Ring Rot Research Articles

Minireview/MinisynthèseProspect for functional eradication of the bacterial ring rot disease of potato

The feasibility of eradicating the bacterial ring rot disease of potato, caused by Clavibacter michiganensis subsp. sepedonicus, must be evaluated in terms of current knowledge pertaining to pathogen ecology and disease epidemiology, as well as in consideration of developments in laboratory methods for pathogen detection, seed certification regulations and plant health legislation. For our purposes here, eradication should be understood in a functional sense as describing the apparent elimination of the disease without really knowing whether or not the causal bacterium still lurks somewhere in the crop production system. The potential for such eradication depends on the causal bacterium's capacity (or incapacity) to persist in the environment and the means by which vertical and horizontal transmission among potato production units can be controlled. Strict application of certification regulations and early detection of infected potato lots are essential not only to maintaining control of the ring rot disease but also underlie progress toward its functional eradication. For an eradication programme to be successful it must include three fundamental elements, namely, early detection, sanitation and prevention. Ultimately, however, the feasibility of achieving success will depend on economic factors and the commitment of the entire potato industry.

Enhancing bioefficacy of Bacillus subtilis with sodium bicarbonate for the control of ring rot in pear during storage

Pear ring rot caused by Botryosphaeria berengeriana is a common disease throughout pear-growing areas. The use of biocontrol agents to control the disease can potentially reduce the negative effects of chemical fungicides on fruit and on the environment. Here, we evaluated the preventative (before pathogen inoculation) and curative (after pathogen inoculation) effects of biocontrol agents (BA) with sodium bicarbonate (SBC) against B. berengeriana alone or in combination on plates and on fruit. The order of treatments applied in the combinations, and two different doses were also evaluated. The inhibition rates were 42.30%, 75.77–85.41%, and 83.00–90.23% in vitro with 2% SBC (w/v), BA (strains 11, 28, 2 and sf 628), and combination treatments, respectively. On fruit, the curative inhibition rates were 23.83%, 12.79%, 71.09%, 48.16%, 74.57%, and 57.90% when treated with BA 11 (30 mL), SBC (30 mL), BA 11 (15 mL) + SBC (15 mL), SBC (15 mL) + BA 11 (15 mL), BA 11 (30 mL) + SBC (30 mL), and SBC (30 mL) + BA 11 (30 mL), respectively; the preventative inhibition rates were 28.72%, 17.31%, 78.25%, 51.06%, 80.91%, and 61.50%, respectively. Combinations were superior to individual treatments and significantly better with SBC following BA than vice versa. Therefore, the proper combination of SBC with BA can be developed as a promising approach against pear ring rot during storage.

Integrated pest management of potatoes

The potato (Solanum tuberosum L.) is the most important dicotyledonous source of human food. It ranks fifth major food crop of the world, exceed only by the grasses such as wheat, rice, maize, and barley. It is characteristically a crop of the cool, temperate regions or of elevation of approximately 2,000 m or more in the tropics. It requires cool nights and well drained soil with adequate moisture and does not produce well in low altitude, warm, tropical environment. Commercial production of most potatoes is primarily through vegetative propagation by means of lateral buds formed on the tuber, a modified stem. Trough such vegetative propagation, many diseases are transmitted from generation to generation. Suppression of such diseases and reduction of yield losses due to disease are a necessary part of increasing the food supply. The principles, strategies, and tactics of plant disease management are important to preventing yield losses. Integrated pest management (IPM) may supply effective control of the potato pests including aphids (vector of some viruses), Verticillium wilt blackleg, bacterial ring rot, Rhizoctonia, Phytophthora infestans (late blight) and several weeds (night shades, pigweeds, lambs quarters, and annual grasses). It includes regular inspection for healthy seed or nursery, crop production, correct identification of the problem, cultural practices (crop rotation, sanitation etc.), biological control, soil fumigation (if necessary), seed or nursery stock treatment and disinfestations of cutting tools. In this review, pest management methods of potatoes included in IPM was summarized.

