This article is from the April 2008 issue of published by The American Phytopathological Society For more information on this and other topics related to plant pathology, we invite you to visit APSnet at www.apsnet.org Identification and Pathogenicity of Lasiodiplodia theobromae and Diplodia seriata, the Causal Agents of Bot Canker Disease of Grapevines in Mexico J. R. Úrbez-Torres, Department of Plant Pathology, University of California, Davis 95616; G. M. Leavitt, University of California Cooperative Extension, Madera 93637; J. C. Guerrero, Departamento de Agricultura, Universidad de Sonora, Hermosillo 83000, Mexico; J. Guevara, Campo Experimental Costa de Ensenada (INIFAP), Baja California 22800, Mexico; and W. D. Gubler, Department of Plant Pathology, University of California, Davis ABSTRACT Úrbez-Torres, J. R., Leavitt, G. M., Guerrero, J. C., Guevara, J., and Gubler, W. D. 2008. Identification and pathogenicity of Lasiodiplodia theobromae and Diplodia seriata, the causal agents of bot canker disease of grapevines in Mexico. Plant Dis. 92:519-529. Perennial cankers and consequent grapevine dieback are a major problem in vineyards of Sonora and Baja California, the most important grape-production areas of Mexico. In order to identify the canker-causing agents, symptomatic arms, cordons, and trunks were collected from 13 and 6 vineyards in Sonora and Baja California, respectively. Two Botryosphaeriaceae spp., Lasiodiplodia theobromae and Diplodia seriata, were isolated frequently from infected wood and identified based on morphological and cultural characters as well as analyses of nucleotide sequences of three genes, the internal transcribed spacer region (ITS1-5.8S-ITS2), a partial sequence of the β-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). Although both L. theobromae and D. seriata were isolated from grapevine cankers in Baja California, only L. theobromae was found in vines in the Sonora region. Pathogenicity of both species was verified by inoculation of rooted cuttings and green shoots of Thompson Seedless and Chardonnay cultivars. Isolates of L. theobromae were more virulent, based on the extent of spread in the secondary wood and green tissue, than those of D. seriata. These findings confirm L. theobromae and D. seriata as the causal agents of dieback and canker formation of grapevines in northern Mexico. Additional keywords: dead arm, Eutypa dieback, Eutypa lata, trunk diseases, Vitis vinifera Grapevine (Vitis vinifera L.), cultivated in over 26,000 hectares, is the most important fruit crop in the Mexican States of Baja California and Sonora. These regions are the largest wine and table grapeproducing areas of Mexico and generate an annual crop valued of US$152 million dollars (39). Grapevine decline in Mexico first was observed in the states of Aguascalientes and Coahuila-Durango in 1979, and was associated with Eutypa lata (Pers.) Tul. & C. Tul., the causal agent of Eutypa dieback (44). Ten years later, grapevine cankers and subsequent dieback were observed in table-grape vineyards of Hermosillo, Sonora. Disease symptoms were characterized by dead arms and cordons, trunk dieback due to canker formation in the vascular tissue, and the total absence of the stunted chlorotic spring growth which is typical of infections by E. lata. Subsequently, these symptoms were associated with Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (23). For many Corresponding author: W. D. Gubler E-mail: wdgubler@ucdavis.edu Accepted for publication 14 November 2007. doi:10.1094 / PDIS-92-4-0519 © 2008 The American Phytopathological Society years, E. lata was thought to be the most important canker- and dieback-causing agent of grapevines not only in Mexico but worldwide. However, recent studies also have identified Botryosphaeriaceae spp. as important grapevine pathogens causing cankers and other dieback symptoms such as wood streaking, shoot dieback, cane bleaching, bud necrosis, and graft failure in all major viticulture regions throughout the world (20,25,29,43,45,47,48). Based on a recent study, the genus Botryosphaeria has been shown to be restricted only to Botryosphaeria dothidea (Moug.) Ces. & De Not. and B. corticis (Demaree & Wilcox) Arx & E. Müll. (12). Consequently, the name Botryosphaeria is no longer acceptable for most of the species with Fusicoccum-like and Diplodialike anamorphs (12,31). Although the genus Neofusicoccum Crous, Slippers & A.J.L. Phillips has been described to accommodate Botryosphaeriaceae with Fusicoccum-like anamorphs; no teleomorph name has been proposed for Botryosphaeriaceae spp. with Lasiodiplodiaand Diplodia-like anamorphs (12). For this reason, fungi such as B. rhodina and B. obtusa will be named by their anamorphs, L. theobromae and Diplodia seriata De Not., respectively (12,31). Along with all species of Botryosphaeriaceae found in grapevines, L. theobromae (=B. rhodina), D. seriata (=B. obtusa), Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (=B. parva), B. dothidea (Moug.) Ces. & De Not., D. mutila (Fr.) Mont. (=B. stevensii), N. luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (= B. lutea), and N. australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (=B. australis) are the most commonly found associated with grapevine dieback worldwide (2,20,29,43,45,47,48). L. theobromae is a pleomorphic and plurivorous Ascomycete, mostly prevalent in tropical and subtropical climate regions (33). L. theobromae is recognized as an important wood pathogen and has been reported to cause cankers, dieback, and fruit and root rots in over 500 different hosts, including perennial fruit and nut trees, vegetable crops, and ornamental plants (32). Vascular cankers and grapevine dieback caused by L. theobromae first were reported in Egypt in 1972 (13). Fifteen years later, a field study conducted in California showed L. theobromae to be an important grapevine pathogen, capable of causing cankers, dieback, and dead arm symptoms on vines (24). The disease has since been referred to as “Bot canker” or “Diplodia cane dieback” and now is known to be prevalent in the warm growing areas of the southern San Joaquin Valley and the Coachella valley in southern California (17,23,45). Recent studies conducted in South Africa and Australia, where L. theobromae was isolated primarily from declining grapevines showing perennial cankers in spurs and cordons, have confirmed the significance of this species as a grapevine pathogen (43,48,50). D. seriata has been observed in temperate areas on most continents and described from over 35 different hosts, including Vitis spp. (34). D. seriata is recognized as an important pathogen of pome and stone fruit trees, causing cankers, leaf spots, and black rot of the fruit (5,6,22,40,41). Unlike L. theobromae, pathogenicity of D. seriata in grapevines remains unclear. Whereas studies in Western Australia and Portugal have considered D. seriata to be a saprophyte or a weak and secondary pathogen of grapevines, respectively (28,43), other studies report D. seriata to be a primary pathogen in grapevines causing vascular Plant Disease / April 2008 519 symptoms such as brown streaking of the wood and cankers in Australia, South Africa, Chile, and France (2,10,20,37,48). Different studies have reported D. seriata to be associated with grapevine decline symptoms such as trunk and bark infections in Herzegovina, Yugoslavia (35), xylem necrosis in Italy (3,36), infectious drying of grapes in Ukraine (18), vine decline in Hungary (16,25), perennial cankers in Spain (1,47), “Black dead arm” in Lebanon (11), and darkened pith in New Zealand (4). However, Koch’s postulates were not accomplished in most of these studies. Therefore, whether D. seriata is acting as a saprophyte or is a pathogen causing grapevine dieback symptoms has not yet been clarified in many grapegrowing regions worldwide. The purpose of this study was to identify and characterize, by means of morphological features, multigene phylogeny, and pathogenicity studies, the causal agents of grapevine dieback in Mexican vineyards. MATERIAL AND METHODS Field survey, disease symptoms, and fungal isolation. Field surveys were conducted in 2004 in 13 and 6 vineyards in Sonora and Baja California regions, respectively (Fig. 1). Diseased plants were characterized by dead arms and spur positions with poor or no shoot development. Depending on the disease progress, death of the entire part of the cordon or different portions could be observed while other parts of the vine showed normal and healthy growth. Canker development was observed to grow basipetaly from putative points of infection. In all, 55 and 180 cankers were collected from infected arms, cordons, and trunks from different winegrape cultivars (Cabernet Sauvignon, Carignane, Chenin Blanc, Sauvignon blanc, and Petit Sirah) in Baja California and from table-grape cultivars (Flame Seedless, Perlette, Sugar One, Rubi Seedless, and Superior) in Sonora, respectively, and brought to the laboratory. Longitudinal and transversal cuts from symptomatic arms, cordons, and trunks were made to observe any internal symptom. Vascular symptoms were characterized by wedgeshaped cankers, which appeared to be associated with pruning wounds (Fig. 2a). In addition to sectioning, diseased samples were surface sterilized in 10% sodium hypochlorite for 10 min. Surface tissue was cut away to expose the canker, and wood chips of approximately 1 cm2 were cut from the margin of the dead tissue and cultured on 4% potato dextrose agar (PDA; Difco, BD Micro Biology Systems, Franklin Lakes, NJ) amended with 0.01% tetracycline hydrochloride (PDA-tet; SigmaAldrich, St. Louis). Cultures were incubated at 25°C until fungal colonies were observed. Botryosphaeriaceae isolates first were separated based on colony morphology by examination of the cultures after 5, 10, and 14 days. Fungal mycelium from Botryosphaeriaceae colonies was transferred to fresh PDA-tet petri plates and incubated at room temperature, and hyphal tips from 2-day-old cultures were aseptically placed on PDA petri plates using a dissecting microscope in order to obtain pure cultures. Isolates were incubated at 25°C in a 12-h fluorescent light-and-dark cycle. Based on colony and pycnidia morphology formed in culture of all Botryosphaeriaceae isolates inspected, 20 were selected for detailed identification to species level (Table 1). Morphological characterization. In all, 15 isolates from both the Baja California and Sonora grapevine regions were used for morphological characterization Fig. 1. Map of Mexico indicating the wine-grape region of Baja California and the table-grape region of Sonora where field surveys were conducted. 520 Plant Disease / Vol. 92 No. 4 (Table 1). Botryosphaeriaceae spp. first were identified based on colony and conidial morphology and by comparing with previous published studies (29,33,34,43, 45,47). Colony morphology of Botryosphaeriaceae isolates from Mexico was compared in the laboratory with previously identified Botryosphaeriaceae spp. from California (45) (Table 2). In order to enhance sporulation, cultures were placed on 2% water agar (Difco, BD Micro Biology Systems) containing autoclaved grapevine wood chips and incubated at 25°C under intermittent light (12 h). Isolates were examined weekly for formation of pycnidia and conidia. Conidial morphology (cell wall, shape, color, and presence or absence of septa) from pycnidia was recorded using a compound microscope (Nikon Inc. Instruments Group, Elville, NY). The length and width of 50 conidia per isolate were measured using the imaging device SPOT RT software (v3.5.1; SPOT; Diagnostic Instruments Inc., MI). Minimum, maximum, mean, standard deviation, and 95% confidence intervals were calculated from measurements using summary statistics in SAS (SAS System, version 8.1; SAS Institute, Cary, NC). Optimum growth temperature and mycelium growth rates were determined in nine isolates (Table 1) by placing a 4-mmdiameter plug from the growing margin of a 3-day-old colony in the center of an 85mm-diameter PDA petri dish. Three replicates of each isolate were incubated separately in the dark at 5 to 40°C at 5°C intervals. Colony diameter was measured at 24h intervals, and data were converted to daily radial growth in millimeters. The experiment was conducted twice. Isolates used in this study are maintained in the collection in the Department