Antifungal activity of bergenin, a constituent of Flueggea microcarpa

The antifungal activity of bergenin against some plant pathogenic fungi, namely, Alternaria alternata , A. brassicae , A. carthami , Fusarium udum , F. oxysporum f.sp. ciceri , Curvularia lunata and Erysiphe pisi , was studied. Bergenin as its monosodium salt was effective against all the fungi and the effective dose for complete inhibition of spore germination varied from 15 μg mL⁻¹ for F. udum to 125 μg mL⁻¹ for E. pisi . Experiments on the effect of bergenin on powdery mildew development under glasshouse conditions revealed that it can control powdery mildew of pea at 2000 μg mL⁻¹ by postinoculation treatment, the results being comparable with those of carbendazim (1000 μg mL⁻¹) and wettable sulfur (2000 μg mL⁻¹). It affected hyphal elongation and the number of primary and secondary branches.     

Effect of 2,6-dichloroisonicotinic acid, its formulation materials and benzothiadiazole on systemic resistance to alternaria leaf spot in cotton

A wettable powder (WP) formulation providing 5–25 μg mL⁻¹ of 2,6-dichloroisonicotinic acid (INA) and 15–75 μg mL⁻¹ of WP applied to cotton cotyledons significantly increased the resistance of the next two leaves to challenge inoculation by Alternaria macrospora . The wettable powder alone at 15–75 μg mL⁻¹ had a lesser effect. A wettable granule (WG) formulation supplying 35 μg mL⁻¹ of benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and 35 μg mL⁻¹ of WG, applied as a cotyledonary treatment, significantly reduced the formation of lesions on the subsequent two leaves when challenged with A. macrospora . The WG control had no effect. Each treatment except for the WG control also raised the activities of β-1,3-glucanase in unchallenged leaf and stem tissue. Each of the components of the wettable powder without INA applied to cotyledons raised enzyme activities in the next leaves. Individual components, as suspensions of silicic acid and kaolin and solutions of the detergent Attisol II, the wetting agent Ultravon W300 and pure INA, applied to cotyledons increased the resistance of the next leaves to A. macrospora . The responsiveness of cotton to BTH and to each of the components of formulated INA is discussed in relation to knowledge of the effects of BTH and INA on other plants and to possible ways in which the other components of the wettable powder may affect the process of signalling for systemic resistance to disease.     

Management of late leaf spot of groundnut (Arachis hypogaea) with chlorothalonil-tolerant isolates of Pseudomonas aeruginosa

Fifteen groundnut-associated bacterial isolates that inhibited by > 90% the in vitro conidial germination of Phaeoisariopsis personata , causal agent of late leaf spot disease of groundnut, were applied as a prophylactic spray (10⁸ cfu mL⁻¹) and tested for control of the disease in the glasshouse. Two groundnut seed-associated bacterial isolates, GSE 18 and GSE 19, identified as Pseudomonas aeruginosa , reduced the lesion frequency (LF) by up to 70%. A 90-day-old peat-based formulation of P. aeruginosa GSE 18 reduced LF measured 15 days postinoculation by up to 60%. Both P. aeruginosa GSE 18 and GSE 19 were tolerant to chlorothalonil (Kavach®) up to 2000  µ g mL⁻¹ in LB broth. In glasshouse trials, GSE 18 and GSE 19 tested in combination with reduced concentrations of chlorothalonil were highly efficient in management of the disease. The disease was completely controlled by chlorothalonil (> 250  µ g mL⁻¹), and in the presence of GSE 18 or GSE 19, 100  µ g mL⁻¹ chlorothalonil was equally effective. Application of rifamycin-resistant mutants of GSE 18 or GSE 19 together with chlorothalonil significantly increased the survival of these isolates in the groundnut phylloplane. In the field, a combination of GSE 18 and 500  µ g mL⁻¹ chlorothalonil reduced disease severity comparable to 2000  µ g mL⁻¹ chlorothalonil alone. Use of chlorothalonil-tolerant pseudomonads together with a quarter concentration of the recommended field dose of chlorothalonil doubled pod yield compared with the untreated unsprayed control.     

