Oxalic Acid Metabolism Contributes to Full Virulence and Pycnidial Development in the Poplar Canker Fungus Cytospora chrysosperma

Poplar Cytospora canker, which is mainly caused by Cytospora chrysosperma, is one of the most destructive and widespread tree diseases worldwide. Although oxalic acid (OA) is demonstrated as an important virulence determinant in several necrotrophic fungi, specific functions of OA during pathogenesis remain controversial. Here, we identified three genes (CcOah, CcOdc1, and CcOdc2) directly involved in OA biosynthesis and catabolism in C. chrysosperma. We demonstrated that CcOah is required for OA biogenesis. All three genes were found to be highly upregulated during early infection stages of the poplar stem. The deletion of any of the three genes led to an obvious reduction of pycnidial production but no abnormality of hyphal growth and morphology. Furthermore, the individual deletion strain exhibited significantly limited lesion sizes on poplar twigs and leaves. Exogenous application of OA or citric acid can complement the virulence defects of ΔCcOah and ΔCcOdc1 strains. We further found that the ΔCcOah strain strongly promoted reactive oxygen species burst of poplar leaves during infection. Finally, induced secretion of OA was observed by monitoring color change of the plates after poplar stem extracts were added in the cultures; however, we failed to quantify OA concentration by high-performance liquid chromatography. Taken together, the present results provide insights into the function of OA acting as an important virulence factor of C. chrysosperma.

Interactions of sapsuckers and Cytospora canker can facilitate decline of riparian willows

Drought has caused large-scale plant mortality in ecosystems around the globe. Most diebacks have affected upland forest species. In the past two decades, a large-scale decline of riparian willows (Salix L.) has occurred in Rocky Mountain National Park, Colorado. We examined whether climatic or biotic factors drive and maintain the willow community decline. We compared annual growth and dieback of willows inside and outside of 14-year-old ungulate exclosures and measured groundwater depth and predawn xylem pressures of stems as indicators of drought stress. We also performed an aerial photo analysis to determine the temporal dynamics of the decline. Aerial photo analysis indicated willow decline occurred between 2001 and 2005 and was best explained by an increase in moose population and a decrease in peak stream flows. A new mechanism for willow stem dieback was identified, initiated by red-naped sapsucker wounding willow bark. Wounds became infected with fungus that girdled the stem. DNA analyses confirmed Valsa sordida (Cytospora chrysosperma) as the lethal fungus. Captured sapsuckers had V. sordida spores on feet and beaks identifying them as one possible vector of spread. Predawn xylem pressure potentials remained high through the growing season on all study willows regardless of depth to ground water. Our results indicate that additional mechanisms may be involved in tall willow decline.

Summer Heat and Low Soil Organic Matter Influence Severity of Hazelnut Cytospora Canker

Cytospora canker, caused by the fungus Cytospora corylicola, is present in hazelnut production areas worldwide. The disease is widespread throughout the main production areas of Italy. The causal agent is considered to be a secondary invader of damaged tissue that attacks mainly stressed plants. However, little is known of disease severity and stress factors that predispose plants to infection. In particular, the role of pedoclimatic factors was investigated. Direct survey indicated that disease severity varied across several study sites. Geostatistics showed a strong positive correlation between disease severity index and summer heat (r = 0.80 and 0.91 for July and August, respectively) and strong negative correlation between disease severity index and soil organic matter (r = –0.78). A moderate positive correlation between disease severity index and magnesium/potassium ratio (r = 0.58) and moderate negative correlations between disease severity index and total soil nitrogen (r = –0.53), thermal shock (r = –0.46), and rainfall (r = –0.53) were determined. No significant correlation between disease severity index and soil aluminum (r = –0.35), soil pH (r = –0.01), and plant age (r = –0.38) was found.

First Report of Branch Canker Caused by Cytospora atrocirrhata on Populus sp. and Salix sp. in China

Cytospora Ehrenb. species and their related teleomorphs are common inhabitants on over 85 species of plants throughout the world, and some of these pathogens have been associated with stem canker and dieback diseases. In July to August of 2011, samples of Cytospora canker were collected from Populus and Salix trees in Aershan City of Xingan League (46.51° N, 120.21° E) and Genhe (50.54° N, 120.30° E) (Inner Mongolia, China), the northeast part of the Chinese mainland, where the forests were frequently stressed by drought and cold springs and seriously suffered from Cytospora canker outbreaks, causing over 150,000 infected trees to die in 1999 (4). Symptoms observed included discoloration of the inner bark, cambium, and sapwood and sunken lesions at the site of active canker growth. The discrete erumpent ostiolar beaks of condimata were visible on the bark. The red spiral tendrils exuded from fruiting bodies when the relative humidity rose above 80%. All isolates were deposited into the China Forestry Culture Collection Center, strain numbers CXY1401, CXY1402, and CXY1403. The colony of single spore isolates on PDA medium was white and conidiomata were produced on autoclaved leaves and segments of Populus tomentosa Carr. and Salix babylonica Linn. The cultural characteristics of the isolates were conidiomatal stromata immersed in bark, discrete, erumpent, leucotorsellioid, and 0.5 to 1.1 × 0.4 to 0.9 mm. Discs were light grey, nearly flat, circular to ovoid, and 0.4 to 0.5 mm diameter, with one central dark grey ostiole. Locules were multi-chambered, subdivided by invaginations into chambers with seperate walls. Conidia were hyaline, eguttulate, elongate-allantoid, aseptate, and 5.5 to 7.0 × 0.8 to 1.2 μm. The ribosomal ITS1-5.8S-ITS2 region was amplified with primers ITS1 and ITS4 from gDNA. BLAST alignments of the consensus sequences of the ITS1 and ITS2 amplicons (JX534242, JX534243, JX534244) revealed 99% identical to the analogous ‘Cytospora atrocirrhata Gvrit.’ sequences reported from Populus spp. and Salix spp. in Iran (EF447305 and EF447306) (2). Pathogenicity tests were carried out using mycelium discs of isolates placed on disinfected 2-year-old P. tomentosa twigs, while the control were inoculated with sterile potato dextrose agar (PDA) discs. Cuttings were incubated at 25°C for 30 days. For 16 of the 20 cuttings, symptoms of brown spot and inner bark discoloration were similar to those observed in the field. Controls did not develop any symptoms, and Koch's postulates were fulfilled with the reisolation of the pathogen from symptomatic tissues. C. atrocirrhata was first reported in the former Soviet Union in 1973 (3) and more recently in Iran (1). To our knowledge, this is the first report of branch canker caused by C. atrocirrhata on Populus sp., and Salix sp. in China. The result provides new information on the geographic distribution of C. atrocirrhata. The appearance of C. atrocirrhata in China seriously threatens the Populus and Salix species, which are widely cultivated for wood production in flat areas. Control measures are needed to prevent further spread of the fungus to new areas. References: (1) K. B. Fotouhifar et al. Rostaniha. 8:129, 2007. (2) K. B. Fotouhifar et al. Mycol. 102:1369, 2010. (3) M. N. Gvritishvili. Miko. Fitopatol. 7:544, 1973. (4) C. L. Wu. China Forest Pest and Disease (in Chinese). 2:36, 1999.

Canker on Bark of Populus spp. Caused by Cytospora tritici, a New Disease in China

Species of Cytospora Ehrenb. and associated teleomorphs cause dieback and canker on over 85 species of angiosperm and gymnosperm plants throughout the world (2). Cytospora tritici Punith. was first observed on Triticum asetivum in Germany in 1980 but may also affect many hardwoods (3). During a survey of landscape trees in 2007, Populus spp. with cankers were found in Fushun, Baoxing, and Luding counties and Chengdu city in Sichuan Province. In these trees, bark canker pathogens discolored the sapwood. During damp weather, conidia were pushed out and formed orange spore horns. Conidiomatal stromata were immersed in bark, prominent, and 1.53 ± 0.33 mm in diameter (n = 10). Discs were white to grey, circular, oval, and 0.59 ± 0.14 mm in diameter (n = 10), with one ostiole per disc. Ostioles were dark grey. Locules were multi-chambered, chambers irregular. Conidia were lelongate-allantoid shaped, hyaline, aseptate, 5.04 ± 0.65 μm long (n = 50), and 1.22 ± 0.13 μm wide (n = 50). Fragments (5 × 5 mm2) of the junction of diseased and healthy tissues were surface sterilized with 1% NaOCl for 30 s and then rinsed twice in sterile distilled water. The pieces were placed on potato dextrose agar (PDA) plates and incubated at 25°C for 7 days. The obtained isolates were cultured on PDA at 25°C in diffuse fluorescent light for 30 days. Upon isolation, the mycelium grew at a rate of 3 to 5 mm per day at 25°C, forming pale white-to-pure white flat colonies. Conidiomata never formed on PDA. ITS1-5.8S-ITS2 sequences were amplified via PCR from genomic DNA obtained from mycelia using universal primers ITS1 and ITS4 (4). The amplification products showed 100% sequence homology with C. tritici isolate DQ243812 from the GenBank database. The ITS sequences were submitted to GenBank (Accession No. JQ277333 to JQ277336). Pathogenicity was confirmed by inoculating 20 disinfected (70% ethanol) Populus tomentosa cuttings. Cuttings were incubated at 25°C for 30 days. Another two cuttings were treated with water agar as controls. In 18 of the 20 cuttings, the cambium developed a brown color and appeared water soaked 15 days later, whereas controls did not develop any symptoms. C. tritici was reisolated from symptomatic tissues. To our knowledge, this is the first report of C. tritici in China causing canker on Populus spp. Cytospora canker is common in practically all countries where poplar are grown. Canker expansion increases when tree defenses are compromised, usually by seasonal dormancy but also by drought, cold injury of wood, sun scald of bark, flooding of root, hail, freezing, or other stress (1). Future spread of C. tritici to western China is considered highly likely. References: (1) G. C. Adams et al. Stud. Mycol. 52:1, 2005. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ March 25, 2012. (3) E. Punithalingam. Nova Hedwigia 32:585, 1980. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Control of Apple Scab and Cytospora Canker with Paclobutrazol

Forty Malus ‘Radiant’ crabapples, a variety known to be highly susceptible to apple scab disease, were field-planted then treated three months later with paclobutrazol (PBZ) as a basal drench, at rates of 0, 0.8, 1.6, and 2.4 g a.i./cm caliper. Apple scab was significantly reduced for two years post treatment by all rates of PBZ applied. However, significant growth regulation occurred through the third and final year of the study. Thirty Picea pungens (Colorado spruce) trees in containers were treated with PBZ, applied as a basal drench, at rates of 0, 1.6, and 3.2 g a.i./cm caliper. Cytospora canker development from subsequent branch inoculations was significantly reduced by both PBZ treatment rates and persisted through the end of the two year monitoring period. Cytospora canker disease control with only moderate growth regulation indicates that a PBZ basal drench could be developed into a viable landscape treatment.