Conidia In Culture Research Articles

An Outbreak of Leaf Spot Caused by Stemphylium solani on Eggplant in Malaysia

In 2011, a severe gray leaf spot was observed on eggplant (Solanum melongena) in major eggplant growing areas in Malaysia, including the Pahang, Johor, and Selangor states. Disease incidence was >70% in severely infected areas of about 150 ha of eggplant greenhouses and fields examined. Symptoms initially appeared as small (1 to 5 mm diameter), brownish-black specks with concentric circles on the lower leaves. The specks then coalesced and developed into greyish-brown, necrotic lesions, which also appeared on the upper leaves. Eventually, the leaves senesced and were shed. Tissue cut from the edges of leaf spots were surface-sterilized in 1% NaOCl for 2 min, rinsed in sterilized water, dried, and incubated on potato dextrose agar (PDA). Fungal colonies were greyish green to light brown, and produced a yellow pigment. Single, muriform, brown, oblong conidia formed at the terminal end of each conidiophore, were each 21.6 to 45.6 μm long and 11.5 to 21.6 μm wide, and contained 2 to 7 transverse and 1 to 4 longitudinal septa. The conidiophores were tan to light brown and ≤220 μm long. Based on these morphological criteria, 25 isolates of the fungus were identified as Stemphylium solani (1). To produce conidia in culture, 7-day-old single-conidial cultures were established on potato carrot agar (PCA) and V8 juice agar media under an 8-h/16-h light/dark photoperiod at 25°C (4). Further confirmation of the identification was obtained by molecular characterization in which fungal DNA was extracted and the internal transcribed spacer (ITS) region of ribosomal DNA amplified using primers ITS5 and ITS4 (2), followed by direct sequencing. A BLAST search in the NCBI database revealed that the sequence was 99% identical with published ITS sequences for two isolates of S. solani (Accession Nos. AF203451 and HQ840713). The amplified ITS region was deposited in GenBank (JQ736023). Pathogenicity testing of a representative isolate was performed on detached, 45-day-old eggplant leaves of the cv. 125066-X under laboratory conditions. Four fully expanded leaves (one wounded and two non-wounded leaflets/leaf) were placed on moist filter paper in petri dishes, and each leaflet inoculated with a 20-μl drop of a conidial suspension containing 1 × 105 conidia/ml in sterilized, distilled water (3). The leaves were wounded by applying pressure to leaf blades with the serrated edge of forceps. Four control leaves were inoculated similarly with sterilized, distilled water. Inoculated leaves were incubated in humid chambers at 25°C with 95% RH and a 12-h photoperiod. After 7 days, symptoms similar to those observed in the original fields developed on both wounded and non-wounded inoculated leaves, but not on control leaves, and S. solani was reisolated consistently from the symptoms using the same method as the original isolations. Control leaves remained asymptomatic and the fungus was not isolated from these leaves. The pathogenicity testing was repeated with similar results. To our knowledge, this is the first report of S. solani on eggplant in Malaysia.

Calonectria metrosideri, a highly aggressive pathogen causing leaf blight, root rot, and wilt of Metrosideros spp. in Brazil

SummaryThe genus Metrosideros includes several tree, shrub and vine species, native to the Pacific Islands. Seedlings from 25 seed lots of Metrosideros polymorpha and two seed lots of M. tremuloides with symptoms of root rot, stem girdling, wilting and round, purple leaf spots were observed in the Forestry Nursery at the Universidade Federal de Viçosa, Brazil. In the original disease site, seedling mortality reached up to 71% in M. polymorpha and 34% in M. tremuloides. Single conidial cultures obtained from infected leaf, root and stem samples of M. polymorpha were used to identify the fungal species. Morphological characters and DNA sequences of four loci, containing partial sequences of β‐tubulin (TUB2), histone H3 (HIS3), calmodulin (CAL) and the elongation factor (tef‐1α) genes of three isolates, indicated that they belong to a new species, described here as Calonectria metrosideri sp. nov. Potting medium infestation and inoculation of seedlings of M. polymorpha with an inoculum suspension at 1 × 104 conidia ml−1 induced typical symptoms of the disease (leaf spots, root rot and wilt), similar to those observed under natural conditions. Calonectria metrosideri was re‐isolated, which fulfilled Koch's postulates, and confirmed its status as a pathogen.

