Browsing by Author "Attanayake, R.N."

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Anti-oxidant activity of selected endo lichenic fungi (ELF) in mangrove ecosystem of Puttalam lagoon.
(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Maduranga, H.A.K.; Attanayake, R.N.; Amarasinghe, M.D.; Weerakoon, G.; Paranagama, P.A.
Natural products based drug development has become an attractive area of research since there are limited options available to treat certain non-infectious diseases such as diabetes. Among these natural products, it has been reported that secondary metabolites of endolichenic fungi (ELF), have the ability to produce promising bioactive compounds. The objectives of this research were to isolate and identify ELF inhabiting mangroves in Puttalam lagoon, Sri Lanka using classical and DNA barcoding approach and to determine anti-oxidant activity of their secondary metabolites. Lichen hosts were collected from Puttalam lagoon in two different sites near, Athathale and around the NARA institute. The ELF were isolated following a standard procedure: a small piece of the thallus was surface sterilized, cut into pieces and dried on sterilized filter papers and then placed on malt extract agar in Petri dishes and incubated at room temperature (28 ⁰C – 30 ⁰C ) . Once pure cultures were obtained, seven isolates were randomly selected for DNA extraction following standard procedures. Quality of DNA was checked by agarose gel electrophoresis. Fungal internal transcribed spacer (ITS) region was amplified using polymerase chain reaction (PCR) with universal ITS 1 and ITS 4 primers and PCR products were sequenced using Sanger dideoxy chain-termination technology. DNA sequences were edited using BioEdit software and compared with the available sequences in the GenBank using Basic Local Sequence Alignment Search Tool (BLAST). In addition, morphological characterization of each fungal isolate was also carried out. Secondary metabolites from each isolate were extracted with ethylacetate separately and the solvent was evaporated under reduced pressure to obtain the crude extract. Free radical scavenging activity of the extracts were evaluated using 2, 2-diphenyl-1-picrylhydrdrazyl (DPPH) assay. Based on the highest sequence similarity to the GenBank sequences, isolates were identified as Diaporthe arengae (98 %), Neurospora crassa (100%), Lasiodiplodia theobromae (100 %), Schizophyllum commune (98 %), Diaporthe musigena (98 %), Hypoxylon anthochroum (98 %) and Nigrospora sphaerica (98%). IC50 values of extracts of Diaporthe arengae, Neurospora crassa and Lasiodiplodia theobromae were 375.9± 0.062μg/mL, 304.9±0.057 μg/mL and 211.2± 0.086 μg/mL respectively. Since percent inhibitions of the rest of the isolates were less than 50 % in the test doses, IC50 values were not calculated. All of the values were compared with standard Butylated Hydroxy Toluene (BHT) (IC50=108.0±0.072). Out of the seven ELF tested, L. theobromae showed the highest DPPH radical scavenging activity. Further testing of the rest of the isolates are being carried out and ELF may provide a good source of antioxidants for biotechnological applications.
Antibacterial Polyketide from Lasiodiplodia theobromae and Pyrenula bahiana on Mangrove Ecosystems in Puttalam Lagoon, Sri Lanka
(Asian Journal of Chemistry, 2022) Paranagama, P.A.; Santhirasegaram, S.; Wickramarachchi, S.R.; Attanayake, R.N.; Weerakoon, G.; Maduranga, K.
Lasiodiplodia theobromae is one of the frequently isolated fast growing endolichenic fungus. This fungus was isolated from the lichen host, Pyrenula bahiana collected from the mangrove ecosystems in Puttlam lagoon and its identification was confirmed based on rDNA-ITS sequence homology. Secondary metabolites of L. theobromae were extracted into ethyl acetate and subjected to antibacterial assay against Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923) and Bacillus subtilis (ATCC6051). Crude extract at a concentration of 6.8 μg/mL showed good antibacterial activity against the bacterial strain S. aureus compared with the activity of the standard azithromycin at a concentration of 5.0 μg/mL. Active crude extract was partitioned to obtain methanol, hexane and chloroform fractions. Chloroform fraction showed the highest activity to S. aureus out of three fractions. This fraction was subjected to bioassay-guided separation on silica gel column chromatography to isolate bioactive pure compounds. The bioactive pure compound was identified as (3R)-de-O-methyllasiodiplodin using LC-MS, 1D and 2D NMR spectroscopy.