of Plant Pathology at the University of California, Davis, and representative isolates of each Botryosphaeriaceae sp. were deposited in the American Type Culture Collection. DNA extraction, polymerase chain reaction amplification, and multigene phylogenetic analysis. Total genomic DNA from 20 Botryosphaeriaceae isolates (Table 1) was extracted from pure culture mycelia using an AquaPure Genomic DNA Kit (Bio-Rad Laboratories; Hercules, CA). Oligonucleotide primers ITS4 and ITS5 were used to amplify the ITS region of the nuclear ribosomal DNA, including the 5.8S gene (49). Oligonucleotide primers Bt2a and Bt2b were used to amplify a portion of the β-tubulin (BT) gene (15). Amplification of part of the translation elongation-factor (EF) gene was done with the primers EF1-728F and EF1-986R (8). Each polymerase chain reaction (PCR) contained 5 µl of 10× PCR buffer containing 15 mM MgCl2, 2 µl of 25 mM MgCl2, 1µl of 10 mM dNTPs, 2 µl of 0.5 mM of each primer, 0.25 µl of Taq DNA polymerase (Taq PCR core kit; Qiagen, Valencia, CA) at 5 units/µl, and 2 µl of template Table 1. Botryosphaeriaceae isolates from Vitis vinifera from Mexico used in this study GenBank accession numbera Isolate UCD1010BCe,f,g,h UCD1015BCe,g UCD1035BCg UCD1038BCe,f,g,h UCD1052BCe,g,h UCD1061BCe,f,g UCD1012BCe,g UCD1014BCe,f,g,h UCD1028BCe,g UCD1030BCe,f,g,h UCD1060BCe,f,g,h UCD810SNe,g UCD881SNg UCD914SNe,f,g,h UCD916SNg UCD917SNg UCD918SNe,f,g,h UCD919SNe,g,h UCD921SNg UCD923SNe,f,g Identityb Diplodia seriata D. seriata D. seriata D. seriata D. seriata D. seriata Lasiodiplodia theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae Host (V. vinifera cvs.) Originc ITS β-tubulin EF1-α ATCCd Cabernet Sauvignon Carignane Chenin Blanc Chenin Blanc Sauvignon Blanc Petit Sirah Carignane Carignane Chenin Blanc Chenin Blanc Petit Sirah Flame Seedless Perlette Rubi Seedless Sugar One Sugar One Sugar One Sugar One Sugar One Sugar One Ensenada Ensenada Ensenada Ensenada Ensenada Ensenada Ensenada Ensenada Ensenada Ensenada Ensenada Hermosillo Hermosillo Hermosillo Hermosillo Hermosillo Hermosillo Hermosillo Hermosillo Hermosillo EU012377 EU012378 EU012379 EU012380 EU012381 EU012382 EU012372 EU012373 EU012374 EU012375 EU012376 EU012363 EU012364 EU012365 EU012366 EU012367 EU012368 EU012369 EU012370 EU012371 EU012429 EU012430 EU012431 EU012432 EU012433 EU012434 EU012424 EU012425 EU012426 EU012427 EU012428 EU012415 EU012416 EU012417 EU012418 EU012419 EU012420 EU012421 EU012422 EU012423 EU012400 EU012401 EU012402 EU012403 EU012404 EU012405 EU012392 EU012393 EU012394 EU012395 EU012396 EU012383 EU012384 EU012385 EU012386 EU012387 EU012388 EU012389 EU012390 EU012391 MYA-4188 MYA-4189 MYA-4190 MYA-4191 MYA-4192 MYA-4193 MYA-4194 MYA-4195 MYA-4196 MYA-4197 MYA-4198 MYA-4199 MYA-4200 MYA-4202 MYA-4203 MYA-4201 MYA-4204 MYA-4205 MYA-4206 MYA-4207 a ITS = internal transcribed spacer and EF1-α = elongation factor. Botryosphaeriaceae spp. from grapevine from Mexico were determined based on morphology and phylogenetic analyses. c Isolates from Ensenada, Baja California, Mexico collected by G. Leavitt and J. Guevara in July 2004 and isolates from Hermosillo, Sonora, Mexico collected by J. R. Úrbez-Torres, G. Leavitt, and J. C. Guerrero in February 2004. d ATCC = American Type Culture Collection. e Isolates used for colony and conidial morphology. f Isolates used for temperature studies. g Isolates used for phylogenetic analyses. h Isolates used for pathogenicity tests. b Table 2. Botryosphaeriaceae isolates used in the phylogenetic study GenBank accession numbera Isolateb UCD191Co* UCD205Co* UCD206Co* CAA006 CMW9074 CMW10130 CBS124.13 CBS115812 UCD244Ma* UCD352Mo* UCD614Tu* UCD710SJ* UCD770St* CMW7774 CMW7775 CMW8230 CMW8232 CBS119049 UCD288Ma UCD1953SB UCD1965SB CMW7060 CBS112554 CBS230.30 JL375 CBS112547 UCD2057Te CBS110299 Identity Lasiodiplodia theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae L. theobromae L.gonubiensis Diplodia seriata D. seriata D. seriata D. seriata D. seriata D. seriata D. seriata D. seriata D. seriata D. seriata Diplodia mutila D. mutila D. mutila D. mutila D. mutila D. mutila D. mutila Diplodia corticola Neofusicoccum luteum N. luteum Hostc Vv cv. Flame Seedless Vv cv. Thompson Seedless Vv cv. Perlette Vitis vinifera Pinus sp. V. donniana Unknown Syzygium cordatum Vv cv. Thompson Seedless Vv cv. Cabernet Sauvignon Vv cv. Muscat Vv cv. Zinfandel Vv cv. French Colombard Ribes sp. Ribes sp. Picea glauca Malus domestica Vitis sp. Vv cv. Thompson Seedless Vv cv. Chenin Blanc Vv cv. Chardonnay Fraxinus excelsior Pyrus communis Phoenix dactylifera Fraxinus excelsior Quercus ilex Vv cv. Cabernet Sauvignon V. vinifera Origin Collectord ITS β-tubulin EF1-α California, USA California, USA California, USA California, USA Mexico Uganda United States South Africa California, USA California, USA California, USA California, USA California, USA New York, USA New York, USA Canada South Africa Italy California, USA California, USA California, USA The Netherlands Portugal California, USA Catalonia, Spain Córdoba, Spain California, USA Portugal J. R. Ú.-T. & G. L. J. R. Ú.-T. & G. L. J. R. Ú.-T. & G. L. T. J. Michailides B. Slippers J. Roux J. J. Taubenhhaus D. Pavlic J. R. Ú.-T. & G. L. J. R. Ú.-T. & G. L. J. R. Ú.-T. & G. L. J. R. Ú.-T. & G. L. J. R. Ú.-T. & G. L. B. S. & G. H. B. S. & G. H. J. Reid W. A. Smith L. Mugnai J. R. Ú.-T. & G. L. J. R. Ú.-T. & G. L. J. R. Ú.-T. & G. L. H. A. van der Aa A. J. L. Phillips L. L. Huillier J. Luque M. E. Sánchez J. R. Ú.-T. & G. L. A.J.L. Phillips DQ008308 DQ008310 DQ008311 DQ458891 AY236952 AY236951 DQ458890 DQ458892 DQ008314 DQ008315 DQ008318 DQ008321 DQ008322 AY236953 AY236954 AY972104 AY972105 DQ458889 DQ008313 DQ233598 DQ233599 AY236955 AY259095 DQ458886 DQ458887 AY259110 DQ233604 AY928043 DQ008331 DQ008333 DQ008334 DQ458859 AY236930 AY236929 DQ458858 DQ458860 DQ008337 DQ008338 DQ008341 DQ008344 DQ008345 AY236931 AY236932 AY972119 AY972120 DQ458857 DQ008336 DQ233619 DQ233620 AY236933 DQ458851 DQ458849 DQ458852 DQ458854 DQ233625 DQ458848 EU012397 EU012398 EU012399 DQ458876 AY236901 AY236900 DQ458875 DQ458877 EU012406 EU012407 EU012408 EU012409 EU012410 AY236902 AY236903 DQ280418 DQ280419 DQ458874 EU012411 EU012412 EU012413 AY236904 DQ458870 DQ458869 DQ458871 DQ458872 EU012414 AY573217 a ITS = internal transcribed spacer and EF1-α = elongation factor. Acronyms of cultures collections: UCD: University of California, Davis, Plant Pathology Department Culture Collection; CMW: Culture Collection Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; CBS: Centraalbureau Schimmelcultures, Utrecht, Netherlands; * indicates isolates used for morphological comparison. c Vv = Vitis vinifera. d J. R. Ú.