Effect of temperature on mycelial growth of Trichoderma , Sclerotinia minor and S . sclerotiorum , as well as on mycoparasitism

Centro Experimental Central do Instituto Biológico, Instituto Biológico, Caixa Postal 70, CEP 13012-970, Campinas, SP, Brazil Laboratório de Química Orgânica de Produtos Naturais, Departamento de Ciências Exatas, ESALQ/USP, Caixa Postal 09, CEP 13418-900, Piracicaba, SP, Brazil Corresponding author: Flávia Rodrigues Alves Patrício (flavia@biologico.sp.gov.br) Data de chegada: 27/11/2015. Aceito para publicação em: 16/02/2016. 10.1590/0100-5405/2146

One of the most important diseases affecting lettuce is lettuce drop, caused by Sclerotinia minor and S. sclerotiorum (8, 9).This disease is favored by mild temperatures and high humidity (11).Managing lettuce drop is complex and involves integrating fungicide applications with several other control methods such as deep plowing, roughing, crop rotation and subsurface-drip irrigation (12 Environmental factors such as soil humidity and temperature can influence the mycoparasitic ability and biocontrol provided by antagonists (4, 7, 10).Partridge et al. (7) showed that the mycoparasitism of sclerotia of S. minor by C. minitans occurred at temperatures ranging from 14 to 22 °C but was suppressed at temperatures above 28 °C.On the other hand, Trichoderma tend to be favored by higher temperatures (3, 10).In the study of Santamarina and Rosselló (10), T. harzianum showed higher mycelial growth at 25 than at 15 °C.Hjeljord et al. (3) found that conidia of commercial products formulated with T. harzianum germinated in 40 to 62 hours at 25 °C but needed 129 to 182 hours to germinate at 12 °C.Similarly, the radial growth of these isolates was higher at 25 °C than at 12 °C.
Considering that biological control is influenced by environmental conditions, this study was carried out to evaluate the effect of temperature on the mycelial growth of three isolates of T. asperellum and one of T. asperelloides obtained in a previous study as antagonists of S. minor and S. sclerotiorum, causal agents of lettuce drop (2).The effect of temperature on the pathogens S. minor and S. sclerotiorum was also evaluated.The mycoparasitism of propagules of the pathogens by the isolate IBLF 914, as well as the disease reduction in lettuce seedlings, was evaluated at different temperatures.These studies were carried out to assess the environmental conditions most favorable for biocontrol of lettuce drop with Trichoderma.

Isolates of S. minor, S. sclerotiorum and Trichoderma
The isolates of S. minor and S. sclerotiorum used in this study were obtained from lettuce plants from the municipality of Mogi das Cruzes, São Paulo State, Brazil.The isolates of T. asperellum (IBLF 897, IBLF 904 and IBLF 914) and T. asperelloides (IBLF 908) were selected as antagonists to S. minor and S. sclerotiorum in a previous study (2).The isolates of S. minor and S. sclerotiorum were grown in wheat grains and the isolates of T. asperellum and T. asperelloides in rice grains, using the methodology described by Elias et al. (2).

Mycelial growth of S. sclerotiorum and S. minor and isolates of Trichoderma at different temperatures
The rate of mycelial growth was measured in colonies grown in Petri dishes containing PDA at the temperatures of 7, 12, 17, 22, 27 and 32 o C for the isolates of S. sclerotiorum and S. minor, and at the same temperatures plus 37 and 42 o C for Trichoderma.A mycelial disc removed from the margin of the colonies of the pathogens and the antagonist was placed in the center of each Petri dish (9 cm diameter) containing PDA.Two days after the onset of the experiments, the average diameter of each colony was measured daily, until the colony reached the edge of the Petri dish.

