First Report of Geosmithia pallida Causing Foamy Bark Canker, a New Disease on Coast Live Oak (Quercus agrifolia), in Association with Pseudopityophthorus pubipennis in California.

Declining coast live oak (Quercus agrifolia) trees have been observed since 2012 throughout urban landscapes in Los Angeles, Orange, Riverside, Santa Barbara, Ventura, and Monterey counties in California. Symptoms causing branch dieback and tree death included a cinnamon-colored gum seeping through multiple 0.95-mm-diameter entry holes on the bole, followed by a prolific, cream-colored foamy liquid. Beneath the outer bark was phloem and xylem necrosis. Fifty 1- to 2.5-mm adult and larval beetles were collected. Adults fit the morphological description of Pseudopityophthorus pubipennis (western oak bark beetle) (R. Rabaglia, personal communication), and ~800 bp of the mitochondrial COI gene was amplified for three beetles using primer pairs and methods previously described (2,3). All three sequences were identical (GenBank Accession Nos. KJ831289 to 91) and a BLAST search confirmed the closest match (94%) as P. pubipennis. Necrotic wood tissues collected from two trees in each county were cultured on potato dextrose agar amended with 0.01% tetracycline (PDA-tet), and incubated at 25°C for 1 week. Ochre-colored cultures with plane or radially furrowed velutinous mycelium were consistently produced. Fifty conidia each measured from two isolates were 3.66 ± 0.04 µm × 1.77 ± 0.03 µm, and arranged in non-persistent conidial chains, at first roughly parallel, becoming tangled with age. These fungal colonies were observed within gallery walls. The rDNA internal transcribed spacer (ITS) was amplified using primer pairs and methods previously described (5). Three isolates were sequenced and matched 100% to known sequences of Geosmithia pallida in GenBank; sequences of two isolates (UCR2208 and UCR2210) were deposited in GenBank (KJ468687 and KJ468688). Pathogenicity tests were performed by inoculating twelve 27.0-cm detached coast live oak shoots for each isolate with a spore suspension of G. pallida (UCR2208 and UCR2210) and sterile distilled water for controls. A 2-mm-wide, 3-mm-deep hole was drilled into the center of each shoot, 20 µl of a 106 conidia/ml spore suspension was pipetted into the hole, and sealed with Vaseline and Parafilm. The experiment was repeated twice. After 4 weeks in a moist chamber at 25°C, lesions produced by G. pallida averaged 8.3 cm and was significantly longer (ANOVA; P < 0.0001) from the control (average 0.4 cm). G. pallida was re-isolated from all inoculated plants and identified by colony morphology. P. pubipennis is a native beetle, common as a secondary agent, and previously not associated with disease. However, cryptic species may be common among bark and ambrosia beetles (4). A larger sample (i.e., populations and loci) is needed to determine the precise taxonomic status of P. pubipennis. G. pallida was shown to inhibit root growth of Q. petraea by 25% in Europe (1), appears to have affinities with a range of subcorticolous insects, and is widely distributed (5), but there is no published record of the fungus occurring in the United States. This is the first report of G. pallida causing foamy bark canker in association with P. pubipennis on Q. agrifolia in California. Results suggest this new disease complex is causing decline of Q. agrifolia throughout the state. References: (1) D. Cizková et al. Folia Microbiol. 50:59, 2005. (2) A. I. Cognato and F. A. H. Sperling. Mol. Phylogenet. Evol. 14:445, 2000. (3) A. I. Cognato et al. Mol. Phylogenet. Evol. 36:494, 2006. (4) B. H. Jordal and M. Kambestad. Mol. Ecol. Res. 14:7, 2014. (5) M. Kolarík et al. Mycol. Res. 108:1053, 2004.

Identification of Species of Botryosphaeriaceae Causing Bot Gummosis in Citrus in California.

