Molecular cloning of an endo-pectate lyase gene from Erwinia carotovora subsp. atroseptica

We report the construction of a clone library of the Erwinia carotovora subsp. atroseptica genome and the isolation of a gene for endo-pectate lyase. The library, inserted into the PstI site of plasmid pBR322, contains approximately 1700 clones. Five of these produce pectolytic enzymes as detected by a plate screening assay. Using a cloned endo-pectate lyase gene from the related bacterium, E. carotovora subsp. carotovora, as a probe, we found that one pectolytic E. carotovora subsp. atroseptica clone had strong homology to the probe. We characterized that clone by restriction endonuclease mapping and studied its pectolytic protein product. The purified enzyme is an endo-pectate lyase with a cofactor preference for Co²⁺. The molecular weight of the protein is 31 000 and it has an isoelectric focusing point of 9·2, corresponding to an endo-pectate lyase produced by E. carotovora subsp. atroseptica, but not to the protein product of the E. carotovora subsp. carotovora probe DNA, which has a pI of 9·5. Restriction endonuclease site polymorphisms in the two cloned endo-pectate lyase genes suggest substantial sequence divergence between these two loci.     

Fusarium moniliforme secretes four endopolygalacturonases derived from a single gene product

Extracellular endopolygalacturonase, purified from the pathogenic fungus Fusarium moniliforme, consists of four molecular forms (38, 41·5, 45, and 48·5 kDa, respectively. Three forms (38, 41·5, and 45 kDa) were purified to homogencity by FPLC on a Mono S column followed by electroelution after SDS-PAGE. The N-terminal amino acid sequences of each of the three forms, and of a mixture containing all four forms were shown to be identical to that predicted from the nucleotide sequence of the endopolygalacturonase gene previously cloned from F. moniliforme. Enzymatic deglycosylation experiments revealed the presence of N-linked, high mannose oligosaccharide side-chains on all four forms of endo polygalacturonase. Hydrogen fluoride catalysed chemical deglycosylation of the polygalacturonase mixture yielded a single polypeptide with an apparent molecular mass of 36·2 kDa. Southern blot analysis, carried out at high stringency with an endopolygalacturonase-specific probe on genomic DNA digested with three different restriction enzymes, showed a single hybridizing restriction fragment in all three digests. A single 2·0 Mb chromosome hybridized with the endo polygalacturonase-specific probe, as shown by Southern blot analysis of F. moniliforme chromosomes separated by CHEF electrophoresis. Northern blot analysis revealed only one mRNA species 1350 nt encoding endo polygalacturonase. These data indicate that a single gene encodes the endopolygalacturonases of F. moniliforme.     

Grouping of Colletotrichum Species in Japan Based on rDNA Sequences

Internal transcribed spacers (ITS) of the ribosomal RNA gene (rDNA) were sequenced for 236 isolates covering 25 Colletotrichum species collected in Japan. The Japanese isolates could be grouped into 20 ribosomal groups (RGs) based on the sequences of ITS1, correlating the species identified by the morphology. Colletotrichum gloeosporioides sensu lato separated into three RGs that were morphologically different. Colletotrichum destructivum, C. linicola and C. higginsianum were possibly conspecific. Colletotrichum dematium sensu lato including C. capsici and other species that produce falcate conidia except for graminicolous ones were separated into three RGs that were difficult to distinguish morphologically. In the phylogenetic study using ITS2 and the 28S rDNA domain 2 region, topologies compiled by neighbor-joining and maximum-parsimony methods were almost the same, reflecting the conidial morphology. The phylogenetic group 1 (PG1) produced conidia with acute ends, e.g., C. acutatum, C. destructivum and C. graminicola; PG2 produced those with obtuse ends, e.g., C. gloeosporioides, and C. orbiculare. Colletotrichum theae-sinensis, which produced the smallest conidia, was grouped as PG3, far from other species, indicating it should not belong to Colletotrichum. Grouping and phylogenetic analysis using ribosomal DNA was an effective tool to classify and identify Colletotrichum species without using morphology. Received 15 July 2002/ Accepted in revised form 12 November 2002     

