Brote Grande, A New Phytoplasma Associated Disease of Chile Peppers in the Desert Southwest

Transcription

1 2011 Plant Management Network. Accepted for publication 10 January Published. Brote Grande, A New Phytoplasma Associated Disease of Chile Peppers in the Desert Southwest Jennifer J. Randall, Department of Entomology, Plant Pathology, and Weed Science, MSC3BE, New Mexico State University, Las Cruces, NM 88003; Paul W. Bosland, Department of Plant and Environmental Sciences, MSC3Q, New Mexico State University, Las Cruces, NM 88003; and Stephen F. Hanson, Department of Entomology, Plant Pathology, and Weed Science, MSC3BE, New Mexico State University, Las Cruces, NM Corresponding author: Stephen F. Hanson. shanson@nmsu.edu Randall, J. J., Bosland, P. W., and Hanson, S. F Brote grande, a new phytoplasma associated disease of chile peppers in the Desert Southwest. Online. Plant Health Progress doi: /php rs. Abstract Chile is one of the most important crops in New Mexico, contributing both to the agricultural economy and cultural identity of the state. Diseases are a major constraint on chile production in New Mexico and across the Desert Southwest. Chile producers in New Mexico recently reported a disorder of unknown etiology that was observed in increasing frequency for the past four years. Affected plants have a bushy appearance, develop overly large green calyces instead of normal flowers, and fail to set fruit. This characteristic phyllody has led the disorder to be referred to as brote grande which is Spanish for big bud. The phyllody is reminiscent of the aberrant flower development associated with tomato big bud, a phytoplasma disease of tomatoes. Microscopic analysis, including light microscopy and TEM along with PCR detection using phytoplasma specific primer sets, indicates that brote grande disease is associated with a novel phytoplasma. Field surveys conducted in 2008 and 2009 indicate that this new disease is widely distributed at low levels across chile production areas in New Mexico and Arizona. Chile (Capsicum annuum) is grown under stressful environmental conditions across the Desert Southwest. Chile is the signature crop of New Mexico, where it contributes more than $500 million per year to the state economy and has long been a central part of the state s agricultural heritage (2). Environmental stresses, such as high temperature, limited water, and poor soil fertility, all combine to make chile an intensively managed crop across the Desert Southwest. The stressful environment and intensive management contribute to making several diseases including Phytophthora sp., Verticillium, Beet curly top virus, Tomato spotted wilt virus, and bacterial leaf spot, the largest constraints on chile production. Growers began reporting an apparent disease of unknown etiology around The disease caused aberrant growth and flower development, leading to a failure in fruit set in affected plants. The aberrant growth symptoms of the disease included vegetative overgrowth combined with witches broom type hyper-proliferation of branch ends that leads to large bushy plants that can be easily identified from a distance (Fig. 1A and 1C). The flower buds of affected plants failed to mature, instead they displayed virescence and phyllody and that developed into very large green buds (Fig. 1B). This characteristic symptom has led to the disease being called brote grande, Spanish for big bud. Other symptoms include thickening and cupping of leaves, calyx deformations including virescence and phyllody, and abortion of flowers. Chlorosis and mosaic symptoms are seen on some but not all affected plants.

2 Fig. 1. Symptoms of brote grande chile disease. Typical symptoms include witches broom-like hyper proliferation of branch ends as shown in panel A and aberrant phyllody type flower development where petals turn green and leafy resulting in large green calyces that never fully develop into flowers as shown in panel B. Panel C is a chile plant in the field that is exhibiting the witches broom hyper proliferation and BCTV symptoms. This plant was positive for both the brote grande phytoplasma and BCTV. For comparison, panel D shows a branch end with flower from a healthy chile plant along side a normal flower and flower bud. Panel E is a chile plant in the field exhibiting witches broom hyper proliferation. The consistent appearance of this disease over the past several years and the failure of affected plants to set fruit has caused concern among chile producers in New Mexico and Arizona. Several phytoplasmas are known to cause similar symptoms in chile and other solanaceous crops (3,10). Preliminary studies on brote grande affected plants showed that the disease is associated with a novel 16Sr group VI phytoplasma (9). Herein, we provide a more detailed description of the disease and results of surveys that indicate brote grande is endemic at low levels throughout the chile production areas of the Desert Southwest. Light microscopy studies revealed differences between healthy and diseased samples. Leaves from symptomatic plants were thicker and had greatly thickened vascular bundles (Fig. 2B) when compared to the cross sections from healthy leaves (Fig. 2A). Cells in the affected leaves were generally smaller,

