Summary. An in vitro bioassay to differentiate pineapple plant resistance levels to Phytophthora nicotianae var. parasitica (heart rot disease) is described here.
In Vitro Cell. Dev. Biol.—Plant 38:613–616, November–December 2002 q 2002 Society for In Vitro Biology 1054-5476/02 $10.00+0.00
DOI: 10.1079/IVP2002346
BIOASSAY FOR IN VITRO DIFFERENTIATION OF PINEAPPLE CULTIVAR RESISTANCE LEVELS TO HEART ROT DISEASE Y. RODRI´GUEZ, M. MOSQUEDA, B. COMPANIONI, M. ARZOLA, O. BORRAS, M. C. PEREZ, J. C. LORENZO,
AND
R. SANTOS*
Laboratory for Plant Breeding, Bioplant Centre, University of Ciego de Avila, 69450, Cuba (Received 25 April 2002; accepted 5 July 2002; editor J. W. Grosser)
Summary An in vitro bioassay to differentiate pineapple plant resistance levels to Phytophthora nicotianae var. parasitica (heart rot disease) is described here. Conditions to cause death of in vitro-cultured plants were defined using a cultivar previously found to be susceptible to this fungus in our Field-Grown Pineapple Germplasm Bank (cv. Smooth Cayenne Serrana). The effects of zoospore concentration, inoculation technique, and disease progress during the course of time after infection were evaluated. The highest rates of plant death were observed with the use of 108 zoospores ml21, and the inoculation technique of needle-mediated leaf base wound. One hundred percent plant death was observed at 144 h after infection. Different susceptible varieties along with a resistant pineapple relative were additionally compared. In vitro results confirmed previous observations obtained under field conditions. The protocol described here may be used for early selection (in vitro ) of new pineapple genotypes showing resistance to this fungus. At present, this protocol is extensively used in the Biotechnology-assisted Cuban Program for Pineapple Breeding. Key words: Ananas comosus (L.) Merr.; Phytophthora nicotianae var. parasitica; early selection.
Bioassays for in vitro selection have been described for alfalfa resistance to Fusarium oxysporum f. sp. medicaginis (Hartman et al., 1984); plantain resistance to Mycosphaerella fijensis (Pino et al., 1996); banana resistance to Fusarium oxysporum (Matsumoto et al., 1995, 1999), Mycosphaerella musicola (Trujillo and Garcı´a, 1996), and Mycosphaerella fijensis (Garcı´a et al., 1997); potato resistance to Alternaria solani Sor. (Dita, 1998); pineapple resistance to Fusarium subglutinans (Borra´s, 2000); and for other crops and plant diseases. However, to our knowledge, such a bioassay has not been described for pineapple heart rot disease. Therefore, the present paper reports the establishment of a protocol for early differentiation (at the in vitro stage) of pineapple cultivar resistance levels to Phytophthora nicotianae var. parasitica.
Introduction Pineapple [Ananas comosus (L.) Merr.] is one the most important crops in Cuba. However, fungal diseases such as heart rot (Phytophthora nicotianae var. parasitica ) dramatically affect its agronomic performance. Plant death is frequently observed after in vitro plant transplantation, causing problems with replacement and rejuvenation of cultivation areas. This disease has also been reported to cause serious economic losses in Australia, Hawaii, Philippines, South Africa and Thailand (Coppens et al., 1997). Therefore, pineapple breeding programs to incorporate disease resistance are well justified. Classical pineapple breeding methods are based on crosses, back-crosses and selection (Botella et al., 2000). These techniques, along with the long generation cycle of pineapple, result in extremely time-consuming breeding programs. Therefore, they can hardly keep pace with the rapid evolution of this pathogenic fungus (Cabot and Lacoevilhe, 1990). Biotechnology has been assisting our Pineapple Breeding Program in several ways to increase its efficiency (Benega et al., 1996a, b, 2000; Daquinta et al., 1996, 1997; Pe´rez et al., 2000). However, the development of bioassays to differentiate (at the in vitro level) resistant from susceptible plants has become one of the most relevant uses of such technology. Despite the preliminary character of in vitro bioassays, they allow research cost reduction because of elimination of a large number of non-resistant clones (Ostry, 1997; Lucas, 1998).
