In vitro sensitivity of Didymella bryoniae, Fusarium oxysporum, Trichoderma spp, Sclerotium rolfsii and Thielaviopsis basicola to the botanical

fungicide Timorex Gold (Melaleuca alternifolia) under the Paper disk diffusion and the Poisoning agar testing methods.
1 1

M. Barbier University of Florida-IFAS, Plant Medicine Program, Gainesville, FL, 32611

BACKGROUND PRODUCT
Timorex Gold is a botanical fungicide which contains tea tree oil (TTO) as active ingredient. TTO is an essential oil steam distilled from the Australian plant Melaleuca alternifolia. TTO contains over 100 components, mostly monoterpenes, sesquiterpenes and their alcohols. TTO as a natural essential oil is known as an effective antiseptic, fungicide and bactericide, and has many safe and effective uses in the health and cosmetics industry. Its use against plant pathogens has very few investigations, mainly done by Biomor Israel Ltd., which did developed an organic product labeled as Timorex Gold, this product contains 23.8% of TTO, and has been reported to be effective against broad spectrum plant diseases in various crops with no phytotoxicity to plant. In vitro tests done by Biomor showed that Timorex Gold at 100 1,000 ppm inhibited spore germination of powdery mildew fungus. Timorex Gold has been evaluated in greenhouse, and open field studies at foliar spray rates of 5,000 10,000 ppm inhibited late blight disease caused by Phytophthora infestans and controlled powdery mildews in various crops such as tomato, cucumber and pepper plants. It was as good as or better than standard treatments. (1)

PATHOGENS
Didymella bryoniae is the causal organism of Gummy stem blight (GSB) in many cucurbits, including watermelon, cantaloupe, cucumber, pumpkin, squash, muskmelon, and other melons. Infection on watermelon and cantaloupe is commonly seen in Florida, and the disease can cause significant production losses when conditions are ideal for the spread of this fungal pathogen. The disease is also known as black rot due to its characteristic appearance on infected fruits. (2) Fusarium oxysporum f. sp. lycopersici (Sacc.) W.C. Snyder and H.N. Hans, a soilborne plant pathogen in the class Hyphomycetes, causes Fusarium wilt specifically in tomato. This disease was first described by G.E. Massee in England in 1895. It is of worldwide importance where at least 32 countries had reported the disease, which is particularly severe in countries with warm climate. At one time, the disease nearly destroyed tomato production in parts of Florida and the southeastern states of United States. However, the development and use of resistant cultivars have nearly eliminated the concern over this disease. Three physiological races of this pathogen have been reported. Race 1 is the most widely distributed and has been reported from most geographical areas. Although, race 2 was first reported in Ohio in

1940, it did not become widespread or of economic concern until its discovery in Florida in 1961. Since then, it was rapidly reported in several of the states and in several other countries, including Australia, Brazil, Great Britain, Israel, Mexico, Morocco, the Netherlands, and Iraq. Race 3 was reported in 1966 in Brazil. Thereafter, it has been found in Australia and in Florida and California. (3) Trichoderma spp are important biological control agents used in plant disease management. They are imperfect fungi, with their teleomorph perfect stage belonging to Hypocreales of Ascomycetes. They are capable of secreting hydrolytic enzymes and cause mycoparasitism on fungal pathogens of plants. Besides, they produce antibiotics and toxins that inhibit the fungal plant pathogens. (3) Sclerotium rolfsii an omnivorous, soilborne fungal pathogen, causes disease on a wide range of agricultural and horticultural crops. Although no worldwide compilation of host genera has been published, over 270 host genera have been reported in the United States alone. Susceptible agricultural hosts include sweet potato (Ipomea batatas), pumpkin (Cucurbita pepo L.), corn (Zea mays), wheat (Triticum vulgare) and peanut (Arachis hypogea). (3) Thielaviopsis basicola (Berk. & Br.) Ferraris is a soil inhabitant that attacks more than 100 plant species in thirty three families. Members of the Fabaceae, Solanaceae, and Cucurbitaceae families are especially affected by this fungus. Thielaviopsis basicola can be found in all regions of the world, especially in regions with cool climates. Causes a disease which is known as 'black root rot' and this name is based on darkly pigmented chlamydospores that form in the root cells of hosts and giving a 'blackened' appearance to the root tip. The black root rot fungus is a member of the Hyphomycetes, order Moniliales, family Dematicaceae. General symptoms are root rot and branch dieback. Black root rot can affect a wide range of woody and herbaceous plants including tobacco, holly, begonia, geranium, poinsettia, and pansy. (3)

