Plant breeding

Plant Breeding Plant Breeding Volume 120 Page 435 October 2001 doi 10 1046/j 1439 0523 2001 00626 x Volume 120 Issue 5 A first attempt to use a Fusarium subglutinans culture filtrate for the selection[.]

Plant Breeding Volume 120 Page 435  - October 2001 doi:10.1046/j.1439-0523.2001.00626.x Volume 120 Issue A first attempt to use a Fusarium subglutinans culture filtrate for the selection of pineapple cultivars resistant to fusariose disease O Borrás1, R Santos2, A P Matos3, R S Cabral3 & M Arzola2 Pineapple, Ananas comosus L Merr., cultivars differing in resistance to fusariose were examined for the phytotoxic effect of culture filtrate (CF), and fusaric acid (FA) isolated from Fusarium subglutinans (Wollew & Reinking) Nelson, Toussoun & Marasas The possibility of using these substances on F1 hybrids as selection agents for resistance was also evaluated The phytotoxic effect of these substances was assessed in pineapple plantlets from tissue culture by placing them on wounded leaf segments The resistance level of the F hybrids and cultivars under natural conditions was measured in greenhouse field tests The susceptible cultivars proved to be the most sensitive in each test, whereas resistant cultivars showed resistance to CF FA affected all the cultivars independently of the degree of resistance of the cultivars The presence of other metabolites in CF was responsible for the screening effect between the varieties Using CF as selection criteria allowed the selection of resistant plants with behaviour quite similar to plants where the fungus itself was used as the selection agent Fusariose, produced by Fusarium subglutinans, constitutes the most serious pineapple disease in the main production area where it was first reported in Brazil Causing losses as high as 80% of marketable pineapple fruits, the pathogen infects approximately 40% of the asexual propagative material and kills about 20% of the pineapple plants prior to harvest The pathogen is able to infect all parts of the pineapple plant and the development of the disease results in the production of a characteristic gum, as well as bending of the stem (usually to the side where the lesion is located), changing the plant phyllotaxis, increasing the number of leaves per spiral, changing the plant architecture, making it funnel-like, shortened stem, death of the apical meristem and chlorosis (Matos 1995) Control of pineapple fusariose has been based mainly on the use of pathogen-free propagating material, on the evasion of the pathogen and the direct protection by chemical control (Matos 1995) Chemical control is costly and often ineffective, and may eventually lead to isolates of the pathogen being resistant to fungicides Moreover, the application of chemicals for crop protection should be reduced because of environmental concerns (Pinho et al 1997) The potential for disease resistance as a control measure for the pineapple fusariose has been suggested in studies based on observations carried out either under field conditions or by using artificial inoculation techniques (Matos et al 1991) Growing resistant cultivars constitutes one of the cheapest and most efficient control measures and the identification of sources of resistance to Fusarium subglutinans is a very important step in obtaining pineapple varieties resistant to this pathogen (Cabral and Matos 1995) A number of plant pathogens produce toxins in association with a number of different diseases ( Durbin 1981, Goodman et al 1986, Hamer and Holden 1997, Lucas 1998) For example, fungi of the genus Fusarium produce a variety of biologically active metabolites in culture filtrate (CF); this CF is reported to be toxic to cotyledons, germinating seeds and plants of many vegetable species (Jin et al 1996, Matsumoto et al 1999a) Fusaric acid (5-n-butylpicolinic acid; FA), produced by several fungi of the genus Fusarium associated with wilt of banana, cotton, pea, tomato, and other plants, affects membrane permeability, increasing leakage of potassium and other electrolytes, and inhibits respiration (McLean 1996, Kuzniak et al 1999) Experiments with FA produced in culture filtrates have