Fusarium eumartii - Springer Link

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Euphytica 80: 63-69, 1994. (~) 1994 Kluwer Academic Publishers. Printed in the Netherlands.

Potential of a Fusarium eumartii culture filtrate on the screening for wilting resistance in potato G r i s e l a L. B o t t a , M a r f a P. D i m a r c o , A l i c i a L. M e l e g a r i , M a r c e l o A. H u a r t e & C a r l o s A. B a r a s s i Unidad lntegrada Balcarce (INTA-UNMP). CC 276, 7620 Balcarce, Argentina Received7 February 1994; accepted 19 August 1994 Key words: Fusarium solani f. sp. eumartii, culture filtrate, electrolyte leakage, potato wilting, resistance, toxin, Solanum tuberosum

Summary Fusarium solani f.sp. eumartii Carp. Snyder and Hansen (Fusarium eumartii) is a soil inhabitant that induces the so-called Potato Wilt and Stem End Rot disease. Prior to wilting, the pathogen induces peculiar small bronze spots on the leaflets. Failure to isolate E eumartii from infected leaflets suggests the involvement of a toxin in the disease. The fungus was grown in liquid Richard's medium and thereafter a filtrate was obtained dialyzing (MW cutoff 12,000-14,000) and sterilizing the culture by filtration (0.22 #m). Potato leaves treated with both the pathogen or the filtrate showed symptoms of bronze spots and significantly higher electrolyte leakage when compared to controls. Tomato leaves showed neither bronze spots nor electrolyte leakage after plant inoculation with the pathogen or with the filtrate treatment. Both, the absence of visible symptoms and the lack of electrolyte leakage in tomato could be associated to a certain degree of host specificity of the F. eumartii filtrate towards potato. The filtrate also induced symptoms similar to infections by E eumartii in adult plants and in vitro plantlets of cultivars Huinkul MAG and Kennebec. Callus responses to the filtrate were related to responses of the cultivars to the pathogen in greenhouse. These results show the potential of the culture filtrate of E eumartii for use in screening for wilting resistance.

Introduction Potato Wilt and Tuber Stem End Rot caused by Fusarium solani f.sp. eumartii Carp. Snyder and Hansen (Fusarium eumartii) is endemic in Argentina and was reported to occur in some regions of Chile, Brazil and USA (Calderoni, 1978; Gerlach & Nirenberg, 1982). Yield losses close to 40% were registered in the susceptible cultivar Huinkul MAG under a severe epidemic outbreak in Argentina (Malamud, 1970). The pathogen is a soil inhabitant that infects potato plants through roots and seed-tuber pieces and its invasion is restricted to the stem base, stolons and tuber stem end. Prior to wilting, the pathogen induces small bronze spots on the leaflets. In tubers, the fungus produces stem end dry rot and brown discoloration of the vascular ring. Failure to isolate E eumartii from infected leaflets and vascular ring suggests the involvement of a toxin in the disease (Goss, 1924; Thomas, 1949). However,

neither the isolation of such a toxin nor the use of culture filtrates for breeding purposes have been reported so far. The objectives of this study were a) to obtain a F. eumartii filtrate able to reproduce in vitro the symptoms observed in vivo after fungal infection, b) to determine the effect of the filtrate on membrane permeability of leaves of the susceptible cultivar Huinkul MAG and its specificity towards potato and, c) to determine whether cultivar responses to the filtrate are related to cultivar responses to the pathogen.

Materials and methods Fungal culture and filtrate preparation A virulent strain of E eumartii (strain A) was cultured on 2% potato dextrose agar (PDA), incubated 1 wk at 250 C in the dark and 2 wk at 180 C 4- 20 C under