Polygalacturonase S31PG1 from <Emphasis Type="Italic">Geotrichum candidum</Emphasis> citrus race S31 expressed in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> versus S31PG2 regarding soft rot on lemon fruit

Gene S31pg1, which encodes a polygalacturonase (PG), was previously isolated from citrus race S31 of Geotrichum candidum, the causal agent of citrus sour rot. We have now isolated and sequenced an additional PG gene, S31pg2, with 95% identity to S31pg1 in the mature proteins. To evaluate the contribution of the two PG genes in the development of citrus sour rot, each gene was expressed in the fission yeast Schizosaccharomyces pombe. Both genes conferred PG activity to the yeast. Crude enzyme solutions containing S31PG1 severely degraded the albedo tissue of lemon peel, but those containing S31PG2 did not. Concentrated crude S31PG1 solutions also caused soft rot on lemon fruit, indicating that not S31PG2 but S31PG1 is an important pathogenicity factor in citrus sour rot. Next, the protopectinase (PP) activity of each PG was measured. Although S31PG1 and S31PG2 are highly homologous, S31PG1 had high PP activity, whereas S31PG2 had much lower activity. PG from G. candidum noncitrus race S63 (nonpathogenic to citrus fruits) was also assayed but did not have any PP activity at all. These results suggest that the different PP activities of the PGs are a key to the pathogenicity of G. candidum to lemon fruit.     

Pathogenic and genetic variation among isolates of Corynespora cassiicola in Japan

In order to develop a method for discrimination of Corynespora cassiicola isolates pathogenic to sweet pepper among Japanese isolates, this study analysed pathogenic variations of 64 Japanese isolates of C. cassiicola on perilla, cucumber, tomato, aubergine and sweet pepper, and their multigene phylogeny. Japanese isolates were divided into seven pathogenicity groups (PG1–PG7). The virulence of isolates in PG1–PG5 was restricted to perilla, cucumber, tomato, aubergine and sweet pepper, respectively. Isolates in PG6 were virulent to sweet pepper, tomato and aubergine. Isolates in PG7 were avirulent to all tested plants. Multigene phylogenetic analysis of the isolates based on β‐tubulin, translation elongation factor 1‐α, calmodulin and actin genes showed three divergent clusters, MP‐A, MP‐B and MP‐C. These clusters included all isolates in PG1, PG2, PG8 and PG9 (MP‐A), PG3 and PG5 (MP‐B) and PG4 and PG6 (MP‐C). Isolates in PG7 were distributed amongst all clusters. Furthermore, random amplified polymorphic DNA (RAPD) analysis using universal primers, Q17 (5′‐GAAGCCCTTG‐3′) and Q13 (5′‐GGAGTGGACA‐3′), facilitated discrimination of isolates virulent on sweet pepper amongst isolates in MP‐B and MP‐C, respectively. Together, a combination of the multigene analysis and the RAPD technique allowed the discrimination of the isolates virulent to sweet pepper.     

Fusarium solani endo-polygalacturonase from decayed muskmelon fruit: purification and characterization

Fusarium solani is one of the more important fungal pathogens involved in pre- and post-harvest decay of muskmelon fruit. Production of polygalacturonase (PG), by F. solani was studied in vitro and in vivo . The fungus produced at least 14 PG isozymes with pIs of 4.5 to 9.5 in shake culture using pectin as the sole carbon source. When glucose and pectin were used in combination as the carbon source, total PG activity decreased substantially as compared to pectin alone, suggesting that glucose may suppress PG production in vitro . Only one PG isozyme, designated as PG1, was detected in extracts from infected fruit tissue. PG1 from decayed fruit was purified to homogeneity by protein extraction, ultrafiltration, gel filtration chromatography, and cation exchange chromatography. The molecular weight of PG1 was estimated at 38 kDa based on SDS-PAGE with a pI of 9.5 according to IEF-PAGE. PG1 exhibited only endo-PG activity based on viscosity reduction and thin layer chromatography analysis of products released by enzymatic action. The optimum pH for PG1 activity was 6. TheK_m and V_maxof PG1 using polygalacturonic acid as the substrate were 1.34 mg ml^-1and 0.30 unit μg protein^-1, respectively. PG1 effectively macerated fruit tissue which suggests that it may play an important role in decay of muskmelon fruit caused by F. solani .     

