A new biopesticide from a local <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">tenebrionis</Emphasis> (Xd3) against alder leaf beetle (Coleoptera: Chrysomelidae)

Use of chemical pesticides in agriculture harms humans, non-target organisms and environments, and causes increase resistance against chemicals. In order to develop an effective bio-pesticide against coleopterans, particularly against Agelastica alni (Coleoptera: Chrysomelidae) which is one of the serious pests of alder leaf and hazelnut, we tested the insecticidal effect of 21 Bacillus isolates against the larvae and adults of the pest. Bacillus thuringiensis var. tenebrionis-Xd3 (Btt-Xd3) showed the highest insecticidal effect based on screening tests. For toxin protein production and high sporulation of Xd3, the most suitable medium, pH and temperature conditions were determined as nutrient broth medium enriched with salts, pH 7 and 30?°C, respectively. Sporulated Btt-Xd3 in nutrient broth medium enriched with salts transferred to fermentation medium containing soybean flour, glucose and salts. After fermentation, the mixture was dried in a spray dryer, and spore count of the powder product was determined as 1.6?×?10¹⁰ c.f.u. g^?1. Moisture content, suspensibility and wettability of the formulation were determined as 8.3, 86% and 21 s, respectively. Lethal concentrations (LC₅₀) of formulated Btt-Xd3 were determined as 0.15?×?10⁵ c.f.u. ml^?1 for larvae at laboratory conditions. LC₅₀ values were also determined as 0.45?×?10⁶ c.f.u. ml^?1 at the field condition on larval stage. Our results showed that a new bio-pesticide developed from B. thuringiensis tenebrionis (Xd3) (Btt-Xd3) may be valuable as a biological control agent for coleopteran pests.     

Kinetics of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">israelensis</Emphasis> growth on high glucose concentrations

The kinetic and general growth features of Bacillus thuringiensis var. israelensis were evaluated. Initial glucose concentration (S ₀) in fermentation media varied from 10 to 152 g/l. The results afforded to characterize four morphologically and physiologically well-defined culture phases, independent of S ₀ values: Phase I, vegetative growth; Phase II, transition to sporulation; Phase III, sporulation; and Phase IV, spores maturation and cell lysis. Important process parameters were also determined. The maximum specific growth rates (μ _X,m) were not affected with S ₀ up to 75 g/l (1.0–1.1 per hour), but higher glucose concentrations resulted in growth inhibition by substrate, revealed by a reduction in μ _X,m values. These higher S ₀ values led to longer Phases III and IV and delayed sporulation. Similar biomass concentrations (X _m = 15.2–15.9 g/l) were achieved with S ₀ over 30.8 g/l, with increasing residual substrate, suggesting a limitation in some other nutrients and the use of glucose to form other metabolites. In this case, with S ₀ from 30.8 to 152 g/l, cell yield (Y _X/S ) decreased from 0.58 to 0.41 g/g. On the other hand, with S ₀ = 10 g/l growth was limited by substrate, and Y _X/S has shown its maximum value (0.83 g/g).     

Biology of <Emphasis Type="Italic">Dermacentor silvarum</Emphasis> (Acari: Ixodidae) under laboratory conditions

The minimum life cycle of Dermacentor silvarum Olenev had a mean duration of 87.5 days (range 74–102 days) under laboratory conditions [(27±1 °C), 70% RH, 6 L: 18 D]. The mean time in (days) for the different stages of its cycle was as follows: incubation period of eggs was 15.3 days; prefeeding, feeding and premoulting periods of larvae and nymphs averaged 5.5, 4.0 and 7.3 days, and 5.2, 5.0 and 14.6 days, respectively; prefeeding, feeding, preoviposition and oviposition periods of female adults lasted for 7.8, 4.5, 4.3 and 14.0 days, respectively. There existed a highly significant correlation between engorged body weight of females and egg masses laid (r = 0.9877, p<0.001). The reproductive efficiency index (REI) and reproductive fitness index (RFI) in females were 11.09 and 9.58, respectively. No relationship between nymphal engorged body weight and resultant sexes was observed. Delayed feeding and non-oviposition (in June and July) existed in females, and low temperature (−10 °C) treatment for 45 days could terminate oviposition diapause. However, the egg masses laid by post-diapause females were significantly smaller than those laid by females engorged in March, April and May.     

