Elucidating "lucidum": Distinguishing the diverse laccate Ganoderma species of the United States.

Ganoderma is a large, diverse and globally-distributed genus in the Basidiomycota that includes species causing a white rot form of wood decay on a variety of tree species. For the past century, many studies of Ganoderma in North America and other regions of the world have used the name G. lucidum sensu lato for any laccate (shiny or varnished) Ganoderma species growing on hardwood trees or substrates. Molecular studies have established that G. lucidum sensu stricto (Curtis) Karst is native to Europe and some parts of China. To determine the species of the laccate Ganoderma that are present in the United States, we studied over 500 collections from recently collected samples and herbarium specimens from hardwoods, conifers, and monocots. A multilocus phylogeny using ITS, tef1α, rpb1 and rpb2 revealed three well-supported clades, similar to previously reported findings. From the U.S. collections, thirteen taxa representing twelve species were identified, including: G. curtisii, G. lucidum sensu stricto, G. martinicense, G. oregonense, G. polychromum, G. ravenelii, G. sessile, G. tsugae, G. tuberculosum, G. cf. weberianum, G. zonatum, and Tomophagus colossus (syn. G. colossus). The species G. meredithiae is synonymized with G. curtisii, and considered a physiological variant that specializes in decay of pines. The designation G. curtisii f.sp. meredithiae forma specialis nov. is proposed. Species such as G. curtisii and G. sessile, once considered as G. lucidum sensu lato, were found to be divergent from one another, and highly divergent from G. lucidum sensu stricto. Morphological characteristics such as context tissue color and features (e.g. melanoid bands), basidiospore shape and size, geographic location, and host preference were found to aid in species identification. Surprisingly, G. lucidum sensu stricto was found in the U.S., but only in geographically restricted areas of northern Utah and California. These collections appear to have resulted from the introduction of this species into the United States possibly from mushroom growers producing G. lucidum outdoors. Overall, this study clarifies the chaotic taxonomy of the laccate Ganoderma in the United States, and will help to remove ambiguities from future studies focusing on the North American species of laccate Ganoderma.

Fungal Planet description sheets: 558-624.

Novel species of fungi described in this study include those from various countries as follows: Australia: Banksiophoma australiensis (incl. Banksiophoma gen. nov.) on Banksia coccinea, Davidiellomycesaustraliensis (incl. Davidiellomyces gen. nov.) on Cyperaceae, Didymocyrtis banksiae on Banksia sessilis var. cygnorum, Disculoides calophyllae on Corymbia calophylla, Harknessia banksiae on Banksia sessilis, Harknessia banksiae-repens on Banksia repens, Harknessia banksiigena on Banksia sessilis var. cygnorum, Harknessia communis on Podocarpus sp., Harknessia platyphyllae on Eucalyptus platyphylla, Myrtacremonium eucalypti (incl. Myrtacremonium gen. nov.) on Eucalyptus globulus, Myrtapenidiella balenae on Eucalyptus sp., Myrtapenidiella eucalyptigena on Eucalyptus sp., Myrtapenidiella pleurocarpae on Eucalyptuspleurocarpa, Paraconiothyrium hakeae on Hakea sp., Paraphaeosphaeria xanthorrhoeae on Xanthorrhoea sp., Parateratosphaeria stirlingiae on Stirlingia sp., Perthomyces podocarpi (incl. Perthomyces gen. nov.) on Podocarpus sp., Readeriella ellipsoidea on Eucalyptus sp., Rosellinia australiensis on Banksia grandis, Tiarosporella corymbiae on Corymbia calophylla, Verrucoconiothyriumeucalyptigenum on Eucalyptus sp., Zasmidium commune on Xanthorrhoea sp., and Zasmidium podocarpi on Podocarpus sp. Brazil: Cyathus aurantogriseocarpus on decaying wood, Perenniporia brasiliensis on decayed wood, Perenniporia paraguyanensis on decayed wood, and Pseudocercospora leandrae-fragilis on Leandrafragilis.Chile: Phialocephala cladophialophoroides on human toe nail. Costa Rica: Psathyrella striatoannulata from soil. Czech Republic: Myotisia cremea (incl. Myotisia gen. nov.) on bat droppings. Ecuador: Humidicutis dictiocephala from soil, Hygrocybe macrosiparia from soil, Hygrocybe sangayensis from soil, and Polycephalomyces onorei on stem of Etlingera sp. France: Westerdykella centenaria from soil. Hungary: Tuber magentipunctatum from soil. India: Ganoderma mizoramense on decaying wood, Hodophilus indicus from soil, Keratinophyton turgidum in soil, and Russula arunii on Pterigota alata.Italy: Rhodocybe matesina from soil. Malaysia: Apoharknessia eucalyptorum, Harknessia malayensis, Harknessia pellitae, and Peyronellaea eucalypti on Eucalyptus pellita, Lectera capsici on Capsicum annuum, and Wallrothiella gmelinae on Gmelina arborea.Morocco: Neocordana musigena on Musa sp. New Zealand: Candida rongomai-pounamu on agaric mushroom surface, Candida vespimorsuum on cup fungus surface, Cylindrocladiella vitis on Vitis vinifera, Foliocryphia eucalyptorum on Eucalyptus sp., Ramularia vacciniicola on Vaccinium sp., and Rhodotorula ngohengohe on bird feather surface. Poland: Tolypocladium fumosum on a caterpillar case of unidentified Lepidoptera.Russia: Pholiotina longistipitata among moss. Spain: Coprinopsis pseudomarcescibilis from soil, Eremiomyces innocentii from soil, Gyroporus pseudocyanescens in humus, Inocybe parvicystis in humus, and Penicillium parvofructum from soil. Unknown origin: Paraphoma rhaphiolepidis on Rhaphiolepsis indica.USA: Acidiella americana from wall of a cooling tower, Neodactylaria obpyriformis (incl. Neodactylaria gen. nov.) from human bronchoalveolar lavage, and Saksenaea loutrophoriformis from human eye. Vietnam: Phytophthora mekongensis from Citrus grandis, and Phytophthora prodigiosa from Citrus grandis. Morphological and culture characteristics along with DNA barcodes are provided.

