Ring Rot Research Articles

Upcoming Plant Pathological Techniques in the Disease Diagnosis

Serodiagnostics for the detection of plant pathogens particularly the plant viruses started in the fifties and had a quantum jump in application after the introduction of enzyme conjugated antibodies in enzyme linked immunosorbent assays EIA in the s Improvements and modifications in the original EIA made its application routine in the large scale indexing of vegetatively cultivated crops It is still the methods of choice for many purposes However its limited sensitivity as compared to nucleic acid based diagnostics particularly PCR restricts its use in critical decision making related to pathogens of quarantine significance like ring rot of potato caused by Corynebacterium sepedonicum tuber necrosis strains of PVY PVYNTN etc Serodiagnostics due to limited sensitivity and specificity in the detection of fungal and bacterial pathogens has not become popular for the detection of many of these pathogens Recent developments in the field of genomics of microorganisms and PCR based assays of microbes have revolutionized diagnostics A combination of immunocapture of pathogen and PCR has made the detection simpler and more sensitive than PCR alone Combination of immunocapture with multiplex PCR further reduces cost of detection Combination of nucleic acid probes and ELISA has the potential of its adoption for large scale indexing of propagating stocks WTO agreements related to trade in agricultural commodities need internationally acceptable diagnostic tools for the detection of pathogens of quarantine significance An internationally recognized monitoring organization is the urgent need The organization will be responsible for the evaluation of reagents and protocols for diagnostics and their regular update The issue was mooted in a recent symposium titled Moving Plant Disease Detection from the Ivory Towers to the Real World where a ring test process was described to validate and standardize new diagnostic procedures As more and more techniques are validated by the scientific community and their reliability and cost effectiveness demonstrated molecular based techniques will play a greater and more important role in the detection of pathogens

Isolation and characterization of Bacillus amyloliquefaciens PG12 for the biological control of apple ring rot

Bacillus spp. are promising candidates for biological control of postharvest diseases. Bacillus amyloliquefaciens PG12 was isolated from apple fruit and exhibited broad-spectrum antifungal activity. Botryosphaeria dothidea was significantly suppressed by PG12 in in vitro and in vivo. Lumpy appearance and abnormal structure of the mycelia from the edge of inhibition zone were observed using scanning electron microscopy (SEM) in in vitro assays. Furthermore, the lipopeptide crude extracts from cell-free supernatant of PG12 had remarkable antifungal activity against B. dothidea, indicating that lipopeptides played a major role in the biological control ability of PG12. PCR detection revealed that PG12 harbored the gene clusters required for the biosynthesis of the two main families of lipopeptide, including iturin and fengycin. One iturin-like compound (Rf 0.4) showed inhibitory activity against B. dothidea using thin layer chromatography (TLC)-bioautography analysis and were further fractionated by semipreparative high performance liquid chromatography (HPLC). The fraction with a molecular weight of 1043.55 m/z was identified as iturin A by electrospray ionization quadrupole-time-of-flight mass spectrometry/mass spectrometry (ESI-Q-TOF MS). Taken together, B. amyloliquefaciens PG12 was an effective biocontrol agent against apple ring rot caused by B. dothidea and iturin A was an important factor in its activity.

Vplyv hnojenia na biologick� aktivitu p�dneho spolocenstva streptomyc�t

The search for new natural mechanisms to inhibit the growth of phytopathogenic microorganisms has become widely widespread. Therefore, the main objective of the present study was determination of antimicrobial activities of actinomycetes isolated from agricultural soil, which was fertilized mainly by organic fertilizers, against 8 selected phytopathogenic strains. Among the actinomycetes, Streptomyces species have been extensively studied, because they have been recognized as an important source of secondary metabolites, which can suppress the growth of undesirable pests in crops. The results indicated that the richest source of Streptomyces colonies was soil fertilized with compost (103 x104 CFU*g-1 dry soil). On the basis of morphological signs, total of 65 isolates were selected and examined for antimicrobial activities. Isolates exhibited the best activity against Gram negative bacterium Clavibacter michiganensis subsp. sepedonicus, disease agent of “ring rot” of potatoes and against fungus Fusarium poae, disease agent of Fusarium head blight of wheat. Twelve isolates exhibited promising broad-spectrum activity against tested organisms. On the basis of results, six of them were selected for further screening. Comparison of polyphasic studies with available literature led to identification of biological active strains S. olivochromogenes (13SC11), S. avermitilis (13SC2), S. rishiriensis (13SC13), S. globisporus (13SC19), S. sampsonii (13SPC10) and S. avidinii (13SPC4). After quantification analysis of various enzymes, tested isolates produced alkaline phosphatase, leucinearylamidase, valinearylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, glucosidase in high values (>40 nmol) and were positive for nitrate reduction, hydrolysis of gelatin, urease, and esculin. These isolates can be used in the development of new biopesticides anf biofertilizers with antibacterial and antifungal effect.

