Canker Formation Research Articles

First Report of Twig and Branch Dieback of English Walnut (Juglans regia) Caused by Neofusicoccum mediterraneum in California.

California produces 99% of the U.S. English walnut crop with more than 30 cultivars on ~89,000 ha. Production for 2008 was ~436,000 tons with a value of $527 million. In early summer of 2009 and 2010, branch and twig dieback of English walnut (Juglans regia L.) was detected in orchards in Yolo County and submitted to our diagnostic laboratory. Disease symptoms included death of twig tips, branch dieback, wood lesions, and canker formation. Pycnidia were embedded within the bark of dead twigs. Conidia from pycnidia were hyaline, fusoid-ellipsoidal, widest usually in the middle, and 21 to 24 (-27) × 5 to 7 μm (n = 30). Isolations from cankers yielded the fungus Neofusicoccum mediterraneum Crous, M.J. Wingf. & A.J.L. Phillips (1). Fungal colonies of N. mediterraneum grew light olive green to gray on potato dextrose agar, becoming dark olive green with age. Identification of fungal isolates was confirmed by sequence comparison of Californian isolates with ex-type (CBS 121558) sequences in GenBank (3) using the internal transcribed spacer region of the rDNA, a portion of the β-tubulin gene, and part of the translation elongation factor. Sequences of Californian isolates (GenBank HM443604-HM443609) were identical to the ex-type sequences for all three genes. Previous studies in California reported the occurrence and pathogenicity of N. mediterraneum into grapevine (Vitis vinifera L.) (3) and almond trees (Prunis dulcis L.) (2). Inderbitzin et al (2) investigated the host range of N. mediterraneum in California and reported the occurrence of pycnidia on English walnut trees. However, this study did not investigate the pathogenicity of N. mediterraneum on this host. In the current study, the pathogenicity of N. mediterraneum in J. regia cvs. Hartley and Chandler was investigated in an orchard at UC Davis using two fungal isolates. Pathogenicity tests were performed by inoculating eight 2- to 4-year-old branches of mature J. regia trees. Inoculations were made in June 2009 with a 5-mm cork borer to remove bark and placing an 8-day-old 5-mm-diameter agar plug bearing fresh mycelium of the fungal isolates directly into the fresh wound, mycelium side down. An additional eight branches of each cultivar were inoculated with noncolonized agar plugs to serve as controls. Inoculated wounds were covered with petroleum jelly and wrapped with Parafilm to retain moisture. Branches were harvested after 10 months of incubation and checked for canker development. The extent of vascular discoloration was measured in each branch and isolations were made from the edge of discolored tissue to confirm Koch's postulates. Statistical analyses were performed with analysis of variance and Dunnett's t-test to assess significant differences in the extent of vascular discoloration between inoculations with N. mediterraneum and the control. Necrosis length for the two isolates averaged 131.5 mm in Hartley branches and 110 mm in the Chandler branches. Average necrosis lengths in the control branches were 18.5 mm and 16.7 mm, respectively, significantly lower (P < 0.05) than the average necrosis length found in branches inoculated with N. mediterraneum. Fungal recovery was 75% in both varieties. To our knowledge, this study is the first report of N. mediterraneum as a pathogen of J. regia trees in California.

Summer heat and an epidemic of cytospora canker of Alnus

An epidemic of cytospora canker [Valsa melanodiscus, anamorph Cytospora umbrina] is associated with dieback and mortality of Alnus incana ssp. tenuifolia in the Southern Rocky Mountains and had begun by the late 1980s. Isolations showed that inoculum was often abundant on bark and bud surfaces even in winter, especially near diseased stems. The pathogen was occasionally isolated from internal tissues of dormant buds and asymptomatic wood and phloem. In infected stems, the pathogen was detected up to 5 cm beyond visible canker margins. The data suggest that the pathogen can cause latent infections, but the infection court remains unclear. Inoculations of healthy stems in the field did not induce canker formation. Fruiting of the anamorph was primarily in late winter and spring, and sexual maturation occurred in late summer and fall. Stem age and growth data support earlier conclusions that the mortality does not represent a steady-state condition with normal attrition of older stems. Canker expansion and killing of branches and stems occurred almost exclusively in the warmest part of summer. We present a hypothesis and supporting evidence suggesting that warm summer temperatures are conducive to the disease. Locally, summer temperatures, and especially maxima, have varied with a dominant oscillation period of approximately 21 years. We speculate that alder populations expand during cool climate phases and shrink during warm phases due to epidemics of cytospora canker. More recently, the oscillation has dampened as an increasing trend of temperature has become dominant, locally and globally. If the trend continues, this already severe epidemic may become more damaging, without intervening opportunities for alder populations to recover.

