Special Report Effect of Benzothiadiazole on Transmission of X-Disease Phytoplasma by the Vector Colladonus montanus to Arabidopsis thaliana, a New Experimental Host Plant Alberto Bressan, Department of Environmental Science, Policy and Management, University of California, Berke- ley 94720-3114, and Università di Padova, Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Viale dell’Università 16, 35020 Legnaro, Italy; and Alexander H. Purcell, Department of Environmental Science, Policy and Management, University of California, Berkeley Systemic acquired resistance (SAR) is ABSTRACT an inducible resistance mechanism in Bressan, A., and Purcell, A. H. 2005. Effect of benzothiadiazole on transmission of X-disease plants that provides resistance against a phytoplasma by the vector Colladonus montanus to Arabidopsis thaliana, a new experimental broad spectrum of plant pathogens: vi- host plant. Plant Dis. 89:1121-1124. ruses, fungi, and bacteria. This mechanism of resistance is activated in several plant Colladonus montanus (Van Duzee), a leafhopper vector of X-disease phytoplasma (Xp), effi- species by natural attacks of pathogens. ciently transmitted the pathogen to Arabidopsis thaliana Columbia wild type. During transmis- Alternatively, chemicals (elicitors) have sion trials, the phytoplasma was inoculated into 22-, 34-, and 40-day-old plants. Phytoplasma been developed to apply directly to plant infections were confirmed by polymerase chain reaction (PCR) using primers specific for Xp. Symptoms in Xp-positive A. thaliana were overall stunting and reduced or no fruit (silques). All tissues (13,15) to activate SAR. To our symptom-free plants were PCR negative. Leafhopper nymphs free of Xp that fed on diseased A. knowledge, no data are available on the thaliana acquired and transmitted Xp to celery plants, a diagnostic host, causing typical X- effect of SAR on phytoplasma infections. disease symptoms. Foliar spray applications of the plant resistance elicitor benzothiadiazole This report describes our use of the (BTH) to A. thaliana 1 week before phytoplasma inoculation significantly reduced phytoplasma leafhopper vector C. montanus and Xp- infection, ranging from an infection rate of 73.7% for untreated plants to 50 and 35% for plants infected celery as a source of inoculum for treated with 1.2 and 4.8 mM BTH, respectively. Vector leafhoppers survival was significantly the leafhopper, to test A. thaliana as an ex- reduced on BTH-treated A. thaliana compared with leafhoppers on nontreated plants, suggesting perimental host of Xp. Additionally using that systemic acquired resistance in this plant may have some detrimental effect on the leafhop- the elicitor benzo-(1,2,3)-thiadiazole-7- per C. montanus. carbothioic acid S-methyl ester (BTH), an inducer of SAR in A. thaliana (15), we Additional keywords: mollicute, SAR assessed the effect of SAR in the Xp infec- tion of A. thaliana inoculated by infective C. montanus. Phytoplasmas are cell-wall-less mem- tally transmitted the western strain of X- MATERIALS AND METHODS bers of the bacterial class Mollicutes that disease causal agent (then incorrectly as- Leafhopper rearing and phytoplasma are obligate parasites of plants and insect sumed to be a virus) from infected celery transmission. The leafhopper vector C. vectors. Phytoplasmas cause hundreds of to 12 different plant species. Chiykowski montanus was reared on healthy celery plant diseases, several of which have and Sinha (3) reported 17 host species of plants in a greenhouse insectary. Infectious worldwide agricultural significance (17). an eastern strain of X-disease vectored by insects were produced by confining groups The plant host range of some phytoplasma Paraphlepsius irroratus (Say). We have (approximately 100 to 200) of mid-instar strains may be very wide. This is the case recorded disease symptoms from numer- nymphs on celery with symptoms of X- for X-disease phytoplasma (Xp). Although ous other plant species inoculated with disease (strain GVX, originally transmitted X-disease is an economic disease of stone infectious leafhopper vectors (A. H. Pur- to celery by C. montanus from sweet fruit trees such as cherry and peach cell, unpublished). cherry, Prunus avium L., near Lodi, CA) (10,18), the pathogen infects a wide range The small and fast-growing crucifer for an acquisition access period of 15 days. of herbaceous plants (3,6,12,14) in both Arabidopsis thaliana has become widely Although we hereafter refer to these as eastern and western North America. used in plant genetic studies, and its ge- “infectious” insects, it was likely that not Using the polyphagous leafhopper nome has been completely sequenced (2). all insects exposed to Xp-infected plants (Hemiptera: Cicadellidae) vector Colla- Moreover, the small-sized and fast- acquired Xp or became capable of trans- donus montanus (Van Duzee) (20), Frazier maturing A. thaliana has numerous eco- mitting it to plants even if internally in- and Jensen (6) and Jensen (12) experimen- types that are available for researchers. A. fected