Chiou-Chu Su 1,5, Che-Ming Chang 1, Chung-Jan Chang 2, 3, Wen-Ying Su 1, Wen-Ling Deng 2, 5, and Hsien-Tzung Shih 4, 5
1 Pesticide Application Division, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Council of Agriculture, Executive Yuan, Taichung, Taiwan, ROC
2 Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan,
3 Department of Plant Pathology, University of Georgia, Griffin, GA, USA ROC
4 Applied Zoology Division, Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, Taichung, Taiwan, ROC
5 Co-corresponding authors: C. C. Su, E-mail: auba@tactri.gov.tw; W. L. Deng, E-mail: wdeng@nchu.edu.tw; H. T. Shih, E-mail: htshih@tari.gov.tw
ABSTRACT
PD of grapes caused by Xylella fastidiosa, a xylem-limited bacterium, has been listed as one of the international quarantine plant diseases. From 2002, the Bureau of Animal and Plant Health Inspection and Quarantine launched a survey project to detect PD in Taiwan. So far, a total of 13,666 grapevines were confirmed to be infected by X.
fastidiosa via direct isolations and PCR protocols. Other than grapes, the following four plants were confirmed to be the alternative hosts of PD in Taiwan: Diplocyclos palmatus (L.) C. Jeffrey, Ampelopsis brevipedunculata (Maxim.) Trautv var. hancei (Planch.) Rehder., Humulus scandens (Lour.) Merr., and Mallotus paniculatus (Lam.) Muell.-Arg. The 16S rRNA sequences of PD strains isolated from tissues of grapevines exhibiting PD symptoms collected from 5 counties and 4 alternative hosts were compared with X. fastidiosa from other hosts. The phylogenetic trees constructed with neighbour-joining method revealed that X. fastidiosa strains from different hosts could be divided into five subgroups and the PD strains from Taiwan were grouped with grapevine and mulberry strains from the Americas. The control strategies recommended for limiting the spread of PD are as follows: (1) to plant the healthy seedlings and eradicate the PD-like grapevines immediately (2) to eradicate the alternative hosts and the weeds that insect vectors favor (3) to control the population of indigenous insect vectors and avoid the invasion of foreign insect vectors.
Keywords: Xylella fastidiosa, Pierce’s disease of grape, alternative hosts, phylogenetic analysis
INTRODUCTION
Pierce’s disease (PD) of grapes, caused by Xylella fastidiosa, a xylem-limited bacterium, was described in California in 1892 (14) as Anaheim disease of gapes. The bacterium inhabits only in xylem tissues and requires specific and enriched media for in vitro growth (40). All strains of X. fastidiosa are classified as one species but infect different host range. Besides PD, X. fastidiosa also causing diseases on citrus (3, 16), pear (19), elm (11), almond (22), oak (4), plum (27), peach (39) and mulberry (18). Ever since Wells et al.(40) named then xylem-limited bacterium as X. fastidiosa in 1987, X.
fastidiosa has been reclassified into five subspecies according to their differences in genetic makeup, host range, physiology, and biochemistry. They are X. fastidiosa subsp.
fastidiosa for strains of grape, almond, alfalfa, and maple, X. fastidiosa subsp.
multiplex for strains of peach, plum, almond, elm, sycamore, and pigeon grape, X.
fastidiosa subsp. pauca for strains of citrus (31), X. fastidiosa subsp. sandyi for strains of oleander, daylily, jacaranda, and magnolia (32), and X. fastidiosa subsp. tashke for strains of Chitalpa tashkentensis, a common ornamental landscape plant (26). However, the last two subspecies have not been officially recognized by the researchers in the community of systematic bacteriology.
Distribution of PD
PD was reported from countries in the Americas including Mexico and the United States in North America, Costa Rica (1) and Venezuela (15) in Central America and Caribbean, and Argentina and Peru (24) in South America. In 1998, Kosovo, former Yugoslavia (2), was the only country that reported a PD incidence in European Continent. In Asian Continent, description of PD-like disorder from China was reported in 2001(7), and PD incidence was confirmed in Taiwan in 2002 (36). In the southeastern United States, PD is endemic and plays an important role in limiting the development of winery industry. During the period from1994 to 2000, PD destroyed more than 1000 acres of vineyards and caused losses of $30 million in northern California.
