Vegetable Pathology
`
`84
`
`COMPATIBILITY OF THE BIOCONTROL AGENT
`TRICHODERMA HARZIANUM C52 WITH SELECTED
`FUNGICIDES
`
`K.L. McLEAN1, J. HUNT2 and A. STEWART1
`
`1Soil, Plant and Ecological Sciences Division P.O. Box 84, Lincoln University,
`Canterbury
`2Agrimm Technologies Ltd, P.O. Box 13245, Christchurch
`Corresponding author: stewarta@lincoln.ac.nz
`
`ABSTRACT
`Trichoderma harzianum C52 is an effective biocontrol agent of the onion
`white rot pathogen Sclerotium cepivorum. For this biocontrol agent to be
`integrated into an existing disease management programme, it must be
`compatible with the fungicides commonly used on onions. The sensitivity
`of T. harzianum spores to the field rate of eight fungicides commonly
`applied to onions was determined in an in vitro assay. Results indicate
`that T. harzianum was least sensitive to procymidone and captan and most
`sensitive to mancozeb, tebuconazole and thiram. A glasshouse pot trial
`confirmed that T. harzianum was sensitive to mancozeb but tolerant of
`captan. This research indicates that in furrow applications of T. harzianum
`would be compatible with a captan and/or benomyl seed treatment for
`control of other seedling diseases.
`Keywords: Sclerotium cepivorum, Trichoderma harzianum, biocontrol,
`onion, fungicide sensitivity.
`
`INTRODUCTION
`Trichoderma harzianum C52 has been identified as a promising biocontrol agent of
`onion white rot disease caused by the soil-borne pathogen Sclerotium cepivorum Berk
`(Kay & Stewart 1994a). Under low to medium disease pressure, the biocontrol fungus
`gave good control of the disease (60-70%) when applied as a soil additive at planting
`time (McLean & Stewart 2000). However, under high disease pressure, efficacy of the
`biocontrol agent declined (30%), necessitating a combination of measures to obtain
`adequate disease control. There is an opportunity to integrate the use of T. harzianum
`C52 with reduced fungicide applications for white rot control. However, the biocontrol
`fungus must be compatible with the fungicides used to control white rot and other onion
`diseases.
`The sensitivity of T. harzianum C52 to the fungicides routinely used on onions was
`determined in in vitro and glasshouse assays to determine its compatibility with fungicide
`applications as part of an integrated disease management programme.
`
`METHODS
`Test fungicides are listed in Table 1. Fungicides were prepared in distilled water and
`applied at the recommended field rate (Table 1).
`In vitro spore sensitivity assay
`A spore suspension was prepared by flooding 5 ml sterile water onto 10 day old T. harzianum
`C52 colonies grown on PDA in 9 cm diameter Petri dishes at 20°C in the light. The stock
`spore suspension was diluted to 1 x 105 spores/ml using potato dextrose broth to provide
`nutrients required for spore germination. Aliquots (0.5 ml) of spore suspension and
`each fungicide (0.5 ml) were added to 1.5 ml conical tubes, with sterile distilled water as
`
`New Zealand Plant Protection 54:84-88 (2001)
`
`© 2001 New Zealand Plant Protection Society (Inc.) www.nzpps.org
`Refer to http://www.nzpps.org/terms_of_use.html
`
`

`

`Vegetable Pathology
`
`85
`
`TABLE 1: Fungicide name, active ingredient, field rate and formulation of
`fungicides tested against Trichoderma harzianum (C52).
`
`Fungicide
`product name
`
`Active
`ingredient
`
`Foliar spray Seed treatment
` (ai/ha)
` (ai/kg seed)
`
`Formulation
`
`Field rate
`
`benomyl
`Benlate ®
`procymidone
`Sumisclex ® 25
`procymidone
`Sumisclex® WP
`triadimenol
`Cereous
`Folicur® 430SC tebuconazole
`Orthocide® 48F
`captan
`Orthocide 80W captan
`Euparen® DF
`dichlofluanid
`Thiram 40F
`thiram
`Thiram technical
`thiram
`Mancozeb 80W mancozeb
`
`250 g
`750 g
` -
`375 g
`376 g
` 6 kg
` -
` 1 kg
` 16 kg
` -
` 1.6 kg
`
`125 g
` -
` 5 g
` -
` -
` -
` 8 g
` -
` -
` 8 g
` -
`
`wettable powder
`suspension concentrate
`wettable powder
`emulsifiable concentrate
`suspension concentrate
`suspension concentrate
`dust
`water dispersible granule
`suspension concentrate
`dust
`wettable powder
`
`a no-fungicide treatment. Each spore/fungicide mix was replicated four times. The
`tubes were fastened to the side of a rotating arm in an oven and slowly rotated at 20-
`25°C. After 24 h, four samples were taken from each tube and germination was recorded
`for 50 spores per sample. Spores were considered germinated when germ tube length
`equalled spore diameter. Significant differences in spore germination between fungicides
`were determined using a one-way ANOVA with fungicide as the factor. Where ANOVA
`indicated a significant treatment effect, this was further explored using a Fisher’s LSD
`test.
`Glasshouse trial
`Plastic pots (6 cm x 6 cm x 8 cm) were filled to within 2 cm of the top with
`Wakanui silt loam soil, and the soil moisture was adjusted to -0.3 bar. Trichoderma
`harzianum C52 was formulated as Trichoboost™ (conidia coated onto an inorganic
`based prill approximately 0.5–1 mm diameter) by Agrimm Technologies Ltd.,
`Christchurch, New Zealand (9.5 x 106 spores/g product), and 0.05 g was added to
`each of six planting holes in each pot. Three pots (replicates) were prepared for
`each treatment. A single onion seed was placed into each planting hole (0.5 cm
`below the soil surface and equidistant from each other in a grid arrangement) and
`lightly covered with soil.
`The glasshouse trial comprised 11 treatments. In the first five treatments, onion seeds
`were treated with one of the following fungicides before planting: captan, thiram,
`procymidone, benomyl or a combination of thiram, procymidone and benomyl (TPB) at
`the recommended field rate (Table 1). In four further treatments, uncoated onion seeds
`were planted as described above, and foliar sprays of dichlofluanid, mancozeb,
`procymidone and triadimenol were applied immediately after planting as soil drenches
`at the field rate (Table 1). The remaining two treatments were a TPB seed treatment
`combined with a soil drench of mancozeb (TPBM), and the T. harzianum formulation
`alone as a no-fungicide treatment.
`Each pot was placed in a separate drip tray and randomly positioned in a glasshouse
`for the duration of the trial. Pots were watered as necessary. A teaspoon was used to
`collect a soil sample from one planting hole and the surrounding soil in each pot,
`before any fungicides were added (0) and 3, 12, 30 and 50 days after fungicide
`application. A colony forming unit (cfu) assay was performed on 1 g of the soil
`sample for each pot and aliquots (0.5 ml) from each dilution were pipetted and spread
`over the surface of four Trichoderma selective medium plates (McLean, 2001). Results
`
`© 2001 New Zealand Plant Protection Society (Inc.) www.nzpps.org
`Refer to http://www.nzpps.org/terms_of_use.html
`
`

