Efectos del quitosano en hongos fitopátogenos y en la germinación de semillas de Jatropha curcas L.

Diana Pabón-Baquero¹; Miguel G. Velázquez-del Valle¹; Silvia Evangelista-Lozano²; Renato León-Rodríguez³; Ana N. Hernández-Lauzardo^*1

¹ Departamento de Interacciones Planta-Insecto, Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional. Carretera Yautepec-Jojutla, km 6, calle CEPROBI núm. 8, col. San Isidro. C. P. 62731. Yautepec, Morelos, México.

² Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional. Carretera Yautepec-Jojutla, km 6, calle CEPROBI núm. 8, col. San Isidro. C. P. 62731. Yautepec, Morelos, México. Correo-e: anhernandez@ipn.mx Tel.: (52) 55 5729 6000 ext. 82511 (*Autora para correspondencia).

³ Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Tercer circuito exterior s/n, edificio C, Ciudad Universitaria. C. P. 04510. Coyoacán, México, D. F.

Received: October 28, 2014.
Accepted: July 7, 2015.

ABSTRACT

Jatropha curcas is a plant with great agricultural and industrial potential. In this study, two fungal species were isolated from ungerminated seeds. The fungal isolates were morphologically and molecularly identified as Fusarium equiseti and Curvularia lunata. Effects of chitosan on mycelial growth, sporulation and spore germination of F. equiseti and C. lunata were evaluated. In addition, its effect on seed germination of J. curcas was studied. The results showed that all tested chitosan concentrations (0.5, 1.0, 2.0 and 4.0 mg·mL^-1) inhibited the mycelial growth of the fungi. Sporulation and spore germination responses differed depending on the fungal species. Chitosan completely inhibited sporulation of C. lunata and spore germination of F. equiseti. Inoculation with F. equiseti and C. lunata reduced seed germination of J. curcas by 20 and 26.6 %, respectively. However, application of chitosan before inoculation inhibited pathogenic activity. Therefore, chitosan did not affect seed germination and caused inhibitory effects on F. equiseti and C. lunata. This is the first report on the effect of chitosan on J. curcas.

Keywords: Fungal diseases, polymer, oil seeds, Fusarium equiseti, Curvularia lunata.

RESUMEN

Jatropha curcas es una planta con gran potencial agrícola e industrial. En este estudio se aislaron dos hongos de semillas no germinadas. Los aislamientos fúngicos se identificaron morfológica y molecularmente como Fusarium equiseti y Curvularia lunata. Los efectos del quitosano se evaluaron sobre el crecimiento micelial, esporulación y germinación de esporas de F. equiseti y C. lunata. Además, se estudió el efecto sobre la germinación de las semillas de J. curcas. Los resultados demostraron que todas las concentraciones probadas de quitosano (0.5, 1.0, 2.0 y 4.0 mg·mL^-1) inhibieron el crecimiento micelial de los hongos. Las respuestas de esporulación y germinación de esporas fueron diferentes dependiendo de la especie fúngica; el quitosano inhibió completamente la esporulación C. lunata y la germinación de esporas de F. equiseti. La inoculación con F. equiseti y C. lunata redujo la germinación de semillas de J. curcas 20 y 26.6 %, respectivamente; sin embargo, la aplicación de quitosano antes de la inoculación inhibió la actividad patogénica. En conclusión, el quitosano no afectó la germinación de las semillas y causó efectos inhibitorios en F. equiseti y C. lunata. Este es el primer reporte del efecto del quitosano en J. curcas.

INTRODUCTION

Mexico has been identified as the center of origin and domestication of Jatropha curcas L. (Dias, Missio, & Dias, 2012). It is a species with many attributes and considerable potential. Its seeds contain oil that can be processed to obtain biodiesel fuel. On the other hand, edible varieties of J. curcas have been identified in Mexico; their seeds have high protein content and can be used for preparation of various traditional dishes and for animal feed (Martínez-Herrera et al., 2012).