by the phytoplasma. About 35 days thaliana may provide a useful tool to in- later, we transferred the leafhoppers to 45- crease knowledge of molecular details of to 60-day-old healthy celery seedlings to Corresponding author: A. H. Purcell plant–phytoplasma interactions (5). To maintain phytoplasma on plants and to E-mail: [email protected] date, the beet leafhopper-transmitted vires- provide sources for later vector acquisi- This research was partially supported by the Fon- cence (BLTV) phytoplasma (5,8) and Spi- tion. The median latent period of Xp in C. dazione Ing. Aldo Gini, University of Padova, Italy. roplasma citri (4,5) are recognized as mol- montanus is 28 to 35 days, depending on licute pathogens of A. thaliana. Because temperature (7). For the transmission trials Accepted for publication 3 June 2005. phytoplasmas have never been cultured, (see below) we used cylindrical cellulose molecular genetic methods have become acetate-butyrate plastic cages (5 cm in DOI: 10.1094/PD-89-1121 indispensable tools for studies of phyto- diameter and 10 cm high) ventilated with © 2005 The American Phytopathological Society plasma biology and pathology. Dacron organdie windows to singly con- Plant Disease / October 2005 1121 fine insects on test plants for an inocula- symptomatic A. thaliana as phytoplasma PCR procedure was used with the primer tion access period of 2 days. At the end of sources to feed 60 C. montanus nymphs pairs fP1 and rWXint (19). For specific each transmission assay, we removed leaf- for an acquisition access period of 7 days, detection of Xp, these primer pairs amplify hoppers from each plant and recorded followed by weekly transfer to celery. a region of approximately 1,600 bp in the insect survival. From this group, we tested 18 leafhoppers 16Sr DNA and the adjacent spacer region Plants. Seeds of A. thaliana Columbia singly for 7 days of inoculation access on of Xp (19). For PCR, each reaction con- wild-type were sowed singly in sterile celery. Plants with the same treatments tained 1× PCR buffer (Perkin-Elmer, Well- potting compost in 5-cm-wide pots and left were spatially randomized during trans- sley, MA), 50 ng of template DNA, 0.5 µM in the dark for 4 days at 4ºC. Trays with mission and during the incubation period. each primer, 150 µM each dNTP, and 1 U pots then were maintained under fluores- After inoculation, every 2 days, plants of AmpliTaq DNA polymerase (Perkin- cent lights (8 h of light and 16 h of dark- were checked carefully for possible symp- Elmer) in a final volume of 30 µl. Amplifi- ness per day) at a temperature of 25 ± 2ºC. toms associated to phytoplasma infection. cation consisted of 30 cycles of denatura- For 20 days following inoculation with Plants with possible symptoms were com- tion for 1 min at 94ºC and annealing for 1 infectious leafhoppers, we maintained the pared with plant controls that had been min at 48ºC, followed by final extension plants in a heated greenhouse with natural inoculated with noninfectious C. mon- for 2 min at 72ºC. PCR products were light and temperature (26 ± 5ºC) to com- tanus. We looked for leaf chlorosis and separated by electrophoresis in 1% agarose plete the disease incubation period (time root necrosis in celery and leaf discolora- gel stained with ethidium bromide and for symptoms to appear). Seeds of celery tion, deformation, stunting of the stems, observed under UV illumination. were germinated in a sand-peat soil in and tissue necrosis for A. thaliana. Statistics. Differences among treat- plastic pots, maintained in the greenhouse, Effect of BTH on phytoplasma trans- ments were tested for significance using χ2 and used when the plants were about 45 mission. BTH was supplied by Syngenta with Yates correction for continuity, and days old. Corporation as 50% active ingredient in a level of significance (P) was fixed at 0.05 Transmission trials and phytoplasma wettable powder formulation. It was di- using Statistica (ver. 6.1; StatSoft Italy, symptoms in A. thaliana. To test the luted with sterile water and applied to Padova, Italy). pathogenicity of Xp to A. thaliana, we upper leaf surfaces to run-off by using a confined single infectious C. montanus on spray bottle. Elicitor application was made RESULTS each of 16 A. thaliana seedlings for each 7 days before phytoplasma inoculation Symptoms of Xp in A. thaliana. Be- of the three separate treatments, using 22-, using infectious leafhoppers. We used 80 ginning 35 to 45 days after vector inocula- 34-, and 40-day-old plants for an inoc- 30-day-old A. thaliana seedlings in the tion during the reproductive phase (in- ulation access period of 2 days. Alterna- transmission trial in four different treat- duced by long days), numerous A. thaliana tively, infective leafhoppers from the same ments: 20 plants each were sprayed with a plants had pronounced stunting, shortened acquisition cohort were confined singly on solution of 1.2 or 4.8 mM BTH, or with internodes, aborted flowers, and fewer and 16 celery seedlings to compare rates of Xp sterile water.