STREPTOMYCES NIGELLUS AS A BIOCONTROL AGENT OF TOMATO DAMPING-OFF DISEASE CAUSED BY PYTHIUM ULTIMUM

Selim Mohsen Helmy¹, Kameil Zeinat², Saad Abd-elnaby Mahmoud ¹, Saad Moataza¹, Morsi Nagwa², Hasabo Amany^1
1 Microbial Chemistry Department, National Research Center, Cairo, Egypt
² Faculty of Science, Cairo University, Cairo, Egypt

ABSTRACT

A collection of about 15 strains of Streptomyces belonging to different spp. were screened for their ability to grow on fragmented Pythium ultimum mycelia and to produce metabolites that inhibit the growth of this plant pathogenic fungi.  Some factors affecting level of growth and fungal cell wall lytic enzymes formation by the most active Streptomyces strains were carried out. On the other hand, Streptomyces  nigellus NRC10 showed a strong in-vitro antagonism against P. ultimum in plate assay by producing antifungal metabolites. Furthermore, plant growth chamber study was also carried out to test suppression of damping off disease by the selected strain. The results showed that percentage of diseased tomato plant was greatly reduced in P.ultimum infested soil when treated with this strain.

Key words: biocontrol, damping-off, Pythium ultimum, Streptomyces nigellus.

INTRODUCTION

Intensified use of fungicides has resulted in the accumulation of toxic compounds potentially hazards to humans and environment [7] and also in the build up of resistance of the pathogens [9]. In order to tackle these national and global problems, effective alternatives to chemical are being investigated and the use of antagonistic microbes seems to be one of the promising approaches [4,5,27]. Actinomycetes represent a high proportion of the soil microbial biomass [3]. They have the capacity to produce a wide variety of extracellular hydrolases [13], that give them an important role in the decomposition of organic matter in the soil. In addition to their active function in decomposition, actinomycetes appear to be of importance among the microbial flora of the rhizosphere. Moreover, actinomycetes of the genus Streptomyces have been used to commercially control plant diseases [23].

Several experiments demonstrated that soil inoculation with specific Streptomyces strains could significantly reduce damage caused by Pythium or Phytophthora species in ornamental [29], legume [21], and horticultural [6,26] productions.

El-Mehalawy et.al., [10] isolated 43 strains of actinomycetes from the rhizosphere associated soil of cotton plant. Four actinomycete isolates were found to have a potent antagonistic activities against Rhizoctonia solani and were identified as Streptomyces erumpens, S.purpureus, S.aurantiacus and S.microflavus.

The β-glucan are the most abundant group of naturally occurring polysaccharides. Among β-glucan type polymer in fungal cell walls those compose of monomers linked by β-1,3 and β-1,4 glucosidic bounds are predominant. In addition, there is some evidence to suggest that  β-glucan may play an important rule in fungal pathogensis in plants [24]. The degradation of fungal  β-glucan cell walls require the enzymes that can hydrolyze polymers of glucose with various glucosidic bounds, and the hydrolysis of these glucans weakness the mechanical strength of cell wall, resulting in fungal cell lysis [16]. Recently, mutual interaction between three strains of Trichoderma and nine phytopathogenic moulds was studied by Piegza et al. [22]. The highest antagonistic ability was demonstrated for Trichoderma harziamum T33, which in many cases caused complete growth inhibition of tested moulds. Also, Streptomyces spp. are often used to produce enzymes for lysing pathogenic fungi. Usually they can digest a large spectrum of host organisms [30]. Extracellular enzymes with glucanase activities are an important component of actinomycetes fungus antagonism [2,12]. Streptomyces sp. EF-14 has been identified as one of the most potent antagonists of Phytophthora spp. and a β-1,6 glucanase (EC3.2.1.75 glucan-endo-1,6- β glucoside) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as a main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of β-1,6 glucanase activity always preceded that of β-1,3 glucanase [12]. El-Tarabily [11] reported that isolates of Microbispora rosea, Micromonospora chalcea and A.philippinesis produced β-1,3 , β-1,4 and β-1,6 glucanases in vitro, caused lysis of Pythium aphanidermatum hyphae in vitro and reduced damping- off disease of cucumber under controlled glass house conditions. These isolates were able to suppress damping-off in soil amended with or without cellulose. The treatment which combined all isolates in soil suplemented with cellulose was superior to all other treatments in suppressing damping-off. The purpose of this study is to evaluate the antagonistic activity of Streptomyces species as a potential biocontrol agent of tomato plant pathogenic Pythium ultimum.