However, there are reports of several diseases affecting J. curcas, such as root rot and collar rot caused by Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Latha et al., 2009), black rot caused by Botryosphaeria diplodia (Moug.) Ces. & De Not. (Rao, Kumari, Wani, & Marimuthu, 2011), root rot caused by Rhizoctonia bataticola (Taubenh.) E. J. Butler (Kumar, Sharma, Pathak, & Beniwal, 2011), anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz. y Sacc. (Kwon, Choi, Kim, & Kwak, 2012), and inflorescence blight caused by Alternaria alternata (Fr.) Keissl. (Espinoza-Verduzco et al., 2012), among others. The presence of some species of phytopathogenic fungi has been found on J. curcas seeds in storage (Dharmaputra, Worang, Syarief, & Miftahudin, 2009). At the beginning of storage, the seed-infecting fungi were field fungi (Cladosporium spp., Colletotrichum sp., and Fusarium spp.). These fungal populations decreased with increased storage time and were replaced by postharvest fungi (Aspergillus spp. and Penicillium spp.). The lipid content, viability and vigor of seeds decreased with increasing storage time (Dharmaputra et al., 2009). Additionally, other authors reported that Aspergillus flavus (Link.) and Rhizopus nigricans (Ehrenb.) affected germination and seedling vigor of J. curcas (Anjorin, Omolewa, & Salako, 2011). Synthetic chemical fungicides are widely used to combat seed pathogenic fungi. However, these chemicals pose risks to the environment and human health (Alavanja, Ross, & Bonner, 2013). Therefore, it is necessary to use natural alternatives for the control of phytopathogenic fungi.

Chitosan is a deacetylated derivative of chitin with excellent antimicrobial properties. It has been used to control phytopathogenic fungi and to enhance plant defenses and development. Positive effects of chitosan application as a seed coating have been reported for various plant species such as wheat (Triticum spp.), rice (Oriza spp.), maize (Zea mays L.), peanuts (Arachis hypogaea L.) and carrots (Daucus carota L.) (El Hadrami, Adam, El Hadrami, & Daayf, 2010). Additionally, Ziani, Ursúa, & Maté (2010) showed that chitosan coating significantly increased seed germination and plant growth of artichoke (Cynara scolymus L.) and resulted in decreased fungal contamination. Other authors have observed that chitosan coating resulted in a lower incidence of fungal infections of chili (Capsicum spp.) seeds, but it did not significantly affect moisture content and germination of seeds (Chookhongkha, Sopondilok, & Photchanachai, 2013). In the case of lentil (Lens culinaris Medikus) seeds, chitosan coating induced the highest germination percentage, hypocotyl length, radical length, hypocotyl dry weight and radical dry weight (Al-Tawaha & Al-Ghzawi, 2013). However, there are no reports about the influence of chitosan on J. curcas. Therefore, the aim of this study is to evaluate the effects of chitosan on phytopathogenic fungi and seed germination of J. curcas.

MATERIALS AND METHODS

Isolation of fungi from J. curcas seeds

Jatropha curcas seeds were disinfected with captan (0.2 %) for 5 min, then washed three times with sterile distilled water and dried on sterilized paper towels. Dry seeds were placed in glass flasks with 50 % Murashige and Skoog (MS) medium and incubated for 6 days at 25 ± 2 °C. Thereafter, fragments of mycelia of 18 fungi from the ungerminated seeds were transferred to Petri plates with potato dextrose agar (PDA) and incubated at 25 ± 2 °C for 7 days.

Mycelial discs (5 mm) of the 18 isolated fungi were placed on J. curcas seeds in glass flasks with 50 % MS medium. The same medium without mycelia was used as a control. The flasks were incubated at 25 ± 2 °C for 6 days. To confirm Koch's postulates, the fungal isolates that grew on ungerminated seeds were re-isolated from the seeds on PDA. The colonies formed were purified by serial dilutions to obtain monosporic cultures.