Isolation and Characterization of a New Virulence Gene (abvA) of Agrobacterium tumefaciens

A mutant (M-1) was isolated by transposon (Tn5) insertion mutagenesis of Agrobacterium tumefaciens (strain A-208, C58 chromosome, nopaline type T37 pTi, virulent). The M-1 mutant exhibited a complete avirulent phenotype on Kalanchoe daigremontiana leaf and Kalanchoe pinnata stem but a very attenuated virulent phenotype on root of Daucus carota. The mutant had one insertion of Tn5 in pTi. A wild-type target segment (2.3 kb) that included the site of Tn5 insertion in M-1 mutant was cloned. Introducing the 2.3 kb segment into M-1 complemented completely the avirulent phenotype, producing galls as big as strain A-208. The 2.3 kb segment was sequenced, identifying three open reading frames, ORF 1 (354 bp), ORF 2 (261 bp) and ORF 3 (801 bp) in the segment. A Tn5 was inserted between the third and fourth nucleotide of ORF 1 in M-1. The ORF 1 had no homology to any reported genes and thus was named the abvA gene. The ORF 3 had the high homology (identities 44%, positive 68%) to the gene of the sarcosine oxidase β subunit (accession no. sp/P40875). Introduction of the DNA segment (743 bp) containing the abvA gene and its promoter region into M-1 partially complemented the avirulent phenotype of the mutant, producing galls smaller than strain A-208. The abvA gene was distributed not only on nopaline-type pTi (T37) but also on octopine-type pTi (A6NC) and chromosome (C58) of A. tumefaciens. M-1, being avirulent on K. daigremontiana and K. pinnata, had a Tn5 insertion only in the abvA gene on pTi but not in the abvA gene on the chromosome, implying that the abvA gene on the chromosome in strain A-208 is not functional. A binary vector, pIG121-Hm, containing the β -glucuronidase (GUS) gene with an intron was introduced into M-1, which was then applied to leaves of K. daigremontiana to assay GUS activity for monitoring T-DNA transfer to the host nucleus. High GUS activity comparable to that in strain A-208 was detected in M-1 in spite of its inability to induce galls, suggesting that M-1 can transfer T-DNA into the host nucleus, but cannot integrate it into the chromosome. Received 25 October 2000/ Accepted in revised form 28 December 2000     

Establishment of heterologous expression of polygalacturonase S63PG1 from nonpathogenic isolate S63 of <Emphasis Type="Italic">Geotrichum candidum</Emphasis>

Previously, we established an expression system of polygalacturonase (PG) S31PG1 and S31PG2 from Geotrichum candidum pathogenic isolate S31 using the fission yeast Shizosaccharomyces pombe and clarified the importance of S31PG1 in the pathogenicity of G. candidum S31 on lemon fruit. In the present study, we established an expression system for S63PG1 from the nonpathogenic isolate S63. When S63PG1 was expressed, only PG activity was detected, whereas both PG and protopectinase (PP) were active when S31PG1 was expressed. Furthermore, S63PG1 had no ability to cause soft rot, while S31PG1 did. These results indicate that the PP activity of PG is a key to the pathogenicity of the fungus.     

