Centraalbureau Voor Schimmelcultures Research Articles

First Report of Crown Rot and Root Rot Caused by Cylindrocladium pauciramosum on Feijoa (Feijoa sellowiana) in Italy.

Feijoa sellowiana (O. Berg) O. Berg is native to South America and grown in Italy as an ornamental plant and for its fruit. During 2006, 2007, and 2008, a severe wilting was noticed on ∼ 10,000 potted 6-month to 3-year-old plants obtained from seeds and produced by three commercial nurseries in Catania Province (southern Italy). Wilting was always associated with root rot and brown discoloration at the base of the stem. Leaf spots or stem lesions were not observed. A Cylindrocladium sp. was isolated consistently from the crown, basal stem, and roots of symptomatic plants on potato dextrose agar (PDA). Ten isolates of a Cylindrocladium sp. were obtained from single-spore colonies made from a 14-day-old culture grown on PDA and subsequently subcultured on carnation leaf agar (CLA). Cultures were incubated at 25°C under near-UV light with a 12-h light/dark regimen and examined after 7 days (1). Only fungal structures occurring on the carnation leaf tissue were examined. Isolates were identified based on morphological traits and mating type responses (2,4). Isolates were mated with opposite mating tester strains of C. pauciramosum (DISTEF-G128 [MAT1-1] and DISTEF-G87 [MAT1-2]) and C. scoparium (DISTEF-GCs7 = CBS 120892 [MAT1-1] and DISTEF-GP0 = CBS 119669 [MAT1-2]) selected from an Italian collection. Plates were parafilmed, sealed in plastic bags, incubated in a chamber at 25 ± 1°C with a photoperiod of 16 h of light and 8 h of darkness, and examined weekly until perithecia developed. Successful crosses were determined after 2 months of incubation and were regarded as those isolate combinations that produced perithecia with viable ascospores. Conidia were observed to be 30 to 60 × 3.5 to 5.0 μm while conidiophores mostly had two to three series of branches and stipes terminated in obpyriform to broadly ellipsoidal vesicles, which is characteristic of C. pauciramosum C.L. Schoch and Crous (teleomorph Calonectria pauciramosa C.L. Schoch and Crous). Fertile perithecia containing viable ascospores were similar to Calonectria pauciramosa. They were obtained only from crosses of the examined isolates with mating type testers of C. pauciramosum. Six isolates (when paired with DISTEF-G128) and four isolates (when mated with DISTEF-G87) provided fertile progeny. No perithecia were obtained in pairings of the 10 isolates with tester strains of C. scoparium. One representative isolate was deposited at the Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures (DISTEF-AS4 = CBS-120618). Koch's postulates were fulfilled by inoculating 30 6-month-old seedlings of feijoa with a spore suspension (105 conidia per ml) of one isolate of the pathogen (DISTEF-AS4) grown on CLA for 14 days. The same number of seedlings was used as a control. Following inoculation, plants were maintained in a growth chamber at 25 ± 1°C and 90 to 95% relative humidity. Wilting, crown rot, and root rot symptoms identical to those observed in the nurseries appeared within 15 to 50 days. Symptoms were not observed on the control plants. C. pauciramosum was previously reported to cause leaf spots on feijoa (3). To our knowledge, this is the first report of wilting due to crown and root rot caused by C. pauciramosum in this host. The disease is a limiting factor for feijoa cultivation in Sicilian nurseries.

Sympodiomycopsis lanaiensissp. nov., a basidiomycetous yeast (Ustilaginomycotina:Microstromatales) from marine driftwood in Hawai‘i

A previously unknown basidiomycetous yeast is described for which the name Sympodiomycopsis lanaiensis is proposed. The type strain, LM418(T), was isolated from driftwood collected on a beach on Lana'i (Hawai'i). On the basis of ribosomal DNA sequence analysis [large subunit (LSU), internal transcribed spacer (ITS) 1 & 2, and 18S], LM418(T) belongs to the order Microstromatales, which includes the genera Sympodiomycopsis, Rhodotorula, Microstroma, Volvocisporium and Quambalaria. The strain is described as a new species in the genus Sympodiomycopsis on the basis of morphological and physiological characteristics and the phylogenetic relationship to Sympodiomycopsis paphiopedili. Sexual reproduction was not observed in LM418(T). GenBank accession numbers for nucleotide sequences of regions of the LSU, ITS and 18S regions of the ribosomal operon in LM418(T) are DQ990016, DQ990017 and DQ990018, respectively. LM418(T) has been deposited in the DSMZ as DSM 18755, in the ATCC as MYA-4092, in the Agricultural Research Service Culture Collection as NRRL Y-48466, and the Centraalbureau voor Schimmelcultures as CBS 10858.