3 packed more densely, and disorganized relative to the cells in healthy leaves (Fig. 2A vs. Fig. 2B). This was especially apparent in the thickened and disorganized vascular bundles of symptomatic leaves (Fig. 2B). The shrunken and disorganized nature of cells in the phloem was also apparent in comparisons of higher magnification light micrographs from healthy and symptomatic tissues (Figs. 2C and 2D). Also apparent in the high magnification light micrographs was the presence of numerous bacterial sized bodies in the sieve elements of symptomatic tissue (Fig. 2D) that were not observed in healthy tissues (Fig. 2C). Low magnification transmission electron micrographs also showed that the sieve elements of symptomatic tissues contained numerous bacterial sized bodies (Fig. 3B), whereas the sieve elements of healthy tissues did not contain the bacteria-like bodies (Fig. 3A). In addition, the low magnification transmission electron micrographs also revealed a general disorganization of phloem tissue and cellular abnormalities that included thickened cell walls and irregular cell shape with cells being smaller and packed more densely (Fig. 3B). Higher magnification transmission electron micrographs of sieve elements from symptomatic leaves showed that the bacterial sized bodies in the sieve elements were consistent with phytoplasma cells in having a plieomorphic globular shape with diameters ranging from ~ µm, being surrounded by a membrane, and lacking cell walls (Fig. 3C). All of these microscopic observations are consistent with the presence of phytoplasmas in symptomatic plants. Fig. 2. Light microscopy of leaf cross-sections from healthy and brote grande affected leaves. Leaf tissue was detached near the base of selected leaves and fixed, embedded, cross sectioned, and stained with methylene blue and azure A as detailed in the methods section. Panels A and B show low magnification (100 ) photomicrographs from healthy and symptomatic plants respectively with arrows pointing to the vascular bundles which are enlarged and disorganized in the symptomatic sample. Panels C and D show high magnification photomicrographs (400 ) from healthy and symptomatic tissue, respectively, with arrows pointing to individual sieve elements that are clear in the healthy sample but contain numerous bacteria like bodies in the symptomatic sample.

4 Fig. 3. Electron microscopy of vascular tissue from healthy and brote grande affected plants. Cross sections of vascular tissue from the base of leaves were fixed, embedded, sectioned and stained as described in the methods section. Panels A and B are low magnification images from healthy and symptomatic tissue, respectively. Scale bars indicate 5 µm and 0.5 µm in low and high magnification photomicrographs, respectively. The arrow in panel B points to a phloem sieve element containing numerous bacteria like bodies. Panel C is a high magnification image of the bacteria like bodies present in the phloem sieve elements of symptomatic tissues. Molecular testing determined that phytoplasma DNA sequences were present in affected plants. PCR amplification of DNA extracted from leaves of 42 symptomatic and two healthy plants with the previously described phytoplasma specific PCR primer pair P1/P7 (12) produced amplicons of the expected size (~1.8 Kb) from 34 symptomatic samples while no amplicons were produced from healthy tissue (Fig. 4). Testing with another phytoplasma specific primer set, P1/Tint (12), also produced positive results with the same 34 symptomatic samples but not from healthy samples (data not shown). Amplicons produced with the P1/P7 primer set, which include the majority of the 16SrRNA gene and the entire ITS region, were directly sequenced and used for database searches and phylogenetic analysis. Comparison of these sequences against each other showed that the ITS regions from all samples were identical and contains a complete trna-ile gene as is typical for phytoplasmas (7). Blast searching against Genbank showed that the brote grande associated phytoplasma (Genbank accession HQ436488) was most closely related to other 16SrVI phytoplasmas like Candidatus phytoplasma trifolii (Genbank accession AY390261) and vinca virescence phytoplasma (Genbank accession AY500817).