64 continuous light (60/~E m -2 s-l). A 0.5 cm diameter agar disc with fungal growth was transferred to a 250 ml flask with 100 ml of liquid Richard's medium containing 10.0 g KNO3, 5.0 g KH2PO4, 2.5 g MgSO4.7H20, 0.02 g FeC13 and 50.0 g sucrose per liter of distilled water (The Commonwealth Mycological Institute, 1985). Cultures were incubated 4 wk at 220 C + 20 C in the dark with shaker agitation at 180 r min- 1 during 9 h per day. Cultures were filtered through Whatman N O 1 paper and dialyzed (MW cutoff 12,000-14,000) against 11 of distilled, deionized water during 24 h at 4 ° C. Initial and final conductivities were 17.4 and 4.5 mmhos cm-1 at 250 C respectively, both in culture and in control filtrates. After pH adjustment to 7.0 with 1 M KOH, the liquid was sterilized by filtration with a SARTORIUS SM 16510 filter unit, provided with a GCFMS 9043 MM pre-filter and a 0.22 #m filter. Non-inoculated Richard's medium was equally dialyzed and filtered to be used in control treatments. Filtrates were stored at 40 C in the dark until used. When required, conidial suspensions were obtained as follows: mycelia were scraped from PDA growth and suspended in sterile, distilled water. Conidia washings were accomplished by successive centrifugations and re-suspensions in sterile distilled water. Final concentration was 3.0 x 10 6 conidiam1-1. Bioassay Non-inoculated whole leaves of Solanum tuberosum cultivar Huinkul MAG and Lycopersicum esculentum cultivar Plautaco INTA, were used to study the effect of the filtrate on membrane permeability. Potato leaves were detached from the middle portion of 8-wk-old healthy plants growing in greenhouse at 180 C + 80 C. Only one leaf was taken from each plant. Tomato leaves were excised from seedlings with two leaves growing in greenhouse under the conditions mentioned above. Only leaf petioles were immersed either in F. eumartii filtrate or in Richard's filtrate and incubated at 22 ° C -42 ° C, 16 h light (90 #E m - 2 s- 1)/8 h dark and humidity close to 100%. After 48 h of incubation, leaves were examined for presence of bronze spots and modified cell permeability. Changes in cell permeability were determined with a conductivity method which measures ion leakage (Sukumaran & Weiser, 1972). All the leaflets of each leaf were placed in an individual flask containing 30 ml of distilled, deionized water and incubated at 22 ° C with shaker agitation at 180 r min -1. After 1 h shaking, initial liquid conductance was measured with a Metrohm 644 Conductometer.

Leaflets were frozen at - 600 C during 24 h to kill the tissues, re-immersed in the original liquid and shaken for 1 h. Final conductivity of the liquid was measured. Percent conductivity, calculated as the ratio of the initial to the final conductivities, was expressed as percent of electrolyte leakage. Electrolyte leakage was also measured in leaves with bronze spot symptoms of cultivar Huinkul MAG inoculated with F. eumartii and in symptomless leaves of controls. In all the experiments each leaf represented one sample. No more than one sample was taken from each plant. Means were compared by Duncan's test (p < 0.025). Confidence limits were constructed for the comparisons. Potato callus and in vitro plants Potato stem cuttings from in vitro plants were cultured in the minimal organic medium of MurashigeSkoog (Murashige & Skoog, 1962), containing 30.0 g sucrose, 8.0 g agar, 2.5 g gibberellic acid and 0.002 g calcium pantothenate per liter of distilled water, and incubated at 22 ° C, 18 h light (70/_rE m - 2 s- 1)/6 h dark. Multiplication was carried out every six weeks. Leaflets of potato cultivars Huinkul MAG, Kennebec and Russet Burbank were used for callus production. Leaf discs (10 mm diam) were successively immersed in 70% ethanol for 10 sec and in NaOC1 (2% C1) for 15 min, rinsed three times in sterile distilled water and dried on sterilized filter paper. Discs were cultured on Murashige-Skoog minimal organic medium, plus 30.0 g sucrose and 8.0 g agar per liter of distilled water. Medium was supplemented with 2 mg l-1 naftalen acetic acid (NAA) and 0.5 mg l6-benzilaminopurine (BPA) (Ochatt & Caso, 1986) for cultivars Huinkul MAG and Russet Burbank, or 5 mg 1-1 NAA and 1 mg 1-1 BAP for cultivar Kennebec (Botta, 1992). Cultures were incubated at 220 C, 18 h light (70 #E m -2 s-1)/6 h dark. Greenhouse experiments Three sets of experiments are included in this section: one was designed to check strain virulence and specificity towards potato and the other two, to evaluate disease incidence (DI) after inoculation with conidia and after inoculation with the fungal filtrate, respectively. Plants of S. tuberosum cultivar Huinkul MAG and L. esculentum cultivar Plautaco INTA growing in autoclaved soil, were inoculated with E eumartii. Inoculation was carried out at stages of four and two devel-