Editorial ‐ Has Clavibacter michiganensis ssp. sepodonicus, causal pathogen of potato ring rot, been reported validly in Egypt?

In October 2014 New Disease Reports (NDR) reported Clavibacter michiganensis ssp. sepodonicus as the cause of ring rot of potato ( Solanum tuberosum ) for the first time in Egypt (and purportedly the first record for Africa) (Seleim et al .,…

Effect of fertilization on biological activity of community of soil streptomycetes

The search for new natural mechanisms to inhibit the growth of phytopathogenic microorganisms has become widely widespread. Therefore, the main objective of the present study was determination of antimicrobial activities of actinomycetes isolated from agricultural soil, which was fertilized mainly by organic fertilizers, against 8 selected phytopathogenic strains. Among the actinomycetes, Streptomyces species have been extensively studied, because they have been recognized as an important source of secondary metabolites, which can suppress the growth of undesirable pests in crops. The results indicated that the richest source of Streptomyces colonies was soil fertilized with compost (103 x104 CFU*g-1 dry soil). On the basis of morphological signs, total of 65 isolates were selected and examined for antimicrobial activities. Isolates exhibited the best activity against Gram negative bacterium Clavibacter michiganensis subsp. sepedonicus, disease agent of “ring rot” of potatoes and against fungus Fusarium poae, disease agent of Fusarium head blight of wheat. Twelve isolates exhibited promising broad-spectrum activity against tested organisms. On the basis of results, six of them were selected for further screening. Comparison of polyphasic studies with available literature led to identification of biological active strains S. olivochromogenes (13SC11), S. avermitilis (13SC2), S. rishiriensis (13SC13), S. globisporus (13SC19), S. sampsonii (13SPC10) and S. avidinii (13SPC4). After quantification analysis of various enzymes, tested isolates produced alkaline phosphatase, leucinearylamidase, valinearylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, glucosidase in high values (>40 nmol) and were positive for nitrate reduction, hydrolysis of gelatin, urease, and esculin. These isolates can be used in the development of new biopesticides anf biofertilizers with antibacterial and antifungal effect.

Natural infection of Clavibacter michiganensis subsp. sepedonicus in tomato (Solanum tuberosum)

