Huanglongbing (greening) disease caused by a nonculturable, phloem-limited bacterium is a severe disease of citrus. On the basis of the influence of temperature on host symptoms and the causal agent, this disease can be categorized as Asian caused by “Candidatus Liberibacter asiaticus”, African caused by “Ca. L. africanus”, and American caused by “Ca. L. americanus”. Kumquat (Fortunella margarita (Lour.) Swingle), a member of the Rutaceae is an economically important crop for export and local consumption in Taiwan. Recently, a Huanglongbing-like disease was found on kumquat in Yilan County, the largest kumquat-producing area in northeastern Taiwan. Even though the disease has been reported on Citrus spp. from Taiwan, it has never been reported on kumquat. Symptoms of infected kumquat were mottling, yellowing, hardening, and curling of leaves followed by premature defoliation, twig dieback, decay of feeder rootlets and lateral roots, and ultimately the death of the entire plant. Typical sieve-tube-restricted bacteria were observed in kumquat cells by electron microscopy (1). In addition, psyllid-transmission tests demonstrated that the Asian psyllid (Diaphorina citri) could transmit this bacterium to healthy kumquats. Positive bud graft transmissions were obtained to F. margarita, F. japonica (Thunb.) Swingle, F. obovata Hort. ex Tanaka, Luchen sweet orange (Citrus sinensis (L.) Osb.), and Wentan pummelo (C. maxima f. sp. butan Hay.). These inoculated plants showed symptoms in 3 to 8 months, and bacteria could be detected by polymerase chain reaction (PCR) using a common primer pair that amplified a 226-bp specific DNA fragment (2). For further molecular identification, the bacterial DNA was extracted from the inoculated plants and PCR was performed by using two sets of primers selected from the 16S rRNA region (GenBank Accession No. L22532) and 16S/23S intergenic spacer region (GenBank Accession No. AB019793). The expected DNA fragments of 1,389 bp and 862 bp were, respectively, amplified from symptomatic plants but not from healthy plants. The PCR products were cloned and sequenced (GenBank Accession Nos. DQ302750 and DQ207841). The 16S rRNA has 98 to 99% identity and 16S/23S intergenic spacer region has 99% identity to the corresponding region of “Ca. L. asiaticus” in GenBank. These molecular analyses confirm the presence of “Ca. L. asiaticus” in kumquat. Since Huanglongbing has been rarely reported naturally on kumquat, further analysis of this bacterium as a special strain of “Ca. L. asiaticus” is needed. References: (1) M. Garnier et al. Ann. Microbiol. 135A:169, 1984. (2) T. H. Hung et al. J. Phytopathol. 147:599, 1999.
“Candidatus phytoplasma asteris” and related strains (i.e., aster yellows group 16SrI) have been associated with diseases of numerous plant species worldwide. Symptoms of aster yellows (AY) have been reported on rapeseed/canola (Brassica napus and B. rapa) crops in Saskatchewan (SK) and Manitoba, Canada since 1953 (2). Symptoms generally include stunting, virescence, leaf yellowing or purpling, phyllody, and formation of bladder-like siliques. A total of 120 mature B. rapa cv. AC Sunbeam plants exhibiting AY symptoms were collected in commercial fields near Medstead, SK during 2003 and 2004 (one field per year). As described previously (4), total genomic DNA was extracted from leaf, stem, roots, and seeds collected from the 120 plants, from seeds from the seed lots sown in 2003 and 2004, and from leaf and stem tissue of 20 greenhouse-grown plants from each seed lot. The latter DNA samples were assayed for phytoplasma DNA by a nested polymerase chain reaction (PCR) assay incorporating phytoplasma universal 16S rRNA primer pairs P1/P6 (1) followed by R16R2/R16F2 (4). Seed samples analyzed from the 2003 and 2004 seed lots and tissues of the 40 greenhouse-grown plants all tested negative for phytoplasma DNA using this assay. Leaf, stem, and/or root tissues of all plants collected in the field in 2003 (60 plants) and 2004 (60 plants) and 71.1% (315 of 443) of seed samples (five seeds per sample) tested positive for the presence of phytoplasma DNA, as evidenced by the presence of an expected band of 1.2 kb on the gels after the second amplification with primers R16R2/R16F2. Nested PCR products from plant samples collected in 2003 were cloned, sequenced, and compared with phytoplasma sequences archived in the GenBank nucleotide database. On this basis, phytoplasmas detected in plants or their seeds collected in 2003 were found to be most similar (98.8%) to CHRY (Accession No. AY180956), a 16SrI-A subgroup strain, or were most similar (98.9%) to isolate 99UW89 (Accession no. AF268407), a known 16SrI-B subgroup strain. Sequences of phytoplasmas detected in plants or their seeds in 2004 were obtained by direct sequencing of rRNA products amplified from samples using PCR incorporating primer pairs P1/P6 and P4/P7 (3). Analysis of sequence data revealed that phytoplasmas in these plants were all most similar (99.5%) to AY-WB (Accession no. AY389828), a 16SrI-A subgroup member. The nucleotide sequences have been deposited with GenBank under Accession nos. DQ404346, DQ404347, and DQ411470. To our knowledge, this is the first report of 16SrI-A and 16SrI-B subgroup phytoplasmas infecting plants and seed of B. rapa in Saskatchewan. References: (1) I.-M. Lee et al. Phytopathology, 83:834, 1993. (2) W. E. Sackston. Can. Plant Dis. Surv. 33:41, 1953. (3) L. B. Sharmila et al. J. Plant Biochem. Biotech. 13:1, 2004. (4) E. Tanne et al. Phytopathology, 91:741, 2001.
