Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD₆₀₀ of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.     

Genetics of resistance to Cry1C toxin from Bacillus thuringiensis in Spodoptera litura (Fab.)

The present study was undertaken to determine the genetics of Cry1C resistance in Spodoptera litura. Selection of S. litura (Fab.) with Cry1C was done for eight generations to develop resistance. Reciprocal crosses between resistant and susceptible populations were made to understand the population genetics of Cry1C resistance in S. litura. Generation wise selection with Cry1C was evaluated for resistance development in S. litura. The LC₅₀ of Cry1C was 0.14 µg/cm² for the first selected generation and it increased to 23.98 µg/cm² after eight selected generations, which is a 285.47-fold increase in resistance compared with the susceptible strain. The estimated realized heritability (h²) after eight generations of selection with Cry1C insecticidal protein was 0.44. The number of generations required for the tenfold increase in LC₅₀ (1/R) was estimated to be 3.33. Response to Cry1C selection in S. litura was 0.30, the estimated selection differential was 0.69 and the pheonotypic standard deviation (dP) was 0.24. Reciprocal crosses between Cry1C resistant and susceptible strain of S. litura showed autosomal resistance.     

Expression of chitinase A (chiA) gene from a local isolate of Serratia marcescens in Coleoptera-specific Bacillus thuringiensis

Aims:  The present study focused on cloning and expression of chiA gene from a highly chitinolytic local isolate of Serratia marcescens in an anti-Coleopteran Bacillus thuringiensis and comparison of the characteristics of the native and recombinant ChiAs.
Methods and Results:  chiA gene from Ser . marcescens was cloned, sequenced and compared with the previously cloned chiA genes. chiA gene was PCR cloned and expressed in anti-Coleopteran B. thuringiensis strain 3023 as verified by Western blot analysis. Specific ChiA activity of the recombinant B. thuringiensis (strain 3023-SCHI) reached its highest level at 21st hour of growth (16·93 U mg⁻¹), which was 5·2- and 1·3-fold higher than that of its parental strain and Ser . marcescens , respectively. Temperature and pH effects on native and recombinant ChiAs were next determined. The recombinant plasmid was quite stable over 240 generations.
Conclusions:  Serratia marcescens ChiA was heterologously expressed in an anti-Coleopteran B. thuringiensis at levels even higher than that produced by the source organism.
Significance and Impact of the Study:  Bacillus thuringiensis 3023-SCHI co-expressing anti-Coleopteran Cry3Aa protein and Ser . marcescens chitinase offers a viable alternative to the use of chitinolytic microbes/enzymes in combination with entamopathogenic bacteria for an increased potency because of synergistic interaction between them.     

Assessment of the effect of biosurfactant produced by Pseudomonas aeruginosa in lethality of Bacillus thuringiensis Berl. against 3rd instars larvae of white cabbage butterfly (Pieris brassicae L.)

Surfactant that is produced from cheap sources like oil sludge by biological agents such as bacteria can be used in various industrial processes. For example, it can be used in environmental processes such as bioremediation and elimination of environmental pollutants, and acts as synergistic agents and distributor pesticides on waxy leaves in agriculture. In this study, biosurfactant which is produced by Pseudomonas aeruginosa (isolated from petroleum sludge) at the intervals of 24, 48, 72 and 96?h, along with chemical surfactant Tween 80 and the biological control agent, Bacillus thuringiensis, in a pilot project for controlling one important cabbage pest (Pieris brassicae), their synergistic properties were evaluated. Statistical analysis of the results showed that B. thuringiensis in combination with biosurfactant produced at different times and B. thuringiensis in combination with chemical surfactant Tween 80 when compared with control treatments like B. thuringiensis alone and B. thuringiensis plus tween 80 as positive controls and distilled water as negative control have significant differences (p?<?0.05). This research showed that surfactant treatment produced at the intervals of 24 and 48?h in combination with B. thuringiensis has the greatest synergistic effect when compared to chemical surfactant treatment. This study concluded that biosurfactant can be used as a distributor and synergistic agent against plant pests and in addition to this, their biological roles in bioremediation can be used as a viable alternative to non-economical chemical surfactants that annually enter millions of tonnes of harmful chemical substances into the fields and underground water.     

