Botryosphaeriaceae Species Associated With Cankers and Dieback Symptoms of Acacia mangium and Pinus caribaea var. hondurensis in Venezuela.

Several species in the Botryosphaeriaceae family cause wood stain, cankers, and dieback of trunks and branches in a wide range of forest tree species. The aim of this study was to characterize the botryosphaeriaceous fungi associated with decline symptoms observed in Acacia mangium and Pinus caribaea var. hondurensis, two economically important forest tree species grown in commercial plantations in Venezuela. Fungi isolated from symptomatic samples collected from both hosts in commercial sites were identified based on their morphology and DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2) and part of the ß-tubulin and translation elongation factor 1-α genes. Lasiodiplodia theobromae and L. venezuelensis were routinely isolated from A. mangium and P. caribaea var. hondurensis. Additionally, the novel species Diplodia guayanensis was isolated and characterized from symptomatic and asymptomatic tissues of A. mangium. Multigene phylogenetic analyses along with restriction fragment length polymorphism studies further supported the identification of these species. A pathogenicity study was conducted under natural conditions and 12 weeks after inoculation all Botryosphaeriaceae spp. were shown to be highly virulent on A. mangium. Contrary, no lesions were observed in the wood of P. caribaea var. hondurensis when inoculated with L. theobromae and L. venezuelensis. However, both species were consistently reisolated from the asymptomatic tissue beyond the inoculation point. This study contributes to a better understand the role that species in the Botryosphaeriaceae play on disease symptoms and dieback of A. mangium and P. caribaea var. hondurensis from plantations in eastern Venezuela.

Cryptosphaeria Dieback of Fremont Cottonwood Caused by Cryptosphaeria pullmanensis and C. multicontinentalis in California.

Fremont cottonwood is a large and tall tree native to riparian areas in the southwestern United States. It grows along streams, rivers, and wetlands and serves many ecological and socioeconomic functions. In recent years, we observed a severe decline of Fremont cottonwood trees in California. Trees showed branches dying back, with dark-brown internal discoloration and decay of the wood of twigs, branches, or trunks. Eventually, the cambium and the bark were killed, causing a canker. The fungus Cryptosphaeria pullmanensis was isolated consistently from the necrotic wood of branches and twigs. On rare occasion, C. multicontinentalis was also isolated from symptomatic wood. Therefore, we investigated the pathogenicity in Fremont cottonwood of C. pullmanensis and C. multicontinentalis. Koch's postulates were completed in saplings and both species appeared highly virulent, producing internal necrosis and staining of the wood. This study is the first to report Cryptosphaeria dieback of Populus fremontii caused by C. pullmanensis and C. multicontinentalis. Symptoms and signs of this new disease are described and illustrated.

First Report of Lasiodiplodia pseudotheobromae Causing Trunk Cankers in Acacia mangium in Venezuela.

In May 2010, canker and wood stain symptoms in trunks and stems of 125 Acacia mangium were observed during a survey conducted in the Uverito plantations, Monagas State, Venezuela. Cankers were 20 to 65 cm long and were brownish on the margins and dark brown in the center. Many of the cankers had swollen margins and in some cases a black exudate could be seen leaking from the most severe cankers. Small pieces (4 to 5 mm) of necrotic tissues from the cankers were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar amended with 0.01% tetracycline hydrochloride (PDA-tet). Plates were incubated at 25°C under near-UV light. Colonies developed from symptomatic tissue and formed a compact mycelium, which was initially white, but became dark gray with age. Based on colony morphology, isolates were tentatively identified as a member of the Botryosphaeriaceae family. Pycnidia were produced on sterilized pine needles on 2% water agar after 5 weeks of incubation at 25°C under continuous near-UV light. Conidia were ellipsoidal, initially hyaline, unicellular, becoming dark brown, and developing a thick wall, a central septum, and longitudinal striations with age. Conidia measured 26 to 31 µm long and 11 to 16 µm wide (n = 60). The conidial morphology matched that of Lasiodiplodia, a member of the Botryosphaeriaceae family (1). Primers ITS4/ITS5, Bt2a/Bt2b, and EF1-688F/EF1-1251R (2) were used to amplify and subsequently sequence the ITS1-5.8S-ITS2 region and parts of the beta-tubulin (BT) and translation elongation factor 1-alpha (TEF1-α) gene regions, respectively. The putative Lasiodiplodia isolates had 98 to 99% homology with Lasiodiplodia pseudotheobromae isolate CBS 116459 for all three loci (EF622077, EF622057, and EU673111) (1). Based on morphological characters and DNA sequencing, the canker isolates from Venezuela (CBS129752 and UCD-A1) were then identified as L. pseudotheobromae (1) and sequences were deposited in GenBank (Accession Nos. JX545091 to JX545092, JX545111 to JX545112, and JX545131 to JX545132). Pathogenicity tests were performed by inoculating 2-year-old A. mangium tree trunks with isolates CBS129752 and UCD-A1. Twenty trees per isolate were inoculated by placing a mycelium plug from the growing margin of 8-day-old colonies upside down directly into a fresh wound made with a 5-mm cork borer. Wounds were sealed with Parafilm. Ten control trees were inoculated with non-colonized PDA plugs. After 12 weeks, all inoculated seedlings showed bark swelling around the inoculation points and a brown necrosis of the wood could be observed when removing the bark. Average length necrosis above and below the point inoculation was 27.2 cm; additionally, a black exudate was observed when the outer bark was removed from inoculation points. L. pseudotheobromae was successfully reisolated from the necrotic tissue observed in symptomatic plants. No symptoms were observed in the control plants and L. pseudotheobromae was not isolated from the controls. L. pseudotheobromae has been reported in Africa, Asia, Europe, and Latin America, where it occurs on forest and fruit trees (1). This study shows L. pseudotheobromae to be highly virulent on A. mangium and, to our knowledge, this is the first report of L. pseudotheobromae on this host in Venezuela. References: (1) A. Alves et al. Fungal Diversity 28:1, 2008. (2) J. R. Úrbez-Torres et al. Plant Dis. 90:1490, 2006.