Validation of PCR–reverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples

A dermatophyte-specific PCR-reverse line blot (PCR-RLB) assay based on internal transcribed sequences was developed. This assay allows the rapid detection and identification of nine clinically relevant species within the three dermatophyte genera Trichophyton, Microsporum and Epidermophyton in nail, skin and hair samples within 1 day. Analysis of 819 clinical samples (596 nail, 203 skin and 20 hair) revealed a positive PCR-RLB result in 93.6% of 172 culture-positive and microscopy-positive samples. PCR-RLB was superior to culture and direct microscopy, in both detection and species identification. Comparison of identification results of 208 PCR-positive and culture-positive clinical samples showed five discrepancies (2.4%) between PCR-RLB identification and classical microscopic/biochemical identification of isolates. Comparison of PCR-RLB identification and classical identification of 98 other isolates (dermatophytes and non-dermatophytes) revealed 13 discrepancies (13.3%) and five incomplete identifications of Trichophyton spp. Sequence analysis of ITS1 regions of 23 samples with discrepant or incomplete identification results (four Centraalbureau voor Schimmelcultures dermatophyte strains, four clinical samples and 15 clinical isolates) confirmed identification results of PCR-RLB in 21 of the 23 analyzed samples. PCR-RLB proved to be extremely suitable for routine detection and identification of dermatophytes directly in nail, skin and hair samples because it is rapid, sensitive, specific and accurate.

First Report of Seedling Damping-Off of Fennel Caused by Alternaria petroselini in the Netherlands.

In April 2005, serious seedling damping-off was noted on fennel (Foeniculum vulgare Mill. cv. Rondo) in a transplant greenhouse facility in Maasdijk, the Netherlands. Symptoms appeared 3 to 4 weeks after sowing and included black, sunken lesions aboveground on the stem and belowground on the hypocotyls. Mortality of seedlings was 6 to 10% (10 to 15 seedlings per 150-plant tray). Following removal of diseased seedlings, further transplant mortality in the field was not evident. Samples of diseased tissue were collected, surface disinfested, and placed in petri dishes containing water agar. After 7 to 10 days of incubation at 25°C under fluorescent lights, an Alternaria sp. was growing from each sample. Single conidium cultures were obtained from representative colonies and cultured on potato dextrose agar (PDA) and potato carrot agar (PCA) for morphological examination. On PDA, colonies were dark olive brown, cottony, subsurface microsclerotia production abundant, and no production of pigments in the medium. On PCA, conidia were darkly pigmented, broadly ellipsoidal to subsphaerical, and produced singly. Mature conidia were 28 to 45 × 20 to 25 μm with two to four transepta and one to three longisepta. Characteristics were consistent with those of Alternaria petroselini (2,3). In subsequent freezer-blotter assays (ISTA blotter method; www.seedtest.org ) of seed lot used in the original planting, the same fungus was recovered at an infestation level of 30%, confirming that it was seedborne. To confirm pathogenicity on fennel, pathogenicity tests were conducted on a common fennel cultivar (Floro F1) in the greenhouse and on fennel stalks in the laboratory. Fennel seeds were soaked in a conidia suspension (106/ml in sterile H2O) for 10 min. Control seeds were soaked in sterile H2O. Seeds were dried on paper, sown in soil plugs, and grown in the greenhouse at 16 to 20°C. After 4 weeks, black lesions were observed on the fennel stems and symptoms were similar to those observed on the original infected material. Control plants remained healthy. A fungus was reisolated from the lesions of symptomatic plants and was identical to the fungus isolated from the original infected material. For the fennel stalk assay, two surface-sterilized fennel stalks were sliced longitudinally and three 4-mm plugs from a 10-day-old culture of each isolate were placed along the fennel stalks. Sterile agar plugs were used as negative controls. After 7 to 10 days of incubation at 25°C in plastic boxes, test isolates grew extensively from agar plugs and resulted in extensive black necrosis of the fennel stalks. No necrosis resulted from control plugs. DNA was extracted from field isolates, and the nuclear internal transcribed spacer region was sequenced using protocol previously described (1). A representative sequence was deposited in GenBank (Accession No. EF636901). A BLAST search of the NCBI database revealed A. petroselini Accession No. AY154685 as the closest match (total score = 1,014, 100% coverage, 99% sequence identity). The next closest match was A. radicina Accession No. DQ394074 (total score 987, 100% coverage, 98% sequence identity). To our knowledge, this is the first report of A. petroselini causing disease of fennel and the fungus being seedborne on fennel seed. An isolate has been deposited at the Centraalbureau voor Schimmelcultures (Accession No. 118228).

Verticillium albo‐atrum and V. dahliae on hop

<i>Verticillium albo‐atrum</i> and <i>V. dahliae</i> on hop

First Report of Phaeoacremonium mortoniae Causing Petri Disease of Grapevine in Spain.