MATERIALS AND METHODS

Streptomyces strains
Fifteen Streptomyces strains obtained from culture collection of Microbial Chemistry Department, National Research Center, Egypt, were screened for their antagonistic activity against Pythium ultimum. In addition, for their abilities to produce glucan hydrolysis enzymes including β-1, 3 and β-1, 4 glucanases. These strains were routinely maintained on Czapex Dox agar slants and stored at 5°C.

Fungal strain:
Plant pathogenic Pythium ultimum was obtained from Plant Pathology Department, National Research Center, Cairo, Egypt. This strain was maintained on Potato Dextrose Agar (PDA) and stored at 5°C.

Media:
Two media were used throughout this study:
1 – Czapex Dox [8]. This medium was solidified by adding 2% of agar when needed. In some experiments carbon or nitrogen sources of this medium were replaced by appropriate ones.
2 – Potato Dextrose agar medium (PDA).

Screening of antagonistic Streptomyces strains:
Strains of Streptomyces were tested for their abilities to grow and produce clear zones around their growth on the sterilized fragmented mycelia of P. ultimum agar plates.

Screening of β-1,3 and β-1,4 glucanase enzymes producing Streptomyces strains:
Streptomyces strains were cultivated in flasks containing Czapex Dox medium with 1 % of laminarin or cellulose as sole source of carbon for β-1,3 and β-1,4 glucanases, respectively. The flasks were incubated at 28°C with shaking. After five days of growth the cells were harvested by centrifugation and glucanase activities were estimated in the supernatants.

Enzymes assay:
β-1,3 and β-1,4 glucanase activities were determined according Miller et al., [19] DNS (dinitrosalicylic acid) method by measuring the amount of reducing sugar, released from laminarin for β-1,3 glucanase or carboxymethylcellulose (CMC) for β-1,4 glucanase. One unit of enzyme activity is defined as the amount of enzyme required to release 1.0 µmol of glucose equivalent per min [16].

In-vitro antagonism test:
A modified procedure of Grawford et al., [14] was used. P. ultimum was grown on PDA plates for 48 h. before the test. A loopful of the chosen Streptomyces strain was streaked on Czapex Dox agar plates to one side of the center and incubated at 28°C for about 7 days. Then 5 mm. diameter agar plug of fungal mycelium was transferred on the center side of uninoculated Dox agar plates. Cultures were examined for inhibition of fungal growth after 2–5 days of incubation. Inhibition was indicated when P.ultimum mycelial growth in the direction of the Streptomyces colony was retarded or prevented.

Green house trials:
The experiment was designed to study the antifungal activity of the most active isolate of Streptomyces against P. ultimum responsible of damping off disease of tomato plants. The pathogenicity test was carried out in plastic pots containing clay sandy soil infested with fungal inoculums. Pots were watered for a week before planting. On the other hand, the chosen Streptomyces was thoroughly dispersed through the soil two weeks before adding the pathogen inoculums and sowing. Relative plant infection cause by the fungus was recorded 2 weeks after planting.

