versión On-line ISSN 0718-1620
Cienc. Inv. Agr. v.36 n.3 Santiago dic. 2009
Cien. Inv. Agr. 36(3):381-390
Effects of Phaeomoniella chlamydospora ana Phaeoacremonium aleophilum on grapevine rootstocks
Efecto de Phaeomonietta chlamydospora y Phaeoacremonium aleophilum sobre portainjertos de vid
Gonzalo A. Díaz, Marcela Esterio, and Jaime Auger1
Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas, Universidad de Chile, Casilla 1004, Santiago, Chile. 1Corresponding author: email@example.com
Cuttings of five grapevine (Vitis vinifera) rootstocks were wounded and immediately inoculated with suspensions (approximately 5x103 conidia- mL-1) of either Phaeomoniella chlamydospora, Phaeoacremonium aleophilum or a mixture of both species. The presence of these endophyte fungi affected the quality of each of the five rootstocks. Among the roostocks investigated in this study, 1103P and 101-14 MG were less susceptible to the infection caused by Pa. chlamydospora wd Pm. aleophillum.
Key words: Endophytes fungi, fungal diseases, Petri disease, Vitis vinifera, wood fungi.
Se inocularon cinco portainjertos de vid con una suspensión conidial (20 uL de aproximadamente 5x103 conidiamL-1) de Phaeomoniella chlamydospora, Phaeoacremonium aleophilum y una mezcla de ambos patógenos, mediante una perforación en la base de cada estaca. Estos hongos endófitos afectaron todo los parámetros de calidad en cada uno de los portainjertos, siendo menos susceptible el 1103 P y 101-14 MG. El portainjerto SO4 fue el que presentó la mayor susceptibilidad a estos hongos endófitos. Los portainjertos Kober 5BB y 3309 C presentaron una susceptibilidad intermedia y los portainjertos 1103 P y 101-14 MG se comportaron como los menos susceptibles bajo las condiciones de este estudio.
Palabras clave: Enfermedad de Petri, enfermedades fungosas, hongos de la madera, hongos endófitos, Vitis vinifera.
Chile is a major exponer of table grapes (Vitis vinifera L.) and wines, and regional viticulture has expanded considerably in recent years. The rapid growth of this industry has considerably increased the demand for nursery plants, and particularly the demand for grafted grapevines, and with increasing demand, the quality of the available propagation material has declined. The problems associated with the production of the grafted grapevines include inhibition of the basal callus formation, decrease in root emission, poor formation of the grafting callus and graft failures, and symptoms of incompatibility.
Petri disease is recognized as one of the fungal diseases related to the decreasing quality of propagation plant materials. This disease affects young grapevine plants and causes plant loss in many countries. Research focused in Chile (Auger et al., 2004b), Italy (Mugnai et al., 1999), USA (Morton, 1995), France (Larignon and Dubos, 1997), South Africa (Ferreira et al., 1994) and Australia (Pascoe and Cottral, 2000) has attributed the symptoms of Petri disease to the presence of vascular endophytic fungi (Mugnai et al., 1999). The causal agents associated with Petri disease are Phaeomoniella (Pa) chlamydospora and species of Phaeoacremonium (Pm) (Mugnai et al., 1999; Eskalen et al., 2001; Whiting et al., 2001; Fourie and Hallen, 2004; Santos et al., 2005). Phaeoacremonium aleophilum is the species of Phaeoacremonium most commonly identified in diseased grapevines (Wallace etal, 2002; Auger et al., 2005a). Numerous studies have suggested that Pa. chlamydospora and Pm. aleophilum are the causal agents of Petri disease (Feliciano et al., 2004; Gaforio et al., 2005; Mostert et al., 2006).
