Cladosporium Research Articles

Multi-Level Characterization of Microbial Consortia Involved in the Biodeterioration of Wooden and Stone Romanian Heritage Churches

The 17th to 19th century wooden and stone churches are an iconic symbol of Romanian national heritage. The present study investigates by qualitative and quantitative methods the microbial communities from the biodeteriorated surfaces of wooden and stone monument churches included in the cultural heritage list of local or national importance. From a total of twelve monuments, samples were taken with cotton sterile swabs, inoculated on specific culture and identified by classical, automated and molecular methods. A total of 133 strains belonging to Ascomycota phylum were identified and confirmed at species level from the wooden churches, amongst which, Penicillium spp. strains (mostly P. corylophylum, P. chrysogenum) were the most frequent, followed by Alternaria alternata and species of Trichoderma, Aspergillus, Rhizopus, Mucor, and Fusarium genera. From the stone churches a total of 100 strains belonging to Aspergillus, Alternaria, Mucor, Penicillium, Aspergillus, Trichoderma, Fusarium and Rhizopus genera were isolated. A total of 55 bacterial strains were isolated and identified as Bacillus, Artrobacter and Pseudomonas species. The microbial load of the samples ranged between 2.18x107 and 3x105 CFU/mL. A very small number of fungal strains (6/77) isolated from wooden churches (mostly A. alternata, followed by P. corylophilum and one Cladosporium spp. strain) and from stone churches (5/55) (mostly A. alternata, followed by A. versicolor, A. nidulans strain) were involved in biofilm formation. The results of this study can help to improve understanding of the microbial deterioration of Romanian heritage churches and allow more reliable decontamination, conservation and preservation tools to be defined.

The characteristics of soil fungal community in degraded alpine meadow in the Three Rivers Source Region, China.

To clarify the variation of species composition, diversity, and functional structure of soil fungi community along alpine meadow degradation,we examined the characteristics and controlling factors of soil fungal communities in non-degraded, lightly degraded, moderately degraded, severely degraded and extremely degraded (black soil beach) alpine meadows at the Three Rivers Source Region, based on the high-throughput gene sequencing and FUNGuild functional prediction. The results showed that the dominant phyla in alpine meadow soil were Ascomycota, Basidiomycetes, and Mortierellomycota. Species composition of soil fungal community varied greatly in alpine meadow under different levels of degradation. The abundance of Cladosporium flabelliforme, Entoloma sodale, Hygrocybe conica, Inocybe sp. and Trichocladium opacum increased, while that of Gibberella tricincta and Dactylonectria macrodidyma decreased following grassland degradation. The meadow under severe degradation had higher soil fungal Chao1 index, while that under light degradation had lower Shannon index and Simpson index. The abundance of pathologic, symbiotic, and saprophytic types of fungi varied among different alpine meadows. Along with the grassland degradation, the abundance of soil symbiotic fungi decreased, while that of pathological fungi increased. The soil fungal community and functional compositions changed obviously with degradation in the alpine meadow. Plant aboveground biomass, soil water content, pH, total organic carbon, total nitrogen, ammonium nitrogen, available phosphate, total potassium and AN/AP (ratio of available nitrogen and available phosphorus) were the main driving factors for the variations in soil fungal community structure.

IDENTIFICACIÓN Y EVALUACIÓN DE LA INCIDENCIA DE INSECTOS Y HONGOS BENÉFICOS ASOCIADOS A Diaphorina citri KUWAYAMA (HEMIPTERA: LIVIIDAE) EN PLANTAS TRASPATIO (Citrus spp. y Murraya paniculata) DEL CANTÓN CATAMAYO (LOJA - ECUADOR)