MATERIALS AND METHODS METHODS

Paper disk diffusion method The paper disk diffusion test is also known as the Kirby-Bauer disk-diffusion method, is a means of measuring the effect of an antimicrobial agent against fungi grown in culture. A filter-paper disks, is soak into the compound to be evaluated for 15 minutes, and later the disk is take it out by letting it dry for an hour. Then, the disk is placed on the surface of the agar. Subsequently, a piece of 5 mm of growing fungi is placed over the paper. The compound diffuses from the filter paper into the agar. If the compound is effective against the fungi at a certain concentration, no growth or reduction on the

growth will be observed. The inhibition of the growth is known as inhibition zone. The diameter of the growth zone of the fungi is measured in millimeters during six days each 24 hours. Thus, the size of the zone of the fungi growth is a measure of the compound's effectiveness: the smaller the area around the filter disk, the more effective the compound. Poison agar method Potato dextrose agar (PDA) is prepared and mixed with the concentrations of the compound to be evaluated (in percentage or parts per million - ppm) and placed to autoclave. Then, the compounds are allowed to warm to 50C. The Poisoning Agar (PA) containing the different concentrations of the evaluating compound are poured into 90 mm Petri dishes to allowed cooling. Afterwards, the cultures of the fungi to be evaluated are cut into 5 mm x 5 mm squares. Each square is placed in the centre of a paper disk which is placed over the surface of the PA. Each treatment is at least replicated three times inside the disk. Subsequently, the plates have to be placed into an incubator at 22-24C for six days. Diameter growth is measured around each block and results are recorded in a daily basis.

MATERIALS
One isolate of each evaluating fungi were tested. These isolates were sub-cultured from cultures stored at the Clinical Trials Program laboratory.

The concentrations of Timorex Gold evaluated were prepared by dissolving in water. The final fungicide concentrations were adjusted to achieve 2,500; 5,000; 10,000 and 20,000 ppm. These concentrations were based on the recommended rates of Timorex Gold label for use in different crops in Central America due to the product is in the registration process in the United States.

For the disk diffusion method, cellulose discs were placed for 15 minutes in four clean petri dishes containing each the test solution that was previously prepared. Later, the discs were placed in the interior side of the petri dishes lid for sixty minutes to evaporate free moisture from the disc surface. After the one hour dry time the moist discs were placed onto the potato dextrose agar so that each plate (three per isolate per concentration) contained two treated discs and one untreated check disc.

For the poisoning agar method, potato dextrose agar (PDA) was prepared using the same solutions that were previously used for the disk diffusion. After taking the PA containing the different concentrations of the evaluating compound from the autoclave, the bottles allowed to warm to 50C. They were poured into 90 mm Petri dishes and allowed to solidify before placing three untreated disks per petri dish. Two disks of each concentration were prepared in order to have a total of six repetitions.

Inoculation The cultures of the fungi to be evaluated were cut into 5 mm x 5 mm squares. Each square was placed in the centre of each paper disks that were placed over the surface of the Disk diffusion and the Poisoning Agar petri dishes.

The inoculated plates were incubated for 144 hours at 25C with 12 hours of light. The radial growth of the test fungi were measured and recorded.

For the Disk diffusion the experiment was conducted three times, because efficacy levels of Timorex Gold over the tested fungi did differ illogically between concentrations. The second time, a modification in the solvent and drying time steps in the methodology was performed to check if the solvent and/or the drying times were the reason of the differences between the treatments. No differences were found in the solvent and drying time steps in the methodology.