Mycoparasitic activity of Trichoderma at different temperatures
The experiments of mycoparasitic activity were carried out by using a method adapted from Partridge et al. (7).Gerboxes were filled with 100 g of the commercial substrate Plantmax (Eucatex TM ) previously humidified with 10 mL of sterile distilled water.Before being used, the substrate was autoclaved for 60 minutes at 121 o C in two consecutive days.Twenty baits colonized with S. minor or S. sclerotiorum, prepared as previously described by Elias et al.
(2), were placed on the surface of the substrate.Twenty sclerotia of S. minor and 10 sclerotia of S. sclerotiorum were also placed over strips of sterile filter paper on the borders of each gerbox (Figures 4).The gerboxes were sprayed with a spore suspension of each isolate of Trichoderma containing 10 6 conidia.mL - and placed in BODs with the temperature adjusted to 12, 17, 22, 27 and 32 °C.After ten days, S. minor or S. sclerotiorum baits were examined under a stereomicroscope and the baits colonized with Trichoderma were counted, as well as the baits containing mycelial growth of the pathogens, which were counted as viable.The sclerotia were removed from the gerboxes, washed in water, superficially sterilized with a 10% NaClO solution for 30 seconds, washed again in sterile distilled water, and placed in Petri dishes containing water-agar medium with 0.2% of a veterinary antibiotic (benzylpenicillin benzathine 350,000 UI g -1 , benzylpenicillin procaine 174,000 UI g -1 , benzylpenicillin potassium 174,000 UI g -1 , dihydrostreptomycin base 145 mg g -1 and streptomycin base 145 mg g -1 ).After the sclerotia were removed, 25 pre-germinated lettuce seedlings were placed in the gerboxes.The seeds were pregerminated in Petri dishes containing two humidified filter papers, which were maintained for 48 hours in a BOD at 20 o C.
The Petri dishes containing the sclerotia were maintained for seven days in BODs at 20 o C. The sclerotia were examined using a stereoscopic microscope, and the sclerotia colonized with Trichoderma spp.were counted, as well as the sclerotia that germinated, which were counted as viable.The gerboxes that contained the lettuce seeds and the baits were maintained in BODs at 12, 17, 22, 27 and 32 °C, during four days, and the number of surviving seedlings was counted.

Experimental design and statistical analysis
The experiments of mycelial growth of Trichoderma isolates and the pathogens were carried out in a completely randomized design with four replicates per temperature, and each replicate was represented by a Petri dish.ANOVA of the data was performed and the means were compared according to Tukey's test at 5% probability.
The experiments of mycoparasitism were carried out in a completely randomized design with four replicates, each replicate represented by a gerbox.The data were subjected to ANOVA and the means were compared according to Tukey's test at 5% probability.A factorial analysis of variance was performed for the data of the surviving lettuce seedlings with S. minor or S. sclerotiorum.The media of the treatments were compared according to Tukey's test at 5% probability.

Rate of mycelial growth of the isolates of Trichoderma, S. minor and S. sclerotiorum at different temperatures
The mycelial growth of all isolates of Trichoderma was inhibited at the temperature of 7 °C, was proportional to the increase in the temperatures ranging from 12 to 27 o C, and decreased until 37 o C, being inhibited at 42 o C (Figure 1).For the isolates of T. asperellum (IBLF 897, IBLF 904 and IBLF 914) the maximum growth rate occurred at 27 o C, but for the isolate of T. asperelloides (IBLF 908) the maximum growth rate occurred at 32 o C (Figure 1).
The pathogens were able to grow at temperatures ranging from 7 to 32 °C for S. sclerotiorum or 27 °C for S. minor and showed maximum growth rate at 22°C (Figure 2).

Mycoparasitic activity at different temperatures
The baits and sclerotia of S. minor were not mycoparasitized by the isolate IBLF 914 at the temperature of 12 o C, but they were colonized by the antagonist and lost their viability at temperatures from 17 to 32°C.For the treatment without the antagonist, the baits were viable at all temperatures, except at 32°C (Table 1, Figure 3).
At 12°C the T. asperellum isolate did not parasitize the baits and sclerotia of S. sclerotiorum, but at 17°C, 61.2 % of the baits were mycoparasitized.At temperatures from 22 to 32°C, all baits and sclerotia were colonized by the antagonist and these temperatures are probably most suitable for biological control with this antagonist.For the control treatment, baits and sclerotia of S. sclerotiorum were viable at temperatures ranging from 12 to 27 °C, but no bait showed mycelial growth at 32 °C.The sclerotia removed from the gerboxes kept at all different temperatures exhibited mycelial growth after being removed from the gerboxes and maintained at 20 o C (Table 2, Figure 4).