Functional Analysis of an ATP-Binding Cassette Transporter Gene in Botrytis cinerea by Gene Disruption

The BMR1 gene encoding an ABC transporter was cloned from Botrytis cinerea. To examine the function of BMR1 in B. cinerea, we isolated BMR1-deficient mutants after gene disruption. Disruption vector pBcDF4 was constructed by replacing the BMR1-coding region with a hygromycin B phosphotransferase gene (hph) cassette. The BMR1 disruptants had an increased sensitivity to polyoxin and iprobenfos. Polyoxin and iprobenfos, structurally unrelated compounds, may therefore be substrates of BMR1. Received 18 December 2000/ Accepted in revised form 18 April 2001     

Vegetative compatibility groups in Colletotrichum coccodes from Turkey and their aggressiveness to potato

Black dot, caused by Colletotrichum coccodes, is a common disease of potato in Turkey, affecting tuber quality and yield. The objectives of the current study were to characterize vegetative compatibility groups (VCGs) of C. coccodes isolates from three regions in Turkey, and to assess the correlation between VCGs and aggressiveness of isolates on potato. A total of 147 C. coccodes isolates were recovered from plants showing typical black dot symptoms on stolons, roots and stems. The frequency of nitrate non‐utilizing (nit) nit1/nit3 and NitM phenotypes were 79% and 21%, respectively. Complementation between nit mutants of the isolates and eight European/Israeli EU/I‐VCG tester isolates was used to characterize the VCGs. Amongst the tested isolates, 33.3% were assigned to EU/I‐VCG6, 21.8% to EU/I‐VCG8, 15.7% to EU/I‐VCG4. EU/I‐VCG1, EU/I‐VCG3, EU/I‐VCG5 and EU/I‐VCG7 were classified at 1.4%, 3.4%, 4.8% and 5.4%, frequency, respectively. No isolate was assigned to EU/I‐VCG2 group, while 21 isolates (14.3%) were not assigned to any of the EU/I‐VCGs. The pathogenicity tests indicated significant differences in aggressiveness of the isolates with respect to sclerotia density on potato tissues. The highest densities of sclerotia on roots and crown were obtained with EU/I‐VCG6 isolates and the lowest with EU/I‐VCG1, EU/I‐VCG3 and EU/I‐VCG5 isolates. The results demonstrate that there is significant VCG diversity among C. coccodes isolates from potato plants in Turkey.     

Field emergence of Lamium amplexicaule L. and L. purpureum L. in relation to the annual seed dormancy cycle

Seedling emergence of Lamium amplexicaule L. and L. purpureum L. was monitored in field plots tilled at various times during the growing season, and the number of viable seeds in the soil was determined. In plots tilled in early spring, only seeds of L. amplexicaule, germinated, but seeds of both species germinated in the same plots in autumn without further disturbance. Additional seeds of L. amplexicaule, but not of L. purpureum, germinated the following spring. In plots tilled in late spring and summer, seeds of L. amplexicaule germinated in autumn and the following spring, whereas seeds of L. purpureum germinated only in autumn. The number of viable seeds in the top 13 mm layer of soil ranged from 189 to 1216 m^?2 for L. amplexicaule and from 131 to 854 for L. purpureum. These field results support those obtained in previous glasshouse-laboratory physiological studies on the annual dormancy cycle in the two Lamium species. Levée au champ de Lamium amplexicaule L. et L. purpureum L. par rapport au cycle annuel de la dormance des graines La levée de jeunes plants de Lamium amplexicaule L. et L. purpureum L. a été observée sur des parcelles cultivées à différentes époques de la saison de végétation et le nombre de graines viables au sol a été déterminé. Sur les parcelles cultivées en début de printemps, seules les graines de L. amplexicaule ont germé, mais des graines des deux espèces ont germé en automne sur les mêmes parcelles, sans cultivation ultérieure. Au printemps suivant, une nouvelle germination a été constatée chez L. amplexicaule mais pas chez L. purpureum. Sur les parcelles cultivées en fin de printemps et en été, des graines de L. amplexicaule ont germé en automne et aussi au printemps suivant, tandis que les graines de L. purpureum n'ont germé qu'en automne. Le nombre de graines viables dans les premiers 13 mm de la surface du sol allait de 189 à 1216 m^?2 pour L. amplexicaule et de 131 à 854 pour L. purpureum. Ces résultats sur le terrain confirment ceux obtenus dans des études physiologiques sur le cycle annuel de dormance chez les deux espèces de Lamium, menées préalablement en serre et au laboratoire. Ueber dus Auflaufen von Lamium amplexicaule L. und L. purpureum L. in Bezug auf den Jährlichen Zyklus der Samenruhe unter Feldbedingungen In Parzellen, die zu verschiedenen Zeitpunkten während der Vegetationsperiode einer Boden-bearbeitung unterworfen worden waren, wurde sowohl das Auflaufen der Keimlinge von Lamium amplexicaule L. und L. purpureum L. registriert, als auch die Anzahl lebenfähiger Samen festgestellt. Nach Bodenbearbeitung im frühen Frühling keimten nur Samen von L. amplexicaule; im Herbst keimten in denselben Parzellen jedoch Samen beider Arten, sofern der Boden nicht mehr bewegt worden war. Weitere Samen von L. amplexicaule, nicht aber von L. purpureum, keimten im folgenden Frühling. In Parzellen, die im späten Frühling und im Sommer bearbeitet worden waren, keimte L. amplexicaule im Herbst und im folgenden Frühling. Während L. purpureum nur im Herbst keimte. Die Anzahl lebensfähiger Samen m^?2 in den obersten 13 mm des Bodens bewegte sich zwischen 189 und 1216 für L. amplexicaule und zwischen 131 und 854 für L. purpureum. Diese Feldresultate bestätigen Ergebnisse von Gewüchshausstudien über den jährlichen Zyklus der Samenruhe der beiden Lamium-Arten.     

Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea

Previously, we cloned a putative osmosensing histidine kinase gene (BcOS1) and revealed that a single amino acid substitution, isoleucine to serine at codon 365, conferred dicarboximide resistance in field isolates of Botrytis cinerea. This point mutation (type I) occurred within the restriction enzyme TaqI site of the wild-type BcOS1 gene. Thus, a procedure was developed for detecting the type I mutation of the BcOS1 gene using a polymerase chain reaction (PCR) in combination with restriction fragment-length polymorphism (RFLP). Diagnosis by PCR-RFLP was conducted on the 105 isolates isolated from 26 fields in Japan. All dicarboximide-sensitive isolates (49 isolates) had the wild-type BcOS1 gene, and the 43 isolates with the type I mutation were resistant to dicarboximides without exception. These data indicate that dicarboximide-resistant isolates with type I mutation are widespread throughout Japan. However, other types of dicarboximide resistance were detected among isolates from Osaka; among the 24 resistant isolates from Osaka, 12 had the BcOS1 gene without the type I mutation. BcOS1 gene sequencing of these resistant isolates classified them into two groups, type II and type III. The type II isolates have three amino acid substitutions within BcOS1p (³⁶⁸Val to Phe, ³⁶⁹Gln to His, and ⁴⁴⁷Thr to Ser). The type III isolates have two amino acid substitutions within BcOS1p (³⁶⁹Gln to Pro and ³⁷³Asn to Ser). These amino acid changes are located on the amino acid repeat domain in BcOS1p. The three types of resistant isolates were all moderately resistant to dicarboximides without significant osmotic sensitivity, and their pathogenicity on cucumber leaves was also very similar to that of the wild-type isolate.     

Genetic variability and population structure of <Emphasis Type="Italic">Grapevine virus A</Emphasis> coat protein gene from naturally infected Italian vines

