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2011 Potomac Division Meeting Abstracts

March 9-11, 2011 - Rehoboth Beach, Delaware

Statistical tools useful in characterizing the molecular response of cacao to Moniliophthora roreri infection
B. A. BAILEY (3), J. Crozier (1), M. D. Strem (3), R. L. Melnick (3), D. Zhang (3), S. Maximova (2), M. J. Guiltinan (2), L. Meinhardt (3)
(1) CABI Caribbean & Latin America – CATIE Office, Centro Agronómico Tropica de Investigación y Enseñanza (CATIE); (2) Department of Horticulture, The Pennsylvania State University, University Park, PA, U.S.A.; (3) Sustainable Perennial Crops Laboratory, Plant Sciences Institute, USDA/ARS, Beltsville Agricultural Research Center-West

Moniliophthora roreri (Mr) causes frosty pod rot, a destructive disease on Theobroma cacao (cacao). Pods were inoculated with Mr spores in the field and assessed for disease. Total RNA was extracted from pods harvested 7, 30, 60, and 90 days after inoculation and QPCR analysis was carried out using primer sets for 5 Mr ESTs and 89 cacao ESTs. Disease symptoms and Mr EST expression levels in infected pods increased rapidly between 7 and 60 DPI. ANOVA consistently identified individual cacao ESTs responsive to development and infection but was dependent on fixed inoculations and harvest times. Multiple regression analysis allowed fixed inoculation and harvest times to be replaced with independent variables such as pod size and fungal gene expression but still only provided information on individual cacao ESTs. The cacao expression data were analyzed across experiments and ESTs using principle coordinates analysis (PCoA) techniques. PCoA allowed consideration of how individual cacao EST expression was altered in response to Mr infection and development relative to all the ESTs studied in a single analysis. A Mantel test of data from 2 independent experiments indicates that the molecular signature for the susceptible response to Mr is highly reproducible. These techniques should allow us to expand our understanding of the Mr/cacao interaction using field samples without fixed treatments, incorporating divergent plant and pathogen genetic backgrounds.

Leaf anthracnose, a new disease of swallow-worts from Russia
D. K. Berner (1), C. A. CAVIN (1)

Black swallow-wort Vincetoxicum nigrum (L.) Moench (=Cynanchum louiseae Kartesz & Gandhi) and pale swallow-wort Vincetoxicum rossicum (Kleopow) Borhidi (=Cynanchum rossicum (Kleopow) Borhidi) are invasive plants belonging to the family Apocynaceae and are the targets of biological control efforts to control their spread in the U.S.A. In 2010, diseased leaves of a related species, V. scandens Sommier & Levier, were collected in the Krasnodar area of Russia and sent to the BLS-3 containment facility at the foreign disease-weed science research unit (FDWSRU) of USDA, ARS in Frederick Maryland. Surface disinfested leaves were placed in sterile moist chambers, and, after several days at 20–25°C, acervuli formed on irregular tan necrotic lesions. Pure cultures (FDWSRU 10-002) were obtained by transferring spore masses with sterile glass needles onto 20% V-8 juice agar. Seeds of V. scandens, collected simultaneously in Russia, were placed in a freezer at –20°C for 6 weeks and then germinated in sterile Petri plates on moist filter paper. The seedlings were then transplanted and grown in a 20°C greenhouse under 12 hours of light. Five two-month-old plants each of V. scandens, V. nigrum, and V. rossicum were inoculated with spores from two-week-old cultures of isolate 10-002. Plants were inoculated by spraying an aqueous suspension of 10*6 spores per ml onto each plant until all leaves were wet. The plants were placed in 20–24°C dew chambers for 18 hours and then placed in a 20°C greenhouse. Two weeks later, diseased leaves of each species were harvested, and the fungus was re-isolated from each species. Spore and appressoria measurements conformed to the description of Colletotrichum lineola Corda, and ITS sequences of this isolate (GenBank # HQ731491) aligned 100% to 15 isolates of C. lineola in GenBank. Voucher specimens of the fungus have been deposited in the U.S. national fungus collection.