Fungal Planet description sheets: 400-468.

Novel species of fungi described in the present study include the following from Australia: Vermiculariopsiella eucalypti, Mulderomyces natalis (incl. Mulderomyces gen. nov.), Fusicladium paraamoenum, Neotrimmatostroma paraexcentricum, and Pseudophloeospora eucalyptorum on leaves of Eucalyptus spp., Anungitea grevilleae (on leaves of Grevillea sp.), Pyrenochaeta acaciae (on leaves of Acacia sp.), and Brunneocarpos banksiae (incl. Brunneocarpos gen. nov.) on cones of Banksia attenuata. Novel foliicolous taxa from South Africa include Neosulcatispora strelitziae (on Strelitzia nicolai), Colletotrichum ledebouriae (on Ledebouria floridunda), Cylindrosympodioides brabejum (incl. Cylindrosympodioides gen. nov.) on Brabejum stellatifolium, Sclerostagonospora ericae (on Erica sp.), Setophoma cyperi (on Cyperus sphaerocephala), and Phaeosphaeria breonadiae (on Breonadia microcephala). Novelties described from Robben Island (South Africa) include Wojnowiciella cissampeli and Diaporthe cissampeli (both on Cissampelos capensis), Phaeotheca salicorniae (on Salicornia meyeriana), Paracylindrocarpon aloicola (incl. Paracylindrocarpon gen. nov.) on Aloe sp., and Libertasomyces myopori (incl. Libertasomyces gen. nov.) on Myoporum serratum. Several novelties are recorded from La Réunion (France), namely Phaeosphaeriopsis agapanthi (on Agapanthus sp.), Roussoella solani (on Solanum mauritianum), Vermiculariopsiella acaciae (on Acacia heterophylla), Dothiorella acacicola (on Acacia mearnsii), Chalara clidemiae (on Clidemia hirta), Cytospora tibouchinae (on Tibouchina semidecandra), Diaporthe ocoteae (on Ocotea obtusata), Castanediella eucalypticola, Phaeophleospora eucalypticola and Fusicladium eucalypticola (on Eucalyptus robusta), Lareunionomyces syzygii (incl. Lareunionomyces gen. nov.) and Parawiesneriomyces syzygii (incl. Parawiesneriomyces gen. nov.) on leaves of Syzygium jambos. Novel taxa from the USA include Meristemomyces arctostaphylos (on Arctostaphylos patula), Ochroconis dracaenae (on Dracaena reflexa), Rasamsonia columbiensis (air of a hotel conference room), Paecilomyces tabacinus (on Nicotiana tabacum), Toxicocladosporium hominis (from human broncoalveolar lavage fluid), Nothophoma macrospora (from respiratory secretion of a patient with pneumonia), and Penidiellopsis radicularis (incl. Penidiellopsis gen. nov.) from a human nail. Novel taxa described from Malaysia include Prosopidicola albizziae (on Albizzia falcataria), Proxipyricularia asari (on Asarum sp.), Diaporthe passifloricola (on Passiflora foetida), Paramycoleptodiscus albizziae (incl. Paramycoleptodiscus gen. nov.) on Albizzia falcataria, and Malaysiasca phaii (incl. Malaysiasca gen. nov.) on Phaius reflexipetalus. Two species are newly described from human patients in the Czech Republic, namely Microascus longicollis (from toenails of patient with suspected onychomycosis), and Chrysosporium echinulatum (from sole skin of patient). Furthermore, Alternaria quercicola is described on leaves of Quercus brantii (Iran), Stemphylium beticola on leaves of Beta vulgaris (The Netherlands), Scleroderma capeverdeanum on soil (Cape Verde Islands), Scleroderma dunensis on soil, and Blastobotrys meliponae from bee honey (Brazil), Ganoderma mbrekobenum on angiosperms (Ghana), Geoglossum raitviirii and Entoloma kruticianum on soil (Russia), Priceomyces vitoshaensis on Pterostichus melas (Carabidae) (Bulgaria) is the only one for which the family is listed, Ganoderma ecuadoriense on decaying wood (Ecuador), Thyrostroma cornicola on Cornus officinalis (Korea), Cercophora vinosa on decorticated branch of Salix sp. (France), Coprinus pinetorum, Coprinus littoralis and Xerocomellus poederi on soil (Spain). Two new genera from Colombia include Helminthosporiella and Uwemyces on leaves of Elaeis oleifera. Two species are described from India, namely Russula intervenosa (ectomycorrhizal with Shorea robusta), and Crinipellis odorata (on bark of Mytragyna parviflora). Novelties from Thailand include Cyphellophora gamsii (on leaf litter), Pisolithus aureosericeus and Corynascus citrinus (on soil). Two species are newly described from Citrus in Italy, namely Dendryphiella paravinosa on Citrus sinensis, and Ramularia citricola on Citrus floridana. Morphological and culture characteristics along with ITS nrDNA barcodes are provided for all taxa.

