Potato Research 40 (1997) 3 9 9 - 406

Assessing potato cultivars in Tunisia for susceptibility to leak caused by Pythiumaphanidermatum SYLVIE P R I O U I. M.A. T R I K I 2.. M. EL M A H J O U B 2. M. F A H E M 3 and M. S O U I B G U I 4 IInternational Potato Center (CIP), Apartado 1558, Lima 12, Peru 2Laboratoire de Phytopathologie. Ecole Sup6rieure d'Horticulture (ESH), 4042 ChottMariem, Tunisia *Present address: Centre de Recherche Phoenicole. 2260 Degache, Tunisia 3Groupement lnterprofessionnel des L6gumes (GIL), 1 rue Ebnour Rachik, 1002 Tunis Belv6d~re, Tunisia 4Centre de Perfectionnement et de Recyclage Agricole (CPRA), 2031 Essa'l'da,Tunisia Accepted for publication: 24 September 1997

Additional kevwords: Solanum tuberosum L., watery wound rot

Summary The susceptibility of 21 potato cultivars to leak (watery wound rot) caused by Pvthium aphanidernlatunl was compared. Whole tubers were inoculated after wounding by dippihg into a water suspension of 10 3 oospores/ml and incubated for 3 days at 25 ~ The mean penetration of tissue calculated from the recording of lesion width and depth and the qualitative assessment of rot extension were highly correlated. For 10 of 12 cultivars tested at least twice, consistent reactions were obtained: cvs Korrigane, Superstar, Safrane and Yesmina were moderately susceptible, whereas Atlas, Diamant, Mondial. Ob61ix, Spunta and Timate were susceptible to highly susceptible. Tubers originated from two locations and/or two cropping seasons: thus. the performance of these cultivars under different production conditions reveals a satisfactory level of agreement with the test. Moreover, ratings assigned are supported by field or store observations.

Introduction Post-harvest losses of potatoes in Tunisia are mainly due to tuber leak (watery wound rot) in the field and during the summer in unrefrigerated storage. Two pathogens are responsible for these rots: Pythium aphanidermatum (Edson) Fitzp. and P. ultimum var. ultimum Trow, the former being the most frequently isolated from diseased tubers, stem lesions and soil (Priou, 1994). These pathogens are seed tubertransmitted and can persist in soil because of their ability to produce resistant organs: oospores for the former (Burr & Stanghellini, 1973), hyphal swellings and resting sporangia for the latter (Stanghellini & Hancock, 1971). Farms in Tunisia are small and thus successive cropping of potato in the same fields is commonplace, leading to increases in soil population levels of Pythium spp. High leak incidence compromises the local potato seed programme. In the absence of effective control, because soil fumigation is forbidden in Tunisia, some degree of management through resistant cultivars may be achieved, as has been reported for beans (Adegbola & Hagedorn, 1970). Moreover, cultivar trials on susceptibility to leak could increase the geographical distribution of less susceptible cultivars

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SYLVIE PRIOU, M.A. TRIKI, M. EL M A H J O U B , M. FAHEM A N D M. S O U I B G U I

wherever soil infestation or other factors enhance disease development. The most frequently used method for pathogenicity tests of Phytophthora erythroseptica, the closely related fungus that causes similar symptoms known as pink rot, consists of inserting an agar culture plug into a hole made with a cork borer (Fernandez-Northcote et al., 1972: Rowe & Schmitthenner, 1977). This method has also been used to compare potato cultivars and lines for susceptibility to a watery pinkish rot caused by P. crvptogea and P. parasitica (Grisham et al., 1983). The quantity of inoculum present in the culture fragment, however, may not be constant and as a result, the testing procedure is not uniform. Lennard (1980) reported differences in susceptibility of potato cultivars inoculated by dipping wounded tubers in a suspension of mycelium and oospores of P. erythroseptica, but the inoculum concentration seemed to be unknown. We thus developed a reproducible testing procedure using a defined concentration of inoculum and allowing the tuber to initiate defense reactions to the pathogen's multiplication. We compared 21 potato cultivars for their susceptibility to tuber leak caused by Pythium aphanidermatum (Pa), and assessed 12 of them at least twice to evaluate the importance of tuber origin and to make conclusions about the reliability of the test. Materials and methods