shown that tissue response correlates with the disease reaction of the host variety (Remotti et al 1997) Thus, in these cases, selection using a phytotoxin might offer an alternative to field selection, as reported for a number of host–pathogen combinations, and it may allow selection of important traits in disease resistance (Daub 1986) Creation of genetic variability and a suitable procedure for the reliable identification of resistant genotypes constitutes two prerequisites for improvement of disease resistance Screening for disease resistance in the field is time-consuming, costly and dependent upon natural fluctuations in inoculum abundance and weather factors that influence pathogen spread, infection, disease development and disease expression (Ostry 1997) The aim of this study was to determine the phytotoxic effect of CF and FA produced by F subglutinans on pineapple leaves of susceptible and resistant cultivars Moreover, the possibility of using of CF and FA as selection tools for disease resistance was evaluated Materials and Methods Culture filtrates and fusaric acid production: An isolate of Fusarium subglutinans (isolated by the Bioplants Centre, Ciego de Avila, Cuba) was grown on potato dextrose agar (PDA) dishes for 7 days at 26 ± 2°C in alternating 12-h light at 4.00 lx and 12-h darkness, provided by warm-white fluorescent tubes Dishes of fungal mycelium were inoculated in Czapek–Dox broth (1 l, with 2 g NaNO3, 1 g K2HPO4, 0.5 g MgSO4.7H2O, 0.5 g KCl, 10 mg FeSO4, and 30 g sucrose, with 1 ml of 1% (w/v) ZnSO4 and 0.5% (w/v) CuSO4) The cultures were grown in 250-ml Erlenmeyer flasks with 100 ml of medium at 26 ± 2°C with the same light conditions described above After 3 weeks of stationary cultivation, mycelium and conidia were filtered through Whatman No filter paper (Whatman, Clifton, NJ, USA) and a 0.2-μm Millipore membrane (Sartorius, NG, Göttingen, Germany) The CF was evaporated under reduced pressure at 40°C to 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100% (v/v) using a rotary evaporator (Heidolph, Bioblock, Paris, France) The isolation, extraction and purification of FA were performed using the methodology of Bacon et al (1996) Bioassay: The phytotoxic effect was determined by placing the CF or FA in different concentrations on wounded leaf segments FA concentrations of each culture filtrate concentrations were used The cultivars were 'Perolera' (resistant), 'Spring' (resistant) 'Perola' (susceptible) and 'Smooth Cayenne' (susceptible) Leaf segments (approximately 20 mm) of 90day-old tissue culture pineapple plantlets were punctured with a fine needle and 5 μl of each concentration from CF and FA were dropped on the wound area The assays were performed using five leaf segments per plant and 25 plants per cultivars All leaf segments were incubated on wet filter paper in Petri dishes under continuous fluorescent light at 22 ± 2°C The reactions of leaves were scored after 35 h of incubation and necrotic spots were measured using the ellipse area (Borrás et al 1998) Selection procedures: The bioassay mentioned above was used to select susceptible and resistant F1 hybrids The CF and FA concentrations used were 80% (v/v) and 0.75 mg/ml, respectively F1 hybrids of 'Spring' × 'Smooth Cayenne', 'Smooth Cayenne' × 'Spring', 'Smooth Cayenne' × 'Perolera' and 'Perolera' × 'Smooth Cayenne' were used The resistance level of the F1 hybrids and cultivars under natural conditions was measured in greenhouse field tests Twenty-five plantlets per cultivars and F1 hybrids were wounded with stainless steel needles A puncture wound was made at the basal region Inoculation was performed by dipping wounded plantlets in an inoculum of 105 conidia/ml for 3 min After inoculation, the plantlets were kept under greenhouse conditions (25–30°C) for 2 months, and then inspected for disease development Evaluation was based on a 0–6 numerical rating system in which 0=no disease development in the stem, 1=to 2% of the stem infected by the pathogen, 2=3–5%, 3=6–10%, 4=11–20%, 5=21–50% and 6=51–100% (Disease Index) A resistant reaction was defined as one in which there was no disease development in inoculated