First Report of Brown Rot Caused by Monilinia fructicola Affecting Peach Orchards in Slovenia.

Monilinia fructicola, the causal agent of brown rot, is a destructive fungal pathogen that affects mainly stone fruits (Prunoideae). It causes fruit rot, blossom wilt, twig blight, and canker formation and is common in North and South America, Australia, and New Zealand. M. fructicola is listed as a quarantine pathogen in the European Union and was absent from this region until 2001 when it was detected in France. In August 2009, mature peaches (Prunus persica cv. Royal Glory) with brown rot were found in a 5-year-old orchard in Goriška, western Slovenia. Symptoms included fruit lesions and mummified fruits. Lesions were brown, round, rapidly extending, and covered with abundant gray-to-buff conidial tufts. The pathogen was isolated in pure culture and identified based on morphological and molecular characters. Colonies on potato dextrose agar (PDA) incubated at 25°C in darkness had an average daily growth rate of 7.7 mm. They were initially colorless and later they were light gray with black stromatal plates and dense, hazel sporogenous mycelium. Colony margins were even. Sporulation was abundant and usually developed in distinct concentric zones. Limoniform conidia, produced in branched chains, measured 10.1 to 17.7 μm (mean = 12.1 μm) × 6.2 to 8.6 μm (mean = 7.3 μm) on PDA. Germinating conidia produced single germ tubes whose mean length ranged from 251 to 415 μm. Microconidia were abundant, globose, and 3 μm in diameter. Morphological characters resembled those described for M. fructicola (1). Morphological identification was confirmed by amplifying genomic DNA of isolates with M. fructicola species-specific primers (2-4). Sequence of the internal transcribed spacer (ITS) region (spanning ITS1 and ITS 2 plus 5.8 rDNA) of a representative isolate was generated using primers ITS1 and ITS4 and deposited in GenBank (Accession No. GU967379). BLAST analysis of the 516-bp PCR product revealed 100% identity with several sequences deposited for M. fructicola in NCBI GenBank. Pathogenicity was tested by inoculating five mature surface-sterilized peaches with 10 μl of a conidial suspension (104 conidia ml-1) obtained from one representative isolate. Sterile distilled water was used as a control. Peaches were wounded prior to inoculation. After 5 days of incubation at room temperature and 100% relative humidity, typical brown rot symptoms developed around the inoculation point, while controls showed no symptoms. M. fructicola was reisolated from lesion margins. Peach and nectarine orchards in a 5-km radius from the outbreak site were surveyed in September 2009 and M. fructicola was confirmed on mummified fruits from seven orchards. The pathogen was not detected in orchards from other regions of the country, where only the two endemic species M. laxa and M. fructigena were present. To our knowledge, this is the first report of M. fructicola associated with brown rot of stone fruits in Slovenia.

ChemInform Abstract: Isolation and Structure Elucidation of Hymatoxins B - E and Other Phytotoxins from Hypoxylon mammatum Fungal Pathogen of Leuce Poplars.

Abstract Two groups of phytotoxic substances were isolated from a culture filtrate of the plant pathogenic fungus Hypoxylon mammatum (Wahl.) Miller, responsible of the canker formation in aspen. The main group consists of a mixture of original diterpenes, hymatoxins, from which have been isolated hymatoxins A, B, C, D and E that belong to the pimarane class. The minor group of toxins is composed of isomeric 3,4,5- and 3,4,8-trihydroxynapthalenones. The structures of the isolated compounds were determined by spectroscopic methods.

First Report of Diplodia corticola Causing Grapevine (Vitis vinifera) Cankers and Trunk Cankers and Dieback of Canyon Live Oak (Quercus chrysolepis) in California.

