Single Ascospore Cultures Research Articles

First Report of a New Postharvest Disease of Pear Fruit Caused by Ceratocystis fimbriata in Kunming, China.

Pear (Pyrus pyrifolia (Burm.f.) Nakai) is widely planted in China and plays a key role in economy. In the autumn of 2016, five pear fruits showing symptoms of brown rot (Fig. 1A) were found in a Suancun farmer market in Kunming, Yunnan Province, China (25°02' N; 102°42' E). The incidence of this disease in postharvest pear fruits ranged from 2 % to 5 % in this city. Three fruit samples were taken to run further tests. The decayed area of the fruit was soft, brown, slightly sunken, and circular. Carrot baiting was used to isolate the pathogen from symptomatic tissue (Moller et al. 1968). Primary isolates were made by transferring ascospore drops from the tips of the perithecia formed on the carrot discs onto PDA plates. Single ascospore cultures were generated by transferring single ascospores to potatoe dextrose agar (PDA) plates. Cultures were incubated 7 days at 25°C with a 12-h light/12-h dark cycle. In culture, mycelium was initially white, turned to a shallow celadon and gradually to grey-greenish later. Measurements were made 10 days after the formation of perithecia. Six pure cultures (lik-1~lik-6) were stored at -80 °C in 15% glycerol and stored at the State Key Laboratory for Conservation and Utilization of Bio-Resources of Yunnan Agricultural University. Four isolates (lik-1~lik-4) produced ascomatal bases that were submerged in the agar. Bases (Fig. 1E) were globose, black, 192.15 to 250.81 µm wide, 192.94 to 251.31 µm long, and had straight necks terminating in ostiolar hyphae (Fig. 1F) that were divergent, hyaline, and 74.19 to 116.33 µm long. Asci were not observed. Ascospores (Fig. 1I) were ovoid, hat-shaped (dimensions 3.2 to 5.1 × 2.3 to 4.6 µm). Conidiogenous cells were with enteroblastic conidium ontogeny, flask-shaped or tubular, 65.3 to 130.6 μm long, and produced cylindrical, straight aseptate conidia (8.5 to 18 × 2.5 to 3.5 µm) (Fig.1 G). All isolates produced dark brown, 10.07 to 13.08 ×8.51 to 11.64 µm aleurioconidia (Fig. 1H). Two (lik-1, lik-3) of six isolates were used for molecular identification and genomic DNA was extracted using the CTAB method (Lee & Taylor 1990). The primers ITS1 and ITS4, EF1F and EF2R were used to amplify and sequence the rDNA-ITS and TEF-1α regions (Thorpe et al. 2005; Jacobs et al. 2004). The sequences of rDNA-ITS of the isolates lik-1 and lik-3 (GenBank Accession Nos: MF153994, MF153993) showed 99.49% similarity to AF395679 (C. fimbriata isolate CMW2219). Additionally, the TEF-1α sequences of isolates lik-1 and lik-3 (GenBank Accession Nos: KY708912, KY708915) showed 100% identify to MF347676 (C. fimbriata isolate CM18). Based on symptoms, morphological characteristics, rDNA-ITS and TEF1-α sequence analysis and pathogenicity, this fungus was identified as C. fimbriata. Pathogenicity tests were conducted using 2 isolates (lik-1, lik-3) and repeated three times. Three fresh pear fruits were disinfected with 75% alcohol, then they were wounded with a 2 mm hole punch and inoculated with 200 μL conidia suspension of the fungus (approximately 2.0 × 106 conidia / mL) on the fruit surface. After inoculation pear fruits were incubated in boxes at 25°C with a relative humidity of 80% and a 12-h light / 12-h dark cycle. Three pear fruits that served as controls were wounded by punching a 2 mm hole into the skin and inoculated with 200 μL sterile distilled water. Symptoms of rot were observed one week after inoculation (Fig.1 B). The diameter of the external lesion varied from 1.5 to 2.5 cm, on average 1.9 cm. When pears were cut, the white pulp had turned black and was rotting (Fig.1 C, D). The pathogen re-isolated from all inoculated symptomatic tissue was identical to the isolates originally obtained from the pear fruits at the market by morphology and ITS analysis. No symptoms developed on the control. The pathogenicity assay showed that C. fimbriata was pathogenic on pears. To our knowledge, this is the first report of C. fimbriata on pear in China. The spread of this disease may pose a threat to pear quality in China and further studies could be performed to determine effective disease management strategies.

