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This article is downloaded from http://researchoutput.csu.edu.au It is the paper published as: Author: W. M. Pitt, R. Huang, C. Steel and S. Savocchia Title: Pathogenicity and epidemiology of Botryosphaeriaceae species isolated from grapevines in Australia Journal: Australasian Plant Pathology ISSN: 0815-3191 1448-6032 Year: 2013 Volume: 42 Issue: 5 Pages: Abstract: Botryosphaeriaceae species are among the most common fungi isolated from grapevine (Vitis vinifera) cankers in Australia. Thirty-eight isolates comprising eight Botryosphaeriaceae species isolated from grapevine cankers throughout New South Wales and South Australia were used in a pathogenicity study on mature wood of 15-year-old Chardonnay grapevines. Experiments showed that all eight species were able to infect grapevines under field conditions causing vascular discoloration and/or staining of the wood (lesions). However, differences in pathogenicity were evident among strains and species (P<0.001). Neofusicoccum parvum and Lasiodiplodiatheobromae were the most pathogenic based on lesion length, followed byneofusicoccum australe, Botryosphaeria dothidea and Diplodia mutila and thereafterby Dothiorella viticola, Dothiorella iberica and Diplodia seriata, which were the least pathogenic. Growth rates also differed significantly among species depending ontemperature (P<0.001). Lasiodiplodia theobromae and B. dothidea grew optimally between 29 and 30- C, Diplodia and Neofusiccocum spp. between 25 and 27- C, anddothiorella spp. at temperatures between 22 and 24- C. Temperature-growth relationships correlated well with previous data on the prevalence and distribution of Botryosphaeriaceae species in Australian vineyards. Due to their pathogenicity,prevalence, distribution and tolerance to a wide range of environmental conditions, the Botryosphaeriaceae pose a significant threat to the Australian wine industry. DOI/URL: http://dx.doi.org/10.1007/s13313-013-0221-3 http://researchoutput.csu.edu.au/r/-?func=dbinjump-full&object_id=45261&local_base=gen01-csu01 Author Address: wpitt@csu.edu.au/ CRO Number: 45261

Manuscript Click here to download Manuscript: AUPP-D-13-00060_revised.docx 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Pathogenicity and epidemiology of Botryosphaeriaceae species isolated from grapevines in Australia W.M. Pitt *, R. Huang, C.C. Steel, S. Savocchia National Wine and Grape Industry Centre, School of Agricultural and Wine Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia. * Corresponding author: wpitt@csu.edu.au Keywords Grapevine trunk disease, grapevine canker, dieback, decline, Botryosphaeria canker, Eutypa, epidemiology. 1

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Abstract Botryosphaeriaceae species are among the most common fungi isolated from grapevine (Vitis vinifera) cankers in Australia. Thirty-eight isolates comprising eight Botryosphaeriaceae species isolated from grapevine cankers throughout New South Wales and South Australia were used in a pathogenicity study on mature wood of 15-year-old Chardonnay grapevines. Experiments showed that all eight species were able to infect grapevines under field conditions causing vascular discoloration and/or staining of the wood (lesions). However, differences in pathogenicity were evident among strains and species (P<0.001). Neofusicoccum parvum and Lasiodiplodia theobromae were the most pathogenic based on lesion length, followed by Neofusicoccum australe, Botryosphaeria dothidea and Diplodia mutila and thereafter by Dothiorella viticola, Dothiorella iberica and Diplodia seriata, which were the least pathogenic. Growth rates also differed significantly among species depending on temperature (P<0.001). Lasiodiplodia theobromae and B. dothidea grew optimally between 29 and 30 C, Diplodia and Neofusicoccum spp. between 25 and 27 C, and Dothiorella spp. at temperatures between 22 and 24 C. Temperature-growth relationships correlated well with previous data on the prevalence and distribution of Botryosphaeriaceae species in Australian vineyards. Due to their pathogenicity, prevalence, distribution and tolerance to a wide range of environmental conditions, the Botryosphaeriaceae pose a significant threat to the Australian wine industry. Introduction Worldwide, more than 20 species in the Botryosphaeriaceae are associated with the disease of grapevines known as Botryosphaeria dieback (Úrbez-Torres 2011), and in Australia 10 species are currently recognised including in addition to the type, Botryosphaeria dothidea, species of Diplodia, Dothiorella, Lasiodiplodia and Neofusicoccum (Pitt et al. 2010, Wunderlich et al. 2011). Botryosphaeria dieback arises when fungi invade the vascular system of the vine, gaining entry via pruning and reworking wounds or other exposed surfaces. Symptoms include perennial cankers, shoot and branch dieback, bud necrosis, graft failure and fruit rot, but wedge shaped lesions in the trunks and cordons of infected vines and a lack of vegetative growth are most characteristic of the disease (Leavitt 1990, Phillips 2002, van Niekerk et al. 2006, Úrbez-Torres 2011). The pathogenicity of Botryosphaeriaceae species on grapevines was until recently, the subject of considerable debate, due in large part to a lack of detailed information on the 2