Germination and growth of Colletotrichum acutatum and Colletotrichum gloeosporioides isolates from coffee in Papua New Guinea and their pathogenicity to coffee berries

The biology and pathogenicity of Colletotrichum acutatum and C. gloeosporioides isolates from infected coffee berries with anthracnose in Papua New Guinea (PNG) was investigated in vitro and in vivo. Optimum germination in vitro occurred at concentrations of 1 x 105 conidia/mL, while germination was inhibited by 1 x 107 conidia/mL. Optimum germination of C. acutatum and C. gloeosporioides conidia occurred at temperatures between 21–29°C and 25–31°C respectively, after 18–24 h incubation periods, between pH5–pH7 and 100% RH. Maximum growth of C. acutatum occurred at 21°C and C. gloeosporioides at 25–31°C. Conidial germination increased in the presence of free water in both species. Colletotrichum acutatum produced abundant secondary conidia in culture. The process of infection of attached and detached coffee berries by conidia of C. gloeosporioides and C. acutatum was investigated and demonstrated to be equivalent for the two species. The optimum temperatures for conidia germination, appressoria formation and anthracnose development ranged between 25–31°C. Conidia germinated after 3–12 h, appressoria were formed after 6–48 h and anthracnose symptoms appeared 6 days after inoculation. Colletotrichum acutatum infected both non-wounded and wounded green and ripe red berries whereas C. gloeosporioides only infected ripe red non-wounded and wounded berries. This is the first report of C. acutatum as an etiological agent of coffee berry anthracnose.

First Report of Dieback on Hybrid Rhododendrons Caused by Neofusicoccum luteum and N. parvum in Spain.

During the conducting of Phytophthora ramorum surveys at Galician public parks (northwestern Spain) in 2010, established Rhododendron spp. plants were observed to be exhibiting leaf spots and necrosis, shoot blight, and cankers and dieback of shoots and branches. Branches and leaves of affected rhododendrons contained pseudothecia with bitunicate asci and hyaline pseudoparaphyses, and pycnidia were observed within the same stromatic masses. Symptomatic samples were disinfested in 0.5% sodium hypochlorite for 3 min. Tissues were cut from the margin of lesions, placed onto malt extract agar amended with streptomycin (25 μg ml-1), and incubated at 25°C in the dark. Cultures displaying morphological characteristics associated with Botryosphaeriaceae species were subcultured on 2% water agar with sterilized Pinus pinaster needles as a substrate and incubated at 25°C under near-UV light to encourage pycnidial production (1). Single conidial cultures gave rise to two distinct colonies on potato dextrose agar (PDA) at 25°C. In type 1, isolates produced a sparse, aerial mycelium and a characteristic yellow pigment that was more intense after 3 days, thereafter becoming violaceous and gradually turning dark gray. Growth occurred in the range of 4 to 38°C with an optimum at 29°C. Conidia were hyaline, fusiform, aseptate, thin walled, and averaged 21.1 (14.3 to 25.0) × 5.7 (4.3 to 6.8) μm with a length/width (L/W) ratio of 3.7 ± 0.4 (n = 100). On the basis of these characteristics, isolates were identified as Neofusicoccum luteum (1,3). Colonies of type 2 produced a dense, white-to-yellowish mycelium that rapidly became gray followed by marked diurnal zonation. Mycelial growth occurred in the range of 6 to 38°C with an optimum at 29 to 30°C. Conidia were hyaline, elliptical or fusiform, aseptate, thin walled, and averaging 18.3 (14.1 to 20.7) × 5.8 (4.6 to 7.0) μm with a L/W ratio of 3.2 ± 0.4 (n = 100). These isolates were identified as N. parvum (1,2). Identity was confirmed by DNA sequences analysis of internal transcribed spacer (ITS) regions. Comparison of the sequences of type 1 and 2 showed 100% homology with N. luteum and N. parvum (GenBank Accession Nos. EU673311 and GU251146, respectively). Representative sequences were deposited at GenBank (Accession Nos. HQ197352 and HQ197351). Pathogenicity of each isolate of N. luteum and N. parvum was confirmed by inoculating four 3-year-old Rhododendron spp. seedlings grown in pots. Shallow cuts were made in three branches of each plant. A colonized 6-mm agar plug, removed from the margin of an actively growing colony, was inserted beneath the flap and sealed with Parafilm. Four control seedlings received only sterile PDA agar plugs. Plants were maintained at 26°C and 70% humidity for 21 days. Inoculated plants began showing symptoms after 3 days. Necrosis progressed quickly and bidirectionally from the wound, resulting in death of leaves and wilting of shoots. N. luteum and N. parvum were reisolated from all inoculated plants but not from the controls. To our knowledge, this is the first report of N. luteum and N. parvum on Rhododendron spp. in Spain.