Phylogenetic insights resolve Dacampiaceae (Pleosporales) as polyphyletic: Didymocyrtis (Pleosporales, Phaeosphaeriaceae) with Phoma-like anamorphs resurrected and segregated from Polycoccum (Trypetheliales, Polycoccaceae fam. nov.)

A phylogenetic analysis of nuLSU and ITS sequences representing genera previously included in Dacampiaceae indicates that the family is strongly polyphyletic and that the type species of Dacampia is placed in Pleosporales. The genus Munkovalsaria s. str. is placed in Didymosphaeriaceae (Pleosporales). Polycoccum s. str. and two species of Clypeococcum are shown to form a new lineage sister to the Trypetheliaceae in Trypetheliales and described here as Polycoccaceae. Other members of Polycoccum s. lat. are included in the Pleosporales and are closely related to lichenicolous Phoma-like species of the family Phaeosphaeriaceae. The genus Didymocyrtis is resurrected for these species and for lichenicolous species previously assigned to Diederichia, Diederichomyces, Leptosphaeria and Phoma. The genera Diederichia and Diederichomyces are synonymized with Didymocyrtis. The new combinations Didymocyrtis bryonthae, D. cladoniicola, D. foliaceiphila, D. infestans, D. kaernefeltii, D. melanelixiae, D. pseudeverniae, D. ramalinae, D. slaptoniensis and D. xanthomendozae are made, and the new name D. epiphyscia is introduced for Phoma physciicola. Some anamorph-teleomorph relationships are resolved, such as Didymocyrtis ramalinae–Phoma ficuzzae and Didymocyrtis consimilis–Phoma caloplacae, the phylogenetic results being supported by single ascospore cultures that lead to the asexual stage producing pycnidia and conidia in culture. Speciation by host switching is assumed to be important in the genus Didymocyrtis. An identification key to Didymocyrtis species is provided.

Mycosphaerella and Teratosphaeria species associated with leaf diseases on Eucalyptus globulus in southern Brazil

Leaf blight and defoliation caused by Teratosphaeria species is one of the most important leaf diseases of Eucalyptus globulus . Due to the importance of this tree species for the production of pulp and paper, and recent reports of severe leaf disease symptoms in Brazil, the present study was conducted to identify the pathogen(s) involved. Symptomatic leaves were collected in the Brazilian states of Parana and Rio Grande do Sul, single ascospore cultures established, and isolates were investigated using DNA-based molecular tools. A species-specific PCR and sequence data from the nuclear ribosomal DNA operon were used for species identification. The following seven species were identified: Mycosphaerella scytalidii , Dissoconium dekkeri (= M. lateralis ), Teratosphaeria ohnowa , T. perpendicularis , T. pseudafricana , T. flexuosa and T.nubilosa . Of the recorded species, T. nubilosa is regarded as the most serious threat to the cultivation of E. globulus in the states surveyed.

Cloning and analysis of the MAT1-2-1 gene from the traditional Chinese medicinal fungus Ophiocordyceps sinensis

The entomopathogenic fungus Ophiocordyceps sinensis has been important in traditional Chinese medicine but has yet to be commercially cultivated. One bottleneck is the very low frequency of stromata formation from artificially infected moth larvae. The mating system of fungi is the determining factor for sexual reproduction, but mating-type genes of O. sinensis have not been previously investigated. In this study, the putative mating-type gene MAT1-2-1 within the MAT1-2 idiomorph was amplified by polymerase chain reaction (PCR) and was determined to consist of 859 nucleotides that encode 249 amino acids; genes within the MAT1-1 idiomorph, however, were not determined. The MAT1-2-1 gene contained the conserved high-mobility group (HMG) box, and MAT1-2-1 flanking sequences were subsequently obtained. Although no putative open reading frames of the MAT1-1 idiomorph were detected within the ca. 8-kb flanking sequences of MAT1-2-1, a putative DNA lyase gene (which is present next to both idiomorphs in some heterothallic fungi) was found ca. 3.0 kb downstream of MAT1-2-1. The intervening distance between MAT1-2-1 and the DNA lyase gene in O. sinensis is larger than that in Cordyceps militaris and Cordyceps takaomontana. In addition, O. sinensis showed low sequence similarities with C. militaris and C. takaomontana in both MAT1-2-1 and the DNA lyase gene. In the phylogenetic tree, different MAT1-2-1 haplotypes of O. sinensis clustered together with high bootstrap support. As a single-copy gene, MAT1-2-1 was detected in all examined O. sinensis isolates including tissue cultures and single-ascospore cultures. This report describes, for the first time, a mating-type gene of O. sinensis.