5 Fig. 4. Phytoplasma polymerase chain reaction products separated on a 1% agarose gel stained with ethidium bromide. Total DNA was extracted from healthy or symptomatic chile plants and tested for the presence of phytoplasma by PCR with the phytoplasma specific primers P1 and P7. Reactions were resolved on a 1% agarose gel that was visualized by ethidium bromide staining. Lane M contains the size standard (1 Kb plus DNA ladder, Invitrogen) with sizes of bands indicated at left. Lanes 1-42 are PCR reactions from extracted DNA from symptomatic chile plants. The arrow denotes the positive PCR band. Lanes (-) contain PCR reactions performed on DNA extracted from healthy chile plants. A 1000 bootstrapped neighbor joining tree that includes Acholeplasma laidlawii (Genbank accession EU925161) as an outgroup is shown in Figure 5a. Examination of this tree shows that the brote grande sequence groups with other members of the 16SrVI phytoplasmas including vinca virescence phytoplasma, potato witches broom phytoplasma, Columbia basin potato purple top phytoplasma, and Candidatus phytoplasma trifolii (Fig. 5b). The next most related branches include the 16SrV elm yellows and 16SrVII ash yellows phytoplasma branches. The brote grande phytoplasma sequence clusters well apart from the 16S type XII stolbur of pepper phytoplasma and the type I and III tomato big bud phytoplasma branches. Taken together the sequence and phylogenetic analysis demonstrate that the brote grande of chile is associated with a novel 16SrVI phytoplasma.

6 Fig. 5. Neighbor joining phylogenetic tree of 16S rrna and ITS region sequences showing relationships between the brote grande associated phytoplasma and previously described phytoplasmas. The tree was made using the Tamuri-Nei distance model with 1000 bootstrapping using Acholeplasma laidlawii (Acho l)16s rrna and ITS region sequence as the outgroup. (A) Phylogenetic tree with representatives of all described classes of phytoplasmas. Phytoplasma names are listed as abbreviations with Genbank accession numbers at branch tips. Bootstrap consensus support values are indicated at nodes. Corresponding 16Sr rrna groupings are indicated at the right. The brote grande branch is shaded in dark grey while stolbur of pepper and tomato big bud branches are shaded in light grey. Phytoplasma abbreviations: Alf WB = alfalfa witches broom, App prolif = apple proliferation, Ast Yel = aster yellows, Ash Yel = ash yellows, Chayote WB = chaote witches broom, Clover Phyl = clover phyllody, Clover YE = clover yellow edge, Elm Yel = elm yellows, Jujube WB = jujube witches broom, Phyto solani = phytoplasma solani, Phyto Tri = phytoplasma trifolii, Pot WB = potato witches broom, Pig pea WB = pigeon pea witches broom, S Pot WB = sweet potato witches broom, Stolbur = stolbur of pepper, Tom BB-Ark = tomato big bud isolate from Arkansas, Vinca vir = vinca virescence, brote grande = brote grande of pepper from New Mexico. (B) Enhanced view of the class VI phytoplasma group including the New Mexico and Arizona phytoplasmas from this study. Phytoplasmas from several different 16Sr groups have been reported to cause big bud of tomato. The 16S rrna gene / ITS sequences of the brote grande associated phytoplasma are highly related to those of BLTVA and Candidatus phytoplasma trifolii, both of which are 16SrVI phytoplasmas reported to cause big bud symptoms on tomato in California (6,12). Interestingly, another 16SrVI clover proliferation type phytoplasma was reported to be associated with one outbreak of a stolbur-like disease on peppers in Spain (3). While similar to several 16SrVI phytoplasmas known to cause big bud symptoms on tomato, the brote grande-associated phytoplasma is distinct from other phytoplasmas causing similar diseases in pepper and tomato, including the 16Sr group I-A isolate reported in Arkansas (8) and Europe, the group I-B isolate reported in Italy (7), group III isolates from Italy (11) and Brazil (1) and the group II-E isolate common in Australia (5). The brote grande phytoplasma is also distinct from several phytoplasmas recently reported on potatoes in Mexico (10). Brote grande affected plants were observed in numerous fields across all of southern New Mexico and Arizona during the past three growing seasons (Fig. 6). Incidence was always low with no field having more than 3% of the chile plants affected. The 16S rrna gene and ITS region sequences were obtained from 34 plants isolated across an area ranging from Las Cruces, NM, to Tucson, AZ, in Examination of these sequences revealed that all were infected with the brote grande-associated phytoplasma demonstrating that this new