晶胞粘连苏云金芽胞杆菌C15杀线虫毒力因子发掘与功能鉴定

苏云金芽胞杆菌（Bacillus thuringiensis,Bt）制剂作为一种高效的微生物杀虫剂,在植物病虫害防控领域有着广泛的应用。Bt制剂的主效成分为杀虫晶体和芽胞,其中,杀虫晶体的环境低持久性是Bt农药应用的重要限制因素之一。自然界中存在着一些Bt菌株,其产生的杀虫晶体位于芽胞外壁和芽胞衣之间,这种特殊的表型被称为晶胞粘连（spore-crystal association, SCA）表型。由于芽胞外壁对晶体的保护作用,SCA表型可以提升晶体抵抗不良环境因素的能力,是开发新型Bt生物囊杀虫剂的有效育种策略。本文选取对线虫具有强毒杀能力的 SCA菌株C15作为研究对象。获得了C15菌株的完整基因组序列,包括一个5 637 049 bp的环状染色体和8个不同大小的环形质粒（240 314 bp到3 188 bp）。C15基因编码了5个杀虫蛋白（Cry蛋白）基因：cry21-99、cry21-67、cry21-66、cry21-46和cry-N。在Bt无晶体突变株BMB171中异源表达cry21-99基因,发现其表达产物形成菱形晶体,且对秀丽隐杆线虫（Caenorhabditis elegans）和南方根结线虫（Meloidogyne incognita）均有毒杀活性。同时,还在全基因组范围内预测了Cry毒素以外的杀线虫毒力因子和次生代谢产物。此外,在C15基因组中预测了本团队已报道的苏云金芽胞杆菌幕虫亚种（B. thuringiensis serovar finitimus）菌株YBT-020 SCA表型决定因子的同源基因,缺失后突变体仍然保留稳定的SCA表型,说明C15菌株的SCA表型形成机制与YBT-020不同,该菌株代表了一种新的SCA表型形成机制。本研究为转基因作物防控线虫提供了新的遗传资源,也为研究SCA表型形成机制,开发新型高效Bt制剂提供了新线索。     

Biological control of pre-harvest aflatoxin contamination in groundnut (Arachis hypogaea L.) with Bacillus subtilis G1

The antagonistic activity of Bacillus subtilis strain G1 was tested against various isolates of Aspergillus flavus in vitro. A talc-based powder formulation of B. subtilis strain G1 was prepared and evaluated to control A. flavus infection and aflatoxin B1 contamination in groundnut under greenhouse and field conditions. The results showed that B. subtilis strain G1 could inhibit the growth of all isolates of A. flavus tested in dual culture assay and the growth inhibition ranged from 93 to 100%. Results of greenhouse and field experiments indicated that B. subtilis strain G1 when applied to groundnut as seed treatment and soil application significantly suppressed A. flavus population in the soil, A. flavus infection and aflatoxin B1 content in kernels and increased the pod yield. These studies show that B. subtilis strain G1 has potential as a biocontrol agent for control of aflatoxin contamination in groundnut.     

Comparative bioassay of different isolates of Bacillus thuringiensis subsp. kurstaki on the third larval instars of diamondback moth,Plutella xylostella (L.) (Lep.: Plutellidae)

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most important pests of cruciferous plants throughout the world. In recent years, this insect has been a serious pest for cabbage fields in Tehran province. Resistance of P. xylostella to all main groups of insecticides has been recorded and it is ranked in the 20 most resistant pest species reported up to now. According to many researchers, to eliminate the problem of pest resistance to chemical pesticides, an integrated pest management programme should be used. In line with this, the uses of microbial control agents (MCAs) are discussed. The bacterium, Bacillus thuringiensis (Bt) is one of microbial control agents of pests. It is characterised by its ability to produce proteic crystalline inclusions during sporulation. Cry1 protein has insecticidal activity and is highly specific to certain insects and not toxic to unrelated insects, plants or vertebrates. In this work, the pathogenicity of some Bt isolates, including Dipel, 20, 29, 79 and 87, was tested against P. xylostella and the lethal concentrations (LC₅₀) of their crystal proteins to P. xylostella third larval instar was determined. The experiment was designed in factorial in randomised complete design with 5 treatments (different concentrations including 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ CFU/ml and 5 replications and with 10 third larval instars. Spore–crystal complex was applied to the surface of natural diets (cabbage leaves) and the mortality of P. xylostella larvae was assessed 120?h after exposure of Bt toxin in each treatment. Results showed that percentage of survival was significantly higher for control treatment. Results also showed that after 5?days, LC₅₀ for isolates of Dipel, 20, 29, 79 and 87 were equal to 1?×?10⁶, 1?×?10⁵, 5?×?10⁵, 4?×?10⁵ and 1?×?10⁴ CFU/ml, respectively. LT₅₀ were equal to 93.71, 48.04, 71, 40.49 and 75.28?h. Of and most the percentage larval mortality relate to attendance 87 and also at least percentage mortality is related to the groom Dipel.     