Grapevine virus A (GVA) is considered one of the viruses associated with rugose wood (RW), one of the most economically important diseases of grapevine. Thirty-seven GVA isolates collected from grapevine cultivars from Marche (central-eastern Italy), Apulia and Campania (southern Italy), were subjected to molecular characterization. The genetic and population diversity was studied in the coat protein (CP) gene by RT-PCR-RFLP analysis with three restriction enzymes (MseI, AluI, and AciI), and nucleotide sequencing. A new primer pair (CP1F/R) allowing amplification of the whole CP gene (621 bp) was developed. RFLP with AciI yielded the highest number of variants in GVA isolates, showing seven different ‘simple’ profiles (A, B, C, D, E, F, and G). ‘Complex’ profiles were also found, and the most common variant combination was A + B in 39% of isolates. The analysis of GVA sequences confirmed the presence of plants infected with more than one GVA variant and suggested that RT-PCR-RFLP is suitable for evaluating population diversity of GVA enabling a screening of different haplotypes. The distribution of RFLP profiles and the phylogenetic analysis were not correlated with the location of infected plants, showing the presence of a GVA population with genetic diversity in the average with those of RNA viruses.     

A rapid qualitative molecular method for the identification of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> and <Emphasis Type="Italic">C. gloeosporioides</Emphasis>

Identification of the causal agent for anthracnose caused by C. acutatum and C. gloeosporioides based on morphological and cultural criteria is problematic as both are morphologically and genetically diverse. To evaluate a qualitative molecular method to readily distinguish between these two species, Restriction fragment length polymorphisms (RFLP) of a 1-kb intron of the glutamine synthetase (GS) gene was evaluated utilizing representative isolates from a world-wide collection. Unique band patterns of the 1-kb GS intron were obtained for C. acutatum (two fragments with 600 and 350 bp) and C. gloeosporioides (four fragments with 238–340, 252–254, 204, and 108–116 bp) based on PstI enzyme digestion of the amplified PCR product. These data were also confirmed by PstI digestion of the intron DNA sequences using BioEdit software. The identification based on RFLPs of the 1-kb GS intron was consistent with the identification based on previously evaluated species-specific primers (CaInt2 and CgInt). In addition, both species can be differentiated by multiplex PCR. CaInt2, CgInt and ITS4 in one PCR will distinguish between C. acutatum and C. gloeosporioides by differences in PCR product fragment size: 490 bp and 470 bp, respectively. Also, a rapid DNA extraction method was developed, which reduced the time for DNA extraction from two hours to five minutes. In summary, RFLP of the 1-kb GS intron is a reliable technique for identification and differentiation between both species, does not require a sequencing step, and may be useful to diagnostic clinics in helping to make disease management recommendations.     

斜纹夜蛾的生物控制研究

应用以作用因子组配的生命表技术及其相应的分析方法,开展了几项生物防治措施单项和联合使用防治斜纹夜蛾(Spodoptera litura Fabricius.)的试验。结果表明,施用斜纹夜蛾核型多角体病毒(Spodoptera litura nuclear polyhedrosis virus)(浓度为3.1×10~7PIBs/ml,即每g虫尸兑水800倍液)和散放叉角厉蝽(Cantheconidea furcellata Wolff)(1.8头/m~2)均能控制斜纹夜蛾种群的增长。采用两项生防措施后,种群趋势指数值分别降至对照的0.0259倍和0.0532倍,当联合使用这两项生防措施后,种群趋势指数值则降至对照的0.0109倍。浇施斯氏线虫需高剂量(40～80万条/m~2)才能控制斜纹夜蛾种群数量的增长,高剂量可使种群趋势指数值下降至对照的0.758～0.094倍。若以短期内对害虫的杀伤作用作为评价依据,散放叉角厉蝽(1.8头若虫/m~2)的效果最好,进入暴食期的虫口密度下降到对照的0.0917倍.     

Class I hydrophobin BcHpb1 is important for adhesion but not for later infection of <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

Hydrophobins are small secreted proteins unique to filamentous fungi. In this study, we cloned and characterized the class I hydrophobin gene BcHpb1 in the necrotrophic pathogen Botrytis cinerea. The BcHpb1 protein consisted of 117 amino acids. Similar to class I hydrophobins from other fungi, BcHpb1 contains eight conserved cysteine residues. The hydropathy plot of the BcHpb1 amino acid sequence was characteristic of a class I hydrophobin. These results indicated that the BcHpb1 gene encodes a class I hydrophobin. Vegetative growth of ΔBcHpb1 strains, null mutants of BcHpb1, was similar to that of the wild-type strain as were the conidiophores, conidia, appressoria and virulence on host plants. However, adherence of ΔBcHpb1 strains to hydrophobic surfaces was greatly reduced, implying that BcHpb1 is important for the hydrophobicity of conidia and that BcHpb1 may be required to adhere to plant surfaces under certain environmental conditions.     