First Report of Colletotrichum boninense, C. capsici, and a Glomerella sp. as Causes of Postharvest Anthracnose of Passion Fruit in Florida.

Anthracnose is an important foliar and fruit disease of passion fruit, Passiflora spp. (3). In 2008, postharvest anthracnose on purple and yellow passion fruits (P. edulis Sims and P. edulis f. flavicarpa O. Degner, respectively) from a commercial planting in Miami-Dade County, FL was examined. Lesions began as light brown areas that became papery, covered much of the fruit surface, and developed pink-to-dark sporulation. Single-conidium isolates from lesions were examined morphologically and with internal transcribed spacer (ITS) sequences. Four taxa were identified: Colletotrichum boninense (GenBank No. GU045516) with felted cream-to-orange colonies and cylindrical conidia; C. capsici (synonym C. truncatum [2]) (GU045515) with sparse, white mycelia, setose acervuli, and falcate conidia; C. gloeosporioides with fluffy white-to-gray colonies and straight, cylindrical conidia; and a Glomerella sp. (GU045517) with darkly pigmented perithecia. In two experiments, four mature, yellow passion fruit were wounded at a single equatorial site with a sterile needle and inoculated with a 15-μl drop of 0.3% water agar that did not contain (noninoculated control) or contained 105 conidia per ml of representative isolates from each taxon. After 21 days at 25°C without light, anthracnose incidence was recorded and the presence of the isolates was confirmed by their recovery from lesion margins on potato dextrose agar. Anthracnose did not develop on noninoculated control fruit. Mean incidences of anthracnose exceeded 50% for isolates of C. boninense (three from passion fruit), C. capsici (two from passion fruit), and a Glomerella sp. (two from passion fruit and one each from papaya and eugenia). Despite its common indictment as a causal agent of anthracnose on passion fruit (3), symptoms developed on only one fruit that was inoculated with an isolate of C. gloeosporioides from passion fruit (13%) and did not develop after inoculation with an isolate from papaya. Work is needed to determine whether host-specific populations of C. gloeosporioides exist on passion fruit that were not assessed during this study or whether the pathogen was misidentified in previous reports on this host. C. boninense was associated previously with postharvest anthracnose of passion fruit in Japan and Colombia, whereas C. capsici was associated with leaf anthracnose of passion fruit in Florida and Japan (4); both species are reported here for the first time as causes of postharvest anthracnose of passion fruit in Florida. Glomerella sp. caused darkly pigmented lesions and produced the teleomorph on symptomatic passion fruit and in single-ascospore cultures. Isolates with ITS sequences that are 99% homologous to those from passion fruit have been recovered in South Florida from eugenia, papaya, and Piper betle (4) and from other locations on several other hosts (GenBank); they are often nonpathogenic endophytes. Almeida and Coêlho (1) reported in Brazil a Glomerella sp. that formed the teleomorph in culture and caused anthracnose on passion fruit, but did not provide ITS sequences. Additional work is warranted on the identity and ecology of these fungi.