En los últimos años, la citricultura mundial se ha encontrado amenazada por la presencia del psílido asiático de los cítricos, Diaphorina citri, un insecto perjudicial por ser portador de la bacteria “Candidatus Liberibacter asiaticus (las)”, causante de la enfermedad Huanglongbing (HLB), que ha provocado la devastación de plantaciones y la reducción de la producción de cítricos en todo el mundo. En el año 2017 se reportó la presencia de D. citri en plantas traspatio del cantón Catamayo, en la provincia de Loja, por lo que la presente investigación tuvo por objetivo identificar los enemigos naturales (depredadores, parasitoides y hongos) asociados a D. citri, y evaluar su incidencia en campo. Para ello, se realizaron muestreos en plantas traspatio (Citrus spp. y Murraya paniculata) del cantón Catamayo. Los muestreos se efectuaron a una altura de 1,5 m de las plantas en los cuatro puntos cardinales de la misma. Se colectaron larvas y adultos de depredadores, ninfas parasitadas y adultos de parasitoides, y adultos de D. citri para el aislamiento de hongos. Como resultado de la identificación en el laboratorio, se determinó la presencia de 11 especies depredadoras (principalmente Cheilomenes sexmaculata y Chrysopa spp.), dos especies de parasitoides (Diaphorencyrtus sp. y Tamarixia radiata) y cuatro especies de hongos (Cladosporium sp., Fusarium sp., Aspergillus sp. y Alternaria sp.). Diaphorencyrtus sp. tuvo una tasa de parasitismo del 7,76 %. Estas especies podrían utilizarse para establecer programas de control biológico, aprovechando su potencial como reguladores de poblaciones de ninfas de Diaphorina citri.

QUALITY AND MYCOBIOTA COMPOSITION OF STORED EGGS

The aim of this study was to monitor the quality and mycobiota composition of table eggs during storage period. The most significant changes in the egg weight and water activity were observed on Day 7. To identify the mycobiota present on the eggshell by PCR method, a newly designed procedure for the extraction of fungal DNA based on a combination of commercial isolation kit, proteinase K and ultrasound was implemented. Identified mold genera included Penicillium spp., Cladosporium spp., Fusarium spp. and Alternaria alternata group. Their ratio varied considerably during storage with the dominance of Penicillium spp. on Day 14.

PHYTONCIDAL ACTIVITY OF ESSENTIAL OILS OF MEDICINAL PLANTS TO SOME STRAINS OF MOLD FUNGI

Herbal antiseptics with phytoncidal action, for example, plant essential oils, can be used indoors in the presence of people. The antifungal activity of essential oils of medicinal plants (anis, basil, clary cage, clove, coriander, jasmine, juniper, lavender, lemon, orchid, rose, rosemary, tea tree, wormwood, ylang ylang) was evaluated by the degree of growth inhibition of mold colonies Alternaria alternata , Aspergillus oryzae, Aspergillus flavus, and Cladosporium cladosporioides. Colonies incubated in Petri dishes with a drop of essential oil on the lid under control petrolatum. All essential oils in the experiment had phytoncidal properties. The oils were divided into groups: 1 with fungicidal action (coriander, jasmine, orchid, rose); 2 with high fungistatic activity to all strains (lavender, basil, tea tree oil); 3 with fungistatic properties against individual strains (C. cladosporioides rosemary and anise, A. alternata and A. flavus lemon, A. oryzae wormwood); 4 with a stimulating effect of the growth of colonies A. oryzae lemon oil.

Screening of fungal contaminants in banana tissue cultures in Jkuat, Kenya

Tissue culture is prone to high costs of production arising from losses incurred from fungal contamination. The aim of the study was to characterise fungal contaminants and elucidate the exhibited mode of resistance to most preferred sterilants. Twenty nine fungal samples were collected at the different stages of tissue culture growth, using purposive sampling technique. Morphology results were confirmed by molecular characterization using fungal 18S rRNA sequences. Biochemical and antibiosis tests, identification of genes for capsulation and ATP binding Cassete (ABC) transporters, were performed to show the relationship between the fungi and sterilants resistance. Amylases and proteases were highly expressed by all isolates while xylanases and lipases were moderately expressed and esterases were lowly expressed. Only fourteen isolates had antagonistic activity for Candida albicans while nine of them had antagonistic activity for Pseudomonas aeruginosa. Three isolates were both antagonistic for Staphylococcus aureus and for Escherichia coli. Cunninghamella bainieri (10R) recorded a unique antibiosis and extra cellular enzymatic activity (p<0.05). All the isolates were positive for mdr1 gene and three isolates had CAP64 capsule genes. Aspergillus sp., Penincillium sp., Cladosporium sp., Cunninghamella sp. and Fusarium sp. were identified to be the major fungal contaminants of tissue culture banana cultures in JKUAT laboratories. Key words: Tissue culture, 18S rRNA, fungal contaminants, banana cultures.