Materials and Methods Effect of zoospore concentration on in vitro-plant survival. A pineapple cultivar found to be susceptible in our Field-Grown Pineapple Germplasm Bank was used: Smooth Cayenne Serrana. Plants were obtained in accordance with the micropropagation procedure of Daquinta and Benegas (1997). Explants were placed in conventional glass containers (300 ml) for in vitro culture where five shoots were fed by 25 ml medium. The medium for shoot proliferation included: MS (Murashige and Skoog, 1962) inorganic salts and vitamins, 100 mg l21 inositol, 30 g l21 sucrose, 0.3 mg l21 (1.6 mM ) naphthaleneacetic acid, and 2.1 mg l21 (9.3 mM ) 6-benzyladenine. The culture medium for rooting included: MS inorganic salts and vitamins, 100 mg l21 inositol, 30 g l21 sucrose, and 0.5 mg l21 (2.7 mM ) naphthaleneacetic acid. Pineapple leaves showing typical symptoms of heart rot disease were sampled from field-grown plants. Lesions were washed with plenty of water and disinfected with calcium hypochlorite (1%, w/v) for 3 min. Then, they were immersed in ethanol (70%, v/v) for 3 min and rinsed with sterile distilled water. The fungus in vitro culture was carried out in potato–dextrose
*Author to whom correspondence should be addressed: Email rsantos@ bioplantas.cu
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RODRI´GUEZ ET AL.
614 TABLE 1
EFFECT OF ZOOSPORE CONCENTRATION ON IN VITRO SURVIVAL OF PINEAPPLE PLANTS AFTER INOCULATION WITH PHYTOPHTHORA NICOTIANAE VAR. PARASITICA Zoospore concentration (zoospores ml21) 0 102 104 106 108
Percentage of in vitro plant survivalz 100.0 100.0 100.0 93.4 26.7
a a a b c
z Results with the same letter are not statistically different (Kruskal– Wallis, Student– Newman–Keuls, P , 0:05). Treatments were replicated five times and 15 plants were used for each replication.
agar culture medium (Petri dishes, 288C, 48 h, darkness). A separated mycelium was re-cultured in the same medium to obtain putatively pure isolates. Phytophthora nicotianae var. parasitica identity was verified in accordance with Erwin and Ribeiro (1996a). Different concentrations of zoospores (0 –108 ml21) were tested for in vitro plant infection. The fungus was suspended in sterile distilled water and a Neubauer chamber was used to fix treatments. A modification of the infection procedure by Ricci et al. (1992) was used. The plant root tips (1 mm long) were cut with a scalpel and discarded. The wounded plants were immersed in zoospore suspensions for 144 h. The complete experiment was conducted under sterile conditions. Treatments were replicated five times and 15 plants were used for each replication. Percentage of plant survival was recorded. Effect of the inoculation technique on in vitro plant survival. The pineapple cultivar, the micropropagation procedure, and the steps for Phytophthora nicotianae var. parasitica isolation and culture were as described above. Two forms for plant wound were compared: wound of roots with a scalpel as previously mentioned; and wound of leaf base with a needle as recommended by Matos (1992). The wounded plants were immersed in a 108 ml21 zoospore suspension for 144 h. Two control treatments (plants immersed into sterile distilled water after wounding) were included. The complete experiment was conducted under sterile conditions. Treatments were replicated five times and 15 plants were used for each replication. Percentage of plant survival was recorded at 144 h after infection. Increase of plant death percentage with the course of time. The experimental conditions were as described above. The technique of leaf base wound and 108 ml21 zoospores were used. Percentages of plant survival were recorded at 0, 24, 48, 72, 96, 120, and 144 h after infection. Treatments were replicated five times and 15 plants were used for each replication. Effect of pineapple genotype on in vitro plant survival. Seven pineapple cultivars from the Bioplant Centre (University of Ciego de Avila, Cuba) Field-Grown Germplasm Bank were micropropagated as described above. Six of them belonged to the Ananas comosus (L.) Merr. species and have been