Hydrolysis of the pyrethroid insecticide fenpropathrin in aqueous media

The hydrolysis of [¹⁴C] fenpropathrin ( I ) [(RS)-α-cyano-3-phenoxybenzyl 2,2,3,3-tetramethylcyclopropanecarboxylate] was studied in buffer solutions at pH 1.9–10.4, and in natural river and sea water at 25, 40, 55 and 65°C under laboratory conditions. The hydrolysis of I proceeded predominantly through neutral (pH independent) and base-catalysed processes in the regions below pH 3.9 and above pH 7.0, respectively, whereas both reactions occurred between pH 3.9 and 7.0. The rates of hydrolysis of I in buffer solutions were similar to those in one sample of river and one sample of sea water. If this obtains generally, it may be expected that the half-life of I in natural waters, normally within the range pH 5–9, will range from 1.54 to 1080 days at 40°C, 11.3 to 8520 days at 25°C and, by extrapolation of the data obtained in buffer solutions, 106 to 83 000 days at 10°C. The rate constants for hydrolysis of I in aqueous media can be expressed by: Where log k_N = 9.60–(5.56 × 10³ T^?1) and log k_B = 7.32–(2.56 × 10³ T^?1). The calculated rate constants were in good accord with the observed values in buffer solutions. Cleavage of the ester linkage was more rapid than hydration of the cyano group at any pH and temperature tested.     

Zur Kenntnis der Blattminen-MotteCameraria ohridella Desch. & Dim. (Lep., Lithocolletidae) anAesculus hippocastanum L. in der Tschenchischen Republik

Cameraria ohridella was recorded first in 1985 in Macedonia. It gradually expanded to north and west and at present it is a serious pest of Horse ChestnutAesculus hippocastanum in the Czech Republic, having been established at about 80 localities. There are 4–5 overlapping generations with sizes of the larvae of 0.4–4.0 mm. The larva develops inside the leaf tissue in the upper parenchym layer of the leaflet and causes a mine, the size of which is broadened with growing larva. First adults start to fly at the end of April. After mating the females lay single eggs on the leaves. Larval development lasts 25–30 days followed by the prepupa I and II. The latter spins a cocoon in which the pupa of the last generation hibernates. The development from L4 to the prepupa lasts for 3–6 days at 22°C. During the summer it is possible to find all larval and prepupal stages in attacked leaves. FourAesculus species:A. parviflora Walk,A. carnea Haey.,A. glabra Walk andA. indica J. Hobb. were found to be resistent toC. obridella. A. lutea H. J. was liftle andA. pavia L. was heavily attacked. The parasitization ofC. o. larvae was very low. Only 2 parasites were found in 1500 mines in the first and second generation ofC. o., and 40 parasites in 1000 mines of the fourth and fifth generation. The highest mortality takes place in moths, eggs and young larvae. It was found in all 4 generation that there were i. m. 50 eggs/leaflet from which i. m. 3 hibernating pupae resulted. Supposing 2 moths (1♀, 1♂)/leaflet emerged in spring which produced 50 eggs (75 eggs/♀—33% moth mortality), the density of eggs in the first generation after hibernating being the same as in the last (fourth) generation before hibernating. As to the whole populations density in this case we can calculate as following: 3 pupae/leaflet on the tree=3000 pupae/leaflets pro m² on the soil=2000 emerging moths pro m²=50,000 moths (25,000 ♀♀) pro tree (namely 25 m² projecting area of the tree crown×2000 moths).