Two 'Candidatus Liberibacter asiaticus' Strains Recently Found in California Harbor Different Prophages.

'Candidatus Liberibacter asiaticus' (CLas), an α-proteobacterium, is associated with citrus Huanglongbing (HLB; yellow shoot disease). In California, two cases of CLas have been detected in Los Angeles County, one in Hacienda Heights in 2012 and the other in San Gabriel in 2015. Although all infected trees were destroyed in compliance with a state mandate, citrus industry stakeholder concerns about HLB in California are high. Little is known about the biology of CLas, particularly the California strains, hindering effective HLB management efforts. In this study, next-generation sequencing technology (Illumina MiSeq) was employed to characterize the California CLas strains. Data sets containing >4 billion (Giga) bp of sequence were generated from each CLas sample. Two prophages (P-HHCA1-2 and P-SGCA5-1) were identified by the MiSeq read mapping technique referenced to two known Florida CLas prophage sequences, SC1 and SC2. P-HHCA1-2 was an SC2-like or Type 2 prophage of 38,989 bp in size. P-SGCA5-1 was an SC1-like or Type 1 prophage of 37,487 bp in size. Phylogenetic analysis revealed that P-HHCA1-2 was part of an Asiatic lineage within the Type 2 prophage group. Similarly, P-SGCA5-1 was part of an Asiatic lineage within Type 1 prophage group. The Asiatic relatedness of both P-HHCA1-2 and P-SGCA5-1 was further presented by single nucleotide polymorphism analysis at terL (encoding prophage terminase) that has been established for CLas strain differentiation. The presence of different prophages suggests that the two California CLas strains could have been introduced from different sources. An alternative explanation is that there was a mixed CLas population containing the two types of prophages, and limited sampling in a geographic region may not accurately depict the true CLas diversity. More accurate pathway analysis may be achieved by including more strains collected from the regions.

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 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.