Survival of lettuce seedlings at different temperatures
At the temperature of 12 °C, parasitism of the baits of S. minor by T. asperellum isolate was not evident (Figure 4), but the viability of lettuce seedlings was higher for the Trichoderma-inoculated treatment than in the non-inoculated one at the same temperature (Table 3).At 32 °C, both inoculated and non-inoculated treatments showed the same number of lettuce seedlings (Table 3).Although the antagonist colonized the pathogen, S. minor was unable to reduce the viability of the lettuce seedlings because its mycelial growth was inhibited at this temperature (Figure 3).
The same tendency was observed for treatments carried out with S. sclerotiorum, except at the temperatures of 27 and 32 °C, when this pathogen was unable to reduce the viability of seedlings (Table 3).The isolate of T. asperellum significantly reduced the disease in seedlings maintained at 17 and 22 °C (Table 3).

DISCUSSION
The mycelial growth of S. minor and S. sclerotiorum occurred at temperatures ranging from 12 to 27 o C, and the pathogens reduced the viability of lettuce seedlings at the same temperatures.Similarly, Imolehin et al. (5) showed that sclerotia of S. minor germinated and exhibited mycelial growth at temperatures ranging from 6 to 30 o C with optimum growth at 18 o C and infection of lettuce plants occurred at temperatures ranging from 6 to 24 o C. For S. sclerotiorum, Young et al. (14) verified that ascospores could cause bottom rot in lettuce at temperatures ranging from 8 to 27 o C, but the disease occurred more rapidly and was more severe at temperatures ranging from 16 to 27 o C, peaking at 22 o C.
The mycelial growth shown herein by the isolates of Trichoderma was proportional to the increase in temperature when the latter ranged from 12 to 27 °C for T. asperellum isolates and from 12 to 32 °C for T. asperelloides isolate.Jackson et al. (6) observed similar results using isolates of T. viride and T. pseudokoningii, which showed mycelial growth at temperatures ranging from 10 to 30 o C, with maximum growth at 25 o C. In another study, Santamarina & Roselló (10) found that the mycelial growth of a T. harzianum strain was higher at 25 than at 15 o C.
The results of this study showed that the T. asperellum isolate was not effective at 12 o C but was favored by temperatures varying      13) with an isolate of T. konigii showed that species of Trichoderma tend to grow and parasitize sclerotia at temperatures higher than those favorable for the occurrence of lettuce drop.In Brazil, commercial products formulated with Trichoderma have been used in combination with other methods for controlling white mold, caused by S. sclerotiorum, in soybeans and beans, but producers usually apply the isolates during warmer months of the year, generally before planting, when conditions are more favorable to the mycoparasitism of sclerotia, and this system could be adapted for the management of lettuce drop.In light of these findings, the antagonist could be applied in lettuce crop during warmer months of the year, aiming to reduce the viability of the sclerotia present in the soil or in lettuce debris before the winter crop, which is most affected by lettuce drop in Brazil, although the disease may occur whenever favorable conditions prevail.
by the same letter do not differ according to Tukey's test at 5% level.2No variance.

1
Means followed by the same letter do not differ at 5% level according to Tukey's test 2 No variance

Table 1 .
Mycelial growth of Sclerotinia minor baits and sclerotia inoculated or not with the isolate IBLF 914 of Trichoderma asperellum and maintained at different temperatures.Temperature Parasitism of S. minor baits S. minor baits with mycelial growth S. minor sclerotia with mycelial growth ( o C) Inoculated with isolate IBLF 914 Inoculated with isolate IBLF 914 Non inoculated Inoculated with isolate IBLF 914 Non inoculated

Table 2 .
Viability of S. sclerotiorum baits and sclerotia inoculated or not with the isolate of Trichoderma asperellum (IBLF 914) and maintained at different temperatures.