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© 2002 Plant Management Network.
Accepted for publication 16 June 2002. Published 2 July 2002.


Infection of Grape Berry and Rachis Tissues by Phomopsis viticola


O. Erincik and L. V. Madden, Department of Plant Pathology, The Ohio State University, Wooster, 44691; D. C. Ferree, Horticulture and Crop Science, The Ohio State University; and M. A. Ellis, Department of Plant Pathology, The Ohio State University


Corresponding author: M. A. Ellis. ellis.7@osu.edu


Erincik, O., Madden, L. V., Ferree, D. C., Ellis, M. A. 2002. Infection of grape berry and rachis tissues by Phomopsis viticola. Online. Plant Health Progress doi:10.1094/PHP-2002-0702-01-RS.


Abstract

Studies were conducted to determine if Phomopsis viticola can infect grape berries directly and cause latent infections, or if fruit infection occurs via mycelial invasion from infected rachis tissues. Examination of inoculated immature berries using scanning electron microscopy showed that alpha spores of P. viticola germinated on the surface of the berry, produced appressoria, and appeared to penetrate the cuticle. Intact clusters of immature berries on ‘Seyval’ grape vines were inoculated with a spore suspension of P. viticola. Clusters were harvested after various periods (12, 18, 24 and 30 hr) of continuous wetness at 20°C. In a separate study, inoculated berries were incubated for 24 hr at 20°C then moved to a greenhouse for incubation periods of 1, 2, and 3 weeks. Immediately after drying (first study) or after the incubation period (second study) berries were separated from rachises and surface disinfested. Each rachis was divided into 10 sections and was also surface disinfested. Berries and rachis sections were then immersed in a 1:32 dilution of 37% paraquat dichloride to kill plant tissues and induce growth of the fungus. Abundant mycelia and pycnidia of P. viticola formed on inoculated berries and rachis sections for all wetness periods and incubation periods. Results demonstrate that direct infection of berries can result in latent infections on green fruit. Whereas the fungus may invade berries via infected rachis tissues, rachis infection is not required for Phomopsis fruit rot development.


Introduction


Fig. 1. Necrotic lesions on a ‘Seyval’ rachis caused by Phomopsis viticola (Click image for larger view).

Phomopsis cane and leaf spot, caused by Phomopsis viticola (Sacc.) Sacc., is an important disease of grapes worldwide (1,7,10,11,12,14). The fungus attacks all green parts of the vine, including canes, leaves, flowers, rachises, and berries. However, most economic damage is caused by rachis (Fig. 1) and berry infection (Fig. 2) (8,13). Berry and rachis infection by P. viticola have been studied in some detail, although there are conflicting results in the literature (6,7,8,13). Gregory (7) reported that ripe Vitis labrusca ‘Niagara’ fruit showed symptoms within 18 days after inoculation, indicating that fruit infection by P. viticola can occur in ripe fruit. Lal and Arya (10) reported that direct berry infection by P. viticola did not occur on ripe V. vinifera ‘Thompson Seedless’ berries. They reported that berries could not be infected unless they were wounded prior to inoculation. Hewitt and Pearson (8) stated that most fruit infections by P. viticola arise from mycelial invasion from lesions on the rachis or pedicel. Pscheidt and Pearson (13) reported that berry infection on ‘Concord’ berries occurs primarily during bloom and shortly after bloom, and that the infections remain latent until berries begin to ripen. As berries ripen, the fungus becomes active and fruit rot develops. They also reported that little or no fruit infection occurs after pea-sized berries are formed. Erincik et al. (6) indicated that both rachis and berry infections by P. viticola on ‘Seyval,’ ‘Catawba,’ and ‘Chambourcin’ grapes can occur throughout the growing season (bloom through véraison).


Fig. 2. Phomopsis fruit rot on ‘Seyval’ grape berries caused by Phomopsis viticola (Click image for larger view).

In Ohio, the occurrence of Phomopsis fruit rot appears to be increasing (5). In 1997, yield losses due to Phomopsis fruit rot were estimated to be as great as 30% in several southern Ohio vineyards (M. A. Ellis, unpublished). At present, it is unclear whether the fungus can infect berries directly, or if the fungus invades the fruit through infected rachis tissues. Studies need to be conducted in order to better understand the etiology of the berry infection phase of this disease.

Paraquat, a widely used contact herbicide, has been used to detect latent infections by fungi in symptomless plant tissues (3,11). Pscheidt and Pearson (13) reported that 2 weeks after treatment with paraquat, symptomless grape berries were covered with sporulating pycnidia of P. viticola. The fungus apparently developed from latent infections in green fruit. The paraquat technique was used in this study to determine if P. viticola is present in green fruit after inoculation and incubation under various wetness periods.