Fungal Planet description sheets: 371-399.

Novel species of fungi described in the present study include the following from Australia: Neoseptorioides eucalypti gen. & sp. nov. from Eucalyptus radiata leaves, Phytophthora gondwanensis from soil, Diaporthe tulliensis from rotted stem ends of Theobroma cacao fruit, Diaporthe vawdreyi from fruit rot of Psidium guajava, Magnaporthiopsis agrostidis from rotted roots of Agrostis stolonifera and Semifissispora natalis from Eucalyptus leaf litter. Furthermore, Neopestalotiopsis egyptiaca is described from Mangifera indica leaves (Egypt), Roussoella mexicana from Coffea arabica leaves (Mexico), Calonectria monticola from soil (Thailand), Hygrocybe jackmanii from littoral sand dunes (Canada), Lindgomyces madisonensis from submerged decorticated wood (USA), Neofabraea brasiliensis from Malus domestica (Brazil), Geastrum diosiae from litter (Argentina), Ganoderma wiiroense on angiosperms (Ghana), Arthrinium gutiae from the gut of a grasshopper (India), Pyrenochaeta telephoni from the screen of a mobile phone (India) and Xenoleptographium phialoconidium gen. & sp. nov. on exposed xylem tissues of Gmelina arborea (Indonesia). Several novelties are introduced from Spain, namely Psathyrella complutensis on loamy soil, Chlorophyllum lusitanicum on nitrified grasslands (incl. Chlorophyllum arizonicum comb. nov.), Aspergillus citocrescens from cave sediment and Lotinia verna gen. & sp. nov. from muddy soil. Novel foliicolous taxa from South Africa include Phyllosticta carissicola from Carissa macrocarpa, Pseudopyricularia hagahagae from Cyperaceae and Zeloasperisporium searsiae from Searsia chirindensis. Furthermore, Neophaeococcomyces is introduced as a novel genus, with two new combinations, N. aloes and N. catenatus. Several foliicolous novelties are recorded from La Réunion, France, namely Ochroconis pandanicola from Pandanus utilis, Neosulcatispora agaves gen. & sp. nov. from Agave vera-cruz, Pilidium eucalyptorum from Eucalyptus robusta, Strelitziana syzygii from Syzygium jambos (incl. Strelitzianaceae fam. nov.) and Pseudobeltrania ocoteae from Ocotea obtusata (Beltraniaceae emend.). Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Fungal Planet description sheets: 281-319.