Previously undescribed phytoplasmas in diseased plants of passion fruit (Passiflora edulis f. flavicarpa Deg.)
R. E. DAVIS (3), Y. Zhao (3), E. L. Dally (3), R. Jomantiene (2), I. Lee (3), E. W. Kitajima (1)
(1) Escola Superior de Agricultura “Luiz de Queiroz”, Piracicaba, Sao Paolo, Brazil; (2) Phytovirus Laboratory, Nature Research Centre, Vilnius, Lithuania; (3) USDA-Agricultural Research Service, Beltsville, MD, U.S.A.

Diseases of passion fruit (Passiflora edulis f. flavicarpa, family Passifloraceae) possibly associated with infections by phytoplasmas have been observed in Brazil, where the plant is widely cultivated and used in food products and beverages. Although the association of phytoplasmas with passion fruit disease has been recognized for over 30 years, little work has been done to characterize the phytoplasmas. In the present study, passion fruit plants exhibiting symptoms of witches’ broom growths in Brazil were investigated for possible infection by phytoplasma. Analysis of 16S ribosomal (r) DNA amplified in the polymerase chain reaction (PCR) indicated that the symptomatic plants were infected by two distinctly different phytoplasma strains. Based on results from actual and virtual (iPhyClassifier) RFLP analysis of 16S rDNA, one strain (PassWB-Br4) represents a previously undescribed subgroup lineage in group 16SrIII (X-disease phytoplasma group); the other strain (PassWB-Br3) represents a previously undescribed subgroup lineage in group 16SrVI (clover proliferation group, ‘Candidatus Phytoplasma trifolii’-related strains). Phylogenetic analyses and nucleotide sequence alignments of 16S rRNA gene sequences revealed that strain PassWB-Br3 differed from all previously described ‘Ca. Phytoplasma’ species and should be recognized as representative of a new ‘Ca. Phytoplasma’ species.

An investigation into homeostasis of gibberellin in potato purple top phytoplasma-infected tomato plants
Y. DING (1), W. Wu (1), W. Wei (1), I. Lee (1), R. E. Davis (1), Y. Zhao (1)
(1) MPPL-ARS-USDA, Beltsville, MD, U.S.A.

Phytoplasmas are a group of phloem-restricted, cell wall-less bacteria responsible for numerous plant diseases. Plants infected by phytoplasmas exhibit various symptoms believed to be the results of hormonal imbalance. Gibberellins (GAs) are essential phytohormones that regulate growth and development of plants. It has been shown that exogenous application of GA can effectively remit dwarf symptoms caused by phytoplasma and spiroplasma infections, a strong indication of the shortage of endogenous GA in mollicute-infected plants. The purpose of the current study was to investigate the phytoplasma-induced changes in expression profiles of GA metabolism genes, and to examine whether exogenous GA application would restore GA homeostasis. Columbia Basin potato purple top (PPT) phytoplasma and Rutgers tomato were used as a model pathogen-host pair in this study. Results revealed that, compared with healthy (mock-inoculated) plants, transcript levels of two GA biosynthesis genes, GA20ox2 (encoding GA 20-oxidase 2) and GA3ox2 (encoding GA 3-oxidase 2), were notably decreased in PPT-infected plants. Transcript levels of a GA catabolism gene, GA2ox1 (encoding GA 2-oxidase 1), were also slightly reduced in PPT phytoplasma infected plants. Exogenous application of gibberellin acid (GA3) significantly attenuated the “big bud” symptoms on PPT-infected plants. Expressions of GA20ox2 and GA3ox2 were significantly down-regulated after GA application in PPT-infected plants. On the other hand, expression of GA2ox1 was dramatically up-regulated upon GA treatment. These results indicated that i) GA homeostasis was maintained by feedback regulation of GA metabolism genes; ii) phytoplasma infection caused a shift in GA homeostasis; and iii) exogenous application of GA3 was able to partially reverse the shift, toward normal GA homeostasis. Findings from the present study will aid our understanding of the role of GA in phytoplasma pathogenesis and exogenous GA-induced phytoplasmal disease symptom remission.