The strain P1 of Pa used for inoculations was isolated from brown vascular necrosis of a plant exhibiting leak symptoms on progeny tubers in 1993. Inoculum was prepared as follows: a 10-day-old culture of P1 grown on oatmeal agar was mixed in an electric blender with 50 ml sterile distilled water. The suspension composed of mycelium and oospores was homogenized and the oospore concentration evaluated with the Malassez counting chamber. The suspension, approximately 105 oospores/ml, was then diluted with sterile distilled water to the desired oospore concentration. Tubers were washed and surfaces sterilized in 1% sodium hypochlorite for 5 min. After rinsing with sterile tap water, tubers were allowed to dry for one hour before inoculation. Concentrations of 1, 10, 102, 103 and 104 oospores/ml were first investigated with tubers of cv. Spunta to evaluate the discriminating inoculum concentration and the infection threshold. Three punctures per tuber to a depth of 5 mm were made equidistant along the long axis on one side of the tuber using the point of an iron wire 2 mm in diameter. Tubers were immediately dipped into the oospore suspensions for 15 min. Tubers were then placed on egg-plugs in plastic trays containing moistened paper on the bottom, wrapped with plastic film, sealed with a rubber band to maintain a high relative humidity and incubated for three days at 25 ~ The tubers were then sliced across the inoculation holes, the maximum width (W, mm) and depth (D, mm) from the surface along the inoculation hole were recorded and the penetration of tissues (Pen, mm) was calculated following the formula described by Lapwood et al. (1984): Pen=(W/2+[D-5])/2. Data were recorded for the three inoculation sites per tuber and the mean 400

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penetration was calculated for each tuber. Individual tubers were taken as replicates for analysis of variance according to a one-factor complete randomized block design (MSTAT-C). Rot extension was also qualitatively assessed by the following fivepoint scale: 0=no necrosis, l=necrosis extended to less than 1/3 of the tuber, 2=rot extended between 1/3 and 2/3 of the tuber, 3=rot spread to more than 2/3 but the tuber was not completely rotted, 4=the whole tuber rotted. The disease index (DI) was then given by the mean of scores recorded for all tubers of each cultivar. According to the mean penetration obtained for each potato variety, a qualitative susceptibility grouping was given: moderately susceptible (MS, Pen_<6), susceptible (S, 6l 1). Tubers of 15 cultivars listed in Table 2, originating from a fall crop, were supplied in December 1994 by the Centre de Perfectionnement et de Recyclage Agricole (CPRA) of Essa'fda (Essa'l'da I). The C P R A also provided tubers of 11 cultivars from a spring crop (Essa'ida 2). Ten tubers per cultivar were tested one month after harvest in January and June 1995 for Essa'fda l and 2, respectively. Twelve cultivars were harvested in June 1995 at the G r o u p e m e n t Interprofessionnel des L6gumes (GIL) of Bousalem and 15 tubers per cultivar were inoculated after three months of refrigerated storage (Bousalem). Results and discussion

Evaluation of infection threshold and discriminating inoculum concentration. Fig. 1 shows the symptoms obtained after inoculation of wounded tubers with the oospore suspension of Pa. After 5 rain. of exposure to air, the sliced tubers showed a characteristic pinkish discouloration. The mean penetration significantly increased as the oospore concentration increased (P=0.001. Table 1). An infection threshold of 10 oospores/ml was found. With up to 102 oospores/m[, many tubers remained uninfected and others rotted only around the inoculation sites, probably because more oospores are needed to colonize and penetrate the wound and overcome defense reactions. The inoculum concentration of 103 oospores/ml was the most discriminating: all tubers were infected, but not entirely rotted, as was observed with a concentration of 104 oospores/ml. This last inoculum dose seemed to be too severe and would probably not allow cultivar comparisons. Moreover. by mixing one O M A plate with 50 ml sterile distilled water, a concentration of approximately 105 oospores/ml was obtained, with one liter of inoculum needed to inoculate 10 tubers by dipping. Thus, the inoculum dose of 103 reproductive organs/ml is readily achieved. Cultivar comparisons. Table 2 presents the mean penetration for each cultivar as well as the disease indices. For the three lots tested, differences between cultivars were significant (P<0.001). There was no significant difference between the sources of tubers except for the Bousalem samples. Variability between tubers seems to be higher for the samples stored for three months at 3 ~ For any of the samples tested, however, we noticed that the standard deviations were high and at least equal to the Potato Research 40 (1997)

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SYLVIE PRIOU, M.A. TRIKI, M. EL M A H J O U B , M. FAHEM A N D M. SOU1BGUI

Fig. 1. (A) Different levels of susceptibility of potato tubers (cv. Spunta) inoculated after wounding by dipping in an oospore suspension of Pvthium aplranidermatum and incubated for 3 days at 25 ~'C. (B) After five days of incubation at 25 '~'C~the characteristic black colour of the tissue beneath the lenticels and the pink discolouration of the potato flesh is intensilied.