Biological control of fusarium crown and root rot of tomato with antagonistic bacteria and integrated control when combined with the fungicide carbendazim

Two bacterial isolates, Bacillus megaterium (c96) and Burkholderia cepacia (c91), demonstrated to be antagonistic against Fusarium oxysporum f.sp. radicis-lycopersici , the causal organism of fusarium crown and root rot of tomato, were evaluated as biocontrol agents alone and when integrated with the fungicide carbendazim. In an initial screening, these isolates reduced disease incidence by 75 and 88%, respectively. In vitro , both biocontrol agents were highly tolerant to the fungicide carbendazim, commonly used to control fusarium diseases. Carbendazim reduced disease symptoms by over 50% when used at > 50  µ g mL⁻¹, but had little effect at lower concentrations. Combination of the bacterial isolates and carbendazim gave significant ( P  ≤ 0·05) control of the disease when plants were artificially inoculated with the pathogen. Application of carbendazim at a low concentration (1  µ g mL⁻¹) in combination with B. cepacia c91 reduced disease symptoms by 46%, compared with a reduction of 20% obtained with the bacterium alone and no control with the chemical treatment alone. A combination of B. megaterium c96 with an increased application rate of 10  µ g mL⁻¹ carbendazim significantly reduced disease symptoms by 84% compared with inoculated controls and by 77% compared with carbendazim treatment alone. In this experiment, the integrated treatment also slightly outperformed application of 100  µ g mL⁻¹ carbendazim, and bacteria applied without fungicide also provided good disease control.     

Sensitivity of Sclerotinia homoeocarpa to demethylation-inhibiting fungicides in Ontario, Canada, after a decade of use

In late 2003, nine populations of Sclerotinia homoeocarpa in Ontario Canada (seven of which had been previously sampled in early 1994, prior to the registration of sterol demethylation-inhibiting (DMI) fungicides for turf disease control in Canada) were sampled and tested for sensitivity to propiconazole. Four of the nine populations had not been treated with DMI fungicides during the intervening years, and isolates from these locations were sensitive to propiconazole (geometric mean EC₅₀ values of 0·005–0·012 µ g mL⁻¹, compared with 0·005–0·008 µ g mL⁻¹ for the original 1994 populations). Among the five populations from 2003 that had been exposed to DMI fungicides, mean EC₅₀ values were significantly greater, ranging from 0·020 to 0·048 µ g mL⁻¹. A significant correlation of determination was found between estimated number of fungicide applications and log EC₅₀ ( R ² = 0·832, P  = 0·0001), and the equation predicted that 42·3 applications of propiconazole would be needed to bring a sensitive population (EC₅₀ < 0·01  µ g mL⁻¹) to a resistant level (EC₅₀ > 0·10  µ g mL⁻¹). Fungicide sensitivity vs. duration of fungicide efficacy was also tested, and it was found that isolates with decreased sensitivity were able to more quickly overcome the inhibitory effects of fungicide application, reducing the duration of control from 3 weeks to 2 weeks.     

Sensitivity of Phytophthora infestans to mandipropamid and the effect of enforced selection pressure in the field

Sensitivity to the new carboxylic acid amide (CAA) fungicide mandipropamid (MPD) in Phytophthora infestans was measured for isolates collected between 1989 and 2002 in Israel prior to the commercial use of MPD (baseline sensitivity, 44 isolates), and from MPD-treated (25 isolates) and untreated fields (215 isolates) in nine European countries and Israel between 2001 and 2005. All isolates were sensitive to MPD, with EC₅₀ values ranging between 0·02 and 2·98  µ g mL⁻¹. Plastic-tunnel (UK), shade-house (Israel) and field experiments (Israel) conducted during 2001–05 showed that enforced selection pressure, applied preventively or curatively, imposed by repeated sublethal (5  µ g mL⁻¹) or excessive (500–1000  µ g mL⁻¹) doses of MPD on mixed isolates of P. infestans produced no isolates resistant to the compound. The results of this study indicate that the probability of a buildup of resistant sub-populations of P. infestans to mandipropamid in the field is low.     

Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan

A total of 651 isolates of cucumber corynespora leaf spot fungus ( Corynespora cassiicola ) collected from cucumber in Japan, either with (438 isolates) or without (213 isolates) a prior history of boscalid use, were tested for their sensitivity to boscalid by using a mycelial growth inhibition method on YBA agar medium. Additionally, seven isolates of C. cassiicola obtained from tomato, soybean, eggplant (aubergine) and cowpea in different locations in Japan were tested before boscalid registration. Minimum inhibitory concentration (MIC) and 50% effective concentration (EC₅₀) values for 220 isolates from crops without a prior history of boscalid use ranged from 0·5 to 7·5 μg mL⁻¹ and from 0·04 to 0·59 μg mL⁻¹, respectively. Two hundred and fourteen out of 438 isolates collected from ten cucumber greenhouses in Ibaraki Prefecture, Japan, which received boscalid spray applications showed boscalid resistance, with MIC values higher than 30 μg mL⁻¹. Moreover, resistant isolates were divided into two groups: a moderately resistant (MR) group consisting of 189 isolates with EC₅₀ values ranging from 1·1 to 6·3 μg mL⁻¹, and a very highly resistant (VHR) group consisting of 25 isolates with EC₅₀ values higher than 24·8 μg mL⁻¹. MR isolates were detected from all ten greenhouses, but VHR isolates were detected from only three. As a result of fungus inoculation tests which used potted cucumber plants, control failures of boscalid were observed against resistant isolates. Efficacy of boscalid was remarkably low against VHR isolates in particular. This is the first known report on boscalid resistance in Japan.     

Control of Phytophthora cryptogea in the hydroponic forcing of witloof chicory with the rhamnolipid-based biosurfactant formulation PRO1

Rhamnolipids, extracellular metabolites of Pseudomonas aeruginosa with surfactant properties, proved to be very effective in controlling the spread of brown root rot disease caused by Phytophthora cryptogea in the hydroponic forcing system of witloof chicory ( Cichorium intybus var. foliosum ). The biosurfactant was applied as the product PRO1, a formulation of 25% rhamnolipids in oil. Both an in vitro screening and in vivo experiments in a mini-hydroponic system demonstrated the ability of PRO1 to control brown root rot. A 25  µ g mL⁻¹ rhamnolipids nutrient solution was enough to obtain good control of an artificial infection with a zoospore suspension of P. cryptogea . The biosurfactant PRO1 performed well in a semicommercial system under growers' conditions. A treatment of 25  µ g mL⁻¹ rhamnolipids (100  µ g mL⁻¹ PRO1) reduced the disease incidence significantly in two independent experiments. However, PRO1 was not effective when a mycelial suspension was used as inoculum. Rhamnolipids have good potential to limit the spread of P. cryptogea in the hydroponic forcing system of witloof chicory, and can be used as a preventive measure against brown root rot.     

Reduced sensitivity of Cercospora beticola isolates to sterol-demethylation-inhibiting fungicides

In a survey conducted during October 1995, single-lesion isolates of the sugar beet leaf-spot fungus, Cercospora beticola , were tested for sensitivity to the sterol demethylation inhibiting fungicides (DMIs) flutriafol and bitertanol. The isolates were collected from fields in three different areas of northern Greece. Fields at Serres and Imathia had been sprayed with DMIs for about 15 years to control sugar beet leaf-spot. At the third site, Amyndeon, DMI fungicides had not been used. From each area 150 isolates were tested. ED₅₀ values were calculated for individual isolates by regressing the relative inhibition of colony growth against the natural logarithm of the fungicide concentration. The mean ED₅₀ values for flutriafol for the Serres, Imathia and Amyndeon populations were 1·07, 0·73 and 0·5 µg mL⁻¹, respectively (significantly different at P  = 0·05). For bitertanol the mean ED₅₀ values for the Serres and Imathia populations were 0·72 and 0·81 µg mL⁻¹, respectively, which were not significantly different at P  = 0·05. The mean ED₅₀ value of the Amyndeon population was 0·48 µg mL⁻¹, which was significantly lower than those of the other two populations ( P  < 0·05). A cross-resistance relationship was found to exist between the two triazole fungicides tested when log transformed ED₅₀ values of 60 isolates were subjected to a linear regression analysis ( r  = 0·81).     

Plant sensitivity to atrazine and chlorsulfuron residues in a soil-free system

The sensitivity of 22 major crops, pastures and weeds from the north-east grain region of Australia to atrazine and chlorsulfuron residues was determined in a glasshouse using a soil-free bioassay system. A logistic equation was fitted to the seedling fresh weights as a function of the logarithm of herbicide concentration by non-linear regression and used to calculate the doses for 10%, 30% and 50% inhibition of seedling growth (ID₁₀, ID₃₀ and ID₅₀). The ID₅₀ for atrazine ranged from 0.03 to 0.04 mg a.i. L^–1 for Salvia reflexa Hornem. and barley to 1.47 mg a.i. L^–1 for sorghum. The ID₅₀ for chlorsulfuron ranged from 0.19 to 0.21 μg a.i. L^–1 for lucerne and snail medic to 102 μg a.i. L^–1 for wheat. Based on ID₅₀ values measured, the predicted responses of each species to a range of concentrations of atrazine and chlorsulfuron were classified into four categories ranging from no damage to severe damage. These sensitivity data will assist in planning cropping sequences in soils previously treated with atrazine or chlorsulfuron.     