First Report of Diplodia seriata Causing Pear Branch Canker Dieback in California.

California produces 26% of the United States pear crop on approximately 5,600 ha. A survey of seven northern California pear orchards (Pyrus communis cv. Bartlett) in summer 2010 revealed the presence of wedge-shaped cankers on 2- to 5-cm diameter branches, equating to 1- to 3-year-old wood. Many of the observed cankers occurred near pruning wounds, and there was decreased foliation on infected branches. Infected wood was surface disinfected with 95% ethanol and briefly flamed. After removing bark, small sections of diseased tissue were plated onto 4% potato dextrose agar (PDA) amended with 0.01% tetracycline and placed on the lab bench at 22°C until fungal growth emerged. Fungal colonies that were consistently isolated were transferred to fresh PDA using hyphal tip isolation. Fungal colonies were dark brown to gray with aerial mycelium and formed pycnidia after 15 days of incubation at 22°C. Conidia were brown, oval to oblong, and measured (16.5-) 20 to 24 (-26) × (7.5) 8.75 to 11 (-12.5) µm (n = 50). DNA from 14- to 21-day-old colonies was extracted and sequences of the rDNA internal transcribed spacer region and part of the ß-tubulin gene were amplified using primers ITS4/ITS5 and Bt2a/Bt2b, respectively (2). The DNA sequences of fungal isolates from California showed 99 to 100% homology with the ex-type Diplodia seriata De Not. (1) CBS112555 deposited in GenBank. DNA sequences from three California isolates were submitted to GenBank with accession numbers KC937062, KC937065, KF481957, KF481598, KF481959, and KF481960. Pathogenicity tests were performed in March 2011 on 3-year-old Bartlett pear trees planted at an experimental farm in Davis, CA. A single, circular, 2-cm pruning wound at the top of the trunk was inoculated on each of three single-tree replications using 2-cm mycelial plugs from 14-day-old colonies growing on PDA. After inoculation, mycelial plugs were covered and sealed with Parafilm and aluminum foil for the duration of the trial. Three control trees were inoculated using sterile PDA plugs. Twelve months after inoculation, UCD103 and UCD105 were consistently re-isolated from the margin between necrotic and healthy tissue using the same methods described for the original isolation, and UCD102 was re-isolated in two out of three plants. The average lesion lengths of UCD102, UCD103, UCD105, and control plants were 12.5, 17.3, 23, and 1 mm, respectively. Control lesions were short and sterile, and seemed to be a physiological reaction from the plant. A second pathogenicity test was completed in 5 months beginning in June 2012. UCD105 was consistently re-isolated, and UCD102 and UCD103 were re-isolated in two out of three plants. The average lesion lengths for UCD102, UCD103, UCD105, and control plants were 2, 3, 5, and 1 mm, respectively. Compared to grapevine (Vitis vinifera), the pathogen grows more slowly in pear tissue under natural conditions. To our knowledge, this is the first report describing D. seriata as a causal agent of pear branch canker in California. Canker diseases can reduce the lifespan of perennial plants, ultimately leading to long term economic losses for growers (3). References: (1) A. J. L. Phillips et al. Fungal Diversity 25:141, 2007. (2) J. R. Urbez-Torres et al. Plant Dis. 90:1490, 2006. (3) J. R. Urbez-Torres and W. D. Gubler. Plant Dis. 93:584, 2009.