52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 pathogenicity and epidemiology of these fungi, which is thought by many to be greatly influenced by climate (Leavitt 1990, Úrbez-Torres et al. 2006, van Niekerk et al. 2006, Pitt et al. 2010). While recent studies conducted both in Australia and overseas have shown many of these species to be pathogenic to grapevines, much of this work has been performed on excised wood, green shoots or potted vines under laboratory or glasshouse conditions (Phillips 1998, Castillo-Pando et al. 2001, Larignon et al. 2001, Taylor et al. 2005, Savocchia et al. 2007, Úrbez-Torres et al. 2008, Úrbez-Torres et al. 2009, Amponsah et al. 2011), and to date, few studies have examined the effect of these fungi on mature vines in the field (Leavitt 1990, van Niekerk et al. 2004, Wood and Wood 2005, Burruano et al. 2008, Úrbez-Torres and Gubler 2009). Moreover, less than half comprise multiple species, and none to date have been conducted with Australian isolates under local conditions. The objectives of this study were to determine the pathogenicity under field conditions, of eight Botryosphaeriaceae species on the wood of mature grapevines. Additionally, growth rates of the various fungi were assessed under laboratory conditions to determine cardinal temperatures for each species. The pathogenicity and temperature-growth relationships of Botryosphaeriaceae species are discussed in context with previous data on their prevalence and distribution in Australian vineyards. Materials and Methods Fungal strains Forty-one fungal strains isolated from a range of grapevine cultivars and locations throughout New South Wales (NSW) and South Australia (SA) were used in a pathogenicity trial conducted at the National Wine and Grape Industry Centre (NWGIC), Wagga Wagga, NSW, Australia (Table 1). Species included B. dothidea, Diplodia mutila, Diplodia seriata, Dothiorella iberica, Dothiorella viticola, Lasiodiplodia theobromae, Neofusicoccum australe, Neofusicoccum parvum and Eutypa lata. All fungal strains used in the study are stored on potato dextrose agar (PDA) at 4 C and are maintained in the collection at the NWGIC and at the Plant Pathology Herbarium (DAR), Orange Agricultural Institute, Department of Primary Industries (DPI), Orange, NSW, Australia. GenBank ITS accession numbers are also provided for each strain used in the study as an assurance of identity (Table 1). Pathogenicity studies In October 2010, wounds were created in the trunks of 15-year-old Chardonnay grapevines approximately 30 cm below the crown using a sterile 10-mm-diameter drill bit. 3

86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 Plugs (3 5-mm-diameter) from the margins of 7 day-old colonies of E. lata and Botryosphaeriaceae species grown on potato dextrose agar supplemented with 100 mg/liter of streptomycin sulfate (PDA-strep), were inserted into the wounds and sealed with woodfilling putty (Selleys Australia, Padstow, NSW, Australia). Sterile non-colonised plugs of PDAstrep were used as controls. In September 2012, 24 months after inoculation, a 50-cm portion of the trunk comprising the inoculation point and 25-cm of tissue above and below the wound, was removed from each grapevine. Trunks were sectioned longitudinally in half through the point of inoculation to expose any staining or discoloration of the wood (lesions). The extent to which each fungal strain was able to cause disease was then determined by cumulating the lengths of lesions above and below the inoculation point. To fulfil Koch s postulates, and to determine the degree to which fungi colonised grapevines, small pieces of wood (~2 mm 2 ) from the edge of each lesion and at 25-mm intervals up to 150 mm above and below the inoculation point were removed from each trunk, surface sterilised for 2 minutes in 2.5% NaOCl, rinsed in sterile distilled water and transferred to PDA-strep. Cultures were incubated under fluorescent light for 7 to 10 days and identified using colony morphology (Carter 1991, Úrbez-Torres et al. 2006, Úrbez-Torres et al. 2007). The furthest distance each fungal strain was reisolated above and below the point of inoculation was then recorded and cumulated to determine total colonisation distances. Growth rates Cardinal temperatures for growth were also determined for each of the 38 Botryosphaeriaceae strains. Five-mm-diameter plugs from the margins of actively growing 4- day-old cultures of each strain were transferred to PDA-strep and incubated in the dark at 5, 10, 15, 20, 25, 30, 35 and 40 C. Colony diameters were measured at daily intervals and growth rates (mm/day) for each strain determined at each temperature. Regression curves were fitted to values of daily growth rate versus temperature, and optimum growth temperatures established for each strain based on relationships described by a third order polynomial (Sánchez et al. 2003). For species comparisons, growth rates at each temperature were first obtained from the mean of the constituent fungal strains, after which curves were fitted as indicated. Data analysis The pathogenicity trial was conducted using a randomised complete block design with 6 blocks (replicates) comprising a total of 42 treatments, including controls. Data was 4

120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 skewed, such that a log transformation was introduced to normalise data and eliminate heteroscedasticity. Bartlett s test was used to check for homogeneity of variance across blocks (to test for replicate effects). One-way analysis of variance (ANOVA) was used to assess differences in lesion lengths among fungal strains and species, with means separated using Tukey's test (P=0.05). Means were back-transformed to the original scale (mm). After a preliminary experiment was conducted to investigate temperature-growth relationships of the fungal strains, temperatures 5 and 40 C were omitted from the study. Duplicate trials comprising three plates per strain were then conducted at temperatures of 10, 15, 20, 25, 30 and 35 C. Data was skewed, such that a log transformation was introduced to normalise data and eliminate heteroscedasticity. Bartlett s test was used to check for homogeneity of variance across trials. ANOVA was used to compare differences in growth rates between strains, species and temperatures as main effects, and between species at each temperature, with means separated using Tukey's test (P=0.05). All statistical analyses were performed using GenStat (Version 14, VSN International Ltd., Hemel Hempstead, United Kingdom). Results Pathogenicity tests Data was normalised by a log transformation (P=0.123), and variances were homogeneous across blocks (replicates) facilitating pooling of data (P=0.341). Twenty-four months after inoculation of Botryosphaeriaceae species into the trunks of 15-year-old Chardonnay grapevines, longitudinal sectioning revealed widespread vascular staining and discoloration of the wood (lesions) proximal to, and at varying distances above and below the wound (Figure 1). Mean lesion lengths ranged from 59 to 202 mm. While significant differences were evident among strains (P<0.001), with the exception of one strain of N. australe, differences were almost solely between, and not within species (P=0.05; Table 2). However, due to the variability among replicates, many strains (34 of 41) produced lesions of a similar length, and statistically only about one third of the strains (14 of 41) produced lesions that were significantly longer than those on controls (P=0.05). When data were pooled, there were significant differences among the species (P<0.001), with mean lesion lengths ranging from 76 mm for D. seriata to 165 mm for N. parvum. While lesions produced by all Botryosphaeriaceae species as well as E. lata were significantly longer than those on controls, fewer differences were apparent among species, with seven of the nine producing lesions of a similar length (P=0.05; Figure 2). 5

