HSIANG PUBLICATIONS  (updated 2014/03/13, Abstracts & PDF links below)

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Supplemental Data to Publications

Abstracts of Publications and links to PDFs

Shi F, Hsiang T. 2014. Pseudonectria buxi causing leaf and stem blight on Buxus in Canada. Eur. J. Plant Pathol 138:763-773. abstract.

Zhou Y, Zhang J, Wang X, Yang L, Jiang DH, Li GQ, Hsiang T, Zhuang WY. 2014. Morphological and phylogenetic identification of Botrytis sinoviticola, a novel cryptic species causing gray mold disease on table grapes (Vitis vinifera) in China. Mycologia 106:43-56. abstract.

Hsiang T, Shi F, Darbyson A. 2014. First report of Sclerotinia homoeocarpa from the sedge Trichophorum cespitosum in eastern Canada which causes dollar spot disease on Lolium perenne and Poa pratensis but not on Agrostis stolonifera. Plant Disease 98:161. abstract.

Zheng L, Shi F, Hsiang T. 2013. Genetic structure of a population of Rhizoctonia solani AG 2-2 IIIB from Agrostis stolonifera revealed by inter-simple sequence repeat (ISSR) markers. Can. J. Plant Pathol. 35:476-481. abstract.

Liu L, Zhao D, Zheng L, Hsiang T, Wei Y, Fu Y, Huang J. 2013. Identification of virulence genes in the crucifer anthracnose fungus Colletotrichum higginsianum by insertional mutagenesis. Microbial Pathogenesis 64:6-17. abstract.

Mo JY, Li Q, Guo TX, Huang SP, Pan ZB, Ning P, Hsiang T. 2013. First report of gummosis caused by Botryosphaeria dothidea on mango trees in Guangxi, south China. Journal of Plant Pathology 95:659. abstract.

Li Q, Mo J, Huang S, Guo T, Pan Z, Ning P, Hsiang T. 2013. First report of leaf spot disease caused by Glomerella magna on Lobelia chinensis in China. Plant Disease 97:1383. abstract.

Tung J, Goodwin PH, Hsiang T. 2013. Chlorophyll fluorescence for quantification of fungal foliar infection and assessment of the effectiveness of an induced systemic resistance activator. Eur. J. Plant Pathology 136:301-315. abstract.

Li Q, Guo T, Pan C, Huang S, Mo J, Ning P, Hsiang T. 2013. An outbreak of gummosis of mango trees caused by Lasiodiplodia theobromae in Guangxi, south China. Plant Disease 97:690. abstract.

Li Q, Huang S, Guo T, Mo J, Ning P, Hsiang T. 2013., First report of leaf spot caused by Corynespora cassiicola on Baphicacanthus cusia in China. Plant Disease 97:690. abstract.

Jewell LE, Hsiang T. 2013. Multigene differences between Microdochium nivale and Microdochium majus. Botany 91:99-106. Microdochium nivale (Fr.) Samuels & Hallett and Microdochium majus (Wollenw.) Glynn & S.G. Edwards are sister species
that cause diseases on grasses and cereals at low temperatures. The DNA sequences of RPB2 (RNA polymerase II), beta-tubulin, EF-1a
(elongation factor), and ITS (rDNA internal transcribed spacer) from these groups were analysed to compare the extent of differences between these species, among isolates from Europe compared with those from North America, and among isolates of M. nivale originally collected from Agrostis spp. compared with isolates from wheat (Triticum aestivum). All of the regions studied
except for ITS resolved M. nivale and M. majus isolates into separate clades. The RPB2 sequences also resolved both the North
American and European M. majus isolates and M. nivale isolates from either turfgrasses or wheat into separate clades. These
results support the recent elevation of M. nivale and M.majus to sister species and also provide some support for the assertion that
there may be host-specific differences among M. nivale, which has a wider host range than M. majus.
Journal Reprint or contact author

Huang CH, Hsiang T, Trevors JT. 2013. Comparative bacterial genomics: defining the minimal core genome. Antonie van Leeuwenhoek 103:385-398. A comparative genomics analysis revealed 702 genes present in the bacterial Gram-negative core gene set (92 species analyzed) and 959 genes in the Gram-positive core gene set (93 species analyzed). Mycoplasma genitalium, which has the smallest known genome (517 genes) of a non-symbiont, was used in a three-way reciprocal analysis with the Gram-negative core genes and the Gram-positive core genes, and 151 common bacterial core genes were found. Of these 151 core genes, 39 were putative genes encoding the 30S and 50S ribosomal subunits, whilst among recognized cell division genes, only one gene, the major ftsZ, was present. In addition, 86 reciprocal matches were identified between the 151 common bacterial genes and a previously determined 2,723 common eukaryotic core gene set. An analysis was also done to optimize the threshold bit score used to declare that genes were homologous, and a bit score cutoff of 40 was selected. Journal Reprint or contact author

Xue M, Yang J, Li Z, Hu S, Yao N, et al. (2012) Comparative Analysis of the Genomes of Two Field Isolates of the Rice Blast Fungus Magnaporthe oryzae. PLoS Genet 8(8): e1002869. Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice worldwide. The fungal pathogen is notorious for its ability to overcome host resistance. To better understand its genetic variation in nature, we sequenced the genomes of two field isolates, Y34 and P131. In comparison with the previously sequenced laboratory strain 70-15, both field isolates had a similar genome size but slightly more genes. Sequences from the field isolates were used to improve genome assembly and gene prediction of 70-15. Although the overall genome structure is similar, a number of gene families that are likely involved in plant-fungal interactions are expanded in the field isolates. Genome-wide analysis on asynonymous to synonymous nucleotide substitution rates revealed that many infection-related genes underwent diversifying selection. The field isolates also have hundreds of isolate-specific genes and a number of isolate-specific gene duplication events. Functional characterization of randomly selected isolate-specific genes revealed that they play diverse roles, some of which affect virulence. Furthermore, each genome contains thousands of loci of transposon-like elements, but less than 30% of them are conserved among different isolates, suggesting active transposition events in M. oryzae. A total of approximately 200 genes were disrupted in these three strains by transposable elements. Interestingly, transposon-like elements tend to be associated with isolate-specific or duplicated sequences. Overall, our results indicate that gain or loss of unique genes, DNA duplication, gene family expansion, and frequent translocation of transposon-like elements are important factors in genome variation of the rice blast fungus. Journal Reprint

Zheng L, Liu J, Liu T, Zhu Z, Jiang D, Huang J, Hsiang T. 2012. Fusarium wilt of Coleus forskohlii caused by Fusarium oxysporum in China. Can. J. Plant Pathology 34:310-314. Since 2005, a wilt disease on Coleus forskohlii of high severity has been observed in Tongcheng County, Hubei province, China, with the annual crop area affected estimated to be over 100 ha. Symptoms of disease, which included leaves wilting, stem brown discolouration and root rot, were observed after seed germination of C. forskohlii during the spring and summer growing seasons. After isolation and pathogenicity testing, the pathogen was identified as Fusarium oxysporum on the basis of morphological and cultural characteristics. Subsequent analysis of the internal transcribed spacer region of ribosomal DNA and the b-tubulin gene sequence confirmed F. oxysporum as the causal agent. This is the first report of F. oxysporum infecting C. forskohlii worldwide. Journal Website Reprint or contact author for reprint

Shi FA, Hsiang T. 2012. Volutella blight of boxwood. Landscape Trades 34(6):16-17. Reprint

Hsiang T. 2012. First report of leaf spot of Kentucky bluegrass (Poa pratensis) caused by Nigrospora oryzae in Ontario. Plant Disease 96:909.

Wang Y, Sun G, Hao B, Zhang Q, Tuo E, Zhang R, Jin S, Wang Y, Hsiang T. 2012. Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis. Fungal Biology 116:522-528. In a search for specific molecular markers for population analysis of Puccinia striiformis f. sp. tritici, the ribosomal DNA intergenic spacer 1 region (rDNA-IGS1, between the 28S and the 5S rDNA genes) was amplified, cloned, and sequenced. It was found to exhibit multiple bands and length polymorphism. Surprisingly, single isolates were found to possess between three to five different IGS1 haplotypes. Bands were cloned and sequenced, and two highly variable regions were found between conserved regions, with repeat units interspersed in both types of regions. There were 14 different repeat units, and these were sometimes grouped further into four combinations of repeat units, with a few individual nucleotides (A or C) inserted between the repeats. Among three geographically dispersed isolates, the variable region was divided into eight types, and the variable region was divided into two types based on repeat units. Most of the 14 repeat units were shared by the variable and the conserved regions. Among the three isolates, there were a total of 12 IGS1 haplotypes, but some of these were shared between isolates such that there were only eight unique haplotypes. The occurrence of multiple haplotypes within single isolates may be useful for analyzing the population structure, tracking the origin of annual epidemics and providing insights into evolutionary biology of this pathogen.  Journal Website Reprint

Li Q, Ning P, Zheng L, Huang J, Li Q, Hsiang T. 2012. Effects of volatile substances of Streptomyces globisporus JK-1 on control of Botrytis cinerea on tomato fruit. Biological Control 61:113-120 Volatile substances produced by Streptomyces globisporus JK-1 grown on autoclaved wheat seed inhibited Botrytis cinerea growth and development both on media and in inoculated tomato fruit. The volatiles suppressed mycelial growth of various plant pathogens in vitro, especially that of Botrytis cinerea and Sclerotinia sclerotiorum. Conidial germination and sporulation of B. cinerea were also suppressed. Disease incidence and severity on wound-inoculated tomato fruit (Lycopersicon esculentum) were inhibited when fumigated with 120 g wheat seed culture of S. globisporus JK-1 per liter of airspace in treatment containers. Suppression of the infection process of B. cinerea on tomato fruit was observed via scanning microscopy, showing inhibition of conidial germination and of appressorial formation on tomato fruit, as well as abnormal morphology of appressoria and conidia. The viability of the conidia obtained from the volatile-treated and non-treated disease lesions was tested with the vital stains fluorescein diacetate (FDA) and propidium iodide (PI). Conidia fumigated with 30 g/L, 60 g/L or 120 g/L wheat seed culture of S. globisporus JK-1 at 20 °C for 6 days showed 46.0%, 69.8%, or 80.9% reduction in viability, respectively. Transmission electron microscopy of fumigated and untreated B. cinerea showed excessive vesiculation or thickened cell walls in exposed conidia and increased vesiculation or strong retraction of plasma membrane in exposed hyphae. These results provide a better understanding of the mode of action of volatiles from JK-1 on B. cinerea. The inhibition growth of B. cinerea both in vitro and in vivo showed that volatiles from S. globisporus JK-1 have the potential for control of postharvest grey mold of tomato fruits through fumigant action. Journal Website Reprint

Lv R, Zheng L, Zhu Z,Pan L, Huang J, Hsiang T. 2011. First report of stem blight of Eleocharis dulcis caused by Phoma bellidis in China. Plant Disease 95:1190. Journal Website Reprint or local preprint.

Lv CC, Hsiang T, Li JQ, Luo LX. 2011. First report of dollar spot of Agrostis stolonifera, Poa pratensis, Festuca arundinacea and Zoysia japonica caused by Sclerotinia homoeocarpa in China. New Disease Reports 23:37. Journal Website Reprint or local preprint

Hsiang T, Goodwin PH, Cortes-Barco AM. 2011. Plant defense activators and control of turfgrass diseases. Outlooks on Pest Management 22:160-164. Plants are known to possess many natural defense mechanisms against stresses including diseases. Under intensive plant maintenance systems, these natural mechanisms may be insufficient to guard against disease outbreaks without significant economic loss. However, there are chemicals that have been observed to stimulate the natural resistance pathways in plants, with most of the research on broadleaf plants. These compounds generally do not have a direct antifungal activity, but act by triggering existing defense mechanisms of the plant. We have been investigating the activity and mechanisms of action of certain new compounds in activating defenses against diseases in plants, particularly turfgrasses, and present here an overview of plant defense activation as well as some research findings. Contact authors for Reprint

Haridas S, Breuill C, Bohlmann J, Hsiang T. 2011. A biologist's guide to de novo genome assembly using next-generation sequence data: a test with fungal genomes. J. Microbiological Methods 86:368-375. We offer a guide to de novo genome assembly  using sequence data generated by the Illumina platform for biologists working with fungi or other organisms whose genomes are less than 100 Mb in size. The guide requires no familiarity with sequencing assembly technology or associated computer programs. It defines commonly used terms in genome sequencing and assembly; provides examples of assembling short-read genome sequence data for four strains of the fungus Grosmannia clavigera using four assembly programs; gives examples of protocols and software; and presents a commented flowchart that extends from DNA preparation for submission to a sequencing center, through to processing and assembly of the raw sequence reads using freely available operating systems and software. Reprint at Journal Website or contact authors for reprint password & then click here for article and here for supplemental data.

Li Q, Jiang Y, Ning P, Zheng L, Huang J, Li G, Jiang D, Hsiang T. 2011. Suppression of Magnaporthe grisea by culture filtrates of Streptomyces globisporus JK-1. Biological Control 58:139-148. An isolate, JK-1, was obtained as a contaminant from a fungal culture, and identified as Streptomyces globisporus. In dual cultures on solid media, this strain suppressed mycelial growth of numerous plant pathogenic fungi, especially that of Magnaporthe oryzae, Bipolaris maydis and Cryphonectria parasitica. Culture filtrates of JK-1 inhibited mycelial growth of M. oryzae, and histological investigations showed that conidial germination and appressorial formation of M. oryzae were suppressed on detached rice leaves. When applied at 15 μl per 5-cm-long detached leaf, washed cells of JK-1 at 108 CFU/ml could suppress disease incidence of rice blast caused by M. oryzae co-inoculated at 5 x 105 spores/ml, but disease incidence was not reduced when washed cells of JK-1 were used at 106 or 107 CFU/ml. Applying the culture filtrate on rice seedlings in the greenhouse at 2 h post inoculation (HPI) with M. oryzae showed 88.3% disease reduction of rice blast, while culture filtrate application before pathogen inoculation showed even higher rates of disease reduction. Suppression of the infection process of M. oryzae on detached rice leaves was observed by light and scanning electron microscopy, showing inhibited conidial germination and reduced appressorial formation on rice leaves sprayed with culture filtrate before 3 HPI. Application of JK-1 as cells or culture filtrates to plant tissues did not cause any noticeable negative effects. These results indicate that the antifungal substance(s) in the culture filtrate of S. globisporus JK-1 can exhibit an inhibitory effect on M. oryzae and a suppressive effect on rice blast disease. Journal Website or contact authors for reprint.

Wijekoon CP, Goodwin PH, Valliani M & Hsiang T. 2011. The role of a putative peroxisomal-targeted epoxide hydrolase of Nicotiana benthamiana in interactions with Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci. Plant Science. 181:177-187. Motif analysis among thirty EH1 and EH2 epoxide hydrolases from Solanaceaeous plants showed differences primarily in the lid region around the catalytic site. Based on in silico models of 3D structures, EH1 proteins lack a catalytic triad because of the orientation of one of the conserved lid tyrosines, while the orientation of that tyrosine in EH2 proteins fomed a catalytic triad inside a hydrophobic tunnel. Two similar EH2 protein genes from Nicotiana benthamiana, NbEH2.1 and NbEH2.2, have a predicted peroxisomal targeting sequence, catalytic triad, and structural similarities to a potato cutin monomer-synthesizing epoxide hydrolase. NbEH2.1 expression increased with infections by the hemibiotrophs, Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci only during their biotrophic phases, while there was only a slight increase during the hypersensitive response to P. syringae pv. tabaci (avrPto). In contrast, among the four pathogens, NbEH2.2 expression increased only in response to P. syringae pv. tabaci. Virus-induced gene silencing of NbEH2.1 significantly affected only the interaction with C. destructivum, resulting in a delay in the appearance of necrosis that may be related to its biotrophic phase being restricted to single epidermal cells, which is unique among these pathogens. These results differed from that of a previously reported EH1 gene of N. benthamiana for these interactions, demonstrating specialization among EH genes in basal resistance. PDF at  Journal Website or contact authors for reprint

Wang F, Zhao L, Li G, Huang J and Hsiang T. 2011. Identification and characterization of Botryosphaeria spp. causing gummosis of peach trees in Hubei Province, Central China. Plant Disease 95:1378-1384. Peach (Prunus persica) is one of the most important and widely grown fruit trees in China; however, perennial gummosis on trunks and branches is a major problem in peach orchards of Hubei Province, one of the most important peach production areas of China. In order to identify the gummosis-causing agents, diseased trunks and branches were collected from 11 peach orchards in Hubei Province. Fungal isolates were obtained from these samples, yielding three species: Botryosphaeria dothidea (anamorph Fusicoccum aesculi), B. rhodina (anamorph Lasiodiplodia theobromae), and B. obtusa (anamorph Diplodia seriata). They were identified based on conidial morphology and cultural characteristics, as well as analyses of nucleotide sequences of three genomic regions: the internal transcribed spacer region, a partial sequence of the β-tubulin gene, and the translation elongation factor 1-α gene. Fusicoccum aesculi was found in all 11 orchards but L. theobromae was found only in Shayang County in the Jingmen region and D. seriata only in Gong'an County in the Jingzhou region. Via artificial inoculation using mycelia on wounded twigs or branches, these three species were all found to be pathogenic, causing dark lesions on the twigs and branches and, sometimes, gum exudation from diseased parts. Isolates of L. theobromae were the most virulent and caused the largest lesions and most copious gummosis, and D. seriata had less gum than the other two species. This report represents the first description of L. theobromae and D. seriata as causal agents of gummosis on peach in China. Journal Website  or Preprint

Zheng L, Lv R, Li Q, Huang J & Hsiang T. 2011. First report of leaf spot of Houttuynia cordata caused by Alternaria alternata in China. Plant Disease 95:359. Journal Website

McLaughlin JM, Hsiang T, Hayden GH, Griefenhagen S. 2011. Abiotic and biotic factors useful to assess decline risk in red pine (Pinus resinosa Ait.) plantations. Forestry Chronicle 87:99-115. This study was conducted to assess causes of unprecedented rates of mortality in maturing, commercial-sized red pine (Pinus resinosa Ait.) plantations in southern Ontario, Canada. Concentrated and diffuse mortality as well as windthrow of living trees were observed in many plantations, while others seemed disease-free. Nine sites exhibiting recent mortality (diseased) plus three not exhibiting disease (healthy) were selected. In sample plots at each site, abiotic site factors, host characteristics, and insect pests and fungal pathogens were assessed. Tree mortality was attributed to Armillaria root disease, annosus root rot, black pineleaf scale, drought, and iron deficiency. The single abiotic factor that distinguished healthy sites from diseased sites was C soil horizon pH, which averaged 8.35 on diseased sites compared to 6.55 on healthy sites. Rooting was also deeper on healthy sites than on diseased sites. We suggest that an alkaline C horizon may result in shallower rooting and greater susceptibility to drought stress, rendering trees less resistant to root disease pathogens and insect pests. The pH of the C horizon and the depth of the A and B horizons may be useful as indicators of the likelihood of red pine mortality and to guide the choice of management objectives for plantations. PDF Reprint

Bertrand A, Castonguay Y, Azaiez A, Hsiang T, Dionne J. 2011. Cold-induced responses in annual bluegrass genotypes with differential resistance to pink snow mold (Microdochium nivale). Plant Science 180:111-119. Greens-type annual bluegrass (Poa annua L.) is susceptible to winter stresses including subfreezing temperatures and pink snow mold (SM). To better understand the mechanisms of SM resistance in annual bluegrass, four SM-resistant and four SM-sensitive genotypes were incubated at low temperature with Microdochium nivale (Fries) Samuels & Hallett", the causal agent of pink snow mold (SM). We assessed the impact of a 6-wk incubation period with SM at 2 C under high humidity (= 98%) on the accumulation of cold-induced metabolites and on freezing tolerance. SM iIncubation of annual bluegrass inoculated with SM lead to a major decrease in concentration of cryoprotective sugars such as sucrose and HDP (high degree of polymerization) fructans. Conversely, major amino acids linked to stress resistance such as glutamine and arginine increased in crowns of annual bluegrass in response to SM inoculation. One of the major differences between resistant and sensitive genotypes was found in the concentration of HDP fructans, which remained higher in SM-resistant genotypes throughout the incubation period. HDP fructans were also more abundant in freeze-tolerant genotypes, reinforcing their positive impact on winter survival of annual bluegrass. The identification of genotypes that are resistant to both SM and freezing shows the possibility of being able to improve both traits concomitantly. PDF Reprint

McLaughlin JA, Hsiang T, Hayden GH, Greifenhagen. 2010. Mortality in southern Ontario red pine plantations: causes, consequences, and management options. Ontario Forest Research Institute Forest Research Note No. 69. 3pp. Red pine is a shade intolerant conifer that is among the most extensively planted tree species in southern Ontario. In the 1920s, reforestation programs were initiated to restore idle marginal and waste land to productive use, prevent soil erosion, and conserve water resources. Red pine was an ideal species for these reforestation programs: it thrives on sites too poor for agriculture, it produces high value timber for example for sawlogs, utility poles, and preservative-treated landscape timbers, it grows well in plantations and can provide economic returns to landowners through occasional thinning treatments, and it is resistant to most pests. Over time, these plantations have become important for providing timber, and for education and recreation opportunities. PDF Reprint   (This note also includes at the end a one-page Simcoe County management recommendation for Red Pine)