First Report of Cytospora punicae Causing Wood Canker and Branch Dieback of Pomegranate (Punica granatum) in the United States.

Pomegranates (Punica granatum L.) are an expanding industry in the United States with California growing approximately 32,000 acres with a crop value of over $155 million (1). During June and July of 2012, we observed severe limb and branch dieback in pomegranate orchards cv. Wonderful located in Contra Costa, Kings, and Kern counties of California. Disease symptoms included yellowing of leaves, branch and limb dieback, wood lesions, and canker formation. Dark brown Cytospora-like cultures were consistently isolated from active cankers on potato dextrose agar (PDA) amended with 100 mg l-1 tetracycline hydrochloride. Three isolates (UCCE1223, UCCE1233, and UCCE1234) representative of each orchard were sub-cultured onto PDA and incubated at 22°C under fluorescent intermittent light (12 h light, 12 h dark). Fungal colonies had whitish mycelia that turned olive green to dark brown with maturity and formed globose and dark brown pycnidia after 12 days. Conidia were hyaline, aseptate, allantoid, and (4) 4.5 to 5 (6) × (1) 1.5 (2) µm (n = 180). Pycnidia formed in culture measured (250) 350 to 475 (650) µm in diameter (n = 40). Identification of the isolates was confirmed by sequence comparison of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rDNA and part of the translation elongation factor 1-α gene (EF1-α) with sequences available in GenBank. Consensus sequences of both genes of all isolates showed 99% homology to the species Cytospora punicae Sacc. (2). All sequences were deposited in GenBank (Accession Nos. KJ621684 to 89). Pathogenicity of the isolates was determined by branch inoculation. In December 2012, 3-year-old branches of P. granatum cv. Wonderful were inoculated by placing 5-mm-diameter mycelium plugs from the growing margin of 14-day-old PDA cultures in fresh wounds made with a 5-mm-diameter cork-borer. Eight branches per isolate were inoculated on eight different trees. Eight control branches were inoculated with non-colonized PDA agar plugs. Inoculations were covered with Vaseline and wrapped with Parafilm to retain moisture. Branches were harvested in August 2013 and examined for canker development and the extent of vascular discoloration spreading downward and upward from the inoculation point. Isolations from the edge of discolored tissue were conducted to fulfill Koch's postulates. C. punicae was re-isolated from 100% of the inoculated branches. Total length of vascular discoloration averaged 30.2 mm in branches inoculated with the three C. punicae isolates and 9 mm in the control branches. No fungi were isolated from the slightly discolored tissue of the controls. To our knowledge, this is the first report of C. punicae as a fungal trunk pathogen of pomegranate trees in the United States. References: (1) California County Agricultural Commissioners' Data, 2010 Crop Year. USDA NASS California field office, retrieved from http://www.nass.usda.gov/Statistics_by_State/California/ Publications/AgComm/201010cactb00.pdf , 2011. (2) P. A. Saccardo. Sylloge Fungorum 3:256, 1884.

Phomopsis Dieback: A Grapevine Trunk Disease Caused by Phomopsis viticola in California.

Field surveys recently conducted in California and in other grape-growing regions in the United States showed Phomopsis viticola to be one of the most prevalent fungi isolated from grapevine perennial cankers in declining vines. The current study has not only confirmed the presence of P. viticola from grapevine cankers in California but also has for the first time revealed the occurrence of Diaporthe ambigua, D. eres, and D. neotheicola in symptomatic grapevine wood in California by means of morphological studies and multi-gene sequence analysis. Pathogenicity trials conducted on mature cordons of Vitis vinifera 'Syrah' and 'Red Globe', as well as on lignified Syrah dormant canes, showed P. viticola isolates from California to be capable of causing perennial cankers. Lengths of vascular discoloration caused by P. viticola were similar to those caused by Eutypa lata and several Botryosphaeriaceae spp., which are well-known grapevine trunk disease pathogens. Additionally, a lack of spring growth was commonly observed in dormant canes inoculated with P. viticola spore suspensions in two pathogenicity trials. As part of this study, V. vinifera 'Cabernet Sauvignon' and 'Zinfandel' wood was shown to be more susceptible to infection by P. viticola than 'Barbera', 'Chardonnay', 'Merlot', and 'Thompson Seedless' wood. After more than 40 years overlooking P. viticola as a grapevine wood pathogen, this study provides strong evidence of the role of P. viticola as a canker-causing organism, and suggests its addition to the fungi involved in the grapevine trunk disease complex. Results from this study suggest D. ambigua and D. neotheicola to be saprophytes or weak pathogens on grapevine wood.

First Report of Neofabraea alba Causing Fruit Spot on Olive in North America.

