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Biological Control of Sclerotinia Stem Rot (S. minor ) of Sunflower Using Trichoderma Species



J. Abdollahzadeh, E. Mohammadi Goltapeh and H. Rouhani
 
ABSTRACT

Based on in vitro results, three isolates: T. harzianum (J1), T. harzianum (J6) and T. virens were selected for greenhouse studies. Biocontrol efficacy of these isolates as well as the combination of T. harzianum (J1) and T. virens on crown and root rot of sunflower were studied in series of experiments using three methods; incorporation of conidial suspension into the soil (107 conidia g-1 soil), incorporation of conidial suspension mixed with compost (107 conidia g-1 compost) into the soil and impregnating of sunflower seeds with conidial suspension (107 conidia mL-1). Experimental data were analyzed with Randomized Complete Block Design (RCBD) and means compared by Duncan`s multiple range test. Greenhouse experiments (in vivo) showed that, the combination of T. harzianum (J1) and T. virens using incorporation of conidial suspension mixed with compost into the soil had the most efficiency on biocontrol of S. minor and reduced disease incidence up to 50%.

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  How to cite this article:

J. Abdollahzadeh, E. Mohammadi Goltapeh and H. Rouhani , 2006. Biological Control of Sclerotinia Stem Rot (S. minor ) of Sunflower Using Trichoderma Species. Plant Pathology Journal, 5: 228-232.

DOI: 10.3923/ppj.2006.228.232

URL: https://scialert.net/abstract/?doi=ppj.2006.228.232

INTRODUCTION

Sclerotinia minor and S. sclerotiorum are important soil borne pathogens of sunflower (Helianthus annuus). Both species can infect the root system and cause a basal stem rot and wilt (Clarke et al., 1990). For the first time, this disease was reported from Western Azarbaijan, Iran by Ale Agha (1971). Control measures are currently limited to the use of long rotations and the early sowing of tolerant hybrids (Clarke et al., 1993), as commercial cultivars with adequate resistance are not yet available. Also, chemical control using dicarboximide fungicides is cost-effective only for crops where disease incidence is likely to reach 20% (Clarke et al., 1990). Furthermore, recently some strains of Sclerotinia with resistance to this fungicides have been detected (Burgess and Hepworth, 1996). Biological control so can be considered as a valuable method to study. Study on different mycoparasites known to infect the sclerotia of S. sclerotiorum showed that Trichoderma virens was one of the most effective antagonist in reducing sclerotia viability in vitro (Whipps and Budge, 1990). Preliminary tests using an isolate of T. virens from a sunflower field in Northern Victoria indicated its potential for biocontrol of S. minor. Trichoderma species such as T. harzianum and T. virens have also been shown attack the sclerotia and mycelium of Sclerotinia spp. (Whipps and Budge, 1990; Knudsen et al., 1991). In order to identification of some effective isolates and use of them against disease caused by S. minor on sunflower, this study was conducted. Different methods of Trichoderma application were also investigated.

MATERIALS AND METHODS

Production of sclerotinia inoculum: S. minor isolate collected from infected sunflower of Khoy. Plates containing PDA medium were inoculated with mycelium from a 3-day-old culture of S. minor. The culture were incubated at 25°C in darkness for 2 weeks and sclerotia were recovered and dried on blotting paper for 30 min and stored in plates at room temperature.

Production of trichoderma inoculum: In experimental tests, three isolates of Trichoderma namly, T. harzianum(J1) isolated from soil of infected field of sunflower and T. harzianum (J6) and T. virens from substrate (compost) of edible fungi (Agaricus bisporus), were collected and used as the biological control agent in this work. Conidial suspension were prepared by adding sterile distilled water (SDW) to a 4-week-old culture of Trichoderma isolates on PDA and gently scarping the surface with a sterile rod. The conidial concentration was adjusted at 108 conidial mL-1 for soil treatment and 107 conidial mL-1 for seed treatment.

In vitro studies
Antagonism by trichoderma:
Antagonism of S. minor was studied in co-culture on PDA by one, two and six isolates of T. virens, T. viride and T. harzianum, respectively. Disks of antagonist isolates and S. minor were cut of the margin of 5-day-old cultures on PDA and placed 6 cm apart on fresh PDA plates. The cultures were incubated at 22°C for two weeks. The mycelia in the interface of antagonist and pathogen colonies were examined by microscope (Magnification, 40x10) after incubation for 2 days. In order to study the colonization ability of antagonist isolates, 3 and 14-day-old cultures of S. minor were inoculated with disks of antagonist and incubated for two weeks. Both experiments were carried out in four replicate.