Control of Lepidopteran insect pests in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) transformed with a synthetic Bacillus thuringiensis cry1C gene

 A synthetic Bacillus thuringiensis cry1C gene was transferred to three Korean cultivars of Chinese cabbage via Agrobacterium tumefaciens-mediated transformation of hypocotyl explants. Hygromycin resistance served as an efficient selective marker. The transformation efficiency ranged from 5% to 9%. Transformation was confirmed by Southern blot analysis, PCR, Northern analysis, and progeny tests. Many transgenic plants of the closed-head types (lines Olympic and Samjin) flowered in vitro. Over 50 hygromycin-resistant plants were successfully transferred to soil. The transgenic plants and their progeny were resistant to diamondback moths (DBM, Plutella xylostella), the major insect pest of crucifers world-wide, as well as to cabbage loopers (Trichoplusia ni) and imported cabbage worms (Pieris rapae). Both susceptible Geneva DBM and a DBM population resistant to Cry1A protein were controlled by the Cry1C-transgenic plants. The efficient and reproducible transformation system described may be useful for the transfer of other agriculturally important genes into Chinese cabbage. Received: 12 June 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

The effect on Orchestia hurleyi (Amphipoda: Talitridae) of a whitey disease caused by Bacillus subtilis

Abstract

Field observations made over 10 years suggested that a bacterial disease of adults of the terrestrial amphipod Orchestia hurleyi Duncan, caused by Bacillus subtilis, is progressing southwards down the eastern side of New Zealand's South Island. As the disease spread, amphipod density appeared to decline and population age structure became truncated. In the vicinity of Dunedin and further south the amphipods are still disease-free. Signs of the disease are a progressive weakening and wasting. The animal cannot jump, and its speed of walking is reduced. Its body becomes opaque white instead of the normal translucent reddish-brown. Diseased females do not brood. There is no evidence that diseased animals moult. Death is caused by general wasting or by predators. The disease-causing organism was isolated, and healthy amphipods were re-infected from the isolate. Signs of the disease were apparent within 7 days of inoculation. The presence of the disease-causing organism in the haemocoel causes host defences to be mobilised, as shown by elevated haemocyte counts (4512 mm^?3, cf. 300 mm^?3 in healthy, disease-free adults), but as the disease progresses the animal's defences are overcome, and haemocyte counts fall to an average of 784 mm^?3 during the later stages of disease. The blood of terminally diseased amphipods is thick and creamy white, packed with motile bacterial cells, and few (if any) haemocytes are present in the circulation. Two populations were studied, one disease-free (at Dunedin) and the other heavily diseased (at Christchurch). The incidence of disease (as measured by a performance test) was about 30%r in Christchurch adults. The disease-causing strain of B. subtilis was found on the body surface of almost all adults in the diseased population. It is possible that the bacterium gains entry to the haemocoel through wounds suffered during ecdysis, conflict, or predator attack. The main differences shown by the diseased population relative to the disease-free population were: lower average density (992 m^?2, cf. 1677 m^?2); lower maximum density (3104 m^?2, cf. 9971 m^?2); smaller average size, with fewer adult instars; a smaller proportion of females brooding in each instar; and much lower egg production. The brood size/mother age relationship was the same for both populations — number of eggs in brood = -4.9 + 0.64(instar number of mother)—because in the diseased population only healthy females breed. Lower egg production in the diseased population reflects the smaller proportion of healthy females, and the number of broods per female is lower since life expectancy is much less. A computer model based on Leslie matrices was used to simulate the ecological effects of the disease. It gave predictions which conformed with the observed population features with respect to age structure and density.     

Evaluation of Streptomyces spp. and Bacillus spp. for biocontrol of Fusarium wilt in chickpea (Cicer arietinum L.)

A study was carried out to test direct and indirect antagonistic effect against Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (FOC), and plant growth-promoting (PGP) traits of bacteria isolated from rhizosphere soils of chickpea (Cicer arietinum L.). A total of 40 bacterial isolates were tested for their antagonistic activity against FOC and of which 10 were found to have strong antagonistic potential. These were found to be Streptomyces spp. (five isolates) and Bacillus spp. (five isolates) in the morphological and biochemical characterisation and 16S rDNA analysis. Under both greenhouse and wilt sick field conditions, the selected Streptomyces and Bacillus isolates reduced disease incidence and delayed expression of symptoms of disease, over the non-inoculated control. The PGP ability of the isolates such as nodule number, nodule weight, shoot weight, root weight, grain yield and stover yield were also demonstrated under greenhouse and field conditions over the non-inoculated control. Among the ten isolates, Streptomyces sp. AC-19 and Bacillus sp. BS-20 were found to have more potential for biocontrol of FOC and PGP in chickpea. This investigation indicates that the selected Streptomyces and Bacillus isolates have the potential to control Fusarium wilt disease and to promote plant growth in chickpea.     