The pathogen of PD is X. fastidiosa, a gram-negative, non-flagellate, nutritionally fastidious, rod-shaped with rippled cell walls. The cell size was measured 0.2–0.4 µm
in width and 1–3 µm in length (Fig. 1). X. fastidiosa only resided in the xylem tissue and the optimal temperature for growth was 26-28℃ and the optimal pH was 6.5-6.9.
X. fastidiosa grows slowly even in specific and enriched media. At 12-day post-subculture, the opalescent circular colonies reached 1 mm in diameter and were convex with smooth margins in morphology.
Diseased grapevines usually begin with scorch symptoms, necrotic tissue with yellow margins, and followed by the systemic development of leaf scorch symptoms in upper and lower leaves (Fig. 2) (12). Severely affected grapevines drop leaves early, leaving petioles remain attached to the canes, decline in vigor, followed by stunting, dieback and eventual death in 2-4 years after initial infection (34, 38).
The primary goal of this report was to describe the occurrence of PD and the potential alternative hosts in Taiwan. Moreover, the epidemiological information of PD would be drawn to develop control strategies to reduce the risk of PD in Taiwan.
The occurrence of PD in Taiwan
Table grapes and winemaking grapes were both cultivated in central Taiwan, including Miaoli County, Changhua County, Nantou County, Taichung City. Table grapes including Kyoho, Italia, Honey red and Himrod seedless are competitive to other imported fruits, and gradually become one of the high economic valued fruits in Taiwan;
Golden Muscat, Black queen and Muscat Bailey A are for winemaking. In 2002, the Bureau of Animal and Plant Health Inspection and Quarantine launched a survey project to investigate the occurrence of PD in central Taiwan. From 2003 to 2012, a total of 399 vineyards and 13,666 grapevines, including table grapes and winemaking grapes, were confirmed to be infected by X. fastidiosa by performing direct isolation and cultivation of the bacterium and PCR (21, 30) ; and hence eradicated except those in Changhua County (Table 1).
The survey result showed the morbidity of vineyards conducted from 2002 to 2012 is 19% in Caoton Township, 57% in Zhushan Township, 69% in Waipu District, 93% in Houli District, and 87% in Tonxiao Township (Fig. 3). The winemaking grapes mainly cultivated in Waipu District, Houli District, and Tonxiao Township showed higher morbidity than the table grapes cultivated in areas where they were managed accurately and effectively. In the case of the vineyards in Zhushan, the abandoned vineyards might conserve the pathogen of PD in the diseased plant and the complex vegetation. Once, the alternative hosts were eradicated around the vineyard the insect vectors were forced
to migrate to the adjacent vineyards and spread PD.
The geographic distribution of diseased vineyards in Taiwan.
According to the survey results obtained from central Taiwan, the geographic distribution of diseased vineyards can be summarized as two characteristics: (1) The vineyards in Tonxiao Township, Waipu District and Houli District were typically located in hilly terrain; (2) The vineyards adjacent to the river or valley were found in Zhusan Township, Caoton Township, Zhuolan Township and Xinshe District. These mentioned counties were considered as high risk areas for PD, and the diseased vineyards were almost found in the margin of these counties where they were surrounded by undeveloped mixed forest which favors the survival of the pathogenic bacteria and the insect vectors.
The vineyards in Xinyi Township, Shuili Township and Shigang District have been free of PD incidence. However, the geographical characteristics of vineyards in these counties were similar to those mentioned above; hence they were classified as moderate risk area of PD that need constant survey of PD incidence.