Members of the Botryosphaeriaceae family are known to cause Bot gummosis on many woody plants worldwide. To identify pathogens associated with Bot gummosis on citrus in California, scion and rootstock samples were collected in 2010 and 2011 from five citrus-growing counties in California. Symptoms observed on citrus included branch cankers, dieback, and gumming. Various fungal species were recovered from necrotic tissues of branch canker and rootstock samples. Species were identified morphologically and by phylogenetic comparison as 'Eureka' lemon, 'Valencia', 'Washington Navel', 'Fukumoto', grapefruit, 'Satsuma', and 'Meyer' lemon. Species were identified morphologically and by phylogenetic comparison of the complete sequence of the internal transcribed spacer regions, ß-tubulin gene, and elongation factor α-1 genes with those of other species in GenBank. A consensus-unrooted most parsimonious tree resulting from multigene phylogenetic analysis showed the existence of three major clades in the Botryosphaeriaceae family. In total, 74 isolates were identified belonging to the Botryosphaeriaceae family, with Neofusicoccum spp., Dothiorella spp., Diplodia spp., (teleomorph Botryosphaeria), Lasiodiplodia spp., and Neoscytalidium dimidiatum (teleomorphs unknown) accounting for 39, 25, 23, 10, and 3% of the total, respectively. On inoculated Eureka lemon shoots, lesion length was significantly different (P < 0.05) among 14 isolates recovered from portions of cankered tissues of the original trees. Lesion lengths were significantly longer (P < 0.05) for shoots inoculated with isolates of Neofusicoccum luteum and shorter for shoots inoculated with isolates of Dothiorella viticola (P < 0.05) than those of other species. Identifying the distribution and occurrence of these fungal pathogens associated with Bot gummosis is useful for management applications during occasional outbreaks in California.

First Report of a Fusarium sp. and Its Vector Tea Shot Hole Borer (Euwallacea fornicatus) Causing Fusarium Dieback on Avocado in California.

Per capita consumption of avocado in the United States has nearly doubled between 2000 and 2010. The California avocado industry supplies almost 40% of U.S. demand and the remaining 60% is supplied by imports from Latin America and New Zealand. The Tea Shot Hole Borer (TSHB) is an ambrosia beetle from Asia that forms a symbiosis with a new, yet undescribed Fusarium sp. and is a serious problem for the Israeli avocado industry (3). The beetle also causes severe damage on the branches of tea (Camelia sinensis) in Sri Lanka and India (1). In California, TSHB was first reported on black locust (Robinia pseudoacacia) in 2003, but there are no records of fungal damage (4). In 2012, nine backyard avocado trees (cvs. Hass, Bacon, Fuerte, and Nabal) exhibiting branch dieback were observed throughout the residential neighborhoods of South Gate, Downey, and Pico Rivera in Los Angeles County. Upon inspection, symptoms of white powdery exudate, either dry or surrounded by wet discoloration of the outer bark in association with a single beetle exit hole, were found on the trunk and main branches of the tree. Examination of the cortex and wood under the exit hole revealed brown discolored necrosis. The TSHB was also found within galleries that were 1 to 4 cm long going against the grain. Symptomatic cortex and sapwood tissues were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet). The TSHB was dissected and plated onto PDA-tet after surface disinfestation following methods described by Kajimura and Hijii (2). After 5 days of incubation at room temperature, regular fungal colonies with aerial mycelia and reddish brown margins were produced. Single spore isolations were used to establish pure culture of the fungus. Fifty conidia were hyaline, clavate with a rounded apex, and initially aseptate (4.1 to 12.0 × 2.4 to 4.1 µm) becoming one- to three-septate (7.6 to 15.1 × 2.8 to 4.5 µm, 9.2 to 17.2 × 3.4 to 4.8 µm, and 13.5 to 17.6 × 4.3 to 4.7 µm, respectively). Identity of the fungal isolates was determined by amplification of the rDNA genes with primers ITS4/5 and EF1/2, respectively. Sequences were deposited into GenBank under Accession Nos. JQ723753, JQ723760, JQ723756, and JQ723763. A BLASTn search revealed 100% similarity to Fusarium sp. (Accession Nos. JQ038020 and JQ038013). Detached green shoots of healthy 1-year-old avocado were wounded to a depth of 1 to 2 mm and 5-mm mycelial plugs from 5-day-old cultures (UCR 1781 and UCR 1837) were placed mycelial side down onto the freshly wounded surfaces and then wrapped with Parafilm. Control shoots were inoculated with sterile agar plugs and five replicates per treatment were used. Shoots were incubated at 25 ± 1°C in moist chambers for 3 weeks. Lesions were observed on all inoculated shoots except for the control. Mean lesion lengths were 10.7 and 12.8 cm for UCR1781 and UCR1837, respectively, and were significantly different (P ≤ 0.05) from the control. Both isolates were reisolated from 100% of symptomatic tissues of inoculated shoots to complete Koch's postulates. This experiment was conducted twice and similar results were obtained. To our knowledge, this is the first report of Fusarium sp. and its vector E. fornicatus causing Fusarium dieback on Avocado in California. References: (1) W. Danthanarayana. Tea Quarterly 39:61, 1968. (2) H. Kajimura and N. Hijii. Ecol. Res. 7:107, 1992; (3) Mendel et al., Phytoparasitica, DOI 10.1007/s12600-012-0223-7, 2012. (4) R. J. Rabaglia. Annals Entomol. Soc. Amer. 99:1034, 2006.