The botryosphaeriaceous fungus Diplodia corticola A. J. L. Phillips, Alves & Luque was shown to be the most prevalent canker and dieback pathogen in cork oaks (Quercus suber L.) in the Iberian Peninsula causing a general decline of the trees as a consequence of canker formation in the trunks (1). In addition, D. corticola has been recently reported as a grapevine pathogen causing cankers in the vascular tissue of 1-year-old canes, spurs, and cordons in Texas (3). In 1998, Jacobs and Rehner reported one isolate of D. corticola from oak in California, but no information regarding the oak species from which the isolate was obtained and its virulence were available in the study (2). In 2009, D. corticola was isolated on potato dextrose agar (PDA) amended with 0.01% tetracycline hydrochloride from symptomatic grapevine cordons and on acidified PDA from the trunk of a canyon live oak tree from Sonoma and Plumas counties, respectively. Two grapevine isolates (UCD1260So and UCD1275So) and one oak isolate (CDFA519) were examined and morphologically compared with previously identified D. corticola isolates CBS678.88 and UCD2397TX from cork oak from Spain and grapevine in Texas, respectively. D. corticola colonies from California were characterized by moderately fast-growing, dark olivaceous, and dense aerial mycelium on PDA. Conidia were obtained from pycnidia formed on pine needles placed on 2% water agar. Conidia were hyaline, contents granular, aseptate, thick walled, ellipsoidal, sometimes becoming dark brown and septate with age. Nucleotide sequences of three genes (ITS1-5.8S-ITS2, a partial sequence of the beta-tubulin gene BT2, and part of the translation elongation factor EF1-α) from D. corticola isolates UCD1260So, UCD1275So, and CDFA519 from California were amplified. All DNA sequences from grapevine and oak tree isolates from California showed 99 to 100% homology with D. corticola isolates previously identified and deposited into GenBank. All DNA sequences obtained from Californian isolates were also deposited into GenBank. Pathogenicity tests were conducted by inoculating detached Vitis vinifera cv. Red Globe dormant canes and canyon live oak branches with agar plugs of isolates UCD1260So, UCD1275So, and CDFA519 (10 inoculations per isolate per host) as described by Úrbez-Torres et al. (3). The same number of grapevine canes and oak branches were inoculated with noncolonized agar plugs as controls. Six weeks after inoculation, the extent of vascular discoloration that developed from the point of inoculation was measured. D. corticola isolates UCD1260So, UCD1275So, and CDFA519 caused an average vascular lesion length of 30.4, 29.6, and 24 mm and 15, 13.2, and 8.6 mm in grapevine dormant canes and oak branches, respectively. Furthermore, D. corticola isolates from grapevine were pathogenic in oak branches and vice versa. Reisolation of D. corticola from discolored vascular tissue of inoculated material was 100%. The extent of vascular discoloration from inoculated grapevine canes and oak branches was significantly greater (P < 0.05) compared with the controls (1.8 and 2 mm, respectively). No fungi were reisolated from the slightly discolored tissue of the controls. To our knowledge, this is the first report of D. corticola causing grapevine cankers and oak trunk cankers in California.

DETERMINATION OF HYPOVIRULENT ISOLATES OF CRYPHONECTRIA PARASITICA BY DSRNA ANALYSIS IN AYDIN PROVINCE, TURKEY

Cryphonectria parasitica (Murr.) Barr isolates which were obtained from the major chestnut growing areas of Aydm, Turkey and selected as hypovirulent according to the canker formation and their cultural characteristics were assayed for the presence of dsRNA. DsRNA was isolated from the mycelia of these isolates which were grown in liquid culture. After purification, the dsRNA's were analyzed by agarose gel electrophoresis. Among the isolates only one was found to contain dsRNA. It was observed that dsRNA banding patterns isolated from the single hypovirulent isolate and from known hypovirulent strains from the USA are similar in number and molecular size which is between 12.0 and 13.0 kbp dsRNA segment.

First report of Holocryphia eucalypti on Eucalyptus grandis in Uganda

In recent years, Uganda has embarked on extensive establishment of plantations of fast growing eucalypts to meet local demand for construction timber. In 2007, stem cankers were observed on a small number of five year-old Eucalyptus grandis trees in a clonal trial in Kifu, near Kampala in Uganda. The cankers comprised shallow fissures with orange fruiting bodies, and a sharply defined necrosis at the canker edge. The cankers were similar to those caused by the fungus Holocryphia eucalypti on eucalypts in South Africa. The morphological characteristics of the conidia (aseptate, hyaline, cylindrical characteristics) and the orange stromata also matched published descriptions of H. eucalypti. Spore masses from orange fruiting bodies on the bark of several trees were aseptically transferred to malt extract agar plates. Using protocols from Gryzenhout et al. (2006), DNA sequences of the internally transcribed spacer regions ITS1 and ITS2 and 5.8S rRNA were obtained. The Ugandan isolate sequences (GenBank GQ292540) gave 100% bootstrap values when compared with known isolates of H. eucalypti. Greenhouse inoculation studies on one E. grandis clone under quarantine conditions in South Africa confirmed that H. eucalypti from Uganda was pathogenic. Stem inoculations with H. eucalypti on young trees (∼5 mm diam) produced lesions within three weeks of inoculation, while trees with control inoculations using sterile agar discs produced no lesions. The fungus was re-isolated from the lesions but not the control plants. Previously, H. eucalypti was known only from Australia and South Africa (Gryzenhout et al., 2006). In Australia, H. eucalypti is associated with more severe stem cankers and is considered an important pathogen of stressed trees especially (Wardlaw, 1999). Symptoms in Uganda were similar to that in South Africa, with no tree mortality observed and only the formation of superficial bark cankers. The implications of this disease for Uganda are unclear and further monitoring of its status is needed.

