Phytophthora Species Are Common on Nursery Stock Grown for Restoration and Revegetation Purposes in California.

Phytophthora tentaculata was detected for the first time in North America in 2012 in a nursery on sticky monkeyflower plant (Diplacus aurantiacus) and again in 2014 on outplanted native plants. At that time, this species was listed as a federally actionable and reportable pathogen by the USDA. As a result of these detections, California native plant nurseries were surveyed to determine the prevalence of Phytophthora species on native plant nursery stock. A total of 402 samples were collected from 26 different native plant nurseries in California between 2014 and 2016. Sampling focused on plants with symptoms of root and crown rot. Symptomatic tissue was collected and tested by immunoassay, culture, and molecular techniques (PCR). Identifications were made using sequences from the internal transcribed spacer (ITS) rDNA region, a portion of the trnM-trnP-trnM, or the atp9-nad9 mitochondrial regions. Phytophthora was confirmed from 149 of the 402 samples (37%), and from plants in 22 different host families. P. tentaculata was the most frequently detected species in our survey, followed by P. cactorum and members of the P. cryptogea complex. Other species include P. cambivora, P. cinnamomi, P. citricola, P. hedraiandra, P. megasperma, P. multivora, P. nicotianae, P. niederhauserii, P. parvispora, P. pini, P. plurivora, and P. riparia. A few Phytophthora sequences generated from mitochondrial regions could not be assigned to a species. Although this survey was limited to a relatively small number of California native plant nurseries, Phytophthora species were detected from three quarters of them (77%). In addition to sticky monkeyflower, P. tentaculata was detected from seven other hosts, expanding the number of associated hosts. During this survey, P. parvispora was detected for the first time in North America from symptomatic crowns and roots of the nonnative Mexican orange blossom (Choisya ternata). Pathogenicity of P. parvispora and P. nicotianae was confirmed on this host. These findings document the widespread occurrence of Phytophthora spp. in native plant nurseries and highlight the potential risks associated with outplanting infested nursery-grown stock into residential gardens and wildlands.

First Detection of Puccinia ballotiflora on Salvia greggii.

Salvia greggii, autumn sage, is grown for its bright red to white flowers that bloom in late summer and fall. In February of 2008, a rust sample was sent to the CDFA plant pathology diagnostics laboratory in Sacramento from a nursery in Santa Barbara County, CA. Pustules were abundant on older leaves causing moderate defoliation of containerized stock. Only the varieties with entirely red or pink flowers were affected. S. greggii 'Hotlips,' a popular white/red bicolor, was unaffected. Amphigenous uredinia were cinnamon brown, round, powdery, and sometimes surrounded by yellow halos. Pustules were found primarily on the leaves, although a few were on the stems. Urediniospores were broadly obovoid, subglobose to broadly ellipsoid, echinulate, and 22 to 27 × 24 to 32 µm (24.9 × 26.9 µm average) with one apical pore and 2 to 3 equatorial pores. Urediniospore walls were cinnamon brown in color and measured 1.0 to 2.0 µm (1.5 µm average). No telia were observed. After the initial detection, this rust was found in additional nursery sites in Santa Cruz, Santa Clara, Santa Barbara, and Ventura counties in 2008 and 2009. In November of 2011, a sample from a landscape planting in Santa Barbara County of a similar rust with telia and teliospores was submitted. Urediniospores and teliospores were present in the same lesions. Lesions with teliospores were located primarily on the stems. Mature teliospores were two-celled, verrucose, chocolate brown, and 25 to 31 × 32 to 40 µm (28.6 × 35.3 µm average) with a pedicel ranging from 8 to 12 × 38 to 104 µm, sometimes attached obliquely. The rust matched the morphological characteristics of Puccinia ballotiflora (Syn = P. ballotaeflora Long) (2). To confirm pathogenicity, three 20-cm-tall plants of S. greggii 'Navajo Red' in 3.8-liter pots were spray inoculated with 10 ml of a 2.5 × 103 urediniospores per ml suspension and incubated in a dew chamber at 23°C for 2 days in the dark. Plants were transferred to a growth chamber maintained at 22°C with a 12-h photoperiod. Three plants were sprayed with sterile distilled water as controls. Uredinial pustules (1 to 2 mm) appeared on the abaxial surface of the leaves after 3 weeks. The pathogenicity test was repeated with similar results. The internal transcribed spacer region of rDNA and a portion of the 28S rDNA were amplified with primer pairs ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3'), Rust1 (5'-GCTTACTGCCTTCCTCAATC-3'), and Rust2inv (5'-GATGAAGAACACAGTGAAA-3'), LR6 (5'-CGCAGTTCTGCTTACC-3') as described by Aime (1) and sequenced using the amplification primers, Rust2 (5'-TTTCACTGTGTTCTTCATC-3') and Rust3 (5'-GAATCTTTGAACGCACCTTG-3'). BLAST query of the assembled sequence, GenBank KF381491, was 91% identical to P. acroptili, JN204194, its closest match of similar length. P. ballotiflora has been found in Colombia on S. cataractarum, S. petiolaris, and S. mayori (3), and in Texas and Mexico on S. ballotiflora (4). To the best of our knowledge, this is the first detection of P. ballotiflora on S. greggii worldwide. P. ballotiflora is already widespread in the nursery trade in California and frequent fungicide applications are necessary to keep plants marketable. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) J. W. Baxter and G. B. Cummins. Lloydia 14:201, 1951. (3) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Botany and Mycology Laboratory, Online publication http://nt.ars-grin.gov/fungaldatabases ARS, USDA, 2014 (4) F. D. Kern et al. Mycologia 25:448, 1933.