Morphological and molecular identification of the fungal isolates

Mycelial discs (5 mm) of each isolate were placed in the center of Petri plates containing PDA. For morphological characterization, the description of mycelia and spores was carried out in accordance with taxonomical keys (Barnett & Hunter, 1998; Leslie & Summerell, 2006), and the information deposited in the MycoBank (2012). For molecular identification, genomic DNA was isolated from fungal mycelium grown on PDA, according to the protocol of Doyle and Doyle (1990). A region of ribosomal DNA was amplified by PCR using the ITS1 and ITS4 primers (White, Bruns, Lee, & Taylor, 1990); the amplification products were examined by electrophoresis and sequenced. The sequences were compared against sequences in databases using the BLAST Basic Local Alignment Search Tool (BLAST, 2012).

Chitosan solutions

To prepare a stock solution (10 mg·mL^-1), 3 g of chitosan (Sigma-Aldrich) of low molecular weight were dissolved in 150 mL of distilled water with 3 mL of acetic acid on a stirrer for 24 h, and the volume was adjusted to 300 mL with distilled water. The pH was adjusted to 5.6 with NaOH (1 M). The chitosan solution was autoclaved for 15 min. Corresponding aliquots were taken to obtain different chitosan concentrations (0.5, 1.0, 2.0 and 4.0 mg·mL^-1).

Inhibition of mycelial growth of the fungal isolates by chitosan

One mycelial disc (5 mm) of phytopathogenic fungal isolates was placed in the center of Petri plates containing PDA with chitosan concentrations of 0.5, 1.0, 2.0 and 4.0 mg·mL^-1. Control Petri plates contained only PDA. The Petri plates were incubated at 28 ± 2 °C. The mycelial growth was measured with a digital vernier (Thomas Scientific model 1235C55, USA) when mycelium reached the edges of the control plates and expressed as an average diameter (mm). The mean diameter of fungal growth in the presence of chitosan was compared with that of the control cultures in order to determine the inhibition percentage of the mycelial growth. All experiments were repeated twice with five replicates each.

Effect of chitosan on sporulation of the fungal isolates

Fungal isolates were incubated on PDA supplemented with chitosan (0.5, 1.0, 2.0 and 4.0 mg·mL^-1) and without chitosan (control) for 21 days. Thereafter, Petri plates were rinsed with 10 mL of distilled water, and the surface was scraped with a sterile glass rod. Spores were counted using a Neubauer hemocytometer under a light microscope (Nikon Alphaphot-2YS2, Japan) at 40x magnification. The data obtained were expressed as the number of spores·mL^-1.

Spore suspensions (~300 spores·mL^-1) were obtained from cultures of the fungal isolates after 15 days of incubation. An aliquot (300 μL) was spread over PDA containing chitosan (0.5, 1.0, 2.0, and 4.0 mg·mL^-1) in a Petri dish. The control treatment contained no chitosan. The plates were incubated for 48 h, and the remaining spores were counted. The data obtained were expressed as a percentage of spore germination (Al-Hetar, Zainal, Sariah, & Wong, 2011). Six repetitions per treatment were made, and each experiment was replicated twice.

Effect of chitosan on seed germination of J. curcas (in vivo study)

J. curcas seeds were disinfected with captan (0.2 %) for 5 min, then washed three times with sterile distilled water and dried on sterilized paper towels. Thereafter, 10 seeds per treatment were placed in humid chambers (70 %) to be treated as follows: control seeds (without chitosan), seeds treated with chitosan (2.0 or 4.0 mg·mL^-1), seeds inoculated with F. equiseti (10⁴ spores·mL¹), seeds inoculated with F. equiseti (10⁴ spores·mL¹) and treated with chitosan (2.0 or 4.0 mg·mL^-1), seeds inoculated with C. lunata (10⁴ spores·mL¹), and seeds inoculated with C. lunata (10⁴ spores·mL¹) and treated with chitosan (2.0 or 4.0 mg·mL^-1). The chambers were put into plastic bags, and the bags were tied up with rubber bands. Ten repetitions were made for each treatment in three replicates. The humid chambers were incubated at 28 ± 2 °C for 3 days, and percentages of seed germination were evaluated.