The symptoms of Petri disease include general weakness, slowed growth, delayed bud burst, short internodes, reduction of the diameter of trunks and arms, small leaves, and early leaf senescence. The formation of dark brown streaks in the vascular tissue of the xylem, close to the pith, is observed in longitudinal sections of the trunk or stems. Cross-sections of trunk or stem material have revealed black punctuated patterns corresponding to the occlusion of the xylem vessels (Scheck et al., 1998; Khan et al., 2000; Eskalen et al., 2001; Stamp, 2001; Whiting et al., 2001; Wallace et al., 2002; Auger et al., 2005b; Díaz, 2008).
Phaeomoniella chlamydospora and Pm. aleophilum were reported recently in Chile and were primarily associated with young grapevine cvs. Several varieties in vineyards located in Regions V, VI, VII and the Metropolitan Región that were grafted on 3309 C and Kober 5BB (Cabernet Sauvignon, Merlot, Pinot Noir, Red Globe, Fíame Seedless, Thompson Seedless and Ruby seedless) were all affected by decline symptoms (Auger et al., 2004b; Auger et al., 2005c). Pa. chlamydospora and Pm. aleophium have occasionally been detected along with different species of Botryosphaeriaceae in Red Globe grapevines (Auger et al., 2004a).
The previously discussed species Phaeoacremonium and Pa. chlamydospora are part of the fungus complex that causes dieback of adult grapevines, increasing the incidence of chlorotic leaf roll associated with Fomitiporella vitis (Auger et al., 2005a). In Europe, Pa. chlamydospora and Phaeoacremonium spp. have also been isolated from grapevines affected by esca and eutipiosis (Larignon and Dubos, 1997).
In Chile, the use of grafted grapevines, primarily SO4 (V. berlandieri x V. riparia), has increased in recent years, but the its behavior or response against Chilean isolates Pa. chlamydospora and Pm. aleophilum is unknown. The rootstock SO4 is known to be very susceptible to these two endophytic fungi (Eskalen et al., 2001). Therefore, the objective of this study was to determine the effects of Pa. chlamydospora and Pm. aleophilum on grafted grapevines on the major rootstock used for wine grapes in Chile.
Materials and methods
The effects of Pa. chlamydospora and Pm. aleophilum on basal callus formation, root emission, grafting callus and bud burst was characterized for V. vinifera cv Carménére grafted on 3309 C (V. riparia x V. rupestris), 1103 P (V. berlandieri x V. rupestris), 101-14 MG (V. riparia x V. rupestris), Kober 5BB (V. berlandieri x V. riparia), and SO4 (V. berlandieri x V. riparia). The effects were studied in 480 30-cm-long woody cuttings of each rootstock.
Inoculum and inoculation
Conidia of Pa. chlamydospora (pachOl-2004) and Pm. aleophilum (pmalOl-2004) were obtained in potato dextrose agar (PDA) incubated at 25°C for 15 days in darkness. The inoculum concentrationwas adjustedto 5xl03 conidia mL1 (Wallace et al., 2002). The inoculation method involved injecting 20 uL of conidial suspensión into the bases of woody cuttings of grapevine rootstocks. The following inoculation treatments were performed: i. Pa. chlamydospora, ii. Pm. aleophilum or iii. a mixture of Pa. chlamydospora and Pm. aleophilum. An equal number of cuttings from each rootstock were injected with 20 uL of sterile deionized water and used as control treatment (Eskalen et al., 2001). This assay was performed twice using 100 cuttings per inoculation treatment (Test 1) and it was repeated (Test 2) using 20 cuttings.
Effects of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum on the root formation, basal callus, grafting callus and bud burst (Test 1)
We arranged 400 previously inoculated cuttings of each rootstock (n = 2000) in 100 L hydration pools during a 48 h period. V. vinifera cv. Carménére (5 cm long) were grafted on to the rootstocks using a graft machine. To avoid dehydration, grafted cuttings were immediately immersed in wax (70-72°C). Cuttings were subsequently placed in a callus growth chamber at 28-31°C and 85% relative humidity (RH) for 20 days. To promote growth and root development, cuttings were maintained for 28 days in a greenhouse at 24- 28°C and 65-75% RH. The basal callus formations as well as the callus formations on the grafting áreas were evaluated after the callus formations process, using the scale described by Fourie and Hallen (2004). Root emissions and bud burst were evaluated at the end of the greenhouse grow-ing period, and were subjectively graded based on the visible proportion of callus formation on the cutting base grafting área (0 = 0%, 1 = 1 to 25%, 2 =26 to 50%, 3 = 51 to 75%, 4 = 76 to 99% and 5 = 100%). Bud burst and root emission were scored from 1 (no bud burst or roots emission) to 2 (bud burst or roots emission > 2 cm in length).