154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 All eight Botryosphaeriaceae species and E. lata were also reisolated from grapevines, with all strains being recovered and identified from inoculated wood. Botryosphaeriaceae species were recovered solely from the margins of necrotic tissues, and were not reisolated from asymptomatic tissue in advance of lesion borders. In contrast, strains of E. lata were consistently recovered from necrotic lesions as well as symptomless tissue several centimetres ahead of the disease margin. Mean lesion lengths for the three strains of E. lata were 69, 74 and 109 mm, respectively, while colonisation distances for the same three strains equated to 121, 117 and 158 mm. When the means for lesion length were subtracted from those of colonisation distance all three strains had cumulated colonisation distances that exceeded lesion length by more than 40 mm, although the furthest distance that any strain of E. lata was isolated from wood in advance of the lesion was 67.5 mm. Growth rates Data was normalised by a log transformation (P=0.166), and variances were homogeneous across trials facilitating pooling of data (P=0.110). Botryosphaeriaceae species grew over a range of temperatures from 5 to 40ºC (Table 3). However, only Do. iberica and Do. viticola exhibited any growth at 5ºC, and growth at 40ºC was limited to L. theobromae and several strains of B. dothidea. Temperatures for each species at which the maximum radial growth were obtained were 29.4 for L. theobromae, 29.7ºC for B. dothidea, 25.8ºC for D. mutila, 26.2, 26.6 and 26.8ºC for N. australe, D. seriata and N. parvum, and 22.9 and 24.0ºC for Do. iberica and Do. viticola, respectively (Figure 3). While differences among strains were not significant, within nor between species (P=0.198), when data were pooled, growth rates differed significantly depending on temperature and fungal species (P<0.001), with 25ºC being significantly more, and 10ºC significantly less, favorable for growth than the other temperatures in the study (P=0.05). Lasiodiplodia theobromae had the highest growth rate and Do. iberica the least, although differences among many species were not statistically significant (P=0.05). When interactions between species and temperature were investigated differences were pronounced at extremes of temperature, such that the growth rates of Do. iberica and Do. viticola at 10ºC and B. dothidea and L. theobromae at 35ºC, were significantly greater than all other species tested (P=0.05). However, at 15, 20, 25 and 30ºC differences between species were not significant. Discussion 6

188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 In this study, all eight Botryosphaeriaceae species including B. dothidea, D. mutila, D. seriata, Do. iberica, Do. viticola, L. theobromae, N. australe and N. parvum acted as pathogens when inoculated into the wood of 15-year-old Chardonnay grapevines. In each case inoculations produced necrotic lesions at sites of infection that were significantly longer than those on controls, and fungi were reisolated from the margins of developing lesions. Neofusicoccum parvum along with N. australe and L. theobromae produced the longest lesions and were among the most pathogenic of the eight species tested. This confirmed reports by Úrbez-Torres and Gubler (2009) who consistently found these species to be among the most aggressive of nine tested in the United States, and also supported virulence rankings Úrbez-Torres (2011) recently proposed for the Botryosphaeriaceae, in which Neofusicoccum and Lasiodiplodia spp. were categorised as highly virulent. These species were originally described from kiwifruit (Actinidia deliciosa) (Pennycook and Samuels 1985), wattle (Acacia sp.) (Slippers et al. 2004), and cacao (Theobroma cacao) (Patouillard and De Lagerheim 1892), but as pathogens of grapevines did not become prominent until the 1990s and 2000s when cankers and episodes of wood dieback began to emerge from the United States and Europe (Leavitt 1990, Phillips 1998, Phillips 2002), and thereafter from South Africa (van Niekerk et al. 2004), Australia (Taylor et al. 2005, Wood and Wood 2005) and other regions (Úrbez-Torres et al. 2008). Likewise, we found B. dothidea and Diplodia spp. to be moderately pathogenic in comparison to the more aggressive species cited above, and this too was supported by the virulence rankings proposed by Úrbez-Torres (2011) for these species, as well as reports by other researchers who also described them as moderately pathogenic (Phillips 1998, Taylor et al. 2005, Luque et al. 2009). Yet, while B. dothidea and D. mutila are recognised pathogens, notorious as the agents of black dead arm in the vineyards of Hungary and France (Lehoczky 1974, Larignon and Dubos 2001), D. seriata is perhaps best known for its controversial status as a pathogen of grapevines. For example, Luque et al. (2009) and van Niekerk et al. (2004) considered the fungus weakly pathogenic, while Phillips (1998) believed it to be a secondary invader of wounds previously infected by more pathogenic species. Still, other reports tell of dark lesions on vines inoculated with the fungus (Castillo-Pando et al. 2001, Larignon et al. 2001), and in recent studies in California, Úrbez-Torres and Gubler (2009) attached a moderately pathogenic status to the fungus. Finally, Úrbez-Torres (2011) considered Dothiorella spp. to be only weakly virulent. In Australia, Do. viticola (=Spencermartinsia viticola) and Do. iberica represent fairly recent discoveries (Pitt et al. 2010), and along with Dothiorella sarmentorum (Martin and Cobos 7