The objective of this study was to determine if P. viticola can infect grape berries directly, without movement of mycelium from infected rachis tissues into the fruit.


Studies with Scanning Electron Microscope and Paraquat

Preparation of plants with intact clusters. Experiments were conducted on greenhouse-grown plants of ‘Seyval’ with intact fruit clusters. One-year-old dormant ‘Seyval’ plants were planted in a mixture of peat, steam disinfected loam, and perlite (1:1:1, v/v/v) in 20-cm plastic pots. Shortly after bud break, vines were cut to a height of approximately 50 cm. One or two buds with clusters were selected and each plant was trained to one or two shoots with one cluster per shoot. Plants were fertilized with 18 g of Osmocote (14-14-14, N-P-K) every other month and were watered as needed with deionized water, taking care not to wet flowers or fruit clusters.

Preparation of inoculum and cluster inoculation. Fifteen isolates of P. viticola were collected from infected grape vines in five commercial Ohio vineyards in 1997. Inoculations were conducted on internodes and leaves of greenhouse-grown ‘Seyval’ grape plants to confirm pathogenicity of all isolates. In addition, spore and mycelium morphology of all isolates was compared to a known isolate of P. viticola obtained from mycologist M. E. Palm, USDA Systematic Botany and Mycology Laboratory, Beltsville, MD. Representative isolates were sent to M. E. Palm and were confirmed as P. viticola. Whereas all isolates were pathogenic and produced typical symptoms on grape, the isolate obtained from M. E. Palm was consistently the most aggressive in preliminary studies and was chosen for use in this study. Pathogenicity of the isolate was maintained by inoculating surface-disinfected disks cut from mature grape berries (‘Thompson Seedless’) placed on potato-dextrose agar (PDA) (2). Plugs cut from the edge of actively growing mycelia were placed in the center of each grape disk, and the pathogen was reisolated from PDA after it had grown through the disk. Cultures were grown on PDA in petri plates at 21°C under continuous fluorescent light at 58 Em-2s-1 for 15 to 20 days. Spores were collected by flooding culture plates with sterile deionized water. Pycnidia and spores were detached by rubbing the surface with a paint brush. The suspension was filtered through four layers of cheesecloth and was adjusted to 1 x 107 alpha spores per milliliter using a hemacytometer. Attached clusters were inoculated at Eichhorn-Lorenz growth stage 29 (Fig. 3) (4). The spore suspension was sprayed to run off onto clusters prior to incubation under various wetness durations. Noninoculated clusters were sprayed with water to serve as controls.


Fig. 3. Eichorn-Lorenz growth stage 29; berries small; bunches begin to hang (Click image for larger view).

Scanning Electron Microscopy (SEM) study. Five inoculated plants were incubated for 12 hr under continuous wetness at 20°C. Plants were removed from the mist chamber and two berries were randomly selected from each cluster. Noninoculated berries served as controls. A section (0.2 to 0.5 mm thick) including intact cuticle and epidermis was cut from the distal surface (away from pedicel) of each berry using a razor blade. These sections served as samples in SEM studies.

Samples were fixed by using standard fixation procedures (9). Briefly, samples were fixed in gluteraldehyde and paraformaldehyde, dehydrated in a series of solutions containing increasing amounts of ethanol, and then coated with platinum in a Polaron E-5180 sputter coater (platinum coating approximately 30 nm thick). Coated samples were examined under a Hitachi S-3500 variable pressure scanning electron microscope.

Paraquat studies to determine berry infection. To determine if the fungus was penetrating grape berry tissues, fifteen attached ‘Seyval’ clusters, comprising three replications of five clusters each per wetness duration, were inoculated with P. viticola at Eichhorn-Lorenz growth stage 29 (Fig. 3) as previously described. Inoculated plants were placed in a growth chamber and maintained at 20°C for wetness durations of 12, 18, 24, and 30 hr. Immediately after completion of each wetness duration, plants were removed from the growth chamber and were allowed to dry in the greenhouse for 30 min. Clusters were then harvested. After harvest, all berries were separated from the rachis (Fig. 4), and thirty randomly selected berries from each cluster were surface disinfested by soaking in 95% ethanol for 10 sec, then in 0.05% sodium hypochlorite for 2 min. Berries were then immersed in a 1:32 dilution of 37% paraquat dichloride (Gramoxone Extra, Zeneca Ag Products Inc, Wilmington, DE) for 1.5 min (13), then placed on an elevated screen within a disinfested moist chamber and incubated at 21 to 22°C for 21 days. Three weeks after paraquat treatment, all fruit were visually evaluated for presence of P. viticola.


