Effects of temperature, pH and water potential on mycelial growth, sporulation and chlamydospore production in culture of Cylindrocarpon spp. associated with black foot of grapevines

37 www.fupress.com/pm ISSN (print): 0031-9465 © Firenze University Press ISSN (online): 1593-2095 Phytopathologia Mediterranea (2012) 51, 1, 37−50 Corresponding author: J. Armengol Fax: +34 963879269 E-mail address: [email protected].es Research Papers Effects of temperature, pH and water potential on mycelial growth, sporulation and chlamydospore production in culture of Cylindrocarpon spp. associated with black foot of grapevines Carlos AGUSTÍ-BRISACH and Josep ARMENGOL Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022-Valencia, Spain Summary. The effects of temperature, pH and water potential (Ψs) on mycelial growth, sporulation and chlamydospore production of Cylindrocarpon liriodendri, C. macrodidymum and C. pauciseptatum isolated from grapevines was studied. Three isolates per species were incubated on potato dextrose agar (PDA) under different temperature, pH, and Ψs conditions. All isolates were able to grow over a range of temperatures from 5 to 30ºC, with an optimum temperature between 20 to 25ºC, but they did not grow at 35ºC. Active mycelial growth was observed over a range of pHs, from 4 to 8. Regarding the effect of Ψs, in general, mycelial growth was greater on amended media at -0.5, -1.0 or/and -2.0 MPa compared with that obtained on nonamended PDA (-0.3 MPa), and was reduced at Ψs values lower than -2.0 MPa. Most of the Cylindrocarpon spp. isolates were sporulated at all temperatures, pHs and water potentials tested. In all studied conditions, C. liriodendri had the greatest sporulation capacity compared with C. macrodidymum and C. pauciseptatum. In general, chlamydospore production was not much affected by temperature, pH and Ψs. Chlamydospores were observed in PDA cultures of all isolates at all pH values studied, while some isolates did not produce chlamydospores at 5 and 10ºC or -4.0 and/or -5.0 MPa. These results improve understanding of the biology of these important grapevine pathogens. Key words: Cylindrocarpon liriodendri, Cylindrocarpon macrodidymum, Cylindrocarpon pauciseptatum, soilborne diseases. Introduction Black foot disease of grapevines, caused by Cylindrocarpon spp., is a serious disease in most wine and grape-producing regions of the world, particularly in nurseries and young vineyards (Halleen et al., 2006a). It was first described in 1961 (Grasso and Magnano Di San Lio, 1975), and over the last decade, black foot disease has been reported in most grapevine production areas of the world, including Portugal (Rego et al., 2000), Argentina (Gatica et al., 2001), Germany (Fischer and Kassemeyer, 2003), New Zealand and South Africa (Halleen et al., 2004), Brazil (Garrido et al., 2004), California (Petit and Gubler, 2005), Chile (Auger et al., 2007), Australia (Whitelaw-Weckert et al., 2007), Spain (Alaniz et al., 2007), Iran (Mohammadi et al., 2009), Uruguay (Abreo et al., 2010), northeastern United States and southeastern Canada (Petit et al., 2011). Vines affected by Cylindrocarpon spp. often show sunken necrotic root lesions with a reduction in root biomass and root hairs. Removal of rootstock bark reveals black discoloration and necrosis of wood tissues which develops from the base of the rootstock. Moreover, affected vines show low vigour with small trunks and short internodes, a reduction in total foliage and leaf size, with leaves depicting interveinal chlorosis and necrosis, frequently leading to death of the plants (Halleen et al., 2006a; Alaniz et al., 2007, 2009; Abreo et al., 2010). Phytopathologia Mediterranea 38 C. Agustí-Brisach and J. Armengol Black foot disease of grapevine is caused by Cylindrocarpon spp. (Cylindrocarpon destructans [Zinnsm.] Scholten, C. liriodendri J.D. MacDonald & E.E. Butler, C. macrodidymum Schroers, Halleen & Crous and C. pauciseptatum Schroers & Crous) and Campylocarpon spp. (Campylocarpon fasciculare Schroers, Halleen & Crous and Campyl. pseudofasciculare Halleen, Schroers & Crous) (Halleen et al., 2004; Halleen et al., 2006a, 2006b; Schroers et al., 2008). In Spain, surveys carried out in recent years in grapevine nurseries and young vineyards have confirmed the importance of Cylindrocarpon spp. affecting this crop. These pathogens were found in grapevine nurseries very early in the planting material production process, in grapevine plants ready to be planted and in young vineyards showing decline symptoms (Aroca et al., 2006; Giménez-Jaime et al., 2006; Alaniz et al., 2007; Gramaje et al., 2010). In all cases, Cylindrocarpon spp. were mostly isolated from rootstocks, especially from the basal ends. To date, C. liriodendri, C. macrodidymum and C. pauciseptatum are the species which have been identified associated with young vines showing symptoms of black foot disease in Spain (Alaniz et al., 2007; Martin et al., 2011). Species of Cylindrocarpon are common and may be isolated as soil inhabitants, saprobes on dead plant material, root colonizers or pathogens, or weak pathogens of various herbaceous and woody plants (Brayford, 1993). The production of chlamydospores may allow Cylindrocarpon spp. to survive for extended periods in soil (Halleen et al., 2004). Given these findings, it could be assumed that black foot disease pathogens could survive in the soil to infest grapevine plants. However, very little information is currently available regarding the basic biology of these pathogens, such as mycelial growth, sporulation and chlamydospore production under various environmental conditions, and the role that chlamydospores might play during the infection processes (Halleen et al., 2006a). In common with all microorganisms, fungi are profoundly affected by physical and physicochemical factors, such as temperature, aeration, pH, water potential (Ψs), and light. These factors not only affect the growth rate of fungi but can also act as triggers in developmental pathways (Deacon, 2006). These factors are known to influence host-pathogen interactions in C. destructans on Ginseng (Rahman and Punja, 2005) and in a number of other soilborne pathogens such as Monosporascus cannonballus Pollack & Uecker on muskmelon and watermelon (Ferrin and Stanghellini, 2006; Armengol et al., 2011), Rhizoctonia solani J.G. Kühn on lupin and potato (Kumar et al., 1999; Ritchie et al., 2006; Ritchie et al., 2009), and diseases caused by Pythium and Phytophthora spp. (Sommers et al., 1970; Abdelzaher et al., 1997). In Cylindrocarpon spp. associated with black foot disease of grapevines, only growth temperature experiments have been conducted so far. These studies were performed when these pathogens were recently described and/or characterized (Halleen et al., 2004; Petit and Gubler, 2005; Halleen et al., 2006b; Alaniz et al., 2007; Schroers et al., 2008). The aim of the present study was to expand knowledge of the effects of temperature, pH and Ψs on mycelial growth, sporulation and chlamydospore production of C. liriodendri, C. macrodidymum and C. pauciseptatum isolated from grapevines. Materials and methods Fungal isolates Three isolates of C. liriodendri (Cy59, Cy89 and Cy100), three isolates of C. macrodidymum (Cy47, Cy14 and Cy81) and one isolate of C. pauciseptatum (Cy593), obtained from roots or the basal ends of rootstocks from grapevines exhibiting symptoms of black foot in Spain were used in this study. Additionally, two isolates of C. pauciseptatum from roots of grapevines in Slovenia (CBS120171 and CBS120173) were obtained from the collection of the Centraalbureau voor Schimmelcultures (CBS, Utrecht, the Netherlands) (Table 1). Single spore isolates were stored in 15% glycerol solution at –80°C in cryovials (1.5 mL capacity). Prior to use, a small plug of the colonized agar from each cryovial was transferred to potato dextrose agar (PDA) (Biokar-Diagnostics, Zac de Ther, France) plates and allowed to grow at 25ºC in darkness for 14 d. Effects of temperature on mycelial growth, sporulation and chlamydospore production of Cylindrocarpon spp. To determine the effect of temperature on mycelial growth, all isolates were maintained and grown on PDA plates at 25°C. Agar plugs (8 mm diam.) were cut from the leading edges of 14-d-old colonies 39 Vol. 51, No. 1, April, 2012 Growth, sporulation and chlamydospore production of Cylindrocarpon spp. and placed in the center of PDA plates (one plug per plate) that were then incubated in the dark at 5, 10, 15, 20, 25, 30 or 35°C. There were four pseudoreplicates for each isolate and temperature combination. After 10 d, colony diameter was measured along two perpendicular axes, and data were converted to radial growth (mm d-1). Colonies grown on PDA were further incubated over 20 days to evaluate sporulation and determine the presence/absence of chlamydospores. The number of conidia produced on mycelia from agar plugs was measured following the method described by Whiting et al. (2001) and Alaniz et al. (2007). After 30 d of incubation, two plugs of agar (4 mm diam.) with mycelia and spores, were cut from the growing edge of each colony, and each was placed in an Eppendorf vial (1.5 mL capacity) containing 1 mL of sterile water. Vials with plugs were vortexed for 5 s, and the number of conidia per mL was counted using a haemocytometer. To observe chlamydospores, a small quantity of fungal material was removed from each colony surface with a sterile needle taking care to minimize disturbance of the fungal structures, and placed in a drop of distilled water in the centre of a clean slide, and a cover slide was carefully lowered on to the drop. Two preparations per Petri dish were observed microscopically at ×100 and ×400 magnification. The experiment was repeated. Effects of pH on mycelial growth, sporulation and chlamydospore production of Cylindrocarpon spp. The effects of pH on mycelial growth and sporulation of Cylindrocarpon spp. in culture was determined on PDA. Mycelial plugs (8 mm diam.) obtained from the growing edges of colonies were transferred to the center of PDA plates (one plug per plate) which were adjusted to pH 4, 5, 6, 7 and 8 with the addition of 50 mM citrate phosphate buffer (pH 4‒7) or 50 mM Tris-HCl buffer (pH 8) (Gomori, 1955). Plates were incubated in the dark at 25ºC. There were two replicates for each isolate and pH combination. Table 1. Sources of isolates of Cylindrocarpon spp. associated with black foot disease and their optimum growth temperatures. Species/Isolate Year Geographical origin Scion/rootstock Optimum growth (°C)b Town Province C. liriodendri Cy59 2003 Tarazona de la Mancha Albacete Cencibel/1103 P 21.9 Cy89 2004 Aielo de Malferit Valencia Garnacha/110R 23.7 Cy100 2004 Alesanco La Rioja Garnacha/110R 24.3 C. macrodidymum Cy14 2002 n.d.cBurgos Tempranillo/110R 24.5 Cy47 2003 Mollina Málaga Pedro Ximénez/1103 P 23.3 Cy81 2003 Beneixama Alicante Tempranillo/161-49C 24.1 C. pauciseptatum Cy593 2009 Tobarra Albacete Syrah/S04 24.4 CBS120171a2005 Krsko (Eslovenia) n.d. n.d. 24.0 CBS120173a2005 Doljenska (Eslovenia) n.d. n.d. 22.9 a Reference isolates of C. pauciseptatum (CBS 120171 and CBS 120173) were obtained from the collection of Centralbureau voor Schimmelcultures inUtrecht, the Netherlands (CBS). b For each Cylindrocarpon spp. isolate, temperature average growth rates were adjusted to a regression curve to estimate the optimum growth temperature. c n.d., Not determined. Phytopathologia Mediterranea 40 C. Agustí-Brisach and J. Armengol Mean mycelial growth rates, the number of conidia produced on mycelia and the presence/absence of chlamydospores were evaluated as