In laboratory experiments were evaluated TRICHOD®, FOLIGUARD®, Trichoderma sp, and watery extracts of pine, marigold and eucalyptus, respectively, against F. oxysporum. There were no significant differences between TRICHOD® and FOLIGUARD®. The extract of marigold of 120 g/L was the best result in reducing the area of the colony of F. oxysporum. In field pots experiment were evaluated the extract of marigold (120 g/L), potassium phosphite (400 g/L), TRICHOD and benomyl (1.5 g/L) to control F. oxysporum. Treatments were applied to the soil, 15 days after inoculation with F. oxysporum. The best obtained heights were of 32 cm with extract of marigold 120 g/L, followed by potassium phosphite with 31 cm, and the control reached 26 cm of height. The treatment with an extract of marigold 120 g/L, obtained the best dry weight average of 6.5 g, statistically different from the control. The extract of marigold presented the best efficiency against the disease, because it reduced the length of the vascular symptom in 88.5%; followed by TRICHOD®, benomyl and potassium phosphite that showed reductions of 86, 85 and 84 percent, respectively.
The diversification in planting ornamental species has been a strategy used by the producers, as an alternative to extending markets, to improve competitiveness and to increase profitability. A step in this respect has been the production and commercialization of new flowers and foliages, as the proteas of the genus Leucadendron.
The cultivation of proteas is relatively new in the market of ornamental plants for the production of flowers, dried flowers and flower pots [
Proteas production has been affected by the incidence of diseases caused by three pathogens of great impor- tance that inhabit the soil: Phytophthora cinnamomi, Rhizoctonia spp. and Fusarium spp. [
In Colombia, F. oxysporum is the causal agent of Leucadendron wilt [
In general, the diseases caused by F. oxysporum are difficult to control. Resistant varieties, planting certified seeds, and healthy seedlings are the best practices to control wilt caused by Fusarium species. Also antagonist biocontrol agents, such as Trichoderma, offer possibilities to control, primarily because it inhibits the growth and development of the disease [
A variety of Trichoderma species has been evaluated to control F. oxysporum in different crops. To control this pathogen in papaya plants (Carica papaya L.) a treatment of Trichoderma at a concentration of 106 conidia wasapplied to the soil artificially infested with F. oxysporum. Its antagonist effect has also been evaluated on tomato plants to wilt caused by F. oxysporum [
In searching for new alternatives to organic plant disease management, the use of plant metabolites has been considered feasible [
In Colombia, the growers apply some chemical fungicides, such as benomyl to wilt control. This product re- duced by 50% the incidence of wilt caused by F. oxysporum on susceptible tomato plants [
The antimicrobial properties of the phosphites were found in studies aimed at controlling diseases caused by oomycetes [
The main objective of this research was to determine the effect of Trichoderma spp, potassium phosphite, benomyl and plant extracts in the control of F. oxysporum in Leucadendron proteas, in laboratory and field pot conditions.
F. oxysporum was isolated from diseased Leucadendron proteas collected in established crops, in a farm located in Subachoque, Department of Cundinamarca, Colombia, at 2600 altitude, and temperature between 15˚C and 19˚C. Wilty Petra variety plants were chosen with external and internal symptoms. The fungus was isolated in acidified Potato Dextrose Agar (PDA + A) with 2500 µL of 50% lactic acid per 1000 mL of medium. Stem samples with visible wilt symptoms were previously washed with tap water to remove soil residues. Of each stem, square 5 mm pieces were cut, and immersed 1 min in 70% ethanol, two minutes in sodium hypochlorite 1% and washed in sterile distilled water for 30 s [
Data from each of the two laboratory experiments, were processed with the SAS statistical program, through which the fulfillment of assumptions prior to the execution of the analysis of variance was tested. In cases where the F-test analysis of variance was significant, the honest Tukey multiple comparison (Tukey HSD) was used to determine the differences within treatments.