First Report of Bot Canker Caused by Diplodia corticola on Coast Live Oak (Quercus agrifolia) in California.

Sharp decline and mortality of coast live oak (Quercus agrifolia) has been observed in San Diego County, CA since 2002. Much of this decline has been attributed to a new pest in California, the goldspotted oak borer (GSOB, Agrilus coxalis) (1). Symptoms include crown thinning, bark cracking and/or peeling, patches of stain (1 to 10 cm in diameter), bleeding on the bole, and tree death and are most often observed on trees with a diameter at breast height (DBH) >30 cm. In 2008, a Botryosphaeria sp. was recovered from necrotic tissue of bleeding bole cankers from GSOB-affected trees in Jamul, CA. Zone lines separated dead and live tissue in affected phloem and xylem. Pycnidia were observed on the bark surface of the infected host. Fifty conidia averaging 32 × 18 µm, one-septate with age, and morphologically similar to conidia described by Úrbez-Torres et al. were observed (4). Oak stands with tree mortality were surveyed in GSOB-infested and -uninfested sites over eight locations throughout San Diego and Riverside counties in 2009 and 2010. Symptomatic tissue or conidia from pycnidia of affected trees, plated onto potato dextrose agar amended with 0.01% tetracycline and incubated at 25°C for 1 week, consistently produced cultures with dense, wooly, olive-green mycelium. Mycelia fit the description of Botryosphaeria corticola A.J.L. Phillips, Alves et Luque (anamorph Diplodia corticola) (2). The resulting amplified ITS4/5 region of two sequences matched 100% to published D. corticola sequences (GU799472 and GU799460) (4). These sequences were deposited with NCBI GenBank (HM104176 and HM104177). Koch's postulates were conducted by inoculating 2-mm-diameter holes on five coast live oak trees with D. corticola. Holes were drilled to the cambium at 2 to 4 locations per tree within 1 to 2 m up the bole using a 0.157-cm portable electric drill. Trees ranged from 3.7- to 32.4-cm DBH. Either single agar plugs from two isolates each of a 7-day-old culture (UCR454 and UCR793) or noncolonized agar plugs as uninoculated controls were inserted into the holes and then covered with petroleum jelly and Parafilm. Average temperature was 10°C, relative humidity of 64%, and no precipitation during inoculation. Inoculations were conducted at a location in San Diego County uninfested by GSOB and repeated twice. After 3.5 months, bark was removed from inoculation sites. Average lesion length was not significantly different between inoculations, thus data were combined (one way analysis of variance [ANOVA]; P = 0.05). Lesions averaged 13.9 × 2.3 cm and were significantly different (n = 30; one way ANOVA; P = 0.05) from controls that measured 0.31 × 0.3 cm. Staining was observed around the inoculation points on all trees and three trees exhibited bleeding. Necrotic tissue was observed in the phloem and 3 mm into the xylem tissue, where the lesion had extended up and down the grain. D. corticola was consistently reisolated from necrotic tissue but not from control treatments. B. corticola was originally described as a canker pathogen on Quercus spp. in the western Mediterranean (2), and is known to contribute to the decline of cork oak (Q. suber) in the region (3). To our knowledge, this is the first report of D. corticola causing bot canker on coast live oak in California. References: (1) T. W. Coleman and S. J. Seybold. U. S. For. Serv. R5-PR-08, 2008. (2) A. Correia et al. Mycologia 96:598, 2004. (3) J. Luque et al. For. Pathol. 38:147, 2008. (4) J. R. Úrbez-Torres et al. Plant Dis. 94:785, 2010.