Greenhouse studies: Studies were done with sunflower (Helianthus annuus) cv. Record in agriculture research center of Urmia (Western Azarbaijan, Iran) in winter of 2001. Four seeds were sown in pots containing clay-loam soils. The pots were maintained in greenhouse condition at 22±5°C and watered every 3 days. All treatments were replicated 3 times and data were analyzed using Randomized Complete Block Design (RCBD) and means separated using Duncan’s multiple range test. Data were transformed before the analysis according to:

Soil incorporation of conidial suspension: In this experiment, a conidial suspension (100 mL) of Trichoderma isolates were added to 1 kg soil on each pot. In order to infect the pots, 100 sclerotia of S. minor were added to 1 kg soil on each pot. Treatments were as the follows:

a) T. harzianum (J1) b) T. harzianum (J6) c) T. virens d) T. harzianum (J1) + T. virens

In the later treatment 50 mL of conidial suspension of each isolate was added to 1 kg soil on each pot. Controls were treated with tap water. After 7 days sunflower seeds were sown. The pots were then observed daily for the development of basal stem lesion.

Soil incorporation of conidial suspension with support: In this experiment, at first 100 g ground pasteuraized button mushroom compost were mixed with 100 mL conidial suspension of Trichoderma isolates and incubated in lab condition for 1 week. Then the compost was added to 1 kg soil on each pot containing 100 sclerotium of S. minor. Treatments were as follows:

a) T. harzianum (J1) b) T. harzianum (J6) c) T. virens d) T. harzianum (J1) + T. virens

In the later treatment, 50 mL-1 of conidial suspension of each isolate was added to 100 g ground pasteuraized button mushroom compost. Controls were treated with uninoculated compost (100 g) and tap water. After 7 days sunflower seeds were sown. The pots were then observed daily for the development of basal stem lesion.

Seed treatment: In this experiment, after preparation of conidial suspension (107 conidium mL-1), the Arabic gum (3 g L) was added in order to stick conidia on seed coat. Then the seeds were impregnated for at least 2 h in the conidial suspension and sown in the soil infected with 100 sclerotium of S. minor. Treatments were as the follows:

a) T. harzianum (J1) b) T. harzianum (J6) c) T. virens d) T. harzianum (J1) + T. virens

The later treatment was containing of 50 mL of conidial suspension of each isolate.

RESULTS

In vitro studies
Antagonism by trichoderma:
Examination of mycelia in the interface of all Trichoderma isolates revealed the absence of cytoplasmic contents due to destruction and endolysis of hyphal tips of S. minor (Fig. 1). Invasion of hyphae of S. minor was not detected. Study on colonization ability of antagonist isolates on 3-day-old cultures of S. minor showed that T. virens and T. harzianum (J1) overgrews the mycelium of S. minor on PDA and colonized sclerotial initials and preventing melanization of sclerotia (Fig. 2 ). Study on colonization ability of Trichoderma isolates on 14-day-old cultures of S. minor showed that T. harzianum (J1) and T. harzianum (J6) overgrews the clony of S. minor and also colonized, sporulated on mature sclerotia and lysed them on PDA (Fig. 3).

Greenhouse studies
Soil incorporation of conidial suspension:
In this experiment, the application of conidial suspension of T. harzianum (J6), T. virens, T. harzianum (J1) and T. virens + T. harzianum (J1) in 1 kg soil on each pot containing sclerotia of S. minor reduced disease incidence by 8.33, 16.66, 25 and 41.66%, respectively (Table 1).

Fig. 1: Light microscopy of S. minor mycelium from the interface with all Trichoderma isolates in co-culture on PDA, showing empty hyphal tip of S. minor (Magnification 40x10)

Fig. 2: Colonization of mycelia and sclerotial initials and preventing melanization of sclerotia of S. minor by T. virens and T. harzianum(J1) on 3-day-old cultures of S. minor

Fig. 3: Colonization and sporulation of mature sclerotia of S . minor by T. harzianum (J1) and T . harzianum (J6) on 14-day-old cultures of S. minor

Table 1: Effect of Trichoderma isolates on crown and root rot of sunflower caused by S. minor in greenhouse condition
X: values averaged over three replicates with four seed per pot., Y: means followed by different letter are significantly different (p>0.01) according to Duncan’s Multiple range test., A*: application method: soil incorporatin of conidial suspension, B*: application method: soil incorporatin of conidial suspension with support

Table 2: Effect of seed treatment with Trichoderma isolates on crown and root rot of sunflower caused by S. minor in greenhouse condition
X: values averaged over three replicates with four seed per pot., Y: means followed by different letter are significantly different (p>0.01) according to Duncan’s Multiple range test

Soil incorporation of conidial suspension with support: In this experiment, the application of conidial suspension of T. harzianum (J6), T. virens, T. harzianum (J1) and T. virens + T. harzianum (J1) with support ( pasteurized compost) in 1 kg soil on each pot containig sclerotia of S. minor reduced disease incidence by 16.66, 25, 33 and 50%, respectively (Table 1).

Seed treatment: In this experiment, the isolates of T. harzianum (J6), T. virens, T. harzianum (J1) and T. virens + T. harzianum (J1) reduced disease incidence caused by S. minor by 0, 16.66, 8.33 and 8.33%, respectively (Table 2).

