Identifying and Characterizing Summer Diseases on Babygold Peach in South Carolina

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2006 Plant Management Network. Accepted for publication 23 December 2005. Published.

1 2006 Plant Management Network. Accepted for publication 23 December Published. Identifying and Characterizing Summer Diseases on Babygold Peach in South Carolina Guido Schnabel, Associate Professor, Wenxuan Chai, Research Assistant, and Kerik D. Cox, Postdoctoral Research Scientist, Department of Entomology, Soils, and Plant Sciences, Clemson University, Clemson, SC Corresponding author: Guido Schnabel. Schnabel, G., Chai, W., and Cox, K. D Identifying and characterizing summer diseases on Babygold peach in South Carolina. Online. Plant Health Progress doi: /php rs. Abstract Summer diseases can cause significant yield losses in processing peach varieties, such as the Babygold lines. In this study we identified and characterized the pathogens responsible for disease outbreaks in two orchards (PH and JC) located in the northern Piedmont area of South Carolina. Three pathogens, Geotrichum candidum, Colletotrichum acutatum, and Botryosphaeria dothidea, the causal agents of sour rot, anthracnose, and Botryosphaeria fruit rot disease respectively, were identified on fruit from orchard PH using symptomology, culture and spore morphology, and ribosomal DNA analysis. G. candidum and C. acutatum were also isolated from symptomatic fruit from orchard JC. The QoI fungicide azoxystrobin and a mixture of pyraclostrobin and boscalid were evaluated for their in vitro efficacy against five isolates of each of the three pathogens to investigate their possible usefulness in designing management strategies. Azoxystrobin inhibited mycelial growth of C. acutatum isolates (EC 50 values of 0.01 to 0.55 mg/liter) but was ineffective against mycelium of G. candidum and B. dothidea isolates (EC 50 values >300 mg/liter). The pyraclostrobin-boscalid mixture was highly effective against mycelium of C. acutatum (EC 50 values of 0.01 to 0.05 mg/liter) and B. dothidea isolates (EC 50 values of 0.02 to 0.03 mg/liter), but only marginally effective against mycelium of G. candidum (EC 50 values to mg/liter). This study provides a diagnostic guide of pathogens that can cause summer diseases on Babygold peaches and reports their in vitro sensitivity to registered respiration inhibitor fungicides. Introduction Babygold peaches (Prunus persica L. Babygold ) are widely used for the production of baby food because the flesh has a desirable orange color with distinct apricot-like taste and remains firm during and after processing. The drawback of growing Babygold peaches includes their high susceptibility to summer diseases, such as Phomopsis fruit rot caused by Phomopsis sp. (5,10) or bacterial spot caused by Xanthomonas arboricola pv. pruni (4). Due to processors demand for tree ripened fruit, Babygold peaches grown in South Carolina face additional threats by disease as susceptibility increases with fruit maturity (15). Babygold producers from South Carolina have suffered yield loss due to summer diseases in the past despite the use of fungicides, especially during years with weather conditions favorable for disease development. The latest disease outbreak occurred in 2004 when diseased fruit exhibited symptoms indicating the presence of more than one disease. Controlling summer diseases on peach in South Carolina is primarily achieved by applying fungicides throughout the growing season because latent infections may develop as early as at pit hardening (17). Peach producers prefer to use broad spectrum, multi-site inhibitor fungicides (e.g., captan or sulfurbased materials) for summer disease control in so called cover sprays because they are generally less expensive than potentially more potent products with single-site action. However, multi-site inhibitors are often not sufficiently effective for disease control under high disease pressure. Other fungicides registered on peach, such as the site-specific demethylation inhibitor (DMI)

$Respiration inhibitor fungicides of Fungicide Resistance Action Committee (FRAC, www.frac.$ info) groups 7 and 11 may be more effective for summer disease control compared to multi-site inhibitor fungicides, but their efficacy against pathogens causing summer diseases on Babygold peach has

info) groups 7 and 11 may be more effective for summer disease control compared to multi-site inhibitor fungicides, but their efficacy against pathogens causing summer diseases on Babygold peach has

The purpose of this study was to identify the causal organisms of the 2004 summer disease outbreak in South Carolina Babygold peach orchards and to determine the efficacy of registered respiration

Information from this study includes detailed descriptions of uncommon fruit diseases of peach and a basis for the development of more effective disease management programs.