Experiment 1. The TRICHOD® (Orius Biotecnologia) and FOLIGUARD® (Live Systems Technology) commercial biological products with the active ingredient Trichoderma harzianum and an isolate of Trichoderma sp uncharacterized, were evaluated against F. oxysporum using spore suspensions at a concentration of 1 × 108 spores/mL. A completely randomized design was followed, with 15 replications, where each treatment cor- responded to a biological product. Two discs of 8 mm diameter, one of the pathogens and one of the antagonists, were transferred to a Petri dish with PDA + A. These discs were located one in front of the other at a distance of 3 cm, and were incubated at 20˚C. The pathogen area growth in each treatment, and the control without treat- ment was measured three times at week for a period of three weeks.
Experiment 2. In order to set the action of plant extracts of pine, eucalyptus and marigold on the develop- ment of colonies of F. oxysporum, the next procedure was followed: Samples of these plant species were col- lected in the campus of UDCA, washed with water, immersed in sodium hypochlorite 1% for two minutes, and finally rinsed with sterile distilled water. Three subsamples of 60 g, 120 g and 180 g, for each one of pine, euca- lyptus and marigold samples, respectively, were evaluated. Each subsample was boiled in a liter of sterile dis- tilled water and subsequently filtered through filter paper, adjusted to 1000 ml of water to prepare a culture me- dium PDA + A + Extract (PDA + A + E).
From the pure culture of F. oxysporum obtained in experiment 1, 15 discs of 8 mm in diameter were extracted and planted in individual petri dishes containing the PDA + A + E culture medium, and incubated at 20˚C. The colony area was measured three times a week for a period of three weeks. It was followed a completely rando- mized design with 15 replications for each extract and concentration.
Leucadendron proteas plants, cultivar “Safari Sunset”, one month old at the time of transplantation, with no in- ternal and external disease symptoms were planted in 5 kg pots, filled with unsterilized soil obtained in the ex- perimental field of UDCA. Once planted they were located according to the experimental design in rows of 10 pot plants per each of the five treatments, spaced 0.30 m apart.
Then, a randomized complete block design was followed with five treatments and five replications. The treat- ments were T. harzianum, marigold plant extract 120 g/L, potassium phosphite, benomyl 50 WP, and the absolute control. The treatments had randomization within the blocks and in the experimental area. Each experimental unit consisted of 10 plants for a total of 250 plants. For data analysis, the Minitab 16 statistical software was used.
To isolate F. oxyporum it was followed the procedure previously described. Ten days after planting, the seedlings were inoculated with a suspension of 106 macroconidia/mL of F. oxysporum [Salazar et al., 2010]. One mL of the suspension was injected into the root collar, using a plastic syringe. Fifteen days after the inocu- lation the following treatments were applied: 1) 5 mL per pot around the plant of a suspension of TRICHOD® at a concentration of 2 × 1010 conidia/mL of T. harzianum; 2) This treatment consisted of the application of 10 mL of marigold extract, around the neck of the plant. The marigold extract was prepared with 120 g of marigold leaves per liter of water, because the best results were obtained in the laboratory study; 3) For the treatment with the fungicide the product benomyl 50 WP was used in doses of 1.5 g per liter of water. Two mL of the solution were used to drench the soil around the stem in each plant; 4) To evaluate the potassium phosphitethe commer- cial product Agrifos 400 SL, was used. The drench had a concentration of 7.5 mL of the commercial product per liter of water. Each plant was drenched with 5 mL of this solution; 5) The control did not receive chemical nor biological treatments.
For six months, every 15 days data were taken in five randomly selected plants in each treatment and replica- tions. a) The height of the plant was measured between the soil surface and the apex of the last leaf of the plant with a ruler; b) The number of side shoots was quantified; c) At the end of six months, no external symptoms of the disease manifested and for this reason destructive sampling was performed in order to observe wilt symp- toms in the vascular bundles of the plant. Dry matter accumulation was determined in the roots of the plants se- lected at random, in the destructive sampling. Fresh weight was taken and then placed in an oven for 48 hours at 60˚C, to obtain dry weight. Five plants per treatment were evaluated, totaling 25 plants. For data analysis the Minitab 16 statistical software was used and the data were subjected to analysis of variance, Tukey test and the examination of contrasts.
Significant differences were found (p < 0.01) among three treatments: TRICHOD®, FOLIGUARD® and Tri- choderma sp, and the control. In general, they reduced the growth of the F. oxysporum colony. Duncan test in- dicates that TRICHOD® reduced significantly the colony area of F. oxysporum, followed by FOLIGUARD® and Trichoderma sp (
The results indicate that Trichoderma inhibits colony growth of F. oxysporum under laboratory conditions. This antagonistic effect has already been described by other researchers to explain the potential of the genus Trichoderma for the management of this important plant pathogen. Cifuentes [
highly effective in controlling Fusarium solani in tomato. Also, when the chemical control and the use of anta- gonists against F. graminearum were compared, it was determined that copper oxychloride inhibited growth of the pathogen, whereas Trichoderma reduced 64% the colony growth and was effective in reducing the severity of the disease in wheat crops [
According to Stefanova et al., [
The tested plant extracts showed significant differences (p < 0.01) among treatments (
There is a possibility of using plant extracts to control plant pathogens. In coffee plants promising results have been obtained in the management of diseases because of their mechanism of direct action on pathogens, and their potential for inducing systemic resistance [
The results explore the possibility of new organic technologies that allow a sustainable management of plant health problems, which counteract the harmful effects of the use of synthetic chemical fungicides. New tech- nologies are a common benefit to produce proteas and other crops where Fusarium attacks generate large losses. Moreover, the results are a significant contribution to knowledge and use of biodiversity, because working with native and introduced plants, and domesticated microorganisms in Colombia generate knowledge to expand their potential uses.
Plant height. The data were transformed, and significant differences (p < 0.001) among treatments and blocks were found. The heights were 31 cm and 32 cm between plants treated with potassium phosphite and plants treated with marigold extract, respectively. These results were statistically different to the control (
enough to allow normal plant development. The control presented a marked delay in growth with 26 cm and development compared to treated plants.
Number of side shoots. There were no significant differences (p ≤ 0.05) among the treatments: TRICHOD, potassium phosphite, marigold extract and benomyl. However, there were differences between these treatments and the control. The greatest number of side shoots was 2.5 occurred with the TRICHOD® treatment (
Treatment with marigold extract 120 g/L, had the best average of 6.5 g and it was statistically different from the control (
The disease did not show any external symptoms after six months of performing the inoculation. However, when the destructive sampling was done, it was found 100% incidence in all treatments. In the destructive sam-
pling, it was evident the manifestation of symptoms in the vascular bundles, characterized by brown color around the xylem, extending from the base of the stem to the lateral side stems.
There were significant differences (p ≤ 0.001) between treatments. The marigold extract presented the best efficacy against the disease, because reduced damage in 88.5%, followed by TRICHOD, benomyl and potassium phosphite, which showed reductions in 86%, 85% and 84%, respectively. Although the treatment of potassium phosphite was not superior to marigold extract, it did show good plant height average (
Overall, marigold extract produced the best results in this investigation. Marigold has a number of compounds, such as sesquiterpenes, sesquiterpenoles, saponins and flavonoids that give certain anti-fungal properties. Mar- tinez [
Treatment with T. harzianum did not provide outstanding averages in the expression of growth traits, except the number of side shoots, which differed from the others; however, presented a significant reduction in lesion length, compared with the control. Possibly, the application of T. harzianum must be done before the infection, for greater efficiency, because, as demonstrated in the results obtained by Perez et al. [
In contrast test was found that the length of the lesion was significantly higher in benomyl treatment versus marigold extract, indicating that the latter was better than the chemical treatment. Studies developed by Amini and Sidovich [
To Universidad de Ciencias Aplicadas y Ambientales UDCA, for supporting this research.