Sensitivity of Phytophthora infestans to flumorph: in vitro determination of baseline sensitivity and the risk of resistance

The sensitivity of 127 Phytophthora infestans isolates to flumorph was determined in 2003 and 2004. The isolates originated from two geographical regions and showed similar levels of sensitivity in both years. Baseline sensitivities were distributed as a unimodal curve with EC₅₀ values for growth of mycelia ranging from 0·1016 to 0·3228  µ g mL⁻¹, with a mean of 0·1813 (± 0·0405) µ g mL⁻¹. There was no cross-resistance between flumorph and metalaxyl. Laboratory studies were conducted to evaluate the risk of P. infestans developing resistance to flumorph. Mutants resistant to metalaxyl or flumorph were obtained by treating mycelium of wild-type isolates with ultraviolet radiation. Metalaxyl-resistant mutants were obtained with a high frequency and exhibited resistance factor values (EC₅₀ resistant/EC₅₀ sensitive phenotypes) of more than 100, while flumorph-resistant mutants were obtained at much lower frequencies and had very small resistance factors (1·5–3·2). There was cross-resistance between flumorph and dimethomorph, but not with azoxystrobin or cymoxanil. Most flumorph-resistant mutants showed decreases in hyphal growth in vitro and in sporulation both in vitro and on detached leaf tissues. These studies suggested that the risk of resistance developing was much lower for flumorph than metalaxyl. However, as P. infestans is a high-risk pathogen, appropriate precautions against resistance development should be taken.     

Growth and photosynthesis of four invasive aquatic plant species in Europe

Crassula helmsii , Hydrocotyle ranunculoides , Ludwigia grandiflora and Myriophyllum aquaticum are four well known invasive aquatic plants in European waters. In this study, plant growth at different nutrient availabilities, regeneration capacity and photosynthesis were investigated. Results show high relative growth rates (RGR) of the species of up to 0.132 ± 0.008 g g⁻¹ dry weight (dw) day⁻¹ ( H. ranunculoides ) and a significant increase in RGR with increasing nutrient availability. All species show a high regeneration capacity and the ability to form new shoots from single nodes, even though it differs between the species. Ludwigia grandiflora and M. aquaticum also show regeneration from single leaves. Species differed in maximal amounts, and in temperature and light optima of net assimilation rates: H. ranunculoides leaves reach maximum photosynthetic rates of up to 3500 μmol CO₂ × h⁻¹ g⁻¹ dw, L. grandiflora (leaves) up to 2200 μmol CO₂ × h⁻¹ g⁻¹ dw, M. aquaticum (shoots) 400   μmol CO₂ × h⁻¹ g⁻¹ dw and C. helmsii (shoots) up to 200 μmol CO₂ × h⁻¹ g⁻¹ dw. Hydrocotyle ranunculoides , L. grandiflora and M. aquaticum preferred high light intensity and high temperatures, whilst C. helmsii was negatively affected by intense sunlight. Summarising, it can be assumed that at least H. ranunculoides , L. grandiflora and M. aquaticum can grow well under current and likely future central European climate conditions.     

Striga asiatica seed conditioning and 1-aminocyclopropane-1-carboxylate oxidase activity

Conditioned seeds of Striga asiatica (L.) Kuntze release ethylene, which elicits germination. We investigated the activity of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and respiration during conditioning. Seeds incubated in vivo with ACC, the substrate for ACC oxidase, produced negligible ethylene at the beginning of conditioning or if they were dormant (i.e. would not germinate after conditioning and treatment with stimulant). Non-dormant seeds produced 3000 ηL of ethylene/600 seeds/24 h after 12 days of conditioning. In vitro ACC oxidase activity at day 0 of conditioning produced 500 ηL of ethylene/μg protein/h and 8000 ηL of ethylene/μg protein/h after 12 days of conditioning. Incubation of seeds in strigol before protein extraction did not enhance enzyme activity. Seeds released 4000 μL/L CO₂ in the first 24 h of conditioning, with the rate increasing to 15 000 μL/L/24 h on day 4 and then remaining roughly unchanged. Maximum in vitro activity of ACC oxidase required ACC, catalase, O₂, Fe²⁺, ascorbate and CO₂. In vivo activity of ACC oxidase required ACC and/or germination stimulant(s), suggesting that stimulants may be involved in providing substrates for the ACC oxidase. No difference was observed in the separation of extracted proteins, which suggests that ACC oxidase is activated during conditioning, perhaps as a result of changes in co-factor concentration. Application of these findings to Striga control is discussed.     