Two new Corollospora species and one new anamorph based on morphological and molecular data

Two new species of the genus Corollospora, namely, C. anglusa sp. nov. with its anamorph Varicosporina anglusa sp. nov. and C. portsaidica sp. nov., which were isolated from the coast of the Mediterranean Sea in Egypt, are described in this article based on morphological and molecular evidence. The two new species have one-septate ascospores. Corollospora anglusa resembles C. gracilis by having narrow one-septate hyaline ascospores; however, they differ in ascomata and ascospore dimensions and in pure culture characteristics. Single-ascospore culture of C. anglusa produces the conidia of its anamorph, whereas an anamorph has not been reported for C. gracilis. Varicosporina anglusa differs from the other two known Varicosporina species by having conidial branches that are filamentous, rectangularly branched, hypha like, and disarticulated into two- or one-celled fragments. Corollospora portsaidica is morphologically similar to C. cinnamomea, but the two species differ in the dimensions, shape, and ornamentation of the ascospores. The new Corollospora species were confirmed to be divergent from other similar Corollospora species based on phylogenetic analyses of partial sequences of the LSU rDNA region.

Two new <i>Corollospora</i> species and one new anamorph based on morphological and molecular data

Two new species of the genus Corollospora, namely, C. anglusa sp. nov. with its anamorph Varicosporina anglusa sp. nov. and C. portsaidica sp. nov., which were isolated from the coast of the Mediterranean Sea in Egypt, are described in this article based on morphological and molecular evidence. The two new species have one-septate ascospores. Corollospora anglusa resembles C. gracilis by having narrow one-septate hyaline ascospores; however, they differ in ascomata and ascospore dimensions and in pure culture characteristics. Single-ascospore culture of C. anglusa produces the conidia of its anamorph, whereas an anamorph has not been reported for C. gracilis. Varicosporina anglusa differs from the other two known Varicosporina species by having conidial branches that are filamentous, rectangularly branched, hypha like, and disarticulated into two- or one-celled fragments. Corollospora portsaidica is morphologically similar to C. cinnamomea, but the two species differ in the dimensions, shape, and ornamentation of the ascospores. The new Corollospora species were confirmed to be divergent from other similar Corollospora species based on phylogenetic analyses of partial sequences of the LSU rDNA region.

Monoclonal Antibody‐based TAS‐ELISA for Quantitative Detection of Mycosphaerella fijiensis Antigens

AbstractBlack Sigatoka caused by Mycosphaerella fijiensis Morelet is the most dangerous and devastating disease of banana around the world. Disease control is carried out by integrating cultural, genetic and chemical measures. Mycosphaerella musicola has been replaced by M. fijiensis wherever Black Sigatoka has been introduced in America and elsewhere, and remains as a significant problem at sites located at relatively high altitudes. To optimize fungicide applications for disease management a presymptomatic quantification of fungal proteins in leaves is desirable. In this study, we describe the generation of a mouse monoclonal antibody (Mab) reactive to a high‐molecular weight antigens from M. fijiensis mycelial single ascospore in vitro culture, but not reactive to mycelial antigens from M. musicola, M. musae and M. minima single ascospore cultures. A rabbit‐specific polyclonal antibody against the same M. fijiensis antigen, reactive also to fungal secreted proteins, was able to discriminate naturally M. fijiensis infected from healthy leaves as well as other concomitant fungi. The Mab was used as a capturing reagent and the polyclonal preparation as a second antibody for a triple antibody enzyme‐linked immunosorbent assay able to quantify mycelial protein antigens in a range of 10–40 μg / ml and differentiate it from healthy banana leaf extracts. The aim of this study was the analytical setting up of a Mab‐based immunoassay for the quantification of mycelial and secreted proteins of M. fijiensis. It is intended for the further establishment and optimization of an asymptomatic leaf assay for an improved forecast and warning system applied to Black Sigatoka disease management.

First Record of the Perfect Stage Calonectria pauciramosa on Mastic Tree in Italy.

