Laboratory studies on benzimidazole resistance in Septoria tritici1

A technique is described for testing isolates of Septoria tritici from winter wheat for resistance to benzimidazole fungicides. Secondary spores from 23 isolates were tested on Czapek Dox V-8 agar amended with benomyl at 1–10 μg ml. Twenty-one isolates were recovered from eight crops in England in 1984 and two (PBI isolates) were obtained in 1973. Thirteen isolates, including both PBI isolates, were sensitive to benomyl at 1 μg ml^?1 and nine were resistant at 10 μg ml^?1. The remaining isolate had a low proportion (1:3.7 x 10⁴) of resistant spores. The minimum inhibitory concentration for sensitive isolates was 0.2–0.4 μg ml ¹ benomyl and for resistant isolates was more than 1000 μg ml ¹. Benomyl-resistant isolates were cross-resistant to carbendazim, thiabendazole and thiophanate-methyl, but not to 12 other fungicides with different modes of action. The implications of these findings are discussed.     

Carbendazim resistance in the eyespot pathogen Pseudocercosporella herpotrichoides

Mycelial isolates (115) of Pseudocercosporella herpotrichoides were obtained from five field sites in England. Carbendazim-resistant isolates were detected by their mycelial growth on agar containing 1 μg/ml carbendazim. Resistant isolates were found at two of the five sites examined and one of these had never been treated with benzimidazole fungicides. Amongst the carbendazim- resistant isolates there was a predominance of isolates with pale mycelium, an irregular colony margin and a relatively slow growth rate; however, this association was not absolute. Large differences in the effects of carbendazim on mycelial growth of sensitive and resistant isolates were demonstrated; growth of sensitive isolates was completely inhibited at 0.5 μg /ml carbendazim whilst five of the six resistant isolates examined grew on agar containing 1000 μg/ml fungicide. The carbendazim-resistant isolates were cross-resistant to benomyl, thiophanate-methyl and to a Icsser degree thiabendazole, but not to prochloraz. Conidia of carbendazim-resistant isolates were as resistant. Carbendazim-resistant isolates were just as pathogenic to wheat as sensitive isolates. The implications of these results and other reports of benzimidazole resistance in P. herpotrichoides are discussed in relation to disease control.     

Sensitivity of Lasiodiplodia theobromae from Brazilian papaya orchards to MBC and DMI fungicides

Stem rot caused by Lasiodiplodia theobromae is an important postharvest disease of papaya in Brazil, responsible for reducing the quality and quantity of fruits. Fungicide use is one of the main disease management measures. However, there are no estimates available of pathogen sensitivity to commonly employed fungicides. Therefore, the EC₅₀ from 120 isolates of L. theobromae from northeastern Brazil, representative of six populations of the pathogen, was estimated in vitro for fungicides of the methyl benzimidazole carbamates—MBC (benomyl and thiabendazole) and demethylation-inhibiting—DMI (imazalil, prochloraz, tebuconazole) groups. Mycelial growth on fungicide-free media and virulence on papaya fruits of the MBC-sensitive and non-sensitive isolates were compared. For MBCs, 8.4% of isolates were non-sensitive to fungicides. For the remaining 91.6%, the mean EC₅₀ ranged from 0.002 to 0.13 μg ml⁻¹ and 0.36 to 1.27 μg ml⁻¹ for benomyl and thiabendazole, respectively. For DMIs, the mean EC₅₀ range for imazalil was 0.001 to 2.27 μg ml⁻¹, 0.04 to 1.75 μg ml⁻¹ for prochloraz, and 0.14 to 4.05 μg ml⁻¹ for tebuconazole. The EC₅₀ values of non-sensitive isolates were significantly (P≤0.05) higher those for the sensitive isolates for each of the DMI fungicides. Differences (P≤0.05) were found in the levels of sensitivity to DMI fungicides among the isolate populations associated with orchards. The populations from two orchards were less sensitive to DMIs. No solid evidence was found for fitness costs relating to MBC non-sensitive isolates because mycelial growth in fungicide-free media and virulence on papaya fruits were similar to those of sensitive isolates.     