In May 2006, symptoms of grapevine decline were observed on 4-year-old grapevines (cv. Cabernet Sauvignon) grafted onto 110 R rootstock in Daimiel (Ciudad Real Province, central Spain). Affected vines had low vigor, reduced foliage, and chlorotic leaves. Cross or longitudinal sections of the rootstock trunk showed black spots and dark streaking of the xylem vessels. Five symptomatic plants were collected and analyzed for fungal isolation. Sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g L-1 of streptomycin sulfate. Plates were incubated at 25 to 26°C in the dark for 14 to 21 days and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were yellowish white on PDA and white-to-pale gray on MEA. Conidiophores were short and unbranched, 12.5 to 37.5 (20.5) μm long, and often consisting of a single subcylindrical phialide. Conidia were hyaline, oblong to ellipsoidal or reniform, 2.5 to 7.5 (4.6) μm long, and 1.2 to 1.9 (1.6) μm wide. On the basis of these characteristics, the isolates were identified as Phaeoacremonium mortoniae (2,3). Identity of isolate Pmo-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region (Phaeoacremonium-specific primers Pm1-Pm2) with the restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the β-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. EF517921). The sequence was identical to the sequence of P. mortoniae (GenBank Accession No. DQ173109). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using two isolates, Pmo-1 and a reference isolate of P. mortoniae (CBS-101585) obtained from the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands). Seedlings were inoculated when two to three leaves had emerged by watering the roots with 25 mL of a conidial suspension (106 conidia mL-1) harvested from 21-day-old cultures grown on PDA. Controls were inoculated with sterile distilled water. There were 20 replicates for each isolate with an equal number of uninoculated plants. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 2 months after inoculation, symptoms developed as reduced growth, chlorotic leaves, severe defoliation, and finally wilting. Control plants did not show any of these symptoms. The fungus was reisolated from internal tissues of the crown area and the stems of all inoculated seedlings, completing Koch's postulates. To our knowledge, this is the first report of P. mortoniae causing young grapevine decline in Spain.

First Occurrence of Calonectria Leaf Spot on Mexican Blue Palm in Italy.

In April 2006, a new leaf disease occurred in a private garden in eastern Sicily (Italy) on young, 2-year-old seedlings of Mexican blue palm, Brahea armata S. Watson, in the Arecaceae. Symptoms were detected on 80% of seedlings. The leaves had minute, brown spots that enlarged into dark brown, circular or elliptical lesions, 3 to 6 mm in diameter, and with a necrotic, gray center. The lesions sometimes were surrounded by a chlorotic halo, and older leaves had larger chlorotic areas between spots. Conidia, conidiophores, and terminal vesicles were examined from diseased leaves. A Cylindrocladium sp. was consistently isolated from leaf lesions on Oxoid (Basingstoke, Hampshire, England) potato-dextrose agar after surface disinfestations with 0.8% NaOCl. Cylindrocladium isolates were cultured on carnation leaf agar (CLA) using single hyphal tips. Five isolates were established and identified as Calonectria pauciramosa C.L. Schoch & Crous based on obpyriform to broadly ellipsoidal terminal vesicles, conidiophore branching pattern, conidia size (52 × 4.6 μm), perithecium morphology, and ascopores size (36 × 6.8 μm). Perithecia were obtained with C. pauciramosa tester strains from Italy (G87 and G128) and South Africa (U 971 and U 1670) (2,3) that confirmed both mating types to be present. Further confirmation was obtained by internal transcribed spacer (ITS) analysis. The sequence of rDNA ITS1-5.8 S-ITS2 regions, obtained after amplification with primer ITS1 and ITS4, revealed that the Brahea isolates showed total homology with the sequence of the C. pauciramosa (STE-U 971 from soil) (= Cylindrocladium pauciramosum) available in GenBank. Isolate CBS 120619 from Mexican blue palm was deposited at Centraalbureau voor Schimmelcultures. Spray inoculations of 10 2-year-old Mexican blue palm seedlings were performed with a spore suspension of the fungus adjusted to 105 conidia per ml obtained from 14-day-old single-spore colonies on CLA at 24°C under cool white fluorescent irradiation on a 12-h light/dark regimen. In addition, the following species were similarly inoculated using 10 1-year-old plants: Arecastrum romanzoffianum (Cham.) Becc., B. edulis H. Wendl. ex S. Watson, Chamaerops humilis L., Howea forsteriana Becc., Phoenix canariensis Hort. ex Chabaud., Trachycarpus fortunei (Hook.) H. Wendl., and Ravenea rivularis Jumelle & Perrier. Inoculated, and 10 control plants were placed in separate plastic bags in a growth chamber at 25 ± 1°C. After 7 to 10 days, foliar symptoms including flecks and spots developed on both species of Brahea and on Chamaerops humilis, and on these hosts, pathogenicity tests were repeated. Other palm species and control plants remained healthy. C. pauciramosa was consistently reisolated from inoculated plants on the basis of vesicle shape and conidia sizes of the anamorph. Cylindrocladium candelabrum, Cylindrocladium colhounii, Cylindrocladium floridanum, Cylindrocladium parasiticum, Cylindrocladium pteridis, Cylindrocladium scoparium, and Cylindrocladium theae have been reported as leaf spots pathogens of Arecaceae (1). To our knowledge, this is the first occurrence of C. pauciramosa on Mexican blue palm and the first report of the pathogen on Arecaceae.

