Fungal Automata

We study a cellular automaton (CA) model of information dynamics on a single hypha of a fungal mycelium. Such a filament is divided in compartments (here also called cells) by septa. These septa are invaginations of the cell wall and their pores allow for the flow of cytoplasm between compartments and hyphae. The septal pores of the fungal phylum of the Ascomycota can be closed by organelles called Woronin bodies. Septal closure is increased when the septa become older and when exposed to stress conditions. Thus, Woronin bodies act as informational flow valves. The one-dimensional fungal automaton is a binary-state ternary neighborhood CA, where every compartment follows one of the elementary cellular automaton (ECA) rules if its pores are open and either remains in state 0 (first species of fungal automata) or its previous state (second species of fungal automata) if its pores are closed. The Woronin bodies closing the pores are also governed by ECA rules. We analyze a structure of the composition space of cell-state transition and pore-state transition rules and the complexity of fungal automata with just a few Woronin bodies, and exemplify several important local events in the automaton dynamics.

Character evolution of modern fly-speck fungi and implications for interpreting thyriothecial fossils

PREMISE OF THE STUDYFossils show that fly-speck fungi have been reproducing with small, black thyriothecia on leaf surfaces for ∼250 million years. We analyze morphological characters of extant thyriothecial fungi to develop a phylogenetic framework for interpreting fossil taxa.METHODSWe placed 59 extant fly-speck fungi in a phylogeny of 320 Ascomycota using nuclear ribosomal large and small subunit sequences, including newly determined sequences from nine taxa. We reconstructed ancestral character states using BayesTraits and maximum likelihood after coding 11 morphological characters based on original observations and literature. We analyzed the relationships of three previously published Mesozoic fossils using parsimony and our morphological character matrix, constrained by the molecular phylogeny.KEY RESULTSThyriothecia evolved convergently in multiple lineages of superficial, leaf- inhabiting ascomycetes. The radiate and ostiolate scutellum organization is restricted to Dothideomycetes. Scutellum initiation by intercalary septation of a single hypha characterizes Asterinales and Asterotexiales, and initiation by coordinated growth of two or more adjacent hyphae characterizes Aulographaceae (order incertae sedis). Scutella in Microthyriales are initiated apically on a lateral hyphal branch. Patterns of hyphal branching in scutella contribute to distinguishing among orders. Parsimony resolves three fossil taxa as Dothideomycetes; one is further resolved as a member of a Microthyriales-Zeloasperisporiales clade within Dothideomycetes.CONCLUSIONSThis is the most comprehensive systematic study of thyriothecial fungi and their relatives to date. Parsimony analysis of the matrix of character states of modern taxa provides an objective basis for interpreting fossils, leading to insights into morphological evolution and geological ages of Dothideomycetes clades.

Fungal microflora biodiversity of healthy and diseased Adansonia digitata and Sclerocarya birrea trees in Kenya

A study was conducted in Eastern Kenya to assess incidence and severity of Adansonia digitata and Sclerocarya birrea diseases under seasonal variations, and to assess associated fungal genera and their distribution. Asymptomatic and symptomatic tissues were sampled from 175 randomly selected trees. Isolations were done from leaves, twigs and bark following laboratory standard procedures. Samples were plated on Malt Extract Agar (MEA) and incubated at 25°C for 7 days. Fungal colonies were evaluated, and pure cultures were obtained using a single hypha. Fungal pathogens were identified based on morphological characteristics of cultures and spores. Statistical analysis were done using GENSTAT version 18. Fungal morphotypes isolated included: Pestalotia (39.0%), Botryosphaeria (41.0%), Fusarium (12.0%), Alternaria (7.9%) and Cladosporium (0.1%). There were no stastically significant differences (p<0.01) in number of isolated fungi among different plant samples and sampling locations. This is the first detailed study on fungal diversity associated with diseased and healthy A. digitata and S. birrea trees in Kenya and it clearly indicates the need for detailed studies of fungal species isolated to develop mitigation strategies.