RESULTS
Statistics For the diffusion disk method for each treatment fungi tested three petri dish were prepared containing each petri dish two treated discs (DD) and one untreated check disk (DD-CTL), giving a total of 6 repetitions for treatments and 3 repetitions for the check controls. For the poisoning agar method for each treatment fungi tested two petri dish were prepared containing each petri dish three untreated discs that were place over the treated agar for the treatments and over the untreated agar for the absolute control, giving a total of 6 repetitions for the treatments and the control. A total of thirteen treatments were analyzed as indicated below: Disks Total disks Petri Treatment inside petri taken as a dishes dishes repetition
Absolute control PDA Disk diffusion 2,500 ppm TG Disk diffusion 2,500 ppm control Poison Agar 2,500 ppm TG Disk diffusion 5,000 ppm TG Disk diffusion 5,000 ppm control Poison Agar 5,000 ppm TG Disk diffusion 10,000 ppm TG Disk diffusion 10,000 ppm control Poison Agar 10,000 ppm TG Disk diffusion 20,000 ppm TG Disk diffusion 20,000 ppm control Poison Agar 20,000 ppm TG
2 3 3 2 3 3 2 3 3 2 3 3 2 3 2 1 3 2 1 3 2 1 3 2 1 3 6 6 3 6 6 3 6 6 3 6 6 3 6

Statistical analyses were performed using SAS 9.2. Fishers Least Significant Difference (LSD) test was used at a confidence level of 95%, therefore, p values <0.05 were considered significant. Didymella bryoniae The evaluation of the disk diffusion (DD) method showed that the four concentrations of Timorex Gold have limited or no inhibition of the radial growth of the isolate tested when compared with each individual control inside the same petri dish (bottom disk of each picture), after 144 hours of fungi were placed in petri dishes the inhibition was negative and growth stimulation was as follows: 2,500 = 0%; 5,000 ppm = 1%; 10,000 ppm = 4%; and 20,000 ppm = 1%.

DD - 2,500 ppm

DD - 5,000 ppm

DD - 10,000 ppm

DD - 20,000 ppm

On the other hand, when the four concentration of Timorex Gold where compared with the absolute control with no product inside the petri dish, a limited inhibition of the radial growth of the isolate was observed. After 144 hours of fungi were placed in petri dishes, inhibition was as follows: 20,000 ppm rate = 18%; 10,000 ppm = 17%; 5,000 ppm = 22%; and 2,500 = 20%.

Absolute control

The evaluation of the poison agar (PA) method showed that the four concentrations of Timorex Gold inhibit the radial growth of the isolate tested when compared with absolute control. After 144 hours of fungi were placed in petri dishes, Didymella bryoniae inhibition was as follows: 20,000 ppm = 93%; 10,000 ppm = 59%; 5,000 ppm = 50%; and 2,500 = 35%;

PA - 2,500 ppm

PA - 5,000 ppm

PA - 10,000 ppm

PA - 20,000 ppm

Treatment
Absolute control PDA Disk diffusion 2,500 ppm TG Disk diffusion 2,500 ppm control Poison Agar 2,500 ppm TG Disk diffusion 5,000 ppm TG Disk diffusion 5,000 ppm control Poison Agar 5,000 ppm TG Disk diffusion 10,000 ppm TG Disk diffusion 10,000 ppm control Poison Agar 10,000 ppm TG Disk diffusion 20,000 ppm TG Disk diffusion 20,000 ppm control Poison Agar 20,000 ppm TG

Means of radial fungi growth 24 h 48 h 72 h

6.2 CDE 6.3 BCDE 7.0 ABC 5.3 EF 6.7 ABCD 6.0 CDEF 5.3 EF 6.8 ABC 7.7 A 5.7 DEF 6.3 BCDE 7.3 AB 5.0 F 25.7 A 17.0 D 17.7 CD 17.7 CD 18.8 BC 18.7 BC 11.5 E 17.8 BCD 19.3 B 7.0 F 17.5 CD 19.0 BC 5.0 G 39.2 A 32.0 DE 33.7 BC 23.7 F 34.2 B 33.0 BC 21.3 G 32.8 CD 33.0 C 13.0 H 31.5 E 33.0 C 5.0 G

96 h
45.0 A 36.2 E 38.0 D 31.0 F 39.2 BCD 40.0 BC 25.0 G 38.5 D 39.7 BC 16.7 H 38.0 D 40.7 B 5.0 I

120 h
51.3 A 43.8 I 45.7 G 36.3 J 47.7 C 48.3 B 27.8 K 46.3 F 47.3 D 20.5 L 44.0 H 47.0 E 5.0 M

144 h
70.0 A 56.0 BCD 56.0 BCD 45.8 E 54.3 CD 54.0 D 35.3 F 58.0 B 56.0 BCD 28.8 G 57.3 B 56.7 BC 5.0 H

Different letters indicate that the means are significantly different.