plantlets F1 hybrids and cultivars in which infection took place but the colonization of the host tissue proceeded slowly, reaching a disease index of or less 2 months after inoculation and which showed significantly less disease severity than the susceptible controls, were considered as tolerant to the pathogen (Matos et al 1991) The experiment was conducted in a completely randomized design Data analysis: The experimental data were analysed by analysis of variance conducted according to Duncan's new multiple range test using SPSS/PC (1992) Results Fig. 1: The effect of culture filtrate (a) and fusaric acid (b) produced by Fusarium subglutinans on pineapple leaves of the cultivars 'Smooth Cayenne' (◊), 'Perola' (□), 'Perolera' (○) and 'Spring' ( ) under laboratory conditions The data points represent the mean values and the vertical bars the standard deviations Figure 1a describes the effect of different CF concentrations on the development of necrotic spots from pineapple leaves The filtrate of F subglutinans induced necrotic spots on pineapple leaves from resistant and susceptible cultivars 35 h after treatment The necrotic spot areas in susceptible pineapple leaves treated with the filtrate were larger than those of the resistant cultivars 'Smooth Cayenne' and 'Perola', after treatment with CF, developed necrotic lesions up to 10 mm2 A significant increase of necrotic spot area was observed on susceptible cultivars; this was directly correlated with the increase in CF concentrations This effect varied between 40% and 100% concentrations, respectively The 'Perolera' and 'Spring' cultivars treated with CF showed lower degrees of necrotic spot area than the susceptible cultivars Significant differences between susceptible and resistant cultivars were observed from 50% concentrations upward However, all cultivars independent of their resistance level were sensitive to FA (Fig. 1b) The sensitivity of the cultivars to FA increased with the increase in the concentration used The FA had a non-specific behaviour on the susceptible and resistant cultivars Table 1: Behaviour of F1 hybrids and cultivars treated with culture filtrate (CF), fusaric acid (FA) and Fusarium subglutinans pathogen in laboratory and greenhouse conditions1 Susceptible F1 hybrid pineapple leaves treated with CF developed necrotic spot areas between and 10 mm2 (Table 1) The CF produced the highest phytotoxic effect on the susceptible F1 hybrids The mean disease index registered in pineapple plants inoculated with the Fusarium subglutinans pathogen was significantly lower in F1 hybrids resistant to the CF than in susceptible ones In total, eight F1 hybrids showed resistance to CF, one F1 hybrid had partial resistance and four were sensitive to it The FA had a phytotoxic effect on all cultivars and F1 hybrids, producing necrotic spot areas of 14 mm2 without significant genotypic difference Discussion This report represents the first attempt to assess the potential of using CF as a selection agent for fusariosis resistance The role of toxins in pathogenesis is usually assessed by evaluating the correlation between toxin production and pathogenicity of the organism, and between the sensitivity of different genotypes to the toxins and their susceptibility to the disease (Yoder 1980) Our results have shown that cultivar responses to the CF of F subglutinans were related to the responses of these cultivars to the pathogen This was also found by Jin et al (1996) who used a phytotoxic culture filtrate from Fusarium solani on wounded leaves and showed that the susceptible cultivars were more sensitive to CF than were resistant cultivars Furthermore, their assay showed that the response of soybean plants to crude fungal CFs was correlated with the severity of sudden death syndrome of soybean (Glycine max L.) in inoculated greenhouse-grown plants Similarly, in a previous study, pineapple varieties differing in resistance to fusariose were examined for their response to the phytotoxic effect of F subglutinans CF Phytotoxic effect was assessed in seedlings by placing the CF on wounded leaves and callus (inhibition of growth) Susceptible cultivars