Mycoflora and Public Health Risks of Smoked Fish Sold in Port Harcourt Markets, Nigeria

Fish is a preferred source of protein globally, especially in developing countries like Nigeria. It is a savoured protein source in the Niger Delta, including Port Harcourt. Smoking is used to preserve fish by reducing its moisture content with a view to improved shelf life. This study aimed at determining the Mycoflora and the Public Health risks of smoked fish sold in Port Harcourt Markets. A total of 54 fish samples were collected from three strategic markets; Mile one, Oil Mill and Creek Road markets. Fish collected consists of 6 different species; Gadus morhua, Pseudotolithus typhus, Lutijanus goreensis, Ethalmosa fimbriata, Pseudotolithus senegalensis and Dasyatis pastinaca. All samples were grouped accordingly. Mycological study of fish samples was done using standard methods on Sabouraud Dextrose Agar. There was a significant difference in the mycoflora counts of smoked fish from different markets (p<0.05). Fungal load ranged from 1.23±0.08 x103 sfu/g in Lutjanus goreensis to 8.89±0.10 x 103 sfu/g in Gadus morhua, at Creek road market. From Mile 1 market, Lutjanus goreensis still hosted the highest population of 13.25±0.7 x 103 sfu/g and Dasyatis pastinaca had the least; 0.66±0.01 x 103 sfu/g. At Oil mill market, Ethalmosa fimbriata hosted 13.23±0.47 x 103 sfu/g while Gadus morhua had 0.77±0.02 x 103sfu/g. The fungal load in all fish from all three markets were significantly high for food and calls for attention. Nine fungal genera; Saccharomyces spp, Rhizopus spp, Penicillium spp, Mucor spp, Fusarium spp, Cladosporium spp, Candida spp, Absidia spp and Aspergillus spp, were isolated. All six fish species studied recorded more than 50 % occurrence of fungal species in all the markets. The mycoflora of smoked fish sold in Port Harcourt markets suggest significant public health risks. The need for improved storage and handling of this important protein source is high towards reduced public health risk. Proper preparation method, such as boiling, is strongly advocated.

Cladosporium hebeiense sp. nov., pathogenic on grape leaves in China

<i>Cladosporium hebeiense</i> sp. nov., pathogenic on grape leaves in China

Endophytic fungal communities of ancient wheat varieties

The fungal community composition and structure of two ancient tetraploid wheat varieties, native to the Sicilian territory of Italy, Perciasacchi (winter wheat) and Tumminia (spring wheat) were investigated using High Throughput Sequencing (HTS). This showed a predominance of Ascomycetes and Basidiomycetes including Alternaria , Fusarium , Mycosphaerella, Filobasidium, Cystofilobasidium, Cryptococcus , Leucosporidium , Dioszegia, Puccinia , Sporobolomyces, Aureobasidium, Cladosporium, Holtermanniella and Gibberella . Principal Coordinates Analysis (PCoA) and Linear discriminant analysis Effect Size (LEfSe) showed that Aureobasidium, Leucosporidium and Puccinia differentiated between the two wheat varieties. In addition, the microbial association analysis suggested that some endophytic taxa play important roles within the wheat fungal community. Genera such as Cryptococcus and Cystofilobasidium were shown to have consistent antagonistic activity against Gibberella spp., while, Acremonium and a group of unidentified ascomycetes had mutual exclusion relationships with Puccinia. Since both Gibberella and Puccinia contain several economically important pathogens of wheat, the detected fungal interactions may indicate microbial-mediated resistance in these wheat varieties.