`

`Vegetable Pathology
`
`86
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`were analysed using a two-way ANOVA with fungicide and sample time as factors.
`Where ANOVA indicated a significant treatment effect, this was further explored using
`Fisher’s LSD test.
`
`RESULTS
`
`In vitro spore sensitivity assay
`The T. harzianum C52 spores in the no-fungicide treatment all germinated after 24 h
`incubation in potato dextrose broth. Trichoderma harzianum spores were least sensitive
`(P<0.05) to procymidone, which failed to inhibit spores from germinating (Table 2),
`and were also relatively insensitive to captan. Trichoderma harzianum spores were
`most sensitive to tebuconazole, thiram and mancozeb, at the rates used.
`
`TABLE 2: Mean percentage inhibition of Trichoderma harzianum C52 spore
`germination when exposed to field rates of selected fungicides.
`
`Fungicide
`
`procymidone
`captan
`dichlofluanid
`triadimenol
`benomyl
`mancozeb
`tebuconazole
`thiram
`
`% Inhibition of
`spore germination
`
`0
`3.5
`17.1
`41.2
`58.3
`100
`100
`100
`
`a1
`b
`c
`de
`f
`g
`g
`g
`
`1Mean values followed by the same letter do not differ significantly (P<0.05).
`
`Glasshouse trial
`The T. harzianum C52 concentration in the no-fungicide and fungicide treatments
`prior to fungicide application (time 0) ranged from 1.4-7.8 x 104 cfu/g soil, with no
`difference (P>0.05) in cfu counts between treatments. Three days after fungicide
`spray application, the T. harzianum concentration in the mancozeb (2.2 x 103 cfu/g
`soil) and the TPBM (2.9 x 103 cfu/g soil) treatments was significantly lower (P<0.05)
`than the T. harzianum concentration in the no-fungicide and other treatments (Table
`3). Twelve days after fungicide application, the T. harzianum concentration in all
`treatments, except the captan seed and procymidone soil drench treatments, were
`significantly lower (P<0.05) than the no-fungicide treatment. The T. harzianum
`concentration in the combined TPBM treatment was significantly less (P<0.05) than
`all other treatments except the mancozeb treatment. Thirty days after fungicide
`application, the T. harzianum concentration was similar for all treatments. Fifty
`days after initial fungicide application, the T. harzianum concentration in the
`dichlofluanid and procymidone soil drenches and the benomyl, thiram, and TPB
`seed treatments were significantly less (P<0.05) than the no-fungicide treatment.
`The T. harzianum concentration in the combined TPBM treatment was significantly
`less (P<0.05) than all other treatments.
`
`DISCUSSION
`The in vitro spore germination assay showed that T. harzianum C52 was highly sensitive
`to mancozeb, tebuconazole and thiram, less sensitive to benomyl, triadimenol and
`dichlofluanid, and relatively insensitive to procymidone and captan. However, the
`glasshouse results were less extreme with no single fungicide or combination of fungicides
`
`© 2001 New Zealand Plant Protection Society (Inc.) www.nzpps.org
`Refer to http://www.nzpps.org/terms_of_use.html
`
`