First Report of Damping-Off of Common Oak Plantlets Caused by Cylindrocladiella parva in Italy

In April 2006, damping-off of common oak (Quercus robur L.) plantlets as much as 3-years-old was observed in a typically declining Q. robur L. forest located in northeastern Italy (Cessalto, VE). Cotyledons on seedlings were chlorotic and wilted and occasionally desiccated. Stems were stunted and discolored with yellowish brown longitudinal streaks starting from the collar. Rootlets were partly rotten and their apices frequently dead (approximately 65%). There were no fruiting bodies on or near the damaged areas. Longitudinal stem sections showed dark brown streaks in the vascular tissue, and microscopic examination revealed that vessels frequently contained mycelium. Twenty plants with these symptoms were selected, and from each of them, isolations were made from surface-sterilized (1% sodium hypochlorite) necrotic margins of stems. Lesions were cut lengthwise into two parts approximately 5 mm long, placed on potato dextrose agar, and incubated at 22 ± 1°C for 5 days in the dark. Among a variety of microorganisms, Cylindrocladiella parva (P.J. Anderson) Boesewinkel was isolated from 12 plants. The epidermis on the collars of 21-day-old, container-grown Q. robur asymptomatic seedlings was surface sterilized with 1% sodium hypochlorite, rinsed in water, and gently scraped (wounded) with a sterile scalpel. After masking the remainder of the plant with a plastic sheet, the wounds were sprayed with a conidial suspension in water (103 conidia/ml) and sealed with Parafilm. Controls were treated the same way but sprayed only with sterile water. Each treatment was replicated on 10 seedlings and incubated in the greenhouse (20 ± 2°C, 80% relative humidity, and 12 h of natural light per day). After 21 days, wounds treated with C. parva developed necrotic lesions that turned into small patches of dead epidermis forming discolored streaks. Radial sections through the stem 15 mm above the inoculation site from five plants showed the presence of mycelium in the vessels from which the fungus was reisolated. Thirty-five days after inoculation, the remaining five plants showed the same symptoms as those observed in the forest and microscopic observations confirmed the presence of the fungus. No disease symptoms or mycelium in the xylem tissue were observed in the control plants. The pathogenicity test was repeated twice with the same results. C. parva is the known causal agent of seedling blight, damping-off, and root rots on a broad host range of monocots and dicots. Detailed information on taxonomic and epidemiological features is available (1). To our knowledge, this is the first report of the disease in Italy where common oak decline is widespread (2). The isolate is preserved in the Centraalbureau voor Schimmelcultures (Baarn, the Netherlands) collection (No. 114857).

First Report of Damping-Off and Leaf Spot Caused by Cylindrocladium scoparium on Different Accessions of Bottlebrush Cuttings in Italy.

In May of 2006, approximately 10,000 cuttings of bottlebrushes (Callistemon cvs. Laevis, Hannah Ray, Kings Park Special, Masotti Mini Red, and Rose Opal with either C. viminalis (Soland. ex Gaertn.) Cheel. [excluded] or C. citrinus (Curtis) Skeels as one parent) grown in a nursery in eastern Sicily (Italy) exhibited severe disease symptoms including damping-off, leaf spots, and collar and root rot. Initially, the infections were detected on approximately 30% of the cuttings, but by late September 2006, 70% of the plants had symptoms. A Cylindrocladium sp. was consistently isolated from the diseased portions of plants onto potato dextrose agar. To determine the species, single-conidial isolates of the fungus were cultured on carnation leaf agar (CLA) for 7 days at 25°C with 12 h of light/dark conditions. Only the mycelia and spores growing on the carnation leaves were examined with a light microscope, and the isolates were identified as Cylindrocladium scoparium Morgan (teleomorph Calonectria morganii Crous, Alfenas & M.J. Wingf.) on the basis of their pyriform to broadly ellipsoidal terminal vesicles, conidiophore branching pattern, and conidia (1). In addition, the ability of the colonies to mate with South African tester strains of C. scoparium (2,3) confirmed the identification. Koch's postulates were fulfilled by inoculating 10 cuttings for each bottlebrush accession with a spore suspension (105 conidia per ml) of one isolate of the pathogen (DISTEF-GCs7) obtained from 14-day-old single-spore colonies grown on CLA at 24°C under fluorescent cool white lights with 12 h of light/dark. Following inoculation, all plants were maintained in plastic bags in a growth chamber at 25 ± 1°C and 90 to 95% relative humidity. The same number of cuttings was used as a control. Damping-off, crown root rot, and leaf spots symptoms identical to those observed in the nursery appeared within 5 to 20 days. No symptoms were detected on the control plants. C. scoparium was reisolated from the artificially infected tissues. The isolate, used in the pathogenicity proof, was deposited at the Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures (Accession No. CBS 120930). The presence of C. scoparium was detected for the first time in Italy on mastic tree in 2005 (3). To our knowledge, this is the first report of C. scoparium on bottlebrush in Italy and it represents the first information about the susceptibility of these Callistemon cultivar accessions to the fungus and confirms the spread of the pathogen in Sicilian ornamental nurseries.