222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 2007, Gramaje et al. 2009) and Dothiorella americana (Úrbez-Torres et al. 2012), are the only species in this genus currently associated with grapevine decline. Described by Luque et al. (2005) and Phillips et al. (2005) respectively, Do. viticola and Do. iberica were first reported to be pathogenic to grapevines by Úrbez-Torres et al. (2007), causing vascular discoloration and necrotic lesions on 15-year-old Zinfandel in California, a status the authors then confirmed in subsequent studies, where the two species were deemed the least pathogenic of nine tested (Úrbez-Torres and Gubler 2009). In this study, Do. viticola and Do. iberica were also among the least pathogenic of those tested, but due to small differences between species, along with B. dothidia and Diplodia spp. they were considered moderately rather than weakly pathogenic. This study also confirmed the pathogenicity of E. lata to grapevines, with this species being similar in virulence to B. dothidea and species of Diplodia and Dothiorella based on lesion lengths. Eutypa lata is well known as a pathogen of grapevines and the causal agent of Eutypa dieback (Carter 1991), and in this study was categorised as moderately or weakly pathogenic. This was slightly different to results of Úrbez-Torres and Gubler (2009), who found that E. lata caused a similar level of disease as L. theobromae, B. dothidea and species of Neofusicoccum on cordons of Red Globe and Shiraz. However, in contrast to the Botryosphaeriaceae, which in this study were reisolated solely from the margins of developing lesions, strains of E. lata were frequently recovered from asymptomatic or otherwise healthy wood several centimetres in advance of disease symptoms. The ability of the fungus to colonise wood ahead of the disease front is thought to result, at least in part, from the production of phytotoxins, that may allow the fungus to adopt a hemibiotrophic lifestyle, and has been observed previously in E. lata and other diatrypaceous species (Sosnowski et al. 2007, Pitt et al. 2013). While lesion length measurements did not take this into account, when considered in terms of colonisation, all three strains of E. lata were similar in pathogenicity to L. theobromae. While Botryosphaeriaceae species are found in all of the major viticultural regions of the world, the prevalence and distribution of species infecting grapevines is thought by many to be greatly influenced by climatic conditions (Leavitt 1990, Úrbez-Torres et al. 2006, van Niekerk et al. 2006, Pitt et al. 2010). In this study L. theobromae and B. dothidea had optimal growth temperatures close to 30 C, similar to that reported by Pennycook and Samuels (1985) and Úrbez-Torres et al. (2006). In Australia these species were quite rare and were predominantly associated with grapevines grown in the hotter dryer regions of northern NSW and Western Australia (WA), but together accounted for less than 3% of the total number of 8

256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 specimens collected in four surveys (Taylor et al. 2005, Pitt et al. 2010, Qiu et al. 2011, Wunderlich et al. 2011). Similarly, Dothiorella spp., were few in number, accounting for less than 7% of specimens, but were absent in WA (Taylor et al. 2005) and most commonly isolated from grapevines grown in the cooler climate regions of NSW and SA (Pitt et al. 2010, Wunderlich et al. 2011). Dothiorella viticola and Do. iberica possessed optimal growth temperatures between 22 and 24 C, the lowest of the eight Botryosphaeriaceae species tested, and had higher growth rates than most other species at 10, 15 and 20 C. While no prior growth rate studies appear to have been conducted on these species, D. sarmentorum, a similar fungus found on grapevines in Spain (Martin and Cobos 2007, Gramaje et al. 2009), and commonly associated with cankers of cork oak (Quercus suber) was also shown to grow optimally in this range, with an ideal temperature of ~22 C (Sánchez et al. 2003). The prevalence of Dothiorella spp. in cooler climate regions thus appears well supported by recent growth rate data. In contrast, Diplodia and Neofusicoccum spp. were the most widely distributed of the eight species isolated by Pitt et al. (2010) in surveys across NSW and SA, and were found in all or most regions. Diplodia seriata was by far the most prevalent, accounting for nearly 80% of isolates collected, and was also the most isolated of all species found by Qiu et al. (2011) in the Hunter Valley and Mudgee regions of NSW and by Taylor et al. (2005) in WA, where this species accounted for 70% and 56% of the total number of isolates, respectively. While D. mutila was also found in most regions of NSW and SA, albeit in fewer numbers, Neofusicoccum spp. were also found in most regions but were less prevalent and accounted for only 4, 16 and 41% of the total number of specimens collected by Pitt et al. (2010), Qiu et al. (2011) and Taylor et al. (2005), respectively. In growth rate studies, species of Diplodia and Neofusicoccum grew optimally at moderate temperatures in the range 25 to 27 C, similar to that reported by Pennycook and Samuels (1985), Sánchez et al. (2003) and Úrbez-Torres et al. (2006), which may help to explain why these species are so prevalent and occur over such large geographic ranges. Why Neofusicoccum spp. are found in lesser numbers than Diplodia however, is unknown as both species appear suited to similar climates. However, dark-spore species may, through the production of melanin, be more robust than their hyaline counterparts (Durrell 1964). Currently, management strategies for Botryosphaeria dieback and other grapevine trunk diseases such as Eutypa dieback rely heavily on remedial surgery to remove infected 9