Statistical analysis

Experiments were conducted using a completely randomized design. The analysis of the data was performed by ANOVA. All experiments were repeated at least twice. Means separation was carried out by use of the Holm-Sidak test (P < 0.05) using the SigmaPlot 11.0 program (Systat Software Inc., 2009).

RESULTS AND DISCUSSION

Pathogenicity test and identification of the fungal isolates

A total of 18 isolates were obtained from ungerminated J. curcas seeds. However, a pathogenicity test indicated that only two isolates (A1 and A2) inhibited the seed germination process (Figure 1). Figure 1c shows the germination of uninoculated J. curcas seeds in 50 % MS. Normal root development can be observed in Figure 1d. Subsequently, the A1 and A2 fungal isolates were re-isolated from ungerminated seeds on PDA. The results of morphological and molecular identification of the fungal isolates obtained from ungerminated J. curcas seeds are shown in Table 1. A relationship between the morphological and molecular identification was found for both A1 and A2 isolates. The BLAST results revealed that two fungal species were isolated from ungerminated seeds. The percentages of identity with GenBank (National Center for Biotechnology Information [NCBI], 2012) sequences are shown in Table 1. Isolate A1 was identified as Fusarium equiseti (Corda) Sacc., and its colonies showed the following characteristics: white to yellow color, cottony texture and no aerial mycelium. The release of a yellow pigment into the media was also observed for this fungal species. On the other hand, isolate A2 was identified as Curvularia lunata (Wakker) Boedjin. This species formed medium brown, velvety colonies and no aerial mycelium. It was demonstrated that both fungi affected seed germination of J. curcas. Fusarium is one of the most important genera of fungi, causing plant diseases, producing mycotoxins, and affecting human health (Summerell & Leslie, 2011). The pathogenicity of F. equiseti has been reported in previous studies on Pinus ponderosa Douglas ex C. Lawson seeds, and it has been demonstrated to cause damping-off and root rot on seedlings (Salerno & Lori, 2007). Additionally, F. equiseti is prevalent in ginseng (Panax quinquefolius L.) soil causing the discoloration of the root surface (Punja et al., 2008). In a recent study, F. equiseti obtained from seeds caused foliar necrosis and wilt on pecan (Carya illinoinensis [Wangenh.] K. Koch) (Lazarotto et al., 2014). On the other hand, C. lunata has been isolated from seeds of plants of economic importance such as rice (Kapse, Bhale, & Jogi, 2012), wheat (Pathak & Zaidi, 2013) and sorghum (Sorghum spp.) (Funnell-Harris, Prom, & Pedersen, 2013). In previous studies, C. lunata caused the deterioration of J. curcas seeds during storage (Srivastava, Sinha, & Srivastava, 2011). However, to date there have been no reports about the presence and potential damage that can be caused by F. equiseti and C. lunata to J. curcas seeds. Taking into account the agricultural and industrial potential of J. curcas, it is necessary to evaluate natural alternatives for the control of phytopathogenic fungi affecting this crop.

The antifungal effects of chitosan on the mycelial growth of F. equiseti and C. lunata are shown in Table 2. The mycelial growth of the two fungal isolates was reduced on media supplemented with chitosan at all tested concentrations. According to the percentages of inhibition of mycelial growth, all treatments showed statistically significant differences (P < 0.05) at all chitosan concentrations evaluated compared to the control. The highest inhibitory effects were observed at chitosan concentrations of 2.0 and 4.0 mg·mL^-1 against both F. equiseti (92.0 and 98.7 %, respectively) and C. lunata (88.8 and 92.3 %, respectively). In earlier studies, antifungal effects of chitosan were demonstrated on mycelial growth of Fusarium lunatum (Ellis & Everhart) Arx, Fusarium oxysporum Schtdl. and C. lunata. However, there were differences in sensitivity between the species (Flores-Flores et al., 2013). Likewise, it was demonstrated that responses of different types of fungal cells to chitosan were different; spores were clearly more sensitive than hyphae (Palma-Guerrero, Hansson, Salinas, & López-Llorca, 2008).