65 80 C. Leaves were periodically examined for the presence of bronze spots. For the following experiments, three-week-old in vitro plants of cultivars Huinkul M A G and Kennebec were individually transplanted into pots containing soil sterilized with methyl bromide (450 g/1.87 m 3 of soil). Plants grew in the greenhouse for 30 days at 180 C 4- 80 C. Prior to inoculation, plants were removed and their roots washed with distilled water. In one experiment, roots were immersed in a conidial suspension containing 3.0 × 10 6 conidia m1-1. Treatments were arranged in a completely randomized design with four replications including 20 plants each. In the other experiment roots were immersed in the filtrate. Treatments were arranged in a completely randomized design with five replications including 20 plants each. Plants with root immersion in distilled water or in Richard's filtrate were used as controls in the first and in the second experiment, respectively. After treatment, plants were transplanted to the same pots. Disease incidence was recorded as the number of wilted plants after 50 days from inoculation. Percentages transformed by the arcsin square root were subject to analysis of variance. Means were compared by Waller's test (p _< 0.05). In vitro e x p e r i m e n t s

Fig. 1. Early symptoms on potato leaves infected by E eumartii or treated with F. eumartii filtrate. Interveinalbronze spots scattered on leaflets from (A) F. eumartii - infected potato plants, and (B) leaves treated with E eumartii filtrate.

oped leaves in potato and tomato, respectively. Prior to inoculation, plants were removed, their roots washed with distilled water and finally immersed for 5 min in distilled water containing 3.0 × 10 6 conidia m l - 1. Plants with root immersion in autoclaved inoculum were included as controls. Inoculated plants were transplanted to the original pots and grown at 180 C 4-

Four-week-old in vitro plants of cultivars Huinkul MAG and Kennebec individually cultured in test tubes, were inoculated with 0.5 ml of filtrate injected in the medium near the roots. Plants inoculated with the same amount of Richard's filtrate were included as controls. Plants were incubated at 220 C, 18 h light (70 # E m -2 s - 1)/6 h dark. Treatments were arranged in a completely randomized design with five replications, including fifteen plants each. Disease incidence was recorded as the number of wilted plants after 12 days from inoculation. Percentages transformed by the arcsin square root were subject to analysis of variance. Means were compared by Waller's test (p < 0.05). Ten-day-old callus of cultivars Huinkul MAG, Kennebec and Russet Burbank selected for uniform size, were cultured in petri dishes containing the medium and the growth regulators used for callus production. In one experiment, calli were inoculated by applying 0.05 ml of the filtrate on the surface. Treatments were arranged in a completely randomized design with five replications, each including five petri dishes and four calli per dish. After 12 days, calli were scored on a scale of 0-3, where 0 = absence of symptoms; 1 =

66 loss o f turgor and necrosis restricted to the inoculation point; 2 = loss o f turgor and partial necrosis; 3 = generalized necrosis. Disease severity (DS) was expressed as: DS = ~(i=0-i=3)Xi(ni)/Xt(n3), where ni = score in the severity scale; Xi = number of callus in class ni; Xt = total number o f callus. In other experiment, calli were cultured on media containing the filtrate. Filtrate was added to the previously autoclaved and cooled medium to reach final concentrations o f 10, 25 and 50% (v:v). Treatments were arranged in a completely randomized design with three replications, each including 62, 96 and 110 calli o f cultivars Russet Burbank, Huinkul and Kennebec, respectively. No more than five calli per dish were included. After 30 days, calli were scored on a scale o f 0 - 2 , where 0 = normal growth and green colour; 1 = impaired growth and restricted necrosis; 2 = absence o f growth and generalized necrosis. Disease severity was expressed as: DS = )](i=0_i=2)Xi(ni)/X t(n2). In both experiments, calli treated with equivalent amounts and concentrations o f Richard's filtrate were used as controls. Cultures were incubated at 220 C, 18 h light (70 # E m - 2 s - l ) / 6 h dark. Means of disease severity were compared with Waller's test (p