On the other hand, Changhua County, where about 1800 hectares of table grapes were cultivated, has been free of PD according to the detection survey and report of Taichung District Agricultural Research and Extension Station, were classified as low risk area. Based on the following assumptions, we think those in Changhua County might avoid PD explosion: (1) the cultivating area were located in plains, and dense planting cultivation were performed; (2) grapevines are fully replaced approximately every seven years; and (3) immediate eradication of PD-like plants to keep the vegetation clean and simple.
Alternative hosts of PD in Taiwan
X. fastidiosa has a broad host range of native plant species, and many of them appear to be symptomless. In California more than 94 species host plants in 28 families are identified as alternative hosts, including Acacia longifolia, Artemesia vulgaris, Avena fatua, Chenopodium ambrodioides, Fuchsia magellanica, Hydrangea paniculota, Lolium multiflorum, Marjorana hortensis, Poa annua, Rosa California, Rosemary officinalis, Rubus vitifolius, Salix spp., Veronica spp. and Vitis califonica
(9,28).
From 2003 to 2012, about 5,404 plant samples (Table 2) were collected from the proximity of PD-confirmed vineyards in central Taiwan and they were classified as 251 species in 72 families. So far, the result showed 5 Diplocyclos palmatus (L.) C. Jeffrey samples in Taichung City and Miaoli county, 8 Ampelopsis brevipedunculata (Maxim.) Trautv var. hancei (Planch.) Rehder. in Taichung City, Nantou county and Miaoli county, 10 Humulus scandens (Lour.) Merr. in Taichung City and Miaoli county, and just 1 Mallotus paniculatus (Lam.) Muell.-Arg in Taichung City were confirmed to be the alternative hosts of PD strains based on the direct isolation and PCR detection (Fig.
4). The pathogenic bacteria isolated from four alternative hosts were used for artificial inoculation to healthy grapevines by xylem-infiltration method (8, 13). The symptomatic leaves appeared from the base near the inoculation sites and moved upwards 1 month post-inoculation.
Upon further analysis, it was revealed that there was a geographic correlation between the distribution of alternative hosts and the diseased grapevines in the vineyard which were indirectly confirmed that the indigenous insects were the vectors in Taiwan.
In previous reports, 39 species of Cicadellinae and 5 species of Cercopoidea were confirmed as the vectors of different strains of X. fastidiosa in the United States and Brazil (29). Results of extensive surveys revealed that one Kolla paulula (Walker), a xylem-feeding leafhopper, was identified as a candidate insect that may transmit X. fastidiosa in central Taiwan (33). Other than Kolla paulula (Walker), we have got positive PCR detection from Bothrogonia ferruginea (Fabricius) and Anatkina horishana (Matsumura). Nevertheless, the transmission protocol by B. ferruginea (Fabricius) and A. horishana (Matsunura) has not been tested, and it awaits further investigations for the fulfillment of the Koch's postulates.
Phylogenetic analysis of the PD strains from Taiwan and from the Americas and X. fastidiosa from other hosts
The 16S rRNA gene (6,16) sequences of PD strains from Taiwan have been deposited in GenBank under the accession numbers indicated in Table 3 and were used as queries for the similarity search. Twenty sequences of Pierce’s disease strains (Table 4) from grapevines and other alternative hosts found in Taiwan were compared with strains from other host plants via multiple sequences alignment by Clustal X program
(17) using Xanthomonas axonopodis pv. citri strain XCW as an outgroup (5, 10, 36).
The phylogenetic tree was constructed with neighbour-joining method and evaluated by bootstrap analysis for 1000 replicates using MEGA 4 program (37) with the orthologous sequences of XCW as outgroups for phylogenetic analyses (20). The neighbour-joining trees showed two distinct monophyletic groups of the 31 strains:
Group 1 contained 2 PLS strains (35) and group 2 contained the other 29 strains. The strains in group 1 and group 2 were closely related to each other with bootstrap probabilities of 88% and 99% for the 16S rRNA phylogenetic tree. Group 2 can be subdivided into 4 subgroups: GM (grape and mulberry), C (coffee and citrus), PS (peach, pecan, plum and sycamore) and O (oleander) (Fig. 5). The PD strains from grapevines and confirmed alternative hosts in Taiwan were grouped with American PD and mulberry strains that belonged to the subspecies fastidiosa (31) which demonstrated that the pathogens might exist in indigenous alternative hosts and from which the indigenous insect vectors spread the disease.