Novel species of fungi described in the present study include the following from South Africa: Alanphillipsia aloeicola from Aloe sp., Arxiella dolichandrae from Dolichandra unguiscati, Ganoderma austroafricanum from Jacaranda mimosifolia, Phacidiella podocarpi and Phaeosphaeria podocarpi from Podocarpus latifolius, Phyllosticta mimusopisicola from Mimusops zeyheri and Sphaerulina pelargonii from Pelargonium sp. Furthermore, Barssia maroccana is described from Cedrus atlantica (Morocco), Codinaea pini from Pinus patula (Uganda), Crucellisporiopsis marquesiae from Marquesia acuminata (Zambia), Dinemasporium ipomoeae from Ipomoea pes-caprae (Vietnam), Diaporthe phragmitis from Phragmites australis (China), Marasmius vladimirii from leaf litter (India), Melanconium hedericola from Hedera helix (Spain), Pluteus albotomentosus and Pluteus extremiorientalis from a mixed forest (Russia), Rachicladosporium eucalypti from Eucalyptus globulus (Ethiopia), Sistotrema epiphyllum from dead leaves of Fagus sylvatica in a forest (The Netherlands), Stagonospora chrysopyla from Scirpus microcarpus (USA) and Trichomerium dioscoreae from Dioscorea sp. (Japan). Novel species from Australia include: Corynespora endiandrae from Endiandra introrsa, Gonatophragmium triuniae from Triunia youngiana, Penicillium coccotrypicola from Archontophoenix cunninghamiana and Phytophthora moyootj from soil. Novelties from Iran include Neocamarosporium chichastianum from soil and Seimatosporium pistaciae from Pistacia vera. Xenosonderhenia eucalypti and Zasmidium eucalyptigenum are newly described from Eucalyptus urophylla in Indonesia. Diaporthe acaciarum and Roussoella acacia are newly described from Acacia tortilis in Tanzania. New species from Italy include Comoclathris spartii from Spartium junceum and Phoma tamaricicola from Tamarix gallica. Novel genera include (Ascomycetes): Acremoniopsis from forest soil and Collarina from water sediments (Spain), Phellinocrescentia from a Phellinus sp. (French Guiana), Neobambusicola from Strelitzia nicolai (South Africa), Neocladophialophora from Quercus robur (Germany), Neophysalospora from Corymbia henryi (Mozambique) and Xenophaeosphaeria from Grewia sp. (Tanzania). Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Species of Mycosphaerellaceae and Teratosphaeriaceae on native Myrtaceae in Uruguay: evidence of fungal host jumps.

Mycosphaerella species are well-known causal agents of leaf diseases on many economically and ecologically important plant species. In Uruguay, a relatively large number of Mycosphaerellaceae and Teratosphaeriaceae are found on Eucalyptus, but nothing is known of these fungi on native Myrtaceae. The aim of this study was to identify Mycosphaerellaceae and Teratosphaeriaceae species associated with leaf diseases on native Myrtaceae in Uruguay and to consider whether host jumps by the pathogen from introduced Eucalyptus to native Myrtaceae have occurred. Several native forests throughout the country were surveyed with special attention given to those located close to Eucalyptus plantations. Five species belonging to the Mycosphaerellaceae and Teratosphaeriaceae clades were found on native Myrtaceous trees and three of these had previously been reported on Eucalyptus in Uruguay. Those occurring both on Eucalyptus and native Myrtaceae included Pallidocercospora heimii, Pseudocercospora norchiensis, and Teratosphaeria aurantia. In addition, Mycosphaerella yunnanensis, a species known to occur on Eucalyptus but not previously recorded in Uruguay, was found on leaves of two native Myrtaceous hosts. Because most of these species occur on Eucalyptus in countries other than Uruguay, it appears that they were introduced in this country and have adapted to be able to infect native Myrtaceae. These apparent host jumps have the potential to result in serious disease problems and they should be carefully monitored.

Host Range Investigations of New, Undescribed, and Common Phytophthora spp. Isolated from Ornamental Nurseries in Minnesota.

Eleven woody landscape plants commonly grown in the upper Midwestern United States were inoculated with up to three unnamed Phytophthora taxa (Phytophthora taxon Pgchlamydo, a Phytophthora alni-like isolate [Phytophthora MN14d], and Phytophthora sp. MN1) to explore their host ranges. In addition, P. cactorum, P. citricola, P. citrophthora, P. hedraiandra, and P. nicotianae were used to inoculate plants to further investigate the susceptibilities of plant genera previously found associated with these pathogens, to explore the susceptibility of important landscape plants (i.e., oak) to common ornamental Phytophthora spp., and to prove Koch's postulates. Koch's postulates were completed on fragrant sumac with P. citricola and P. nicotianae and on common lilac with P. citrophthora. A nonwound or wound inoculation technique were used to determine host susceptibility. Phytophthora sp. MN1 caused symptoms on American cranberrybush, bur and red oak, common lilac, fragrant sumac, Norway maple, and 'P.J.M.' rhododendron. The newly described organism P. hedraiandra caused disease on American cranberrybush, common lilac, red oak, and 'Snowdrift' crabapple. Fragrant sumac and common lilac generally were the most susceptible hosts to all Phytophthora spp. This study demonstrated that many ornamental Phytophthora pathogens have larger potential host ranges than previously known. The biology and ecology of P. hedraiandra and Phytophthora sp. MN1 must be further investigated, and methods for rapid identification should be developed.