DISCUSSION

The results of this study showed that all isolates exhibited antibiosis, evidenced by distortion and endolysis of hyphal tips of S. minor in co-culture. Antibiosis is well documented for T. virens (Lewis et al., 1991; Robert and Lumsden, 1990) and parasitism of hyphae of S. sclerotiorum has been also reported (Tu, 1980). In present studies, hyphal invasion of S. sclerotiorum by all above mentioned Trichoderma isolates, was observed (In press). However, parasitism of S. minor hyphae by T. virens has not yet reported and was not also detected by any isolates in our investigation. In our studies, colonization of sclerotial initials and preventing melanization of sclerotia on 3-day-old cultures as well as colonization, sporulation and lysis of melanized sclerotia on 14-day-old cultures of S. minor were observed and supported with results of other (Burgess and Hepworth, 1996 ). The results indicated that distortion and endolysis of S. minor hyphal tips by antibiosis and parasitism of sclerotia were two important antagonistic mechanisms of Trichoderma species on S. minor. Jones and Stewart (1999) reported that the soil incorporation of T. harzianum (C52) was more effective than pellet information and transplant root spore dip. It reduced disease incidence of S. minor by 50% in lettuce.

Burgess and Hepworth (1996) also reported that application methods of T. virens (i.e., soil incorporation and seed treatment) reduced disease incidence of sclerotinia stem rot (S. minor) in sunflower by 44.2 and 14.8% in greenhouse condition, respectively. Present results, in consistent with previous authors showed that soil incorporation was more effective than seed treatment. Rouhani (2000) proved that out of 10 Trichoderma isolates only one could adjust to rhizosphere condition. The reason for little or no influence of Trichoderma isolates in the seed treatment could be due to slow stabilization of Trichoderma in rhizosphere as well as in crown of the host. In other words, rhizosphere and crown condition were not suitable to stablize Trichoderma isolates. Furthermore, in this method, the number of conidia carried to the soil was greatly smaller in compare with other methods. In addition, these results showed that method of soil incorporation of conidial suspension with support(pasteurized compost) was more effective than application of them without support. May be it is due to that most of conidia were probably lysed without first germinating, or they germinated in response to some nutrients released from organic matter and subsequently lysed in the absence of food base adequate enough to sustain further growth and sporulation (Papavizas, 1985). In conclusion, the application of Trichoderma isolates by soil incorporation of conidial suspension with organic matter was more effective in crown and root rot disease incidence reduction caused by Sclerotinia on sunflower in greenhouse condition. But, it is necessary to evaluate the results of this study in the field.

REFERENCES
Ale Agha, N., 1971. Crown rot of sunflower. Annual Report of Study of Important Disease in Crops.

Burgess, D.R. and G. Hepworth, 1996. Biocontrol of sclerotinia stem rot (Sclerotinia minor) in sunflower by seed treatment with Gliocladium virens. Plant Pathol., 45: 583-592.

Clarke, R.G., I.J. Porter and M. Woodroofe, 1993. Effect of sowing date on the incidence of sclerotinia stem rot caused by Sclerotinia minor and yield a long- and a short-season sunflower cultivar. Aust. Plant Pathol., 22: 8-13.
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Clarke, R.G., I.J. Porter and M. Woodroofe, 1990. Potential strategies for control of sclerotinia stem rot in sunflowers. Proceedings of the Australian Sunflower Association 7th Workshop, Moama, 1990 NSW, Australia: Australian Sunflower Association, Queensland.

Jones, E.E. and A. Stewart, 1999. Biological control of Sclerotinia minor in lettuce using Trichoderma species. Proceedings of the 50th New Zealand Plant Protection Conference, (NZPPC'1999), Lincoln, New Zealand, pp: 154-158.

Knudsen, G.R., D.J. Eschen, L.M. Dandurand and L. Bin, 1991. Potential for biocontrol of Sclerotinia sclerotiorum through colonization of sclerotia by Trichoderma harzianum. Plant Dis., 75: 466-470.
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Lewis, J.A., D.P. Roberts and M.D. Hollenbeck, 1991. Induction of cytoplasmic leakage from Rhizoctonia solani hyphae by Gliocladium virens and partial characterization of a leakage factor. Biocontrol Sci. Technol., 1: 21-29.

Papavizas, G.C., 1985. Trichoderma and Gliocladium: Biology, ecology and potential for biocontrol. Annu. Rev. Phytopathol., 23: 23-54.
CrossRef  |  Direct Link  |  

Roberts, D.P. and R.D. Lumsden, 1990. Effect of extracellular metabolites from Glicladium virens on germination of sporangia and mycelial growth of Pythium ultimum. Phytopathology, 80: 461-465.
Direct Link  |  

Rouhani, H., 2000. Minor pathogen of wheat root and their biological congress. Proceeding of the 7th Arab Congress of Plant Pathology, (ACPP'2000), IEEE Xplore, London, pp: 402-402.

Tu, J.C., 1980. Gliocladium virens, a destructive mycoparasite of Sclerotinia sclerotiorum. Phytopathology, 70: 670-674.

Whipps, J.M. and S.P. Budge, 1990. Screening for sclerotial mycoparasites of Sclerotinia sclerotiorum. Mycol. Res., 94: 607-612.

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