RESULTS AND DISCUSSION

Screening of antagonistic Streptomyces strains:
Streptomyces strains exhibiting the ability to produce clear zones on fungal mycelium agar plats were considered to be antagonistic. I this study  86% of Streptomyces strains collection exhibited growth surrounded with clear zone on mycelium plates (Table 1). According to Valois et. al., [28] antagonistic Streptomyces strain that produced clear zones on mycelium agar were shown to produce glucanases cleaving β-1,3 and β-1,4 bounds. In addition El-Tarabily [11] reported that the mycelia fragments not only provide the substrate for the production of these enzymes but may also provide the signal necessary for the induction of these enzymes.

So, the  ability of antagonistic Streptomyces strains to produce β-1,3 and β-1,4 glucanases  were tested in Dox medium supplemented with laminarin or cellulose as  inductor of β-1,3 and β-1,4 glucanases, respectively. Results presented in Table (2) shows that the highest production of both enzymes was noticed for  Streptomyces nigellus NRC 10 and therefore it was used for further studies. S. nigellus NRC 10 was tested for its in-vitro antagonism against P.ultimum. Obtained results shown on figure (Fig. 1) indicate that fungal growth was inhibited by Streptomyces and the antagonistic effect was more active after 5 days of incubation.

Effect of some carbon sources on β-1, 3 and β-1,4 glucanases production
In order to study the nature of β-1, 3 and β-1, 4 glucanases synthesis by S. nigellus NRC 10 (i.e. inducible or constitutive), the strain was cultivated in a medium supplemented with different carbon sources (each was added separately). Obtained results  showed that β-1,3 glucanase was  not constitutively synthesized (Table 3). Some substrates induce synthesis of β-1,3 glucanase and the highest enzyme formation (94 units/ml) was obtained with yeast glucan as sole carbon source (Fig. 2) at 1% in medium (Table 4). Induction of β-1,3 glucanases of Trichoderma hamatum was reported by Maj et al. [17].

On the other hand β-1,4 glucanase   was found to be constitutively synthesized (Table 3) and cellulose was the most suitable source followed by maltose, fructose, sucrose, lactose, cellobiose and starch. It gives 25, 22, 15.4, 13.9, 12 and 12 units/ml respectively. According to this data it was necessary to establish the most suitable concentration of these carbons. Lactose and cellobiose at 1.5% (final concentration in medium)  were correlated with maximum enzyme production:  60 and 54 units/ml, respectively (Fig. 3).  At higher concentrations  a slight decrease of enzymes activity was observed. Those results were in agreement with that reported by  Saha [25] for  Mucor circinelloids NRRL26519, Mandels and Reese [18] for  Trichoderma viride and Morikawa et. al. [20] on Trichoderma  reesei.

Effect of some  nitrogen sources on β-1,3 and β-1,4 glucanases production
Regarding the effect of organic or inorganic nitrogen sources on growth and production of β-1,3 and β-1,4 glucanases by S. nigellus NRC 10 ten compounds were tested as N source (Table 5). Therefore, nitrogen source of medium was replaced by other nitrogen in such amount that final concentration of nitrogen in medium remained unchanged. Obtained results  showed that tribasic ammonium phosphate was the most favorable nitrogen source for the highest production of both enzymes and concentration  of 0.08% (i.e.0.02% as N base) was found to be the most suitable  for enzyme production (Fig. 4).  The use of  ammonium phosphate in medium for production of these enzymes  was reported by Ahuga et. al., [1] for bacteria Terredinobacter turnirae, and also by El-Katatny et. al., [9] for T. harzianum. 

Enzyme activity on fungal mycelia:
The ability of S. nigellus NRC 10 to lyse fungal mycelia of P. ultimum was investigated by growing Streptomyces in medium containing fungal mycelia as a source of carbon. Result in Table 6, indicate that S. nigellus NRC 10 could utilize fungal mycelia and secreted β-1,3 and β-1,4 glucanases into the growth medium. In addition, 7 days incubation was correlated with maximum enzymes formation.

Green house experiment:
Results of green house experiment in Table 7 showed that percentage of diseased plants was considerably  reduced, from 48.9% of pathogen infected  plants to 22.7% of pathogen infected  and Streptomyces treated plants. This experiment could be considered as the first record of the biocontrol of wilt disease caused by P. ultimum using S. nigellus NRC 10. In addition, seed germination plant growth were increased, this phenomenon indicate that Streptomyces may act as a plant growth stimulants as recorded by Yuan and Crawford [28] and Hassanein et. al. [15].

CONCLUSIONS

The report focuses on screening for Streptomyces isolates  as a biocontrol agent against plant pathogenic fungi. A total of 15 strains of Streptomyces were screened for their ability to grow on fragmented Pythium ultimum mycelia and to produce metabolites that inhibit the growth of this plant pathogenic fungus. S. nigellus NRC 10 seems to be the most active strain. In plant growth chamber studies (carried out to test suppression of damping off disease by the selected strain) was demonstrated that percentage of diseased tomato plants  in P. ultimum infested soil was greatly reduced when  soil was supplemented  with this Streptomyces. Therefore, the present study demonstrated that S. nigellus NRC 10  is a new potential biocontrol agent against some plant pathogenic fungi.

REFERENCES

1. Ahuga S.K., Ferreira  G.M., Moreira A.R., 2003. Production of an endoglucanase by the shipworm bacterium, Teredinobacter turnirae. J. Ind. Microbiol. Biotechnol 31, 41–47.

2. Anitha A., Rebeeth,M., 2009.In vitro antifungal activity of Streptomyces griseus against Phytopathogenic fungi of Tomato field. Academic J. of Plant Science 2(2), 119–123.

3. Atlas R. M., Bartha R., 1993. Microbial ecology: fundamental and applications, 3^rd ed. The Bengamin/cummings Co., Redwood City, Calif.

4. Aziz A., Trotel-Aziz P., Dhuicq L., Jeandet P., Couderchet M., Vernet G., 2006. Chitosan oligomers and cooper sulfate induce grapevine defense reactions and resistance to gray mild and downy mildew. Phytopathology, 96, 1188–1194.

5. Bargabus R.L., Zidack N.K., Sherwood J.W., Jacobsen B.J., 2004. Screening for the identification of potential biological control agents that induce systemic acquired resistance in sugar beet. Biological Control. 30, 342–350.

6. Cao L., Qui Z., You J., Tan H., Zhou S., 2005. Isolation and characterization of endophytic Streptomycete antagonists of Fusarium wilt pathogen from surface-sterilized banana roots. FEMS Microbiology Letters, 247, 147–152.

7. Cook R.J., Baker  K.F., 1983. The nature and practice of  biological control of plant pathogens. American Phytopathological Society, St Paul, MN, PP539.

8. Difco, Manual 1972. "Difco Manual of dehydrated culture, media and reagents" 9^th ED, 245. Difco Laboratories, Detroit, Michigan, USA.

9. El-Katatny M.H., Somitsch W., Robra K.H., El-Katatny M.S., Gubitz G.M., 2000. Production of chitinase and 1,3-glucanase by Trichoderma harzianum for control of the phytopathogenic fungus Sclerotium rolfsii. Food Technol. Biotechnol. 38, 173–180.

10. EL-Mehalawy A.A., Hassanin S.M., Hassanin N.M., Zaki S.A., 2007. Induction  of resistance biocontrol of Rhizoctonia in cotton damping –offdisease by rhizosphere microorganisms. New Egyptian Journal of Microbiology. 17, 148–168.

11. El-Tarabily K. A., 2006. Rhizosphere – competent isolates of Streptomycete and non-Streptomycete actinomycetes capable of producing cell wall degrading enzymes to control Pythium aphanidermatum damping-off disease cucumber. Canadian Journal of Botany , 84, 211–222.

12. Fayad  K.P., Simao-Beaunoir A.M., Gauthier  A., Leclerc  C., Mamady  H., Beaulicu C., Brzezinski R., 2001. Purification and properties of a b-1,6 glucanase from Streptomyces sp. EF-14, an Actionomycete antagonistic to Phytophthora spp. Appl. Microbiol. Biotechnol. 57, 117–123.

