EVALUATION OF XANTHOMONAS ARBORICOLA pv. PRUNI INOCULATION TECHNIQUES TO SCREEN FOR BACTERIAL SPOT RESISTANCE IN PEACH AND APRICOT

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1 015_COST(Patohi)_S :58 Pagina 91 Journal of Plant Pathology (2012), 94 (1, Supplement), S1.91-S1.96 Edizioni ETS Pisa, 2012 S1.91 EVALUATION OF XANTHOMONAS ARBORICOLA pv. PRUNI INOCULATION TECHNIQUES TO SCREEN FOR BACTERIAL SPOT RESISTANCE IN PEACH AND APRICOT D. Soquet-Juglard 1,2, A. Patohi 1, J.F. Pothier 1, D. Christen 3 and B. Duffy 1 1 Agrosope Changins-Wädenswil (ACW) Researh Station, Phytopathology, Shloss 1, 8820 Wädenswil, Switzerland 2 Swiss Federal Institute of Tehnology Zürih (ETH-Z), Phytopathology, Universitätstrasse 2, 8092 Zürih, Switzerland 3 Agrosope Changins-Wädenswil (ACW) Researh Station, Conthey Researh Centre, Fruit growing and reeding group, 1964 Conthey, Switzerland SUMMARY Corresponding author: A. Patohi Fax: andrea.patohi@aw.admin.h Sreening for resistane to aterial spot of apriot (Prunus armeniaa) and other stone fruit speies suh as peah (P. persia) and plum (P. domestia), aused y the quarantine aterium Xanthomonas aroriola pv. pruni requires an inoulation method whih is rapid, effiient and whih permits to differentiate levels of resistane to this disease. Four inoulation tehniques were ompared under greenhouse onditions: (i) infiltration in the leaves using a needleless syringe; (ii) injuries aused y a needle; (iii) injuries aused y sissors; (iv) dipping shoots of the plant into the inoulum. All inoulation tehniques indued symptoms: infiltration was very effiient and quikly aused severe nerosis and leaf drop; the use of needle was also very effiient ut the symptoms were very weak, while the sissors tehnique was not as effiient as the first two approahes and the symptoms were also very weak. Finally dipping peah shoots into the inoulum was the least effiient in symptom indution, ut this tehnique permitted to otain more quantitative data suitale for evaluating the level of resistane of a ultivar than all the other tehniques (e.g. numer of spots per leaf, or damaged surfae, alulated disease inidene and resistane index). For this reason, the inoulation tehnique y immersion was applied to six apriot ultivars to e sreened for resistane against aterial spot. Plants were assessed 15, 28, 38 and 64 days post inoulation for spot numer, leaf surfae damaged, disease inidene and severity of symptoms. Cv. Orangered was the most resistant, showing low levels of damage to the leaves (10% +/- 3.54) and only a few symptomati leaves per plant ( /- 0.56). Cv. Goldar was the most suseptile with / % of symptomati leaves and high levels of damage per leaf (85 +/ %). Four other vs., Bergarouge, Bergeron, Goldrih and Kioto showed intermediate levels of suseptiility to the aterium. Results of resistane testing were omparale to those oserved under field onditions as reported in the literature. This indiates that shoot immersion into the inoulum is an effiient and reliale tehnique to sreen for resistane against aterial spot of stone fruits under greenhouse onditions. Key words: aterial spot, Prunus, inoulation tehniques, sreening for resistane. Xanthomonas aroriola pv. pruni (Xap), the ausal agent of aterial spot of stone fruits, is a quarantine pathogen for the European Union (EU) and the European Plant Protetion Organisation (EPPO). It mainly auses neroti lesions on leaves and fruits of peah, plum, apriot and netarine, ut in more severe ases, ankers, defoliations and death of the trees an e oserved (du Plessis, 1988). This aterium has een reported from all ontinents, and is still expanding its range (EPPO, 2006). In Europe, aterial spot has een identified first in northern Italy in 1934 (Petri, 1934) where it is now onsidered as endemi (Battilani, 1999). The disease ontinues to spread in Europe and outreaks have ourred more reently in Frane, Bulgaria, Romania and Ukraine (EPPO, 2006). In Switzerland the disease was deteted in 2005 on apriot trees (Pothier et al., 2010), and it is now onsidered as loally estalished. Due to lak of effiient hemial or iologial ontrol methods, host resistane might e the most promising solution to omat the disease. Different levels of tolerane against