Drinking water. Microscopic fungi. Mycotoxins (literature review)

We have analized available literature on the capability of micro- scopic fungi (micromycetes) to exist and reproduce in the water (i.e. tap water) that is provided to consumers. The following mi- cromycetes, most typically present in tap water, were identified, namely: Aspergillus, Fusarium, Penicillium, Alternaria, Clad- osporium and Candida. The average number of micromycetes ranges from 8 to 18 CFU/100 cm3 , with yeast fungi dominantly present. Toxic substances (mycotoxins) that produce microscop- ic fungi are described, as well as their effects on the human body. It is noted that most mycotoxins stimulate formation of excessive free radicals and oxidative stress, which leads to impaired func- tion of the body’s antioxidant system, namely the production of a prooxidant enzyme in liver cells, immune organs and in the gastrointestinal tract. Therefore, , the presence of processes of apoptosis that lead to little sorption syndrome, immunosuppres- sion. The potential water routes of mycotoxins into the human body have been identified. The ability of mycotoxins, which form micromycetes – most often found in tap water, to cause cancer, have been analyzed in detail, using available literature on the subject. Mainly the effect of aflatoxins, trichothecenes, ochratox- ins and gliotoxins was examined. The presence of microscopic fungi in water supplied to the consumer, as well as their ability to form toxins necessitates systematic studies of water samples for the detection of micromycetes and the toxins present in it. And, finally, an important avenue for further research is outlined, which is to develop a simple and affordable method for the de- tection of mycotoxins in the water, based on the use of a sensitive test objects. Key words: drinking water‚ micromycetes‚ mycotoxins‚ human health For citation: Zabolotnyi DI, Rudenko AV, Honcharuk VV, Saprykina MM, Bolgova OS. Drinking water. Microscopic fungi. Mycotoxins (literature review). Journal of the National Academy of Medical Sciences of Ukraine. 2019;25(3):253–59

Diaporthe as the main cause of hazelnut defects in the Caucasus region

Production of hazelnuts is affected by defects, the incidence of which is year-dependent but also economically damaging. The objectives of the present study were to quantify the incidence of different fungal genera in hazelnuts, and define the causal agent/agents of nut defects. A 4-year study (2013 to 2016) was carried out in the Caucasus region, in an orchard planted in 2008 with hazelnut cv. Anakliuri. Hazelnuts were sampled at early and full ripening stages, observed for defects, and then associated fungi were isolated. Nuts with necrotic spots and internal browning were commonly found, and some collected in 2016 were completely rotted, wet and almost black. Alternaria, Aspergillus, Cladosporium, Colletotrichum, Diaporthe, Fusarium, Penicillium, Pestalotiopsis and Phoma spp. were regularly isolated from diseased and symptomless nuts. Diaporthe spp. was the dominant genus, with increasing incidence from early to full ripening, and were more isolated from defected compared to healthy kernels and in the years with the greatest incidence of defects. Rainfall was associated with the incidence of nut defects. The role of Diaporthe as a key cause hazelnut defects was confirmed by pathogenicity tests. Three isolates from the Diaporthe population were identified as D. eres on the basis of EF, ACT, TUB and ITS loci. This supports the crucial role of climate during the crop-growing season for the development of defects in hazelnuts caused by Diaporthe.

Physiological and sanitary quality of seeds de Acacia mearnsii De Wild. – Fabaceae

Acacia mearnsii has great economic importance and representation in the forestry sector. The aim of this study was to evaluate the physiological and sanitary quality of two lots of black wattle seeds. The seeds were obtained in Fepagro Forests and Mercosul seeds, respectively in the Santa Maria and Ijui counties in the Rio Grande do Sul state. For sanity used to detect fungus on potato-dextrose-agar (PDA) and substrate blotter-test (BT). For each test were four replicates of 25 seeds, evaluation, for both health testing was performed observing the fungal structures in stereoscopic and optical microscope. For the germination test, were used 200 seeds of each lot, divided into eight replicates of 25 seeds each, sowing was carried out in previously disinfected gerbox with 70% alcohol and sodium hypochlorite 1%, as substrate was used two filter paper sheets sterilized and moistened with distilled water. Fungi associated with seeds independent of utilized method were Penicillium sp., Cladosporium sp., Aspergillus sp. Mercosul’s lot, showed a greater number of healthy seeds on blotter-test (97.5%) and PDA (86%). The fungus with the highest incidence in the two methods compared (PDA and BT) was Cladosporium sp. in the Fepagro’s lot. For germination Mercosul’s lot showed 79.5% differing of Fepagro’s lot which showed 26.5%.