Biological aspects of <Emphasis Type="Italic">Amblyomma brasiliense</Emphasis> (Acari: Ixodidae) under laboratory conditions

Although Amblyomma brasiliense Aragão 1908 has been reported as one of the most aggressive ticks to humans in Brazil, information about the biology of this tick species is virtually inexistent. This work reports data on the life cycle of A. brasiliense fed on rabbits and pigs and maintained in an incubator at 20°C, 90% RH and 12 h of light for off-host development. Tick yield of adult females fed on pigs and rabbits was 81.2% and 58.3%, respectively. Females fed on pigs had mean engorgement weight of 862.3 mg and egg mass of 208 mg, while females fed on rabbits had mean engorgement weight of 606.1 mg and egg mass of 160 mg; these values did not differ statistically between host species. Feeding period of female ticks fed on pigs (10 days) was significantly shorter than that on rabbits (17 days). Mean preoviposition period was slightly longer (35.9 days) for ticks fed on pigs than on rabbits (30 days). The minimum incubation period of eggs of ticks from both host species was similar and over 100 days. Egg production efficiency was low for females fed on both hosts (less than 30% and 20% for ticks from pigs and rabbits, respectively). More than 55% of larvae and 79% of nymphs fed on rabbits, set free inside the feeding chambers, engorged successfully. These ticks attained an engorgement weight of 1.3 and 18.2 mg, respectively, and fed for approximately 5 days. The minimum pre-molt period was 30 days for engorged larvae and over 44 days for nymphs. Molting success was low, less than 50% in the case of larvae and less than 20% for nymphs. Further studies are required to better determine the off-host requirements of this tick species.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Novel roles of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> to control plant diseases

Bacillus thuringiensis is well known as an effective bio-insecticidal bacterium. However, the roles of B. thuringiensis to control plant diseases are not paid great attention to. In recent years, many new functions in protecting plants from pathogen infection have been discovered. For example, acyl homoserine lactone lactonase produced by B. thuringiensis can open the lactone ring of N-acyl homoserine lactone, a signal molecule in the bacterial quorum-sensing system. This in turn, significantly silences bacterial virulence. This finding resulted in the development of a new strategy against plant bacterial diseases by quenching bacterial quorum sensing. Another new discovery about B. thuringiensis function is zwittermicin A, a linear aminopolyol antibiotic with high activity against the Oomycetes and their relatives, as well as some gram-negative bacteria. This paper summarized the relative progresses of B. thuringiensis in plant disease control and its favorable application prospects.     

Chitinase production by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>: Their potential in antifungal biocontrol

Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na⁺, Mg²⁺, Cu²⁺, and Ca²⁺ caused enhancement of enzyme activities whereas they were markedly inhibited by Zn²⁺, Hg²⁺, and Ag⁺. In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.     

Hyaloscyphaceae in Japan (7): <Emphasis Type="Italic">Hyaloscypha albohyalina</Emphasis> var. <Emphasis Type="Italic">monodictys </Emphasis>var. nov.

 Hyaloscypha albohyalina var. monodictys, a new variety in the family Hyaloscyphaceae, Helotiales with Monodictys anamorph is described and illustrated. Received: June 26, 2002 / Accepted: July 27, 2002 Present address: Strategic Product Portfolio Department, Sankyo Co., Ltd., 3-5-1 Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8426, Japan Tel. +81-3-5255-7040 (Ext. 2528); Fax +81-3-5255-7086 e-mail: hosoya@hq.sankyo.co.jp Correspondence to:T. Hosoya     

Screening of <Emphasis Type="Italic">cry2</Emphasis> genes in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> isolates from Argentina

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identification of cry2 genes from Bacillus thuringiensis (Bt) was established. Strains from different sources of Argentina were analyzed to study the distribution of cry2 genes. The results showed that cry2Aa/cry2Ab profile was the most abundant irrespective of source and represented 56 of 59 Bt isolates (94.9%). Three different cry2 profiles were found in this collection, one of which was novel.     

Chitosanase activity in<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The ability to produce extracellular chitosanase (EC 3.2.1.132) was found by plate assays in 18 (23%) out of 77 crystalliferous strains of Bacillus thuringiensis. The best chitosanase producer was selected after the growth chosen in a liquid medium with colloidal chitosan as carbon source. Enzyme production was optimized (a 4-d incubation at 32 degrees C with shaking in a medium of pH 6.5 with 4% colloidal chitosan) and the enzyme was partially characterized. This is the first report on the chitosanase of B. thuringiensis.     

Dissection of <Emphasis Type="Italic">cry</Emphasis> Gene Profiles of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Isolates in Taiwan

On the basis of the newly revised nomenclature system of cry genes, the PCR amplification method has been adopted to resolve the cry gene combinations of 294 Bacillus thuringiensis isolates from five selected areas of Taiwan. Our results indicate that cry1 (especially cry1A + 1B + 1F) and cry2 were the most abundant cry genes in Taiwan. In contrast, cry3 and cry6 genes were detected only on Yang Ming Mountain, while the cry13 gene was found only on Snow Mountain. In addition, some distinctive combinations of cry genes were detected in distinct areas of Taiwan, such as cry1C, cry1D, cry1C + 1D, cry4, cry1 + 4, cry1 + 11, cry4 + 11, and cry1 + 4 + 11 in the Taipei area; cry1A + 1C + 1F in the Taichung area; cry1E and cry1A + 1B + 1I on Yang Ming Mountain; cry1 + 13, cry1 + 2 + 11, and cry1 + 2 + 13 on Snow Mountain; and cry1 + 5 and cry1 + 2 + 5 on Jade Mountain. These data clearly indicate that the distribution of cry gene combinations of B. thuringiensis isolates seems to be geographically related.     

Biological characterization of two <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains toxic against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Two Bacillus thuringiensis strains isolated from diseased Spodoptera frugiperda larvae collected in the northwest of Argentina were molecularly and phenotypically characterized. Insecticidal activity against Spodoptera frugiperda larvae was also determined. Both strains were highly toxic against first instar larvae. One strain (Bacillus thuringiensis LSM) was found to be even more toxic than the reference strain Bacillus thuringiensis var. kurstaki 4D1. This strong biological effect was represented by both a higher mortality which reached 90%, and a shorter LT₅₀. Molecular characterization showed that Bacillus thuringiensis LSM carried a cry gene profile identical to that of Bacillus thuringiensis var. kurstaki 4D1. Evaluation of length polymorphism of the intergenic transcribed spacers between the 16S and 23S rDNA genes revealed an identical pattern between native strains and Bacillus thuringiensis var. kurstaki 4D1. In contrast, phenotypic characterization allowed differentiation among the isolates by means of their extracellular esterase profiles. Lytic activity that would contribute to Bacillus thuringiensis effectiveness was also studied in both strains. Analyses like those presented in the current study are essential to identify the most toxic strains and to allow the exploitation of local biodiversity for its application in biological control programmes.     

Synergistic interaction between sublethal doses of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Campoletis chlorideae</Emphasis> in managing <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) is an important solitary larval endoparasitoid of the tomato fruit borer Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in India. The interaction between Bacillus thuringiensis subspecies kurstaki (Btk) HD-1 and C. chlorideae was studied under laboratory condition to explore their compatibility in managing H. armigera. The results had indicated that the growth and development of H. armigera was affected in a dose-dependent manner upon feeding on sublethal doses of Btk HD-1-treated diets. There were no larval survivors in lethal doses of Btk HD-1 (LC₇₀ and LC₉₀). The growth and survival of the parasitoid were normal when the host larvae were fed with sublethal doses or subjected to short time exposure to lethal doses of Btk HD-1. However, the parasitoid offsprings developed slowly and pupal as well as adult period, adult weight and adult emergence rate were reduced significantly if the parasitoid was developing inside a severely Bt intoxicated host larvae. There were no evident differences in longevity of parasitoid adults that were fed on honey solution containing different concentrations of Btk HD-1 as compared to adults fed only on honey solution. This indicates no direct adverse effect of Btk HD-1 on C. chlorideae. Further, the gravid female parasitoid did not discriminate Btk HD-1 intoxicated and normal H. armigera larvae for oviposition. The result implies that spore crystal formulation of Btk HD-1 can be effectively used in a synergistic manner along with existing natural or prereleased population of C. chlorideae in organic farming or as components in biointensive IPM module for managing H. armigera.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

<Emphasis Type="Italic">In vivo</Emphasis> fluorescence observation of parasporal inclusion formation in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A recombinant gene expressing a Cry1Ac-GFP fusion protein with a molecular mass of approximately 160 kD was constructed to investigate the expression of cry1Ac, the localization of its gene product Cry1Ac, and its role in crystal development in Bacillus thuringiensis. The cry1Ac-gfp fusion gene under the control of the cry1Ac promoter was cloned into the plasmid pHT304, and this construct was designated pHTcry1Ac-gfp. pHTcry1Ac-gfp was transformed into the crystal-negative strain, HD-73 cry⁻, and the resulting strain was named HD-73⁻(pHTcry1Ac-gfp). The gfp gene was then inserted into the large HD-73 endogenous plasmid pHT73 and fused with the 3′ terminal of the cry1Ac gene by homologous recombination, yielding HD-73Φ(cry1Ac-gfp)3534. Laser confocal microscopy and Western blot analyses showed for the first time that the Cry1Ac-GFP fusion proteins in both HD-73⁻(pHTcry1Ac-gfp) and HD-73Φ(cry1Ac-gfp)3534 were produced during asymmetric septum formation. Surprisingly, the Cry1Ac-GFP fusion protein showed polarity and was located near the septa in both strains. There was no significant difference between Cry1Ac-GFP and Cry1Ac in their toxicity to Plutella xylostella larvae.     