First Report of Botryosphaeria dothidea Causing Canker and Shoot Blight of Eucalyptus in China.

Eucalyptus spp. are grown on approximately 2 million ha in China and is the most important genus in forest plantations worldwide. An outbreak of cankers and shoot blight was observed for the first time on Eucalyptus grandis in China during May of 2008. Symptoms included dieback of shoots and branches, lesions and canker formation on the stems, and clear or yellow-to-red exudates on stems and branches. Isolations were made from affected trees in Shilin County from May to July of 2008. Diseased samples were plated on potato dextrose agar (PDA) and incubated at 26°C. Fungal isolates developed copious, white, aerial mycelium that became dark gray after 5 to 6 days and formed black pycnidia after 14 days. Conidia were hyaline, aseptate, thin walled, fusiform, and measured 19 to 28 × 4 to 6 μm. Ascospores were hyaline, aseptate, and widest from the middle to upper third (17 to 28 × 6 to 13 μm). Identity was confirmed by analysis of the rDNA internal transcribed spacer region (ITSI-5.8S-ITS2) with primers ITS1 and ITS4. BLAST searches showed 99 to 100% identity with Botryosphaeria dothidea isolates from GenBank (Accession Nos. FJ358703 and EU080916). Representative sequences of B. dothidea from eucalyptus from China were deposited into GenBank (Accession Nos. FJ517657 and FJ517658). On the basis of morphological and molecular results, the fungus isolated from diseased eucalyptus wood was confirmed to be B. dothidea. Pathogenicity tests were conducted by stem inoculation of 10-month-old E. grandis seedlings. Two experiments were conducted using two inoculation techniques. In the first experiment, 2-mm-diameter actively growing mycelium plugs of B. dothidea were applied to 2-mm-long bark wounds on the middle point of the stems, and control seedlings were inoculated with sterile PDA plugs in a similar fashion as above. Inoculated and control seedlings were inoculated in a greenhouse and watered as needed. In the second experiment, segments of branches (averaging 18 mm in diameter and 30 cm long) were inoculated with 5-mm-diameter plugs of actively growing mycelium. Control segments of branches were inoculated as previously described. The branches were incubated at 26°C in moist chambers. There were five replicate seedlings per inoculation technique. After 20 days, all E. grandis seedlings showed leaf wilting, Dark, vascular stem tissue was observed. Symptoms were more abundant on the segment of branches. After 6 days, vascular necroses that developed on the inoculated plants were 5.2 ± 1.2 cm. B. dothidea was reisolated from all inoculated symptomatic tissue; no symptoms were visible in the control seedlings and no fungus was isolated from them. These results confirm previous reports of B. dothidea causing canker and dieback symptoms of Eucalyptus species in Australia (2), the United States (1), and South Africa (3). To our knowledge, this is the first report of B. dothidea causing canker disease on eucalyptus in China.

Identification and Pathogenicity of Lasiodiplodia theobromae and Diplodia seriata, the Causal Agents of Bot Canker Disease of Grapevines in Mexico

Perennial cankers and consequent grapevine dieback are a major problem in vineyards of Sonora and Baja California, the most important grape-production areas of Mexico. In order to identify the canker-causing agents, symptomatic arms, cordons, and trunks were collected from 13 and 6 vineyards in Sonora and Baja California, respectively. Two Botryosphaeriaceae spp., Lasiodiplodia theobromae and Diplodia seriata, were isolated frequently from infected wood and identified based on morphological and cultural characters as well as analyses of nucleotide sequences of three genes, the internal transcribed spacer region (ITS1-5.8S-ITS2), a partial sequence of the β-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). Although both L. theobromae and D. seriata were isolated from grapevine cankers in Baja California, only L. theobromae was found in vines in the Sonora region. Pathogenicity of both species was verified by inoculation of rooted cuttings and green shoots of Thompson Seedless and Chardonnay cultivars. Isolates of L. theobromae were more virulent, based on the extent of spread in the secondary wood and green tissue, than those of D. seriata. These findings confirm L. theobromae and D. seriata as the causal agents of dieback and canker formation of grapevines in northern Mexico.

Phenotypic and Genetic Analysis of Epiphytic Pseudomonas syringae Populations from Sweet Cherry in Michigan.

A severe outbreak of bacterial canker occurred on sweet cherry in Michigan in 2002. Blossom infection and subsequent canker formation was observed following a prolonged freeze event during bloom. Epiphytic blossom isolates of Pseudomonas syringae were recovered from 39 orchards from the three major cherry-growing areas (southwest [SW], west-central [WC], and northwest [NW]) of Michigan in 2003 and 2004. Average P. syringae populations over 2 years were 4.0, 5.1, and 4.8 log10 CFU/g of blossom tissue from the SW, WC, and NW areas, respectively. In 2003, copper-resistant P. syringae comprised 47.4, 21.1, and 3.1% of the total populations from the SW, WC, and NW areas, respectively, and levels of copper resistance were similar in 2004. Identification of 10 randomly chosen isolates from each orchard using polymerase chain reaction (PCR) assays indicated that 75 and 52% of the isolates from 2003 and 2004, respectively were P. syringae pv. syringae and that 1% and 23% of the isolates from 2003 and 2004, respectively, were P. syringae pv. morsprunorum. In addition, we were unable to determine the pathovar status of approximately 25% of the isolates each year, suggesting that a third P. syringae pathovar also was present in Michigan sweet cherry orchards. Pathogenicity on immature cherry fruit was confirmed for all P. syringae isolates. The frequency of ice nucleation was assessed for 44 individual P. syringae pv. syringae isolates, and the mean number of cells per active ice nucleus was 1,883. Extrapolating from this result, we estimated that active ice nuclei are present on most sweet cherry blossoms in Michigan orchards. Genetic fingerprinting of P. syringae pv. syringae using arbitrarily primed PCR indicated a high level of diversity and a clear differentiation of these organisms from the P. syringae isolates of unknown pathovar. A 2-year field trial evaluating the effect of dormant copper applications in spring and reduced-rate copper applications prior to bloom showed that these treatments were inconsistent in reducing P. syringae populations on blossoms.

First Report of Canker and Dieback Disease of Grevillea robusta in East Africa Caused by Botryosphaeria spp.

An outbreak of a canker and dieback disease was observed for the first time on Grevillea robusta in Uganda during October of 2001. Disease symptoms included dieback of shoots and branches, lesions and canker formation on the stems, clear or yellow-to-red exudates on stems and branches, formation of clusters of small and deformed leaves, and sapling and tree mortality. Isolations were made from the inner bark and wood of stems and branches in the Masaka and Rakai districts of Uganda in March and August to October of 2003. Samples from the border between healthy and necrotic tissues were plated on potato dextrose agar, vegetable juice agar, and water agar as well as on selective Phytophthora medium. Fungal isolates from several of the symptomatic trees were identified as belonging to the genus Botryosphaeria on the basis of morphological characteristics. Botryosphaeria-like fungi were not isolated from healthy trees. The internal transcribed spacer regions of the ribosomal DNA, as well as parts of the beta-tubulin and elongation factor 1-alpha genes, were sequenced from eight of these isolates. Five isolates matched Botryosphaeria parva in GenBank and one isolate matched B. rhodina. Two isolates matched an unidentified Botryosphaeria sp. previously isolated from Eucalyptus grandis in Uganda (GenBank Accession Nos. AY228102 and AY226852). Pathogenicity tests were performed by inoculating 10 18-month-old G. robusta seedlings with each isolate. A 10-mm3 malt agar plug with mycelium was inserted into a cut in the bark and the wound was sealed using grafting tape. One month after inoculation, the number of seedlings displaying death from the point of inoculation to the top (TM) and whole seedling mortality (SM) were recorded. Six of the isolates produced pathogenic responses in the seedlings: Botryosphaeria sp. isolate 1 (TM 10, SM 2), Botryosphaeria sp. isolate 2 (TM 10, SM 1), B. parva isolate 1 (TM 6, SM 1), B. parva isolate 2 (TM 3, SM 1), B. parva isolate 3 (TM 3, SM 0), and B. parva isolate 4 (TM1, SM 0). Inoculation with B. parva isolate 5 and B. rhodina, as well as sterile agar plugs, resulted in no TM or SM. The canker and dieback disease seems to be widespread in the East African Region since typical symptoms have been observed on G. robusta in Kenya (February 2004) and Ethiopia (February 2006). Botryosphaeria spp. are known to have multiple hosts and have been isolated from several other species in this region, including Eucalyptus spp. (1,2). From these findings, it appears that the same species of Botryosphaeria, which are previously known to be pathogenic on Eucalyptus, also cause serious disease on G. robusta.

First Report of Botryosphaeria iberica and B. viticola Associated with Grapevine Decline in California.

Grapevine decline symptoms in California include dead spurs and cordon and trunk dieback due to canker formation in the vascular tissue. Seven Botryosphaeria spp. are known to be associated with grapevine cankers in California, viz. Botryosphaeria australis, B. dothidea, B. lutea, B. obtusa, B. parva, B. rhodina, and B. stevensii (3). Recently, B. iberica and B. viticola also were isolated from grapevine cankers in a field survey that was conducted throughout California. Identification was based on morphological comparisons along with DNA analyses with previously identified isolates from Spain (1,2): B. iberica (CBS115035, ex-type) and B. viticola (CBS117006 and CBS117009, ex-type). DNA sequences of the rDNA internal transcribed spacer region (ITSI-5.8S-ITS2), part of the β-tubulin gene (BT2), and part of the translation elongation factor 1-α gene (EF1-α) from B. iberica and B. viticola isolates from California were amplified using primers ITS4/ITS5, Bt2a/Bt2b, and EF-728F/EF-986R, respectively. All DNA sequences of B. iberica and B. viticola from California showed 99 to 100% homology with those previously identified and deposited in GenBank. B. iberica, isolated from grapevine cankers from San Luis Obispo County (central coast), formed colonies on potato dextrose agar (PDA) that were dark green with aerial mycelium, optimum growth at 20 to 25°C, and formed pycnidia after 15 days of incubation at 25°C. Conidia were brown, one-septate, oblong to ovoid with a rounded apex, and measured (20.1-) 22.5 to 23.5 (-27.1) × (8.1) 9.3 to 9.8 (-11.2) μm, length/width ratio = 2.4 (n = 60). B. viticola, isolated from grapevine cankers in Sonoma (north coast), San Luis Obispo, Santa Barbara (south coast), Riverside (southern California), and Yolo (Sacramento Valley) counties, formed colonies on PDA that were dark green to grayish with aerial mycelium, optimum growth at 25°C, and formed pycnidia after 2 weeks. Conidia were brown, one-septate, oval to oblong, and measured (16.6-) 19.3 to 20.3 (-23.5) × (8.1) 9.3 to 9.6 (-11.1) μm, length/width ratio = 2.1 (n = 60). Two isolates of each species were used to complete pathogenicity tests (B. iberica: ATCC MYA-4110, ATCC MYA-4111; B. viticola: ATCC MYA-4115, ATCC MYA-4116). Ten fresh pruning wounds on 15-year-old cv. Zinfandel vines were inoculated per isolate using 50 μl of a 5 × 106 conidia per ml suspension. Twenty control pruning wounds were inoculated with the same amount of sterile water. Twelve months after inoculation, all wood inoculated with B. iberica and B. viticola showed internal necrosis extending 35 to 50 and 30 to 35 mm from the point of inoculation, respectively. Necrosis and extent of vascular discoloration in infected wounds was significantly greater (P < 0.05) than in control inoculations (6.5 mm). B. iberica and B. viticola were reisolated from the necrotic region surrounding all inoculation sites. Representative isolates of B. iberica and B. viticola from California were deposited at the American Type Culture Collection (B. iberica: MYA-4110, MYA-4111; B. viticola: MYA-4112 to MYA-4116). Sequences from the studied DNA regions of all isolates were deposited at GenBank. To our knowledge, this is the first report implicating either species as a cause of grapevine decline in California and B. iberica as a pathogen of Vitis vinifera anywhere in the world.

PATHOLOGICAL ANATOMY OF NECTRIA CANKER ON FRAXINUS MANDSHURICA VAR. JAPONICA

The anatomical characteristics of Nectria canker on Fraxinus mandshurica var. japonica were analyzed. Typical cankers were conspicuous, round to oval, with uniform concentric rings of affected xylem in a target-like structure. Each concentric annual growth ring was wider than the corresponding annual rings lateral to the cankers. The xylem elements were extremely disoriented. The cambial zone became discontinuous and disappeared. An inoculation test with the causal fungus, Nectria galligena, produced similar anatomical abnormalities and revealed the process of canker formation. Fewer and narrower vessels were formed, and water conduction took place only in the large vessels of the current year in the cankers.

First Report of Aspergillus Vine Canker of Table Grapes Caused by Aspergillus niger.

