Epicoccum Species Research Articles

High Diversity of Epicoccum Species Associated with Leaf Spot on Italian Ryegrass in Southwestern China: Six New Records and Three New Species.

Italian ryegrass is widely cultivated for the production of forage, hay, and silage because of its high nutritional value and good palatability. Leaf spots caused by fungi pose a serious threat to forage crops. In order to expand the knowledge of fungi causing leaf spots in ryegrass (Lolium multiflorum) in Sichuan, Yunnan, Chongqing, and Guizhou of southwestern China, a comprehensive survey was undertaken from 2015 to 2022. The survey discovered that Epicoccum leaf spot (ELS) was a common and widespread disease, more serious at the late stage of growth (after late May). Symptomatic leaf samples collected from the four different provinces were analyzed, and a total of 202 Epicoccum isolates were obtained. Based on both multilocus phylogeny (ITS, LSU, TUB2, and RPB2) and morphology, 10 Epicoccum species were finally identified, including three novel species (E. endololii sp. nov., E. lolii sp. nov., and E. loliicola sp. nov.), six new host records (E. draconis, E. endophyticum, E. oryzae, E. plurivorum, E. thailandicum, and E. tobaicum), and an unknown species (Epicoccum sp.1). Pathogenicity tests showed that E. endophyticum, E. endololii, and Epicoccum sp.1 were nonpathogenic to Italian ryegrass, which were confirmed as endophytes in this study; the other six species could infect Italian ryegrass and cause leaf lesions to different degrees, of which E. draconis was more aggressive (P ≤ 0.05). Coupled with the isolation rates and geographical distributions of these species, it was found that E. plurivorum was the predominant pathogen in Yunnan while E. oryzae and E. tobaicum were the predominant pathogens in the other three provinces. This work provides an initial understanding of the taxonomy, virulence, and distribution of Epicoccum species associated with ELS in southwestern China and lays a solid foundation for the diagnosis in the field and scientific control of ELS on Italian ryegrass.

First report of leaf spot caused by Epicoccum latusicollum on Hemerocallis citrina in China.

Hemerocallis citrina is a popular vegetable crop. Its eatable flower buds contain abundant nutrients, especially lecithin (Guo et al., 2022). In March 2021, leaf spot disease was observed on 90% cultivated H. citrina seedlings in Dazhou city (31°17'56″ N, 107°31'59″ E), Sichuan, China. Totally, 15 diseased seedlings were sampled (three samples per 666 m2). The symptomatic leaves were cut into pieces (5 × 3 mm), superficially disinfected with 70% ethanol for 20 s and 1% Sodium hypochlorite (NaClO) for 40 s, and washed with sterile distilled water six times. The disinfected tissues were incubated on PDA amended with streptomycin sulfate (50 mg/L) in dark at 25 ℃. Two days later, hyphal tips from the edges of growing colonies were transferred to fresh PDA plates. Finally, 40 purified isolates were obtained. Using primer pairs ITS1/ITS4 (Glass & Donaldson, 1995), amplified rDNA internal transcribed spacer (ITS) regions indicated that these isolates belonged to different genera, mainly including Epicoccum, Fusarium and Colletotrichum. Six isolates of Epicoccum genus similar in morphology, named HHC46, HHC47, HHC491, HHC492, HHC51 and HHC58, were selected for identification. Cultured on oatmeal agar for 7 days, colonies were initially white and villose. Fourteen days later, mycelia started to secrete scarlet pigment. The NaOH spot test showed color changed from green to red, identical to that in Epicoccum species (Boerema et al., 2004). Meanwhile, colonies produced abundant conidia. Conidia were ellipsoidal, aseptate, and 4.1 to 6.5 × 1.3 to 2.9 µm (n = 30). Chlamydospores were also observed, globose to subglobose. The morphological features were similar to those of Epicoccum latusicollum (Xu et al., 2022). The DNA sequences of Beta-tubulin (TUB2) and DNA-directed RNA polymerase II second largest subunit (RPB2) of six isolates were amplified and sequenced, using primer pairs Bt2a/Bt2b (Glass & Donaldson, 1995), and RPB2-5f2/RPB2-7cr (O'Donnell et al., 2012), respectively. BLASTN searches indicated our ITS (OP107240 - OP107245), TUB2 (OP131865 - OP131870) and RPB2 (OP131871 - OP131876) sequences except one TUB2 (OP131867), showed 100% identity to the corresponding sequences of E. latusicollum CGMCC:3.18346 (KY742101, KY742343 and KY742174, respectively). There was a nucleotide divergence between OP131867 and reference sequence. Based on concatenated ITS, TUB2 and RPB2 sequences, the constructed phylogenetic tree of Epicoccum species, confirmed that our isolates were E. latusicollum. To test pathogenicity, 2-year-old healthy seedlings of cultivar "chuanhuanghua No.1" were sprayed with conidial suspension of HHC51 (105 conidia/mL), with controls treated with sterile distilled water. Each treatment (biological replicates = 3) was incubated in a greenhouse (at 25°C under 90% relative humidity, 16/8 h light/dark cycle). The experiment was repeated twice. After 18 days, leaf spot symptom in inoculated seedlings appeared. Whereas, non-inoculated controls showed no symptom. The pathogens were re-isolated from diseased leaves and identified as E. latusicollum, based on morphology and molecular methods described above. E. sorghinum was previously reported as causal agent of leaf spot in H. citrina (Ma et al., 2021). To our knowledge, this is the first report of E. latusicollum causing leaf spot in H. citrina worldwide. Our study will assist with monitoring disease distribution in H. citrina and host diversity of E. latusicollum (Chen et al., 2017).