Antioxidant activity and chemical constituents of methanolic extract of Durio zibethinus Murr. (durian) peels
(MEDICINAL PLANTS - INTERNATIONAL JOURNAL OF PHYTOMEDICINES AND RELATED INDUSTRIES, 2021) Perera, P.J.; Binuwangi, A.K.D.M.; Silva, A.A.G.; Attanayake, R.N.; Wickramarachchi, S.R.; Rajapakse, C.S.K.
This study aimed to determine the DPPH free radical scavenging activity, total phenolic content (TPC) and total flavonoid content (TFC) of methanolic extract of Durio zibethinus Murr. (durian) peels and its fractions. The chemical constituents of durian peels extracted into methanol by soxhlet extraction were sequentially extracted into hexane, dichloromethane and aqueous methanol. Among the fractions, the dichloromethane fraction showed the highest DPPH free radical scavenging activity (IC50 179.9 ± 6.6 μg/ml) with high TPC and TFC (85.82 ± 12.11 mg gallic acid equivalent/g of dried weight of extract and 12.66 ± 1.94 mg of quercetin equivalent/g of dried weight of extract, respectively). A very strong positive correlation (r = 0.9677) was observed between the DPPH free radical scavenging activity and the TPC of fractions and a strong positive correlation (r = 0.7858) was noticed between the DPPH free radical scavenging activity and TFC of the fractions indicating that phenolic compounds in durian peels may contribute to their strong antioxidant activity. As the dichloromethane fraction had constituents with the highest antioxidant activity, it was analyzed by Gas chromatography-Mass spectrophotometry to identify its volatile constituents. The results revealed that the dichloromethane fraction was rich in [1,2-Benzenedicarboxylic acid, bis (2-ethylhexyl) ester], [2,3-diphenylquinoxaline], [2-coumaranone], [4-((1E)-3-hydroxy-1-propenyl)-2-methoxyphenol], [7,9-di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione] and [phenol, 2,4-bis(1,1-dimethylethyl)], which are known to exhibit antioxidant activity.
Antioxidant, a-Amylase Inhibitory Activities and Photoprotective Properties of Peels of Nephelium Lappaceum Linn. (Malwana Special)
(Oriental Journal of Chemistry, 2021) Binuwangi, A.K.D.M.; Perera, M.P.J.; Silva, A.A.G.; Attanayake, R.N.; Rajapakse, C.S.K.
This study focused on evaluation of antioxidant, α-amylase inhibitory activities and photo protective properties of peels of Nephelium lappaceum Linn. (rambutan); Malwana special. Methanolic extract of peels was sequentially partitioned in hexane, dichloromethane (DCM) and aqueous methanol. The methanol extract showed a significantly (p greater then 0.05) higher DPPH radical scavenging activity than that of butylated hydroxytoluene. Among the fractions, the highest total phenolic content (TPC) was found in the aqueous methanol fraction. DCM and aqueous methanol fractions were rich in flavonoids. In vitro α-amylase inhibitory activity of the aqueous methanol fraction was also significantly higher than the standard drug, acarbose. Partially purified aqueous methanol fraction of rambutan peels exhibited UV-B absorption with a moderate solar protection factor. The results revealed that the peels of Nephelium lappaceum Linn., Malwana special can be considered as a promising source for the development of natural antioxidant, cosmeceutical sunscreen and antidiabetic agents.
Can Anaerobic Soil Disinfestation (ASD) be a Game Changer in Tropical Agriculture?
(Pathogens, 2021) Priyashantha, A.K.H.; Attanayake, R.N.
Anaerobic soil disinfection (ASD) has been identified as an alternative soil-borne pathogen control strategy to chemical fumigation. ASD involves the application of an easily liable carbon source followed by irrigation to field capacity and maintenance of an anaerobic condition for a certain period. A literature search undertaken on ASD found that more than 50 comprehensive research projects have been conducted since its first discovery in 2000. Most of these studies were conducted in the USA and in the Netherlands. Though the exact mechanism of ASD in pathogen control is unknown, promising results have been reported against a wide range of pathogens such as fungi, nematodes, protists, and oomycetes. However, it is interesting to note that, except for a few studies, ASD research in the developing world and in the tropical countries has lagged behind. Nevertheless, with soil quality depletion, reduction in arable lands, and exponential population growth, a drastic change to the current agricultural practices should be adapted since yield gain has reached a plateau for major staple crops. Under such circumstances, we identified the gaps and the potentials of ASD in tropical agricultural systems and proposed promising biodegradable materials.