Effects of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum on the vascular system (Test 2)
In this test, 80 cuttings from each rootstock (n = 400) were inoculated with the conidial suspensions described above. The cuttings were placed in an incubation chamber with 12 h of light and 12 h of darkness, immersed in water (5 cm from the base) at 25-28°C, and continuously agitated for 49 days. This procedure was designed to fasterbud burst and the roots emission in the cuttings, and to stimulate the production of auxins synthesized by leaves (Hartman and Kester, 1997), a process which occurs generally 21 days post-budburst. After this treatment period, cuttings were inoculated according to Eskalen et al. (2001). We measured the length of the vascular tissue damage (streak) 7 months post-inoculation. To re-isolate the inoculated pathogens, samples of diseased tissue were isolated from the margins of the necrotic lesions and were plated in PDA and incubated at 25°C in continuous darkness for 14 days. Pathogen identification was based on the description and taxonomic key described by Crous et al. (1996) and Crous and Gams (2000).
Design and statistical analysis
Inoculated treatments were distributed as a completely randomized design with 100 and 20 replicates, in Tests 1 and 2, respectively. A two-factor analysis of variance (ANOVA) was performed (rootstock x inoculated treatments), and when data was non-parametric, a Kruskal-Wallis test was applied. Means were assessed using Tukey's múltiple range test (p < 0.05). When a significara (p < 0.05) interaction between inoculated treatments and rootstocks was obtained, Tukey's múltiple range test was used to assess rootstocks.
Effects of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum on root formation, basal callus, grafting callus and bud burst (Test 1)
Inoculation of the cuttings with Pa. chlamydospora andP/w. aleophilum negatively affected the quality of grapevine rootstock cuttings. For the base callus development, the interaction between inoculated treatments and rootstocks was not statistically significara (p = 0.5652). In the case of the parameter grafting callus, we identified a significara interaction between the rootstock and inoculated treatment factors (p = 0.0076) (Table 1). With respect to the development of the basal callus, the ANOVA showed significara effects of inoculation treatment and rootstock type (p < 0.0001) (Table 2). For root emission, interaction between the treatments and the rootstock factors was not significara (p = 0.9652). The ANOVA showed significara differences between inoculated treatments (p < 0.0001) and rootstocks (p < 0.0001) in the root emission (Table 3). For bud burst, inoculation treatments and rootstock types showed no significara interaction (p = 0.3271). The ANOVA showed a significara effect of both treatments (p < 0.0001) and rootstocks (p = 0.0002) (Table 4).
Effect of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum on the vascular system (Test 2)
The cuttings inoculated with Pa. chlamydospora, Pm. aleophilum or a mixture of both pathogens showed a reddish brown to dark brown vascular discoloration. In each rootstock, the presence of these fungi was consistently associated with the treated sample and was consistently absent from the cuttings that had not been inoculated. The results indicated a significant interaction (p < 0.0001) between the rootstocks and inoculation treatments.
Our study results confirmed that Pa. chlamydospora and Pm. aleophilum partially or completely inhibited the formation of the basal callus and the development of the grafting callus (Figure 1), as previously reported for other grapes cultivars (Khan et al., 2000). However, we found that Pm. aleophilum showed little effect on callus formation in rootstocks of Kober 5BB, 1103 P, 101-14 MG, SO4, Ramsey, and 99 Richter grapevines (Wallace etal, 2002). These differential responses may be due to the use of less virulent strains of Pm. aleophilum. Differences in virulence have been demonstrated by Santos et al., (2005); theses authors compared different strains of Phaeoaremonium spp., and Pa. chlamydospora and detected different levéis of aggressiveness.
