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Item 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.Item 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.Item 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.Item Potential alternative hosts for a powdery mildew on pea(Phytopathology, 2009) Attanayake, R.N.; Glawe, D.A.; Dugan, F.M.; Chen, W.Powdery mildew of pea (Pisum sativum) is an important disease in the field and in the greenhouse. The most widely documented powdery mildew on pea is Erysiphe pisi, but E. trifolii and E. baeumleri have also been reported. From greenhouse-grown peas, we obtained powdery mildew samples with rDNA ITS sequences nearly identical to previously deposited sequences of E. trifolii. Because detailed studies on host range of this pea powdery mildew in the US Pacific Northwest were lacking, we tested common legume plants from the region as potential alternative hosts. Eleven species were used in greenhouse cross inoculation studies: Lens culinaris, Glycine max, Melilotus albus, M. officinalis, Medicago polymorpha, M. lupulina, M. scutellata, Lathyrus latifolius, Trifolium pratense, Vicia cracca, and V. faba. Except for Glycine max, all the plant species tested developed powdery mildew lesions in 10?14 days after inoculation. Susceptibilities of two of these species (L. culinaris and M. albus) were also confirmed with detached leaf assays. Results showed that all the above legumes (except soybean) are potential alternative hosts for the E. trifolii found on pea. Powdery mildews found on wild legumes (Meliotus albus and Medicago lupulina) were also confirmed to be E. trifolii, suggesting that the wild legumes could be inoculum sources of powdery mildew on greenhouse pea plants during winter months. These findings have implications in managing powdery mildew of pea.Item Taxonomic complexity of powdery mildew pathogens found on lentil and pea in the U.S. Pacific Northwest(Phytopathology, 2008) Attanayake, R.N.; Glawe, D.A.; McPhee, K.E.; Dugan, F.M.; Chen, W.and field production conditions in the U.S. Pacific Northwest was investigated using morphological and molecular characters. Isolates collected from lentil plants grown in the greenhouse or field displayed morphologies in substantial agreement with descriptions of Erysiphe trifolii, but sometimes with more extensively branched chasmothecial appendages resembling those of E. diffusa. ITS sequences of the lentil fungi were identical to each other, and more similar to GenBank accessions of E. trifolii (99.5%) than of E. diffusa (97%). The data suggest there may be more than one Erysiphe species causing lentil powdery mildew. The fungus on field-grown pea plants was determined to be E. pisi. However, powdery mildew samples obtained from greenhouse pea plants were either E. pisi or E. trofolii depending on the time of sampling and greenhouse location. Therefore, the powdery mildews infecting lentil and pea are more diverse than previously assumed. Powdery mildews from black medic (Medicago sp.) and sweet clover (Melilotus sp.) found near the greenhouses exhibited ITS sequences with 99.9 to 100% similarity to isolates from lentil and pea in the greenhouses, and to isolates from lentil from the field. These weedy legumes could be inoculum sources for powdery mildew of lentil and pea in the greenhouses, and serve as alternative hosts for cultivated legumes.Item Pair-wise linkage disequilibrium decay among linked loci suggests meiotic recombination in natural populations of Sclerotinia sclerotiorum(Asilomar Conference Grounds, 2013) Attanayake, R.N.; Chen, W.Both clonal and recombining population structures have been reported in Sclerotinia sclerotiorum populations around the world. Association of independent and putatively unlinked markers indicates clonal population structure, whereas random association of the markers suggests recombination and outcrossing. However, high mutation rates of markers used for inference, like certain microsatellite markers, could interfere and compromise the inferences of recombination and outcrossing. To test if the recombination is due to outcrossing or mutation, we used 12 microsatellite loci distributed over four chromosomes to genotype 230 isolates sampled from seven populations in the USA and China from a variety of crops. All the isolates produced single alleles for each of the loci tested, indicating all the isolates were homokaryotic for the microsatellite loci in consideration. Pair-wise linkage disequilibrium (LD) tests (Hedrick?s D, Fishers exact test and IA) between physically linked loci showed relationships ranging from linked to random association with increasing distance between loci on three chromosomes. For the three loci on chromosome four, LD decay with increasing physical distance between loci was found in six of the seven populations.Item 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.Item 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.Item Population structure and mating type distribution of the chickpea blight pathogen Ascochytarabiei form Pakistan and the United States(Journal of plant pathology, 2012) Ali, H.; Alam, S.S.; Attanayake, R.N.; Rahman, M.; Chen, W.Ascochyta blight caused by the fungus Ascochyta rabiei (AR) depresses chickpea production in Pakistan and worldwide. Thirty two AR isolates representing six geographical regions of Pakistan were compared with a US-AR population for mating type frequency and genetic variation. Mating type results showed that the 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. The US population showed a 1:1 distribution of the two mating types. The uneven distribution of mating types indicates that sexual reproduction among the PAR is rare due to either unavailability of both mating types or lack of conducive environment, but statistical analysis showed that panmixia is there reflecting past recombinational events. Genetic variation at six microsatellite loci was assessed and each isolate was assigned to a microsatellite haplotype. Population structure of the isolates was inferred using Bayesian analyses implemented in the structure software which differentiated isolates into three distinct clusters, two clusters of PAR and one of the US isolates. However, few isolates from the US shared the same genetic background with one cluster of the PAR isolates, providing a link of inter-continental migration of the pathogen. Additionally, the two clusters of PAR-isolates are not strictly associated with geographic locations in Pakistan, suggesting frequent gene flow of AR among different locations. Future studies should extend the sampling of representative populations to overcome the limitations of the small sample size for more accurate assessment of population structure.Item The Importance of Reporting New Host-Fungus Records for Ornamental and Regional Crops(Peer-Reviewed by Plant Health Progress, 2009) Dugan, F.M.; Glawe, D.A.; Attanayake, R.N.; Chen, W.Accurate and timely reports of new host-fungus records are essential for diagnostics and identification, management, and prevention of plant diseases. Important also are venues to publish these reports in a timely manner and the ability to rapidly search for the information contained in these reports. Presented herein are examples of first reports of fungal pathogens on regional crops, including ornamentals and turf grasses, which illustrate how first reports contribute to preparedness, accurate diagnostics, and knowledge of biogeography and host range. We provide a guide to sources of host-fungus records, discuss venues for publishing new records, and review the information important in a new record, including deposition of voucher specimens. We appeal to plant health professionals to increase their efforts of discovering, documenting, and reporting new records.