Carbon Source dependent - anaerobic Soil Disinfestation (ASD) Mitigates the Sclerotial Germination of Sclerotinia Sclerotiorum
(Tropical Plant Pathology (2020), 2020) Mahalingam, T.; Rajapakse, C.S.K.; Somachandra, K.P.; Attanayake, R.N.
Though Sclerotinia sclerotiorum IS a well-studied plant pathogen that causes significant economic damage worldwide, sustain able and environmental friendly control methods are difficult to establish due to it wide host range, cosmopolitan distribution and production of recalcitrant structures that can survive in soil for a long time. The pathogen was found causing a severe disease incidence on cabbage in 2016 in Sri Lanka. It was hypothesized that a) isolates of the recent disease outbreak display cross resistance to commonly applied fungicides and b) carbon (C) source supplemented Anaerobic Soil Disinfestation (ASD) is effective in mitigating the germination of sclerotia. In vitro fungicide sensitivity assays showed large variation in mycelial growth inhibition indicating high adaptability of the population towards environmental fluctuations and management practices. Signatures of cross resistance were evident. ASD was carried out using cabbage (Brassica oleracea) and leek (Allium ampeloprasum) cull piles, durian (Durio zibethinus) peels and grass cuttings (Axonopus compressus) as C sources and determined the sclerotial viability. Cabbage and leek cull piles at rates of 60—100 mg/g soil completely inhibited sclerotial germination. Maintaining anaerobic condition along with C source amendments was found to be a critical step in mitigating the sclerotial germination. GC-MS analysis of the volatiles of cabbage leaves, leeks and durian further confirmed the presence of various bioactive compounds with potential antifungal activity. Therefore, in addition to elevated microbial activity in treatments, the volatiles of C sources may have helped mitigating sclerotial germination.
Characterization of Agrobacteriu/ll strains from agricultural soils of Bandarawela, Sri Lanka
(First National Symposium of Sri Lanka Association for Mycology and Plant Pathology (SLAMPP), 2019) Somarathna, G.M.T.K.; Somachandra, K.P.; Jayalath, W.G.H.; Attanayake, R.N.
Agrobacterium is a Gram negative, rod shaped, aerobic and motile soil inhabiting bacterium of the family Rhizobiaceae. It is well known as the causative agent of crown gall disease of many plant species around the world. However, not all the Agrobacterium strains are pathcvenic and can cause galls. Only the virulent strains cause crown gall disease on number of plant species and are found only in contaminated soils. These virulent strains of A. tumefaciens harbor Ti plasmids with transfer DNA (T-DNA) region and virulence (vir) genes that are responsible for the pathogenicity. virD2 gene codes for virD2 protein and the endonuclease domain of the virD2 protein cleaves T-DNA border sequences. The ipt gene is the T-DNA borne cytokinin synthesis gene. Therefore, the presence of virD2 gene and ipt gene are useful in identifying pathogenic strains of Agrobacterium. The major objective of this research was to determine whether agricultural soils of Bandarawela were contaminated with virulence strains of A. tumefaciens. Soil samples were collected and bacteria were isolated using soil dilution method, and cultured on Yeast Mannitol Acar supplemented with Congo red and on Yeast Extract Peptone Affar. Five pure cultures of putatively Agrobacterium were further characterized using morphological and biochemical tests including Gram staining, catalase test, citrate utilization test, sugar fermentation test and 3-ketolactose test. These testes were often used for the species level identification of A. tumefaciens. Out of five isolates four were rod shaped with rounded ends and were either single or in pairs. However, the other isolate was in chains and Iono rod shaped. Interestingly, all the isolates were positive for all the biochemical tests. However, these tests do not help differentiating the virulence strains. Molecular characterization of all the soil isolates were carried out using universal 16s rRNA primers and Agrobacterium specific primers targeting virD2 and ipt genes. PCR amplification with virD2 primers successfully amplified the targeted band of 224 bp in all five isolates while ipt produced the expected fragment of about 427 bp in three of the isolates. virD2 cene sequences of selected soil isolates were 100-99% similar to the tumefaciens of the GenBank accession CP032925 and CP032929 reported from Taiwan. According to morphological, biochemical, and molecular Characterization using virD2 and ipt genes it was confirmed that the soil in the inspected field of Bandarawela is contaminated with pathogenic strains of A. tumefaciens. Therefore, farmers should maintain awareness when cultivating susceptible plant varieties in these fields.
Characterization of Ascochyta rabiei for population structure, mating type and pathogenic variability from Pakistan and United States
(III international Ascochyta workshop, Cordoba, Spain, 2012) Hinaali, A.S.S.; Attanayake, R.N.; Rahman, M.; Chen, W.
Chickpea production is greatly hampered by blight causing fungal pathogen Ascochyta rabiei (AR) in chickpea growing regions of the world. Genetic variability and mating type frequency of thirty two AR isolates from six geographical regions of Pakistan were compared with a US-AR population. Pakistani AR (PAR) population had an apparent skewed (3 Mat1-2: 1 Mat1-1) distribution, although Chi-square tests showed non significant deviation from equal distribution due to small sample sizes and the US-population showed a 1:1 distribution. The results showed that sexual reproduction is rare in PAR due to either unavailability of both mating types or lack of conductive environment but statistical analysis showed panmixia which may be due to past recombinational events. Genetic variation at six microsatellite loci was assessed and each isolate was assigned to a microsatellite haplotype. Population structure using Bayesian analyses differentiated isolates into three distinct clusters, two clusters of PAR and one of the US isolates. However, few isolates from US shared same genetic background with one cluster of the PAR isolates, providing a link of inter-continental migration of the pathogen due to import of seeds. Additionally, the two clusters of Pak-isolates are not strictly linked to the geographic locations in Pakistan, suggesting frequent gene flow of AR among different locations. Pathogenic variability of nineteen PAR collected from two different provinces was assessed. The results based on the reaction of isolates with differential lines showed that aggressive and highly aggressive pathotypes II and III respectively are prevalent in Pakistan as compared to least aggressive pathotype I. It is interesting to note that highly aggressive pathotypes III and IV have only beenreported from Syria and Pakistan where we assume less frequency of sexual reproduction due to predominance of one mating type, in contrast to other countries where both mating types are present in equal ratio hence, this issue needs further investigations.
Cloning and characterization of anonymous regions of Ascochyta lentis and a. Fabae genomes and suitability of these regions for phylogenetic analysis of Ascochyta species
(Proceedings of the Second International Ascochyta Workshop, 2009) Peever, T.L.; Drader, T.; Njambere, E.N.; Attanayake, R.N.; Stewart, J.E.
Ascochyta species cause blights on a number of wild and cultivated cool?season legume hosts, including chickpea (Cicer arietinum L.), faba bean (Vicia faba L.), lentil (Lens culinaris Medik.), pea (Pisum sativum L.), and vetches (Vicia spp.). Ascochyta blight of faba bean and lentil are caused by the host?specific fungi A. fabae Speg. and A. lentisVassiljevsky, respectively. Identification of these species has been primarily based on host specificity because they are morphologically indistinguishable (Gossen et al.1986). Previous studies have demonstrated that each species have distinct RAPD?PCR banding patterns (Kaiser et al. 1997), and each form a monophyletic group in a combined phylogeny estimated from glyceraldehyde?3?phospatedehydrogenease (G3PD), translation elongation factor alpha (EF), and chitin synthase (CHS) genes (Peever et al. 2007). No additional fast?evolving markers have been identified for these fungi that would facilitate research at the population/species interface. Therefore, the objective of this research was to develop sequence characterized anonymous region (SCAR) markers for identification of A. fabae and A. lentis, for estimating genetic variation within and among species, and for inferring phylogenetic relationships.
Contarinia maculipennis as an emerging threat to Dendrobium in Sri Lanka - A case study.
(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Dias, M.A.; Amarasinghe, L.D.; Jayalath, W.G.H.; Attanayake, R.N.
Blossom midge, Contarinia maculipennis which belongs to Order Diptera: Family Cecidomyiidae is considered as one of the major threat to ornamental and several crop plant species due to its wide host range. For the first time C. maculipennis was recorded from Dendrobium sp. in 1992 from Florida, but the origin of this species is considered as Southeast Asian region. In Korea, it is officially nominated as a quarantine pest since 2007 due to it’s sever economic impact on vegetable crops and ornamental plants. For the first time in Sri Lanka, completely damaged Dendrobium cultivation was found in an ornamental plant nursery at Gampaha district in 2017. It was noted that the symptoms were similar to that of blossom midge damage. Maggots were found to be feeding inside unopened flower buds, causing deformed, discolored buds and blossoms causing premature bud or blossom drop. Floral buds were often found to be rotted. Samples from immature bud stage to fully opened flowers were randomly collected from infected fields into polythene bags. To identify causative agent, floral buds with larval stages were kept in glass containers to allow them to complete their life cycle and thereby morphological characters were studied to confirm the pest species. In addition, yellow color grease sheets were kept inside the greenhouses to trap any adult stages of the pest species. Samples were collected and preserved using 70% ethanol for identification. Since all the damaged flower buds displayed symptoms of bacterial rots, to determine if any bacterial infection is also associated with the symptoms, bacterial isolation procedure was carried out. Different stages of floral samples were separately surface sterilized for two minutes using 70% ethanol and three serial washings with sterilized distilled water. Tissue macerate was prepared and kept for 3 hours before culturing on Nutrient Agar (NA) plates, Potato Dextrose Agar (PDA) and Luria-Bertani (LB) plates. Each sample had three replicates and ten samples were cultured. Growth from the tissues were observed and pure cultures were obtained. Relative length of the first and second flagellomeres, wing length and pattern, larval sternal spatula and its associated papillae and larval eighth abdominal segments were compared with identification keys which were used to identify the genera, Contarinia. The adult stages of trapped insects and adult stages of insect immerged from the larval stages were useful in confirming the species as C. maculipennis. Basic biochemical tests and Gram’s staining assisted in identifying the bacterium as belonging to the genera, Erwinia sp. and it appears that the bacterial infection occurs as a secondary infection after larval stages of C. maculipennis damage the floral tissues. This is the first record of C. maculipennis infecting orchid nurseries in Sri Lanka and if proper care is not taken it will invade other crop species as the pest has a broad host range. It is not clear whether the pest was a recent introduction through the imports of plant material or whether it is a result of host jump and therefore, it warrants further research.
Decaying Hardwood Associated Fungi Showing Signatures of Polyethylene Degradation
(BioResources, 2021) Perera, P.; Deraniyagala, A.S.; Mahawaththagea, M.P.S.; Herath, H.; Rajapakse, C.S.K.; Wijesinghe, P.; Attanayake, R.N.
The involvement of wood decay fungi and the importance of their enzymes in polyethylene degradation is well documented. Therefore, decay-resistant hardwood associated fungi should be better degraders with their versatile enzymatic systems. In the current study, decaying hardwood associated fungi were isolated and their ability to degrade low-density polyethylene (LDPE) was assessed. Thirty-three isolates were identified by sequencing the internal transcribed spacer region of nuclear ribosomal DNA. Randomly selected isolates were tested for laccase producing abilities. Three species were selected to test their potentials in LDPE sheet degradation. Fungi were incubated in Czapek-Dox broth containing 20-micron LDPE sheets at room temperature for 60 days. The biodegradation signatures were assessed by analyzing the changes in structural characteristics of LDPE using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), percent reduction of tensile properties, and weight loss. FTIR analysis revealed changes in certain functional groups compared with the control, indicating chemical changes resulting from the treatment. LDPE sheets incubated with fungi showed cracks and holes under SEM analysis, percent reduction in tensile properties, and weight loss, which are the signatures of degradation. This study revealed that the hardwood decaying basidiomycetes, Phlebiopsis flavidoalba, Schizophyllum commune, and Phanerodontia chrysosporium have the potential for in vitro LDPE degradation.
Detection of intrachromosomal recombination in Sclerotinia sclerotiorum populations
(Phytopathology, 2012) Attanayake, R.N.; Chen, W.