First report of Phytophthora capsici on cucurbits and peppers in British Columbia

Phytophthora capsici is a devastating pathogen of several commercial cucurbit and solanaceous crops. In August 2004, P. capsici was isolated and identified for the first time in British Columbia from an infected pumpkin (Cucurbita pepo var. medullosa) purchased from a market garden in Kelowna. Visual symptoms of fruit rot were also evident on several varieties of squash (Cucurbita spp.), pepper (Capsicum annuum), and eggplant (Solanum melongena var. esculentum) from the same area. Further characterization of the causal agent was needed to confirm its identification, pathogenicity, and ability to overwinter. Eight isolates obtained from infected pumpkin and from five different squash varieties grew on selective media for P. capsici. Single-spore isolates were produced from these cultures, and DNA was extracted from them. On the basis of sequence analysis, the eight isolates were P. capsici, differing by two or fewer nucleotides in the ribosomal spacer (internal transcribed spacer 2 (ITS2)) regions compared with previously identified strains. In pathogenicity studies, isolates 2256 and 2289, originally from butternut squash (Cucurbita moschata) and pumpkin, respectively, infected sweet pepper (Capsicum annuum var. frutescens) and butternut squash, killing pepper plants in 8 days. The isolates were less pathogenic on squash, according to the area under the disease progress curve, which was approximately six times higher in pepper than squash. Phytophthora capsici was recovered from both sweet pepper and butternut squash, as well as from infected zucchini (Cucurbita pepo var. melopepo) and watermelon (Citrullus lanatus) plants. Mating studies showed that isolates 2256 and 2289 were probably the A1 mating type because they produced oospores only if they were paired with the A2 tester strain obtained from the Centraalbureau voor Schimmelcultures culture collection. A hypothesis on how the disease developed and spread in British Columbia is presented.

First Record of Crown and Root Rot Caused by Cylindrocladium pauciramosum on California Lilac in Italy.

California lilacs, or Ceanothus, are evergreen, drought tolerant, colorful ornamental shrubs belonging to the Rhamnaceae family. Ceanothus thyrsiflorus var. repens is the most common species grown in Italy. In October 2005, a severe wilting was noticed on approximately 3,000 potted, 6-month-old plants obtained from cuttings produced by a commercial nursery in Lecco Province (northern Italy). Wilting was always associated with root rot and brown discoloration at the base of the stem. No leaf spots or stem lesions were detected. A Cylindrocladium sp. was isolated consistently from crown, basal stems, and roots of symptomatic plants with potato dextrose agar. Although many crown rots are caused by Phytophthora spp., this pathogen was not found associated with rotten roots and crown plating small pieces of diseased root tissue and crowns onto selective medium. A random selection of five Cylindrocladium sp. isolates was obtained from the infected crown and basal stem. Subsequently, they were identified on carnation leaf agar (CLA) as Cylindrocladium pauciramosum C.L. Schoch & Crous on the basis of their obpyriform to broadly ellipsoidal terminal vesicles, conidiophore branching pattern, conidium, and perithecial morphology, as well as mating type with tester strains of C. pauciramosum selected isolates (1,4). All single-conidial isolates were mated with tester strains DISTEF-G87 (MAT1-1) and DISTEF-G128 (MAT1-2) of C. pauciramosum on CLA, which confirmed both mating types to be present. Two of the isolates were deposited at Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures (CBS-120145 and CBS-120146). Pathogenicity tests were performed by macerating CLA cultures of C. pauciramosum, obtained from a single-spore isolate and incorporating them on the soil surface of 20 8-month-old C. thyrsiflorus var. repens potted plants. The same number of plants was used as the control. Following inoculation, plants were well irrigated and kept in a growth chamber at 25 ± 1°C. All inoculated plants developed crown rot, basal stem rot, and root rot 25 days after inoculation. Uninoculated control plants remained healthy. C. pauciramosum was reisolated from the artificially inoculated plants. C. pauciramosum causes considerable damage to the ornamental industry in Italy, where projects have been carried out since the first record of the fungus in Europe (3). C. pauciramosum was previously detected on Ceanothus sp. in the United Kingdom (2), where foliar and stem lesions were described. To our knowledge, this is the first record of the pathogen on C. thyrsiflorus var. repens and the first report of wilting due to crown and basal stem rot.