First Report of Pyrenophora seminiperda in Turkey and Greece.

Pyrenophora seminiperda (Brittleb. & D.B. Adam) Shoemaker (anamorph Drechslera campanulata (Lév.) B. Sutton) is a generalist seed pathogen that can cause high mortality in the seed banks of annual and perennial grasses and a minor leaf spot disease. Its current reported distribution is mainly temperate grasslands, deserts, and winter cereal-growing regions in Argentina, Australia, Canada, Egypt, New Zealand, South Africa, and the United States (2). P. seminiperda was originally described in France in the mid-1800s, but there are no recent reports from eastern Europe or Asia (2). In May of 2008, we observed what appeared to be P. seminiperda on seeds from seed bank samples collected in Turkey. Evidence of disease was observed as macroscopic black stromata protruding from the seed. The characteristic club-shaped stromata were collected from a Taeniatherum caput-medusae seed near Pamukkale, Turkey and six Bromus tectorum seeds in Love Valley near Goreme, Turkey. An additional collection from a single undispersed B. tectorum seed was obtained from Perissa, Greece. Identity of the pathogen was tentatively established by evaluating morphological characteristics for nine isolates in V8 agar culture. After 4 days of incubation at 20°C with a 12-h photoperiod, the cultures produced white mycelium. Following wounding, the mycelium produced black, club-shaped stromata (2 to 8 × 0.4 to 0.9 mm) in a radial pattern. These produced branched conidiophores bearing crescent-shaped, multicellular conidia (79 to 125 μm long). These attributes are consistent with those of the anamorph of P. seminiperda as described by Shoemaker (4) and Campbell et al. (1). The teleomorph was not observed. The identity of the isolates as P. seminiperda was confirmed with ribosomal DNA internal transcribed spacer (ITS) genetic sequencing analysis. ITS sequences obtained were identical to sequences for North American haplotypes of this species. Four of the Love Valley isolates, (representative isolate: GQ168725, BPI 879142, NRRL 54032) matched the HTA haplotype (GQ168724), while the other four (representative isolate: GQ168736, BPI 879143, NRRL 54033) matched the HTJ haplotype (GQ168735). The isolate from Perissa, Greece (GQ168728, BPI 879144, NRRL 54034) matched the HTC haplotype (GQ168727). Pathogenicity of several Love Valley isolates was confirmed by producing conidia in culture, dusting nondormant B. tectorum seeds with 0.003 g of conidial inoculum per 50 seeds, and incubating for 14 days at 10/20°C with a 12-h photoperiod. Stromata developed on >90% of inoculated seeds and mortality as high as 34% was observed. Morphological similarities combined with ITS sequence data provide conclusive evidence that P. seminiperda occurs in Turkey and Greece. The discovery of this pathogen in these countries indicates that it may be widespread in Eurasia and that it could have arrived in North America on seeds of B. tectorum (3) rather than representing a novel pathogen for this important weed in its North American range.

Characteristics of Hypovirulent Strains of Chestnut Blight Fungus, Cryphonectria parasitica, Isolated in Korea

Chestnut blight disease caused by Cryphonectria parasitica is widely distributed throughout chestnut tree plantations in Korea. We surveyed 65 sites located at 9 provinces in South Korea, and isolated 248 virulent and 3 hypovirulent strains of chestnut blight fungus. Hypovirulent strains had dsRNA virus in the cytoplasm, which is one of the typical characteristics of hypovirulent strains. In addition, they showed more characteristics of hypovirulent strains, i.e., suppressed conidiation, reduced pigmentation in colony color, and reduced phenol oxidase activity as well as reduced pathogenicity. Hypovirulent strains, KCPH-22, KCPH-135 and KCPH-136, had a genomic dsRNA band with the molecular weight of 12.7 kb, which is the L-dsRNA of CHV1. They also had a 2.7 kb defective dsRNA band. Single conidia isolated from hypovirulent strains were cultured and various phenotypes and absence of dsRNA bands were obtained from single conidial cultures, which means that hypovirulenee transmission is unstable in asexual reproduction and variations in viral heredity by asexual reproduction. Bioeontrol trial using hypovirulent strains was also carried out in the chestnut tree plantations, and canker expansion in the treated trees was stopped and healed by callus formation at the margin of the canker. These results show the potentials in successful biocontrol of chestnut blight if the vegetatively compatible hypovirulent strains could be directly used around the canker formed by compatible virulent strains.