The potential of antagonistic moroccan Streptomyces isolates for the biological control of damping‐off disease of pea (Pisum sativum L.) caused by Aphanomyces euteiches

Three hundred and fifty‐nine isolates of actinobacteria collected from different Moroccan soils were evaluated for their in vitro antimicrobial activity against the oomycete pathogen Aphanomyces euteiches, the causal agent of damping‐off of pea and other legumes. Eighty‐seven isolates (24%) had an inhibitory in vitro effect against A. euteiches. Fourteen bioactive isolates with the greatest inhibitory effect against A. euteiches and no inhibitory effect on plant beneficial rhizobia were tested for their ability to protect pea seeds and seedlings against the damping‐off disease using culture supernatants or spore suspensions as treatments. The two most protective isolates, OB21 and BA15, significantly reduced, compared to untreated control plants, damping‐off by 33% and 47%, respectively. The two bioactive isolates were classified as species of the genus Streptomyces based on 16S rDNA analysis and morphological and chemical characteristics.     

Microbiological differences between limed and unlimed soils and their relationship with cavity spot disease of carrots (Daucus carota L.) caused by Pythium coloratum in Western Australia

Application of lime (4000 kg ha^-1) to a soil used for commercial carrot production (pH 6.9) significantly (p<0.05) reduced the incidence of cavity spot disease of carrots compared to unlimed soil (pH 5.1). It significantly (p<0.01) increased soil microbial activity as measured by the hydrolysis of fluorescein diacetate and arginine ammonification. The application of lime resulted in a significant (p<0.01) increase in the total numbers of colony forming units (efu) of aerobic bacteria, fluorescent pseudomonads, Gram negative bacteria, actinomycetes and a significant (p<0.01) decrease in the cfu of filamentous fungi and yeasts compared to unlimed soil. Liming also increased the cfu of non-streptomycete actinomycetes rarely reported in similar studies. These non-streptomycete actinomycetes were estimated and isolated using polyvalent Streptomyces phages and the dry heat technique to reduce the dominance of streptomycetes on isolation plates. The non-streptomycete actinomycetes isolated included species of Actinoplanes, Micromonospora, Streptoverticillium, Nocardia, Rhodococcus, Microbispora, Actinomadura, Dactylosporangium and Streptosporangium. The numbers of actinomycetes antagonistic to Pythium coloratum, a causal agent of cavity spot disease of carrots increased in soil amended with lime. Application of lime also reduced the isolation frequency of P. coloratum from asymptomatic carrot roots grown in soil artificially infested with the pathogen, 3, 4 and 5 weeks after sowing.     

Identification of powdery mildew (Erysiphe graminis sp. tritici) and take-all disease (Gaeumannomyces graminis sp. tritici) in wheat (Triticum aestivum L.) by means of leaf reflectance measurements

The ability to identify diseases in an early infection stage and to accurately quantify the severity of infection is crucial in plant disease assessment and management. A greenhouse study was conducted to assess changes in leaf spectral reflectance of wheat plants during infection by powdery mildew and take-all disease to evaluate leaf reflectance measurements as a tool to identify and quantify disease severity and to discriminate between different diseases. Wheat plants were inoculated under controlled conditions in different intensities either with powdery mildew or take-all. Leaf reflectance was measured with a digital imager (Leica S1 Pro, Leica, Germany) under controlled light conditions in various wavelength ranges covering the visible and the near-infrared spectra (380–1300 nm). Leaf scans were evaluated by means of L*a*b*-color system. Visual estimates of disease severity were made for each of the epidemics daily from the onset of visible symptoms to maximum disease severity. Reflectance within the ranges of 490₇₈₀ nm (r² = 0.69), 510₇₈₀nm (r² = 0.74), 516₁₃₀₀nm (r² = 0.62) and 540₁₃₀₀ nm (r² = 0.60) exhibited the strongest relationship with infection levels of both powdery mildew and take-all disease. Among the evaluated spectra the range of 490₇₈₀nm showed most sensitive response to damage caused by powdery mildew and take-all infestation. The results of this study indicated that disease detection and discrimination by means of reflectance measurements may be realized by the use of specific wavelength ranges. Further studies have to be carried out, to discriminate powdery mildew and take-all infection from other plant stress factors in order to develop suitable decision support systems for site-specific fungicide application.