Resistance of Erysiphe graminis on barley and wheat to sterol C-14-demethylation inhibitors1

Isolates of Erysiphe graminis f. sp. hordei and tritici with decreased sensitivity to triadimefon showed cross-resistance to other inhibitors of sterol C-14-demethylation, such as triadimenol, propiconazol, diclobutrazol, prochloraz and nuarimol. The isolates exhibited a moderate degree of resistance to these compounds. No cross-resistance was detected to tridemorph, fenpropimorph and pyrazophos. The resistant hordei isolates were more sensitive to ethirimol than the sensitive isolate. The competitive abilities of resistant hordei and tritici isolates were inferior to that of the sensitive isolates. In the presence of the fungicides no differences in germination, appressorium formation and penetration between the sensitive and resistant isolates were observed; 48 h after inoculation the sensitive isolate showed several morphological alterations and further fungal development was arrested. At four to five times higher doses of triadimefon, similar morphological alterations were detected in the resistant isolate. Low concentrations of triazole fungicides which slightly affected mycelium growth of both the sensitive and the resistant isolate of f.sp. hordei severely inhibited development of conidiophores of the sensitive isolate whereas that of the resistant isolate was hardly affected.     

The occurrence of thiabendazole-resistant isolates of Polyscytalum pustulans and Helminthosporium solani on seed potato tubers in relation to fungicide treatment and storage

Tubers infected with thiabendazole-sensitive isolates of Polyscytalum pustulans and Helminthosporium solani were treated annually for 4 years with benomyl, thiabendazole or a formulated mixture of thiabendazole and imazalil, and grown at three farms in Scotland. The proportion of thiabendazole-resistant isolates of H. solani increased, and isolates producing black colonies became more common with successive annual applications of thiabendazole or benomyl. Silver scurf was not reduced after three annual applications. When these fungicides were applied once to untreated seed the incidence of resistant isolates of H. solani was much less in 1988 than in 1991 when tubers had been grown on farms for 3 years from untreated seed.
The proportion of resistant isolates of P. pustulans increased with the number of successive applications of thiabendazole or benomyl but at differing rates on each farm. At one farm, skin spot was not reduced by three annual applications of these fungicides whereas at the other farms it was reduced by 90–100% by four annual applications.
A smaller proportion of resistant isolates of P. pustulans and H. solani was obtained after applying the mixture of thiabendazole and imazalil than after benomyl or thiabendazole alone. Their occurrence was not related to the number of fungicide applications. The mixture also reduced both diseases by more than 75% over the 4 years.
At one farm where resistant isolates of H. solani were present, tubers were infected when stored on trays but not when stored in bags.     

《植物保护学报》1985年第12卷总目录

第1期河南省水稻狐期稻纵卷叶螟的危害损失与防治指标的研究·······························...··……张桂芬刘芹轩(1)麦蚜远距离迁飞和传毒规律的研究··一张向才周广和史明方建中赵争平李淑华董庆周魏凯(9)四川棉区棉铃虫发生规律的研究····································································……李锦华李兹新(17)花角蚜小蜂桔园发生规律研究································…     

A mini-bag technique for evaluation of fungicide effects on Trichoderma spp in mushroom compost