First Report of Gummosis Disease of Japanese Apricot Caused by Botryosphaeria dothidea in Taiwan.

Japanese apricot (Prunus mume Sieb. et Zucc.) is an economically important fruit crop grown on more than 10,000 ha in Taiwan. During May 2008, twigs of Japanese apricot trees in the commercial farms of Renai Region (Nantou County) showed symptoms of gummosis disease, with 12 to 18% of the trees affected. The disease was more severe on trees weakened by drought stress. Limb and twig infections began around lenticles as small, sunken, discolored lesions at the margins of wounds. Following infection, cortical cells collapsed, bark became depressed, and blisters developed, which were often cracked with whitish gummy exudation. Necrotic areas were seen on the cortical tissues. Leaves showed yellowing and drooping. In winter months, numerous black pycnidia or perithecia formed on infected twigs. Single conidial isolates of the pathogen were obtained from diseased twigs on acidified potato dextrose agar (PDA) incubated at 25 ± 1°C for 3 days. On the basis of morphological characteristics, the fungus was identified as Botryosphaeria dothidea (3). Conidia (17 to 22.6 × 4.3 to 6.0 μm) were hyaline, unicellular, and spindle shaped. Asci (78 to 125 × 15 to 17 μm) were hyaline, bitunicate, clavate, and eight spored. Ascospores (18 to 22 × 7.0 to 8.2 μm) were hyaline and spindle shaped or fusoid. The pathogen identity was further confirmed by PCR amplification and sequencing of ribosomal DNA internal transcribed spacer from the fungus with the primers ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (4), and a representative sequence was deposited in NCBI GenBank (Accession No. GU594225). The sequence showed 99 to 100% homology with previously characterized strains of B. dothidea (GenBank Accession Nos. EU441944, DQ177876, and AY786320). Pathogenicity tests were conducted with inoculum prepared by culturing the fungus on PDA under a continuous photoperiod of 128 ± 25 μE·m-2·s-1 at 25°C for 3 days. Shallow cuts (3 × 3 × 3 mm) were made on 12- to 15-month-old healthy twigs with a scalpel and inoculated with either a 5-mm mycelial disc or 0.5 ml of conidial suspension (105 conidia/ml) of the fungus. Two twigs on each of six trees were inoculated. Inoculated areas were covered with moist, sterile cotton and the entire twigs were enclosed in plastic bags. Twigs were inoculated with 5-mm PDA discs or sterile water for controls. The symptoms described above were observed on all inoculated twigs 14 days after inoculation, whereas control twigs remained healthy. Reisolation from the inoculated twigs consistently yielded B. dothidea. In Taiwan, B. dothidea has been reported as the causal agent of gummosis of peach (1) and fruit ring rot of pear (2); however, to our knowledge, this is the first report of B. dothidea causing gummosis on Japanese apricot.

Effect of aminoethoxyvinylglycine application on the occurrence of ring rot on fruit of Japanese pear

Aminoethoxyvinylglycine (AVG), also known as aminoethoxyvinylglycine hydrochloride, is a biochemical plant regulator. A field experiment was conducted during the 2006 season using trees of Japanese pear ‘Kosui’ to investigate the effect of AVG on the occurrence of ring rot on fruit. A spray of AVG at 150 ppm was applied about 1 month before commercial harvest was expected. The result confirmed that such a pre-harvest spray delayed the occurrence after harvest of ring rot on fruit of Japanese pear.