289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 wood and inoculum sources from the vineyard (Úrbez-Torres 2011). However, due to the propensity of E. lata and other diatrypaceous species to colonise grapevine tissues ahead of disease symptoms (Sosnowski et al. 2007, Pitt et al. 2013), recommendations advocate the removal of an additional 10 cm of healthy tissue in advance of the disease margin (Sosnowski et al. 2007). While this did not appear to be necessary for grapevines infected with Botryosphaeriaceae species, our results confirm that this recommendation would be sufficient for the management of E. lata considering the distance that strains of the fungus colonised grapevine wood ahead of the disease margin. The use of fungicides and paints to protect pruning wounds against infection has also been successful in reducing the incidence both of Botryosphaeria and Eutypa dieback (Sosnowski et al. 2008, Rolshausen et al. 2010, Pitt et al. 2012, Diaz and Latorre 2013). However, recent studies to evaluate fungicides for the control of Botryosphaeriaceae species in vitro have shown that not all fungicides are equally effective against all species (Bester et al. 2007, Amponsah et al. 2012, Pitt et al. 2012), and greater knowledge of the epidemiology of these fungi including their prevalence and distribution may improve efforts to develop more effective control strategies. For instance, while strains of L. theobromae and B. dothidea have been shown to be highly pathogenic to grapevines (Phillips 1998, Burruano et al. 2008, Úrbez-Torres et al. 2008, Úrbez-Torres and Gubler 2009), in Australia these species were quite rare and in surveys, occurred within a narrow geographic range associated with high temperatures (Taylor et al. 2005, Wood and Wood 2005, Pitt et al. 2010, Qiu et al. 2011). Hence, despite their virulence, they likely pose less risk to grapevines in this country than to those in other parts of the world where climatic conditions may be more favourable for their growth. In contrast, Diplodia and Dothiorella were ubiquitous, and Australian climatic conditions clearly favoured the growth of these species over some of their more aggressive counterparts (Pitt et al. 2010). Could these species circumvent their limited virulence through shear prevalence and distribution? Regardless, future studies will continue to evaluate promising agents for the control of Botryosphaeria dieback, but given this knowledge, perhaps we might best focus our attention on the management of the most prevalent rather than pathogenic of species. Acknowledgements This work was funded by the Winegrowing Futures Program, a joint initiative between the NWGIC and the Grape and Wine Research and Development Corporation (GWRDC). We thank Renaud Travadon for providing ITS sequence data for strains of E. lata. 10

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377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 24. Pitt WM, Trouillas FP, Gubler WD, Savocchia S, Sosnowski MR (2013) Pathogenicity of diatrypaceous fungi on grapevines in Australia. Plant Disease (In press). 25. Qiu Y, Steel CC, Ash GJ, Savocchia S (2011) Survey of Botryosphaeriaceae associated with grapevine decline in the Hunter Valley and Mudgee grape growing regions of New South Wales. Australasian Plant Pathology 40: 1 11. 26. Rolshausen PE, Úrbez-Torres JR, Rooney-Latham S, Eskalen A, Smith RJ, Gubler WD (2010) Evaluation of pruning wound susceptibility and protection against fungi associated with grapevine trunk diseases. American Journal of Enology and Viticulture 61: 113 119. 27. Sánchez ME, Venegas J, Romero MA, Phillips AJL, Trapero A (2003) Botryosphaeria and related taxa causing oak canker in southwestern Spain. Plant Disease 87: 1515 1521. 28. Savocchia S, Steel CC, Stodart BJ, Somers A (2007) Pathogenicity of Botryosphaeria species from declining grapevines in sub tropical regions of Eastern Australia. Vitis 46: 27 32. 29. Slippers B, Fourie G, Crous PW, Coutinho TA, Wingfield BD, Wingfiled MJ (2004) Multiple gene sequences delimit Botryosphaeria australis sp. nov. from B. lutea. Mycologia 96: 1028 1039. 30. Sosnowski MR, Creaser M, Wicks T, Lardner R, Scott ES (2008) Protection of grapevine pruning wounds from infection by Eutypa lata. Australian Journal of Grape and Wine Research 14:134 142. 31. Sosnowski MR, Lardner R, Wicks TJ, Scott ES (2007) The influence of grapevine cultivar and isolate of Eutypa lata on wood and foliar symptoms. Plant Disease 91: 924 931. 32. Taylor A, Hardy GEStJ, Wood P, Burgess T (2005) Identification and pathogenicity of Botryosphaeria species associated with grapevine decline in Western Australia. Australasian Plant Pathology 34: 187 195. 33. Úrbez-Torres JR (2011) The status of Botryosphaeriaceae species infecting grapevines. Phytopathologia Mediterranea 50: S5 S45. 34. Úrbez-Torres JR, Adams P, Kamas J, Gubler WD (2009) Identification, incidence, and pathogenicity of fungal species associated with grapevine dieback in Texas. American Journal of Enology and Viticulture 60: 497 507. 35. Úrbez-Torres, JR, Gubler WD (2009) species isolated from grapevine cankers in California. Plant Disease 93: 584 592. 36. Úrbez-Torres JR, Gubler WD, Luque J (2007) First Report of Botryosphaeria iberica and B. viticola associated with grapevine decline in California. Plant Disease 91: 772. 37. Úrbez-Torres JR, Leavitt GM, Guerrero JC, Guevara J, Gubler WD (2008) Identification and pathogenicity of Lasiodiplodia theobromae and Diplodia seriata, the causal agents of bot canker disease of grapevines in Mexico. Plant Disease 92: 519 529. 38. Úrbez-Torres JR, Leavitt GM, Voegel TM, Gubler WD (2006) Identification and distribution of Botryosphaeria species associated with grapevines cankers in California. Plant Disease 90: 1490 1503. 39. Úrbez-Torres JR, Peduto F, Striegler RK, Urrea-Romero KE, Rupe JC, Cartwright RD, Gubler WD (2012) Characterization of fungal pathogens associated with grapevine trunk diseases in Arkansas and Missouri. Fungal Diversity 52: 169 189. 40. van Niekerk JM, Crous PW, Groenewald JZ, Fourie PH, Halleen F (2004) DNA phylogeny, morphology and pathogenicity of Botryosphaeria species on grapevines. Mycologia 96: 781 798. 41. van Niekerk JM, Fourie PH, Halleen F, Crous PW (2006) Botryosphaeria spp. as grapevine trunk disease pathogens. Phytopathologia Mediterranea 45: S43 S54. 42. Wood PM, Wood CE (2005) Cane dieback of dawn seedless table grapevines (Vitis vinifera) in Western Australia caused by Botryosphaeria rhodina. Australasian Plant Pathology 34: 393 395. 43. Wunderlich N, Ash GJ, Steel CC, Raman H, Savocchia S (2011) Association of Botryosphaeriaceae grapevine trunk disease fungi with the reproductive structures of Vitis vinifera. Vitis 50: 89 96. 12