Phytophthora Species Associated with Diseased Woody Ornamentals in Minnesota Nurseries.

Phytophthora species are responsible for causing extensive losses of ornamental plants worldwide. Recent international and national surveys for the detection of P. ramorum have led to the finding of previously undescribed Phytophthora species. Since no previous Phytophthora surveys have been carried out in Minnesota, surveys of ornamental nurseries were performed over 4 years to isolate and identify the Phytophthora species causing diseases of woody plants in Minnesota. Species were identified by direct sequencing of internal transcribed spacer (ITS) rDNA, ß-tub, and mitochondrial coxI genes. Species associated with diseased ornamental plants include P. cactorum, P. cambivora, P. citricola, P. citrophthora, P. hedraiandra, P. megasperma, P. nicotianae, and the previously identified but undescribed taxon P. Pgchlamydo. The most common species encountered were P. cactorum, P. citricola, and P. citrophthora. Two additional isolates obtained did not match known species. One was similar to P. alni subsp. alni, and the other appeared to be a new species and is referred to as P. sp. MN1. In addition, species are reported for the first time from several hosts. Results indicated that several Phytophthora species were more widespread in the nursery industry than previously thought, and undescribed species were causing disease in Minnesota ornamental nurseries.

First Report of Dieback and Leaf Lesions on Rhododendron sp. Caused by Phytophthora hedraiandra in the United States.

Surveys for Phytophthora ramorum in Minnesota nurseries revealed the presence of P. hedraiandra de Cock & Man in't Veld and several other Phytophthora species but not P. ramorum. Symptomatic leaf and stem tissues from diseased Rhododendron and Quercus species were cultured on PARP, a selective growth medium for Phytophthora (3). The Phytophthora isolates obtained were later identified by sequencing the internal transcribed spacer (ITS) region of the rDNA and comparing the sequences with those in GenBank using BLAST searches (1). The ITS sequences of six cultures (GenBank Accession Nos. DQ139804-DQ139809), isolated during 2003 from various Rhododendron cultivars exhibiting leaf lesions and shoot dieback, showed 100% identity with the ITS sequence of P. hedraiandra (GenBank Accession No. AY707987) (2). This is a recently described pathogenic species from the Netherlands responsible for causing leaf spots on Viburnum spp. Since the ITS sequence of P. hedraiandra differs little from that of P. cactorum (2), we verified one isolate to be P. hedraiandra by sequencing the mitochondrial cytochrome c oxidase subunit I gene (cox1) (GenBank Accession No. DQ139810). Comparison of this sequence with the P. hedraiandra voucher specimen in GenBank (Accession No. AY769115) showed 99% identity, which was the closest match. Reproductive structures were measured on V8 juice agar. The average oogonium diameter for three isolates was 29 µm with a range of 26 to 32 µm, while the average antheridium length was 13 µm (11 to 15 µm). Sporangium length and width averages on crushed hemp seeds were 32 µm (28 to 36 µm) and 26 µm (21 to 30 µm), respectively, with the average length to width ratio of 1.25 (1.23 to 1.29). Pathogenicity tests on Rhododendron cv. Mikkeli were carried out using three of our P. hedraiandra isolates. Spore suspensions of 2 × 104 zoospores per ml were used to mist-spray shoots of five, 3-year-old plants for each isolate. Five controls were mist sprayed with water. After inoculation, plants were placed in plastic bags in a dark growth chamber (22°C) for 7 days and then moved to a greenhouse. Leaf blotches and shoot dieback were apparent 5 days after inoculation, and P. hedraiandra was reisolated from those symptomatic tissues and identified by an exact match of the ITS sequence. Necrotic areas lengthened from the shoot tips to the main stems of the plants while expanding into petioles and leaves. No symptoms were observed on control plants. To our knowledge, this is the first report of P. hedraiandra in the United States as well as the first report of Koch's postulates performed with P. hedraiandra on Rhododendron cv. Mikkeli. The significance of this disease to other woody plants in nurseries or the landscape is unknown, and further study is needed to determine the host range and extent of the disease that may occur from this introduction. References: (1) S. F. Altschul et al. J. Mol. Biol. 215:403, 1990. (2) A. W. A.M de Cock and C. A. Lévesque. Stud Mycol 50:481, 2004. (3) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996.

Etiology of Bronze Leaf Disease of Populus.