Five tomato plants cv. ‘Merlice’ on Maxifort rootstock were received late April 2014 from a substrate crop in Flanders, Belgium. They exhibited yellowing and necrosis of the leaf mesophyll, withering of leaflets and wilting of whole leaves. Vascular tissues were probed at various nodes of the stem because explicit discolouration of the xylem was not observed. The tissues were fragmented in sterile 10 mM phosphate buffer (PB) and the dilution was plated on Pseudomonas Agar F containing 5g/l of sucrose. Creamy white colonies developed from the diluted extracts after seven days incubation at room temperature (Fig. 1). A pure culture from each diseased tomato plant (designated GBBC 1958 to GBBC 1962) was identified as Clavibacter michiganensis subsp. sepedonicus (Cms) in taxon-specific conventional PCR (Pastrik & Rainey, 2) and TaqMan real-time PCR (Schaad et al., 3). They were also assigned to the gyrB sequence cluster of Cms (Fig. 2) with unique subspecies signatures and displayed specific biomarker proteins of Cms in MALDI-TOF (Fig. 3) (Zaluga et al., 4). The vascular tissue extracts tested positive for Cms in an immunofluorescence test with polyclonal antiserum (Prime Diagnostics, The Netherlands) and with the monoclonal antibody 9A1 (Agdia Biofords, France). They tested negative for C. m. subsp. michiganensis (Cmm) in an immunofluorescence test with polyclonal antiserum (Prime Diagnostics, The Netherlands). Pathogenicity of the five isolates was tested in tomato and potato using suspensions of about 108 cells/ml in PB. Tomato plantlets with two fully developed leaves were inoculated by stem infiltration (Zaluga et al., 5) and placed in a growth chamber at 20-25°C. High-grade seed tubers cv. ‘Fontane’ were inoculated by dipping a potato knife in the cell suspension and making a longitudinal cut from the heel end over about two-thirds of the tuber. After overnight retention at 16°C, they were hand planted in preformed ridges in a contained field plot at ILVO. Buffer-inoculated controls separated each test object. The type strains of Cmm and Cms were used as pathogenic controls. In tomato plantlets, the five Cms isolates displayed flaccidity and chlorosis of leaf margins, wilting or necrosis of individual leaf parts and finally wilting of whole leaves (Fig. 4). The first symptoms appeared 10-12 days after inoculation and progressed slowly. In potato plants, the five Cms isolates caused a general burned appearance with rolling and necrosis of leaf margins, mottling and yellowing between veins (Fig. 5). Ring rot symptoms were identified in the progeny tubers and confirmed by TaqMan PCR. Each isolate was recovered from infected test plants and re-identified as Cms by the gyrB barcode. Although tomato is considered a host upon artificial inoculation, this is to our knowledge the first report of a natural infection of C. m. subsp. sepedonicus in tomato plants. The incidence in the crop was limited to ten successively arranged plants in one row, suggesting transmission from a primary infected plant but at a low rate. The origin of the infection is unknown. Inspection of tomato crops issued from the same seed lot did not result in additional findings. The official status of the pathogen in Belgium is reported as under eradication on tomato and absent on potato (EPPO, 1). The five isolates are deposited at the LMG collection (strain numbers 28446-28450). The corresponding gyrB sequences are available at NCBI (GenBank Accession Nos. KP899559-KP899663).

Wood decomposition by porodaedalea pini from different regions of Poland

Red ring rot (Porodaedalea pini (Brot.) Murrill., previously Phellinus pini) is a fungus frequently found in pure and mixed pine stands and in tree plantings partly composed of pines. Porodaedalea pini sensu lato has been documented as occurring on a wide variety of coniferous and some hardwood hosts, including the following genera: Abies, Acer, Betula, Calocedrus, Cedrus, Chamaecyparis, Crataegus, Larix, Picea, Pinus, Pseudotsuga, Taxus, Thuja and Tsuga [Gilbertson, Ryvarden 1986; Larsen, Cobb-Poulle 1990]. The range of P. pini covers the whole of Europe, northern Asia and North America [Gilbertson, Ryvarden 1987]. In Poland, this fungus infests approx. 8% of Scots pine wood [Szewczyk 2008]. Porodaedalea pini decomposes heartwood (affecting stands starting from age class II), forming white pocket rot as a result [Ezhov, Konyushatov 2001]. The older the stand, the greater the number of infested pines. It is estimated that in a 100-year-old stand, approx. 15–35% of the pines are infested, while in a 160-year-old stand, it may be as much as 100% of the trees. The range of rot

Streptomyces rochei A-1 induces resistance and defense-related responses against Botryosphaeria dothidea in apple fruit during storage

Apple ring rot caused by Botryosphaeria dothidea is an important disease in most apple-growing regions in China. It results in severe losses during fruit storage. Induced resistance against plant pathogens is a promising alternative to traditional control strategies. In the present study, a potential biocontrol agent, Streptomyces rochei A-1, was evaluated for its induction of resistance against B. dothidea in postharvest apple fruit, and the defense mechanisms involved were investigated. The results indicated that S. rochei A-1 treatment could effectively induce the resistance of apple fruit to ring rot caused by B. dothidea. The lesion area and disease incidence were 65.4% and 27.1% lower than those of the control fruit after storage at 25°C for 7d. S. rochei A-1 treatment significantly increased the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and phenylalanine ammonia-lyase (PAL) and highly inhibited lipid peroxidation. β-1,3-glucanase and chitinase activities and H2O2 generation were also markedly promoted by the biocontrol agent. Moreover, the expression levels of three pathogenesis-related proteins genes (PR-8, GLU and CHI) were enhanced. The most significant increase was observed in apple fruit pretreated with S. rochei A-1 and inoculated with B. dothidea. Therefore, our results suggest that S. rochei A-1 treatment induces resistance against B. dothidea in apple fruit by stimulating a series of defense responses.