Table 1. Details of fungi isolated from grapevines (V. vinifera) and used in pathogenicity and growth rate studies x. Species Strain Origin Cultivar Collector Collection date Herb. Acc. y GenBank ITS Botryosphaeria dothidea BMV14 Mittagong, NSW Sauvignon Blanc W.M. Pitt Dec. 2007 DAR79239 FJ176567 B. dothidea BMV15 Mittagong, NSW Sauvignon Blanc W.M. Pitt Dec. 2007 DAR79240 FJ176568 B. dothidea GE14 Young, NSW Chardonnay W.M. Pitt Sep. 2007 DAR79241 FJ176569 B. dothidea TS16 Hunter Valley, NSW Chardonnay A. Somers Mar. 2007 DAR79242 FJ176570 B. dothidea VT1 Canowindra, NSW Chardonnay W.M. Pitt Sep. 2007 DAR79244 FJ176572 Diplodia mutila FF18 Adelaide Hills, SA Shiraz W.M. Pitt Oct. 2007 DAR79137 EU919686 D. mutila PG15 Wagga Wagga, NSW Tinto Cao W.M. Pitt Oct. 2007 DAR79131 EU919687 D. mutila VP11 Cowra, NSW Chardonnay W.M. Pitt Sep. 2007 DAR79132 EU919688 D. mutila TSE17 Jaspers Brush, NSW Malbec W.M. Pitt Dec. 2007 DAR79138 EU919690 D. mutila CG15 Tumbarumba, NSW Pinot Noir W.M. Pitt Nov. 2007 DAR79135 EU919691 Diplodia seriata II12 Barossa Valley, SA Shiraz W.M. Pitt Oct. 2007 DAR79238 FJ176560 D. seriata J10 Eden Valley, SA Chardonnay W.M. Pitt Jul. 2007 DAR79140 FJ176561 D. seriata DO4 Murrumbateman, NSW Shiraz W.M. Pitt Nov. 2007 DAR79133 FJ176562 D. seriata ME21 Mittagong, NSW Sauvignon Blanc W.M. Pitt Dec. 2007 DAR79237 FJ176564 D. seriata YVW2 Murrumbateman, NSW Chardonnay W.M. Pitt Nov. 2007 DAR79142 FJ176566 Dothiorella iberica J4 Eden Valley, SA Chardonnay W.M. Pitt Jul. 2007 DAR78992 EU768874 Do. iberica J24 Eden Valley, SA Chardonnay W.M. Pitt Jul. 2007 DAR78991 EU768875 Do. iberica L5 Loxton, SA Cabernet Sauvignon W.M. Pitt Jul. 2007 DAR78993 EU768876 D. iberica M21 Barossa Valley, SA Gamay W.M. Pitt Jul. 2007 DAR78994 EU768877 Do. iberica N19 Adelaide Hills, SA Chardonnay W.M. Pitt Jul. 2007 DAR78995 EU768878 Dothiorella viticola CV11 Orange, NSW Sauvignon Blanc W.M. Pitt Sep. 2007 DAR78873 EU603288 D. viticola K16 Eden Valley, SA Riesling W.M. Pitt Jul. 2007 DAR78866 EU603291 Do. viticola N23 Adelaide Hills, SA Chardonnay W.M. Pitt Jul. 2007 DAR78872 EU603292 Do. viticola L19 Loxton, SA Cabernet Sauvignon W.M. Pitt Jul. 2007 DAR78870 EU603293 Do. viticola M11 Barossa Valley, SA Gamay W.M. Pitt Jul. 2007 DAR78868 EU603294 Lasiodiplodia theobromae MW643 Griffith, NSW Chardonnay M.A. Weckert Nov. 2006 DAR82122 KC825336 L. theobromae C2 Port Macquarie, NSW Pinot Noir W.M. Pitt Feb. 2007 DAR79507 FJ422895 L. theobromae G31A Hunter Valley, NSW Cabernet Sauvignon Y. Qiu Nov.2005 DAR77824 GU944821 Neofusicoccum australe DNW8 Tenterfield, NSW Mataro W.M. Pitt Dec. 2007 DAR79501 FJ422897 N. australe ME10 Mittagong, NSW Sauvignon Blanc W.M. Pitt Dec. 2007 DAR79504 FJ422899 13