Genetic structure and reproductive mode of the homothallic fungal pathogen Sclerotinia sclerotiorum have been widely studied using linkage disequilibrium (LD) tests with putatively unlinked molecular markers. We previously observed random association between linked loci in S. sclerotiorum populations suggesting intrachromosomal recombination or high mutation rates at these loci. This study was aimed at testing intrachromosomal recombination using 12 microsatellite loci distributed over four chromosomes. Two hundred thirty isolates sampled from seven populations in the USA and China from a variety of crops were genotyped. Each isolate carried a single allele for each of the 12 loci suggesting the isolates were haploid and homokaryotic. Pairwise LD tests of all the intrachromosomal loci showed relationship ranged from linked to random association, and in many cases LD declined with increasing physical distance between loci. Thus the random associations of alleles cannot be simply attributed to random mutation. Majority of the isolates were mycelially incompatible, likely minimizing the possibility of heterokaryon formation and mitotic recombination. Thus the observed high intrachromosomal recombination is most likely due to meiotic recombination following outcross in these populations.
Diversity of decaying wood associated fungi in Dimbulagala forest of Sri Lanka
(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Shashikala, M. M. P.; Deraniyagala, A.; Attanayake, R.N.
Sri Lanka is one of the 34-biodiversity hotspots in the world bearing a tremendous diversity in flora and fauna. Therefore, it should hold true for fungal species diversity as well. In Sri Lanka, tropical wet evergreen rain forest reserves, mainly Sinharaja forest is well studied for the macro and micro fungal diversity. However, studies in the dry zone and intermediate zone forests are neglected though 83% of the country’s forest cover belongs to this category. The current study was initiated to describe the decaying wood associated fungal diversity in a dry mixed evergreen forest reserve. Decaying wood samples were collected from Dimbulagala forest reserve. Decaying hard wood pieces of at least 6 inches length were collected randomly. Fungal strains associated with the decaying woods were isolated into PDA or semi-selective medium and pure cultures were obtained. Macroscopic and microscopic features were observed. Total genomic DNA was isolated from a modified CTAB method and Polymerase Chain Reaction (PCR) was conducted targeting rDNA-ITS region using universal ITS primers and Sanger dideoxy sequencing was carried out to determine the nucleotide sequence of the region. Sequences were manually edited and compared with the GenBank using Basic Local Sequence Alignment Search Tool (BLAST). Phylogenetic relationships among decaying wood associated fungi were determined using MEGA (version 7.0) and according to the phylogenetic analysis well-defined clusters of fungi that belongs to divisions Ascomycota and Bascidiomycota were found. Fungal cultures were maintained at the Department of Botany using dry filter papers and in sterilized distilled water. A total of 55 fungal isolates were obtained from 36 decaying wood pieces and 35 fungal species were successfully identified. Results indicated that Sri Lankan dry mixed evergreen forests are rich in species of Paecilomyces, Daldinia, Trametes, Perenniporia, Phanerochaete, Hypoxylon, Schizophyllum, Lentinus, Fusarium, Coriolopsis, Phlebia, Coprinellus, Gymnopilus, Scytalidium, Trichoderma, Xylogone, Lasiodiplodia, Neoscytalidium and Pleurostoma. Species of Trichoderma and Lasiodiplodia were the most abundant species. T . harzianum T. lixii, T . longibrachiatum, and T . erinaceus were also found. Out of six Lasiodiplodia isolates, three were L . crassispora, and the rest belonged to the species of L . pseudotheobromae and L . theobromae. Some of the isolated fungi were already known plant pathogens and some were well-known biodegraders. The results indicated that the least studied Sri Lankan dry mixed evergreen forests are rich in various fungal species and could serve as another source in finding biotechnologically important fungal species.
Erysiphe trifolii Causing Powdery Mildew of Lentil (Lens culinaris)
(Plant Disease, 2009) Attanayake, R.N.; Glawe, D.A.; Dugan, F.M.; Chen, W.
