Plant Science

‘Candidatus Liberibacter africanus’ is associated with citrus greening (huanglongbing [HLB]) in South Africa. Various unpublished reports have suggested that the related bacterium ‘Ca. L. asiaticus’ associated with HLB in citrus might be seed transmissible based on real-time PCR results. Seed transmission poses a risk of long distance disease spread, especially with the dissemination of rootstock seed. Therefore, it was essential to determine whether ‘Ca. L. africanus’ is seed transmitted in citrus. Fruit from 26 ‘Ca. L. africanus’-infected branches of six citrus cultivars showing greening symptoms were collected and the seed was harvested. Cultivars included were Minneola tangelo (Citrus reticulata × C. paradisi); sweet oranges (C. sinensis) Premier midseason, Clanor midseason, and Olinda Valencia; Eureka lemon (C. limon) and Troyer citrange (Poncirus trifoliata × C. sinensis) rootstock variety. Branches bearing each fruit were collected and confirmed to contain ‘Ca. L. africanus’ by real-time PCR testing using Taqman probe HLBp and HLBaf and HLBr primers as described by Li et al. (3). The seed of each sample was sorted into five categories ranging from healthy looking to totally aborted based on their appearance before planting. Germination was done in seed trays under vector-free conditions at 24 to 28°C. Thereafter the seedlings were planted in small, plastic bags and monitored for greening-like symptoms or other abnormalities for up to 2 years. A slow-release, balanced fertilizer was applied and supplemented with micro-nutrient sprays for plant maintenance. Plants showing abnormal symptoms were potted into larger pots and closely monitored. These samples and a number of other seedlings showing growth abnormalities were tested for ‘Ca. L. africanus' by real-time PCR as described above. In total, 1,570 seedlings were obtained. Some abnormal symptoms such as small chlorotic leaves, interveinal chlorosis, yellow veins, and stunting were seen in some seedlings. Most symptoms resembled deficiencies, and no blotchy mottle typical of ‘Ca. L. africanus’ infection was noted on any of the seedlings. Abnormal seedlings arose from normal and abortive seed. One hundred and eighteen of these seedlings (8 Minneola tangelo; 24 Premier midseason, 42 Clanor midseason, 33 Olinda Valencia, and 11 Troyer citrange seedlings) were individually tested using real-time PCR for ‘Ca. L. africanus’ detection. These seedlings had germinated from essentially healthy-looking seed (category 1) to seeds with severe abnormalities (category 5) and 33, 24, 23, 30, and 8 seedlings, respectively, were tested from each of the five seed categories. No samples tested positive with real-time PCR based on a positive/negative threshold Cq value of 35. Buds of some seedlings that yielded the lowest Cq values above 35 were grafted onto healthy ‘Madam vinous’ sweet orange (C. sinensis) seedlings and monitored for symptom development for 3 months. No symptoms developed and all these indicators also tested negative for ‘Ca. L. africanus’, indicating the absence of a transmissible agent. Just as other researchers (1,2) have recently indicated a lack of evidence for seed transmission of ‘Ca. L. asiaticus’, no seed transmission of ‘Ca. L. africanus’ could be demonstrated in this experiment either. References: (1) U. Albrecht and K. D. Bowman. HortScience 44:1967, 2009. (2) J. S. Hartung et al. Plant Dis. 94:1200, 2010. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006.

In January 2011, tomato (Solanum lycopersicum) plants exhibiting stunting, yellow mosaic, short, chlorotic leaves, aborted flowers, and reduced-size fruits, symptoms similar to those exhibited by plants infected by ‘Candidatus Liberibacter solanacearum’ (2), were observed in approximately 5% of tomato plants in greenhouses in Jocotitlan in the State of Mexico, Mexico. Occasional plant recovery was also observed. Tomato plants in this facility were previously shown to be infected by Mexican papita viroid (MPVd), Pepino mosaic virus (PepMV), and aster yellows phytoplasma. Eight symptomatic leaf samples (designated MX11-01 to MX11-08) were collected and screened against selected tomato viruses and pospiviroids by reverse transcription (RT)-PCR using purified plant RNA or for ‘Ca. L. solanacearum’ by PCR using purified plant DNA. As expected, both PepMV and MPVd were detected in these samples. However, two ‘Ca. L. solanacearum’-specific PCR products (1,168 and 669 bp) were also amplified in two samples (MX11-02 and MX11-05) using primers OA2 (2) and OI2c (1) or CL514F/CL514R (3), respectively. Each ‘Ca. L. solanacearum’-specific PCR product was gel purified with Geneclean (Q-Biogene, Carlsbad, CA) and cloned into pCR2.1 using TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced (Functional Biosciences, Madison, WI). Sequences of 16S rRNA (1,168 bp) in both isolates (GenBank Accession Nos. JF811596 and JF811597) were identical. However, the 669-bp 50S rRNA sequences in these two isolates (GenBank Accession Nos. JF811598 and JF811599) contained two single nucleotide polymorphism (SNP) mutations. BLASTn searches showed that both 16S rRNA and 50S gene sequences in MX11-05 were identical to the ‘Ca. L. solanacearum’ previously identified on potato in Chihuahua (GenBank Accession Nos. FJ829811 and FJ829812) and Saltillo (GenBank Accession Nos. FJ498806 or FJ498807) in eastern Mexico. These ‘Ca. L. solanacearum’ isolates were recently classified as the “b” haplotype (4). Alignment analysis of the ‘Ca. L. solanacearum' 16S rRNA sequences also revealed the conserved SNP mutations (g.212T > G and g.581T > C) in MX11-02 and MX11-05 as previously identified for other “b” haplotype isolates (4). ‘Ca. L. solanacearum’ was first identified in greenhouse tomatoes in 2008 in New Zealand (2). It has also been identified in greenhouse and field tomatoes in the United States. ‘Ca. L. solanacearum’ was previously reported to infect field tomatoes in Sinaloa, Mexico (3), which was recently considered as the “a” haplotype (4). To our knowledge, this is the first report of ‘Ca. L. solanacearum’ naturally infecting tomatoes in Jocotitlan in the State of Mexico, Mexico. The greenhouse tomato ‘Ca. L. solanacearum’ may be transmitted from infected solanaceous plants by potato psyllids (Bactericera cockerelli), which were observed in this facility. References: (1) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009 (4) W. R. Nelson et al. Eur. J. Plant Pathol. 130:5, 2011.