An in vivo technique was developed to observe colonisation of mushroom compost by Trichoderma spp. Isolates of T. harzianum (Th2), T. harzianum (Th1), T. koningii (Tk) and T. viride (Tv) were artificially introduced into compost using a mini-bag system. Wheat grains, colonised by Trichoderma spp, were placed centrally on a layer of compost at the bottom of 1-litre polythene bags which were then filled with 350 g of spawned or un-spawned compost, and partially sealed. After 14 and 21 days incubation at 27 degrees C, the bags were assessed for recovery of Trichoderma from middle and top zones using a needle stab re-isolation technique and a visual colonisation scoring system. Visible green mould contamination, similar to that observed in practice, developed within 21 days. The visual colonisation scoring was reliably related to the re-isolation success. In this evaluation, Trichoderma spp showed considerable differences in their relative abilities to colonise spawned and un-spawned compost, with Th2 isolates being consistently superior to the other isolates of Th1, Tk and Tv in colonising spawned compost. This technique was employed to evaluate the effects of fungicides on the colonisation of mushroom compost by three Trichoderma spp: Th2, Th1 and Tk, using 1-litre and 5-litre mini-bag systems. Aqueous suspensions of benomyl, carbendazim, thiabendazole, prochloraz and prochloraz+carbendazim incorporated into the compost at 50 mg litre(-1), or applied to spawn at 50 mg kg(-1), reduced the colonisation by Trichoderma spp. Prochloraz and prochloraz+carbendazim were superior to benomyl, carbendazim or thiabendazole in reducing compost colonisation by Th2, Th1 and Tk, with Th2 being the most persistent type, capable of colonising treated compost in the presence of all five fungicides. The prochloraz+carbendazim mixture, not normally used in mushroom production, was equal to or better than prochloraz alone. The incidence of green mould colonisation by Th2 was as extensive in the 5-litre compost bags as in the 1-litre bags, but colonisation by Th1 and Tk was more apparent in the 5-litre bags. The in vivo mini-bag evaluations using wheat grain Trichoderma inoculum and needle stab re-isolation procedures proved an efficient method for studying colonisation and screening for effectiveness of fungicides applied to mushroom compost or spawn.     

Resistance to thiabendazole in isolates of Helminthosporium solani, the cause of silver scurf disease of potatoes

Isolates of Helminthosporium solani were obtained from potato tubers and cultured on agar containing thiabendazole. Some grew on agar containing 100 mg/l and these were classified as resistant, whereas sensitive isolates produced no mycelial growth on agar containing 5 mg/l. Isolates were also found that were intermediate in their sensitivity to the fungicide. All isolates from Scottish virus tested stem cutting stocks (VTSC) on two farms were resistant whereas those from another farm were sensitive to the fungicide. The proportion of resistant isolates from eight English once-grown seed stocks ranged from 0 to 96%. There has been no previous report of fungicide resistance in H. solani and the possible reasons for these results and their implications are discussed.     

甜菜褐斑病菌对苯并咪唑类杀菌剂抗药性的研究

1980～1981年期间,自各地甜菜褐斑病病叶样本上分离出563个单孢菌株,并测定了各菌系对甲基托布津的敏感性。结果表明,山东高密、江苏东辛农场等地单独使用苯并咪唑杀菌剂3～4年以后,病菌产生了严重的抗药性,黑龙江巴彦、呼兰、山东薛城等地因没有或极少用药防治,病菌仍然表现敏感。山东菏泽、滕县等地已有一定数量的抗性菌株,但分布尚不普遍。试验证实,抗性菌株对甲基托布津、多菌灵和苯来特等三种苯并咪唑杀菌剂具有交互抗性,百菌清、醋苯锡和毒菌锡对抗性及敏感病菌均有良好效果。目前可选择百菌清作为苯并咪唑类的主要替换药剂。初步观察,停止用药后三年以内抗性菌株的数量及抗性程度仍然保持稳定。此外,甜菜频繁调种加速了抗性病菌的扩散。建议未发现抗性的地区应尽量避免从抗性严重的地区调种。     

Negatively correlated cross-resistance to phenylcarbamate fungicides in benomyl-resistant Venturia inaequalis and V. pirina