Endophytic fungi isolation and identification from Memecylon species of Western Ghats, India

Endophytic fungi have been documented as sources for novel secondary metabolites with useful medicinal properties. Interest in fungal endophytes is mainly due to their chemical diversity. These signify a virtually untapped source of chemical reservoir that finds applications in agriculture and therapeutics. Sampling and characterization of fungal endophyte diversity is an emerging challenge, which leads to the discovery of new species producing new compounds and a better understanding of their role in ecosystems. In the present study fungal endophytes were isolated from surface sterilized leaf and stem segments of five Memecylon species such as M. umbellatum, M. edule , M. talbotianum, M. malabaricum and M. wightii . A total of 156 isolates of endophytic fungi were obtained from 2000 tissue segments of five Memecylon species being investigated. Of the 156, endophytic isolates recovered, 86 sporulated and belonged to 10 genera, Alternaria (12.2%), Pleosporales (6.4%), Stagonosporopsis (3.8%), Cladosporium (8.2%), Fusarium (7.3%), Aspergillus (12.2%), Pestalopsis (7.5%), Collectotrichum (21.6%), Phoma (4.4%) and fungal mycelia (19.1), which are identified based on morphological characteristics and the dominant endophytes such as C. gleosporiades , Pleosporales sp., Stagonosporopsis cucurbitacearum and other fungal endophytes are further confirmed based on PCR amplification and sequencing of 5.8srRNA gene region and their accessions are obtained KT375578, KT375576.1, KT375577.1 and KT375579.1. The study provides the first report on the isolation and identification of endophytic fungi from Memecylon species.

Microbial Estimation and Characterization of Wastewater and Sludge in Awka Metropolis, Nigeria

This study focuses on the estimation and characterization of microorganisms isolated from wastewater and sludge in Awka metropolis, Nigeria. The samples were collected randomly from four (A, B, C and D) respective areas, analyzed microbiologically by homogenizing the samples under aseptic conditions to obtain a homogenous mixture, the isolation techniques used was pour plate methods as described by Cheesbrough, (2010); Joanne et al, (2011); APHA, (1989). The samples were diluted serially, inoculated on to MacConkey agar and Sabouraud Dextrose agar media each and microorganisms were isolated; wastewater had a range from 44 to 100 colonies and total of 284 (71 ± 24.30), CFU / ml (10-4) of 1.0 × 107 to 8.0 × 106 with total count of 19.4 (4.85 ± 2.96) × 106, while sludge was ranges from 60 to 120 colonies with a total of 358 (89.5 ± 29), CFU / ml (10-4) ranged from 1.0 × 107 to 7.0 × 106 with total count of 15.2 (3.8 ± 3.1) × 106. The colony and the morphology of the isolates were recorded, the bacterial isolates were characterized by motility tests, Gram staining and identified by biochemical tests. The fungal isolates were microscopically examined using a wet mount procedure. From the results obtained 8 bacterial and 7 fungal isolates were identified from both the wastewater and the sludge; Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Micrococcus leteus, Enterococcus faecalis, Serratia mercescens and Yersinia enterocolitica. Acrophialophora fusispora, Epicoccum Purpurascens, Rhizopus microsporus, Exophiala spinifera, Cladosporium cladosporiodes, Aspergillus niger and Phaeoacremonium parasiticum. Results also showed that staphylococcus aureus have the highest percentage of occurrence in wastewater, while in sludge Micrococcus species have the highest percentage of occurrence.

Cladosporium species, a new challenge in strawberry production in Iran.