Paecilomyces farinosus destroys powdery mildew colonies in detached leaf cultures but not on whole plants

Since 2001, several isolates of Blumeria graminis, the causal agent of cereal powdery mildew, maintained on detached leaves at the John Innes Centre, Norwich, UK, have spontaneously become infected with an unknown filamentous fungus whose mycelia have quickly overgrown the powdery mildew colonies and destroyed them completely. A total of five isolates of the contaminant were obtained and identified as Paecilomyces farinosus based on morphological characteristics and rDNA ITS sequence data. To determine whether these P. farinosus isolates can be considered as biocontrol agents (BCAs) of powdery mildews, we studied the interactions between P. farinosus and the following four powdery mildew species: B. graminis f.sp. hordei infecting barley, Oidium neolycopersici infecting tomato, Golovinomyces orontii infecting tobacco and Podosphaera fusca infecting cucumber. The powdery mildew colonies of all these four powdery mildew species were quickly destroyed by P. farinosus in leaf cultures but neither conidial suspensions nor cell-free culture filtrates of P. farinosus isolates could suppress the spread of powdery mildew infections on diseased barley, tomato, tobacco or cucumber plants in the greenhouse. It is concluded that P. farinosus cannot be considered as a promising BCA of powdery mildew infections although it can destroy powdery mildew colonies in detached leaf cultures and can be a menace during the maintenance of such cultures of cereal, apple, cucurbit and tomato powdery mildew isolates.

Molecular cloning ofAbGst1encoding a glutathione transferase differentially expressed during exposure ofAlternaria brassicicolato isothiocyanates

The AbGst1 gene encoding a glutathione transferase from the necrotrophic pathogen Alternaria brassicicola was cloned from a benzyl isothiocyanate-treated conidial culture using differential display reverse transcription. The deduced amino-acid sequence of AbGst1p showed a significant degree of similarity to glutathione transferase-I from Saccharomyces cerevisiae and glutathione transferase-III from Schizosaccharomyces pombe. The transcription of AbGst1 was significantly enhanced by isothiocyanates, heavy metals and 1-chloro-2,4-dinitrobenzene. However, no significant transcript response was obtained with superoxide-generating menadione and paraquat. Recombinant AbGst1p expressed in Escherichia coli exhibited high transferase activity with allyl and benzyl isothiocyanates as substrate compared with 1-chloro-2,4-dinitrobenzene, but no peroxidase activity was detected. AbGst1 was upregulated in planta during the first day postinfection, suggesting the potential involvement of this enzyme in isothiocyanate detoxification mechanisms during host plant infection.

Cordyceps bassiana and Production of Stromata in vitro Showing Beauveria Anamorph in Korea

A Cordyceps species was found with a Beauveria anamorph state on larval insect cadavers on Obong mountains in Gangwon Provinces, Republic of Korea. Cultures from discharged aseospores formed an anamorph identifiable as Beauveria bassiana. This teleomorph-anamorph connection was also confirmed by the in vitro production of fertile aseomata from conidial cultures with morphology like that of lield-eolleeted specimen. This is the first report of in vitro production of a teleomorph for any Beauveria species. The Cordyceps species has been eonspeeilied as Cordyceps bassiana, a species described from China with B. bassiana anamorph.

Botryosphaeria species from Eucalyptus in Australia are pleoanamorphic, producing Dichomera synanamorphs in culture

Species within the genus Botryosphaeria include some of the most widespread and important pathogens of woody plants, and have been the focus of numerous taxonomic studies in recent years. It is currently accepted that anamorphs of Botryosphaeria belong to two distinct genera, Fusicoccum and Diplodia. Species within the genus Fusicoccum commonly produce aseptate, hyaline conidia. In the present study, fungi were isolated from foliage and wood of Eucalyptus in native forests and plantations in Australia. Although these fungi produced Dichomera anamorphs in culture, they clustered within the Fusicoccum clade of Botryosphaeria based on their ITS sequence data. Four species, Botryosphaeria dothidea, B. parva, B. ribis and B. australis produced Dichomera conidia in culture. The Dichomera synanamorphs are described for these four species of Botryosphaeria. In addition, falling within the Fusicoccum clade of Botryosphaeria, two species were found to be distinct from previously described Botryosphaeria spp. based on their ITS sequences, but synonymous with D. versiformis and D. eucalypti. These observations are currently unique to isolates from host trees within the genus Eucalyptus in Australia, and the pleoanamorphic nature of these species is discussed.

Classification of the guava wilt fungus Myxosporium psidii, the palm pathogen Gliocladium vermoesenii and the persimmon wilt fungus Acremonium diospyri in Nalanthamala

Psidium guajava wilt is known from South Africa, Malaysia and Taiwan. The fungus causing this disease, Myxosporium psidii, forms dry chains of conidia on surfaces of pseudoparenchymatous sporodochia, which develop in blisters on bark. Similar sporodochia are characteristic of Nalanthamala madreeya, the type species of Nalanthamala. Nalanthamala, therefore, is the appropriate anamorph genus for Myxosporium psidii, while Myxosporium is a nomen nudum (based on M. croceum). For M. psidii the combination Nalanthamala psidii is proposed. Nalanthamala psidii, the palm pathogen Gliocladium (Penicillium) vermoesenii, another undescribed anamorphic species from palm, two species of Rubrinectria and the persimmon pathogen Acremonium diospyri are monophyletic and belong to the Nectriaceae (Hypocreales) based on partial nuclear large subunit ribosomal DNA (LSU rDNA) analyses. Rubrinectria, therefore, is the teleomorph of Nalanthamala, in which the anamorphs are classified as N. vermoesenii, N. diospyri or Nalanthamala sp. Nalanthamala squamicola, the only other Nalanthamala species, has affinities with the Bionectriaceae and is excluded from this group. Rubrinectria/Nalanthamala species form dimorphic conidiophores and conidia in culture. Fusiform, cylindrical, or allantoid conidia arise in colorless liquid heads on acremonium-like conidiophores; ovoidal conidia with somewhat truncated ends arise in long, persistent, dry chains on penicillate conidiophores. No penicillate but irregularly branched conidiophores were observed in N. diospyri. Conidia of N. psidii that are held in chains are shorter than those of N. madreeya, of which no living material is available. Nalanthamala psidii and N. diospyri are pathogenic specifically to their hosts. They form pale yellow to pale orange or brownish orange colonies, respectively, and more or less white conidial masses. Most strains of Rubrinectria sp., Nalanthamala sp. and N. vermoesenii originate from palm hosts, form mostly greenish or olive-brown colonies and white-to-salmon conidial masses. They form a mono-phyletic clade to which Nalanthamala psidii and N. diospyri are related based on analyses of the internal transcribed spacer regions and 5.8S rDNA (ITS rDNA), LSU rDNA, and partial β-tubulin gene. Few polymorphic sites in the ITS rDNA and β-tubulin gene indicate that Nalanthamala psidii comprises two lineages, one of which has been detected only in South Africa.

Long-term effects on other fungi are studied in biological and chemical stump treatments in the fight against Heterobasidion annosum coll

The effects on mycoflora of Norway spruce stumps of biological and chemical treatments in the fight against Heterobasidion annosum coll. were investigated two yr after applications of these treatments. The biological treatments were Hypholoma fasciculare, Phanerochaete velutina, Vuilleminia comedens, Trichoderma harzianum and both the conidial suspension and culture filtrate of Verticillium bulbillosum; propiconazole was used as chemical treatment. Samplings were performed on 130 stumps, including controls with (C1) and without (C2) an autologous wood disk. Forty-nine fungal taxa were isolated, and most were Deuteromycetes. Trichoderma harzianum significantly reduced the number of taxa versus controls (three versus 25), while the other treatments showed more limited qualitative and quantitative effects. Cluster and correspondence analysis differentiated three groups of treatments: one including the three Basidiomycetes, V. bulbillosum and C1; one comprising propiconazole and C2; and one composed of the treatment with T. harzianum only. Because the same stumps already had been sampled one yr after treatments in a similar study, comparisons between data were possible and were very useful in the investigation of the temporal evolution of the effects of each treatment. Multivariate analysis showed that the strong effects of T. harzianum on stump mycocenoses increased over time. Transient effects were shown in most treatments (i.e., the three Basidiomycetes), whereas V. bulbillosum had the least impact on naturally occurring mycoflora.