Leaf spots, stem lesions, crown rot, and root rot, caused by heterothallic fungus Cylindrocladium pauciramosum C.L. Schoch & Crous,, on young plants in a nursery represent an important problem in many representative ornamental plant-growing areas of Italy since the disease was first reported in 1993 (2). Symptoms observed during surveys in Italy from 1993 to 2005 were attributed solely to the asexual stage, and the teleomorph (Calonectria pauciramosa C.L. Schoch & Crous) was never found. In October 2005, orange-to-red brown fungal fruiting bodies pertaining to the genus Calonectria were observed in belowground regions and at the soil line on collar and stem tissues of seedlings of the mastic tree (Pistacia lentiscus L., Anacardiaceae) affected by damping off in two nurseries of eastern Sicily. The sexual structures were solitary or in groups, orange-to-red brown, subglobose to ovoid. On the basis of 50 microscopic observations of leaf tissues, they ranged from 180 to 280 μm in diameter and from 270 to 400 μm high. In the presence of adequate moisture, ascospores were discharged from mature perithecia in visible, viscous, and white droplets. Asci containing eight ascospores were clavate and tapering to a long thin stalk. Fusoid ascospores were hyaline and guttulate with rounded ends, slightly curved, 1-septate, not or slightly constricted at the septum, and measured 33 to 39 μm long and 6 to 8 μm wide. However, measurements show that perithecia, asci, and ascospores also fall within the range described by Schoch et al. (3). Single ascospore cultures did not produce perithecia. On the basis of these descriptions and the ability of single-ascospore cultures to mate with two Italian (DISTEF-G87 and DISTEF-G128) and two South African (STE-U 971 and STE-U 1670) selected tester strains of Cylindrocladium pauciramosum (2), the perithecia were identified as C. pauciramosa. To our knowledge, this is the first natural occurrence of the perfect stage of Cylindrocladium pauciramosum in Italy. The presence of the teleomorph is very important because it represents a second means of spread after conidia. Furthermore, the occurrence of the teleomorph also could lead to a 1:1 mating type ratio. In this case, lower percentages of hermaphrodites should be expected (1,4).

Mating and Pseudothecial Development in Mycosphaerella citri, the Cause of Citrus Greasy Spot

ABSTRACT Greasy spot, caused by Mycosphaerella citri, is a serious disease of citrus in the Caribbean basin. M. citri is a loculoascomycete and produces pseudothecia in decomposing leaves after intermittent wetting and drying. A new in vitro mating technique was developed for production of pseudothecia on sterilized leaf disks in petri dishes. Of the single-ascospore cultures that were recovered from individual asci, four were one mating type and four were a second mating type (tentatively designated mat+ and mat-), indicating that M. citri probably is heterothallic and bipolar like most other loculoascomycetes. Most populations of ascospores recovered from individual leaves or from leaves from groves of different citrus species and various locations had a 1:1 ratio of mating types consistent with random mating. Cytological studies demonstrated that the ontogeny of pseudothecial development was similar to other loculoascomycetes. The formation of mature pseudothecia required 30 to 45 cycles of wetting and drying of infected, dead leaves which required approximately 60 to 90 days. The in vitro system for pseudothecial production and the knowledge of the mating system in M. citri will facilitate genetic studies of this important pathogen.

Detection of the "Togninia" Teleomorph of "Phaeoacremonium aleophilum" in Australia

Moist incubation of grapevine wood infected with both Phaeoacremonium aleophilum and Phaeomoniella chlamydospora yielded an ascomycete referable to the genus Togninia (Ascomycota, Calosphaeriales). Single ascospore cultures were morphologically identical to Pm. aleophilum. The rDNA ITS sequence of single ascospore isolates was identical to published sequences for the majority of Pm. aleophilum isolates. Comparison with the morphology of other wood staining Togninia species confirms that the teleomorph of Pm. aleophilum is Togninia minima.

Heterothallic life cycle in the white root rot fungus Rosellinia necatrix

To prepare homologous DNA fragments as restriction fragment length polymorphism (RFLP) markers, the genes encoding phenol oxidase, chitinase, and xylanase were amplified from genomic DNA of Rosellinia necatrix strains. RFLP analysis using the amplified DNA fragments as probe was carried out, with segregation of the markers among two sets of F1 progenies isolated from an independent perithecium. RFLP was frequently found using rpo1 as the RFLP marker among strains of R. necatrix, which was isolated from single ascospores and the circumference of the perithecium. In each set, RFLPs of some F1 progenies were different from that of the parent strain. Random amplified polymorphic DNA (RAPD) also revealed that several strains, which were of different genotypes from the parent strain, were contained in the single ascospore culture isolated from the same perithecium. From these results, it is suggested that another strain, which was genetically different, was required for mating and development of the ascus in R. necatrix. Therefore, the life cycle in R. necatrix was presumed to be heterothallism. This is the first report about a heterothallic life cycle in R. necatrix.