Epicoccum sp. as the causative agent of a reddish-brown spot defect on the surface of a hard cheese made of raw ewe milk

Colour defects can affect the appearance of cheese, its flavour, the safety of its consumption, and the price it can demand. This work reports the identification of five fungal isolates from a dairy plant where the surface of most cheeses was affected by patent, reddish-to-brown stains. One of these isolates was obtained from cheese, two from brine, and two from a bulk tank containing ewe milk. Molecular identification by partial amplification, sequencing, and database comparison of the concatenated sequence of the genes coding for the largest subunit of RNA polymerase II (RPB2), β-tubulin (β-TUB), and the large subunit of the rRNA molecule (LSU), plus the internal transcribed sequence (ITS) regions, assigned the isolates to Epicoccum layuense, Epicoccum italicum, and Epicoccum mezzettii. Features of the growth of these different species on different agar-based media, and of the morphology of their conidia following sporulation, are also reported. The strain isolated from cheese, E. layuense IPLA 35011, was able to recreate the reddish-brown stains on slices of Gouda-like cheese, which linked the fungus with the colour defect. In addition, two other strains, E. italicum IPLA 35013 from brine and E. italicum IPLA 35014 from milk, also produced stains on cheese slices. Epicoccum species are widely recognized as plant pathogens but have seldom been reported in the dairy setting, and never as human or animal pathogens.

First Report of Brown Leaf Spot Caused by Epicoccum tobaicum on Oat (Avena sativa) in Korea.

Oat (Avena sativa) is one of the Korean winter crops, and oat consumption has been increasing because it is widely perceived as a superfood. Recently, various fungal diseases have been reported likely due to climate changes during the winter season in Korea (Choi et al., 2018; Kim, 2020). During the 2020-2021 winter to spring, we surveyed new fungal diseases among cereals, including oats, in the southern region of Korea. In April 2021, brown leaf spots on oat leaves were observed in Gangjin, Jeollanam-do, Korea. These brown spots were irregular circles, ranging from 2-7 mm in diameter. Samples from three infected leaves were surface sterilized by treating them with 70% ethanol for 1 min and 1% NaOCl for 1 min. The samples were subsequently rinsed at least twice with distilled water and dried with a sterile paper towel before being placed on 1.5% water agar supplemented with 100 ppm streptomycin. Hyphal tips derived from infected tissues after incubation at 25C for 7 days were transferred to a fresh potato dextrose agar (PDA). Three isolates, labeled as KJO-AN2-S1, KJO-AN2-S2 and KJO-AN2-S3, were obtained via single hyphal tip purification. Colonies on PDA were pigmented vermilion and subsequently turned to saffron color with irregular margins after 7 days. Conidia produced on PDA were golden to dark brown, globose to subglobose, solitary, and measured 15.5-21.5 μm in diameter (n=50). Cultural and morphological characteristics suggested that these isolates belong to Epicoccum species (Chen et al. 2017). For identification by sequencing, the ITS (MW691866-68), tub2 (MW691872-74), and rpb2 (MW691869-71) regions of three isolates were amplified using the primer pairs ITS5/ITS4 (White et al., 1990), Btub2Fd/ Btub4Rd (Woudengerg et al., 2009), and RPB2-5F2 (Sung et al., 2007)/fRPB2-7cR (Liu et al., 1999), respectively. A maximum likelihood phylogenetic analysis based on the concatenated ITS, tub2, and rpb2 sequences placed the three isolates within a clade comprising E. tobaicum CBS 384.36. A mycelial plug (5 mm diameter) was inoculated onto wounded and unwounded leaves of healthy 12-day-old oat (cv. Choyang) seedlings. The control leaves were inoculated with a sterile PDA plug. All inoculated and control plants were placed in a plastic box and incubated at 20℃ in darkness with 100% humidity. After 1 day, the inoculated mycelial plug or sterile PDA plug from plants was removed; the plants in plastic boxes were then transferred to a growth chamber set at 20℃ with 12 h light and 60-70% humidity. While brown spot lesions were observed in both unwounded and wounded leaves 7 days post-inoculation, both wounded and unwounded control leaves remained asymptomatic. The pathogen was recovered from all symptomatic leaf tissues but could not be isolated from control leaves. The re-isolated pathogen was identified as E. tobaicum through morphological characterization and sequence-based identification, fulfilling Koch's postulates. This study is the first to report a causal relationship between E. tobaicum and brown leaf spot disease of oat in Korea. Identification of this newly emerging fungal disease on oats will help prepare for effectively managing this disease.