Resting spores for long-term storage of Synchytrium solstitiale, a candidate for biological control of yellow starthistle
F. M. ESKANDARI (1), W. L. Bruckart III (1), T. L. Widmer (1)
(1) USDA, ARS, FDWSRU, Ft. Detrick, MD, U.S.A.

An isolate of Synchytrium solstitiale from France has been evaluated recently for biological control of yellow starthistle (YST, Centaurea solstitialis). Protocol was needed for long-term storage of S. solstitiale for research and archival purposes. In greenhouse studies, germination of mature resting spores of S. solstitiale resulted after storage in dried YST leaves for >2.5 years. Dried YST leaves were surface sterilized, and resting spores galls were removed by scraping or grinding leaf tissue. Spores were placed on 2% water agar in Petri dishes that were wrapped with Parafilm and aluminum foil and incubated at 10/15°C (night/day temperatures). One vesicle per resting spore, each with a single sporangium (= sorus), developed in 7–20 days. Zoospores were released from sori in sterile distilled water with 100 ppm Streptomycin. Plants also inoculated with sori from resting spores were incubated in moist plastic bags at 10/15°C (night/day temperatures) and an 8-hour photoperiod. Plants were removed from the growth chamber after 10 days, placed in a 20°C greenhouse, and observed for symptom development. Successful germination and plant infection occurred from inoculation by resting spore galls following this protocol. A test to measure viability and virulence of resting spores after 1.4 or 2.6 years of storage resulted in successful germination and infection of YST plants using the protocol described. Thus, long-term storage and maintenance protocol for S. solstitiale has been achieved.

The rice blast fungus, Magnaporthe oryzae, copes with plant-generated reactive oxygen species through the virulence factor MoHYR1
K. HUANG (3), K. J. Czymmek (3), J. L. Caplan (1), J. A. Sweigard (2), N. M. Donofrio (3)
(1) Delaware Biotechinolodge Institute; (2) Stine-Haskell Lab; (3) University of Delaware

During plant-pathogen interactions, the plant may mount several types of defense responses to either block the pathogen completely or ameliorate the amount of disease. One such response is the production of reactive oxygen species (ROS). On the other hand, a successful pathogen will likely have its own ROS detoxification mechanisms to cope with this inhospitable host environment. We focused on one potential fungal virulence factor, MoHYR1 (MGG_07460), from the rice blast fungus Magnaporthe oryzae, and its role in ameliorating effects of plant-produced ROS. MoHYR1 has a glutathione peroxidase domain, and its yeast homologue specifically detoxifies phospholipid peroxides by forming an inter-molecular disulfide bond with a transcription factor YAP1. Deleting MoHYR1 from the fungal genome led to a decreased ability to tolerate both H2O2 in the medium and ROS generated by a susceptible plant, including ROS associated with cell wall appositions (CWAs). Moreover, the deletion of this gene caused a virulence defect in M. oryzae, which we believe is associated with the mutant’s inability to detoxify plant-generated ROS. Expression analysis on ten fungal genes revealed that the MoHYR1 mutants became less sensitive to exogenously applied H2O2. Together, our data suggest that HYR1 is a virulence factor in the rice blast pathogen, and its role in virulence is directly related to sensing and managing ROS generated during early infection events. We are also working on understanding how HYR1 and YAP1 interact to manage ROS, and data will be presented on initial interactions.

Plant hormones as potential biomarkers of early phytoplasma infection
Y. JIANG (2), W. Wei (2), R. E. Davis (2), I. Lee (2), R. W. Hammond (2), D. L. Nuss (1), Y. Zhao (2)
(1) IBBR-University of Maryland, College Park, MD, U.S.A.; (2) MPPL-ARS-USDA, Beltsville, MD, U.S.A.

Phytoplasmas, a diverse group of unculturable cell wall-less bacteria, are causative agents of numerous plant diseases affecting a broad range of agriculturally and environmentally important plant species worldwide. Phytoplasma-infected plants often exhibit symptoms suggestive of a disturbed hormonal balance. The current study was designed to investigate changes in the endogenous levels of four major plant hormones in infected plants, and to determine whether plant hormones can be exploited as biomarkers of phytoplasma infection. Columbia Basin potato purple top (PPT) phytoplasma (a member of subgroup 16SrVI-A) and its alternate host Rutgers tomato were used as a model pathogen-host pair in our study. Paraffin-embedded tissue sections prepared from various parts of PPT phytoplasma-infected and healthy tomato plants were subjected to a semi-quantitative immunohistochemical assay optimized in our laboratory. Results from the study revealed that, in PPT-infected plants, levels of abscisic acid (ABA) and cytokinins (N6-benzyladenosine, trans-zeatin riboside, and cis-zeatin riboside) were elevated, and the levels of auxin (IAA) and gibberellin acid (GA3) were decreased. A time-course analysis indicated that changes in hormonal level were most evident in the specimens from samples collected at 10 days post graft-inoculation, a time point when no disease symptom was visible and no PPT phytoplasma DNA was detectable using nested polymerase chain reactions. Our findings suggest that endogenous levels and ratios of plant hormones are potential pathological markers of early phytoplasma infection. Findings from the study will aid our understanding of the roles of plant hormones in phytoplasma pathogenesis and disease symptom expression.

A novel way to prepare plant DNA using the GenomiPhi DNA Amplification Kit
B. D. KARGER (1)
(1) GE Healthcare Life Sciences

Plant species vary widely in their genome size and tissue composition. The tissues of many plants contain high levels of polyphenols and polysaccharides which can interfere with DNA extraction. Polyphenolic and polysaccharide substances can also interfere with restriction enzymes and DNA polymerases which can make the DNA unusable for molecular analysis and research applications. The GenomiPhi™ DNA Amplification Kit has been successfully validated to amplify extracted DNA from a variety of plant seeds and leaves. We present data that shows the generation of microgram amounts of DNA from small amounts of input DNA. Using a simple extraction method, such as alkaline lysis, or a more detailed method, such as the CTAB method, the amplification of plant DNA was equally successful using the GenomiPhi DNA amplification method.

Gene expression profiling in Phytophthora phaseoli during the infection of lima bean
S. G. KUNJETI (1), N. M. Donofrio (1), A. G. Marsh (1), N. F. Gregory (1), S. Kunjeti (1), B. C. Meyers (1), T. A. Evans (1)
(1) University of Delaware, Newark, DE, U.S.A.

Lima bean is an important legume crop to the state of Delaware. This crop is susceptible to an oomycete pathogen, Phytophthora phaseoli, which caused significant crop loss in the year 2000. In this study we have used Illumina RNA-seq to identify genes in P. phaseoli orthologous to several effector genes in P. infestans, a close relative of P. phaseoli. To study the function of these effector proteins, we selected ten candidates with similarity to RxLRs, elicitins, NPP1 and crinklers, all of which are different classes of effectors. Full-length sequences of most of the candidates showed more than 90 percent identity to amino acid sequences in P. infestans. The above effectors genes were validated by performing in planta RT-PCR. Phylogenetic analyses of candidate effector genes from other oomycete pathogens confirm a close relationship of P. phaseoli and P. infestans for all the corresponding effector genes. Selected effectors were cloned into Agrobacterium and injected into Nicotiana benthamiana leaves for transient assays. Three elicitins (Pp_INF1, Pp_INF4 and Pp_06908) showed a hypersensitive response. Currently, we are performing functional characterization of RxLR effectors and other effector genes, which will help us to gain a better understanding of this pathosystem and will serve as a basis for future research.

Seasonal distribution of SI fungicide resistance in apple scab in Virginia
S. C. MARINE (2), D. G. Schmale (1), K. S. Yoder (2)
(1) PPWS Dept., Virginia Tech, Blacksburg, VA; (2) Virginia Tech AHS AREC, Winchester, VA

Venturia inaequalis is the casual organism of apple scab, an economically devastating disease of apples that occurs wherever apples are grown. Management has predominantly relied on chemical applications, with sterol inhibitor (SI) fungicides being one of the dominant systemic fungicides used in commercial apple production. Unfortunately, Virginia populations of V. inaequalis are developing resistance to myclobutanil and other SIs. We evaluated fungicide resistance in 266 single-spored V. inaequalis isolates collected in Winchester, VA between 2006 and 2010. Within a given season, the mean colony growth of V. inaequalis isolates was significantly different (P < 0.001) among assay treatments (0, 0.1, 0.5 or 1 ppm myclobutanil) and assay times (7, 14, 21 or 28 days). Whether the V. inaequalis isolate came from a treated or non-treated tree was significant in 2006 (P < 0.001) and 2008 (P = 0.002), but not in other years. Sampling interval was significant (P < 0.001) in 2007 and 2008. When analyzed concurrently, all factors were significant (P < 0.001) including collection year. Percent growth suppression (PGS) – the difference in colony growth on 0 and 1 ppm myclobutanil at 28 days – was used to assess fungicide resistance. Generally, a range of resistance was seen at each sampling interval, and the average PGS was similar for treated and non-treated trees of the same cultivar. In the May and June sampling intervals, the average PGS hovered around 60% regardless of cultivar or tree treatment. In contrast, the average PGS in the July sampling interval was around 30% (i.e. more resistant to myclobutanil). The average PGS in the August sampling interval (around 50%) was more similar to that seen early in the summer. High levels of fungicide resistance in populations of V. inaequalis suggest that replacement programs should be considered. Future research may rely on DNA-based methodologies to determine fungicide resistance and employ appropriate disease management strategies.

The molecular interaction of Theobroma cacao and Moniliophthora perniciosa, causal agent of witches’ broom, during infection of young pods
R. L. MELNICK (2), J. Marelli (1), B. A. Bailey (2)
(1) Mars Center for Cocoa Science, Fazenda Almirante, CP 55 Itajuipe Bahia 4630-000 BRAZIL; (2) Sustainable Perennial Crops Lab, PSI, USDA/ARS, Beltsville, MD, U.S.A.

Infection of Theobroma cacao (cacao) flower cushions with the basidiomycete Moniliophthora perniciosa (Mp) can result in the formation of parthenocarpic fruits resembling cherimoya. Young green cherimoya-like fruits and healthy young cacao pods were obtained from 7 clones of different genetic backgrounds in Bahia, Brazil. QPCR analysis was conducted to determine how the infection process impacts the expression of both cacao and Mp ESTs. The effect of genotype was significant in the expression level of nearly all cacao ESTs tested. Mp induced the expression of ESTS related to defense (TcChi1, TcChi4, TcMAPK3, TcMKK4, TcNPR1), although expression of ESTs with a putative function of PR1 were either repressed or were not differentially expressed. TcESTs previously shown to be upregulated in Mp infected stems, such as caffeine synthase, peroxidase, hevein, and beta-1,3-glucanase, were not induced in infected pods. Key Mp ESTs related to synthesis of GA, such as GA oxidase, GA4 desaturase geranylgeranyl diphosphate synthase, and IAA, such as DOPA and copper amine oxidase, were expressed by Mp in infected cherimoya-like pods. The expression of phytohormones by the fungus could potentially induce parthenocarpy. Additionally, Mp expressed ESTs related to pectin degradation in infected pods. Overall, this study underpins the mechanisms involved in to the interaction between Mp and cacao as well as potential pathogenicity factors of Mp.

Within-field temporal and spatial dynamics in the Soybean mosaic virus – aphid and Bean pod mottle virus - beetle soybean pathosystems
F. W. NUTTER (1), E. Byamukama (1), S. Eggenberger (1)
(1) Department of Plant Pathology, Iowa State University, Ames, IA

Soybean mosaic virus (SMV) and Bean pod mottle virus (BPMV) are two of the most prevalent viruses affecting soybean production worldwide, resulting in losses of up to 35% (SMV) and 52% (BPMV). Both viruses are vectored by insects (aphids and bean leaf beetles, respectively). Little is known about the temporal and spatial dynamics of within-field spread of these two viruses. A quadrat-based plot design, with and without SMV- or BPMV-infected point sources of inoculum, was used to quantify the temporal and spatial dynamics of these two viruses. The rate of spread of SMV within field plots ranged from 0.06 (slow) to 0.13 logits per day (moderately fast), which translates into doubling times from 14.1 to 5.4 days. Rates of temporal spread for BPMV were very similar and ranged from 0.5 to 0.13 logits per day (with doubling times of 14.1 to 5.4 days). Spatial analyses (ordinary runs and black-white joins counts) were used to determine if the spatial patterns of virus-infected quadrats over time were random or clustered. Spatial patterns for these two viruses were quite different, with SMV-infected quadrats being predominantly random throughout each growing season, whereas BPMV exhibited highly aggregated spatial patterns over time. These results indicate that a random sampling design should be used to quantify SMV incidence, whereas a systematic sampling design should be used to quantify BPMV incidence. However, to quantify the incidence of both viruses in the same study, a systematic sampling design is recommended.

Stability of QoI resistance of grapevine powdery mildew in competition experiments and in the field
L. E. RALLOS (1), A. Baudoin (1)
(1) PPWS, Virginia Tech, Blacksburg, VA, U.S.A.

Quinone outside inhibitors (QoIs) became one of the most commonly used fungicide groups against grapevine powdery mildew. However, their effectiveness diminished in recent years due to resistance development. Some types of fungicide resistance incur a fitness penalty which can cause the resistant fraction of the population to diminish when the selection pressure is alleviated. To examine this possibility, competition assays were conducted by growing mixtures of QoI resistant and sensitive isolates on fungicide-free plants. The resistant fraction was monitored by real-time PCR using the G143A mutation in the fungal cytochrome b gene as the resistance marker. Most (71%) of the inoculations resulted in an increase in the resistant proportion. Rarely did the %G143A decrease (6%) or remain unchanged (23%). Monitoring in one vineyard that has stopped using QoIs for the past four years revealed the presence of resistant isolates or populations in all survey years, and a continued high frequency of resistance after 3 and 4 years. Altogether, these results indicate that resistant strains are competitive, and QoI resistance can remain in the population even in the absence of the selection pressure.

Molecular genetic identification of virulence determinants of Pantoea ananatis, a causal agent of onion center rot
J. P. Sinn (1), B. K. Gugino (1), M. A. Mansfield (1), L. M. Wittmer (1), M. R. Ebner (1), T. W. MCNELLIS (1)
(1) Penn State, University Park, PA, U.S.A.

Center rot is a serious disease of onions that has recently been reported in the northeastern United States. The gram-negative bacterium Pantoea ananatis is one of two Pantoea species associated with onion center rot. Although the complete genome of a South African P. ananatis strain isolated from eucalyptus has been sequenced and published, little functional genetic information is available about the mechanisms of pathogenesis of this organism. We conducted a Tn5 transposon mutagenesis of two Pennsylvania isolates of P. ananatis and screened the resulting mutants on onion bulb tissue to identify those with altered virulence. To date, 30 mutants with altered virulence phenotypes have been identified and further characterized. Several mutations are in genes predicted to be involved in exopolysaccharide production and quorum sensing. Predicted functions of some of the putative virulence genes identified include a histidine kinase two-component sensor, a quorum sensing phosphorelay protein, and an ATP-dependent protease. The potential relevance of these and other genes to onion center rot disease development will be discussed.

Inoculum density effects on infection of selected Eastern U.S. forest species by Phytophthora ramorum
P. W. TOOLEY (1), M. Browning (1)
(1) USDA-ARS, FDWSRU, Ft. Detrick, MD, U.S.A.

Inoculum threshold information can be used to better understand the epidemiology of P. ramorum should it become established in the Eastern US. Detached leaves from Quercus prinus, Q. rubra, Acer rubrum, Kalmia latifolia ‘Hoffman’s K’, and Rhododendron ‘Cunningham’s White’ were exposed to sporangia concentrations ranging from 0 to 3000 sporangia/ml. Three leaves per species per experiment were dip-inoculated and positioned between layers of moistened paper towels in sealed plastic containers at 20°C for five days. Experiments were also conducted using stems of 2–3 year old seedlings of Q. prinus, Q. rubra and A. rubrum. Treatments ranging from 0 to 3000 sporangia/ml were applied to wounded and unwounded stem tissue. Detached leaf inoculations resulted in disease in all five species at all concentrations tested. Wounding was required for infection of stems to occur. With both stems and detached leaves, disease was observed at the lowest sporangia concentrations utilized; 100 sporangia/ml for stems and 10 sporangia/ml for detached leaves. The results indicate that P. ramorum is capable of infecting some major Eastern U.S. forest species at low inoculum levels. This information can be combined with knowledge of understory host distribution and sporulation capacity to predict epidemic potential in new areas, and enhance development of risk assessment models.