Olive (Olea europaea) is a widely planted evergreen tree primarily grown for its oil, fruit for pickling, and landscape appeal in Mediterranean and temperate climates. California produces most of the olives grown in the United States; its industry was valued at $53 million in 2011 (4). In 2005 and 2008, fruit spotting occurred on coratina and picholine cultivars in two commercial orchards in Sonoma County. The spots were scattered, slightly sunken and brown, and surrounded by a green halo. Many of the spots were associated with lenticels. A slow to moderate growing, cream to rose-colored fungus was isolated from the spots onto potato dextrose agar (PDA) amended with 0.01% tetracycline hydrochloride. Sporulation was observed in vitro on PDA after 40 days under near-UV light. Macroconidia, produced from conidiomata, were hyaline, aseptate, cylindrical to fusiform-allantoid, slightly curved, and 17 to 27 × 2.5 to 3.5 µm (average 21.1 × 2.9 µm). Microconidia were aseptate, strongly curved, hyaline, and 14 to 18 × 0.75 to 1 µm (average 16.1 × 0.9 µm). rDNA sequences of the internal transcribed spacer (ITS) region of the isolate (GenBank KC751540), amplified using primers ITS1 and ITS4, were 99.8% identical to Neofabraea alba (E.J. Guthrie) Verkley (anamorph Phlyctema vagabunda) (=Gloeosporium olivae) (AF141190). Pathogenicity was tested on detached, green fruit (cv. frantoio). Olives were surface sterilized in 10% sodium hypochlorite for 5 min and air dried. Five olives were wounded with a needle and 10 µl spore suspension (105 spores/ml) was placed on each wound. An equal amount of spore suspension was placed on five unwounded olives. Water was also placed on wounded and unwounded olives to serve as a control. The olives were placed on racks in 22.5 × 30 cm crispers lined with wet paper towels and incubated at 23°C. After 21 days, the olives began to turn red. Olives wounded and inoculated with N. alba had a distinct green ring around the inoculation point where maturity was inhibited. Control olives uniformly turned red. After 35 days, wound-inoculated olives began to form a sunken, brown lesion at the inoculation point where aerial mycelium was visible. After 51 days, lesions were visibly sunken and immature conidiomata began to form in concentric rings giving a bull's eye-like appearance. Unwounded fruit exhibited uneven maturity and green spots associated with the lenticels throughout the experiment but did not develop sunken lesions. Control fruit showed no symptoms and ripened normally. After 56 days, fruit was surface sterilized in 10% sodium hypochlorite for 5 min and plated onto PDA. N. alba was isolated from the sunken and green areas of all of the wounded and unwounded fruit. No fungi grew from the control fruit. The experiment was repeated once with similar results. N. alba has been reported to cause an anthracnose disease on fruit and leaves of olives in Spain and Italy (1,2). In North America, N. alba causes a bull's eye rot on fruit of Malus and Pyrus spp. in the Pacific Northwest and coin canker of Fraxinus spp. in Michigan and Canada (3). To our knowledge, this is the first report of N. alba causing disease on olive in North America. References: (1) J. Del Maral de la Vega et al. Bol. San Veg. Plagas. 12:9. 1986. (2) S. Foschi. Annali. Sper. Agr., n.s. 9:911. 1955. (3) T. D. Gariepy et al. Can. J. Plant Pathol. 27:118. 2005. (4) United States Department of Agriculture, National Agricultural Statistics Service, California Field Office, California Agriculture Statistics, Crop Year 2011.

Olive Twig and Branch Dieback: Etiology, Incidence, and Distribution in California.

Eighteen different fungal species were isolated from symptomatic wood of olive trees (Olea europaea) affected by twig and branch dieback in California and identified by means of morphological characters and multigene sequence analyses of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), a partial sequence of the ß-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). These species included Diaporthe viticola, Diatrype oregonensis, Diatrype stigma, Diplodia mutila, Dothiorella iberica, Lasiodiplodia theobromae, Phaeomoniella chlamydospora, Phomopsis sp. group 1, Phomopsis sp. group 2, and Schizophyllum commune, which are for the first time reported to occur in olive trees; Eutypa lata, Neofusicoccum luteum, Neofusicoccum vitifusiforme, and Phaeoacremonium aleophilum, which are for the first time reported to occur in olive trees in the United States; and Botryosphaeria dothidea, Diplodia seriata, Neofusicoccum mediterraneum, and Trametes versicolor, which have been previously reported in olive trees in California. Pathogenicity studies conducted in olive cultivars Manzanillo and Sevillano showed N. mediterraneum and Diplodia mutila to be the most virulent species and Diatrype stigma and D. oregonensis the least virulent when inoculated in olive branches. Intermediate virulence was shown for the rest of the taxa. This study demystifies the cause of olive twig and branch dieback and elucidates most of the fungal pathogens responsible for this disease in California.

