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

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.