First Report of Bacterial Fruit Blotch on Melon Caused by Acidovorax citrulli in California.

In July 2013, a melon plant sample (Cucumis melo cv. Saski) with disease symptoms resembling bacterial fruit blotch (BFB), was collected from a 10-acre field located in Yolo County, California, and submitted to the Plant Pest Diagnostics Center of the CDFA. Melon leaves had small (5 to 10 mm in diameter), tan to dark reddish-brown, angular lesions surrounded by yellow halos, and larger V-shaped lesions that extended from the leaf margins to the midrib. Bacterial streaming was observed at 400× magnification. The bacterium isolated from a leaf tissue wet mount formed smooth, round, cream-colored, non-fluorescent colonies on Pseudomonas F agar, was gram-negative, rod-shaped, aerobic, and oxidase-positive. The strain grew at 41°C and produced a strong hypersensitive response on tobacco (Nicotiana tabacum) 24 h after tissue infiltration. Based on a positive immunoassay test for Acidovorax citrulli (Eurofins STA Lab, Inc., Longmont, CO) and positive real-time PCR assays using species-specific primer sets, BX-L1/BX-S-R2 (1) and ZUP2436/2437 (4), the strain was identified as A. citrulli. A 360-bp fragment of the 16S ribosomal RNA gene was amplified using conventional PCR with primers WFB1 and WFB2 (3). The fragment, GenBank Accession No. KJ531595, showed 100% identity with the corresponding regions of A. citrulli (CP000512) strain AAC00-1 by BLAST query. Pathogenicity tests were performed by injecting 0.5 to 1 ml suspensions of the bacteria (106 CFU/ml) under the rind of three mature honeydew fruit (Cucumis melo var. indorus), three watermelon fruit (Citrullus lanatus cv. Sugar Baby), and into the cotyledons of ten, 10-day-old watermelon seedlings (cv. Sugar Baby). The fruit and seedlings were incubated in plastic bags at 30°C and similar treatments with sterile water served as negative controls. After 4 days, the seedlings inoculated with the suspect strain exhibited dark brown necrotic lesions with yellow halos that later coalesced, causing the cotyledons to collapse. Seven days after inoculation, the honeydew fruit exhibited dry, rotten gray cavities (4 to 6 cm in diameter) in the pericarp tissue below the rind. In contrast, the watermelon fruit had completely collapsed in a watery rot after 7 days. No symptoms were observed on the negative control fruits and seedlings treated with water. The pathogen was re-isolated from the inoculated fruit and seedlings and confirmed as A. citrulli by species-specific PCR and immunoassay as described above. The melon seed lot used to plant the field in Yolo County, CA, also tested positive for A. citrulli using species-specific real-time PCR assays (1,4). DNA fingerprinting by pulse field gel electrophoresis of Spe I-digested whole cell genomic DNA showed that all of the California A. citrulli strains were members of subgroup II (haplotype C strain) (3). These haplotypes normally occur on watermelon. BFB is a seed-transmitted disease of cucurbits and a major concern for national and global seed trade. First observed in United States commercial watermelon fields in 1989, BFB can cause economic losses up to 90% for commercial watermelon, cantaloupe, and honeydew growers (1,2). While BFB routinely occurs in the southeastern United States, this is the first official record of the disease in California. References: (1) O. Bahar et al. Plant Pathol. 57:754, 2008. (2) R. Walcott et al. J. Phytopathol. 152:277, 2004. (3) R. Walcott et al. Plant Dis. 84:470, 2000. (4) B. Woudt et al. Phytopathology 99:S143, 2009.