`

`Vegetable Pathology
`
`87
`
`TABLE 3: Mean number of Trichoderma harzianum C52 colony forming units
`per gram of soil at 3, 12, 30 and 50 days after fungicide application in
`the glasshouse trial.
`
`Fungicide
`
`3
`
`12
`
`30
`
`50
`
`Days after fungicide application
`
`No-fungicide
`thiram1
`procymidone1
`captan1
`benomyl1
`TPB1
`procymidone2
`triadimenol2
`mancozeb2
`dichlofluanid2
`TPBM1+2
`
`3.1 x 104 a3
`4.2 x 103 a
`4.7 x 103 a
`3.8 x 104 a
`1.2 x 104 a
`3.4 x 104 a
`6.5 x 103 a
`6.0 x 103 a
`2.2 x 103 b
`6.1 x 103 a
`2.9 x 103 b
`
`6.5 x 105
`a
`3.5 x 104 cd
`3.9 x 104 cd
`1.3 x 105 ab
`3.5 x 104 cd
`2.8 x 104 cd
`9.8 x 104 ab
`5.9 x 104 bc
`5.8 x 103 de
`8.3 x 104
`b
`1.8 x 103
`e
`
`6.8 x 105 a
`3.8 x 104 a
`4.5 x 104 a
`6.4 x 104 a
`3.4 x 104 a
`6.0 x 104 a
`5.5 x 104 a
`2.3 x 105 a
`4.5 x 104 a
`2.1 x 105 a
`2.4 x 104 a
`
`3.1 x 105
`a
`4.2 x 104
`c
`7.7 x 104
`ab
`6.2 x 104 abc
`2.4 x 104
`c
`8.1 x 104
`ab
`5.7 x 104
`bc
`4.0 x 104
`c
`7.0 x 104
`ab
`5.5 x 104
`c
`4.0 x 103
`d
`
`1Seed treatment 2Foliar spray applied as a soil drench 3Mean values followed by the
`same letter do not differ significantly within each column (P<0.05), n = 3.
`
`completely suppressing the activity of T. harzianum C52 in the soil. The glasshouse
`results are a more realistic assessment of the compatibility of T. harzianum C52
`with the various fungicides, since it is unlikely that the level of direct contact between
`fungus and fungicide observed in the in vitro assay would occur in the field
`environment given the strong buffering capacity of the soil. However, the in vitro
`results do allow us to better explain the trends detected in the glasshouse trial. For
`example, Trichoderma colonisation was lowest in the combination seed treatment
`plus mancozeb foliar spray (TPBM) and the single mancozeb treatment. This can be
`explained on the basis of the strong inhibition of Trichoderma spore germination
`exhibited by mancozeb and thiram. Similarly, there was almost no suppression of
`Trichoderma colonisation of the soil in the captan treatment, which was the fungicide
`that showed low inhibitory activity against spore germination in the in vitro assay.
`In many of the treatments, in particular the TPBM treatment, there was an initial
`decline in Trichoderma cfu counts and then a gradual recovery over time. It is possible
`that the fungicides reduced the germination capability of the initial spore inoculum
`but, subsequently, the germinated spores established and sporulated in the soil to
`bring the cfu counts back up to 104-105/g soil. A previous study showed that T. harzianum
`C52 mycelial growth was insensitive to thiram and mancozeb, which supports this
`hypothesis (Kay & Stewart 1994b).
`Although further trials are needed to determine the sensitivity of T. harzianum to
`repeated fungicide applications, these preliminary results indicate that integrated
`control of onion white rot is possible. Trichoderma harzianum C52 could be applied
`at planting with captan and/or benomyl treated onion seed, the seed treatments
`providing control of other seedling diseases, but it may not be advisable to use
`thiram in combination with a Trichoderma treatment at planting time. If additional
`control of onion white rot is required, dichlofluanid or procymidone could be applied
`as foliar sprays towards the end of the growing season. This integration of fungicides
`and biological control agents may enable the number of fungicide sprays to be
`reduced, while still providing control of onion white rot.
`
`© 2001 New Zealand Plant Protection Society (Inc.) www.nzpps.org
`Refer to http://www.nzpps.org/terms_of_use.html
`
`

`

`Vegetable Pathology
`
`88
`
`REFERENCES
`Kay, S.J.; Stewart, A. 1994a: Evaluation of fungal antagonists for control of onion white
`rot in soil box trials. Plant Path. 43: 371-377.
`Kay, S.J.; Stewart, A. 1994b: The effect of fungicides on fungal antagonists of onion
`white rot and selection of dicarboximide-resistant biotypes. Plant Path. 43: 863-
`871.
`McLean, K.L.; Stewart, A. 2000: Application strategies for control of onion white rot by
`fungal antagonists. N. Z. J. Crop Hort. Sci. 28: 115-122.
`McLean, K.L. 2001: Biological control of onion white rot using Trichoderma harzianum.
`PhD thesis, Lincoln University, Canterbury.
`
`© 2001 New Zealand Plant Protection Society (Inc.) www.nzpps.org
`Refer to http://www.nzpps.org/terms_of_use.html
`
`