Publications

4373

The diseases huanglongbing [HLB, associated with Candidatus Liberibacter asiaticus (CLas)] and Asian citrus canker [ACC, caused by Xanthomonas citri (Xcc)] are widespread in Florida and many other citrus-growing areas, presenting unprecedented challenges for citrus breeding. Because HLB and ACC weaken trees and compromise cropping, breeding is much less efficient using seed parents that have been exposed to these diseases. Therefore, it would be highly desirable to use unique disease-exposed selections only as pollen parents with pollen applied to disease-free trees. However, there may be a risk of introducing these diseases using such pollen sources. To assess this potential, abundance of the pathogens associated with these diseases was assessed in anthers and flowers using quantitative polymerase chain reaction. Because CLas is systemic, levels on mature leaves from the flower source trees were assessed to see if the presence of CLas in flowers was associated with leaf levels. Disease-exposed trees were tested in 10 genotypes from each of three broad genotypic categories, which reflect different levels of susceptibility to the diseases associated with the pathogens studied: Poncirus trifoliata hybrids (most resistant to HLB), Citrus maxima and hybrids (susceptible to both diseases), and C. reticulata and hybrids (considerable resistance to ACC). Of the 30 samples of each tissue type analyzed for CLas, 88% of mature leaves, 69% of flowers, and 88% of anthers had one or more CLas bacterium per sample. The trifoliate genotypic group had significantly lower levels of CLas than the pummelo and mandarin groups in mature leaf samples, but CLas levels were more similar between groups in anther and flower samples, and the pathogen was present in most of the trifoliate hybrids tested. Mean numbers of CLas detected per nanogram nucleic acid were 100 to 800 times higher in mature leaf samples, most characteristic of HLB symptoms, compared with anther samples. Xcc DNA was detected in 30% of flower samples and 23% of anther samples. No significant differences in Xcc levels were found between tissue type or genotypic group. However, regressions between Xcc levels in flowers and percent of plant pedigree derived from mandarin had a negative correlation and an r2 of 0.159 (P = 0.029). The biology of CLas is consistent with the pathogen being present in anthers from unopened flowers, whereas the ACC pathogen detected inside flowers was likely the result of contamination despite great care in sample collection and handling. Where exceptional diligence to exclude HLB and ACC is appropriate, results suggest that there may be a risk of spreading these pathogens through use of pollen from trees on infected farms.

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.

A novel aerobic, chemoheterotrophic bacterium, strain YO-36T, isolated from the rhizoplane of an aquatic plant (a reed, Phragmites australis) inhabiting a freshwater lake in Japan, was morphologically, physiologically and phylogenetically characterized. Strain YO-36T was Gram-negative and ovoid to rod-shaped, and formed pinkish hard colonies on agar plates. Strain YO-36T grew at 20–40 °C with optimum growth at 30–35 °C, whilst no growth was observed at 15 °C or 45 °C. The pH range for growth was 5.5–8.5 with an optimum at pH 6.5. Strain YO-36T utilized a limited range of substrates, such as sucrose, gentiobiose, pectin, gellan gum and xanthan gum. The strain contained C16 : 0, C16 : 1, C14 : 0 and C15 : 0 as the major cellular fatty acids and menaquinone-12 as the respiratory quinone. The G+C content of the genomic DNA was 62.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YO-36T belonged to the candidate phylum OP10 comprised solely of environmental 16S rRNA gene clone sequences except for two strains, P488 and T49 isolated from geothermal soil in New Zealand; strain YO-36T showed less than 80 % sequence similarity to strains P488 and T47. Based on the phylogetic and phenotypic findings, a new genus and species, Armatimonas rosea gen. nov., sp. nov., is proposed for the isolate (type strain YO-36T  = NBRC 105658T  = DSM 23562T). In addition, a new bacterial phylum named Armatimonadetes phyl. nov. is proposed for the candidate phylum OP10 represented by A. rosea gen. nov., sp. nov. and Armatimonadaceae fam. nov., Armatimonadales ord. nov., and Armatimonadia classis nov.