proved to be the most sensitive in each test, whereas resistant cultivars showed resistance to the CF Resistant callus grew in the presence of high concentrations that were completely toxic to susceptible cultivars (Borrás et al 1998, Borrás-Hidalgo et al 1999) In our study, the FA produced phytotoxic effects on all cultivars The response of different pineapple genotypes was not correlated with the response to F subglutinans under natural conditions (Table 1) However, since FA has been classified as a non-selective toxin (Gäumann 1957, Matsumoto et al 1995), it is possible that there are other components present in the CF that are responsible for the selectivity observed Extracellular molecules as fungal elicitors of plant resistance have been reported in a number of studies of host–pathogen interactions (Knogge 1996, Bailey et al 1997, Abad et al 1997, Ellis et al 2000) There are no effective chemicals to prevent this disease The best way forward is to develop cultivars resistant or at least tolerant to F subglutinans In cross-breeding programmes, new resistant cultivars can be developed using resistant cultivars or wild species as breeding material Reactions of pineapple plants to Fusarium subglutinans infection can vary within and among cultivars, indicating that differences in resistance to fusariose disease exist in pineapple genotypes under some conditions (Cabral and Coppens 1997) Our results show that it is possible to select resistant plants by the use of CF, and this trait could also be expressed under natural conditions A number of efficient protocols have been developed to select for resistance to various pathogens in plants by using CF and purified toxins in selection experiments (Litz and Lavi 1997, Alarcon et al 1998, Matsumoto et al 1999b) A prerequisite for success is the sensitivity of the plant to the toxin, reflecting the susceptibility of the intact plant to the pathogen Resistance selection can be done in several ways One method is one-step selection, in which a lethal concentration of the selective agent is used; another method is stepwise selection, in which the concentration of the selective agent is increased gradually until it reaches a lethal concentration (McLean 1996) These results suggest that the selection approach could have two major limitations: (1) a lack of knowledge about genotypic dependence of toxins or extracellular metabolites that play a role in the disease; and (2) the confidence that the susceptibility and/or resistance of cultured tissues to the CF or toxins will reflect those of the whole plant Therefore, the evaluation of genotypes produced within pineapple breeding programmes using CF could offer a first step towards a simplification of selection procedures that should be complemented by a second step through selection using artificial inoculation with the pathogen as previously reported by Matos et al (1991) References 1    2    3    4    5    6    7    8    9    10    11    12    13    14    15    16    17    Abad, M S., S M Hakimi, W K Kaniewski, C M T Rommens, V Shulaev, E Lam, D M Shah, 1997: Characterisation of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin Mol Plant Microbe Interact 10,635—645 Alarcon, C., J Castro, F Muños, P Arce-Johnson, J Delgado, 1998: Protein (s) from the Gram-positive bacterium Clavibacter michiganensis ssp michiganensis induces a hypersensitive response in plants Phytopathology 88,306—310 Bacon, C W., J K Porter, W P Norred, J F Leslie, 1996: Production of fusaric acid by Fusarium species Appl Environ Microbiol 64,4039—4043 Bailey, B A., J C Jennings, J D Anderson, 1997: The 24-kDa protein from Fusarium oxysporum Schlechtend: Fr f. sp erythroxyli: occurrence in related fungi and the effect of growth medium on its production Can J Microbiol 43,45—55 Borrás, O., A P Matos, R Cabral, R Tapia, M Arzola, R Santos, M Pérez, 1998: Phytotoxic effect of culture filtrate from Fusarium subglutinans the causal agent of fusariose of pineapple (Ananas comosus, L Merr) Euphytica 104,73—77 Borrás-Hidalgo, O., R Santos, R T Tussel, A Pires de Matos, R S Cabral, M Arzola, M C Pérez, 1999: Phytotoxicity of Fusarium subglutinans culture