described above. The experiment was repeated. Effects of water potential (Ψs) on mycelial growth, sporulation and chlamydospore production of Cylindrocarpon spp. The effect of Ψs on mycelial growth and sporulation of Cylindrocarpon spp. in culture was determined on PDA. Mycelial plugs (8 mm in diam.) obtained from the growing edges of colonies were transferred to the center of PDA plates (one plug per plate) amended with KCl or NaCl prior to sterilization to obtain six Ψs values: -0.5, -1.0, -2.0, -3.0, -4.0 and -5.0 MPa, according to Robinson and Stokes (1959). Non-amended PDA (-0.3 MPa) was used as and experimental control. Plates were incubated in the dark at 25ºC. There were two replicates for each isolate, type of solute and Ψs combination. Mean mycelia growth rates, the number of conidia produced on mycelia and the presence/absence of chlamydospores were evaluated as described above. The experiment was repeated. Statistical analyses Data from each Cylindrocarpon spp. were analyzed separately. Two way analyses of variance (ANOVA) were conducted with radial growth and sporulation data obtained from temperature and pH experiments, and a three way ANOVA was conducted for Ψs experiments using Statgraphics Plus 5.1 software (Manugistics Inc., Rockville, MD, USA). For all Cylindrocarpon spp., ANOVA analyses indicated that the radial growth and sporulation data between the two repetitions in temperature, pH and Ψs experiments were similar (P>0.05). Thus, in all cases, data from both experiments were combined. For each Cylindrocarpon spp. isolate, temperature, pH or Ψs average mycelial growth rates were adjusted to a regression curve using Statgraphics Plus 5.1 software, and the best polynomial model was chosen based on parameter significance (P<0.05) and coefficient of determination (R2). Previous to this analysis, data of mycelial growth from the Ψs experiment were converted to relative growth rate as a percentage of the experimental controls. Additionally, the polynomial models in the temperature experiments were used to estimate the optimum growth temperature for each isolate. Results Effects of temperature on mycelial growth, sporulation and chlamydospore production of Cylindrocarpon spp. Statistically significant effects of the isolate on radial growth were observed for C. macrodidymum (P=0.0019) and C. pauciseptatum (P=0.0073), but not for C. liriodendri (P=0.4999). The effect of isolate × experiment was also not significant in all of the species studied (P>0.05) (Table 2). The effects of temperature on mycelial radial growth and sporulation of the nine Cylindrocarpon spp. isolates are shown in Figure 1. All isolates were able to grow on PDA over a range of temperatures from 5 to 30ºC, and no growth was obtained at 35ºC. At 5ºC, C. liriodendri isolates showed growth rates between 0.038 cm day-1 for isolate Cy89 and 0.043 cm day-1 for isolate Cy59, while the growth rates of the C. macrodidymum and C. pauciseptatum isolates were almost negligible at this temperature. Optimum growth temperatures for all isolates ranged between 21.9ºC for isolate Cy59 (C. liriodendri) and 24.5ºC for isolate Cy14 (C. macrodidymum) (Table 1). Regarding sporulation, significant effects of the isolate on sporulation were observed for C. macrodidymum (P<0.001) and C. pauciseptatum (P<0.001), but not for C. liriodendri (P=0.9328). The effect of isolate × experiment was also not significant in all species studied (P>0.05) (Table 2). Most of the Cylindrocarpon spp. isolates produced conidia at all temperatures, showing broad variation. In general, the three C. liriodendri isolates sporulated more abundantly than C. macrodidymum and C. pauciseptatum isolates in all studied temperatures, with values greater than 104 conidia mm-2. In C. macrodidymum, there was more variability among the isolates, isolate Cy14 being the only one for which values greater than 104 