Formamidine structure and detachment of the cattle tick Boophilus microplus

Evaluation of the effectiveness of topically applied chlordimeform, 14 other formamidines, and 5 sulfur-containing related nonformamidine compounds in causing female Boophilus microplus ticks to detach from mice enabled activity to be related to structure. Five compounds were inactive and 15, including 2 sulfur-containing nonformamidines and 1 sulfur-containing formamidine were active at doses ranging from 0.0005 to 2.0 μg/tick. The most active compounds were the N-monomethyl formamidines, BTS-27271 [N′-(2,4-dimethylphenyl)-N-methylformamidine], and C-8520 (demethylchlordimeform). Among the analogs of chlordimeform tested, those with alkyl substitutions at the amino nitrogen decreased in effectiveness in the order, monomethyl (demethylchlordimeform), monoethyl, mono-n-butyl, mono-i-butyl, mono-i-propyl, dimethyl (chlordimeform), and di-n-propyl. Inactive compounds resulted from the replacement of the chlorine at ring position 4 of the aryl moiety of chlordimeform by bromine or hydrogen or by the conversion of the NCH amidino moiety to the NHCSN < thiourea moiety.Detachment due to chlordimeform was antagonized by piperonyl butoxide but that due to its N-monomethyl analog, a known metabolite of chlordimeform in ticks, was synergized by the same compound. These effects on the detaching response parallel those reported elsewhere concerning synergism and antagonism of toxic responses of B. microplus to formamidines.BTS-27271, which was the most effective formamidine in causing detachment after topical application to ticks was moderately effective when injected into mice but its potency relative to chlordimeform was considerably reduced; when sprayed onto cattle BTS-27271 was somewhat more effective in depressing percentage survival of ticks of all stages than chlordimeform.     

Induction of hyperactivity in larvae of the cattle tick Boophilus microplus by formamidines and related compounds

The effect of chlordimeform and 18 related compounds on the aggregation behavior of the negatively geotactic larvae of the cattle tick, Boophilus microplus was investigated. Aggregations were treated and hyperactivity in the forms of immediate dispersal, delayed dispersal, and prolonged leg waving evaluated. A marked structure-activity relationship with delayed dispersal resulted; most active were the N-monomethyl formamidines, N′-(2,4-dimethylphenyl)-N-methylformamidine and demethylchlordimeform. Other compounds, including chlordimeform, with structures compatible with metabolism to N-monomethyl formamidines were also active. Delayed dispersal caused by those possessing the N,N-dimethyl moiety was antagonized after inhibition of oxidative N-demethylation to N-monomethyl analogs by piperonyl butoxide. Since the N-monomethyl moiety had already been reported as important in the killing action of the formamidines in cattle tick larvae, the possibility of a relationship between delayed dispersal and acaricidal effectiveness was examined and percentage mortality at 24 hr found to correlate positively with the rate of onset of dispersal.Delayed dispersal was not characteristic of the responses to other acaricides such as lindane, allethrin, carbaryl, and coumaphos. In addition, the monoamine oxidase inhibitors, tranylcypromine, pargyline, and nialamide, did not induce delayed dispersal and showed no lethal effects.     

A comparative study of the reactivity of acetylcholnesterases of the cattle tick Boophilus microplus and cattle erythrocytes with organophosphorus and carbamate inhibitors

The kinetic constants, K_a (affinity) k₊₂ (acylation), k_a (second order, acylation), and k₊₃ (deacylation) were determined for the reaction of a number of organophosphorus (OP) and carbamate inhibitors with acetylcholinesterase (AChE) from inhibitor-sensitive Yeerongpilly (Y) OP susceptible ticks, relatively insensitiveBiarra (B) and Ridgelands (R) OP-resistant ticks and bovineerythrocytes (Bov). B, R, and Bov AChE exhibited lower affinity for inhibitors than Y AChE. K₊₂ (carbamylation and phosphorylation) decreased in the order Bov ? Y ? B ? R. For inhibitors with markedly different leaving groups there was no clear-cut linear relationship between rates of alkaline hydrolysis and enzymatic hydrolysis (acylation). Decarbamylation and dephosphorylation were rate limiting. The ratios of Y to B to R AChE for V_max and catalytic center activity (substrate hydrolysis) and k₊₃ (carbamate hydrolysis) were very similar. This was a requirement if a lowered deacylating ability was the cause of the reduced catalytic hydrolysis of substrate observed for B and R AChE. Changes to the catalytic sites of B and R AChE are discussed. A strong parallel of these results is drawn with those obtained for spider mite AChE.     

A simple binding mechanism accounts for the temperature-dependent toxicity of cis-permethrin to larvae of the cattle tick, Boophilus microplus

The action of cis-permethrin, which evinced a strong negative correlation of toxicity with temperature in larvae of the pyrethroid-susceptible Yeerongpilly and pyrethroid-resistant DDT-R and Malchi strains of the cattle tick, was accounted for by a simple binding mechanism. The mechanism assumed that a single pyrethroid molecule binds to a single site and inactivates it, and that the equilibrium constant which describes the ensuing equilibrium between concentrations of free pyrethroid, unoccupied site, and inactivated site then varies with temperature according to the van't Hoff equation. From van't Hoff plots of the data the enthalpies of binding of cis-permethrin to sites in larvae of each of the above strains were calculated to be ?87, ?92, and ?102 kJ mol^?1, respectively. It was concluded from these results that the kdr-type insensitivity (≈6×, in terms of resistance) observed in DDT-R and Malchi ticks could not be explained by altered binding sites at the site of action. It was suggested that the insensitivity might be explained by altered access to the site of action.     

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification.     

Substrate-specificity and toxicological significance of pyrethroid-hydrolyzing esterases of mouse liver microsomes

An esterase or esterases in acetone powder preparations of mouse liver microsomes hydrolyze the cyclopropanecarboxylate ester linkage of pyrethroid insecticide chemicals derived from primary alcohols. The rate of cleavage of (+)-trans-chrysanthemates with various alcohol moieties decreases in the following order: 5-propargyl-2-furylmethyl; 5-benzyl-3-furylmethyl (bioresmethrin); 3-phenoxybenzyl; tetrahydrophthalimidomethyl esters. The hydrolysis rate of benzylfurylmethyl esters with various acid moieties decreases in the order: (+)- or (?)-trans-chrysanthemate; (+)-trans-ethanochrysanthemate; tetramethylcyclopropanecarboxylate; (+)- or (?)-cis-chrysanthemate or (+)-cis-ethanochrysanthemate. The trans-isomers of chrysanthemates and ethanochrysanthemates are hydrolyzed from 2.6- to more than 50-fold more rapidly than the corresponding cis-isomers. This enzyme system does not hydrolyze secondary alcohol esters, i.e., allethronyl (+)-trans- and (+)-cis-chrysanthemates.On intraperitoneal administration to mice, the (+)-trans-chrysanthemate and -ethanochrysanthemate of benzylfurylmethanol are of very low toxicity relative to the corresponding (+)-cis-isomers and the tetramethylcyclopropanecarboxylate. S,S,S-tributyl phosphorotrithioate (DEF) pretreatment increases the toxicity of these five compounds by 2.6- to more than 188-fold, with the exception of bioresmethrin whose toxicity is not altered. When the toxicity is increased, it is probably the result of esterase inhibition since DEF strongly inhibits the esterase activity of fresh liver microsomes while the mixed-function oxidase system remains active. The oxidase system metabolizes the chrysanthemates more rapidly than the ethanochrysanthemates of benzylfuryl-methanol. Depending upon the pyrethroid involved, the esterase or the mixed-function oxidase system, or both may be responsible for limiting the toxicity of these pyrethroids to mice.     

Comparative inhibition of the juvenile hormone esterases from Trichoplusia ni,Tenebrio molitor, and Musca domestica

Twenty-seven compounds were screened as potential inhibitors of juvenile hormone esterases. Of these compounds O-ethyl-S-phenyl phosphoramidothiolate provided the best inhibition for the cabbage looper, Trichoplusia ni (Hubner), and the yellow mealworm, Tenebrio molitor L., while the juvenile hormone esterases of the house fly, Musca domestica L., were best inhibited by a juvenoid carbamate (1-(m-phenoxy-N-ethyl carbamate)-3,7-dimethyl-7-methoxy-2E-octene). The inhibition patterns of T. ni and T. molitor are similar, while those of M. domestica are relatively different. Further studies on the juvenile hormone and α-napthyl acetate esterases of T. ni showed that they could be differentially inhibited. Diisopropyl phosphorofluoridate and an alkyl trifluoromethyl ketone selectively inhibit the hydrolysis of α-naphthyl acetate and juvenile hormone, respectively, while O-ethyl-S-phenyl phosporamidothiolate inhibits both enzymes. The juvenile hormone esterases of T. ni also appear to be unique enzymes that are selective for juvenile-hormone-like molecules. The in vivo inhibition of T. ni juvenile hormone esterases by O-ethyl-S-phenyl phosphoramidothiolate slows the in vivo hydrolysis of juvenile hormone and results in delayed pupation and malformed larvae that resemble larval-pupal intermediates. Thus, the esterases involved in juvenile hormone metabolism appear to be important in juvenile hormone regulation.     

A standardised in vivo and in vitro test method for evaluating tick repellents

The threat of transmission of Lyme borelliosis and tick-borne encephalitis by ixodid ticks has resulted in an increasing number of tick repellents coming onto the market. To allow proper evaluation of the efficacy of different types of compounds and their formulations, there is a need for standardised methods for testing ticks repellents. Ticks show a marked negative geotactic response following contact with a potential host, i.e., they climb up in order to locate attachment and feeding sites, whereas exposing ticks to repellents induces positive geotaxis, i.e., ticks walk downwards or drop off the treated host or substrate. We describe here complementary tests that employ these geotactic responses to evaluate repellents: one in vitro on a warm glass plate and the other on the lower human leg (shin). The compounds tested were DEET, EBAAP, icaridin, capric acid, lauric acid, geraniol, citriodiol, citronella essential oil and lavender essential oil, all non-proprietary ingredients of widely distributed tick repellent formulations.     

Mechanism of action of clenpyrin on the cattle tick Boophilus microplus

Clenpyrin is effective against all stages of development of one-host cattle ticks (Boophilus spp.) including strains resistant to organophosphorus compounds and carbamates. Ticks treated with clenpyrin are quickly immobilised due to a slackening of the muscles. Manometric measurements revealed a marked depression in respiratory gas exchange and a rise in the R Q within 24 h after treatment. Clenpyrin inhibits NADH oxidation and, as a result, all synthesising processes in the tick. The breakdown of proteins taken up with the blood meal is also inhibited. Clenpyrin, however, does not interfere with carbohydrate degradation. These results have been corroborated in detail by determining various substances in treated and untreated ticks: proteins, haematin, guanine, glycogen, glucose, lactate, ATP, lipoids and cuticular wax. Clenpyrin does not inhibit tick proteinase, tick and other acetylcholinesterase, or rat monoamine oxidase; therefore, the mode of action of clenpyrin differs from that of other known tickicides, especially those involving the acetylcholinesterase system.     

Effects of the avermectin, MK-243, on ovary development and salivary gland degeneration in the ixodid tick, Amblyomma hebraeum

Injection of the avermectin analogue, MK-243, into engorged female Amblyomma hebraeum Koch resulted in reduced ovary weight, oocyte length, and ovary vitellin content. There was no significant reduction in hemolymph vitellogenin concentration in MK-243 treated ticks. Although MK-243 was previously shown to markedly reduce hemolymph 20E-concentration, injection of 20E, the vitellogenic hormone in this tick, did not reverse the effects of MK-243 on ovary development. These data suggest that MK-243 may exert its inhibition of egg development more at the level of vitellogenin uptake than vitellogenin synthesis. MK-243 also reversed salivary gland degeneration slightly, probably via its inhibitory effect on 20E-synthesis.     

Inhibition of oviposition in the cattle tick Boophilus microplus by certain acaricides

Doses of ten acaricides, ranging from 2.5 to 10 μg, induced about 10-60% mortality and inhibited oviposition in engorged Boophilus microplus as well as preventing larval hatching from the eggs. The effects of the acaricides were dose dependent. The efficacy of acaricides in reducing the reproductive potential (inhibition of oviposition × inhibition of hatching/100) was in the following order: dimethoate > dioxathion > naled > diazinon > chlordimeform > carbaryl > trichlorphon > phosphamidon > gamma-HCH > fenitrothion. Most of the acaricides also retarded the oviposition cycle, delaying the peak activity by 4-8 days. The ovarian development in control ticks reached its peak between the 5th and 7th days before decreasing gradually and ceasing completely between the 16-20th days after engorgement. The protein content of the ovaries and the rate of incorporation of [¹⁴C]glycine into the tissues followed a similar pattern. Carbaryl, fenitrothion and naled inhibited these activities. Chlordimeform stimulated [¹⁴C]glycine incorporation and increased the ovarian protein content up to the 13th day before resorption of the oocytes. The eggs from treated ticks were mostly non-viable and contained more protein; those from dioxathion, carbaryl and chlordimeform treatments had a higher dry weight than the control.     

The purification and characterization of esterases from insecticide-resistant and susceptible house flies

Four major esterases in one susceptible (CSMA) and two resistant (Hirokawa, E₁) house fly strains were separated by chromatofocusing. Of the four esterases, those with pI's of 5.1 and 5.3 accounted for 90% of the p-nitrophenyl butyrate hydrolyzing activity in the three house fly strains. They also accounted for 70% (Hirokawa, E₁) and 40% (CSMA) of the paraoxon-hydrolyzing activity as well as 87% (Hirokawa), 39% (E₁) and 66% (CSMA) of the malathion-hydrolyzing activity in microsomes as measured by esterase-antibody interaction. In the Hirokawa strain, the pI 5.1 esterase was the predominant esterase and was more active than that of the the CSMA strain. Different substrate specificities and a different K_m toward acetylthiocholine, as well as different rates of malathion and paraoxon hydrolysis between the Hirokawa and CSMA strains, suggest a qualitative difference in the pI 5.1 esterase. For the pI 5.1 esterase from the E₁ strain, a different substrate specificity, a different K_m for p-nitrophenyl butyrate, a different sensitivity to inhibitors, and a different rate of paraoxon hydrolysis suggest that it is a modified esterase. This esterase is not a phosphorotriester hydrolase, nor does it lack nonspecific esterase activity. It is a modified esterase which has a different substrate specificity when compared to the esterases from the other strains. The molecular weight of the esterases studied was approximately 220,000, with pH optima of about 7.0.The ratio of malathion α-monoacid to β-monoacid formation was about 9.0 for the pI 5.1 and 5.3 esterases and 1.5 for the pI 4.8 and 5.6 esterases. The existence of a higher $\text{α}\text{β}$ ratio for the pI 5.1 and 5.3 esterases and their significant rate of malathion hydrolysis in the Hirokawa strain indicate that an increase in the $\text{α}\text{β}$ ratio in house flies reported was due to the increase in the pI 5.1 esterase in the resistant strain.     

In vitro inhibition of Sclerotinia sclerotiorum by mixtures of azoxystrobin, SHAM, and thiram

The necrotrophic fungal phytopathogen Sclerotinia sclerotiorum (Lib.) de Bary has a broad host range and frequently causes destructive diseases. The extensive use of common fungicides to control these diseases has selected for resistance in populations of S. sclerotiorum. In this study, 105 isolates of S. sclerotiorum from different geographical regions in Jiangsu Province of China were characterized for baseline sensitivity to azoxystrobin, and the average EC₅₀ value was 0.2932 μg/mL for mycelial growth. Of the mixtures of the fungicides thiram and azoxystrobin that were tested using an in vitro mycelial growth assay, the 1:4 ratio provided the greatest inhibition of S. sclerotiorum. When tested against nine isolates, the 1:4 mixture resulted in a mean synergy ratio of 2.31, indicating synergistic inhibition. Mycelial respiration was inhibited for about 2 h by azoxystrobin alone but for 48 h by the mixture of thiram and azoxystrobin. Salicylhydroxamic acid (SHAM, a known inhibitor of alternative respiration) also increased the inhibition of mycelial growth and respiration caused by azoxystrobin. These results suggest the need for further study of effects of combinations of azoxystrobin with thiram or SHAM in planta to evaluate their potential for management of diseases caused by S. sclerotiorum.     

Influence of three diets on susceptibility of selected insecticides and activities of detoxification esterases of Helicoverpa assulta (Lepidoptera: Noctuidae)

The oriental tobacco worm, Helicoverpa assulta Guenée, is one of the most destructive pests of tobacco and peppers in China. We determined the susceptibility of H. assulta reared on an artificial diet, chili pepper and tobacco to four insecticides (fenvalerate, phoxim, methomyl, indoxacarb) under laboratory conditions associated with the activities of acetylcholinesterase (AChE), carboxylesterase (CarE) and glutathione S-transferase (GST) in its larvae. H. assulta larvae that were fed with chili pepper were more susceptible to fenvalerate, indoxacarb, and phoxim than those that were fed with tobacco and the artificial diet, but not to methomyl. The larvae that were fed with chili pepper were 3.65-, 2.49-, 1.92- and 2.44-fold more susceptible to fenvalerate, phoxim, methomyl, and indoxacarb than those fed with tobacco, respectively. The AChE activities of H. assulta larvae that were fed with chili pepper and tobacco were 2.12 and 1.07 μmol mg⁻¹ 15 min⁻¹, respectively, almost 2-fold difference. The CarE activity of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 4.12, 7.40 and 7.12 μmol mg⁻¹ 30 min⁻¹, respectively. Similarly, the GST activities of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 52.02, 79.37 and 80.02 μmol mg⁻¹ min⁻¹, respectively. H. assulta larvae that were fed with chili pepper were more resistance to the tested insecticides. The low activities of AChE and the high activities of CarE and GST lead to H. assulta become more susceptible to the tested insecticides.     

Molecular characterization of two acetylcholinesterase genes from the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

Acetylcholinesterase (AChE), which is encoded by the ace gene, catalyzes the hydrolysis of the neurotransmitter acetylcholine to terminate nerve impulses at the postsynaptic membrane. AChE is a primary target of many insecticides including organophosphates (OP) and carbamates (CB). In this study, full-length cDNA sequences of two ace genes (Nlace1 and Nlace2) were sequenced from the brown planthopper (BPH) Nilaparvata lugens, the most destructive insect pest of rice crops. Nlace1 cDNA is 2842 nucleotides long and contains an ORF potentially encoding a 790 amino acid peptide. Nlace2 cDNA is 2852 bp in length and contains an ORF that potentially encodes a 672 amino acid peptide. NlAChE1 has an identity of 40% with NlAChE2 at the amino acid sequence level. Phylogenetic analysis of 59 AChEs from 32 animal species showed that NlAChE1 is most closely related to AChE1s from Blattella germanica and Nephotettix cincticeps, while NlAChE2 is most closely related to AChE2 from N. cincticeps. Quantitative RT-PCR analysis showed that Nlace1 is expressed at a much higher level than Nlace2 in all developmental stages and tissues, demonstrating that NlAChE1 may be the dominant AChE form of the two enzymes. This result will help reveal the resistance mechanism of N. lugens to organophosphorous and carbamate insecticides and promote development of more selective insecticides targeting the main NlAChE1.     

Toxicology and metabolism of some synthetic pyrethroids in larvae of susceptible and resistant strains of the cattle tick boophilus microplus (Can.)