13. Gilbert M., Morosoli R., Schareck F., Kluepfel D., 1995. Production and secretion of proteins by Streptomycetes. Crit. Rev. Biotechnol 15, 13–39.

14. Grawford D.N., James M., John M., Margaret  A., 1993. Isolation and  characterization of actinomycete antagonists of a fungal root pathogen. Appl. Microbiol. 59 (11), 3899–3905.

15. Hassanein  N.M., El-Mehalawy A.A., Khater  H.M., Karam El-Din E.A., Youssef Y.A., 2002. The potential of selected rhizosphere actinomycetes and yeast fungi for the biological control of late wilt disease of maize caused by Cephalosporium maydis. Proceeding of the third International conference of fungi. Cairo, 30^th–31^th Oct. 2002 Vol. 1, 167–188.

16. Hong T.Y., Meng  M., 2003. Biochemical characterization and antifungal activity of an endo- β-1,3 glucanase of paenibacillus sp. isolated from garden  soil.  Appl. Microbiol. Biotechnol. 61, 472–478.

17. Maj A., Witkowska D., Robak M., 2002. Biosynthesis and properties of beta 1,3 glucanases of Trichoderma hamatum, EJPAU, Biotechnology, 5(2), 1–8 

18. Mandels M., Reese E.T., 1960. Induction of cellulase in fungi by cellobiose. J. Bacteriol. 79, 816–826.

19. Miller G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428.

20. Morikawa Y., Ohashi T., Mantani O., Okada H., 1995. Cellulase induction by lactose in Trichoderma reesei PC-3-7. Applied Microbiology and Biotechnology. 44, 106–111.

21. Nassar  A.H., El-Tarabily  K.A., Sivasithamparam K., 2003. Growth promotion of bean, (Phaseolus vulgaris L.) by a polyamine-producing isolate of Streptomyces griseolueus. Plant. Growth Regulation, 40, 97–106.

22. Piegza M., Stolaś J., Kancelista A., Witkowska D., 2009. Wpływ grzybów rodzaju Trichoderma na wzrost patogennych grzybów strzępkowych w testach biotycznych na nietypowych źródłach węgla. [Influence of Trichoderma strains on the growth of pathogenic moulds in biotic test on untypical carbon sources]. Acta Sci. Pol., Biotechnol. 8(1), 4–14.

23. Prapagdee B., Kuekulvong C., Mongkolsuk S., 2008. Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytogenic fungi. Int. J. Biol. Sci. 4, 330–337.

24. Ruel K., Joseleau J., 1991. Involvement of an extracellular glucan sheath during degradation of populous wood by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 57, 374–384.

25. Saha B.C., 2004. Production, purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides. Process Biochemistry 39: 1871–1876.

26. Sutherland E.D., Papavizas G.C., 1991. Evaluation of oospore hyperparasites for the control of Phytophthora crown rot of pepper. J. Phytopathol. 131, 33–39.

27. Tjamos S.E., Flemetakis E., Paplomatas E.J., Katinakis P., 2005. Induction of resistance to Verticillium dahliae in Arabidopsis thaliana by biocontrol agent k-165 and pathogenesis-related proteins gene expression. Mol. Plant. Microbe Interact., 18, 555–561.

28. Valois D., Fayad K., Barasubiye T., Garon M., Dery C., Brzezinski R., Beaulieu C., 1996. Glucanolytic actinomycetes antagonistic to Phytophthora fragariae var. rubi, the causal agent of raspberry root rot. Appl. Environ. Microbiol. 62, 1630–1635.

29. Yuan  W., Crawford D., 1995. Characterization of Streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots. Appl. Environ. Microbiol. 61, 3119–3128.

30. Zoog H., Jäggi W., 1974. Soil-Borne plant pathogens. Phytopath. Z. 81, 160-169.

Accepted for print: 21.10.2010

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