found to be susceptible to Phytophthora nicotianae var. parasitica under field conditions: Smooth Cayenne Serrana, Red Spanish Enana, Red Spanish Nozera´n, Red Spanish Colorada Caney, Red Spanish Pinaren˜a, and Red Spanish Colorada Ramo´n. Bromelia pinguin was used as a resistant control (100% plant survival in highly infected field areas where other cultivars died). The technique of leaf base wound and 108 ml21 zoospores was used to infect these species. Treatments were replicated five times and 15 plants were used for each replication. Percentage of plant survival was recorded at 144 h after infection. Statistical analysis. The experimental design was completely randomized. The Statistical Package for Social Sciences was used (version 8.0 for Windows, SPSS Inc.). Non-parametric tests were performed: Kruskal–Wallis and Student– Newman–Keuls ðP , 0:05Þ:
Results and Discussion The highest zoospore concentration (108 ml21) caused the most dramatic reduction of susceptible pineapple plant in vitro survival after 144 h of infection with Phytophthora nicotianae var. parasitica (Table 1). The technique involving the use of a needle to wound the leaf base before infection was more effective at causing plant death than the root wounding technique (Table 2). The control treatments did not produce any damage to the plants. Plants started dying at 96 h after infection with the fungus and all of them were dead at 144 h (Table 3). In vitro comparison of pineapple cultivar resistance status confirmed previous observations made in our Field-Grown Pineapple Germplasm Bank (Table 4). Different selection agents have been described to develop bioassays for in vitro selection and to study plant– fungus interactions: fungal toxins, fungal in vitro culture filtrates, and the fungus itself. The use of toxins has been reported for barley – Fusarium spp. (Chawla and Werzel, 1987), carnation – Fusarium oxysporum (Masayoshi and Masayuki, 1993), banana – Fusarium oxysporum (Matsumoto et al., 1995), and banana– Mycosphaerella fijensis (Pino et al., 1996) interactions. Fungal filtrates have been reported in studies on interactions between alfalfa – Fusarium oxysporum f. sp. medicaginis (Hartman et al., 1984), plantain – Mycosphaerella fijensis (Pino et al., 1996), banana – Fusarium oxysporum f. sp. cubense (Matsumoto et al., 1999), banana – Mycosphaerella musicola (Trujillo and Garcı´a, 1996), potato – Alternaria solani Sor. (Dita, 1998), and pineapple – Fusarium subglutinans (Borra´s, 2000). In vitro trials involving the fungus itself have been recommended to study plantain – Mycosphaerella fijensis (Lepoivre et al., 1986) and banana –Fusarium oxysporum (Matsumoto et al., 1999) interactions. Even though toxins and fungal in vitro culture-derived filtrates
TABLE 2 EFFECT OF INOCULATION TECHNIQUE ON IN VITRO SURVIVAL OF PINEAPPLE PLANTS AFTER INOCULATION WITH PHYTOPHTHORA NICOTIANAE VAR. PARASITICA Inoculation technique Technique
Inoculation with
Scalpel-mediated root wound Needle-mediated leaf base wound Scalpel-mediated root wound Needle-mediated leaf base wound
Sterile distilled water Sterile distilled water 108 ml21 zoospores in sterile distilled water 108 ml21 zoospores in sterile distilled water
z
Results with the same letter are not statistically different (Kruskal–Wallis, Student– Newman –Keuls, P , 0:05). Treatments were replicated five times and 15 plants were used for each replication.
Percentage of in vitro plant survivalz 100.0 100.0 40.0 0.0
a a b c
615
DIFFERENTIATION OF PINEAPPLE CULTIVAR RESISTANCE LEVELS TABLE 3 EFFECT OF THE COURSE OF TIME ON IN VITRO SURVIVAL OF PINEAPPLE PLANTS AFTER INOCULATION WITH PHYTOPHTHORA NICOTIANAE VAR. PARASITICA Time after inoculation (h)
Percentage of in vitro plant survivalz
0 24 48 72 96 120 144
100.0 100.0 100.0 100.0 93.4 60.0 0.0
a a a a b c d
z Results with the same letter are not statistically different (Kruskal– Wallis, Student–Newman– Keuls, P , 0:05). Treatments were replicated five times and 15 plants were used for each replication.