The taxonomy of the powdery mildew fungus infecting lentil in the Pacific Northwest (PNW) of the United States was investigated on the basis of morphology and rDNA internal transcribed spacer (ITS) sequences. Anamorphic characters were in close agreement with descriptions of Erysiphe trifolii. However, teleomorphs formed chasmothecial appendages with highly branched apices, whereas E. trifolii has been described as producing flexuous or sometimes loosely branched appendages. Branched appendages have been described in Erysiphe diffusa, a fungus reported from species of Lens, Glycine, and Sophora, raising the possibility that the PNW fungus could be E. diffusa. Examination of morphological characters of an authentic specimen of E. trifolii from Austria determined that it included chasmothecial appendages resembling those seen in PNW specimens. Furthermore, ITS sequences from five powdery mildew samples collected from lentils in PNW greenhouses and fields from 2006 to 2008 were identical to one another, and exhibited higher similarity to sequences of E. trifolii (99%) than to those of any other Erysiphe spp. available in GenBank. Parsimony analysis grouped the lentil powdery mildew into a clade with Erysiphe baeumleri, E. trifolii, and E. trifolii?like Oidium sp., but indicated a more distant relationship to E. diffusa. In greenhouse inoculation studies, the lentil powdery mildew fungus did not infect soybean genotypes known to be susceptible to E. diffusa. The pathogenicity of E. trifolii on lentil was confirmed using modified Koch's postulates. This is the first report of E. trifolii infecting lentil. E. diffusa and E. trifolii have different host ranges, so the discovery of E. trifolii on lentil has implications both for determining species of powdery mildews on cool-season grain legumes, and in disease management.
Erysiphe trifolii? a newly recognized powdery mildew pathogen of pea
(Plant Pathology, 2010) Attanayake, R.N.; Glawe, D.A.; McPhee, K.E.; Dugan, F.M.; Chen, W.
Diversity of powdery mildew pathogens infecting pea (Pisum sativum) in the US Pacific Northwest was investigated using both molecular and morphological techniques. Phylogenetic analyses based on rDNA ITS sequences, in combination with assessment of morphological characters, defined two groups of powdery mildews infecting pea. Group I (five field samples and three glasshouse samples) had ITS sequences 99% similar to those of Erysiphe pisi in GenBank and exhibited simple, mycelioid type of chasmothecial appendages typical of E. pisi. Erysiphe pisi is normally considered as the powdery mildew pathogen of pea. Group II (four glasshouse samples and two field samples) had ITS sequences 99% similar to those of E. trifolii and produced chasmothecia with dichotomously branched appendages similar to those of E. trifolii. There are fourteen nucleotide differences in the ITS region between the two groups. The correlation of rDNA ITS sequences with teleomorphic features for each of the two groups confirms their identity. Repeated samplings and artificial inoculations indicate that both E. pisi and E. trifolii infect pea in the US Pacific Northwest. Erysiphe trifolii is not previously known as a pathogen of pea. The existence of two distinct powdery mildew species infecting pea in both glasshouse and field environments may interfere with the powdery mildew-resistance breeding programmes, and possibly explains putative instances of breakdown of resistance in previously resistant pea breeding lines.
Evaluation of phenetic diversity of selected orchid cultivars with ornamental value
(The Institute of Biology, Sri Lanka, 2016) Farook, F.; Attanayake, R.N.; Senanayake, S.P.
First Report of Powdery Mildew of Chickpea (Cicer arietinum) Caused by Leveillula taurica in Washington State
(Plant Health Progress, 2008) Chen, W.; Attanayake, R.N.; Glawe, D.A.; McPhee, K.E.; Dugan, F.M.
In Oct. 2007, powdery mildew was found in chickpea fields in an experimental farm near Pullman, Whitman County, Washington. Although disease signs were observed on all chickpea cultivars in the fields, high incidence was seen only on cvs. Dwelley and Spanish White. To our knowledge this is the first record of powdery mildew caused by Leveillula taurica on chickpea in WA. The pathogen has also been reported from chickpea in California and elsewhere, e.g., Ethiopia, India, Iran, Morocco, Pakistan, Sudan, Turkey, and the former USSR.
Genetic and phenotypic divergence of the Sclerotiniasclerotiorum populations from China and USA
(Phytopathology, 2013) Attanayake, R.N.; Carter, P.A.; Jiang, D.H.; Río-Mendoza, L.D.; Chen, W.