Fusarium oxysporum The evaluation of the disk diffusion (DD) method showed that the four concentrations of Timorex Gold have limited or no inhibition of the radial growth of the isolate tested when compared with each individual control inside the same petri dish (bottom disk of each picture). After 144 hours of fungi were placed in petri dishes, the inhibition was negative and growth stimulation was as follows: 2,500 = 8%; 5,000 ppm = -1%; 10,000 ppm = 8%; and 20,000 ppm = 6%.

DD - 2,500 ppm

DD - 5,000 ppm

DD - 10,000 ppm

DD - 20,000 ppm

On the other hand, when the four concentration of Timorex Gold where compared with the absolute control with no product inside the petri dish, a limited inhibition of the radial growth of the isolate was observed. After 144 hours of fungi were placed in petri dishes, inhibition was as follows: 20,000 ppm rate = 4%; 10,000 ppm = -2%; 5,000 ppm = 18%; and 2,500 = 17%.

Absolute control

The evaluation of the poison agar (PA) method showed that the four concentrations of Timorex Gold inhibit the radial growth of the isolate tested when compared with absolute control. After 144 hours of fungi were placed in petri dishes, Fusarium oxysporum inhibition was as follows: 20,000 ppm = 78%; 10,000 ppm = 47%; 5,000 ppm = 29%; and 2,500 = 21%;

PA - 2,500 ppm

PA - 5,000 ppm

PA - 10,000 ppm

PA - 20,000 ppm

Treatment
Absolute control PDA Disk diffusion 2,500 ppm TG Disk diffusion 2,500 ppm control Poison Agar 2,500 ppm TG Disk diffusion 5,000 ppm TG Disk diffusion 5,000 ppm control Poison Agar 5,000 ppm TG Disk diffusion 10,000 ppm TG Disk diffusion 10,000 ppm control Poison Agar 10,000 ppm TG Disk diffusion 20,000 ppm TG Disk diffusion 20,000 ppm control Poison Agar 20,000 ppm TG

Means of radial fungi growth 24 h 48 h 72 h

6.3 BCD 6.8 BC 8.3 A 5.5 DE 5.8 CDE 7.0 B 5.5 DE 7.2 B 7.0 B 5.8 CDE 5.7 DE 6.3 BCD 5.0 E 21.0 A 17.3 BCD 20.3 A 17.2 CD 15.7 DE 16.7 CD 13.7 E 18.0 BC 19.3 AB 9.7 F 17.0 CD 17.0 CD 5.8 G 33.8 A 28.7 BC 27.7 CD 26.7 D 27.7 CD 26.7 D 22.0 E 30.2 B 30.0 B 12.3 F 29.7 B 29.0 BC 7.5 G

96 h
41.3 A 33.5 CDE 30.0 FG 29.7 FG 31.8 DEF 31.3 EF 26.8 G 37.5 B 36.7 BC 17.0 H 35.8 BC 35.0 BCD 9.5 I

120 h
45.5 A 36.5 CDE 35.0 DEF 32.5 FG 34.5 EF 34.7 DEF 30.2 G 41.0 B 39.3 BC 20.5 H 37.8 BCD 36.0 DE 10.2 I

144 h
48.3 A 40.3 CD 37.3 DE 38.2 DE 39.7 D 40.3 CD 34.2 E 49.2 A 45.7 AB 25.7 F 46.5 AB 44.0 BC 10.8 G

Different letters indicate that the means are significantly different.

Trichoderma spp The evaluation of the disk diffusion (DD) method showed that the four concentrations of Timorex Gold have limited or no inhibition of the radial growth of the isolate tested when compared with each individual control inside the same petri dish (bottom disk of each picture). After 144 hours of fungi were placed in petri dishes the inhibition was as follows: 20,000 ppm = 2%; 10,000 ppm = 5%; 5,000 ppm = 15%; and 2,500 = 3%.

DD - 2,500 ppm

DD - 5,000 ppm

DD - 10,000 ppm

DD - 20,000 ppm

On the other hand, when the four concentration of Timorex Gold where compared with the absolute control with no product inside the petri dish, a limited inhibition of the radial growth of the isolate was observed. After 144 hours of fungi were placed in petri dishes, inhibition was as follows: 20,000 ppm = 14%; 10,000 ppm = 17%; 5,000 ppm = 17%; and 2,500 = 20%.

Absolute control

The evaluation of the poison agar (PA) method showed that the four concentrations of Timorex Gold inhibit the radial growth of the isolate tested when compared with absolute control. After 144 hours of fungi were placed in petri dishes, Trichoderma spp inhibition was as follows: 20,000 ppm = 87%; 10,000 ppm = 65%; 5,000 ppm = 35%; and 2,500 = 0%.

PA - 2,500 ppm

PA - 5,000 ppm

PA - 10,000 ppm

PA - 20,000 ppm

Treatment
Absolute control - PDA Disk diffusion 2,500 ppm TG Disk diffusion 2,500 ppm control Poison Agar 2,500 ppm TG Disk diffusion 5,000 ppm TG Disk diffusion 5,000 ppm control Poison Agar 5,000 ppm TG Disk diffusion 10,000 ppm TG Disk diffusion 10,000 ppm control Poison Agar 10,000 ppm TG Disk diffusion 20,000 ppm TG Disk diffusion 20,000 ppm control Poison Agar 20,000 ppm TG

Means of radial fungi growth 24 h 48 h 72 h

5.3 A 5.0 A 5.0 A 6.0 A 5.0 A 5.0 A 5.0 A 5.0 A 5.0 A 5.2 A 5.0 A 5.2 A 5.0 A 8.3 A 6.2 BCDE 6.7 BCDE 7.5 AB 6.0 CDE 6.3 BCDE 5.5 DE 6.8 BCD 7.3 ABC 5.3 E 7.2 ABC 7.3 ABC 5.3 E 14.2 A 9.8 BC 11.7 AB 10.3 B 10.3 B 11.0 B 9.2 BC 9.7 BC 10.0 BC 7.5 CD 11.3 B 10.7 B 6.2 D

96 h
31.0 A 24.3 B 25.7 AB 25.0 B 25.5 B 24.0 B 14.7 C 26.5 AB 27.3 AB 9.8 CD 27.2 AB 26.3 AB 6.5 D

120 h
40.8 AB 34.3 B 37.0 AB 35.2 AB 35.0 AB 37.0 AB 22.0 C 36.0 AB 39.3 AB 13.5 D 41.5 A 39.3 AB 7.3 D

144 h
56.5 AB 45.3 D 46.7 D 58.3 A 46.8 D 55.0 ABC 37.0 E 46.7 D 49.0 BCD 19.7 F 48.8 CD 50.0 BCD 7.3 G

Different letters indicate that the means are significantly different.

Sclerotium rolfsii The evaluation of the disk diffusion (DD) method showed that the four concentrations of Timorex Gold have limited or no inhibition of the radial growth of the isolate tested when compared with each individual control inside the same petri dish (bottom disk of each picture). After 48 hours of fungi were placed in petri dishes the inhibition was as follows: 2,500 ppm = 12%; 5,000 ppm = 14%; 10,000 ppm = 9%; and 20,000 ppm = 8%. After 72 hours the growth of all treatment reached the borders of the petri dish and no differences were recorded when compared with the control.

72 hours

48 hours

DD - 2,500 ppm

DD - 5,000 ppm

DD - 10,000 ppm

DD - 20,000 ppm

When the four concentration of Timorex Gold where compared with the absolute control with no product inside the petri dish, a similar limited inhibition of the radial growth of the isolate was observed. After 48 hours of fungi were placed in petri dishes, inhibition was as follows: 20,000 ppm = 6%; 10,000 ppm = 5%; 5,000 ppm = 10%; and 2,500 = 19%. After 72 hours the growth of all treatment reached the borders of the petri dish and no differences were recorded when compared with the control.

Absolute control 48 h

Absolute control 72 h

Absolute control 96 h

Absolute control 144 h

The evaluation of the poison agar (PA) method showed that the four concentrations of Timorex Gold inhibit the radial growth of the isolate tested when compared with absolute control. After 96 hours of fungi were placed in petri dishes, Sclerotium rolfsii inhibition was as follows: 20,000 ppm = 86%; 10,000 ppm = 38%; 5,000 ppm = 10%; and 2,500 = 2%. After 96 hours the growth of treatments 2,500 ppm and

5,000 ppm reached the borders of the petri dish and no differences were recorded when compared with the control. Treatments 10,000 ppm and 20,000 ppm showed that these concentrations of Timorex Gold inhibited the radial growth of the isolate tested when compared with absolute control until 144 hours after fungi were placed in petri dishes. Sclerotium rolfsii inhibition was as follows: 20,000 ppm = 74% and 10,000 ppm = 6%.

144 hours

96 hours

DD - 2,500 ppm

DD - 5,000 ppm

DD - 10,000 ppm

DD - 20,000 ppm

Treatment
Absolute control - PDA Disk diffusion 2,500 ppm TG Disk diffusion 2,500 ppm control Poison Agar 2,500 ppm TG Disk diffusion 5,000 ppm TG Disk diffusion 5,000 ppm control Poison Agar 5,000 ppm TG Disk diffusion 10,000 ppm TG Disk diffusion 10,000 ppm control Poison Agar 10,000 ppm TG Disk diffusion 20,000 ppm TG Disk diffusion 20,000 ppm control Poison Agar 20,000 ppm TG

Means of radial fungi growth 24 h 48 h 72 h

9.8 A 6.8 CD 7.3 BCD 9.3 AB 7.7 ABCD 9.0 ABC 6.3 D 7.8 ABCD 8.7 ABCD 6.8 CD 7.3 BCD 8.3 ABCD 6.8 CD 47.8 C 38.7 H 44.0 F 29.8 I 43.1 G 50.0 A 23.5 J 45.5 D 50.0 A 13.0 K 44.7 E 48.7 B 7.0 L 70.0 A 70.0 A 70.0 A 51.3 B 70.0 A 70.0 A 42.3 C 70.0 A 70.0 A 27.7 D 70.0 A 70.0 A 7.8 E

96 h
70.0 A 70.0 A 70.0 A 68.5 B 70.0 A 70.0 A 63.3 C 70.0 A 70.0 A 43.2 D 70.0 A 70.0 A 10.0 E

120 h
70.0 A 70.0 A 70.0 A 70.0 A 70.0 A 70.0 A 70.0 A 70.0 A 70.0 A 50.2 B 70.0 A 70.0 A 12.3 C

144 h
70.0 A 70.0 A 70.0 A 70.0 A 70.0 A 70.0 A 70.0 A 70.0 A 70.0 A 65.5 B 70.0 A 70.0 A 18.2 C

Thielaviopsis basicola The evaluation of the disk diffusion (DD) method showed that the four concentrations of Timorex Gold have limited or no inhibition of the radial growth of the isolate tested when compared with each individual control inside the same petri dish (bottom disk of each picture). After 144 hours of fungi were placed in petri dishes was growth inhibition for the 2,500 = 14% and 20,000 ppm = 4%, being negative and growth stimulation was observed for the 5,000 ppm = 23% and 10,000 ppm = 3%.

DD - 2,500 ppm

DD - 5,000 ppm

DD - 10,000 ppm

DD - 20,000 ppm

Same behavior was observed when the four concentration of Timorex Gold where compared with the absolute control with no product inside the petri dish. After 144 hours of fungi were placed in petri dishes was growth inhibition for the 2,500 = 5% and no growth for 20,000 ppm = 0%, being negative and growth stimulation was observed for the 5,000 ppm = 20% and 10,000 ppm = 7%.

Absolute control

The evaluation of the poison agar (PA) method showed that the four concentrations of Timorex Gold inhibit the radial growth of the isolate tested when compared with absolute control. After 144 hours of fungi were placed in petri dishes, Thielaviopsis basicola inhibition was as follows: 20,000 ppm = 83%; 10,000 ppm = 78%; 5,000 ppm = 50%; and 2,500 = 38%.

PA - 2,500 ppm

PA - 5,000 ppm

PA - 10,000 ppm

PA - 20,000 ppm

Treatment
Absolute control - PDA Disk diffusion 2,500 ppm TG Disk diffusion 2,500 ppm control Poison Agar 2,500 ppm TG Disk diffusion 5,000 ppm TG Disk diffusion 5,000 ppm control Poison Agar 5,000 ppm TG Disk diffusion 10,000 ppm TG Disk diffusion 10,000 ppm control Poison Agar 10,000 ppm TG Disk diffusion 20,000 ppm TG Disk diffusion 20,000 ppm control Poison Agar 20,000 ppm TG

Means of radial fungi growth 24 h 48 h 72 h

5.0 B 5.0 B 5.0 B 5.0 B 5.0 B 5.0 B 5.0 B 5.0 B 5.0 B 5.0 B 5.2 A 5.0 B 5.0 B 6.3 ABC 7.7 AB 7.3 AB 5.2 C 8.2 A 7.3 AB 6.0 BC 6.7 ABC 5.3 C 5.0 C 7.7 AB 6.7 ABC 5.0 C 11.2 CD 13.0 BC 17.0 A 7.3 E 12.7 BC 14.0 B 7.2 EF 10.2 D 12.3 BCD 5.0 F 13.3 BC 12.3 BCD 5.0 F

96 h
17.0 C 20.5 AB 23.0 A 9.0 D 20.0 B 21.3 AB 7.5 DE 22.0 AB 22.0 AB 5.5 E 20.2 AB 21.7 AB 5.0 E

120 h
22.3 B 24.0 AB 25.3 AB 12.5 C 26.0 A 23.7 AB 9.7 C 24.7 AB 25.0 AB 6.0 D 23.7 AB 24.0 AB 5.0 D

144 h
28.8 B 27.3 B 31.7 AB 18.0 C 34.7 A 28.3 B 14.3 C 30.7 AB 29.7 AB 6.3 D 28.8 B 30.0 AB 5.0 D

Different letters indicate that the means are significantly different.

OBSERVATIONS, CONCLUSIONS AND RECOMMENDATIONS

Observations The major components of tea tree oil (active ingredient of Timorex Gold) are monoterpenes which area characterized by a very high vapor pressure ranging from 5.7 Pa ( Terpineol) to 544 Pa ( pinene). In the disk diffusion method, the growths measurements of the evaluated fungi were very similar between the treated disks with Timorex Gold and the check disks (no impregnated) in all evaluated treatments. When compared the check disks with the absolute control of each treatment there were statistically significant differences between all inside check disks and the absolute control. In Fusarium oxysporum also some depigmentation were observed for the 10,000 and 20,000 ppm treatments. In the poison agar method, significant differences were observed between all treatments; the absolute control and the treatments with the disk diffusion method. These significant differences were observed in the four evaluated rated in the five evaluated fungi.

Conclusions Our research indicates that In Vitro evaluations of Timorex Gold under the disk diffusion method gives inconsistent data regarding the efficacy of the product. Under the poison agar method, all rates showed some efficacy, but just the 10,000 and 20,000 ppm rates showed effective inhibition of the five fungi isolations after the 144 hours evaluation period. Recommendations The efficacy of Timorex Gold should be evaluated in greenhouse and then in open field. There is a fact that the high vapor pressure of the product components must be taken into account to avoid inadequate use of dilution volume and spraying pressure.

For further information contact Marcel Barbier to any of the following contact information: e-mails: marcelbarbier@ufl.edu or marcelbarbier65@gmail.com Phone: + 1 (800) 576-5134 or Fax: + 1 (800) 803-2790 Acknowledgements The authors would like to thank Zena Armella for assisting in the photographical activities of this study. References 1. Timorex Gold Central America label. Biomor Israel Ltd. 2008 2. Management of Gummy Stem Bliht (Black Rot) on Cucurbits in Florida. Mathews L. Paret, Nicholas S. Dufault, and Stephen M. Olson. Plant Pathology Department, Florida Cooperative Extension Service, IFAS, University of Florida. 2011. 3. Soilborne Plant Pathogens. North Carolina State University. NCSU Department of Plant Pathology, 2518 Gardner Hall, Raleigh, NC 27695-7616.