Li Q, Ning P, Zheng L, Huang J, Li G, Hsiang T. 2010. Fumigant activity of volatiles of Streptomyces albolongus JK-1 against Penicillium italicum on Citrus microcarpa. Postharvest Biology and Technology 58:157-165. Antifungal activity against Penicillium italicum of volatile substances from Streptomyces globisporus JK-1 grown on autoclaved wheat seed was studied in vitro and in planta. Fungal spore germination and mycelial growth of P. italicum cultures as well as sporulation and disease incidence on fungal-inoculated fruit were suppressed in the presence of the volatiles. For naturally infected fruit, disease incidence was reduced from 25% to 7.5%. Suppression of the infection process of P. italicum on Shatang Mandarin fruit (Citrus microcarpa) was observed via scanning electronic microscopy, showing inhibited spore germination on the Shatang Mandarin, and abnormal morphology for conidiophores and hyphae exposed to the volatiles. Based on gas chromatography/mass spectrophotometric analyses, 41 volatile organic compounds were identified from the volatiles of S. globisporus JK-1, and the most abundant compound was trans-1,10-dimethyl-trans-9-decalol (geosmin), an earthy smelling substance. Among these, technical grade formulations of eight were chosen for further study: phenylethyl alcohol, caryophyllene, dimethyl disulfide, dimethyl trisulfide, acetophenone, d-limonene, isoledene, and aromadendrene. D-limonene, isoledene and aromadendrene showed no observable antifungal activity in vitro and in planta at tested concentrations. Both phenylethyl alcohol and caryophyllene showed weak inhibitory activity in vitro but no significant efficacy against P. italicum on Shatang Mandarin. Dimethyl disulfide or dimethyl trisulfide showed antifungal activity in vitro and efficacious control on Shatang Mandarin at a concentration of 100 uL/L of airspace in treatment containers. Acetophenone showed antifungal activity in vitro at a concentration of 100 uL/L, and efficacious control on Shatang Mandarin at the highest concentration of 1000 uL/L. Volatiles from S. globisporus JK-1 have potential for control of blue mold of citrus species through fumigant action. PDF Reprint

Ni M, Liu T, Ding Y, Zheng L, Huang J, Hsiang T. 2010. A leaf spot of figwort (Scrophularia ningpoensis) caused by Phoma sp. Canadian Journal of Plant Pathology 32:493-495. Since 2006, a new disease caused by Phoma sp. has been identified on figwort (Scrophularia ningpoensis) in Hubei province, China. The pathogen was isolated from diseased S. ningpoensis leaves growing under field conditions, and attempts were made to identify it by cultural and morphological characteristics as well as analysis of the internal transcribed spacer region of ribosomal DNA. There were no identical matches of the DNA sequence on GenBank (highest match less than 98% identity), and morphological and cultural characteristics placed it closely with Phoma congesta, but the host species did not match. Another two species with striking similarities, P. pooiensis and P. nebulosa, are also found on Scrophulariaceae, but there were significant morphological or cultural differences from these species. Pathogenicity of the isolate was tested by inoculating 20 S. ningpoensis plants under greenhouse conditions. Koch’s postulates were fulfilled by re-isolating the pathogen from the inoculated plants. This is the first report of leaf spot of S. ningpoensis caused by Phoma sp., but the exact species remains to be determined. PDF Reprint

Zheng L, Lv R, Huang J, Jiang D, Hsiang T. 2010. Isolation, purification and biological activity of a phytotoxin produced by Stemphylium solani. Plant Disease 94:1231-1237. Stemphylium solani, the causal agent of leaf blight of garlic (Allium sativum), produced phytotoxic metabolites in culture. A non-host-specific phytotoxin from culture filtrate of S. solani isolate DY-5, named SS-toxin, was extracted by ethyl acetate, isolated by bioassay-guided thin layer chromatography on silica gel, and purified by preparative liquid chromatography. SS-toxin produced necrotic lesions on detached garlic leaves, similar to that caused by S. solani. When wounded leaves were each treated with a 10 ul droplet of toxin. Changbanpo, a garlic cultivar susceptible to leaf blight, showed necrotic lesions at 11 ug/ml toxin, while a resistant cultivar, Qingganruanye, showed symptoms at 44 ug/ml. The EC50 values for root growth and shoot elongation of the susceptible cultivar were 64.9 and 178.5 ug/ml, respectively. The SS-toxin significantly inhibited mitotic activity of root tip cells of the susceptible cultivar from 22 ug/ml and higher causing an abnormally high frequency of cells in interphase. Concentrations over 50 ug/ml of SS-toxin were found to be significantly toxic to total chlorophyll, both chlorophyll a and b, of the susceptible cultivar. The plasma membrane-cell wall interface, nuclear membranes, mitochondria and chloroplasts were affected by SS-toxin in susceptible leaf cells. This is the first report of the purification and testing of a phytotoxin produced by S. solani. PDF Reprint

de la Cerda K, Hsiang T, Joshi V. 2010. Waitea circinata from turfgrass in British Columbia. Plant Disease 94:277. In Canada, Waitea circinata was first identified from buckwheat (Fagopyrum sp.) in 1965 in Ontario (4). In 2004, the fungus was found on diseased putting greens of Poa annua and Agrostis stolonifera near Toronto, Ontario (2). In late July 2009, symptoms on A. stolonifera resembling those of brown ring patch were seen at a golf course in Kelowna, British Columbia. Brown rings with light-colored, cottony growth were observed on a putting green with mixed P. annua and A. stolonifera, originally seeded with A. stolonifera cv. Penncross. Following a short incubation of the diseased grass at 25 C, hyphae of a Rhizoctonia-like fungus, not matching the characteristics of R. cerealis or R. solani, were seen. Symptomatic leaves were surface sterilized in 1% hypochlorite and plated onto potato dextrose agar (PDA) amended with streptomycin. After 1 week at 23 C, the plates contained white colonies that were 5 cm across. DNA was extracted and amplified with primers ITS1/ITS4 and sequenced with ITS1. The 600-bp sequence (deposited in GenBank as GU176409) from the internal transcribed spacer (ITS) region of ribosomal DNA showed a 100% match in the overlapping range with sequence FJ755879 from GenBank, which is annotated as W. circinata var. circinata. Pathogenicity was tested at 23 C by inoculating 3-week-old A. stolonifera 'Penncross' plants grown in Magenta boxes and incubated for 15 days after inoculation with ground wheat seed inoculum of W. circinata. Within 1 week, significant blighting of leaves and sheaths was observed as well as spherical orange brown sclerotia that were 2 to 5 mm in diameter on sheaths. These sclerotial features match characteristics of W. circinata var. circinata (1). Symptomatic leaves were plated on PDA and fungal growth characteristic of W. circinata was recovered. W. circinata was previously reported as the causal agent of brown ring patch on A. stolonifera in Japan (3), as a pathogen of P. annua in the United States (1), and as a pathogen of both species in Ontario, Canada (2). To our knowledge, this is the first report of W. circinata from turfgrass in western Canada.

Lv C, Luo L, Li J and Hsiang T. 2010. First report of dollar spot of seashore Paspalum (Paspalum vaginatum) caused by Sclerotinia homoeocarpa in South China. Plant Disease 94:373. Seashore paspalum (Paspalum vaginatum Swartz) is a warm season perennial grass, native to tropical and subtropical regions of North and South America (Carrow, 2000). Its fine texture and tolerance to low mowing and hypersaline environments make it a commercially promising turfgrass species for coastal regions of south China. In late March 2009, disease symptoms were observed from two golf courses fairways, in Shenzhen and Foshan, Guangdong province, China. Small, round patches from 25 to 75 mm diameter were found, consisting of bleached, straw color leaf lesions bounded by reddish-brown margins. Similar patches had previously been observed on seashore paspalum since 1997 in Guangdong province, but this turf species has been grown in southern China only since the early 1990s. These symptoms were observed when daytime temperatures were above 25 C and with heavy dew formation at night. Greatest severity was seen in spring and fall. Several contact and systemic fungicides applied after first symptoms were observed, and they were usually successful in suppressing disease. To confirm the disease as dollar spot, isolates from Shenzhen and Foshan were obtained by plating diseased leaf blades of P. vaginatum (cv. Salam) on potato dextrose agar media. Isolates produced white, fluffy, aerial mycelium, columnar when mature, and usually with a cinnamon base and dark brown or black substratal stroma on and in the agar. One representative isolate from each location was chosen for pathogenicity testing. Six-week-old P. vaginatum (cv. SalamTM) grown from seed in pots was inoculated with 5-mm-diameter agar plugs with hyphae from 5-day-old cultures, by direct placement onto leaves, with three replicate pots per isolate. Plants treated with sterile agar plugs served as controls. Inoculated turf was incubated at 25 C under 12 hour light/dark conditions. A plastic film was also placed over the pots to retain moisture. Chlorotic leaf lesions developed starting 4 days after inoculation and became a bleached straw color. The same fluffy white fungus was re-isolated from lesions, while no disease was observed on controls, thus completing Koch's postulates. The internal transcribed spacer region of the ribosomal DNA (ITS) was amplified from DNA extracted from two isolates using the primers ITS5 and ITS4 (White et al.,1990), and the 610-bp sequences showed 98% similarity with Sclerotinia homoeocarpa F.T. Bennett in GenBank and have been deposited as accessions GQ386985 and GU002301. Dollar spot on P. vaginatum has been commonly observed in the U.S.A. (Carrow, 2000). To our knowledge, this is the first confirmed report of dollar spot on P. vaginatum in China, or from any host plant in China.

Zheng L, Lv R, Li Q, Liu T, Huang J & Hsiang T. 2010. Effect of SS-toxin, a metabolite of Stemphylium solani, on H+-ATPase activity and standard redox system in plasma membranes from garlic (Allium sativum) seedling leaves. European Journal of Plant Pathology 127:419-425. Leaf blight of garlic (Allium sativum) is a severe disease in garlic growing regions. SS-toxin is a newly described non-proteinaceous toxin produced by the phytopathogenic fungus, Stemphylium solani, the cause of garlic leaf blight. In this study, the effects of SS-toxin on the H+-ATPase activity and standard redox activity in the plasma membranes isolated by aqueous polymer two-phase partitioning from garlic seedling leaves were studied in vitro. The H+-ATPase activity, NADH oxidation rate and Fe(CN)63- reduction rate of the plasma membranes isolated from susceptible and resistant cultivars were all inhibited in a dose-dependent manner. Our results suggest that, under in vitro conditions, the plasma membrane H+-ATPase and standard redox system can be both the cellular targets of SS-toxin. PDF Reprint

Zheng L, Lv R, Huang J, Jiang D, Liu X, Hsiang T. 2010. Integrated control of garlic leaf blight in China. Can. J. Plant Pathol. 32:135-145. Leaf blight caused by Stemphylium solani is a major fungal disease of garlic (Allium sativum) in central China where it has caused severe crop losses during the winter growing season from the end of autumn to the middle of spring. Epidemiology, cultivar resistance, and chemical controls were investigated during the 2006 to 2008 growing seasons in Dangyang County to improve disease control methods. Disease severity monitoring revealed that the activity of S. solani was variable between growing seasons, and this may have been due to weather conditions. Disease severity was positively correlated with increasing temperatures, but no consistent relationship was found between total rainfall and disease. Additionally, the study demonstrated that conidia and mycelium of S. solani could survive in garlic debris for long periods and serve as the primary inoculum source for the subsequent season. Relatively few of the commonly grown cultivars had high levels of resistance to leaf blight. Garlic cultivars ‘Qingganruanye’, ‘Ruanruanye’ and ‘Zixuan-2’ were among the most resistant, but except for ‘Zixuan-2’, did not produce sufficient harvestable bolts which is desirable for the local market. All fungicide treatments applied to cloves used as planting material seemed to promote seedling emergence, but significant effects (P = 0.05) were observed only with fludioxonil (0.05 g kg-1) and thiram (1.25 g kg-1). Fungicide applications in the field were effective in controlling leaf blight, and flusilazole (50 g ha-1), flusilazole plus famoxadone (50 g plus 104 g ha-1) or mancozeb (350 g ha-1) had the highest efficacy in reducing leaf blight severity. PDF Preprint

McLaughlin JM and Hsiang T. 2010. Identification protocol for six Armillaria species from northeastern North America. Canadian Journal of Forest Research 40:536-548. DNA sequences (~3 kb long) extending from the intergenic spacer (IGS) 1 region to the 18S gene were obtained for isolates of Armillaria ostoyae, A. calvescens, A. gallica, and A. sinapina. Additional investigation of 16 A. ostoyae, 11 A. gemina, 21 A. calvescens, 18 A. gallica, and 15 A. sinapina isolates produced 117 sequences spanning the 3' end of the IGS1 through the 5S gene and into the 5' end of the IGS2 region. Additional sequences spanning the 3' IGS2 to 5' 18S gene region were obtained for two A. ostoyae, three A. gemina, two A. calvescens, two A. gallica, and three A. sinapina isolates. This is the first report of complete IGS2 sequences from Armillaria species. A species identification protocol involving species-specific primers and restriction fragment length polymorphism analysis was devised based on species-specific polymorphisms. The protocol successfully identified all 16 A. ostoyae, 11 A. gemina, 3 of 3 A. mellea, 18 A. gallica, as well as 14 of 15 A. sinapina (11/12 diploid and 3/3 haploid), and 14 of 21 A. calvescens, (13/15 diploid and 1/6 haploid) isolates included in this study. To the best of our knowledge, this success rate has not been matched by other methods. PDF Reprint

Cortes-Barco AM, Goodwin PH, Hsiang T. 2010. Induced systemic resistance against three foliar diseases of Agrostis stolonifera by (2R,3R)-butanediol or an isoparaffin mixture. Annals of Applied Biology 157:179-189. Induced systemic resistance (ISR) is a type of plant defense mechanism typically activated by nonpathogenic root-associated microorganisms and systemic priming of gene expression in response to subsequent pathogen challenge. ISR was found to be activated by PC1, a mixture of food-grade synthetic isoparaffins, and (2R,3R)-butanediol, a volatile organic compound produced by bacteria. In controlled environment tests, application of PC1 or (2R,3R)-butanediol to the soil reduced the diseased leaf area of Agrostis stolonifera by 20 to 40% for the fungal pathogens, Microdochium nivale, Rhizoctonia solani or Sclerotinia homoeocarpa compared to the water control. In A. stolonifera, expression of the jasmonate synthesis-related genes, AsAOS1, encoding an allene oxide synthase, and AsOPR4, encoding a 12-oxo-phytodienoic acid reductase, and expression of a pathogenesis-related protein gene, AsGns5, encoding an acidic alpha-1,3-glucanase, were primed for increased expression by PC1 or (2R,3R)-butanediol when M. nivale was inoculated seven days later. However, the compounds differed in their ability to induce expression prior to pathogen challenge. PC1 induced AsAOS1 expression upon treatment, whereas (2R,3R)-butanediol induced expression of AsOPR4 and AsGns5 upon treatment. These results indicate that both (2R,3R)-butanediol and PC1 can produce ISR in A. stolonifera but may do so through different mechanisms. PDF Preprint

Cortes-Barco AM, Goodwin PH, Hsiang T. 2010. A comparison of induced resistance activated by benzothiadiazole, (2R,3R)-butanediol and an isoparaffin mixture against anthracnose of Nicotiana benthamiana. Plant Pathology 59:643-653. Resistance in Nicotiana benthamiana was activated by benzothiadiazole (BTH), (2R,3R)-butanediol or PC1, an isoparaffin-based mixture, against anthracnose caused by the hemibiotrophic fungus, Colletotrichum orbiculare. BTH, (2R,3R)-butanediol and PC1 reduced the number of lesions per leaf area caused by C. orbiculare by 98%, 77% and 81%, respectively. Foliar application of BTH induced expression of genes for the acidic pathogenesis-related (PR) proteins, NbPR-1a, NbPR-3Q and acidic NbPR-5. In contrast, soil application of (2R,3R)-butanediol or PC1 primed expression of genes for the basic PR proteins, NbPRb-1b, basic NbPR-2 and NbPR-5dB. These results are consistent with the activation of systemic acquired resistance (SAR) by BTH and induced systemic resistance (ISR) by (2R,3R)-butanediol or PC1, and show that (2R,3R)-butanediol and PC1 can affect gene expression similarly to plant growth-promoting rhizobacteria. However, the effects of (2R,3R)-butanediol and PC1 were not identical. In addition to priming, (2R,3R)-butanediol induced expression of basic NbPR-2, whereas PC1 treatment induced expression of both NbPRb-1b and basic NbPR-2. Although a number of microbial products, such as (2R,3R)-butanediol, have been shown to produce ISR, this is the first demonstration that an isoparaffin-based mixture, which is not derived from a microorganism, can produce ISR. PDF Preprint

Xue AG, Voldeng HD, Savard ME, Fedak G, Tian X, Hsiang T. 2009. Biological control of Fusarium head blight of wheat with Clonostachys rosea strain ACM941. Can. J. Plant Pathol. 31:169-179. Fusarium head blight (FHB), caused by Gibberella zeae, is a devastating disease of wheat. A strain of Clonostachys rosea, ACM941 (American Type Culture Collection ATCC 74447), was evaluated for antibiosis against G. zeae in vitro and for control of FHB under greenhouse and field conditions in comparison to the registered fungicide Folicur (tebuconazole). Strain ACM941 reduced mycelial growth of the pathogen by 52.6% in dual-culture after 6 days and completely suppressed spore germination for6hwhen cocultured with a macroconidial suspension of G zeae. Strain ACM941 reduced G zeae perithecial production by more than 99% in a leaf disk assay, 60% - 77% on infected corn kernels, and 32% - 57% on spikelet debris in the field. These effects were significant (P < 0.05) and not statistically different from those produced by tebuconazole. When strain ACM941 was sprayed onto wheat heads 2 days prior to inoculation with G. zeae, it significantly reduced infected spikelets (IS) by 64% and Fusarium-damaged kernels (FDK) by 65% in greenhouse experiments. Under simulated disease epidemic conditions during 2005 - 2007, strain ACM941 reduced the FHB index by 58%, IS by 46%, FDK by 49%, and deoxynivalenol (DON) in kernels by 21%. These effects were significant but lesser in magnitude than those achieved by tebuconazole, which reduced FHB index by 97%, IS by 82%, FDK by 73%, and DON by 62%. Results from this research suggest that strain ACM941 of C. rosea is a promising biocontrol agent against G. zeae and may be used as a control measure in an integrated FHB management program. PDF Reprint

Hsiang T. 2009. All you ever wanted to know about Fusarium patch / Microdochium patch / pink snow mold or whatever that disease is called. GreenMaster 44(4):18-19 [July/August 2009]. PDF Reprint

Hsiang T, Tian LX, Sopher C. 2009. Non-native hosts and control of Rhytisma acerinum causing tar spot of maple. SDU Faculty of Forestry Journal Serial: A, Special Issue, Pages 189-193. Tar spot of maple is an increasingly common disease in eastern North America. Rhytisma acerinum, causing large tar spot, was apparently introduced from Europe, and causes the most extensive problems on introduced maple species such as Acer platanoides (Norway maple). However, this pathogen was recently confirmed via molecular methods on native North American species of maple, A. saccharum and A. negundo. This raises the possibility of pathogenic adaptation to native species that will result in more widespread epidemics. Ascospore production on Norway maple was observed to occur over a relatively short period annually (May 25 - June 22, 2006; May 25 - July 3, 2007; and May 21 - June 13, 2008). The duration of spore dispersal was dependent on the frequency of rainfall from the start of dispersal. Field studies on fungicidal control of tar spot on Norway maple were conducted in summer 2008, using nine chemicals and a water control. All nine chemicals were found to be effective if applied between late May and early June. A single application was sufficient to control disease in summer 2008. PDF Reprint

Benedetto D and Hsiang T. 2009. Effect of azoxystrobin on dollar spot disease development in creeping bentgrass and Kentucky bluegrass. International Turfgrass Society Research Journal 11:151-163. Dollar spot disease, caused by the fungus Sclerotinia homoeocarpa F.T. Bennett is common on intensively managed creeping bentgrass (Agrostis stolonifera L.) and annual bluegrass (Poa annua L.), and other turfgrasses. Frequent chemical applications during the growing season are made to control this disease. Previous observations state that application of fungicides such as azoxystrobin can increase the level of disease within the same growing season. The purpose of this study was to quantify the extent of disease increase caused by an application of azoxystrobin the previous year. In three field tests on creeping bentgrass between 2005 and 2007, a single application of azoxystrobin in the late fall was found to increase the incidence of dollar plot several fold on treated plots in the following summer. In addition, this study also found a slight enhancement of dollar spot incidence on Kentucky bluegrass (Poa pratensis L.), which has not been previously reported. Although the cause of this disease enhancement is not known, the physical and chemical characteristics of azoxystrobin, along with turfgrass cultural practices and the finding of the longer term effect from this study, implies that the phenomenon stems from non-target effects on microbial populations of leaves or soil rather than a direct long term effect on the plants. Studies on phyllosphere communities may shed light on this phenomenon and point out directions for future research. PDF Reprint