Potatoes (Solanum tuberosum) are one of the most important crops in China following rice, wheat, and corn. Aster yellows phytoplasma appeared to be widespread in China; it was found to cause diseases on alfalfa, oranges, peaches, periwinkles, bamboo (1), and cactus (4). However, scant information of this pathogen on potatoes is available except for a few short reports published during the 1950s. During the potato disease surveys conducted from 2005 to 2010 in Yunnan and Inner Mongolia, 10 to 35% of potato plants exhibited symptoms of yellowing or purpling of apical leaves, with the top leaves rolling inward and aerial tubers formation. Total DNA was extracted from midveins of leaves and roots of 125 diseased and asymptomatic plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. A nested PCR was carried out with the first round primer pair P1/P7 followed by the second round primer pair R16F2n/R16R2 (2,3). A PCR product of approximately 1.2 kb was amplified from diseased plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting a 1.2-kb product using restriction enzymes AluI, BfaI, BstUI, HhaI, HpaI, KpnI, MseI, and RsaI. Comparing the RFLP patterns with previously published phytoplasma strains (2), aster yellows phytoplasma found on potato plants in Yunnan and Inner Mongolia belong to group I, subgroup B (16SrI-B). The PCR product from P1/P7, diluted 1:30, was amplified by using primer pair P1A/P7A (3) and P1A/16S-SR (3). The nested-PCR products from P1A/P7A and P1A/16S-SR were cloned into pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by the Core Lab of the University of Alaska–Fairbanks and GENEWIZ (South Plainfield, NJ). The nucleotide sequence (GenBank Accession No. HQ599228) was analyzed by iPhyClassifier software and had 99.53% sequence identity to the reference strain (GenBank Accession No. M30790) for ‘Candidatus Phytoplasma asteris’. The RFLP similarity is identical (coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank Accession No. NC_005303). To our knowledge, this is the first report revealing the molecular characteristics of a phytoplasma associated with aster yellows-diseased potatoes in China. References: (1) H. Cai et al. Plant Prot. 31:38, 2005. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (4) W. Wei et al. Plant Dis. 91:461, 2007.

Elm yellows phytoplasmas (EY) belonging to the 16SrV‐A subgroup were recently proposed as a new candidate species ‘ Candidatus Phytoplasma ulmi’. These pathogens infect elm trees, causing leaf yellowing and premature drying. In this study, 25 isolates originating from localities in northeast, east and southwest Serbia were characterized by means of RFLP analysis and DNA sequencing of four genomic loci: 16S rRNA, ribosomal protein rpl22 ‐ rps3 , secY and map . In total, five different genotypes were identified based on collective sequencing of all four genes. Four of these genotypes showed significant nucleotide changes compared with the EY1 T reference strain. Phylogeny based on parsimony analyses of ribosomal protein, secY and map genetic loci indicated a single monophyletic origin of EY1 T and the new ‘ Ca . Phytoplasma ulmi’ strains. Unlike phylogenetic clustering, DNA sequence comparison of EY1 T and the novel strains revealed mutations in oligonucleotide signature sequences for all three genes (16S, rpl22 ‐ rps3 and secY ) used for the characterization and assignment of 16SrV‐A phytoplasmas to the ‘ Ca.  Phytoplasma ulmi’ species in the original description. Based on their high degree of genetic variability, the Serbian strains were assigned to four different subtypes of ‘ Ca.  Phytoplasma ulmi’ (EY‐S1, EY‐S2, EY‐S3 and EY‐S4). New diagnostic enzymes for practical use in ‘ Ca.  Phytoplasma ulmi’ identification are proposed for the 16S rRNA, ribosomal protein and secY genes. The implications of genetic variability within signature sequences for taxonomy and identification of ‘ Ca . Phytoplasma’ species, as well as the importance of geographic variability and number of strains characterized for species description, are discussed.

Prophages are important genetic elements of bacterial genomes and are involved in lateral gene transfer, pathogenicity, environmental adaptations, and interstrain genetic variability. In this study, the sequence of a prophage terminase gene of ‘Candidatus Liberibacter asiaticus’, a bacterium associated with citrus Huanglongbing (HLB), was selected as a molecular marker to assess the genetic variation in two ‘Ca. L. asiaticus’ populations from geographically distinct provinces (Guangdong and Yunnan) in China. The frequency of the prophage terminase gene was 15.8% (19/120) in Guangdong (altitude <500 m) and 97.4% (38/39) in Yunnan (altitude >2,000 m). The difference was highly significant (P < 0.0001) based on χ2 analysis. However, the partial prophage terminase gene sequences obtained from 10 Guangdong strains and 6 Yunnan strains were identical or highly similar, suggesting that at least some bacterial strains in the two locations shared a common recent origin. This is the first report on population variation of ‘Ca. L. asiaticus’ in China, where HLB was first described. The population variation of ‘Ca. L. asiaticus’ in the two geographical regions and the related HLB epidemiology were discussed.