Corroborating previous studies, the basal callus was consistently larger in grapevines inoculated with Pa. chlamydospora than with those inoculated with Pm. aleophilum (Bertelli et al., 1998; Khan et al., 2000) (Table 2). With the exception of rootstock 3309 C, we detected significant differences in grafting callus formation between all control plants and inoculated plants (Table 1). In terms of the developing grafting callus (Table 1), we found that the interface was formed when the rootstock and graft have a close interaction. Previous studies by Hartmann and Kester (1997) demonstrated that the entire graft interface is complex, and that the formation of the interface becomes increasingly complicated with the introduction of any interfering factor. The presence of Pa. chlamydospora, Pm. aleophilum or other endophytic fungi may faster the accumulation of tylosis and phenolic compounds in the colonized tissues. Further, the toxic metabolites (exopolysacarides) produced by Pa. chlamydospora and Pm. aleophilum can adversely affect the formation of the grafting callus (Sparapano et al., 2000; Lorena et al., 2001). These results were similar to those obtained in South Africa using rootstock 99-Richter grafted with 'CheninBlanc' (Ferreira et al., 1994).
Santos et al. (2005) examined callus formation under laboratory conditions and demonstrated the inhibitory effect of Pa. chlamydospora and Phaeoacremonium spp. for the rootstock 3309 C, the Baga and María Gomes cultivars. Ad-ditionally, they determined that this inhibition was different for different cultivars and root-stocks, demonstrating the differential susceptibility between the grapevine genotypes. We found similarly diverse results, and showed that the rootstock SO4 was consistently more susceptible than other rootstocks (Table 2). The rootstock 1103 P and the rootstock 101-14 MG developed the best basal and grafting calluses. The rootstock that showed the least susceptibility to the pathogens was 101-14 MG (Table 2).
Inoculation with Pa. chlamydospora greatly inhibited root emission (Table 3). Our results with this fungus differ from the previous findings of Khan et al. (2000), which did not conclusively demónstrate differences in root emissions between Pa. chlamydospora and species de Phaeoacremonium. The rootstock SO4 showed the most susceptibility with respect to root emissions, and emissions were significantly lower than the rest of the rootstocks (Table 3).
The inoculated treatments showed severely reduced grafting bud bursts when rootstocks were inoculated withPa. chlamydospora orPm. aleophüum (Table 4). Significant differences in bud bursts were not detected between SO4, 3309 C, Kober 5BB and 101-14 MG, but the rootstock 1103 P showed significantly higher rates of bud burst for treated samples (Table 4). Notably, during the process of bud burst, symptoms of incompatibility were observed in the inoculated treatments, and the joint between the rootstock and the scion was observed to be very weak.
Significant differences in the stem streaks were observed between the control samples and samples inoculated with fungi, consistent with the results obtained by Ferreira et al. (1994), Khan et al. (2000), Eskalen et al. (2001), Laukart et al. (2001) and Feliciano et al. (2004) (Figure 2). The rootstock SO4 inoculated with a mixture of Pa. chlamydospora and Pm. aleophüum showed a shorter streak length compared to inoculation with either Pa. chlamydospora or Pm. aleophilum. Independently of the rootstock type, cuttings inoculated withPa. chlamydospora or Pm. aleophilum developed streaks similar in length. The rootstock S04 showed a longer streak than the other tested rootstocks, demonstrating the high susceptibility to Pa. chlamydospora isolate and Pm. aleophilum isolate (Table 5). The results suggest that there are considerable differences between rootstock types with respect to streak formation inside the cuttings. These results are similar to the observations made by Eskalen et al. (2001), and may be due to genetic differences between the rootstocks. Similar response has been observed in grapevine cultivars (Khan et al. 2000; Feliciano et al. 2004).
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Received 8 October 2008. Accepted 19 March 2009.