N. australe VP13 Cowra, NSW Chardonnay W.M. Pitt Sep. 2007 DAR79506 FJ422902 N. australe SDW4 Mittagong, NSW Shiraz W.M. Pitt Dec. 2007 DAR79505 FJ422903 N. australe FF10 Adelaide Hills, SA Shiraz W.M. Pitt Oct. 2007 DAR79502 FJ422904 Neofusicoccum parvum B12 Port Macquarie, NSW Cabernet Sauvignon W.M. Pitt Feb. 2007 DAR78997 EU919694 N. parvum B19A Port Macquarie, NSW Cabernet Sauvignon W.M. Pitt Feb. 2007 DAR78998 EU919695 N. parvum B7A Port Macquarie, NSW Cabernet Sauvignon W.M. Pitt Feb. 2007 DAR78999 EU919700 N. parvum B7B Port Macquarie, NSW Cabernet Sauvignon W.M. Pitt Feb. 2007 DAR79000 EU919701 N. parvum TS24A Hunter Valley, NSW Chardonnay A. Somers Mar. 2007 DAR82123 EU919705 Eutypa lata z B003 Barossa Valley, SA Shiraz M.L. Creaser 2000 DAR79088 TBA E. lata WB052 Eden Valley, SA Shiraz M.R. Sosnowski 2003 DAR82124 TBA E. lata OR039 Coonawarra, SA Shiraz M.R. Sosnowski 2003 DAR82125 TBA Control Ctrl - - - - - - x Growth rate studies were not conducted for strains of E. lata. y Herbarium accession number (Plant Pathology Herbarium (DAR), Orange Agricultural Institute, DPI, Orange, NSW, Australia). z Isolates provided by Mark Sosnowski, South Australian Research and Development Institute (SARDI). 14

Table 2. Mean lesion lengths caused by Botryosphaeriaceae species and Eutypa lata 24 months after inoculation of 15-year-old Chardonnay grapevines at the National Wine and Grape Industry Centre, Wagga Wagga, NSW, Australia; October 2010 to September 2012. Species Strain Mean lesion length (mm) y Reisolation z Botryosphaeria dothidea TS16 (88.9 ) 104.1 ( 121.8) a-h 6 B. dothidea GE14 (70.2 ) 89.7 ( 114.5) b-i 6 B. dothidea BMV14 (64.3 ) 88.9 ( 123.0) b-i 5 B. dothidea VT1 (58.2 ) 83.5 ( 119.8) b-i 6 B. dothidea BMV15 (68.1 ) 77.9 ( 89.0) c-i 6 Diplodia mutila CG15 (96.4 ) 113.1 ( 132.8) a-h 5 D. mutila PG15 (70.0 ) 103.8 ( 153.8) a-h 5 D. mutila TSE17 (70.4 ) 81.7 ( 94.9) c-i 5 D. mutila FF18 (56.7 ) 70.7 ( 88.1) f-i 4 D. mutila VP11 (45.6 ) 68.9 ( 104.1) g-i 6 Diplodia seriata J10 (70.4 ) 82.7 ( 97.2) b-i 5 D. seriata YVW2 (60.4 ) 76.7 ( 97.5) c-i 6 D. seriata DO4 (64.5 ) 76.2 ( 89.9) c-i 6 D. seriata II12 (59.5 ) 73.0 ( 89.7) e-i 6 D. seriata ME21 (54.0 ) 71.5 ( 94.7) e-i 5 Dothiorella iberica J24 (67.0 ) 101.5 ( 153.7) a-h 5 Do. iberica M21 (61.2 ) 83.5 ( 114.1) b-i 6 Do. iberica N19 (61.6 ) 75.5 ( 92.5) d-i 6 Do. iberica L5 (47.3 ) 70.1 ( 103.9) f-i 4 Do. iberica J4 (48.1 ) 65.9 ( 90.3) g-i 5 Dothiorella viticola N23 (84.0 ) 90.0 ( 96.5) b-i 6 Do. viticola CV11 (70.9 ) 88.3 ( 110.1) b-i 6 Do. viticola M11 (65.5 ) 82.2 ( 103.1) c-i 4 Do. viticola L19 (63.3 ) 79.9 ( 100.8) c-i 5 Do. viticola K16 (66.4 ) 76.6 ( 88.3) c-i 5 Lasiodiplodia theobromae G31A (132.4 ) 149.1 ( 167.9) a-e 5 L. theobromae MW643 (122.9 ) 140.5 ( 160.6) a-g 6 L. theobromae C2 (70.9 ) 83.1 ( 97.5) b-i 6 Neofusicoccum australe SDW4 (88.1 ) 123.4 ( 172.7) a-g 5 N. australe DNW8 (74.5 ) 115.9 ( 180.5) a-h 6 N. australe ME10 (62.2 ) 87.7 ( 123.5) b-i 6 N. australe FF10 (71.3 ) 84.4 ( 100.1) b-i 6 N. australe VP13 (48.7 ) 58.8 ( 70.9) hi 5 Neofusicoccum parvum B7A (154.3 ) 202.0 ( 264.4) a 6 N. parvum B12 (146.8 ) 172.4 ( 202.5) ab 6 N. parvum B19A (118.9 ) 158.6 ( 211.7) a-c 6 N. parvum TS24A (141.3 ) 153.2 ( 166.1) a-d 6 N. parvum B7B (103.0 ) 146.0 ( 206.9) a-f 6 Eutypa lata B003 (80.9 ) 103.9 ( 133.5) a-h 6 E. lata OR039 (48.1 ) 68.2 ( 96.7) g-i 6 E. lata WB052 (59.4 ) 68.2 ( 78.3) g-i 6 Control Ctrl (35.8 ) 43.7 ( 53.2) i 0 y Mean lesion lengths, with upper and lower 95% confidence intervals in parenthesis. All values are back-transformed to the original scale (mm). Means followed by the same letters are not significantly different according to Tukey s test (P=0.05). z Number of samples from which fungi were reisolated out of 6 replicates. 15