Isolates ofVenturia inaequalis and ofV. pirina sensitive (S) or resistant (R) to benomyl were examined in vitro on media amended with two phenylcarbamate fungicides. There was a negatively correlated cross-resistance (NCCR) to both methyl N-(3,5-dichlorophenyl) carbamate (MDPC) and isopropyl N-(3,4-diethoxyphenyl) carbamate (NPC) in some benomylresistant isolates. InV. inaequalis, isolates with low benomyl resistance (LR) did not show NCCR to MDPC, whereas isolates with medium (MR), high (HR) and very high (VHR) resistance to benomyl were more sensitive to MDPC than were the benomyl-sensitive isolates. To NPC, MR and VHR isolates showed NCCR whereas LR and HR isolates reacted similarly as sensitive isolates. InV. pirina only HR and VHR isolates showed NCCR to MDPC. The VHR isolates were sensitive to NPC, whereas the reactions of S, LR, MR and HR to NPC were similar.Crosses between benomyl-sensitive and benomyl-resistantV. pirina as well as between different resistant isolates showed that NCCR is inheritable and controlled by a single Mendelian gene.Samenvatting Benomyl-gevoelige en-resistente isolaten vanVenturia inaequalis enV. pirina werden in vitro onderzocht op media met de fungiciden methyl N-(3,5-dichlorophenyl) carbamate (MDPC) en isopropyl N-(3,4-dichlorophenyl) carbamate (NPC). Een aantal benomyl-resistente isolaten van deze pathogeen bleken een negatief gecorreleerde kruisresistentie (NCCR) te vertonen ten opzichte van MDPC en NPC.Isolaten vanV. inaequalis met matige (MR), hoge (HR) en zeer hoge (VHR) benomyl-resistentie vertoonden NCCR. Ten opzichte van NPC vertoonden alleen MR en VHR isolaten NCCR, en niet de LR en HR isolaten. InV. pirina vertoonden HR en VHR isolaten NCCR ten opzichte van MDPC, maar alleen de VHR isolaten ten opzichte van NPC.Kruisingen tussen benomyl-gevoelige en-resistenteV. pririna, en tussen verschillende benomyl-resistente isolaten onderling, toonden aan dat NCCR erfelijk is en berust op een enkel gen.Contribution No. 1712-E, 1986 series, from the ARO.     

Resistance to thiabendazole in isolates of Polyscytalum pustulans (skin spot) and Fusarium solani var. coeruleum (dry rot) in Scotland

Between 1988 and 1991, isolates of Polyscytalum pustulans were obtained from potato tubers grown in Scotland and tested for sensitivity to thiabendazole (TBZ) on agar. Sensitive isolates did not grow at 1 mg TBZ/1, whereas growth of resistant isolates was unaffected at 1 mg and reduced by 10–30% at 10 mg/1. Resistant isolates were present on all classes of seed potatoes, and were also recovered from the air of stores during grading. Isolates from a stock of seed tubers from Northern Ireland were resistant. All 44 isolates of Fusarium solani var. coeruleum collected in 1991 were sensitive to TBZ.     

Variations among field isolates of Botrytis cinerea in their sensitivity to antifungal compounds

Seventeen field isolates of Botrytis cinerea were compared by determining their radial growth on synthetic media containing various amounts of 21 antifungal compounds. Twelve of these compounds were fungicides that are recommended for the control of Botrytis infections. There were marked differences between the isolates in their sensitivity to the compounds. Individual isolates displayed high levels of resistance to some of the fungicides, including benomyl, carbendazim, iprodione, thiabendazole, thiophanate-methyl, vinclozolin and zineb. The most potent growth inhibitors were benomyl and carbendazim (ED₉₅ values for most isolates <0.1 μg fungicide ml^?1 media), dichlofluanid, iprodione, nystatin, thiabendazole, thiophanatemethyl and vinclozolin (ED₉₅ values for most isolates < 1.0 μg ml^?1), and captan, chlorothalonil, dicloran and thiram (ED₉₅ values for most isolates < 6.0 μg ml^?1). Zineb was much less potent than the other recommended anti-Botrytis fungicides; it was no more effective than carboxin, dinocap and mancozeb (ED₉₅ values for most isolates > 25 μg ml^?1).     