2 fungicides, are recommended for pre-harvest brown rot but not for summer disease control (3) to reduce the risk of selection for resistance in the brown rot pathogen Monilinia fructicola (13). Respiration inhibitor fungicides of Fungicide Resistance Action Committee (FRAC, groups 7 and 11 may be more effective for summer disease control compared to multi-site inhibitor fungicides, but their efficacy against pathogens causing summer diseases on Babygold peach has not been investigated. The purpose of this study was to identify the causal organisms of the 2004 summer disease outbreak in South Carolina Babygold peach orchards and to determine the efficacy of registered respiration inhibitor fungicides against the causal agents. Information from this study includes detailed descriptions of uncommon fruit diseases of peach and a basis for the development of more effective disease management programs. Collection and Identification of Causal Agents from Infected Babygold Fruit Symptomatic fruit (Fig. 1) was only found in orchards containing Babygold peaches but not in adjacent orchards with other varieties. A total of 171 diseased fruit were collected from orchard PH in Greenville Co., SC, and 125 fruit were collected from orchard JC in Spartanburg Co., SC, on July 20, 2004 when fruit was tree ripe. Spores from sporulating lesions or ruptured ascostroma (Fig. 2) were used to obtain one single-spore isolate from each collected fruit. Fig. 1. Symptoms of summer diseases on Babygold peach: (A) Anthracnose caused by Colletotrichum acutatum; (B) Botryosphaeria fruit rot caused by Botryosphaeria dothidea; (C) Sour rot caused by Geotrichum candidum; and (D) A combination of anthracnose and Botryosphaeria fruit rot symptoms. Scale bar in A through D is 1 cm.

Fig. 2. (A) Conidia of Colletotrichum acutatum and (B) Geotrichum candidum, and (C) ascospores of Botryosphaeria dothidea from ruptured ascoma. Scale bar in A through C is 30 µm.

Five representative isolates of each culture morphology were randomly selected to identify the pathogens using ribosomal DNA analysis and culture and spore morphology.

8S-ITS2 region was amplified using universal primers ITS1-F (5 -CTTGGTCATTTAGAGGAAGTAA-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) (6,16).

3 Fig. 2. (A) Conidia of Colletotrichum acutatum and (B) Geotrichum candidum, and (C) ascospores of Botryosphaeria dothidea from ruptured ascoma. Scale bar in A through C is 30 µm. Three different fungal culture morphologies were observed corresponding to three different lesion types on the collected peach fruit (Table 1). Five representative isolates of each culture morphology were randomly selected to identify the pathogens using ribosomal DNA analysis and culture and spore morphology. Genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA). The ribosomal ITS1-5.8S-ITS2 region was amplified using universal primers ITS1-F (5 -CTTGGTCATTTAGAGGAAGTAA-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) (6,16). PCR reactions were performed in a total volume of 50 µl containing ng of DNA, 1 µm primers, 200 µm each dntp, 1.25 U of Taq DNA polymerase (Promega Corp., Madison, WI), 2.5 mm MgCl 2, 50 mm KCl, and 0.1% Triton X-100. Cycling parameters were 94 C for 2 min, 35 cycles of 94 C for 30 s, 50 C for 40 s, and 72 C for 30 s. The final elongation step was 72 C for 7 min. All PCR reactions were performed in an icycler thermal cycler (Bio-Rad Laboratories, Hercules, CA). Amplification products were subjected to gel electrophoresis using 1% agarose gels containing 0.5 µg/ml ethidium bromide in 0.5 X TBE. Amplification products were purified using the QiaQuick Gel Extraction Kit (Qiagen, Valencia, CA) and sequenced using BigDye Terminator v3.1 (Applied Biosystems, Inc., Foster City, CA) at the Clemson University Sequencing Facility. Sequences were assembled using DNASTAR software (DNASTAR, Inc., Madison, WI). Complete ITS1-5.8S-ITS2 sequences were searched using BLAST at the NCBI website and selected sequences (one for each species identified) were submitted to GenBank (accession numbers DQ to DQ177877).