Table 3. Cardinal temperatures of Botryosphaeriaceae species used in pathogenicity studies. Species Strain Growth at 5 C w Growth at 10 C x Growth at 35 C x Growth at 40 C w Growth Opt. C x R 2 Botryosphaeria dothidea GE14 y + slight 30.2 0.9295 z B. dothidea VT1 + 29.0 0.8999 B. dothidea BMV15 + slight 30.4 0.9494 B. dothidea BMV14 + slight 30.1 0.9680 B. dothidea TS16 + slight 30.3 0.9440 Diplodia mutila FF18 slight slight 25.9 0.9655 D. mutila PG15 slight 26.3 0.9006 D. mutila VP11 slight 25.9 0.9540 D. mutila TSE17 slight 26.1 0.9514 D. mutila CG15 slight slight 25.4 0.9436 Diplodia seriata J10 + 26.4 0.9796 D. seriata DO4 slight + 26.5 0.9709 D. seriata YVW2 slight + 26.1 0.9820 D. seriata II12 slight + 26.2 0.9369 D. seriata ME21 slight + 26.2 0.9867 Dothiorella iberica M21 slight + 22.5 0.7733 Do. iberica N19 slight + 23.1 0.7855 Do. iberica L5 slight + 23.2 0.7642 Do. iberica J4 slight + 22.9 0.7980 Do. iberica J24 slight + 23.6 0.8307 Dothiorella viticola K16 + 24.0 0.8583 Do. viticola M11 slight + slight 24.1 0.9085 Do. viticola L19 + 24.5 0.8521 Do. viticola N23 slight + 23.9 0.9307 Do. viticola CV11 + 24.2 0.8843 Lasiodiplodia theobromae MW643 + slight 29.7 0.9888 L. theobromae C2 + slight 29.7 0.9963 L. theobromae G31A + slight 29.4 0.9844 Neofusicoccum australe ME10 + 26.1 0.9369 N. australe VP13 slight + 26.0 0.9635 N. australe SDW4 + 26.8 0.9396 N. australe FF10 slight + 25.7 0.9188 N. australe DNW8 + 26.3 0.9098 Neofusicoccum parvum B12 slight + 26.3 0.9761 N. parvum B19A + 26.7 0.9473 N. parvum B7A + 27.3 0.9348 N. parvum B7B + 26.7 0.9411 N. parvum TS24A slight + 26.6 0.9486 w Data from preliminary trial comprising temperatures 5, 10, 15, 20, 25, 30, 35 and 40 C. x Combined data from duplicate trials comprising temperatures 10, 15, 20, 25, 30 and 35 C. y slight indicates that the strain grew to a diameter (including inoculum plug) of no more 10 mm after 72 hours, indicates no growth occurred, and + indicates growth to a diameter > 10 mm occurred. z R 2 values of polynomial regression equations used to determine optimal temperatures for radial mycelial growth (mm). 16

Figure Captions Fig. 1 Lesions produced on the trunks of 15-year-old Chardonnay grapevines 24 months after inoculation with Neofusicoccum parvum B19A. Lane 1 = control. Fig. 2 Mean lesion length caused by Botryosphaeriaceae species and Eutypa lata 24 months after inoculation of 15-year-old Chardonnay grapevines at the National Wine and Grape Industry Centre, Wagga Wagga, NSW, Australia; October 2010 to September 2012. All values are back-transformed to the original scale (mm). Means followed by the same letters are not significantly different according to Tukey s test (P=0.05). Bars represent the 95% confidence interval of the mean. Fig 3 Temperature growth relationships and estimated optimal growth temperatures for Botryosphaeriaceae species used in pathogenicity studies. Lines represent the average growth rate of five isolates from data combined over two trials. 17

colour figure 1 Click here to download colour figure: Fig.1.eps

line figure 2 Click here to download line figure: Fig.2.eps 200 a Lesion length (mm) 160 120 80 40 ab bc bc bc c c c c d 0 N. parvum L. theobromae N. australe B. dothidea D. mutila D. viticola E. lata D. iberica D. seriata Control

colour figure 3 Click here to download colour figure: Fig.3.eps B. dothidea, 29.7 C D. seriata, 26.6 C L. theobromae, 29.4 C N. parvum, 26.8 C D. mutila, 25.8 C D. viticola, 24.0 C D. iberica, 22.9 C N. australe, 26.2 C