Evaluate the antifungal effect of chitosan against Rhizoctonia solani in vitro and the possible mechanisms of its induced activity in potato tubers to control black scurf disease.
The in vitro influence of chitosan at different concentrations on mycelial growth of R. solani was tested by using the poisoned food technique in PDA medium. The effect of these concentrations on the development of lesion diameters in tubers inoculated with R. solani mycelium was assayed for 30 days. The concentration that showed the greatest inhibitory effect on lesion diameters was tested to assess the induced activity of defense-related enzymes in the infected tubers.
In the poisoned food technique, chitosan at 1% completely inhibited the growth of R. solani mycelium. In vivo tests showed that chitosan treatment at 0.5% effectively controlled the black scurf in tubers inoculated with R. solani mycelium. Chitosan increased the activities of defense-related enzymes such as Peroxidase (POD), Polyphenol Oxidase (PPO) and Phenylalanine Ammonia-lyase (PAL) in treated tubers of tested cultivars.
This work demonstrated that chitosan directly inhibited the growth of R. solani, and potentially elicited defense reaction in potato tubers.
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* Address correspondence to this author at the Plant Protection Department, Faculty of Agriculture, Saratov State Vavilov Agrarian University, Saratov, 410012, Russia; Tel: +79026665260; E-mail: email@example.com
Inhibition of Mycelial Growth of Rhizoctonia Solani by Chitosan in vitro and in vivo
Rhizoctonia solani Kühn [teleomorph: Thanatephorus cucumeris (Frank) Donk] AG-3 PT, the pathogen of stem canker and black scurf in potato (Solanum tuberosum L.), is a worldwide plant disease [1Banville GJ, Carling DE, Otrysko BE. Rhizoctonia disease on potato Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control 1996; 321-30. [http://dx.doi.org/10.1007/978-94-017-2901-7_29] ]. Yield losses from R. solani can reach 50%, resulting in marked economic losses for farmers [2Keiser A. Rhizoctonia solani a fungal disease with multiple symptoms: means of preventive and curative control. Potato Research for a Production of Quality Information Day 2008.]. Control of R. solani relies mainly on fungicides application [3El Bakali AM, Martín MP. Black scurf of potato. Mycologist 2006; 20(4): 130-2. [http://dx.doi.org/10.1016/j.mycol.2006.03.006] ] such as azoxystrobin, flutolanil, and pencycuron [4Campion C, Chatot C, Perraton B, Andrivon D. Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France. Eur J Plant Pathol 2003; 109(9): 983-92. [http://dx.doi.org/10.1023/B:EJPP.0000003829.83671.8f] , 5Djébali N, Belhassen T. Field study of the relative susceptibility of eleven potato (Solanum tuberosum L.) varieties and the efficacy of two fungicides against Rhizoctonia solani attack. Crop Prot 2010; 29(9): 998-1002. [http://dx.doi.org/10.1016/j.cropro.2010.06.012] ] which are not always efficient due to the development of fungicide resistant communities. Furthermore, pesticides are environmental and human health concern. Consequently, more momentum has gained in search of sustainable solutions to chemical control. Chitosan (CH) (poly-β-(1 4) N-acetyl-d-glucosamine) is a modified, natural safe biopolymer derived by deacetylation of chitin, the second most abundant natural polymer in the world [6Rabea EI, Badawy ME, Steurbaut W, Stevens CV. In vitro assessment of N-(benzyl) chitosan derivatives against some plant pathogenic bacteria and fungi. Eur Polym J 2009; 45(1): 237-45. [http://dx.doi.org/10.1016/j.eurpolymj.2008.10.021] , 7El Hadrami A, Adam LR, El Hadrami I, Daayf F. Chitosan in plant protection. Mar Drugs 2010; 8(4): 968-87. Available at: http://www.ncbi.nlm. nih.gov/pubmed/20479963 [http://dx.doi.org/10.3390/md8040968] [PMID: 20479963] ]. Chitosan is very useful for several industries, such