Alta Crown: A New Russet Potato Cultivar with Resistance to Common Scab and A Low Incidence of Tubers Deformities

Alta Crown (CV92028-1) is a new russet potato cultivar with resistance to common scab and a low incidence of tuber deformities. It was selected at the Lethbridge Research Centre of Agriculture and Agri-Food Canada, from a cross made at Colorado State University, San Luis Valley Research Center between breeding clones AC83172-1RU and C086030-1RU. Alta Crown has semi-erect, short, and compact vines. The cultivar has uniform, oblong to long, medium to large tubers with a medium to dark russet skin, cream- colored flesh and shallow eyes. In trials across western Canada and Ontario, Alta Crown produced similar or lower total yields than did Russet Burbank and Shepody, but a higher percentage of marketable tuber yield at maincrop harvest, and at early harvest. The average weight of marketable tubers was superior to that of Russet Burbank at all sites, and to that of Shepody at some sites. Alta Crown exhibited resistance to tuber deformities, hollow heart and internal necrosis defects. Specific gravity of Alta Crown in western Canadian regional trials was similar to that of Shepody and Russet Burbank. Fry colour was also similar to Russet Burbank and Shepody in product quality evaluations. Alta Crown was identified as having resistance to common scab and was moderately resistant to late blight tuber rot and foliar early blight, but was susceptible to foliar late blight. It shows moderate foliar and tuber symptoms when infected with bacterial ring rot. A post-harvest laboratory testing is recommended to complement visual inspection.

Erratum to: Freedom Russet—A Dual Purpose Russet Potato Cultivar with Resistance to Common Scab and Good Fry Quality

Freedom Russet was developed from a cross between ND14-1, a parent with scab resistance, and W1005rus, a parent with good processing quality. It is a mid-season cultivar and can be used for dual purposes for both fresh market and processing. The tubers of Freedom Russet are oblong-long with medium-dark russet skin, and have an attractive appearance. Freedom Russet tubers have better external and internal qualities than Russet Burbank tubers. The total and US No. 1 yield of Freedom Russet is equal to or higher than Russet Burbank in most environments. The specific gravity of Freedom Russet is similar or 1–3 unit less than Russet Burbank. Freedom Russet produced better fry color than Russet Burbank when processed after storage at 10, 8.9, and 7.2°C. It showed similar susceptibility to both early blight and late blight compared to Russet Burbank. Freedom Russet is resistant to common scab and moderately resistant to powdery scab. It is similarly susceptible to soft rot and pink rot as Russet Burbank, and clearly expresses bacterial ring rot symptoms. Freedom Russet is tolerant to Verticillium wilt and showed better yield potential than Russet Burbank under early dying pressure.

Comprehensive bioeconomic modelling of multiple harmful non-indigenous species

Harmful non-indigenous species (NIS) introductions lead to loss of biodiversity and serious economic impacts. Government agencies have to decide on the allocation of limited resources to manage the risk posed by multiple NIS. Bioeconomic modelling has focused on single species and little is known about the optimal management of multiple NIS using a common budget. A comprehensive bioeconomic model that considers the exclusion, detection and control of multiple NIS spreading by stratified dispersal and presenting Allee effects was developed and applied to manage the simultaneous risk posed by Colorado beetle, the bacterium causing potato ring rot and western corn rootworm in the UK. A genetic algorithm was used to study the optimal management under uncertainty. Optimal control methods were used to interpret and verify the genetic algorithm solutions. The results show that government agencies should allocate less exclusion and more control resources to NIS characterised by Allee effects, low rate of satellite colonies generation and that present low propagule pressure. The prioritisation of NIS representative of potential NIS assemblages increases management efficiency. The adoption of management measures based on the risk analysis of a single NIS might not correspond to the optimal allocation of resources when other NIS share a common limited budget. Comprehensive bioeconomic modelling of multiple NIS where Allee effects and stratified dispersal are considered leads to a more cost-effective allocation of limited resources for the management of NIS invasions.