Fig. 4. Berries were separated from rachis before paraquat treatment (Click image for larger view).

Each rachis was divided into 10 approximately equal sections and surface disinfested and treated with paraquat as previously described for berries. Two weeks after paraquat treatment, all rachis pieces were evaluated for the presence of P. viticola. The experiment was repeated a total of three times. Equal numbers of noninoculated berries and rachises were treated as described and served as controls. The experimental design was a randomized complete block.

To determine if the fungus established latent infections in grape berries, fifteen attached ‘Seyval’ clusters, consisting of three replications of five clusters each, were inoculated as previously described at Eichhorn-Lorenz growth stage 29 (Fig. 3). Inoculated and noninoculated plants with attached clusters were placed in a moist chamber immediately after inoculation in order to maintain continuous wetness at 20°C for 24 hr. Plants were then returned to the greenhouse. Plants were watered without wetting clusters. Clusters were harvested at 1, 2, and 3 weeks after inoculation.

After harvest, berries were separated from the rachis (Fig. 4), and berries and rachises were treated and evaluated as previously described. Experimental design was a randomized complete block. The experiment was performed in 1999 and 2000.

Data Analysis. Data were analyzed to determine the effect of wetness period (first study) and length of incubation period (second study) and on disease incidence by using analysis of variance (ANOVA) with the Minitab Statistical package (Minitab Inc., State College, PA). The arcsine-square root transformed proportion of berries and rachis pieces with P. viticola signs and symptoms was the dependent variable. Experimental factors were wetness period (first study), length of the incubation period (second study), and replication. Individual clusters or rachises were treated as subsamples. Means were separated using Fishers least significant difference (LSD; P=0.05) if the F test for wetness period and incubation period indicated a significant effect.


Summary and Conclusions


Fig. 5. Scanning electron micrograph showing germinated alpha conidia of Phomopsis viticola on the distal surface of a ‘Seyval’ grape berry. A) appressoria. B) hypha penetrating berry cuticle. Bar represents 5 μm (Click image for larger view).

Scanning electron microscopy studies showed that alpha spores of P. viticola germinated on the distal surface of green ‘Seyval’ berries, produced appressoria, and penetrated the cuticle of the fruit within 12 hr after inoculation and incubation (Fig. 5). No fungal growth was observed on the surface of noninoculated control berries. Although these observations alone do not demonstrate that infections were established in inoculated berries, they do indicate that the surface of green grape berries can be penetrated by P. viticola.

Paraquat killed berry and rachis tissues and induced the development of mycelia and fruiting structures (pycnidia and cirrhi) of P. viticola on inoculated berries and rachises (Fig. 6). White cottony mycelia of P. viticola developed on inoculated rachis tissues and berries at 5 to 7 days after paraquat treatment. Black pycnidia with cream-colored cirrhi developed on the surface of inoculated rachises and berries within 7 and 10 days, respectively, after treatment with paraquat. The use of paraquat allowed early detection of the fungus in symptomless berry tissues. Phomopsis viticola was recovered from 33% of inoculated berries at 12 hr after inoculation (Table 1). At 30 hr after inoculation, P. viticola was recovered from 79% of inoculated berries. No P. viticola was recovered from noninoculated control berries. There was a significant increase in the level of berry infection from 12 to 30 hr, and there was no significant difference in the level of berry infection between 12, 18, and 24 hr. In the second study, P. viticola was recovered from 87% of inoculated berries and 99% of inoculated rachises after a 24-hr wetness period at 20°C, followed by one week under ambient conditions in the greenhouse (Table 2). There was no effect on the level of berry or rachis infection when time held in the greenhouse was extended from one to three weeks (Table 2). Without the paraquat treatment, berries did not show any disease symptoms or signs of the pathogen. These results combined with observations using SEM suggest that P. viticola can directly infect grape berry tissues resulting in latent infections.


Fig. 6A. White colored cottony mycelia on rachis section (Click image for larger view).

Fig. 6B. Cirrhi exuding from pycnidia on berries after paraquat treatment (Click image for larger view).


Table 1. Recovery of Phomopsis viticola from berries and rachises from noninoculated or inoculated ‘Seyval’ grape clusters following wetness periods of 12, 18, 24, and 30 hr at 20°C, then treatment with paraquat.