Inhibitory activity of Athyrium sinense extracts against Clavibater michiganense subsp. sepedonicum

Clavibater michiganense subsp. sepedonicum , the causal agent of a serious disease called bacterial ring rot of potato, is widespread under both greenhouse and field cultivation. In order to find a promising method for the control of this disease, Athyrium sinense extracts were investigated in the study presented here. Results showed that the A. sinense extracts had significantly inhibitory activity against C. michiganense subsp. sepedonicum . Eight organic solvents were used to obtain A. sinense extracts, and ethanol was selected as an ideal extraction solvent. Based on single factor experiment results and L 27 3 (13) orthogonal experiment results, optimum extraction conditions were obtained. They were A 2 B 3 C 3 , solid to liquid ratio 1:20 (g:mL), temperature 97°C and extraction time 10h. Stability assays revealed that Zn 2+ , Fe 3+ , acidic and neutral conditions enhanced inhibitory activity, and extracts were stable under ultraviolet (UV). Transmission electron microscopy (TEM) showed drastic changes caused by A. sinense extracts, including lack of cytoplasmic materials, formation of vacuoles and bacterial deformation. These results revealed A. sinense extracts have strong inhibitory activity against C. michiganense subsp. sepedonicum , and can be used as a potent phytochemical fungicide.

Влияние заражения клубней кольцевой гнилью и обработки монойодацетатом натрия совместно с прогреванием на хранение картофеля

В статье обсуждается влияние на проращивание клубней монойодацетата натрия (МИА) совместно с прогреванием (45 °C, 1 ч) в качестве потенциального агента для борьбы с Clavibacter michiganensis ssp. sepedonicus (Cms) перед закладкой картофеля на хранение. В работе использованы клубни картофеля (Solanum tuberosum L.) сорта Лукьяновский. В эксперименте присутствовало четыре варианта обработки клубней, в каждом варианте было 30 клубней. После проведенных обработок клубни помещали в условия овощехранилища на 8 месяцев. Весной подсчитывали количество ростков, измеряли длину проростков у клубней и проводили клубневой анализ. Полученные результаты статистически обрабатывали с использованием пакета программ Excel. Показано, что заражение клубней Cms снижало способность клубней к прорастанию. Обработка МИА и прогревание здоровых клубней картофеля перед закладкой на хранение подавляло прорастание клубней, что полезно для продовольственного картофеля. Результаты клубневого анализа показали, что прогревание снижало общее количество больных клубней примерно на 39 %, клубни были менее поражены кольцевой и бурой гнилями. Обработка МИА также способствовала снижению количества клубней, пораженных бурой и кольцевой гнилями. Обработка клубней МИА совместно с прогреванием снижала общее количество больных клубней, пораженных проволочником и пустотелых, кольцевой и сухой гнилями, а также паршой. Обработка МИА и прогреванием инфицированных клубней картофеля перед закладкой на хранение снимала подавляющий эффект бактерий на прорастание. Полученный результат актуален для обработки семенного картофеля, так как кольцевая гниль является латентным заболеванием.

Efficacy of agricultural disinfectants on biofilms of the bacterial ring rot pathogen, Clavibacter michiganensis subsp. sepedonicus

Susceptibility of biofilms of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato, to three common agricultural disinfectants was tested. The MBECTM assay device was used to evaluate optimum parameters for growing artificial biofilms. These conditions were determined to be 7 days of growth at 23°C in a yeast extract-glucose-mineral salts medium. As expected, the bacteria in the biofilm state were more resistant to disinfection by chemical treatment with sodium hypochlorite, quaternary ammonium, and hydrogen peroxide when compared with planktonic cells. Artificial biofilms were also grown on five different surface materials typically found in commercial potato storage facilities (concrete, mild steel, rubber, polycarbonate and wood) using the BESTTM assay device to test the effect of surface type on biofilm susceptibility to disinfection. Sodium hypochlorite was the most effective disinfectant on the wood surface and hydrogen peroxide was best on the mild steel surface. Efficacies of the various disinfectants were not significantly different on concrete, rubber and polycarbonate surfaces. When artificially grown biofilms and those grown naturally in potato tissue were transferred to, and dried onto, coupons of the different surface materials, they were significantly more difficult to inactivate than in situ grown biofilms. The resistance of plant pathogenic bacteria in the biofilm state, particularly when spread and dried onto surfaces of agricultural machines and other equipment, to commonly used disinfectants, has important implications for disease control strategies that depend largely on strict sanitary and hygienic practices.