Distribution and fitness of isolates of Botrytis cinerea with multiple fungicide resistance in Spanish greenhouses

Forty-nine greenhouses of vegetable crops were surveyed in southeast Spain at the beginning of the disease season in December 1992 to estimate frequencies of resistance to benzimidazoles, dicarboximides and N -phenylcarbamates (NPC) in B. cinerea . Out of 261 isolates collected, 28% were sensitive to both benzimidazoles and dicarboximides, 15% were benzimidazole-resistant and dicarboximide-sensitive, 8% were benzimidazole-sensitive and dicarboximide-resistant and 46% were benzimidazole- and dicarboximide-resistant. Resistance to benzimidazole, dicarboximide and N -phenylcarbamate was determined by measuring the ability of each isolate to grow in the presence of carbendazim, procymidone and diethofencarb fungicides respectively. Carbendazim- or procymidone- resistant isolates were found in all surveyed greenhouses. Three isolates were found with resistance to carbendazim, procymidone and diethofencarb collected in two adjacent greenhouses that were sprayed with the carbendazim and diethofencarb mixture. All other isolates were sensitive to the mixture because they were either sensitive to carbendazim and resistant to diethofencarb or vice versa. Fitness of 31 isolates of B. cinerea was determined in vivo by measuring their sporulation and lesion growth rate on leaf disks. No fitness costs were associated with resistance to iprodione (dicarboximide) and benomyl (benzimidazole). Isolates with EC₅₀ values higher than 101 mg/L for benomyl and 1.6 mg/L for iprodione were considered to be field resistant (they caused visible lesions on cucumber leaf disks treated with each fungicide).     

Sensitivity of populations ofBotrytis cinerea to triazoles,benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC₅₀s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6g ml^–1, respectively, and were log-normally distributed. The variation factor (ratio between EC₅₀s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC₅₀ of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.     

Resistance to organomercury in Pyrenophora graminea

Isolates of Pyrenophora graminea resistant to organomercury were obtained from barley crops in England and Scotland in 1984–6. Resistant isolates were approximately 100 times less sensitive in vitro than sensitive isolates to phenyl mercuric acetate (PMA), but the difference was only four times for methoxyethyl mercuric acetate (MEMA). In small-plot field trials, seed treatment with either PMA or MEMA gave good control of leaf stripe disease caused by organomercury-sensitive strains of P. graminea but neither compound gave commercially acceptable control of resistant strains. Fungicides with different modes of action (triadimenol + fuberidazole, flutriafol + ethirimol + thiabendazole, carboxin-+- thiabendazole + imazalil, and guazatine + imazalil) were effective against organomercury-sensitive and organomercury-resistant strains. The few cases of resistance found do not justify a change from the use of organomercury as the standard seed treatment for barley.     

Evolution of fungicide resistance in the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis in France

Field isolates of the cereal eyespot pathogen can be divided into two groups which are now considered as two species: Tapesia yallundae and Tapesia acuformis. In both species the first case of acquired resistance was observed with benzimidazole fungicides in the early 1980s. At the same time, a number of sterol C-14 demethylation inhibitors (DMIs), such as the imidazole prochloraz and several triazoles, including flusilazole, were introduced. Surprisingly T. acuformis appeared intrinsically resistant to the triazole derivatives in comparison to T. yallundae, but both species were sensitive to prochloraz. The intensive use of these DMIs led to the development of acquired resistance towards triazoles in T. yallundae and towards prochloraz in T. acuformis. Today all the strains in both species appear equally sensitive to the anilinopyrimidine cyprodinil. ©1997 SCI     

Development of resistance to thiabendazole in Helminthosporium solani (silver scurf) as a result of potato seed tuber treatment

Applying thiabendazole to potato seed tubers affected with silver scurf caused by Helminthosporium solani sensitive to thiabendazole decreased the severity of disease on progeny tubers at harvest, but about 50% of the isolates from these were resistant to the fungicide. The disease was not decreased when samples of the progeny tubers were treated with thiabendazole and planted in the following year, and the incidence of resistant isolates increased. Resistant isolates continued to be present when tubers were planted in the next 2 years without fungicide treatment. Treatment with a mixture of thiabendazole and imazalil also decreased the disease and fewer isolates were resistant than when treated with thiabendazole alone, although the proportion increased after treatment with the mixture in the following year. When seed tubers were infected with thiabendazole-resistant H. solani , silver scurf on progeny tubers was not affected by thiabendazole applied to the seed tubers but was decreased by the mixture of thiabendazole and imazalil. Imazalil was equally effective against H. solani sensitive or resistant to thiabendazole.
Some isolates of H. solani had grey aerial mycelium and of 516 of these isolates obtained in 4 years 29% were resistant to the fungicide. Other isolates produced small, black colonies and their frequency increased with thiabendazole treatment of seed tubers. Of 244 of these isolates, 62% were resistant.     