as cosmetology, biotechnology, food, pharma- cology and medicine [8Hamed I, Özogul F, Regenstein JM. Industrial applications of crustacean by-products (chitin, chitosan, and chitooligosaccharides): A review. Trends Food Sci Technol 2016; 48: 40-50. [http://dx.doi.org/10.1016/j.tifs.2015.11.007] , 9Choi C, Nam J-P, Nah J-W. Application of chitosan and chitosan derivatives as biomaterials. J Ind Eng Chem 2016; 33: 1-10. [http://dx.doi.org/10.1016/j.jiec.2015.10.028] ]. Since the 1980s, farmers have used chitosan as biopesticide, biofertilizer and agricultural film in seed and fruit coating [10Malerba M, Cerana R. Chitosan Effects on Plant Systems. Int J Mol Sci 2016; 17(7): 996. Available at: http://www.ncbi.nlm.nih.gov/pubmed/27347928 [http://dx.doi.org/10.3390/ijms17070996] [PMID: 27347928] ]. Chitosan has been demonstrated to control postharvest diseases on several horticultural commo- dities such as apples, pears, kiwifruit, strawberries, tomato, raspberries, table grape and others [11Du J, Gemma H, Iwahori S. Effects of chitosan coating on the storage of peach, Japanese pear, and kiwifruit. J Jpn Soc Hortic Sci 1997; 66(1): 15-22. [http://dx.doi.org/10.2503/jjshs.66.15] -15Meng X, Li B, Liu J, Tian S. Physiological responses and quality attributes of table grape fruit to chitosan preharvest spray and postharvest coating during storage. Food Chem 2008; 106(2): 501-8. [http://dx.doi.org/10.1016/j.foodchem.2007.06.012] [PMID: 2604 9245] ]. Chitosan protects rice, tomato, tobacco, and lettuce plants against R. solani infection [16Abd-El-Kareem F, El-Mougy NS, El-Gamal NG, Fotouh Y. Use of chitin and chitosan against tomato root rot disease under greenhouse conditions. Res J Agric Biol Sci 2006; 2: 147-52.-19Kurzawińska H. Potential use of chitosan in the control of lettuce pathogens. Polish Chitin Society. Monograph 2007; 12: 173-8.]. However, to our recent knowledge, there is no available information about the antifungal activity of chitosan against potato black scurf pathogen R. solani. This study aims to evaluate the antifungal activity and the induced effect of chitosan treatment on potato tuber resistance to R. solani under in vitro and in vivo conditions.
2. MATERIALS AND METHODS
2.1. Potato Tubers
Tubers of cultivars Sante and Kolobok were harvested from Moiseev farm, Bazarno-Karabulaksky District, Saratov region, Russia. The tubers based on size and showing no visible signs of disease infection or physical injuries were packed in net bags, transported to the laboratory, and stored at (16 ± 2) °C. Before treatment, the tubers were superficially disinfected with 2% sodium hypochlorite for 3 min, washed several times with sterilized water to wash out the remaining disinfectant solution and then air-dried.
Tubers with typical symptoms of black scurf were used to isolate the pathogen. Diseased tissue was cut into small pieces (1 cm2) and surface-disinfected in 70% ethanol for 30 s, followed by treatment with 2% (vol/vol) sodium hypochlorite for 3 min. Superficially sterilized pieces were rinsed several times in sterilized water to wash out the remaining disinfectant solution and cut aseptically into two pieces. These pieces were then dried between sterilized filter papers and then were placed on Petri dishes containing fresh Potato Dextrose Agar medium (PDA) (Difco Laboratories, St. Louis) supplemented with streptomycin sulphate 5 mg L-1. The plates were incubated at 24°C for 4 days, and single tips of fungal mycelium were transferred to sterile PDA for purification [20Papavizas G, Davey C. Isolation of Rhizoctonia solani Kuehn from naturally infested and artificially inoculated soils. Plant Dis Rep 1959; 43: 404-10.]. Several pure cultures of Rhizoctonia solani isolates were identified by light microscopy as described by Sherwood [21Sherwood R. Physiology of Rhizoctonia solani 1970.]. Rhizoctonia mycelia were stored at 4°C.