Role of lenticels and microcracks on susceptibility of apple fruit to Botryosphaeria dothidea

Apple ring rot, caused by Botryosphaeria dothidea, is one of the most serious diseases affecting apple industry in East Asia. To study the correlation between fruit natural exocarp structures and susceptibility to B. dothidea, lesion length of Malus domestica cultivars and Malus germplasm accessions was measured 14 days after in vitro inoculation with B. dothidea mycelia at different developmental stages. Area per fruit surface (APFS) of lenticels and microcracks and thickness of cuticular wax were observed. Lesion length, lenticel APFS and cuticular wax thickness varied significantly among the 11 cultivars studied. A positive correlation between lesion length and lenticel APFS and a negative correlation between lesion length and cuticular wax thickness were detected among cultivars and during fruit development. In addition, similar correlations were validated in nine Malus germplasm accessions. Numerous microcracks were observed on surfaces of Golden Delicious and Golden Spur fruit, accounting for 90 % of the natural openings. The hyphae of B. dothidea penetrated into exocarp through both lenticels and microcracks. Fruit bagging with double-layered paper bags and exogenous GA4+7 reduced lenticel APFS, thickened cuticular wax and elevated resistance to disease. Natural openings on fruit exocarp are an important factor affecting susceptibility of apple to B. dothidea.

Varietal Susceptibility of Current Shoots and Fruits to Apple Ring Rot

Varietal Susceptibility of Current Shoots and Fruits to Apple Ring Rot

Przydatność przeciwciał króliczych uzyskiwanych dwiema metodami do wykrywania Clavibacter michiganensis subsp. sepedonicus testem DAS-ELISA

Clavibacter michiganensis subsp. sepedonicus (Cms) bacterium is responsible for ring rot of potato, one of the most dangerous disease that affects potato plants and tubers. The best method to limit the disease propagation is quick and sensitive diagnostic of Cms bacteria. The deciding factor for the effectiveness of the immunologic tests is the quality of antibodies that detect the pathogen. In order to examine the influence of isolation of the antibodies aimed at entire bacterium cells (Cms) on their quality, two different methods of ion-exchange chromatography allowing isolation of IgG from rabbit blood serum were assessed. Separations were performed in columns filled with DEAE cellulose (diethylaminoethyl cellulose) and thiophilic Sepharose beds respectively. The stage that preceded chromatographic separations in both methods comprised an initial purification by precipitation of proteins from the serum in presence of salt solution. Using both matrices a preparative isolation of antibodies and removal of other impurities were achieved. The applied methods were compared in terms of quality of the antibodies obtained as well as time and difficulty of their accomplishment. Sensitivity of the antibodies to detect Cms bacteria was examined by application of DAS-ELISA assay (doubleantibody sandwich enzyme linked immunosorbent assay).

Bacilysin overproduction in Bacillus amyloliquefaciens FZB42 markerless derivative strains FZBREP and FZBSPA enhances antibacterial activity.

Bacillus amyloliquefaciens strains FZBREP and FZBSPA were derived from the wild-type FZB42 by replacement of the native bacilysin operon promoter with constitutive promoters P repB and P spac from plasmids pMK3 and pLOSS, respectively. These strains contained two antibiotic resistance genes, and markerless strains were constructed by deleting the chloramphenicol resistance cassette and promoter region bordered by two lox sites (lox71 and lox66) using Cre recombinase expressed from the temperature-sensitive vector pLOSS-cre. The vector-encoded spectinomycin resistance gene was removed by high temperature (50 °C) treatment. RT-PCR and qRT-PCR results indicated that P repB and especially P spac significantly increased expression of the bac operon, and FZBREP and FZBSPA strains produced up to 170.4 and 315.6% more bacilysin than wild type, respectively. Bacilysin overproduction was accompanied by enhancement of the antagonistic activities against Staphylococcus aureus (an indicator of bacilysin) and Clavibacter michiganense subsp. sepedonicum (the causative agent of potato ring rot). Both the size and degree of ring rot-associated necrotic tubers were decreased compared with the wild-type strain, which confirmed the protective effects and biocontrol potential of these genetically engineered strains.

Identification of a xyloglucan-specific endo-(1-4)-beta-d-glucanase inhibitor protein from apple (Malus × domestica Borkh.) as a potential defense gene against Botryosphaeria dothidea

Botryosphaeria dothidea is the causal agent of apple ring rot which is a highly destructive apple disease in China. Here, a putative xyloglucan-specific endo-(1-4)-beta-d-glucanase inhibitor protein from Malus×domestica (designated as MdXEGIP1) was found to be involved in defense against B. dothidea infection. MdXEGIP1 shares high amino acid sequence identity with other apple XEGIPs, but exhibited significantly different responses to B. dothidea infection. Quantitative real-time PCR revealed that MdXEGIP1 expression was significantly induced in shoot bark of apple plant by B. dothidea and showed different expression pattern in resistant and susceptible apple cultivars. In resistant cultivar, MdXEGIP1 expression was elevated with larger amplitude than that in susceptible cultivar after B. dothidea infection. MdXEGIP1 expression was also significantly enhanced by treatment with exogenous methyl jasmonate and salicylic acid in apple plantlets. Further investigation revealed that recombinant MdXEGIP1 has significant inhibitor activity to XEGs from family 12 and 74 of glycoside hydrolase. More importantly, recombinant MdXEGIP1 inhibited crude enzyme solution of XEG from B. dothidea, suggesting that MdXEGIP1 might protect apple plant from B. dothidea infection by inhibiting XEG activity. Taken together, the results indicated that MdXEGIP1 is a potential defense gene against B. dothidea in apple.

Lateral flow immunoassay for rapid detection of potato ring rot caused by Clavibacter michiganensis subsp. sepedonicus

A lateral flow immunoassay for the rapid detection of Clavibacter michiganensis subsp. sepedonicus bacteria causing potato ring rot was developed. Multimembrane composites (test strips) containing poly� clonal antibodies against the bacteria and gold nanoparticleantibody conjugates were used for the analysis. The test strips are suitable for the analysis of potato tuber and leaf extracts within 10 min; the detection limit of bacteria is 4 × 10 5 cells/mL. No crossreactivity with strains of Clavibacter michiganensis subsp. michigan� ensis, Pectobacterium carotovorum subsp. carotovorum and saprophytes of healthy potato plants was detected. The results of analysis of 26 potato samples by the developed tests were compared with those obtained by the PCR method and using the commercial enzyme immunoassay kits. The results of lateral flow immunoassay were confirmed in 96.2% of cases, which supports the high correlation with other analytical approaches. The developed immunoassay may be considered as a promising means of phytosanitary control.

Multiple locus genealogies and phenotypic characters reappraise the causal agents of apple ring rot in China

Apple ringrot inflictssevereeconomiclosses inthe main apple producing areas of East Asia. The causal agent of the disease has been variously identified as Macrophoma kuwatsukai, Physalospora piricola and Botryosphaeria berengeriana f. sp. piricola ,a lthoughB. dothidea is currently the most widely accepted pathogen name. The taxonomic uncertainty has delayed research that is needed to manage effectively this destructive disease. In the present study, gene- alogical concordance phylogenetic species recognition (GCPSR) was applied to pathogenic fungal isolates from apple and pear from several locations in China, along with several reference isolates. Phylogenetic results based on se- quences of four nuclear loci (ITS, EF-1α, HIS and HSP) revealed the existence of two species within the examined isolates. One includes an ex-epitype isolate ofB. dothideaand the other includes an isolate that was previously designated as B. berengeriana f. sp. piricola. Morphologically, the latter taxon presented an appressed mycelial mat on PDA whereas B. dothidea displayed columns of aerial mycelia reaching the lids, and conidia of the latter species were longer than B. dothidea. Botryosphaeria dothidea had a faster growth rate than the latter taxon under relatively high temperatures. Path- ogenicity tests showed that on pear stems the latter taxon caused large-scale cankers along with blisters whereas B. dothidea was non-pathogenic, but on apple shoots the two fungi induced large and small wart-like prominences, respectively. Overall, this cryptic species demonstrated suffi- cient genetic variations and biological differences from B. dothidea. As a result of taxonomic study, we described here the latter taxon in a new combination, Botryosphaeria kuwatsukai and designate an epitype. Both B. kuwatsukai and B. dothidea are considered to be the main causal agents for apple ring rot in China and Japan.