-T. & G. L. = J. R. Úrbez-Torres & G. Leavitt and B. S. & G. H. = B. Slippers & G. Hudler. b Plant Disease / April 2008 521 DNA adjusted with purified water (Mili-Q Water Systems; Millipore, Billerica, MA) to a final volume of 50 µl. Amplification reactions were carried out in a thermal cycler (PTC 200; M. J. Research Company, Watertown, MA). ITS and BT temperature profiles were as follows: an initial preheat for 2 min at 94°C, followed by 35 cycles of denaturation at 94°C for 60 s, annealing at 58°C for 60 s, and extension at 72°C for 90 s. Translation EF temperature profiles were one cycle of initial denaturation at 95°C for 3 min; followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 60 s; with a final extension at 72°C for 5 min. The PCR amplification products were separated by electrophoresis in 1.5% agarose gels in 1.0× Tris-boric acid-EDTA (TBE) buffer and photographed under UV light after staining with ethidium bromide for 15 min. PCR products were purified using a QIAquick PCR purification kit (Qiagen). ITS, BT2, and EF1-α regions were sequenced in both directions by the University of California, Davis, Division of Biological Sciences sequencing facility. Sequences were edited and assembled using Sequencher (version 4.1; Gene Codes, Ann Arbor, MI). Sequences were aligned using the computer software BioEdit Sequencer Alignment Editor (version 7.0.0; Tom Hall, Isis Pharmaceuticals, Inc, Carlsbad, CA) and alignment gaps were treated as missing data. Sequences of Botryosphaeriaceae from Mexico were compared with those of Botryosphaeriaceae from previous studies available in GenBank (Table 2). The alignment was corrected by visual inspection, and any ambiguously aligned characters were deleted using BioEdit. A partition Fig. 2. Disease symptoms and Lasiodiplodia theobromae and Diplodia seriata colony and conidial morphology. a, Cross section of a 14-year-old Thompson Seedless grapevine cordon. Wedge-shaped canker was the primary vascular symptom observed in grapevines in both Baja California and Sonora grapegrowing regions. b, Colony morphology of 21-day-old L. theobromae (UCD919SN). c, Conidiogenous cells and young hyaline and thick-walled L. theobromae (UCD918SN) conidia. Nonseptate paraphyses are indicated by black arrows. d, Dark-brown mature L. theobromae (UCD918SN) conidia. Longitudinal striations and central septum can be observed. e, Colony morphology of 21-day-old D. seriata (UCD1061BC). f, Light-brown mature D. seriata (UCD1061BC) conidia. Conidial photographs were taken at ×100 (immersion oil) from pycnidia formed on grapevine wood. Scale bar = 10 µm. 522 Plant Disease / Vol. 92 No. 4 homogeneity test was performed with PAUP (version 4.0b10; Sinauer Associates, Inc., Publishers, Sunderland, MA; 42) to determine whether the ITS, β-tubulin, and EF1-α datasets could be combined together. Separate phylogenetic analyses also were performed for the ITS dataset alone, β-tubulin dataset alone, and EF1-α dataset alone, and tree topologies were compared. Maximum parsimony analyses was performed in PAUP using the heuristic search option branch swapping, and 1,000 random addition sequences replicates. Bootstrap values were calculated using 1,000 replicates to test branch strength. Tree length, consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) also were recorded. Resulting trees were printed in PAUP version 4.0b10. N. luteum sequences from GenBank were used as outgroup (Table 2). The ITS, β-tubulin, and EF1-α sequences from Botryosphaeriaceae from Mexico used in this study were deposited into GenBank. Pathogenicity tests. Nine Botryosphaeriaceae isolates were used in two separate tests to determine the pathogenicity of L. theobromae and D. seriata from Mexico on grapevines (Table 1). An initial pathogenicity test was conducted in 1-year-old grapevine cuttings of cvs. Chardonnay and Thompson Seedless. In all, 100 dormant cuttings of each cultivar were cut into uniform lengths containing six to seven buds. A basal cut was made just below the lower bud and the upper cut 2 cm above the top bud. In order to enhance callusing and rooting, dormant cuttings were buried into a 3:1 soil:vermiculite mix in plastic boxes, and placed in a callusing room at 35°C and 80% humidity for 4 weeks at the Viticulture Experimental Station of the University of California, Davis. After callusing and rooting, cuttings were wounded at the uppermost internode with a 4-mm cork borer. A 4-mm mycelium agar plug from a 1-week-old culture was placed in the wound. Wounds first were covered with 100% pure Vaseline petroleum jelly (Unilever, Greenwich, CT) and wrapped with parafilm. In all, 10 cuttings per fungal isolate were used for each cultivar. Ten cuttings of each cultivar were inoculated with 4-mm noncolonized PDA agar plugs from two different plates for negative controls. Inoculated cuttings were planted immediately in individual pots and placed in a lathhouse at the University of California Experimental Station in Davis during the last week of April. Plants were arranged in a completely randomized design. Cuttings were maintained under ambient environmental conditions and watered every 3 days or as needed. Cuttings were collected after 20 weeks and inspected for lesion development. Extent of vascular discoloration was measured upward and downward from the point of inoculation. Small pieces (0.5 to 1 cm) of necrotic tissue from the edge of each lesion were cut and placed on PDA-tet petri plates in an attempt to recover the inoculated fungus and complete Koch’s postulates. Fungal identity was verified by its colony and conidial morphology. A second pathogenicity test, using the same nine Botryosphaeriaceae isolates from Mexico (Table 