Didymella rabiei, the teleomorph of Ascochyta rabiei, found on chickpea stubble in Western Australia

Ascospores of Didymella rabiei were trapped from chickpea stubble containing naturally occurring pseudothecia. Ascospores produced typical Ascochyta rabiei cultures on potato-dextrose agar plus gentamicin. Ascochyta blight developed on chickpea plants inoculated with conidia derived from single-ascospore cultures. This is the first record of the teleomorph, D. rabiei, in Australia. Its presence has implications in the long distance dispersal and epidemiology of ascochyta blight.

First report of apothecia of Tapesia yallundae occurring on the wild grass Holcus lanatus (Yorkshire Fog) in New Zealand

Tapesia yallundae is a causal agent of eyespot, a damaging stem base disease of cereal crops. The pathogen has also been detected on many wild and cultivated grasses even if characteristic eyespot symptoms are not visible (Lucas et al ., 2000). The pathogen exhibits a heterothallic mating system and apothecia of the sexual stage have been detected on straw stubble in many European countries, Australasia, southern Africa and North America (Dyer et al ., 2001). During field studies in New Zealand in 1995, apothecia resembling those of T. yallundae were discovered on decaying stem bases of Holcus lanatus (Yorkshire Fog) at the margin of one wheat field near Carterton, Wairarapa, North Island. To verify that apothecia were indeed T. yallundae , ascospore discharge was induced and single ascospore cultures established. Resultant colonies were confirmed as T. yallundae on the basis of conidia and colony morphology, and growth rate (2·3 ± 0·1 mm day − 1 ) on potato dextrose agar (Dyer et al ., 1996). Isolates from H. lanatus also produced a 1·05-kb PCR product characteristic of T. yallundae following amplification with the speciesspecific primers Ty 16F and Ty 16R (Nicholson et al ., 1997). No product was obtained with primers specific to the closely related species T. acuformis . Furthermore, amplification with RAPD primer OPA-10 resulted in the production of 0·68and 1·32-kb bands, again characteristic of T. yallundae (Dyer et al ., 1996). Finally, inoculation of wheat seedlings (cv. Avalon) with conidial suspensions resulted in production of characteristic eyespot lesions. This represents the first report of apothecia of T. yallundae occurring on H. lanatus and the first evidence of infection of H. lanatus by T. yallundae . The sexual stage of T. yallundae has also been detected on the wild grasses Bromus diandrus and Hordeum leporinum (barley grass) (Wallwork, 1987). Should the sexual cycle occur regularly on wild grasses then these species may provide an important reservoir of eyespot disease able to infect nearby cereal crops as a result of the dispersal of airborne ascospores. This problem may be exacerbated by the recent establishment of grassy islands (‘beetle banks’) as wildlife refuges in UK crops.

Isolation and genotype analyses of ascospores produced between genetically different Arthroderma benhamiae strains.

Thirty-one single ascospore cultures were obtained from one ascoma produced in mating of RV 26678 (+) (Institut de Medicine Tropicale, Antwerp, Belgium) and KMU 4169 (-) (Dept. Dermatology, Kanazawa Medical University, Uchinada, Japan), which are genetically different strains of Arthroderma benhamiae. The isolation was performed with the aid of an inverted microscope and a binocular microscope. The internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene of all the ascospore cultures were analyzed by restriction fragment length polymorphism (RFLP) with the restriction enzyme HinfI. The mating types of all the single-ascospore cultures were also checked. Eight cultures had mating type (+) and RV 26678 genotype, 10 had mating type (-) and RV 26678 genotype, 6 had mating type (+) and KMU 4169 genotype and 7 had mating type (-) and KMU 4169 genotype. There was no linkage between the mating types and the genotypes, implying that the genes control the mating behavior and the genes of ribosomal RNA are on different chromosomes from each other. The hybrids comprised half of the isolates and so they were actually from the ascospores and not from the microconidia or the peridial hyphae.