Bronze leaf disease is a potentially destructive disorder of the Populus section of the genus Populus. The causal agent has been reported to be Apioplagiostoma populi (anamorph: Discula sp.). Based on etiological and symptomological studies, field observations of symptom development suggest that the pathogen moves systemically in the host. This was verified by graft experiments where symptoms progressed from the scion into the elongating stem. A bronze-pigmented vascular discoloration was observed in symptomatic leaves and branches. Dieback of affected stems also was common. Spore-trap studies elucidated the timing and necessary weather conditions of A. populi ascospore dispersal in relation to infection and symptom development. Exposure-tree experiments revealed that ascospores of A. populi are the primary inoculum and resulting infection causes distinctive disease symptoms on affected trees. Perithecia of A. populi were observed on overwintered symptomatic leaves, but were not observed on asymptomatic leaves. Acervular conidiomata were observed on symptomatic leaves during August and September. Although A. populi ascospores germinated in vitro, A. populi was not recovered from symptomatic tissue. Isolations from diseased leaves consistently yielded Epicoccum nigrum, but the role of this species is unclear. Inoculations of susceptible plants with E. nigrum conidia failed to reproduce symptoms, but inoculations with ascospores of A. populi produced symptoms typical of bronze leaf disease and Koch's postulates were performed.

Nitrogen cycling by wood decomposing soft-rot fungi in the "King Midas tomb," Gordion, Turkey.

Archaeological wood in ancient tombs is found usually with extensive degradation, limiting what can be learned about the diet, environment, health, and cultural practices of the tomb builders and occupants. Within Tumulus Midas Mound at Gordion, Turkey, thought to be the tomb of the Phrygian King Midas of the 8th century B.C., we applied a stable nitrogen isotope test to infer the paleodiet of the king and determine the nitrogen sources for the fungal community that decomposed the wooden tomb, cultural objects, and human remains. Here we show through analysis of the coffin, furniture, and wooden tomb structure that the principal degrader, a soft-rot fungus, mobilized the king's highly (15)N-enriched nutrients, values indicative of a diet rich in meat, to decay wood throughout the tomb. It is also evident from the delta(15)N values of the degraded wood that the nitrogen needed for the decay of many of the artifacts in the tomb came from multiple sources, mobilized at potentially different episodes of decay. The redistribution of nutrients by the fungus was restricted by constraints imposed by the cellular structure of the different wood materials that apparently were used intentionally in the construction to minimize decay.

Bacterial biodegradation of extractives and patterns of bordered pit membrane attack in pine wood.

Wood extractives, commonly referred to as pitch, cause major problems in the manufacturing of pulp and paper. Treatment of nonsterile southern yellow pine chips for 14 days with Pseudomonas fluorescens, Pseudomonas sp., Xanthomonas campestris, and Serratia marcescens reduced wood extractives by as much as 40%. Control treatments receiving only water lost 11% of extractives due to the growth of naturally occurring microorganisms. Control treatments were visually discolored after the 14-day incubation, whereas bacterium-treated wood chips were free of dark staining. Investigations using P. fluorescens NRRL B21432 showed that all individual resin and fatty acid components of the pine wood extractives were substantially reduced. Micromorphological observations showed that bacteria were able to colonize resin canals, ray parenchyma cells, and tracheids. Tracheid pit membranes within bordered pit chambers were degraded after treatment with P. fluorescens NRRL B21432. P. fluorescens and the other bacteria tested appear to have the potential for biological processing to substantially reduce wood extractives in pine wood chips prior to the paper making process so that problems associated with pitch in pulp mills can be controlled.

Biological Processing of Pine Logs for Pulp and Paper Production with Phlebiopsis gigantea.

Phlebiopsis gigantea (=Phanerochaete gigantea) is a white rot fungus that rapidly colonizes cut stumps, stems, and branches of pine. Two laboratory and several field studies showed that inoculation of red pine logs, Pinus resinosa, with P. gigantea reduced the pitch content of wood, facilitated bark removal, modified wood cells, and controlled detrimental sapstain. Isolations from inoculated logs revealed up to 100 and 80% colonization of the sapwood by P. gigantea after 8 weeks in the field and 32 days in the laboratory, respectively. Logs colonized by P. gigantea in both the laboratory and field showed a 9 to 71% reduction in pitch content, as well as a significant enhancement of bark removal. Examination with Simons' stain of refined wood fibers from inoculated logs revealed an increase in cell wall porosity. Blue stain fungi that cause dark discoloration of the sapwood were inhibited by inoculation with P. gigantea. These studies demonstrate that biological processing of logs with P. gigantea can result in substantial benefits to the pulp and papermaking process.