Genetic and phenotypic diversity and population differentiation of Sclerotinia sclerotiorum isolates infecting canola from China and the United States were investigated. Genetic diversity was assessed with eight microsatellite markers and mycelial compatibility groups (MCGs). Phenotypic diversity was assessed with sensitivity to three fungicides, production of oxalate and sclerotia, growth rate, and virulence on two canola cultivars. No shared MCGs or multilocus haplotypes were detected between the two populations, and populations differed significantly (P < 0.001). Recombination was detected in both populations but was greater in the Chinese population. A polymerase chain reaction detection assay showed that ~60% of the isolates were inversion-plus at the mating type locus. The two populations differed significantly (P < 0.05) for all of the phenotypic traits except for sensitivity to fungicide fluazinam and virulence. Isolates in the Chinese population were unique in several aspects. Despite the phenotypic differentiation, heritabilities of the phenotypic traits were similar for both populations. Significant correlations were found among five phenotypic traits. Cross resistance to benomyl and iprodione was detected. Virulence was not significantly correlated with any other phenotypic trait and had the least heritability. However, both populations were equally virulent on either a susceptible or a moderately resistant canola cultivars.
Genetic and phenotypic diversity and random association of DNA markers of isolates of the fungal plant pathogen Sclerotinia sclerotiorum from soil on a fine geographic scale
(Soil Biology and Biochemistry, 2012) Chen, W.; Johnson, D.A.; Porter, L.; Attanayake, R.N.
Sclerotia of the soil-borne plant pathogen Sclerotinia sclerotiorum were collected from 1 m2 area of the top 1.27 cm layer of soil in an alfalfa field in southeastern Washington state of the US. Out of 272 sclerotia collected, 40 were randomly selected and analyzed for genetic diversity in terms of microsatellite loci, mycelial compatibility groups (MCGs) and phenotypic diversity using five phenotypic traits (fungicide sensitivity, oxalic acid production, growth rate, colony color and virulence). Sixteen microsatellite haplotypes and 15 MCGs were found among the 40 isolates. The isolates showed three colony colors (beige, dark and white) on Difco PDA and exhibited significant differences in growth rate, oxalic acid production, and sensitivity to three fungicides, benomyl, fluazinam and iprodione. However, these isolates did not show differences in their ability to colonize detached pea leaves. No apparent relationship among the neutral genetic markers and the phenotypic traits was detected. Two of the haplotypes accounted 40% of the isolates, suggesting isolates of these haplotypes might be better adapted to the environmental conditions in this alfalfa field. Several lines of evidence indicated high levels of genetic diversity and potential outcrossing within the population of S. sclerotiorum: 1) high likelihood of five genetic populations based on Bayesian probability and the presence of admixed isolates; 2) random association of alleles in every pair-wise linkage disequilibrium test among eight independent microsatellite loci; 3) discordances between microsatellite haplotypes and MCGs and 4) lack of correspondence among the genetic markers and phenotypic traits. Multilocus Index of Association test suggested that outcrossing occurs only within interbreeding subpopulations of S. sclerotiorum.
Genetic diversity and population differentiation of Sclerotinia sclerotiorum collected from canola in China and in U.S.A.
(Phytopathology, 2011) Attanayake, R.N.; del Río-Mendoza, L.; Chen, W.; Jiang, D.
Sclerotinia sclerotiorum is an important pathogen of canola and many other crops worldwide. Genetic diversity and population differentiation of S. sclerotiorum collected from canola fields in Anhui Province, China (30 isolates) and in North Dakota, U.S.A. (29 isolates) were investigated in terms of genetic variation in 8 simple sequence repeat (SSR) marker loci, mycelial compatibility groups (MCGs) and three phenotypic traits: sensitivity to fungicides benomyl, iprodione and fluzinam, oxalic acid production, and pathogenicity. Significant genetic differences were observed; there were no shared SSR haplotypes and no shared MCGs between the two populations. Population differentiation was significant (p = 0.000) indicating lack of gene flow between the two populations. There were also significant differences between the two populations in oxalic acid production and in fungicide sensitivity. The Chinese population displayed high levels of insensitivity (faster growth rate) to benomyl and fluzinam and higher levels of oxalic acid production per unit dry weight of mycelium than did the U.S. population. However, there was no significant difference in pathogenicity between the two populations as measured by colonization of detached canola leaves. Data Vol. 101, No. 6 (Supplement), 2011 S11 suggest that despite geographic and genetic isolation the two populations of S. sclerotiorum were equally adapted to colonizing canola plants, and pathogenicity is under different selection pressure than the other genetic and phenotypic traits.