filtrate on in vitro plantlets and calli of resistant and susceptible pineapple (Ananas comosus.) Plant Pathol 48,756—758 Cabral, J R S & G Coppens, 1997: Segregation for resistance to fusariose, leaf margin type, and leaf colour from the EMBRAPA pineapple hybridisation programme Acta Hort 425,193—197 Cabral, J R S & A P Matos, 1995: Pineapple breeding for resistance to Fusariosis in Brazil Revista Agron– UCV 21,137—145 Daub, M., 1986: Tissue culture and the selection of resistance to pathogens Annu Rev Phytopathol 24,159 —186 Durbin, R D., 1981: Toxins in Plant Disease, 38—72 Academic Press, New York Ellis, J., P Dodds, T Pryor, 2000: The generation of plant disease resistance gene specificities Trends Plant Sci 5,373—379 Gäumann, E., 1957: Fusaric acid as a wilt toxin Phytopathology 47,342—357 Goodman, R N., A Kiraly, K R Wood, 1986: The Biochemistry and Physiology of Plant Disease, 318—346 Univ of Missouri Press, Columbia Hamer, J E C & D W Holden, 1997: Linking approaches in the study of fungal pathogenesis: a commentary Fungal Genet Biol 21,11—16 Jin, H., G L Hartman, C D Nickell, J M Widholm, 1996: Phytotoxicity of culture filtrate from Fusarium solani, the causal agent of Soybean Sudden Death Syndrome Plant Dis 80,922—927 Knogge, W., 1996: Fungal infection of plants Plant Cell 8,1711—1722 Kuzniak, E., J Patykowski, H Urbanek, 1999: Involvement of the antioxidative system in tomato response to fusaric acid treatment J Phytopathol 147,385—390.10.1046/j.1439-0434.1999.00392.x 18    Litz, R E & U Lavi, 1997: Biotechnology In: R E Litz (ed.), The Mango: Botany, Production and Uses, 401 —423 CAB Int., Wallingford 19    Lucas, J A., 1998: Plant Pathology and Plant Pathogens, 3rd edn, 222—248 Blackwell Sci., Oxford Matos, A P., 1995: Pathological aspects of the pineapple crop with emphasis on the fusariose Revista Agron–UCV 21,179—197 Matos, A P., X Maurichon, F Lapeyre, 1991: Reaction of pineapple accessions to inoculation with Fusarium moniliforme var subglutinans Fruits 46,647—652 Matsumoto, K., M L Barbosa, L A C Souza, J B Teixeira, 1995: Race Fusarium with tolerance on banana plants selected by fusaric acid Euphytica 84,67—71 Matsumoto, K., L A C Souza, M L Barbosa, 1999a: In vitro selection for Fusarium wilt resistance in banana I: Co-cultivation technique to produce culture filtrate of race Fusarium oxysporum f. sp cubense Fruits 54,97—102 Matsumoto, K., M L Barbosa, L A C Souza, J B Teixeira, 1999b: In vitro selection for resistance in banana II: Resistance to culture filtrate of race Fusarium oxysporum f. sp cubense Fruits 54,151—157 McLean, M., 1996: The phytotoxicity of Fusarium metabolites: an update since 1989 Mycopathologia 133,163 —179 Ostry, M E., 1997: In vitro screening and selection for disease resistance In: N B Klopfenstein, Y W Chun, M S Kim, and M R Ahuja (eds), Micropropagation, Genetic Engineering, and Molecular Biology of Populus, 155—160 Rocky Mountain Forest and Range, Fort Collins, CO Pinho, N M., L Zambolim, J A Ventura, 1997: Protoplast isolation of (Ananas comosus (L.) Merr.) cv Perolera Acta Hort 425,259—266 Remotti, P C., H J M Löffler, L Vloten–doting, 1997: Selection of cell-line and regeneration of plant resistant to fusaric acid from Gladiolus × grandiflorus cv 'Peter Pear' Euphytica 96,237—245 SPSS/PC, 1992: SPSS/PC (Statistics Package for Social Science) User's Guide, Version 4.0 The Microsoft Office Corporation, Chicago 20    21    22    23    24    25    26    27    28    29    30    Yoder, O C., 1980: Toxins in Pathogenesis Annu Rev Phytopathol 18,103—129  ... pineapple plantlets were punctured with a fine needle and 5 μl of each concentration from CF and FA were dropped on the wound area The assays were performed using five leaf segments per plant and 25 plants... subglutinans In cross -breeding programmes, new resistant cultivars can be developed using resistant cultivars or wild species as breeding material Reactions of pineapple plants to Fusarium subglutinans... D., 1981: Toxins in Plant Disease, 38—72 Academic Press, New York Ellis, J., P Dodds, T Pryor, 2000: The generation of plant disease resistance gene specificities Trends Plant Sci 5,373—379 Gäumann,