Identification of Epicoccum poaceicola causing eggplant leaf spot and its cross-infection potential to other solanaceous vegetable crops

This study characterised and identified the causative agent of irregular necrotic leaf spots in eggplant. The culture characteristics and morphology of three fungal isolates indicate that they are from the Didymellaceae group. Using a polyphasic approach—combined morphological, cultural, pathogenicity, and molecular characterisation (concatenated sequences of three gene loci: ITS, TUB2, LSU), the fungal isolates MBELIQ03A and MBELIQ03D were identified as Epicoccum poaceicola. Isolate MBELIQ02 was identified as Epicoccum sp. due to a lack of clear grouping with a single Epicoccum species. In repeated trials, the three Epicoccum isolates were pathogenic to eggplant leaves in detached leaf assays. Similar fungi were consistently re-isolated from the inoculated leaves and thus, establishing Koch’s postulates. Inoculation of the three isolates on solanaceous leaves and fruits (tomato, eggplant, and pepper) revealed their varied pathogenicity. This is the first confirmed scientific report of Epicoccum poaceicola causing eggplant leaf spots. This is also the first record of this fungal species in the Philippines. Inoculum from leaf infection could serve as inoculum for fruit infection and other solanaceous vegetable crops grown nearby a field planted with eggplant.

Epicoccum spp. Causing Maize Leaf Spot in Heilongjiang Province, China.

Maize leaf spot occurs worldwide and affects maize production. Maize can be infected by several pathogens causing leaf spot, such as Bipolaris zeicola, Bipolaris maydis, Curvularia species, Alternaria species, etc. In the current study, 30 Epicoccum isolates recovered from symptomatic maize leaves were identified based on morphological characteristics, pathogenicity, and multilocus sequence analyses of nuLSU, ITS, tub2, and rpb2. These maize isolates were grouped into five Epicoccum species, including E. nigrum, E. layuense, E. sorghinum, E. latusicollum, and E. pneumoniae. Pathogenicity tests showed that all five Epicoccum species could produce small ellipse- and spindle-shaped spots on maize leaves. The lesion center was grayish yellow to dark gray and surrounded by a chlorotic area. Furthermore, the Epicoccum isolates exhibited high pathogenicity to 20 main maize varieties of Heilongjiang Province but showed different sensitivities to the commonly used fungicides carbendazim and tebuconazole. In addition, these Epicoccum isolates showed different production capacity of pectinase, cellulase, protease, amylase, laccase, and gelatinase, but all showed high lipase activity. This is the first report globally of E. layuense, E. latusicollum, and E. pneumoniae as causal agents of maize leaf spot. E. pneumoniae was first reported as a plant pathogen.

First Report of Leaf Spot of Weigela florida Caused by Epicoccum layuense in China.