Xap have een deteted in peah, netarine, apriot and plum ased on field oservations (Simeone, 1985; Topp et al., 1989; Layne and Hunter, 2003; Garin et al., 2005; Garin and Bresson, 2011). The availaility of an artifiial inoulation tehnique allowing evaluations in a standardized quantitative manner of the degree of suseptiility or resistane (tolerane) of different ultivars under greenhouse onditions may foster the seletion or identifiation of resistant or tolerant ultivars. Few tehniques for leaf inoulation have een desried, mostly on peah (Prunus persia) and to a less extent on plum (P. domestia) and apriot (P. armeniaa). For pratial aspets, detahed leaf assays have often een used for pathogeniity assays. For example, Boudon et al. (2005) immersed detahed

2 015_COST(Patohi)_S :58 Pagina 92 S1.92 Inoulation tehniques for resistane sreening to Xap Journal of Plant Pathology (2012), 94 (1, Supplement), S1.91-S1.96 peah leaves in a aterial suspension and applied a moderate vauum pressure whereas Randhawa and Civerolo (1985) as well as Hammershlag (1988) used a syringe without a needle to infiltrate the aterial suspension into peah leaves. These tehniques permitted to oserve neroti lesions 2 weeks after inoulation (Boudon et al., 2005) and to otain differenes etween lones and ultivars 3 weeks after inoulation (Hammershlag, 1988). On-tree experiments have een reported for peah and plum, i.e. Miles and o-workers (1977) used a wind-rain mahine to expose trees in the field to a onentration of CFU ml -1 and Civerolo (1974) sprayed a seleted irular area of peah leaves and otained lesions 2 days post inoulation (dpi) with a onentration of 10 8 CFU ml -1. A system using pressure with a vauum pump was used y Sortihini et al. (1996) on peah, apriot, Japanese plum and sweet herry. Depending on the plant speies and ultivars tested, the time for otaining symptoms with this tehnique ranged from 20 to 25 days. Other systems using an artist s airrush (Zehr and Shepard, 1996; Shepard et al., 1999) or a spray gun onneted to a ompressed air supply (du Plessis, 1988) permitted to otain symptoms 4 weeks after inoulation on peah, plum, and apriot (du Plessis, 1988) or 3 dpi in peah at 30 C (Zehr and Shepard, 1996). Du Plessis (1987) used a needle to punture a droplet diretly into the vasular system via the petiole, main vein or seondary vein of plum shoots to study anker development. Finally, the immersion of an atively growing shoot of plum trees in the inoulum has suessfully een used in the open field to detet differenes in suseptiility etween ultivars, and therefore to determine a seletion index (Topp and Sherman, 1995). The ojetives of this study were to evaluate different inoulation tehniques under greenhouse ondition, identify the one that would allow a quantitative evaluation of the resistane level of a genotype to Xap and to test the tehnique on a set of apriot ultivars for whih field resistane data were availale. Two-year-old saplings of the rootstok GF305 (hereafter referred as peah plants ) were grown in 5 litre pots ontaining a mixture of peat and loam and were pruned efore short term storage at 4 C in a old hamer. Six different apriot ultivars were hosen for their range of resistane or suseptiility mainly ased on field oservations made y Garin and Bresson (2005), with two to eight repliates per ultivar. These plants were grafted on the plum rootstok Saint-Julien 655-2, potted in 2 litre pots and kept in a old hamer at 2 C for long term storage. Peah plants were otained from the Pépinières de Saxon (Switzerland), and apriot sions were from the Agrosope Changins-Wädenswil nursery at Conthey (Switzerland). All peah plants were grown under greenhouse onditions at 23 C, 60% relative humidity under natural onditions of light for one month and apriots for two weeks efore eing transferred to a quarantine greenhouse. No fertilization was applied. Four strains of Xap were used, two of whih were olleted in Valais, (Switzerland) in 2005 (XA1.29) and in 2007 (XA1.51), respetively. The other two strains were the pathotype strain NCPPB 416 isolated from P. saliina in New Zealand in 1953 and the sequened strain CFBP 5530 (Pothier et al., 2011) isolated from P. persia in Italy in For long-term