conidia mm-2 were obtained at 15, 20, 25 and 30ºC. Isolate Cy47 only sporulated at 15, 20 and 25ºC. Sporulation of C. pauciseptatum isolates was also variable among isolates, isolate Cy593 being the only one in which values greater than 104 conidia mm-2 were obtained at 5, 20, 25 and 30ºC. Isolate CBS120173 did not sporulate at 5ºC. Chlamydospores were observed in PDA cultures of all isolates from 15 to 30ºC. No chlamydospores were observed at 5 or 10ºC. (Continued) 41 Vol. 51, No. 1, April, 2012 Growth, sporulation and chlamydospore production of Cylindrocarpon spp. Table 2. Analysis of variance for the effects of temperature, pH and Ψs on radial growth and sporulation of Cylindrocarpon liriodendri, C. macrodidymum and C. pauciseptatum isolates. Parameter C. liriodendri C. macrodidymum C. pauciseptatum d.f.aMSbP < Fcd.f. MS P < Fd.f. MS P < F Temperature Radial growth Experiment (A) 1 0.00012 0.8911 1 <0.0001 0.9894 1 0.00007 0.9199 Isolate (B) 2 0.00427 0.4999 2 0.04492 0.0019 2 0.03480 0.0073 A × B 2 0.00019 0.9688 2 0.00011 0.9839 2 0.00001 0.9980 Residual 326 0.00615 329 0.00703 330 0.00697 Sporulation Experiment (A) 1 17.5208 0.1418 1 17.2798 0.0629 1 5.17642 0.3420 Isolate (B) 2 0.56193 0.9328 2 379.699 <0.001 2 195.172 <0.001 A × B 2 4.69997 0.5596 2 2.11654 0.6532 2 4.39406 0.4645 Residual 330 8.08063 330 4.96344 330 5.71707 pH Radial growth Experiment (A) 1 <0.0001 0.9790 1 0.00005 0.8122 1 <0.0001 0.9793 Isolate (B) 2 0.00318 0.0895 2 0.04015 <0.001 2 0.00557 0.0006 A × B 2 0.00017 0.8787 2 0.00005 0.9459 2 <0.0001 0.9987 Residual 226 0.00131 221 0.00089 225 0.00073 Sporulation Experiment (A) 1 0.02486 0.8548 1 <0.0001 0.9132 1 <0.0001 0.2186 Isolate (B) 2 2.26406 0.0492 2 <0.0001 <0.001 2 <0.0001 <0.001 A × B 2 0.09818 0.8759 2 <0.0001 0.9882 2 <0.0001 0.2346 Residual 212 0.74085 210 <0.0001 208 <0.0001 Ψs Radial growth Experiment (A) 1 0.00004 0.8891 1 0.00002 0.9411 1 0.00002 0.9329 Isolate (B) 2 0.02469 <0.001 2 0.05107 <0.001 2 0.05301 <0.001 Salt type (C) 1 0.05410 <0.001 1 0.05925 <0.001 1 0.16224 <0.001 A × B 2 0.00020 0.8982 2 0.00016 0.9547 2 <0.0001 0.9992 A × C 2 0.00247 0.2742 2 0.02014 0.0026 2 0.00127 0.6735 B × C 1 0.00182 0.3290 1 0.00021 0.8009 1 0.00005 0.9011 A × B × C 2 0.00042 0.8029 2 0.00019 0.9449 2 0.00006 0.9817 Residual 639 0.00191 658 0.00335 were used to estimate the optimum growth temperature for each isolate. Results Effects of temperature on mycelial growth, sporulation and chlamydospore production of Cylindrocarpon spp. Statistically significant effects of the isolate on radial growth were observed for C. macrodidymum (P=0.0019) and C. pauciseptatum (P=0.0073), but not for C. liriodendri (P=0.4999). The effect of isolate × experiment was also not significant in all of the species studied (P>0.05) (Table 2). The effects of temperature on mycelial radial growth and sporulation of the nine Cylindrocarpon spp. isolates are shown in Figure 1. All isolates were able to grow on PDA over a range of temperatures from 5 to 30ºC, and no growth was obtained at 35ºC. At 5ºC, C. liriodendri isolates showed growth rates between 0.038 cm day-1 for isolate Cy89 and 0.043 cm day-1 for isolate Cy59, while the growth rates of the C. macrodidymum and C. pauciseptatum isolates were almost negligible at this temperature. Optimum growth temperatures for all isolates ranged between 21.9ºC for isolate Cy59 (C. liriodendri) and 24.5ºC for isolate Cy14 (C. macrodidymum) (Table 1). Regarding sporulation, significant effects of the isolate on sporulation were observed for C. macrodidymum (P<0.001) and C. pauciseptatum (P<0.001), but not for C. liriodendri (P=0.9328). The effect of isolate × experiment was also not significant in all species studied (P>0.05) (Table 2). Most of the Cylindrocarpon spp. isolates produced conidia at all temperatures, showing broad variation. In general, the three C. liriodendri isolates