Chitosan, edible level with an average molecular weight of 150 kDa and degree of deacetylation 80%, was purchased from Chitosan Technologies Limited Company, Engels city, Russia.
2.4. Influence of Chitosan Treatment on Mycelial Growth of Rhizoctonia solani In Vitro
The effect of chitosan on mycelial growth was assessed by inoculating mycelial disks (5 mm in diameter) from the edge of a 4-day-old-culture of the fungus on the center of 90-mm Petri dishes containing 20 ml PDA medium amended with chitosan at (0, 0.125, 0.25, 0.5, or 1%). Plates were incubated in the dark at 25 °C and the mycelial growth was determined by measuring the colony diameter when the mycelium reached to the edges of the control plate. Each treatment was replicated using three plates, and the experiment was performed three times.
2.5. Influence of Chitosan Treatment On Lesion Diameters of Tubers Inoculated with Rhizoctonia Solani
Tubers were wounded (3 mm deep and 3 mm wide) at the equator with a sterile dissecting needle and then mycelial plugs of 4-day-old R. solani culture were inoculated into wounded sites with the hyphal side down. After 3 h of inoculation, tubers were treated with different concentrations of chitosan 0, 0.125, 0.25, 0.5, or 1% (w/v) dissolved in 0.5 mol L-1 glacial acetic acid, and the pH of CH solutions was adjusted to 5.5-6.0 with 1M aqueous NaOH. The treated tubers were incubated in plastic boxes (190 mm× 157 mm × 90 mm) with sterile water to maintain high relative humidity and stored at room temperature (20±2°C). The diameter of the lesions was measured after 30 d of inoculation. Each treatment was applied to three replicates of 15 tubers. The experiment was repeated twice.
2.6. Influence of Chitosan Treatment on Defense-Related Enzymes
Three grams of fresh weight were taken from 3-4 mm below the treated sides (Rhizoctonia with chitosan) and untreated ones (Rhizoctonia lacking chitosan) with a stainless-steel cork borer after 0, 1, 2, 3, 4, 5, 6, and 7 d of treatment with 0.5% chitosan and then were ground to a fine powder in liquid N2 and used for extraction of Peroxidase (POD), Polyphenol Oxidase (PPO) and Phenylalanine Ammonia-lyase (PAL), which are three defense-related enzymes in potato tubers. POD activity was assayed as described by Venisse et al. [22Venisse JS, Malnoy M, Faize M, Paulin JP, Brisset MN. Modulation of defense responses of Malus spp. during compatible and incompatible interactions with Erwinia amylovora. Mol Plant Microbe Interact 2002; 15(12): 1204-12. Available at:
http://www.ncbi.nlm.nih.gov/ pubmed/12481992 [http://dx.doi.org/10.1094/MPMI.2002.15.12.1204] [PMID: 1248 1992] ] by measuring the absorbance at 470 nm. PPO activity was estimated according to Jiang et al. [23Aili J, Shiping T, Yong X. Effect of CA with high-O2 or high-CO2 concentrations on postharvest physiology and storability of sweet cherry. Acta Bot Sin 2002; 44(8): 925-30.] by measuring the absorbance at 420 nm. PAL activity was assayed according to the method of Assis et al. [24Assis JS, Maldonado R, Muñoz T. Escribano MaI, Merodio C. Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit. Postharvest Biol Technol 2001; 23(1): 33-9. [http://dx.doi.org/10.1016/S0925-5214(01)00100-4] ] by measuring the absorbance at 290 nm. The content of total protein was determined by the method of Bradford [25Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72(1-2): 248-54. [DOI]. [http://dx.doi.org/10.1016/0003-2697(76)90527-3] [PMID: 942051] ] using bovine serum albumin as standard.