Between April and August 2013, two new fungal pathogens were isolated from strawberry foliage in strawberry production regions of Iran. Based on morphology and translation elongation factor 1-alpha (TEF) gene sequence analysis, the pathogens were identified as Cladosporium macrocarpum and C. limoniforme . Pathogenicity tests were performed on strawberry ( cv . Parous) fruits and leaves and the original C. macrocarpum and C. limoniforme were re-isolated complying with Koch’s postulates. This is the first report of C. macrocarpum and C. limoniforme as causal agents of a strawberry disease in Iran.

A combination of two rarities: Idiopathic CD4+ lymphocytopenia and cerebellar phaeohyphomycosis

Cladosporium bantianum is a neurotropic, dematiaceous fungus that causes deep-seated phaeohyphomycosis in humans. Idiopathic CD4+ T-lymphocytopenia is termed as severe unexplained HIV seronegative immunosuppression. We report a case of Cladosporium cerebellar abscess in a 40-year-old man with idiopathic CD4+ T-lymphocytopenia.

Diversity and distribution of fungal communities within the hot springs of soda lakes in the Kenyan rift valley

Fungi are highly diverse and versatile, with members growing under different environmental conditions including extreme environments. Although fungal communities in some extreme environments have been investigated in recent years, little is known about their structure and richness within the hot springs of soda lakes in Kenya. The aim of the study was to determine the biogeography and diversity of fungi from the hot springs of four Soda lakes. Water, sediment and microbial mat samples were collected in triplicates from Lakes Bogoria, Magadi, Elmenteita and Little Magadi in Kenya. 454-Pyrosequencing was used to sequence amplicons of Internal Transcribed Spacer (ITS) gene region of the total community DNA in order to explore the fungal community composition in twenty four samples collected. Sequences were analyzed using QIIME pipeline Version 1.8.0, while hierarchical clustering, non-metric dimensional scaling (NMDS) and diversity indices were carried out using the R programming language and the Vegan package. A total of 139,023 quality sequence reads were obtained from which, 2,179 operational taxonomic units (OTUs) were realized at 3% genetic distance. Three known phyla (Ascomycota [83.3%], Basidiomycota [15.8%], Glomeromycota [0.02%]) were identified. Richness, abundance and taxonomic analyses identified Agaricomycetes as the most abundant and diverse class within Basidiomycota. Sequences matching with Ascomycota had high affinities with seven known classes, with Sordariomycetes and Dothideomycetes being the most abundant and diverse classes. The most abundant OTUs showed the highest sequence similarity to Cladosporium sp., Cladosporium cladosporioides, Pleosporales sp., Aureobasidium pullulans and Aspergillus oryzae. Key words: Hot springs, fungi, 454 pyrosequencing, diversity, extreme environments.

Effects of essential oil from Cymbopogon citratus leaves and its susceptibility on the quality of fresh orange juice during storage

The present study aims to evaluate the effect of essential oil (EO) from Cymbopogon citratus leaves against the spoilage flora of fresh orange juice. Thus, the EO was extracted by hydrodistillation from fresh leaves of Cymbopogon citratus collected in southern Benin and its chemical composition was determined by gas chromatography, coupled to mass spectrometry (GC/MS). Orange samples were collected from large production areas of South and Central Benin and juices were extracted by mechanical pressing. After identification of spoilage flora of fresh orange juice, antimicrobial tests were carried out with the EO of Cymbopogon citratus to evaluate its antimicrobial activity on spoilage flora of fresh orange juice. Results indicate that the spoilage flora of fresh orange juice is mainly composed of fungi belonging to the genera of Cladosporium, Penicillium and Fusarium. Bacteria such as Enterobacter cloacae and Enterobacter aerogenes were also identified in some samples. The major compounds identified in the EO by GC/MS are Neral (33.0%) and geranial (41.3%) with a predominance of oxygenated monoterpenes (85.5%). Antimicrobial tests have revealed a high antibacterial activity of the EO, with minimum bactericidal concentrations (MBC) between 0.1 and 0.15 μL.mL-1. Antifungal tests revealed that fungi are also susceptible to this EO with minimum fungicidal concentration (MFC) between 0.15 and 0.25 μL.mL-1. Results obtained during the evaluation of the physicochemical characteristics of the orange juice stored by adding EO, indicated a significant decrease in the pH and vitamin C content. However, with EO concentration of 0.250 μL.mL-1, the pH of stored juice was 6.4 ± 0.1 after 15 days of preservation, with a best vitamin C content of 28.06 ± 0.03 mg / 100mL. The EO of Cymbopogon citratus, with high antimicrobial activity, could be used as an alternative in the preservation of fruit juices, replacing antimicrobials from chemical synthesis.