First Published Report of Rust on White Alder Caused by Melampsoridium hiratsukanum in the United States.

White alder (Alnus rhombifolia) is a fast-growing tree native to the western United States and is planted frequently in landscapes. In September 2010, mature leaves of white alder with small, orange-yellow pustules were collected in a commercial nursery in Santa Cruz County, CA. Approximately 25 white alder trees were affected. Collected leaves were sent to the California Department of Food and Agriculture Plant Pest Diagnostics Laboratory. Young uredinial pustules were bullate, with urediniospores emerging from a single pore in the pustule. Spiny cells lined the ostiole. With age, pustules broke open to release more spores. Urediniospores were obovate to oval and measured from 14 to 20 × 27 to 41 µm (17.1 × 32.2 µm average, n = 62). Spores were uniformly echinulate and contained a nearly hyaline cell wall measuring from 1 to 2 µm (1.5 µm average) in thickness. A portion of the 28S ribosomal subunit (GenBank Accession No. KC313888) and the internal transcribed spacer regions (KC313889) were amplified and sequenced from DNA extracted from urediniospores using primers LR6 and rust2inv (1) and ITS1-F and ITS4-B (2), respectively. Our ITS sequence had 99% identity to GenBank accession EF564164, Melampsoridium hiratsukanum. In September 2011, white alder leaves with similar symptoms were collected from a commercial nursery in Santa Barbara County, CA. The spore morphology matched the white alder sample previously collected in Santa Cruz County, CA, in 2010. At that time, pathogenicity assays were conducted on three 1-year-old, 61-cm white alder trees planted in 3.8-liter pots. Six detached leaves with visible rust pustules were rubbed gently onto both the apical and distal side of moistened leaves of the healthy alders. Each infected leaf was used to inoculate a total of 6 to 10 healthy leaves by rubbing two leaves per tree before moving to the next tree. Leaves on three additional white alder trees were rubbed with healthy leaves as controls. Trees were incubated in a dew chamber for 3 days in darkness at 24°C, then placed in a growth chamber at 22°C with a 12-h photoperiod. Twelve days after inoculation, small lesions were visible on a few of the leaf undersides of each inoculated tree. Not all inoculated leaves developed pustules. No lesions developed on the control trees. M. hiratsukanum has been reported in Canada, Europe, and eastern Asia (3). There are no published reports of this rust in the United States, but there is an unpublished specimen from white alder in the USDA Systematic Mycology Herbarium (BPI 028048) collected from California in 1931, which was identified as M. hiratsukanum by G. B. Cummins using morphological criteria. We are unaware if older specimens of this rust exist because we were unable to search other herbaria in the United States. To the best of our knowledge, this rust has been present in California since 1931, but has only recently been found causing disease in nursery plants. There have been no reports of the serious foliar disease symptoms on trees in California wild lands as have been reported in Europe, presumably due to dry summer and fall seasons in white alder's natural habitat. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (3) J. Hatula et al. Mycologia 101:622, 2009.

First Report of Candidatus Liberibacter asiaticus Associated with Citrus Huanglongbing in California.