Larvae of the Malchi and DDT-R pyrethroid-resistant strains of ticks, compared with larvae of the Yeerongpilly (susceptible) strain, exhibited knock-down resistance equally to (1 RS)-cis- and (1 RS)-trans-permethrin but little resistance to either (1 RS)-cis- or (1 RS)-trans-cypermethrin. Kill resistance was higher in Malchi than in DDT-R ticks and more pronounced against the permethrin isomers. The cis-isomers were more potent overall than the trans-isomers. Cypermethrin isomers penetrated more slowly than permethrin isomers, especially into Malchi larvae. Metabolic detoxication, mainly esteratic, was fastest in Malchi larvae, trans-isomers consistently being attacked faster than cis-isomers. The greatei resistance of Malchi ticks is therefore probably due to a combination of these two differences. Dual mechanisms of the toxic action of pyrethroids are discussed.     

Involvement of esterases in diazinon resistance and biphasic effects of piperonyl butoxide on diazinon toxicity to Haematobia irritans irritans (Diptera: Muscidae)

Resistance to insecticides remains a major problem for the successful control of the horn fly, Haematobia irritans irritans (L.), one of the most important pests of cattle in many countries including the United States. The organophosphate (OP) insecticide diazinon has been used to control pyrethroid-resistant populations of the horn fly. There are only a few reported cases of horn fly resistance to diazinon in the United States and Mexico. Piperonyl butoxide (PBO) has been used successfully as a synergist of pyrethroid insecticides to control horn flies. PBO-synergized diazinon products are also available for horn fly control in the United States, although PBO is known to inhibit the bio-activation of certain OP insecticides including diazinon. A study was conducted to evaluate the effect of PBO on diazinon toxicity to horn flies using a filter paper bioassay technique. These bioassays in both the susceptible and diazinon-resistant horn fly strains revealed a biphasic effect of PBO on diazinon toxicity to horn flies. PBO inhibited diazinon toxicity when the PBO concentration used was high (5%), and no effect was observed when PBO concentration was intermediate (2%). However, at low concentrations (1% and lower), PBO significantly synergized diazinon toxicity. We demonstrated that enhanced esterase activity was associated with survivability of horn flies exposed to diazinon alone. PBO has been shown to inhibit esterase activity in other insect species. However, results of biochemical assays with esterases from this study suggest that PBO did not have significant effect on the overall esterase activity in the horn fly. The observed synergistic effect of PBO at lower concentrations on diazinon toxicity to horn flies could not be explained by reduced esterase activity due to PBO inhibition. It is likely that PBO synergized diazinon toxicity at lower concentrations by facilitating penetration of diazinon through the cuticle and/or inhibiting the oxidative detoxification of diazinon, and reduced diazinon toxicity at high PBO concentration by inhibiting the bio-activation of diazinon.     

Isolation and partial characterization of an antifungal protein from the endophytic Bacillus subtilis strain EDR4

An antifungal protein E2, from the culture filtrate of the endophytic Bacillus subtilis strain EDR4 of wheat with a high activity against numerous fungal species in vitro and take-all in wheat caused by Gaeumannomyces graminis var. tritici in vivo, was purified by (NH₄)₂SO₄ precipitation, hydrophobic-interaction chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis (PAGE). The molecular mass of the protein was about 377.0 kDa determined by gel permeation chromatography (GPC) using a Superdex 200 10/300 GL pre-packed column and the pI value of the protein detected by isoelectric focusing PAGE was 6.59. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the antifungal protein showed a band with a molecular mass of 39.1 kDa, which suggest that the native protein consists of multi-subunits. The amino acid sequences of three peptides from the antifungal protein were obtained by using a nano-ESI-MS/MS (Q-TOF2) System. The protein isolated may be regarded as a new protein according to amino acid sequences of three peptides. The purified protein exhibited inhibitory activity on mycelium growth of e.g. Fusarium graminearum, Macrophoma kuwatsukai, Rhizoctonia cerealis, Fusarium oxysporum f.sp. vasinfectum, Botrytis cinerea and G. graminis var. tritici (Ggt). Scanning electron microscopy showed that hyphae of Ggt treated with the antifungal protein were severely deformed. The antifungal protein E2 exhibited ribonuclease and hemagglutinating activities as well as a trifle protease activity. However, no β-1,3-glucanase, β-1,4-glucanase, chitinase or protease inhibitory activities were detected.