DISCUSSION
Even though the PD of grapes in Taiwan was not reported in a respected journal until this year, it still represents the very first PD in Taiwan as well as in the entire Asian Continent (36). We did take immediate action from 2002 onward to survey the vineyards using direct isolation and PCR detection and to eradicate diseased grapevines, PD however has not been successfully controlled. In Taiwan, the diseased vineyards were usually in hilly terrains, and therefore it is possible that the vegetation in the vineyards serves as the natural bacterial reservoirs. More than 94 species of plants in 28 families were reported as hosts of PD bacterium in California, and most of them were symptomless hosts. This study represents the first report confirming that D. palmatus, A.
brevipedunculata, H. scandens and M. paniculatus serve as alternative hosts of X. fastidiosa in Taiwan. Because of the availability of the alternative hosts that reserve X. fastidiosa in its vegetation, the insect vectors could acquire X.
fastidiosa at any time and inoculate grapevines. Understanding the stages of X.
fastidiosa transmission by insect vectors will provide information for PD control in Taiwan.
At last, we should understand thoroughly the epidemiology of PD in order to develop successful control measures. The control strategies include the follows:
(1) Enter Quarantine: The pathogen of PD may be brought in with diseased branches or insect vectors. Therefore, the government must enhance the quarantine of grapevines imported for use as planting material originated from counties where PD occurs and must strongly restrict smuggling.
(2) We should always survey the grapevines for suspicious PD-like symptoms which should be marked up immediately and experts should be contacted for further identification in the high risk area.
(3) Integrated control:
(a) Plant healthy seedlings to prevent the pathogens being brought into the vineyard.
(b) Eradicate the abnormal grapevines immediately.
(c) Monitor the indigenous insect vector population.
(d) Eradicate the confirmed alternative hosts.
(e) Eradicate the weeds that the insect vectors favor.
(f) Pesticides should be applied to the weeds and insects in the vineyards on weekly basis or as recommended. Pesticides with different inhibition mechanisms should be used and changes of pesticides should be done after one type is being used consecutively for 2-3 times.
LITERATURE CITED
1. Aguilar, E., L. Moreira, and C. Rivera. 2008. Confirmation of Xylella fastidiosa infecting grapes Vitis vinifera in Costa Rica. Tropical Plant Pathology 33:444-448.
2. Berisha, B., Chen, Y. D., Zhand, G. Y., and Chen, T. A. 1998. Isolation of Pierce’s disease bacteria from grapevines in Europe. European Journal of Plant Pathology 104(5):427-433.
3. Chang, C. J., Garnier, M., Zreik, L., Rossetti, V., and Bove, J. M. 1993. Culture and serological detection of the xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain of Xylella fastidiosa. Curr.
Microbiol. 27:137-142.
4. Chang, C. J., and Walker, J. T. 1988. Bacterial leaf scorch of northern red oak:
isolation, cultivation, and pathogenicity of a xylem-limited bacterium. Plant Dis.
72:730-733.
5. Chen, J., Hartung, J. S., Chang, C. J., and Vidaver, A. K. 2002. An evolutionary
perspective of Pierce's disease of grapevine, citrus variegated chlorosis, and mulberry leaf scorch diseases. Curr. Microbiol. 45:423-428.
6. Chen. J., Bank, D., Jarret, R. L., Chang, C. J., and Smith, B. J. 2000. Use of 16S rDNA sequences as signature characters to identify Xylella fastidiosa. Current Microbiology 40:29-33.
7. Chu, Y. J. 2001. Pierce's disease of grape and control techniques. Yantai Fruits 4:11–12 (in Chinese)
8. Davis, M. J., Thomson, S.V. and Purcell, A. H. 1980. Etiological role of a xylem-limited bacterium causing Pierce’s disease in almond leaf scorch.
Phytopathology 70:472-475.