First Report of Ilyonectria macrodidyma Causing Root Rot of Olive Trees (Olea europaea) in California.

The California olive industry produces 99% of the U.S. olive crop, which represented a value of over $113 million in 2010. During the 2008 and 2009 growing seasons, decline of young super-high-density olive cvs. Arbequina, Arbosana, and Koroneiki trees (<4 years old) was observed in orchards throughout Glenn, Yolo, and San Joaquin Counties. Symptomatic trees showed stunted growth and chlorotic leaves with roots having black, sunken, necrotic lesions, which frequently prolonged into the base and crown of the tree. Twenty-five trees were collected from different orchards and necrotic roots as well as infected trunk tissue were plated onto potato dextrose agar amended with 0.01% tetracycline hydrochloride. Cultures were incubated at room temperature (23 ± 2°C) until fungal colonies were observed. In 17 out of 25 trees collected (68%), light yellow fungal colonies were observed from the symptomatic tissue after 7 to 10 days. Colonies turned dark yellow to orange with age and showed an orange-dark brown reverse. Both microconidia (hyaline, ellipsoidal to ovoidal and aseptate (n = 60) (6.5) 11.5 to 13.5 (17.1) × (3) 3.4 to 4.5 (5.6) µm) and macroconidia (hyaline, cylindrical, straight and/or slightly curved with one, two or three septa (n = 60) (12.5) 26.5 to 38.5 (44.1) × (4) 5.5 to 7.5 (8.5) µm) were observed. Culture and conidial morphology were in concordance with previous published description of Ilyonectria macrodidyma (Halleen, Schroers & Crous) P. Chaverri & C. Salgado (1,3,4). Identification to species level was confirmed by sequence comparison of four Californian isolates (UCCE958, UCCE959, UCCE960, and UCCE961) with sequences available in GenBank using the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rDNA (primers ITS1/ITS4), a portion of the ß-tubulin gene (BT1a/BT1b), and a partial sequence of the mitochondrial small subunit rDNA (NMS1/NMS2) (4). Fungal sequences of isolates from olive from California (GenBank JQ868543 to JQ868554) showed 99 to 100% homology with previously identified and deposited I. macrodidyma isolates in Genbank for all three genes. Pathogenicity of I. macrodidyma in olive cvs. Arbequina, Arbosan, and Koroneiki was investigated using two fungal isolates (UCCE958 and UCCE960) as reported by Petit and Gubler (4). The roots of 10 1-year-old trees per fungal isolate for each olive cultivar were individually inoculated with 25 ml of a 106 conidia/ml spore suspension and placed in a lath house at the UC Davis field station. Additionally, 10 trees per cultivar were inoculated with sterile water as controls. Six months after inoculation, most of the inoculated olive plants showed chlorotic leaves similar to those observed in commercial orchards. Root necrosis for each cv. was expressed as the percentage of root length having lesions (2). No significant difference was observed between isolates and average root necrosis was 29.4, 35.6, and 38.3% in Koroniki, Arbosana, and Arbequina, respectiveley. I. macrodidyma was recovered from symptomatic roots in each of the cvs. and identified based on morphology. No root rot symptoms were observed in the controls. To our knowledge, this is the first report of I. macrodidyma causing root rot of olive trees not only in California but anywhere in the world. References: (1) P. Chaverri et al. Stud. Mycol. 68:57, 2011. (2) M. Giovanetti and B. Mosse. New Phytol. 84:489, 1980. (3) F. Halleen et al. Stud. Mycol. 50:421, 2004. (4) E. Petit and W. D. Gubler. Plant Dis. 89:1051, 2005.

Calosphaeria Canker of Sweet Cherry Caused by Calosphaeria pulchella in California and South Australia.

California is the second largest sweet cherry producer in the United States with annual revenues up to $200 million. The South Australian cherry industry generates about 10% of Australia's overall production with approximately 1,500 metric tons of cherries produced yearly. In California, perennial canker diseases and subsequent branch dieback are responsible for extensive damage throughout sweet cherry orchards, reducing annual yields and tree longevity. Surveys of cherry orchards and isolation work were conducted in California to identify the main canker-causing agents. Calosphaeria pulchella was the main fungus isolated from cankers, followed by Eutypa lata and Leucostoma persoonii, respectively. Preliminary surveys in cherry orchards in South Australia documented C. pulchella and L. persoonii in cankers. The pathogenicity of C. pulchella in sweet cherry was confirmed following field inoculations of 2- to 3-year-old branches. C. pulchella was able to infect healthy wood and produce cankers with as much virulence as E. lata or L. persoonii. Spore trapping studies were conducted in two sweet cherry orchards in California to investigate the seasonal abundance of C. pulchella spores. Experiments showed that rain and sprinkler irrigation were important factors for aerial dissemination. Finally, this study illustrates the symptoms and signs of the new disease Calosphaeria canker.