Negative cross resistance between benzimidazole and N-phenylcarbamate fungicides and control of Botrytis cinerea on grapes

Isolates of Botrytis cinerea resistant to benzimidazoles (Ben^R), dicarboximides (Dic^R), or both (Ben^R Dic^R) were found on table grapes in vineyards in Israel. In vineyards treated for one or two seasons with benomyl and dicarboximides, 41% of the isolates were benomyl-resistant, 18% were dicarboximide-resistant and 21 % were resistant to both fungicides. The frequency of resistant strains was very low in non-treated vineyards. Negatively correlated cross resistance (NCCR) was manifested by 46 Ben^R isolates which were sensitive to isopropyl N -(3,4-diethoxyphenyl) carbamate (NPC) and 73 benomyl-sensitive (Ben^s) isolates which were insensitive to NPC; NCCR was not influenced by sensitivity to dicarboximides. A mixture of Ben^s Dic^R plus Ben^R Dic^s isolates, inoculated on rose petals, was inhibited by mixtures of benzimidazoles plus NPC but not by benomyl, NPC or iprodione alone. Grey mould, incited on maturing grape berries by a vineyard population composed of Ben^s and Ben^R strains, was not controlled by benomyl, while the mixture of NPC plus carbendazim was effective.     

Resistance to benzimidazole fungicides in the gummy stem blight pathogen Didymelta bryoniae on cucurbits

Benzimidazoles were used successfully against gummy stem blight of cucurbits for only one growing season (1980). It was soon noticed in experiments in vivo that they were not effective against some isolates of the pathogen. In a survey during the 1981 crop season, 76 out of 102 isolates collected from 20 plastic greenhouses all over the main cucurbit-growing areas of Crete were resistant to benomyl. The average benomyl concentrations that reduced mycelial growth rates of seven resistant and seven sensitive isolates by 50% were 716 ± 49 and 1.2 ± 0.34μg/ml respectively. The average growth rate of resistant isolates was lower than that of sensitive isolates but there were no differences in spore production and spore germination. The pathogenicity of resistant isolates on young cucumber plants was lower than that of sensitive isolates. The application of benomyl, carbendazim, cypendazole and thiophanate methyl to young cucumber plants at the rate of 300μg a.i./ml 1 day before inoculation with 500 000 spores/ml of the resistant isolates did not affect the infection rate of the disease after 4 days. Sensitive isolates caused no onslight symptoms on similarly treated plants.     

Uptake and translocation of fungicides in wheat after seed treatment,as measured by disease response to Fusarium culmorum

The toxicities of five systemic fungicides [benomyl, carbendazim, methyl 4-(2- aminophenyl)-3-thioallophanate (NF48), thiabendazole and thiophanate-methyl] and of two non-systemic fungicides (guazatine and phenylmercury acetate) against Fusarium culmorum were compared on agar plates; their performance as seed treatments was measured by inoculating the shoot bases of wheat seedlings with F. culmorum in pot experiments. The two most effective compounds, benomyl and thiabendazole, and the less effective thiophanate-methyl, were evaluated in further seed treatment experiments in which leaf sheaths and roots of slightly older plants were infected. The three fungicides protected the first leaf sheath for more than 5 weeks, but thiophanate-methyl was least effective. Against root disease, they were effective when infection was mild, but only thiabendazole significantly controlled severe infection. Bioautography confirmed that fungicide levels in shoots were greater after benomyl and thiabendazole, than after carbendazim and thiophanate-methyl treatments, and that concentrations of fungicide after benomyl, carbendazim and thiophanate-methyl treatments generally declined between 5-13 weeks after treatment. Thiabendazole produced a second fungitoxic component detectable in plants after 34 days.