Wetness periodw Mean % berries
with P. viticolax
Mean % of rachis sections
with P. viticolay
Noninoculated 0.0 cz 0.0 c
12 hr 32.7 b 98.0 a
18 hr 51.3 ab 98.0 a
24 hr 54.3 ab 100.0 a
30 hr 79.0 a 99.0 a

w  ‘Seyval’ vines with attached clusters were inoculated at Eichhorn-Lorenz growth stage 29 and incubated under continous wetness for 12, 18, 24, and 30 hr prior to treatment with paraquat to kill tissues.

x  Mean percentage of berries with P. viticola based on 30 randomly selected berries from each of 5 clusters for each of three replications.

y  Mean percentage of rachis sections with P. viticola based on 10 rachis sections from each of 5 clusters for each of three replications.

z  Means followed by different letters within a column are significantly different (P = 0.05) based on Fisher's least significant difference (LSD), calculated based on arcsine-transformed percentage data. Only the untransformed mean percentages are shown.


Table 2. Recovery of Phomopsis viticola from symptomless berries and rachises from ‘Seyval’ clusters after treatment with paraquat at 1, 2 and 3 weeks after inoculation in 1999 and 2000.

Length of 
the incubation
periodw
Mean % berris
with P. viticolax
Mean % rachis sections
with P. viticolay
1999 2000 1999 2000
1 week 87.1az 85.1a 98.7a 98.7a
2 weeks 85.1a 85.6a 96.0a 98.1a
3 weeks 85.5a 83.3a 96.7a 98.7a

w  Inoculated ‘Seyval’ plants with attached clusters were inoculated at Eichhorn-Lorenz growth stage 29 (4), then incubated for 1, 2, and 3 weeks in the greenhouse before the paraquat treatment.

x  Mean percentage of berries with P. viticola based on 30 randomly selected berries from each of 5 clusters for each of three replications.

y  Mean percentage of rachis sections with P. viticola based on 10 rachis sections from each of 5 clusters for each of three replications.

z  Means followed by same letters within a column are not significantly different (P = 0.05) based on analysis of arcsine square-root transformed percentage data. Only untransformed mean percentages are shown.


Rachis tissues appear to be much more susceptible than berry tissues. After 12 hr of leaf wetness, 98% of rachis sections were infected by P. viticola compared to 33% of the berries (Table 1). The incidence (percentage of infected sections) of rachis infection did not significantly increase with increasing wetness duration. Virtually all rachis tissues were infected with P. viticola after 12 hr incubation.

The paraquat technique has been used for the detection of several other pathogenic fungi in symptomless plant tissues (3,11,13). Cerkauskas and Sinclair (3) reported that paraquat treatment induced the development of fungal mycelia and fruiting structures of Phomopsis sojae, Cercospora kikuchii, Fusarium spp., and Colletotrichum dematium var. truncata on symptomless soybean pods. Pscheidt and Pearson (13) reported that 2 weeks after treatment with paraquat, symptomless immature grape berries were covered with sporulating pycnidia of P. viticola. Use of the paraquat technique in these studies provides additional evidence that P. viticola directly infects immature grape berries and causes latent infection in symptomless immature grape berries. In addition, previous studies have shown that fruit infection can occur throughout the growing season (prebloom through véraison) (6).

In conclusion, the results of this study indicate that green grape berries are susceptible to direct infection by P. viticola. Furthermore, the abundant recovery of P. viticola from inoculated green fruit indicates that direct infection of grape berries can result in latent infections in immature fruit. As fruit matures, the fungus becomes active resulting in development of Phomopsis fruit rot at or near harvest. Our results also demonstrate that rachis tissues are also highly susceptible to infection and are probably more susceptible than fruit. Although the fungus may invade berries through infected rachis tissues (especially during fruit maturation or in ripe fruit), rachis infection is not required for development of Phomopsis fruit rot.

Results from these studies provide new information on the etiology of the fruit rot phase of this disease. From a practical perspective, the fact that all portions of the grape cluster (berries and rachises) are susceptible to infection further emphasizes the need for complete fungicide coverage of the entire cluster in order to obtain effective disease control. This is especially true when protectant fungicides are used. At present, the recommended fungicides for control of P. viticola on grapes in Ohio are mancozeb, captan, or ziram. All of these are protectant fungicides.


Acknowledgements

Salaries and support provided by state and Federal funds (especially grants from the Ohio Grape Industries Program and the USDA Viticulture Consortium-East through a subcontract with Cornell University, NYSAES, under agreement #34360-7382 appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, Wooster).


Literature Cited

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3. Cerkauskas, R. F., and Sinclair B. C. 1980. Use of paraquat to aid detection of fungi in soybean tissues. Phythopathology 70:1036-1038.

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