storage, the strains were kept in 40% glyerol at -80 C. A loopful of a pure ulture of eah aterial strain was separately grown on peptone yeast extrat glyerol agar (NYGA) (Turner et al., 1984) at 28 C for 36 h. Cultures were then suspended in 1% sterile KCl and adjusted using a Genesys 10 UV spetrophotometer (Spetroni Uniam, USA) to an optial density of 0.5 at 600 nm orresponding to CFU ml -1, as estimated y ounting on YPGA plates. After adjusting the onentration of the different strains separately, equal volumes of the aterial suspensions were mixed just efore inoulation. Four different inoulation tehniques were tested on two peah plants per tehnique, presenting 5 to 10 atively growing shoots. The same tehniques were repeated with sterile KCl (resuspension uffer of X. aroriola pv. pruni strain mix) on different plants to serve as negative ontrol for omparison. The first tehnique onsisted in dipping sissors into the inoulum efore utting 4 to 10 leaves per ranh in the middle of the lamina. With the seond tehnique 8 wounds per leaf were made on 4 to 8 leaves per ranh with a needle, and droplet of inoulum of a. 5 µl was plaed on eah wound. With the third tehnique the inoulum was infiltrated with a 1 ml syringe without needle in 8 sites of the aaxial side of 4 to 8 leaves per shoot. Infiltration was stopped when a lear water-soaked spot appeared around the inoulation point. Finally, the fourth inoulation tehnique onsisted in the immersion of 4 to 5 atively growing shoots with 3 to 6 leaves in the aterial suspension and gentle agitation for aout 5 seonds until the leaf surfaes were fully wetted on oth sides. Following inoulations, onditions were set at 85% relative humidity and 23 C during daytime and 18 C during the night. No extra light nor fertilization were applied. The following data were reorded from inoulated peah plants, aording to the inoulation tehnique used: (i) total numer of leaves with symptoms (sissors); (ii) numer of neroti spots per leaf developing from the 6 to 8 inoulation points (needle and syringe); (iii) numer of symptomati leaves per shoot (dipping). All data were then expressed as perentage. Assessments were made 1, 2 and 3 weeks post inoulation. For sreening apriot ultivars, four assessments were made 15, 28, 38 and 64 days post inoulation, taking into aount four different traits: (i) numer of spots per leaf on four leaves per plant presenting most symptoms (ref-

3 015_COST(Patohi)_S :58 Pagina 93 Journal of Plant Pathology (2012), 94 (1, Supplement), S1.91-S1.96 Soquet-Juglard et al. S1.93 erene leaves); (ii) perentage of surfae damaged y the disease on those four leaves; (iii) disease inidene determined y ounting the numer of leaves per plant showing symptoms and y dividing y the total numer of leaves per ranh; and (iv) resistane index alulated with the following formula: where n 1 is the numer of leaves presenting 0 to 25% damage, n 2 26 to 50%, n 3 51 to 75%, n 4 76 to 100% and N is the total numer of leaves per plant. Student s t-tests were alulated for eah assessment using software JMP (SAS Institute, USA). The area under disease progress urve (AUDPC) was alulated for disease inidene and resistane index with Mirosoft Exel (Mirosoft Corporation, USA) using the following formula: where t is the time in days of eah measurement, y is the variale at eah measurement and n is the numer of measurements. Inidene and severity of aterial spot on inoulated peah plants varied greatly depending on the inoulation tehnique used. The first symptoms ould e oserved etween one and two weeks post inoulation (wpi). Water-soaked spots aused y infiltration eame neroti after already 1 wpi, resulting rapidly in high infetion perentages (100%, Tale 1). Symptoms were severe and many leaves dropped from 1 to 2 wpi, so that no quantitative results, e.g. size of the spots, ould e sored three weeks after inoulation. Yellow halos were present on all leaves around the water-soaked spot aused y the syringe 2 wpi. In average, 23% of the wounds aused per leaf y infeted needles eame neroti 2 wpi, and 3 wpi typial neroti spots were oserved at all inoulation points (Tale 1). As for the infiltration tehnique, yellow halos around the inoulation point were present on all leaves 