Survival, dispersal, and potential soil-mediated suppression of Phytophthora ramorum in a California redwood-tanoak forest.

Because the role of soil inoculum of Phytophthora ramorum in the sudden oak death disease cycle is not well understood, this work addresses survival, chlamydospore production, pathogen suppression, and splash dispersal of the pathogen in infested forest soils. Colonized rhododendron and bay laurel leaf disks were placed in mesh sachets before transfer to the field in January 2005 and 2006. Sachets were placed under tanoak, bay laurel, and redwood at three vertical locations: leaf litter surface, litter-soil interface, and below the soil surface. Sachets were retrieved after 4, 8, 20, and 49 weeks. Pathogen survival was higher in rhododendron leaf tissue than in bay tissue, with >80% survival observed in rhododendron tissue after 49 weeks in the field. Chlamydospore production was determined by clearing infected tissue in KOH. Moist redwood-associated soils suppressed chlamydospore production. Rain events splashed inoculum as high as 30 cm from the soil surface, inciting aerial infection of bay laurel and tanoak. Leaf litter may provide an incomplete barrier to splash dispersal. This 2-year study illustrates annual P. ramorum survival in soil and the suppressive nature of redwood-associated soils to chlamydospore production. Infested soil may serve as primary inoculum for foliar infections by splash dispersal during rain events.

Detection, Distribution, Sporulation, and Survival of Phytophthora ramorum in a California Redwood-Tanoak Forest Soil.

ABSTRACT Recovery of Phytophthora ramorum from soils throughout sudden oak death-affected regions of California illustrates that soil may serve as an inoculum reservoir, but the role of soil inoculum in the disease cycle is unknown. This study addresses the efficacy of soil baiting, seasonal pathogen distribution under several epidemiologically important host species, summer survival and chlamydospore production in soil, and the impact of soil drying on pathogen survival. The efficacy of rhododendron leaves and pears as baits for detection of soilborne propagules were compared. Natural inoculum associated with bay laurel (Umbellularia californica), tanoak (Lithocarpus densiflorus), and redwood (Sequoia sempervirens) were determined by monthly baiting. Summer survival and chlamydospore production were assessed in infected rhododendron leaf disks incubated under bay laurel, tanoak, and redwood at either the surface, the litter/soil interface, or in soil. Rhododendron leaf baits were superior to pear baits for sporangia detection, but neither bait detected chlamydospores. Most inoculum was associated with bay laurel and recovery was higher in soil than litter. Soil-incubated inoculum exhibited over 60% survival at the end of summer and also supported elevated chlamydospore production. P. ramorum survives and produces chlamydospores in forest soils over summer, providing a possible inoculum reservoir at the onset of the fall disease cycle.