Host range, biological variation, and phylogenetic diversity of Eutypa lata in California.

The objectives of this study were to investigate the host range of Eutypa lata in the major grape-growing regions in California and to analyze the phenotypic variation and phylogenetic diversity of E. lata isolates. Perithecia of E. lata were found on grapevines, in apricot, almond, cherry, apple, and pear tree orchards, and on ornamentals (oleander) and native plant species (California buckeye, big leaf maple, and willow). Multigene phylogenies of ribosomal DNA internal transcribed spacer, ß-tubulin, and RPB2 genes confirmed the identity of E. lata recovered from the various host plants but also revealed sequence differences among isolates. The intraspecific phylogenetic diversity of E. lata did not correspond to geography or source of isolation, and intraspecific groups were not consistent across the different DNA phylogenies. Significant phenotypic variation also was detected among E. lata isolates, including ascospore and conidium length as well as level of aggressiveness on grapevines. Pathogenicity studies proved that all isolates were infectious to grapevine, suggesting that the native vegetation surrounding vineyards can serve as inoculum sources that may constitute an important element in the epidemiology of Eutypa dieback in grapevines.

Pathogenicity of Diatrypaceae Species in Grapevines in California.

Diatrypaceous fungi have been isolated from cankered wood of grapevines (Vitis vinifera) in California. However, the pathogenicity of these fungi and their potential to cause diseases in grapevine was unknown. A series of pathogenicity tests were conducted to determine if these fungi were grapevine pathogens. In all experiments, species of the family Diatrypaceae were re-isolated from the margins of developing lesions, although frequency of re-isolation and average length of vascular discolorations varied among isolates. Cryptovalsa ampelina, Diatrype stigma, and Eutypa leptoplaca were capable of colonizing dormant canes and causing vascular necrosis. Cryptosphaeria pullmanensis, C. ampelina, D. stigma, Diatrype whitmanensis, and E. leptoplaca infected and caused lesions in green shoots of the new vegetative growth. Diatrype oregonensis and Diatrypella verrucaeformis isolates did not produce lesions that were significantly different from those produced in the control shoots and canes. This suggests that D. oregonensis and D. verrucaeformis fungi may be saprophytic rather than pathogenic to grapevine.

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 ß-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. References: (1) P. W. Crous et al. Fungal Planet 19, 2007. (2) P. Inderbitzin et al. Mycologia. Online publication. doi:10.3852/10-006, 2010. (3) J. R. Úrbez-Torres et al. Plant Dis. 94:785, 2010.

First Report of Calosphaeria pulchella Associated with Branch Dieback of Sweet Cherry Trees in California.

California is the second largest sweet cherry producer in the United States with approximately 10,800 ha and an average annual crop value of approximately $150 million. Perennial canker diseases constitute major threats to the cherry industry productivity by reducing tree health, longevity, and yields. During the course of summer 2006, we observed severe limb and branch dieback of sweet cherry (Prunus avium L.) in San Joaquin, San Benito, Contra Costa, and Stanislaus counties of California. Isolation from diseased branches repeatedly yielded the fungus Calosphaeria pulchella (Pers.: Fr.) J. Schröt. (1,2). Cankers and vascular necroses had developed in tree limbs and branches, generally initiating from the heart wood and later spreading into the sapwood. External symptoms of disease may be unapparent throughout the early stages of infection, particularly in large diameter shoots. Older infections often appeared as wilted leaves. Branches and trunks affected with cankers from which C. pulchella was isolated also generally bore perithecia of C. pulchella beneath the periderm. Perithecia were nonstromatic and arranged in dense, circinate groups, with elongated necks converging radially and fissuring the periderm. Asci were unitunicate, clavate, and 45 to 55 × 5 to 5.5 µm. Ascospores were allantoid to suballantoid, hyaline, and 5 to 6 × 1 µm. Colonies on potato dextrose agar (PDA) were dark pink to red in their center with a white margin. Conidia were hyaline, allantoid to oblong-ellipsoidal, and (3-) 4 to 6 (-9) × 1.5 to 2 (-2.5) µm. Identification of C. pulchella isolates also was confirmed by sequence comparison in GenBank database using the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rDNA. Sequences of California isolates shared 100% similarity with C. pulchella reference isolate CBS 115999 (EU367451) (2). ITS sequences of the California isolates used in this study were deposited into GenBank (Nos. HM237297 to HM237300). Pathogenicity of four isolates recovered from the margin of active cankers was determined by branch inoculations. In December 2006, 2- to 4-year-old twigs of P. avium cv. Bing were inoculated with a 5-mm cork borer to remove bark and by placing an agar plug from the growing margin of 8-day-old colonies directly into the fresh wound, mycelium side down. Ten branches per isolate were inoculated. Ten control shoots were inoculated with noncolonized agar plugs. Inoculations were covered with vaseline and wrapped with Parafilm to retain moisture. Branches were harvested in July 2007 and taken to the laboratory to be examined for canker development, and the extent of vascular discoloration in each branch was assessed. Isolations from the edge of discolored tissue were conducted to fulfill Koch's postulates. After 8 months, C. pulchella was reisolated from 100% of the inoculated branches. Length of vascular discoloration averaged 62.5 mm in branches inoculated with the four C. pulchella isolates and 16.5 mm in the control twigs. No fungi were reisolated from the slightly discolored tissue of the controls. To our knowledge, this study constitutes the first report of C. pulchella as a pathogen of sweet cherry trees in California. References: (1) M. E. Barr. Mycologia 77:549, 1985. (2) U. Damm et al. Persoonia 20:39, 2008.