Effects of chitosan on sporulation of F. equiseti and C. lunata

Sporulation is an important stage in the development of fungi. The results obtained in this study were different depending on the fungal species (Table 2). Chitosan effects on the sporulation of F. equiseti were concentration-dependent, and the highest effect was observed for the chitosan concentration of 4 mg·mL^-1. However, chitosan completely inhibited the sporulation of C. lunata at all concentrations tested. Therefore, C. lunata demonstrated greater sensitivity to chitosan than F. equiseti, although the sporulation of both phytopathogenic fungi was affected in its presence. Similar results were obtained for F. oxysporum treated with chitosan at 8 mg·mL^-1 (Al-Hetar et al., 2011).

Effects of chitosan on spore germination of F. equiseti and C. lunata

Spore germination of F. equiseti was completely inhibited in all chitosan treatments (Table 2). There were statistically significant differences (P < 0.05) between the C. lunata control and the chitosan treatments at all concentrations used, and complete inhibition of spore germination was observed at 2.0 and 4.0 mg·mL^-1 of chitosan. The germination of F. equiseti spores was more affected than that of C. lunata. It was demonstrated earlier that responses of different types of fungal cells to this polymer were different. For example, hyphae and spores of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. behaved differently in the presence of chitosan (Hernández-Lauzardo et al., 2008). Some authors pointed out that chitosan was transported into conidia of F. oxysporum through an energy-dependent process and caused an ultrastructural damage. In other cases, the germination of spores was completely inhibited by chitosan (Palma-Guerrero et al., 2008). There are no previous reports showing an effect of chitosan on spore germination of C. lunata. In general, the use of chitosan for inhibiting the development of phytopathogenic fungi without affecting the germination of J. curcas seeds would be considered a favorable alternative.

Studies on J. curcas seeds

The results demonstrated that chitosan did not affect seed germination (Table 3). Seed germination of J. curcas inoculated with F. equiseti and C. lunata was inhibited in this study by 20.0 and 26.6 %, respectively. The chitosan treatments did not show statistically significant differences, regardless of fungal inoculation. Thus, chitosan did not affect the seed germination but showed an inhibitory effect against the pathogenic activities of F. equiseti and C. lunata (Figure 2). Total inhibition of seed germination or inhibition of root growth and the oxidation process were observed in the presence of F. equiseti. Similar effects were caused by Fusarium graminearum Schwabe, on barley seeds (Yang, Svensson, & Finnie, 2011). In other reports, it was demonstrated that A. flavus and R. nigricans affected seed germination and seedling vigor of J. curcas (Anjorin et al., 2011). It should be noted that the above species were obtained from fungal collections and not directly from J. curcas seeds, and even though each inhibited germination, greater effects on seed germination were obtained when the two species were inoculated together. The presence of phytopathogenic fungi in seeds should not only be considered in terms of seed viability; it also causes changes in the content of chemical compounds such as lipids. In particular, Worang, Dharmaputra, Syarief and Miftahuddin (2008) reported that J. curcas seeds, when colonized by fungi during storage, showed a significant decrease in the content of lipids and an increase in the content of free fatty acids and in lipase activity. Recent studies indicated that species of the genera Fusarium and Curvularia produced significant quantities of extracellular lipases (Iftikhar et al., 2011) In particular, it was demonstrated that F. equiseti produced elevated levels of extracellular lipases under certain environmental conditions (Kakde & Chavan, 2011). This enzymatic activity can negatively affect biodiesel production from J. curcas. Therefore, it may be of significance that the treatments with chitosan (2 and 4 mg·mL^-1) before the fungal inoculation prevented unfavorable effects on seed germination caused by the phytopathogenic fungi. To date, there have been no reports of chitosan applications on J. curcas seeds. Based on the results obtained in this research, chitosan has high potential as a control agent of F. equiseti and C. lunata in J. curcas seeds.

CONCLUSIONS

ACKNOWLEDGMENTS

The authors are grateful to the Instituto Politécnico Nacional (IPN) of Mexico for financial support.

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