Biodiversity of soil-inhabiting fungi

This survey is concerned only with filamentous fungi living in the soil layer. The observed diversity of soil fungi largely depends on the method used and the numbers of isolates obtained. Particle-plating usually yields higher numbers of taxa than dilution plating. The Centraalbureau voor Schimmelcultures (CBS) preserves a great diversity of soil fungi. The CBS database contained 2,210 species of soil fungi in 2001, an estimated 70% of the known species available in culture. Thus, the current estimate for described culturable soil fungi is approximately 3,150 species, many of which have a cosmopolitan distribution. Adding the ca. 150 spp. of nonculturable Glomerales results in 3,300 species of currently known soil fungi. Molecular studies in such groups as Fusarium, Trichoderma, Penicillium, and Aspergillus are finding a number of more narrowly distributed cryptic species. Thus the number of species of soil fungi is expected to be considerably higher than the 3300 species currently known.

Optimisation of membrane area and energy requirement in tubular membrane modules

Distillers’ grain (DG) is one of the lignocellulosic agricultural waste streams having a high potential for biofuel production, however it is causing health and environmental problems in Southwest region of China because of improper treatment before being used for landfill. Here, we aimed to identify fungal strains that efficiently hydrolyze DG and explored the possibility to further improve the hydrolysis of DG and other agricultural wastes by co-culturing these fungi. The tested fungal strains included 11 strains that were isolated from DG (Maotai Town, Guizhou, China) and 19 strains that were selected from the CBS collection of Westerdijk Fungal Biodiversity Institute. Aspergillus niger CBS 110.42 and CBS 554.65 and Phanerochaete chrysosporium CBS 246.84 showed overall best cellulose and xylan degrading activity, whereas high laccase and peroxidase activities were detected for Oidiodendron echinulatum CBS 113.65 and Bjerkandera adusta CBS 143380, respectively. Furthermore, A. niger CBS 554.65 co-cultured with Trichoderma reesei CBS 383.78 and A. niger CBS 110.42 with P. chrysosporium CBS 246.84 showed the best improvement (up to 2- and 3-fold higher of β-glucosidase and β-xylosidase activities). These fungi and the combinations have strong potential to be further developed for local bioenergy production.

Annual Meeting, Québec, Quebec, 2006, The Canadian Phytopathological Society / Réunion annuelle, Québec, Québec, 2006, La Société canadienne de phytopathologie

Annual Meeting, Québec, Quebec, 2006, The Canadian Phytopathological Society / Réunion annuelle, Québec, Québec, 2006, La Société canadienne de phytopathologie

Polimorfismos genéticos de aislamientos del género Malassezia obtenidos en Colombia de pacientes con lesión dermatológica y sin ella.

Malassezia spp. yeasts are opportunistic and newly emergent diseases. One or more species have been isolated in association with dermatological pathology and systemic disease. Their pathological role has not been fully elucidated since Malassezia spp. are common in normal skin flora. In the current study, a search was undertaken for genetic markers in the Malassezia spp. isolates that correlate with each type of dermatologic lesion. A total of 103 strains of Malassezia spp were isolated from patients with pytiriasis versicolor, seborrheic dermatitis, seborrheic dermatitis HIV (+), and atopic dermatitis, as well as from healthy individuals. Eight isolates from CBS Centraalbureau voor Schimmelcultures (Netherlands) were used as controls. Fingerprinting was done using random amplification of polymorphic DNA technique (RAPD) with three primers (OPA2, OPA4, OPA13). The data were analyzed with Diversity Database and SYN-TAX-PC programs. Intraspecies genetic heterogeneity in M. furfur, M. globosa, M. restricta, M. slooffiae, M. obtusa was observed, whereas M. sympodialis showed the greatest homogeneity. The dermatological disease caused by these different species was not associated with distinctive RAPD fingerprints.

Outbreak of Pitch Canker Caused by Fusarium circinatum on Pinus spp. in Northern Spain.