9. Freitag, J. H. 1951. Host range of Pierce’s disease virus of grapes as determined by insect transmission. Phytopathology 41:920-934.
10. Hauben L., Vauterin L., Swings J., and Moore E.R. 1997. Comparison of 16S ribosomal DNA sequences of all Xanthomonas species. Int. J. Syst. Bacteriol.
47:328-335.
11. Hearon, S. S., Sherald, J. L., and Kostka, S. J. 1980. Association of xylem-limited bacteria with elm, sycamore and oak leaf scorch. Can. J. Bot.
58:1986-1996.
12. Hill, B.L., and Purcell, A. H. 1995. Multiplication and movement of Xylella fastidiosa within grapevine and four other plants. Phytopathology 85:1368-1372.
13. Hopkins, D. L. 1984. Variability of virulence in grapevine among isolates of the Pierce’s disease bacterium. Phytopathology 74:1395-1398.
14. Hopkins, D. L. 2001. Pierce’s disease. In:Encyclopaedia of Plant Pathology Volume II, Maloy, O. C. and Murray, T. D.(eds), John Wiley and Sone Inc., New York, pp71-772.
15. Jimenez, A. 1985. Immunological evidence of Pierce’s disease of grapevine in Venezuela. Turrialba 35:243-247.
16. Jagoueix S, Bove J. M., and Garnier M. 1994. The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria. Int. J. Syst. Bacteriol. 44:379-386.
17. Jeannmougin F., Thompson J. D., Gouy M., Higgins D. G., and Gibson T. J.
1998. Multiple sequence alignment with Clustal X. Trends Biochem Sci 23:403-405.
18. Kostka, S. J., Tattar, T. A., Sherald, J. L., and Hurtt, S. S. 1986. Mulberry leaf scorch, a new disease caused by a fastidious xylem-inhabiting bacterium. Plant Dis. 70:690-693.
19. Leu, L. S. and Su, C. C. 1993. Isolation, cultivation, and pathogenicity of Xylella fastidiosa, the causal bacterium of pear leaf scorch disease in Taiwan.
Plant Dis. 77:642-646.
20. Mehta, A., and Rosato, Y. B. 2001. Phylogenetic relationships of Xylella fastidiosa strains from different hosts, based on 16S rRNA and 16-23S intergenic spacer sequences. International Journal of Systematic and Evolutionary Microbiology 51:311-318.
21. Minsavage, G. V., Thompson C. M., Hopkins, D. L., Leite, R. M. V. B. C., and Stall, R. E. 1994. Development of polymerase chain reaction protocol for detection of Xylella fastidiosa in plant tissue. Phytopathology 84:456-461.
22. Mircetich, S. M., Lowe, S. K., Moller, W. J. and Nyland G. 1976. Etiology of almond leaf scorch disease and transmission of the causal agent.
Phytopathology 66:17-24.
23. Pooler, M. R., and Hartung, J. S. 1995. Specific PCR detection and identification of Xylella fastidiosa strains causing citrus variegated chlorosis.
Current Microbiology 31:377-381.
24. Purcell, A. H. 1997. Xylella fastidiosa, a regional problem or global threat?
Journal of Plant Pathology 79(2):99-105.
25. Purcell, A. H. and Saunders, S. R. 1999a. Glassy-winged sharpshooters expected to increase plant disease. California Agriculture 53(2):26-27.
26. Randall, J. J., Goldberg, N. P., Kemp, J. D., Radionenko, M., French, J. M., Olsen, M. W., and Hanson, S. F. 2009. Genetic analysis of a novel Xylella fastidiosa subspecies found in the Southwestern United States. Appl. Environ.
Microbiol. 75:5631-5638.
27. Raju, B. C., Wells, J. M., Nyland, G., Brlansky, R. H., and Lowe, S. K. 1982.
Plum leaf scald: isolation, culture, and pathogenicity of the causal agent.
Phytopathology 72:1460-1466.