sporulated more abundantly than C. macrodidymum and C. pauciseptatum isolates in all studied temperatures, with values greater than 104 conidia mm-2. In C. macrodidymum, there was more variability among the isolates, isolate Cy14 being the only one for which values greater than 104 conidia mm-2 were obtained at 15, 20, 25 and 30ºC. Isolate Cy47 only sporulated at 15, 20 and 25ºC. Sporulation of C. pauciseptatum isolates was also variable among isolates, isolate Cy593 being the only one in which values greater than 104 conidia mm-2 were obtained at 5, 20, 25 and 30ºC. Isolate CBS120173 did not sporulate at 5ºC. Chlamydospores were observed in PDA cultures of all isolates from 15 to 30ºC. No chlamydospores were observed at 5 or 10ºC. (Continued) Phytopathologia Mediterranea 42 C. Agustí-Brisach and J. Armengol Effects of pH on mycelial growth, sporulation and chlamydospore production of Cylindrocarpon spp. Statistically significant effects of the isolates on radial growth were observed for C. macrodidymum (P<0.001) and C. pauciseptatum (P=0.0006), but not for C. liriodendri (P=0.0895). The effect of isolate × experiment was also not significant in the three species studied (P>0.05) (Table 2). The effects of pH on mycelial radial growth and sporulation of the nine Cylindrocarpon spp. isolates are shown in Figure 2. All isolates were able to grow on pH-adjusted PDA at all pH values studied. In general, for each Cylindrocarpon spp. all isolates showed similar growth rates from pH 4 to pH 8, although in C. liriodendri and C. macrodidymum radial growth increased slightly as pH increased. In C. liriodendri, mycelial radial growth was greatest at pH 8 for all isolates, and in C. macrodidymum, mycelial growth of Cy47 and Cy81 isolates was also greatest at pH 8, while for Cy14 this was greatest at pH 6. In C. pauciseptatum, mycelial growth of isolates CBS120171 and CBS120173 was greatest at pH 6, while for isolate Cy593 this was greatest at pH 8. Regarding sporulation, significant effects of the isolate on sporulation were observed for C. liriodendri (P=0.0492), C. macrodidymum (P<0.001) and C. pauciseptatum (P<0.001). The effect of isolate × experiment was not significant for all Cylindrocarpon spp. (P>0.05) (Table 2). Most of the isolates produced conidia at all pH values, showing a broad range of variation among Cylindrocarpon spp. In general, the three C. liriodendri isolates sporulated more abundantly than C. macrodidymum and C. pauciseptatum at all studied pH values, producing more than 105 conidia mm-2. In C. macrodidymum, there was variability among the isolates, isolate Cy14 being the only one for which values greater than 105 conidia mm-2 were obtained at pH 6 and 8. Isolates Cy47 did not sporulate at pH 5 and Cy81 did not sporulate at pH 6. Sporulation of C. pauciseptatum isolates was also variable among isolates, isolate Cy593 being the only one for which values greater than 105 conidia mm-2 were recorded at pH 6, but this isolate did not sporulate at pH 4. Isolate CBS120171 only sporulated at pH 5, 6 and 7, and isolate CBS120173 did not sporulate at pH 5. Chlamydospores were observed in PDA cultures of all isolates at all pH values studied. Effects of water potential (Ψs) on mycelial growth, sporulation and chlamydospore production of Cylindrocarpon spp. Statistically significant effects of the isolate and salt type on mycelial growth were observed for all three species (P<0.001). All the interactions were not Parameter C. liriodendri C. macrodidymum C. pauciseptatum d.f.aMSbP < Fcd.f. MS P < Fd.f. MS P < F Sporulation Experiment (A) 1 4.06421 0.1092 1 2.37550 0.5960 1 0.52946 0.8002 Isolate (B) 2 0.88494 0.5723 2 68.3923 0.0003 2 91.4964 <0.001 Salt type (C) 1 0.67427 0.0391 1 1.26907 0.6983 1 118.754 0.0002 A × B 2 1.70771 0.3409 2 1.70329 0.8175 2 3.05828 0.6908 A × C 2 10.0494 0.0019 2 297.890 <0.001 2 52.2602 0.0019 B × C 1 3.36179 0.1452 1 0.62988 0.7848 1 15.4059 0.1721 Residual 2 0.06835 0.9578 2 7.15412 0.4293 2 8.13652 0.3742 A × B × C 660 1.58416 660 8.44962 660 8.26433 a Degrees of freedom. b Mean square. c Probabilities associated with individual F-tests. Table 2. Continues. 43 Vol. 51, No. 1, April, 2012 Growth, sporulation and chlamydospore production of Cylindrocarpon spp. Temperatures (°C) Radial growth (cm day-1) Sporulation [log 10 (conidia mm-2 + 1)] CP CM CL CL CL ! (Cy59) y= -0.00002x3-0.0005x2 + 0.0107x - 0.0331, R2=0.8925 ! (Cy89) y= -0.00004x3+0.0016x2 + 0.0084x - 0.0423, R2=0.9409 ! (Cy100) y= -0.00005x3 - 0.002x2 + 0.0159x - 0.0687, R2=0.9871 ! (Cy14) y= -0.00004x3 + 0.0018x2 - 0.0151x - 0.0446, R2=0.7615 ! (Cy47) y= -0.00003x3 + 0.0011x2 + 0.0033x - 0.0379, R2=0.9494 ! (Cy81) y= -0.00004x 3 + 0.0017x 2 - 0.0081x - 0.0042, R 2 =0.8302 CM ! (Cy593) y= -0.00004x3 + 0.0015x2 - 0.0084x - 0.0076, R2=0.9179 ! (CBS120171) y= -0.00005x3 + 0.0018x2 + 0.0059x - 0.0112, R2=0.9719 ! (CBS120173) y= -0.00003x3+ 0.0008x2 - 0.0082x - 0.0569, R2=0.9656 CP A B Figure 1. A, mean colony radial growth rates (cm day-1) of three isolates of C. liriodendi (CL), C. macrodidymum (CM) and C. pauciseptatum (CP), after 10 days of incubation in darkness on PDA at 5, 10, 15, 20, 25, 30 or 35ºC; B, mean amounts of sporulation [log10(conidia mm-2 + 1)] of three isolates of C. liriodendri (CL), C. macrodidymum (CM) and C. pauciseptatum (CP), after 30 days of incubation in darkness on PDA at 5, 10, 15, 20, 25, 30 or 35ºC. Results are the mean of two independent sets of four pseudoreplicates for each temperature. Vertical bars are the standard error of the means. Phytopathologia Mediterranea 44 C. Agustí-Brisach and J. Armengol Figure 2. A, mean colony radial growth rates (cm day-1) of three isolates of C. liriodendi (CL), C. macrodidymum (CM) and C. pauciseptatum (CP), after 10 days of incubation in darkness on PDA at pHs 4, 5, 6, 7 or 8; B, mean amounts of sporulation [log10(conidia mm-2 + 1)] of three isolates of C. liriodendri (CL), C. macrodidymum (CM) and C. pauciseptatum (CP), after 30 days of incubation in darkness on PDA at pHs 4, 5, 6, 7 or 8. Results are the mean of two independent sets of four pseudoreplicates in each pH.Vertical bars are the standard error of the means. B A pH Radial growth (cm day-1) Sporulation [log 10 (conidia mm-2 + 1)] ! (Cy593) y= - 0.0027x2 + 0.0399x + 0.0794, R2=0.3507 ! (CBS120171) y= - 0.0087x2 + 0.1048x - 0.0624, R2=0.3632 ! (CBS120173) y= - 0.0085x 2 + 0.1031x - 0.0729, R 2 =0.4469 CP ! (Cy14) y= - 0.0094x2 - 0.1295x - 0.1454, R2=0.5203 ! (Cy47) y= - 0.0002x2 + 0.0156x - 0.1487, R2=0.7905 ! (Cy81) y= - 0.002x2 - 0.0366x - 0.0934, R2=0.958 CM ! (Cy59) y= 0.0135x2 - 0.1423x + 0.5737, R2=0.8288 ! (Cy89) y= 0.0076x2 - 0.0817x + 0.4365, R2=0.6599 ! (Cy100) y= 0.0086x2 - 0.0836x - 0.3978, R2=0.8066 CL CL CM CP 45 Vol. 51, No. 1, April, 2012 Growth, sporulation and chlamydospore production of Cylindrocarpon spp. significant with the exception of experiment × salt type for C. macrodidymum (P<0.0026) (Table 2). The effect of Ψs on mycelial growth of the nine Cylindrocarpon spp. isolates is shown in Figure 3, and on sporulation is shown in Figure 4. The patterns of the mycelial radial growth responses of the isolates to decreasing Ψs were similar for the two osmotica tested, but, in general, Cylindrocarpon spp. were more tolerant to NaCl than KCl. Mycelial growth generally increased compared with mycelial growth on nonamended PDA (-0.3 MPa) at -0.5 MPa and -1.0 Mpa by the addition of KCl and NaCl. The exception was isolate Cy89 which showed the greatest mycelial growth at -2.0 MPa. At lower water potentials, mycelial growth decreased as Ψs reduced, showing the lowest percentages at -5.0 MPa. Regarding sporulation, a significant effect of the isolate on sporulation was observed for C. macrodidymum (P=0.0003) and C. pauciseptatum (P<0.001), but not for C. liriodendri (P=0.5723). The effect of salt type was significant for C. liriodendri (P=0.0391) and C. pauciseptatum (P=0.0002), but not for C. macrodidymum (P=0.6983). All the interactions were not significant with the exception of experiment × salt type for all Cylindrocarpon spp. (Table 2). Most of the isolates were able to produce conidia at all Ψs values, showing a broad range of variation. In general, the three C. liriodendri isolates sporulated more abundantly than C. macrodidymum and C. pauciseptatum isolates in all studied Ψs values for each salt tested, with values greater than 105 conidia mm-2 in KCl and 104 conidia mm-2 in NaCl. In C. macrodidymum, there was more variability among the isolates, and isolates Cy47 and Cy81 did not sporulate at -5.0 MPa in NaCl. In C. pauciseptatum, sporulation was also variable among isolates. Isolate CBS120171 did not sporulate at -1.0 MPa in KCl and at -0.5 in NaCl, and isolate Cy593 did not sporulate at -5.0 MPa in NaCl. Chlamydospores were observed in PDA cultures of all isolates at almost all Ψs values in both salts. No chlamydospores were observed in isolate Cy593 (C. pauciseptatum) at -4.0 MPa in NaCl and at -5.0 MPa both in NaCl and KCl, and in Cy59 (C. liriodendri) at -4.0 and -5.0 MPa in KCl. Discussion This study has identified differences in the effects of temperature, pH and Ψs on mycelial growth, sporulation and chlamydospore production of C. liriodendri, C. macrodidymum and C. pauciseptatum. In general, these Cylindrocarpon spp. were able to grow over a range of temperatures from 5 to 30ºC, with optimum temperatures for growth between 20 to 25ºC, but they did not grow at 35ºC. These results are in agreement with those obtained in previous studies (Halleen et al., 2004; Petit and Gubler, 2005; Halleen et al., 2006b; Alaniz et al., 2007; Schroers et al., 2008), and indicate that Cylindrocarpon spp. associated to black foot disease of grapevine are mesophilic, as most fungi, which commonly grow within the range 10‒40ºC (Deacon, 2006). In addition, our study showed differences among Cylindrocarpon spp. in the effect of low temperatures on mycelial growth. Growth of C. macrodidymum and C. pauciseptatum at 5 and 10ºC was almost negligible compared with that of C. liriodendri. This is in agreement with the results obtained by Alaniz et al. (2007), who demonstrated that C. macrodidymum can be differentiated from C. liriodendri by growing more slowly at 5 and 10ºC. Previous research has suggested that species belonging to the genus Cylindrocarpon are calcicolous, with optimal pH around 7.0 and being poorly represented in acid soils (Matturi and Stenton, 1964a). Nevertheless, in our study Cylindrocarpon spp. associated with black foot of grapevine showed broad pH tolerance for mycelial growth, although with slight differences among isolates. All isolates were able to grow between pH 4.0 to 8.0. This corresponds with the optimum pH range indicated by Deacon (2006) for mycelial growth of most fungi. Regarding the effect of Ψs, the response of Cylindrocarpon spp. isolates was reduced mycelial growth as Ψs decreased. Our results indicate that the isolates may have benefited from small to modest additions of solutes. In general, mycelial growth was greater on amended media at -0.5, -1.0 and/or -2.0 MPa compared with that on nonamended PDA (-0.3 MPa), and was reduced at Ψs values less than -2.0 MPa. Moreover, the effect of Ψs on mycelial growth was similar whether KCl or NaCl was used as the osmoticum, indicating that the observed responses were caused by changes in Ψs rather than by toxicity of the osmotic. These results, together with those obtained in the pH experiments, suggest that Cylindrocarpon spp. pathogenic to grapevine are likely to proliferate in most vineyard soils. In all studied conditions, C. liriodendri was the species with the greatest capacity for sporulation