First Report of Root Rot of Chicory Caused by Phytophthora cryptogea in Chile

Chicory (Cichorium intybus L. var sativum Bisch.), a relatively new high-value crop in Chile, was introduced for commercial production of inulin. Inulins are polysaccharides extracted from chicory tap roots that are used in processed foods because of their beneficial gastrointestinal properties. Approximately 3,000 ha of chicory are grown for local processing in the BioBio Region near Chillan in south central Chile. Recently, a severe rot of 1 to 3% of mature roots in the field and after harvest has been observed in most fields, which caused yield and quality losses. Typical symptoms include a brown discoloration and a soft, watery decay of the root. Tissue pieces from symptomatic roots were placed on water agar and clarified V8 juice agar medium amended with antibiotics (1) for isolation of the causal pathogen. A Phytopthora sp. had been consistently isolated from root lesions, and axenic cultures were obtained using single-hypha transfers. The species was provisionally identified as Phytopthora cryptogea (Pethybridge and Lafferty, 1919) on the basis of morphological and cultural characteristics (1). Mycelia grew between 5 and 30°C with optimal growth at 20 to 25°C and no growth at 35°C. All isolates produced hyphal swellings and nonpapillate, persistent, internally proliferating, and ovoid to obpyriform sporangia with mean dimensions of 45 × 31 μm in sterile soil extract. The isolates were of A1 mating type because they produced oospores only when paired with reference isolates of P. cinnamomi A2 on clarified V8 juice agar amended with thiamine, tryptophan, and β-sitosterol (1) after 20 days at 20°C in the dark. On the basis of morphological and sequence data from cytochrome c oxidase subunit 1 and 2, internal transcribed spacer 2, and β-tubulin (GenBank Accession Nos. JQ037796 to JQ037798, respectively), the pathogen was identified as P. cryptogea. Pathogenicity tests were conducted using three isolates of P. cryptogea by placing a 7-mm-diameter disk from a 1-week-old V8 agar culture on 10 wounded and nonwounded healthy chicory roots (2). Control roots were mock inoculated with agar plugs. The inoculated roots were incubated at 20°C in a moist chamber. Root rot symptoms, identical to those observed both in field and storage, developed after 4 to 6 days only on wounded sites inoculated with the pathogen, and P. cryptogea was reisolated from these inoculated plants. Mock-inoculated roots remained healthy. This experiment was completed twice and similar results were obtained. To our knowledge, this is the first report of Phytophthora root rot of chicory caused by P. cryptogea in Chile. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) M. E. Stanghellini and W. C. Kronland. Plant Dis. 66:262, 1982.

First Report of a Decline and Wilt of Young Olive Trees Caused by Simultaneous Infections of Verticillium dahliae and Phytophthora palmivora in Sicily

In summer 2008, leaf chlorosis, defoliation, exceptional fruit set, twig dieback, and wilt were observed on 4-year-old olive (Olea europea L.) trees cv. Tonda Iblea in a drip-irrigated orchard in eastern Sicily. Rot of fine roots was associated with these symptoms and on ~15% of symptomatic trees rot extended to the crown and basal stem. Trees declined slowly or collapsed suddenly with withered leaves still attached. Incidence of affected trees was ~10%. A fungus identified as Verticillium dahliae Kleb. was isolated from the xylem of main roots and basal stem. An oomycete identified as Phytophthora palmivora (Butler) Butler was isolated from roots and basal trunk bark. Both pathogens were recovered from symptomatic trees with mean frequency of positive isolations per tree of 80 and 30% for V. dahliae and P. palmivora, respectively. To isolate V. dahliae, wood chips were surface disinfested in 0.5% NaOCl for 1 min and plated onto potato dextrose agar (PDA). The fungus was identified on the basis of microsclerotia, verticillate arrangement of phialides on conidiophores, and hyaline single-celled conidia. Ten monoconidial isolates were characterized by PCR using primer pairs INTND2f/INTND2r and DB19/espdef01 (3). Only 824-bp amplicons, diagnostic of the virulent, nondefoliating V. dahliae pathotype, were obtained. P. palmivora was isolated on selective medium (2) and pure cultures were obtained by single-hypha transfers. Colonies grew on PDA between 10 and 35°C (optimum at 27°C). Chlamydospores and elliptical to ovoid, papillate, caducous (mean pedicel length = 5 μm) sporangia (length/breadth ratio of 1.8) were produced on V8 juice agar. All isolates were paired with reference isolates of P. nicotianae and produced gametangia only with isolates of the A2 mating type. PCR amplicons of a representative isolate generated using primers ITS 6 and ITS 4 (1) were sequenced and found to be identical to those of a reference isolate of P. palmivora (GenBank No. AY208126). Pathogenicity of V. dahliae (IMI 397476) and P. palmivora (IMI 397475) was tested on 6-month-old rooted cuttings of olive cv. Tonda Iblea. Ten cuttings were transplanted into pots with steam-sterilized soil and inoculum of P. palmivora (4% vol/vol) produced on wheat kernels. Ten olive cuttings were inoculated with V. dahliae by injecting the stem with 150 μl of a conidial suspension (107 conidia ml–1) and 10 cuttings were stem inoculated with V. dahliae and transplanted into soil infested with P. palmivora. Controls were 10 noninoculated cuttings transplanted into steam-sterilized soil. Pots were kept in a greenhouse (25 ± 3°C) for 4 months. No aerial symptoms were observed on cuttings transplanted into soil infested with P. palmivora. However, root dry weight was reduced by 40% in comparison with the controls. Cuttings inoculated solely with V. dahliae had a 15% reduction in height compared with the controls but only four cuttings wilted. All cuttings inoculated with P. palmivora and V. dahliae wilted, indicating a synergism between the two pathogens. Controls remained healthy. Each pathogen was reisolated solely from inoculated cuttings and both pathogens were reisolated from cuttings with double inoculations. A similar syndrome ‘seca’ (drying) was reported in Spain (4). References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) H. Masago et al. Phytopathology 67:425, 1977. (3) J. Mercado-Blanco et al. Plant Dis. 87:1487, 2003. (4) M. E. Sánchez-Hernández et al. Eur. J. Plant Pathol. 104:34, 1998.