First report of the pitch canker fungus, Fusarium circinatum, on pines in Chile

Pitch canker, caused by Gibberella circinata (Fusarium circinatum), is currently regarded as one of the most serious diseases of pines in the world. The disease was first discovered in the south-eastern United States in the early 1900s and subsequently, has caused sporadic occurrences of severe disease in that area. More recently, the disease appeared in California where it is currently causing a serious epidemic on native Pinus radiata, which is of international concern (Gordon et al., 2001). Pinus radiata forms the basis of extensive areas of exotic forest plantation in many countries in the Southern Hemisphere. For this reason, pitch canker is viewed as one of the greatest threats to globally relevant forest industries in these areas. Consequently, various international programmes have been established in an attempt to reduce this threat (Devey et al., 1999). During the course of recent surveys in the Concepción area of Chile, dying P. radiata plants have been discovered in both containerized and open rooted clonal hedges. Plants appear to die rapidly and typically display resin exudation from the root collar areas and pitch-soaked wood associated with these lesions. Isolations from symptomatic tissue have consistently yielded cultures of Fusarium belonging to the Fusarium subglutinans species complex. Isolates have sterile hyphal coils strongly reminiscent of F. circinatum. Single conidial cultures paired with mating tester strains of the ‘H’ mating population (Britz et al., 1999) consistently showed sexual compatibility. To further confirm their identity, a PCR-RFLP test based on histone H3 gene sequences (Steenkamp et al., 1999) was used on six isolates to compare them with members of the F. subglutinans species complex. This technique reliably distinguishes F. circinatum from other species in this complex. Results confirmed morphological comparisons and mating compatibility tests showing that the fungus associated with dying plants in Chile is F. circinatum. Although F. circinatum is causing damage to P. radiata plants in Chilean nurseries, it has as yet, not been found on trees in plantations. In this sense, the situation is similar to that in South Africa, where the pathogen causes serious damage to seedlings in nurseries but is not associated with typical symptoms of pitch canker. The fact that P. radiata is highly susceptible to infection by F. circinatum and that this tree forms the basis of the local forestry is of significant concern. This concern is heightened by the fact that various insect pests of P. radiata are present in Chile that are capable of providing infection sites for the pitch canker fungus. Consequently, various strategies linked to tree breeding and selection have been established to minimise the long-term threat of pitch canker in Chile.

Report of Leaf Spot of Spinach Caused by Stemphylium botryosum in Maryland and Delaware.

In October 2000, leaf spot symptoms were observed on spinach (Spinacia oleracea L. 'Seven R') at the University of Maryland Lower Eastern Shore Research and Education Center in Salisbury. In April 2001, a similar leaf spot disease was observed in two commercial spinach fields (cv. Vancouver) in Dorchester County, MD, and Sussex County, DE. Symptomatic plants occurred in foci, and overall disease incidence in the research and commercial fields was <10% of plants with lesions. However, low disease incidence may reduce the value of a spinach crop by requiring additional hand-sorting (fresh market) or lowering the grade (processing). Leaf spot lesions were small (0.2 to 0.7 cm), circular, tan, and papery and lacked visual signs of fungal infestation. Lesions resembled a new leaf spot of spinach reported in California (1) caused by Stemphylium botryosum Wallr. Plating surface-disinfested lesion margins on 0.25-strength potato dextrose agar consistently yielded S. botryosum. Single conidial cultures of three isolates were grown on V8 agar in a growth chamber with a 12 h light/dark regime at 21°C and were used for the pathogenicity test. Conidia were collected from 7-day-old colonies to test pathogenicity. Conidia were suspended in distilled water (1.1 × 105 conidia per milliliter), and sprayed on 4-week-old spinach plants (with four to six true leaves) of cvs. Seven R, Vancouver, and Melody. Noninoculated control plants were sprayed with deionized water. Plants were incubated for 72 h in a dew chamber (18°C, 9 to 15 h light/dark regime where dew formed during the dark periods) and then placed on a greenhouse bench (23°C) for 2 weeks. Plants that had been inoculated with any of the three isolates developed the aforementioned leaf spot lesions after 4 days in the greenhouse. Plants sprayed with deionized water were symptomless. One week after inoculation, more lesions were observed on 'Seven R' and 'Vancouver' than on 'Melody' (41, 39, and 1 lesion per plant, respectively; P< 0.0030), and the lesions were 1.5, 1.2, and 0.5 mm in diameter, respectively (P< 0.0001). S. botryosum was consistently reisolated from leaf spot lesions. The pathogenicity test was repeated with similar results. Isolates grown on V8 agar and incubated for ≈10 days produced conidia with mean dimensions of 31 × 19 μm. To our knowledge, this is the first report of leaf spot of spinach caused by S. botryosum in Maryland and Delaware. Reference: (1) S. T. Koike et al. Plant Dis. 85:126, 2001.