Proper adjuvant selection to enhance the activity of triclopyr combined with metsulfuron on the control of Hedyotis verticillata

A study was conducted to evaluate the combined activity of a tank mixture of triclopyr plus metsulfuron with non-ionic surfactant (NIS), crop oil concentrate (COC), and organosilicon (OS) adjuvants on the control of Hedyotis verticillata under glasshouse and field conditions. The results of both the glasshouse and field experiments showed that 160 g ai ha⁻¹ triclopyr plus 0.2 g ai ha⁻¹ metsulfuron and 320 g ai ha⁻¹ triclopyr plus 0.4 g ai ha⁻¹ metsulfuron, with the addition of 0.25% NIS, 0.05% COC, or 0.05% OS, were effective in controlling H. verticillata . A comparison of the cost revealed that the most cost-effective combination for controlling H. verticillata is 160 g ai ha⁻¹ triclopyr plus 0.2 g ai ha⁻¹ metsulfuron combined with 0.25% NIS.     

Efficient, long-lasting resistance against the soft rot bacterium Pectobacterium carotovorum in calla lily provided by the plant activator methyl jasmonate

The potential of three externally applied chemical plant activators, Bion, BABA and methyl jasmonate, known to act only through the plant defence system and not on the pathogen directly, to induce resistance against wild-type Pectobacterium carotovorum was examined in white-flowered calla lily ( Zantedeschia aethiopica ). Following a 24-h induction period, plants were challenge-inoculated with P. carotovorum , originally isolated from calla lily or potato plants, previously transformed using a gfp broad-host-range promoter-probe vector. After another 24 h, Bion treatment (10  µ g mL⁻¹, as a drench) reduced disease symptoms more than sixfold and bacterial proliferation by four orders of magnitude. BABA treatment (5–10  µ g mL⁻¹, also as a drench) reduced the rate of infection by 75–85%. However, the protection afforded by both inducers did not persist. Also, at higher concentrations both displayed a phytotoxic effect. By contrast, methyl jasmonate (10 m m , applied as a leaf spray) completely inhibited P. carotovorum development in calla lily leaves and afforded a long-lasting effect. It is suggested that the defence response of calla lily against P. carotovorum involves the SA-signalling pathway in the short term, but the jasmonate/ethylene-signalling pathway is required for durable protection.     

Genetic characteristics of Fusarium verticillioides isolates from maize in Costa Rica

Thirty-nine isolates of Fusarium verticillioides from maize seeds from three regions of Costa Rica were classified on fertility, fumonisin production, vegetative compatibility and pathogenicity. The identity of the isolates was verified by sexual crosses with standard tester strains and by isozyme analysis. Twenty-three isolates (59%) were mating type A⁻ and 16 (41%) were A⁺; 29 (74%) were female fertile. The isolates produced high amounts of fumonisin B₁ when grown on sterilized maize grits, 32 isolates producing more than 1000 μg g⁻¹, as determined by TLC, and 7 less than 1000 μg g⁻¹. Vegetative compatibility tests by pairing nit mutants identified 34 vegetative compatibility groups (VCGs), of which 29 had one member and 5 had two members. Isolates belonging to the same VCG were obtained from the same seed sample. Two pathogenicity tests with different inoculation methods were performed: on toothpick inoculation of 7-week-old maize stalks, 71% of the isolates were pathogenic according to the length of the necrosis formed in the stalk, and on sand inoculation of maize seedlings all the isolates were pathogenic, according to shoot length and dry weight production. Differences in aggressiveness between some of the isolates were recorded. It is concluded that natural populations of F. verticillioides in Costa Rica consist of genetically diverse, highly fertile and pathogenic isolates that represent a potential risk for disease development and fumonisin accumulation in maize crops.     