Disease Progression of Phytophthora ramorum and Botryosphaeria dothidea on Pacific Madrone.

Infection by Phytophthora ramorum was associated with stem and leaf lesions of Pacific madrone (Arbutus menziesii) seedlings and saplings. In addition, a common and native pathogen, Botryosphaeria dothidea, caused similar leaf and stem lesions. When exposed to natural levels of inoculum in forests infested with P. ramorum, 50 to 66% of madrone saplings used as bait died. Recovery of P. ramorum from colonized plant tissue on culture media was generally low. From initial infection, P. ramorum was not culturable from leaf tissue after a mean of 3.5 weeks or from stem tissue after a mean of 8 weeks. Generally, B. dothidea was recovered more frequently from necrotic stems and leaves than was P. ramorum. Experimental inoculations of madrone seedlings showed that leaf and stem lesion lengths were, on average, greater on tree seedlings inoculated with P. ramorum than on those inoculated with B. dothidea. P. ramorum and B. dothidea appear to coexist in stem and leaf tissue, forming a novel pathogen complex, affecting growth and reproduction of Pacific madrone.

Laparoscopic nephrectomy in children.

BACKGROUND: Laparoscopic nephrectomy in the adult population is reported with increased frequency. We present our initial experience with laparoscopic nephrectomy in children. METHODS: Over a 2-year period, 11 nephrectomies were performed in nine children aged 16 months to 16 years (mean, 6.5 years). All patients were referred due to complications of a nonfunctioning kidney. Seven patients had recurrent urinary tract infections, and two had refractory hypertension. Two patients underwent bilateral laparoscopic nephrectomy. The operation was performed using four access ports measuring 3.5 to 10 mm. RESULTS: All kidneys were removed successfully using a laparoscopic technique. The average length of the operation was 163 min per kidney (range, 90-420). The estimated blood loss was <10-150 ml (mean, 45). No patient required transfusion. Seven patients were discharged home by postoperative day 2. The two patients with the longest operating times were discharged home on postoperative days 4 and 5 due to delay in return of bowel function. Narcotic use was minimal, and all patients enjoyed a rapid return to full activity. CONCLUSION: Laparoscopic nephrectomy is a viable alternative to open nephrectomy in children. Further experience with this technique is required to establish its efficacy and reduce the operating time

Laparoscopic evacuation of a subcapsular renal hematoma causing symptomatic hypertension.

We report the case of a subcapsular hematoma following extracorporeal shockwave lithotripsy which presented as symptomatic hypertension. When medical therapy proved ineffective, laparoscopic decompression of the hematoma corrected the hypertension.

Dressing for surgical wounds of the penis.

We describe a simple tubular elastic gauze dressing for surgical wounds of the penis. The amount of pressure placed on the penis is consistent and reproducible. The material is elastic enough to avoid vascular occlusion and is easily applied with a plastic tube. The dressing stays in place, can be used with stents or catheters, and is easily removed by the patients at home.

Pseudosarcomatous fibromyxoid tumor of the prostate. A case report with review of the literature.

A previously healthy 42-year-old white male presented with urinary obstruction. Radiographic evaluation revealed a 4-cm prostatic mass extending into the bladder. Transrectal biopsies revealed a sarcomatoid histology with atypical spindle cells suspicious for possible sarcoma. A transurethral resection of the prostate was performed revealing a benign fibromyxoid lesion with spindle cell proliferation. Postoperatively, the patient voided normally with no evidence of recurrence on follow-up of over 1 year. The clinical presentation and histologic features are consistent with pseudosarcomatous fibromyxoid tumor, a rare but benign lesion which has previously been mistaken for a malignant prostatic sarcoma. It is important for the urologist to recognize this benign process so that radical procedures are not performed.

Induction of DNA repair in HeLa S3 carcinoma cells by the N-nitroso derivatives of 1-(N-L-tryptophan)-1-deoxy-D-fructose and 1-(5-hydroxytryptamino)-1-deoxy-D-fructose.