First Report of Colletotrichum acutatum in Strawberry in Norway.

Anthracnose caused by Colletotrichum acutatum J. H. Simmonds was detected in strawberry (Fragaria × ananassa Duch.) for the first time in Norway in 1999. Symptoms were found in greenhouse grown strawberries in the cultivar Korona. Symptoms were typical of strawberry anthracnose: sunken, brown, and firm lesions appeared on maturing fruits. Masses of conidia were produced in acervuli in the center of lesions. The fungus was isolated on acidified potato dextrose agar. Colonies grown on potato dextrose agar (PDA) were pale to mouse gray and became dark greenish to blackish in reverse. Conidia were formed in orange to salmon pink masses in the center of the culture. Conidia in cultures were 16.5 (13.8 to 18.8) × 4.5 (3.8 to 5) μm, and were hyaline, cylindrical, with pointed ends, and aseptate. Setae were never observed in culture or on fruits. The fungus did not form an ascigerous stage in culture. Mycelial growth rate at 25 to 26°C on PDA was 8.1 to 8.4 mm per day. Morphological characters and growth rate were in accordance with previous reports on C. acutatum (1,2). The isolated fungus was confirmed to be C. acutatum by both the International Mycological Institute, Egham, England, and Centraalbureau voor Schimmelcultures, Baarn, the Netherlands. Koch's postulates were fulfilled by inoculating ripe and unripe fruits on strawberry plants with the isolated fungus. Fruits were either sprayed with a conidial suspension (106 conidia per ml) or slightly wounded with a needle that had been dipped in a conidial mass from a pure culture of C. acutatum. Symptoms appeared after 4 days at 20°C, and after 5 days, brown, sunken, circular lesions reached a size of 1 cm in diameter on wounded, ripe fruits. In unripe fruits the lesions developed more slowly, and in unwounded fruits sprayed with a conidial suspension, large, irregular spots developed. Leaves were inoculated by placing a small block of agar at the base of petioles on intact strawberry plants. The tissue underneath the agar was either unwounded or slightly wounded with a needle. After 20 days (at 20 to 25°C) some necrosis developed on both unwounded and wounded petioles. No symptoms were observed in the crown tissue where the inoculated petioles were attached. The fungus was readily reisolated from both fruits and petioles, after which typical morphological characters developed in culture as described above.

Hyperdermium: a new clavicipitalean genus for some tropical epibionts of dicotyledonous plants

The morphological characteristics of two tropical clavicipitalean fungi on species of the plant family Asteraceae were examined. These species were found to be superficial on the stems of living plants but early stages of infection involve necrotrophy of scale insects. These fungi are distinct from other Clavicipitaceae by possession of anamorphs producing multiseptate conidia. A new genus, Hyperdermium, was proposed to accommodate these two species. In cultures on potato dextrose agar overlaid with cellulose acetate sheets, both species produced colonies resembling those on plant tissues. Hyperdermium bertonii produced conidia in culture but not perithecia. Hyperdermium pulvinatum produced both conidia and perithecia in culture. Phylogenetic analysis using large subunit rDNA sequences placed Hyperdermium in the subfamily Cordycipitoideae. A study was made of perithecial ontogeny in H. pulvinatum. Centrum development was of the Epichloë-type.