The combined use of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Nesidiocoris tenuis</Emphasis> against the tomato borer <Emphasis Type="Italic">Tuta absoluta</Emphasis>

Since Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) was first detected at the end of 2006 in the Mediterranean Basin, several endemic natural enemies have been reported to prey on this exotic pest. The predator Nesidiocoris tenuis Reuter (Hemiptera: Miridae) can regulate T. absoluta populations, because it is able to prey efficiently on T. absoluta eggs. Furthermore, previous studies have demonstrated that first-instar larvae of T. absoluta are highly susceptible to Bacillus thuringiensis (Bt) treatments. In this work, we tested the combination of both approaches under greenhouse conditions. B. thuringiensis formulations were sprayed weekly for two months, three months or throughout the growing cycle, and in all cases, one N. tenuis per plant was also released. Control plants were completely destroyed by the infestation levels reached by T. absoluta. In contrast, all treatments based on B. thuringiensis treatments and releases of N. tenuis reduced leaf damage by more than 97% when compared to the untreated control, with no significant differences among them. Furthermore, yield in the control plants was significantly reduced when compared with all Bt–N. tenuis treatments. Our results demonstrate that when B. thuringiensis treatments are applied immediately after the initial detection of T. absoluta on plants, they do not interfere with N. tenuis establishment in the crop because T. absoluta eggs are available. According to our data, treatments with B. thuringiensis later in the growing season would no longer be necessary because mirids alone would control the pest.     

Postembryonic development of oribatid mites <Emphasis Type="Italic">Cepheus cepheiformis</Emphasis> and <Emphasis Type="Italic">Conchogneta traegardhi</Emphasis> (Acari,Oribatida)

The morphology of all the juvenile stages of Cepheus cepheiformis and Conchogneta traegardhi is described. The structure of the gnathosoma, ovipositor, and legs is studied in adults of both species. Developmental rates of C. traegardhi in the laboratory conditions are established.     

Efficient Transformation of <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> by Electroporation and Conjugation with <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The conjugative self-transmissible plasmid pHT73, harbored in Bacillus thuringiensis var. kurstaki, was demonstrated to be transferred to Cellulomonas flavigena, a cellulolytic bacterium. Both conjugation and transformation procedures yielded resistant colonies; however, chromosomal integration was observed only when bacterial conjugation occurred. The efficiency of conjugation was 10% of recipient strain, which is considered a very efficient process. When the plasmid pHT73 was introduced by transformation, erythromycin-resistant cells contained the plasmid as an episome with no arrangements, as assayed by Southern blot analysis. In contrast, conjugated-resistant cells harbor the plasmid integrated into the chromosome. These data suggest a common mechanism of cell communication between nonrelated bacterial species with similar ecological habitats, and also that both electroporation and conjugation can be used to transform C. flavigena efficiently.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

Characterization of the <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Isolates Virulent Against Rice Leaf Folder, <Emphasis Type="Italic">Cnaphalocrocis medinalis</Emphasis> (Guenee)

Soil samples were collected from different rice fields of Singur, Hooghly, West Bengal, India. Spore forming bacteria were isolated from the soil samples and among them, two isolates (BUSNC25 and BUSNC26) were larvicidal against third, fourth and fifth instar larvae of rice leaf folder, Cnaphalocrocis medinalis. The phenotypic, biochemical characterization and 16S rDNA analysis of the two isolates were done. On the basis of phenotypic, biochemical and phylogenetic analysis, the selected bacterial isolates (BUSNC25 and BUSNC26) were identified as Bacillus thuringiensis. The antibiotic sensitivity tests of these two isolates against selected doses of some standard antibiotics were done. Against the 3rd, 4th and 5th instar larvae of C. medinalis, the LC₅₀ values of BUSNC25 were 2.45 × 10⁴, 1.325 × 10⁴ and 2.35 × 10⁴ cfu/ml and of BUSNC26 were 3.375 × 10⁴, 1.9 × 10⁴ and 3.325 × 10⁴ cfu/ml, respectively.