Abscisic acid influences progression of a tomato disease caused by potato purple top phytoplasma
W. WEI (2), Y. Jiang (2), R. E. Davis (2), I. Lee (2), R. Hammond (2), D. L. Nuss (1), Y. Zhao (2)

Phytoplasmas are small, cell wall-less bacteria that are associated with numerous plant diseases. Plants infected by phytoplasmas often exhibit an array of symptoms consistent with hormone disorders. Significant changes in the endogenous levels of plant hormones upon phytoplasma infection suggest a role of plant hormones in the interactions of phytoplasmas with their plant host. Abscisic acid (ABA), one of the major plant hormones, is involved in various plant physiological processes. Conflicting evidence has been presented that ABA either promotes or represses plant resistance to pathogen infections. Results from our study using Columbia Basin potato purple top (PPT) phytoplasma and Rutgus tomato, as a model pathogen-host pair, revealed that i) endogenous levels of ABA surged at the early stage of phytoplasma infection and ii) exogenous administration of ABA delayed symptom development in PPT phytoplasma-infected tomato. Compared with control (ABA-untreated, PPT-infected) plants, appearance of the characteristic “big bud” symptom was significantly delayed and the number of “big buds” was significantly less in ABA-treated, PPT-infected plants. In addition, “little leaf” symptoms became unnoticeable in most of the ABA-treated, PPT-infected plants. Results of quantitative real time PCR analysis showed that, in scions, phytoplasma DNA titers remained higher in ABA-treated plants as compared to ABA-untreated plants 10 days post graft inoculation. One explanation of such phenomenon could be that ABA impeded phytoplasma movement from infected parts (scions) to healthy parts (root stocks). Similar results were also obtained from parallel experiments using an ABA biosynthesis deficient tomato mutant as root stock. Our findings provide new insight into the role of ABA in phytoplasma-host interaction.

Gibberellin acid (GA3) treatment attenuates tomato floral deformation caused by potato purple top phytoplasma infection
W. WU (1), Y. Ding (1), W. Wei (1), I. Lee (1), R. E. Davis (1), Y. Zhao (1)
(1) MPPL-ARS-USDA, Beltsville, MD, U.S.A.

One of the most striking symptoms exhibited by potato purple top (PPT) phytoplasma-infected tomato plants is the formation of an aberrant floral structure known as “big bud”. Due to early abortion of pistils, stamens, and petals, the “big bud” structure is mainly composed of enlarged and fused sepals. It is apparent that PPT phytoplasma infection disrupts sequential expression of genes crucial for normal flower development. Since gibberellins are essential plant hormones that regulate flower morphogenesis, we were interested to learn whether exogenous application of gibberellin acid (GA3) would reprogram floral gene expressions and alleviate the “big bud” symptom. PPT-infected tomato plants were treated with 100 µM GA3 via leaf spraying two days after graft-inoculation. Developing bud samples were collected at four time points corresponding to four landmark stages in normal tomato flower development (stage 6, 9, 12 and 15) for morphological profiling and floral homeotic gene expression pattern comparisons. The morphological data from bud morphometric measurement and histological assay showed that GA3 treatment restored pistil, petal, and stamen development in PPT-infected tomato plants to levels comparable to those of healthy control plants. However, GA3 treatment did not significantly reduce sepal hypertrophy. Quantitative RT-PCR analysis of homeotic gene transcripts revealed that the expressions of class B and C genes LeAP3, FA and TAG1 were up-regulated at stages 6 and 9 in GA3-treated, PPT-infected plants (compared with PPT-infected plants without GA3 treatment), whereas the expression of class A gene was not substantially changed following GA3 treatment. In summary, treatment of PPT phytoplasma-infected tomato plant with 100 µM GA3 can partially restore floral organ development through transcriptional reprogramming of key floral homeotic genes.