Accessory conidium production in three species of Harposporium and an evaluation of nematophagous members of the genus

Harposporium arcuatum, H. dicorymbum and H. subuliforme were isolated from nematodes and grown in pure culture. All three species produced accessory conidia in culture and two species also produced arthroconidia. A comparative study of spore production within nematophagous members of the genus Harposporium was made.

Bipolaris multiformis (Jooste) Alcorn Pathogenic on Leaves of Cucumis sativus in Italy

AbstractA destructive disease was observed in August 1981 in a field of Cucumis sativus cv. Radar. Sincc then sporadic spots on leaves of the same cultivar were recorded in 1985, 1988 and 1993. The causal agent was identified in Bipolaris multiformis (Jooste) Alcorn. The fungus produces frequently irregular (mostly Y‐shaped) conidia in culture but rarely on leaves. The pathogen germinates bipolarly in distilled water at 20–25°C (95 and 90% respectively), and on leaves it produces appressoria;, entry points are either stomata or the intact surface. Pathogenicity tests carried out on various cucurbitaceous plants showed symptom intensity decreasing with the following order: C. sativus cv. Radar and Eeballium elaterium &gt; Brionia cretica subsp. dioica &gt; Cueurbita pepo L. var. pommiformis and C. pepo var. patissonina f. radiata &gt; C. moscata and Sechium edule. The other two tested cultivars of Cucumis sativus (Mezzolungo Marketer and Corto per sottaceti) and C. pepo cv. Verde scuro di Milano showed sterile, very small necrotic dot. Control plants, Citrullus lanatus cv. Sugar baby, Cucumbis melo and Lagenaria siceraria showed no symptoms.

Virulence of Four Fungal Pathogens to Coptotermes formosartus (Isoptera: Rhinotermitidae)1

Four species and 10 isolates of entomogenous fungi were tested for virulence against the Formosan subterranean termite, Coptotermes formosanus Shiraki. Five isolates of Beauveria bassiana (Balsamo) Vuillemin and three of Metarhizium anisopliae (Metschnikoff) Sorokin were virulent to C. formosanus. The isolates of these two fungal species were chosen because they originated from different phylogenetic groups of host insects. The B. bassiana isolates originated from hosts in the Orthoptera, Lepidoptera, Homoptera, Dermaptera, and Isoptera; the M. anisopliae isolates came from hosts in the Coleoptera, Isoptera, and Homoptera. The B. bassiana isolate from an isopteran was most virulent to C. formosanus, when LD50 and mean time until death were both considered. There was no other pattern in virulence of isolates relative to the phylogeny of their original insect hosts. All five B. bassiana isolates and one of the three M. anisopliae isolates produced full growth of external mycelium and conidia on the dead termites. Termites exposed to fungal doses killing &amp;gt; 67% of the insects died more quickly than those exposed to doses killing &amp;lt; 67%. Metarhizium flavoviride Gams and Rozsypal was pathogenic to C. formosanus but had low virulence (1% infection in bioassays). Termites that walked across a hyphal mat of Conidiobolus coronatus (Constantin) Batko suffered 100% mortality within 2d, but this fungus did not produce conidia in culture on media.

Cultural studies on the genus Termitomyces in South Africa. I. Macro- and microscopic characters of basidiome context cultures

Characteristics of basidiome context cultures of five South African species of Termitomyces were investigated, namely T. umkowaani, T. reticulatus, T. sagittaeformis, T. clypeatus and T. microcarpus. Microscopic characters of the cultures were very similar but macroscopic characters differed markedly. It was possible to distinguish between different species by relying strictly on macroscopic characters. Growth characters did not change when the nutrient medium and incubation conditions were standardised, and proved to be a reliable taxonomic criterion for the species under investigation. With the exception of T. microcarpus, all the species produced conidiophores and holoarthric conidia in culture with numerous aged, inflated, ungerminated conidia (sphaerocysts). With the exception of T. clypeatus, conidiophores were aggregated into spherical, farinaceous sporodochia which resembled comb sporodochia. Conidiophores of T. clypeatus were closely compacted to form synnematous structures. Cultures of T. microcarpus exhibited typical basidiomycetous growth characters. However, they differed significantly from cultures of the other species which, unlike T. microcarpus, formed conidiophores and produced a raised, tough, cerebriform mycelium mat which could be considered a stroma. It is suggested that T. microcarpus should be transferred to the genus Podabrella Singer.