Mineralization of alachlor by lignin-degrading fungi.

White rot fungi were able to mineralize the aromatic ring carbon of alachlor to CO2. After 122 days, 14 and 12% of the alachlor that was initially present in malt extract cultures supplemented with a wood substrate was mineralized at room temperature by Ceriporiopsis subvermispora and Phlebia tremellosa, respectively. Although Phanerochaete chrysosporium mineralized alachlor at 25 degrees C, it did so more slowly than the other two white rot fungi. The brown rot fungus Fomitopsis pinicola did not mineralize alachlor.

Effect of white rot basidiomycetes on chemical composition and in vitro digestibility of oat straw and alfalfa stems.

Five white rot basidiomycetes were evaluated for their potential to improve ruminal degradation of oat straw and alfalfa stems. Phanerochaete chrysosporium (PC), Scytinostroma galactinum (SG), Phlebia tremellosa (PT), Phellinus pini (PP), and Pholiota mutabilis (PM) were incubated on oat straw and alfalfa stems for 30 d at 28 degrees C and 90% relative humidity. Detergent fiber and total fiber components (neutral sugars, uronic acids, Klason lignin [KL], and ester- and ether-linked non-core lignin phenolics), core lignin nitrobenzene oxidation products, and IVDMD were determined. Electron microscopy of KMnO4-stained and cellulase/colloidal gold-labeled sections was used to monitor fungal activity. Large losses of DM were noted for all fungal species on both substrates. Lignin (KL and ADL) was removed (P less than .05) from oat straw by PC and PT treatment, but no net loss of lignin was observed for fungal treatment of alfalfa stems. Cell-wall polysaccharides were removed from both substrates by fungal activity. Only PC increased (P less than .05) IVDMD of oat straw, and SG, PT, PP, and PC treatment decreased (P less than .05) IVDMD of alfalfa stems, presumably because the fungi removed the most readily fermentable polysaccharides. Transmission electron microscopy using KMnO4 staining showed a nonselective white rot attack. Cytochemical studies using colloidal gold-labeled exo- and endocellulases were used to map the location of cellulose in the cell wall before and after decay by the white rot fungi. All the white rot fungi tested had eroded and thinned cell walls. Residual cell walls were well-labeled; both endo- and exocellulose-colloidal gold identified the cellulosic wall material that remained.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative studies of delignification caused by ganoderma species.

Isolates of six species of Ganoderma in the G. lucidum complex were evaluated for their ability to decay wood of Quercus hypoleucoides A. Camus and Abies concolor (Gord. and Glend.) Lindl. ex. Hildebr. by using in vitro agar block decay tests. Morphological, ultrastructural, and chemical studies of decayed wood were used to determine the extent of delignification or simultaneous decay caused by each species of Ganoderma. All species decayed both white fir and oak wood; however, less percent weight loss (%WL) occurred in white fir than oak. In white fir, isolates of two undescribed Ganoderma species (RLG16161, RLG16162, JEA615, and JEA625) caused significantly higher%WL (21 to 26%) than that in G. colossum, G. oregonense, G. meredithiae, and G. zonatum (10 to 16%). Only Ganoderma sp. isolates JEA615 and JEA625 caused delignification, with JEA615 causing a lignin-to-glucose gram loss ratio of 1.6:1. Morphological and ultrastructural studies confirmed delignification by this fungus and showed that some delignification had occurred by all of the species, although areas of delignification were limited to small regions adjacent to simultaneously decayed cells. In oak, G. colossum caused significantly less%WL (22 to 35%) than the other species (38 to 52%). All of the species, except G. meredithiae, caused delignification with lignin-to-glucose gram loss ratios ranging from 1.4 to 4.9:1. Extensive delignification by isolates of G. colossum and G. oregonense was observed; moderate delignification was caused by the other species. Ganoderma meredithiae caused a simultaneous decay, with only small localized regions of cells delignified, while delignification by G. zonatum was irregular, with specific zones within the cell wall delignified. The thermophilic and chlamydosporic G. colossum has the capacity to cause extensive delignification and appears ideally suited for use in lignin degradation studies and biotechnological applications of lignin-degrading fungi.

Characterization of palo podrido, a natural process of delignification in wood.