4 Table 1. Characteristics of pathogens isolated from Babygold orchards PH and JC. Orchard (no. of isolates) Disease Sour rot Anthracnose Botryosphaeria fruit rot Organism Geotrichum candidum Colletotrichum acutatum Botryosphaeria dothidea PH JC Symptoms Lesions (1-4 cm) are circular and brown becoming moist, soft, and covered with mycelium and spores within a few days Lesions (1-4 cm) are circular, sunken, firm to touch, and contain concentric rings of colored sporulation which begin in the center and expand outward Lesions (2-10 cm or whole fruit) are circular, occasionally sunken, and may resemble anthracnose except that the lesions are softer to touch, frequently larger, and lack obvious sporulation. Signs Colonies on lesions consist of white, cottony, yeast-like mycelium that give rise to single-celled, short, cylindrical, conidia ( µm on average). Colonies on lesions consist of concentric rings of mycelium and salmon to orange conidial masses. Elongate, single-celled, hyaline, conidia ( µm on average) are borne singly in acervuli which are often obscured by abundant sporulation. Small black ascostromata were observed throughout the lesions in the current study. The ascostromata (cushioned mass) contain locules in which asci develop directly. Gentle agitation of ascostroma liberates single-celled, hyaline ascospores ( µm on average). Three different fungal pathogens, Geotrichum candidum Link, Colletotrichum acutatum J.H. Simmonds, and Botryosphaeria dothidea (Moug.:Fr.) Ces & De Not, were identified based on BLAST search results (Table 1). Single-spore isolates with similar culture and spore morphology had identical ITS1-5.8S-ITS2 sequences and therefore were considered to belong to the same species. Nucleic acid-based identification was substantiated by culture and spore morphologies consistent with published descriptions (2,7). Koch s postulates were fulfilled by inoculating mature peaches with spore suspensions of each pathogen individually and verifying sign and symptom development. These pathogens cause diseases of minor importance on peach in the southeastern United States, namely sour rot, anthracnose, and Botryosphaeria fruit rot (Table 1). Because of the low impact of these diseases on other cultivars, no management strategies have been developed for sour rot or Botryosphaeria fruit rot. Our descriptions and pictures of disease symptoms and signs should enable growers and extension specialists to more easily identify pathogens causing summer diseases of Babygold peaches (Table 1).

5 In Vitro Sensitivity of Fungal Isolates to Respiration Inhibitors of FRAC Fungicide Groups 7 and 11 Azoxystrobin, pyraclostrobin, and boscalid are respiration inhibitor fungicides registered for disease control on peach in the United States. Azoxystrobin is marketed as Abound (Syngenta, Greensboro, NC) and pyraclostrobin and boscalid are marketed as a mixture under the trade name Pristine (BASF Corp., Research Triangle Park, NC). The two components of Pristine are currently not marketed individually for stone fruits. Azoxystrobin and pyraclostrobin are QoI fungicides that belong to group 11 with identical mode and site of action, whereas boscalid is a carboximide belonging to group 7 with a different mode and site of action (1). Both commercial products are labeled for various summer diseases including anthracnose, Alternaria leaf spot, powdery mildew, peach scab, and shothole caused by Colletotrichum sp., Alternaria sp., Sphaerotheca sp. and Podosphaera sp., Fusicladosporium carpophilum, and Wilsonomyces carpophilus, respectively. The in vitro sensitivity of the causal organisms of the Babygold summer diseases to both formulated products was determined using amended agar tests. An agar plug (6 mm) containing fungal mycelium was placed onto fungicide-amended PDA at final concentrations of 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 30, 100, and 300 mg/liter azoxystrobin or pyraclostrobin, and fungicide sensitivity was expressed as the concentration required to suppress radial growth of mycelium by 50% (EC 50 ) as described previously (18). Salicylhydroxamic acid (SHAM) was added to the fungicide-amended and control media at a final concentration of 100 mg/liter as described previously (14). The experiment was conducted with six replicates per concentration and was completely repeated once. Both independent experiments produced comparable results that were not statistically different (P > 0.05). Therefore both experiments are shown as a combined dataset in Table 2. Table 2. In vitro sensitivity of mycelial growth of C. acutatum, G. candidum, B. dothidea isolates to respiration inhibitor fungicides. Species Isolate Origin Azoxystrobin EC 50 (mg/liter)* Pyraclostrobin and boscalid C. acutatum 27 JC JC JC PH PH G. candidum 41 JC > JC > JC > PH > PH > B. dothidea 33 PH > PH > PH > PH > PH > * The 50% effective dose (EC 50 ) was determined in mycelial growth tests. Isolates from the three fungal species reacted very differently when grown on amended media (Table 2). C. acutatum isolates were susceptible to both formulated products with all five isolates being highly susceptible to the