Although no loss of crown shape or unusual growth were evident on two mature Chinaberry trees (Melia azedarach L.) located near the citadel in central Hué city, Vietnam, leaves on both trees displayed distinctive interveinal yellowing during September 2003. This symptom was reminiscent in appearance to foliar discoloration previously observed on mature Chinaberry trees in El Torno, Santa Cruz, Bolivia that was subsequently attributed to phytoplasma infection of these trees (2). Eight samples of yellowed leaves were collected from affected trees and preserved by pressing and drying for later analysis. Total nucleic acids were extracted from 0.5 g of each leaf sample and assayed for phytoplasma DNA using a polymerase chain reaction (PCR) assay with phytoplasma universal rRNA primer pair P1 and P7 (4). No visible product was generated from any Chinaberry sample while a product of expected size (1.8 kb) was obtained from DNA of a periwinkle plant (Catharanthus roseus (L.) G. Don) infected with “Candidatus Phytoplasma asteris”-related strain eastern aster yellows (EAY) and included as a known positive control in the assay. After P1/P7-primed products were reamplified by PCR with nested phytoplasma universal 16S rRNA primer pair R16mF2/R16mR1 (1), a 1.4-kb product of predicted size was obtained from the eight samples and EAY positive control, whereas no product was obtained from DNA of seed-grown healthy periwinkle included as a negative control. Digests of nested PCR products (1.4 kb) with HaeIII or MseI endonuclease, and electrophoresis of digests through 8% polyacrylamide gels, revealed no apparent differences in restriction fragment patterns among products from Chinaberry samples. However, HaeIII and MseI patterns differed from those obtained by digestion of nested PCR products from EAY, a known 16SrI-A subgroup phytoplasma (3), with these enzymes. Chinaberry phytoplasmas were definitively identified as group 16SrI strains after reevaluation of samples by a PCR incorporating ribosomal protein (rp) gene primer pair rpF1/rpR1 and reamplification of resulting products with nested 16SrI group-specific primer pair rp(I)F1A/rp(I)R1A (3). A 1.2-kb product of expected size was obtained from all Chinaberry samples and EAY positive control only. Restriction fragment length polymorphism patterns produced by DraI or SspI endonuclease digests of nested PCR products (1.2 kb) revealed no differences among Chinaberry samples, although patterns associated with each enzyme differed from those observed for the EAY positive control. Sequence comparison and phylogenetic analysis of Chinaberry yellows phytoplasma (CbY-V) 16Sr DNA (GenBank Accession No. AY863003) determined this strain to be most closely related (99.65%) to Epilobium phyllody phytoplasma, a 16SrI-B subgroup strain (3). However, based on analysis of rp gene sequences (GenBank Accession No. DQ321823), strain CbY-V was judged most similar (99.59%) to cabbage proliferation, a well characterized 16SrI-B subgroup, rpI-B subgroup phytoplasma (3). To our knowledge, this is the first record of phytoplasma infection of Chinaberry, a common urban shade tree in Vietnam. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) N. A. Harrison et al. Plant Pathol. 52:147, 2003. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004 (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.