7 phytoplasma and the disease are widely distributed across this major chile production area. Fig. 6. Map of southern New Mexico and Arizona where samples with brote grande disease symptoms were collected. The counties in New Mexico and Arizona that were surveyed for brote grande are denoted with an asterisk. The map was adapted from a free U.S. geological survey. Most phytoplasmas are known to be transmitted by leafhoppers. Beet leafhoppers (Circulifer tenellus) have been established as a vector for several other 16Sr group VI phytoplasmas including BLTVA (7). Beet leafhoppers are common in New Mexico chile fields where they contribute to epidemics of Beet curly top virus (BCTV) (4,13). Interestingly, field surveys over the past three years revealed that BCTV is nearly always present in fields affected by brote grande. Further, about 20% of the brote grande plants identified also tested positive for BCTV with the double infected plants displaying the typical brote grande witches broom symptoms accompanied by mosaic symptoms (Fig. 1C). While BCTV and brote grande commonly co-occur on plants in the same fields the incidence of brote grande is usually substantially lower than that of BCTV. For instance, brote grande symptomatic plants were never observed over 3% in the during the past three years while some of the same fields had 20% of the plants affected by BCTV. The disparity in frequency could be explained by the brote grande phytoplasma having a smaller reservoir from which to be acquired, having a lower acquisition or transmission efficiency, or by having a shorter persistence in transmitting vector. While brote grande has not yet been seen at levels above 3% in chile fields other phytoplasmas have historically caused severe epidemics on other solanaceous crops such as potato and tomato. The widespread occurrence of the brote grande phytoplasma across the southwest suggests that brote grande has potential to become a major disease of chile peppers in the future. Thus, continued monitoring for brote grande in chile production areas across the Desert Southwest is recommended. Acknowledgments We thank Patricia Kysar of the Electron Microscopy Laboratory, Department of Medical Pathology and Laboratory Medicine, School of Medicine, University of California at Davis, and Dr. Soumitra Ghoshroy from the University of South Carolina for excellent assistance in microscopy. This work was supported by USDA grants # and # and by the New Mexico State University Agricultural Experiment Station. Literature Cited 1. Amaral-Mello, A. P., Bedendo, I. P., Kitajima, E. W., Ribeiro, L. F., and Kobori, R Tomato big bud associated with a phytoplasma belonging to group 16Sr III in Brazil. Int. J. Pest Manage. 52: Bosland, P. W The wonders of New Mexico chile. N. M. J. Sci. 34:46-47.

8 3. Castro, S., and Romero, J The association of clover proliferation phytoplasma with stolbur disease of pepper in Spain. J. Phytopathol.-Phytopathol. Z. 150: Creamer, R., LuqueWilliams, M., and Howo, M Epidemiology and incidence of beet curly top geminivirus in naturally infected weed hosts. Plant Dis. 80: Davis, R. I., Schneider, B., and Gibb, K. S Detection and differentiation of phytoplasmas in Australia. Aust. J. Agric. Res. 48: Gundersen, D. E., Lee, I. M., Rehner, S. A., Davis, R. E., and Kingsbury, D. T Phylogeny of mycoplasma-like organisms (Phytoplasmas): A basis for their classification. J. Bacteriol. 176: Lee, I. M., Davis, R. E., and Gundersen-Rindal, D. E Phytoplasma: Phytopathogenic mollicutes. Annu. Rev. Microbiol. 54: Lee, I. M., Gundersen-Rindal, D. E., Davis, R. E., Bottner, K. D., Marcone, C., and Seemuller, E 'Candidatus Phytoplasma asteris', a novel phytoplasma taxon associated with aster yellows and related diseases. Int. J. Syst. Evol. Microbiol. 54: Randall, J. J., Bosland, P. W., and Hanson, S. F Brote grande, a new phytoplasma-associated disease of chile peppers. Plant Dis. 93: Santos-Cervantes, M. E., Chavez-Medina, J. A., Mendez-Lozano, J., and Leyva- Lopez, N. E Detection and molecular characterization of two little leaf phytoplasma strains associated with pepper and tomato diseases in Guanajuato and Sinaloa, Mexico. Plant Dis. 92: Serrone, P. D., Merzachi, C., Bragaloni, M., and Galeffi, P Phytoplasma infection of tomato in central Italy. Phytopathol. Mediterr. 40: Shaw, M. E., Kirkpatrick, B. C., and Golino, D. A The beet leafhoppertransmitted virescence agent causes tomato big bud disease in California. Plant Dis. 77: Velasquez-Valle, R., Medina-Aguilar, M. M., and Creamer, R First report of Beet mild curly top virus infection of chile pepper in north-central Mexico. Plant Dis. 92: