Abstract: Five herbicides commonly used in rapeseed crop, canola (Brassica napus var Olifera) fields in Iran were studied with an eye to their antifungal activities against six isolates of Trichoderma species and also the phytopathogenic fungus Sclerotinia sclerotiorum. The doses tested were the same as are recommended for applications in canola crops. The results showed that the dinitro aniline herbicides (trifluralin and ethalfluralin) had the highest antifungal activities in vitro on the toxified CDA media; also three other herbicides behaved differently in relation to the fungi investigated. Cycloxydim had the most toxicity against Trichoderma sp. and sethoxydim had the least deleterious effects. But, with, Sclerotinia sclerotiorum, sethoxydim had the most toxicity together with cycloxydim with an intermediate level of anti-fungal activity between sethoxydim and haloxy fop ethoxy ethyl. This may be the first global report of the antifungal activities of the haloxy fop ethoxy ethyl, ethanol fluralin and cycloxydim.
INTRODUCTION
Diseases caused by Sclerotinia sclerotiorum occur in numerous plant hosts. The broad host range of this fungus is important to the control of the disease in agricultural crops because it restricts the number of non-host crops that can be included in crop rotations designed to reduce the concentration of sclerotia in infested soils. Furthermore, inoculum produced on alternative hosts such as dandelions and clover in noncultivated areas can contribute to susceptible crops (Abawi and Grogan, 1975). Little information is available on the relative importance of these sources of inoculum to outbreaks of disease (Boland and Hall, 1994).
Based on an index of plants reported to be susceptible to Sclerotinia sclerotiorum, the fungus attacks 42 subspecies or varieties, 408 species, 278 genera and 75 families, most of them are herbaceous plants from the subclass Dicotyledonae of the Angiospermae but several hosts also occur in the subclass Monocotyledonae and only one species from the Peridophyta, leather leaf fern [Rumohra adiantiformis (G. Forst.) Ching] from the Polypodiaceae is infected by the pathogen. At least four species of Pinaceae, class Gymnospermae have been known as hosts for this fungus (Boland and Hall, 1994).
In Iran, rapeseed (Brassica napus var Olifera) is the most commonly cultivated oilseed crop and the most destructive and harmful disease which is widespread in the most important areas of oilseed production, especially in the northern marginal flats of Caspian sea, is the white stem rot, caused by the S. sclerotiorum.
With an eye to the importance of the disease and the difficulty of its control through the prevalent methods, it seems that an integrated control by a combination of several methods will be useful in disease management. Considering the broad host range of the pathogen and their significant influences on the epidemiological aspects of the disease, it may be possible to use a chemobiologically combined method including herbicides and biocontrol agents. Especially this method can decrease the volume of agricultural practices causing soil erosion and reduce several applications of other chemicals of ecological danger. This study is to solve a problem of those associated to the sustainable agriculture. As for effective biocontrol of soil-borne plant pathogens, hyphal growth of Trichoderma through soil is important for colony extension and colonization of target propagules after introduction into soil (Dandurand and Knudsen, 1993; Knudsen and Bin, 1990; Knudsen et al., 1991), therefore, we focused on the effects of commonly used rapeseed crop prevalent herbicides and their effects on the hyphal growth of Trichoderma as the first step.
MATERIALS AND METHODS
To study the effects of herbicides commonly used in rapeseed (canola) production areas in Iran, five herbicides including trifluralin (Treflan®, EC 48% w/v), ethanol fluralin (Sonalan®, EC 33% w/v), sethoxydim (Nabu S®, OEC 12.5% w/v), cycloxydim (Focus®, EC 10% w/v) and haloxy fop ethoxy ethyl (Gallant®, EC 12.5% w/v) were selected. Trifluralin and ethal fluralin are of soil herbicides, with broad herbicidal range of activities and others are selectively applied in canola fields to control unwanted inter-grown plants.
The fungi chosen for study were three isolates of Trichoderma sp. isolated from the soils (T-5, T-7 and T-35), three other Trichoderma isolates from the plant
shoots (T-26, T-94, P-24) and one isolate of Sclerotinia sclerotiorum, pathogenic on the canola plants.
The experiment was made in two sets; one including terrestrial isolates of Trichoderma sp., Sclerotinia sclerotiorum and soil herbicides and the other including shoot isolates of Trichoderma sp., Sclerotinia sclerotiorum and selective herbicides.
Cultures of the fungi on Czapek’s Dox Agar (CDA), incubated in dark at 25°C for 5 days were used. The studied doses of individual herbicides were those recommended for canola crop. The studied doses were as Treflan® (2000, 3000 and 4000 ppm), Sonalan® (6000 and 6500 ppm), Nabu S® (1500, 5000, 7500 and 10000 ppm), Focus® (3000, 6500, 10000 and 16000 ppm) and Gallant® (1500, 6500 and 10000 ppm). No herbicide was added to the media considered for checks.
The total volume considered for each plate was 20 mL of the final medium. The experiment was performed in triplicates for each isolate and herbicides. The fungi were cultured by plating a 5 mm disc of the active culture per plate. The incubation in the case of the first part of the experiment performed under dark conditions at 25±1°C, however, with the second experiment, it was carried out in light conditions at the room temperature 25±1°C.
The results were recorded when the Fungal colony diameter had reached to 90 mm in control plates recorded. This time for Trichoderma sp. was 3 days after culture, but for Sclerotinia sclerotiorum was 10 days. To compensate the significant difference, daily growth rate (mm/day) was considered. Also, the cultures of S. sclerotiorum were inspected from the view point of sclerotia development. The data were statistically analysed based on the completely random design and the comparison of the means were carried out through Duncan’s test using SAS software.
RESULTS AND DISCUSSION
The decrease in disease after herbicide application due to the decreased growth of the pathogen has been suggested earlier (Altman and Campbell, 1977). All the herbicides studied here, have been favoured (Razavi, 1995) and officially recommended for applications in rapeseed (canola) crop in Iran. Therefore, the post-emergence herbicides used in this study are not phytotoxic under usual conditions recommended for field applications.
With no exception, no fungal isolate could grow on the media including soil herbicides trifluralin and ethal fluralin belonged to the group dinitro anilines. Infact, these herbicides had shown really the highest antifungal activities and did not permit any growth of fungi on CDA media under incubation conditions. Even after several additional days, no fungus could begin to grow. These results are in contrast to the results obtained by other workers (Tyunyaeva et al., 1974; Fontana et al., 1976) but in agreement with the results of Grinstein et al. (1976) and Makawi et al. (1979). Anicuta (1985) reported the favored growth of both Fusarium oxysporum f. phaseoli and F. solani f. phaseoli using Treflan® in bean (Phaseolus vulgaris) cultivation. It has been reported that Treflan® is utilized by Penicillium waksmani and Alternaria alternata as sole C and N source (Abushady et al., 1983). However, Grinstein et al. (1981) have reported the effect of trifulralin as a sensitizer for Fusarium resistance in tomatoes.
Trifluralin and ethalfluralin although effective against the germlings and active mycelia of white stem rot pathogen in soil, they can never impose their suppressive effect against Sclerotinia sclerotiorum when it is active inside weed plants or plant debris, because as members of dinitro aniline herbicides, they can not move inside the plant (Gunsolus and Curran, 1999). The action site of these herbicides is tubulin protein involved in plant cell division, however, it is not clear if the same mechanism of action prevents fungal growth.
Johnson (1994) has proposed a model of dose-response relationship, stating that the degree of disease control obtained with a biological agent depends on the density of the agent, the density of the pathogen, how efficiently individual units of the agent render units of the pathogen ineffective and on the proportion of the pathogen population potentially affected by the agent. The reduction of Sclerotinia sclerotiorum biocontrol efficacy of Trichoderma due to increased interactions between Trichoderma and soil microorganisms and the favored shift from hyphal growth to sporulation because of the microbial competition in soil has been indicated most recently (Bae and Knudsen, 2005). As the biocontrol efficacy of Trichoderma depends mainly on the initial hyphal growth originating from the pellets introduced to soil (Knudsen et al., 1991), therefore, in the case of soil herbicides, their application may help to control the disease in the field with the previous history of disease occurrence and additionally, as these herbicides are of a broad range, therefore they can undoubtedly create a partial biological vacuum in the soil and favor the establishment of certain exogenously introduced or indigenous Trichoderma isolates, so that diseases may be suppressed (Baker, 1981; Papavizas, 1985). However, it seems that the antifungal activity of these herbicides is still a controversial subject and there are different texts in literature (Anicuta, 1985; Abushady et al., 1983; Makawi et al., 1979; Fontana et al., 1976; Tyunyaeva et al., 1974) dependant on the kind and species of the microorganisms and conditions.
Sethoxydim, cycloxydim and haloxy fop ethoxy ethyl had shown different levels of antifungal activities with three Trichoderma isolates and an isolate of Sclerotinia sclerotiorum tested (F = 409.26, p = 0.0001>F). The daily growth mean was significantly influenced by treatments (herbicide-dose components) (F = 1145.83, p = 0.0001>F) and the fungal factor (isolate or species) (F = 1514.97, p = 0.0001>F). Additionally, it was under the meaningful interactive effect of treatment and fungal factors (F = 71.59, p = 0.0001>F).
The first two herbicides are of cyclohexanediones (DIMs) and the latter is of propionic acid derivatives, aryloxyphenoxypropionates (FOPs), (http:\\wwwweedresearch.com/summary/chemfamilySum.asp?lstActive=29&btnSub1=Go&lstHRAC=).
In the case of sethoxydim, the isolate P-24 had more growth rate than other isolates of Trichoderma on the CDA media amended with this herbicide (Table 1). There was a significant difference among the fungal isolates tested and the isolate P-24 was of the most fast daily growth rate. All three Trichoderma isolates were of more daily growth rates compared to the isolate of Sclerotinia sclerotiorum (Table 1).
In the case of haloxy fop ethoxy ethyl, with all doses tested, the isolate P-24 had more growth rate than other Trichoderma isolates studied (Table 2).
Finally, with cycloxydim, no isolate of Trichoderma could grow on the media, which contained higher doses of the herbicide (10000 and 16500 ppm), but in the case of two lower doses (3000 and 6500 ppm), the isolate P-24 supported highest growth compared to other Trichoderma isolates investigated.
| Table 1: | Comparison of fungal isolates based on their daily growth
means (mm) in vitro |
| Table 2: | The effect of different herbicide-dose components on the daily
growth means of Trichoderma isolates and an isolate of Sclerotinia
sclerotiorum |
Generally with all herbicides, higher the dose tested, the lower growth rate (Table 2). Collectively, the growth rate of the most tolerant isolate of Trichoderma, P-24 was the highest on the media with sethoxydim and higher on the media involved haloxy fop ethoxy ethyl compared to the media contained cycloxydim (Table 2).
At the dose of 10000 ppm, three herbicides were comparable considering their antifungal activities, so that cycloxydim had shown the most antifungal activity on Trichoderma isolates and sethoxydim had shown the least antifungal influence on these fungi of importance in biological control of plant diseases (Table 2).
With cycloxydim and haloxy fop ethoxy ethyl, the levels of antifungal activities were confirmed considering their suppressive effects at the dose of 6500 ppm (Table 2 and 3).
With Sclerotinia sclerotiorum, sethoxydim had the highest antifungal activity and haloxy fop ethoxy ethyl had the least activity against the fungus. Also, while haloxy fop ethoxy ethyl and cycloxydim induced the formation of sclerotia, sethoxydim prevented the development of these resistant bodies in all the doses tested. The induction of sclerotium formation by Haloxy fop ethoxy ethyl was observed with all of the doses, however, cycloxydim had a suppressive effect on the sclerotium development when it was used at the rate of 16500 ppm.
| Table 3: | The total effect of different herbicide-dose treatments on
the fungal daily growth mean |
In an experimental program of the IOBC/WPRS working group, Hassan et al. (1994) found that sethoxydim is one of the herbicides more toxic for some of 25 species of beneficial organisms including 3 entomogenous fungi and concluded that more work should be carried out with this herbicide in semi-field and field experiments.
The results of present study are promising that application of sethoxydim will eliminate weeds in canola fields and omit their harmful competitive effects and their roles in disease epidemiology; meanwhile, will impose a preventive effect on the growth of Sclerotinia sclerotiorum with less deleterious effects on the growth of Trichoderma sp. with saprophytic competition abilities more than those of Sclerotinia sclerotiorum (Fig. 1) and thus able to take advantage from the dead weeds residues to raise their own populations and control the disease.
| Fig. 1: | Effect of sethoxydim (Nabu S) herbicide on the growth of Sclerotinia
sclerotiorum and Trichoderma isolate in vitro: (A) Suppressed
growth of Scl. sclerotiorum on Potato Dextrose Agar (PDA) medium
including the herbicide (5000 ppm); (B) Growth of Scl. sclerotiorum
on PDA medium as control; (C) Growth of Trichoderma on the PDA medium
with sethoxydim compared with it’s growth on PDA medium (control) (D) |
Sethoxydim as a herbicide from cyclohexanediones belongs to lipid synthesis inhibitors, i.e., it prevents the enzyme acetyl coenzyme A carboxylase (ACCase) involved in the formation of fatty acids which are the essential components for the in plant production of lipids. Lipids are vital to the integrity of cell membranes and to a new plant growth. The lipid synthesis inhibitor herbicides inhibit a single key enzyme involved in fatty acid biosynthesis. These herbicides are taken up by the foliage and move in the phloem to areas of new growth (Gunsolus and Curran, 1999), therefore, sethoxydim might be potentially much effective in the control of rapeseed white stem rot disease, as it may have its controlling effect against the pathogen even inside the treated weed plants infected by Sclerotinia sclerotiorum.
Also, as a post-emergent herbicide, its persistence might be lengthened by the shading of the soil surface underneath the plant canopy which would reduce herbicide volatility and photo degradation (Klingman, 1961). Such an increased persistence has been explained as the reason for the sufficiency of a single post-emergence application of the dinitrophenol herbicides for the suppression of Sclerotinia blight as effectively as multiple applications (Porter and Rud, 1980).
On the other hand, if the same mechanism of action is involved in the antifungal activity of sethoxydim against the pathogen like what occurs in planta, it may act synergistically together with Trichoderma antibiotic peptides, peptaibiotics, or peptaibols. This is expectable as the antimicrobial activity of peptaibols arise from their membrane activity and their ability to form pores in lipid membranes. The pores so formed are able to conduct ionic species; this conductance leads to the loss of osmotic balance and cell death (Chugh and Wallace, 2001; El Hajji et al., 1989; Le Doan et al., 1986; Molle et al., 1987). The last promising point with sethoxydim can be regarded here, is its suppressive impression on the development of sclerotia as observed in this study. Indeed, the inhibitory effect of sethoxydim on the Sclerotinia sclerotiorum acetyl-CoA carboxylase might be the true reason for its effect on the fungus, as with Aspergillus fumigatus, the enzyme has been found essential for survival. Essential genes are those required for growth (metabolism, division, or reproduction) and survival of an organism. It is known that fungal biosynthesis of fatty acids takes place in the cytosol and starts with carboxylation of acetyl-CoA to malonyl-CoA. From this malonyl-CoA consecutive C2 units are added to acetyl-CoA or the growing fatty-CoA ester chain by an intericate fatty acid synthase complex harboring seven different enzymatic activities (http://www.wipo.int/cgi-pct/guest/getbykey5?KEY=00/39287.000706&ELEMENT_SET=DECL).
Therefore, present results prove that ACCase can be regarded as a new target for the production of a new generation of antimycotics and fungicides that will be of more importance considering the medical problems encountered with the control of the fungal diseases caused by the strains resistant to the applied fungicides and antimycotics. However, it should not be ignored that such a presumptive generation of fungicides and antimycotics shall be applied in a well-planned manner, as because of their single target site of antifungal effect, the probability of fungicide resistance development is expected high. The results from our experiment confirm that there are at least some fungal species that are naturally more resistant.