibited growth of all three fungus isolates. Mycorrhizoplane-associated actinomycetes show potential for use as coinoculants with selected ectomycorrhizal fungi to optimize the soil microflora for developing seedlings.

A comparison of mycorrhizal and saprotrophic fungus tolerance to creosote in vitro

Abstract Twenty-six isolates (18 species) of mycorrhizal fungi and 13 isolates (13 species) of saprotrophic decay fungi were grown on agar amended with 25, 50 and 100 ppm creosote. All saprotrophic fungi exhibited some tolerance to creosote; 11 of 13 saprotrophic fungal isolates (85%) had intermediate, high or complete tolerance to creosote. However, only 12 of 26 mycorrhizal fungal isolates (46%) had comparable tolerance to creosote. Of the saprotrophic fungi, Irpex lacteus , Neolentinus lepideus , Ouedemansiella radicata , Phanaerochaete chrysosporium , Postia placenta and Trametes versicolor exhibited the greatest tolerance to creosote. Of the mycorrhizal fungi, Cenococcum geophilum , Laccaria bicolor (one isolate), Laccaria laccata (one isolate) and Suillus granulatus exhibited the highest tolerance to creosote. Mycorrhizal fungi with either very low or no tolerance (killed) by creosote were Amanita flavoconia , Astraeus hygrometricus , Lactarius rufus , Pisolithus tinctorius , and Scleroderma citrinum .

Stimulating spalting in sugar maple using sub-lethal doses of copper

Copper II is a common fungicide, especially in wood preservative formulations. Published research has noted fungal stimulation when fungi are subjected to sub-lethal amounts of copper ions. In this paper, the use of copper salts to stimulate spalting, as measured by fungal pigment production is investigated. At 1.0 kg/m3 copper sulfate, Xylaria polymorpha increased external zone lines when grown on sugar maple (Acer saccharum). Use of this experimental method to produce spalted wood should yield increased external spalting without an associated increased loss in machinability.ZusammenfassungKupfer II ist ein herkömmliches Fungizid, das insbesondere in Holzschutzmitteln verwendet wird. Aus der Literatur ist bekannt, dass Pilze angeregt werden, wenn sie einer subletalen Dosis an Kupferionen ausgesetzt werden. Im vorliegenden Artikel wurde untersucht, wie Kupfersalze pilzbedingte Verfärbungen stimulieren. Die Pilzpigmentproduktion wurde bestimmt. Bei einer Kupfersulfatmenge von 1,0 kg/m3 nahmen die durch Xylaria polymorphia auf Zuckerahorn (Acer saccharum) erzeugten Grenzlinien zu. Mit diesem Verfahren sollte es möglich sein, ausgeprägtere Holzverfärbungen zu erhalten ohne die damit in der Regel schlechtere Bearbeitbarkeit in Kauf nehmen zu müssen.

Differential sensitivity of fungi to lithium chloride in culture media.

Forty species of fungi, representing a range of ecological and taxonomic groups, were tested for their ability to grow on agar media amended with lithium chloride (LiCl) at 1.5, 3 and 6 g l(-1). Species of Trichoderma varied considerably in their sensitivity to LiCl; at one week on 6 g l(-1) LiCl medium, the growth of seven species of Trichoderma was considerably inhibited; however, by three weeks at this level, four of the species tested were able to attain > or =30% of control growth. Of the seven species tested, an isolate of T. viride was the most sensitive to LiCl in agar. Eleven other imperfect fungi also showed a range of ability to grow on agar amended with LiCl, from total inhibition to complete lack of inhibition. Six ascomycete fungi were greatly inhibited by LiCl at all levels; however, an isolate of Chaetomium globosum was highly tolerant of LiCl. Seven basidiomycete wood-decay fungi were quite sensitive to LiCl in agar, showing total to nearly total inhibition even at the lowest level; however, after three weeks, an isolate of Postia placenta was nearly uninhibited except at 6 g l(-1). Five ectomycorrhizal basidiomycete fungi were totally inhibited by all levels of LiCl; however, one ectomycorrhizal imperfect fungus (Cenococcum graniforme) was able to grow at 3 g l(-1) and was uninhibited at 1.5 g l(-1). Four zygomycete fungus isolates were nearly unaffected in their growth by all levels of LiCl.

Pure culture synthesis of Pinus resinosa ectomycorrhizae with Scleroderma aurantium

Numerous techniques and many variations on them have been reported for the synthesis of ec? tomycorrhizae in vitro (e.g., Chu-Chou, 1979; Danielson, 1984; Duddridge and Read, 1984a; Fortin, 1966; Molina, 1979; Palm and Stewart, 1984; Yang and Wilcox, 1984). Many of these techniques involve elaborate manipulations and exacting cultural conditions. In our study, lab? oratory procedures for the pure culture forma? tion of ectomycorrhizae were simplified as much as possible. We chose the gasteromycete Scleroderma aurantium (Vaill.) Pers. for testing because of its association with red pine (Pinus resinosa Ait.) on a variety of sites that we sampled in Michi? gans Upper Peninsula, and because of its vigorous growth in culture. The agarics, Cantharellula umbonata (Gmel.: Fr.) Sing. and Hygrophoropsis aurantiaca (Wulf.: Fr.) Maire in Martin-Sans, were chosen for similar reasons. Red pine was selected as test host because of its importance in the Lake States forest industry. Wide-mouth quart Mason jars, about one liter capacity, were used as synthesis vessels. The in? ner lids ofthe jars were inverted within the outer ring to prevent sealing and to allow gas exchange. The outer ring was screwed on until firm, and then loosened about one-quarter turn. As a rooting substrate, 250 ml of a vermiculite-peat mix? ture (10:1, v/v) were added to each jar. The peat used in this mixture was ground sphagnum peat moss. One hundred twenty-five ml of modified Melin-Norkrans (MMN) nutrient medium (Marx, 1969) were then added and the jars were auto? claved at 121 C for 45 min. The autoclaved jars were then stored for 2-7 da before planting. Red pine seeds were surface-sterilized in 30% H202 for 30 min, rinsed in sterile distilled water several times and plated on water agar. Three axenically germinated seedlings, 2-3 wk old, were planted in each jar. Jars were placed on a northeast-facing wi dow sill and seedlings were allowed to be? come established for 2-3 wk.