Leptosphaeria maculans causing stem canker of oilseed rape in China

Leptosphaeria maculans occurs on oilseed rape (Brassica napus) as two types: the aggressive A-group and less damaging B-group (West et al., 1999). The disease occurs worldwide, but little is known of L. maculans from China. Stems with early stem lesion symptoms were collected from three field-sites in central China, Wuhan, Hefei and Guiyang, in April 1999. Pseudothecia appeared on stem surfaces after three months incubation in natural conditions. The ascospores produced in the pseudothecia (Figure 1) fit the description of Leptosphaeria maculans (Knashnobish & Shearer, 1996; Punithalingham & Holliday, 1972) and were similar to ascospores produced from UK debris (Figure 2). Pseudothecia did not develop on stems from Wuhan. Eight single ascospore cultures were made from two stems collected in Hefei, and ten were made from two stems collected in Guiyang. All isolates were classified in the B-group of L. maculans, based on colony growth rate and production of a yellow pigment (Williams & Fitt, 1999). Leaves two and four of oilseed rape plants cv. Lipton, grown in 9 cm pots, with 4 leaves expanded, were wounded with three adjacent pinpricks. Conidial suspensions (2x107 ml-1) of two isolates from Hefei (HefA1 and HefB2), two from Guiyang (Gui2b3 and Gui2a2), a UK B-group isolate (UK11) and a UK A-group isolate (96M5) were sprayed onto two plants, per isolate. Plants were grown at 15°C, initially under polyethylene to maintain high humidity. Small lesions appeared at the wound sites after 12 days on all inoculated leaves and pin-point lesions appeared on unwounded areas. Isolations from wound-site lesions confirmed the presence of the inoculated pathogen. After 14 days, some pin-point lesions expanded (Figure 3) occasionally coalescing and producing pycnidia, with a bright pink cirrus. In central China in April 1999, stem canker was found rarely but only the less aggressive, B-group was found. This, and cultural practices that result in the removal of most potential inoculum, suggests why stem canker is not currently considered to be a problem in central China.

An improved technique for obtaining single ascospore cultures of ascomycetes

A revised method for obtaining axenic, single spore cultures of ascomycetes is described. The proposed method greatly simplifies the technique for picking up single ascospores, helps to clearly distinguish different taxa occurring on the same substrate, and also enables mycologists to easily compare spore germination patterns.

The Relationship between Diaporthe perjuncta and Phomopsis viticola on Grapevines

A species of Diaporthe occurring on grapevines in Portugal was identified as D. perjuncta. The fungus was self-fertile and the teleomorph was produced on sterilized grapevine twigs inoculated with single ascospore cultures. Asci and ascospores in culture were indistinguishable from those found on the host in the field. Apart from its association with Vitis, the anamorph bears little resemblance to Phomopsis viticola but appears to be more closely related to Phomopsis oblonga. The taxonomy of the anamorph is discussed.

The relationship between Diaporthe perjuncta and Phomopsis viticola on grapevines

A species of Diaporthe occurring on grapevines in Portugal was identified as D. perjuncta. The fungus was self-fertile and the teleomorph was produced on sterilized grapevine twigs inoculated with single ascospore cultures. Asci and ascospores in culture were indistinguishable from those found on the host in the field. Apart from its association with Vitis, the anamorph bears little resemblance to Phomopsis viticola but appears to be more closely related to Phomopsis oblonga. The taxonomy of the anamorph is discussed.

A Taxonomic Reassessment of Phyllachora proteae, a Leaf Pathogen of Proteaceae

Phyllachora proteae is a well known leaf pathogen of Protea spp. In the present study this fungus was recollected from several genera and species of Proteaceae in the Western Cape province of South Africa, and its taxonomy was reassessed. Single ascospore cultures produced a Fusicoccum anamorph in culture, described here as F. proteae. A microconidial synanamorph with narrowly ellipsoidal, brown, thick-walled conidia was commonly associated with F. proteae in culture. Based on its bitunicate asci, as well as pseudothecial and ascospore morphology, a new combination for P. proteae is proposed in Botryosphaeria, as B. proteae.