Bertrand A, Castonguay Y, Thibault G, Rochette P, Hsiang T and Dionne J. 2009. Greens-type annual bluegrass resistance to abiotic and biotic stresses during winter. International Turfgrass Society Research Journal 11:723-736. Unseeded annual bluegrass (Poa annua L.) is an important component of golf greens in many regions of northern countries. Although this turfgrass species has desirable playing attributes, it suffers from susceptibility to environmental and biological stresses including subfreezing temperatures, anoxia, and snow molds. During the last decade (1999-2008), we undertook a series of experiments to better understand the mechanisms of adaptation and resistance of annual bluegrass to winter biotic and abiotic stresses. When assessing freezing tolerance of three genotypes originating from North Eastern Canada and United States, we observed that the genotypes differed significantly in their freezing tolerance with an unexpected lower level of tolerance for the genotype originating from the most northern latitude, in Québec, where the depth and duration of snow cover are important factors affecting winter survival. After assessing the level of organic reserves, the results showed that the genotype originating from Québec maintained higher levels of reserves than the two other genotypes. Our studies also showed that annual bluegrass is sensitive to anoxic conditions and that its susceptibility could be linked to the reduction of organic reserves under these conditions. We observed large genetic variability for pink snow mold resistance among 29 genotypes collected on golf greens located in Québec and Ontario and concluded that snow mold disease exerts major selection pressure for the generation of genetic diversity among annual bluegrass genotypes in northern climates. Our results suggest that high level of reserves is a characteristic of annual bluegrass genotypes adapted to deep, long lasting snow cover and could be more critical for winter survival than freezing tolerance to allow plants to survive long winter period without new carbohydrate synthesis. PDF Reprint

Zheng L, Lv RJ, Hsiang T, and Huang JB. 2009. Host range and phytotoxicity of Stemphylium solani, causing leaf blight of garlic (Allium sativum) in China. Eur. J. Plant Pathology 124:21-30. Since 2004, a new leaf blight disease on garlic of high severity has been observed in Dangyang County, Hubei province, China. Initial symptoms consisted of multiple, small, irregular to oval, white leaf spots, which enlarge to produce sunken purple lesions, sometimes surrounded by a bright yellow margin. As the disease progressed, lesions expanded and merged, resulting in withering of leaf tips. After isolation and pathogenicity testing, the causal agent of leaf blight of garlic was identified as Stemphylium solani from cultural and morphological characteristics, and subsequent analysis of the internal transcribed spacer region of ribosomal DNA. When fungal plugs of two S. solani isolates were inoculated onto 11 garlic cultivars and 20 other crop species, leaf spots appeared on all inoculated plants, but two garlic cultivars (Qingganruanye and Ruanruanye) and three crop species (Capsicum annuum, Brassica napus and Amaranthus mangostanus) showed the smallest leaf spots. In cross-inoculation experiments, no indications of host specificity were observed, but S. solani isolated from garlic was generally the most virulent on five plant species, while the isolate from leek (Allium odorum) was generally the least virulent. Toxicity testing of the crude culture filtrates indicated that garlic isolates produced toxin(s) that were not heat-labile and induced different levels of phytotoxicity toward various garlic cultivars and crops. PDF Reprint

Wang Y, Zhu M, Zhang R, Yang H, Wang Y, Sun G, Jin S, Hsiang T. 2009. Whole genome amplification of the rust Puccinia striiformis f. sp. tritici from single spores. Journal of Microbiological Methods 77:229-234. Rust fungi are obligate parasites and cannot be routinely cultured to obtain sufficient biomass for DNA extractions. Multiple displacement amplification (MDA) was demonstrated in this study for whole genome amplification from single spores of the rust fungus, Puccinia striiformis. The genomic DNA coverage and fidelity of this method was evaluated by PCR amplification and sequencing of two genetic markers: portions of the multi-copy nuclear ribosomal DNA internal transcribed spacer region (ITS) and the single copy alpha- tubulin gene from two geographical diverse isolates. Our results show that MDA is a valuable tool for whole genome amplification from single spores, and we propose that MDA-amplified DNA can be used for molecular genetic analysis of the wheat yellow rust fungus. PDF Reprint

Li X, Nie J, Ward L, Madani M, Hsiang T, Zhao Y, De Boer SH. 2009. Comparative genomics-guided loop-mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicola. J Appl Microbiol. 107:717-726. Aims: To design and evaluate a loop-mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae. pv. phaseolicola (PSP), the causal agent of halo blight disease of bean (Phaseolus vulgaris L.). Methods and Results: Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar-specific region encoding pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium, Erwinia and Pantoea. Conclusion: A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant-associated bacteria. Significance and Impact of the Study: This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences. PDF Reprint

Long Y, Hsiang T, Huang J. 2009. First Report of Leaf Spot of Smilax china L. Caused by Alternaria longipes in China. Plant Pathology 58:800. NDR Reprint

Zhang L, Zheng L, Hsiang T, Lv R, Huang J. An outbreak of head rot of cabbage caused by Rhizoctonia solani AG2-1 in Central China. Plant Disease 93:109. Journal Reprint

Bertrand A, Castonguay Y, Cloutier J, Couture L, Hsiang T, Dionne J, Laberge S. 2009. Genetic diversity for pink snow mold resistance in greens-type annual bluegrass. Crop Science 49:589-599. Unseeded annual bluegrass (Poa annua L.) is an important component of golf greens in many regions of Canada and the United States. Although this turfgrass species has desirable playing attributes, it suffers from susceptibility to environmental and biological stresses including subfreezing temperatures and snow molds. In this study, we compared 29 genotypes collected from golf greens located in Québec and Ontario for their resistance to pink snow mold (SM). Plants were inoculated with Microdochium nivale [(Fries) Samuels & Hallett], causal agent of SM, and incubated under controlled conditions. High levels of variation in SM resistance were detected within the collection and between genotypes. Analysis of the relationship between climatic parameters at the sites of origin and SM susceptibility revealed that level of resistance was positively correlated to the duration of snow cover. Genetic diversity within the Poa collection was estimated using the sequence related amplified polymorphism (SRAP) technique. The UPGMA (unweighted-pair group method arithmetic average) dendrogram yielded two main clusters that differed markedly in their proportion of SM-resistant genotypes. Our results show that SM disease is a major selection pressure for the generation of genetic diversity among annual bluegrass biotypes that evolved on golf greens in northern climates. SRAP polymorphisms between bulked genotypes with contrasting resistance to SM were identified and could be used as markers for SM resistance in annual bluegrass. PDF Reprint

Hsiang T, Tian XL. 2008. Sporulation and Identity of Tar Spot of Maple in Canada Acta Silv. Lign. Hung. Spec. Edn, Pages 71-74. Tar spot of maple has been increasing in incidence and severity in the Great Lakes region of eastern North America since the 1990's. The purpose of this work was to examine tar spot on native and imported maple species to determine the fungal species involved. By extracting and sequencing DNA from tar spot samples obtained from Europe and across Canada, we have found that giant tar spot on Norway maple (Acer platanoides) is caused by Rhytisma acerinum, the same fungus found in Europe, whereas native maple species in North America have the large tar spot caused by R. americanum (e.g. on silver maple, A. saccharinum) or the speckled tar spot caused by R. punctatum (on big-leaf maple, A. macrophyllum). We also found that ascospore release from tar spots on Norway maple in southern Ontario occurred over a three-week period, the start of which coincided with full leaf expansion in Norway maple. PDF Reprint

Jaime MDLA, Hsiang T, McDonald MR. 2008. Effects of Glomus intraradices and onion cultivar on Allium white rot development in organic soils in Ontario. Commercial products containing formulations of the vesicular-arbuscular mycorrhiza (AM) Glomus intraradices were assessed for their effectiveness in suppressing Allium white rot (WR) on onions (Allium cepa) in organic soils and compared with the fungicide Folicur 3.6F (430 g a.i./L tebuconazole) under field conditions. The trials were conducted during 2000 and 2001 in commercial onion fields in the Holland-Bradford Marsh in Ontario. The AM product MIKRO-VAM, which is used in transplanted onions, reduced the incidence of WR by almost 50% compared with the untreated control and was comparable with that of the fungicide treatment, Folicur 3.6F, applied according to label recommendations. This is one of the few studies to demonstrate season-long disease suppression with AM under field conditions, and it is the first to show that the AM products can be as effective as a fungicide treatment under commercial production practices. A consistent difference in incidence of WR was found between the cultivars ‘Hoopla’ and ‘Fortress’ onions. ‘Hoopla’ was more susceptible to WR than ‘Fortress’ in 10 of 13 field trials and all trials where WR incidence on ‘Hoopla’ was 4%. There was a significant negative correlation between disease incidence and AM root colonization, suggesting that AM colonization was an important factor in the reduction of WR observed in this study. PDF Reprint

Wijekoon CP, Goodwin PH, Hsiang T. 2008. The involvement of two epoxide hydrolase genes, NbEH1.1 and NbEH1.2, of Nicotiana benthamiana in the interaction with Colletotrichum destructivum, Colletotrichum orbiculare or Pseudomonas syringae pv. tabaci. Epoxide hydrolase hydrates epoxides to vicinal diols in the phyto-oxylipin peroxygenase pathway resulting in the production of epoxy alcohols, dihydrodiols, triols and epoxides, including many lipid epoxides associated with resistance. Two epoxide hydrolase genes from Nicotiana benthamiana L., NbEH1.1 and NbEH1.2, were amplified from cDNA of leaves during a susceptible response to the hemibiotrophic pathogens, Colletotrichum destructivum O’Gara,C. orbiculare Berk. & Mont. von Arx. or Pseudomonas syringae pv. tabaci Wolf & Foster, or the hypersensitive resistance response to P. syringae pv. tabaci expressing avrPto. Increases in expression of NbEH1.1 generally occurred during the late biotrophic and necrotrophic stages in the susceptible responses and prior to the hypersensitive response. NbEH1.2 expression was not significantly induced by C. orbiculare but was induced by C. destructivum, P. syringae pv. tabaci and P. syringae pv. tabaci expressing avrPto, although to a lesser degree than NbEH1.1. Virus-induced gene silencing of NbEH1.1 delayed the appearance of lesions for C. destructivum, reduced populations of P. syringae pv. tabaci and increased populations of P. syringae pv. tabaci expressing avrPto. The importance of epoxide hydrolase during pathogen attack may be related to its roles in detoxification, signaling, or metabolism of antimicrobial compounds. PDF Reprint

Murphy J, Wong F, Tredway L, Crouch JA, Inguagiato J, Clarke B Hsiang T, Rossi F. 2008. Best management practices for anthracnose on annual bluegrass turf. Golf Course Management August 2008, pages 93-104. PDF Reprint   or the 2009 BCGSA Dogwood version  or the 2010 OSGA Green is Beautiful version

Wijekoon CP, Goodwin PH, Hsiang T. 2008. Quantifying fungal infection of plant leaves by digital image analysis using Scion Image software. Journal of Microbiological Methods 74:94-101. A digital image analysis method previously used to evaluate leaf color changes due to nutritional changes was modified to measure the severity of several foliar fungal diseases. Images captured with a flatbed scanner or digital camera were analyzed with a freely available software package, Scion Image, to measure changes in leaf color caused by fungal sporulation or tissue damage. High correlations were observed between the percent diseased leaf area estimated by Scion Image analysis and the percent diseased leaf area from leaf drawings. These drawings of various foliar diseases came from a disease key previously developed to aid in visual estimation of disease severity. For leaves of Nicotiana benthamiana inoculated with different spore concentrations of the anthracnose fungus Colletotrichum destructivum, a high correlation was found between the percent diseased tissue measured by Scion Image analysis and the number of leaf spots. The method was adapted to quantify percent diseased leaf area ranging from 0 to 90% for anthracnose of lily-of-the-valley, apple scab, powdery mildew of phlox and rust of golden rod. In some cases, the brightness and contrast of the images were adjusted and other modifications were made, but these were standardized for each disease. Detached leaves were used with the flatbed scanner, but a method using attached leaves with a digital camera was also developed to make serial measurements of individual leaves to quantify symptom progression. This was successfully applied to monitor anthracnose on N. benthamiana leaves. Digital image analysis using Scion Image software is a useful tool for quantifying a wide variety of fungal interactions with plant leaves. PDF Reprint

Xie J, Fu Y, Jiang D, Li G, Huang J, Li B, Hsiang T and Peng Y. 2008. Intergeneric transfer of ribosomal genes between two fungi. BMC Evolutionary Biology 8:87. Background: Horizontal gene transfer, also called lateral gene transfer, frequently occurs among prokaryotic organisms, and is considered an important force in their evolution. However, there are relatively few reports of transfer to or from fungi, with some notable exceptions in the acquisition of prokaryotic genes. Some fungal species have been found to contain sequences resembling those of bacterial genes, and with such sequences absent in other fungal species, this has been interpreted as horizontal gene transfer. Similarly, a few fungi have been found to contain genes absent in close relatives but present in more distantly related taxa, and horizontal gene transfer has been invoked as a parsimonious explanation. There is a paucity of direct experimental evidence demonstrating the occurrence of horizontal gene transfer in fungi. Results: We found a fungal field isolate from rice (Oryzae sativa) that contains ribosomal DNA sequences from two species of fungal rice pathogens (Thanatephorus cucumeris and Ceratobasidium oryzae-sativae). This field isolate has four types of ribosomal DNA internal transcribed spacers (ITS), namely pure ITS of C. oryzae-sativae, which was dominant in this field isolate, pure ITS of T. cucumeris, and two chimeric ITS, with ITS1 derived from C. oryzae-sativae and ITS2 from T. cucumeris, or ITS1 from T. cucumeris and ITS2 from C. oryzae-sativae. The presence of chimeric forms indicates that the intergeneric hybrid was not merely composed of nuclei from the parental species, but that nuclear fusion and crossing over had taken place. Conclusions: Hyphae of T. cucumeris and C. oryzae-sativae are vegetatively incompatible, and do not successfully anastomose. However, they parasitize the same host, and perhaps under the influence of host enzymes targeted to weaken pathogen cells or in dying host plant tissue, the fungal hyphae lost their integrity, and normal vegetative incompatibility mechanisms were overcome, allowing the hyphae to fuse. Based on the presence of other similarly anomalous isolates from the field, we speculate that these types of intergeneric hybridization events and occurrences of horizontal gene transfer may not be so rare in the field. Journal reprint

Hsiang T, Tian LX, Sopher C. 2008. Tar spot of maple: where did it come from and is it getting worse? Horticulture Review 26(1):35-37. Tar spot of maple caused by species of Rhytisma is becoming worse in the Great Lakes Region. This article looks at the history of this disease in Ontario, and speculates on some factors that might be responsible for the increased incidence, mentions some methods of management, and states some research needed. PDF reprint. A similar article in Ontario Arborist

Zheng L, Huang J.B., Hsiang T. 2008. First report of leaf blight of garlic (Allium sativum) caused by Stemphylium solani in China Plant Pathology 57:380. Leaf blight of garlic is a destructive disease in Hubei province, China. From fall 2004 to spring 2007, symptoms were observed on infected leaves in Dangyang County, with diseased area estimated at over 7000 ha. Garlic yield was reduced by 30% on average with up to 70% yield losses in some fields (Fig. 1). Lesions were initially small and white (Fig. 2), and these enlarged to produce apical necrosis, extending until the leaves withered. Isolations were made onto potato sugar agar (PSA) giving white colonies. After 4 days on PSA, the centers turned gray, and the agar became yellow brown throughout (Fig. 3). Single spores were cultured onto 2% water agar, and pieces of autoclaved filter paper were placed on the agar to induce sporulation. Conidiophores were up to 170 μm long. Conidia were pointed at the swelled apex of each conidiophore, clavate, with 1-3 transverse septa, 2-7 longitudinal or oblique septa, and 34~55x17~27 μm (Fig. 4). The pathogen was identified as Stemphylium solani based on Ellis (1971). Genomic DNA was extracted from three isolates, and sequences of rDNA-ITS were obtained. Comparison with sequences in GenBank showed 99% similarity with S. solani (AF203450). To test pathogencity, a conidial suspension (1x106 conidia/ml) containing 0.1% Tween-20 was sprayed until runoff (200 m1 per plant) onto upper and lower surfaces of 20 garlic leaves of seven 14-day-old 20-cm tall plants in the lab. Plants were incubated with a 12 h photoperiod at 25¡æ and 90% relative humidity. Five days after inoculation, white spots were observed on inoculated leaves but no symptoms were seen on water-treated control plants. Koch’s postulates were fulfilled by re-isolating S. solani from diseased leaves. Leaf blight of garlic is caused by S. botryosum, S. vesicarium, (Boiteux et al., 1994) or Cladosporium echinulatum (Pal, 1976). In China, only S. vesicarium has been reported as a pathogen of garlic (Shang et al., 1997). To our knowledge, this is the first report of S. solani infecting garlic. References Boiteux LS, Lima MF, Menezes Sobrinho JA, Lopes CA, 1994. A garlic (Allium sativum) leaf blight caused by Stemphylium vesicarium in Brazil. Plant Pathology 43,412-14. Ellis MB, 1971. Dematiaceous Hyphomycetes. Kew: Commonwealth Mycological Institute. Pal AK, Basuchaudhary KC, 1976. A new leaf blight of garlic caused by Cladosporium echlnulatum (Berk) de Vries, from Varanasi, Uttar Pradesh. Current Science 45,739. Shang HS, Wang SQ, Zuo JZ, Zhao JY, 1997. The causal agent of white spot and rot of garlic bolt. Acta Agriculturae Boreali-occidentalis Sinica 6,73-6. (In Chinese)

Hao L., P.H. Goodwin and T. Hsiang. 2007. Expression of a metacaspase gene of Nicotiana benthamiana after inoculation with Colletotrichum destructivum or Pseudomonas syringae pv. tomato and the effect of silencing the gene on the host response. Plant Cell Reports 26:1879-1888 Metacaspases are cysteine proteinases that have homology to caspases, which play a central role in signaling and executing programmed cell death in animals. A type II metacaspase cDNA, NbMCA1, was amplified from Nicotiana benthamiana infected with Colletotrichum destructivum. It showed a peak in expression at 72 h postinoculation corresponding with the switch to necrotrophy by C. destructivum. Inoculation of N. benthamiana with an incompatible bacterium, Pseudomonas syringae pv. tomato, which should induce a non-host hypersensitive response (HR), did not result in an increase in NbMCA1 expression at the time of necrosis development at 20 to 24 h postinoculation. Virus-induced silencing of NbMCA1 resulted in three to four times more lesions due to C. destructivum compared with leaves inoculated with the PVX vector without the cloned metacaspase gene or inoculated with water only. However, virus-induced silencing of NbMCA1 did not affect the HR necrosis or population levels of P. syringae pv. tomato. Although this metacaspase gene does not appear to be involved in the programmed cell death of non-host HR resistance to P. syringae, it does affect the susceptibility of N. benthamiana to C. destructivum indicating a function in a basal defense response. Possible roles of NbMCA1 could be in degrading virulence factors of the pathogen, processing pro-proteins involved in stress responses, eliminating damaged proteins created during stress, and/or degrading proteins to remobilize amino acids to fuel de novo synthesis of proteins involved in stress adaptations. PDF Reprint

Hsiang, T., A. Liao and D. Benedetto. 2007. Sensitivity of Sclerotinia homoeocarpa to demethylation-inhibiting fungicides 10 years after first use. Plant Pathology 56:500-507. In previous research, eight populations of Sclerotinia homoeocarpa were sampled in early 1994 prior to the registration of sterol demethylation-inhibiting (DMI) fungicides for turf disease control in Canada, and seven of these were found to contain only wild-type isolates with high sensitivity to DMI fungicides such as propiconazole and myclobutanil. In late 2003, seven of the eight original populations plus two new populations were tested for sensitivity to propiconazole. Four of the nine populations had not been treated with DMI fungicides during the intervening years, and isolates from these locations were sensitive to propiconazole (geometric mean EC50 values from 0.005 to 0.012 g ml-1 compared to 0.005 to 0.008 g ml-1 for the original 1994 populations). Among the five populations from 2003 that had been exposed to DMI fungicides, mean EC50 values were significantly greater, ranging from 0.020 to 0.048 g ml-1. A significant correlation was found between estimated number of fungicide applications and logEC50 (R2 = 0.832, p = 0.0001), and the equation predicted that 42.3 applications of propiconazole would be needed to bring a sensitive population (EC50 < 0.01 g ml-1) to a resistant level (EC50 > 0.10 g ml-1). We also tested fungicide sensitivity vs. the duration of fungicide efficacy, and found that isolates with decreased sensitivity were able to more quickly overcome the inhibitory effects of fungicide application, reducing the duration of control from 3 weeks to 2 weeks. PDF Preprint

Huang, J.B., K.F. Fang, and T. Hsiang. 2007. First report of brown leaf spot caused by Bipolaris australiensis on Cynodon in China. Plant Pathology 56:349. Bipolaris leaf spot affects many turfgrasses. Bipolaris cynodontis and B. hawaiiensis were reported to be pathogens of Cynodon spp. in Hainan (Liu & Pu, 2004) and Guangdong Provinces (Xiang & Zhong, 1999), respectively. From the summer of 2003 to the winter of 2005, a new disease was observed on C. dactylon x C. transvaalensis cultivar ‘Tifgreen’ at the Wuhan and E-Zhou golf courses in Hubei province. The disease which caused discoloration of up to 25% of affected patches, was characterised by irregularly shaped, brown, water-soaked spots on leaves and sheaths. Severely infected leaves became straw-coloured. A fungus was isolated on quarter strength potato dextrose agar (PDA) which was then purified by single sporing. Cultures were initially white, but after three days acquired an olive green to black cast found to contain cylindrical conidia with three pseudosepta. Sizes ranged from 13-35 mm x 8-10 mm. Conidiophores were brown, simple or branched, geniculate and sympodial. The hilum was characterised by a flattening of the basal cell, slightly protruding at the tip. Morphological characterstics matched those of B. australiensis. Sequencing of the internal transcribed spacer region of rDNA showed a 100% match with Cochliobolus australiensis (telemorph of B. australiensis) GenBank accession AY923860. To confirm pathogenicity of B. australiensis, 20 day-old, pot-grown ‘Tifgreen’ leaves were treated with plugs or spores. Mycelial plugs from PDA (6-mm-diameter) were placed on leaves for 48 h and then removed. Small circular brown spots from 3 to 5 mm in diameter appeared three days later. Other leaves were sprayed with a conidial suspension of 2x105 spores per ml until run off. Deep brown spots similar to those seen at the golf courses appeared after five days. Leaves with attached sterile agar plugs or sprayed with sterile water, remained healthy. We re-isolated and confirmed the presence of B. australiensis on induced symptoms. B. australiensis has been reported previously to be pathogenic on Chloris and Pennisetum species in Australia, Kenya and India (Smith et al., 1989). To the best of our knowledge, this is the first report of brown leaf spot caused by Bipolaris australiensis on Cynodon spp. in China. REFERENCES: Liu XM, Pu JJ, 2004. Initial report on turfgrass occurrence in Hainan Province. Pratacultural Science 21, 73-74 (in Chinese). Smith JD, Jackson N, Woolhouse AR, 1989. Fungal Diseases of Amenity Turfgrasses, Third Edition. New York, USA: E & F.N. Spon Ltd.  Xiang MM, Zhong XP, 1999. The pathogen, identification and chemical control in vitro of leaf blight disease on Cynodon hybrid in the Huizhou Golf Course, Guandong Province. Pratacultural Science 16, 58-61 (in Chinese).