During the winter of 2003-2004, dieback symptoms were observed on Pinus radiata and P. pinaster in pine nurseries in Asturias (northern Spain). Small groups of affected seedlings appeared randomly distributed throughout the nurseries. The seedlings died rapidly, showing basal needle dieback, stem lesions, resin exudations, and wilting. Isolations from infected material onto potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate and Komada's medium consistently yielded Fusarium sp. cultures. The isolates were transferred to PDA and Spezieller Nährstoffarmer agar and incubated at 25°C for 10 days with a 12-h photoperiod. The cultures were identified as Fusarium circinatum Nirenberg & O'Donnell (= Fusarium subglutinans Wollenweb. & Reinking), causal agent of pitch canker disease, on basis of the presence of polyphialides and characteristic sterile, coiled, hyphae (2). To further confirm their identity, a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) based on histone H3 gene sequences (4) and a test based on the F. circinatum-specific primers, CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment of the intergenic spacer region of the nuclear ribosomal operon (3), were used. Results obtained with both techniques confirmed the morphological identification of the cultures. A representative culture has been placed in the Centraalbureau voor Schimmelcultures (CBS 117843). The pathogen was isolated only from seedlings of P. radiata and P. pinaster. Other species such as P. nigra, P. sylvestris, and Pseudotsuga menziesii, which were also grown in these nurseries, did not show symptoms. Pathogenicity was confirmed by inoculating 6- to 9-month-old P. radiata and P. pinaster seedlings. Small strips of bark (10 × 1 mm) were cut from the stems and similar sized pieces of PDA colonized by F. circinatum were placed in contact with the open wounds and covered with parafilm. Basal needle dieback was observed 10 days after inoculation that resulted in wilting of the seedlings. F. circinatum was reisolated from the affected stems fulfilling Koch's postulates. Later in the year, symptoms of pitch canker were also observed on 20-year-old P. radiata in one forest plantation in Cantabria (northern Spain). Infected branches and shoots of the trees exudated abundant resin, resulting in resinous cankers. The needles, distal to branch tip infections, wilt, fade to yellow then red, and fall from the tree. Affected trees showed noticeable crown dieback. The isolations from the cankers also yielded F. circinatum cultures that were identified as described above. Although a nonrefereed report appeared in 1998 (1), to our knowledge, this is the first report of F. circinatum on P. radiata and P. pinaster in Spain and in Europe.

Kazachstania aquatica sp. nov. and Kazachstania solicola sp. nov., novel ascomycetous yeast species

The unidentified strains AS 2.0706(T), preserved in the China General Microbiological Culture Collection Center (CGMCC), Academia Sinica, Beijing, China, and CBS 6904(T), preserved in the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands, were shown to represent two novel ascomycetous yeast species of the genus Kazachstania by 18S rDNA, internal transcribed spacer (ITS) region (including 5.8S rDNA) and 26S rDNA D1/D2 domain sequence analysis and electrophoretic karyotype comparison. The names Kazachstania aquatica sp. nov. and Kazachstania solicola sp. nov. are proposed for strains AS 2.0706(T) and CBS 6904(T), respectively. Phylogenetically, the two novel species are closely related to Kazachstania aerobia, Kazachstania servazzii and Kazachstania unispora.

First Report of Bacterial Blight of Strelitzia augusta Caused by Pseudomonas syringae pv. lachrymans

White bird of paradise tree (Strelitzia augusta Thunb.), originally from South Africa, is a tender perennial cultivated as an ornamental plant and is used in gardens in Italy. During February of 2004, a new blight disease was noticed on potted S. augusta at different ages (6 months to 4 years) in several commercial nurseries of eastern Sicily. Field inspections revealed disease incidences as high as 40%. Initial symptoms were small, water-soaked leaf spots that expanded throughout the veins in dark brown streaks. Stem cross sections revealed browning of the vascular tissues, which might involve the entire stem. In some cases, the necrosis extended to the apical bud, causing death of the plant. Thirty explants from infected tissues were washed in sterile water and plated on plate count agar (PCA) from which two types of bacterial colonies were consistently isolated. Pathogenicity tests were performed on S. augusta plants. Twenty-four plants were inoculated (12 per bacterial isolate) using two different procedures: spray with a bacterial suspension (106 CFU/ml) and wounding with an infected needle on the midribs. The same number of noninoculated plants was used as controls. All plants were maintained at 24 to 26°C with 95 to 100% relative humidity until symptoms occurred 4 days later. Just one of the two tested bacterial types was pathogenic. The symptoms were similar to those previously observed in the field. No symptoms were observed in the plants spray inoculated with the bacterial suspension, proving that the bacteria were unable to infect in the absence of a wound. The controls showed no symptoms. Koch's postulates were fulfilled by the reisolation of the infective strain which was sent to the CBS (Centraalbu-reau voor Schimmelcultures) and identified as Pseudomonas syringae pv. Lachrymans/pisi using the Biolog MicroLog3 4.01C program (Biolog Inc., Hayward, CA). Further pathogenicity tests have been carried out on zucchini and pea pods to characterize the pathovar using 48SR1 of P. syringae pv. syringae and B4 of P. syringae pv. pisi as reference strains. Necrotic, sunken, water-soaked spots surrounded by a chlorotic halo, reported in the literature as typical symptoms of P. syringae pv. lachry-mans (Smith & Bryan) Young, Dye & Wilkie (1), were observed on zucchini when inoculated with our strain. Our P. syringae strain did not cause the typical symptoms of P. syringae pv. pisi on inoculated pea pods. The results of the pathogenicity tests and the inability of the P. syringae strain isolated from S. augusta to utilize homoserine, used to discriminate pv. pisi from other pathovars of P. syringae, allowed us to identify the strain as P. syringae pv. lachrymans. Low temperature damage and late transplant may have promoted the spread of the disease in the nurseries. Under these conditions, the economic importance of this disease for the crop can be considered high. To our knowledge, this is first report of P. syringae pv. lachrymans on S. augusta. Reference: (1) K. Pohronezny et al. Plant Dis. Rep. 62:306, 1978.