1), was conducted on cvs. Chardonnay and Thompson Seedless green shoots. In all, 100 green shoots of approximately 30 cm in length from each cultivar were cut from vines at the Experimental Station of the University of California, Davis, and immediately inoculated as previously described. Ten green shoots of each cultivar were used per fungal isolate. Ten green shoots of each cultivar were inoculated with 4-mm noncolonized PDA agar plugs from two different plates for negative controls. Green shoots were placed in sterile water into 50-ml screw-cap tubes (Sarstedt, Inc., Newton, NC) and maintained for 10 days at room temperature on the laboratory bench. Afterward, green shoots were sectioned longitudinally, vascular discoloration was recorded, and green tissue reisolations were made as described above. One-way analyses of variance (ANOVA) in SAS (SAS System, version 8.1; SAS Institute) was performed in order to assess differences in the extent of vascular discoloration induced by L. theobromae and D. seriata for both hardwood and green tissue pathogenicity tests in both cultivars. To satisfy the assumptions of the ANOVA, the log10 transformation of the data was used. Homogeneity of variance was assessed using Levene’s test. The Tukey’s test was used for comparison of treatment means at P < 0.05. A two-way ANOVA was performed to determine significant differences between cultivars in inoculation of both 1year-old cuttings and green shoots treatments. Table 3. Morphological description of Lasiodiplodia theobromae and Diplodia seriata isolates from Mexico. Conidial dimensions Identity, isolate Conidial size (µm)a Temperature study L/W ratiob Opt. (°C)c Growth rated (29.2–) 23.6–16.8 (–12.6) × (12.8) 10.7–8.4 (–7.4) (26.7–) 22.6–17.6 (–14.5) × (12) 11.4–8.4 (–7.9) (26.6–) 23.6–18 (–13.2) × (12.7) 11.1–8.7 (–7.2) (24.2–) 22.1–18.5 (–16.7) × (12.9) 10.6–8.3 (–7.1) (28.7–) 22.7–17.1 (–13.1) × (11) 10.1–8.3 (–7.1) 2.1 2.03 2.1 2.13 2.16 25–30 … 25–30 … 25–30 17.5 … 26.3 … 19.8 (28.5–) 26.7–22.5 (–19.9) × (17.8) 14.6–12.4 (–11.5) (37.3–) 28.7–22.1 (–14.9) × (16.3) 13.9–11.9 (–10) (28.3–) 26.2–22.6 (–19.7) × (16.4) 14.7–12.3 (–11.4) (31.6–) 26.8–22.2 (–17.7) × (16.6) 14.5–12.3 (–11.2) (29.3–) 27.2–23.4 (–21.7) × (15.7) 14.1–12.3 (–11.1) (30.1–) 27.8–23 (–20.5) × (14.9) 13.8–11.8 (–10) (30.9–) 27.6–22.8 (–19.8) × (15.2) 13.8–11.2 (–9.8) (29.1–) 27.4–24 (–21.8) × (14.9) 13.9–11.7 (–10.8) (34.5–) 28.3–23.1 (–20.1) × (16.7) 13.5–10.7 (–9.1) (34.4–) 28.8–23.6 (–21.4) × (15) 13.1–10.5 (–9.3) 1.8 1.9 1.8 1.8 1.9 2 2 2 2.1 2.2 … 30–35 … 30–35 35 … 35 30–35 … 35 … 42.5 … 37.2 42.5 … 36.9 42.5 … 42.5 seriatae D. UCD1010BC UCD1015BC UCD1038BC UCD1052BC UCD1061BC L. theobromaef UCD1012BC UCD1014BC UCD1028BC UCD1030BC UCD1060BC UCD810SN UCD914SN UCD918SN UCD919SN UCD923SN a Data are represented as the lower and upper 95% confidence limits, with maximum and minimum dimensions in parenthesis. L/W = length/width. c Optimum temperature is defined as the temperature that produced maximum radial growth after 48 h. d Maximum mycelium radial growth in millimeters after 48 h. e Colony morphology: Moderate aerial mycelium. Light-green colonies that became dark green with age. Colonies produced many black, single, small (0.5-1 mm in diameter), and ostiolate pycnidia. Conidial morphology: Elliptical-rounded with one of the bases truncated. Immature conidia initially hyaline and thick-walled. Light brown to dark brown when mature. Aseptate conidia. f Colony morphology: Abundant aerial mycelium. White colonies with a light-green center. Entire colony became dark-green with age. Colonies produced black, grouped, large (up to 7 mm diameter), ostiolate, and globose pycnidia, embedded in stroma. Conidial morphology: Oval with rounded and pointed ends. Immature conidia hyaline, thick-walled, aseptate and densely granulated. Aseptate paraphyses. Conidia dark brown with longitudinal striations and one septum when mature. b Plant Disease / April 2008 523 RESULTS Field survey. Botryosphaeriaceae spp. were the most common fungi isolated from grapevine perennial cankers in both Baja California and Sonora grape-growing regions. D. seriata was found in all vineyards surveyed in Baja California and was isolated from 21 of 55 perennial cankers (38%). L. theobromae was found in four of six vineyards in Baja California and was isolated from 9 of 55 grapevine cankers (16%). In this study, D. seriata and L. theobromae were not isolated together from the same canker in any of the samples from Baja California. Other sporadically isolated fungi from cankers in Baja California (5%) were species of Alternaria and Aspergillus. L. theobromae was the only fungus isolated from grapevine cankers in Sonora region. L. theobromae was found in all vineyards sampled in Sonora and was isolated from 140 of 180 grapevine cankers (80.5%). Morphological characterization. Isolates of Botryosphaeriaceae from Baja California and Sonora were separated into two groups based on their appearance in culture, conidial morphology and size, and optimum growth temperature (Table 3; Fig. 2). Based on the morphological characters observed and by comparing them with those previously reported (2,20,29, 33,34,43,45,47), isolates from Mexico were identified as L. theobromae (Fig. 2b– d) and D. seriata (Fig. 2e and f). Phylogenetic analysis. ITS, β-tubulin, and EF1-α sequences of L. theobromae and D. seriata from grapevines from Mexico (Table 1) were aligned with GenBank ITS, β-tubulin, and EF1-α sequences of Botryosphaeriaceae spp. from grapevines and other hosts from different countries (Table 2). After alignment, a partition homogeneity test showed a value of P = 0.09, indicating that the ITS, β-tubulin, and EF1-α datasets were congruent (P > 0.05) and could be combined in a single phylogenetic analysis. The combined dataset consisted of 1,246 characters, of which 954 were constant, 52 were parsimony uninformative, and 240 were parsimony informative. The maximum parsimony analyses yielded one most parsimonious tree (length = 477, CI = 0.786, RI = 0.955, RC = 0.751, and HI = 0.214; Fig. 3). The combined data set phylogenetic tree included two well-separated clades. L. theobromae isolates formed a strongly