28. Raju, B. C., Goheen, A. C. and Frazier, N. W. 1983. Occurrence of Pierce’s disease bacteria in plants and vectors in California. Phytopathology 73(9):1309-1313.
29. Redak, R. A., Purcell A. H., Lopes J. R. , Blua M. J., Mizell R. F. III, and
Andersen, P. C. 2004. The biology of xylem fluid-feeding insect vectors of Xylella fastidiosa and their relation to disease epidemiology. Annu. Rev.
Entomol. 49: 243-270.
30. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular cloning: A laboratory manual. 2nd edition. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press)
31. Schaad, N. W., Postnikova, E., Lacy, G., Fatmi, M., and Chang, C. J. 2004.
Xylella fastidiosa subspecies: X. fastidiosa subsp. piercei, subsp. nov., X.
fastidiosa subsp. multiplex subsp. nov., and X. fastidiosa subsp. pauca subsp.
nov. Syst. Appl. Microbiol. 21:290-300.
32. Schuenzel, E. L., Scally, M., Southammer, R., and Nunney, L. 2005. A multigene phylogenetic study of clonal diversity and divergence in North American strains of the plant pathogen Xylella fastidiosa. Appl. Environ.
Microbiol. 71:3832-3839.
33. Shih, H. T., Su, C. C., Feng, C. Y., Fanjiang, C. C., Hung W. F., and Hung, L. Y.
2009. Studies on the morphology, ecology, and host range for Kolla paulula (Walker, 1858) (Hemiptera: Membracoidea: Cicadellidae: Cicadellinae).
Formosan Entomol. 29(4): 353 (abstract) (in Chinese).
34. Smith, I. M., McNamara, D. G., Scott, P. R. and Harris, K. M. 1997. Xylella fastidiosa. In:Quarantine Pests for Europe, CAB International, Wallingford, UK, pp845-851.
35. Su, C. C., Chang, C. J., Yang, W. J., Hsu, S. T., Tzeng, K. C., Jan, F. J. and Deng, W. L. 2012. Specific characters of 16S rRNA gene and 16S-23S rRNA internal transcribed spacer sequences of Xylella fastidiosa pear leaf scorch strains. Eur. J. Plant Pathol. 132:203-216.
36. Su, C. C., Chang, C. J., Chang, C. M., Shih, H. T., Tzeng, K. C., Jan, F. J. and Deng, W. L. 2013. Pierce’s Disease of Grapevines in Taiwan: Isolation, Cultivation and Pathogenicity of Xylella fastidiosa. J. Phytopathol 161:389-396.
37. Tamura, K., Dudley, J., Nei, M., and Kumar, S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599.
38. Varela, L.G., Purcell, A. H., and Smith, R. J. 2000. University of California Cooperative Extension and Statewide IPM Project, Pierce’s Disease in the North Coast. http://www.cnr.berkeley.edu/xylella/pd97.html
39. Wells, J. M., Raju, B. C., and Nyland, G. 1983. Isolation, culture, and pathogenicity of the bacterium causing phony disease of peach. Phytopathology 73:859-862.
40. Wells, J. M., Raju, B. C., Hung, H.-Y., Weisburg, W. G., Mandelco-Paul, L., and Brenner, D. J. 1987. Xylella fastidiosa gen. nov., sp. nov: Gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp. Int. J. Syst.
Bacteriol. 37:136-143.
Table 1. Survey results of Pierce’s disease incidence: total numbers of diseased plants and vineyards in various counties conducted from 2002 to 2012
Table 2. Survey results of other possible host plants for Xylella fastidiosa Pierce’s disease (PD) strains: total number of various plant species collected from the proximity of PD-confirmed vineyards and sample sizes conducted from 2003 to 2012
Table 3. Detection of Xylella fastidiosa Pierce’s disease (PD) strains in four confirmed alternative hosts collected from the proximity of PD-confirmed vineyards conducted from 2003 to 2012
*: Positive isolation and PCR detection.
ND: Non-detection
Table 4. Source of Xylella fastidiosa: Pierce’s disease strains from grapevines and other alternative hosts in Taiwan