Detection of Ralstonia solanacearum (biovar 2A) in stems of symptomless plants before harvest of the potato crop using post‐enrichment DAS‐ELISA

The detection of Ralstonia solanacearum (biovar 2A) in stems of symptomless plants before harvest of the potato crop, instead of tubers, would not only save highly valued planting material but would be less time‐consuming and would also enhance farmers’ market decisions. Although pathogen detection in stems has been proven efficient for ring rot, this has never been investigated for bacterial wilt (BW). Therefore the possibility of detecting BW latent infection in stem pieces about three weeks before harvest was assessed in 57 fields of the Andean highlands of Peru. Two sensitive, specific and user‐friendly serological methods were used to detect the pathogen in latently infected tubers and stems: double‐antibody sandwich (DAS)‐ELISA and indirect ELISA on nitrocellulose membrane (NCM) after enrichment of the plant extracts in a semi‐specific broth. Optimum sample sizes of stems and tubers were evaluated for 37 potato crops showing between 0 and 0·1% BW incidence using a binomial distribution model to calculate the detection probabilities. Although results of detection using the two serological techniques had 100% concordance, detection probabilities were higher using DAS‐ELISA, whatever the plant part tested. BW detection probabilities were higher for tubers than for stems; a 99% detection probability was obtained by analysing 400 stems sections or 250 tubers using DAS‐ELISA. Detection of BW infection in symptomless plants 20 days before harvest using post‐enrichment DAS‐ELISA is a reliable and user‐friendly technique that can easily be used by national plant protection services and seed programmes in developing countries.

First report of Clavibacter michiganensis subsp. sepedonicus causing bacterial ring rot of potato in Turkey

During the 2007 seed potato monitoring programme in Turkey, 336 tuber samples from Kayseri province were tested for the presence of ring rot and brown rot diseases according to the EC Directives 93/85/EEC (Anon., 1993) and 98/57/EC (Anon., 1998).An intensive survey in 1988-1989 had shown that the potato growing areas of Turkey were free of the ring rot pathogen (Benlioglu et al., 1991). This is the first report of Cms in Turkey. Strict control measures have been taken in contaminated and potentially contaminated fields since Cms is a regulated quarantine organism in Turkey.

The Putative Secreted Serine Protease Chp-7 Is Required for Full Virulence and Induction of a Nonhost Hypersensitive Response by Clavibacter michiganensis subsp. sepedonicus

Molecular biological studies on Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato, have gained greater feasibility due to the recent availability of whole genomic sequences and genetic tools for related taxa. Here, we describe the first report of construction and characterization of a transposon (Tn) mutant library of C. michiganensis subsp. sepedonicus sp. strain R10. Since virulence of R10 in potato has been shown previously to be associated with elicitation of a nonhost hypersensitive response (HR), the mutant library was screened initially for loss of HR in tobacco. The screen identified two HR-negative mutants containing Tn insertions within the same gene, CMS2989 (chp-7), although at distinct locations. chp-7 is one of 11 pat-1 homologs in C. michiganensis subsp. sepedonicus. HR-negative mutants of R10 multiplied to the same extent as wild type in planta but were less virulent in potato. Complementation with chp-7 restored virulence as well as the HR phenotype. Together, these findings demonstrate a role for chp-7 in C. michiganensis subsp. sepedonicus-plant interactions.

A Real-Time PCR Assay for the Detection of Clavibacter michiganensis subsp. sepedonicus Based on the Cellulase A Gene Sequence.

Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot (BRR) of potato (Solanum tuberosum), is a globally important quarantine pathogen that is managed in North America using zero tolerance regulations in the certified seed industry. C. michiganensis subsp. sepedonicus is well documented to cause symptomless infections in potato, contributing to its persistence in certified seed stocks. Reliable laboratory methods to detect symptomless infections with a high degree of sensitivity could assist in the reduction of inoculum in certified seed potato stocks. A real-time polymerase chain reaction (PCR) assay was developed using the cellulase A (CelA) gene sequence as the basis for primer design. CelA primers were specific to C. michiganensis subsp. sepedonicus grown in vitro and did not detect any other coryneform bacteria or potato pathogenic bacteria but did detect 69 strains of C. michiganensis subsp. sepedonicus. The CelA real-time PCR assay was more sensitive than immunofluorescence (IFA) and Cms50/72a PCR assays in detecting C. michiganensis subsp. sepedonicus in infected potato tuber cores blended with healthy tuber cores in simulated seed lot contamination experiments. CelA primers detected nonmucoid and mucoid strains with equivalent sensitivity. In naturally infected seed lots, CelA PCR primers also were more sensitive in detecting symptomless infections of C. michiganensis subsp. sepedonicus in seed tubers prior to planting compared to Cms50/72a PCR primers, IFA, and enzyme-linked immunosorbent assay. A real-time PCR format using the newly developed CelA primers proved to be a very robust detection tool for C. michiganensis subsp. sepedonicus with the added advantage of detecting only virulent strains of the ring rot bacterium.