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive citrus diseases worldwide and is seen as a major threat to the multimillion dollar citrus industry in California. The vector of the two bacterial species associated with this disease, Candidatus Liberibacter asiaticus and Ca. L. americanus, is the Asian citrus psyllid (ACP), Diaphorina citri (4). ACP was detected in California in August of 2008 and has since been detected in nine counties in southern California. As part of a long term survey and testing program for the ACP carrying the HLB associated bacteria, groups of ACP nymphs and adults were submitted to the Jerry Dimitman Citrus Research Board/Citrus Pest and Disease Prevention Program Laboratory in Riverside, CA. In March 2012, DNA extracted using the Qiagen MagAttract 96 DNA plant kit (QIAGEN Inc., 27220 Turnberry Lane, Suite 200, Valencia, CA 91355) from a group of three ACP adults tested positive for Ca. L. asiaticus with the real-time PCR assay developed by Li et al. (4). ACP adults were collected from a residential citrus tree located in the Hacienda Heights area of Los Angeles County, California. The approximately 1.8 meter tall lemon tree had 23 graft unions, primarily of lemon (Citrus × meyeri) and pomelo (Citrus maxima) varieties. The tree was unthrifty, with yellow shoots and chlorotic leaves. Symptoms on the lemon and pomelo leaves included asymmetrical blotchy mottling, yellowing, and corking of the leaf veins, with the blotchy mottle more prominent in the pomelo leaves. Pomelo leaves appeared crinkled along the thickened veins. Lemon leaves had yellow veins and a few had islands of green tissue completely surrounded by yellow tissue. The entire tree was removed, cut into sections, bagged, and transported to the CDFA Plant Pest Diagnostics Lab for analysis. Two hundred milligrams of petiole and midrib tissue from leaves apical to each graft union was collected, and DNA from each sample was extracted using the Qiagen DNeasy plant mini kit. DNA extracted from both lemon and pomelo leaves tested positive for Ca. L. asiaticus using real-time PCR (4). A 1,160-bp fragment of the 16S ribosomal RNA gene was amplified from the insect and plant DNA extracts using conventional PCR with primers Ol1 and OI2c (2). A 703-bp fragment of the ß-operon gene was amplified from the insect and plant extracts with primers A2 and J5 (1). The 16S rDNA fragments from the insect and plant respectively (GenBank Accession Nos. JX430434 and JX455745) and the ß-operon fragments (JX430435 and JX455746) showed 100% identity with the corresponding regions of Ca. L. asiaticus (CP001677) strain psy 62. Our 16S rDNA sequence showed 98% identity with Ca. L. africanus (EU921620), 97% identity with Ca. L. solanacearum (HM246509), and 96% with Ca. L. americanus (FJ036892). In response to the detection of HLB, a 241 km2 quarantine area around the detection site was established. Surveys for ACP and symptomatic host plants within the HLB quarantine area are ongoing. To date, there have been no additional positive detections. In the United States, HLB was first detected in Florida in 2005 (4) and in Texas in January of 2012 (3). To our knowledge, this is the first confirmed report of Ca. L. asiaticus associated with HLB in California. References: (1) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) M. Kunta et al. Phytopathology 102:S4.66, 2012. (4) W. Li et al. J. Microbiol. Methods 66:104, 2006.

The effect of force, timing, and location on bone-to-implant contact of miniscrew implants.

This study was conducted to evaluate the effect of timing and force of loading, as well as implant location, on bone-to-implant contact (BIC) of loaded and control miniscrew implants (MSI). Using seven skeletally mature male beagle dogs, 1-2 years of age, followed over a 110 day period, a randomized split-mouth design compared immediate versus delayed loading, 50 versus 25 g loading, and 25 g loads in the maxilla versus the mandible. Mobility was evaluated using a 0-3 point scale before the MSIs were prepared for histological analysis. Histomorphometric analyses were performed under light microscopy using Metamorph software on undecalcified sections. The percentage BIC was measured at three levels (coronal, middle, and apical) of the MSI. BIC was compared statistically using pairwise Wilcoxon signed-rank tests. Mobility was detected in three of the 56 (5.4 per cent) MSIs. The mobile implants were all unloaded controls and showed no BIC. All remaining stable MSIs showed some BIC. However, variation in BIC was large, ranging from 2.2 to 100 per cent. There were no significant (P > 0.05) differences in BIC associated with timing of force application, amount of force applied, or implant location. There was a tendency for less BIC at the coronal level, but the differences between levels were not statistically significant. Within the limits of this study, it is concluded that the timing and amount of force at loading and location of implant placement do not affect BIC. Moreover, it appears that only limited amounts of osseointegration are necessary to ensure implant stability.

Determining amalgam marginal quality: effect of occlusal surface condition.

Currently there is no "standard" finishing and polishing procedure for dental amalgam restorations. This investigation evaluated the effect of four different finishing techniques on the durability of dental amalgam restorations as determined by the marginal breakdown. Burnishing immediately before carving or as part of the condensation process seems to improve the carving characteristics of the amalgam.