are used extensively in early selection systems, there are particular plant – pathogen interactions not showing in vitro symptoms corresponding to those appearing in the field environment (Trujillo and Garcı´a, 1996). We previously studied the effect of Phytophthora nicotianae var. parasitica culture filtrates on pineapple in vitro plants (data not shown) and no difference between Smooth Cayenne Serrana (susceptible) and Bromelia pinguin (resistant) was observed. This reason – along with the complexity of toxin production, isolation and purification – encouraged us to use the pathogen itself to develop the selection system described in this paper. Moreover, we are not aware of previous research on in vitro differentiation of pineapple plant resistance levels to heart rot disease with the use of the fungus. The zoospore concentration has been found to be crucial to differentiate plant lines with disease resistance in microorganisminvolving selection systems (Binarova´ et al., 1990). Different densities have been recommended: 105 ml21 conidia of Fusarium subglutinans for pineapple (Matos, 1992); 105 ml21 spores of Pseudoperonospora cubensi for pumpkin (Lebeda, 1991); and 107 ml21 conidia of Fusarium oxysporum for banana (Matsumoto
et al., 1999). The optimal density we observed (108 ml21 zoospores; Table 1) was also recommended by Erwin and Ribeiro (1996b) to test the pathogenic potential of Phytophthora cinnamomi on pineapple plants. Another factor affecting in vitro selection effectiveness is the inoculation strategy, and several organs and explants have been recommended: inoculation of leaf disks (Krikorian, 1990), roots (Kroon et al., 1991; Ricci et al., 1992; Dita, 1998), leaves (Robin and Guest, 1994; Pino et al., 1996), and decapitated stems (Candela et al., 2000). We produced higher mortality by wounding the leaf base with a needle in comparison with the root-wounding technique (Table 2). In the first case, inoculation was performed in the place where this disease normally starts (leaf). The fungus penetrates and rapidly invades the leaf tissues (Matos, 1992). On the contrary, penetration through the roots delays the colonization of the leaves. Ex vitro progression of plant diseases has been generally found to be related to the time course, but symptom development under an in vitro environment has not been well studied. Only a few reports are available: Kroon and Elgersma (1993) reported the highest tomato plant mortality at 7 d after inoculation with Fusarium oxysporum f. sp. lycopersici; Rodrı´guez et al. (1998) recommended evaluations at 72 h after pineapple inoculation with Fusarium subglutinans; and Dita (1998) reported potato mortality by Alternaria solani Sor. at 72 h after inoculation. In our experiment on pineapple– Phytophthora nicotianae var. parasitica interaction, the highest mortality was found at 144 h after fungal inoculation (Table 3). Contrasting results have been observed under the field environment where plant death starts at 45 d (Erwin and Ribeiro, 1996b). Explanation of such a difference might be related to the fragile status of all in vitro plants (Debergh, 1983) that allows a more dramatic action of the fungus. Correlation between in vitro and ex vitro resistance has been described for banana– Fusarium oxysporum f. sp. cubense (Matsumoto et al., 1995), plantain – Mycosphaerella fijensis (Pino et al., 1996), and potato – Alternaria solani Sor. (Dita, 1998) interactions. Results presented in Table 4 indicate such a correlation in our research. Therefore, the bioassay described here should be useful to select, at the in vitro stage, new pineapple genotypes with putative resistance to Phytophthora nicotianae var. parasitica. Even though the new genotype resistance still requires
TABLE 4 EFFECT OF GENOTYPE ON IN VITRO SURVIVAL OF PINEAPPLE PLANTS AFTER INOCULATION WITH PHYTOPHTHORA NICOTIANAE VAR. PARASITICA Pineapple genotype Species Bromelia pinguin Ananas comosus (L.) Ananas comosus (L.) Ananas comosus (L.) Ananas comosus (L.) Ananas comosus (L.) Ananas comosus (L.) z
Merr. Merr. Merr. Merr. Merr. Merr.
Cultivar
Classificationz
– Smooth Cayenne Serrana Red Spanish Enana Red Spanish Nozera´n Red Spanish Colorada Caney Red Spanish Pinaren˜a Red Spanish Colorada Ramo´n
Resistant Susceptible Susceptible Susceptible Susceptible Susceptible Susceptible
Classification in accordance with observations made in the Bioplant Centre Field-Grown Germplasm Bank. Results with the same letter are not statistically different (Kruskal–Wallis, Student–Newman–Keuls, P , 0:05). Treatments were replicated five times and 15 plants were used for each replication.
y
Percentage of in vitro plant survivaly 100.0 0.0 26.7 40.0 13.4 20.0 0.0
a c bc b bc bc c
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