Detection of extracellular proteinases in the potato ring rot pathogen

Detection of extracellular proteinases in the potato ring rot pathogen

Maritime Regional Meeting, 2008

Analysis of gene expression of two potato cultivars during common scab infection using microarrays C. Goyer, D. De Koeyer, H. Tai, V. Gustafson, R. Griffith, and C. Rothwell. Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, NB E3B 4Z7, CAnada; and (V.G., C.R.) BioAtlantech, P.O. Box 636, Station A, 921 College Hill Road, Fredercton, NB E3B 5A6,Canada Does ethylene play a role in tomato inresponse to viroid invasion? X. Hu, H. Tai, and X. Nie. (X.H., H.T., X.N.) - Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada; and (X.H.) College of Horticulture and LAndscape, Hunan Agricultural University, Changsha, Hunan 410128, China Current research at he Potato Research Centre — resistance to golden nematode A.M. Murphy, D. De Koeyer, S. Wood, K. Douglass, T. Dalton, and D.H. Wilson. Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada; and (S.W.) Plant Health Laboratory, Canadian Food Inspection Agency, 10 Barter's Hill, St. Johns, NL A1C 5X1, Canada Management of potao seed-piece decay caused by fungicide-resistant strains of Fusarium spp. R.D. Peters, K.A. Drake, and I.K. Macdonald. Crops and Livestock Research Centre, Agriculture and Agr-Food Canada, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada Dynamics of phage and Streptomyces scabiei populations in soil and their efficacy in controlling common scab. M. Ramirez and C. Goyer. Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada A single procedure for the isolation of viruses, Potato spindle tuber viroid, and bacterial ring rot from potato plants for detection by reverse transcription - polymerase chain reaction R.P. Singh, and A. Sullivan. Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada; (M.S.) Agricultural Certification Services Inc., 1030 Lincoln Road Fredericton, NB E3B 8B7, Canada; and (A.S.) Plant Propagation Centre, New Brunswick Department of Agriculture and Aquaculture, 598 Lincoln Road, Fredericton, NB E3B 5H1, Canada

Quantification of viable cells of Clavibacter michiganensis subsp. sepedonicus in digester material after heat treatment by TaqMan® BIO-PCR

With bacterial ring rot affected potato lots must be decontaminated under official control without causing a risk for the dissemination of the disease. Therefore, at the Bavarian State Research Center for Agriculture (LFL) in Freising a research project is conducted to investigate, which characteristics (temperature and retention period) during anaerobic digestion must be given to eliminate Clavibacter michiganensis sub-sp. sepedonicus. Most biogas plants are run at temperatures between 35 and 55°C. Hence, at first a method was developed to study and describe thermosensitivity of the organism in digester material in the relevant temperature range. For this purpose the streptomycin resistant mutant Cms 4053 strep (NCPPB collection, UK) was used. Viable organisms could be exactly quantified after being incubated for ten minutes at different temperatures by combining an enrichment step on growing medium supplemented with streptomycin with real-time PCR (ABI Prism 7000) using TaqMan® probes (TaqMan® BIO-PCR). It could be shown, that the number of colony forming units (cfu) linearily declines between 35 and 55°C with rising temperatures during equal incubation time periods. Every temperature increase of 8°C reduced the number of cfu by a log10 unit.