HeLa S3 cells, when incubated at 37 degrees C with the N-nitroso derivative of the Amadori compounds 1-(N-L-tryptophan)-1-deoxy-D-fructose (FRU-TRP) or 1-(5-hydroxytryptamino)-1-deoxy-D-fructose (FRU-SEROT) in the presence of a six-fold molar excess of sodium nitrite, exhibit increased intracellular DNA synthesis. Sodium nitrite alone, at identical levels, elicits a similar response, albeit to a much lesser degree. No response whatsoever is produced when the cells are incubated with the parent Amadori compounds. The observed stimulation of DNA replication is DNA repair. Two major routes are suggested by which nitrosated FRU-TRP (NO-FRU-TRP) and nitrosated FRU-SEROT (NO-FRU-SEROT) could damage intracellular DNA.

The influence of 1-(N-L-tryptophan)-1-deoxy-D-fructose [Fru-Trp] and its N-nitrosated analogue [NO-Fru-Trp] on the viability and intracellular synthetic activity (DNA, RNA, and protein synthesis) of HeLa S3-carcinoma cells.

Exposing HeLa S3 cells at 37 degrees C to varied concentrations of, respectively, Fru-Trp (0.1 microM - 1 mM), NO-Fru-Trp (0.1 microM - 1 mM), and NaNO2 (0.6 microM - 6 mM) for varied periods of time (1 - 36 hr) does neither affect their viability (trypan blue dye exclusion test) nor capability to synthesize RNA or protein but is of considerable influence on DNA synthesis in the case of NO-Fru-Trp and NaNO2, but not in the case of Fru-Trp which continues to be ineffective. None of the three compounds tested is of significant influence on cell number. Both NO-Fru-Trp and NaNO2 stimulate DNA synthesis: a maximum of activity [( 3H] thymidine incorporation) exists at the 24 hr time point of incubation, with NO-Fru-Trp, for instance, generating a 2.5-fold increase (over control) at 1 mM concentration in the medium while NaNO2, at comparable concentration, increases DNA synthesis by a factor of 1.6 over control. The increase in DNA synthesis is not due to stimulatory influences on (semi-conservative) DNA replication but represents DNA repair. This was verified by keeping the cells under conditions that prevent normal (semi-conservative) replication but permit repair ("unscheduled DNA synthesis"). Two major routes are suggested by which NO-Fru-Trp could impart DNA damage and, thus, assume mutagenic properties.

Mutagenic activity of a nitrosated early Maillard product: DNA synthesis (DNA repair) induced in HeLa S3 carcinoma cells by nitrosated 1-(N-L-tryptophan)-1-deoxy-D-fructose.

HeLa S3 cells in suspension were incubated at 37 degrees C with various concentrations of the Amadori compound 1-(N-L-tryptophan)-1-deoxy-D-fructose (Trp-Fru), of its nitrosated analogue NO-Trp-Fru and of sodium nitrite, for varying periods of time, and were assayed for viability (trypan blue exclusion test) and for intracellular DNA, RNA and protein synthesis. None of the compounds tested had any effect on cell viability, or on RNA and protein synthesis apart perhaps from a slightly inhibitory action. While Trp-Fru remained ineffective also as far as intracellular DNA synthesis was concerned, both NO-Trp-Fru and NaNO2 had a major effect on DNA synthesis. With NaNO2, stimulation of DNA synthesis occurred at concentrations above 1 mM in the growth medium, but with NO-Trp-Fru synthesis increased at concentrations below 1 microM. The excess DNA synthesis (i.e. synthesis above control activity) observed with NO-Trp-Fru and also with NaNO2 was due to DNA repair. This was verified by keeping the cells under conditions that prevented normal semi-conservative replication but permitted DNA repair ('unscheduled DNA synthesis'). Two major routes are suggested by which NO-Trp-Fru could damage DNA.