Table 1. Mean penetration of tissues (Pen) and percentage of tubers infected (%TI) after inoculation of 15 potato tubers with each of live concentrations of oospores of Pvthium

aphanidernlatum. lnoculum concentration (oospores/ml)

Pen* mm

SD**

I 10 102 103 104

0.5 d 3.4 c 5.1 c 12.1 b 17.2 a

f).0 3.4 3.2 5.3 2.7

%TI 0115 8/15 I 1/15 15/15 15/15

* Means followed by the same letters are not signillcantly different at P=0.01 as determined by Duncan's multiple range test. ** SD. standard deviation of penetration.

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Potato Research 40 (1997)

ASSESSING

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Potato Research 40 (1997)

403

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means, Table 2. Indeed, for any cultivar, many tubers remained uninfected, with the frequency of healthy tubers depending on the relative susceptibility of the cultivar. This could be the result of tuber resistance to multiplication of the pathogen probably caused by physiological differences among tubers. A similar variability of tuber responses occurs following inoculation with soft rot erwinias. The level of susceptibility to leak might be the result of an interaction between genetic resistance and physiological characteristics of the tuber caused by nutrition, age and storage conditions as demonstrated for soft rot (Hidalgo & Echandi, 1983: Ibrahim et al., 1979; Abgrall et al., 1990; McGuire & Kelman, 1984; W o r k m a n et al., 1976). A high correlation between Pen and DI was found for the values listed in Table 2 (r=0.96, P<0.001). Thus, visual evaluation of the rotted part of the tuber seems to be an accurate means of assessing rot extension. Moreover, this notation method is quick and simple and would be useful for screening large numbers of tubers. For the 12 cultivars tested at least twice, no change in susceptibility grouping could be observed except for Baraka and Claustar. Cvs Korrigane, Superstar, Safrane and Yesmina were found to be moderately susceptible, whereas Atlas, Diamant, Mondial, Ob61ix, Spunta and Timate were susceptible to highly susceptible cultivars. Correlations between tuber origins and mean penetration were calculated for the cultivars tested at both sites (Table 3). The six potato cultivars tested in common at Bousalem and Essaida 1 or at Bousalem and Essaida 2 had similar susceptibility ranks as attested by high correlations (Table 3). But the testing of eight common cultivars from Essa'l'da 1 and Essai"da 2 showed contradictions for cvs Baraka and Claustar. Thus, cropping season (spring or autumn) could change tuber physiology or components that lead to differences in susceptibility to leak for certain cultivars. Cv. Spunta exhibited a higher rot extension at Essai"da 2 than at Essa~'da 1, but its susceptibility grouping remained unchanged. At Essaida 2, the mean penetration was greater when compared with the two other tuber origins since the range of lesion sizes was larger for most of the tubers, whereas at Bousalem it was lower than Essaida 1 and 2, with a higher frequency of tubers remaining healthy. Unfortunately, we cannot conclude whether this is the result of particular cultural practices such as fertilization, watering] soil quality, dates of planting or harvest. Differences in manure application and nitrogen fertilization, which were much higher at Essa'l'da 2 than at Bousalem, and higher at Essai"da 2 (spring crop) than at Essai"da 1 (autumn

Table 3. Correlations between tuber origins and mean penetration for cultivars tested at both sites.

Number of cultivars in common Mean penetration Correlation coefficient

Essalda 1/Bousalem

Essaida 2/Bousalem

Essafda 1/Essaida 2

8 8.35 0.214

6 9.80 0.830*

6 11.70 0.806*

12.95

5.90

7.25

* Significant at P<0.05. 404

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A S S E S S I N G POTATO SUSCEPTIBILITY TO LEAK

crop), could explain a higher tuber susceptibility. Moreover, the growing season was 100 days at Essa'l'da 2 and 120 days at Bousalem; thus, tuber physiological age was greater at Bousalem. This lower susceptibility of tubers from Bousalem might also be due to the three months refrigerated storage before testing. For another storage disease, such as soft rot, however, Ibrahim et al. (1979) observed an increase in susceptibility to soft rot, with an increasing age of tubers, and Otazu & Secor (1981) and Hidalgo & Echandi (1983) reported increasing susceptibility to Erwinia chrysanthemi with increasing length of storage at 4 ~ because of higher reducingsugar content.

