Plant Science

Australian lucerne yellows (ALuY), a phytoplasma-associated disease, is a major problem in Australia that causes the pasture seed industry millions of dollars in losses annually (3). Samples were collected from lucerne (Medicago sativa L.) plants exhibiting symptoms indicative of ALuY (4) in a seed lucerne paddock (cv CW 5558) at Griffith, southwestern New South Wales (NSW), Australia, in November 2005 and again in January 2006. Samples were kept at 4°C and processed within 36 h of collection. Total DNA was extracted from approximately 0.3 g of leaf midribs and petioles of each plant sample and used as template in a nested PCR assay with phytoplasma universal primer pairs P1/P7 and fU5/m23sr. PCR products resulting from the first amplification were diluted (1:30) with sterile distilled water (SDW) before reamplification with fU5/m23sr. DNA of Australian tomato big bud (TBB) phytoplasma and SDW were used as positive and negative assay controls, respectively. Ten of fifteen plant samples collected in November tested positive for phytoplasma DNA. Restriction digestion profiles of nested PCR amplicons with HpaII endonuclease were the same for all symptomatic plants but differed from the control. Phytoplasma identity was determined by sequencing two nested PCR products that yielded identical sequences. One was deposited in the GenBank database (Accession No. DQ786394). BLAST analysis of the latter sequence revealed a >99.6% similarity with “Candidatus Phytoplasma australiense” (L76865) and related strains papaya dieback (Y10095), phormium yellow leaf (U43570), strawberry green petal (AJ243044), and strawberry lethal yellows (AJ243045). Direct PCR with primers FP 5′-GCATGTCGCGGTGAATAC-3′ and RY 5′-TGAGCTATAGGCCCTTAATC-3′ designed to specifically amplify DNA of “Ca. P. australiense” detected the phytoplasma in 8 of 40 samples collected in January. Whether this phytoplasma is the etiological agent solely responsible for ALuY is currently under investigation. “Ca. P. asteris” and stolbur group (16SrXII) phytoplasmas have been reported in lucerne in the United States (2) and Italy (1), respectively. Within the stolbur group 16SrXII, “Ca. P. australiense” and stolbur phytoplasma are regarded as separate species and both are distinct from “Ca. P. asteris”, a group 16SrI strain. To our knowledge, this is the first report of a “Ca. P. australiense” related strain in lucerne. References: (1) C. Marzachi et al. J. Plant Pathol. 82:201, 2000. (2) R. D. Peters et al. Plant Dis. 83:488, 1999. (3) L. J. Pilkington et al. Australas. Plant Pathol. 28:253, 1999. (4) L. J. Pilkington et al. First report of a phytoplasma associated with ‘Australian lucerne yellows’ disease. New Disease Report. Online publication at http://www.bspp.org.uk/ndr/jan2002/2001-46.asp .

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.