2.7. Statistical Analysis
All statistical analyses were performed using the CoStat 6.45 software program. To test the effect of chitosan treatment, the data were Analyzed by One-way Analysis of Variance (ANOVA). Mean separations were performed by the least significant difference (LSD) test. Significance was defined as P< 0.05.
3.1. Influence of Chitosan Treatment on Mycelial Growth of Rhizoctonia solani In Vitro
Chitosan significantly inhibited the growth of R. solani mycelium in a concentration-dependent manner. The mycelial growth was completely inhibited by chitosan at 1% (Fig. 1).
3.2. Influence of Chitosan Treatment On Lesion Diameters of Tubers Inoculated with Rhizoctonia Solani
Treatment with chitosan significantly reduced the lesion diameter of R. solani AG-3 PT inoculated tubers after 30 d of incubation and the reduction enhanced with increasing the concentration. However, no significance was found between 0.5 and 1% concentrations in both cultivars (Fig. 2A, B). The CH treatment at 0.5% reduced the lesion diameter by 67.1 and 62.8%, respectively, in Kolobok and Cante cultivars.
Fig. (1) Influence of chitosan concentrations on mycelial growth of Rhizoctonia solani. Bars represent standard deviations of the means. Values followed by different letters are significantly different according to LSD test at P < 0.05.
Fig. (2) Influence of chitosan at different concentrations on lesion diameters of inoculated tubers of tow cultivars. A, Kolobok. B, Cante. Data are expressed as a mean of triplicate samples (± SD). Significant differences (P> 0.05) as indicated by LSD test are shown by different letters.
Fig. (3) Influence of chitosan at 0.5% on the activity of defense-related enzymes POD (A, D), PPO (B, E) and PAL (C, F) in two cultivars (A, B, and C, Kolobok. D, E, and F, Cante). Data is presented as mean (± SD) based on three replicates.
3.3. Influence of Chitosan Treatment on Defense-Related Enzymes
POD was activated following treatment with CH 0.5% in both cultivars Kolobok and Cante. In Kolobok cultivar, a significant increase in POD activity was early observed after 1 day. This response was followed by a strong increase in the activity up to the 4th day (Fig. 3A). In contrast, this response was weaker and delayed in Cante cultivar, the first peak of POD activity appeared only after 3 days of treatment (Fig. 3D). The induction in POD activity reached its higher increase 4 and 5 days after treatment in Kolobok and Cante cultivars, respectively (Fig. 3A, D).
PPO activity pattern was higher in the treated tubers than the control in both cultivars. In Kolobok cultivar, PPO activity showed a different pattern between CH treated and non-treated tubers. Peaks were observed at 3 and 6 days in treated tubers (Fig. 3B). In Cante cultivar, the PPO activity followed the same trend and fluctuation in both CH-treated and untreated control tubers. However, the PPO activity showed a higher induction in CH-treated samples (range of 1.3-1.9 fold increase) than that in the untreated control samples (Fig. 3E).
PAL activity increased with incubation time in CH-treated tubers of both cultivars, reaching maximum values on 3 and 6 days in Kolobok and Cante cultivars, respectively (Fig. 3C, F).