Botryosphaeriaceae Species Spore-Trapping Studies in California Vineyards.

The seasonal abundance of Botryosphaeriaceae spp. spores was studied in California vineyards by using glass microscope slides covered with petroleum jelly placed on grapevine cordons and Burkard volumetric spore traps at seven and two different locations, respectively. Correlation analysis was used to determine which meteorological variables (precipitation, relative humidity, temperature, and wind speed) influenced Botryosphaeriaceae spp. spore release. Among all variables, regression analysis resulted in a strong relationship between spore release and precipitation. Additionally, a positive relationship between irrigation and spore release was also observed in the Riverside County vineyard. During the study period, spore discharge of Botryosphaeriaceae spp. occurred from the first fall rain through the last spring rains, coinciding with September to April. However, based on the results obtained from the spore traps, most spores (over 60%) were trapped following rain events during the winter months of December, January, and February, which coincides with the grapevine pruning season. Botryosphaeriaceae spp. spore release was much lower in fall and early spring (22%) and very few or no spores were trapped in late spring and summer (3%). This work suggests that a delay of pruning time in California may be warranted to reduce grapevine infection because the current timing coincides with the greatest period of spore discharge.

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. References: (1) A. Alves et al. Mycologia 96:598, 2004. (2) K. A. Jacobs and S. A. Rehner. Mycologia 90:601, 1998. (3) J. R. Úrbez-Torres et al. Am. J. Enol. Vitic. 60:497, 2009.

First Report of Grapevine Cankers Caused by Lasiodiplodia crassispora and Neofusicoccum mediterraneum in California.

Several species in the Botryosphaeriaceae family cause perennial cankers in the vascular tissue of grapevines and are responsible for the disease known as bot canker in California (3). Tissue from grapevine vascular cankers from samples submitted to our laboratory in the summer of 2009 were plated onto potato dextrose agar (PDA) amended with 0.01% tetracycline hydrochloride. Lasiodiplodia crassispora (Burgess & Barber) and Neofusicoccum mediterraneum (Crous, M.J. Wingf. & A.J.L. Phillips) were identified based on morphological and cultural characters as well as analyses of nucleotide sequences. L. crassispora isolates were characterized by a fast-growing, white mycelium that turned dark olivaceous with age on PDA. Conidia from pycnidia formed in cultures were thick walled and pigmented with one septum and vertical striations when mature. Conidia measured (25.8-) 27.5 to 30.5 (-33.4) × (12.1) 14.3 to 16.8 (-18.2) µm (n = 60). Pycnidia contained septate paraphyses. N. mediterraneum was characterized as having moderately fast-growing, light green mycelia on PDA. Pycnidia formation was induced with pine needles placed on 2% water agar. Conidia from pycnidia were hyaline, ellipsoidal, thin walled, unicellular, and measured (18.2-) 20.5 to 27.8 (-29) × (5.1) 5.9 to 6.5 (-7.2) µm (n = 60). DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2), part of the ß-tubulin gene (BT2), and part of the translation elongation factor 1-α gene (EF1-α) from L. crassispora (UCD23Co, UCD24Co, and UCD27Co) and N. mediterraneum (UCD695SJ, UCD719SJ, UCD720SJ, UCD749St, and UCD796St) grapevine isolates from California were amplified and sequenced. Consensus sequences from L. crassispora and N. mediterraneum from California showed 99 to 100% homology with L. crassispora and N. mediterraneum isolates previously identified and deposited in GenBank (1,2). Sequences from the examined DNA regions of all isolates were deposited at GenBank (GU799450 to GU799457 and GU799473 to GU799488). Pathogenicity tests using three isolates per species were conducted on detached dormant canes of cv. Red Globe. Ten canes per isolate were inoculated by placing a 7-day-old 5-mm-diameter agar plug from each fungal culture into a wound made with a drill on the internode (4). Twenty shoots were inoculated with noncolonized PDA plugs for negative controls. Six weeks after inoculations, necrosis was measured from the point of inoculation in both directions. One-way analysis of variance was performed to assess differences in the extent of vascular discoloration and means were compared using Tukey's test. L. crassispora isolates caused an average necrotic length of 21.1 mm, which was significantly lower (P < 0.05) than the average necrotic length of 35.6 mm caused by the N. mediterraneum isolates. Reisolation of L. crassispora and N. mediterraneum from necrotic tissue was 100% for each species. The extent of vascular discoloration in infected canes was significantly greater (P < 0.05) than in control inoculations (8 mm) from which no fungi were reisolated from the slightly discolored tissue. To our knowledge, this is the first report of L. crassispora and N. mediterraneum as pathogens of Vitis vinifera and as a cause of grapevine cankers in California. References: (1) T. I. Burgess et al. Mycologia 98:423, 2006. (2) P. W. Crous et al. Fungal Planet. No. 19, 2007. (3) J. R. Úrbez-Torres and W. D. Gubler. Plant Dis. 93:584, 2009. (4) J. R. Úrbez-Torres et al. Am. J. Enol. Vitic. 60:497, 2009.