2 wpi. Also in this ase no quantitative data ould e olleted 3 wpi, eause the spots remained too small. The tehnique using sissors did not permit to distinguish more than 4 spots per leaf developing losed to the wound area on the 50 leaves, even at 3 wpi, eause the spots were so small that the damaged surfae ould not e quantified. Nevertheless, at 3 wpi disease inidene reahed 48%. Finally, dipping the shoots into the inoulum permitted to otain muh more quantitative information pertaining to: (i) numer of symptomati leaves per ranh; (ii) numer of spots per leaf; (iii) perentage of damaged leaf area aused y the disease on the leaves. Dipped shoots presented in average aout 11% and 38% of symptomati leaves 2 and 3 wpi, respetively (Tale 1). This method was onsequently Tale 1. Disease responses to Xanthomonas aroriola pv. pruni in peah otained after inoulation y wounding with a needle, y infiltration with a syringe, y utting the leaf with infeted sissors and y dipping the plants into the inoulum. Standard deviations, when relevant, are indiated in rakets. Days Post Inoulation Needle 1 (n = 50) Infiltration 2 (n = 50) Sissors 3 (n = 50) Immersion 3 (n = 30) (18.78) (8.53) (12.43) 1 perentage of neroti spots per leaf that have developed from the inoulation point 2 perentage of nerosis per leaf otained from the inoulation point 3 disease inidene (%) Tale 2. Mean and standard deviation (std. dev.) of eah trait measured from the apriot varieties 64 days post inoulation. Means sharing the same letter were not signifiantly different using a t-test (p < 0.05). Cultivar N. of Spot numer 1 Surfae damaged (%) 1 Disease inidene (%) Resistane index repliates mean std. dev. mean std. dev. mean std. dev. mean std. dev. Bergarouge Bergeron a Goldar Goldrih a Kioto Orangered a a a a Calulated with data from the four most damaged leaves per plant

4 015_COST(Patohi)_S :58 Pagina 94 S1.94 Inoulation tehniques for resistane sreening to Xap Journal of Plant Pathology (2012), 94 (1, Supplement), S1.91-S1.96 hosen for a further test to assess the suseptiility to Xap of different apriot ultivars. Negative ontrols of all inoulation tehniques did not develop Xap symptoms. All apriot ultivars exhiited symptoms during the experiments, and a great variation of resistane levels to aterial spot was oserved (Tale 2). The first symptoms appeared 1 wpi on v. Goldar, whereas all other varieties remained symptomless (data not shown). Goldar reahed rapidly (28 dpi) 70% of symptomati leaves and up to 76% of infeted leaves at the end of the time-ourse experiment (64 dpi). The disease developed less rapidly in all other ultivars, and at the end of the experiment, vs Bergarouge, Goldrih and Kioto had aout 65% of their leaves showing symptoms, and v. Bergeron 50%. Only v. Orangered had less than 20% symptomati leaves during the whole experiment (Fig. 1A). Regarding the four leaves per plant presenting the strongest reation (referene leaves), only v. Orangered had signifiantly less leaf surfae damaged and v. Goldar had signifiantly more than the other varieties, ut when the average spot numer was taken into aount, v. Orangered was the only variety with signifiantly less spots than the other 5 (Tale 2). Although vs Kioto, Bergarouge and Bergeron had a relatively high disease inidene 64 dpi, the resistane index (Tale 2 and Fig. 1B) shows that their leaves remained moderately damaged, opposite to vs Goldrih and Goldar, for whih a high disease inidene was aompanied y a heavier damages on the leaves. Orangered was the most resistant ultivar in our experiment, taking oth disease inidene and resistane index into onsideration. Regarding the AUDPC of the disease inidene, no signifiant differene ould e oserved etween vs Bergeron, Kioto, Goldrih and Bergarouge. Only v. Orangered was signifiantly more resistant [AUDPC of /-737 standard deviation (std dev)] than the other ultivars, whih ranged from AUDPCs of 4583 (+/-2186 std dev) for v. Bergeron to 7524 (+/-1655 std dev) for v. Bergarouge, to (+/-2746 std dev) for v. Goldar, whih was signifiantly more suseptile (Fig. 2). When onsidering the AUDPC of the resistane index, however, v. Goldrih was signifiantly more suseptile (AU- d a a d d Fig. 2. AUDPC of the disease inidene (DIaudp, in grey) and AUDPC of the resistane index (RIaudp, in white) for the six apriot ultivars. Means (+standard error) sharing a letter were not signifiantly different using a t-test (p < 0.05). Fig. 1. A. and B. Disease inidene (a) and resistane index () for six apriot ultivars 15, 28, 38 and 64 days post inoulation. DPC of 196 +/-31 std dev) than the vs Bergarouge, Kioto and Bergeron, whih ranged from AUDPCs of 92 (+/-47 std dev) for v. Bergeron to 102 (+/-33 std dev) for v. Kioto (Fig. 2). Orangered was again signifiantly more resistant (AUDPC of 23, +/-7 std dev) and v. Goldar was signifiantly more suseptile (AUDPC of 299, +/-140 std dev). It was possile to indue Xap symptoms with all four inoulation tehniques tested. However, this study shows that quantitative expression of symptoms aused y X. aroriola pv. pruni may vary greatly in peah depending on the inoulation tehnique used. The high inoulum pressure indued y infiltration resulted rapidly and onsistently in a high level of damage ut the symptoms were so severe that often the inoulated leaves fell. Wounds aused y sissors and needles re-

5 015_COST(Patohi)_S :58 Pagina 95 Journal of Plant Pathology (2012), 94 (1, Supplement), S1.91-S1.96 Soquet-Juglard et al. S1.95 sulted into symptom development although the reations were not suffiiently quantitative. In oth the latter ases, small neroti points were visile after three weeks ut neither their exat numer nor the surfae of the leaf damaged ould e estimated. Inoulation y dipping the shoots into inoulum resulted in a larger array of symptoms and therefore in more variales useful to measure or ount. This proved to e the method that offered the est reproduiility without approahing the values zero or 100%, whih would not differentiate various levels of suseptiility/resistane of the host. This last tehnique an e onsidered as a more natural and less invasive method of inoulation than infiltration via a syringe (Kvitko et al., 2009). For these reasons we seleted this method to assess the levels of resistane to Xap of several apriot ultivars and to ompare them with field resistane evaluations. Garin and Bresson (2005, 2011) have assessed resistane to aterial spot of different apriot ultivars in naturally ontaminated orhards oserving important differenes in suseptiility that were partly dependent on the yearly limates. The six ultivars that we used in this study were also evaluated y Garin and Bresson (2011) for disease inidene over 5-7 years in the field. Their study lassified vs Goldar and Goldrih as very suseptile, vs Bergeron and Kioto as intermediate, and vs Bergarouge and Orangered as the least suseptiles, whih is in aordane with our findings. Measurements on the four referene leaves for spot numer and severity has proved not to e preise enough for an appropriate omparison etween the different varieties. Goldrih varied for disease reation depending on the puliation, i.e. Garin and Bresson (2011) lassified it as suseptile, whereas Simeone and Sortihini (2005) found it to e the most tolerant among 33 ultivars tested. In our study, v. Goldrih had a medium disease inidene, ut the resistane index showed that symptomati leaves were more damaged than those of vs Kioto, Bergarouge and Bergeron, so that in our experiment v. Goldrih was retained as rather suseptile. Finally, v. Bergarouge had a high disease inidene sore ut most of the leaves were almost undamaged, showing a potential of tolerane to the disease. In all ases, with sores etween 17 and 70% of symptomati leaves per ultivar, our artifiial inoulations on young plants in a greenhouse led to a muh higher disease inidene than that reported (2 and 38%) from field trials y Garin and Bresson (2011). We antiipate that this ould proaly e due to the fat that working with small plants in greenhouse permits to sore also small lesions, whih under field onditions may remain unnotied or masked y lesions due to other ioti or aioti fators. In this study, a dip inoulation tehnique was used to identify apriot ultivars showing a high resistane level to Xap. The results otained largely overlap with field oservations reported in the literature (Zaardelli, 1998). Although this tehnique may require, depending on the ountry, a