As a natural elicitor and antifungal agent, chitosan is a promising alternative for the management of postharvest plant diseases [26Bautista-Baños S, Hernandez-Lauzardo AN, Velazquez-Del Valle MG, et al. Chitosan as a potential natural compound to control pre and postharvest diseases of horticultural commodities. Crop Prot 2006; 25(2): 108-18. [http://dx.doi.org/10.1016/j.cropro.2005.03.010] ]. In our present study, chitosan was effective in inhibition R. solani mycelial growth. These observations confirmed similar data on the antifungal effect of chitosan on mycelial growth of R. solani [27Freddo ÁR, Mazaro SM, Brun EJ, Wagner Júnior A. Chitosan as fungistatic mycelial growth of Rhizoctonia solani Kuhn. Cienc Rural 2014; 44(1): 1-4. [http://dx.doi.org/10.1590/S0103-84782014000100001] , 28Hirano S, Nagao N. Effects of chitosan, pectic acid, lysozyme, and chitinase on the growth of several phytopathogens. Agric Biol Chem 1989; 53(11): 3065-6.], and other several phytopathogenic fungi, such as Fusarium solani [29Eweis M, Elkholy SS, Elsabee MZ. Antifungal efficacy of chitosan and its thiourea derivatives upon the growth of some sugar-beet pathogens. Int J Biol Macromol 2006; 38(1): 1-8. [http://dx.doi.org/10.1016/j.ijbiomac.2005.12.009] [PMID: 16413607] ], Colletotrichum sp [30de Oliveira KAR, Berger LRR, de Araújo SA, Câmara MPS, de Souza EL. Synergistic mixtures of chitosan and Mentha piperita L. essential oil to inhibit Colletotrichum species and anthracnose development in mango cultivar Tommy Atkins. Food Microbiol 2017; 66: 96-103.http://www.ncbi.nlm.nih.gov/pubmed/28576378 [http://dx.doi.org/10.1016/j.fm.2017.04.012] [PMID: 28576378] ]. This recent study also showed that chitosan can effectively manage black scurf in potato tubers, by induction of defense-related enzymes such as POD, PPO. These results support the findings of CH-enhanced activities of POD and PPO against Sclerotinia sclerotiorum in carrots [31Qing W. ZUO J-h, Qian W, Yang N, GAO Lp. Inhibitory effect of chitosan on growth of the fungal phytopathogen, Sclerotinia sclerotiorum, and sclerotinia rot of carrot. J Integr Agric 2015; 14(4): 691-7. [http://dx.doi.org/10.1016/S2095-3119(14)60800-5] ], Fusarium sulphureum in potato [32Yang B, LI YC, HAN RF, GE YH. Postharvest chitosan treatment induces resistance in potato against Fusarium sulphureum. Agric Sci China 2008; 7(5): 615-21. [http://dx.doi.org/10.1016/S1671-2927(08)60060-7] ], and Alternaria kikuchiana and Physalospora piricola in pear [33Meng X, Yang L, Kennedy JF, Tian S. Effects of chitosan and oligochitosan on growth of two fungal pathogens and physiological properties in pear fruit. Carbohydr Polym 2010; 81(1): 70-5. [http://dx.doi.org/10.1016/j.carbpol.2010.01.057] ]. POD participates in the cell wall building processes, for instance, suberization, phenols oxidation, and lignification of host plant cells during the defense reaction against pathogenic agents [34Sommer A, Ne’eman E, Steffens JC, Mayer AM, Harel E. Import, targeting, and processing of a plant polyphenol oxidase. Plant Physiol 1994; 105(4): 1301-11. [http://dx.doi.org/10.1104/pp.105.4.1301] [PMID: 7972497] ]. PPO is involved in the oxidation of polyphenols into quinones (antimicrobial compounds) and cells lignification in the infected plants [35Kolattukudy P, Mohan R, Bajar MA, Sherf BA. Plant peroxidase gene expression and function 1992. [http://dx.doi.org/10.1042/bst0200333] ]. In this experiment, the activity of PAL has been increased in CH-treated tubers. PAL is the first enzyme in the phenylpropanoid pathway and is involved in the syntheses of phytoalexins, phenols, and lignin which have defense functions in the host plants [36Pellegrini L, Rohfritsch O, Fritig B, Legrand M. Phenylalanine ammonia-lyase in tobacco. Molecular cloning and gene expression during the hypersensitive reaction to tobacco mosaic virus and the response to a fungal elicitor. Plant Physiol 1994; 106(3): 877-86. [http://dx.doi.org/10.1104/pp.106.3.877] [PMID: 7824656] ].