Conclusions Further studies are needed to determine the role of tuber physiology in susceptibility to leak because tuber components are influenced by planting season, fertilization, watering, soil quality, storage length and temperature. No relation between differences in earliness of cultivars and their susceptibility to leak was apparent. The performance of the four reference cultivars, Korrigane, Spunta, Timate and Yesmina, under the three different production conditions reveals a satisfactory level of agreement of the test. Moreover, ratings assigned are supported by field or store experiences: cv. Superstar being moderately susceptible to Pa as observed in greenhouses and field trials (data not shown): Mondial, Nicola, Spunta and Timate, the most common cultivars planted in Tunisia, frequently exhibiting high rates of infection. Thus, validity of this test method as a means for assessing susceptibility to leak is demonstrated for these potato cultivars.

Acknowledgements We thank the technicians of the Phytopathology Laboratory of the Ecole Sup6rieure d'Horticulture, Chott-Mariem (Tunisia), for technical assistance and Dr Edward French (CIP-Lima) for valuable comments on the manuscript and Aiain Baudry from the Laboratoire National de Protection des V6g6taux (LNPV), Villenave d'Ornon, France, for characterizing the Pythium isolates.

References Abgrall, J.Y., B. Jouan & E. Guillery, 1990. Incidence de quelques facteurs agronomiques sur I'expression de la jambe noire dans les cultures de pomme de terre. Proceedings of the llth Triennial Conference of the European Association for Potato Research (EAPR), Edinburgh. pp. 297-298. Adegbola, M.O.K. & D.J. Hagedorn, 1970. Host resistance and pathogen virulence in Pythium blight of bean. Phytopathology 60:1477-1479. Burr. T.J. & M.E. Stanghellini, 1973. Propagule nature and density of Pythium aphanidermatltm in soil. Phytopathology 63: 1499-1501. Fernandez-Northcote, E.N., E. Huapaya & H. Torres, 1972. La podredumbre rosada de la papa: btisqueda de fuentes de resistencia en tub6rculos. Fitopatologia 6: 18-27.

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Grisham, M.P., R.A. Taber & L.W. Barnes, 1983. Phytophthora rot of potatoes in Texas caused by Phytophthora parasitica and P. cryptogea. Plant Disease 67: 1258-1261. Hidalgo, O.A. & E. Echandi, 1983. Influence of temperature and length of storage on resistance of potato to tuber rot induced by Erwinia chrysanthemi. American Potato Journal 60: 1-15. Ibrahim, M., B. Jouan, R. Samson, F. Poutier & M. Sailly, 1979. Prospect of a pathogenicity test concerning Erwinia carotovora var. atroseptica and var. carotovora on half potato tubers. Proceedings of the 4th International Conference of Plant Pathogenic Bacteria, Angers, France, pp. 591-602. Lapwood, D.H., P.J. Read & J. Spokes, 1984. Methods for assessing the susceptibility of potato tubers of different cultivars to rotting by Erwinia carotovora subspecies atroseptica and carotovora. Plant Pathology 33: 13-20. Lennard, J.H., 1980. Factors influencing the development of potato pink rot (Phytophthora erythroseptica). Plant Pathology 29: 80-86. Mc Guire, R.G. & A. Kelman. 1984. Reduced severity of Erwinia soft rot in potato tubers with increased calcium content. Phytopathology 74: 1250-1256. Otazu, V. & C.A. Secor, 1981. Soft rot susceptibility of potatoes with high reducing sugar content. Phytopathology 71: 290-295. Priou, S., 1994. CIP (International Potato Center) Program Report 1993-1994. Lima, Peru. Rowe, R.C. & A.F. Shmitthenner, 1977. Potato pink rot in Ohio caused by Phytophthora erythroseptica and Phytophthora crvptogea. Plant Disease Reporter 61: 807-810. Stanghellini, M.E. & J.G. Hancock, 1971. The sporangium of Pythium ulthnum as a survival structure in soil. Phytopathology 61: 157-164. Workman, M., E. Kerschner & M. Harrison, 1976. The effect of storage factors on membrane permeability and sugar content of potatoes and decay by Erwinia carotovora subsp. atroseptica and Fusarium roseum vat. sambucflTtan. American Potato Journal 53:191-204.

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