Pathogenicity of Botryosphaeriaceae Species Isolated from Grapevine Cankers in California.

Fungal species in the family Botryosphaeriaceae have been recently recognized as the most common fungi isolated from grapevine (Vitis vinifera) cankers in California. However, the role of these fungi in causing grapevine dieback as well as their status as canker-causing agents was unknown. Therefore, pathogenicity studies were conducted to determine their importance as grapevine pathogens in California. A total of 72 isolates representing all nine Botryosphaeriaceae species isolated from grapevine cankers from California were used in five different pathogenicity studies. Overall, experiments showed all nine Botryosphaeriaceae species able to infect both young and mature tissues as well as green shoots of the new vegetative growth causing cankers, vascular discoloration, and/or otherwise dark streaking of the wood. However, virulence varied among species. Lasiodiplodia theobromae was the most virulent species followed by Neofusicoccum luteum, N. parvum, and N. australe, all categorized as highly virulent. Botryosphaeria dothidea was considered intermediately virulent and Diplodia mutila, D. seriata, Dothiorella iberica, and D. viticola were shown to be weakly virulent. This study shows species of Botryosphaeriaceae to be much more important pathogens on grapevines than originally thought, and some of them, in view of their virulence, should be considered high risk for causing severe and rapid canker and dieback diseases in the grapevine industry in California.

Microsatellite markers for the grapevine pathogen, Eutypa lata.

We isolated and characterized nine polymorphic microsatellite markers for Eutypa lata, a fungal pathogen responsible for Eutypa dieback of grapevine, in populations from two California vineyards (24 isolates per vineyard). Allele frequency ranged from two to 11 alleles per locus and haploid gene diversity ranged from 0.33 to 0.83. All samples comprised unique haplotypes. Our results suggest that there is sufficient allelic polymorphism to estimate fine-scale spatial structure, and to identify possible sources of inoculum from habitats outside of vineyards.

Pathogenesis of Eutypa lata in grapevine: identification of virulence factors and biochemical characterization of cordon dieback.

Eutypa lata is a vascular pathogen of woody plants. In the present study we (i) determined which component(s) of the cell wall polymers were degraded in naturally infected grapevines and in artificially inoculated grape wood blocks; (ii) compared the pattern of wood decay in the tolerant grape cv. Merlot versus the susceptible cv. Cabernet Sauvignon; and (iii) identified secondary metabolites and hydrolytic enzymes expressed by E. lata during wood degradation. Biochemical analyses and a cytochemical study indicated that glucose-rich polymers were primary targets of E. lata. Structural glucose and xylose of the hemicellulose fraction of the plant cell wall and starch were depleted in infected woods identically in both cultivars. Moreover, the more tolerant cv. Merlot always had more lignin in the wood than the susceptible cv. Cabernet Sauvignon, indicating that this polymer may play a role in disease resistance. In vitro assays demonstrated the production by E. lata of oxidases, glycosidases and starch degrading enzymes. Phytotoxic secondary metabolites were also produced but our data suggest that they may bind to the wood. Finally, we demonstrated that free glucose in liquid cultures repressed primary but not secondary metabolism.