Weigela florida (Bunge) A. DC. is a dense, rounded, deciduous shrub commonly planted in landscapes. It is also used in Chinese medicine to treat sore throat, erysipelas, cold, and fever (Zheng et al. 2019). In May 2019, leaf spots were observed on approximately 50% of W. florida plants grown in the Wisdom Plaza Park of Anhui Agricultural University in Hefei, Anhui Province, China. Leaf spots begun as small light brown and irregular lesions, enlarged, turned reddish brown, coalesced to form large blighted areas, and eventually covered the entire leaf surface. Five pieces of tissues were removed from the lesion margins of each diseased leaf (five leaves from five different plants), chopped into several 3-4 mm2 pieces, disinfected with 1.5% NaOCl for 2 min, rinsed 3 times with sterile distilled water for 1 min, plated onto Potato Dextrose Agar (PDA) medium containing 50 μg/ml of ampicillin and kanamycin, and incubated at 25°C with a 12-hour photoperiod for 5 days. One segment of the fungal growth from the growing edge of the colony was transferred onto a fresh PDA plate for purification and incubated under the same conditions for another 5 days. The colony morphology of one representative isolate (AAU0519) was characterized by a pale orange cushion in the center surrounded by irregular pink margin, diffusing red orange pigments into the PDA medium. Isolate AAU0519 was cultured on PDA medium for 7 days at 25°C in the dark to induce sporulation. The produced conidia were globose, subglobose to pyriform, golden brown to brown, and with a diameter of 7.7 - 23.8 μm. Both cultural and morphological characteristics suggested that isolate AAU0519 was an Epicoccum species, according to the description by Chen et al. 2017. Amplification and sequencing of the internal transcribed spacer (ITS), beta-tubulin, and 28S large subunit ribosomal RNA (LSU) gene fragments from the extracted genomic DNA of AAU0519 were performed using primer sets ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and LSU1Fd/LR5 (Crous et al. 2009; Vilgalys and Hester 1990), respectively. A phylogenetic tree was constructed by the maximum-likelihood method with 1,000 bootstrapping replications based on the concatenated ITS, beta-tubulin, and LSU sequences from isolate AAU0519 and representative strains of 22 species of the genus Epicoccum (Chen et al. 2017). Isolate AAU0519 clustered with ex-holotype CGMCC 3.18362 of Epicoccum layuense Qian Chen, Crous & L. Cai (Chen et al. 2017). All obtained sequences were deposited into GenBank under accession numbers MK983497 (ITS), MN328723 (beta-tubulin), and MN328724 (LSU). A pathogenicity test was conducted on leaves of five 3-year-old W. florida cultivar "Red Prince" planted in the field (five leaves for each treatment and control per plant) by spraying 30 ml of a spore suspension (106 spores/ml) of isolate AAU0519 as treatment or sterilized distilled water as control. Before the inoculation, the leaves were disinfected with 70% ethanol. After inoculation, the leaves were wrapped with a plastic bag to keep high relative humidity. The average air temperature was about 28°C during the period of pathogenicity test. The experiment was repeated once. Ten days after inoculation, the fungal-inoculated leaves developed light brown lesions resembling those of naturally infected leaves, control leaves did not develop any symptoms. E. layuense was recovered from leaf lesions and its identity was confirmed by morphological and sequence analyses as described above. To our knowledge, E. layuense has been previously reported as a pathogen of Perilla sp. (Chen et al. 2017), oat (Avena sativa) (Chen et al. 2019), and tea (Camellia sinensis) plants (Chen et al. 2020), but this is the first report of E. layuense causing leaf spot on W. florida in China. This pathogen could pose a threat to the ornamental value of W. florida plants. Thus, it is necessary to adopt effective management strategies against leaf spot on W. florida.

Epicoccum species: ubiquitous plant pathogens and effective biological control agents

Epicoccum species are ubiquitous ascomycetes. Several species are known to cause plant diseases and some species act as biological control agents against a range of plant pathogens. Accurate identification of Epicoccum species is paramount to a successful disease management program and successful development of biological control products. There are 18 Epicoccum species that are associated with diseases of 46 plant species. The diseases occur in 20 countries with leaf spot as the most commonly reported disease symptom. There are a further five Epicoccum species (E. nigrum, E. layuense, E. dendrobii, E, mezzettii and E. minitans) that have biological control activity against various plant pathogens. Of the five species, Epicoccum nigrum is the most promising and has been shown to reduce incidence and severity of a wide range of plant diseases. The use of bioactive metabolites (e.g. flavipin and epicolactone) from Epicoccum spp. may offer growers a cheap and safe alternative to conventional pesticides which are widely used to manage plant diseases. We review the current knowledge of Epicoccum species, both those species that are plant pathogens, and those species that are biological control agents for plant disease.

<p><strong>Morphological and phylogenetic characterisation of endophytic fungi associated with the grapevine flowers in China</strong></p>

The harvest from grapevines is berries which are derived from the flowers. Even though numerous studies have been conducted to understand the pathogens associated with grapes, less is known about the fungi associated with the flowering stage and their potential for infecting fruit as latent pathogens. In the present study, 99 endophytic strains were isolated from the calyptra of grape flowers of four different grape cultivars (Italia, Jingxiangyu, Kyoho and Zexiang). The isolates were Alternaria, Cladosporium, Diaporthe and Epicoccum species. Alternaria was the most abundant genus with 88 strains of A. alternata. Four Cladosporium species were identified including two new host records (Cladosporium ramotenellum and C. anthropophilum) and one new species (Cladosporium endoviticola sp. nov.). Diaporthe eres and a novel Epicoccum species (Epicoccum endophytica sp. nov) were also identified. This study provides evidence that some grape fruit pathogens occur as endophytes in flowers. The study enhances the current knowledge of grape pathogens and can be used to develop sustainable management strategies.

Culture-based identification to examine spatiotemporal patterns of fungal communities colonizing wood in ground contact

ABSTRACTTimber durability is often assessed using small wood stakes exposed in direct soil contact, and the assessment generally emphasizes effects on wood rather than organisms involved. Understanding fungal colonization patterns can help identify key decay agents under varying conditions and use these patterns to improve wood protection strategies. Fungal colonization of red alder (Alnus rubra), Douglas-fir (Pseudotsuga menziesii) heartwood/sapwood, and western redcedar (Thuja plicata) field stakes was assessed over 2 y in western Oregon. Spatiotemporal fungal community variations were identified via culturing and DNA sequencing, where 814 isolates were identified from 84 stakes. Forty-six ascomycete genera were identified, with Phialophora, Trichoderma, and Epicoccum species occurring most frequently. Twenty-three basidiomycete genera were identified, with Trametes and Phanerochaete being the most common. Douglas-fir and western redcedar stakes contained the highest and lowest diversity levels, respectively, reflecting natural durability differences of these species. Fungal species abundance was higher below ground than in the above ground and groundline zones, likely reflecting more stable moisture regimes, proximity to soil-based fungi, and potential nutrient migration into wood beneath the soil surface. Ascomycetes were proportionally more abundant early in the exposure period, but basidiomycetes were also observed early in the process, and there appeared to be no consistent colonization pattern.

Epicoccum Species as Potent Factories for the Production of Compounds of Industrial, Medical, and Biological Control Applications

Epicoccum Species as Potent Factories for the Production of Compounds of Industrial, Medical, and Biological Control Applications

Characterization of community structure of culturable endophytic fungi in sweet cherry composite trees and their growth-retarding effect against pathogens.

Endophytic fungi have the potential to protect their host plants in stress situations. Characterizing the ecology and complex interaction between these endophytes and their host plants is therefore of great practical importance, particularly in horticultural plants. Among horticultural plants, fruit trees form a special category because of their longevity and because they are composites of rootstock and scion, which often belong to different plant species. Here we present the first characterization of culturable endophytic fungal community of sweet cherry. Samples from the Hungarian cultivar 'Petrus' grafted on 11 different rootstocks were collected in autumn and in spring in a bearing orchard and the dependence of colonization rate and endophyte diversity on rootstock, organ and season was analysed. On the basis of their ITS sequences 26 fungal operational taxonomic units were identified at least down to the genus level. The dominant genus, comprising more than 50% of all isolates, was Alternaria, followed by different Fusarium and Epicoccum species. We observed some organ-specificity amongst endophytes, and organs showed more sizeable differences in colonization rates and endophyte diversity than rootstocks. Most dynamic endophyte populations, strongly influenced by environmental conditions and crop management, were observed in leaves. The potential of selected endophytes to confer protection against Monilinia laxa was also analysed and 7 isolates were found to inhibit the growth of this pathogen in vitro.

미곡종합처리장 곡류시료 내 곰팡이 오염과 Fusarium 독소 발생

Rice samples including paddy, husk, brown rice, blue-tinged rice, broken rice, discolored rice and polished rice were collected from rice processing complexes(RPC) nationwide to determine the contamination of fungi and mycotoxins on rice during 2010-2013. Among the samples, paddy rice had the highest frequencies of fungal and Fusarium occurrence, and the frequencies decreased along with milling as husk was the next. Blue-tinged rice or discolored rice was similar with brown rice for fungal occurrence, and polished rice showed the lowest frequency. Among Fusarium species, F. graminearum species complex occupied 87% in 2012 but did 35-39% in 2011 and 2013. Aspergillus and Penicillium species appeared at low frequency in most samples but occurred at higher frequency in certain RPC samples. Alternaria, Nigrospora, and Epicoccum species occurred similarly to the pattern of total fungi. The rice samples from 2010-2012 were analyzed for the occurrence of Fusarium mycotoxins including deoxynivalenol, nivalenol, zearalenone, and other trichothecenes. The most highly con- taminated sample was discolored rice in terms of frequency, level, ratio of simultaneous contamination with multiple toxins, followed by blue-tinged rice.

Multimycotoxin analysis of sorghum (Sorghum bicolor L. Moench) and finger millet (Eleusine coracana L. Garten) from Ethiopia

Abstract The natural contamination of sorghum and finger millet by toxigenic fungi and associated mycotoxins has been studied. All the tested sorghum and finger millet samples were found to be contaminated by Fusarium and Aspergillus species. Sorghum was considerably more likely to be contaminated by both genera than finger millet. Penicillium, Alternaria, Rhizopus and Epicoccum species were also present in both grains albeit at lower frequencies. Multimycotoxin analysis using LC–MS/MS revealed the contamination of sorghum and finger millet by 84 and 62 metabolites, respectively. The prevalence of major mycotoxins was lower than 15% in sorghum except zearalenone that occurred in one third of the samples at average level of 44 μg/kg. In finger millet major mycotoxins occurred at a prevalence of 6–52% with zearalenone being the dominant and occurring at average level of 76 μg/kg. Aflatoxins B 1 , B 2 , G 1 , G 2 and M 1 were detected in at least one sorghum sample while only aflatoxins B 1 and G 1 were present in finger millet samples. The average aflatoxins B 1 and G 1 concentrations in sorghum have been higher than European standards. But the level of B 2 , G 2 and M 1 in sorghum and that of B 1 and G 1 in finger millet have been lower. Apart from aflatoxin precursors and other Fusarium metabolites, a broad range of additional metabolites were detected in sorghum and finger millet.

Polyphasic analysis of intraspecific diversity in Epicoccum nigrum warrants reclassification into separate species.

Background Epicoccum nigrum Link (syn. E. purpurascens Ehrenb. ex Schlecht) is a saprophytic ascomycete distributed worldwide which colonizes a myriad of substrates. This fungus has been known as a biological control agent for plant pathogens and produces a variety of secondary metabolites with important biological activities as well as biotechnological application. E. nigrum produces darkly pigmented muriform conidia on short conidiophores on sporodochia and is a genotypically and phenotypically highly variable species. Since different isolates identified as E. nigrum have been evaluated as biological control agents and used for biocompound production, it is highly desirable that this species name refers to only one lineage. However, according to morphological and genetic variation, E. nigrum present two genotypes that may comprise more than one species.Methodology/Principal FindingsWe report the application of combined molecular (ITS and β-tubulin gene sequence analysis, PCR-RFLP and AFLP techniques), morphometric, physiological, genetic compatibility and recombination analysis to study the taxonomic relationships within an endophytic population that has been identified as E. nigrum. This combined analysis established two genotypes showing morphological, physiological and genetic divergence as well as genetic incompatibility characterized by colony inhibition, strongly indicating that these genotypes correspond to different species. Genotype 1 corresponds to E. nigrum while genotype 2 represents a new species, referred to in this study as Epicoccum sp.Conclusions/SignificanceThis research contributes to the knowledge of the Epicoccum genus and asserts that the classification of E. nigrum as a single variable species should be reassessed. In fact, based on the polyphasic approach we suggest the occurrence of cryptic species within E. nigrum and also that many of the sequences deposited as E. nigrum in GenBank and culture collection of microbial strains should be reclassified, including the reference strain CBS 161.73 sequenced in this work. In addition, this study provides valuable tools for differentiation of Epicoccum species.

Endophytic fungi in European aspen (Populus tremula) leaves—diversity, detection, and a suggested correlation with herbivory resistance

Fungal endophytes are found in most seed plants, but their ecological function mainly remains elusive, except in pooid (or clavicipitalean) systems. The diversity and dynamics of endophytes in non clavicipitalean plants make studies of their ecological function challenging. This paper describes the advantage of using molecular techniques to survey the ecological function of endophytes in Populus tremula clones. About 1,000 endophyte isolates were distinguished using traditional methods; these isolates represented approximately 100 morphologically distinct groups. We generated 73 DNA-sequences (18S and ITS rDNA) from these groups and determined 33 distinct taxa. They represented the Basidiomycota and Ascomycota, including diverse Sordariomycetes and Dothideomycetes, and three sequences that were identified, by their ITS sequences, as a species of Epicoccum (a genus of supposedly unknown relation within the Ascomycota) were placed within the Pleosporales, in the 18S phylogeny. Primer pairs were designed for eleven of the fungi. Of these, three primers produced bands for a subset of Aspen samples. The primer pairs allowed endophytes in field samples to be readily identified, with a detection limit of 0.15 percent fungal DNA. The presence of fungi in Aspen clones was related to field damage by herbivores and the pathogen Venturia tremula. A negative association was found in two separate surveys between Aureobasidium sp. and herbivore damage, but we found no evidence that endophyte presence was related to a history of Venturia symptoms. This approach promises to enhance greatly the scope for qualitative and quantitative detection of endophyte communities, and to improve our ability to elucidate the ecological function of non clavicipitalean endophytes.

Suppression of rice blast by phylloplane fungi isolated from rice plants

Rice phylloplane fungi were evaluated for their potential as biocontrol agents for rice blast disease caused by Magnaporthe grisea. A total of 1923 fungal isolates were obtained from rice plants in fields at Ishigaki and Iwama and from potted plants placed in a cedar woods in Iwama as bait. Although 82.9% of isolated fungi could not be identified, species of Epicoccum were the most prevalent among identified isolates. Of the 1923 isolates, 967 were randomly selected for screening against rice leaf blast. Nine isolates (MKP5111B, MKP5112, J2JMR3-2, K2J131-2, I5R3-1, NOP541, K1KM134-1, NOP5112, MKP33222) suppressed the disease when a conidial or hyphal suspension of both the phylloplane fungus and pathogen were simultaneously used to inoculate rice plants cultured in pots in a growth chamber. Five of the isolates originated from potted plants in the woods and four from Ishigaki, a subtropical island. Five (MKP5111B, MKP5112, NOP541, NOP5112, MKP33222) of the nine isolates strongly suppressed conidial germination of M. grisea (≦0.7%) and formed inhibition zones (3–5 mm width) in dual cultures with the pathogen. Methanol extracts from the isolates also inhibited mycelial growth of the pathogen. These results suggest that the five isolates produced antibiotic(s). These five isolates are likely identical or closely related fungal species because the sequence of their ITS regions were 100% similar. ITS sequence analysis also suggested that J2JMR3-2 was associated with a species of Fusarium. Under field conditions, J2JMR3-2 reduced both leaf and panicle blast severity, and three other isolates (MKP5111B, K1KM134-1, K2J131-2) suppressed leaf blast in one of the three experiments.

Microbial population dynamics on Golden Delicious apples from bud to harvest and effect of fungicide applications

SummaryThe microbial population dynamics on apples cv. Golden Delicious were analysed every 15 days between bud and harvest in a fully replicated experiment in northern Spain in 1994 and 1995. The total microbial populations varied with developmental stage, and with prevailing climatic conditions. The predominant mycroflora were the filamentous fungi Cladosporium and Alternaria spp. and white and pink yeasts. Other genera isolated included mainly species of Epicoccum, Fusarium and Acremonium. However, the most important post‐harvest pathogens Penicillium expansum and Botrytis cinerea were seldom isolated from ripening apples. Maximum total filamentous fungal populations occurred after fruit set and during early ripening [2 × 104cfu (colony‐forming units) g‐1 approximately] while those of bacteria were maximum at bud stage (3.5 × 105and 3.0 × 104 cfu g‐1 in 1994 and 1995 respectively). White yeasts were more numerous than pink yeasts. Endophytic infection of apple buds by Alternaria spp., responsible for core rot, was found in almost all bud tissue. By contrast, Cladosporium spp. were initially isolated later from 12.5–50% of tissue samples during blooming and fruit set. The impact of a four‐spray fungicide regime during apple development significantly decreased the total filamentous fungal populations in both years, and that of Cladosporium spp. in 1994. However, bacterial populations were often higher on apples from fungicide‐treated plots. Fungicide sprays decreased populations of Cladosporium, Alternaria and white yeasts for a maximum of up to 15–30 days after application. Fungicide application had little effect on endophytic infection of apples by Alternaria spp. between bud and harvest.