6 pyraclostrobin-boscalid mixture. Azoxystrobin did not inhibit mycelial growth of B. dothidea or G. candidum isolates at concentrations up to 300 mg/liter, but the fungicide mixture inhibited growth of B. dothidea isolates. Isolates of G. candidum were tolerant to the fungicide mixture with EC 50 values between and mg/liter. The results indicate that fungicides from group 11 (e.g., azoxystrobin and pyraclostrobin) may not be suitable for controlling all pathogens of the summer diseases reported in this study. Conversely, the pyraclostrobin-boscalid mixture was effective against C. acutatum and B. dothidea with EC 50 values between 0.01 and 0.05 mg/liter, but its performance against G. candidum may indicate a weakness for sour rot control. The tolerance of G. candidum to respiration fungicides is not surprising because this yeast is known to be tolerant to other site-specific fungicides (8,9,11,12). During the disease outbreak in 2004 captan and sulfur-based products were applied to both sites in 10 to 14 day intervals. Pristine or any other respiration inhibitor fungicides were not used during that time. Based on our in vitro sensitivity tests, the inclusion of Pristine may improve cover sprays due to its efficacy against the pathogens causing anthracnose and Botryosphaeria fruit rot. During the 2005 growing season Pristine was applied as part of the cover spray program in orchards JC and PH and no disease outbreak was observed despite weather conditions conducive to summer disease development (G. Schnabel, personal observation). G. candidum primarily affects mature fruit and needs skin injuries for infection. The ability of G. candidum spores to induce disease may be increased by the presence of spores from other pathogens. Synergistic effects of spores of G. candidum and Penicillium digitatum were observed in infected citrus fruit (11). It would be interesting to investigate possible synergism between G. candidum spores and spores from the other pathogens of the Babygold summer diseases. If verified, such interactions could have significant impact on disease management strategies. For example, if spore mixtures of the here described causal agents do increase the ability of G. candidum to cause disease, a reduction of C. acutatum and B. dothidia inoculum with respiration fungicides may decrease sour rot incidence as well. Acknowledgments This work was supported by Gerber Products Company, Ft. Smith, AR. We thank P. Karen Bryson for technical assistance. Technical Contribution No of the Clemson University Experiment Station. This material is based upon work supported by the CSREES/USDA, under project number SC Literature Cited 1. Anonymous Pristine Fungicide. Tech. Info. Bull. BASF Corp., Raleigh, NC. 2. Barnett, H. L., and Hunter, B. B Illustrated Genera of Imperfect Fungi. American Phytopathological Society, St. Paul, MN. 3. Brannen, P. M., Horton, D., Bellinger, B., and Ritchie, D Southeastern Peach, Nectarine and Plum Pest Management and Culture Guide. Univ. of Georgia, Athens. 4. Ellis, M. A Bacterial spot on stone fruit. Ohio State Univ. Ext. Fact Sheet, HYG Fenn, P., Barczynska, H., and Miller, P Field evaluation of Benlate and Orbit for control of latent Phomopsis infection of developing peach fruit. Pages in: Horticultural Studies. J. R. Clark and M. D. Richardson, eds. Univ. of Arkansas, Fayetteville. 6. Gardes, M., and Bruns, T. D ITS primers with enhanced specificity for basidiomycetes: Application to the identification of mycorrhizae and rusts. Mol. Ecol. 2: Hanlin, R. T Illustrated Genera of Ascomycetes. American Phytopathological Society, St. Paul, MN. 8. Kaul, J. L., Sharma, R. L., and Shandilya, T. R Efficacy of different chemicals against Geotrichum candidum causing rour rot of citrus. Indian J. Mycol. Plant Pathol. 7:78-79.

7 9. Kuramoto, T., and Yamada, S DF-125, a new experimental fungicide for the control of satsuma mandarin postharvest decays. Plant Dis. Rep. 60: Li, X. Y., Gonzales, A. R., and Fenn, P Effects of Phomopsis peach rot on pulp losses and puree quality. J. Food Qual. 19: Morris, S. C Synergism of Geotrichum candidum and Penicillium digitatum in infected citrus. Phytopathology 72: Pritchard, F. J., and Porte, W. S Watery rot of tomato fruits. J. Agric. Res. 24: Schnabel, G., Bryson, K. P., Bridges, W., and Brannen, P. M Reduced sensitivity in Monilinia fructicola to propiconazole in Georgia and implications for disease management. Plant Dis. 88: Schnabel, G., Dai, Q., and Paradkar, M. R Cloning and expression analysis of the ATP binding cassette transporter MFABC1 gene and the alternative oxidase gene MfAOX1 from Monilinia fructicola. Pest Manag. Sci. 59: Watkins, C. B Fruit maturity. Pages in: Concise Encyclopedia of Temperate Tree Fruit. T. A. Baugher and S. Singha, eds. Haworth Press, Inc., Binghamton, NY. 16. White, T. J., Bruns, T., Lee, S. W., and Taylor, G Amplification and direct sequencing of fungal ribosomal RNA genes for phytlogenetics. Pages in: PCR Protocols: A Guide to Methods and Applications. T. J. White, M. Innis, D. H. Gelfand, J. J. Sninsky and M. A. Innis, ed. Academic Press, San Diego, Calif. 17. Zaitlin, B., Zehr, E. I., and Dean, R. A Latent infection of peach caused by Colletotrichum gloeosporioides and Colletotrichum acutatum. Can. J. Plant Pathol. 22: Zehr, E., Luszcz, L. A., Olien, W. C., Newall, W. C., and Toler, J. E Reduced sensitivity in Monilinia fructicola to propiconazole following prolonged exposure in peach orchards. Plant Dis. 83:

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