Plant Disease

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with ‘Candidatus Liberibacter’ species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named ‘Candidatus Liberibacter solanacearum’. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

In São Paulo State, Brazil, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ are associated with huanglongbing (HLB). Affected municipalities occur mainly in the central and southern regions, where the annual number of hours above 30°C is two to five times lower than that in the extreme northern and western regions. The influence of temperature on sweet orange trees infected with ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’ was studied in temperature-controlled growth chambers. Symptom progression on new shoots of naturally infected and experimentally graft-inoculated symptomatic sweet orange trees was assessed. Mottled leaves developed on all infected trees at 22 to 24°C, but not on any ‘Ca. L. americanus’–infected trees at 27 to 32°C. Quantitative, real time-PCR was used to determine the liberibacter titers in the trees. After 90 days, ‘Ca. L. asiaticus’–infected trees had high titers at 32 and 35°C, but not at 38°C, while ‘Ca. L. americanus’–infected trees had high titers at 24°C, but at 32°C the titers were very low or the liberibacters could not be detected. Thus, the multiplication of ‘Ca. L. asiaticus’ is not yet affected at 35°C, while a temperature of 32°C is detrimental to ‘Ca. L. americanus’. Thus, ‘Ca. L. americanus’ is less heat tolerant than ‘Ca. L. asiaticus’. The uneven distribution of these two liberibacters in São Paulo State might be in relation with these results.

Zebra chip (ZC), an emerging disease causing economic losses to the potato chip industry, has been reported since the early 1990s in Central America and Mexico and in Texas during 2000 (4). ZC was subsequently found in Nebraska, Colorado, New Mexico, Arizona, Nevada, California, and Kansas (3). Severe losses to potato crops were reported in the last few years in Mexico, Guatemala, and Texas (4). Foliar symptoms include purple top, shortened internodes, small leaves, enlargement of the stems, swollen axillary buds, and aerial tubers. Chips made from infected tubers exhibit dark stripes that become markedly more visible upon frying, and hence, are unacceptable to manufacturers. Infected tubers may or may not produce plants when planted. The causal agent of ZC is not known and has been the subject of increased investigation. The pathogen is believed to be transmitted by the potato psyllid, Bactericera cockerelli, and the association of the vector with the disease is well documented (3). Following the report of a potential new liberibacter species in solanaceous crops in New Zealand, we sought to identify this liberibacter species in plants with symptoms of the ZC disease. Six potato plants (cv. Russet Norkota) exhibiting typical ZC symptoms were collected in Olton, TX in June of 2008. DNA was extracted from roots, stems, midribs, and petioles of the infected plants using a FastDNA Spin Kit and the FastPrep Instrument (Qbiogene, Inc., Carlsbad, CA). Negative controls from known healthy potato plants were included. PCR amplification was carried out with ‘Candidatus L. asiaticus’ omp primers (1), 16S rDNA primers specific for ‘Ca. L. asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ (1), and 16S rDNA primers OA2 (GenBank Accession No. EU834130) and OI2c (2). Amplicons from 12 samples were directly sequenced in both orientations (McLab, San Francisco CA). PCR amplifications using species-specific primers for the citrus huanglongbing liberibacter were negative. However, 1.1- and 1.8-kb amplicons were obtained with the OA2/OI2C and omp primers, respectively. The sequences for the rDNA were submitted to NCBI GenBank (Accession Nos. EU884128 and EU884129). BLASTN alignment of the 16S rDNA sequences obtained with primers OA2 and OI2c revealed 99.7% identity with a new species of ‘Ca. Liberibacter’ identified in New Zealand affecting potato (GenBank Accession No. EU849020) and tomato (GenBank Accession No. EU834130), 97% identity with ‘Ca. L. asiaticus’, and 94% with ‘Ca. L. africanus’ and ‘Ca. L. americanus’. The neighbor-joining phylogenetic tree constructed using the 16S rDNA fragments delineated four clusters corresponding to each of the liberibacter species. These results confirm that ‘Ca. Liberibacter’ spp. DNA sequences were obtained from potatoes showing ZC-like symptoms, suggesting that a new species of this genus may be involved in causing ZC disease. To our knowledge, this is the first report of the detection of ‘Ca. Liberibacter’ spp. in potatoes showing ZC disease in the United States. References: (1) C. Bastianel et al. Appl. Environ. Microbiol. 71:6473, 2005. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007. (4) G. A. Secor and V. V. Rivera-Varas. Rev. Latinoamericana de la Papa (suppl.)1:1, 2004.