supported clade, with bootstrap value of 100% (Fig. 3). L. theobromae isolates from Mexico had nearly identical sequences and were grouped together with L. theobromae isolates from California (Fig. 3). D. seriata isolates from Mexico resided in a wellsupported separate clade with bootstrap value of 100% (Fig. 3). D. seriata isolates from Mexico grouped together with D. seriata isolates from California, New York, Canada, South Africa, and Italy, showing almost no variation in the DNA sequences 524 Plant Disease / Vol. 92 No. 4 (Fig. 3). Analyses of the ITS, β-tubulin, and EF1-α datasets alone yielded the same tree topology as the combined dataset (trees not shown), and the only differences between trees generated from different datasets were changes in the positions of some isolates within one of the main clades. Results from both the combined and single datasets of the ITS, β-tubulin, and EF1-α DNA sequences verified the morphological identifications of L. theobromae and D. seriata from grapevines from Mexico. Pathogenicity tests. Mean lengths of the extent of vascular discolorations caused by L. theobromae and D. seriata isolates from Mexico on inoculated 1-yearold cuttings and green shoots are shown in Figure 4. L. theobromae and D. seriata produced dark-brown, black necrotic lesions on both 1-year-old cuttings and green shoots, which extended upward and downward from the point of inoculation (Fig. 5b, e, and f). Dark wood streaking or discoloration also was observed in green shoots and 1-year-old cuttings of both cultivars inoculated with L. theobromae (UCD914SN, UCD921SN, and UCD 1014BC) and D. seriata (UCD1015BC, UCD1038BC, and UCD1061BC) (Fig. 5eI). Differences in susceptibility between L. theobromae and D. seriata in 1-year-old Chardonnay cuttings were evident. Chardonnay cuttings inoculated with L. theobromae isolates rarely developed spring growth and, when developed, young shoots, petioles, and leaves died back and dried out very rapidly. Chardonnay cuttings inoculated with D. seriata isolates developed healthy spring growth which did not differ from the control (Fig. 5a). Wedge-shaped cankers and necrotic tissue produced by both species were observed when cross sections were made in 1-yearold cuttings and green shoots, respectively (Fig. 5c, d, g, and h). Pycnidia were observed on both wood and green shoot surfaces in plants inoculated only with all L. theobromae isolates but not with the D. seriata isolates (Fig. 5i). L. theobromae and D. seriata isolates used in this study were pathogenic and caused longer basipetal than acropetal lesions in all treatments (Fig. 4). L. theobromae isolates from Mexico were more virulent and produced significantly longer basipetal lesions in all inoculated plants than those of D. seriata (Figs. 4 and Fig. 5a and f). D. seriata isolates produced substantially smaller lesions than those caused by L. theobromae in all inoculated 1-year-old cuttings and green shoots but still differed significantly in symptom expression from control treatments (Fig. 4). However, two D. seriata isolates (UCD1010BC and UCD1038BC) did not cause lesions in 1-year-old Chardonnay cuttings (Fig. 4A). L. theobromae caused significantly (F = 40.89, DF = 9, P < 0.0001) longer lesions (range from 301.1 to 338.3 mm) in 1-year-old Chardonnay cuttings than those in Thompson Seedless (range from 71.2 to 183.1 mm) (Fig. 4A and B). Lesions caused by L. theobromae in Thompson Seedless green shoots ranged from 58.6 to 85.4 mm and differed significantly (F = 5.1, DF = 9, P = 0.0254) from the lesions caused in Chardonnay (range from 20.9 to 45.5 mm) (Fig. 4C and D). D. seriata isolates from Mexico showed no significant differences (F = 0.35, DF = 9, P = 0.555) in the extent of vascular discoloration between cultivars in both 1-yearold cuttings and green shoots (Fig. 4A–D). Lesions caused by D. seriata in 1-year-old Chardonnay and Thompson Seedless cuttings varied from 21.8 to 50.9 and 36.8 to 49.6 mm, respectively. D. seriata caused lesions on green Chardonnay and Thompson Seedless shoots that ranged from 4.2 to 6.2 and 3.7 to 8.7 mm, respectively. L. theobromae was reisolated from infected tissue in both 1-year-old cuttings and green shoots in 100% of the samples. D. seriata was reisolated in 100% of the samples from 1-year-old cuttings and between 80 to 90% from green shoots. No Botryosphaeriaceae fungi were reisolated from the control treatments. DISCUSSION Botryosphaeriaceae spp. recently have been shown to be a more prevalent and important wood pathogen on grapevines than originally thought. In total, 14 different Botryosphaeriaceae spp. have been described associated with grapevine dieback from the most important grapegrowing areas of North and South America (2,14,21,24,27,38,45,46), Europe (1,20,25, 26,29,47), Africa (13,48), Asia (19), and Oceania (4,10,37,43,50). The present study has shown that two Botryosphaeriaceae spp., L. theobromae and D. seriata, also are associated with grapevine cankers in Mexico. Perennial cankers and grapevine dieback have been known to occur in Mexico since the late 1970s, when Eutypa dieback was associated with grapevine decline in old and abandoned vineyards in the grapegrowing areas of Coauhila-Durango and Aguascalientes (44). However, this association was based mainly on the discovery of E. lata perithecia in old pruning wounds, and no confirmation of the fungus isolated from declining vines and cankers was reported. Therefore, future field surveys and canker isolations from those grapevine areas may reveal the presence of other fungal pathogens (in addition to E. lata), such as species of Botryosphaeriaceae associated with the dieback symptoms reported in the past. Because Eutypa dieback of grapevines has been known and well studied for more than 30 years and because cankers caused by both E. lata and Botryosphaeriaceae spp. are indistinguishable, E. lata has been considered as the primary canker causing agent of grapes worldwide. Our surveys in Sonora and Baja California have shown cankers, dead spurs with lack of spring growth, and dead cordons as the primary symptoms on declining vines. Characteristic Eutypa dieback symptoms such as stunted shoots, shortened internodes, and chlorotic and abnormal growth of the leaves were not observed during our sample collections in either the Sonora or Baja California regions. Furthermore, L. theobromae and D. seriata were the main fungi isolated from canker lesions in the current study whereas E. lata was not recovered from any of the cankers collected. Cankers produced by E. lata are rarely observed in vineyards younger than 10 years old due to the slow movement of the fungus through the vascular system following infection of a pruning wound (9). In the current study, we isolated L. theobromae from cankers collected from vineyards 5 to 7 years old in the Sonora region. This result agrees with a previous study conducted by Leavitt, in which L. theobromae commonly was isolated from cankers on 5-year-old grapevines in Southern California (23). These results suggest that L. theobromae is a faster wood colonizer than E. lata. The dominance of Botryosphaeriaceae spp. and the absence of E. lata from grapevines in Baja California and Sonora may be due to climatic conditions. E. lata perithecia commonly are found in old vines from grape regions with an annual precipitation over 600 mm but rarely are found in areas with precipitation lower than 300 mm per year (9). Weather data recorded during the last 50 years show much lower annual precipitation in both the Ensenada (283 mm) and Hermosillo (240 mm) surveyed grapevine areas than in Aguascalientes (537 mm), from which E. lata perithecia mainly were reported to occur in Mexico. Similarly, field surveys conducted throughout the warm and desert areas of Southern California and Western and Eastern Australia have reported isola- Fig. 3. Most equally parsimonious tree with bootstrap values obtained from the combined internal transcribed spacer, β-tubulin, and elongation factor-1 sequence data using 1,000 replicates generated in PAUP 4.0b10. Asterisks show Botryosphaeriaceae spp. from Vitis vinifera. Isolates from Mexico are indicated in bold. Plant Disease / April 2008 525 tion of Botryosphaeriaceae spp. but no E. lata from cankers and necrotic lesions (10,23,37,43,45). However, other environmental factors, the lack of many of the susceptible hosts to E. lata in desert areas, as well as the possibility that E. lata has not been yet introduced in Baja California and Sonora also could explain the dominance of Botryosphaeriaceae spp. in these regions. Morphological characteristics combined with analyses of DNA sequences allowed us to identify and characterize L. theobromae and D. seriata from grapevine cankers from Mexico. Colony and conidial morphology were the most helpful features with which to identify and distinguish L. theobromae from D. seriata. L. theobromae isolates used in this study grew much faster than those of D. seriata and were able to fully colonize an 85-mm-diameter petri plate in 48 h. Furthermore, isolates of L. theobromae showed a higher optimum growth temperature than those of D. seriata. These results are in agreement with a previous temperature study conducted with Botryosphaeriaceae isolates from California, in which L. theobromae and D. seriata isolates achieved maximum growth rates at 31 and 26°C, respectively (45). Conidial shape and color and the presence of septa and longitudinal striations were robust characteristics for identification and separation of L. theobromae from D. seriata. The utility of these morphological distinctions is in agreement with previous literature (23,32,43,45,50). Another important morphological characteristic for the identification of L. theobromae was the presence of aseptate paraphyses observed in immature pycnidia. This feature was reported in a recent study, in which aseptate paraphyses along with conidial size were the primary morphological characteristics to separate L. theobromae from other Lasiodiplodia spp. found in the tropics (7). DNA sequence comparisons allowed us to verify the morphological identification of L. theobromae and D. seriata from grapevines in Mexico. Results of the single as well as the combined ITS, β-tubulin, and EF1-α phylogenetic analyses clearly separated L. theobromae isolates from D. seriata isolates. The remaining Botryosphaeriaceae isolates with Diplodia-like anamorphs that were included in the analyses, such as D. mutila and D. corticola, clustered together within the D. seriata clade. In our study, L. theobromae from Mexico grouped together with L. theobromae isolates from the desert area known as the Coachella Valley in California, reinforcing the idea that this fungus is prevalent in regions with high temperatures (17,23,32,45). Furthermore, the similarity between L. theobromae sequences from grapevines in Mexico and California could support the hypothesis that this species was introduced accidentally in Mexico from California or vice versa. However, more intensive sampling and, perhaps, also more sensitive measures of interisolate relationships, such as microsatellites or vegetative compatibility studies, would be required to test this hypothesis. L. theobromae isolates from Mexico and California differed from isolates CMW9074 and CMW10130 from South Africa and isolate CBS124.13 from an unknown origin in the United States. Host and geographical differences could explain the variation in the DNA sequences between these isolates. In contrast, D. seriata isolates from Mexico grouped together with D. seriata isolates from other geographical regions and different hosts showing almost no variation in the DNA sequences. This result agrees with previous phylogenetic studies conducted with D. seriata isolates from California (45) and may explain the cosmopolitan distribution of D. seriata worldwide. Pathogenicity tests conducted in 1-yearold cuttings and green shoots of cvs. Chardonnay and Thompson Seedless confirmed that L. theobromae and D. seriata isolates from Mexico were pathogenic. In these tests, L. theobromae isolates produced much larger lesions than those of D. seriata in all inoculated 1-year-old cuttings and green shoots. These results are consistent with previous pathogenicity trials Fig. 4. Pathogenicity study of Lasiodiplodia theobromae and Diplodia seriata in 1-year-old cuttings and green shoots of cvs. Chardonnay and Thompson Seedless. Mean lesion length is based on 10 replicates per isolate. Means followed by different letters differ significantly (P < 0.05) according to Tukey’s test. Bars represent standard error of the mean. 526 Plant Disease / Vol. 92 No. 4 Fig. 5. Pathogenicity of Lasiodiplodia theobromae and Diplodia seriata on a, 1-year-old Chardonnay cuttings 8 weeks after inoculation (aI = L. theobromae UCD914SN, aII = D. seriata UCD1052BC, and aIII = control). White arrows show the point of inoculation. b, Vascular lesion caused by L. theobromae (UCD1060BC) in 1-year-old Chardonnay cutting 8 weeks after inoculation. Dark-brown lesion can be observed upward and downward from the point of inoculation causing the death of the wood and leaf dieback at the last internode. Canker continues growing basipetaly throughout the second internode as shown by the dashed white arrow. c, Cross-section in 1-year-old Chardonnay cutting showing a young wedge-shaped canker caused by D. seriata (UCD1052BC) 20 weeks after inoculations. Black arrow shows the edge of the canker. d, Cross-section in 1-year-old Chardonnay cutting showing the canker caused by L. theobromae (UCD919SN) 20 weeks after inoculations. Light-gray area shows dead wood caused by the total vascular colonization of the fungus. Dark-brown area shows the fungus still growing and colonizing healthy vascular tissue. e, Lesions caused in 1-year-old Thompson Seedless cuttings 20 weeks after inoculation (eI = L. theobromae UCD914SN, eII = D. seriata UCD1052BC, and eIII = control). White arrow shows black wood streaking caused by L. theobromae moving downward the point of inoculation. No lesions were observed on the noncolonized inoculated controls. f, Lesions caused on Thompson Seedless green shoots 10 days after inoculation (fI = L. theobromae UCD918SN, fII = D. seriata UCD1038BC, and fIII = control). g, Crosssection in a Thompson Seedless green shoot showing the necrotic tissue caused by L. theobromae (UCD1060BC) 10 days after inoculation. h, Cross-section in a Thompson Seedless green shoot showing a smaller necrotic lesion caused by D. seriata (UCD1052BC) 10 days after inoculation. i, Pycnidia of L. theobromae (UCD914SN) formed on Chardonnay green shoot surface 10 days after inoculation. Plant Disease / April 2008 527 conducted in California and South Africa, in which L. theobromae was shown to be one of the most virulent species tested on mature grapevine canes (23,48). However, differences in isolates of L. theobromae also have been reported from a recent study conducted in Western Australia, where only one of seven L. theobromae isolates produced lesions significantly different from the noninoculated controls (43). In the current study, 1-year-old Chardonnay cuttings were more susceptible to infection by L. theobromae than Thompson Seedless. The table-grape cv. Thompson Seedless is recommended in grape regions with more than 4,000 degree-days per year. Consequently, Thompson Seedless has long been planted in the warm and desert grape-growing areas of Southern California and Northern Mexico where L. theobromae commonly is found. Similar results were obtained in a pathogenicity study in Western Australia, in which L. theobromae isolates were avirulent or only weakly pathogenic in inoculated Red Globe mature canes (43). Red Globe is another table-grape cultivar widely planted in warm regions with more than 4,000 degree-days per year. Thompson Seedless green shoots were more susceptible to infection by L. theobromae than were Chardonnay shoots. The reason for this difference in susceptibility is still unknown. Furthermore, the lack of multicultivar Botryosphaeriaceae pathogenicity tests limits comparison of our results to those of previous studies. More field inoculations of diverse cultivars are needed to better characterize the range of susceptibilities. D. seriata isolates from Mexico were much less pathogenic than those of L. theobromae but still capable of causing larger lesions than were evident in the noninoculated controls. This result agrees with previous pathogenicity studies conducted in South Africa, the New South Wales region in Australia, Eastern Australia, Chile, and France, in which D. seriata has been reported to be pathogenic in grapevines (2,10,20,37,48). In contrast, D. seriata isolates from grapevines in Western Australia were found to be nonpathogenic (43). We found D. seriata isolates from Mexico to be more pathogenic on 1-yearold cuttings than on green shoots. Differences in pathogenicity and virulence also have been reported among D. seriata isolates in South Africa, Eastern Australia, and France (20,37,48). Furthermore, in France, Chile, and Lebanon, D. seriata has been associated with a disease called “Black dead arm” characterized by lightbrown wood streaking and red patches at the margin of leaves, with large areas of chlorosis and deterioration between the veins (2,11,20). The symptoms observed in Baja California and Sonora were not those associated with Black dead arm. This 528 Plant Disease / Vol. 92 No. 4 agrees with previous studies conducted in California and Portugal (28,45), which found that there were no foliar symptoms associated with this pathogen. Variable reports on the pathogenicity of D. seriata from different grape-growing regions throughout the world may reflect differences in (i) virulence of the isolate used, (ii) cultivar susceptibilities, (iii) inoculation methods, or (iv) incubation times, among other possibilities. Results from this study have confirmed L. theobromae and D. seriata as the primary causal agents of Bot canker of grapevines in Mexico, indicating the important role that these fungi can play in grapevine health, in general. ACKNOWLEDGMENTS We thank T. Michailides (Department of Plant Pathology, University of California, Kearney Agricultural Center) and T. Gordon (Department of Plant Pathology, University of California, Davis) for providing valuable advice on the writing of this manuscript; and M. Davis (Department of Plant Pathology, University of California, Davis) for his assistance in the phylogenetic analyses. LITERATURE CITED 1. 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