Hsiang, T. and P. Masilamany. 2007. First report of Rhizoctonia zeae on turfgrass in Ontario. Plant Pathology 56:350. In May 2004, a disease appeared on Poa annua and Agrostis stolonifera at a golf course near Toronto. Narrow yellow rings enclosing areas up to 30 cm across appeared after air temperatures reached 25  C. The disease resembled yellow patch caused by Rhizoctonia cerealis, but the weather was too warm for normal occurrences of that disease. The rings persisted until the end of July. In late May 2005, the disease appeared again after the weather became hot. A mixture of azoxystrobin and chlorothalonil was applied which seemed to suppress the disease within a week, until it reappeared in July. Samples were collected, and symptomatic leaves were surface sterilized in 1% hypochlorite, and plated onto potato dextrose agar (PDA) amended with streptomycin. After one week at 25C, the plates contained white colonies 5 cm across. Sequencing of the ITS region of ribosomal DNA showed a 99.6% match with a R. zeae sequence in GenBank. Pathogenicity was tested by inoculating 2-wk-old plants with mycelial plugs of R. zeae. Within one week at 25  C, significant blighting on leaves and sheaths was observed, as well as spherical orange sclerotia on sheaths. Symptomatic leaves were plated on PDA, and hyphae of R. zeae were recovered. In Canada, R. zeae has been reported from turfgrass samples in B.C. (Joshi, 2004), but this is the first report from Ontario. In the USA, this fungus has been reported to cause diseases of several turfgrass species (Smiley et al., 2005), which have been called hot weather brown patch, leaf and sheath spot/rot/blight or brown ring patch among others. We propose that it should be referred to as sheath spot. The taxonomy of this organism is similarly confused since R. zeae is considered to be a subspecies of Waitea circinata which contains at least two other subspecies including R. oryzae (Oniki et al., 1985; Leiner & Carling, 1994). More work is required to clarify the taxonomic disposition of R. zeae. REFERENCES: Joshi V, 2004. Diseases diagnosed on commercial crops submitted to the BCMAFF Plant Diagnostic Lab in 2003. Canadian Plant Disease Survey 84, 7-13.  Leiner RH, Carling DE, 1994. Characterization of Waitea circinata (Rhizoctonia) isolated from agricultural soils in Alaska. Plant Disease 78, 385-388.  Oniki M, Ogoshi A, Araki T, Sakai R, Tanaka S, 1985. The perfect state of Rhizoctonia oryzae and R. zeae and the anastomosis groups of Waitea circinata. Transactions of the Mycological Society of Japan 26, 189-198.  Smiley RW, Dernoeden PH, Clarke BB, 2005. Compendium of Turfgrass Diseases, Third Edition. St. Paul, MN, USA: APS Press.

Hsiang T. 2006. Anthracnose: terrorizing turfgrass. GreenMaster 41(6):8-9. HTML version with pictures

Hsiang T. 2006. Neurotic about necrotic ring spot. GreenMaster 41(5):20-21. HTML version with pictures

Kaminski, J. and T. Hsiang. 2006. First Report of Ophiosphaerella agrostis infecting creeping bentgrass in Canada. Plant Disease 90:1114. Reprint on Journal Website or locally

Hsiang, T. D. Olds and R. Gowan. 2006. A new Rhizoctonia fungus on turfgrass in Ontario. GreenMaster 41(3):28-30. Preprint

Hsiang, T. & Baillie, D.L. 2006. Issues in comparative fungal genomics. Applied Mycology and Biotechnology, Bioinformatics Issue Volume 6:100-122. Biologists face an overwhelming richness of nucleotide and protein sequence data. By the middle of 2005, there were almost 300 complete genomes that were publicly accessible. Most of these were archeal or bacterial since prokaryotic genomes are much smaller than eukaryotic genomes. Among eukaryotes, fungi, particularly yeasts, have some of the smallest genome sizes and hence represent the highest number of complete or almost complete genomes sequenced. By mid-2005, there were over 43 fungal genomes that were completely or almost completely sequenced and publicly accessible. What are the relationships among fungi and between fungi and other organisms? What type of genes and pathways are required for pathogenicity and other fungal lifestyles? Researchers are addressing these types of questions with data from high-throughput genomic sequencing. This review examines some recent uses of fungal genomic data in comparative genome analyses. Comparative genomics can facilitate research into the following areas: evolution, phylogenetics, targeted drugs, gene discovery, and gene function. Each of these is discussed as well as the availability and ownership of the genomic data, and the concepts of homology (homologs, orthologs, paralogs) and similarity. PDF Reprint

Hao, L., Hsiang, T. & Goodwin, P.H. 2006. Role of two cysteine proteinases in the susceptible response of Nicotiana benthamiana to Colletotrichum destructivum and hypersensitive response to Pseudomonas syringae pv. tomato. Plant Science 170:1001-1009. Two cysteine proteinase genes of the papain family, NbCYP1 and NbCYP2, were amplified from cDNA of Nicotiana benthamiana leaves infected with the hemibiotrophic fungus Colletotrichum destructivum. Both genes showed peak expression corresponding with the switch from biotrophic to necrotrophic growth by C. destructivum at 72 h post-inoculation (HPI). For N. benthamiana inoculated with the incompatible bacterium, Pseudomonas syringae pv. tomato, expression of NbCYP1 significantly decreased at 12 HPI, whereas NbCYP2 expression increased at 3 HPI. Expression of both genes then returned to near pre-inoculation levels and remained constant as necrosis later appeared due to a non-host hypersensitive response (HR). Virus-induced gene silencing of NbCYP1 and NbCYP2 resulted in increased susceptibility of N. benthamiana to C. destructivum but did not affect the HR necrosis or population levels of P. syringae pv. tomato. These two cysteine proteinase genes do not appear to be involved in the programmed cell death of the HR resistance to P. syringae pv. tomato, but they are involved in limiting the host’s susceptibility to C. destructivum. PDF Reprint

Yang, Y, F. Chen, and T. Hsiang. 2006. Fertile sporophore production of Typhula phacorrhiza in the field is related to temperatures near freezing. Canadian Journal of Microbiology 52:9-15. Two field tests and one lab test were conducted to examine the environmental factors affecting sporophore production in T. phacorrhiza, and to compare these results with those documented for T. ishikariensis and T. incarnata. In the 2001 lab test where lighting, soil moisture, and soil-sand media were tested in 50 mL screw cap tubes incubated at 4 C, the limiting factor for Typhula sporophore production was found to be moisture. In the fall 2001 field test, 100 sclerotia of six isolates from three Typhula spp. were placed into pots filled with a sand and soil mixture. The pots were monitored weekly, and maximum sporophore production for all six isolates and for both watered and unwatered pots was observed at 11 weeks which was soon after mean daily temperatures fell below 0 C. In the second field test in fall 2003, five isolates of the three species were tested with similar procedures, but peak sporophore production was observed after six weeks, and again only after mean daily temperatures fell below 0 C. In the field, sporophore production of T. phacorrhiza seems to require the same environmental cues as those of T. ishikariensis or T. incarnata, namely high moisture and temperatures near freezing. PDF Reprint

Hsiang T and Tian L. 2005. Turf Disease Research Update: Sugar with your Tea? Turf Line News 193(4/5):10-11 or Sports Turf Managers 18(2):10. Reprint

Dean JD, Goodwin PH, Hsiang T. 2005. Induction of glutathione S-transferase genes of Nicotiana benthamiana following infection by Colletotrichum destructivum and C .orbiculare and involvement of one in resistance. Journal of Experimental Botany 56:1525-1533. Four glutathione S-transferase (GST) genes, NbGSTU1, NbGSTU2, NbGSTU3 and NbGSTF1, were amplified from cDNA of Nicotiana benthamiana leaves infected with Colletotrichum destructivum using primers based on conserved regions of N. tabacum GST sequences. Expression of NbGSTU1 and NbGSTU3 increased progressively during infection by either C. destructivum or C. orbiculare, except for a slight decrease by NbGSTU1 late in the infection, whereas NbGSTU2 and NbGSTF1 expression remained relatively constant. Each of the four genes was cloned into a PVX vector for virus-induced gene silencing, and reduced expression of the four genes was detected by RT-PCR. A statistically significant increase in susceptibility of N. benthamiana to infection following gene silencing was found only for NbGSTU1-silenced plants, which had 130% more lesions and 67% more colonization by C. orbiculare compared to control plants. These results demonstrate that that different GST genes respond in different ways to fungal infection, and at least one plant GST gene has an important role in disease development. PDF Reprint

Hsiang, T. and D.L. Baillie. 2005. Comparison of the yeast proteome to other fungal genomes to find core fungal genes. Journal of Molecular Evolution 60:475-483. The purpose of this research was to search for evolutionarily conserved fungal sequences to test the hypothesis that fungi have a set of core genes that are not found in other organisms, as these genes may indicate what makes fungi different from other organisms. By comparing 6355 predicted or known yeast (Saccharomyces cerevisiae) genes to the genomes of thirteen other fungi using Standalone TBLASTN at an e-value < 1E-5, a list of 3340 yeast genes was obtained with homologs present in at least 12 of 14 fungal genomes. By comparing these common fungal genes to complete genomes of animals (Fugu rubripes, Caenorhabditis elegans), plants (Arabidopsis thaliana, Oryza sativa) and bacteria (Agrobacterium tumefaciens, Xylella fastidiosa), a list of common fungal genes with homologs in these plants, animals and bacteria was produced (938 genes), as well as a list of exclusively fungal genes without homologs in these other genomes (60 genes). To ensure that the 60 genes were exclusively fungal, these were compared using TBLASTN to the major sequence databases at GenBank: NR (nonredundant), EST (expressed sequence tags), GSS (genome survey sequences), and HTGS (unfinished high-throughput genome sequences). This resulted in 17 yeast genes with homologs in other fungal genomes, but without known homologs in other organisms. These 17 core fungal genes were not found to differ from other yeast genes in GC content or codon usage patterns. More intensive study is required of these 17 genes and other common fungal genes to discover unique features of fungi compared to other organisms. PDF Reprint

Huang JB, Zheng L, Hsiang T. 2005. First report of leaf spot caused by Curvularia verruculosa on Cynodon sp. in Hubei, China. Plant Pathology 54:253. PDF Reprint

Celetti MJ, Hsiang T, and Llewellyn J. 2004. Daylily Rust. Ontario Ministryof Agriculture and Food Factsheet Agdex 631. Online Article

Hsiang T, Cook S and Zhao Y. 2004. Studies on biology and control of daylily rust in Canada. The Daylily Journal 59(1):47-57. PDF Reprint

Huang JB, Zheng L, Hsiang T. 2004. First Report of Leaf Spot Caused by Curvularia affinis on Festuca arundinacea in Hubei, China. Plant Dis. 88:1048. Reprint

Goodwin, P.H., R.P. Oliver and T. Hsiang. 2004. Comparative analysis of expressed sequence tags from Malva pusilla, Sorghum bicolor, and Medicago truncatula infected with Colletotrichum species. Plant Science 167:481-489 To assess relative gene expression, expressed sequence tag (EST) redundancy was compared between EST collections from susceptible Malva pusilla and Medicago truncatula inoculated with Colletotrichum gloeosporioides f. sp. malvae and C. trifolii, respectively, and resistant and susceptible Sorghum bicolor inoculated with C. graminicola (= C. sublineolum). EST redundancies from the fungal-inoculated S. bicolor and M. truncatula were also compared to healthy S. bicolor and M. truncatula. Several of the more redundant plant ESTs in the C. gloeosporioides f. sp. malvae - M. pusilla interaction represented genes encoding pathogenesis-related proteins, such as beta-1,3-glucanase, osmotin and chitinase, but a number of other ESTs, such as those for cysteine proteinase, heat shock protein and glutathione S-transferase, were also relatively abundant. Differences in EST redundancy between different interactions included a greater abundance of heat shock protein ESTs in the susceptible S. bicolor interaction, and a greater abundance of cysteine proteinase ESTs in the resistant S. bicolor and susceptible M. truncatula interactions. Using EST redundancy to compare gene expression between different host plants interacting with Colletotrichum species provides a useful basis for selecting genes for further study in plant- Colletotrichum interactions.  PDF Reprint

Hsiang, T. and D.L. Baillie. 2004. Recent progress, developments and issues in comparative fungal genomics. Canadian Journal of Plant Pathology 26:19-30. Biologists face an overwhelming richness of nucleotide and protein sequence data. As of the end of 2003, there were over 100 complete or almost complete non-viral genomes in publicly available databases. Most of these were bacterial, since prokaryotic genomes are generally much smaller in size than eukaryotic genomes. Among eukaryotes, fungi have some of the smallest genome sizes and hence represent the highest number of complete or almost complete genomes sequenced, with most of these released within the last two years. What are the genes that fungi have in common? Among these genes, which ones have homologs in plants, animals or bacteria, and which ones are only found in fungi? Researchers are just beginning to be able to address these types of questions with data from high-throughput genomic sequencing. This review examines some recent and possible future uses of fungal genomic data in comparative genome analyses, particularly as they relate to the study of fungal plant pathogens. Comparative genomics can facilitate research into the following areas: phylogenetics (via whole genome comparisons), targeted drugs (via unique target sites in pests), gene discovery (via conserved sequences), and gene function (via guilt by association). Each of these is discussed as well as the availability and ownership of the genomic data, and the concepts of homology and similarity.  PDF Reprint

Khan, A. and T. Hsiang. 2003. The infection process of Colletotrichum graminicola and relative aggressiveness on four turfgrass species. Canadian Journal of Microbiology 49:433-442. Detached 3-wk-old leaves of Agrostis palustris, Lolium perenne, Poa annua, and P. pratensis were inoculated with conidial suspensions of two isolates of Colletotrichum graminicola obtained from A. palustris. Inoculated leaves were incubated at 23 C under high relative humidity (>95%). The infection process was investigated by light microscopy from 2 to168 h after inoculation. Spore germination was observed within 2 h after inoculation (hai), appressoria within 6 hai, and penetration pores within 8 hai on all four hosts. Infection hyphae were observed inside epidermal cells within 24 hai on all four hosts, but significantly greater infection was observed in A. palustris and P. annua than in L. perenne or P. pratensis at 96 and 120 hai. Acervuli appeared on leaves of A. palustris at 72 hai and on L. perenne at 96 hai, but were not found on either P. annua or P. pratensis during the first 168 hai. The infection process was similar to that reported for C. graminicola from other hosts; however, disease development of two isolates of C. graminicola fromA. palustris was faster or fungal growth more extensive on detached leaf tissue of A. palustris than on other turfgrass species tested.  PDF Reprint

Chen, N., P.H. Goodwin and T. Hsiang. 2003. The role of ethylene during the infection of Nicotiana tabacum by Colletotrichum destructivum. Journal of Experimental Botany 54:2449-2456. Two periods of increased ethylene production were observed after inoculation of Nicotiana tabacum by Colletotrichum destructivum. This pathogen exhibits an intracellular hemibiotrophic infection process, with a biotrophic phase followed by a necrotrophic phase. Ethylene production first increased during the biotrophic phase with a peak at 24 hours before the necrotrophic phase. A second increase in ethylene occurred late in the necrotrophic phase when the lesions were expanding. Two different 1-aminocyclopropane-1-carboxylic acid synthase genes showed increased expression after the first ethylene peak with a maximum at 24 hours before the second ethylene increase. Expression of an 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) gene increased during the first ethylene peak and then declined beginning with the second ethylene increase. A second ACO gene showed relatively little change in expression during infection with slightly higher expression at 24 hours before the second ethylene increase, and a third ACO gene showed a progressive decline in expression with a major decrease occurring before the second ethylene increase.  Inoculation of ethylene-insensitive tobacco with C. destructivum revealed that it was more susceptible than the wild-type. The changes in ethylene production and associated gene expression as well as the increased disease susceptibility of ethylene-insensitive tobacco indicate that ethylene plays a role in this interaction, perhaps as a signaling molecule to trigger defense mechanisms. PDF Reprint

Dean, J. Doug, Paul H. Goodwin and Tom Hsiang. 2003. Colletotrichum gloeosporioides infection induces differential expression of glutathione S-transferase genes in Malva pusilla. Functional Plant Biology 30:821-828. Among a collection of 840 expressed sequence tags of Malva pusilla leaves infected with Colletotrichum gloeosporioides f. sp. malvae (Cgm), a total of four different glutathione S-transferase (GST) (EC 2.5.1.18) genes were identified, each showing a different pattern of expression following infection. MpGSTU1 and MpGSTU2 were members of the class tau GSTs, MpGSTF1 was a member of the class phi GSTs, and MpGSTZ1 belonged to the class zeta GSTs. Infection by Cgm occurs by a hemibiotrophic process with an initial biotrophic phase preceding the necrotrophic phase and the appearance of symptoms. Expression of MpGSTZ1 progressively increased during infection, corresponding directly with the growth of the pathogen. Expression of MpGSTU2 was similar to that of MpGSTZ1, except for a greater increase during the late necrotrophic phase. MpGSTU1 expression remained relatively constant throughout the infection, whereas MpGSTF1 expression was induced primarily during the conversion from the biotrophic to necrotrophic phases of infection. Incubation of healthy mallow leaves in the dark resulted in decreased expression of MpGSTF1 and MpGSTU1, but not MpGSTZ1 and MpGSTU2. The differential expression patterns indicate that these mallow GST genes play a variety of roles in healthy and fungal-infected leaf tissue.  PDF Reprint
 

Hsiang, T. and P.H. Goodwin. 2003. Distinguishing plant and fungal sequences in ESTs from infected plant tissues. Journal of Microbiological Methods 54:339-351. Expressed sequence tags (ESTs) from fungal-infected plant tissues are composed of a mixture of plant and fungal sequences. Using freely available software and tools, a novel procedure is described for distinguishing plant and fungal DNA sequences. Although the GenBank non-redundant database is larger and therefore one would presume that BLASTX analysis of it would be more accurate, superior resolution of 700 randomly selected fungal ESTs was found with Standalone TBLASTX analyses with a local matching database composed of a plant and a fungal genome. Standalone TBLASTX analyses of 3983 ESTs from nine different fungal-infected plant EST libraries also proved to be superior in identifying the origin of sequences as either plant or fungal compared to GenBank BLASTX analysis. Standalone TBLASTX with a matching database comprised of a single plant and a single fungal genome appears to be a faster and more accurate method than BLASTX searches of the GenBank non-redundant database to distinguish fungal and plant sequences in mixed EST collections.   PDF Reprint    [Perl scripts can be found at www.uoguelph.ca/~thsiang/est/]

Hsiang, T., F. Chen and P.H. Goodwin. 2003. Detection and phylogenetic analysis of mating type genes of Ophiosphaerella korrae. Canadian Journal of Botany 81:307-315. Portions of the mating type genes from Ophiosphaerella korrae (= Leptosphaeria korrae), a pathogenic fungus of grasses, were examined by PCR. For nine isolates of O. korrae from North America, both mating type genes were amplified demonstrating that both MAT idiomorphs are detectable in this homothallic ascomycete. Amplified fragments from three isolates were sequenced, and parsimony analyses of MAT1 nucleotide and protein sequences placed O. korrae in the basal position of a clade of Phaeosphaeriaceae and Pleosporaceae, whereas the MAT2 nucleotide and protein data placed O. korrae in a clade with Pleosporaceae. The internal transcribed spacer (ITS) and 18S ribosomal DNA of O. korrae were also sequenced. The 18S sequences had insufficient variability to resolve the placement of O. korrae, while the ITS data placed it in Phaeosphaeriaceae. A total evidence analysis of Dothideomycetes with 18S, ITS and MAT data placed O. korrae alongside Phaeosphaeria species, with moderate bootstrap support. However, the Kishino-Hasegawa test did not demonstrate this topology to be significantly different from one where O. korrae was placed with Pleosporales. Although O. korrae does not belong in Leptosphaeria based on ITS data, MAT data do not strongly support its placement in Phaeosphaeriaceae.  PDF Reprint

Chen, N., T. Hsiang and P.H. Goodwin. 2003. Use of green fluorescent protein (GFP) to quantify the growth of Colletotrichum during infection of tobacco. Journal of Microbiological Methods 53:113-122. To develop a quantitative assay of fungal growth inside plant tissues, strains of Colletotrichum destructivum and C. orbiculare were transformed with a modified green fluorescent protein (GFP) gene fused with a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus nidulans.  Transformants expressed GFP in culture and had the same growth rate and general appearance as the wild type. GFP was observed in all fungal structures during infection of leaves of Nicotiana benthamiana, except for the melanized appressoria and setae. The timing and appearance of the fungal structures in the host appeared to be identical to that of the wild type.  GFP accumulation in inoculated leaves of N. benthamiana was quantified in leaf extracts using a fluorescence microplate reader, and the quantity of fluorescence was strongly correlated with the growth of the fungus as measured by the amount of fungal actin gene expression using northern blot hybridizations. These results demonstrated that assaying green fluorescence levels from a GFP-transformed fungus is an accurate, fast and easy means of quantifying fungal growth inside host plant cells.  PDF Reprint

Dean, J.D, P.H. Goodwin, and T. Hsiang. 2002. Comparison of relative RT-PCR and northern blot analyses to measure expression of b-1,3-Glucanase in Nicotiana benthamiana infected with Colletotrichum destructivum. Plant Molecular Biology Reporter 20:347-356. Although northern blot analysis is effective for quantifying gene expression, reverse transcription-polymerase chain reaction (RT-PCR) is much more sensitive. Obtaining quantitative RT-PCR results, however, can be challenging. Relative RT-PCR uses standard PCR techniques but permits the comparison of transcript quantities between samples by coamplifying the gene of interest with a housekeeping gene that acts as an internal control. To analyze the expression of a plant gene encoding a pathogenesis-related protein, such as b-1,3-glucanase, a translation elongation factor 1a (EF-1a) gene was selected as an internal control. Northern blot analysis demonstrated constitutive expression of the plant EF-1a gene following infection of Nicotiana benthamiana by Colletotrichum destructivum. Primers for the gene of interest and internal control were compatible, and 35 cycles of amplification gave reproducible relative RT-PCR results for b-1,3-glucanase gene expression. A high correlation was observed between the relative quantification of b-1,3-glucanase gene expression as determined by northern blot and relative RT-PCR analyses, demonstrating the validity of relative RT-PCR with a plant EF-1a gene as a control.  PDF Reprint

Chen, F., P.H. Goodwin, A. Khan and T. Hsiang. 2002. Population structure and mating type genes of Colletotrichum graminicola from Agrostis palustris. Canadian Journal of Microbiology 48:427-436. Eighty-seven isolates of Colletotrichum graminicola were collected from grass fields, most of which were from Agrostis palustris in Ontario, Canada. Specific primers were designed to amplify the mating type (MAT) genes, and among 35 isolates tested, all yielded a band of the expected size for MAT2. For six isolates, the MAT2 PCR products were sequenced and found to be similar to that reported for MAT2 of C. graminicola from maize. Based on 119 polymorphic bands from 10 random amplified polymorphic DNA primers, analyses of genetic distances were found to generally cluster isolates by host and geographic origin. Among 42 isolates from a grass field in Ontario, significant spatial autocorrelation was found to occur within a 20 m distance, implying that this is the effective propagule dispersal distance. Although clonal propagation was observed in the 87 isolates with 67 unique genotypes, the extent of genetic variation in local populations implies some occurrence of sexual or asexual recombination.  PDF Reprint

Deckert, R.J., T. Hsiang and R.L. Peterson. 2002. Genetic relationships of endophytic Lophodermium nitens isolates from needles of Pinus strobus L. Mycological Research 106:305-313. The foliage of Pinus strobus (eastern white pine), like that of all other conifers examined to date, is occupied by endophytic fungi, the most frequent of which is Lophodermium nitens.   The number and extent of endophytic infections and the genetic relationship of individual isolates within living needles as well as their relationship to isolates from forest floor needles is unknown.  To examine these and related questions, forest floor isolates and foliar endophytes from needle segments were obtained for ribosomal DNA sequencing and randomly amplified polymorphic DNA (RAPD) analysis.  Molecular and morphological data were compared and infection frequency determined as a function of position along the needle.  Ribosomal DNA sequences of foliar and ascospore isolates showed high levels of genetic similarity (>97% identity) for the internal transcribed spacer region.  RAPD profiles were able to distinguish ascospore siblings from non-siblings, and also revealed that many needle isolates belonged to the same genotype as adjacent neighbour isolates, as would be expected from mycelial spread within the needle.  Morphotype evaluation and RAPD profiles showed similar patterns: identical morphotypes grouped together and showed little or no genetic difference under RAPD analysis.  Both morphological and molecular data indicated that the majority of infections were contained within 1 mm needle segments but could extend to about 4 mm in length.  Infection frequency increased along the length of the needle from the proximal (shoot) end to the distal tip, with markedly higher rates in the distal quarter.  Thus, endophytic infections of L. nitens in white pine needles consist of many localized, discrete infections, originating from ascospores and differentially distributed along the length of the needle.  In the course of this work, it was found that GenBank accession number AF203470 under the name Meloderma desmaszieresii appeared not to be that species but L. nitens on the basis of the ITS sequences.  PDF Reprint

Chen, C.C., T. Hsiang, F.L. Chiang and C.A. Chang. 2002.  Molecular characterization of Tuberose mild mosaic virus and preparation of its antiserum to the coat protein expressed in bacteria. Botanical Bulletin of Academia Sinica 43:13-20.  A 2-kb DNA product was amplified from purified Tuberose mild mosaic virus (TMMV) virions as well as from infected tissues of tuberose by the use of degenerate primers for potyvirus. The PCR product was subsequently cloned and its sequence analyzed, It was found comprised of 1947 nucleotides (nts) corresponding to the 3'-terminal region of potyviruses. The deduced amino acid sequence contained 598 residues encoding part of the 3'-terminal region of Nlb gene (319 residues) and the complete sequence of coat protein (CP) gene (279 residues). A 136 nts of non-coding region (NCR) was found located at the V-terminal region of the DNA. A genetic code for aphid transmissibility of potyviruses, DAG triplet, was found at the 19-21 residues from the N-terminus of CP gene. Compared to the known sequences of potyviruses, the percent of nucleotide identities of the CP gene and the NCR were less than 62% and 39%, respectively. Similarly, percent identities of TMMV's CP amino acid sequence to those of other known potyviruses were all below 58%, confirming our previous finding that TMMV is a new species of Potyvirus. Using directional cloning technology, a 39-kDa fusion protein containing a complete CP sequence of TMMV and a partial sequence encoded by the expression vector plasmid (pET-30b, Novagene) was highly expressed and purified from E. coli cell cultures. The antigenicity of the fusion protein was determined to be indistinguishable from the viral CP. Antiserum prepared against this fusion protein showed comparable reactivities in the serological detection of TMMV with the conventional antibodies against purified virus particles.  

He, C.Y., T. Hsiang and D.J. Wolyn. 2002. Induction of systemic disease resistance and pathogen defence responses in Asparagus officinalis inoculated with nonpathogenic strains of Fusarium oxysporum. Plant Pathology 51: 225-230. The ability of nonpathogenic isolates of Fusarium oxysporum (npFo)  to induce systemic resistance and defence responses against subsequent  challenge with a pathogenic strain of F. oxysporum f. sp. asparagi ( Foa) was examined in Asparagus officinalis. In a split-root experiment, roots inoculated with npFo exhibited a hypersensitive  response and those subsequently inoculated with Foa displayed  resistance. Induction of systemic resistance in npFo-treated plants  led to significantly fewer necrotic lesions (P = 0.05) and reduced  Foa disease severity compared with plants not treated with npFo . In  hyphal-sandwich root inoculation experiments, activities of peroxidase  and phenylalanine ammonia-lyase and lignin content were higher in np  Fo-treated plants and increased more rapidly than in npFo-untreated  plants after Foa inoculation. Antifungal activity (inhibition of  fungal spore germination and germ-tube growth) from exudates of roots  inoculated with Foa were observed for npFo-treated plants but not  for npFo-untreated plants. Thus, isolates of npFo may function as  inducers of systemic acquired resistance (SAR) and defence responses  against Foa invasion in A. officinalis.  PDF Reprint

Gossen, B.D., T. Hsiang and T. Murray. 2001. Managing snow mold diseases of winter cereals and turf. pp.182-192 in: N. Iriki, D.A. Gaudet, A.M. Tronsmo, N. Matsumoto, M. Yoshida and A. Nishimune (eds). Low temperature Plant Microbe Interactions under Snow. Hokkaido National Experiment Station, Sapporo, Japan. PDF Reprint

Tronsmo, A.M., T. Hsiang, H. Okuyama and T. Nakajima. 2001. Low temperature diseases caused by Microdochium nivale. pp. 75-86 in: N. Iriki, D.A. Gaudet, A.M. Tronsmo, N. Matsumoto, M. Yoshida and A. Nishimune (eds). Low temperature Plant Microbe Interactions under Snow. Hokkaido National Experiment Station, Sapporo, Japan. PDF Reprint

Goodwin, P.H., S. Shen and T. Hsiang. 2001. Hemibiotrophic infection and identity of the fungus, Colletotrichum destructivum, causing anthracnose of tobacco. Mycological Research 105:1340-1347. The causal agent of tobacco anthracnose was identified as Colletotrichum destructivum based on the morphology of the fungus and a comparison of the sequence of  the rDNA ITS with those of other Colletotrichum species. The infection process on tobacco (Nicotiana tabacum and N. benthamiana) was examined by light microscopy, which revealed that the pathogen acted as an intracellular hemibiotroph. Penetration occurred preferentially at the anticlinal walls of epidermal cells by an appressorium and penetration peg. An infection vesicle formed in the penetrated host cell by 48 h after inoculation, and out of this, a multi-lobed infection vesicle grew which remained limited to the initially infected cell. The interaction at this point was biotrophic, which was confirmed by plasmolysis and accumulation of a vital stain by the infected host cells. Thin secondary hyphae arose from multi-lobed infection vesicles at 60 h after inoculation, which then penetrated the host cell wall and began the necrotrophic phase of the infection.  Acervuli formed on the plant surface by 96 h after inoculation, typically with a single melanized seta. In addition to tobacco, the fungus could infect alfalfa, cowpea, and Medicago truncatula, but not soybean.  The process of infection of C. destructivum in tobacco was very similar to that previously reported in alfalfa and cowpea.  PDF Reprint

Shen, S., Goodwin, P.H., Hsiang, T. 2001. Infection of Nicotiana species by the anthracnose fungus, Colletotrichum orbiculare.  European Journal of Plant Pathology 107:767-773. Colletotrichum gloeosporioides f. sp. malvae, isolate Biomal(R), ATCC 20767, was originally isolated from round-leaved mallow (Malva pusilla) and developed as a weed biocontrol agent. Ribosomal DNA sequence analysis was recently used to re-classify this fungus as C. orbiculare, which is an aggregate species with a number of formae speciales. Several morphological features of ATCC 20767 were examined that were consistent with those described for C. orbiculare, and inoculation of a number of Nicotiana species and several cultivars of N. tabacum showed that this fungus was pathogenic to many of these previously undescribed hosts. Spore germination and appressorium formation were higher on tobacco than previously observed on round- leaved mallow. The pathogen produced melanized appressoria on N. tabacum leaves that formed preferentially at the anticlinal epidermal cell wall. A symptomless phase of infection persisted for 72-96 h postinoculation, during which time the fungus first produced a spherical infection vesicle from an infection peg, and then large primary hyphae which grew through the epidermal cells. The large primary hyphae were highly constricted at the points of penetration of the host cell walls. Thin secondary hyphae appeared at 96-120 h postinoculation coinciding with the appearance of light green, water-soaked spots and the formation of acervuli. The infection of tobacco by C. orbiculare ATCC 20767 is not a non- specific interaction but appears to follow an intracellular hemibiotrophic infection process that is very similar to that established for the C. orbiculare infection of round-leaved mallow, cucurbits and beans.  PDF Reprint

Hsiang, T., Hsieh, T. F., and Chastagner, G. A. 2001. Relative sensitivity to the fungicides benomyl and iprodione of Botrytis elliptica from Taiwan and the Northwestern U.S.A.  Plant Pathology Bulletin 10:93-95. Isolates of Botrytis elliptica were tested for growth on potato dextrose agar as well as potato dextrose agar amended with discriminatory concentrations of benomyl (1 ug a.i./ ml) or iprodione (10 ug a.i./ ml).  Fourteen of these isolates originated from Washington or Oregon State in the USA, and the remaining 48 isolates were from Taiwan.  Relative fungicide sensitivity was assessed by dividing the extent of growth after 96 h on amended media over the growth on unamended medium. There was no significant difference in growth rates on unamended PDA between isolates from Taiwan compared to those from the U.S.A.; however isolates from Taiwan showed higher relative growth, 4-fold or 2-fold greater, respectively, on benomyl or iprodione-amended media than isolates from the U.S.A. More research is needed to determine whether these Taiwan isolates showing less sensitivity to benomyl or iprodione also exhibit resistance in the field to normal fungicide application rates.  PDF Reprint

Hsiang, T. and P.H. Goodwin. 2001. Ribosomal DNA sequence comparisons of Colletotrichum graminicola from turfgrasses and other hosts. European Journal of Plant Pathology 107:593-599. The 5.8S ribosomal gene and the flanking internal transcribed spacers (ITS) 1 and 2 from Colletotrichum graminicola isolates causing anthracnose disease of Agrostis palustris and Poa species were sequenced. Although bootstrap support was not high, two major groups were observed with both UPGMA and parsimony algorithms, one containing isolates from A. palustris and another with isolates from Poa spp. The ITS sequences were also compared with those of isolates of C. graminicola and C. sublineolum from Sorghum spp., Zea mays and Rottboellia cochinchinesis as well as other Colletotrichum species. Except for one isolate from P. annua in Texas, the ITS1 and ITS2 sequences of turfgrass isolates always grouped separately from C. graminicola or C. sublineolum from non-turfgrass hosts with high bootstrap support. ITS sequences of the turfgrass isolates were more similar to those of other species of Colletotrichum, such as C. coccodes and C. dematium, than they were to C. graminicola isolates from other hosts. Turfgrass isolates have ITS sequences which are not identical to those of isolates from Zea mays and Sorghum species demonstrating diversity among fungi conventionally classified as C. graminicola.  PDF Reprint

Hsieh, T.F., J.W. Huang and T. Hsiang. 2001. Light and scanning electron microscopy studies on the infection of oriental lily Leaves by Botrytis elliptica.  European Journal of Plant Pathology 107: 571-581. Light, scanning electron and fluorescent microscopy were used to observe the infection process of Botrytis elliptica on leaves of oriental lily (cv. Star Gazer). At 20  C and 100% relative humidity, conidia germinated on both adaxial and abaxial foliar surfaces, but germ tubes failed to invade epidermal cells on the adaxial surface. On abaxial surfaces, short (< 20 mm) swollen germ tube appressoria penetrated through stomatal openings (19%), through the epidermis near guard cells (52%), or directly through epidermal cells (29%). Esterase activity was detected on germ tubes and conidia after 6 h of incubation, and deformation of the cuticle on abaxial surfaces of lily was observed surrounding infection sites. By 3 h after inoculation, almost 70% of the conidia had germinated, but no penetration was observed. At 6 h after inoculation, almost one-third of germinated conidia had penetrated epidermal cells, and water-soaked lesions were associated with 20% of the penetrations. By 9 h after inoculation, approximately 60% of the germinated conidia had penetrated plant tissues, and water-soaked lesions were associated with 60% of the infections. Fluorescent microscopy with a specific fungal stain allowed assessment of successful infection and visualization of sub-epidermal hyphae. We conclude that penetration of abaxial foliar surfaces of oriental lilies by B. elliptica occurs via short swollen germ tube appressoria mostly near stomata. PDF Reprint

Huang, J.B., T.F. Hsieh, G.A Chastagner, and T. Hsiang.  2001. Clonal and sexual propagation in Botrytis elliptica. Mycological Research 105:833-842. Genetic variation was found between isolates of Botrytis elliptica from the USA and Taiwan based on 22 polymorphic markers from five RAPID primers. Among these 69 isolates, there were 43 different genotypes. One clonal genotype was found spanning Oregon and Washington, USA, and in a population from Hsinshe, Taiwan, clonal genotypes were frequent, demonstrating the importance of asexual spread of this pathogen. However, when only unique genotypes were considered, gametic disequilibrium among putative RAPD loci of the genotypes from Taiwan (33) or Hsinshe (23) was not detected, suggesting an underlying sexual recombination system for this population of B. elliptica. A weak level of cultivar specialization was detected among B. elliptica isolates from the Hsinshe population based on analysis of genetic distances between isolates.  PDF Reprint  [disequilibrium software can be found here]

He, C., T. Hsiang and D. Wolyn. 2001. Activation of defense responses to Fusarium infection in Asparagus densiflorus. European Journal of Plant Pathology 107: 473-483.  Defense responses to Fusarium oxysporum f. sp. asparagi and F. proliferatum were compared after root inoculation of the asparagus fern, Asparagus densiflorus vars. Myersii and Sprengeri, and cultivated asparagus, A. officinalis cv. Guelph Millennium. Both varieties of A. densiflorus exhibited a hypersensitive response with rapid death of epidermal cells within 8-24 h and restricted the fungal growth. In A. officinalis roots, rapid cell death was not found, and necrotic lesions were observed 8-14 d after fungal inoculation. Peroxidase and phenylalanine ammonia-lyase activities increased significantly in inoculated A. densiflorus but not A. officinalis plants. Local and systemic induction of peroxidase activity was detected after pathogen inoculation in root and spear tissues, respectively, of A. densiflorus. POX activity decreased in roots of inoculated A. officinalis by 8 d post-inoculation. Germination and germ tube growth were inhibited when spores of F. oxysporum f. sp. asparagi were incubated in root exudates and on root segment surfaces of inoculated A. densiflorus plants exhibiting hypersensitive cell death. Spore germination of F. proliferatum and three fungi non-pathogenic to cultivated asparagus was inhibited as well. Rapid induction of hypersensitive cell death in A. densiflorus was associated with restriction of fungal growth, and activation of peroxidase and phenylalanine ammonia-lyase, two defense enzymes thought to be important for plant disease resistance.  PDF Reprint

Hsiang, T. and J.D. Dean. 2001. DNA sequencing for anastomosis grouping of Rhizoctonia solani isolates from Poa annua. International Turfgrass Society Research Journal 9:674- 678.  The internal transcribed spacer region of the ribosomal DNA from four isolates of Rhizoctonia solani Kuehn from Poa annua L. and one from Agrostis palustris Huds. were sequenced, and compared to sequences from other R. solani isolates representative of 12 anastomosis groups (AG), with multiple sequences from AG1, AG2-1, AG2-2 and AG4.  The sequence alignment of the 789 bp region was subjected to distance and parsimony analyses.  Both analyses showed that representatives of the different anastomosis groups clustered separately, although branching patterns were not exactly the same in the two dendrograms.  Isolates from P. annua all clustered with AG4 HGII. An isolate from A. palustris in Ontario clustered with AG2-1, while an isolate from the same host in Wisconsin clustered with AG1-IB.  Screening of a larger number of turfgrass isolates from a wider geographic collection may reveal whether P. annua is always associated with AG4 HGII, and the group or subgroup with which A. palustris is most frequently associated.  PDF Reprint

Hsiang, T. and S. Cook. 2001. Effect of Typhula phacorrhiza on winter injury in field trials across Canada. International Turfgrass Society Research Journal 9:669-673.  An isolate of the fungus Typhula phacorrhiza (Reich:Fr.) Fr. was evaluated in field tests across Canada for suppression of winter injury which was rated based on combined abiotic and biotic symptoms after snowmelt. This isolate, TP94671, has been identified in previous tests as being suppressive to gray snow mold, and a specific purpose of these trials was to evaluate the effect on pink snow mold.  Out of 11 trials across Canada, six did not show significant differences in winter injury among the treatments (untreated control, fungicide treatment, or T. phacorrhiza treatment).  In these locations, abiotic winter injury may have confounded the results or disease pressure was so low such that the effects on winter injury, including pink and gray snow molds, could not be assessed. In two trials at Montreal, Quebec (1999) and Waskeiu, Saskatchewan (1999), the -treated plots showed more winter injury than the fungicide-treated plots, but less than the untreated control plots.  In three trials at Guelph, Ontario (1999), Barrie, Ontario (1999) and Panorama, British Columbia (2000), there were significant differences among treatments, with the T. phacorrhiza treatment suppressing winter injury as much as or even more than the fungicide control.  In these trials, both pink snow mold and gray snow mold were commonly observed on the untreated plots and surrounding areas, and the T. phacorrhiza treatment was able to suppress winter injury in all three locations to an acceptable level (<10% affected area).  PDF Reprint

Smith, J.D., B.D. Gossen, and T. Hsiang. 2001. First Report of Dollar Spot, Caused by Sclerotinia homoeocarpa F.T. Bennett, on Poa pratensis in Saskatchewan.  Plant Disease 85:803. Dollar spot disease affects many species of grasses in North America but has not been previously reported from Saskatchewan.   In July 2000, symptoms were observed on golf course fairways in Saskatoon. No dollar spot disease was observed on adjacent putting greens or tees composed of creeping bentgrass (Agrostis palustris), perhaps because the tees and greens were grown under a higher nitrogen fertility regime. Fungicide treatments are usually not required for turf disease control during the warm, dry summer growing season and no fungicides had been applied at this location.  Daytime temperatures near 25 C and heavy dew at night preceded the disease outbreak which affected about 5% of the turf across large areas of several fairways.  The fairways were originally seeded to Festuca rubra subsp. rubra (common creeping red fescue) and Poa pratensis (Kentucky bluegrass), but also contained P. annua (annual bluegrass).  The disease was observed on leaf blades of all three species.  In addition to 5-cm-diameter circular patches of brown grass, sharply delimited individual leaf lesions and cobweb-like aerial mycelia on the grass were observed. Fungal isolates were obtained by plating infected P. pratensis leaf blades on potato dextrose agar and then transferring to oat agar.  On oat agar, isolates produced white, fluffy aerial mycelium, columnar when mature, and usually with a cinnamon base and dark brown stromata or sclerotial flakes on and in the agar. DNA was extracted from an isolate and amplified using the primers ITS1 and ITS4 (1). A 500-bp fragment presumed to contain the internal transcribed spacer region of the ribosomal DNA (ITS) was purified and sequenced (Mycol. Res. 104:16), and it showed showed 96% identity with the ITS of a Sclerotinia homoeocarpa isolate, GENBANK accession AF067640. To test Koch's postulates, P. pratensis cv. Loft 1757 was grown from seed in 15 ml of sand at 20 C under constant fluorescent light. Two-wk-old turf was inoculated with 5-mm- diameter mycelial plugs of the fungus from 1-wk-old cultures on potato dextrose agar, by placing inoculum plugs on the sand. Inoculated turf was incubated in a in a loosely lidded clear plastic container at 20 C under constant fluorescent lighting. After 1 wk, lesions and white aerial hyphae were observed on the leaf blades, while no disease was observed on the uninoculated controls.  The fungus was reisolated from foliar lesions to complete Koch's postulates. In addition to P. pratensis, both P. annua and A. palustris cv. Penncross® were also inoculated and showed disease symptoms, with greater disease severity on P. annua.

Inglis, D., M. Derie and T. Hsiang. 2001. Stem Canker of Cabbage Seed Stalks Caused by Botrytis cinerea in Western Washington.  Plant Disease 85:559.  Stem cankers were observed during 1998 on bolting stalks of cabbage (Brassica oleracea var. capitata L.) in seed production fields in western Washington.  In 1999, approximately 4 ha of cabbage hybrid 'Wk 121', was severely affected.  Lesions occurred at the base of seed stalks after they emerged from heads of plants overwintered in the field, or on flower branches and seed-bearing stalks that developed during the growing season.  Lesions girdled a branch or stalk, and killed or weakened it so that it broke during pod fill.  Isolates of Botrytis cinerea Pers.:Fr. (Bc) were obtained by plating spores from lesions onto potato dextrose agar.  To confirm pathogenicity, stems of 12-day-old seedlings of 'Wk 121' were scraped with a razor blade or left intact, atomized with sterile 0.01 % Tween 80 or a suspension of Bc at 1.0 x 106 conidia/ml, and kept at 20oC in a dew chamber in plastic bags for 72 hr.  The fungus was re-isolated from small lesions on wounded stems inoculated with Bc.  No lesions developed on non-wounded or wounded control plants.  Bc is reported to cause storage rot of cabbage (2) and grey mold on Brassica oleracea L. (cabbage, kale, kohlrabi, wild cabbage) in Washington (1), but not stem canker.  This new seed crop disease may be the result of pre-disposition to infection by freezing injury or mechanical damage on a highly susceptible cultivar grown under cool, wet weather.

Li, J., S. Jin, T. Hsiang & P.H. Goodwin. 2001. A novel actin-related protein gene of Colletotrichum gloeosporioides f. sp. malvae shows altered expression corresponding with spore production. FEMS Microbiology Letters 197:209-214.  A novel actin-related protein (arp) was found in the plant pathogenic fungus, Colletotrichum gloeosporioides f. sp. malvae (Cgm) which causes anthracnose disease of round-leaved mallow (Malva pusilla).  Sequence comparisons showed that this gene, arpA, belongs to the highly divergent "other arps" category in the current arp classification system. ArpA is most similar to the arp11 gene of Mus musculus but has a unique structure with deletions at the C terminus similar to that of the arp10 gene of  Saccharomyces cerevisae.  A portion of another putative arp gene, arpB, was found immediately downstream of arpA. Expression of arpA was compared to the constitutively expressed Cgm actin gene, actA.  In culture, the relative expression of arpAincreased when growth conditions favored sporulation. During infection, arpA expression was greatest at the late necrotrophic phase, when sporulation occurred. Arps have been shown to be important in nuclear migration in fungal hyphae, and the expression pattern of arpA indicates that it may have a particular role during sporulation.  PDF Reprint

Hsiang, T. 2000. Biological control of turfgrass snow molds.  GreenMaster 35(5):12-15.
 View the text and images of the disease trials used in this article.

Hsiang, T and J. Huang. 2000. The use of RAPD markers to distinguish juniper and cedar cultivars. Canadian Journal of Botany 78:655-658.  Two species of Chamaecyparis (Cupressaceae) and six cultivars each of Juniperus chinensis and J. scopulorum (Cupressaceae) were subjected to RAPD analysis using seven primers.  UPGMA and principal component analyses of genetic distances between cultivars showed that 42 polymorphic RAPD bands could distinguish among all cultivars and properly group them by species and genera. Where the origin of a specific juniper cultivar is uncertain, analysis of genetic distance can pinpoint close relatives.  For example, we were unable to trace the origin of the J. chinensis'Alps', and we initially thought it was a mislabeled J. chinensis'Blue Alps'.  However, we found 'Alps' to be closer to J. chinensis'Fairview' and 'Mountbatten' than to 'Blue Alps'.  Similarly, 'Wichita Blue' has an unknown origin, but it had the highest genetic similarity with 'Medora'.  PDF Reprint

Hsiang, T., X.L. Ma, L. Yang and S. Cook. 2000. Analyses of RAPD data for detection of host specialization in Sclerotinia homoeocarpa. Plant Pathology 49:269-275. UPGMA analysis, principal component analysis, genetic diversity analysis, and genetic distance analysis of RAPD data were used to assess the extent of host specialization in fifty isolates of S. homoeocarpa from five turfgrass hosts. In UPGMA analysis and principal component analysis, the occurrence of host specialization was not readily apparent based on visual inspection. Genetic diversity analysis showed significant differentiation among isolates from different host species (Gst = 0.34, p<0.001). The strongest evidence for some degree of host specialization came from the statistical analysis of genetic distances among isolates. By grouping pairwise genetic distances between isolates based on their host species, and analysing for average distance within the same host species and among different host species, we found that average distance within species was less than among species (p<0.0001). An analysis of molecular variance of the genetic distances among isolates found that 32.3% of the total variation was attributable to host species. We conclude that these isolates of S. homoeocarpa showed a weak level of host specialization, which was not readily apparent by UPGMA or principal component analyses, but was revealed by genetic diversity analysis and statistical analysis of genetic distances among isolates. Inoculation tests on different host species and tests using a greater number of isolates are required to confirm the extent of specialization.  PDF Reprint

Snider, C.S., Hsiang, T., Zhao, G. and Griffith, M. 2000. Role of ice nucleation and antifreeze activities in pathogenesis and growth of snow molds.  Phytopathology 90:354-361.  We examined the ability of snow molds to grow at temperatures from -5 to 30C, and to influence the growth of ice through assays for ice nucleation and antifreeze activities. Isolates of Coprinus psychromorbidus (low temperature basidiomycete variant), Microdochium nivale, Typhula phacorrhiza, T. ishikariensis, T. incarnata, and T. canadensis all grew at 5C, whereas Sclerotinia borealis and S. homoeocarpa did not grow at temperatures below 4C. The highest threshold ice nucleation temperature was 7C. Because snow molds are most damaging to their hosts at temperatures above this, our results imply that the pathogenesis of these fungi is not dependent on ice nucleation activity to cause freeze-wounding of host plants. All snow molds that grew at subzero temperatures also exhibited antifreeze activity in the growth medium and in the soluble and insoluble hyphal fractions, with the exception of Microdochium nivale and one isolate of T. canadensis. The lack of high ice nucleation activity combined with the presence of antifreeze activity in all fungal fractions indicates that snow molds can moderate their environment to inhibit or modify intra- and extracellular ice formation, which helps explain their ability to grow at subzero temperatures under snow cover.  PDF Reprint

Hsiang, T., J. Huang, L. Yang, and S. Cook. 2000. Occurrence of Kabatina juniperi in Ontario and genetic analysis using RAPD markers. Canadian Journal of Plant Pathology 22:79-88.  A survey conducted in 1992 and 1993 revealed that the major cause of juniper twig blight in Ontario was Kabatina juniperiPhomopsis juniperovora was found in 3 of 26 samples received in 1992 and in none of 23 samples in 1993.  Analysis of acervuli of K. juniperi collected from spring 1997 through spring 1999 showed that spore production was high in spring with lower levels in summer gradually declining in the fall to very low levels in winter.  Using RAPD markers, we found molecular variation between K. juniperi isolates from five locations within a 50 km radius.  RAPD analysis of 24 isolates from five different cultivars of Juniperus scopulorum did not reveal host specialization.  Among 49 isolates sampled from two adjacent rows of J. scopulorum 'Grey Gleam', there were 36 different RAPD haplotypes. Genetic diversity analysis of this population showed significant gametic linkage disequilibrium between putative RAPD loci, and indicated that asexual propagules such as conidia are the major form of dispersal of this pathogen.  A practical implication of this work is that pruning operations should be timed so that they do not coincide with peak spore production.  PDF Reprint  [disequilibrium software can be found here

Goodwin, P.H., T. Hsiang and L. Erickson. 2000. A comparison of stilbene and chalcone synthases including a new stilbene synthase gene from Vitis riparia cv. Gloire de Montpellier. Plant Science 151:1-8. A stilbene synthase gene was cloned from Vitis riparia cv. Gloire de Montpellier after PCR amplification with primers designed to include the start and stop codons of stilbene synthase genes of V. vinifera. The exon was highly similar to that of other stilbene synthases, particularly those from V. vinifera (99% nucleotide identity). An intron was found which interrupted the predicted codon for cysteine in the same location as in other stilbene and chalcone synthase genes. The intron showed high nucleotide identity (86%) with an intron from a stilbene synthase gene of V. vinifera. The V. riparia sequence was used in an evaluation of the relatedness of stilbene and chalcone synthases of plants. Five procedures involving distance, parsimony and maximum likelihood methods were used for constructing phylogenetic trees, and they yielded slightly to considerably different results. However, none of the trees were consistent with a previous hypothesis that stilbene and chalcone synthases cluster solely based on the genetic relatedness of the species, implying that stilbene synthase genes arose independently in plant families. In our analyses, stilbene and chalcone synthases of Vitis always clustered separately. The relatedness and origin of stilbene and chalcone synthases appears to be more complex than originally believed. PDF Reprint

Hsiang, T. and C. Wu. 2000. Genetic relationships of pathogenic Typhula species assessed by RAPD, ITS-RFLP and ITS sequencing. Mycological Research 104:16-22. Several species of Typhula cause diseases of cereals and grasses at low temperatures. Taxonomic separation of T. phacorrhiza, T. incarnata, and T. ishikariensis was investigated using RAPD (randomly amplified polymorphic DNA method), ITS-RFLP (restriction fragment length polymorphisms of the internal transcribed spacer regions of genomic rDNA), and nucleotide sequencing of the ITS region. Using 10 RAPD primers on genomic DNA or eight restriction enzymes on PCR-amplified ITS regions, banding patterns were generated for each of 19 isolates from five different species or varieties.Polymorphisms were found between species and varieties, with fewer among isolates within species or varieties. RAPD analysis suggested that T. ishikariensis var. idahoensis may be distinct from the other varieties (var. ishikariensis and var. canadensis), although this was not supported by ITS-RFLP. To confirm relationships, ITS nucleotides sequences were obtained from single isolates of each of the five Typhula taxa, and sequence alignments were subjected to distance and parsimony analysis. Although there were differences in branching between dendrograms derived from sequencing vs. RAPD or ITS-RFLP, sequence data supported the distinction of the three main Typhula species, with the T. ishikariensis varieties grouping together and showing differences at a sub-specific level. ITS sequence data also indicated the closer taxonomic affinity of Typhula species to the agaricoid Tricholoma sejunctum compared to the clavarioid Thelephora terrestris.  PDF Reprint

Hsiang, T., C. Wu and S. Cook. 1999. Residual efficacy of Typhula phacorrhiza as a biocontrol of grey snow mould on creeping bentgrass. Canadian Journal of Plant Pathology 21:382-387. Isolates of the biocontrol fungus, Typhula phacorrhiza (TP), were evaluated in field tests over a 5-year period for suppression of grey snow mould caused by T. incarnata (TINC) and T. ishikariensis (TISH). Out of hundreds of isolates of TP collected from across southern Ontario in the spring 1994, 42 were cultured on mixed grains and applied in the late fall to creeping bentgrass at a rate of 200 g/m2 (4 x 105 cfu/m2) along with inocula of TINC or TISH at 10 g/m2 (2 x 104 cfu/m2). In late fall of each year from 1995 to 1998, TINC and TISH were reapplied to the same plots, but no TP was re-applied. Plots were rated for disease after snow melt in each year from 1995 to 1999. High positive correlations of winter injury were found between the suppression trials during the first three years, with several isolates showing significant control of grey snow mould in the first three years. By the fourth year after inoculation, suppression of snow mould in plots treated with the most efficacious TP isolates had severely declined and there were no significant differences in winter injury between plots treated with TP isolates in the fourth and fifth years. PDF Reprint

Hsiang, T., N. Matsumoto and S.M. Millett. 1999. Biology And Management of Typhula Snow Molds of Turfgrass. Plant Disease 83:788-798. Feature Article in Plant Disease. View the figures used in this manuscript. PDF Reprint

Hsiang, T. and J. Richter. 1999. Juniper Rust Species in an Ontario Nursery, 1998. Canadian Plant Disease Survey 79:142-143.  View the text and images of juniper rust used in this article.

Goodwin, P.H. and T. Hsiang. 1999. Anthracnose basal rot of creeping bentgrass. GreenMaster 34(3):38-42. View the text and images of anthracnose disease used in this article.

Wu, C. and T. Hsiang. 1999. Mycelial growth, sclerotial production and carbon utilization of three Typhula species. Canadian Journal of Botany 77:312-317. The mycelial growth, sclerotial production and carbon utilization of the snow mould biocontrol agent Typhula phacorrhiza were compared to the two grey snow mould fungi, T. ishikariensisandT. incarnata. Variation was observed among the four isolates for each species, but there was greater variation among species. All three species were able to grow at the lowest temperature (0C), but temperature optima differed with T. ishikariensis lowest, and T. phacorrhiza highest. On potato dextrose agar or potato malt agar at 10C, T. phacorrhiza had greater radial growth than T. ishikariensis but less than T. incarnata. All species could utilize microcrystalline cellulose, bacto-cellulose and glucose as carbon sources, but radial growth of T. phacorrhiza was significantly greater than T. incarnata and T. ishikariensis on these defined carbon sources tested, except for Indulin-AT, which was inhibitory to T. incarnata and T. phacorrhiza. This greater ability to utilize these structural and storage carbohydrates, combined with mycelial growth and sclerotial production over a wider range of temperatures, may help explain how some isolates of T. phacorrhiza are able to out-compete grey snow mould in field tests. PDF Reprint

Hsiang, T. and G.S. Mahuku. 1999. Genetic variation within and between southern Ontario populations of Sclerotinia homoeocarpa. Plant Pathology 48:83-94. Restriction fragment length polymorphisms (RFLP) of the intergenic spacer region (IGS) of rDNA and random amplified polymorphic DNA (RAPD) markers were used to survey genetic variability among 181 isolates of Sclerotinia homoeocarpa from Ontario and 10 from Japan. RAPD and IGS-RFLP analyses revealed polymorphisms within and between populations of S. homoeocarpa, resulting in 151 genotypes. Both types of markers gave similar results in phenetic analysis of genetic distances between populations. Cluster analysis showed that Japanese isolates of S. homoeocarpa were genetically distinct from Ontario isolates demonstrating significant intraspecific differentiation. An average genetic similarity of 0.66 was found between Japanese isolates. Among Ontario isolates, average genetic similarity was 0.86, and genotypic diversity analysis showed that 49.3% of the total genetic variation observed within Ontario populations was found among individuals within populations compared to 50.7% between populations. Gametic linkage disequilibrium analysis within Ontario populations found an average 15.6% significant non-random associations between putative RAPD loci, and that half of the populations showed signs of significant linkage disequilibrium. These results suggest that both clonal propagation and recombination events occurred in local populations of S. homoeocarpa.The high level of genetic similarity between populations and the low levels of intraspecific genetic variation may reflect a small founding population for southern Ontario isolates of S. homoeocarpa. PDF Reprint

Hsiang, T. and J. Huang. 1998. Identity of ectotrophic black runner hyphae on roots of Poa annua. Canadian Plant Disease Survey 78:116-117. PDF Reprint.

Hsieh, T., J.W. Huang and T. Hsiang. 1998. Light and scanning electron microscopy studies on the infection of oriental lily leaves by Botrytis elliptica. European Journal of Plant Pathology 107:571-581. Light, scanning electron and fluorescent microscopy were used to observe the infection process of Botrytis elliptica on leaves of oriental lily (cv. Star Gazer).  At 20C and 100% relative humidity, conidia germinated on both adaxial and abaxial foliar surfaces, but germ tubes failed to invade epidermal cells on the adaxial surface. On abaxial surfaces, short (<20 m) swollen germ tube appressoria penetrated through stomatal openings (19%), through the epidermis near guard cells (52%), or directly through epidermal cells (29%). Esterase activity was detected on germ tubes and conidia after 6 h of incubation, and deformation of the cuticle on abaxial surfaces of lily was observed surrounding infection sites. By 3 h after inoculation, almost 70% of the conidia had germinated, but no penetration was observed. At 6 h after inoculation, almost one third of germinated conidia had penetrated epidermal cells, and water-soaked lesions were associated with 20% of the penetrations. By 9 h after inoculation, approx. 60% of the germinated conidia had penetrated plant tissues, and water-soaked lesions were associated with 60% of the infections. Fluorescent microscopy with a specific fungal stain allowed assessment of successful infection and visualization of sub-epidermal hyphae. We conclude that penetration of abaxial foliar surfaces of oriental lilies by B. elliptica can occur via short swollen germ tube appressoria mostly near stomata.  PDF Reprint

Wu, C. and T. Hsiang. 1998. Pathogenicity and formulation of Typhula phacorrhiza, a biocontrol agent of gray snow mold. Plant Disease 82:1003-1006. In previous testing, we found that gray snow mold, caused by Typhula ishikariensis or T. incarnata, could be controlled by applying T. phacorrhiza onto turfgrass prior to snowfall. To test for phytopathogenicity of this biocontrol agent, the five most disease-suppressive isolates were cultured on mixed grains and applied to 12 turfgrass cultivars and two winter wheat cultivars in December 1995 and 1996. After snow melt in April 1996 and 1997, significantly greater winter injury was visible on plots treated with the pathogens compared to T. phacorrhiza-treated plots or untreated plots. Except for one cultivar in 1996, there were no significant differences between T. phacorrhiza-treated plots and untreated plots. Pelletized alginate formulations of T. phacorrhiza containing kaolin clay with various nutritional amendments were tested for viability and efficacy. After 64 wk of storage, viability remained >85% at -15C, and >70% at 4C, but <30% at 10C and 25C. Significant control of gray snow mold by T. phacorrhiza, using small numbers of pellets (20 g/m2), was equivalent to using larger amounts of mixed grain inoculum (200 g/m2) or wheat bran inoculum (100 g/m2) in two years of field testing. Preprint

Wu, C., T. Hsiang, L. Yang and L. X. Liu. 1998. Efficacy of Typhula phacorrhiza as a Biocontrol Agent of Grey Snow Mould of Creeping Bentgrass. Canadian Journal of  Botany 76:1276-1281. Isolates of the fungus Typhula phacorrhiza (TP) were evaluated in field tests over a 3-year period for suppression of grey snow mould caused by T. ishikariensis (Tish) and T. incarnata(Tinc). Isolates of TP were collected across southern Ontario in the spring of 1994. In December, 1994, 46 of these isolates which had been cultured on mixed grains, were applied to creeping bentgrass at a rate of 200 g/m2 (4 x 105 cfu/m2) with inoculum of Tish or Tinc at 10 g/m2 (2 x 104 cfu/m2). In December, 1995, 30 selected TP isolates were inoculated onto a new set of plots along with grey snow mould fungi. In November, 1996, 22 of these isolates were re-inoculated onto the 1995 plots. All plots were rated for injury after snowmelt, 1995-1997. Isolates of TP varied significantly in their ability to suppress disease. No strong correlations were found between in vitro growth characteristics and field performance; however, significant positive correlations were found between the disease suppression trials for the three years, with several isolates showing statistically significant control of grey snow mould equal to a fungicide treatment.  PDF Reprint

Raffle, V.L. and T. Hsiang. 1998. Low level of DNA polymorphisms in Leptsophaeria korrae detected with random amplified polymorphic DNA markers. Canadian Journal of Plant Pathology 20:48-54. The genetic diversity of the turfgrass pathogen, Leptosphaeria korrae was examined with random amplified polymorphic DNA (RAPD) markers at the sibling, population, regional and cross-continental levels using 71 isolates. The seven RAPD primers tested showed polymorphisms within and between regions in North America. No polymorphisms were observed between sibling spores nor between twenty-one isolates from a single field. Based on differences observed with 48 polymorphic RAPD markers, only six unique but closely related haplotypes were identified among 33 geographically separated field isolates from British Columbia, Ontario, and Quebec in Canada, and Washington State in the USA. Although there was clustering of isolates of a haplotype, some haplotypes had wide distributions across different regions. We hypothesize that very few genotypes were introduced into these regions from the centre of origin.  PDF Reprint

Hsiang, T., L. Yang and W. Barton. 1998. Relative virulence of isolates of Sclerotinia homoeocarpa with varying sensitivity to propiconazole. European Journal of Plant Pathology 104:163-169. Isolates of Sclerotinia homoeocarpa were collected from across southern Ontario from areas where demethylation inhibiting fungicides reportedly had never been used. Forty of these isolates with propiconazole EC50 values ranging from 0.003 to 0.069 µg ml-1 were inoculated onto creeping bentgrass (Agrostis palustris) in summer 1995 and summer 1996 to assess virulence. Each isolate was grown on mixed cereal grains, dried, ground up and applied separately at a rate of 10 g/m2 to 0.25 m2 plots with three replicates per isolate. For both years, no spots were visible on the plots at time of inoculation; however, by the end of each experiment, there were up to 180 spots per plot with significant differences between isolates. Growth rate in culture was not significantly related to fungicide sensitivity (log-EC50 values). Statistically significant negative relationships were found in both years between AUDPC and log-EC50 values. These significant relationships imply that in the absence of propiconazole use, isolates that are more sensitive to propiconazole may out-compete isolates that are less sensitive. However, further study is required to determine the time frame for this to occur, and whether DMI-resistance prevention strategies can feasibly be based on the existence of resistance-related fitness costs.  PDF Reprint

Mahuku, G.S, T. Hsiang and L. Yang. 1998.Genetic variation within Microdochium nivale isolates from turfgrass. Mycological Research 102:559-567. Conserved primers were used in a polymerase chain reaction to amplify the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) of 100 Microdochium nivale isolates collected from different turfgrasses in southern Ontario. The profile of the restriction digestion of the amplified ITS region revealed that all of the M. nivale isolates analyzed belonged to var. nivale. Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs) of the intergenic spacer (IGS) region of rDNA revealed extensive genetic diversity within var. nivale. With RAPD markers, the average similarity coefficient was 66% and the estimate of genotypic diversity was 0.179. Population subdivision analysis showed that 92.2% of the total genetic diversity was found among individuals within populations compared to 7.8% among populations. In dendrograms derived from genetic distances using RAPD and IGS-RFLP markers, there was some evidence for host specialization. However, most RAPD markers were shared by individuals from different turfgrasses, and the populations were not highly differentiated. The high level of genotypic diversity detected within populations and the low level of genetic differentiation among populations show that recombination and migration are likely playing important roles in the population biology of M. nivale var. nivale. PDF Reprint

Wu C. and Hsiang T. 1997. Mechanisms Involved in Biocontrol of Grey Snow Mould of Turfgrass with Typhula phacorrhiza. Phytopathology 87:S104-105.  In three years of field studies, we have found that biocontrol of grey snow mold caused by Typhula ishikariensis (Tish) or T. incarnata (Tinc) can be obtained by applying T. phacorrhiza (Tp) onto turf. The mode of suppression has not been previously elucidated. We found that parasitism was not involved in the interaction between Tp and either Tish or Tinc. Using the cellophane membrane technique, we found that mutual inhibition was common in all combinations between Tp, Tish and Tinc, but we could not detect the presence of any antibiotics or staling products in liquid cell-free extracts, and in modified dual culture. Studies of population dynamics from all dual combinations of the three Typhula species on turf thatch medium suggested that Tp was more competitive than Tinc, and Tish was the least competitive. Tp had also greater ability in utilizing carbon sources than Tinc and Tish. We conclude that competition for nutrient/space plays a major role in grey snow mold disease suppression by Tp. More studies are needed to determine the substrate/site for which these organisms compete, and other possible mechanisms such as induced resistance. [APS Abstract]

Hsiang, T. and K. Carey, B. He and J.L. Eggens. 1997. Compposition of mixtures of four turfgrass species four years after seeding under non-wear conditions. International Turfgrass Society Research Journal 8:671-679. Twenty-four mixtures of four turfgrass species were seeded in 1 m x 12 m plots in 1988. Plots had low maintenance cultural conditions similar to those used for athletic fields but were not subjected to wear. In 1992, the plots were rated for frequency of occurrence of species by examining sampled plants (-1000 per mixture) under a dissecting microscope. When averaged across all plots, relative frequency of both Perennial Ryegrass and Kentucky Bluegrass was higher in 1992 (stem count) than in the seed mixture (seed weight) in 1988, while fine fescue was present at the same frequency, and tall fescue was at a lower frequency than in the seed mixture. Perennial Ryegrass was the most competitive among the four species and increased in relative frequency in nearly all plots in which it was seeded. For tall fescue, the higher the initial seeding rate, the greater the decrease in frequency four years later. In graphs showing changes in relative frequency (1992 minus 1988) against frequency by weight or number in the seed mixture (1988), there were equilibrium points toward which Kentucky Bluegrass or fine fescue populations tended: frequencies of 35 to 42% for Kentucky Bluegrass and 24 to 25% for fine fescue. PDF Reprint

Hsiang, T. and G.A Chastagner. 1997. Resistance of Kentucky Bluegrass Cultivars to Necrotic Ring Spot. International Turfgrass Society Research Journal 8:893-904. Our analysis indicates that the top Kentucky bluegrass cultivars had significantly lower levels of necrotic ring spot disease compared to the most highly susceptible cultivars. Among these, the top ten were: NE 80-88, Princeton 104 (=P-104), Washington, Alpine, Mystic, Joy, Miranda, Aldephi, Bristol, and Unique. It is possible that use of any of the top 10 or 20 cultivars would effectively minimize the potential for NRS. In areas where NRS is a problem, the selection of cultivars of Kentucky bluegrass should be based on their tolerance to NRS, the local adaptability of the cultivar and its susceptibility to other locally important diseases. This assessment should aid in that selection.  PDF Reprint

Mahuku, G.S., P.H. Goodwin, R. Hall and T. Hsiang. 1997. Variability in the highly virulent type of Leptosphaeria maculans within and between oilseed rape fields. Canadian Journal of Botany 75:1485-1492. Pathogenicity and random amplified polymorphic DNA (RAPD) markers were used to assess genetic diversity among 93 highly virulent isolates of Leptosphaeria maculans (Desm.) Ces. & de Not. collected from two oilseed rape (Brassica napus L.) fields located 20 km apart in southern Ontario. Using three differential host cultivars of B. napus(Westar, Quinta, and Glacier), isolates were separated into three pathogenicity groups (PG). Eighty percent of the isolates were in PG4, virulent on all cultivars, and 11% were in PG3, virulent on two cultivars. The remaining 9% were tentatively placed into a new PG, PG5, because they showed intermediate virulence on the three cultivars. There was no relationship between RAPD pattern and either PG or isolate collection site. Cluster analysis of RAPD patterns based on estimates of genetic distance, grouped isolates into two populations corresponding to the field of collection. Analysis of molecular variance attributed 45.5% of the total variance to differences between populations and 54.5% to differences among isolates. Every isolate was genetically distinct, suggesting that the populations of the fungus were produced mostly by sexual reproduction. We conclude that inoculum in the two fields consisted principally of ascospores, derived almost equally from sources that were common to both fields and sources that were distinct for each field. Preprint

Hsiang, T., L. Yang and W. Barton. 1997. Baseline sensitivity and cross-resistance to demethylation-inhibiting fungicides by Ontario isolates of Sclerotinia homoeocarpa. European Journal of Plant Pathology 103:409-416. Four hundred and thirty-five isolates of Sclerotinia homoeocarpafrom eight populations in southern Ontario were tested for sensitivity to the demethylation-inhibiting (DMI) fungicides, propiconazole, myclobutanil, fenarimol and tebuconazole. The isolates were collected in summer 1994 just prior to legal DMI fungicide use on turfgrass in Ontario. There were wide variations in sensitivities, and seven of the eight populations were very sensitive to the fungicides. Based on mean EC50 and the distribution of DMI sensitivity, one population near the U.S. border was suspected of having been previously exposed to DMI fungicide. Pairwise comparisons of EC50 values for the different fungicides showed low to moderate correlations between fungicides. EC50values of myclobutanil and propiconazole had the best correlation, followed by the pair of tebuconazole and fenarimol. Other pairwise comparisons were not statistically significant except for a barely significant relationship between EC50 values of myclobutanil and tebuconazole. For field populations of plant pathogens, cross-resistance to different DMI fungicides may not be as strong as conventionally thought. The data collected here will allow comparison to subsequent years to look for detectable shifts in S. homoeocarpasensitivity to DMI fungicides as they become more frequently used in Ontario. PDF Reprint

Liu, L.X. and T. Hsiang. 1996. Estimating benzimidazole residues in thatch and turfgrass by bioassay. Pesticide Science 46:139-143. Bioassays using pellets of agar, thatch-agar and turfgrass-agar were developed using benzimidazole-sensitive Penicillium expansum, to detect the fungicide methyl 2-benzimidazole carbamate (MBC) which is the major fungitoxic degradation product of benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate] in thatch and turfgrass clippings. These bioassays were used to estimate the amount of fungicide that was biologically available and hence by subtraction from that applied, the amount that remained bound and biologically unavailable. The limit of quantitation was 0.5 mg kg-1. From 19.9% to 93.2% of the applied fungicide was bound by thatch and 46.2% to 56.9% was bound to turfgrass clippings depending on the concentrations used. In vitro degradation studies showed that MBC had a half life of approximately 2.5 weeks at 23 C in non-sterilized thatch. Preprint

Raffle, V.L. and T. Hsiang. 1995. Intersterility groups of Heterobasidion annosum in Canada. Phytopathology 85:1141. Annosus root rot was first recognized in red pine (Pinus resinosa) in southern Ontario in 1955. The incidence of this disease has increased as most of the first rotation of red pine in southern Ontario is now at or approaching the age of first commercial thinning. The intersterility group (S or P) had thus far been determined for only three isolates of H. annosum from Ontario and they all belonged to the P group (Commun. Inst. Forest. Fenn. 94(6):1-25). Using crossplating with known tester strains and random amplified polymorphic DNA (RAPD) analyses, we have been investigating which intersterility group or groups of H. annosum are present on conifers in southern Ontario and western hemlock (Tsuga heterophylla) in British Columbia. All of 36 isolates collected in southern Ontario from various coniferous hosts belonged to the P group. Five isolates from western hemlock in British Columbia belonged to the S group.

Goodwin, P.H., T. Hsiang, B.G. Xue and H.W. Liu. 1995. Differentiation of Gaeumannomyces graminis from related turf fungi with oligonucleotide primers from ribosomal internal transcribed spacers. Plant Pathology 44:384-391. A region comprising the 5.8S RNA gene and internal transcribed spacers 1 and 2 of the take-all patch fungus, Gaeumannomyces graminis var. avenae, was cloned and sequenced using primers from the flanking 17S and 26S ribosomal RNA genes. The sequenced region had 99% similarity between the two G. graminis isolates, and from 70% to 80% similarity between these two isolates and several other species of fungi. From the sequence, oligonucleotide primers were selected which permitted specific amplification of DNA from G. graminis vars. avenae and graminis using the polymerase chain reaction (PCR). The assay could detect DNA of G. graminis strains obtained from a wide variety of hosts but did not amplify DNA from many other fungi including the important turfgrass root pathogens Magnaporthe poae and Leptosphaeria korrae. The primers also did not amplify DNA from G. graminis var. tritici,M. rhizophila or Phialophora graminicola. The PCR-based assay shows promise as a diagnostic tool for the take-all pathogen in turfgrass pathology. Preprint

Liu, L.X., T. Hsiang and J.L. Eggens. 1995. Core cultivation and efficacy of benomyl applied to creeping bentgrass. Agronomy Journal 87:272-275. Hollow tine core cultivation is practiced for the management of creeping bentgrass (Agrostis palustris Huds.) golf course greens and fairways to increase water infiltration, increase turf root and shoot growth, and control thatch. Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate] is commonly used on turf as a soil drench for the control of disease such as dollar spot (caused by Sclerotinia homoeocarpa Bennett). Our objective was to determine the effect of core cultivation on the movement of a systemic pesticide in thatch and soil and the uptake by turfgrass using benomyl as a model. Core cultivation was conducted 1, 7, and 14 days before benomyl application in a field trial during 1992 and 1993 to study the effect of coring and time of coring on the movement and uptake of benomyl and control of dollar spot disease. Fungicide levels in turfgrass clippings, thatch and soil were determined by bioassay. Core cultivation 1 day before benomyl treatment provided the longest-lasting uptake of benomyl and control of dollar spot disease compared to 7 and 14 days before benomyl treatment. Clipping, thatch, and soil samples from areas close to the coring holes had significantly higher (P = 0.05) levels of fungicide residues than those farther away from the coring holes. This study suggests that core cultivation shortly before benomyl application can increase movement of benomyl into thatch and soil, increase uptake of benomyl by turfgrass, and therefore improve disease control. Preprint

Liu, L.X., T. Hsiang, K.C. Carey and J.L. Eggens. 1995. Microbial populations and suppression of dollar spot disease of creeping bentgrass with inorganic and organic amendments. Plant Disease 79:144-147. Alginate(reg), ammonium nitrate, Bovamura(reg), Milorganite(reg), Ringer Lawn Restore(reg), Ringer Greens Super(reg), Ringer Turf Restore(reg), Sandaid(reg), sewage sludge and sulphur-coated urea were evaluated from 1991 to 1993 on a creeping bentgrass (Agrostis palustris) green and a Kentucky bluegrass (Poa pratensis) lawn for their effects on soil bacterial and fungal populations and dollar spot disease incidence. Over the 3 yr, fertilizers were applied every 4 wk at recommended rates from early June to September, and once again in November. Application of Ringer fertilizers, ammonium nitrate and sulfur-coated urea gave rise to significantly higher microbial populations on turfgrass leaves and in thatch and soil than most other fertilizers. In most experiments, Ringer fertilizers also improved water retention in thatch compared to other treatments. Ringer Greens Super(reg), Ringer Turf Restore(reg), or ammonium nitrate on the creeping bentgrass green significantly suppressed dollar spot disease compared to the other amendments or the untreated control, but for most of the season, they did not control disease as well as the fungicide chlorothalonil. PDF Reprint

Liu, L.X., T. Hsiang & J.L. Eggens. 1995. Effect of a wetting agent on adsorption, movement and uptake of benomyl applied to creeping bentgrass. Journal of Turfgrass Management 1(3):77-89. Laboratory and field experiments showed that the wetting agent Aqua-Gro (AG) (polyoxyethylene ester and ether of cyclic acid and alkylated phenols) significantly reduced the adsorption of the fungicide benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate] by creeping bentgrass (Agrostis palustris Huds.). With AG, significantly (P = 0.05) less fungicide was initially adsorbed and significantly more fungicide was desorbed from the thatch layer after 20 mm of water irrigation. Aqua-Gro increased movement, uptake, and biological availability of the fungicide and resulted in a higher residue level of fungicide in the grass clippings. For dollar spot disease, benomyl applied at 1 kg ha-1 with AG gave as good control as the recommended rate of 1.5 kg ha-1 without AG. Preprint

Hsiang, T., C. Wu, L. Yang and L. Liu. 1995. Pythium root rot associated with cool-season dieback of turfgrass in Ontario and Quebec. Canadian Plant Disease Survey 75:191-195. Pythium species were isolated from diseased samples of creeping bentgrass and annual bluegrass. Among fourteen isolates from decayed roots and crowns, seven were P. graminicola, five were P. torulosum and two were P. ultimum. These species were identified by comparison of cultural and morphological characteristics to literature and reference isolates. In a tissue culture plate assay for quantifying virulence of Pythium spp., significant differences in seedling emergence were found among isolates. In general, isolates produced more severe root rot on perennial ryegrass than on creeping bentgrass, and isolates of P. ultimum were more virulent than those of P. graminicola or P. torulosum. Furthermore, greater virulence was generally found at 15 C than at 30 C. Virulence differences were confirmed in greenhouse pot tests. Oospores and sporangia were recovered from inoculated symptomatic samples. We conclude that Pythium species are a cause of cool-season dieback of golf course turfgrass in Ontario and Quebec. PDF Reprint

O'Gorman, D., B. Xue, T. Hsiang & P.H. Goodwin. 1994. Detection of Leptosphaeria korrae with PCR and primers from the ribosomal internal transcribed spacers. Canadian Journal of Botany 72:342-346. Leptosphaeria korrae, the causal agent of necrotic ring spot, is a destructive patch disease of Kentucky bluegrass. To develop a rapid molecular test for the detection of this pathogen, an assay based on the polymerase chain reaction was developed utilizing the internal transcribed spacer region 1 (ITS 1) of L. korrae ribosomal DNA. DNA sequence comparison showed 94.8% similarity of the ITS 1 region among L. korraeisolates, and only 45-50% similarity between L.korrae and other fungal species. Based on ITS 1 sequence differences, a pair of oligonucleotide primers, LK17S and 5.8SC, were selected. With the polymerase chain reaction, the primers specifically amplified L. korrae DNA and did not amplify DNA isolated from 15 other fungal species or healthy Kentucky bluegrass. The assay could also specifically detect L. korrae in diseased turfgrass samples. PDF Reprint

Liu, L.X. & T. Hsiang. 1994. Bioassays for benomyl adsorption and persistence in soil. Soil Biology and Biochemistry 26:317-324. Two bioassay methods were modified to evaluate persistence, adsorption and biological availability of the fungicide benomyl in soil. The amount of biologically-available fungicide was assayed using the fungus Penicillium expansum Link ex Gray. The bioassay results were not significantly different from those obtained by spectrophotometry. The bioassays detected methyl 2-benzimidazole carbamate (MBC), which is the major toxic degradation compound of benomyl, to a limit of detection of 0.2 ug g-1 and a limit of quantitation of 0.5 ug g-1 in soil and leachate. The bioassays could be completed within two days. The amount of MBC adsorbed by soil varied, depending on soil type and fungicide concentration. In vitro soil degradation studies showed a half life for MBC of around four weeks. Preprint

Hsiang, T. and G.A Chastagner. 1993. Variation in Melampsora occidentalis rust on poplars in the Pacific Northwest. Canadian Journal of Plant Pathology 15:175-181. We examined the variation in virulence of Melampsora occidentalis on poplar clones. Thirty isolates of M. occidentalis from coastal or interior areas of the Pacific Northwest were tested in vitro on leaf disks from five poplar clones (four Populus trichocarpa, and one P. trichocarpa x P. deltoides). Significant differences were found among clones and among isolates with respect to uredial number, disease severity and disease incidence. Most importantly, isolates from the interior east side of the Cascade Mountains show a significant clone x isolate interaction for uredial number, indicating physiological specialization of the pathogen on different poplar host genotypes. For the 20 coastal west-side isolates, the clone x isolate interaction was not significant. For the 10 interior M. occidentalis isolates, which showed greater overall virulence on the five poplar clones tested than did coastal isolates, the occurrence of a significant clone x isolate interaction could indicate that the pathogen has adapted to the less rust-favourable environment of the interior through greater physiological specialization. Although these data indicate the potential for physiological specialization in M.occidentalispopulations, the selection pressure required to cause a change in the virulence structure of this essentially wild and discontinuous pathosystem in the coastal Pacific Northwest would have to be intense, and possibly would result only from widespread domestication of the host species. PDF Reprint

Hsiang, T., G.A Chastagner, J.M. Dunlap and R.F. Stettler. 1993. Genetic variation and productivity of Populus trichocarpa T&G and its hybrids. VI. Field susceptibility of seedlings to Melampsora occidentalis jacks. leaf rust. Canadian Journal of Forest Research 23:436-441. A three-year study on the incidence and severity of Melampsora rust on hybrid poplars began in 1987 when inter- and intra-specific crosses were made with 20 P. trichocarpa (T), eight P. maximowiczii (M), and three P. trichocarpa x P. deltoides(TD) parents.  A total of 5100 seedlings from 67 families were initially surveyed for rust in 1988; however, this number declined to less than 3700 by 1990, and rust infection likely contributed to seedling mortality.  There was significantly greater rust incidence and severity in TxT crosses compared to TxM, MxTD, TDxM, or MxM crosses.  For the 12 MxM crosses, and the six three-way crosses (MxTD, TDxM) less than 1% of the seedlings had any rust in the three years. In the 20 TxM crosses, over 65% of the seedlings showed no rust and less than 1% showed heavy rust. For TxT seedlings from 29 crosses, less than 3% had no rust. Reciprocal crosses of P. trichocarpa from east or west of the Cascade Mountains in Washington showed that the female parent contributes more to rust susceptibility than the male parent.  In regression analyses, P. trichocarpa midparent rust ratings were negatively correlated with family survival, and positively correlated with the mean progeny rust rating.  Narrow-sense family heritabilities were moderate to high for rust susceptibility (h2: 0.43 in 1988, 0.53 in 1989, and 0.79 in 1990). Preprint

Hsiang, T. and G.A Chastagner. 1992. Production and viability of sclerotia of fungicide-resistant and sensitive isolates of three Botrytis species. Plant Pathology 41:600-605.  Production of sclerotia by isolates of three Botrytis spp. differing in resistance to benzimidazole and dicarboximide fungicides were compared in vitro.  Sensitive isolates of B. cinerea and B. tulipae produced fewer sclerotia than benzimidazole-resistant isolates, but there were no differences in the size of sclerotia within each species.  For B. elliptica, sclerotia of dicarboximide-resistant isolates were larger and less numerous than those of sensitive isolates.  Sclerotia from fungicide- resistant and sensitive isolates of B. elliptica, B. tulipae, and B. cinerea were produced in vitro, placed into nylon bags, set in loam soil at 0, 10 and 20 cm soil depths, and recovered periodically after 1 to 18 months.  Sclerotial viability declined during the 18 months and was lowest at the soil surface for sclerotia of B. cinerea and B. tulipae.  For B. elliptica, sclerotial viability of fungicide-sensitive isolates was reduced to 50% after 18 months, compared to 21% for dicarboximide-resistant isolates, when averaged over all depths.  Sensitive isolates of B. tulipae maintained a trend of higher viability than benzimidazole-resistant ones, and fungicide-sensitive isolates showed greater viability at 18 months (49%) than did benzimidazole-resistant ones (27%).  No differences in sclerotial viability were apparent between the fungicide-resistant and sensitive isolates of B. cinerea with an average viability of 77% after 18 months.  After 18 months of field exposure, all isolates retained their original fungicide-resistance groupings, indicating the persistence of fungicide resistance in sclerotia. Preprint

Hsiang, T. and G.A Chastagner. 1991. Growth and virulence of fungicide-resistant isolates of three Botrytis species. Canadian Journal of Plant Pathology 13:226-231. Fungicide-sensitive and fungicide-resistant isolates of Botrytis cinerea, B. elliptica and B. tulipae were compared in the absence of fungicides with respect to lesion development and sporulation on host tissue and growth rate, spore size, and sporulation on potato dextrose agar (PDA).  Multiple resistance to benzimidazole (tested as benomyl) and dicarboximide (tested as iprodione) in B. cinerea was associated with reduced sporulation on tulip leaf disks and on PDA, indicating that multiple fungicide-resistance in this species may be associated with reduced parasitic fitness.  Compared to fungicide-sensitive isolates, benzimidazole-resistant isolates of B. tulipae produced larger spores on PDA but smaller leaf lesions and fewer spores on tulip leaves and PDA, again indicating that fungicide-resistance was associated with reduced fitness.  In B. elliptica, spores of dicarboximide-resistant isolates were longer and almost twice as wide as those of fungicide- sensitive isolates.  However, most measures indicated resistance to fungicide was associated with reduced fitness in B. elliptica:  fungicide-resistant isolates grew more slowly on PDA and produced smaller lesions on lily leaves than sensitive isolates; and dicarboximide-resistant isolates and multiple-resistant isolates produced fewer spores on PDA and were less successful in establishing and developing lesions on leaves than sensitive isolates. PDF Reprint

Hsiang T and Edmonds RL. 1989. Physiological specialization of Heterobasidion annosum on its conifer hosts. Canadian Journal of Botany 67:2396-2400. Eight conifer hosts belongin*g to five conifer species were inoculated in vitro with conidial suspensions of eight isolates of Heterobasidion annosum from Washington and Califomia. The conifer trees were represented in inoculation tests by 1 cm diameter branch disks of 0.7 cm thickness. The ability of H. annosum to colonize dying woody tissue was assessed in terms of the number of conidiophores produced on the disks, measured 2 to 3 weeks after inoculation. Analysis of variance showed that there was a great range in conidiophore production with most of this variation attributable to host differences rather than to differences between the pathogen isolates. A second analysis involving four Tsuga heterophylla trees and five isolates showed similar results. In both the interspecific analysis with eight trees and the intraspecific analysis with four T. heterophylla trees, there were significant differential interactions between the isolates and the trees. This indicated that physiological specialization exists in this natural disease system at both the host-interspecific and host-intraspecific levels. PDF Reprint

Hsiang T and Edmonds RL and Driver CH. 1989. Conidia of Heterobasidion annosum from Tsuga heterophylla in Western Washington. Canadian Journal of Botany 67:1262-1266. Heterobasidion annosum produces conidia abundantly in culture; however, since conidiophores are rare in nature, conidia are usually considered to have little or no role in dispersal. Heterokaryotic mycelia of H. annosum produce both heterokaryotic and homokaryotic conidia, whereas basidiospores are homokaryotic. This difference was exploited to assess the relative prevalence of these spore types in western hemlock forests of western Washington state. Two out of 10 spores trapped on selective media were found to give rise to heterokaryotic mycelia identified by the presence of clamp connections. However, homokaryotic conidia could not be distinguished from basidiospores by this method, so two approaches were taken in the laboratory: examining conidia for number of nuclei and determining frequency of clamp connections in conidial cultures. Both methods indicated that from a single heterokaryotic mycelium, half of the conidial progeny were homokaryotic and the other half heterokaryotic. Thus the presence of two heterokaryotic conidia in 10 spores implied that conidia may make up a third to a half of the aerial spore load of H. annosum in western hemlock forests of western Washington state. PDF Reprint

Edmonds, R.L., D.C. Shaw T Hsiang and C.H. Driver. 1989. Impact of precommercial thinning on the development of Heterobasidion annosum in western hemlock. pp. 85-94 in: W.J. Ostrosina and R.F. Scharpf (eds). Symposium on Research and Management of Annosus Root Disease (Heterobasidion annosum) in Western North America. USDA For. Serv. Gen. Tech. Rep. PSW-116. The impact of precommercial thinning of western hemlock (Tsuga heterophylla) on the development of Annosus root and butt rot (caused by Heterobasidion annosum) in coastal Washington has been followed for more than 20 years. Infection of stumps and wounds was high following thinning and there was a high probability of residual tree infection. Eleven years after precommercial thinning tree infection was high, but after 20 years levels of infection were low in both thinned and unthinned stands (averaging <5 percent). Volume losses were even lower (<1 percent). Host tree defense mechanisms, including wetwood, appear to be effective in minimizing losses due to H. annosum. Precommercial thinning did not appreciably increase the incidence of H. annosum in the current rotation but problems could occur in future rotations. Borax treatment of precommercially thinned stumps was not effective in reducing the incidence of H. annosum. PDF

Edmonds RL and Hsiang T. 1987. Forest floor and soil influence on response of Douglas-fir to urea. Soil Science Society of America Journal 51:1332-1337. Data from the Regional Forest Nutrition Research Project (RFNRP) in Washington and Oregon were analyzed to improve stand-specific prediction of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] response to urea fertilization. The response variable (relative difference in volume growth between fertilized and control plots 4 yr after fertilization with 448 kg N/ha) was regressed against 28 stand and site variables (e.g., age, elevation, forest floor C/N ratio, soil cation exchange capacity, etc.) using stepwise multiple regression analysis. Data from 120 installations were stratified by thinning level (thinned or unthinned), geographic location (provinces), and site quality (site index and class). Forest floor C/N ratio was the dominant variable related to response. In thinned installations of high site quality (site classes 1 and 2), 60% of variation in response was explained by the forest floor C/N, and 75% of the variation in response was explained with inclusion of surface soil exchangeable K. In thinned, low site quality stands, response was not as well related to forest floor C/N. Analysis of the data by province indicated that S may be limiting in southwest Oregon and P in coastal Washington.

Hsiang, T. and B.J. van der Kamp. 1985. Variation in rust virulence and host resistance of Melampsora on black cottonwood. Can. J. Plant Pathol. 7: 247-252. Disease severity, as expressed by spore production rate, was compared in a test of 14 clones of black cottonwood (Populus trichocarpa) inoculated with 10 isolates of melampsora rust (Melampsora occidentalis), both the clones and the isolates were collected from their natural pathosystem. Spore production rate was measured by average daily production on leaf disks over a period 2 * the latent period. The overall average spore production during the time from inoculation through a period equal to 2 * the latent period was 747 spores/disk/day. The latent period ranged from 6 to 12 days with a median of 8 days. Clones as well as isolates differed significantly in their contributions to spore production, but there was no differential interaction between clones and isolates. Analysis of variance of total spore production, total pustules, and latent period gave the same results. The number of urediniospores per uredinium varied significantly between clones but not between isolates. The absence of qualitative resistance and virulence indicates that qualitative interactions do not play a major role in disease in this natural pathosystem. This suggests that the resistance of cultivated black cottonwood to melampsora rust is unlikely to be readily overcome in genetically uniform plantations. PDF Reprint


Areas of Research
Bioinformatics

13a Core bacterial gene set

12f Comparative genomics of Magnaporthe oryzae

12b Multiple IGS haplotypes in Puccinia striiformis

11g Denovo genome assembly

09d Comparative genomics & loop mediated isothermal amplification

06c Issues in Comparative Fungal Genomics

05b Comparative genomics and core fungal genes

04b Comparison of Colletotrichum-infected plant EST libraries

04a Review on fungal genomics

03c Distinguishing plant and fungal sequences in ESTs from infected plant tissues

Fungal population biology

09e Whole genome PCR amplification of multicopy and single copy fungal genes

08b Horizontal Gene Transfer between fungal rice pathogens

02d Population structure and mating type genes of Colletotrichum graminicola

02c Genetic relationships of endophytic Lophodermium nitens isolates from white pine

01g Clonal and sexual propagation in Botrytis elliptica

00c Occurrence of Kabatina juniperi in Ontario and genetic analysis

99a Genetic variation within and between southern Ontario populations of Sclerotinia homoeocarpa

98a Genetic variation within Microdochium nivale isolates from turfgrass

97b Variability in the highly virulent type of Leptosphaeria maculans

93b Variation in Melampsora occidentalis rust on poplars in the Pacific Northwest.

85a Host-parasite interaction between Melampsora occidentalis and Populus trichocarpa

Molecular characterization & systematics

11e Epoxide hydrolase II and Colletotrichum spp.

10a Induced systemic resistance by a mineral oil against tobacco anthracnose

08e Involvement of epoxide hydrolases of Nicotiana benthamiana in fungal or bacterial interactions

07d Metacaspase of Nicotiana benthamiana in a compatible fungal or incompatible bacterial interaction

05d Cysteine proteinases of Nicotiana benthamiana in a compatible fungal or incompatible bacterial interaction

05c GST induction following infection of Nicotiana benthamiana by Colletotrichum spp.

03e Role of ethylene in Colletotrichum gloeosporioides infection of Nicotiana benthamiana

03d Colletotrichum gloeosporioides infection induces differential expression of glutathione S-transferase genes in Malva pusilla

03b Mating type genes of Ophiosphaerella korrae

03a Green fluorescent protein to quantify Colletotrichum growth in tobacco

02e RT-PCR and northern blot analyses to measure expression of b-1,3-glucanase in Nicotiana benthamiana infected with Colletotrichum destructivum

02b Molecular characterization of Tuberose mild mosaic virus

01e DNA sequencing for anastomosis grouping of Rhizoctonia solani isolates from Poa annua

01a A novel actin-related protein gene of Colletotrichum gloeosporioides

00f The use of RAPD markers to distinguish juniper and cedar cultivars

00b A comparison of stilbene and chalcone synthases

00a Genetic relationships of pathogenic Typhula species assessed by RAPD, ITS-RFLP and sequencing

95e Differentiation of Gaeumannomyces graminis from related fungi by PCR

94b Detection of Leptosphaeria korrae with PCR and primers

Woody plant diseases

12d Volutella blight of boxwood

11b Red pine decline

11b Red pine decline

10c Armillaria species in North America

09i Tar spot of maple, control

08g Tar spot of maple, cause

08a Tar spot of maple, review

02c Genetic relationships of endophytic Lophodermium nitens isolates from white pine

00f The use of RAPD markers to distinguish juniper and cedar cultivars

00c Occurrence of Kabatina juniperi in Ontario and genetic analysis

99d Juniper rust species in an Ontario nursery

95f P group of Heterobasidion annosum in Ontario

93b Variation in Melampsora occidentalis rust on poplars in the Pacific Northwest.

93a Genetic variation and productivity of Populus

89c Heterobasidion annosum physiological specialization

89b Heterobasidion annosum conidia

89a Annosus root rot and precommercial thinning

87a Douglas-fir and urea

85a Host-parasite interaction between Melampsora occidentalis and Populus trichocarpa

Turfgrass diseases
  dollarspot

11i Dollar spot on cool season grasses in China

10f Dollar spot on seashore paspalum

10b Induced systemic resistance by a mineral oil against turf diseases

09h Resurgence of dollar spot disease after strobilurin application

07c DMI fungicide resistance of dollar spot 10 years after first use

01c First report of dollar spot, caused by Sclerotinia homoeocarpa in Saskatchewan

00e Analyses of RAPD data for detection of host specialization in Sclerotinia homoeocarpa

99a Genetic variation within and between southern Ontario populations of Sclerotinia homoeocarpa

98b Relative virulence of isolates of Sclerotinia homoeocarpa with varying fungicide sensitivity

97a Baseline sensitivity and cross-resistance to demethylation-inhibiting fungicides in dollarspot

95c Microbial populations and suppression of dollar spot disease

  anthracnose

06g Anthracnose: terrorizing turfgrass

03f The infection process of Colletotrichum graminicola on turfgrasses

02d Population structure and mating type genes of Colletotrichum graminicola

01i Ribosomal DNA sequence comparisons of Colletotrichum graminicola from turfgrasses

99c Anthracnose basal rot of creeping bentgrass

  necrotic ring spot

06f Neurotic about necrotic ring spot

03b Mating type genes of Ophiosphaerella korrae

98c Low level of DNA polymorphisms in Leptsophaeria korrae

97c Resistance of Kentucky bluegrass to Necrotic Ring Spot

94b Detection of Leptosphaeria korrae with PCR and primers

  other turf diseases

12c Nigrospora leaf spot of Kentucky bluegrass

10g Waitea circinata on turf in British Columbia

07b Bipolaris leaf spot on Cynodon

07a Rhizoctonia zeae (Waitea circinata) on turf in Ontario

06e Ophiosphaerella agrostis in Ontario

06d Rhizoctonia zeae in Ontario

05e Sugar, compost tea, Brassica glucosinolates and Turf Diseases

05d Turf Diseases in Central China

05a Curvularia verruculosa on Cynodon in China

04c Curvularia affinis on Festuca arundinacea in China

01e DNA sequencing for anastomosis grouping of Rhizoctonia solani isolates from Poa annua

98g Dark runner hyphae on annual bluegrass roots

98a Genetic variation within Microdochium nivale isolates from turfgrass

95e Differentiation of Gaeumannomyces graminis from related fungi by PCR

95a Pythium root rot associated with cool-season dieback of turfgrass

Snow molds

13b Microdochium species differences

11a Cold induced responses to pink snow mold by annual bluegrass

09j Fusarium Patch vs. Pink Snow Mold

09g Annual bluegrass and winter stresses

09a Annual bluegrass resistance to pink snow mold

06a Clavula production of Typhula species

01n Managing snow mold diseases of winter cereals and turf

01m Low temperature diseases caused by Microdochium nivale

01d Effect of Typhula phacorrhiza on winter injury in field trials across Canada.

00g Biological control of turfgrass snow molds

00d Role of ice nucleation and antifreeze activities in snow mold pathogenesis

00a Genetic relationships of pathogenic Typhula species assessed by RAPD, ITS-RFLP and sequencing

99f Residual efficacy of Typhula phacorrhiza as a biocontrol of grey snow mould

99e Biology And management of Typhula snow molds of turfgrass

99b Mycelial growth, sclerotial production and carbon utilization of three Typhula species.

98e Pathogenicity and formulation of Typhula phacorrhiza, a biocontrol agent of gray snow

98d Efficacy of Typhula phacorrhiza as a biocontrol agent of grey snow mould

97e Biocontrol mechanisms of Typhula phacorrhiza against snow moulds

Plant diseases

12f Fusarium oxysporum on Coleus

12a Streptomycetes against Botrytis

11j Stem blight of Eleocharis dulcis

11h Induced resistance in plants: review

11f Streptomyces against Magnaporthe oryzae

11d Peach gummosis

11c leaf spot of Houttuynia cordata

10j Streptomyces volatile against citrus Penicillium

10i Phoma on figwort

10h Properties of Stemphylium phytotoxin

10e Target site of Stemphylium phytotoxin

10d Integrated control of Stemphylium blight of garlic

09k Clonostachys biocontrol of Fusarium head blight

09f Stemphylium solani on garlic

09c Alternaria on Smilax in China

09b Rhizoctonia head rot of cabbage

08f Effects of Vesicular arbuscular mycorrhizae on onion white rot

08c Use of Scion Image to estimate disease severity

07e Stemphylium on onion in China

04d Biology and control of daylily rust in Canada

02a Induction of systemic disease resistance and pathogen defence responses in asparagus

01l Hemibiotrophic infection and identity of the fungus, Colletotrichum destructivum

01k Infection of Nicotiana species by the anthracnose fungus, Colletotrichum orbiculare

01f Activation of defense responses to Fusarium infection in Asparagus

01b Stem canker of cabbage seed stalks caused by Botrytis cinerea in Western Washingston

00c Occurrence of Kabatina juniperi in Ontario and genetic analysis

99d Juniper rust species in an Ontario nursery

97b Variability in the highly virulent type of Leptosphaeria maculans

Fungicide biology and resistance

07c DMI fungicide resistance of dollar spot 10 years after first use

01j Relative sensitivity to benomyl and iprodione by Botrytis elliptica

98b Relative virulence of isolates of Sclerotinia homoeocarpa with varying fungicide sensitivity

97b Variability in the highly virulent type of Leptosphaeria maculans

97a Baseline sensitivity and cross-resistance to demethylation-inhibiting fungicides

96a Estimating benzimidazole residues in thatch and turfgrass by bioassay

95d Core cultivation and efficacy of benomyl applied to creeping bentgrass.

95b Effect of a wetting agent on adsorption, movement and uptake of benomyl

94a Bioassay for benomyl adsorption and persistence in soil.

92a Production and viability of sclerotia of fungicide-resistant and sensitive Botrytis species

91a Growth and virulence of fungicide-resistant isolates of three Botrytis species.

Botrytis elliptica

01j Relative sensitivity to benomyl and iprodione by Botrytis elliptica

01h Light and scanning electron microscopy studies on the infection of lily by Botrytis elliptica

01g Clonal and sexual propagation in Botrytis elliptica

01j Relative sensitivity to benomyl and iprodione by Botrytis elliptica

98f Botrytis elliptica infection of lily

92a Production and viability of sclerotia of fungicide-resistant and sensitive Botrytis species

91a Growth and virulence of fungicide-resistant isolates of three Botrytis species.


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