A new ‘Candidatus Liberibacter’ species was recently identified in tomato, capsicum, and potato in New Zealand. The tomato/potato psyllid, Bactericera cockerelli, is thought to be the vector of this species of liberibacter. During studies to determine additional host plants of the pathogen, leaves of Solanum betaceum (tamarillo, also known as tree tomato) and leaves and stems of Physalis peruviana (cape gooseberry) were collected from a home garden in South Auckland, New Zealand in July of 2008. These plants were not showing any obvious disease symptoms. They were located close to a commercial glasshouse site containing known liberibacter-infected tomatoes, and many psyllids were observed on the tamarillo tree over the summer and until late autumn. Total DNA was extracted from four tamarillo and two cape gooseberry samples with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Samples from tamarillo that were used for the extraction were taken from the midveins of old and young leaves and from young petioles. For cape gooseberry, samples were from the leaf midveins and the stems. The samples were tested by PCR using primers OA2 (GenBank Accession No. EU834130) and OI2c (1). These primers amplify a 1,160-bp fragment of the 16S rRNA gene of the new liberibacter species. Amplicons of the expected size were obtained from all four tamarillo samples, with no amplification from negative control tamarillo plants grown from seed in an insect-proof glasshouse. Almost the entire length of the 16S rRNA gene was amplified using primer pairs fD2 (3)/OI2c and OA2/rP1 (3), and the 16S-23S rRNA intergenic spacer was amplified with primer pair OI2/23S1 (2). These amplicons, along with that from the OA2/OI2c primer pair, were directly sequenced, and overlapping fragments were assembled using the SeqMan software of the LaserGene package (DNASTAR, Inc., Madison, WI) (GenBank Accession No. EU935004). A 650-bp fragment of the β operon was also amplified and sequenced directly (GenBank Accession No. EU935005). BLAST analysis showed 100% nt identity to the liberibacter of tomato (GenBank Accession Nos. EU834130 and EU834131) and potato (GenBank Accession Nos. EU849020 and EU919514). The two cape gooseberry samples produced amplicons of the expected size with the 16S rRNA and β operon primers and the origin of the fragments were confirmed by direct sequencing with BLAST analysis showing 100% nt identity to isolates from tomato, potato, and tamarillo. To determine the distribution of disease, 53 samples of 10 leaves each (representing two leaves from five plants) were collected randomly from a commercial tamarillo crop in South Auckland. Small sections of the midveins were removed from each of the 10 leaves, bulked, and DNA was extracted as described above. The samples were tested by PCR using primer pair OA2/OI2c. Amplicons of the expected size were obtained from 2 of the 53 samples. To our knowledge, this is the first report of a liberibacter in tamarillo and cape gooseberry. It is unknown if the liberibacter induces symptoms in these species or if they act as symptomless reservoirs of the pathogen. The infected plants will be observed for symptom development over the course of a growing season. References: (1) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (2) S. Jagoueix et al. Int. J. Syst. Bacteriol. 47:224, 1997. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