Wilt and Collapse of Cuphea ignea Caused by Phytophthora tropicalis in Italy

The genus Cuphea (Lythraceae) includes approximately 250 species of annual, evergreen perennials and short shrubs native to Central and South America. During the springs of 2003 and 2004, 10% of the nursery stock of approximately 12,000 potted cigar-flowers (C. ignea A. DC) grown in a screenhouse at a commercial ornamental nursery near Piedimonte Etneo, Sicily, had symptoms of wilt, defoliation, and rapid collapse of the entire plant. These foliar symptoms were associated with a reduced root system, browning of the collar, and dark brown discolored roots. A Phytophthora species was consistently recovered by plating small pieces of rotted roots of symptomatic plants onto selective medium (3); pure cultures were obtained by single-hypha transfers. On potato dextrose-agar (PDA), cardinal temperatures for growth were 10 to 35°C and the optimum was 28 to 30°C. Sporangiophores were umbellate or in a close monoclasial sympodium and mean dimensions of sporangia were 52 × 26 mm, with a mean length/width ratio of 2:1. Sporangia produced on V8 juice agar (VJA) were ellipsoid, fusiform, or limoniform with a tapered base. They were papillate, occasionally bipapillate, caducous, with a long pedicel (as much as 150 μm). All isolates were mating type A1 determined by pairing with A2 reference isolates of P. palmivora (Butl.) Butl. and P. nicotianae Breda de Haan. Oogonia with amphigynous antheridia were formed on VJA after 10 to 15 days at 24°C in the dark. Occasionally, 10 of 15 isolates formed small chlamydospores on VJA. Electrophoretic patterns of total mycelial proteins and four isozymes (acid and alkaline phosphatase, esterase, and malate dehydrogenase) on polyacrylamide slab gels (3) of all Cuphea isolates were very similar to those of reference isolates of P. tropicalis M. Aragaki & J. Y. Uchida from Convolvulus cneorum L. (IMI 391714) and Rhamnus alaternus L., respectively. In addition, the Cuphea isolates were clearly distinct from reference isolates of other species including P. capsici Leon., P. citricola Sawada, P. citrophthora (R. E. Smith & E. H. Smith) Leon., P. nicotianae, and P. palmivora. On the basis of morphological cultural characters and the electrophoretic phenotype, the isolates were identified as P. tropicalis. Internal transcribed spacer (ITS) regions of rDNA sequences (2) confirmed the identification. Koch's postulates were fulfilled by testing three cigar-flower isolates, including isolate IMI 391709, on 10 6-month-old potted cuttings of Cuphea inoculated by applying a 10-ml zoospore suspension (2 × 104 zoospores/ml) to the crowns, incubated for 24 h at 100% relative humidity, and maintained in the greenhouse at 20 to 24°C. After 10 days, crowns and stems were brown and all plants wilted within 20 days. Ten control plants treated with water remained healthy. P. tropicalis was reisolated from infected tissues. The test was repeated with similar results. In Europe, P. tropicalis has been reported on Cyclamen persicum Mill. in Germany (4) and C. cneorum and R. alaternus in Italy (1), indicating a broad host range and spreading in ornamental nurseries. References: (1) S. O. Cacciola et al. Boll. Acc. Gioenia Sci. Nat. 31:57, 1999. (2) S. O. Cacciola et al. For. Snow Landsc. Res. 76:387, 2001. (3) D. C. Erwin and O. K. Ribeiro. Pages 39–41, 138–139 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul MN. 1996. (4) W. W. P. Gerlach and A. Schubert. Plant Dis. 85:334, 2001.