Grain Mold Resistance and Polyphenol Accumulation in Sorghum

Ten sorghum [Sorghum bicolor (L) Moench] genotypes with differences in phenolic compound concentrations and grain mold resistance were evaluated at West Lafayette, IN, over three crop seasons (1989, 1990, and 1992) to assess changes in phenolic compounds during seed development and how these changes influence grain molding. Samples were collected for 9 weeks at 7-day intervals starting 7 days after anthesis. Acidified methanol extracts of the seeds were assayed to determine concentrations of 3-deoxyanthocyanidins, flavan-4-ols, and proanthocyanidins. Seeds were also plated on biological media to observe the level of seed infection by mold-causing fungi. Flavan-4-ol concentrations were high and similar for both the mold-resistant and mold-susceptible genotypes at early stages of seed development. In susceptible genotypes, the flavan-4-ol concentration dropped by 67% between the third and the last sampling dates compared with a 20% decline for the resistant genotypes in the same period. In addition, the resistant genotypes (P954255, P932062, IS15346, IS7822, P013931) had high concentrations of proanthocyanidins throughout the season compared with susceptible lines, which lacked or had negligible amounts of this material. Although significant differences occurred among genotypes for 3-deoxyanthocyanidins, the presence of these pigments did not differentiate mold-resistant and mold-susceptible genotypes. The results also showed that the highest incidence of seed infection by fungi occurred between 25 and 35 days after anthesis. Alternaria, Fusarium (especially F. moniliforme), Cladosporium, and Epicoccum species were the major fungi isolated from the seeds. Keywords: Sorghum; polyphenol; 3-deoxyanthocyanidin; flavan-4-ols; proanthocyanidin; apigeninidin; luteolinidin; grain mold

Phyllosticta Beaumarisii Sp.nov.: A Cause of Leafspot on Muehlenbeckia Adpressa.

Muehlenbeckia adpressa (Labill.) Meissn. is a vigorous climber or ground cover which scrambles over other plants. It is a widespread coastal plant endemic to Southern Australia where it is commonly known as "Climbing lignum" or "Macqaurie vine"(2). The species produces rounded leaves, 2 to 6 cm in length, often cordate at the base and crinkled around the margins. Insignificant yellowgreen flowers are borne in short axillary racemes. Plants are grown from seed or can be easily propagated by stem cuttings. M. adpressa grows vigorously during spring and autumn, slowly during winter and is virtually dormant during a hot summer. Because of its rapid growth and hardiness, M. adpressa is useful for fence or embankment cover in coastal situations (5 ) . In July 1984, a patch of badly diseased M. adpressa was observed at Beaumaris, Victoria on a cliff top, adjacent to Port Phillip Bay. Virtually all mature leaves of plants in an area of 10 sq, m. contained distinctive necrotic spots. Spots were roughly circular to elliptical in shape and were tan in colour with a maroon margin. Individual spots reached a diameter of 11 mm but frequently coalesced to form a larger area of necrosis. Spots appeared to be randomly distributed, were visible from both sides of leaves and sometimes contained numerous pycnidia barely visible to the naked eye (Fig. 1). Numerous lesions were excised, washed, surface sterilized and placed on agar media. A variety of fungi developed in culture including species of Epicoccum, Alternaria, Penicillium, Aspergillus, Fusarium and several unidentified yeasts. The only fungi producing pycnidia in culture were Phoma macrostoma Mont. and a slow growing Phyllosticta sp. which had pycnidia similar to those present within the necrotic lesions. Extractions from very tiny lesions, or from pycnidia in diseased leaf tissue, yielded pure cultures of this Phyllosticta sp.. Fig. 1. Pycnidia of Phyllostica beaumarisii sp. nov. and necrotic spots on leaf of Meuhlenbeckia adpressa. (x2)