Symptoms resembling “zebra chip” disease (3) were observed in potato (Solanum tuberosum) tubers harvested from a breeding trial in South Auckland, New Zealand in May 2008. The tubers had necrotic flecking and streaking that became marked when the potatoes were fried. Affected plants generally senesced early, at the beginning of April. The mean yield was approximately 60% less than expected and harvested tubers had less dry matter (13%) than normal (19%). Large numbers of the psyllid Bactericera cockerelli were observed on the crop during the summer. Total DNA was extracted from the vascular tissue of five symptomatic tubers and seven volunteers collected from the affected field with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Samples were tested by PCR using primers OA2 (GenBank Accession No. EU834130) and OI2c (2). These primers amplify a 1,160-bp fragment of the 16S rRNA sequence of a ‘Candidatus Liberibacter’ species identified in tomato and capsicum in New Zealand. No fragment was amplified from healthy plants, but amplicons of the expected size were obtained from all symptomatic tubers and one plant. A 650-bp fragment of the β operon was also amplified from symptomatic tubers. The amplicons were directly sequenced (GenBank Accession Nos. EU849020 and EU919514). BLAST analysis showed 100% identity to the tomato/capsicum liberibacter (GenBank Accession Nos. EU834130 and EU834131). From a commercial potato field adjoining the breeding trial, groundkeeper tubers were collected and separated into those that were asymptomatic and those that exhibited a range of symptoms. Total DNA was extracted and tested by PCR using the OA2/OI2c primers. In the first category, 6 of 10 tubers tested positive, whereas the 10 tubers in the second category tested negative. Two phytoplasmas seem to be involved in the “zebra chip” disease complex (4) but were not detected in the samples in this study. To our knowledge, this is the first report of a liberibacter associated with disease in potato. From transmission electron microscope observations, previous researchers have hypothesized that a bacterium-like organism may cause “zebra chip” (1) and B. cockerelli is associated with the disease (3). “Zebra chip” was first reported in Mexico in 1994, since then it has caused significant economic damage in Guatemala, Mexico, and the southwestern United States. The economic impact of the disease in New Zealand is yet to be determined. References: (1) S. H. De Boer et al. Page 30 in: New and Old Pathogens of Potato in Changing Climate. A. Hannukkala and M. Segerstedt, eds. Online publication. Agrifood Research Working Paper 142, 2007. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007. (4) G. A. Secor et al. Plant Dis. 90:377, 2006.

In recent years, lily (Lilium spp.) has become an important ornamental crop in diverse regions of Mexico. Since 2005, unusual symptoms have been observed on lily plants grown from imported bulbs in both greenhouse and production plots at San Pablo Ixayo, Boyeros, and Tequexquinauac, Mexico State. Symptoms included a zigzag line pattern on leaves, dwarfism, enlargement of stems, shortened internodes, leaves without petioles growing directly from bulbs, air bulbils, death of young roots, atrophy of flower buttons, and flower abortion. Symptoms were experimentally reproduced on healthy lily plants by graft inoculation. Total DNA was extracted from 50 diseased, 10 symptomless, and 10 graft-inoculated plants by the method of Dellaporta et al. (2). DNA samples were analyzed for phytoplasma presence by two different nested PCR assays. One assay employed ribosomal RNA gene primer pair P1/P7 followed by R16F2n/R16R2 (1), whereas ribosomal protein (rp) gene primer pairs rpF1/rpR1 and rp(I)F1A/rp(I)R1A (4) were used in a second assay. A DNA fragment approximately 1.2 kb long was consistently amplified from all symptomatic plant samples only by both assays. A comparative analysis of 16S rDNA sequences (Genbank Accession Nos. EF421158–EF421160 and EU124518–EU124520) and rp gene sequences (EU277012–EU277014), derived from PCR products, revealed that phytoplasma infecting lily were most similar (99.9% to 16S rDNA and 99.7% to rp) to carrot phytoplasma sp. ca2006/5 and also were similar (99.8% to 16SrDNA and 99.2% to rp) to broccoli phytoplasma sp. br273. Both carrot and broccoli phytoplasmas were classified as members of aster yellow 16S rDNA restriction fragment length polymorphism subgroup 16SrI-B (3). Although infection of lilies by aster yellows (‘Ca. phytoplasma asteris’) subgroup 16SrI-B and 16SrI-C was reported from the Czech Republic and Poland, to our knowledge, this is the first report of ‘Ca. phytoplasma asteris’-related strains associated with lily plants in Mexico. References: (1) R. F. Davis et al. Microbiol. Res. 158:229, 2003. (2) S. L. Dellaporta et al. Plant Mol. Biol. Rep. 1:19, 1983. (3) B. Duduk et al. Bull. Insectol. 60 2:341, 2007. (4) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004.