Incidence and etiology of postharvest diseases of fresh fruit of date palm ( Phoenix dactylifera L.) in the grove of Elx (Spain)

The incidence and etiology of postharvest diseases affecting fresh date fruit in the palm grove of Elx (Spain) were determined under local environmental conditions. Latent and wound pathogens were assessed for two consecutive seasons on fruit from two important commercial cultivars, ‘Boufeggous’ and ‘Medjool’, grown in different orchards. Healthy dates were either surface-disinfested or artificially wounded in the rind and placed in humid chambers at 20oC for up to 7 weeks. Irrespective of cultivar, season, orchard, and type of infection, the most important causal agents of disease were Penicillium expansum , Alternaria alternata , Cladosporium cladosporioides , and a black aspergillus species belonging to the Aspergillus niger clade. These fungi were identified by macroscopic and microscopic morphology and/or DNA amplification and sequencing. Their pathogenicity was demonstrated by fulfilling Koch’s postulates. Disease development at 20 and 5oC was characterized on artificially inoculated dates.

FIRST REPORT OF CLADOSPORIUM OXYSPORUM CAUSING LEAF SPOT OF ROSA INDICA IN PAKISTAN

In December 2014, necrotic leaf spots were observed on more than fifty rose ( Rosa indica ) plants in a private garden at Lahore, Pakistan, all showing the same symptoms The infected plants were 1-2 year old and on an average 90% of the leaves were covered with 2-5 mm circular black spots, with irregu- lar margins without chlorosis around the margins. Initially, infected leaves remained attached to the plants but dropped when the spots coalesced and covered more than 80% leaf area. No symptoms were observed on flowers. For pathogen isolation, one spot per leaf from six symptomatic leaves of dif- ferent plants were cut into pieces, surface sterilized, inoculated on 2% malt extract agar (MEA) and incubated at 25 ± 2oC. Morphological observations were made on 7-day-old pure cul- tures. Colonies were greenish black and attained the diameter of 4 mm. Conidiophores were macronematous, smooth walled, up to 500μm long and 4-5μm wide, with intercalary and termi- nal swellings. Conidia were pale brown arising from terminal swelling of conidiophores, in the form of simple or branched chains. Conidia were spherical, subspherical or limoniform, 3-6 μm in diameter; ramoconidia were 2-4×7-3 μm in size. A representative pure culture of the fungus was deposited to First Fungal Culture Bank of Pakistan under the accession No.FCBP1517. Based on morphology, the fungus was identi- fied as Cladosporium oxysporum (Bensch et al ., 2010). For se- quencing of ITS region, a DNA fragment of ca . 600 bp was amplified using universal primer pair ITS1/ITS4 and total ge- nomic DNA as template. BLASTn results indicated that ITS nucleotide sequence of this strain (KT283681) had 99% simi- larity with many other isolates of C . oxysporum in GenBank, including KT936546, LC040920, KJ475816 and JQ775499. Pathogenicity test was performed three times on young pot- ted rose plants using two different isolates. Since the inoculum of this fungus persists in soil, conidia from pure cultures were suspended in sterilized water (10 7 spores/ml) and sprayed in the soil of three healthy plants; three control plants were treated similarly with sterilized water. All plants, covered with poly- thene bags, were kept in a growth chamber at 23 ± 2oC. Ne- crotic spots similar to those described above started appearing on leaves only on inoculated plants after 21 days of incubation. Re-isolation of same fungus fulfilled Koch’s postulates. To our knowledge, this is the first report of R . indica leaf spot caused by C . oxysporum in Pakistan.