First report of potato bacterial ring rot caused by Clavibacter michiganensis subsp. sepedonicus in Africa

In 2014, potato tubers (cv. Diamant and cv. Spunta) showing typical symptoms of bacterial ring rot disease caused by Clavibacter michiganensis subsp. sepedonicus (Cms) were observed in Gharbia, Menoufia, Minia, Assiut and Sohag governorates, Egypt, in 10 out of 10,000 samples examined at the time of planting. Splitting infected tubers toward the stem end revealed a yellow to brown discolouration of the vascular tissue. The discoloured area had a cheesy appearance and exuded a milky ooze when squeezed (Fig. 1). Samples were tested for the presence of ring rot disease, according to the EC Directive 93/85/EEC (Anon., 1). Extracts from 1 cm tuber heel-end cores were streaked onto NCP-88 medium (de la Cruz et al., 2), yeast peptone glucose agar (Anon., 1) and yeast peptone glucose agar with the addition of neomycin (3.5 mg/l), polymyxin B sulphate (16250 IU) and clotrimazole (10 mg/l). The resultant colonies (white to cream, round to irregular with entire margins, raised, smooth, mucoid and glistening) were selected and purified. Suspensions of isolates in distilled water (106 cfu/ml) were injected (EU, 4) into stems of eight Solanum melongena plants (cv. Black Beauty) per isolate at the three leaf stage. Plants were incubated at 21 ± 2°C and 70-80% RH. Control plants were inoculated with sterile water. After 14 days plants began to wilt, wilted tissue appeared dark green or mottled but turned paler before becoming necrotic. Inter-veinal wilts had a greasy water-soaked appearance. Necrotic tissue had a bright yellow margin. Some plants were killed completely. No symptoms were observed in control plants. Bacteria were re-isolated from symptomatic inoculated plants and identified. Pure cultures of ten Gram-positive isolates hydrolysed aesculin, utilized acetate and produced catalase. They produced acid from mannitol and sorbitol, but not from glucose, glycerol, lactose and salicin. They were negative for oxidase activity, urease activity, hydrogen sulphide production, indole production, growth at 37°C, tolerance of 7% NaCl, and gelatin liquefaction. They gave negative or weak reactions for starch hydrolysis (Anon., 1). Identification of the isolates was confirmed by PCR using primers Cms50F/Cms50R (Gudmestad et al., 3) that yield an amplicon size of 192 bp. This is the first report of potato bacterial ring rot caused by Cms in Egypt. To our knowledge, this is the first report of Cms in Africa.

Antibacterial activity of petroleum ether fraction from Laminaria japonica extracts against Clavibacter michiganensis subsp. sepedonicus

We prepared an ethanol extract from Laminaria japonica, and obtained five fractions from the extract using various solvents. We investigated the antibacterial activities of the fractions against Clavibacter michiganensis subsp. sepedonicus (Spieckermann & Kotthoff) Davis et al., the causal agent of bacterial ring rot of potato (BRR). Among all the fractions, the petroleum ether fraction showed the strongest (P < 0.05) antibacterial activity against C. michiganensis subsp. sepedonicus. We analyzed the petroleum ether fraction to detect the components with antibacterial activity, and identified 24 compounds. The main constituents of the petroleum ether extract were esters (35.28 %) and acids (44.84 %). Cell membrane permeability assays, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealed that the petroleum ether fraction caused drastic ultrastructural changes in the cells of the pathogen. The petroleum ether fraction caused envelope disruption, cell deformation, the separation of the cell wall from the cell membrane, and the formation of vacuoles. These results clearly indicate that the petroleum ether fraction obtained from L. japonica shows strong antibacterial activity against the causal agent of BRR.