Citrus huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The disease is associated with three different species of fastidious α-proteobacteria, namely ‘Candidatus Liberibacter asiaticus’, Ca. L. americanus, and Ca. L. africanus (1). ‘Ca. L. asiaticus’ was first detected in South Florida in 2005 and has spread throughout the citrus-growing areas of Florida. ‘Ca. L. asiaticus’ is transmitted naturally by the Asian citrus psyllid, Diaphorina citri, and can also be transmitted by graft propagation and via various species of dodder (Cuscuta). HLB affects most if not all citrus and citrus relatives within the family Rutaceae (2), including the ornamental shrub Murraya paniculata (4). In addition, ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’ can infect tobacco (Nicotiana xanthi) and periwinkle (Catharanthus roseus) (1,4). Here we report that ‘Ca. L. asiaticus’ can infect tomato (Lycopersicon esculentum) cvs. Manapal and FL47. Manapal and FL 47 plants grown from seed were placed adjacent to ‘Ca. L. asiaticus’-infected sweet orange (Citrus sinensis) plants with dodder (Cuscuta pentagona) already well established on them. Young dodder shoots still connected to the citrus were draped over the tomato plants and subsequently also became attached to the tomato stems. After 1 month, the tomato plants were detached from the citrus and most of the dodder removed. One month later, these tomato plants started to show vein clearing and subsequently the mature leaves became thicker and leathery. Some leaves showed blotchy mottle symptoms and some fruits became lopsided in a manner similar to HLB symptom expression on citrus. PCR amplification of the ‘Ca. L. asiaticus’ 16S rDNA with primers OI1/OI2c and the β-operon with primers A2/J5 (1) revealed the presence of ‘Ca. L. asiaticus’ DNA. Sequence analysis confirmed that the sequences of the cloned amplicons were identical to those from the HLB-infected citrus source plant. Both conventional and quantitative real-time PCR (3) revealed a much lower abundance of ‘Ca. L. asiaticus’ DNA in tomato as compared with ‘Ca. L. asiaticus’-infected citrus or periwinkle, indicating that ‘Ca. L. asiaticus’ bacteria multiplied at a lower titer in these tomato cultivars. References: (1) J. M. Bové, J. Plant Pathol. 88:7, 2006. (2) S. E. Halbert et al. Fla. Entomol. 87:330, 2004. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006. (4) L. Z. Zhou et al. Plant Dis. 91:227, 2007.

During a survey in 2003, safflower plants (Carthamus tinctorius) with phyllody symptoms were observed in production fields in several districts of Fars and Yazd provinces in Iran. Affected plants showed floral virescence, phyllody, proliferation of axillary buds, and little leaf symptoms. Incidence of the disease was less than 10%. Direct and nested PCR assays were used to verify association of phytoplasma with the disease. Total DNA was extracted from fresh, fine roots of eight phyllody-affected safflower plants and one symptomless plant. With phytoplasma universal primer pair P1/P7 (5′-AAGAGTTTGATCCTGGCTCAGGATT-3′/5′-CGTCCTTCATCGGCTCTT-3′), target DNA fragments of approximately 1.8 kb were amplified by direct PCR from phyllody-affected plants and Iranian cabbage yellows (ICY) phytoplasma used as a positive control. Reamplification of P1/P7 products with 16S rRNA gene primer pair R16F2n/R16R2 (5′-GAAACGACTGCTAAGACTGG-3′/5′-TGACGGGCGGTGTGTACAAACCCCG-3′) yielded fragments of the expected size (1.2 kb) from all eight diseased plants and the ICY-positive control. No products were amplified from the symptomless plant by either assay. R16F2n/R16R2 products were subjected to restriction fragment length polymorphism (RFLP) analysis by separate digestion with AluI, HaeIII, HhaI, HinfI, HpaII, MseI, RsaI, Sau3AI, or TaqI endonuclease. Comparison of resulting RFLP patterns with published patterns of other phytoplasmas (2) tentatively identified safflower phyllody (SP) phytoplasma as a member of clover proliferation group 16SrVI, subgroup C. HhaI digests also differentiated SP from ICY phytoplasma, a previously reported subgroup 16SrVI-A strain (3). After sequencing of the 16S rDNA fragment (GenBank Accession No. DQ88948), a BLAST search determined that SP phytoplasma shared closest homology with 16SrVI group members (‘Candidatus Phytoplasma trifolii’) and related strains (4). Furthermore, phylogenetic analysis of 16S rDNA sequences revealed SP phytoplasma to be most similar (99.7%) to brinjal little leaf (BLL) phytoplasma (GenBank Accession No. X83431). Analysis of putative restriction sites in 16S rRNA gene sequences revealed that SP and BLL shared identical restriction profiles and that both differed from the ‘Ca. Phytoplasma trifolii’ reference strain (GenBank Accession No. AY390261) because of the absence of a single HhaI site and the presence of an additional MseI site. Although safflower phyllody disease has been previously reported in Israel, the associated phytoplasma was classified as a strain of the aster yellows subgroup 16SrI-B (1). To our knowledge, this is the first report of safflower as a host of a ‘Ca. Phytoplasma trifolii’-related strain. References: (1) M. Klein. Plant Dis. Rep. 54:735, 1970. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (3) M. Salehi et al. Plant Dis. 91:625, 2007. (4) K. Wang and C. Hiruki, Phytopathology 91:546, 2001.

Atalantia buxifolia (Poir.) Oliv., synonym Severinia buxifolia (Poir.) Ten. as commonly found in literature, is a common landscape plant and a popular Chinese medicinal herb known as Jiubingle or Dongfengjie. It remains unclear if this rutaceous plant could host ‘Candidatus Liberibacter asiaticus’, the pathogen of citrus Huanglongbing (HLB) in Guangdong, P. R. China. This information is important for HLB control in citrus because infected A. buxifolia could serve as a source of inoculum. In August of 1994, three A. buxifolia plants adjacent to a citrus experimental orchard of the South China Agricultural University at Guangzhou were found showing leaf mottle/yellowing symptoms. Two buds from each plant were grafted onto three mandarin trees (Citrus reticulata cv. Pongan) in a psyllid-proof screenhouse for indexing. By October of 1995, typical leaf mottle symptoms were observed in all three grafted trees compared with a healthy control. In March of 1996, one of the A. buxifolia plants was transferred to a screenhouse and has been maintained there. The leaf mottle/yellowing symptoms persisted but did not significantly affect plant growth. DNA was extracted from leaf samples in October 2006 by using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR using the general bacterial 16S rDNA primer set fDl/rD1 as the first round of amplification and primer set OI1/OI2c as second round amplification (1,3). After agarose gel electrophoresis and staining with ethidium bromide, an approximate 1.1-kb DNA band was detected in symptomatic samples but not healthy leaf samples of A. buxifolia and C. reticulata. XbaI digestion of the amplicons yielded approximate 500- and 600-bp fragments, characteristic of ‘Ca. L. asiaticus’. Similarly, a standard PCR with primer set A5/J2 (3) yielded an approximate 700-bp DNA band characteristic of ‘Ca. L. asiaticus’ from symptomatic samples only. To our knowledge, this is the first report of graft transmission and PCR detection of ‘Ca. L. asiaticus’ from A. buxifolia in Guangdong, P. R. China. This work also confirms the findings from Taiwan (2) that A. buxifolia could serve as a source of ‘Ca. L. asiaticus’. References: (1) X. Deng et al. Online publication. doi:10.1094/PHP-2007-0419-01-BR. Plant Health Progress, 2007. (2) T.-H. Hung et al. Eur. J. Plant Pathol. 107:183, 2001. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996.

Little is known about ‘Candidatus Liberibacter americanus’, a causal agent of huanglongbing or greening disease in Brazil, or its interaction with citrus trees. Greenhouse experiments were conducted with the objective of determining conditions favorable for transmission from field affected trees to young potted plants, to evaluate the reaction of multiple citrus species to the disease, and to determine the efficiency of pathogen propagation from individual buds. Single buds or bark pieces of various sizes or bark plus wood (budstick) that were removed from symptomatic or asymptomatic branches were used as sources of inoculum. Transmission success was evaluated through polymerase chain reaction analysis of total DNA extracted from leaf samples. Beginning at 4 to 5 months after inoculation, infected plants manifested leaf mottling and symptoms similar to those of iron, manganese, and zinc deficiencies. Blotchy mottled leaves appeared only on sweet oranges and Murcott tangor. Pathogen transmission was higher for these citrus cultivars and species (31.2 to 65.2%) than for limes, mandarins, or Swingle citrumelo (2.0 to 25.0%). Deformed small fruits with brownish columellae also developed on sweet oranges. Only buds and budsticks served as sources of inoculum and the larger the piece of tissue, the higher the transmission efficiency. Experiments initiated during the winter showed lower rates of graft tissue survival but relatively higher percentages of pathogen transmission.