Developing Common Spray Schedules for Pest Control with Combination of Pesticides and Fungicides under Mixed Conditions of Apple and Pear Orchards 1. Screening of Fungicides and Examining Efficacy of Spray Schedules for Control of Pear Ring Rot Caused by Botryosphaeria berengeriana

Developing Common Spray Schedules for Pest Control with Combination of Pesticides and Fungicides under Mixed Conditions of Apple and Pear Orchards 1. Screening of Fungicides and Examining Efficacy of Spray Schedules for Control of Pear Ring Rot Caused by <i>Botryosphaeria berengeriana</i>

The role of peroxidases weakly bound to the cell walls in potato resistance to ring rot infection

The role of peroxidases weakly bound to the cell walls in potato resistance to ring rot infection

Intracellular localization of “soluble” adenylate cyclase in potato plants

In animal cells, a key enzyme of the adenylate cuclase system, adenylate cyclase (AC), is represented by its transmembrane (tmAC) and soluble (sAC) forms [1]. Earlier, we have established that both forms of this enzyme are present in plants as well (potato), in nuclei and chloroplasts [2]. Using immunoprecepitation and Western blotting with antibody against animal sAC, we have found five sAC isoforms with mol wts of 225, 193, 92, 68, and 60 kD [3]. However, it is not clear until now, whether sAC is present only in nuclei and chloroplasts or it is localized also in other cell compartments, which could provide for more rapid transduction of external signals. Therefore, the objective of our work was to analyze intracellular sAC localization at the level of the cell ultrastructure. In this study, we applied for the first time the method of immuno-electronic cytochemistry for the studying of sAC intracellular localization in various potato tissues and its re-dislocation in the response to biotic stress. Tube-grown potato plantlets of two cultivars contrasting in their resistance to the causal agent of the ring rot were used in experiments: cv. Lugovskoi, resistant, and cv. Luk’yanovskii, susceptible. The root segments and leaf pieces from the upper part of the shoot of control plants (with roots not treated with extracellular polysaccharides (EPS) of the pathogen) and experimental plants kept for 2 h in the 0.1% EPS solution were fixed for 2 h in the mixture of 4% formaldehyde and 1% glutaraldehyde (Sigma, United States) prepared in 0.1 M Na-phosphate buffer, pH 7.2. Thereafter, samples were postfixed in 0.8% OsO 4 in the same buffer (2 h), dehydrated by the routine procedure, and embedded in the mixture of epoxide resins, Epon and Araldite. Immunocytochemistry was performed on ultrathin sections mounted on nickel grids, using primary rabbit polyclonal antibodies (PAB) against animal sAC at 1 : 200 dilution conjugated with 10-nm colloidal gold particles and secondary goat antibodies against rabbit immunoglobulins (SAB, Sigma, United States) at 1 : 30 dilution. All procedures were performed as described in [4], with the exception of the usage of the hoarse serum at 1 : 5 dilution as a blocking solution. Two control treatments were applied: (a) ultrathin sections not treated with PAB and (b) treatment with nonimmune rabbit serum instead PAB and then with SAB saturated with such serum. Specimens stained with uranyl acetate were examined with an LEO 906E electron microscope (Carl Zeiss, Germany), and the images were shown on the computer display. To evaluate sAC partition between cell compartments, we counted the number of gold particles in them. In each treatment, we analyzed 25 images; the total number of particles on the area of each compartment was calculated per 1 µ m 2 . The results were processed statistically. We observed the presence of SAB, reflecting sAC localization, in many intracellular compartments. Thus, in the cells of the root meristem and elongation zone, gold particles were seen in the cytosol, mitochondria,

An Internal Reaction Control for Routine Detection of Clavibacter michiganensis subsp. sepedonicus Using a Real-Time TaqMan PCR-Based Assay.