quarantine greenhouse and other ontainment failities, we ame in three months time to very similar onlusions to those ased on field trials under natural onditions over a period of several years. Our tehnique is onsequently time-effiient and permits to onsider the total amount of leaves per plant as well as the progression of the disease on the same leaves without the risk of onfusion with other damages provoked y other pathogens or aioti fators. ACKNOWLEDGEMENTS Finanial support was provided y the Swiss State Seretariat for Eduation and Researh (SBF C and SBF C ). We thank Rolf Blapp, Jürgen Krauss and Verena Knorst for plant grafting and greenhouse support. REFERENCES Battilani P., Rossi V., Saardi A., Development of Xanthomonas aroriola pv. pruni epidemis on peahes. Journal of Plant Pathology 81: Boudon S., Maneau C., Nottéghem J.L., Struture and origin of Xanthomonas aroriola pv. pruni populations ausing aterial spot of stone fruit trees in western Europe. Phytopathology 95: Civerolo E.L., Quantitative aspets of pathogenesis of Xanthomonas pruni in peah leaves. Phytopathology 65: du Plessis H.J., Canker development on plum shoots following systemi movement of Xanthomonas ampestris pv. pruni from inoulated leaves. Plant Disease 71: du Plessis H.J., Differential virulene of Xanthomonas ampestris pv. pruni to peah, plum, and apriot ultivars. Phytopathology 78: Garin A., Rouzet J., Nottéghem J.L., Xanthomonas des arres fruitiers à noyau. Editions CTIFL, Paris, Frane. Garin A., Bresson J., Sensiilité des arres à noyau au Xanthomonas Bilan de huit ans d expérimentation. L aroriulture Fruitière 653: Hammershlag F.A., Seletion of peah ells for insensitivity to ulture filtrates of Xanthomonas ampestris pv. pruni and regeneration of resistant plants. Theoretial and Applied Genetis 76: Kvitko B.H., Park D.H., Velásquez A.C., Wei C.F., Russell A.B., Martin G.B., Shneider D.J., Collmer A., Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III seretion effetor genes reveal funtional overlap among effetors. PLoS Pathogens 5: e Layne R.E.C., Hunter D.M., AC Harostar Apriot. HortSiene 38:

6 015_COST(Patohi)_S :58 Pagina 96 S1.96 Inoulation tehniques for resistane sreening to Xap Journal of Plant Pathology (2012), 94 (1, Supplement), S1.91-S1.96 Miles W.G., Daines R.H., Rue J.W., Presymptomati egress of Xanthomonas pruni from infeted peah leaves. Eology and Epidemiology 67: Petri L., Rassegna dei asi fitopatologii osservati nel Bollettino della Stazione di Patologia Vegetale di Roma, NS 14: Pothier J.F., Pelludat C., Bünter M., Genini M., Vogelsanger J., Holliger E., Duffy B., First report of the quarantine pathogen Xanthomonas aroriola pv. pruni on apriot and plum in Switzerland. Plant Pathology New Disease Reports 20:15. Pothier J.F., Smits T.H.M., Blom J., Voröhlter F.-J., Goesmann A., Pühler A., Duffy B., Complete genome sequene of the stone fruit pathogen Xanthomonas aroriola pv. pruni. Phytopathology 101: S144-S145. Randhawa P.S., Civerolo E.L., A detahed-leaf ioassay for Xanthomonas ampestris pv. pruni. Phytopathology 75: Sortihini M., Janse J.D., Rossi M.P., Derks J.H.J., Charaterization of Xanthomonas ampestris pv. pruni strains from different hosts y pathogeniity tests and analysis of whole-ell fatty aids and whole-ell proteins. Journal of Phytopathology 144: Simeone A.M., Study on peah and netarine ultivars suseptiility to the main fungi and ateria. Ata Hortiulturae 173: Simeone A.M., Sortihini M., Susettiilità di ultivar di alioo nei onfronti della atteriosi da Xanthomonas aroriola pv. pruni nell Agro romano (Prunus armeniaa L.; Lazio). Rivista di Fruttioltura e di Ortoflorioltura 67: Topp B.L., Heaton J.B., Russell D.M., Mayer R., Field suseptiility of Japanese-type plums to Xanthomonas ampestris pv. pruni. Australian Journal of Experimental Agriulture 29: Topp B.L., Sherman W.B., Plum aterial spot resistane in leaves and stems measured y a seletion index. Ata Hortiulturae 403: Zaardelli M., Malaguti S., Bazzi C., Biologial and epidemiologial aspets of Xanthomonas aroriola pv. pruni on peah in Italy. Journal of Plant Pathology 80: Zehr E.I., Shepard P.D., Baterial spot of peah as influened y water ongestion, leaf wetness duration, and temperature. Plant Disease 80: