Plant Science

Zebra chip disease (ZC), putatively caused by the fastidious bacterium ‘Candidatus Liberibacter solanacearum’, is a threat to potato growers worldwide. However, little is known about biochemical shifts in different potato genotypes in response to ‘Ca. L. solanacearum’ infection. To address this, ‘Red La Soda’, ‘Russet Norkotah’, and ‘FL 1867’ potato were infected with ‘Ca. L. solanacearum’ 4, 3, 2, and 1 weeks before harvest to observe variability in cultivar responses to ‘Ca. L. solanacearum’ infection. ZC symptoms, ‘Ca. L. solanacearum’ titers, and tuber biochemistry were assessed. Red La Soda tubers exhibited greater symptoms when infected for 4 weeks than Russet Norkotah or FL 1867 tubers. ‘Ca. L. solanacearum’ titers did not vary among cultivars. Tuber levels of amino acids, carbohydrates, and phenolics varied among cultivars but no consistent trends were observed. Individual amino acids and phenolics were greater in FL 1867 than Red La Soda, whereas others were greater in Red La Soda or Russet Norkotah than FL 1867. Most amino acids, carbohydrates, and phenolics were positively associated with infection duration and symptoms regardless of cultivar. Associations between most of the evaluated compounds and ‘Ca. L. solanacearum’ titer were positive in Red La Soda. However, no associations between ‘Ca. L. solanacearum’ quantity and compounds were observed in FL 1867 and Russet Norkotah.

‘Candidatus Phytoplasma australiense’ is associated with a number of plant diseases in New Zealand. The only known vector of this pathogen was Zeoliarus atkinsoni, a planthopper considered to be monophagous on New Zealand flax (Phormium spp.). The work carried out shows that Z. oppositus, which is polyphagous, is able to vector ‘Ca. P. australiense’ to both Coprosma robusta (karamu) and Cordyline australis (New Zealand cabbage tree). Although transmission was achieved to both these species, the disease symptomatology was more evident in C. australis. Two approaches were taken to achieve transmission. First, insects were collected from areas around symptomatic Coprosma plants and caged directly on test plants. Second, insects were collected from grasses and sedges in areas where disease was less evident and were fed on known infected Coprosma plants prior to being caged on test plants. Transmission was achieved using both approaches, although transmission was far greater (30% compared with 4%) from insects that were directly applied. Phytoplasma DNA was detected in 12% of Z. oppositus individuals tested during all the trials. This work identifies a new vector for ‘Ca. P. australiense’ and contributes to our understanding of the ecology of Cordyline sudden decline and Coprosma lethal decline.

Huanglongbing (HLB) disease, associated with ‘Candidatus Liberibacter asiaticus’ (CLas), is primarily spread via infected citrus nursery trees and by infective Asian citrus psyllid, the insect vector. Recently, the Texas Department of Agriculture initiated regulations requiring commercial and retail citrus nurseries in Texas to transition from traditional open-field to enclosed facilities with insect-resistant screens to mitigate the risk of nurseries serving as sources of CLas. Although several nursery production facilities have adopted this regulation, non-enclosed nurseries persist and pose a significant threat to the citrus industry as potential sources of CLas. A systematic survey for HLB was embarked on in a semi-open nursery facility in South Texas in April 2014. Leaf tissue samples taken from 94 trees representing 5% of the total number of potted trees in the nursery were tested for CLas by quantitative and conventional PCR assays. Of 94 trees tested, 3.2% (3 trees) were positive for CLas by both assays. The presence of CLas in the PCR-positive samples was confirmed by multi-locus sequence analyses. The results represent the first report of HLB in a nursery facility in Texas, and underscore the need for more intensive surveillance for HLB in citrus nursery stock as an integral component of HLB mitigation efforts in Texas. Accepted for publication 27 August 2014. Published 15 December 2014.

Elm yellows (EY) is a lethal disease of American (Ulmus americana L.) and other elm species (1). On the Pennsylvania State University campus, EY, together with Dutch elm disease, has killed 82 of about 400 mature elms since 2007, the year of first EY detection. Candidatus Phytoplasma ulmi, associated with EY, has been reported to be transmitted by the whitebanded elm leafhopper Scaphoideus luteolus Van Duzee, the meadow spittlebug Philaenus spumarius L., and the leafhopper Allygus atomarius Fabricius (1) in North America, but correlation of these insects with EY in the eastern United States has not been reported. Three Cicadellidae collections using sweep nets and aspirators were performed from July to September 2012 on branches of an EY infected red elm (U. rubra Muh; 40°48.408′N, 77°52.208′W) and on vegetation within a 0.5 km radius. The red elm is in close proximity to trees, shrubs, and a managed meadow and has repeatedly tested positive for EY since 2007. During each collection, about 200 cicadellids were captured in BioQuip No-See-Um catch bags with cups, and the bags were hung around the red elm branches, forcing the insects to feed on the infected tree for 24 h. Insects were transferred to BugDorm rearing tents containing wild grasses, elm seedlings, cowpeas, celery, carrots, and basil, all grown from seed, and were kept for 3 weeks in a controlled environment chamber at 28°C and 70% humidity with a 16-h photoperiod. Insects easily recognized in the same species or individual insects of uncertain identity were then isolated for about 1 week in cages each containing one 6-month-old healthy American elm seedling (grown from seed in growth chamber). Up to 10 morphospecies were found in each collection, with 1 to 20 individuals per morphospecies. The total number of unique morphospecies used in the three transmission trials and later identified as different species was 8. Dead insects collected daily were stored in 80% ethanol and later identified to genus or species level. About 70% insect mortality was recorded, but about 60 individuals from each collection survived the change of diet and environment. After 3 months, individual elm seedlings were tested by RT-PCR (3) for the presence of phytoplasmas using universal primers fU5/rU3 (2). PCR products were visualized on 1.5% agarose gel, and if DNA was amplified, it was cloned and sequenced. Three of 30 seedlings tested positive for phytoplasmas and sequencing of the cloned products (24 clones were sequenced per transformation, per each of the three positive seedlings) confirmed that only Ca. P. ulmi was present in the 3 infected seedlings but not in the remaining 27 or in 46 unexposed control seedlings. The 3 seedlings were each exposed to a single insect and the same insects that were used in the transmission trial were identified. One spittlebug (Cercopidae) Lepyronia quadrangularis Say, one P. spumarius, and one leafhopper in the genus Latalus (Cicadellidae: Deltocephalinae) were identified as vectors. The phytoplasma-positive seedlings showed stunting and yellowing, and died shortly after testing. Other insects captured and identified in the survey were A. atomarius, Neophilaenus lineatus L., Metcalfa pruinosa Say, Amblysellus curtisii Fitch and individuals in the genera Draeculacephala, Elymana, Empoasca, Mesamia, Stroggylocephalus, and Ceratagallia. S. luteolus was not captured during this sampling but was captured on yellow sticky traps and in light traps in previous years at other locations on the campus. This is the first report suggesting that L. quadrangularis and Latalus sp. can serve as natural vectors of EY. References: (1) P. Herath et al. Plant Dis. 94:1355, 2010. (2) H. Lorenz et al. Phytopathology 85:771, 1995. (3) P. Margaria et al. Plant Dis. 91:1496, 2007.

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’. Infection with ‘Ca. L. asiaticus’ is incurable; therefore, knowledge regarding ‘Ca. L. asiaticus’ biology and pathogenesis is essential to develop a treatment. However, ‘Ca. L. asiaticus’ cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of ‘Ca. L. asiaticus’ in vitro, ‘Ca. L. asiaticus’ inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima ‘Mato Buntan’) was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air–liquid interface of juice cultures contained ‘Ca. L. asiaticus’ cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining ‘Ca. L. asiaticus’ viability in vitro, which will contribute to future development of a culture medium for ‘Ca. L. asiaticus’.

In April and May of 2012, bell pepper (Capsicum annuum) plants exhibiting symptoms that resembled those of the bacterium ‘Candidatus Liberibacter solanacearum’ infection (2,4) were observed in commercial pepper fields in several departments in Honduras, including Francisco Morazán, Ocotepeque, El Paraíso, and Olancho. Many of the fields were infested with the psyllid Bactericera cockerelli, a vector of ‘Ca. L. solanacearum’ (3). The plants exhibited chlorotic or pale green apical growth and leaf cupping, sharp tapering of the leaf apex, shortened internodes, and overall stunting (2,4). All cultivars grown were affected and 20 to 75% of plants in each field were symptomatic. Pepper (var. Nataly) plant samples were collected from a total of eight affected fields (two fields per department). Total DNA was extracted from the top whole leaf tissue of a total of 19 plants, including 14 symptomatic and 5 asymptomatic pepper plants, with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (1). The DNA samples were then tested by PCR using specific primer sets OA2/OI2c and OMB 1482f/2086r to amplify a portion of 16S rDNA and the outer membrane protein (OMB) genes, respectively, of ‘Ca. L. solanacearum’ (1,2). OMB gene and 16S rDNA fragments of 605 and 1,168 bp, respectively, were amplified from the DNA of 7 of 14 (50%) symptomatic plants with each primer set, indicating the presence of ‘Ca. L. solanacearum.’ No ‘Ca. L. solanacearum’ was detected in the five asymptomatic plants with either primer sets. DNA amplicons with both primer sets were cloned from the DNA of plant samples collected from each of the three departments: Francisco Morazán (in the locality of Zamorano), Ocotepeque (municipality of Plan del Rancho in Sinuapa), and El Paraíso (municipality of Danlí), and four clones of each of the six amplicons were sequenced. BLASTn analysis of the 16S rDNA resulted in a single consensus sequence for all three locations (deposited in GenBank as Accession Nos. KF188226, KF188227, and KF188228) and showed 100% identity to numerous 16S rDNA sequences of ‘Ca. L. solanacearum’ in GenBank, including accessions HM245242, JF811596, and KC768319. Similarly, identical OMB consensus sequences were observed in all three locations (deposited in GenBank as KF188230, KF188231, and KF188233) that are 100% identical to several ‘Ca. L. solanacearum’ sequences in GenBank (e.g., KC768331 and CP002371) along with a second consensus sequence (deposited in GenBank as accession KF188232) from Ocotepeque that was 99% identical to the consensus sequence from the three locations and sequences in GenBank. To our knowledge, this is the first report of ‘Ca. L. solanacearum’ associated with pepper crops in Honduras, where pepper constitutes an economically important commodity. This bacterium has also caused millions of dollars in losses to potato and several other solanaceous crops in United States, Mexico, Central America, and New Zealand (1,2,3,4). Furthermore, ‘Ca. L. solanacearum’ has been reported to severely damage carrot crops in Europe, where it is transmitted to carrot by the psyllids Trioza apicalis and Bactericera trigonica (3). Monitoring this pathogen and its vectors will prevent serious damage they cause to economically important crops. References: (1) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012. (4) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009.

In May of 2012, eggplant (Solanum melongena) plants in an experimental research plot located at Zamorano in the Department of Francisco Morazán, Honduras, were observed with symptoms that included leaf chlorosis and cupping, overall stunting, and production of small and malformed fruits. The research plot was planted next to a commercial tomato field heavily infested with the psyllid Bactericera cockerelli, a vector of ‘Candidatus Liberibacter solanacearum’ (1,2,3). This bacterium severely affects potato and other solanaceous species and is the putative causal agent of zebra chip disease (2,3). The plot was planted with the eggplant variety ‘China’ and about 25% of the plants were symptomatic. A total of 10 eggplant samples, including five symptomatic and five asymptomatic plants, were collected from the plot. Total DNA was extracted from the leaf tissue of each of the collected plants with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (1). The DNA samples were then tested by PCR using specific primer sets OA2/OI2c and OMB 1482f/2086r to amplify a portion of 16S rDNA and the outer membrane protein (OMB) genes, respectively, of ‘Ca. L. solanacearum’ (1,2). OMB gene and 16S rDNA fragments of 605 and 1,168 bp, respectively, were amplified from the DNA of two of the five (40%) symptomatic plants with each primer set, indicating the presence of ‘Ca. L. solanacearum.’ No ‘Ca. L. solanacearum’ was detected in the five asymptomatic plants with either primer sets. DNA amplicons with both primer sets were cloned from the DNA of the two ‘Ca. L. solanacearum’-positive plant samples and four clones of each of the four amplicons were sequenced. BLASTn analysis of the 16S rDNA resulted in two independent but related consensus sequences (deposited in GenBank as Accession Nos. KF188224 and KF188225) and were 99% similar to each other. The two sequences showed 99 to 100% identity to a number of 16S rDNA sequences of ‘Ca. L. solanacearum’ in Genbank, including accessions HM245242, FJ811596, and KC768319. For the OMB amplicons, a single consensus sequence was obtained following clone sequencing and was deposited in GenBank as accession KF188229. BLASTn analysis of the sequence indicated that it was 100% identical to several OMB sequences of ‘Ca. L. solanacearum’ in GenBank, including accessions KC768331 and CP002371. To our knowledge, this is the first report of ‘Ca. L. solanacearum’ associated with eggplant in Honduras. Eggplant is an economically important commodity in Central America and serious damage to this crop due to this plant pathogen could expand throughout the region, especially if its insect vector B. cockerelli is not properly managed. ‘Ca. L. solanacearum’ has also caused millions of dollars in losses to potato and several other solanaceous crops in the United States, Mexico, Central America, and New Zealand (2,3). In addition, this bacterium severely damages carrot crops in Europe, where it is transmitted to carrot by the psyllids Trioza apicalis and B. trigonica (3,4). It is imperative that both ‘Ca. L. solanacearum’ and its insect vectors be effectively monitored and managed to minimize their threat to economically important vegetable crops in many parts of the world. References: (1) J. M. Crosslin et al. Southwest. Entomol. 36:125, 2011. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012. (4) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010.

Abstract Background Huanglongbing (HLB) is a highly destructive citrus disease which threatens citrus production worldwide and ‘Candidatus Liberibacter asiaticus’ (Las), a non-culturable phloem-limited bacterium, is an associated causal agent of the disease. To better understand the physiological and molecular processes involved in host responses to Las, 2-DE and mass spectrometry analyses, as well as ICP spectroscopy analysis were employed to elucidate the global protein expression profiles and nutrient concentrations in leaves of Las-infected grapefruit plants at pre-symptomatic or symptomatic stages for HLB. Results This study identified 123 protein spots out of 191 spots that showed significant changes in the leaves of grapefruit plants in response to Las infection and all identified spots matched to 69 unique proteins/peptides. A down-regulation of 56 proteins including those associated with photosynthesis, protein synthesis, and metabolism was correlated with significant reductions in the concentrations of Ca, Mg, Fe, Zn, Mn, and Cu in leaves of grapefruit plants in response to Las infection, particularly in symptomatic plants. Oxygen-evolving enhancer (OEE) proteins, a PSI 9 kDa protein, and a Btf3-like protein were among a small group of proteins that were down-regulated in both pre-symptomatic and symptomatic plants in response to Las infection. Furthermore, a Las-mediated up-regulation of 13 grapefruit proteins was detected, which included Cu/Zn superoxide dismutase, chitinases, lectin-related proteins, miraculin-like proteins, peroxiredoxins and a CAP 160 protein. Interestingly, a Las-mediated up-regulation of granule-bound starch synthase was correlated with an increase in the K concentrations of pre-symptomatic and symptomatic plants. Conclusions This study constitutes the first attempt to characterize the interrelationships between protein expression and nutritional status of Las-infected pre-symptomatic or symptomatic grapefruit plants and sheds light on the physiological and molecular mechanisms associated with HLB disease development.