A vine canker was first observed in the San Joaquin Valley, CA, in fall 1989, on exceptionally vigorous 1-year-old cv. Redglobe vines (Vitis vinifera) when vines were trained up the stakes. Since 1989, the same canker symptoms have been observed in Tulare, Kern, Fresno, and Riverside (Coachella Valley, CA) counties on cv. Redglobe, Crimson Seedless, Chardonnay, and Grenache vines. In affected vineyards, the disease resulted in the retraining of 2 to 6.1% of vines the following spring, using a shoot originating from below the canker. In a sample of 54 infected vines collected in 1997, 65% of cankers were found at the branching (crotch) of the vine, 24% along the shoot, or both (11%). All infections started through wounds caused by removing lateral shoots or leaves when the vine was topped to form cordons or possibly through growth cracks that occur on rapidly growing 1-year-old shoots. The first symptoms usually appear in August as red pinhead-size drops of sap on the surface of discolored tissue. By October to November, the canopies of vines girdled by the canker prematurely display fall colors and are very distinct from healthy vines. The trunk is slightly swollen and spongy where the canker occurs. Internal canker tissue is discolored and dead. Black spores are abundant within the canker, on the surface of the canker, or both. Callous tissue is often associated with the canker as the vine attempts to repair the damage with new tissue. Canker length can range from 3.5 to 26.5 cm (average 7.0 cm) and can affect the shoot's cross section from 0.4 cm to completely girdling the shoot (up to 9.0 cm in circumference). Isolations from cankers or black sporulation inside the canker on acidified potato dextrose agar (APDA) consistently yielded Aspergillus niger van Tiegh. Six well-matured current-season canes of cv. Redglobe in an experimental vineyard at Kearney Agricultural Center were inoculated by inserting a 7-mm plug of mycelium from actively growing cultures on APDA in a cut made with a 7-mm cork borer or by brushing spores of the culture over the surface of six canes wounded with a sterile razor. Six canes were inoculated with a 7-mm plug of APDA and used as noninoculated controls. Inoculated sites were sealed with Parafilm to avoid dehydration. Inoculation of grapevines with A. niger resulted in cankers similar to those observed in commercial vineyards 5 months after inoculation. Cankers ranged from 2.4 to 4.2 cm for mycelial-plug inoculation (100% of canes infected) and 2.3 to 7.3 cm for spore-brushing inoculation (67% infected). Noninoculated control canes were not infected. In another experiment, inoculation of 10 canes each with A. niger on 17 May, 10 June, 2 July, 21 July, and 16 August resulted in 50, 60, 90, 90, and 100% canker formation, respectively, 5 to 8 months after inoculation, suggesting summer inoculations were more effective than spring inoculations. Reisolation from infected canes on APDA revealed A. niger. Aspergillus species in section Nigri have been reported to be among the pathogens involved in the bunch rot complex (1,2), but to our knowledge, this is the first report of A. niger causing a serious canker of vigorously growing grape vines.

Formation and growth of stem cankers caused by Gremmeniella abietina on young Pinus contorta

From 1990 to 1995, the formation and growth of stem cankers caused by Gremmeniella abietina on Pinus contorta var. latifolia was studied in three stands in northern Sweden. The stands were planted in 1976–80. The total number of cankers on 756 trees that were individually followed increased from 233 to 477 during the 5‐year period. With 42.0% of the cankers, the pathogen entered through or from the base of diseased branches, and 33.6% through visually undamaged bark. Most of the cankers were within 100 cm of the ground. In one of the three areas, the cankers were evenly distributed within 180 cm of the ground. The frequency of cankers facing north exceeded those facing south. The average vertical length of cankers had increased, 55.6% of cankers had increased their percentage coverage of the stem girth; 13.8% had fully girdled the stem. At two of the sites, there was a negative correlation between canker coverage of the stem circumference and tree height increment.

Differentiating A and B groups of Leptosphaeria maculans, causal agent of stem canker (blackleg) of oilseed rape

Stem canker or blackleg of brassicas, caused by Leptosphaeria maculans, is one of the most damaging diseases of winter oilseed rape in the UK. Airborne ascospores, released in autumn and winter, initiate leaf infections which may lead to colonization of the petiole and, later in the season, formation of stem lesions and cankers. Although isolates of the pathogen differ in ability to cause damaging stem cankers, this is not readily apparent from leaf spotting or stem lesion symptoms. However, several cultural, biochemical and genetic characteristics appear to be associated with the ability to form damaging stem cankers and isolates can be assigned to one of two groups, termed A and B, on the basis of differences in these characteristics. To investigate the relationship between leaf spotting symptoms and subsequent stem canker formation, and to improve understanding of the epidemiology of this pathogen, it is desirable to differentiate between the stem canker forming A group and the less damaging B group of L. maculans. Characterization of isolate type is also important in seed testing and crop breeding programs, particularly in countries such as Canada and Poland where the A type is not ubiquitous. This article reviews methods, including plant assays, assessments of growth characteristics in vitro, isozyme analyses, secondary metabolite profiling, serology, and nucleic acid analyses, that can be used to differentiate the A and B groups.

Comparison of Nucleotide Sequences of Canker-forming and Non-canker-forming pthA Homologues in Xanthomonas campestris pv. citri.

Three DNA regions in Xanthomonas campestris pv. citri which were homologous to the avrBs3 gene family commonly found in Xanthomonas spp. were cloned and their nucleotide sequences were determined. The sequences from 247bp upstream of the translational start to 111bp downstream from the termination site of the open reading frames in these regions were identical except in the tandem repeats of 102bp in the middle of their coding regions. Thus, they contain the same leucine zipper, nuclear localizing signals and inverted repeats as noted by De Feyter et al. Two of them enabled a nonpathogenic mutant missing one of these avrBs3 homologous regions to form a canker, but the remaining one did not even 30 days after inoculation. Comparison of the tandem repeats provided us more information on its organization required for canker formation.

Eutypa canker and dieback of almonds

Eutypa canker and dieback of almonds was first recorded in commercial plantations in Greece in mid‐1980 and confirmed as due to the ascomycetous fungus Eutypa lata. The disease manifests itself by the formation of cankers at the point of the junction of the branches and the trunk and is associated with pruning wounds made by the growers in order to confront the‘non‐infectious bud failure’disorder or to remove unwanted limbs close to the crotch. Pathogenicity tests made on young trees of cv. Texas with different isolates originated from grapevine, almond, apricot and pear showed significant differences in virulence. Pathogenicity tests carried out on 20 different almond cultivars gave a significant indication of the existence of tolerant and susceptible cultivars.

Shoot‐feeding Aphids Promote Development of Gremmeniella abietina, the Fungal Pathogen Causing Scleroderris Canker Disease in Conifers

AbstractWe tested experimentally whether shoot feeding aphids Cinara pinea (Mordv.) can promote the development of Gremmeniella abietina (Lagerb.) Morelet, a fungus which causes Scleroderris canker disease in conifers. Pine seedlings were infested with aphids at two different times, and subsequently inoculated with conidia of G. abietina at two different times. The degree of infestation was classified into three groups based on the number of aphids/seedling: none, one (low level), and three (high level). Because of parthenogenetic reproduction, the number of aphids increased during the summer, so we also used aphid numbers to explain the development of the symptoms. Necrosis caused by G. abietina was more prevalent in seedlings infested by aphids in June, and inoculated with fungus in July. Expressed as percents of the total length of the shoots, the necrosis had advanced 50%, 70%, and 95% with no, low, and high aphid levels, respectively. Canker formation was significantly greater when G. abietina conidia were inoculated in July, compared to August. In August, there were no statistically significant differences in the intensity of disease between aphid infestation levels, but the mean number of aphids was related to disease intensity. In the spring following infection, seedlings with aphids had more dead and fewer healthy terminal buds compared to seedlings without aphids.

Cloning and targeted disruption of enpg-1, encoding the major in vitro extracellular endopolygalacturonase of the chestnut blight fungus, Cryphonectria parasitica.

The gene enpg-1, encoding the major extracellular endopolygalacturonase (endoPG) purified from culture filtrates of the chestnut blight fungus, Cryphonectria parasitica, was cloned and characterized. The deduced mature enpg-1 protein product, ENPG-1, had a calculated molecular mass of 34.5 kDa and a pI of 7.2, consistent with empirically derived values for the purified enzyme, and had 66% identity with an endoPG from the maize pathogen Cochliobolus carbonum. Targeted disruption of enpg-1 was accomplished by homologous recombination with a cloned copy of the gene that contained the Escherichia coli hygromycin B phosphotransferase gene (hph) inserted into exon 1. enpg-1 disruption resulted in no reduction in canker formation on dormant American chestnut stems. Unexpectedly, the level of polygalacturonase (PG) activity measured in cankered bark tissue infected with enpg-1 disruptants was indistinguishable from that found in canker tissue infected with virulent strain EP155. Isoelectric focusing and activity gel analysis of PG activity extracted from canker bark tissue revealed ENPG-1 to be a minor (less than 5%) activity component in tissue infected with the virulent strain and to be absent in tissue infected with the disruption mutants. The predominant activity in both canker samples consisted of two previously undetected acidic PG forms that appear absent in C. parasitica culture filtrates. We conclude from these results that the major C. parasitica extracellular endoPG produced in culture, ENPG-1, does not play a significant role in fungal virulence. However, the identification of two acidic PG activities expressed predominantly, if not exclusively, in planta provides new opportunities for examining the importance of PGs in C. parasitica pathogenesis.