Damping-Off of Beech Seedlings Caused by Fusarium avenaceum in Italy.

During May of 2004, damping-off of an estimated 70% of beech (Fagus sylvatica L.) seedlings was observed in a bare-root forest nursery located in northern Italy. Twenty-eight days after sowing, cotyledonary leaves were chlorotic, wilted, and occasionally desiccated; stem and collars appeared stunted and discolored with yellowish gray-to-brownish longitudinal streaks arising from the foot region, and rootlets were partially to completely rotten. No fruiting bodies were present on or near the damaged regions. Seedling establishment was poor within the disease foci that gradually increased in size. Longitudinal sections through the damaged stems showed dark brown streaks in the vascular tissue, and microscopic examination revealed that vessels frequently contained mycelium. Ten symptomatic plants were selected, and isolations were made from the necrotic margins of stems previously surface sterilized with 1% sodium hypochlorite and thoroughly rinsed, longitudinally cut into two parts 5-mm long, placed on potato dextrose agar (PDA), and incubated at 22 ± 1°C for 5 days in the dark. Although a variety of microorganisms were isolated, Fusarium avenaceum (Fr.:Fr.) Sacc. (1) was always recovered. Artificial inoculations with the fungus were made on 20-day-old, container-grown beech seedlings. The epidermis surrounding the collar was surface sterilized with 1% sodium hypochlorite, rinsed, and gently scraped with a sterile scalpel. After masking the rest of the plant with a plastic sheet, the wound was sprayed with a conidial suspension containing 103 macroconidia per cm3 in water and sealed with Parafilm. Controls were treated the same way but with sterile water. Each treatment was applied to 10 seedlings and incubated in the greenhouse (20 ± 2°C, 80% relative humidity, and 12 h of natural light per day). After 20 days, wounds treated with F. avenaceum showed necrotic lesions that developed into small patches of dead epidermis. Radial sections through the stem 2 cm above the inoculation site from five plants showed the presence of mycelium in the vessels, from which the fungus was reisolated. Thirty days postinoculation, the remaining five plants showed the same symptoms observed in the nursery, and microscopic observations confirmed the presence of the fungus. No disease symptoms or mycelium were observed in the inner tissues of control plants. The pathogenicity test was repeated twice with the same results. The fungus was not detected by culturing 100 surface-sterilized seeds from the same stock that had been sown in the nursery. F. avenaceum has a broad host range including angiosperms and gymnosperms, and the described symptoms are fairly typical (2). To our knowledge, this is the first report of this disease in Italy. Further research on fungal survivability in nursery soil and plant debris is in progress. The isolate is preserved in the Centraalbureau voor Schimmelcultures (NL) collection as no. 115957.

First Report of Phoma exigua as a Pathogen of Salal (Gaultheria shallon) in British Columbia, Canada.

Salal (Gaultheria shallon Pursh.) is an ericaceous, evergreen, and rhizomatous shrub that competes for nutrients and moisture with young conifers in low elevation, coastal British Columbia (BC). A survey was conducted on southern Vancouver Island, BC during the summer of 1999 to find fungal pathogens of salal that might serve as biocontrol organisms (3). Phoma exigua Desmaz. (isolate PFC2705) near Parksville, BC proved to be pathogenic on salal. Identification of PFC2705 at the Centraalbureau voor Schimmelcultures was based on morphology and ITS sequences (GenBank Accession No. AY927784). Pathogenicity was determined with 24 salal seedlings (3-month-old) by inoculating with mycelial suspensions (20% v/v) or conidial suspensions (1 × 106 conidia per ml in 0.5% potato dextrose broth). Inoculated seedlings were placed in plastic bags and incubated in a greenhouse (16 to 23°C with natural light). Plastic bags were removed after 2 days. Initial disease symptoms were observed 2 days after inoculation. Brown, sunken lesions appeared on the surface of young leaves and stems and extended quickly. All seedlings were killed within 14 days. Twelve control plants showed no disease symptoms. With diseased salal leaves incubated at 23°C with 12-h fluorescent light/dark and 100% relative humidity, pycnidia appeared on leaf surfaces within 5 days. Conidia were hyaline, ellipsoid, one-celled, sometimes two- to three-celled, 2.5 to 3.8 × 5 to 12.5 μm, with a rounded base; the colony was gray or dark gray on potato dextrose agar after 5 to 7 days. Reisolation from the inoculated diseased leaves produced a mycelial colony that shared the same growth and morphological characteristics as the initial isolate. Phyllosticta gaultheriae Ellis & Everh., a widely reported foliar pathogen of salal, is distinct morphologically from P. exigua (1). To our knowledge, this is the first report of P. exigua as a pathogen of salal in Canada (2). A voucher specimen has been deposited at the Pacific Forestry Center Herbarium (DAVFP No. 28735).