Collar and Root Rot of Olive Trees Caused by Phytophthora megasperma in Sicily

Olive (Olea europea L.) is grown on about 154,000 ha in Sicily (southern Italy). In the summer of 1999, a few 3-year-old olive trees with decline symptoms were observed in a recently planted commercial orchard in the Enna province (Sicily). The trees were propagated on wild olive (O. europea L. var. sylvestris Brot.) rootstock. Aerial symptoms, consisting of leaf chlorosis, wilting, defoliation, and twig dieback followed in most cases by plant death, were associated with root rot and basal stem cankers. A Phytophthora sp. was consistently isolated from rotted rootlets and trunk cankers using the BNPRAH (benomyl, nystatin, pentachloronitrobenzene, rifampicin, ampicillin, and hymexazol) selective medium. Pure cultures were obtained by single-hypha transfers. The species isolated from symptomatic olive trees was identified as P. megasperma Drechsler on the basis of morphological and cultural characteristics. All isolates were homothallic, with paragynous antheridia. The diameter of oospores varied from 28 to 42 μm (mean ± SE = 36.3 ± 0.4) when they were produced on potato-dextrose agar (PDA) and from 30 to 43 μm (mean ± SE = 37.8 ± 0.4) when they were produced in saline solution. Sporangia were non-papillate. Optimum and maximum temperatures for radial growth of the colonies on PDA were 25 and 30°C, respectively. At 25°C, radial growth rate was about 6 mm per day. The identification was confirmed by the electrophoresis of mycelial proteins on a polyacrylamide slab gel. The electrophoretic banding patterns of total soluble proteins and three isozymes (esterase, fumarase, and malate dehydrogenase) of the isolate from olive were identical to those of two isolates of P. megasperma obtained from cherry and from carrot in Italy and characterized previously (1). Conversely, they were clearly distinct from the electrophoretic patterns of four isolates of P. megasperma var. sojae Hildebr. from soybean (= P. sojae Kauf. & Ger.), from those of three isolates from asparagus tentatively identified as P. megasperma sensu lato (1) and from those of reference isolates of various species producing non-papillate sporangia, including P. cambivora (Petri) Buisman, P. cinnamomi Rands, P. cryptogea Pethybr. & Laff., P. drechsleri Tucker, and P. erythroseptica Pethybr. Pathogenicity of the isolate from olive was tested in the greenhouse at 18 to 25°C using 18-month-old rooted cuttings of olive cv. Biancolilla. Cuttings were inoculated on the lower stem by inserting a 3-mm plug taken from actively growing colonies on PDA into an incision made with a sterile scalpel. The wound was sealed with waterproof tape. Agar plugs with no mycelium were placed into the stem of cuttings used as a control. The bark was stripped and lesion areas were traced and measured 60 days after inoculation. The isolate from olive produced a brown necrotic lesion (mean size = 500 mm2) around the inoculation wound and was reisolated from the lesion. Conversely, the wound healed up on control plants. P. megasperma has previously been recognized as a pathogen of olive in Greece and Spain (3). However, this is the first report of P. megasperma causing root and collar rot of olive in Italy. References: (1) S. O. Cacciola et al. Inf. Fitopatol. 46:33, 1996. (2) D. C. Erwin and O. K. Ribeiro, 1996. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN. (3) M. E. Sánchez-Hernádez et al. Plant Dis. 81:1216, 1997.