Chemical and morphological changes of incipient to advanced stages of palo podrido, an extensively delignified wood, and other types of white rot decay found in the temperate forests of southern Chile were investigated. Palo podrido is a general term for white rot decay that is either selective or nonselective for the removal of lignin, whereas palo blanco describes the white decayed wood that has advanced stages of delignification. Selective delignification occurs mainly in trunks of Eucryphia cordifolia and Nothofagus dombeyi, which have the lowest lignin content and whose lignins have the largest amount of beta-aryl ether bonds and the highest syringyl/guaiacyl ratio of all the native woods included in this study. A Ganoderma species was the main white rot fungus associated with the decay. The structural changes in lignin during the white rot degradation were examined by thioacidolysis, which revealed that the beta-aryl ether-linked syringyl units were more specifically degraded than the guaiacyl ones, particularly in the case of selective delignification. Ultrastructural studies showed that the delignification process was diffuse throughout the cell wall. Lignin was first removed from the secondary wall nearest the lumen and then throughout the secondary wall toward the middle lamella. The middle lamella and cell corners were the last areas to be degraded. Black manganese deposits were found in some, but not all, selectively delignified samples. In advanced stages of delignification, almost pure cellulose could be found, although with a reduced degree of polymerization. Cellulolytic enzymes appeared to be responsible for depolymerization. A high brightness and an easy refining capacity were found in an unbleached pulp made from selectively delignified N. dombeyi wood. Its low viscosity, however, resulted in poor resistance properties of the pulp. The last stage of degradation (i.e., decomposition of cellulose-rich secondary wall layers) resulted in a gelatinlike substance. Ultrastructural and chemical analyses of this substance showed the matrix to have no microfibrillar structure characteristic of woody cell walls but to still be rich in glucan.

Colloidal Gold Cytochemistry of Endo-1,4-beta-Glucanase, 1,4-beta-D-Glucan Cellobiohydrolase, and Endo-1,4-beta-Xylanase: Ultrastructure of Sound and Decayed Birch Wood.

Colloidal gold coupled to endo-1,4-beta-glucanase II (EG II) and 1,4-beta-D-glucan cellobiohydrolase I (CBH I), isolated from Trichoderma reesei (QM9414), and endo-1,4-beta-xylanase from Aureobasium pullulans (NRRLY-2311-1) was used successfully to determine the ultrastructural localization of cellulose and xylan in sound birch wood. In addition, these enzyme-gold complexes demonstrated the distribution of cellulose and xylan after decay by three white rot fungi, Phanerochaete chrysosporium, Phellinus pini, and Trametes versicolor, and one brown rot fungus, Fomitopis pinicola. Transverse sections of sound wood showed that EG II was localized primarily on the S(1) layer of the secondary wall, whereas CBH I labeled all layers of the secondary wall. Oblique sections showed a high concentration of gold labeling, using EG II or CBH I. Preference for the sides of the microfibrillar structure was observed for both EG II and CBH I, whereas only CBH I had a specificity for the cut ends of microfibrils. Labeling with the xylanase-gold complex occurred primarily in the inner regions of the S(2) layer, S(1), and the middle lamella. In contrast, little labeling occurred in the middle lamella with EG II or CBH I. Intercellular regions within the cell corners of the middle lamella were less electron dense and labeled positively when EG II- and xylanase-gold were used. Wood decayed by P. chrysosporium or P. pini was delignified, and extensive degradation of the middle lamella was evident. The remaining secondary walls labeled with EG II and CBH I, but little labeling was found with the xylanase-gold complex. Wood decayed by T. versicolor was nonselective, and erosion of all cell wall layers was apparent. Remaining wall layers near sites of erosion labeled with both EG II and CBH I. Erosion troughs that reached the S(1) layer or the middle lamella had less xylanase-gold labeling in the adjacent cell wall that remained. Brown-rotted wood had very low levels of gold particles present in sections treated with EG II or xylanase. Labeling with CBH I had the lowest concentrations in the S(2) layer near cell lumina and corresponded to sites with the most extensive degradation.

Detection of lignin peroxidase and xylanase by immunocytochemical labeling in wood decayed by basidiomycetes.

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.

Ultrastructural Aspects of Wood Delignification by Phlebia (Merulius) tremellosus.

Wood from aspen and birch that had been decayed for 12 weeks by Phlebia tremellosus had averages of 30 and 31% weight loss, respectively, and 70% lignin loss. Digestibility increased from averages of 21 and 13% for sound aspen and birch to 54 and 51% for decayed aspen and birch. Individual wood sugar analyses of decayed birch blocks indicated an average loss of 10% glucose, 45% xylose, and 19% mannose. Micromorphological studies demonstrated the removal of middle lamellae and separation of cells. Vessels also separated at perforation plates. Electron microscopy with OsO(4)-glutaraldehyde-fixed and KMnO(4)-fixed wood showed that lignin was progressively removed first from the secondary cell wall layers, beginning at the lumen surface, and later from the compound middle lamella. Extensive degradation of lignin was found throughout the secondary wall and middle lamella region between cells. In cells with advanced decay, the middle lamella between cells was completely degraded, but cell corner regions remained.