Weed control in summer-sown soybeans with flumioxazin plus acetochlor and flumiclorac-pentyl plus clethodim

Field trials were conducted in Taigu, Shanxi province, China, to evaluate the efficacy of flumioxazin plus acetochlor and flumiclorac-pentyl plus clethodim applied to summer-sown soybeans at pre- and postemergence. It was demonstrated that tank-mixing flumioxazin at 50 g ai ha^-1 and acetochlor at 800 g ai ha^-1 created an effective soil-applied herbicide for weed control in soybean crops. The control efficacy was better than when the herbicides were applied individually, and no injury was caused to the soybeans. Flumiclorac-pentyl at 50 g ai ha^-1 plus clethodim at 70 g ai ha^-1 suppressed both broad-leaved weeds and grass weeds with an increased efficacy of more than 90%. Flumiclorac-pentyl applied alone or tank-mixed caused some injury to soybean seedlings, but the soybeans recovered 2–3 weeks after treatment and there was no reduction in the yield.     

PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band

A 24-mer primer pair was generated by sequencing a URP-PCR fingerprinting-derived polymorphic band that is uniquely shared in Pectobacterium carotovorum ssp . carotovorum strains (Pcc). The primer set (EXPCCF/EXPCCR) amplified a single band of expected size (0·55 kb) from genomic DNA obtained from 29 Pcc strains and three Pectobacterium carotovorum ssp. wasabiae (Pcw) strains, but not from other P. carotovorum subspecies atrosepticum , betavasculorum or odoriferum , or from other Erwinia spp. or bacterial genera. The Rsa I digestion profile of the amplified bands divided Pcc strains into five groups with a unique profile from Pcw strains. First-round PCR detected between 5 × 10² and 1 × 10³ colony forming units (CFU) mL⁻¹ and detection sensitivity was increased to as few as 2–4 CFU mL⁻¹ after second-round (nested) PCR. This PCR protocol was used directly to detect Pcc strains in infected plant tissues.     

Berry infection and the development of bunch rot in grapes caused by Aspergillus carbonarius

The effects of different levels of inoculum of Aspergillus carbonarius and time of inoculation on berry infection and the development of aspergillus bunch rot on grapevines (cv. Sultana) were studied under field conditions. Inflorescences at full bloom were inoculated with aqueous spore suspensions of A. carbonarius containing 0 or 1 × 10⁶ spores mL⁻¹ in 2004/05 and 0, 1 × 10² or 1 × 10⁵ spores mL⁻¹ in 2005/06. In both years, the incidence of infection in inoculated berries was significantly higher than in uninoculated berries. Incidence of infection in berries from veraison until harvest was higher than at earlier stages of bunch development (berry set to berries that were still hard and green). Inoculation of bunches at veraison did not significantly increase A. carbonarius infection prior to harvest, at harvest, 6 days after harvest or when berries were over-ripe. Bunches inoculated at harvest did not significantly increase infection 6 days after harvest or when berries were over-ripe. Aspergillus carbonarius was isolated more frequently from the pedicel end (53·1%) than from the middle section (37·5%) and distal end (35·0%) of berries that were inoculated with 10⁵ spores mL⁻¹.     

A qualitative quick-test for detection of herbicide resistance to tribenuron-methyl in Papaver rhoeas

A qualitative seed-based method useful for the detection of resistance to the herbicide tribenuron-methyl in Papaver rhoeas L. is described. Seeds were germinated on 35 mL of a 1.3% agar medium containing 2 g KNO₃ L^–1 in 8.5 cm Petri dishes in a growth chamber under 20 μmol s^–1 m^–2 of fluorescent light. When 0.24 μM tribenuron-methyl or more was added, growth in susceptible plants stopped after the cotyledon stage and they turned chlorotic. The resistant plants continued developing new leaves. The same effect was achieved when 0.2 g gibberellin (GA₃) L^–1 and 7.68 μM tribenuron-methyl or 0.5 g GA₃ L^–1 and 61.44 μM tribenuron-methyl were added. Germination percentage rose with gibberellin in the presence or absence of the herbicide. Plants developed rapidly, with only about 14 d needed to finish the test but sometimes root growth was reduced because of the addition of gibberellin. In the absence of gibberellin but in the presence of the herbicide, plants grew more slowly and developed smaller leaves with a 17-d evaluation period requirement. The test was validated with pot experiments in a greenhouse and also with field trials. The best combination was found to be 0.2 g GA₃ L^–1 and 7.68 μM tribenuron-methyl, assuring homogenous germination and testing of dormant seeds but avoiding root inhibition associated with too much gibberellin.