This study indicated that chitosan directly inhibited the R. solani growth, and potentially elicited defense reaction in potato tubers.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.
CONSENT FOR PUBLICATION
AVAILABILITY OF DATA AND MATERIALS
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
The authors are grateful to the Biotechnology Lab., Agronomy Faculty, Saratov State Vavilov Agrarian University for the support and technical assistance of this research.
Campion C, Chatot C, Perraton B, Andrivon D. Anastomosis groups, pathogenicity and sensitivity to fungicides of Rhizoctonia solani isolates collected on potato crops in France. Eur J Plant Pathol 2003; 109(9): 983-92. [http://dx.doi.org/10.1023/B:EJPP.0000003829.83671.8f]
Djébali N, Belhassen T. Field study of the relative susceptibility of eleven potato (Solanum tuberosum L.) varieties and the efficacy of two fungicides against Rhizoctonia solani attack. Crop Prot 2010; 29(9): 998-1002. [http://dx.doi.org/10.1016/j.cropro.2010.06.012]
Hamed I, Özogul F, Regenstein JM. Industrial applications of crustacean by-products (chitin, chitosan, and chitooligosaccharides): A review. Trends Food Sci Technol 2016; 48: 40-50. [http://dx.doi.org/10.1016/j.tifs.2015.11.007]
Abd-El-Kareem F, El-Mougy NS, El-Gamal NG, Fotouh Y. Use of chitin and chitosan against tomato root rot disease under greenhouse conditions. Res J Agric Biol Sci 2006; 2: 147-52.
Rodríguez AF, Menéndez DC, Delgado EO, Díaz OL, Pino JC. Evaluation of chitosan as an inhibitor of soil-borne pathogens and as an elicitor of defence markers and resistance in tobacco plants. Span J Agric Res 2007; 5(4): 533-41. [http://dx.doi.org/10.5424/sjar/2007054-274]
Aili J, Shiping T, Yong X. Effect of CA with high-O2 or high-CO2 concentrations on postharvest physiology and storability of sweet cherry. Acta Bot Sin 2002; 44(8): 925-30.
Assis JS, Maldonado R, Muñoz T. Escribano MaI, Merodio C. Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit. Postharvest Biol Technol 2001; 23(1): 33-9. [http://dx.doi.org/10.1016/S0925-5214(01)00100-4]
Bautista-Baños S, Hernandez-Lauzardo AN, Velazquez-Del Valle MG, et al. Chitosan as a potential natural compound to control pre and postharvest diseases of horticultural commodities. Crop Prot 2006; 25(2): 108-18. [http://dx.doi.org/10.1016/j.cropro.2005.03.010]
Qing W. ZUO J-h, Qian W, Yang N, GAO Lp. Inhibitory effect of chitosan on growth of the fungal phytopathogen, Sclerotinia sclerotiorum, and sclerotinia rot of carrot. J Integr Agric 2015; 14(4): 691-7. [http://dx.doi.org/10.1016/S2095-3119(14)60800-5]
Meng X, Yang L, Kennedy JF, Tian S. Effects of chitosan and oligochitosan on growth of two fungal pathogens and physiological properties in pear fruit. Carbohydr Polym 2010; 81(1): 70-5. [http://dx.doi.org/10.1016/j.carbpol.2010.01.057]
Pellegrini L, Rohfritsch O, Fritig B, Legrand M. Phenylalanine ammonia-lyase in tobacco. Molecular cloning and gene expression during the hypersensitive reaction to tobacco mosaic virus and the response to a fungal elicitor. Plant Physiol 1994; 106(3): 877-86. [http://dx.doi.org/10.1104/pp.106.3.877] [PMID: 7824656]