An internal reaction control was integrated into a TaqMan polymerase chain reaction (PCR) assay for the detection of Clavibacter michiganensis subsp. sepedonicus, the causal organism of bacterial ring rot of potato. The reaction control, cloned into plasmid pCmsC4, consisted of a sequence unrelated to C. michiganensis subsp. sepedonicus flanked by the primer sequences used in the TaqMan PCR, thus eliminating the need for multiplexing. Inclusion of the reaction control plasmid in the TaqMan assay had no effect on either the limit of detection or the specificity of the method. Addition of SYBR Green permitted melt analysis of PCR products. The 242-bp reaction control amplicon, with a melt temperature of approximately 94.5°C, could easily be distinguished from the 152-bp primary diagnostic target amplicon, which had a melt temperature of about 85.5°C. Electrophoretic analysis showed that appearance of either melt peak correlated well with the presence of the appropriate amplicon. Two different substances, guanidine-HCl and humic acid, inhibited the amplification of the reaction control at concentrations lower than those that inhibited the primary diagnostic target, demonstrating the reaction control's effectiveness in detecting inhibition or reaction failure. Using the reaction control plasmid, a quantitative threshold for inhibitor detection was established. This permitted the validation of negative results, and thus facilitated the use of TaqMan real-time PCR in the routine testing of diagnostic samples for C. michiganensis subsp. sepedonicus.

First Report of Gummosis Disease of Plum (Prunus salicina) Caused by a Botryosphaeria sp. in Taiwan.

Plum (Prunus salicina Lindell) is grown on more than 3,870 ha in Taiwan. In 2004, a gummosis disease was observed on plum in the Ming Jian Region of Nantou County (120.675°E, 23.919°N), with 15% of the trees affected. Infections started on the current year's growth, primarily through lenticels, and formed small, sunken, discolored lesions. At later stages, white gum exuded from the lesions. Circular to oval, brown, necrotic areas were seen on the inner bark. Severely infected twigs showed defoliation and dieback. During the winter months, numerous black pycnidia or perithecia formed on infected twigs. Single conidial isolates of the pathogen were obtained from diseased twigs on acidified potato dextrose agar (PDA) and incubated at 25 ± 1°C for 3 days. On the basis of morphological traits, the fungus was identified as a Botryosphaeria sp. according to the CMI descriptions of Botryosphaeria ribis (3). Conidia (14.2 to 26.8 × 4.3 to 7.2 μm) were single celled, hyaline, and spindle shaped. Asci (105 to 135 × 12.5 to 15.5 μm) were hyaline, clavate, and bitunicate. Ascospores (18 to 22 × 7.0 to 8.2 μm) were hyaline and spindle shaped or fusoid. For pathogenicity tests, inoculum was prepared by culturing the fungus on PDA under continuous fluorescent light (128 ± 25 μE·m-2·s-1) at 25°C for 3 days. Two twigs on each of six trees were inoculated. Sharp incisions (3 × 3 × 3 mm) were made on healthy twigs (12 to 15 months old) with a sterilized scalpel and inoculated with either a 5-mm mycelial disc or 0.5 ml of a conidial suspension (105 conidia/ml) of the fungus. Inoculated areas were covered with moist, sterile cotton and the entire twigs were enclosed in plastic bags. Twigs inoculated with 5-mm PDA discs or sterile water alone served as controls. The symptoms described above were observed on all inoculated twigs 14 days after inoculation, whereas control twigs did not develop any disease symptoms. Reisolation from the inoculated twigs consistently yielded the Botryosphaeria sp., thus fulfilling Koch's postulates. Botryosphaeria spp. have been reported to cause stem blight of many plants in temperate and tropical regions of the world (4). In Taiwan, B. dothidea has been reported as the causal agent of gummosis disease of peach (1) and fruit ring rot of pear (2); however, to our knowledge, this is the first report of a Botryosphaeria sp. causing gummosis of plum.